Document 1150902

Luminal-derived stimuli as triggers of
irritable bowel syndrome-like states in the rat:
role of neurotrophic factors
by
Ferran Jardí Pujol
A dissertation submitted in partial fulfilment of the requirements
for the degree of Doctor of Philosophy
Doctoral Programme in Pharmacology
Department of Cell Biology, Physiology and Immunology
Universitat Autònoma de Barcelona
Advisors:
Patrocinio Vergara Esteras
Vicente Martínez Perea
Veterinary School
Bellaterra, Barcelona
2014
Patrocinio Vergara Esteras,
Professor of Physiology; Department of Cell Biology, Physiology and Immunology;
Veterinary School; Universitat Autònoma de Barcelona,
and
Vicente Martínez Perea,
Associate Professor of Physiology; Department of Cell Biology, Physiology and
Immunology; Veterinary School; Universitat Autònoma de Barcelona,
We hereby certify that the thesis entitled: “Luminal-derived stimuli as triggers of
irritable bowel syndrome-like states in the rat: role of neurotrophic factors” submitted by
FERRAN JARDÍ PUJOL in partial fulfilment of the requirements for the degree of
Doctor of Philosophy was carried out under our supervision and we authorise its
submission for oral defence.
Bellaterra, Barcelona, March 2014
Dr. Patrocinio Vergara Esteras
Dr. Vicente Martínez Perea
Ph.D. Advisor
Ph.D. Advisor
Acknowledgements
En primer lloc, m’agradaria donar les gràcies als meus directors de tesi. A la Dra. Patri
Vergara, per introduir-me al món de la investigació, per confiar plenament en el meu
criteri i les meves capacitats i per l’excel·lent tracte humà que sempre m’ha donat. Al
Dr. Vicente Martínez, per estar amb mi al laboratori en les primeres etapes del doctorat,
per trobar sempre la manera més idònia d’encaminar la investigació i per la seva
inestimable ajuda a l’hora d’escriure.
També vull esmentar l’ajuda rebuda per la resta d’investigadors principals de la Unitat
de Fisiologia Animal de la Facultat de Veterinària: la Dra. Ester Fernández, el Dr.
Marcel Jiménez i la Dra. Maite Martín.
Gràcies a tots els doctorands i residents que em van rebre quan vaig arribar al
departament, bona part del quals ja no hi són. Al meu estimat amic Burguer, per ser
immensament generós tant dins com fora del laboratori, sempre disposat a donar-te un
cop de mà si insisteixes una mica. Per lo bé i malament que ho hem passat junts, pels
berenars que m’ha patrocinat i per ser el “fan” principal de la meva vessant creativa. Al
“crack” Joan Antoni Fernández, per ensenyar-me tots el secrets del paràsit mil·lenari i
per la seva àmplia visió de la vida que tant m’ha ajudat en èpoques en què els resultats
del laboratori no han estat favorables. A la Sandra, per ser una bona companya de feina i
viatge, gràcies pels bons moments que vam compartir a l’Índia. Al Paco, per ser,
bàsicament, un “bon jan”. A l’Esther Jorge, per tenir una empenta contagiosa. A
l’Estefania, per aguantar amb bon humor i “savoir-faire” tota la conya que li hem fet
amb el Burguer. A la Carol i la Glòria, per aportar vida al departament participant en tots
els esdeveniments. Al Javier Benito, per lo disposat que sempre estava a “pringar”. A la
Lina, per ser un clar exemple de superació personal. A la Diana, per la tranquil·litat que
es respira al seu voltant. A la meva tècnica preferida, la pantera, per ser una especialista
del bany d’òrgans i portar la festa i la conya per allà on passa. Al Víctor, per la seva
senzillesa i el seu particular sentit de l’humor. A la Bego, l’Álvaro, la Clàudia i la resta
de companys de l’Hospital de Mataró, per l’aire nou que suposen per al departament.
De la mateixa manera, no puc oblidar-me de donar les gràcies a tots els companys/es que
es van incorporar després de mi. A la Tapir, per ensenyar-me la importància de “tenir
salsa”, per l’alta estima que em professa, per la seva paciència i picaresca i per lo que
riem quan estem plegats. A la meva “alumna” Sepi, per no queixar-se mai de patir el
meu mal humor i per seguir-nos sempre la conya a mi i al Burguer. A la Mònica, per
participar en el segon capítol de la tesi. A les meves companyes de despatx: a la Míriam
i l’Assun, per suportar els meus moments més delirants, i a l’Elena per la seva bondat. A
la Noe, per ser un sol, per no alterar-se “quasi” mai i per descobrir-me la Francis. Als
meus companys de grup: al senyor Estévez, per ser un exemple d’eficàcia i estar receptiu
a la broma; al Sergio, per seguir el relleu de les pràctiques del curs d’animals, i a la
Marina i al Jakub per adaptar-se tan ràpid al grup.
Vull agrair la feina de totes les persones de la UAB que m’han ajudat en qüestions
tècniques i administratives. A l’Antonio, gràcies per estar amb mi cada matí quan
sondava els animals durant els primers experiments i per no enfadar-se quan li repetia les
coses varies vegades. A David, per preocupar-se de que els becaris no ens quedéssim
sense cobrar. Al Pepe, per entretenir el departament amb els seus mil-i-un canvis de
“look”. A la Núria i la Mar de l’Institut de Neurociències, per la seva amabilitat i
disposició a ajudar-me. A les tècnics d’Anatomia Patològica de la Facultat de
Veterinària, per revelar-me els secrets del desenmascarament antigènic.
Gràcies al nostres veïns de Farmacologia: a la Dra. Rosa Torres, per tots els favors que
m’ha fet durant aquest període de formació. I al Dr. Fernando de Mora, per acceptar
ràpidament ser el meu tutor de tesina.
Special thanks to Dr. Gebhart and his research group in the Pittsburgh Center for Pain
Research, for their guidance and support. I would also like to thank Adora and Steve for
hosting me at their home in Pittsburgh and making my stay unforgettable.
També m’agradaria agrair el tribunal d’aquesta tesi per acceptar la invitació i l’esforç
que suposa.
Vull agrair el finançament que ha fet possible la realització d’aquest treball: el projecte
2009SGR708 de la Generalitat de Catalunya i els projectes BFU2007-6279, BFU200908229 i BFU2010-15401 del Ministerio de Ciencia e Innovación.
Per últim, gràcies a la meva família, per haver-ho donat tot per mi i pel seu suport
incondicional.
“Above all, don’t fear difficult moments. The best comes from them”
– Rita Levi-Montalcini
Italian neurologist who won the Nobel Prize in
1986 for the discovery of nerve growth factor
INDEX
ABBREVIATIONS
3
SUMMARY
7
RESUMEN
8
INTRODUCTION
11
Functional Organization of the Colon
11
Innervation of the Colon
12
Colonic Motility
14
The Intestinal Immune System
15
Functional Gastrointestinal Disorders: Irritable Bowel Syndrome
15
Animal Models of Irritable Bowel Syndrome
17
Mast Cells
19
Neurotrophic Factors
21
HYPOTHESIS AND OBJECTIVES
27
CHAPTER 1 NGF is involved in oral ovalbumin-induced altered colonic
contractility in rats: evidence from the blockade of TrkA receptors
with K252a
31
CHAPTER 2 Colonic motor alterations associated to a luminal antigen are
enhanced during a dysbiotic-like state in rats
63
CHAPTER 3 Plasticity of dorsal root ganglions neurons in a rat model of postinfectious gut dysfunction: potential implication of nerve growth
factor
93
CHAPTER 4 Persistent alterations in colonic afferent innervation in a rat model
of post-infectious gut dysfunction: role for changes in peripheral
neurotrophic factors
117
DISCUSSION
147
CONCLUSIONS
157
CONCLUSIONES
158
REFERENCES
163
APPENDIX
Publications derived from this work
179
Biosketch
193
ABBREVIATIONS
ABBREVIATIONS
ABBREVIATIONS
Actb: B-Actin
AUC: Area under the curve
BDNF: Brain-derived neurotrophic factor
BSA: Bovine serum albumin
CCh: Carbachol
CGRP: Calcitonin gene-related peptide
CPA: Metalloprotease carboxypeptidase A
Ct: Threshold cycle
CTMC: Connective tissue mast cell
Cy3: Carbocyanine 3
DRG: Dorsal root ganglion
EMS: Electrical mucosal stimulation
ENS: Enteric nervous system
FGD: Functional gastrointestinal disorder
FISH: Fluorescence in situ hybridization
GCM: Gut commensal microbiota
GDNF: Glial cell line-derived neurotrophic factor
GFRα: GDNF family receptor α
GMC: Giant migrating contraction
HFLA: High-frequency and low-amplitude contraction
IBD: Inflammatory bowel disease
IBS: Irritable bowel syndrome
IFN: Interferon
IgE: Type E immunoglobulin
IL: Interleukin
iNOS: Inducible nitric oxide synthase
LFHA: Low-frequency and high-amplitude contraction
L-NNA: NG-nitro-L-arginine
LPS: Lipopolysaccharides
LS: Lumbosacral
MC: Mast cell
MMC: Mucosal mast cell
NGF: Nerve growth factor
3
ABBREVIATIONS
NO: Nitric oxide
NT-3: Neurotrophin-3
NT-4/5: Neurotrophin-4/5
NTF: Neurotrophic factor
OVA: Ovalbumin
PAR-2: Protease-activated receptor-2
PI: Post-infection
PI-IBS: Post-infectious-irritable bowel syndrome
RELM: Resistin-like molecule
RMCP: Rat mast cell protease
RMCPII: Rat mast cell protease II
RMCPVI: Rat mast cell protease VI
RPC: Rhythmic phasic contraction
RT-qPCR: Reverse transcription-quantitative polymerase chain reaction
TC: Tonic contraction
TL: Thoracolumbar
Trk: Tropomyosin-receptor-kinase
TrkA: Tropomyosin-receptor-kinase A
TrkB: Tropomyosin-receptor-kinase B
TRPV1: Transient receptor potential vanilloid channel-1
4
SUMMARY
SUMMARY
In the gastrointestinal tract, luminal-derived stimuli have been implicated in the pathophysiology
of irritable b owel syndrome (IBS). A lthough t he pa thways through w hich l uminal factors
contribute to th e o nset/maintenance of IBS -like sy mptoms remain u nclear, a r ole for a
neuroimmune circuitry i mplicating mast cells (M Cs) and neurotrophic f actors (N TFs) has b een
suggested. This work a ims to c haracterize th e a bility o f lu minal-derived stim uli, in cluding gu t
commensal microbiota (GCM), in testinal infections and f ood a ntigens, to tri gger IBS -like
alterations in the colon of the rat, with special emphasis on the potential role of NTFs. For this, we
used tw o a ccepted models o f mucosal MC (M MC)-dependent intestinal dysfunction in ra ts:
chronic exposure to oral ovalbumin (OVA) and infection with Trichinella spiralis. Both, exposure
to o ral OVA for a 6-week p eriod and infection w ith T. sp iralis activated MMCs of t he colon,
although cell r ecruitment was only obs erved dur ing T. sp iralis infection. A 6 -week, but not 1 week, e xposure to oral OVA resulted in a colonic contractile dysfunction, observed both in vivo
and in vitro. Nevertheless, short (1-week) OVA exposure led to similar colonic motor alterations
when combined with a dysbiotic-like state (treatment with bacterial lipopolysaccharides), thereby
indicating a facilitatory interaction between microbiota and food-derived antigens. Treatment with
K252a, a n a ntagonist of tropomyosin-receptor-kinase (Trk) receptors, decreased spontaneous
colonic m otility and prevented p art of the motor a lterations associated to OV A exposure, thus
suggesting that the n eurotrophins-Trk receptors axis participates in t he maintenance o f basal
contractility in the rat colon and in the contractile dysfunction associated to exposure to oral OVA.
In the presence of an infectious-related luminal-derived stimulus (T. spiralis infection), changes in
thoracolumbar dorsal root ganglia (DRG) neurons’ morphology and NTFs content was observed,
pointing t o an extended afferent remodeling th rough th e g astrointestinal tract. Moreover, in
infected animals, altered responses of DRGs (down-regulation of TRPV1 receptors) after colonic
afferent stimulation with capsaicin indicated a persistent change in afferent sensitivity. Alterations
in afferent functionality during T. spiralis infection coincided with time- (early vs. late phases) and
site-specific (j ejunum vs. c olon) c hanges in NT Fs expression. Within th e jeju num, a n ear ly
increase in nerve growth f actor (NGF), g lial cell li ne-derived NT F and a rtemin levels was
observed. In the colon, however, a general down-regulation of NTFs was observed between days
6-14 post-infection. T hese re gional differences might be re lated to th e l ocal degree o f
inflammation; since, c ompared t o t he j ejunum, t he c olon s howed a n a ttenuated inflammatory
response. Within the colon, NGF was located in the epithelium and the enteric nervous system, but
not in MMCs. Nevertheless, MMCs expressed TrkA and treatment with K252a tended to increase
the levels of rat mast cell protease II, thus indicating that colonic MMCs are a target for NGF. In
summary, this work shows that the axis luminal-derived stimuli-MMCs-NTFs is implicated in the
colonic f unctional alterations o bserved in food a ntigen- and i nfection-related models o f IBS in
rats. These results highlight a p otentially im portant role for NT F-related p athways in th e
onset/maintenance of the symptomatology in, at least, a subset of IBS patients in which the disease
is related to luminal dietary- or infectious-related factors. NTFs represent a therapeutic target for
the trea tment of g astrointestinal disorders characterized b y a ltered c olonic sensitivity a nd
dysmotility.
7
RESUMEN
En el tracto gastrointestinal, los estímulos de origen luminal se han implicado en la patofisiología
del síndrome d el intestino irri table (IBS). Aunque l os mecanismos por los cuales contribuyen al
inicio/mantenimiento d e la sintomatología permanecen p oco d efinidos, se h a propuesto la
implicación de un circuito neuroinmune dependiente de mastocitos (MCs) y factores neurotróficos
(NTFs). Este trabajo p rofundiza en l a cap acidad d e los estímulos lu minales, in cluyendo factores
relacionados con la microbiota comensal, las infecciones intestinales y los antígenos alimentarios,
para producir alteraciones de tipo IBS en el colon de la rata, con especial énfasis en la implicación
de lo s NTFs. Con e ste p ropósito, se ha n utilizado dos m odelos de disfunción intestinal
dependientes de m astocitos de mucosa (M MCs): la e xposición oral a o voalbúmina (OV A) y l a
infección p or Trichinella sp iralis. T anto la exposición oral a O VA d urante 6 se manas como la
infección por T. spiralis activaron los MMCs del colon, si bien el reclutamiento celular sólo tuvo
lugar durante la in fección. La e xposición a O VA dur ante 6 s emanas alteró la c ontractilidad
colónica, tanto in v ivo como in v itro, un e fecto q ue n o se o bservó e n trata mientos cortos (1
semana). Sin em bargo, la e xposición durante una semana a O VA combinada con un estado d e
disbiosis, inducido po r el tratamiento c on li popolisacáridos bacterianos, produjo al teraciones
motoras si milares, sugiriendo una in teracción f acilitadora e ntre la microbiota y lo s antígenos
alimentarios. El trata miento c on K252a, u n a ntagonista de lo s re ceptores tropomiosina-receptorquinasa, disminuyó la motilidad espontánea del colon y previno parte de las alteraciones motoras
asociadas a OVA. Esto s re sultados apuntan a que, en la rata, el eje neurotrofinas-receptores Trk
participa en e l mantenimiento d e la contractilidad basal del colon y en la disfunción c ontráctil
asociada a OV A. En p resencia de u n e stímulo lu minal de origen infeccioso ( T. spiralis), se
observaron cambios morfológicos y en el contenido de NTFs en las neuronas de los ganglios de la
raíz dorsal (DRGs) de los segmentos espinales toracolumbares, indicando una remodelación difusa
de l as v ías aferentes entéricas. Los animales in fectados presentaban, además, alteraciones en la
respuesta a la e stimulación aferente (disminución d e T RPV1 en neuronas de l os DRG s),
sugiriendo cambios p ersistentes en la se nsibilidad. Estas alteraciones coincidieron c on c ambios
temporales (fases tempranas vs. tardías) y regionales (yeyuno vs. colon) en la expresión de NTFs.
En e l yeyuno, s e obs ervó un i ncremento t emprano e n l a expresión d e f actor de cr ecimiento
nervioso (NGF), de NTF d erivado d e la línea c elular glial y de artemina. Por el contrario, en el
colon se observó un descenso general en la expresión de NTFs entre los días 6-14 post-infección.
Estas diferencias regionales podrían estar relacionadas con el grado de inflamación local ya que el
colon, comparado con e l yeyuno, m ostró una re spuesta in flamatoria atenuada. En e l colon, se
detectó la presencia de NGF en el epitelio y en el sistema nervioso entérico pero no en los MMCs.
Sin embargo, la expresión de receptores TrkA sí se localizó en MMCs. Además, el tratamiento con
K252a tendió a aumentar los niveles de la proteasa mastocitaria de rata de tipo II, sugiriendo que
los receptores TrkA mastocitarios son funcionales. Por tanto, los MMCs podrían representar una
diana p ara el NGF. Este trab ajo demuestra que el eje e stímulos lu minales-MMCs-NTFs e stá
implicado e n las a lteraciones funcionales c olónicas observadas en m odelos de IB S en la rata
relacionados c on antígenos a limentarios y procesos in fecciosos. Las v ías dependientes de NTFs
podrían desempeñar u n p apel cl ave en e l inicio/mantenimiento de l a sintomatología del IBS , al
menos en aquellos pacientes cuya enfermedad es tá r elacionada co n f actores dietéticos o
infecciosos. Los N TFs representan u na d iana tera péutica p ara e l tratamiento d e lo s desórdenes
gastrointestinales caracterizados por alteraciones sensoriales y motoras.
8
INTRODUCTION
INTRODUCTION
Functional Organization of the Colon
The large intestine is the final part of the digestive tract and can be divided in two parts:
cecum and colon. From a morphological point of view, the development of these two
parts shows high species-related variability (1). Nevertheless, cytokinetic and histologic
studies reveal many similarities between the colon of rats and humans at the
ultrastructural level (2). In both species, the colon is composed by four broad layers,
including the mucosa, the submucosa, the muscularis externa, and the serosa (or
adventitia) (3,4).
In contrast to the small intestine, the colonic mucosa has no villi although it shows
numerous invaginations of the epithelium known as crypts of Lieberkühn. It is lined by
an epithelium composed of simple columnar cells with a thin brush border (colonocytes)
and numerous interspersed goblet cells (Fig. 1). Colonocytes are the most abundant cells
of the epithelium and are implicated in the terminal digestion and absorption of water
and nutrients. Goblet cells produce components of the mucus layer, which protects and
lubricates the mucosal surface. Dispersed among these cell types, the colonic epithelium
also contains enteroendocrine cells. These are secretory cells that produce regulatory
substances such as serotonin (5-hydroxytryptamine, 5-HT), vasoactive intestinal
polypeptide (VIP), or somatostatin (Fig. 1) (3,4). Near the base of the colonic crypts,
resides a population of intestinal stem cells, which can differentiate into the mature cell
types composing the intestinal epithelium, accounting for the continuous epithelial cell
renewal within the colon (2).
Supporting the epithelium, there is a layer of reticular connective tissue, highly
vascularized and rich in immune cells, that constitutes the lamina propria. Directly
beneath the lamina propria and separating it from the submucosa, lies the muscularis
mucosae, which is a thin layer of smooth muscle that imparts fine movements to the
mucosa (3,4).
The submucosa is an irregular fibroelastic connective tissue layer that contains blood
and lymphatic vessels as well as a nerve fiber plexus known as the submucosal plexus
(Fig. 1). This plexus, which is part of the enteric nervous system (ENS), is associated to
the secretomotor control of the mucosa, including local blood flow control (5).
11
INTRODUCTION
The colonic muscularis externa, which is responsible for the motor activity of the colon,
consists of two layers of smooth muscle: an inner circular coat and an outer longitudinal
coat that, in some species, forms three separate longitudinal bands called taenia coli.
Between these two muscle layers resides the other plexus of the ENS, the myenteric
plexus, involved in the control of the activity of the smooth muscle layers (5).
Finally, depending on the region of the colon, the muscularis externa is bounded by the
serosa, a single layer of mesothelial cells together with connective tissue, or the
adventitia, which blends with the connective general tissue of the region.
Figure 1. Tissue layers of the colon and epithelial cell types. Image adapted from
Gray 2005 (6).
Innervation of the Colon
The colon is innervated by both the ENS and extrinsic projections, including
sympathetic and parasympathetic neurons as well as visceral afferents (5).
The neurons of the ENS are usually classified according to their functional properties,
thereby distinguishing between motor neurons, interneurons and primary afferent
neurons (7,8). The smooth muscle receives projections of both excitatory and inhibitory
motor neurons, terms referring to the pattern of neurotransmitters they express. For
instance, the excitatory component of the muscle innervation is predominantly due to the
12
INTRODUCTION
release of acetylcholine and tachykinins while purines (mainly ATP) and nitric oxide
(NO) are the main inhibitory transmitters (7,8). In addition, a subgroup of motor neurons
project to the mucosa and the local blood vessels and are involved in secremotor and
vasomotor control (7,8). As it refers to interneurons, their function is to contribute to the
spreading of reflexes up or down the gut, depending if they are orally (ascending
interneurons) or anally (descending interneurons) directed (8,9). Finally, within the
submucosal and myenteric plexuses, there are also intrinsic primary afferent neurons
(IPANs). These are sensory neurons that respond to chemical and mechanical stimuli
applied to the mucosa or the muscle layers, thereby initiating gut reflexes (8,9).
Figure 2. Extrinsic innervation of the human colon. Image modified from
Sleisenger 2010 (10).
The activity of the ENS is subjected to modulation by the extrinsic, autonomic
innervation (Fig. 2). Sympathetic postganglionic fibers emerge from the prevertebral
ganglia and inhibit intestinal motility by acting on the enteric ganglia (Fig.2) (11). As it
relates to the parasympathetic innervation, preganglionic fibers descend either via the
vagus nerve or the pelvic nerves and synapse on postganglionic neurons located in the
myenteric and submucosal plexuses (11) (Fig. 2). Finally, the colon also receives the
projections of extrinsic primary afferent neurons (EPANs), with their cell body located
either in the nodose (vagal afferents) or dorsal root ganglia (DRGs; spinal and sacral
afferents) (12) (Fig. 2). Vagal afferent innervation of the colon is less dense than in the
13
INTRODUCTION
upper parts of the gastrointestinal tract, with neuronal endings present only in the colonic
mucosa, muscular layers and ganglionic plexuses of the proximal part of the colon
(13,14). The exact role of these fibers in reflex control and transmission of pain
sensation is not clear. In the rat, spinal afferent innervation of the mid and distal colon
has been shown to arise mainly from thoracolumbar (TL, T13-L2) and lumbosacral (LS,
L6-S1) DRGs (15,16). These fibers are well known for carrying nociceptive information
(17).
Colonic Motility
The coordinated contractile activity of the two muscle layers of the colon is required for
the water extraction from the non-digestible waste and for defecation (18).
Colonic motor activity is orchestrated mainly by the ENS although, as mentioned above,
it is highly modulated by the extrinsic innervation. The circular muscle layer of the colon
is the main responsible for the contractility that mixes and propels the content.
Colonic smooth muscle generates three t ypes o f co ntractions: rhythmic phasic
contractions (RPCs), giant migrating contractions (GMCs) and tonic contractions (TCs).
Colonic RPCs are short-amplitude contractions, usually below 50 mmHg, that depending
on their duration can be classified into: short-duration RPCs (2-3 seconds), which occur
more frequently, a nd l ong-duration R PCs ( 15-20 s econds), less f requent and n ormally
organized in bursts (19,20). RPCs mix and propel the colonic content at relatively slow
rates so that adequate time is available for water absorption (19,20). On the other hand,
GMCs ar e characterized b y t heir lar ge amplitude and lo ng-duration, m igrating
uninterruptedly over long distances to produce mass movements and defecation (19,20).
The frequency o f th ese colonic co ntractions varies widely b etween s pecies, o ccurring
between 2-5 t imes a day i n healthy humans while in rodents t hey s how up to 45 times
per h (19). Finally, TCs decrease the luminal diameter and, although their effects alone
in mixing and propelling the content are insignificant, they potentiate the action of RPCs
in propulsion (19).
14
INTRODUCTION
The Intestinal Immune System
The intestinal mucosa is an enormous surface exposed to the external environment,
representing a major gateway for potential pathogens. Thus, its immune system needs to
be ready to respond to these threats, at the same time that must remain tolerant to food
antigens and the gut commensal microbiota (GCM) (21).
The immediate response to enteric pathogens is mediated by the activation of the innate
immune system by pattern recognition receptors (PRRs), which are found both in the
intestinal epithelium and immune cells of the lamina propria (22). This innate response
of the gut is unspecific and implies the secretion of antimicrobial molecules, the
activation of the complement system and the recruitment of phagocytic cells (22).
Additionally, the intestine harbors a large number of adaptive immune cells, including
lymphocytes T and B, which provide specificity and memory to the intestinal immune
response. Depending on the cytokine pattern expressed by T helper (Th) cells after the
activation of the adaptive immune system, these responses can be classified into Th1,
Th2 or Th17 (23).
The intestinal immune system shows a dynamic equilibrium between the ability to
respond rapidly to invading pathogens and the maintenance of tolerance to the GCM and
food antigens (24). The breakdown of this tolerance results into an exacerbated and
persistent activation of the local immune system, leading to chronic inflammatory states
of the gut (24). Thus, it has been suggested that environmental and/or genetic factors
affecting these tolerogenic mechanisms are implicated in the induction of inflammatory
and functional disorders of the gastrointestinal tract (25).
Functional Gastrointestinal Disorders: Irritable Bowel Syndrome
Functional gastrointestinal disorders (FGDs) are defined as a variable combination of
chronic or recurrent symptoms affecting different regions of the gastrointestinal tract and
that are not explained by structural or biochemical abnormalities, at least according to
the methodologies currently available (26). Irritable bowel syndrome (IBS) is the most
frequent FGD and is characterized by abdominal pain, alterations of the bowel habits
and visceral hypersensitivity, in the absence of apparent organic alterations (27). IBS is
15
INTRODUCTION
highly prevalent in industrialized countries, may affect up to 10-20% of the population,
particularly women, who are twice as likely to suffer IBS as men, and the
symptomatology appears for the first time between the ages of 30 and 50 years (28). As
there are no specific diagnostic tests, the identification of IBS patients relies on the
application of symptom-based criteria (Rome III criteria), leading to potential diagnostic
confusion (27). As a result, IBS therapeutics has a limited development. Therefore, the
process has a tremendously important economic burden on health care resources for
direct (e.g. diagnosis, therapy) and indirect (e.g. work absenteeism) costs.
Although the underlying pathophysiology of IBS remains unclear, it is accepted that the
symptomatology
observed
arises
from
a
dysregulation
of
the
bidirectional
communication between gut and brain (the so-called brain-gut axis), modulated by
various psychosocial and environmental factors (e.g. external stressors, dietary
constituents, genetics, altered GCM or intestinal infections). In addition, there is
evidence that IBS patients consistently present a low grade inflammation in the intestinal
mucosa and that IBS-like symptomatology is observed during remission phases of
inflammatory bowel disease (IBD) (29,30).
The potential role of adverse food reactions in IBS has been recently reviewed (31).
Food ingestion has been related to exacerbation of IBS symptomatology in a large
number of IBS patients, thereby making it feasible to hypothesize an implication of food
intolerance or alimentary allergy in the onset of IBS symptomatology (32). Supporting
this hypothesis, it has been demonstrated an increase in the prevalence of atopic
conditions in diarrhea-predominant IBS patients (33). Moreover, the prevalence of IBS
is higher in patients with bronchial asthma compared to patients with other pulmonary
disorders (34). Finally, several studies demonstrate usefulness of oral disodium
cromoglycate, an inhibitor of mast cell (MC) degranulation, and elimination diets in
diarrhea-predominant IBS patients, thus suggesting that part of the symptomatology
observed could be related to food allergy or food intolerance (33,35). However, the
intestinal food allergy-related mechanisms in IBS seem to involve local mucosal
responses to dietary antigens rather than classical type-1 hypersensitivity reactions, as
revealed by observations showing that IBS patients with positive results to colonoscopic
provocation with dietary allergens have low titers of specific type E immunoglobulins
(IgEs) in serum (36).
16
INTRODUCTION
Compelling data demonstrate a strong association between intestinal infections and
development of IBS-like symptomatology. Indeed, studies suggest that the probability of
developing IBS is increased about six-fold after an acute gastrointestinal infection and
remains significantly increased for up to 3 years thereafter (37). Thus, the term postinfectious-IBS (PI-IBS) has been used to refer to those patients that, after undergoing an
acute and normally self-limiting gastrointestinal infection, develop chronic IBS-like
symptomatology (38). Although the odds of developing PI-IBS have been associated to
some risk factors, such as the duration and severity of the illness and the type of
pathogen implicated, the exact mechanisms involved in the onset and perpetuation of the
symptomatology have not been elucidated (39). Referring to the pathophysiology,
increased counts of enterochromaffin cells, lymphocytes and MCs together with high
levels of proinflammaory cytokines are common findings in intestinal biopsies of PI-IBS
patients, thereby representing an underlying inflammatory basis for the functional
alterations observed (38).
Finally, several lines of evidence indicate that GCM-dependent host-bacterial
interactions play an important role in the pathogenesis of IBS (40). For instance, it has
been reported that patients with IBS have an altered GCM and that treatments targeting
GCM
can
ameliorate
the
symptomatology
(41).
However,
the
underlying
pathophysiological mechanisms elicited by GCM in the generation of symptoms in IBS
remain unknown.
Animal Models of Irritable Bowel Syndrome
To better understand IBS pathophysiology, results from human and animal studies have
to be integrated in a comprehensive manner. The selection of animal models for IBS is
limited, in part, because of the large, undefined, spectrum of underlying mechanisms of
the disease. Thus, the models described are focused on the development of one or a
limited set of symptoms of the disease.
Based on the type of the initiating stimulus, animal models of IBS can de classified
mainly in two groups: those induced by a central nervous system (CNS)-directed
stimulus (psychosocial) and those induced by a gut-directed stimulus (physical) (42).
However, due to the bidirectional interaction of the brain-gut axis, these models are not
17
INTRODUCTION
restricted to either CNS or the gut but are likely to include both peripheral and central
elements.
Animal Models Induced by CNS-Directed Stimuli (Psychosocial)
These models reproduce to some extent the impact of psychosocial stressors as a
component related to the induction and/or exacerbation of the symptomatology in
patients with IBS.
The models used include early life stress (neonatal maternal separation) (43,44) as well
as chronic (45,46) and acute (47,48) stress during adulthood. In either case, long-lasting
alterations in the homeostasis of the gastrointestinal tract, with functional changes in
epithelial function, motility and visceral sensitivity, similar to those observed in IBS
patients, can be observed.
Animal Models Induced by Primary Gut-Directed Etiologies (Physical)
These models are triggered by stimuli (mechanical, chemical or infectious) targeting the
intestine and are applied during early life stages or in adulthood (42,49). They have been
used to study alterations in the motor and epithelial barrier function of the intestine and,
with more emphasis, to explore changes in gut perception (42,49). Frequently, and
taking into consideration the multifactorial characteristics of IBS, CNS- and gut-directed
stimuli are combined to generate more predictive models, at least from a theoretical
point of view (50).

Chronic Exposure to Oral Ovalbumin in Rats as a Model of Irritable Bowel
Syndrome Related to Food Allergens
In order to study the suggested role for dietary constituents on IBS pathophysiology,
it could seem feasible to use validated animal models of food allergy showing
functional gastrointestinal alterations similar to those observed in IBS. However, the
mechanisms of hypersensitivity to food antigens in IBS seem to be different from
those implicated in classical alimentary allergies. Food allergies are IgE-mediated
type-I reactions while the aberrant responses of the gut to dietary antigens
implicated in IBS are IgE-independent (31,36). Therefore, classical animal models
of food allergy are not suitable to study the relationship between food antigens and
IBS.
18
INTRODUCTION
Exposure to oral ovalbumin (OVA) in allergy-prone rats (i.e. Brown Norway) is an
accepted model of food allergy with consistent IgE-mediated systemic responses
(51-53). From this model, our group has shown that a similar chronic exposure to
oral OVA in non-allergy-prone rats (i.e. Sprague-Dawley) induces an IgEindependent intestinal dysmotility related to a state of activation/excitation of
intestinal mucosal MCs (MMCs) (54,55). This model, therefore, might represent a
valid approach to study the potential role of food allergens in IBS

Intestinal Infection with Trichinella spiralis as a Model of Post-infectious-Irritable
Bowel Syndrome
The experimental infection with Trichinella spiralis in rodents is a gut-directed
model that has been extensively used to study the pathogenesis of PI-IBS (56-60).
The life cycle of the nematode T. spiralis begins after the host ingests meat
contaminated with larvae cysts. Larvae are released from the cysts by gastric juices
of the stomach (61). Subsequently, the parasites invade the enteric epithelium of the
small intestine to mature into adult individuals and reproduce (61). Before expulsion
by the host immune system, day 14 post-infection (PI) in the rat, females produce a
great amount of newborn larvae, which will make their way through the circulatory
system of the host to reach the muscle fibers, where they encyst (muscle phase of
infection) (61). The enteric phase of the infection in rodents results in long-lasting
functional disturbances of the gut, persisting long after the expulsion of the parasites
(56,57). At these late stages of the infection, the intestinal inflammation has been
resolved except for the presence of a persistent MMC infiltrate in the mucosa (56).
In these conditions, a long-term increase in epithelial permeability and contractility
dysfunction are observed, reminiscing the functional alterations described in PI-IBS
patients (56,59). The presence of sensory alterations related to visceral sensitivity in
this model has received little attention.
