barriera emato

La barriera ematoencefalica
• Circa 100 anni fa fu scoperto che se un
colorante blu veniva iniettato nel sangue di
un animale, tutti i tessuti cerebrali tranne il
cervello ed il midollo spinale, diventavano
blu.
• Per spiegare questa osservazione, gli
scienziati immaginarono una "barriera ematoencefalica" in grado di impedire alle sostanze
presenti nel sangue di entrare nel cervello.
Complessi di giunzione
• La barriera emato-encefalica è semipermeabile: si
lascia attraversare da alcune sostanze, ma non da
altre.
• Nelle maggior parte del corpo, i vasi ematici più
piccoli, i capillari, sono ricoperti soltanto da cellule
endoteliali.
• Normalmente, fra le cellule endoteliali esistono
piccoli spazi che consentono a molte sostanze di
muoversi facilmente attraverso la parete dei capillari
stessi.
• Ma, nel cervello, le cellule endoteliali sono molto
attaccate le une alle altre (complessi di giunzione) e
le varie sostanze non possono attraversare la parete
capillare
Barriera emato-encefalica
• Le cellule gliali (astrociti) si
dispongono a formare uno strato
continuo intorno ai capillari cerebrali.
Sembra, però, che gli astrociti non
siano essenziali per costituire la
barriera emato-encefalica, ma
sarebbero importanti per il trasporto
degli ioni dal cervello al sangue.
Funzioni della barriera emato-encefalica
•
•
•
Proteggere il cervello da "sostanze
estranee" presenti nel sangue, che
potrebbero danneggiarlo
Proteggere il cervello da ormoni e
neurotrasmettitori liberati per agire in altre
parti del corpo.
Mantenere un ambiente costante per il
cervello.
Proprietà generali della barriera ematoencefalica
•
•
•
Le grosse molecole non passano
attraverso la barriera.
Le molecole scarsamente solubili nei lipidi
non penetrano nel cervello. Le molecole
solubili nei lipidi (come i barbiturici e
l'alcool) attraversano, invece, molto bene la
barriera.
Le molecole con elevata carica elettrica
sono rallentate
La barriera emato-encefalica può essere
annullata o ridotta dalle seguenti cause:
•
•
•
•
•
Ipertensione
Sviluppo: la barriera non è completamente formata
alla nascita.
Iperosmolarità: una sostanza presente nel sangue
ad elevata concentrazione può attraversarla.
Microonde.
Radiazioni.
Infezioni.
•
Traumi, Ischemia, Infiammazioni
•
Schematic representation of mechanisms available for endogenous substrates to cross the
BBB. A, small, lipid-soluble substrates are able to diffuse across the membrane, although are
subject to efflux back into the circulation by transporters as discussed in...
An understanding of the physiology of the blood-brain barrier (BBB) is
crucial when addressing complex issues such as drug delivery,
pathogenesis of chronic neurological diseases and biodefense.
1. Studies performed using small animals such as rodents cannot be directly
extrapolated to human brain tissue.
2. Furthermore, most of the promising CNS drugs that proved effective in vitro
have failed in clinical trials due to misleading predictive permeability data
extrapolated from models that were not capable of fully reproducing the
functional properties of the BBB in vivo.
A great effort has been made to develop new in vitro models able to
reproduce the physiological, anatomical and functional characteristics of
the BBB allowing for a better prediction of drug penetration across the
BBB,
How to make a good BBB model:
There is an increasing interest in establishing in vitro BBB cell culture models for a
number of reasons:
(i) to enable prediction of the penetration of drug candidates across the BBB;
(ii) to provide an understanding of how dysfunction in the BBB is involved in
the pathogenesis of various neurological diseases;
(iii) to enable pre-screening and optimization of new drug and gene delivery
formulations before performing experiments on animals or humans
The primary goal of any study of BBB physiology or biology in vitro is to
reproduce as many aspects as possible of the in vivo brain
microvasculature by developing a model that could accelerate the
design of drugs which selectively target the CNS.