Mast Cells
MCs are resident granulocytes derived from pluripotent hematopoietic cells that, after an
initial development in the bone marrow, enter the blood circulation and complete their
differentiation within various tissues (62). The maturation of MCs in the resident tissue
is influenced by a variety of cytokines and growth factors, although the most crucial one
19
INTRODUCTION
is the stem cell growth factor, the ligand for the tyrosine kinase receptor (c-kit) (63).
Therefore, depending upon the microenvironment, different MC phenotypes can develop
in different tissues, and even in different locations of the same tissue (64).
Although mature MCs are ubiquitous in the body, they are specially located in the
proximity to surfaces that interface with the external environment, such as the skin and
the mucosa of the gastrointestinal and respiratory tract (65). In particular, within these
tissues, MCs reside in close association with structures such as blood and lymphatic
vessels and nerves.
Mature MCs are characterized by their ability to synthesize and store in their
intracitoplasmatic granules a wide range of neuroimmune mediators that are released
upon cell activation (63). Among these mediators, MC specific proteases, including
serine proteases (tryptases and chymases) and the metalloprotease carboxypeptidase A
(CPA), have been widely used to distinguish between distinct subsets of MC populations
and as biomarkers of MC activation (66). For instance, in humans, MCs are classified in
two groups, MCT and MCTC, according to their protease profile. MCT only express
tryptases and are localized in mucosal surfaces. MCTC express all types of MC proteases
(tryptases, chymases and CPA) and are located in the skin and intestinal submucosa (67).
In rodents, MCs have been divided into two populations defined by their anatomical
localization: MMCs, which are preferentially located in mucosal surfaces such as the
intestinal mucosa, and connective tissue MCs (CTMCs) located in the skin and the
peritoneal cavity (65). MMCs and CTMCs can be differentiated also by their
proteinases. In rats, the quimase rat MC protease II (RMCPII) has been used as a
specific marker for MMCs while the quimase rat MC protease I (RMCPI) and the
tryptase rat MC protease VI (RMCPVI) have been used as specific markers for CTMCs
(67).
Activation of Mast Cells
Activation of MCs results in the release into the extracellular environment of a broad
array of mediators. MCs mediators can be divided into preformed (stored in the
secretory granules and released within minutes upon activation) and synthesized de-novo
after cell stimulation (68). Preformed mediators include histamine, MC proteases,
proteoglycans and small amounts of tumor necrosis factor alpha (TNF-α), while the
20
INTRODUCTION
molecules produced upon activation comprise phospolipid metabolites, such as
prostaglandin D2 and different leukotrienes, a wide array of cytokines (interleukin-3, 4,
5, 6, 8, 10, 13, IL) and growth factors (69). Upon release, MC mediators are implicated
in the induction and regulation of inflammatory processes and in tissue remodeling,
including neuronal remodeling.
The best characterized mechanism of MC activation is via crosslinking of IgEs bound to
high-affinity receptors for IgE (FcɛRI) located on the cell surface (63). IgE-dependent
MC activation is particularly important during Th2 allergic disorders or in parasitic
responses, which are characterized by high concentrations of circulating IgEs (70). In
addition, a large number of IgE-independent triggers have been described to potentially
activate MCs, including ligands of Toll-like receptors (TLRs), complement system
factors, cytokines, neuropeptides, neurotrophins (mainly nerve growth factor, NGF) and
external stressors (71).
Mast Cells in Irritable Bowel Syndrome
Several observations support an involvement of MCs in the pathophysiology of IBS.
First, many studies have evidenced a MC infiltration in the colon of IBS patients and
high levels of their mediators (mainly proteases) in colonic supernatants and faecal
samples (72-75). Second, the number of activated MCs in close proximity to colonic
nerve terminals is enhanced in IBS patients compared with healthy controls (72). More
interestingly, the severity and the frequency of abdominal pain in IBS patients positively
correlate with the apposition MCs-nerves (72). Indeed, supernatants from colonic
biopsies of IBS patients, containing a variety of MC mediators, sensitize cultured
sensory neurons and elicit functional responses in animals (visceral hypersensitivity and
alterations in colonic barrier function), similar to those observed in IBS patients (73-75).
Thus, overall, these data point towards an important role of MC-derived mediators and
the interaction MCs-nerve fibers on the disturbed secretomotor and sensory functions
that characterize IBS.
Neurotrophic Factors
Neurotrophic factors (NTFs) are peptides that act directly on specific neuronal
populations to support their survival, maintenance and regeneration, both in the
21
INTRODUCTION
developing and mature nervous system (76). For instance, the survival of virtually all
small-diameter sensory neurons depends on the retrograde transport of NTFs during
embryonic life (77). In adulthood, NTFs promote neuronal survival, phenotype
maintenance and injury- and degenerative-plasticity, thereby representing a potential
therapeutic target for the treatment of some nervous system diseases (78).
NTFs have been grouped into four different families of structurally and functionally
related molecules: NGF family, glial cell line-derived NTF (GDNF) family, neurokine
family and non-neuronal growth factors family (78).
Figure 3. Receptors for the NGF and GDNF family members. A: The members of the
NGF family bind specifically to cognate Trk receptors. NT-3 can also potentially interact
with TrkB and TrkA receptors. The low-affinity neurotrophin receptor p75 promiscuously
binds all of the neurotrophins. B: GDNF family members (GDNF, NTN, ART and PSP)
bind a specific GFRα coreceptor and activate the common signaling receptor RET. Solid
arrows indicate the preferred functional ligand-receptor interactions, whereas dotted arrows
indicate putative crosstalk. ART: Artemin; BDNF: Brain-derived neurotrophic factor;
GDNF: Glial cell line-derived neurotrophic factor; GFRα: GDNF family receptor α; NGF:
Nerve growth factor; NT-3: Neurotrophin-3; NT-4/5: Neurotrophin-4/5; NTN: Neurturin;
PSP: Persephin; Trk: Tropomyosin-receptor-kinase. Image adapted from Krieglstein 2008
(79).
The NGF family includes NGF, brain-derived NTF (BDNF), neurotrophin-3 (NT-3) and
neurotrophin-4/5 (NT-4/5) (80). These products interact with two entirely distinct classes
of receptors; the low-affinity receptor p75, and the high-affinity tropomyosin-receptorkinase (Trk) receptors that comprise three subtypes: TrkA, TrkB and TrkC (80) (Fig.
5A). The p75 is a non-selective receptor that binds to all members of the NGF family
with similar affinity. On the other hand, Trk receptors are peptide-specific, with NGF
22
INTRODUCTION
binding TrkA, BDNF and NT-4/5 binding TrkB, and NT-3 binding TrkC (80) (Fig.5A).
NGF, the first member of the family to be discovered, has been particularly studied (81).
The neurotrophin is produced mainly in target tissues of sensory and sympathetic fibers.
NGF interacts with TrkA receptors in axon terminals and the NGF-TrkA complex is
internalized and retrogradely transported to the neuronal body, where it is required for
the survival and maintenance of the neurons (82). Transgenic mice lacking NGF (NGF
knockout) have been reported to suffer severe sympathetic and sensory deficits, thereby
reinforcing the role of NGF on modulating the survival of these neuronal populations
(83).
The GDNF family includes GDNF and three structurally related members called
artemin, neurturin and persephin. These NTFs exert their biological effects by signaling
through a unique multicomponent receptor system consisting of a RET tyrosine kinase
receptor and a glycosyl-phosphatidylinositol-anchored coreceptor (GDNF family
receptor α1–4, GFRα) (84) (Fig.5B). While the RET receptor represents a common
signaling component for all the GDNF family members, each member uses a GFRα
coreceptor as the preferred ligand-binding protein (84) (Fig.5B). For instance, GNDF,
neurturin, artemin and persephin activate GFRα1, GFRα2, GFRα3 and GFRα4,
respectively, although some cross-over might occur under certain conditions (82)
(Fig.5B). Similarly to NGF, a mechanism of receptor-mediated internalization and
retrograde transport to the neuronal soma has been observed for GDNF and neurturin in
sensory and motor neurons, but not in sympathetic neurons (85). GDNF and the related
family members influence a broad spectrum of neurons, both in the peripheral and CNS
(86). Studies in knockout mice for either GDNF or neurturin have demonstrated the
crucial role of these NTFs on neuronal-related processes of the gut during development,
as these animals lack enteric neurons and show severe reduction of the intestinal
parasympathetic cholinergic innervation (87,88).
Neurotrophic Factors in the Gut
Since NTFs are important on neural remodeling, several studies have explored their role
in the physiology of highly innervated organs, such as the gut. Wide data support a role
for NTFs maintaining extrinsic enteric innervation (89-91); however, the exact source(s)
of NTFs within the intestine remains unclear. For instance, NGF-like immunoreactivity
has been described in enteric plexuses and also in non-neural cells, such as epithelial and
smooth muscle cells (45,92-94). Moreover, results suggest that NGF, or at least a NGF
23
INTRODUCTION
precursor (pro-NGF), is synthesized and released by MCs (43,92,94). Overall, the exact
source(s) o f NGF within t he intestine r emains o bscure, with e nteric neurons, ep ithelial
cells and resident immune cells (mainly MCs) as the main candidates.
The fact that NGF is detected outside the nervous system could seem surprising as NTFs
have been tr aditionally d efined as
neural-related m olecules. However, g rowing
evidences reveal that NTFs also exert a large broad of effects in non-neural tissues (95).
Supporting e xtra-neuronal effects o f NGF, t he NGF h igh-affinity re ceptor T rkA h as
been described in non-neuronal structures. In particular, within the intestine, apart from
the ENS, TrkA r eceptors have been d escribed in ep ithelial and la mina propria ce lls
(92,96,97). In vitr o studies support the functionality o f these r eceptors, s uggesting,
overall, an influence of NGF on both neural and non-neural enteric cells (98).
Other NT Fs th at have been d etected in th e intestine include GDNF, NT -3 an d B DNF
(99-101).
Neurotrophic Factors in Inflammatory and Functional Gastrointestinal Disorders
NTFs h ave been s uggested to co ntribute to t he functional alterations obs erved i n
inflammatory and FGDs. Results obtained in IBD and IBS patients and relevant animal
models r eveal an e nhanced expression o f NT Fs, n amely, NGF an d GNDF family
members (58,93,102,103). As NTFs are well known by their ability to sensitize sensory
afferents, it has been hypothesized that the increased expression of these mediators could
account for th e altered v isceral h ypersensitivity o bserved in I BS (45,104,105). T his is
supported by the positive correlation found between abdominal pain and colonic content
of BDNF in IBS patients (106). Signaling of NGF and GNDF family members has also
been i mplicated i n o ther key features of i nflammatory and FGD s, such a s al tered
epithelial barrier f unction a nd in testinal dysmotility (43,58,107) For in stance, upregulated levels of enteric NGF have been associated to the T. spiralis–induced jejunal
hypermotility and the n eonatal m aternal s eparation-related alter ed g ut permeability i n
rats (43,58). Although th ese evidences, as mentioned ab ove, th e source(s) o f i ntestinal
NTFs remains elusive.
24
HYPOTHESIS AND OBJECTIVES
HYPOTHESIS AND OBJECTIVES
From th e previous background, it is ass umed th at luminal f actors, intestinal mast ce lls
(MCs) and neurotrophic f actors ( NTFs) participate in the pathophysiological
mechanisms implicated in the functional alterations that characterize IBS.
Thus, t his work is based on t he HYPOTHESIS that changes in MCs, NT Fs, and t heir
association, are key elements of the neural remodeling observed in abnormal responses
of th e gut to lu minal-derived s timuli, leading to lo ng-term f unctional changes.
In
particular, during colonic conditions characterized by an excited-activated state of MCs,
NTFs, mainly NGF, would modulate the alterations in the motor and sensory functions
of the colon.
In order to demonstrate this hypothesis, two validated animal models of IBS, based on
luminal-derived factors a s i nductive stimuli (chronic e xposure to o ral OV A a nd
intestinal in fection with T. spiralis), together with a new protocol of OVA e xposure
associated to a dysbiotic-like s tate, were used i n r ats. Using th ese models, potential
changes i n colonic motor a nd s ensory functions were determined. I n ad dition, th e
relationship between th e observed co lonic dysfunction and the dynamics of MC s an d
NTFs was also explored.
Therefore, the specific OBJECTIVES of this work were as follows:

To characterize the effects of the exposure to the dietary-related luminal antigen
OVA on co lonic motility, with e mphasis on the potential i mplication o f M Cs
and the neurotrophins-Trk receptors pathway.

To determine the relationship between colonic MCs and NGF and, in particular,
if colonic MCs are a source of NGF.

To d etermine if gut microbiota-derived f actors i nteract with o ral OVA to
mediate functional colonic alterations.

To characterize plastic changes in the sensory afferent innervation of the colon
associated to infectious (T. spiralis)-related luminal stimuli.

To determine if infectious ( T. spiralis)-related luminal stimuli are able to alter
sensory properties of the colon, with emphasis on the potential role of NTFs.
27
CHAPTER 1
CHAPTER 1
CHAPTER 1
NGF I S I NVOLVED I N O RAL O VALBUMIN-INDUCED AL TERED
COLONIC C ONTRACTILITY IN RAT S: EVIDENCE FROM THE
BLOCKADE OF TRKA RECEPTORS WITH K252
F. Jardí 1, V. Martínez *,1,2,3, P. Vergara 1,2,3
1
Department o f C ell Bi ology, Phys iology and
Autònoma d e Barcelona, Spain.
Autònoma de
Immunology, Universitat
2
Instituto de N eurociencias, U niversitat
Barcelona.3 Centro de Investigación Biomédi ca en
Red de
Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud
Running title: Effects of K252a on colonic contractility
Keywords: Colonic contractility; K252a; M ucosal mast ce lls; N erve growth
factor; Ovalbumin; TrkA
Neurogastroenterol Motil. 2012;24:e580-90
31
CHAPTER 1
Abstract
Background Nerve growth factor ( NGF)-mucosal mast cells ( MMCs) i nteraction has
been i mplicated in th e r emodeling o f e nteric circuitries a nd th e as sociated f unctional
changes. We investigated the involvement of NGF and its r eceptor TrkA in the altered
colonic contractile activity o bserved in t he model of o ral o valbumin ( OVA)-induced
MMC h yperactivity i n r ats. We also s tudied th e role of c olonic MMCs as a source o f
NGF. Methods Rats were exposed to o ral OVA, alone or w ith t he TrkA an tagonist
K252a. Co-expression o f NG F/TrkA a nd r at mast cell protease II ( RMCPII) ( double
immunofluorescence), RMCPII content (ELISA) and expression of NGF, brain-derived
neurotrophic factor ( BDNF) an d T rkA/B ( RT-qPCR) were assessed in co lon. C olonic
contractile activity was determined in vivo and in vitro. Key Results TrkA, but not NGF,
was lo calized in co lonic MMCs ( RMCPII-positive ce lls). OVA ex posure increased
colonic RMCPII lev els but did n ot change th e p ercentage of TrkA-positive MM Cs.
Neither OV A nor K2 52a, alone or co mbined, alter ed NGF, B DNF o r T rkA/B
expression. Spontaneous co lonic ac tivity in vivo and in vit ro was altered b y OV A, an
effect p revented b y K252a. Electrical stimulation-induced contractile r esponses in vivo
and carbachol r esponses in vitr o were in creased by OVA in a K252a-independent
manner. In OVA-treated animals, inhibition of NO synthesis with L -NNA significantly
enhanced s pontaneous colonic activity in vitr o, a response completely p revented b y
K252a. Conclusions & I nferences These r esults s uggest th at NGF-TrkA-dependent
pathways ar e implicated in co lonic contractile alterations o bserved d uring OVA
exposure in r ats. NGF-TrkA system might r epresent a potential tar get for tr eatment o f
gastrointestinal disorders characterized by colonic motor alterations.
33
CHAPTER 1
Introduction
Colonic dysmotility is a common finding in functional gastrointestinal disorders (FGDs).
For in stance, irritable bowel syndrome (IBS), th e main FGD, is ch aracterized b y
abdominal p ain-discomfort a ssociated with d ysmotility a nd alter ed b owel habits (1).
Moreover, in a large n umber o f I BS patients, food in gestion h as b een a ssociated with
symptomatology exacerbation, suggesting a role for food allergy in its pathogenesis (2).
Intestinal f ood aller gy-related mechanisms in I BS seem to in volve local mucosal
responses to d ietary a ntigens rather than cla ssical t ype-1 hypersensitivity r eactions (3).
We have shown that chronic exposure to oral ovalbumin (OVA) in Sprague Dawley rats
induces a non-IgE mediated alteration of smooth muscle colonic contractility resembling
that observed by others and us in IBS and animal models of the disease (4-8).
Several observations support an involvement of mast cells in the pathophysiology of IBS
(9). Morphological and functional studies with colonic biopsies from IBS patients point
towards an important role of mast cell-derived mediators and the interaction mast cellsnerve fibers o n t he disturbed s ecretomotor an d sensory functions ch aracterizing I BS
(10,11). In this line, we have demonstrated that in the rat model of chronic exposure to
oral OVA, mucosal mast cells (MMCs) are implicated in the altered colonic contractile
activity; thus suggesting t hat OV A-induced co lonic motor alter ations in r ats ar e
somehow M MC-dependent (4). Therefore, ex posure to o ral OVA i n r ats reproduces
some pathophysiological components o f I BS; at least the MMC h yperactivity an d t he
changes in colonic contractility, thus representing a valid model for studying IBS-related
altered colonic contractile responses and their potential relationship with MMCs.
Recent data suggest that, within th e gut, neurotrophins, m ainly nerve growth f actor
(NGF), interact with MMCs generating a neuroimmune circuit likely to play a potential
role in the pathophysiology of FGDs. For instance, evidences obtained in animal models
of IBS have implicated NGF in the neuronal remodeling of enteric circuitries and MMCs
recruitment, as basis f or th e functional changes o bserved. I n p articular, an ti-NGF
treatment completely b locked in testinal hypermotility i n Trichinella spiralis-infected
rats, an accepted model of post-infectious-IBS (12), and reduced the interaction MMCsnerve fibers in the rat maternal separation model (13). A recent study in colonic biopsies
from IBS patients supports these observations, showing an increased neuronal sprouting
35
CHAPTER 1
within t he mucosa, an e ffect associated to NGF in creased levels, possibly of mast ce ll
origin (14). However, the exact origin of colonic NGF and the potential role for MMCs
as the peptide source are still unclear.
NGF interacts with two classes of cell surface receptors: the TrkA high-affinity receptor,
a selective NGF receptor; and the p75 low-affinity receptor, which presumably binds to
all n eurotrophins (15). To further un derstand t he role of NGF in co lonic motor
alterations we have investigated the effects of the pharmacological blockade of TrkA on
OVA-induced changes in colonic contractility in the rat by using K252a, which has been
widely u sed in t he rat as a TrkA an tagonist (16-20). In a ddition, we evaluated th e
expression lev els o f NGF and TrkA in t he rat colon d uring ex posure to o ral OVA,
complementing this d ata by d etermining t he colonic ex pression of t he related
neurotrophin brain-derived neurotrophic factor ( BDNF) a nd its p referential receptor
TrkB. Fin ally, we evaluated the interplay b etween MM Cs-NGF/TrkA s ystem,
characterizing, i n p articular, if M MCs r epresent a cellular s ource o f t he neurotrophin
and/or express TrkA receptors.
Materials and Methods
Animals
Adult (5 week-old at arrival), specific pathogen free (SPF), Sprague-Dawley (SD) male
rats were used ( Charles R iver, L es Oncins, Fra nce). Animals had free ac cess to water
and a standard pellet diet, free of traces of OVA or any other egg derivative (A04; Safe,
Augy, Fra nce). Rats were m aintained u nder co nventional conditions in a l ight
(12h/12h light-dark cycle) and temperature controlled (20-22ºC) room, in groups of two
per cage. Animals were acclimatized to the new environment for 1 week before starting
any experimental procedure. All the experimental protocols were approved by the Ethics
Committee of the Universitat Autònoma de Barcelona and the Generalitat de Catalunya
(protocols 1010 and 5351, respectively).
36
CHAPTER 1
Experimental design
Rats received OVA by oral gavage (1mg mL-1, 1 mL/rat, n=24), on a daily basis during a
6-week p eriod (21). A group o f r ats receiving v ehicle ( 1 m L/rat, n= 21) was u sed a s
control. After th e third week, 1 1 of the animals receiving OVA an d 1 0 of th e animals
receiving vehicle were treated subcutaneously with K252a (50 μg kg-1). Treatment with
K252a was p erformed daily and last ed until the d ay before an imals were e uthanized,
outlasting for 10 da ys O VA exposure. This antagonist, at th e dose and p attern o f
administration followed h ere, has a lready been used, s howing effective b lockade o f
TrkA a nd biological e ffects in vivo (18-20). The rest of t he animals ( 13 OV A- and 11
vehicle-exposed) were used a s co ntrol groups in which t he tr eatment p rotocol was the
same but K252a was replaced by the corresponding vehicle (1 mL kg-1, sc). Except for
the in vivo experiments, a t t he time of eu thanasia, tissue s amples from the co lon were
obtained an d eith er u sed f or o rgan b ath s tudies, fixed i n 4 % paraformaldehyde for
immunohistochemical studies o r f rozen i n liq uid nitrogen a nd s tored at -80ºC unt il
analysis.
Organ bath
Full thickness preparations, o btained from the mid portion of the colon, were cut 1 c m
long and 0.3 cm wide and hung for organ bath study oriented to record circular muscle
activity. Strips w ere mounted u nder 1 g t ension i n a 10-mL muscle b ath co ntaining
carbogenated Kr ebs s olution ( 95% O2 – 5% CO 2) maintained at 37 ± 1 ºC. The
composition o f Kr ebs s olution was ( in mmol L-1): 10.10 g lucose, 115. 48 N aCl, 21. 90
NaHCO3, 4.61 KCl, 1.14 NaH2PO4, 2.50 CaCl2, and 1.16 MgSO4 (pH 7.3–7.4). One strip
edge was tied to the bottom of the muscle bath using suture silk and the other one to an
isometric force tr ansducer ( Harvard VF -1 Har vard A pparatus I nc., Ho lliston, MA ,
USA). Ou tput from th e tr ansducer was fed to a PC through an a mplifier. Data were
digitalized ( 25 Hz ) u sing Dat a 2001 s oftware (Panlab, B arcelona, Sp ain). Strips were
allowed to equilibrate for about 1h. After this period, contractile responses to carbachol
(CCh; 0 .1–10 μM) and the NO inhibitor NG-nitro-L-Arginine (L-NNA; 1 m M) w ere
assessed. Fo r C Ch, c umulative co ncentration-response c urves, with a 5 -min i nterval
between consecutive concentrations, were constructed. For L-NNA, spontaneous activity
was recorded during a 10-min period after the addition of the drug.
37
CHAPTER 1
To determine the spontaneous contractile activity, the preparation tone was measured for
15 min and the mean value (in g) determined. To test the effects of CCh, the maximum
peak, from the basal tone, was measured after each concentration tested. To measure the
response to L -NNA, th e 10-min me an of the s trip tone b efore th e d rug administration
was d etermined an d co mpared w ith t he 10-min mean o f the strip to ne after L -NNA
addition.
Strain-gauge recordings
After a f asting p eriod o f 6 h , a nimals were placed i n an induction ca mera and
anesthetized by i nhalation o f 4 % isofluorane (Isoflo®; Esteve, B arcelona) in 2 L m in1
oxygen to allo w ca nnulation o f a lateral vein o f th e tail. Thereafter, r ats w ere
maintained in level III of anesthesia by intravenous thiopental sodium, as required, and
exposed to mask d elivery o f 1 L min-1oxygen during all t he procedure. A l aparatomy
was pe rformed, th e colon localized an d a s train-gauge ( F-04IS, Star Medical, T okyo,
Japan) was sutured to its wall (2 cm from de cecum) to record circular muscle activity.
The strain gauge was connected to a high-gain amplifier (MT8P; Lectromed, Herts, UK),
and signals were sent to a recording unit (PowerLab/800; ADInstruments) connected to a
computer. Finally, an electrode holder with two platinum electrodes (WPI, Sarasota, FL,
USA) wa s inserted into the colonic lumen at 1 cm distally to the strain-gauge to induce
ascending e xcitation o f t he peristaltic r eflex b y e lectrical mucosal s timulation ( EMS).
EMS was applied by duplicate at 30 V, 0.6 ms and 4 Hz during 30 s each time, and the
polarity of the stimulating electrodes was reversed at 15 s.
To analyze in vivo colonic motility, co ntractions o f the colon were classified in to lowfrequency an d h igh-amplitude (LFHA) co ntractions a nd h igh-frequency a nd l owamplitude (HFLA) contractions, as previously described (22). HFLA were identified by
having a frequency within the r ange o f 10–15 co ntractions min-1, w hile LFHA were
defined as contractions of an amplitude >300% of that of HFLA contractions at the same
recording s ite (22). HFLA a nd L FHA were assessed over a 15-min p eriod an d the
frequency and amplitude expressed as the mean. When assessing the responses to EMS,
the recording analyzed corresponded to the 30 s of stimulation and the expressed value
was the mean of the duplicates. All analysis was performed using Chart 5 software for
Windows (both from AD Instruments, Castle Hill, NSW, Australia).
38
CHAPTER 1
Immunohistochemistry (IHC)
Immunodetection o f rat mast ce ll protease II ( RMCPII) and NGF was carried o ut on
paraformaldehyde-fixed co lonic samples u sing a monoclonal antibody an ti-RMCPII
(Moredun, E dinburgh, UK) and a polyclonal rabbit a nti-NGF (ab1526; C HEMICON
International, T emecula, US A). Antigen r etrieval for NGF was ac hieved b y processing
the slides in a pressure cooker, at full pressure, for 10 min in 10 mM citrate solution. The
secondary a ntibodies included b iotinylated horse antimouse IgG (BA-2000; Vector
Laboratories, Burlingame, C A, US A) a nd b iotinylated swine antirabbit Ig ( E0353;
DAKO, Carpinteria, CA, U SA). Dete ction was p erformed with avidin/peroxidase kit
(Vectastain ABC kit; Vector L aboratories, Burlingame, C A, US A) and counterstaining
with haematoxylin. Specificity of the staining was confirmed by omission of the primary
antibody. W hen p erforming I HC f or NGF, mouse submaxillary g lands were used as a
positive control. Slides were v iewed with an Ol ympus B H-2 microscope. Fo r MM C
quantification, at least 2 0 non-adjacent X4 00 fields o f co lonic mucosa were randomly
selected and the number of RMCPII-immunopositive cells counted. All procedures were
carried out using coded slides to avoid bias.
Immunofluorescence staining
For an alyses o f co -localization o f NG F, proNGF o r T rkA with R MCPII, d ouble
immunofluorescence was u sed. After 1 h o f b locking with 1 0 % normal goat serum at
room temperature, colonic sections were incubated with a mixture of anti-RMCPII and
anti-NGF or anti-ProNGF (ab5583; CHEMICON International, Temecula, USA) or antiTrkA (s c-118; Santa Cruz Biotechnology, C A, US A) o vernight. Thereafter, s ections
were incubated with a secondary antibody cocktail consisting of fluorescence-conjugated
Alexa Fluor 4 88 goat anti-mouse IgG ( A11029; Mo lecular Probes, Eugene, O R, US A)
and C y3 g oat anti-rabbit I gG (PA-43004; Am ersham-Pharmacia, Buckinghamshire,
UK). Af ter washing, t he slides were coverslipped with Vec tashield Mo unting Me dium
(Vector L aboratories, B urlingame, C A, USA) a nd e xamined un der a n Axioskop 40
fluorescence microscope (Carl Zeiss, J ena, Ger many). Merging o f the images was
analyzed with ImageJ Software (U. S. National Institutes of Health, Bethesda, Maryland,
USA). To assess the percentage of cells with RMCPII and TrkA co -localization, Alexa
Fluor 488- and Alexa Fluor 488-Cy3-stained cells were counted randomly using a 100x
39
CHAPTER 1
objective. Specificity of the staining was confirmed by omission of the primary antibody
and/or th e secondary an tibodies. The absence o f cr oss-reactivity was co nfirmed in
control single-labeled preparations.
ELISA
Protein was extracted f rom c olonic tissue s amples using lysis buf fer ( 50 mM HEPES,
0.05 % Triton X -100, 0.0625 mM P MSF and the Mi ni Complete p rotease inhibitor
Roche) a nd RMCPII co ncentration was d etermined b y E LISA using a commercial kit
(Moredun). T otal protein was determined u sing th e Bradford ass ay k it (BIO-RAD,
Hercules, CA, USA).
RNA extraction and quantitative real-time PCR
Total R NA was ex tracted f rom co lonic samples u sing R ibopure RNA I solation Kit
(Applied Biosystems, CA, USA) and quantified by Nanodrop (Nanodrop Technologies,
Rockland, DE, USA). For cDNA synthesis, 1 µg of RNA was reverse-transcribed in a 20
µl reaction v olume using a h igh ca pacity cD NA r everse transcription kit ( Applied
Biosystems, CA, USA). Expression of NGF, BDNF, TrkA and TrkB was determined by
quantitative r eal-time P CR p erformed with s pecific T aqman p robes (Applied
Biosystems; NGF: Rn01533872_m1, B DNF: Rn00560868, T rkA: R n00572130_m1,
TrkB: R n01441749_m1, B-Actin: R n00667869_m1) mixed with T aqman Un iversal
Master Mix II for 40 c ycles ( 95ºC for 15 s , 60º C for 1 min) on a 7900 r eal-time P CR
system ( Applied B iosystems). Rat submaxillary gland a nd n eocortex were used as
positive controls for th e gene expression o f T rkA, T rkB an d NGF an d B DNF,
respectively. B -Actin e xpression s erved as a n e ndogenous co ntrol for n ormalizing t he
mRNA levels of the target genes. Expression levels were analyzed by the 2 -∆∆CT method.
Chemicals
Ovalbumin (Grade V; A5503) was purchased from Sigma-Aldrich (St.Louis, MO, USA)
and was d issolved in s aline solution. K2 52a [(9S,10R,12R)-2,3,9,10,11,12-Hexahydro10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4i][1,6]benzodiazocine-10-carboxylic acid methyl e ster; T ocris Bioscience, Ellisville,
40
CHAPTER 1
MO, USA] was reconstituted in 8.75 % ethanol in milli-q water. CCh (Sigma-Aldrich)
stock s olution a nd f urther d ilutions were prepared in d istilled water. L -NNA ( SigmaAldrich) was prepared directly in carbogenated Krebs solution.
Statistics
All data are expressed as mean ± SEM. Motility results are presented as raw data (g of
force) or frequency of contractions (number min-1; in vivo recordings). EC50 for CCh was
calculated b y non-linear regression to a sigmoidal equation (GraphPad Prism 4.01, San
Diego, C alifornia, U SA). C omparisons b etween multiple groups were performed with
two-factor ANOVA. W hen the two way ANOVA revealed s ignificant ef fects o f
treatments, data were reanalyzed with one-way ANOVA followed, when n ecessary, by
a Stu dent-Newman-Keuls multiple comparison test to
detect differences b etween
experimental groups. P values < 0.05 were considered statistically significant.
Results
Colonic mucosal mast cell count and RMCPII content
The number of R MCPII-positive cells in th e co lon showed n o s ignificant differences
between e xperimental groups d espite th e tr eatment r eceived ( Fig. 1A). Nev ertheless, a
two-way ANOVA a nalysis r evealed an e ffect of OV A tr eatment in creasing RMCPII
content (P=0.022), although only the OVA-K252a group achieved statistical significance
(P<0.05 vs. vehicle-vehicle; Fig. 1B). K252a, per se, showed a tendency to increase the
levels of RMCPII, although statistical significance was not achieved (P=0.13).
Localization of NGF by immunohistochemistry
Within the colon, immunoreactivity for NGF was detected mainly in the submucosal and
myenteric plexuses (Fig. 2C-D). A diffuse staining was observed in the epithelium, both
on the villi and, occasionally, in the crypts. Within the villi, there were scarce cells, of
undetermined type, s howing NGF -like i mmunoreactivity ( Fig. 2A-B). No lab eling was
detected in the muscle layers. No d ifferences i n th e s taining p attern o r in tensity were
observed between OVA- and vehicle-treated animals or associated to the treatment with
41
CHAPTER 1
K252a. Immunoreactivity was ab sent in s ections in which th e primary a ntibody was
omitted, t hus co nfirming t he specificity o f th e s taining. S taining was i ntense and well
B
8
6
4
2
0
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
K 252a
K 252a
R M C P I I ( n g /µ g p r o t e in )
A
M M C s ( p e r f ie ld , X 4 0 0 )
localized in positive controls from mouse submaxillary glands.
0 .8
*
0 .6
0 .4
0 .2
0 .0
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
K 252a
K 252a
Figure 1. Colonic density of MMCs (A) and RMCPII content (B) in the different
experimental g roups. Data are m ean ± SEM; n = 5-10 pe r g roup. * : P<0.05 v s.
vehicle-vehicle.
Co-localization of RMCPII and NGF, proNGF or TrkA
In ve hicle-treated an imals, d ouble immunofluorescence studies d emonstrated that the
vast majority ( 63.6±10.4%) of
RMCPII-positive ce lls ( identified as MM Cs) co -
expressed T rkA-like immunoreactivity ( Fig. 2G). Si milar ratio o f R MCPII-TrkA c olocalization was observed a fter OVA e xposure (75.0±5.7%). A part from RMCPIIpositive cells, o ther s carce cells within t he lamina propria showed T rkA-labeling,
indicating that not only MMCs express the receptor in the rat colonic mucosa.
No RMCPII-positive cells (MMCs) showed co-staining for NGF or ProNGF.
NGF, BDNF, TrkA and TrkB expression in the colon
Overall, colonic expression levels of NGF, TrKA and TrkB were relatively low, with no
significant differences i n ex pression le vels among t he different e xperimental groups
(Fig. 3 ). Ho wever, it was noticeable that K252a and OVA , p er s e, i ncreased T rkA
expression levels by 32% and 26% respectively when compared to the expression levels
in the control g roup although these effects were not ev ident in OVA-K252a-treated
animals (Fig. 3B).
42
CHAPTER 1
Figure 2. Immunohistochemistry for NGF and double immunofluorescence for TrkA
and RM CPII (M MCs). Photomicrographs sh owing NGF-like imm unoreactivity in
colonic ti ssues of a vehicle-vehicle-treated (A, C ) a nd OVA-vehicle-treated (B, D) rats.
Labeling for NGF was detected in the submucosal and myenteric plexuses, surface of the
villi a nd c rypts, a nd in sc arce, u nidentified c ells within th e v illi. In sert in (B) sh ows a
higher magnification of these NGF-immunoreactive positive cells within the crypts. Note
that no differences in th e sta ining p attern o r in tensity a re o bserved b etween OV A- and
vehicle-treated a nimals. E -G: Dual label immunofluorescence sh owing th e p resence o f
TrkA in MMCs of the rat colon. E: Representative image of anti-RMCPII labeling (green)
of c ells (identified a s M MCs) in th e c olonic mucosa. T he arrows in dicate positively
labeled cells. F: Same field as in E showing labeling for TrkA (red). The arrows indicate
positively lab eled c ells. G: Merged im age o f E and F sh owing e xtensive c olocalization
(yellow) o f RMCPII (M MCs) and TrkA i mmunoreactivities. The arrows indicate double
labeled MMCs, whereas the arrowhead indicates a MMC negative for TrkA.
43
CHAPTER 1
In co ntrol tissues (rat submaxillary g land) e xpression lev els o f NGF, T rkA a nd T rkB
were, r espectively, 6 -, 1 4- and 100 -fold higher t han t hose observed in the colon i n
control conditions.
BDNF was not detectable in th e colon ( ct values higher th an 4 0), alth ough h igh
expression levels were found in the positive control (rat neocortex).
T r k A m R N A fo ld c h a n g e
B
N G F m R N A f o ld c h a n g e
A
2 .5
2 .0
1 .5
1 .0
0 .5
0 .0
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
K 252a
K 252a
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
K 252a
K 252a
T r k B m R N A f o ld c h a n g e
C
2 .5
2 .0
1 .5
1 .0
0 .5
0 .0
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
K 252a
K 252a
2 .5
2 .0
1 .5
1 .0
0 .5
0 .0
Figure 3. R eal-time PCR analysis of mRNA for NGF (A), TrkA (B) and TrkB (C).
Data are mean ± SEM; n=5-9 per group.
Colonic contractility in vitro
Spontaneous colonic contractile activity, as assessed in vitro, was similar in vehicle- and
OVA-exposed an imals ( vehicle: 0.53±0.06 g ; OVA: 0.51±0.03 g ; P>0.05; Fig. 4 A).
K252a decreased s pontaneous ac tivity in similar p roportion in vehicle- (0.40±0.05 g ;
P=0.07 vs. vehicle-vehicle; Fig.4A) or OVA -exposed animals (0.35±0.05 g, P<0.05 vs.
OVA-vehicle; Fig. 4A).
In control conditions, CCh elicited a concentration-dependent contractile response with
an est imated E C50 of 1. 8±1.3 mM. Overall, a two-way ANOVA a nalysis r evealed a n
OVA effect (P=0.031) enhancing the contractile responses to CCh, leading to a left-shift
of th e concentration-response cu rve an d a 5 -fold r eduction in th e estimated EC50
44
CHAPTER 1
(0.39±0.1 m M; Fig. 4 B). Treatment with K252a did not af fect t he responses to CCh,
neither in vehicle- nor in O VA-exposed animals ( EC50; v ehicle-K252a: 1. 7±1.0 mM;
OVA-K252a: 0.16±0.04 mM; Fig. 4B).
In colonic strips obtained from vehicle-vehicle animals, blockade of NO synthesis by the
addition of L-NNA to the organ bath increased spontaneous activity over pre-treatment
values (P=0.036; Fig. 4C). Similar effects were observed in tissues from OVA-vehicleor v ehicle-K252a-treated r ats, although motor ef fects o f L -NNA were enhanced i n
OVA-vehicle-treated an imals ( Fig. 4 C). Ho wever, in an imals tr eated with OV A plus
K252a, L -NNA-induced increased s pontaneous ac tivity was no l onger observed ( Fig.
A
C o n t r a c t ile a c t iv it y ( g )
C
B
0 .8
0 .6
0 .2
0 .0
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
K 252a
K 252a
5
M a x im a l e f f e c t ( g )
*
#
0 .4
 C o n t r a c t ile a c t iv it y ( g )
4C).
#
1 .0
0 .8
0 .6
***
*
**
**
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
K 252a
K 252a
0 .4
0 .2
0 .0
V e h ic le
V e h ic le
4
V e h ic le
K 252a
3
OVA
V e h ic le
2
OVA
K 252a
1
0
-6
-5
-4
-3
L o g C C h D o se (m M )
-2
Figure 4 . Effe cts of oral OVA a nd K 252a o n c olonic contractility in v itro. A:
Colonic spontaneous contractile activity in the different experimental groups. Data are
mean ± SEM; n=5-10 per group. *: P<0.05 vs. OVA-vehicle; #: P=0.074 vs. vehiclevehicle. B: Concentration-response c urves for c arbachol. No te that oral exposure t o
OVA leads to a left-shift of the concentration-response curve, an effect not modified
by tre atment with K2 52a. Da ta represent mean values (symbols) a nd non -linear
regression c urves. n= 5-10 p er group. C: Effect of NO b lockade w ith L -NNA on
spontaneous contractility in t he d ifferent experimental g roups. Data represent
spontaneous contractility before (open bars) and after the addition of L-NNA (closed
bars). Data are mean ± SEM; n=5-10 per group. *: P<0.05, **: P<0.01, ***: P<0.001
vs. respective contractile activity b efore th e a ddition o f L -NNA (P aired t -test); # :
P<0.01 vs. other L-NNA-treated groups (ANOVA).
45
CHAPTER 1
0 .8
C
-1
L F H A -A m p litu d e
C o n t r a c t io n s m in
*
*
3
2
1
0 .0
D
0
H F L A -A m p litu d e
0 .6
g
5
4
1 .0
0 .5
B
L F H A -F r e q u e n c y
g
1 .5
0 .4
0 .2
0 .0
E
P e a k re sp o n se (g )
C o n t r a c t io n s m in
-1
A
4
EM S
20
H F L A -F r e q u e n c y
15
10
*
5
0
V e h ic le - V e h ic le
3
2
*
1
O V A -V e h ic le
V e h ic le - K 2 5 2 a
O V A -K 2 5 2 a
0
F
V e h ic le - V e h ic le
1 g
1 m in
EM S
O V A -V e h ic le
EM S
O V A -K 2 5 2 a
EM S
Figure 5. A-D: Effects of oral OVA and K252a treatment on colonic motility in
vivo. A-B: Frequency ( A) and a mplitude (B) o f LFHA colonic contractions in th e
different experimental groups. Note that oral exposure to OVA tends to increase the
frequency o f L FHA c olonic contractions and trea tment with K252a reduces it in
both v ehicle- and OV A-treated ra ts. *: P<0.05 v s. respective v ehicle. C-D:
Frequency (C) a nd a mplitude ( D) of HFL A colonic contractions in t he d ifferent
experimental g roups. Note that oral exposure to OV A lea ds to an increase o f th e
amplitude o f HFL A c olonic contractions, an e ffect prevented by tre atment with
K252a. Data are mean ± SEM; n=3-5 per group. E: Colonic response to EMS (30 V,
4 Hz, 30 s) showing that exposure to OVA increases EMS-elicited motor responses
in a K 252a-independent m anner. D ata are m ean ± SEM; n= 3-5 pe r g roup. F:
Representative tracings showing spontaneous colonic motility and response to EMS
in a v ehicle-vehicle-, OV A-vehicle- and O VA-K252a-treated a nimals. T he ar rows
indicate L FHA contractions. Note h ow OV A e xposure in creases the f requency o f
LFHA contractions, an effect prevented by K252a treatment.
46
CHAPTER 1
Colonic motility in vivo
As previously described (22), strain-gauge recordings of spontaneous colonic motility in
vehicle-vehicle-treated rats ex hibited t wo d ifferent types of co ntractions; LFHA
contractions with superimposed HFLA contractions (Fig. 5F). Overall, exposure to OVA
altered s pontaneous co lonic motility b y a ffecting b oth t ypes of co ntractions ( Fig. 5 ).
Treatment with OVA te nded to increase the frequency of LFHA contractions ( vehicle:
0.67±0.14 contractions min-1; OVA: 0.93±0.08 contractions min-1; P=0.08; Fig. 5A) and
also enhanced the amplitude ( vehicle: 1 .92±0.25 g; OVA: 2.78±0.20 g; Fig. 5 B). As it
refers to HFLA contractions, exposure to OVA did not alter the frequency but increased
the amplitude (vehicle: 0.37±0.05 g; OVA: 0.59±0.03 g; Fig. 5D). However, in animals
treated with K252, p art of th ese effects o f OV A ex posure on s pontaneous co lonic
motility was no lo nger o bserved. K2 52a inhibited th e OVA-increased f requency o f
LFHA co ntractions (0.49±0.14 co ntractions min-1; P<0.05 v s. OV A-vehicle; Fi g. 5A)
although it did not affect the amplitude. A similar effect of K252a on the frequency but
not the a mplitude o f LFHA contractions was o bserved in vehicle-exposed animals
(0.28±0.09 contractions m in-1; P<0.05 v s. v ehicle-vehicle; Fig . 5A). Concerning t o
HFLA contractions, treatment with K252a showed a tendency to reverse the increase in
the amplitude of HFLA contractions after OVA exposure, as suggested by a significant
interaction between treatments in a two-way ANOVA (0.34±0.05 g; P<0.05; Fig. 5D).
In control co nditions, EMS elicited a LFHA-type r esponse th at coincided with the
stimulation time (Fig. 5F). The contractile response to EMS was increased by exposure
to OV A ( vehicle: 1.24±0.23 g ; OVA: 3. 05±0.23 g ; P= 0.05; Fig. 5 E) in a K252aindependent manner (2.80±0.54 g; Fig. 5E).
Discussion
This s tudy s hows t hat, i n t he r at model o f ch ronic e xposure to o ral OVA , ch anges i n
colonic motility might b e related to an alter ed ac tivity of t he NGF-TrkA pa thway.
Although NGF e xpression l evels were not changed, motor responses f ollowing the
treatment with K2 52a suggest th at NGF-dependent s ignaling p athways ar e involved in
colonic spontaneous motor a ctivity a nd mediate OVA-induced en hancement o f NOdependent inhibitory tone in vitro. Moreover, our results suggest that colonic NGF is not
47
CHAPTER 1
MMC-derived, alth ough th ese cells ex press T rkA r eceptors an d, th erefore, r epresent a
target for NGF within the colonic mucosa.
The results o f t his study co nfirm t hat oral OVA ac tivates MMCs i n th e colon, a s
indicated b y t he increase in RMCPII lev els within the colonic wall, similarly to t hat
described p reviously i n t he same model (4). Data d erived f rom a nimal models o f IBS
have demonstrated th e importance o f MM Cs as effector ce lls mediating th e array o f
pathophysiological changes that characterize IBS in humans. For instance, degranulation
of MMCs seems to be a key step in the onset of visceral hyperalgesia and the alterations
of epithelial barrier function observed both in animal models and the human disease (2326). In the model of chronic exposure to oral OVA in rats, although these salient features
of IBS have not been characterized, observations reveal that MMCs also play a role on
the changes within co lonic smooth muscle contractility, t hus s upporting the validity o f
the model as an appropriate approach to IBS-like altered colonic motor responses.
Results o btained s how t hat OVA-exposed rats h ave colonic contractility dysfunction,
including in creased r esponses to ca rbachol and L -NNA in vitr o and enhanced
spontaneous contractility a nd E MS-elicited responses in vivo . T hese results confirm
previous o bservations in t his model (4), r esembling that o bserved in I BS patients and
other an imal models of t he disease (5-8). T issue histological examination ex cluded
muscle hypertrophy as a potential cause for this carbachol and EMS enhanced responses
(data not shown), t hus s uggesting a n in creased e xcitability o f t he smooth muscle to
cholinergic s timulation. A p ossible ex planation f or t hese OVA-induced co lonic motor
alterations could be related to an excited-activated state of MMCs. This is suggested by
the higher tis sue concentration o f R MCPII o bserved in OV A-treated animals a nd
supported b y e vidences in vivo implicating mast cell degranulation in t he onset of
cecocolonic motor alter ations in r ats (27). MM C mediators ac t on nerve endings of
intrinsic and extrinsic primary afferent neurons forming neural networks within both the
submucosal an d myenteric plexuses, lead ing to a
local amplification o f ef fector
responses (28). Therefore, OVA-induced colonic motor alterations might arise as a result
of altered afferent nerve input into myenteric motor circuits due to a tonic activation of
MMCs.
48
CHAPTER 1
In a nimal models of I BS, both mast cells a nd NG F have b een i mplicated in co lonic
epithelial b arrier f unction, propulsive motor ac tivity a nd s ensitivity to co lorectal
distension (12,24). However, the exact source(s) of colonic NGF remains elusive and the
potential role of mast cells as the main source of intestinal NGF is controversial (24,29).
In o rder to elu cidate these points, i mmunohistochemistry f or t he neurotrophin was
performed in colonic tissues. Although we were able to see specific NGF staining, with
similar d istribution p atterns as those previously r eported (17), we did n ot f ind a ny
obvious difference in staining, intensity or distribution, among the different experimental
groups. This was further confirmed by real time PCR, showing similar expression levels
of NGF among experimental groups. Moreover, during double labeling studies, we were
unable to d etect NGF i mmunoreactivity i n MM Cs, id entified as RMCPII-positive ce lls
within t he colonic mucosa. This co ntrasts with a nimal data suggesting t hat NGF is
released by mast cells upon degranulation (24,30) and with data from colonic biopsies of
patients with functional and i nflammatory g astrointestinal disorders lo calizing NGF in
MMCs (14,31). Ho wever, o ur d ata agree with a recent s tudy i n a rat co litis model i n
which NG F immunoreactivity was not associated to M MCs (29). W hether o r no t t his
represents a s pecies-related d ifference (human v s. rat) an d/or experimental m odeldependent variations in the colonic source of NGF warrants further studies. Interestingly,
Stanzel et al. (2008) evidenced that NGF was synthesized mainly by epithelial cells and
hypothesized t hat MMCs co uld r epresent a source o f p ro-NGF, i n ag reement to th at
suggested also by studies on cultured rat peritoneal mast cells (32). Based on these data,
we also atte mpted, alth ough u nsuccessfully, to lo calize proNGF in co lonic MMCs.
Overall, our observations indicate that MMCs are not a cellular source of NGF in the rat
colon. Nevertheless, results obtained suggest a functional link between MMCs and NGF
and, in p articular, i ndicate th at MMCs ar e a target for N GF. First, we were able to
demonstrate the presence of TrkA receptors on a high proportion of colonic MMCs (by
60%). Seco nd, K2 52a treatment tended to i ncrease colonic RMCPII lev els, t hus
suggesting th at these r eceptors ar e functional and might mediate MMC d egranulation
upon stimulation with NGF. Indeed, the NGF ability to degranulate mast cells has been
previously demonstrated, both in vivo (33) and in vitro (34).
In th is s tudy, we aimed als o to elu cidate the functional i mplication o f N GF in t he
alterations o f co lonic smooth muscle contractility t hat characterize oral OVA e xposure
in rats. A role for NGF o n IBS-like gastrointestinal motor alterations in animal models
49
CHAPTER 1
has been previously suggested (12). In order to further assess this involvement of NGF
we used a pharmacological approach b ased on th e blockade of th e NGF h igh-affinity
receptor, TrkA, with K252a (35). In our conditions, treatment with K252a resulted in a
decrease of spontaneous colonic motor activity both in vivo and in vitro and prevented
the enhancement of t he nitrergic in hibitory to ne secondary to OVA ex posure in vitr o.
Interestingly, d irect addition o f K2 52a to th e organ b ath also d ecreased s pontaneous
colonic co ntractility ( data not shown), t hus r einforcing t he results o btained with t he
treatment with K2 52a and suggesting a nd e ffective b lockade of T rkA in in v ivo
conditions. From t hese o bservations, it is f easible to s peculate that a tonic NGFdependent stimulation might be necessary to maintain basal spontaneous contractility at
optimal conditions. Taking into consideration that rat enteric neurons express the highaffinity r eceptor T rkA, as previously d escribed an d also co nfirmed in this s tudy b y
immunohistochemistry (data not shown; 36), we can hypothesize that K252a is likely to
bind to T rkA receptors o n myenteric n eurons p reventing NGF-mediated e ffects within
the ENS and thus, affecting motor activity.
The dose and p attern o f ad ministration o f K2 52a followed here has already b een used,
showing b iological effects in vivo indicative of an e ffective b lockade of TrkA (18-20).
Therefore, it is f easible to as sume that the responses observed h ere are related to an
effective blockade of TrkA receptors. However, K252a not only binds to TrkA but also
to other neurotrophins receptors, mainly TrkB and TrkC, and other kinases, such as the
Ca2+/calmodulin k inase II (37) or th e myosin lig ht ch ain kinase (38), which a re
implicated in t he contractile ac tivity o f in testinal smooth muscle (39). Fro m o ur
observations, it cannot be ruled out that the effects observed might be associated, at least
partially, to K2 52a effects o n th ese targets. Nev ertheless, s everal observations s uggest
that the responses to K252a are likely to be associated to the blockade of TrkA receptors.
First, a recent study d emonstrated th at the pharmacological blockade of T rkA with
K252a, th e treatment with T rkA a ntisense oligonucleotides and t he in vivo
immunoneutralization of NGF were equally effective preventing chronic stress-induced
visceral hypersensitivity to co lorectal d istension i n r ats (17). Seco nd, in vivo
immunoneutralization of NGF normalized post-infectious gut dysmotility in T. spiralisinfected rats (a model of post-infectious-IBS) (12), as observed here in the OVA model
with K252a. Overall, these observations suggest that K252a-mediated effects within the
gastrointestinal tract are related to the modulation of NGF-TrkA-dependent mechanisms.
50
CHAPTER 1
Although up -regulation o f NGF a nd its h igh-affinity receptor T rkA has b een
demonstrated during colonic acute inflammation (29,31,40), expression results in animal
models of IBS are discordant (24,26,41). In the present study, only marginal changes in
TrkA, and n o ch anges in N GF expression lev els were observed a mong e xperimental
groups. This co uld s eem surprising as we demonstrated th at NGF /TrkA-dependent
pathways are implicated in the OVA-induced colonic dysmotility in the rat. However, an
interesting study in mice followed the increase in mRNA neurotrophins levels during the
inflammation o f t he colon a nd r evealed th at these ar e back to co ntrol conditions oneweek a fter th e induction, s uggesting a rapid a nd s hort-term secretion t ype of t hese
molecules d uring p athological conditions (42). Taking i nto co nsideration that
neurotrophins have both ac ute and lo ng-term biological e ffects (43), it is feasible to
speculate that NGF/TrkA mRNA colonic levels in the OVA-exposed rats at the time of
euthanasia may be n ot be r epresentative o f those d uring the ch ronic-treatment, ev en
when NGF-dependent co lonic dysmotility i s s till p resent. In ad dition, th ere is also th e
possibility t hat the OVA-induced in crease in MMC m ediators release exerts its effects
on the ENS through pathways i nvolving N GF/TrkA-dependent mechanisms alt hough
these are not up-regulated by OVA exposure. In any case, NGF/TrkA expression results
should b e interpreted ca utiously s ince t he interaction b etween T rkA and o ther
neurotrophin receptors (namely the p75 and the neurotrophin receptor homolog) leads to
an enhanced activity of the signaling pathways, without increasing the amounts of NGF
and/or T rkA r eceptors p er s e (44). It is feasible to assume th at th e marginal c hanges
observed in T rkA ex pression af ter OVA o r K2 52a treatment (32% and 2 6% increase,
respectively) might h ave consequences at the protein le vel yet to b e demonstrated. On
the o ne h and, K252a-induced ch anges i n T rkA ex pression co uld r epresent a
compensatory mechanism to th e receptor b lockade. On th e other h and, OV A-induced
changes might b e secondary to the OVA-mediated s timulation o f MM Cs and/or o ther
cell types, including enteric neurons, as discussed above. Interestingly, these changes in
TrkA ex pression were no lo nger o bserved in an imals receiving OV A an d K2 52a.
Although we cannot explain the mechanisms behind this effect, this observation further
supports an interplay between OVA effects and the NGF-TrkA pathway.
In s ummary, t he present study suggests th at NGF-TrkA-dependent mechanisms ar e
implicated i n b asal co lonic co ntractility a nd al so in OVA-induced co lonic motor
alterations in rats. In addition, our results show that MMCs express TrkA receptors and,
51
CHAPTER 1
therefore, represent a target for NGF, rather than being a source of the peptide, in the rat
colon. Ov erall, th is study highlights a
potentially i mportant role f or NGF -TrkA-
dependent signaling pathways on colonic motor alterations, as observed for instance in
FGDs. NGF receptors antagonists could represent a therapeutic target for the treatment
of gastrointestinal disorders characterized by altered colonic motility.
52
CHAPTER 1
Acknowledgments
We would lik e to th ank A. A costa for an imal care and E . Ma rtinez f or tech nical
assistance. T his work was s upported b y grant 2009SGR708 f rom t he Generalitat d e
Catalunya and B FU2007-6279, B FU2009-08229 a nd B FU2010-15401 f rom Mi nisterio
de Ciencia e Innovación.
Disclosures
F. J. d esigned an d p erformed ex periments, a nalyzed d ata and wrote t he paper. V. M.
designed an d p erformed ex periments a nd wrote the paper. P. V. d esigned ex periments
and wrote the paper.
53
CHAPTER 1
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CHAPTER 2
CHAPTER 2
CHAPTER 2
COLONIC M
OTOR
ALTERATIONS
SENSITIZATION T O A L
ASSOCIATED
UMINAL ANT IGEN ARE
TO TH
E
ENHANCED
DURING A DYSBIOTIC-LIKE STATE IN RATS
F. Jardí 1, M. Aguilera1, 2, P. Vergara 1,2,3, V. Martínez 1,2,3
1
Department o f C ell Bi ology, Phys iology and
Autònoma d e Barcelona, Spain.
Autònoma de
Immunology, Universitat
2
Instituto de N eurociencias, U niversitat
Barcelona.3 Centro de Investigación Biomédi ca en
Red de
Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III,
Madrid, Spain.
Running title: Microbiota and responses to food antigens
Key words: Food allergy; Functional gastrointestinal disorders; Gut commensal
microbiota; Irritable bowel syndrome; Ovalbumin
Submitted to Life Sciences
63
CHAPTER 2
Abstract
Background: Enteric dysbiosis is suggested a s a risk f actor for d ietary p roteinsassociated in testinal alter ations, co ntributing to t he development of f ood aller gies an d
the symptomatology o f f unctional gastrointestinal d isorders, mainly ir ritable bowel
syndrome (IB S). W e explored if a d ysbiotic-like state, s imulated b y ad ministration o f
bacterial lipopolysaccharides (LPS), fa cilitates t he sensitization to th e luminal a ntigen
ovalbumin (OVA), in rats. Methods: Rats were exposed to oral OVA for 1 week, alone
or with LPS. Thereafter, colonic histology, goblet cell density, mucosal eosinophils and
mucosal ( MMCs) a nd co nnective tissue mast cells (CTMCs) were ev aluated. Colonic
expression ( RT-qPCR) of interleukins, i nterferon alpha-1 and integrins was assessed to
determine lo cal immune r esponses. Luminal and wall ad hered m icrobiota were
characterized by fluorescence in situ hybridization. Colonic contractility (in vitro) served
to assess functional changes associated to OVA and/or LPS. Results: Neither OVA nor
LPS, alone or co mbined, lead to structural alterations, except for a reduced goblet ce ll
density in OV A-LPS-treated r ats. MM C d ensity was unaffected, while C TMC counts
increased within t he submucosa of OV A-LPS-treated an imals. Marginal i mmune
activation, with u p-regulation o f I L-6 an d i nterferon-alpha-1, was o bserved in OVALPS-treated rats. LPS induced a dysbiotic-like state characterized by decreased luminal
bacterial counts, with a specific loss of clostridia. LPS facilitated Clostridium spp. wall
adherence, an effect prevented by OVA. Colonic contractility was altered in OVA-LPStreated an imals, s howing in creased b asal activity a nd en hanced motor r esponses to
OVA. Conclusions: Changes in
gut microbiota might en hance/facilitate local
neuroimmune responses to food an tigens lead ing to motor alter ations similar to t hose
observed in IBS.
65
CHAPTER 2
Introduction
Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder
and is c haracterized b y ab dominal p ain-discomfort associated with d ysmotility a nd
altered b owel habits (1). Although its underlying p athophysiology r emains unclear,
recent e vidences s uggest th at I BS is d ue to a dysregulation o f t he brain-gut a xis, with
various p eripheral alterations co ntributing to th e exacerbation o f t he symptomatology
(2). For instance, both changes in gut microbiota and adverse reactions to food allergens
have been implicated in the pathogenesis of the disease (2-5).
Intestinal f ood aller gy-related mechanisms in I BS seem to in volve local mucosal
responses to d ietary an tigens with ac tivation o f r esident mast cells ( MCs), r ather t han
classical t ype-1 hypersensitivity r eactions (4,6). In t his lin e, we have p reviously
demonstrated t hat long-term ex posure to o ral ovalbumin ( OVA), without adjuvants,
results in a non-IgE mediated alteration of colonic motility in rats, an effect related to an
excited-activated state of t he tissue mucosal mast cells ( MMCs) (7). These OV Amediated ch anges are reminiscent of t hose observed in I BS patients an d in an imal
models of the disease (7-11).
Gut co mmensal microbiota (GCM) h as b een i mplicated in t he maintenance o f the
normal gastrointestinal hyporesponsive state to f ood an tigens. Fo r in stance, r esults
obtained in s tudies in i nfants with food aller gy s how a d isturbed b alance b etween
beneficial and potentially harmful bacteria in the large intestine and that supplementation
with probiotics ap pears to alleviate the allergic inflammation (12,13). Supporting these
observations, r esults from animal models demonstrate a
cause-effect r elationship
between d ysbiotic states with reduced GCM and the development of allergic responses
to oral antigens (14,15). Overall, these data suggest that a disruption of the GCM might
cause an impairment of the intestinal tolerogenic mechanisms, increasing the risk of food
protein-induced immune activation and the development of food allergy and/or IBS-like
alterations.
The aim of the present study was to further explore the impact of dysbiotic states of the
colon in the generation of abnormal responses to dietary antigens. For this purpose, we
treated r ats with lo w d oses o f b acterial lip opolysaccharides (LPS), s imulating a
67
CHAPTER 2
dysbiotic-like s tate (16). Simultaneously, a nimals were exposed o rally to the al lergic
protein OVA. The potential facilitatory effects of LPS towards OVA sensitization were
studied in v itro by a ssessing spontaneous co lonic contractility a nd th e co ntractile
responses elicited by the presence of the antigen (OVA). To further understand the role
of GCM in the functional alterations observed, changes in the colonic microbiome and
bacterial wall adherence were determined b y fluorescence in situ hybridization (FISH).
Finally, to g ain i nsight into the i mmune nature o f the I BS-like responses r elated to
dietary a ntigens, we assessed lo cal changes in c ytokines ex pression, t he potential
involvement of eosinophils and the dynamics of colonic MC populations.
Materials and methods
Animals
Adult (9 week-old), specific pathogen free (SPF), Sprague-Dawley (SD) male rats were
used (Charles River, Les Oncins, France). Animals had free access to water and were fed
with a standard diet (145 g/kg protein, 40 g/kg fat, 45 g/kg fiber and 13.4 kJ/g; free of
traces of o valbumin o r a ny other eg g d erivative; Teklad Global 14% Protein R odent
Maintenance Diet 2014, Harlan Interfauna Iberica S.A.). During all t he experiment, rats
were maintained under conventional conditions in a light (12h/12h light-dark cycle) and
temperature controlled ( 20-22 ºC) r oom, i n g roups o f t wo p er ca ge. An imals were
acclimatized to t he new e nvironment for 1 week b efore starting an y e xperimental
procedure. All the experimental protocols were approved by the Ethics Committee of the
Universitat Autònoma d e Barcelona and the Generalitat de C atalunya (protocols 1 010
and 5351, respectively).
Exposure to oral ovalbumin and treatment with LPS
Rats were exposed to OVA by a daily oral gavage (10 mg/mL, 1 mL/rat, n=15) during a
one w eek period. A group o f r ats were used a s co ntrols, i n which t he same ex posure
protocol was followed b ut OVA solution was replaced b y s aline (1 mL/rat, n= 15). I n
parallel, a subgroup o f v ehicle- and O VA-exposed r ats (n= 8 f or ea ch) was treated
intraperitoneally with L PS (100 μg/kg/day for 7 days). T he rest of th e r ats (7 vehicle-
68
CHAPTER 2
and 7 OVA - exposed an imals) r eceived the vehicle for LPS (1 mL/kg/day for 7 d ays).
Animals were examined for clinical signs and body weight changes on a daily basis (at
the time of treatment) and were euthanized by decapitation 24 h after the last treatment.
At t he time of e uthanasia, ti ssue samples from th e co lon were collected for functional
studies (organ bath studies) or either fixed in 4 % paraformaldehyde or Carnoy’s
solution, for immunohistochemical a nd fluorescent in s itu hybridization ( FISH) studies
respectively, or frozen in liquid nitrogen and stored at -80ºC until analysis.
Organ bath studies
Full t hickness p reparations were obtained from t he mid p ortion o f t he colon, cut 1 cm
long a nd 0 .3 cm wide and h ung, o riented to r ecord cir cular m uscle activity, for o rgan
bath studies. Strips were mounted u nder 1 g tension in a 10-mL o rgan b ath c ontaining
carbogenated Kr ebs s olution ( 95% O2 – 5% CO 2) maintained at 37 ± 1 ºC. The
composition o f Kr ebs s olution was (in mmol/L): 10.10 g lucose, 1 15.48 NaCl, 2 1.90
NaHCO3, 4.61 KCl, 1.14 NaH2PO4, 2.50 CaCl2, and 1.16 MgSO4 (pH 7.3–7.4). One strip
edge was tied to the bottom of the bath and the other one to an isometric force transducer
(Harvard VF-1 Har vard A pparatus I nc., Ho lliston, MA , USA ). O utput from t he
transducer was fed to a PC t hrough a n a mplifier. Data were digitalized ( 25 Hz) u sing
Data 2 001 s oftware (Panlab, Barcelona, Sp ain). Strip s were allowed to eq uilibrate for
about 1h. After this period, contractile responses to carbachol (CCh; 0.1-10 μM), bovine
serum alb umin ( BSA; 0.1 %) and OV A ( 0.1 %) were assessed. Fo r C Ch, cu mulative
concentration-response curves, with a 5 min i nterval between co nsecutive d oses, were
constructed. For BSA o r OVA, spontaneous ac tivity was r ecorded d uring a 15-min
period after the addition of the protein.
To determine the spontaneous contractile activity, the area under the curve (AUC) was
measured (in g) over a 15 min period. To assess the effects of CCh, the peak response,
from the b asal tone, was measured a fter ea ch co ncentration test ed. For ass essing t he
response to BSA or OVA exposure, the AUC was measured for a 10 min period before
the addition of the peptide and compared with the 10 min AUC measured during t he 5
min to 15 min period post-addition.
69
CHAPTER 2
Histology, immunohistochemistry and cell counting
Paraformaldehyde-fixed ti ssue samples were processed r outinely for p araffin
embedding, and 5-µm thick sections were obtained for haematoxylin and eosin (H&E),
toluidine or alcian blue staining or for immunohistochemistry. All the preparations were
viewed with an Olympus CH30RF200 microscope.
H&E-stained co lonic sections were used f or h istological examination o f t he tissue and
also f or as sessing eo sinophil i nfiltration, as determined by co unting t he number o f
eosinophils in 20 non-adjacent fields of colonic mucosa (X600).
MMCs were identified by immunodetection of rat mast cell protease II (RMCPII) using
a monoclonal an tibody anti-RMCPII ( Moredun Animal Health, E dinburgh, UK) . T he
secondary a ntibody was a b iotinylated horse antimouse IgG (BA-2000; Vec tor
Laboratories, Burlingame, CA, USA). Detection was performed with avidin/peroxidase
kit (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA). Specificity of the
staining was co nfirmed b y o mission o f t he primary an tibody. Sect ions were
counterstained with 1 % toluidine blue solution ( pH 0.5) f or 2 0 m in, which served to
identify C TMCs. Fo r MM C quantification, at least 2 0 n on-adjacent fields ( X400) of
colonic mucosa were randomly selected an d t he number o f R MCPII-immunopositive
cells d etermined manually. C TMCs were identified b y t he p resence o f to luidine bluestained metachromatic granules in t heir c ytoplasm. T otal n umber o f to luidine bluestained cells in the preparation was determined and then normalized for the surface area
of the tissue section. T o id entify goblet ce lls, co lonic tissue sections were stained with
Alcian Blue pH 2.5/Periodic Acid Schiff (AB 2.5/PAS kit; Bio-Optica, Milano, Italy) in
order to specifically s tain neutral (pink) and ac idic ( blue) mucins. Colonic goblet ce lls
were counted in 20 longitudinally-oriented villus-crypt units. Length of the villus-crypt
unit was determined to obtain goblet cell density (number of cells/mm). In all cases, cell
counting was carried out on coded slides to avoid bias.
RMCPII quantification
Protein was extracted f rom c olonic tissue s amples using lysis buf fer ( 50 mM HEPES,
0.05 % Triton X -100, 0.0625 mM P MSF and the Mi ni Complete p rotease inhibitor
70
CHAPTER 2
Roche) an d R MCPII co ncentration was d etermined b y E LISA using a commercial kit
(Moredun). T otal protein was determined u sing th e Bradford ass ay k it (BIO-RAD,
Hercules, CA, USA).