Other important reasons for the use of in vitro systems include :
•Less expensive compared with in vivo studies,
•Ability to perform multiple tests at the same time,
•Lack of limitation to any particular cell type (eg, endothelium,epithelium, etc),
REQUISITI PER L’ALLESTIMENTO DI UNA BBB
The expression of tight junctions between ECs (determining a restrictive
paracellular permeability) and low permeation to sucrose or any other polar
molecules that do not have a specific carrier mediated transport system to pass into
the CNS parenchyma.
Moreover, in a functional BBB model, selective permeability to molecules, based on
their oil/water partition co-efficient and molecular weight, must be guaranteed.
Furthermore, these models must demonstrate selective and asymmetric permeability
to physiologically relevant ions, such as Na+ and K+, and the functional expression
and/or maintenance of active extrusion proteins which occurs in vivo must be
established.
Cell-cell interactions, leading to physiologically realistic cell architecture, and
relative exposure to 'permissive' or 'promoting' factors released by the surrounding
glia are also required.
Most importantly, the in vitro BBB must be easy to culture and data reproducibility
must be assured.
In silico models: Data requirements
in silico models is to predict the BBB permeation of a new drug, by relying on
the physicochemical parameters (eg, solubility, lipophilicity, molecular size,
hydrogen-bonding capacity and charge) of the novel compound with respect to
passive diffusion and active transport mechanisms.
in vitro assessment of BBB penetration, mainly based on models with twochamber systems, but also including the use of cell monolayers and
artificial membranes, require validation against appropriate in vivo data and
the brain penetration data itself must also be considered
In vivo studies usually comprise intravenously administering a radiolabeled
compound to an anesthetized rat, which is then exsanguinated after radioactivity in
the blood reaches a plateau. The brain is then removed and the concentration of the
compound is determined using a scintillation counter.
This experiment provides the most reliable permeation parameter, the so
called logBB value, which is defined as the ratio of the compound in the brain
(cbrain) versus that in the plasma (cplasma) under steady-state conditions
Equation 1. LogBB = log(cbrain/cplasma)
Monodimensional models of the BBB: Endothelial cells
The most common and easy-to-culture model allowing study of the BBB in vitro comes
comprises the 'Transwell' apparatus.
This model is characterized by a side-by-side diffusion system (Figure 1A), in
which primary cultures of brain vascular Ecs derived from various sources
(bovine, mouse, rat, porcine, non-human primate and human) are grown on
porous semipermeable membranes and immersed in their respective growth
media (Figure 1B).
Attractive features of this model are its simplicity (ie, ease of culture) and the ability to
perform multiple experiments at the same time (one type of drug per well or different concentrations of the same
drug in different wells), while minimizing cost and experimental time.
Brain microvessel ECs can be isolated from brain microvessels in culture by
mechanical dispersion (homogenization, filtration, sieving or centrifugation), by
enzymatic procedures using collagenase or by a combination of both
mechanical dispersion and enzymatic digestion.
MONOLAYER Model limitations
the absence of the natural physiological stimuli present in vivo (such as perivascular glia, sheer
stress or interaction with blood cells present in the cerebral circulation under normal conditions),
the ECs lose their BBB properties. This is a major pitfall for such a system, since BBB properties
(eg, the expression of tight junctions) are bestowed on ECs by the surrounding cellular
environment in vivo (eg, astrocytes and pericytes) .
Endothelial cells grown under these in vitro conditions may lack the expression of specific
transporters, which confer on them the BBB phenotype, thus leading to abnormal permeability
across the EC layer,
The addition of hydrocortisone or dexamethasone to the culture media. appears to
provide significant BBB 'tightening' effects, even in the absence of glia, thus improving
the barrier function
ECs at the BBB level in vivo present a functional specialization of the apical and basolateral
membranes. This polarity between the luminal and abluminal membranes, is reflected in the
preferential expression and distribution of membrane transporters and enzymes that function to
protect, as well as to promote, substrate delivery to the brain parenchyma and to maintain brain
homeostasis
Finally, ECs grown as a monodimensional layer upon a porous membrane are exposed to
serum, both on the luminal (intravascular) and abluminal (parenchymal) sides, while in vivo, only
the luminal side is exposed to serum proteins and the abluminal part is either exposed to glial
influence or cerebrospinal fluid. This non-physiological condition may further accelerate the dedifferentiating process that the ECs experience and may enhance the loss of BBB
characteristics.