Bacterial identification by fluorescence in situ hybridization
For FISH, o ligonucleotide probes (Biomers, Ulm/Donau, Germany a nd T ib Mo lbiol,
Mannheim, Germany) with a 5’-Cy3 (c arbocyanine 3) d ye were used ( 5 ng/μL). The
bacterial groups characterized and the specific probes used are specified in Table 1.
In situ hybridization of bacteria in the luminal content was performed on glass slides, as
previously described by us (17-19). Samples were hybridized for 16 h by addition of the
hybridization buffer (20 mM Tris-HCl, 0.9 M NaCl, 0.1% SDS at pH 7.2) and thereafter
washed with the washing buffer (20 mM Tris-HCl, 0.9 M NaCl at pH 7.2) for 30 min.
4',6-diamidino-2-phenylindole (DAPI) s erved as
a control signal in all s amples.
Hybridized slides were viewed under oil immersion, using a Carl Zeiss Axioskop 40 FL
epifluorescence microscope (filter f or C y3) eq uipped with a digital camera (Zeiss
AxioCam M Rm) for o btaining d igital images ( Zeiss AxioVision Release 4 .8.1; C arl
Zeiss, Jena, Germany). For quantification of bacteria, 20 randomly selected fields were
photographed, the number of hybridized cells counted using the CellC software (20) and
the mean value obtained.
To ass ess b acterial ad herence to th e colonic wall, s ections f rom C arnoy-fixed tis sues
were hybridized in the same conditions. Slides were viewed under oil immersion and 20
randomly s elected f ields were photographed. A nalysis o f th e images was performed
manually b y th ree i ndependent researchers th at observed th e pictures and lo calized
hybridized b acteria within t he mucus la yer o r attac hed to th e epithelial surface. A
coincidence between two out of the three observers in bacterial location in at least three
out o f t he 20 p ictures observed was r equired to d ecide that there was bacterial
attachment to the epithelium (18). All procedures were performed o n co ded s lides, to
avoid bias.
71
CHAPTER 2
Table 1. Pro bes used fo r flu orescent in situ hybridization (FIS H) and hybridization
conditions
Probe
Sequence (5’3’)
Target
EUB 338
GCTGCCTCCCGTAGGAGT
NON 338
ACATCCTACGGGAGGC
Hybridization
Temp (ºC)
Bacteria
50
Non bacteria
50
(negative control)
EREC
GCTTCTTAGTCAGGTACCG
Clostridium
50
coccoides
482
cluster XIVa
ENT-D
TGCTCTCGCGAGGTCGCTT-
Enterobacteria
50
Bifidobacterium spp
50
CTCTT
BIF 164
CATCCGGCATTACCACCC
RNA extraction and quantitative real-time PCR
Total R NA was extracted from frozen co lonic s amples by h omogenization i n T rizol
reagent ( Ambion, Austin, T exas, USA) followed b y is opropanol precipitation a nd
quantification by Nanodrop (Nanodrop Technologies, Rockland, DE, USA). For cDN A
synthesis, 1 µg of RNA was reverse-transcribed in a 20 µl reaction volume using a high
capacity cDN A reverse transcription k it ( Applied B iosystems, Foster City, CA, U SA).
Real-time q uantitative P CR (RT-qPCR) w as performed to d etermine mRNA le vels of
inflammatory markers, integrins and resistin-like molecule-beta (RELMb) in the colon.
The Ta qMan® probes listed in T able 2 were used ( Applied B iosystems). T he PCR
reaction mixture was incubated on a 7900 real-time P CR system (Applied Biosystems)
for 40 c ycles (95ºC f or 15 s , 60 ºC f or 1 m in). B -Actin ex pression s erved as an
endogenous co ntrol for n ormalizing t he mRNA le vels of t he target genes. E xpression
levels were analyzed by the comparative Ct method (2-∆∆CT) using the vehicle-vehicle
group as the calibrator.
72
CHAPTER 2
Table 2. TaqMan® gene expression assays
Protein
Gene symbol Assay reference
Inflammatory markers
Interleukin 6 (IL-6)
Il6
Rn01410330_m1
Interleukin 13 (IL-13)
Il13
Rn00587615_m1
Interleukin 10 (IL-10)
Il10
Rn00563409_m1
Interleukin 12 (subunit beta) (IL-12)
Il12b
Rn00575112_m1
Interferon alpha-1 (IFN-alpha-1)
Ifna1
Rn02395770_g1
Integrin beta-1
Itgb1
Rn00566727_m1
Integrin alpha-2
Itga2
Rn01489315_m1
Resistin-like molecule beta (RELM-beta)
Retnlb
Rn01439306_m1
Actb
Rn00667869_m1
Markers of host-microbial interactions
Reference gene
B-Actin
Chemicals
OVA ( Grade V; A5503), B SA ( A9085) and LPS (LPS from Escherichia Coli 055:B5;
62326) were purchased from Sigma-Aldrich (St.Louis, MO, USA) and were dissolved in
saline solution. Carbachol (Sigma-Aldrich) was dissolved in distilled water at a 10 -1 M,
further dilutions were prepared in distilled water.
Statistics
All data are expressed as mean ± SEM; except for bacterial counts that are expressed as
media (interquartile r ange) ± SD. A r obust a nalysis ( one iteration) was used to o btain
mean ± SEM for RT-qPCR data. Comparisons between multiple groups were performed
by a on e-way o r a two-way A NOVA, as appropriate, followed w hen n ecessary b y a
Fisher’s least significant difference (LSD) post hoc test to detect differences between
experimental groups. P values < 0.05 were considered statistically significant.
73
CHAPTER 2
Results
Animal monitoring
Exposure to o ral OVA alo ne during o ne week d id not af fect body weight co mpared to
animals r eceiving saline. Addition o f LPS produced a transitory lo ss o f b ody weight,
evident only during the first day of treatment. LPS-induced body weight loss was similar
in v ehicle-LPS (5.24 ± 1 .01 %; P < 0 .05 v s. v ehicle-vehicle group) an d OV A-LPStreated groups (4.05±0.77%; P < 0.05 vs. OVA-vehicle group). Thereafter, the repeated
administration o f LPS did not longer af fect b ody weight a nd s imilar weight gain was
observed across experimental groups. No other clinical signs were observed.
Colonic histology
Histological examination o f the colon r evealed n o s ubstantial effects as sociated to
treatments, without evidences o f tis sue damage or in flammatory-like changes. T he
number o f eo sinophils i nfiltrating th e colonic mucosa was similar ac ross ex perimental
groups ( Fig. 1 A). Qu antification o f goblet ce lls i n AB-PAS-stained s ections r evealed
that LPS treatment was associated to a diminished cell density, as assessed by a two-way
ANOVA, reaching s tatistical significance in OV A-LPS-treated rats ( 122.7±3.8
cells/mm; P<0.05 vs. vehicle-vehicle: 153.8±4.0 cells/mm; Fig. 1B). Animals exposed to
OVA alo ne also s howed a tendency for a decrease in th e relative abundance o f g oblet
cells (133.8±6.75 ce lls/mm; P= 0.05 v s. vehicle-vehicle; Fig. 1 B). I n vehicle-vehicletreated animals, co mbined AB-PAS staining r evealed that nearly al l the goblet ce lls o f
the colonic mucosa contained ac idic mucins ( blue staining), with very few goblet ce lls
showing mixed o r n eutral mucins ( purple and p ink s taining, r espectively). Relative
abundance of acidic, mixed and neutral mucins was not affected by OVA or LPS or their
combination.
Mast cells counts and RMCPII content
Colonic MMC counts remained unaltered after one week exposure to oral OVA (OVAvehicle: 5.26±0.90 cells/field; P>0.05 vs. vehicle-vehicle: 5.80±2.18 cells/field; Fig. 1C).
74
CHAPTER 2
Addition o f L PS had n o e ffect o n th e colonic d ensity o f MM Cs ( Fig. 1 C). Si milarly,
colonic content of R MCPII w as u naffected b y OVA ( OVA-vehicle; 0.55±0.21 n g/µg
protein; vehicle-vehicle: 0.56±0.23 ng/µg protein; P>0.05) and remained unaltered after
LPS treatment (Fig. 1D).
CTMCs, id entified b y its
methacromatic g ranules, were mainly lo calized in t he
submucosa of t he rat co lon, with a
relatively lo w d ensity i n co ntrol conditions
(2.73±0.29 cells/field; Figs. 1E and 2). Neither OVA nor L PS, per se, affected CTMCs
counts. However, in OVA-LPS-treated animals the counts of CTMCs were increased by
2-fold when co mpared with th e separate treatments o r th e vehicle-vehicle gr oup
(P<0.05; Figs. 1E and 2).
G o b le t C e lls ( p e r m m o f c r y p t )
B
E o s in o p h ils ( p e r f ie ld , X 6 0 0 )
A
10
8
6
4
2
0
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
LPS
LPS
200
&
150
+
100
*
50
0
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
LPS
LPS
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
LPS
LPS
M M C s ( p e r f ie ld , X 4 0 0 )
R M C P I I ( n g /µ g p r o t e in )
D
C
10
8
6
4
2
0
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
LPS
LPS
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
C T M C s ( p e r f ie ld , X 1 0 0 )
E
10
8
*
6
4
2
0
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
LPS
LPS
Figure 1 . Co lonic density o f eosinophils (A), goblet cells (B ), mucosal mast c ells
(C), RMCPII c ontent (D) a nd c onnective tis sue mast c ells (E) in t he different
experimental g roups. Da ta are m ean±SEM o f 4 -8 a nimals pe r group. *: P<0.05 v s.
vehicle-vehicle g roup. & : P= 0.05 v s. vehicle-vehicle g roup. + : P= 0.09 v s. vehiclevehicle group.
75
CHAPTER 2
Figure 2 . Co nnective tiss ue mast cells. Representative microphotographs sh owing
connective ti ssue m ast c ells (CTMCs) in to luidine blue-stained colonic slice s f rom
vehicle-vehicle- (A), OVA-vehicle- (B), LPS-vehicle- (C) and LPS-OVA-treated (D) rats.
The arrows indicate CTMCs in the submucosa, as identified b y the presence of toluidine
blue-stained m etachromatic g ranules in th eir c ytoplasm. No tice th e in creased density o f
the CTMC population in the colonic submucosa of LPS-OVA-treated animals (D).
Characterization of luminal and wall-adhered microbiota
In ve hicle-vehicle-treated an imals, to tal b acterial co unts i n th e luminal content of t he
colon, determined by FISH as EUB338-positive cells and confirmed by DAPI staining,
were within t he margins p reviously d escribed b y us (17,19) (3.74×1010±1.16×1010
cells/mL; Fig. 3). Total bacterial counts were not altered by the administration of OVA
alone (2.90×1010±6.10×109 cells/mL; P>0.05 v s. v ehicle-vehicle; Fi g. 3). Ho wever,
treatment with LPS diminished by 50% total bacterial counts vs. vehicle-vehicle values,
irrespective of the coadmnistration of OVA (LPS-vehicle: 1.75×1010±3.10×109 cells/mL;
LPS-OVA: 1.62×1010±1.84×109 cells/mL; both P<0.05 vs. vehicle-vehicle; Fig. 3). This
effect was further confirmed by similar results obtained in DAPI-positive nuclei counts
(Fig. 3).
76
CHAPTER 2
Independently of the changes in total bacterial counts, LPS and OVA resulted in states of
dysbiosis, a ffecting s electively th e Clostridium cluster XI V or coccoides group
(EREC482 probe). Overall, Clostridium spp. was the most abundant strain, irrespective
of th e treatment considered. E xposure to o ral OVA alo ne or L PS alone decreased
Clostridium spp. co unts in s imilar p roportion ( OVA-vehicle: 7.83×109±1.41×109
cells/mL; v ehicle-LPS: 7.53×109±1.19×109 cells/mL; P= 0.07 an d P = 0.05 v s. v ehiclevehicle, r espectively; Fi g. 3). T he combination o f b oth tr eatments r esulted in a f urther
reduction i n Clostridium spp. c ounts t o 4.26×109±4.03×108 cells/mL ( P<0.05 v s.
vehicle-vehicle; Fi g. 3). Other b acterial g roups a ssessed, n amely Bifidobacterium spp.
(BIF164 p robe) an d E nterobacteria ( ENT-D p robe) w ere not affected b y ei ther OV A,
LPS or their combination.
T o ta l b a c te r ia
*
1 0 11
C e ll s /m L
T o ta l c e lls - D A P I
1 0 10
1 0 12
*
1 0 11
C e ll s /m L
1 0 12
10 9
10 8
10
1 0 10
10 8
10 6
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
LPS
LPS
C lo s tr id iu m sp p .
9
10 8
LPS
1 0 10
10 9
10 8
10 6
OVA
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
LPS
LPS
V e h ic le
V e h ic le
LPS
LPS
1 0 11
1 0 10
10 9
8
10 7
10 6
V e h ic le
OVA
V e h ic le
OVA
V e h ic le
V e h ic le
LPS
LPS
w a ll- a d h e r e n c e ( % )
V e h ic le
E n te r o b a c te r ia
C e ll s /m L
OVA
LPS
10 7
7
12
10
V e h ic le
V e h ic le
1 0 11
*
I n c id e n c e o f b a c t e r ia l
C e ll s /m L
+
C e ll s /m L
&
1 0 10
10
OVA
V e h ic le
1 0 12
1 0 11
10 6
V e h ic le
B if id o b a c te r iu m s p p .
1 0 12
10
*
10 9
10 7
7
10 6
10
*
100
V e h ic le -V e h ic le
80
O V A -V e h ic le
60
O V A -L P S
V e h ic le -L P S
40
20
0
Ent
.
B if . s p p . C l o s t . s p p .
Figure 3. Composition of th e lu minal microbiota and in cidence o f b acterial
wall adherence, a s assessed b y FISH. Bacterial c ounts are m edia (interquartile
range) ± SD, 7-8 animals per group. *: P<0.05 vs.vehicle-vehicle group. +: P=0.05
vs. v ehicle-vehicle g roup. & : P = 0.07 v s. vehicle-vehicle g roup. I ncidence of
bacterial wall a dherence (b ottom rig ht corner): data represent t he percentage o f
animals showing bacterial wall adherence for the different bacterial groups assessed.
Ent: Enterobacteria; Bif. spp: Bifidobacterium spp; Clost. spp: Clostridium spp.
77
CHAPTER 2
In v ehicle-vehicle-treated an imals, b acterial ad herence to th e colonic ep ithelium was
relatively lo w and similar for all t he bacterial groups assessed (by 20% incidence; Fig.
3). Treatment with OV A, d id n ot affect the incidence o f b acterial wall ad herence.
However, L PS showed a te ndency to favour t he adherence o f Bifidobacterium spp.
(incidence: 3 5%) and Clostridium spp. (incidence: 5 0%) (Fig. 4 ). T he combination o f
OVA and LPS completely prevented the adherence of Clostridium spp. (0% incidence)
without affecting the adherence of Bifidobacterium spp. (incidence: 35%).
Expression of inflammatory markers and markers of host-bacterial interactions
In control co nditions (vehicle-vehicle-treated an imals), c olonic mRNA e xpression o f
pro-inflammatory ( IFN-alpha-1, IL-6, IL-12 and IL-13) an d anti-inflammatory m arkers
(IL-10) was relatively low, but within detectable ranges in all s amples. Overall, relative
expression of cytokines was: IFN-alpha-1>IL-10>IL-12-beta>IL-6~IL-13.
Vehicle-Vehicle
OVA-Vehicle
Vehicle-LPS
OVA-LPS
Figure 4. Representative colonic tissue images showing wall adherence of Clostridium spp
(EREC 482 probe) in the different experimental groups.
78
CHAPTER 2
IL -6
m R N A f o ld c h a n g e
3
2
1
V e h ic le
OVA
3
OVA
m R N A f o ld c h a n g e
3
V e h ic le
OVA
LPS
LPS
I F N - a lp h a -1
#
1
V e h ic le
OVA
3
V e h ic le
OVA
LPS
LPS
+
&
1
0
V e h ic le
OVA
V e h ic le V e h ic le
V e h ic le
OVA
LPS
LPS
V e h ic le
OVA
LPS
LPS
V e h ic le
OVA
LPS
LPS
V e h ic le
OVA
LPS
LPS
IL -1 3
2
1
0
V e h ic le
OVA
3
I n te g r in b e t a -1
2
1
0
V e h ic le
OVA
V e h ic le V e h ic le
I n te g r in a lp h a -2
2
3
OVA
V e h ic le V e h ic le
*
V e h ic le V e h ic le
m R N A f o ld c h a n g e
V e h ic le V e h ic le
2
0
0
V e h ic le
1
V e h ic le V e h ic le
1
LPS
I L - 1 2 - b e ta
V e h ic le
2
LPS
2
0
IL -1 0
OVA
m R N A f o ld c h a n g e
m R N A f o ld c h a n g e
V e h ic le V e h ic le
3
V e h ic le
m R N A f o ld c h a n g e
0
m R N A f o ld c h a n g e
m R N A f o ld c h a n g e
Exposure to o ral OV A alo ne did n ot affect cytokines ex pression; with t he
exception o f I L-12-beta, which was s lightly u p-regulated alth ough s tatistical
significance was not reached. Similarly, LPS alone had minor effects on cytokines
expression, with only a tendency (P=0.06) to increase colonic IL-6 levels (Fig. 5).
However, co mbined treatment with OVA and LPS resulted in a two-fold increase
in the expression of IFN-alpha-1 (Fig. 5).
5
R E L M -b e ta
4
3
2
1
0
V e h ic le
OVA
V e h ic le
LPS
LPS
V e h ic le V e h ic le
OVA
Figure 5. Colonic e xpression of inflammatory m arkers and m arkers of
host-bacterial i nteractions. Each s ymbol represents a n in dividual animal, th e
horizontal lines with errors represent the mean±SEM. *: P<0.05 vs. vehicle-LPS
and O VA-vehicle g roup. # : P= 0.08 v s. vehicle-vehicle g roup. + : P= 0.09 v s.
vehicle-vehicle group. &: P=0.07 vs. vehicle-LPS group.
79
CHAPTER 2
In co ntrol conditions, markers o f h ost-bacterial in teractions, n amely i ntegrin b eta-1,
integrin alpha-2 and RELM-beta, were moderately expressed. Relative expression levels
were: integrin b eta-1>integrin a lpha-2>RELM-beta. OV A tended to d own-regulate
integrin alpha-2, particularly in animals co-treated with LPS (Fig. 5).
Colonic contractility in vitro
Spontaneous co lonic contractile activity was similar in v ehicle-vehicle- and OV Avehicle-treated animals ( OVA-vehicle: 3 9.81±4.88 g ; vehicle-vehicle: 34.19±2.39 g ;
P>0.05; Fig. 6A). A two way ANOVA analysis revealed that the challenge with LPS had
a significant ef fect ( P= 0.04) en hancing co ntractile ac tivity b oth i n vehicle-LPS- and
OVA-LPS-treated rats ( Fig. 6A). H owever, f urther post hoc test f ound n o differences
between specific groups.
In vehicle-vehicle-treated animals, CCh elicited contractile responses in a concentrationdependent manner. Ne ither OVA nor LPS, alone o r in co mbination, a ffected t he
contractile responses to CCh (Fig. 6B).
The s pontaneous co ntractile activity o f ti ssue samples o btained from vehicle-vehicletreated an imals was n ot af fected b y t he direct addition of OV A to t he organ b ath.
Similarly, i n tis sues from a nimals exposed to o ral OVA d uring o ne week, d irect
exposure to OVA was without effect. However, OVA challenge on tissues obtained from
OVA-LPS-treated r ats resulted in an i ncrease in t he frequency an d magnitude of
spontaneous co ntractile ac tivity ( P<0.05). Nev ertheless, s ome variability was observed
in t he responses to d irect OVA ex posure, with 6 0% of th e animals being clea rly
“responders”, as determined by a response to OVA challenge above two standard errors
from t he mean r esponse in the vehicle-vehicle group ( Fig. 6C). T he frequency o f
“responders” in the other experimental groups oscillated between 0% and 25% (Fig. 6C).
Regardless the experimental group considered, direct addition of a BSA solution to the
organ bath did not affect colonic spontaneous contractile activity.
80
CHAPTER 2
Figure 6. Colonic contractility in vitro. (A) Colonic spontaneous contractile activity in
the d ifferent experimental g roups. Data are mean ± SEM; n = 4 -6 p er group. (B)
Concentration-response curves for carbachol. Data represent mean values (symbols) and
non-linear re gression curves. n = 4 –6 pe r group. (C) C olonic c ontractile re sponses to
OVA challenge. Ea ch s ymbol represents an in dividual animal, th e h orizontal lines with
errors represent the mean±SEM. The broken horizontal line corresponds to the mean+2x
SEM.*: P <0.05 v s. vehicle-LPS group. #: P= 0.09 v s. OVA-vehicle group. (D)
Representative tracings showing the effects of OVA and BSA challenge on spontaneous
colonic contractility in an OVA-LPS-treated animal. Notice the increase in the magnitude
and f requency o f c olonic contractions after addition of O VA to t he or gan ba th, a n
effected non-observed after the addition of BSA.
Discussion
Dietary an tigens an d g ut microbiota are potential factors co ntributing to th e
pathophysiology of IBS. In this study, we show an interaction between a dietary antigen
(OVA) and microbial components that leads to a situation of dysbiosis together with an
altered colonic contractility, mimicking some of the features described in IBS patients.
Our r esults indicate that LPS f avors th e colonic sensitization to OV A, t hus s uggesting
that al terations o f t he commensal microbiota an d the gut host-microbiotal in teractions
81
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might be factors facilitating the intestinal (colonic) sensitization against dietary antigens
and the development of IBS-like states.
We previously s howed t hat r ats exposed to o ral OV A, without ad juvants, d uring a 6week p eriod, d evelop a colonic contractility d ysfunction, resembling that obs erved i n
IBS patients a nd o ther an imal models of the disease (7-11,21). However, r epeated
exposure to lo w d oses of a dietary a ntigen might lead to a process o f o ral tolerance
(22,23). Therefore, in t he present work, we ass essed t he ef fectiveness o f a s horter
exposure period to high d oses of o ral OVA i n t he induction o f I BS-like co lonic motor
alterations. Results obtained show that colonic contractility remained unaltered after 1 week e xposure to 1 0 m g o f OV A, t hus s uggesting t hat, in co ntrast to lo ng-term
exposure, sensitization to oral antigens induced by few administrations might require the
presence o f f acilitatory e lements, such a s ad juvants (24). We also ex plored i f G CM
might r epresent a p otential factor f avoring the induction of ad verse reactions to f ood
antigens. Administration of low-doses of LPS, which mimics a gram-negative bacterial
overload, resulted in a specific dysbiosis of the colon. In addition, when combined with
oral O VA, LPS induced an e nhanced co lonic co ntractility to th e direct ad dition o f th e
antigen to th e organ b ath, th us i ndicating a sensitization o f th e tissue to OV A. T hese
altered r esponses of th e colon ar e reminiscent of th e exacerbated s ymptomatology
reported in a subgroup of IBS patients after the ingestion of certain foods, reinforcing the
hypothesis that adverse reactions to dietary components might be a contributing factor to
the pathophysiology of the disease (25). Moreover, results shown here support the view
that gut microbiota might facilitate the sensitization process to luminal antigens, thereby
representing a factor lead ing to th e development or th e exacerbation o f s ymptoms i n
IBS.
Dietary-related I BS-like responses have been as sociated, b oth in p atients an d an imal
models, to local mechanisms of the colonic mucosa rather than a systemic reaction, more
characteristic o f food aller gies (4,6,7). In ag reement with t hese o bservations, in th is
study, OVA-induced altered contractility in OVA-LPS-treated rats was neither related to
the presence o f cir culating s pecific IgEs ( unpublished r esults) nor to th e altered
expression of pro- (IL-6 and IL-13) or anti-allergic (IL-12 and IL-10) cytokines (26,27).
However, these animals showed a specific up-regulation of IFN-alpha-1, similar to that
observed in IBS-like states (28;29).
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Colonic motor changes after long term exposure to oral OVA are related to an excitedactivated state of MM Cs (21). Moreover, MM Cs have been d irectly i mplicated i n the
pathophysiology o f I BS, although th eir exact role r emains unclear (30-32). Here, no
changes i n MM C d ensity o r their e xcited-activated s tate was o bserved a fter a 1-week
OVA e xposure, with o r without LPS. However, CTMC c ounts were increased i n th e
submucosa of OVA-LPS-treated animals, while unaffected in animals treated only with
OVA o r L PS. Although t he p otential involvement of CTMCs in I BS has been le ss
studied, several observations suggest that they could act also as effector cells leading to
functional alterations within the gut. For instance, we have described a similar increase
in C TMCs in t he jejunum o f Trichinella s piralis-infected r ats, an ac cepted m odel of
post-infectious-IBS that al so courses with d ysmotility (33). Moreover, CTMC co unts
are increased in mice with food allergy (34,35) and the degranulation of CTMCs excites
the intestinal smooth muscle in vitro (36). Although further in deep studies are required,
these observations s uggest t hat submucosal CTMCs could b e taking p art in t he altered
contractile responses observed in OVA-LPS-treated rats.
Gut
microbiota
is a
dynamic
component o f g astrointestinal h omeostasis.
Epidemiological and clinical data support the hypothesis that significant perturbations of
the GCM can disrupt the mechanisms of oral tolerance leading to allergic responses (37).
In agreement, in the current study, low doses of LPS facilitated the sensitization to oral
OVA at th e same time that i nduced ch anges in GCM (an o verall d ecrease in lu minal
bacterial counts with a specific dysbiotic state characterized by a loss of clostridia). At
the same ti me, LPS favored the adherence o f Clostridium spp. to t he colonic wall. A
cause-effect relationship b etween ad herence and lu minal microbiota can b e suggested
from these changes. It is feasible to speculate that increased host-bacterial interactions,
driven b y th e ad herence o f cl ostridia, might tr igger a local r esponse that results i n t he
dysbiotic-like s tate o bserved when assessing t he luminal microbiota. The apparent
mismatch b etween ad herence an d l uminal bacterial co unts is similar to that o bserved
previously in other models of d ysbiosis and suggests that, besides the absolute number
of bacteria, other factors modulate host-bacterial interactions (18). Nevertheless, we did
not observe changes in t he overall ex pression o f ad hesion molecules o r R ELM-beta,
important co mponents facilitating host-bacterial in teractions within th e gut. From t he
present studies, we cannot state that the observed changes in the microbiome are relevant
in the facilitatory effects of LPS towards OVA sensitization. However, this hypothesis is
83
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supported b y p revious r esults th at demonstrate a direct relationship b etween alter ed
states of GCM and increased susceptibility to sensitization to oral dietary antigens (14).
Abnormal intestinal responses to dietary antigens have been related in part to an altered
epithelial barrier f unction (38). Indeed, alter ed g ut microbiota is ab le to f avor
sensitization towards dietary antigens throughout changes in epithelial permeability (38).
Here, although not directly assessed, it is feasible to assume the presence o f an altered
barrier function as the doses and pattern of administration of LPS were similar to other
studies demonstrating a n i ncreased ep ithelial permeability (39,40). In ad dition,
adherence o f Clostridium spp. to th e colonic epithelium, f avored b y LPS, could b e a
contributing factor i n t he alteration o f t he barrier f unction a nd t he subsequent
sensitization to OV A. Surprisingly, i n O VA-LPS-treated r ats no ad herence o f
Clostridium spp. was observed (0% incidence). This might be associated to the presence
of co lonic hypercontractility g enerated b y th e p resence of OV A i n a state of LPSinduced en hanced s ensitization. I n th ese conditions, in creased co lonic motility might
difficult the interaction between luminal bacteria and the epithelium, preventing bacterial
adherence. In agreement with this hypothesis, we observed that direct addition of OVA
to the organ bath only increased colonic contractile activity in tissues from OVA-LPStreated animals. Moreover, this effect on bacterial dynamics might be potentiated by the
enhanced mucus release present in OVA-LPS-treated rats, as suggested b y the reduced
density o f g oblet ce lls (41). Thus, i n O VA-LPS-treated a nimals, the mucus d ischarge
could f acilitate the trapping of b acteria and p revent their attac hment to th e epithelial
surface, while t he altered motor r esponses would ass ist in th eir s ubsequent ex pulsion
(42).
In s ummary, th is s tudy s hows a n in teraction b etween l uminal dietary a ntigens an d
components o f th e g ut microbiota leading to p otential a lterations i n co lonic motor
activity. In particular, we show that a dysbiotic situation favors the sensitization against
a luminal dietary antigen, namely OVA, and the generation of abnormal motor responses
against that antigen. Si milar m echanisms might contribute to th e pathophysiology o f
IBS, where a significant proportion o f p atients s how an al tered m icrobiota an d
sensitivity to ce rtain f ood co mponents. Alterations of th e microbiota might
enhance/facilitate the local neuroimmune responses to specific food antigens leading to
motor alterations reminiscent of those observed in IBS.
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CHAPTER 2
Acknowledgments
We thank A. Acosta and E . Ma rtínez f or th eir tech nical ass istance. T his work was
supported b y g rant 2009SGR708 f rom t he Generalitat de Catalunya and B FU200908229 and BFU2010-15401 from Ministerio de Ciencia e Innovación.
Disclosures
F. J. designed an d p erformed ex periments, a nalyzed d ata and wrote t he paper. M . A.
designed and performed experiments. P . V. designed experiments and wrote the paper.
V. M. designed and performed experiments, and wrote the paper.
85
CHAPTER 2
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CHAPTER 3
CHAPTER 3
PLASTICITY O F DO RSAL RO OT G ANGLION NE URONS IN A R AT
MODEL O F P OST-INFECTIOUS GUT DYSFUNCTION: POTE NTIAL
IMPLICATION OF NERVE GROWTH FACTOR
F. Jardí 1, J.A. Fernández-Blanco1, V. Martínez *,1,2,3, P. Vergara 1,2,3
1
Department o f C ell Bi ology, Phys iology and
Autònoma de Barcelona, Barcelona, Spai n.
Immunology, Universitat
2
Instituto de N eurociencias,
Universitat Autònoma de Barcelona, Barcelona, Spain.3 Centro de Investigación
Biomédica en Red
de E nfermedades Hepáticas y D igestivas (CIBERehd),
Instituto de Salud Carlos III, Madrid, Spain.
Running title: Nerve growth factor and plasticity of enteric afferent neurons
Keywords: Irritable bowel syndrome; Mucosal mast cells; Nerve growth factor;
Trichinella spiralis; TrkA; visceral afferents
Submitted to Neuroscience Letters
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Abstract
Background Intestinal infections are suggested as a risk factor for the development of
irritable b owel s yndrome (IBS)-like visceral hypersensitivity. T he mechanisms
implicated might i nvolve long-term c hanges in v isceral afferents, with i mplication o f
nerve growth factor (NGF). We explored plastic changes in dorsal root ganglion (DRG)
neurons innervating the gut, and the potential implication of NGF, in a rat model of IBSlike post-infectious gut dysfunction. Methods Rats were infected with T. spiralis larvae.
30 days post-infection, inflammatory markers, including interleukins (ILs) and mucosal
mast cell ( MMC) i nfiltration [rat mast ce ll protease II ( RMCPII)], and NGF and T rkA
expression was determined in the jejunum and colon (RT-qPCR). In the same animals,
morphometry ( neuronal body size) an d NGF co ntent (immunofluorescence) were
assessed in th oracolumbar D RG neurons. Results In in fected an imals, a low grade
inflammatory-like response, ch aracterized b y up-regulated levels of R MCPII an d I L-6,
was observed in the jejunum and colon. TrkA expression was increased in the jejunum
while in the colon showed a slight reduction. NGF le vels remained unaltered regardless
the gut region. Overall, the mean cross-sectional area of DRG neurons was increased in
T. spiralis-infected an imals, with a
reduction i n b oth T rkA an d NG F staining.
Conclusions & Inferences Results suggest that during T. spiralis infection in rats, there
is a remodeling o f sensory af ferents t hat might imply a NGF -mediated m echanism.
Plastic changes in sensory afferents might mediate the long-lasting functional alterations
that characterize this model of IBS. Similar mechanisms might be operating in patients
with post-infectious-IBS.
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Introduction
Alterations in ep ithelial, neural and i mmune f unctions ar e lik ely to co ntribute to t he
initiation and perpetuation of functional gastrointestinal disorders (FGDs). For instance,
remodeling of enteric sensory afferents is suggested to be implicated in the development
of th e visceral hypersensitivity o bserved in ir ritable bowel s yndrome (IBS) (1). In th is
line, it has been shown that intestinal infections evoke hyperexcitability of colonic dorsal
root ganglion ( DRG) n eurons an d in crease afferent nerve activity o f th e jejunum,
suggesting a potential role for these processes on the modulation of afferents’ properties
(2-4).
Data suggest t hat neurotrophins, m ainly ne rve growth f actor ( NGF), ar e implicated in
the pathophysiology o f I BS-like sensorial alter ations. For in stance, NGF co ntent is
increased i n r ectal mucosa o f d iarrhea-predominant I BS children (5). I n ad dition, t he
ability o f N GF to in duce long-lasting c hanges in th e morphological and
electrophysiological properties of DRG neurons has been widely reported in vitro (6,7)
and in vivo (8). T hus, it is feasible to hypothesize that N GF might co ntribute to t he
remodeling o f en teric afferents an d t he onset and/or maintenance o f t he visceral
hypersensitivity states that characterize IBS.
In th e model of e xperimental infection with Trichinella s piralis in r ats, a validated
model of pos t-infectious-IBS (PI-IBS), we h ave demonstrated th e p resence o f a lo nglasting j ejunal epithelial barrier d ysfunction as sociated to a persistent state o f lo cal
immune activation a nd mucosal mast cell (MMC) in filtration (9,10). I n th e present
study, we used the same model with the aim of characterizing changes in enteric sensory
afferents a nd th e potential implication o f NGF. At 3 0 d ays p ost-infection ( PI), w e
assessed the presence of a persistent inflammatory-like state in the jejunum and colon. In
the same animals, morphometric changes in thoracolumbar DRGs (innervating the small
intestine and colon) were used as a surrogate marker for alterations in sensory afferents.
Finally, to assess the potential implication of NGF we determined the expression of the
peptide and its high-affinity receptor TrkA within the gut and in DRGs.
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Materials and Methods
Animals
Adult ( 6 we ek-old at arrival), s pecific pathogen free ( SPF), Sp rague-Dawley male r ats
were used ( Charles River, L es Oncins, Fra nce). Rats were maintained u nder
conventional conditions in a light ( 12h/12h light-dark cycle) an d te mperature controlled
(20-22ºC) r oom, in groups of t wo to th ree p er ca ge, with ac cess to tap water a nd
laboratory rat chow (A04; Safe, Augy, France) ad libitum. Animals were acclimatized to
the new en vironment for o ne week b efore starting a ny experimental p rocedure. All
experimental p rocedures were ap proved by the E thics Committee o f the U niversitat
Autònoma d e B arcelona and the Ge neralitat de Catalunya (protocol numbers 1013 a nd
5352, respectively).
Trichinella spiralis infection
Muscle-stage larvae of T. spiralis were obtained from infected CD1 mice as previously
described (9,10). Rats were infected at 7–8 weeks of age by ad ministration o f 7.500 T.
spiralis larvae, suspended in 1 mL of saline, by oral gavage. Studies were performed on
days 30±2 post-infection (PI). Age-matched rats dosed orally with 1 mL of saline were
used as controls. After the infection, animals were regularly monitored for clinical signs
and body weight changes. Normal course of the infection was confirmed by a significant
decrease of b ody weight in in fected an imals c ompared with co ntrols, with a peak
reduction o n d ays 8 -to-10 P I and a subsequent linear i ncrease over ti me, a s p reviously
described by us (9,10).
Experimental procedures and tissue sampling
Non-infected co ntrols o r an imals at 30 d ays P I were euthanatized b y d ecapitation. A
laparotomy was performed and jejunal and middle colon samples were obtained, frozen
in liq uid nitrogen a nd s tored at -80ºC u ntil analysis. At th e s ame time, th e s pinal
thoracolumbar segment was removed and fixed for 24 h in cold 4 % paraformaldehyde.
Thereafter, dorsal root ganglia (DRGs) from T10 to L2 (T10-L2) were dissected, pooled
together an d p rocessed f or m orphological and i mmunohistochemical studies. T 10-L2
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DRGs contain afferent fibers innervating both the small intestine and the colon of the rat
(11-13).
NGF and TrkA immunofluorescence staining in DRGs
Immunodetection o f NGF a nd TrkA was carried o ut separately o n p araformaldehydefixed DR G sections ( 5 µ m) b y o vernight incubation at 4ºC with a rabbit polyclonal
antibody against NGF (ab1526; Chemicon International, Temecula, CA, USA) or TrkA
(sc-118; Santa Cruz Biotechnology, Sa nta Cruz, C A, U SA). An tigen r etrieval was
achieved b y microwave processing o f th e slides (10mM Tris B ase, 1 mM EDTA
Solution, 0.05% Tween 20, pH 9.0; 2 cycles of 5 min, 800W). Expression was visualized
using a Cy3 go at anti-rabbit IgG ( 1 h, room temperature; P A-43004; A mershamPharmacia, B uckinghamshire, UK) . T he slides were coverslipped an d o bserved with a
Zeiss Axioskop 4 0 m icroscope (Carl Zeiss, J ena, Ge rmany) eq uipped with a digital
camera (Zeiss AxioCam MRm). At least 25 photographs (original magnification X400)
were taken per animal. All procedures were carried out using coded slides to avoid bias.
Image analysis o f D RG neurons: Morphometry a nd q uantification o f NGF a nd T rkA
expression
Intensity o f s taining f or NG F and T rkA a nd ce ll body ar ea o f DR G neurons were
determined i n 1 00 r andomly s elected n eurons from ea ch an imal, u sing t he ImageJ
software. The neuronal cross-sectional area together with the mean pixel density of NGF
and T rkA lab eling were determined f or ea ch n euron an d a mean v alue obtained p er
animal.
Neuronal cr oss-sectional areas were determined b y o utlining t he perimeter o f clea rly
defined n euronal cell bodies. A ccording to t heir s ize, c ells were classified as: s mall
(cross-sectional area ≤500 μm2), medium ( 500-1000 μm2), lar ge (1000-1500 μm2) a nd
very large (>1500 μm2) (14,15).
When measuring NGF - and T rkA-signal intensity, f luorescent intensity i n n egative
controls ( omission o f t he primary antibody) s erved to es tablish a mean b ackground
value. Immunofluorescence intensity in T. spiralis-infected animals was expressed as the
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relative change compared to th e mean i mmunofluorescence in tensity i n n on-infected
controls, tak en as 100%. Comparisons were performed b etween DR G neurons with a
similar cross-sectional area. All measurements were carried out in a blinded manner to
avoid bias.
RNA extraction and quantitative real-time PCR
Total RNA was extracted from frozen jejunal and colonic samples by homogenization in
Trizol reagent (Ambion, Austin, Texas, USA) followed by isopropanol precipitation and
quantification by Nanodrop (Nanodrop Technologies, Rockland, DE, USA). For cDN A
synthesis, 1 µg of RNA was reverse-transcribed in a 20 µl reaction volume using a high
capacity cDN A reverse transcription k it ( Applied B iosystems, Foster City, CA, U SA).
Real-time PCR was performed to determine mRNA le vels o f inflammatory m arkers,
NGF and TrkA in the jejunum and colon. For this purpose, the Taqman® probes listed in
Table 1 were used (Applied Biosystems). The PCR reaction mixture was incubated on a
7900 real-time PCR system (Applied Biosystems) for 40 cycles (95ºC for 15 s, 60ºC for
1 min). B-Actin expression served as an endogenous control for normalizing the mRNA
levels of the target genes. Expression levels were analyzed by the 2-∆∆CT method.
Table 1. TaqMan® gene expression assays
Protein
Gene symbol
Assay reference
Il6
Il13
Il10
Mcpt2
Nos2
Rn01410330_m1
Rn00587615_m1
Rn00563409_m1
Rn00756479_g1
Rn00561646_m1
Ngf
Rn01533872_m1
Ntrk1
Rn00572130_m1
Actb
Rn00667869_m1
Inflammatory markers
Interleukin 6 (IL-6)
Interleukin 13 (IL-13)
Interleukin 10 (IL-10)
Rat mast cell proteinase II (RMCPII)
Inducible nitric oxide synthase (iNOS)
Neurotrophins and receptors
Nerve growth factor (NGF)
High affinity nerve growth factor receptor
(TrkA)
Reference gene
B-Actin
100
CHAPTER 3
Statistical analysis
All data are expressed as mean ± SEM . A r obust analysis ( one iteration) w as u sed to
obtain mean ± SEM f or R T-qPCR da ta. C omparisons be tween groups were performed
using Student’s unpaired t test o r nonparametric Mann–Whitney te st, as ap propriate. P
values <0.05 were considered statistically significant.
Results
Jejunal and colonic expression of inflammatory markers
In v ehicle-treated r ats, ex pression o f i nflammatory ( IL-6, I L-13 an d iNOS) and an tiinflammatory markers ( IL-10) was d etected at similar, r elatively lo w, le vels b oth i n
jejunum a nd colon. I L-13 content in the jejunum was particularly lo w co mpared to the
other markers, with 2, out of the 10 samples analyzed, below levels of detection. Overall,
relative expression f or th e jejunum was IL-10>iNOS>IL-6>IL-13; while f or th e colon
was IL-10>IL-6~IL-13>iNOS.
In the jejunum of infected animals, IL-6 and IL-13 expression was up-regulated by 2.5fold o ver co ntrols ( P<0.05; Fig. 1 ). I n th e colon, ch anges were restricted to I L-6
expression, which was up-regulated b y 2 -fold (P<0.05; Fig. 1). Other markers assessed
(iNOS, IL-10) were not affected in infected animals.
T. s piralis infection in duced a 7-fold an d 4 -fold in crease in R MCPII ex pression in th e
jejunum and colon, respectively (both P<0.05 vs. non-infected controls; Fig. 1).
Jejunal and colonic expression of NGF and TrkA
In no n-infected an imals, NG F and T rkA ex pression was detected at moderate-to-low
levels in b oth j ejunum a nd c olon. I n t he jejunum o f T. s piralis-infected a nimals, T rkA
expression showed a 2-fold increase, without changes in NGF expression (P<0.05; Fig.
2). Ho wever, i n t he same animals, co lonic expression o f TrkA was r educed b y 3 0%
(P<0.05; Fig. 2) while NGF expression was unaffected.
101
*
3
*
2
1
0
iN O S
4
3
2
1
0
JE JU N U M
4
3
2
1
0
JE JU N U M
COLON
m R N A f o ld c h a n g e
m R N A f o ld c h a n g e
JE JU N U M
IL -1 0
COLON
m R N A f o ld c h a n g e
m R N A f o ld c h a n g e
IL -6
4
m R N A f o ld c h a n g e
CHAPTER 3
IL -1 3
4
3
*
2
1
0
JE JU N U M
COLON
COLON
R M C P II
10
C o n tr o l
*
8
*
6
4
T . s p ir a lisin f e c t e d
2
0
JE JU N U M
COLON
Figure 1 . Jejunal and c olonic expression o f in flammation-related markers i n noninfected controls and T. spiralis-infected animals at day 30 PI. Data are mean±SEM of
4-6 animals per group. *: P<0.05 vs. non-infected controls.
Morphometric characterization of DRG neurons
In non-infected animals, the majority of DRG neurons were of small (<500 μm2, 51%) or
medium size (500-1000 μm2, 33%) (Fig. 3C). In T. spiralis-infected animals this pattern
of d istribution was alter ed, with a r eduction in the relative abundance o f s mall-sized
neurons (11%) and an increase in the number of very lar ge-sized neurons (>1500 µm 2,
8%) (Fig. 3 ). O verall, i nfected-animals s howed a 27% increase in the mean cr oss-
3
NGF
m R N A f o ld c h a n g e
m R N A f o ld c h a n g e
sectional area of DRG neurons (P<0.05; Fig. 3D).
2
1
0
JE JU N U M
COLON
3
T rkA
*
2
C o n tr o l
T . s p ir a lisin f e c t e d
*
1
0
JE JU N U M
COLON
Figure 2 . J ejunal and c olonic e xpression o f NG F and Tr kA in n on-infected
controls and T. spiralis-infected animals at day 30 PI. Data are mean±SEM of 4-6
animals per group. *: P<0.05 vs. non-infected controls.
102
CHAPTER 3
NGF- and TrkA-immunoreactivity in DRG neurons
In DRGs f rom no n-infected animals, n eurons s howing N GF- or T rkA-labeling w ere
distributed throughout the ganglia (Fig. 4). Both NGF and TrkA-signal were particularly
abundant in the cytoplasm of small/medium-sized DRG neurons (area ≤1000 µm2) while
large neurons (area >1000 µm 2) showed immunoreactivity at background levels (Fig. 4).
Labeling for T rkA was clea rly v isualized in g lial satellite ce lls s urrounding D RG
neurons (Fig. 4).
In T. s piralis-infected animals, th e pattern o f e xpression o f NG F or T rkA was
maintained. However, signal intensity for both markers was reduced, particularly within
small/medium-sized neurons (Fig.4).
A
D
P e r c e n ta g e o f
D R G n eu ro n s (% )
2
C
C r o s s - s e c t io n a l a r e a (  m )
B
100
C o n tro l
T . s p i r a l i s - in fe c te d
80
60
40
*
*
20
0
 500
5 0 0 -1 0 0 0 1 0 0 0 -1 5 0 0
1500 >
2
C r o s s - s e c t io n a l a r e a (  m )
1000
800
*
600
400
200
0
C o n t r o l T . s p ir a li s in f e c t e d
Figure 3. Morphometric c hanges in
DRG neurons. A-B: Re presentative
photomicrographs of th oracolumbar DRGs from a n on-infected (A) a nd a T. sp iralisinfected rat at day 30 PI (B). Scale bar: 100 µm. C: Size-distribution of thoracolumbar
DRG neurons in non-infected and T. spiralis-infected animals. D: Mean cross-sectional
area of thoracolumbar DRG neurons. Data are mean±SEM, 4-5 animals per group and
100 neurons per animal. *: P<0.05 vs. non-infected controls.
103
CHAPTER 3
TS
T . s p ir a li s - in f e c t e d
C o n tro l
T . s p ir a li s - in f e c t e d
( % f r o m n o n - in fe c t e d )
S t a i n in g in t e n s it y
T rk A
NGF
C o n tro l
100
80
T rkA
NGF
*
*
*
500 -
>1000
*
*
60
40
20
0
<500
<500
1000
500 -
>1000
1000
C r o s s - s e c t io n a l a r e a  m
2
Figure 4. N GF- and T rkA-immunoreactivity in DR G n eurons. Photomicrographs
of N GF and T rkA immunofluorescence i n t horacolumbar DRG neurons from a non infected and a T. spiralis-infected rat at day 30 PI. Note that labeling intensity within
neurons w as c learly decreased after T. spiralis infection. Arrows point g lial sa tellite
cells with TrkA immunoreactivity, surrounding DRG neurons. Scale bar: 50 µm. The
bar g raph s hows the q uantification of N GF a nd T rkA i mmunofluorescence in tensity
(according to th e c ross-sectional area o f DR G neurons) expressed a s the re lative
change c ompared to th e m ean imm unofluorescence in tensity in n on-infected an imals
(taken as 100, broken horizontal line). Data are mean±SEM, 4-5 animals per group and
100 neurons per a nimal. *: P<0.05 v s. staining in tensity in n on-infected c ontrols
(100%).
104
CHAPTER 3
Discussion
The present study evidences morphological changes in the intestinal afferent innervation
in a model of post-infectious gut dysfunction in rats. The altered expression of NGF and
TrkA in DR Gs s uggests an i mplication o f neurotrophins i n th e plastic remodeling o f
sensory n eurons. Si milar c hanges i n g ut afferents might b e taking p lace i n P I-IBS
patients, as basis for the sensorial alterations observed.
Persistent in flammatory s tates o f t he gut have b een ass ociated to t he remodeling o f
sensory neurons innervating the affected region (16,17). In the present report, a ch ronic
inflammatory-like response of th e intestine (up to d ay 3 0 PI) was observed, as
determined by an up-regulation of inflammatory cytokines (primarily IL-6) and RMCPII
(a MMC marker). This is in agreement with previous observations in T. spiralis-infected
animals revealing a lo ng-term i ncrease in t he tissue expression o f i nflammatory
mediators an d a persistent i nfiltrate of MMCs (9,10,18). R MCPII an d I L-6 lev els
showed a positive correlation (unpublished data), suggesting that the increased cytokine
transcript content might result from th e ac tivated s tate o f MMCs, a s s upported b y in
vitro observations (19,20). Interestingly, we show that this inflammatory state extends to
intestinal areas not directly affected by the parasite. Indeed, a similar inflammatory-like
reaction was demonstrated i n th e small i ntestine (jejunum) an d in t he colon. Si nce t he
colon is regarded as the main affected organ in IBS, these observations further support
the validity of the T. spiralis infection model as a good approach to IBS in general, and
PI-IBS in p articular. F urther in d eep studies ar e required to assess if t he colon o f T.
spiralis-infected an imals al so p resents functional alter ations r eminiscent o f those
observed in IBS patients.
Plasticity of afferent sensory pathways during intestinal inflammation might imply both
functional and morphological adaptations (2,16,17,21,22). Here, the cross-sectional area
of t horacolumbar DRG n eurons of T. spiralis-infected an imals was i ncreased b y 2 7%
when co mpared with n on-infected co ntrols. Si milar n euronal changes were observed
during TNBS-induced enteritis in guinea pigs or after partial obstruction of the intestine
in rats (16,22). In these studies, hypertrophy of DRG neurons was accompanied with an
active i nflammation of the gut, lead ing the a uthors to propose th at the r etrograde
transport o f inflammatory mediators f rom t he intestine into DR Gs contributed to t he
105
CHAPTER 3
observed changes. Similar mechanisms might b e operating d uring T. spiralis infection,
since morphological alterations o f DR Gs were associated to an ex tended en teric
inflammatory-like response, as discussed above.
NGF is regarded as an active mediator in inflammation-related neuronal remodeling o f
afferent innervation (7,23). For instance, hypertrophy of DRG neurons has been related
to increased levels of the target-tissue NGF and is prevented by immunoneutralization of
the peptide (24-26). In the present studies, although DRG neurons showed an increase in
size during in testinal inflammation, NG F levels, b oth in th e jejunum a nd co lon,
remained unchanged. T his ap parent discrepancy might be explained b y the rapid an d
short-term up-regulation that neurotrophic factors exhibit during pathological conditions
(7). For instance, in T. spiralis-infected animals, an early peak in jejunal NGF levels has
been described by day 3 PI, followed by normalization at later stages (27). Taking into
account t he long-term b iological effects o f neurotrophins (28), it
is f easible to
hypothesize that the hypertrophy o f s ensory neurons o bserved h ere is a persisting
phenotypic effect o f an ea rly up-regulation o f enteric NGF expression. S upporting t his
hypothesis, changes in the afferent innervation of the bladder of streptozotocin-diabetic
rats were associated to a n i ncrease in DR G neuronal s ize t hat was t wo weeks d elayed
relative to the peak levels of NGF (26). Alternatively, up-regulation of jejunal levels of
TrkA r eceptors, as observed in the j ejunum, ca n also b e in terpreted as an e nhanced
NGF-dependent afferent signaling ar ising from in flamed tis sues, without changes i n
NGF ex pression, th us co ntributing to th e induction an d maintenance o f morphological
changes within D RG neurons. Ho wever, T rkA immunoreactivity i n DR G neurons was
reduced d uring i nfection. T his might suggest a compensatory mechanism co nstraining
the neuroplastic effects a ssociated to th e enhanced NGF -TrkA-dependent s ignaling. I n
fact, since NGF is uptaken b y T rkA r eceptors in ax on ter minals, DRG n eurons co uld
limit NGF-signaling by down-regulating their TrkA expression (29).
It is f easible to s peculate that the morphological changes described h ere in D RG
neurons, together with the expression changes in the NGF-TrkA axis, are associated to
an alter ed s ignaling o f i ntestinal afferents. I n fact, compelling e vidences r eveal that,
during intestinal inflammation, both electrophysiological properties of DRG neurons and
intestinal afferent nerve activity are altered, showing states of sensitization (2-4,16,22).
Moreover, NGF has been directly implicated in the induction of long-term phenotypical
106
CHAPTER 3
changes in sensory neurons leading to states of central and peripheral sensitization (30).
Therefore, an d ev en t hough t he descriptive characteristic o f o ur s tudy, we can
hypothesize that the changes o bserved might be associated to alter ations in visceral
sensitivity, similar to that o bserved in o ther a nimals models o f I BS or i n I BS patients.
Further functional studies are required to confirm such a hypothesis.
In summary, we show the presence of a long-lasting extended low grade inflammation in
the gut of T. spiralis-infected rats. In these conditions, there are persisting morphological
changes in th e intestinal afferent innervation, n amely a hypertrophy o f D RG n euronal
cell bodies. Alterations in N GF-TrkA-depending s ignaling might participate in t hese
plastic changes. Overall, t hese observations s uggest that lo ng-term ac tivation o f t he
enteric immune s ystem d uring i ntestinal infections might account f or c hanges i n gut
afferents p roperties, th ereby providing a neuroimmune basis for th e states of ch ronic
visceral hypersensitivity.
107
CHAPTER 3
Acknowledgments
We thank A . A costa and E. Ma rtínez f or their technical assistance. This work w as
supported b y grant 2009SGR708 f rom t he Generalitat d e Catalunya and B FU200908229 and BFU2010-15401 from Ministerio de Ciencia e Innovación.
Disclosures
F. J. designed and performed experiments, analyzed data and wrote the paper. J.A. F-B
designed and performed experiments. V. M. designed and performed experiments, and
wrote the paper. P. V. designed experiments and wrote the paper.
109
CHAPTER 3
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CHAPTER 4
CHAPTER 4
PERSISTENT
ALTERATIONS I
N CO
LONIC A
FFERENT
INNERVATION IN A RAT
MODEL O F P OST-INFECTIOUS G UT
DYSFUNCTION: RO
OR CH
LE F
ANGES
IN
PERIPHERAL
NEUROTROPHIC FACTORS
F. Jardí 1, J.A. Fernández-Blanco1, V. Martínez *,1,2,3, P. Vergara 1,2,3
1
Department o f C ell Bi ology, Phys iology and
Autònoma d e Barcelona, Spain.
Autònoma de
Immunology, Universitat
2
Instituto de N eurociencias, U niversitat
3
Barcelona. Centro de Investigación Biomédi ca en
Red de
Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III,
Madrid, Spain.
Running title: Neurotrophic factors and remodeling of colonic afferents
Keywords: Colonic afferent innervation; Mucosal m ast ce lls; N euronal
remodeling; Neurotrophic factors; Trichinella spiralis; TRPV1
Submitted to Neuroscience
117
CHAPTER 4
Abstract
Background Visceral hypersensitivity in the inflamed gut is related partly to the effects
of peripheral n eurotrophic f actors (NTFs) on local a fferent n eurons. However,
alterations in sensory afferents of distant areas remain unexplored. Using the Trichinella
spiralis infection m odel, w hich courses primarily w ith a jejunitis, w e investigated the
remodeling o f co lonic af ferents a nd t he potential role of NTFs. Methods Rats w ere
infected w ith T. s piralis. I nflammatory-like changes, mucosal mast ce lls (MMCs)
dynamics, a nd expression o f nerve growth f actor an d g lial cell line-derived NTFs
(GDNF, ar temin a nd n eurturin) were determined i n th e co lon u p to d ay 3 0 p ostinfection. F unctionality o f co lonic afferents was determined ass essing ch anges in t he
expression o f sensory-related markers i n t horacolumbar/lumbosacral DRGs (TL, LS)
following in tracolonic ca psacin. Results T. s piralis induced an i nflammatory-like
response within the colon, partly r esolved at day 3 0 p ost-infection, ex cept for a
persistent MMC in filtrate. While th e jejunum o f in fected an imals s howed an u pregulation in the expression of NTFs, a transitory down-regulation was observed in the
colon. Overall, T .spiralis effects on DRGs gene expression were restricted to a transient
down-regulation o f T PRV1. Stimulation with in tracolonic ca psaicin in duced a downregulation o f T RPV1 lev els in T L an d LS DRGs, a n e ffect en hanced i n LS DRGs o f
infected an imals, r egardless th e post-infection ti me considered. Conclusions &
Inferences During in testinal inflammation, s pread m orphological and f unctional
alterations, including remodeling of visceral afferents, are observed outside the primary
region af fected b y t he insult. Similar mechanisms might be operating in s tates o f
widespread alterations of visceral sensitivity.
119
CHAPTER 4
Introduction
Inflammatory p rocesses of t he intestine have been as sociated to th e remodeling o f
sensory afferents innervating the inflamed area, leading to the development of functional
alterations, f requently manifested as visceral hypersensitivity (1,2). Fo r in stance, a
persistent state of low grade inflammation has been suggested as a contributing factor for
the development of visceral hypersensitivity in irritable bowel syndrome (IBS) patients
(3). Inflammation-related neuroplastic changes al so o ccur at in testinal regions not
directly af fected b y t he primary i nflammatory in sult (4). T he exact pathophysiological
mechanisms involved in this phenomenon remain largely unknown. Different hypothesis
have been s uggested, s uch a s the release to th e blood s tream o f in flammation-related
mediators from the primary inflamed area, the activation of pro-inflammatory cascades
in the distant regions or the implication of neurally-mediated responses (4,5).
Data ac cumulated d uring th e last years s uggest that neurotrophic factors (NTFs),
including n erve growth factor ( NGF) an d th e family o f g lial cell line-derived NTFs
(GDNF, ar temin a nd n eurturin), ar e implicated i n th e sensorial alter ations o bserved i n
inflammatory a nd f unctional gastrointestinal d isorders ( FGDs). Fo r in stance, ev idences
obtained f rom an imal models o f I BS an d g ut inflammation s how a n u p-regulation i n
NTFs expression an d suggest th eir i mplication i n t he sensitization o f e nteric afferent
neurons, as basis for the functional changes observed (6,7). In particular, knockout mice
for th e glial ce ll line-derived neurotrophic factor family receptor α3 (GFRα3), a
component of the receptor of the growth factor artemin, showed reduced visceral painrelated responses during TNBS-induced colitis, in agreement with observations in vitro
showing that colorectal afferents of GFRα3 knockout mice also failed to sensitize when
exposed to inflammatory mediators (8). Moreover, a recent study in IBS patients showed
a positive correlation b etween ab dominal pain s cores and co ntent of b rain-derived
neurotrophic factor ( BDNF) in th e colonic mucosa, t hus f urther s upporting a role for
NTFs in the sensitization of visceral afferents (9).
Special atten tion h as r eceived th e dynamic interplay b etween NG F and i ntestinal
mucosal mast cells ( MMCs). I n p articular, o bservations d erived f rom an imals models
suggest that, within the intestine, MMCs are a target for NGF and that NGF is involved
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CHAPTER 4
in t he recruitment an d ac tivation o f MM Cs a nd mediates t he interaction b etween mast
cells and nerve fibers (10-12).
In the present s tudy, we explored th e potential implication o f NTFs in th e afferent
neuronal remodeling associated to gut inflammation. We used the model of Trichinella
spiralis infection in r ats, a validated model of MM C-associated p ost-inflammatory g ut
dysfunction resembling post-infectious-IBS (PI-IBS) (13,14). In this model, we assessed,
up to 30 days post-infection (PI), inflammatory-like changes and mast cell (mucosal and
connective type) d ynamics in th e co lon, a site not directly af fected d uring T. spiralis
infection, which courses primarily with a jejunitis. To study the potential implication of
NTFs we determined t he expression o f NGF, G DNF, ar temin an d neurturin a nd t heir
receptors (TrkA and GFRα3) in colonic tissues as well as in thoracolumbar (TL) and
lumbosacral (LS) dorsal root ganglion ( DRG) n eurons, s ite of o rigin o f th e afferent
innervation o f th e co lon. F unctionality o f co lonic afferents was d etermined as sessing
changes in the expression o f s ensory-related markers an d NTFs’ receptors in DRG
neurons following stimulation of the ion channel transient receptor potential vanilloid 1
(TRPV1) with intracolonic capsaicin.
Materials and Methods
Animals
Adult (6 week-old at arrival), specific pathogen free (SPF), Sprague-Dawley (SD) male
rats were used ( Charles R iver, L es O ncins, Fra nce). Rats were maintained u nder
conventional conditions in a light ( 12h/12h light-dark cycle) an d te mperature controlled
(20-22ºC) r oom, i n groups of t wo t o t hree pe r c age, with ac cess to tap water a nd
laboratory rat chow (A04; Safe, Augy, France) ad libitum. Animals were acclimatized to
the new en vironment for o ne week b efore starting a ny experimental p rocedure. All
experimental procedures were approved b y th e Ethics Committee o f the U niversitat
Autònoma d e B arcelona and the Ge neralitat de Catalunya (protocol numbers 1013 a nd
5352, respectively).
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CHAPTER 4
Trichinella spiralis infection
Muscle-stage larvae of T. spiralis were obtained from infected CD1 mice as previously
described (13,14). Rats were infected at 7–8 weeks of age by administration of 7.500 T.
spiralis larvae, suspended in 1 mL of saline, by oral gavage. Studies were performed on
days 2, 6, 10, 14, 23 a nd 30 pos t-infection (P I). Age-matched r ats dosed o rally with 1
mL o f s aline were used a s co ntrols. After the i nfection, a nimals were regularly
monitored for clin ical s igns a nd b ody weight ch anges. Normal course of t he infection
was co nfirmed b y a significant d ecrease of b ody weight i n in fected a nimals compared
with controls, with a peak reduction on days 8-to-10 PI and a subsequent linear increase
over time, as previously described by us (13,14).
Experimental procedures and tissue sampling
Non-infected co ntrols o r a nimals a t 2, 6, 10, 14 , 23 a nd 30 da ys P I were deeply
anesthetized with isoflurane followed by euthanasia by decapitation. A laparotomy was
performed and jejunal and middle colon samples were obtained and either fixed in ice cold 4 % paraformaldehyde or Lana's fixative (4% paraformaldehyde, 14% picric acid in
0.4 M phosphate buffer) for histological and immunohistochemical studies, or frozen in
liquid nitrogen and stored at -80ºC until analysis. In addition, paired thoracolumbar (TL,
T12-L2) an d lu mbosacral ( LS, L 6-S2) d orsal r oot ganglia ( DRGs) were removed a nd
processed immediately for gene expression studies (see below). TL and LS DRGs were
selected in order to distinguish the influence of the gut region (small intestine vs. colon)
in the changes observed. In the rat, TL DRGs contain both small intestine and colonic
afferents f ibers w hile L S DRGs are mostly devoided of n eurons innervating th e upper
gastrointestinal tract (15-17).
In s ome cases, b efore tissue sampling, co lonic sensory a fferents were stimulated w ith
intracolonic capsaicin. Fo r th is, n on-infected co ntrols o r a nimals at 14 an d 3 0 d ays P I
were used. Animals were anesthetized with isoflurane and capsaicin (0.1 mL/rat, 0.1% in
ethanol:Tween 8 0:saline; 1:1:8, v :v:v; Si gma Aldrich, St. Louis, MO , U SA) wa s
administered in tracolonically (2 cm f rom the anus) u sing a
18 g auge catheter.
Thereafter, rats were maintained u nder an esthesia for 2 5 m in a nd s ubsequently
123
CHAPTER 4
euthanized by decapitation. Colonic tissue samples and TL and LS DRGs were obtained
as described above.
Histopathological studies
Paraformaldehyde-fixed co lonic tissue samples were processed r outinely f or p araffin
embedding, an d 5 -µm s ections w ere obtained for haematoxylin and eo sin ( H&E)
staining. H &E s lides were evaluated an d a histopathological s core ( ranging from 0,
normal, to 9 , maximal alterations) was a ssigned to ea ch a nimal. P arameters s cored
included: ep ithelial structure (0: n ormal; 1 : mild alter ations o f th e epithelium; 2 : lo cal
epithelium destruction; 3: generalized epithelium destruction); presence of inflammatory
infiltrate (0: normal; 1: mild localized infiltrate; 2: mild generalized infiltrate; 3: severe
generalized infiltrate) and relative density of goblet cells (0: normal; 1: mild increase in
cell density; 2: moderate/zonal increase in cell density; 3: severe/generalized increase in
cell d ensity). The same samples were also u sed for morphometric studies in which t he
thickness of mucosa, submucosa and muscularis externa layers was measured. For this,
digital images were acquired at X200 magnification (Nikon Eclipse 90i, equipped with a
digital camera, DXM 1200F; Nikon corporation, Japan) and were analyzed with the NIH
ImageJ software. At least 10 random measurements per sample (from 2 slices) were used
to obtain a mean thickness value. All procedures were performed in a blinded manner on
coded slides to avoid bias.
Immunohistochemistry for Rat Mast Cell Proteinases II and VI and MC counts
Immunodetection o f r at mast cell proteinases I I ( RMCPII) a nd V I (R MCPVI) w as
carried out o n pa raformaldehyde-fixed co lonic samples u sing a monoclonal antibody
anti-RMCPII ( Moredun, E dinburgh, UK) or a goat polyclonal a nti-mast cell tr yptase
(RMCPVI) an tibody ( sc-32473; Santa C ruz Biotechnology, Da llas, T exas, USA),
respectively. Antigen retrieval for RMCPVI was achieved by processing the slides in a
microwave (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0; 2 cycles
of 5 min, 800W). T he secondary antibodies included biotinylated horse antimouse IgG
(BA-2000; Vector Laboratories, Burlingame, CA, USA) and biotinylated rabbit anti-goat
IgG (s c-2774;
Santa
Cruz
Biotechnology). Dete ction
was p erformed
with
avidin/peroxidase k it (Vectastain ABC kit; Vector L aboratories) and co unterstaining
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CHAPTER 4
with haematoxylin. Specificity of the staining was confirmed by omission of the primary
antibody. Slides were viewed with a Zeiss Ax ioskop 4 0 m icroscope (Carl Z eiss, J ena,
Germany). Mu cosal mast cells ( MMCs), id entified as RMCPII-immunopositive ce lls,
were quantified in, at least, 20 non-adjacent, randomly selected, fields of colonic mucosa
(X400), co vering t he whole mucosal thickness. Den sity o f co nnective tissue mast ce lls
(CTMCs), id entified as RMCPVI-immunopositive cells, was d etermined b y co unting
the total number of cells in the submucosa, external smooth muscle and serosa areas in
two co mplete tissue sections of the colon for each animal. All procedures were carried
out using coded slides to avoid bias.
Immunofluorescence for RMCPII
Following fixation for 4 h i n L ana's fixative, co lonic samples were cryoprotected b y
sequential overnight incubation with 10% and 20% sucrose, embedded in OCT (Sakura
Finetek, Japan), frozen, and sectioned at 20 μm thickness. Tissue sections were
incubated w ith t he monoclonal an tibody a nti-RMCPII overnight a t 4 °C and t hen w ith
Alexa Fluor 4 88 goat anti-mouse IgG ( A11029; Mo lecular Probes, Eugene, O R, US A)
for 1 h at room temperature. Images of the immunostained tissue were acquired at X400
magnification using a scanning confocal microscope (Zeiss LSM 700; Carl Zeiss, Jena,
Germany) and subsequently analyzed using the ImageJ software. Microphotographs of at
least 5 0 randomly selected RMCPII-immunopositive ce lls were obtained, th e
immunofluorescence intensity q uantified ( ImageJ) a nd a mean i ntensity o btained for
each an imal. Immunofluorescence i ntensity i n T. s piralis-infected animals is e xpressed
as the relative change compared to th e mean i mmunofluorescence in tensity i n noninfected an imals, tak en as 100%. In all ca ses, sections from co ntrol and T. s piralisinfected an imals were processed s imultaneously. Specificity o f th e s taining was
confirmed by omission of the primary antibody. Analysis of the data was carried out in a
blinded manner to avoid bias.