Bidimensional models of the BBB: Co-culture of ECs and glia
Bidimensional models (or co-culture) of the BBB have been established as an evolutionary step in
BBB studies compared with monodimensional culture systems, due to the addition of glia . This
model is also based on the Transwell system. The presence of glia and the establishment of
glialendothelial interactions has been demonstrated to increase the expression of brain
endothelial marker enzymes (such asγ-glutamyl transpeptidase (γGTP), alkaline phosphatase,
acetylcholinesterase and Na+-K+-ATPase, transporters such as facilitative glucose transporter
type-1 (GluT-1) and tight junctions, and has helped to produce a phenotype which mimics the in
vivo phenotype more closely.
The co-culture can be established to enable cell-cell contact through astrocytic end-feet by
seeding astrocytes and Ecs on either side of the porous support, or can be arranged without any
contact by seeding the astrocytes at the bottom of the well and the ECs on the porous support.
The main advantage of the bidimensional model, compared with the monodimensional, is the
establishment of conditions that mimic more closely the in vivo situation. Under these co-culture
conditions, ECs retain some of the in vivo BBB characteristics, including initiated tight junctions,
higher TEER values and decreased permeability for hydrophilic molecules such as sucrose or
inulin.
LIMITAZIONI DEL Modello BILAYER
The bidimensional modeling still ignores the presence of intraluminal blood cells and
blood flow, lacking the presence of shear stress, which has been demonstrated to
determine further differentiation of ECs and to play a crucial role in the
cerebrovascular system promoting the differentiation and maintenance of the BBB
phenotype
Tridimensional dynamic models of the BBB (flow-based)
This apparatus is characterized by a pronectin-coated polypropylene
hollow-fiber structure that enables co-culturing of Ecs (intraluminally) with glia
(extraluminally). This newly developed in vitro system allows quasi-physiological
experimental conditions for culturing ECs and astrocytes in a capillarylike structure
and is able to functionally and anatomically mimic the brain microvasculature. The
entire system is connected to a media reservoir via gas-permeable silicon tubes that
allow for the exchange of O2 and CO2.
The BBB properties induced in ECs grown in hollow fibers under dynamic
conditions include low permeability to intraluminal potassium, negligible
extravasation of proteins and the expression of a glucose transporter and BBBspecific ion channels with recent trends in the medical field and expanding
interests in the study of the CNS, we have found this model to be of limited
applicability. Specifically, its design cylindrical in nature, does not allow for
the visualization of either compartment to assess morphologic and/or
phenotypic changes in the cells of interest. Additionally, the cell inoculation
volume and media requirements are enormous, considering the fact that
current models are not reusable. Finally, the volume and physical access to
the extraluminal space in current models
allow only for introduction of cell suspensions and not tissue slice
preparations.
The NDIV-BBB apparatus consists of a rectangular polycarbonate hollow chamber
The chamber contains an adjustable number of artificial polypropylene capillaries.
These capillaries have an inner diameter of 600 μm, and contain transcapillary pores
of 0.64 μm, which allow free diffusion of solutes from the extraluminal compartment to
the intraluminal space and vice versa (Figure 6).
This model in particular has several advantages compared with the previous DIV-BBB
model; the ability to expose and remove single or multiple fibers during the course of
an experiment offers a tremendous advantage over single use models, allowing, for
example, a detailed time-course of EC changes in response to a particular stimulus to
be observed. In a typical experiment, ECs are seeded in the model and exposed to a
particular stimulus. Capillaries can be removed at predetermined times during the
course of an experiment, allowing assessment of morphologic and/or phenotypic
studies in cells cultured on the luminal and/or abluminal surface of the hollow fibers
Furthermore, the new model permits visualization of cell-cell interactions with
inverted light microscopy as well as techniques involving fluorescence imaging. In
contrast to other available models of the BBB, the NDIV-BBB respects the anatomical
aspects of the in situ EC-astrocyte interactions.