RNA extraction and quantitative real-time PCR
Total RNA was e xtracted f rom f rozen co lonic samples and f resh D RGs by
homogenization i n T rizol reagent (Ambion, Austin, T exas, USA) followed b y
isopropanol precipitation an d q uantification b y Na nodrop ( Nanodrop Technologies,
125
CHAPTER 4
Rockland, DE, USA). For cDNA synthesis, 1 µg of RNA was reverse-transcribed in a 20
µl reaction v olume using a h igh ca pacity cD NA r everse transcription kit ( Applied
Biosystems, Foster City, CA, USA). Real-time PCR was performed to determine mRNA
levels o f inflammatory markers an d NTFs in t he colon and ex pression o f p roteinaseactivated r eceptors, NTFs’ receptors, neuropeptides and cation channels in TL a nd L S
DRGs. Fo r th is p urpose, th e T aqman® probes listed in T able 1 w ere used ( Applied
Biosystems). The PCR reaction mixture was incubated on a 7900 real-time PCR system
(Applied Biosystems) for 40 cycles (95ºC for 15 s, 60ºC for 1 min). B -Actin expression
served as an endogenous control for normalizing the mRNA levels of the target genes.
Expression levels were analyzed by the 2-∆∆CT method.
Table 1. Taqman® gene expression assays
Protein
Gene symbol
Assay reference
Inflammatory markers
Mcpt2
Il13
Nos2
Rn00756479_g1
Rn00587615_m1
Rn00561646_m1
F2rl1
Rn00588089_m1
Ngf
Artn
Gdnf
Rn01533872_m1
Rn01761472_g1
Rn01402432_m1
Nrtn
Rn01527513_g1
Ntrk1
Rn00572130_m1
Gfra3
Rn01760829_m1
Calcitonin gene-related peptide (CGRP)
Calca
Rn01511354_m1
Transient r eceptor
channel-1 (TRPV1)
Trpv1
Rn00583117_m1
Actb
Rn00667869_m1
Rat mast cell proteinase II (RMCPII)
Interleukin 13 (IL-13)
Inducible nitric oxide synthase (iNOS)
Proteinase-activated receptors
Protease-activated receptor 2 (PAR-2)
Neurotrophic factors
Nerve growth factor (NGF)
Artemin
Glial ce ll line-derived neurotrophic f actor
(GDNF)
Neurturin
Neurotrophic factors’ receptors
High a ffinity n erve growth factor r eceptor
(TrkA)
GDNF family receptor alpha 3 (GFRα3)
Neuropeptides and Cation channels
potential v anilloid
Reference gene
B-Actin
126
CHAPTER 4
Statistical analysis
All data are expressed as mean ± SEM . C omparisons b etween multiple groups were
performed b y a on e-way o r a two-way ANOVA, as appropriate, f ollowed when
necessary by a Fisher’s least significant difference (LSD) post h oc test to detect
differences b etween experimental groups. P values < 0.05 were considered statistically
significant.
Results
Histopathology and colonic expression of inflammatory markers
Compared w ith n on-infected co ntrols, co lonic microscopic h istological s core w as
significantly i ncreased at 14 days P I, d ue to th e presence o f an in flammatory in filtrate
and an in creased goblet ce ll density ( Table 2 ; Fig. 1 A-B). B y da y 30 P I, n o s igns of
histological alterations were observed ( Table 2; Fig. 1 C). T hickness o f t he mucosa,
submucosa o r muscularis ex terna was n ot altered b y t he infection, r egardless t he day
considered (Table 2). RT-qPCR analysis revealed a rapid up-regulation of colonic IL-13
expression after T. spiralis infection (by 9-fold increase over control values at day 2 PI;
P<0.05; Fig. 2). IL-13 up-regulation persisted up to day 14 PI (by 6 -fold increase over
control values; P<0.05), d eclining towards t he expression o bserved in non-infected
animals by day 30 PI (Fig. 2). Although iNOS mRNA levels showed at day 2 a nd 6 PI,
respectively, a 3 an d 4 -fold in crease over b aseline, no s tatistical significance was
achieved (Fig. 2).
Characterization of colonic mast cell populations
T. spiralis infection was ass ociated to a h yperplasia of MM Cs ( identified as R MCPIIpositive cells) in the mucosa of the colon (Fig. 3A-C). MMC counts started to increase
on da y 10 P I, r eaching a pe ak value on da y 14 P I ( by 6. 5-fold i ncrease o ver co ntrol
values, P<0.05) and persisted at similar levels up to day 30 PI (Fig. 3D). Similarly, gene
expression for RMCPII was up-regulated in T. spiralis-infected animals, with a peak of
expression on day 14 PI (by 32-fold increase over control values, P<0.05; Fig. 2). At day
30 P I, RMCPII ex pression lev els were still i ncreased ( by 10-fold) o ver co ntrol v alues,
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CHAPTER 4
although s tatistical significance was n ot r eached. RMCPII i mmunofluorescence
intensity, as assessed b y co nfocal microscopy, was s imilar in co ntrol and T. s piralisinfected animals, regardless the time PI considered (Fig. 4).
Table 2. Histological changes in the colon after T. spiralis infection
T. spiralis-infected
Control
Day 14 PI
Day 30 PI
0
0
0
Inflammatory
infiltrate (0-3)
0.25±0.07
1.02±0.15*
0.35±0.16
Goblet cell density
(0-3)
0.36±0.12
0.88±0.07*
0.30±0.10
Total (0-9)
0.6±0.12
1.91±0.19*
0.65±0.23
280.2±10.1
280.2±12.8
262.4±0.5
Submucosa
58.5±6.0
49.6±7.0
68.7±6.1
Muscularis
externa
259.9±25.8
253.0±20.6
262.6±24.7
Epithelial
structure (0-3)
Histological
score
Mucosa
Thickness
(µm)
Data are mean±SEM of 6-8 animals per group. *: P<0.05 vs. respective control group.
In no n-infected animals, CTMCs ( identified as RMCPVI-positive ce lls) were r arely
found and, when present, were located mainly in the submucosa of the colon. T. spiralis
infection d id n ot affect the density o r th e pattern o f d istribution o f CTMCs ( control:
8.6±1.7 cells/tissue section; day 30 PI: 10.4±2.8 cells/tissue section)
Colonic and jejunal expression of neurotrophic factors
Expression o f NG F, GDNF, artemin an d neurturin was detected, at similar lev els, i n
colonic and jejunal samples f rom n on-infected co ntrols. Relative expression le vels
were similar in both areas ( colon: ar temin~GDNF>NGF>neurturin; j ejunum:
artemin~GDNF>neurturin>NGF). I n T. spiralis-infected an imals, s pecific site(jejunum vs. colon) a nd time-related changes in expression were observed depending
on the NTF considered.
128
CHAPTER 4
Figure 1. Colonic histology. Representative microphotographs showing haematoxylin and
eosin-stained colonic slices from a control (A) and previously infected rats at days 14 (B)
and 3 0 PI (C). N otice th e p resence o f a n in flammatory in filtrate without evidence o f
epithelium damage at 14 days PI. Scale bar: 100 µm. D: Magnification of the area indicated
in B. Scale bar: 100 µm.
m R N A f o ld c h a n g e
50
C o n tro l
day 2
day 6
day 14
day 30
40
30
*
T . s p ir a lisin fe c te d
20
10
*
**
0
IL -13
iN O S
R M C P II
129
Figure 2. Effects of T. spiralis
infection o
nc
olonic
expression o f inflammationrelated m arkers. Relative
expression of IL-13, iNOS and
RMCPII mRNA in th e c olon
of non -infected controls a nd
previously infected rats at days
2, 6 , 1 4 a nd 30 PI. D ata are
mean±SEM of 4-5 animals per
group. *: P<0.05 vs. respective
control group.
CHAPTER 4
In the colon of infected animals, NGF showed a time-related down-regulation from days
2 to 1 4 P I, w ith minimum e xpression le vels r eached at d ay 1 4 P I ( by 5 0% reduction,
p<0.05 vs. control) and a recovery to normal levels b y day 30 PI (Fig. 5). Other NTFs
showed o nly a transient reduction in e xpression b y d ay 1 4 P I (25% - 40% r eduction)
with the exception of artemin, which showed a biphasic response with an up-regulation
(by 30%) at day 30 PI (Fig. 5).
B
A
D
F
60
( p e r fie ld , X 4 0 0 )
R M C P I I p o s i t iv e c e ll s
C
*
40
*
*
20
0
*
C o n tr o l da y 2
da y 6
da y 1 0 da y 1 4 da y 2 3 da y 3 0
T . s p ir a li s - in f e c t e d
Figure 3. Immunohistochemistry for colonic mucosal mast cells. A-C: Representative
microphotographs showing RMCPII immunopositive c ells (corresponding to M MCs) in
the colonic mucosa of a control (A) and previously infected rats at days 14 (B) and 30 PI
(C). Scale bar: 50 µm . D: Qu antification o f c olonic MMCs (number p er field, X 400).
Data are mean±SEM of 4-6 animals per group. *: P<0.05 vs. control group.
In t he jejunum, c hanges i n NTFs expression i n r esponse to T. s piralis infection w ere
more v ariable, depending upon the NTF considered. NGF s howed a sustained increase
from day 2 to 30 PI, although no statistical significance was achieved, probably because
of the relatively large variability observed (Fig. 5). Artemin and GDNF showed a similar
pattern, w ith an ea rly, tr ansitory, up-regulation ( 75 % -100 % increase) b y da y 2 P I
followed by a normalization in expression (Fig. 5). On the other hand, neurturin showed
130
CHAPTER 4
a p rogressive d own-regulation be tween da ys 6 a nd 14 PI and a recovery b y da y 30 PI
(Fig. 5).
C
D
in te n s ity (p e r M M C )
B
R e la t iv e R M C P I I -s ig n a l
A
1 .5
1 .0
0 .5
0 .0
C o n tr o l
da y 1 4
da y 3 0
T . s p i r a l i s - in fe c t e d
Figure 4 . I mmunofluorescence fo r c olonic mucosal mast cells. A-C: Representative
confocal microphotographs s howing RMCPII imm unopositive cells (corresponding to
MMCs, g reen-stained c ells) in the c olonic mucosa o f a c ontrol (A) a nd p reviously
infected ra ts at days 14 (B) a nd 3 0 PI (C). Scale bar: 50 µm . D: Qu antification o f
RMCPII immunofluorescence in tensity (p er cell) expressed a s the re lative c hange
compared to th e mean i mmunofluorescence in tensity in n on-infected a nimals (tak en a s
1.0) (se e m ethods for details o f the q uantification p rocedure). Ea ch p oint represents a n
individual animal, the horizontal line with error represents the mean±SEM.
Effects of intracolonic capsaicin on colonic neurotrophic factors
In no n-infected co ntrols, i ntracolonic capsaicin i nduced a specific up-regulation o f
artemin, w ithout changes in t he expression of th e other NTFs assessed ( Fig. 6). In T.
spiralis-infected an imals, a similar s ignificant increase in th e expression lev els o f
artemin was observed at day 14 PI; however, this effect was totally absent at day 30 PI
131
CHAPTER 4
(Fig. 6 ). E xpression o f o ther NTFs in T. s piralis-infected animals was not af fected b y
intracolonic capsaicin, regardless the PI time considered.
A R T E M IN
2 .5
2 .5
m R N A fo ld c h a n g e
m R N A fo ld c h a n g e
NGF
2 .0
1 .5
1 .0
*
0 .5
*
COLON
1 .0
COLON
JE JU N U M
JE JU N U M
N E U R T U R IN
*
2 .0
1 .5
*
2 .5
m R N A fo ld c h a n g e
m R N A fo ld c h a n g e
*
0 .5
GDNF
2 .5
0 .5
*
1 .5
0 .0
0 .0
1 .0
*
2 .0
0 .0
2 .0
1 .5
C o n tro l
day 2
T . s p ir a lisday 6
day 14
in fe c te d
day 30
1 .0
*
0 .5
0 .0
COLON
JE JU N U M
COLON
JE JU N U M
Figure 5 . Effe cts of T. spiralis infection o n c olonic and jejunal expression o f
neurotrophic fa ctors. Relative expression o f NG F, a rtemin, GD NF and n eurturin
mRNA in the colon of control and previously infected rats at days 2, 6, 14 and 30 PI.
Data are mean±SEM o f 4 -10 a nimals pe r g roup. *: P <0.05 v s. respective c ontrol
group.
Expression of neurotrophic factors’ receptors and sensory-related markers in TL and LS
DRGs: effects of intracolonic capsaicin
mRNA f or T RPV1, C GRP, PAR-2, TrkA and GFRα3 was detectable in all samples
assessed. Expression levels of these genes in control conditions were similar in LS and
TL DRGs, being the expression levels of TRPV1, TrkA and GFRα3 relatively high
compared to those of PAR-2 and CGRP.
Overall, T. spiralis infection did not affect the expression of the different genes assessed,
neither in TL nor LS ganglia; with the exception of TPRV1 in TL DRGs that showed a
132
CHAPTER 4
down-regulation th at r eached s tatistical s ignificance at day 14 PI ( 30 % decrease over
control values; P<0.05; Fig.7).
Intracolonic capsaicin d own-regulated T RPV1 ex pression in b oth T L an d L S DRGs.
Relative down-regulation of TRPV1 expression in TL DRGs was similar across groups
(30% - 40% r eduction; Fig. 7). However, r esponses in LS DRGs were enhanced in T.
spiralis-infected an imals, p articularly at 3 0 d ays P I ( change in e xpression: co ntrol, 16±9%; day 30 P I, -52±4.15%; P<0.05; Fig.7). R egardless o f th e ex perimental group
considered, th ere was a negative correlation b etween t he relative capsaicin-induced
down-regulation o f T RPV1 in LS DRGs and th e changes in co lonic expression o f
artemin (R2=0.37; P=0.04; Fig. 7).
NGF
A R T E M IN
2 .0
m R N A fo ld c h a n g e
m R N A fo ld c h a n g e
2 .0
1 .5
1 .0
*
0 .5
0 .0
C o n tr o l
day 14
1 .5
*
0 .5
C o n tr o l
GDNF
2 .0
m R N A fo ld c h a n g e
m R N A fo ld c h a n g e
2 .0
1 .5
*
0 .5
0 .0
C o n tr o l
day 14
day 14
day 30
T . s p i r a l i s - in fe c t e d
T . s p ir a li s - in f e c t e d
1 .0
*
+
1 .0
0 .0
day 30
+
T . s p i r a l i s - in fe c t e d
C o n tro l
C a p s a ic in
1 .5
1 .0
*
0 .5
0 .0
day 30
N E U R T U R IN
C o n tr o l
day 14
day 30
T . s p i r a l i s - in fe c t e d
Figure 6 . Effe cts of i ntracolonic c apsaicin o n neurotrophic fa ctors e xpression i n
the c olon. Relative e xpression o f N GF, a rtemin, G DNF a nd n eurturin m RNA in th e
colon f rom c ontrol and p reviously in fected ra ts a t days 14 a nd 3 0 PI. Da ta are
mean±SEM of 3-10 animals per group. *: P<0.05 vs. control-control group +: P<0.05
vs. respective control group.
Regardless t he experimental g roup co nsidered, in tracolonic ca psaicin h ad a
clear
tendency to d own-regulate the expression of TrkA and GFRα3. These effects were
133
CHAPTER 4
particularly evident in TL DRGs at day 30 PI (Fig. 7). Similar effects were observed for
PAR-2, which was down-regulated by capsaicin particularly in TL DRGs in T. spiralisinfected animals (Fig. 7). Expression of CGRP was similar across groups, independently
of the region considered, and was not affected by intracolonic capsaicin (Fig. 7).
LS
1 .5
1 .0
+
*
0 .5
0 .0
+
+
+
e x p r e s s io n ( % )
2 .0
C h a n g e in a r t e m in
m R N A f o ld c h a n g e
TRPV1
TL
C ont D 14 D 30 C ont D 14 D 30
T . s p ir a li s
T . s p ir a li s
80
60
40
20
0
-2 0
20
1 .5
1 .0
+
#
0 .5
0 .0
C ont D 14 D 30
m R N A f o ld c h a n g e
m R N A f o ld c h a n g e
2 .0
LS
C ont D 14 D 30
TL
2 .0
1 .0
0 .5
0 .0
C ont D 14 D 30
$
1 .0
$
+
0 .5
0 .0
C ont D 14 D 30 C ont D 14 D 30
T . s p ir a li s
C ont D 14 D 30
T . s p ir a li s
CGRP
LS
1 .5
+
&
T . s p ir a li s
m R N A f o ld c h a n g e
m R N A f o ld c h a n g e
2 .0
80
GFR3
LS
P A R -2
TL
60
1 .5
T . s p ir a li s
T . s p ir a li s
40
C h a n g e in T R P V 1 e x p r e s s io n ( % )
T rk A
TL
2
R = 0 .3 7
P = 0 .0 4
2 .0
1 .5
LS
TL
C o n tr o l
C a p s a ic in
1 .0
0 .5
0 .0
C ont D 14 D 30
T . s p ir a li s
T . s p ir a li s
C ont D 14 D 30
T . s p ir a li s
Figure 7 . Effe cts of T. spiralis infection a nd in tracolonic capsaicin on
neurotrophic fa ctors’ receptors and sensory-related markers in TL and LS
DRGs. Relative expression of TRPV1, TrkA, GFRα3, PAR-2 and CGRP in TL and LS
DRGs from control and previously infected rats (T. spiralis) at days 14 (D 14) and 30
(D 30) PI. Data are mean±SEM of 3-5 animals per group. *: P<0.05 vs. control-control
group +, #, & and $: P<0.05, P=0.07, P=0.06 and P=0.09 vs. respective control group.
The rig ht panel in th e u pper ro w shows the correlation b etween c hanges in c olonic
expression of artemin and TRPV1 expression in LS DRGs after intracolonic capsaicin.
Each p oint represents a n i ndividual animal; th e br oken l ines r epresent the 9 5%
confidence interval.
134
CHAPTER 4
Discussion
The present study evidences a remodeling of the colonic afferent innervation in a model
of pos t-infectious g ut dysfunction in r ats. R esults o btained s how th e presence o f a n
inflammatory r eaction within th e colon c haracterized b y a persistent MMC i nfiltrate,
although th is o rgan was not directly af fected b y T. s piralis larvae. I n p arallel, lo nglasting ch anges in co lonic s ensory af ferents p roperties w ere revealed b y i ntracolonic
capsaicin administration, suggesting that both inflammation and neuronal remodeling can
be spread widely after localized injury of the intestine.
The in fection with T. spiralis in r odents is a n ac cepted m odel of post-infectious gut
dysfunction, which reproduces some of the alterations observed in patients with PI-IBS
(13,14). Mo st of th e studies u sing t his model focus o n the jejunum, t he main ar ea
affected b y t he larvae invasion, which s hows s tructural and functional alterations
directly r elated to t he inflammatory r esponse induced b y the presence o f the p arasite
(18). Ho wever, en teric dysfunction has also b een r eported in worm-free r egions o f th e
gastrointestinal tract (19). Here, we focused on the rat colon, observing the development
of an inflammatory response resembling that previously described by us in the jejunum
(14). The main characteristic of the jejunal response was the presence of a long-lasting
mast cell infiltrate, with increases in the counts of both MMCs and CTMCs (13,14). In
the colon, a similar long-lasting infiltration of MMCs was observed, but CTMC density
was unaffected. Moreover, presence and migration of the parasite resulted in significant
alterations o f t he epithelium an d muscle layers o f th e j ejunum, while n o structural
alterations were observed in the colon, consistent with the absence of larvae. However,
despite th is larvae absence, th e colon d eveloped a Th2-like c ytokine pattern ( upregulation o f I L-13), which h as been r elated to th e expulsion o f t he parasites (18).
Overall, th ese observations suggest the existence o f an ex tended in flammatory-like
response to the in fection, with some r egion-specific features likely as sociated to th e
presence and migration of the parasite. The affection of the colon, as demonstrated here,
further increases the validity of T. spiralis infection in rats as a model of IBS, since the
colon is regarded as the main affected organ in the disease.
Motor an d s ensorial alter ations o bserved i n th e inflamed g ut have been as sociated, at
least partially, to a n up-regulation i n NTFs expression (10,20,21). Data o btained f rom
animal models and intestinal biopsies of patients with intestinal inflammatory disorders
135
CHAPTER 4
confirm an e nhanced ex pression o f NG F and GDN F family members d uring ac tive
enteric inflammation (20,22,23). In agreement with these data, here we show changes in
the pattern of expression of NGF, GDNF, artemin and neurturin along the course of T.
spiralis infection in rats. Within the jejunum, the early up-regulation of NGF, GDNF and
artemin might be associated to the intestinal response to the presence of the larvae in the
lumen a nd th e mucosa. I ndeed, ea rly i ncreases in NG F have been r elated to th e
hypermotility r esponses ass ociated to th e e xpulsion o f t he parasite (20). Ho wever, the
most striking changes in NTFs expression during T. spiralis infection were observed in
the colon. Conversely to th at o bserved in t he jejunum, i n the colon, NTFs expression
was r educed b etween d ays 6 -14 P I. The reason f or t his d own-regulation is not clea r,
since an i nflammatory-like r esponse was also o bserved in th e colon. I ndeed, d uring
states o f chemically-induced co litis, co lonic contents o f NTFs increase, following a
pattern similar to the observed here for the jejunum (24). Thus, it is feasible to speculate
that these differences might be related to the degree of inflammation observed. During T.
spiralis infection, t he primary site o f i nflammation is th e jejunum, with the co lon
showing, co mparatively, an atten uated i nflammatory-like response. T herefore,
inflammation-dependent modulation o f NTFs might depend u pon t he intensity of t he
inflammatory r esponse elicited. Sin ce NTFs are active in flammatory co mponents,
mediating f or i nstance neural sensitization, a lo cal down-regulation m ight help to
constrain the effects a nd ex tend o f in flammation, p articularly i n ar eas not directly
affected by the inflammatory insult.
Inflammatory s tates of t he colon h ave been as sociated to th e development of v isceral
hypersensitivity, s ubsequent to th e s ensitization o f co lonic afferents (1,2,25). I n
particular, o bservations i n a nimal models reveal that, d uring i nflammation, co lonic
afferents s how alter ed elec trophysiological properties to gether with c hanges in th e
expression p rofile o f s ensory-related markers, i ncluding NTFs’ receptors (1,2,8,24,26).
Here, ef fects of T. spiralis infection o n DR Gs gene expression were restricted to a
transient TPRV1 down-regulation. Interestingly, this effect was observed in TL but not
LS DRGs. T aking in to ac count the distribution o f the afferent innervations alo ng t he
spinal ganglia, this suggests that, rather than related to the colon, the effect resulted from
the T. spiralis–induced in flammation o f the small intestine, which originates mainly i n
the TL region (16). Both up- and down-regulation of TRPV1 expression in DRGs have
been described during intestinal inflammation, depending upon the experimental model
136
CHAPTER 4
considered (24,27,28). Ov erall, levels of T RPV1 s eem to r eflect a balance b etween
expression of the transcript and its transport from the soma of DRG neurons to central
and peripheral axonal terminals (29). Therefore, a reduced content on transcript in DRGs
might be indicative of an increased expression of the channel at the periphery and/or at
spinal cord with subsequent ch anges in a fferent sensitivity, t hus co ntributing to t he
development of afferent sensitization.
TRPV1 ch annels have been related to th e d evelopment a nd maintenance o f co lonic
inflammatory h ypersensitivity (30,31). Her e, m odulation o f T RPV1 m RNA le vels in
DRGs was d etermined as
a way to ass ess p otential changes in co lonic af ferent
excitability. Stimulation of afferents by intracolonic capsaicin caused down-regulation of
TRPV1 mRNA levels in TL and LS DRGs, similarly to that observed in previous studies
(32,33). I nterestingly, ca psaicin ef fects were enhanced in T. spiralis-infected an imals,
particularly i n LS DRGs (main o rigin o f co lonic afferents), u p to d ay 3 0 P I, th ereby
suggesting a long-lasting s ensitization o f co lonic afferents b y t he in fection. Ma ny
mediators released during inflammatory processes have been described to participate in
the sensitization o f T RPV1 channels, in cluding MM C-derived p roteases and g rowth
factors (24,34). In this line, we had previously described that, during T. spiralis infection
in r ats, t here is a n i ncrease i n lo cal ( intestinal) and s ystemic (serum) le vels o f MM Cderived p roteases (13). These o bservations, to gether with t he results o btained here,
showing a persistent MMC infiltrate and an up-regulation of RMCPII transcripts in the
colon, s upport the view t hat MMC-derived p roteases m ight participate in th e
sensitization of visceral afferents. However, in the present conditions, we were unable to
demonstrate an ac tivated state of MM Cs, at least as it relates to ch anges in RMCPII
content per cell.
The early down-regulation of NTFs observed in the colon of T. spiralis-infected animals
might r eflect a compensatory mechanism to th e development o f i nflammation, th ereby
constraining afferent hyperexcitability. Similarly, late (by day 30 PI) increase in artemin
might co ntribute to th e lo ng-lasting maintenance o f afferent hypersensitivity, a s
suggested b y o bservations from GFRα3 knockout m ice (8). A r ole for ar temin
modulating a fferent functionality is further supported b y t he correlation o bserved
between t he changes in ar temin an d T RPV1 ex pression u nder a fferent stimulation,
suggesting t hat TRPV1 ex pression in LS DRGs might act r estraining artemin up-
137
CHAPTER 4
regulation in t he colon an d, th erefore, th e excitability o f afferents. F inally, we have
observed that cultured rat MMCs express GFRα3 (data not shown), s uggesting t hat the
interaction ar temin-MMCs might be a p otential mechanism contributing to v isceral
afferent sensitization.
In s ummary, we show t hat c hanges consistent with an i nflammatory-like r esponse ar e
present in the colon of T. spiralis-infected rats. In these conditions, there are long-lasting
changes in a fferent sensitivity, as suggested b y t he responses to intracolonic capsaicin.
The neuroimmune bases underlying this remodeling of colonic afferents might be related
to ch anges in NTFs levels an d MM C mediators, d erived f rom a persistent MMC
infiltrate. O verall, th ese observations suggest that, d uring in testinal inflammation, a
spread o f morphological an d f unctional alterations, including r emodeling o f v isceral
afferents leading to altered sensitivity, can be observed outside the primary site of action
of the insult. Similar mechanisms might be operating in states of widespread alterations
of visceral sensitivity.
138
CHAPTER 4
Acknowledgments
We would like to thank Dr. Gebhart and his research group in the Pittsburgh Center for
Pain R esearch f or t heir g uidance an d support. Special th anks to A. Acosta and E .
Martínez for their technical assistance. This work was supported by grant 2009SGR708
from th e Generalitat d e Catalunya and B FU2009-08229 a nd B FU2010-15401 f rom
Ministerio de Ciencia, Investigación e Innovación.
Disclosures
F. J. designed and performed experiments, analyzed data and wrote the paper. J.A. F -B
designed and p erformed e xperiments. V. M. d esigned a nd performed experiments, a nd
wrote the paper. P. V. designed experiments and wrote the paper.
139
CHAPTER 4
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DISCUSSION
DISCUSSION
IBS, the m ain functional gastrointestinal d isorder, affects primarily the colon an d i s
characterized by recurrent abdominal pain/discomfort associated with changes in bowel
habits and dysmotility (27). Although the pathogenesis behind this disorder is not clearly
understood, s ymptoms g eneration has been r elated to ce rtain l uminal-derived s timuli,
including gut co mmensal microbiota (GCM), in testinal infections an d food an tigens
(31,37,40,41).
Several authors suggest that the presence of a continuous immune activation, leading to
a s tate o f persistent low grade inflammation, h as to b e considered as a characteristic
feature of IBS (108). Accordingly, evidences suggest an activation of intestinal MCs as
the common pathophysiological pathway through which luminal factors contribute to the
initiation/perpetuation of IBS symptomatology (109,110). For instance, colonic biopsies
of p atients with IBS, either related to d ietary co mponents o r to in testinal infections,
show a n e xcited-activated s tate of e nteric MCs (36,109,110). Mo reover, s tudies in
animal models of t he disease indicate th at MCs ar e key ef fector ce lls mediating th e
changes in v isceral sensitivity an d th e intestinal dysmotility t hat characterize IBS
(55,111,112).
In ad dition, locally r eleased NT Fs ar e suggested to p articipate in t he neuroimmune
circuits t hat characterize the p athophysiology o f t he disorder. I ndeed, the ability o f
NTFs, mainly NGF, to degranulate MCs has been widely described (113-116). Similarly,
NGF is ab le to p roduce lo ng-lasting p henotypical changes i n a fferent s ensory ne urons
that might account for th e sensory d ysregulation o bserved i n I BS patients (117).
Evidences obtained from colonic biopsies of patients with IBS reveal an up-regulation in
the intestinal expression o f n eurotrophins, na mely N GF and B DNF; w ith BDNF
showing a p ositive co rrelation with the s ymptomatology o bserved (103,106). Although
these e vidences, th e intestinal sources o f NT Fs a nd its ca use-effects r elationship with
IBS remain unknown. Thus, here, we hypothesized that NTFs, in conjunction with MCs,
might b e collaborating i n t he onset of I BS-like changes i n colonic motility a nd
sensitivity.
In th e present work, we aimed to ch aracterize the ability o f lu minal-derived s timuli
(GCM, in testinal infections a nd f ood antigens) to tr igger IBS-like co lonic functional
alterations a nd t he potential implication o f NT Fs. For th ese p urposes, two validated
147
DISCUSSION
animal models of MMC-dependent intestinal dysfunction in rats were used: chronic oral
exposure to the food antigen OVA and experimental infection with T. spiralis, together
with a new protocol of OVA exposure in rats that included the coadministration of t he
dietary antigen and bacterial components (LPS).
Mucosal mast ce lls o f t he colon ar e activated d uring t he exposure to a food antigen o r
the infection with T. spiralis
Our results demonstrate that both oral exposure to the food antigen OVA and intestinal
infection w ith T. spiralis stimulate MCs o f th e rat colon (Fig. 4 ). These ef fects ar e
reminiscent of th e ex cited-activated s tate of MCs o bserved in co lonic biopsies of
patients with d ietary-related an d P I-IBS (36,109,110). Ho wever, d ifferences i n M C
responses can b e observed d epending upon th e luminal stimuli considered. T. s piralis
infection was characterized by an abundant, long-lasting, MMC infiltrate, while chronic
exposure to o ral OVA r esulted in a state of e xcitement/activation o f M MCs without
changes in cell density. These responses might be related to the changes that the original
stimuli (parasite vs. food antigen) induce in the tissue microenvironment (64). During T.
spiralis infection, t here is a strong i mmune response, mainly T h2-like, which d rives a
large recruitment o f MMCs t o the intestinal mucosa (118). O n t he other ha nd, c hronic
exposure to oral OVA is not associated to an overt immune response (55), leading to a
limited recruitment of MMCs. Nevertheless, in both cases, MMC activation is observed,
indicating that these cells ar e important in t he functional ch anges o bserved in these
models. T hus, d epending o n th e degree o f i nflammation ass ociated to th e luminalderived stimuli, the MMC response observed (activation vs. recruitment and activation)
might differ. This phenomenon would explain, at least in part, the contradictory results
in MM C co unts r eported f or I BS patients (72,103,119), and s uggests t hat the initial
trigger o f th e disease might determine the relative MMC p articipation i n its
development.
Interestingly, during a s hort-term e xposure to o ral OVA (1-week), MC d ynamics were
unaffected. However, combination of OVA with a dysbiotic-like state for the same time
period resulted in increased counts of CTMCs in the submucosa of the colon. Although
the potential implication o f C TMCs i n I BS has r eceived litt le atten tion, these
observations, to gether with p revious d ata (120), suggest t hat CTMCs could ac t also as
148
DISCUSSION
effector ce lls in I BS, contributing to th e functional alte rations that ch aracterize the
disease.
Figure 4 . Schematic representation o f t he putative l uminal stimuli-MMCs-NGFdependent mechanisms implicated in OVA- and T. spiralis-induced colonic dysfunctions in
rats. In th e c olon, OVA a nd/or OVA d igested fragments a nd th e e xtended in flammatory-like
response d erived f rom T. sp iralis infection act ivate a n euroimmune ci rcuitry th at includes, at
least, MMCs a nd peripheral NTFs. Local actions o f MMC-derived mediators a nd NTFs a re
likely to alter th e colonic motility and epithelial barrier function (not addressed in the p resent
work). NGF, of a yet to be determined source, might bind to TrkA receptors located on MCs,
maintaining a n e xcited-activated sta te, and a lso to T rkA re ceptors located in e nteric se nsory
afferents. Re trograde tran sport of N GF-TrkA c omplexes to DRG n eurons is li kely to in duce
long-term ne uronal phenotypical changes that contribute to th e se nsory d ysfunction th at
characterizes IBS. Luminal microbiota-derived factors (bacterial LPS) facilitate OVA effects.
Exposure to OVA-derived food antigens induces IBS-like motor alterations
Digestive motility disturbances are a common finding in alimentary allergies (121,122).
Here, lo ng-term (6-week) exposure to o ral OVA was ass ociated to a contractile
dysfunction o f t he colon, b oth in vivo and in vitr o. T hese motor ch anges resemble the
alterations d escribed in I BS patients a nd a nimal models o f t he disease, t hereby
149
DISCUSSION
supporting a role for dietary antigens in the development of IBS-like changes in colonic
motility (55,123-126). Moreover, as mentioned ab ove, o ral OVA i nduced a n ex citedactivated state of colonic MMCs. Since MC degranulation has been related to the onset
of colonic motor alterations (127), it is feasible to speculate that, during OVA exposure,
excited-activated MM Cs might be part of th e underlying mechanisms mediating th e
observed m otor ch anges (Fig. 4 ). I n o ur c onditions, th e ab ility o f OV A to d evelop
colonic dysmotility was affected by the duration of the exposure period and the state of
the colonic commensal microbiota. In co ntrast to th at observed af ter a 6-week OV A
exposure, co lonic contractility was u naffected af ter a short-term ( 1-week) ex posure.
Therefore, th ese results suggest that the sensitization to o ral allergens induced b y few
administrations might require th e p resence o f facilitatory elements, such as adjuvants
(128). Fu rthermore, in a nimals in which a dysbiotic-like state was generated (LPS coadministration), s hort-term a dministration o f OVA was e ffective in i nducing alter ed
contractile responses to t he an tigen. T hus, o verall, t hese observations support the
hypothesis th at adverse food r eactions ar e implicated i n th e pathogenesis o f I BS and
suggest that concomitant alterations o f the microbiota, co mmonly observed d uring IBS
(41), might facilitate abnormal responses towards dietary antigens (Fig. 4).
NGF-TrkA-dependent mechanisms modulate OVA-induced colonic dysmotility
NTFs, mainly neurotrophins, have been suggested to play a role in the development of
IBS-like motor alter ations (58,129). Si milarly, we s how t hat the co lonic dysmotility
associated to lo ng-term e xposure to a d ietary a ntigen i s mediated, at least p artially, b y
NGF-TrkA-dependent p athways. I ndeed, tr eatment with K 252a, an an tagonist o f T rkA
(45,130-133), p revented p art of th e in vivo and in vitr o colonic m otor alterations
associated to OVA exposure. These results agree with a previous study in which NGF in
vivo immunoneutralization was able to b lock th e motor alter ations as sociated to T.
spiralis infection in rats (58). Overall, these observations support a role for NGF-TrkAdependent mechanisms o n t he onset/maintenance o f IBS-like gastrointestinal m otor
alterations. Since e nteric neurons ex press T rkA, as p reviously d escribed an d a lso
confirmed by u s (134, and data not shown), we ca n hypothesize that K252a binding to
TrkA r eceptors o n myenteric n eurons p revents NGF -mediated effects w ithin th e ENS
and, thus, modulates motor activity. In addition, K252a could affect the ability of NGF
to in teract with T rkA r eceptors ex pressed o n MM Cs, th us modulating th eir
150
DISCUSSION
excited/activated sate and, therefore, the release of MM C mediators (Fig. 4). Ho wever,
despite th e i mplication o f N GF in t he motor alter ations o bserved, lo ng-term OV A
exposure was n ot associated to ch anges in NGF tissue expression. According to th at
reported, in flammation-dependent ch anges i n NGF e xpression s eem to b e short-lasting
and take place in early phases of the inflammatory course (104). In agreement, in the T.
spiralis infection model in rats, we observed an early peak in jejunal NGF expression at
day 2 PI. Sin ce tr ansitory i ncreases in NGF a vailability have been as sociated to lo nglasting phenotypical changes in the target tissue (43), the persistent colonic dysmotility
observed after the 6-week exposure period to OVA could be related to changes in NGF
content at early times, not ass essed in the present s tudies. We ca nnot ex clude the
possibility that effects on the ENS associated to OVA-induced MMC mediators release
are mediated through p athways i nvolving NGF/TrkA-dependent m echanisms, although
NGF is not directly up-regulated by OVA.
Infection with T. spiralis induces long-term changes in the morphological and functional
properties of enteric afferents
Remodeling o f visceral afferents has been s uggested to b e implicated i n I BS-like
visceral hypersensitivity s tates (135). Moreover, i n s ome patients, first appearance o f
altered gut perception has been related to the resolution of a transient intestinal infection
(the so-called PI-IBS) (136). Thus, we explored if morphological and functional changes
in sensory neurons are also present in the T. spiralis infection model in rats.
At late phases o f the in fection ( day 3 0 PI), s ensory neurons o f T 10-L2 D RGs, which
contain a fferent f ibers in nervating b oth t he small intestine and t he colon o f t he rat
(14,15,137), s howed a
significant increase in t heir cross-sectional area. T hese
observations i ndicate a wide ef fect of t he infection within t he gastrointestinal tract,
covering b oth r egions p rimarily a ffected b y t he parasite (jejunum) and also worm-free
areas (colon) (Fig. 4 ). T o o ur k nowledge, t his i s t he first ev idence o f r emodeling of
sensory a fferents o utside the regions o f the g ut p rimarily a ffected b y a h armful i nsult.
This observation agrees with some opinions considering IBS an alteration affecting the
whole intestine vs. a disease affecting solely the colon (138).
151
DISCUSSION
In o rder to elu cidate if t hese morphological ch anges translate into an al tered af ferent
function, we determined ch anges in T RPV1 lev els in TL ( T12-L2) a nd LS ( L6-S2)
DRGs following intracolonic stimulation of TRPV1 receptors with capsaicin. TL and LS
DRGs were s elected i n o rder to d istinguish t he gut area potentially a ffected b y th ese
changes (small i ntestine vs. co lon) (14,15,137). Stimulation o f s ensory a fferents with
intracolonic ca psaicin i nduced a down-regulation o f T RPV1 ex pression i n T L an d L S
DRGs, similarly to that observed in previous studies (139,140). Interestingly, capsaicin
effects were significantly e nhanced in LS DRGs of T. s piralis-infected r ats, regardless
the PI time considered. These observations agree with data obtained in murine models of
PI-IBS showing a
persisting hyperexcitability o f enteric DRG neurons a fter the
resolution of the infectious process (141,142). T hus, taken together, the results support
the view t hat structural and f unctional remodeling o f e nteric afferents ar e a significant
component of I BS, at least in th ose cases in which th e d isease is tr iggered b y a n
intestinal in fection (PI-IBS). T hese ch anges might serve as a basis for th e altered
visceral sensitivity that appears as a characteristic feature in a significant proportion of
IBS patients.
Neurotrophic factors ar e implicated in th e remodeling o f the enteric sensory a fferent
innervation associated to post-infectious states
As mentioned ab ove, NT Fs have b een r elated to t he remodeling o f visceral afferents
observed in states of inflammation in different organs, including the gastrointestinal tract
(105,143). In our conditions, the infection with T. spiralis was associated to time- (early
vs. late phases of t he infection) an d s ite-specific (jejunum vs. co lon) ch anges in NT Fs
content. Within the jejunum, an early (day 2 PI) increase in the levels o f NGF, GDNF
and artemin was observed. However, in the colon, a general down-regulation of NTFs,
including ne urturin, was o bserved between days 6 -14 P I. These r egional d ifferences in
expression might be related to the local degree of inflammation; since, compared to the
jejunum, t he colon o f T. s piralis-infected r ats s howed an atte nuated in flammatory
response.
Exposure to NTFs, particularly NGF, has been related to long-term neuroplastic changes
in sensory ne urons (117). Therefore, it is f easible to h ypothesize that there is a ca useeffect relationship between the early peak in jejunal NGF expression and the increase in
152
DISCUSSION
the mean cross-sectional area of T10-L2 DRG neurons observed at day 30 PI (Fig. 4). A
similar r ole for NGF i n t he d evelopment of D RG neuronal hypertrophy has been
suggested d uring urethral o bstruction in r ats (144,145). In ad dition, T rkA
immunoreactivity i n DR G neurons was also r educed alo ng th e infectious p rocess, th us
suggesting a compensatory mechanism to constraint the effects of an increased level of
ligand. Sin ce NT Fs ar e uptaken i n a fferents ax on ter minals after b inding to th eir
receptors (146), their signaling in DRG neurons can be limited by diminishing neuronal
receptor expression. Local down-regulation of NTFs at peripheral sites, as observed here
in t he colon o f i nfected-rats in ea rly p hases, can al so contribute to constraint t hese
effects o n visceral a fferents. Su pporting t his hypothesis, th e correlation o bserved
between t he changes in ar temin a nd T RPV1 ex pression u nder af ferent stimulation
suggests that TRPV1 expression in LS DRGs might act restraining artemin up-regulation
in the colon and, therefore, the excitability of afferents. Thus, overall, the results shown
here support the view that locally released NT Fs are modulating the changes in enteric
sensory n eurons o bserved d uring i ntestinal inflammation and s uggest the presence o f
regulatory mechanisms, b oth within t he gut an d in its a fferent innervation, to limit
NTFs’ effects on neuronal remodeling.
Mucosal mast cells of the rat colon are not a source of NGF
Effects of MCs on the development of IBS-like symptomatology have been suggested to
be mediated, at least in part, by NGF (43). Indeed, studies in colonic biopsies of patients
with I BS and i n a nimal models reveal an u p-regulation in the expression le vels o f th e
peptide (43,45,103). Ho wever, th e exact source(s) o f NG F in the colon, i ncluding t he
potential role of MCs, is controversial. In order to elucidate these points, we assessed the
expression of NGF o r its precursor, proNGF, in the colon and, in particular, in resident
MMCs. Our results confirm previous observations showing specific NGF staining in the
epithelium, E NS and s carce ce lls, o f u nidentified t ype, within th e colonic cr ypts (45).
However, we have been u nable to d etect NGF o r its p recursor in MM Cs. T his is i n
agreement with a study in a rat colitis model describing co lonic NGF as an ep ithelialderived, b ut not MC-derived, p roduct (94) and s upports the hypothesis t hat MMCs d o
not r epresent a source o f NG F in the r at colon. I t is worthy to mention t hat in co lonic
biopsies of patients with IBD, NGF has been localized in MMCs (92). Therefore, taking
into consideration that MCs show a high interspecific variability (147), it is feasible to
153
DISCUSSION
speculate t hat these variations might reflect s pecies-related d ifferences ( human vs. r at)
and that the possibility that human MMCs synthesize NGF cannot be ruled out.
Mucosal mast cells of the rat colon are a target for NGF
Both in vivo and in v itro studies have demonstrated th e a bility o f NG F to d egranulate
MCs (113-116). Thereby, it is f easible to h ypothesize the i mplication o f NGF i n th e
excited-activated s tate of co lonic MM Cs th at c haracterize the p athophysiology of IBS
(43,148, present o bservations) (Fig. 4 ). Ou r r esults d emonstrate the presence o f T rkA
receptors on co lonic MMCs of t he rat, i n ag reement with h uman d ata (92) (Fig. 4 ).
Presence of TrkA on MMCs could explain, at least in part, the up-regulation in receptor
levels during co lonic s tates o f MM C ac tivation, as observed in lo ng-term e xposure to
oral OVA. I n ad dition, tr eatment with t he TrkA a ntagonist K252a tended to in crease
colonic RMCPII levels, suggesting that these receptors are functional and might mediate
MMC d egranulation upon s timulation with NGF. I ndeed, th e functionality o f T rkA
receptors ex pressed b y MCs has been p reviously d emonstrated in vitr o (98,113,149).
Thus, overall, the present results suggest that, in the rat colon, MMCs represent a target
for NGF, rather than being a source of the peptide. Therefore, the characteristic excitedactivated s tate of t hese ce lls during I BS co nditions could be modulated by i ncreased
levels of NGF binding to TrkA receptors (Fig. 4).
In s ummary, we show t hat the axis l uminal-derived s timuli-NTFs is i mplicated i n t he
alterations in sensory and motor functions observed in MMC-dependent models of IBS
in rats. Our results highlight a potentially important role for NTF-related pathways in the
onset/maintenance o f the symptomatology i n, at least, a subset o f I BS patients. T hus,
NTFs represent a therapeutic target f or t he tr eatment of gastrointestinal d isorders
characterized by altered colonic sensitivity and dysmotility.
154
CONCLUSIONS
CONCLUSIONS
1. Luminal-derived stimuli comprising intestinal infection with Trichinella spiralis and
exposure to the food a ntigen ovalbumin ac tivate colonic mast cells. Di fferences i n
mast cell responses are observed d epending upon th e inductor s timulus. T. s piralis
infection r esults in a
long-lasting mast cell infiltrate while o ral e xposure to
ovalbumin stimulates mucosal mast cell activity without changes in cell density.
2. Oral ex posure to the food an tigen o valbumin lead s to a colonic motor d ysfunction.
The ab ility o f o valbumin to alter co lonic contractility d epends o n th e time of
exposure and the gut commensal microbiota homeostasis.
3. Colonic motor alter ations induced b y o ral exposure to o valbumin ar e neurotrophinmediated, as d emonstrated by t he pharmacological blockade of t ropomyosinreceptor-kinase receptors with K252a.
4. The neurotrophins-tropomyosin-receptor-kinase receptors pathway participates in the
maintenance of colonic basal contractility in rats.
5. Experimental infection with T. spiralis in rats, although primarily affecting the small
intestine, induces long-term morphological and functional changes in the colon, the
main a ffected o rgan in ir ritable bowel syndrome. This r einforces t he validity o f T.
spiralis infection in rats as a post-infectious-irritable bowel syndrome model.
6. During T. s piralis-induced i ntestinal in flammation, th ere is a morphological and
functional remodeling o f intestinal sensory afferents, manifested as ch anges i n
morphology, neurotrophin co ntent in d orsal r oot ganglia neurons a nd r esponses t o
peripheral stimulation.
7. During T. s piralis infection, a transient u p-regulation o f n eurotrophic factors is
observed at early times in the primary site affected by the parasite (jejunum) and is
followed by an extended down-regulation affecting both jejunum and colon.
8. In r ats, co lonic mucosal mast cells ar e not a source o f nerve growth factor, but
express tr opomyosin-receptor-kinase A r eceptors a nd ar e, therefore, a target f or
nerve growth factor.
9. The lu minal-derived s timuli-mucosal mast ce lls-neurotrophic f actors axis is
implicated i n t he co lonic functional a lterations o bserved in
infection-related
models o
f ir
ritable
157
bowel s
food an tigen and
yndrome
in r
ats.
CONCLUSIONES
1. Los e stímulos d e origen lu minal, en co ncreto la infección intestinal p or Trichinella
spiralis y la exposición al antígeno de la dieta ovoalbúmina, activan los mastocitos
del colon. L as diferencias en las respuestas mastocitarias dependen d el estímulo
inductor. L a infección p or T. s piralis produce un i nfiltrado m astocitario de lar ga
duración mientras que la exposición oral a ovoalbúmina estimula los mastocitos d e
mucosa sin cambiar su densidad.
2. La exposición oral al antígeno de la dieta ovoalbúmina altera la actividad motora del
colon. Los cambios en la contractilidad colónica dependen del tiempo de exposición
al antígeno y de la homeostasis de la microbiota comensal.
3. Las alteraciones de la motilidad del co lon debidas a la ex posición a o voalbúmina
están mediadas por neurotrofinas, tal y como demuestra el bloqueo farmacológico de
los receptores tropomiosina-receptor-quinasa con K252a.
4. La vía n eurotrofinas-receptores tr opomiosina-receptor-quinasa p articipa en
el
mantenimiento de la contractilidad basal del colon en la rata.
5. La infección ex perimental p or T. s piralis en la rata, a unque afecta de manera
primaria al yeyuno, i nduce ca mbios morfológicos y funcionales a lar go p lazo en e l
colon, el órgano q ue se considera principalmente afectado en el síndrome del
intestino irritable. Tal observación refuerza el valor de la infección por T. spiralis en
la r ata como modelo experimental del síndrome de l intestino ir ritable postinfeccioso.
6. En el cu rso d e la i nflamación i ntestinal asociada a la infección p or T. s piralis, se
produce una remodelación morfológica y funcional de las vías aferentes sensoriales
del intestino. Esta remodelación se manifiesta en forma de cambios morfológicos y
en el contenido en neurotrofinas en las neuronas de los ganglios de la raíz dorsal de
la médula espinal, así como en la respuesta a la estimulación periférica.
7. Durante las fases tempranas de la infección por T. spiralis, se observa un incremento
en el co ntenido de factores n eurotróficos en la región in testinal primariamente
afectada ( yeyuno), seguida d e u na r egulación a la baja q ue af ecta tanto al yeyuno
como al colon.
158
CONCLUSIONES
8. En la rata, los mastocitos de mucosa del colon no son una fuente celular de factor de
crecimiento n ervioso. S in e mbargo, expresan r eceptores tropomiosina-receptorquinasa A y, por tanto, representan una diana para factor de crecimiento nervioso.
9. El ej e estímulos luminales-mastocitos d e mucosa-factores n eurotróficos está
implicado en las alteraciones f uncionales d el colon observadas en modelos d el
síndrome del intestino irritable asociados a la exposición a antígenos de la dieta y a
infecciones entéricas en la rata.
159
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APPENDIX
APPENDIX
PUBLICATIONS DERIVED FROM THIS WORK
PAPERS
 F. Jardí, V. Ma rtínez, P . Ver gara. NGF i s i nvolved i n o ral ovalbumin-induced
altered colonic contractility in rats: Evidence from the blockade of TrkA receptors
with K252a. Neurogastroenterol Motil 2012, 24, e580–e590.
 F. Jardí, M. A guilera, P . Ver gara, V. Ma rtínez. Colonic motor alter ations
associated to the sensitization to a luminal antigen are enhanced during a dysbioticlike state in rats. Submitted to Life Sciences.
 F. Jardí, J.A. Fernández-Blanco, V. Martínez, P. Vergara. Plasticity of dorsal root
ganglion neurons in a rat model o f p ost-infectious gut d ysfunction: p otential
implication of nerve growth factor. Submitted to Neuroscience Letters.
 F. Jardí, J.A. Fernández-Blanco, V. Martínez, P. Vergara. Persistent alterations in
colonic afferent innervation in a rat model of post-infectious gut dysfunction: role
for changes in peripheral neurotrophic factors. Submitted to Neuroscience.
ABSTRACTS
 F. Jardí, J.A. Fernández-Blanco, V. Martínez, P. Vergara. Remodeling of colonic
sensory af ferents i n a rat model of p ost-infectious gut d ysfunction: i mplication o f
neurotrophic factors. Poster.
Gastroenterology (in press).
Digestive Disease Week 2014. May 4-6, 2014. Chicago, USA.
 F. Jardí, J .A. Fer nández, E . C epeda, V. Ma rtínez, P . Ver gara. Are mast ce lls a
source o f neurotrophins i n the inflamed g ut? Mis match b etween n eurotrophin
expression and mast cells in a model of enteritis in rats. Poster.
EMBRN-COST International Mast Cell and Basophil Meeting 2013. August 28-30,
2013. Udine, Italy.
 F. Jardí, J .A. Fer nández-Blanco, V. Ma rtínez, P . Ver gara. Mucosal mast ce lls
maintain n ormal contractility o f th e colon in a rat model o f p ost-infectious g ut
dysfunction. Poster.
EMBRN-COST I nternational Ma st Cell a nd B asophil Meeting 2 012. No vember
26-27, 2012. Berlin, Germany.
 F. Jardí, J.A. Fernández-Blanco, V. Martínez, P. Vergara. Neuronal plasticity of
lumbosacral DRG in a model of p ost-infectious gut dysfunction i n r ats. Poster.
Neurogastroenterol Motil 2012; 24 (Sppl. 2):66-67.
Joint International Neu rogastroenterology a nd Mo tility Me eting 2 012. Sep tember
6-8, 2012. Bologna, Italy.
179
APPENDIX
 F. Jardí, J .A. Fer nández-Blanco, V. Ma rtínez, P . Ver gara. Mucosal mast ce lls
maintain normal contractility of the colon through NGF-dependent mechanisms in
a rat model of post-infectious IBS. Poster.
Gastroenterology 2012; 142 (Sppl. 1):S895-S896.
Digestive Disease Week 2012. May 19-22, 2012. San Diego, USA.
 F. Jardí, M. Aguilera, V. Martínez, P. Vergara. Lipopolysaccharides interact with
oral ovalbumin to i nduce food-antigen-related I BS-like alter ations o f co lonic
motility in rats. Oral communication.
Gastroenterology 2011; 140 (Sppl. 1):S-24.
Digestive Disease Week 2011. May 7-10, 2011. Chicago, USA.
 F. J ardí, V. Ma rtínez, P . V ergara. Implication o f nerve growth factor in i ntestinal
mucosal mast cell activity and colonic motor alterations in a model of ovalbumininduced gut dysfunction in rats. Oral communication.
Gut 2010; 59 (Sppl. 3): OP136.
18th United E uropean Gastro enterology W eek. Octo ber 2 3-27, 2010. B arcelona,
Spain.
 F. Jardí, V. Ma rtínez, E . T raver, P. Ver gara. NGF is i mplicated in th e
hypercontractile r esponses o f th e colon in a model of o valbumin-induced gut
dysfunction in rats. Oral communication.
Gastroenterology 2010; 138 (Sppl. 1): S-45.
Digestive Disease Week 2010. May 1-15, 2010. New Orleans, USA.
180
Neurogastroenterology & Motility
Neurogastroenterol Motil (2012) 24, e580–e590
doi: 10.1111/nmo.12027
NGF is involved in oral ovalbumin-induced altered
colonic contractility in rats: evidence from the blockade
of TrkA receptors with K252a
F. JARDÍ ,* V. MARTÍNEZ *, ,à
&
P. VERGARA *, ,à
*Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Barcelona, Spain
Instituto de Neurociencias, Universitat Autònoma de Barcelona, Barcelona, Spain
àCentro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III,
Madrid, Spain
Abstract
Background Nerve growth factor (NGF)-mucosal mast
cell (MMC) interaction has been implicated in the
remodeling of enteric circuitries and associated functional changes. We investigated the involvement of
NGF and its receptor TrkA in the altered colonic
contractile activity observed in the model of oral
ovalbumin (OVA)-induced MMC hyperactivity in rats.
We also studied the role of colonic MMCs as a source
of NGF. Methods Rats received oral OVA, alone or
with the TrkA antagonist K252a. Colonic co-expression of NGF/TrkA and rat mast cell protease II
(RMCPII) (double immunofluorescence), RMCPII
content (ELISA) and expression of NGF, Brain-derived
neurotrophic factor (BDNF) and TrkA/B (QT-PCR)
were assessed. Colonic contractile activity was determined in vivo and in vitro. Key Results TrkA, but not
NGF, was localized in colonic MMCs (RMCPII-positive). Oral ovalbumin exposure increased colonic
RMCPII levels but did not change the percentage of
TrkA-positive MMCs. Neither OVA nor K252a, alone
or combined, altered NGF, BDNF or TrkA/B expression. Spontaneous colonic activity in vivo and in vitro
was altered by OVA, an effect prevented by K252a.
Electrical stimulation-induced contractile responses
in vivo and carbachol responses in vitro were
increased by OVA in a K252a-independent manner. In
OVA-treated animals, inhibition of NO synthesis
with L-NNA significantly enhanced spontaneous
colonic activity in vitro, a response completely
prevented by K252a. Conclusions & Inferences These
results suggest that NGF-TrkA-dependent pathways
are implicated in colonic contractile alterations
observed during OVA exposure in rats. NGF-TrkA
system might represent a potential target for treatment of gastrointestinal disorders characterized by
colonic motor alterations.
Keywords colonic contractility, K252a, mucosal mast
cells, nerve growth factor, ovalbumin, TrkA.
Abbreviations: BDNF, brain-derived neurotrophic factor;
CCh, carbachol; EMS, Electrical Mucosal Stimulation;
ENS, enteric nervous system; FGD, functional gastrointestinal disorder; HFLA, high-frequency and low-amplitude
(contractions); IBS, irritable bowel syndrome; IHC,
immunohistochemistry; LFHA, low-frequency and highamplitude (contractions); L-NNA, NG-nitro-L-arginine;
MMC, mucosal mast cell; NGF, nerve growth factor;
OVA, ovalbumin; RMCPII, rat mast cell protease II.
INTRODUCTION
Colonic dysmotility is a common finding in functional
gastrointestinal disorders (FGDs). For instance, irritable bowel syndrome (IBS), the main FGD, is characterized by abdominal pain-discomfort associated with
dysmotility and altered bowel habits.1 Moreover, in a
large number of IBS patients, food ingestion has been
associated with symptomatology exacerbation, suggesting a role for food allergy in its pathogenesis.2
Intestinal food allergy-related mechanisms in IBS seem
to involve local mucosal responses to dietary antigens
rather than classical type-1 hypersensitivity reactions.3
Address for Correspondence
V. Martı́nez, Department of Cell Biology, Unit of PhysiologyVeterinary School, Physiology and Immunology Universitat
Autònoma de Barcelona, 08193-Bellaterra, Barcelona, Spain.
Tel: +34 93 581 3834; fax: +34 93 581 2006;
e-mail: [email protected]
Received: 23 March 2012
Accepted for publication: 17 September 2012
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Volume 24, Number 12, December 2012
Effects of K252a on colonic contractility
to oral OVA, complementing this data by determining
the colonic expression of the related neurotrophin brainderived neurotrophic factor (BDNF) and its preferential
receptor TrkB. Finally, we evaluated the interplay
between MMCs-NGF/TrkA system, characterizing, in
particular, if MMCs represent a cellular source of the
neurotrophin and/or express TrkA receptors.
We have shown that chronic exposure to oral ovalbumin (OVA) in Sprague–Dawley (SD) rats induces a nonIgE-mediated alteration of smooth muscle colonic
contractility resembling that observed by others and
us in IBS and animal models of the disease.4–8
Several observations support an involvement of mast
cells in the pathophysiology of IBS.9 Morphological and
functional studies with colonic biopsies from IBS
patients point towards an important role of mast cellderived mediators and the interaction mast cells-nerve
fibers on the disturbed secretomotor and sensory
functions characterizing IBS.10,11 In this line, we have
demonstrated that in the rat model of chronic exposure
to oral OVA, mucosal mast cells (MMCs) are implicated in the altered colonic contractile activity; thus
suggesting that OVA-induced colonic motor alterations in rats are somehow MMC-dependent.4 Therefore, exposure to oral OVA in rats reproduces some
pathophysiological components of IBS; at least the
MMC hyperactivity and the changes in colonic contractility, thus representing a valid model for studying
IBS-related altered colonic contractile responses and
their potential relationship with MMCs.
Recent data suggest that, within the gut, neurotrophins, mainly nerve growth factor (NGF), interact with
MMCs generating a neuroimmune circuit likely to
play a potential role in the pathophysiology of FGDs.
For instance, evidences obtained in animal models of
IBS have implicated NGF in the neuronal remodeling
of enteric circuitries and MMCs recruitment, as basis
for the functional changes observed. In particular, antiNGF treatment completely blocked intestinal
hypermotility in Trichinella spiralis-infected rats, an
accepted model of postinfectious IBS,12 and reduced the
interaction MMCs-nerve fibers in the rat maternal
separation model.13 A recent study in colonic biopsies
from IBS patients supports these observations, showing
an increased neuronal sprouting within the mucosa, an
effect associated with NGF increased levels, possibly of
mast cell origin.14 However, the exact origin of colonic
NGF and the potential role for MMCs as the peptide
source are still unclear.
Nerve growth factor interacts with two classes of cell
surface receptors: the TrkA high-affinity receptor, a
selective NGF receptor; and the p75 low-affinity receptor, which presumably binds to all neurotrophins.15 To
further understand the role of NGF in colonic motor
alterations we have investigated the effects of the
pharmacological blockade of TrkA on OVA-induced
changes in colonic contractility in the rat by using
K252a, which has been widely used in the rat as a TrkA
antagonist.16–20 In addition, we evaluated the expression
levels of NGF and TrkA in the rat colon during exposure
2012 Blackwell Publishing Ltd
MATERIALS AND METHODS
Animals
Adult (5 weeks old at arrival), specific pathogen free (SPF), SD
male rats were used (Charles River, Les Oncins, France). Animals
had free access to water and a standard pellet diet, free of traces of
OVA or any other egg derivative (A04; Safe, Augy, France). Rats
were maintained under conventional conditions in a light (12
h/12 h light–dark cycle) and temperature controlled (20–22 C)
room, in groups of two per cage. Animals were acclimatized to the
new environment for 1 week before starting any experimental
procedure. All the experimental protocols were approved by the
Ethical Committee of the Universitat Autònoma de Barcelona and
the Generalitat de Catalunya (protocols 1010 and 5351,
respectively).
Experimental design
Rats received OVA by oral gavage (1 mg mL)1, 1 mL per rat,
n = 24), on a daily basis during a 6-week period.21 A group of rats
receiving vehicle (1 mL per rat, n = 21) was used as control. After
the third week, 11 of the animals receiving OVA and 10 of the
animals receiving vehicle were treated subcutaneously with
K252a (50 lg kg)1). Treatment with K252a was performed daily
and lasted until the day before animals were euthanized, outlasting for 10 days OVA exposure. This antagonist, at the dose and
pattern of administration followed here, has already been used,
showing effective blockade of TrkA and biological effects
in vivo.18–20 The rest of the animals (13 OVA- and 11 vehicleexposed) were used as control groups in which the treatment
protocol was the same but K252a was replaced by the corresponding vehicle (1 mL kg)1, s.c.). Except for the in vivo experiments, at
the time of euthanasia, tissue samples from the colon were
obtained and either used for organ bath studies, fixed in 4%
paraformaldehyde for immunohistochemical studies or frozen in
liquid nitrogen and stored at )80 C until analysis.
Organ bath
Full thickness preparations, obtained from the mid portion of
the colon, were cut 1 cm long and 0.3 cm wide and hung for
organ bath study oriented to record circular muscle activity.
Strips were mounted under 1 g tension in a 10-mL muscle bath
containing carbogenated Krebs solution (95% O2 – 5% CO2)
maintained at 37 ± 1 C. The composition of Krebs solution was
(in mmol L)1): 10.10 glucose, 115.48 NaCl, 21.90 NaHCO3, 4.61
KCl, 1.14 NaH2PO4, 2.50 CaCl2, and 1.16 MgSO4 (pH 7.3–7.4).
One strip edge was tied to the bottom of the muscle bath using
suture silk and the other one to an isometric force transducer
(Harvard VF-1 Harvard Apparatus Inc., Holliston, MA, USA).
Output from the transducer was fed to a PC through an
amplifier. Data were digitalized (25 Hz) using Data 2001
software (Panlab, Barcelona, Spain). Strips were allowed to
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Neurogastroenterology and Motility
equilibrate for about 1 h. After this period, contractile responses
to carbachol (CCh; 0.1–10 lmol L)1) and the NO inhibitor NGnitro-L-arginine (L-NNA; 1 mmol L)1) were assessed. For CCh,
cumulative concentration-response curves, with a 5-min interval between consecutive concentrations, were constructed. For
L-NNA, spontaneous activity was recorded during a 10-min
period after the addition of the drug.
To determine the spontaneous contractile activity, the preparation tone was measured for 15 min and the mean value (in g)
determined. To test the effects of CCh, the maximum peak, from
the basal tone, was measured after each concentration tested. To
measure the response to L-NNA, the 10-min mean of the strip
tone before the drug administration was determined and compared with the 10-min mean of the strip tone after L-NNA
addition.
with avidin/peroxidase kit (Vectastain ABC kit; Vector Laboratories) and counterstaining with hematoxylin. Specificity of the
staining was confirmed by omission of the primary antibody.
When performing IHC for NGF, mouse submaxillary glands were
used as a positive control. Slides were viewed with an Olympus
BH-2 microscope (Olympus, Hamburg, Germany). For MMC
quantification, at least 20 non-adjacent ·40 fields of colonic
mucosa were randomly selected and the number of RMCPIIimmunopositive cells counted. All procedures were carried out
using coded slides to avoid bias.
Immunofluorescence staining
For analyses of co-localization of NGF, proNGF or TrkA with
RMCPII, double immunofluorescence was used. After 1 h of
blocking with 10% normal goat serum at room temperature,
colonic sections were incubated with a mixture of anti-RMCPII
and anti-NGF or anti-ProNGF (ab5583; Chemicon International)
or anti-TrkA (sc-118; Santa Cruz Biotechnology, Santa Cruz, CA,
USA) overnight. Thereafter, sections were incubated with a
secondary antibody cocktail consisting of fluorescence-conjugated
Alexa Fluor 488 goat anti-mouse IgG (A11029; Molecular Probes,
Eugene, OR, USA) and Cy3 goat anti-rabbit IgG (PA-43004;
Amersham-Pharmacia, Buckinghamshire, UK). After washing,
the slides were coverslipped with Vectashield Mounting Medium
(Vector Laboratories) and examined under an Axioskop 40 fluorescence microscope (Carl Zeiss, Jena, Germany). Merging of the
images was analyzed with ImageJ Software (U. S. National
Institutes of Health, Bethesda, MD, USA). To assess the percentage of cells with RMCPII and TrkA co-localization, Alexa Fluor
488- and Alexa Fluor 488-Cy3-stained cells were counted randomly using a 100· objective. Specificity of the staining was
confirmed by omission of the primary antibody and/or the
secondary antibodies. The absence of cross-reactivity was confirmed in control single-labeled preparations.
Strain-gauge recordings
After a fasting period of 6 h, animals were placed in an induction
camera and anesthetized by inhalation of 4% isofluorane
(Isoflo; Esteve, Barcelona, Spain) in 2 L min)1 oxygen to allow
cannulation of a lateral vein of the tail. Thereafter, rats were
maintained in level III of anesthesia by intravenous thiopental
sodium, as required, and exposed to mask delivery of 1 L min)1
oxygen during all the procedure. A laparatomy was performed,
the colon localized and a strain-gauge (F-04IS; Star Medical,
Tokyo, Japan) was sutured to its wall (2 cm from the cecum) to
record circular muscle activity. The strain gauge was connected
to a high-gain amplifier (MT8P; Lectromed, Herts, UK), and
signals were sent to a recording unit (PowerLab/800; ADInstruments, Castle Hill, NSW, Australia) connected to a computer.
Finally, an electrode holder with two platinum electrodes (WPI,
Sarasota, FL, USA) was inserted into the colonic lumen at 1 cm
distally to the strain-gauge to induce ascending excitation of the
peristaltic reflex by electrical mucosal stimulation (EMS). Electrical mucosal stimulation was applied by duplicate at 30 V,
0.6 ms and 4 Hz during 30 s each time, and the polarity of the
stimulating electrodes was reversed at 15 s.
To analyze in vivo colonic motility, contractions of the colon
were classified into low-frequency and high-amplitude (LFHA)
contractions and high-frequency and low-amplitude (HFLA) contractions, as previously described.22 High-frequency and lowamplitude were identified by having a frequency within the range
of 10–15 contractions min)1, while LFHA were defined as contractions of an amplitude >300% of that of HFLA contractions at the
same recording site.22 High-frequency and low-amplitude and
LFHA were assessed over a 15-min period and the frequency and
amplitude expressed as the mean. When assessing the responses to
EMS, the recording analyzed corresponded to the 30 s of stimulation and the expressed value was the mean of the duplicates. All
analysis was performed using Chart 5 software for Windows (both
from ADInstruments).
ELISA
Protein was extracted from colonic tissue samples using
lysis buffer (50 mmol L)1 HEPES, 0.05% Triton X-100,
0.0625 mmol L)1 PMSF and the Mini Complete protease inhibitor
Roche) and RMCPII concentration was determined by ELISA
using a commercial kit (Moredun). Total protein was determined
using the Bradford assay kit (Bio-Rad, Hercules, CA, USA).
RNA extraction and quantitative real-time PCR
Total RNA was extracted from colonic samples using Ribopure
RNA Isolation Kit (Applied Biosystems, Carlstad, CA, USA) and
quantified by Nanodrop (Nanodrop Technologies, Rockland, DE,
USA). For cDNA synthesis, 1 lg of RNA was reverse-transcribed in
a 20 lL reaction volume using a high capacity cDNA reverse
transcription kit (Applied Biosystems). Expression of NGF, BDNF,
TrkA, and TrkB was determined by quantitative real-time PCR
performed with specific Taqman probes (Applied Biosystems; NGF:
Rn01533872_m1, BDNF: Rn00560868, TrkA: Rn00572130_m1,
TrkB: Rn01441749_m1, B-Actin: Rn00667869_m1) mixed with
Taqman Universal Master Mix II for 40 cycles (95 C for 15 s, 60 C
for 1 min) on a 7900 real-time PCR system (Applied Biosystems).
Rat submaxillary gland and neocortex were used as positive
controls for the gene expression of TrkA, TrkB and NGF, and
BDNF, respectively. B-Actin expression served as an endogenous
control for normalizing the mRNA levels of the target gens.
Expression levels were analyzed by the 2)DDCT method.
Immunohistochemistry (IHC)
Immunodetection of RMCPII and NGF was carried out on
paraformaldehyde-fixed colonic samples using a monoclonal
antibody anti-RMCPII (Moredun, Edinburgh, UK) and a polyclonal
rabbit anti-NGF (ab1526; Chemicon International, Temecula, CA,
USA). Antigen retrieval for NGF was achieved by processing the
slides in a pressure cooker, at full pressure, for 10 min in
10 mmol L)1 citrate solution. The secondary antibodies included
biotinylated horse antimouse IgG (BA-2000; Vector Laboratories,
Burlingame, CA, USA) and biotinylated swine antirabbit Ig
(E0353; Dako, Carpinteria, CA, USA). Detection was performed
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Effects of K252a on colonic contractility
A
Chemicals
Ovalbumin (Grade V; A5503) was purchased from Sigma-Aldrich
(St. Louis, MO, USA) and was dissolved in saline solution. K252a
[(9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3¢,2¢,1¢-kl]pyrrolo[3,4-i][1,6]
benzodiazocine-10-carboxylic acid methyl ester; Tocris Bioscience, Ellisville, MO, USA] was reconstituted in 8.75% ethanol in
milli-q water. CCh (Sigma-Aldrich) stock solution and further
dilutions were prepared in distilled water. NG-nitro-L-arginine
(Sigma-Aldrich) was prepared directly in carbogenated Krebs
solution.
B
Statistics
All data are expressed as mean ± SEM. Motility results are
presented as raw data (g of force) or frequency of contractions
(number min)1; in vivo recordings). EC50 for CCh was calculated
by non-linear regression to a sigmoidal equation (GraphPad Prism
4.01, San Diego, CA, USA). Comparisons between multiple
groups were performed with two-factor ANOVA. When the two
way ANOVA revealed significant effects of treatments, data were
reanalyzed with one-way ANOVA followed, when necessary, by a
Student–Newman–Keuls multiple comparison test to detect differences between experimental groups. P values < 0.05 were
considered statistically significant.
Figure 1 Colonic density of mucosal mast cells (A) and rat mast cell
protease II content (B) in the different experimental groups. Data are
mean ± SEM; n = 5–10 per group. *P < 0.05 vs vehicle-vehicle.
RESULTS
firming the specificity of the staining. Staining was
intense and well localized in positive controls from
mouse submaxillary glands.
Colonic mucosal mast cell count and RMCPII
content
The number of RMCPII-positive cells in the colon
showed no significant differences between experimental groups despite the treatment received (Fig. 1A).
Nevertheless, a two-way ANOVA analysis revealed an
effect of OVA treatment increasing RMCPII content
(P = 0.022), although only the OVA-K252a group
achieved statistical significance (P < 0.05 vs vehiclevehicle; Fig. 1B). K252a, per se, showed a tendency to
increase the levels of RMCPII, although statistical
significance was not achieved (P = 0.13).
Co-localization of RMCPII and NGF, proNGF
or TrkA
In vehicle-treated animals, double immunofluorescence studies demonstrated that the vast majority
(63.6 ± 10.4%) of RMCPII-positive cells (identified as
MMCs) co-expressed TrkA-like immunorreactivity
(Figs. 2E–G). Similar ratio of RMCPII-TrkA co-localization was observed after OVA exposure (75.0 ± 5.7%).
Apart from RMCPII-positive cells, other scarce cells
within the lamina propria showed TrkA-labeling,
indicating that not only MMCs express the receptor
in the rat colonic mucosa.
No RMCPII-positive cells (MMCs) showed co-staining for NGF or ProNGF.
Localization of NGF by immunohistochemistry
Within the colon, immunoreactivity for NGF was
detected mainly in the submucosal and myenteric
plexuses (Fig. 2C,D). A diffuse staining was observed in
the epithelium, both on the villi and, occasionally, in
the crypts. Within the villi, there were scarce cells, of
undetermined type, showing NGF-like immunoreactivity (Fig. 2A,B). No labeling was detected in the
muscle layers. No differences in the staining pattern or
intensity were observed between OVA- and vehicletreated animals or associated with the treatment with
K252a. Immunoreactivity was absent in sections in
which the primary antibody was omitted, thus con-
2012 Blackwell Publishing Ltd
NGF, BDNF, TrkA, and TrkB expression in the
colon
Overall, colonic expression levels of NGF, TrKA and
TrkB were relatively low, with no significant differences in expression levels among the different experimental groups (Fig. 3). However, it was noticeable that
K252a and OVA, per se, increased TrkA expression
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B
D
C
E
Figure 2 (A–D) Photomicrographs showing
nerve growth factor (NGF)-like immunoreactivity in colonic tissues. (A, C) vehiclevehicle-treated rat. (B, D) Oral ovalbumin
(OVA)-vehicle-treated rat. Labeling for NGF
was detected in the submucosal and myenteric plexuses, surface of the villi and crypts,
and in scarce, unidentified cells within the
villi. Insert in (B) shows a higher magnification of the NGF-immunoreactive cells
observed within the colonic crypts. Note
that no differences in the staining pattern or
intensity are observed between OVA- and
vehicle-treated animals. (E–G) Dual label
immunofluorescence showing the presence
of TrkA in mucosal mast cells (MMCs) of
the rat colon. (E) Representative image of
anti- rat mast cell protease II (RMCPII)
labeling (green) of cells (identified as MMCs)
in the colonic mucosa. The arrows indicate
positively labeled cells. (F) Same field as in E
showing labeling for TrkA (red). The arrows
indicate positively labeled cells. (G) Merged
image of E and F showing extensive
colocalization (yellow) of RMCPII (MMCs)
and TrkA immunoreactivities. The arrows
indicate double labeled MMCs, whereas the
arrowhead indicates a MMC negative for
TrkA.
F
G
In control conditions, CCh elicited a concentration-dependent contractile response with an
estimated EC50 of 1.8 ± 1.3 mmol L)1. Overall, a
two-way ANOVA analysis revealed an OVA effect
(P = 0.031) enhancing the contractile responses to
CCh, leading to a left-shift of the concentrationresponse curve and a fivefold reduction in the
estimated EC50 (0.39 ± 0.1 mmol L)1; Fig. 4B). Treatment with K252a did not affect the responses to
CCh, neither in vehicle- nor in OVA-exposed animals (EC50; vehicle-K252a: 1.7 ± 1.0 mmol L)1; OVAK252a: 0.16 ± 0.04 mmol L)1; Fig. 4B).
In colonic strips obtained from vehicle-vehicle animals, blockade of NO synthesis by the addition of
L-NNA to the organ bath increased spontaneous activity over pretreatment values (P = 0.036; Fig. 4C).
Similar effects were observed in tissues from OVAvehicle- or vehicle-K252a-treated rats, although motor
effects of L-NNA were enhanced in OVA-vehicletreated animals (Fig. 4C). However, in animals treated
with OVA plus K252a, L-NNA-induced increased
spontaneous activity was not longer observed (Fig. 4C).
levels by 32% and 26% respectively when compared to
the expression levels in the control group although
these effects were not evident in OVA-K252a-treated
animals (Fig. 3B).
In control tissues (rat submaxillary gland) expression
levels of NGF, TrkA and TrkB were, respectively, 6-,
14- and 100-fold higher than those observed in the
colon in control conditions.
Brain-derived neurotrophic factor was not detectable
in the colon (ct values higher than 40), although high
expression levels were found in the positive control (rat
neocortex).
Colonic contractility in vitro
Spontaneous colonic contractile activity, as assessed
in vitro, was similar in vehicle- and OVA-exposed
animals (vehicle: 0.53 ± 0.06 g; OVA: 0.51 ± 0.03 g;
P > 0.05; Fig. 4A). K252a decreased spontaneous activity
in similar proportion in vehicle- (0.40 ± 0.05 g; P = 0.07
vs vehicle-vehicle; Fig. 4A) or OVA-exposed animals
(0.35 ± 0.05 g, P < 0.05 vs OVA-vehicle; Fig. 4A).
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Effects of K252a on colonic contractility
A
A
B
B
C
C
Figure 3 Real-time PCR analysis of mRNA for NGF (A), TrkA (B) and
TrkB (C). Data are mean ± SEM; n = 5–9 per group.
Colonic motility in vivo
Figure 4 Effects of oral ovalbumin (OVA) and K252a on colonic contractility in vitro. (A) Colonic spontaneous contractile activity in the
different experimental groups. Data are mean ± SEM; n = 5–10 per
group. *P < 0.05 vs OVA-vehicle; #P = 0.074 vs vehicle-vehicle. (B)
Concentration-response curves for carbachol. Note that oral exposure
to OVA leads to a left-shift of the concentration-response curve, an
effect not modified by treatment with K252a. Data represent mean
values (symbols) and non-linear regression curves. n = 5–10 per group.
(C) Effect of NO blockade with NG-nitro-L-arginine (L-NNA) on spontaneous contractility in the different experimental groups. Data represent spontaneous contractility before (open bars) and after the
addition of L-NNA (closed bars). Data are mean ± SEM; n = 5–10 per
group. *P < 0.05, **P < 0.01, ***P < 0.001 vs respective contractile
activity before the addition of L-NNA (Paired t-test); #P < 0.01 vs other
L-NNA-treated groups (ANOVA).
As previously described,22 strain-gauge recordings of
spontaneous colonic motility in vehicle-vehicle-treated rats exhibited two different types of contractions;
LFHA contractions with superimposed HFLA contractions (Fig. 5F). Overall, exposure to OVA altered spontaneous colonic motility by affecting both types of
contractions (Fig. 5). Treatment with OVA tended to
increase the frequency of LFHA contractions (vehicle:
0.67 ± 0.14 contractions min)1; OVA: 0.93 ± 0.08 contractions min)1; P = 0.08; Fig. 5A) and also enhanced
the
amplitude
(vehicle:
1.92 ± 0.25 g;
OVA:
2.78 ± 0.20 g; Fig. 5B). As it refers to HFLA contractions, exposure to OVA did not alter the frequency but
increased the amplitude (vehicle: 0.37 ± 0.05 g; OVA:
0.59 ± 0.03 g; Fig. 5D). However, in animals treated
with K252, part of these effects of OVA exposure on
spontaneous colonic motility was no longer observed.
K252a inhibited the OVA-increased frequency of LFHA
contractions (0.49 ± 0.14 contractions min)1; P < 0.05
2012 Blackwell Publishing Ltd
vs OVA-vehicle; Fig. 5A) although it did not affect the
amplitude. A similar effect of K252a on the frequency,
but not the amplitude, of LFHA contractions was
observed on vehicle-exposed animals (0.28 ± 0.09 contractions min)1; P < 0.05 vs vehicle-vehicle; Fig. 5A).
Concerning to HFLA contractions, treatment with
K252a showed a tendency to reverse the increase in
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D
E
F
Figure 5 (A–D) Effects of oral ovalbumin
(OVA) and K252a treatment on colonic
motility in vivo. (A, B) Frequency (A) and
amplitude (B) of low-frequency and highamplitude (LFHA) colonic contractions in
the different experimental groups. Note that
oral exposure to OVA tends to increase the
frequency of LFHA colonic contractions and
treatment with K252a reduces it in both
vehicle- and OVA-treated rats. *P < 0.05 vs
respective vehicle (C, D) Frequency (C) and
amplitude (D) of high-frequency and lowamplitude (HFLA) colonic contractions in
the different experimental groups. Note that
oral exposure to OVA leads to an increase of
the amplitude of HFLA colonic contractions,
an effect prevented by treatment with
K252a. Data are mean ± SEM; n = 3–5 per
group. (E) Colonic response to electrical
mucosal stimulation (EMS) (30 V, 4 Hz,
30 s) showing that exposure to OVA
increases EMS-elicited motor responses in a
K252a-independent manner. Data are mean
± SEM; n = 3–5 per group. (F) Representative
tracings showing spontaneous colonic
motility and response to EMS in a vehiclevehicle-, OVA-vehicle- and OVA-K252atreated animal. The arrows indicate LFHA
contractions. Note how OVA exposure increases the frequency of LFHA contractions,
an effect prevented by K252a treatment.
OVA: 3.05 ± 0.23 g; P = 0.05; Fig. 5E) in a K252aindependent manner (2.80 ± 0.54 g; Fig. 5E).
the amplitude of HFLA contractions after OVA exposure, as suggested by a significant interaction between
treatments in a two-way ANOVA (0.34 ± 0.05 g; P < 0.05;
Fig. 5D).
In control conditions EMS elicited a LFHA-type
response that coincided with the stimulation time
(Fig. 5F). The contractile response to EMS was
increased by exposure to OVA (vehicle: 1.24 ± 0.23 g;
DISCUSSION
This study shows that, in the rat model of chronic
exposure to oral OVA, changes in colonic motility
might be related to an altered activity of the NGF-TrkA
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Effects of K252a on colonic contractility
into myenteric motor circuits due to a tonic activation
of MMCs.
In animal models of IBS, both mast cells and NGF
have been implicated in colonic epithelial barrier
function, propulsive motor activity and sensitivity to
colorectal distension.12,24 However, the exact source(s)
of colonic NGF remain elusive and the potential role of
mast cells as the main source of intestinal NGF is
controversial.24,29 In order to elucidate these points,
immunohistochemistry for the neurotrophin was performed in colonic tissues. Although we were able to
see specific NGF staining, with similar distribution
patterns as those previously reported,17 we did not find
any obvious difference in staining, intensity or distribution, among the different experimental groups. This
was further confirmed by real time PCR, showing
similar expression levels of NGF among experimental
groups. Moreover, during double labeling studies, we
were unable to detect NGF immunoreactivity in
MMCs, identified as RMCPII-positive cells within
the colonic mucosa. This contrasts with animal data
suggesting that NGF is released by mast cells upon
degranulation 24,30 and with data from colonic biopsies
of patients with functional and inflammatory gastrointestinal disorders localizing NGF in MMCs.14,31
However, our data agree with a recent study in a rat
colitis model in which NGF immunoreactivity was not
associated with MMCs.29 Whether or not this represents a species-related difference (human vs rat) and/or
experimental model-dependent variations in the colonic source of NGF warrants further studies. Interestingly, Stanzel et al. evidenced that NGF was
synthesized mainly by epithelial cells and hypothesized that MMCs could represent a source of pro-NGF,
in agreement to that suggested also by studies on
cultured rat peritoneal mast cells.32 Based on these
data, we also attempted, although unsuccessfully, to
localize proNGF in colonic MMCs. Overall, our observations indicate that MMCs are not a cellular source of
NGF in the rat colon. Nevertheless, results obtained
suggest a functional link between MMCs and NGF
and, in particular, indicate that MMCs are a target for
NGF. Firstly, we were able to demonstrate the presence
of TrkA receptors on a high proportion of colonic
MMCs (by 60%). Secondly, K252a treatment tended to
increase colonic RMCPII levels, thus suggesting that
these receptors are functional and might mediate
MMC degranulation upon stimulation with NGF.
Indeed, the NGF ability to degranulate mast cells has
been previously demonstrated, both in vivo 33 and
in vitro.34
In this study, we aimed also to elucidate the
functional implication of NGF in the alterations of
pathway. Although NGF expression levels were not
changed, motor responses following the treatment
with K252a suggest that NGF-dependent signaling
pathways are involved in colonic spontaneous motor
activity and mediate OVA-induced enhancement of
NO-dependent inhibitory tone in vitro. Moreover, our
results suggest that colonic NGF is not MMC-derived,
although these cells express TrkA receptors and,
therefore, represent a target for NGF within the colonic
mucosa.
The results of this study confirm that oral OVA
activates MMCs in the colon, as indicated by the
increase in RMCPII levels within the colonic wall,
similarly to that described previously in the same
model.4 Data derived from animal models of IBS have
demonstrated the importance of MMCs as effector
cells mediating the array of pathophysiological changes
that characterize IBS in humans. For instance, degranulation of MMCs seems to be a key step in the onset of
visceral hyperalgesia and the alterations of epithelial
barrier function observed both in animal models and
the human disease.23–26 In the model of chronic
exposure to oral OVA in rats, although these salient
features of IBS have not been characterized, observations reveal that MMCs also play a role on the changes
of colonic smooth muscle contractility, thus supporting the validity of the model as an appropriate approach
to IBS-like altered colonic motor responses.
Results obtained show that OVA-exposed rats have
colonic contractility dysfunction, including increased
responses to carbachol and L-NNA in vitro and
enhanced spontaneous contractility and EMS-elicited
responses in vivo. These results confirm previous
observations in this model,4 resembling that observed
in IBS patients and other animal models of the
disease.5–8 Tissue histological examination excluded
muscle hypertrophy as a potential cause for this
carbachol and EMS enhanced responses (data not
shown), thus suggesting an increased excitability of
the smooth muscle to cholinergic stimulation.
A possible explanation for these OVA-induced colonic
motor alterations could be related to an excitedactivated state of MMCs. This is suggested by the
higher tissue concentration of RMCPII observed in
OVA-treated animals and supported by evidences in
vivo implicating mast cell degranulation in the onset
of cecocolonic motor alterations in rats.27 MMC
mediators act on nerve ending of intrinsic and extrinsic
primary afferent neurons forming neural networks
within both the submucosal and myenteric plexus,
leading to a local amplification of effector responses.28
Therefore, OVA-induced colonic motor alterations
might arise as a result of altered afferent nerve input
2012 Blackwell Publishing Ltd
e587
F. Jardı́ et al.
Neurogastroenterology and Motility
these observations suggest that K252a-mediated effects
within the gastrointestinal tract are related to the
modulation of NGF-TrkA-dependent mechanisms.
Although up-regulation of NGF and its high-affinity
receptor TrkA has been demonstrated during colonic
acute inflammation,29,31,40 expression results in animal models of IBS are discordant.24,26,41 In the present
study, only marginal changes in TrkA, and no changes
in NGF expression levels were observed among experimental groups. This could seem surprising, as we
show that NGF/TrkA-dependent pathways are implicated in the OVA-induced colonic dysmotility in the
rat. However, a previous study assessing changes in
mRNA neurotrophins levels in mice with colitis
showed an increase during the early phases, returning
to control levels one-week after the induction of
inflammation, thus suggesting a rapid and short-term
regulation of these factors during pathological conditions.42 Taking into consideration that neurotrophins
have both acute and long-term biological effects,43 it is
feasible to speculate that NGF/TrkA mRNA colonic
levels in the OVA-exposed rats at the time of euthanasia may be not be representative of those along the
full period of treatment, even though the persistence of
the colonic dysmotility. In addition, there is also the
possibility that the OVA-induced increase in MMC
mediators release exerts its effects on the ENS through
pathways involving NGF/TrkA-dependent mechanisms although these are not directly up-regulated by
OVA. In any case, NGF/TrkA expression results
should be interpreted cautiously since the interaction
between TrkA and other neurotrophin receptors
(namely the p75 and the neurotrophin receptor homolog) leads to an enhanced activity of the signaling
pathways, without increasing the amounts of NGF
and/or TrkA receptors per se.44 It is feasible to assume
that the marginal changes observed in TrkA expression
after OVA or K252a treatment (32% and 26% increase,
respectively) might have consequences at the protein
level yet to be demonstrated. On the one hand, K252ainduced changes in TrkA expression could represent a
compensatory mechanism to the receptor blockade. On
the other hand, OVA-induced changes might be secondary to the OVA-mediated stimulation of MMC and/
or other cell types, including enteric neurons, as
discussed above. Interestingly, these changes in TrkA
expression were not longer observed in animals receiving OVA and K252a. Although we cannot explain the
mechanisms behind this effect, this observation further supports an interplay between OVA effects and
the NGF-TrkA pathway.
In summary, the present study suggests that NGFTrkA-dependent mechanisms are implicated in basal
colonic smooth muscle contractility that characterize
oral OVA exposure in rats. A role for NGF on IBS-like
gastrointestinal motor alterations in animal models
has been previously suggested.12 In order to further
assess this involvement of NGF we used a pharmacological approach based on the blockade of the NGF
high-affinity receptor, TrkA, with K252a.35 In our
conditions, treatment with K252a resulted in a
decrease of spontaneous colonic motor activity both
in vivo and in vitro and prevented the enhancement of
the nitrergic inhibitory tone secondary to OVA exposure in vitro. Interestingly, direct addition of K252a to
the organ bath also decreased spontaneous colonic
contractility (data not shown), thus reinforcing the
results obtained with the treatment with K252a and
suggesting and effective blockade of TrkA in in vivo
conditions. From these observations, it is feasible to
speculate that a tonic NGF-dependent stimulation
might be necessary to maintain basal spontaneous
contractility at optimal conditions. Taking into consideration that rat enteric neurons express the highaffinity receptor TrkA, as previously described and also
confirmed in this study by immunohistochemistry
(data not shown),36 we can hypothesize that K252a is
likely to bind to TrkA receptors on myenteric neurons
preventing NGF-mediated effects within the ENS and
thus, affecting motor activity.
The dose and pattern of administration of K252a
followed here has already been used, showing biological effects in vivo indicative of an effective blockade of
TrkA.18–20 Therefore, it is feasible to assume that the
responses observed here are related to an effective
blockade of TrkA receptors. However, K252a not only
binds to TrkA but also to other neurotrophins receptors, mainly TrkB and TrkC, and other kinases, such as
the Ca2+/calmodulin kinase II37 or the myosin light
chain kinase,38 which are implicated in the contractile
activity of intestinal smooth muscle.39 From our
observations, it cannot be ruled out that the effects
observed might be associated, at least partially, to
K252a effects on these targets. Nevertheless, several
observations suggest that the responses to K252a are
likely to be associated with the blockade of TrkA
receptors. Firstly, a recent study demonstrated that the
pharmacological blockade of TrkA with K252a, the
treatment with TrkA antisense oligonucleotides and
the in vivo immunoneutralization of NGF were
equally effective preventing chronic stress-induced
visceral hypersensitivity to colorectal distension in
rats.17 Secondly, in vivo immunoneutralization of NGF
normalized postinfectious gut dysmotility in T. spiralis-infected rats (a model of postinfectious IBS),12 as
observed here in the OVA model with K252a. Overall,
e588
2012 Blackwell Publishing Ltd
Volume 24, Number 12, December 2012
Effects of K252a on colonic contractility
colonic contractility and also in OVA-induced colonic
motor alterations in rats. In addition, our results show
that MMCs express TrkA receptors and, therefore,
represent a target for NGF, rather than being a source
of the peptide, in the rat colon. Overall, this study
highlights a potentially important role for NGF-TrkAdependent signaling pathways on colonic motor alterations, as observed for instance in FGDs. Nerve growth
factor receptors antagonists could represent a therapeutic target for the treatment of gastrointestinal
disorders characterized by altered colonic motility.
FUNDING
This work was supported by grant 2009SGR708 from the Generalitat de Catalunya and BFU2007-6279, BFU2009-08229 and
BFU2010-15401 from Ministerio de Ciencia e Innovación (Spain).
DISCLOSURE
No competing interests declared.
AUTHOR CONTRIBUTIONS
FJ designed and performed experiments, analyzed data and wrote
the paper; VM designed, performed experiments, analyzed data
and wrote the paper; PV designed experiments and wrote the
paper.
ACKNOWLEDGMENTS
We would like to thank A. Acosta for animal care and E. Martinez
for technical assistance.
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2012 Blackwell Publishing Ltd
APPENDIX
Ferran Jardí
 EDUCATION/TRAINING
- Doctor in Veterinary Medicine (2002-2007)
Universitat Autònoma de Barcelona, Bellaterra, Spain.
- Ph.D. Research Training (Suficiència Investigadora) (2008-2010)
Doctoral Program in Pharmacology
Universitat Autònoma de Barcelona, Bellaterra, Spain.
 POSITIONS AND EMPLOYMENT
- Post-graduate researcher (2008-2013)
Department o f C ell Biology, P hysiology a nd I mmunology, U nit of P hysiology,
Veterinary School
Universitat Autònoma de Barcelona, Bellaterra, Spain.
 PUBLICATIONS
F. Jardí, V. Martínez, P. Vergara. NGF is involved in oral ovalbumin-induced altered
colonic contractility in rats: Evidence from the blockade of TrkA receptors with
K252a. Neurogastroenterol Motil 2012, 24, e580–e590.
F. Jardí, P. Ver gara. Interacción h uésped-intestino. In “Tratado de
Neurogastroenterología y Mo tilidad Dig estiva: Tomo I - Neurogastroenterología
básica para clínicos” Editorial Panamericana, 2014.
F. Jardí, M. Aguilera, P. Vergara, V. Martínez. Colonic motor alterations associated
to the sensitization to a luminal antigen are enhanced during a dysbiotic-like state
in rats. Life Sciences (submitted).
F. Jardí, J.A. Fernández-Blanco, V. Martínez, P. Vergara. Plasticity o f dorsal root
ganglion neurons in a rat model o f p ost-infectious gut d ysfunction: p otential
implication of nerve growth factor. Neuroscience Letters (submitted).
F. Jardí, J.A. Fer nández-Blanco, V. Ma rtínez, P . Ver gara. P ersistent alter ations in
colonic afferent innervation in a rat model of post-infectious gut dysfunction: role
for changes in peripheral neurotrophic factors. Neuroscience (submitted).
 ABSTRACTS AND MEETINGS
F. Jardí, J .A. Fernández-Blanco, V. Ma rtínez, P . Ver gara. Remodeling o f co lonic
sensory afferents in a rat model of post-infectious gut d ysfunction: implication of
neurotrophic factors. Poster. Gastroenterology (in press).
Digestive Disease Week 2014. May 4-6, 2014. Chicago, USA.
F. Jardí, J.A. Fernández, E. Cepeda, V. Martínez, P. Vergara. Are mast cells a source
of neurotrophins i n t he inflamed g ut? Mis match b etween neurotrophin e xpression
and mast cells in a model of enteritis in rats. Poster.
EMBRN-COST International Mast Cell and Basophil Meeting 2013. August 28-30,
2013. Udine, Italy.
S. Barbosa, M. Aguilera, F. Jardí, M. Ferrer, P Vergara. Gut commensal microbiota
and T LR ex pression v ariations in r ats depending o n c ommercial origin. Oral
communication.
12th FELASA-SECAL 2013. June 10-13, 2013. Barcelona, Spain.
F. Jardí, J .A. Fer nández-Blanco, V. Ma rtínez, P . Ver gara. Mucosal mast ce lls
maintain n ormal contractility o f th e colon in a rat model o f p ost-infectious g ut
dysfunction. Poster.
193
APPENDIX
EMBRN-COST I nternational Ma st Cell a nd B asophil Meeting 2 012. No vember
26-27, 2012. Berlin, Germany.
F. Jardí, J .A. Fernández-Blanco, V. Ma rtínez, P . Ver gara. Neuronal plasticity o f
lumbosacral DRG in a model of p ost-infectious gut dysfunction in r ats. P oster.
Neurogastroenterol Motil 2012;24(Sppl. 2):66-67.
Joint I nternational Ne urogastroenterology an d Mo tility Me eting 2 012. September
6-8, 2012. Bologna, Italy.
J. A. Fernández-Blanco, F. Jardí, T. Shea-Donohue, V. Martínez, P. Vergara. PAR-2:
A feasible target to modulate intestinal barrier f unction a nd motility d ysfunctions
related to a rat model of intestinal nematode infection. Oral communication.
Woods H ole Immunoparasitology C onference, 2012. A pril 22 -25, 2012. W oods
Hole, USA.
J. A. Fernández-Blanco, F. Jardí, M. D. Hollenberg, V. Martínez, P. Vergara. Altered
protease-activated receptor-2-associated control of barrier function and motility in
a r at model of mast ce ll-dependent p ostinfectious g ut dysfunction. Oral
communication. Gastroenterology 2012;142(Sppl. 1):S-4-S-5.
Digestive Disease Week 2012. May 19-22, 2012. San Diego, USA.
F. Jardí, J .A. Fernández-Blanco, V. Ma rtínez, P . Ver gara.
Mucosal m ast cells
maintain normal contractility of the colon through NGF -dependent mechanisms in
a rat model of post-infectious IBS. Poster. Gastroenterology 2012;142(Sppl. 1):S895-S-896.
Digestive Disease Week 2012. May 19-22, 2012. San Diego, USA.
F. Jardí, M. Aguilera, V. Martínez, P. Vergara. Lipopolysaccharides interact with
oral ovalbumin to in duce food-antigen-related I BS-like alter ations o f colonic
motility in rats. Oral communication. Gastroenterology 2011;140(Sppl. 1):S-24.
Digestive Disease Week 2011. May 7-10, 2011. Chicago, USA.
F. Jardí, V. Ma rtínez, P . Ver gara. Implication o f n erve g rowth f actor in intestinal
mucosal mast cell activity and colonic motor alterations in a model of ovalbumininduced gut d ysfunction in rats. Oral communication. Gut 2010, 59 (Sppl. 3): OP
136.
18th United E uropean Gastro enterology W eek. October 2 3-27, 201 0. B arcelona,
Spain.
F. Jardí, V. Ma rtínez, E . Traver, P . Ver gara.
NGF is i mplicated in t he
hypercontractile responses of th e colon in a model of o valbumin-induced gut
dysfunction i n r ats. Or al c ommunication. Gastroenterology 138 (Sppl. 1): S -45,
2010.
Digestive Disease Week 2010. May 1-15, 2010. New Orleans, USA.
E. Teran, MT. Martin, F. Jardí, P. Vergara, V. Martínez. Gut microbiota affects Tolllike receptors (TLR) and cannabinoid 2 receptor (CB2) expression in the intestine
of rats. Poster. Gastroenterology 136 (Sppl 1): 1075, 2009.
Digestive Disease Week 2009. May 30-June 4, 2009. Chicago, USA.
 PARTICIPATION IN RESEARCH GRANTS
- Influence of intestinal microbiota in visceral nociceptive mechanisms and intestinal
barrier function (BFU2009-08229).
Ministerio de Ciencia e Innovación
PI: V. Martínez
 PERSONAL FELLOWSHIPS
- Pre-doctoral fellowship for research stages abroad
194
APPENDIX
Universitat Autònoma de Barcelona, Bellaterra, Spain. 2012
- Travel Grant
18th United E uropean Gastro enterology W eek. Octo ber 2 3-27, 2010, B arcelona,
Spain.
- Ph.D scholarship (PIF)
Universitat Autònoma de Barcelona, Bellaterra, Spain. 2009
Declined acceptance in July 2009
 COURSES
- Training course for the use of laboratory animals (2009)
(FELASA level C – Scientist responsible for directing animal experiments)
Universitat Autònoma de Barcelona, Bellaterra, Spain.
- Course of ex perimental d esign i n b iomedical research with an imals. P ractical
approach and common mistakes (2010)
ICLAS, Spanish committee
Universitat Autònoma de Barcelona, Bellaterra, Spain.
 RESEARCH STAGES
The University of Pittsburgh Center for Pain Research
Dr.Gebhart laboratory, Pittsburgh, Pennsylvania, USA.
October-December, 2012
 THEACHING ACTIVITES
- Certificate course in laboratory animal science
Jointly organized by TANUVAS, CPCSEA, NIAW and LASA
Teacher of theory and practical classes
September 14-23, 2013. Chennai, India.
- Animal physiology. Lab assistant
2nd year veterinary students and 1st year genetic students
2009-10, 20 10-11, 201 2-2013. Un iversitat Autònoma de Barcelona, B ellaterra,
Spain.
- Training course for the use of laboratory animals (2009)
Teacher and coordinator (2012) of the practical classes
2009-2012. Universitat Autònoma de Barcelona, Bellaterra, Spain.
- Master in science and laboratory animal welfare
Teacher and lab assistant of the practical classes
2011 and 2013. Universitat Autònoma de Barcelona, Bellaterra, Spain.
- ARGO program, Institut d e Ciències de l’Educació, Universitat Autònoma de
Barcelona. Collaborator
195