"Understanding the Physiology of the Blood-Brain Barrier In Vitro"
Understanding the Physiology of the Blood-Brain Barrier: In Vitro Models Gerald A. Grant, N. Joan Abbott, and Damir Janigro Endothelial cells exposed to inductive central nervous system factors differentiate into a blood-brain barrier phenotype. The blood-brain barrier frequently obstructs the passage of chemotherapeutics into the brain. Tissue culture systems have been developed to reproduce key properties of the intact blood-brain barrier and to allow for testing of mechanisms of transendothelial drug permeation. T he modern view of the mammalian blood- brain barrier (BBB) has shifted from a purely anatomic concept to a more physiological and channels, or disruption of the endothelial cell membrane. Of course, these pathways may open in combination and are not mutually exclusive. dynamic deﬁnition. Morphologically, the BBB is Irrespective of whether the BBB disruption is the formed by specialized endothelial cells (ECs) lin- main pathogenic factor, or an inevitable conse- ing the intraluminal portion of brain capillaries. quence of the disease itself, our understanding of These ECs are characterized by specialized the cellular mechanisms that lead to the disruption “. . .the BBB is formed regions of intercellular contact (tight junctions) of the BBB is limited. This may be due, in part, to by specialized that prevent leakage of blood-borne substances the lack of available models of BBB. Any such in endothelial cells. . . .” into the brain parenchyma. The kinetic aspects of vitro models must reproduce important features of the passage of ions and molecules from the brain EC (Table 2) while allowing for manipula- blood into the brain and vice versa better tions aimed at mimicking the disease process itself. approximate the physiological function of the For example, the vascular permeability changes BBB. It is clear, however, that the distinct mor- associated with neoplasia and inﬂammation are phological properties of the BBB, as seen at both clearly manifest and of practical importance with the light microscopic and ultrastructural levels, regard to the clinical application of diagnostic and account for the “restraining” nature of brain cap- therapeutic measures. However, a suitable model illary ECs. In contrast, the often-neglected fact to study tumor (or pathogen)-BBB interactions has that the BBB does not act as an absolute barrier, yet to be developed. together with the asymmetry of its permeation Several approaches have been attempted to properties, is less evident from a simple morpho- investigate the unique characteristics of the BBB logical investigation of brain microvessels. endothelium in both the normal and disease The failure of BBB structural integrity and func- state. Experimental observations ﬁrst made by tion plays a pivotal role in the pathogenesis of Paul Ehrlich in 1885 and Edwin Goldman in 1901 many diseases of the central nervous system (CNS) (that the CNS is not stained by intravascular (Table 1). Thus, during ischemia, inﬂammation, water-soluble dyes) provided the ﬁrst demonstra- trauma, neoplasia, hypertension, and epilepsy, tion of a BBB to polar compounds. Pioneering altered BBB permeability is commonly observed. studies of the BBB were performed in vivo using In barrier pathology, it is useful to take into account intracarotid injection single-pass techniques (11). not only the endothelial dysfunction but also dam- Further characterization of the BBB at the cellular age to the basal lamina, pericytes, astrocytes, level has led more recently to the development of vascular innervation, and components of the in vitro experimental approaches. Isolated brain immune system. The extravasation of plasma pro- capillary preparations as well as tissue culture teins associated with BBB dysfunction may occur systems using brain ECs have proven to be a through a number of different transcellular or para- promising methodology to deﬁne the characteris- cellular routes, including altered tight junctions, tics of the brain capillary endothelium at the induction of ﬂuid-phase or nonspeciﬁc pinocytosis molecular and cellular level. The purpose of this and transcytosis, formation of transendothelial review is to describe the virtues and pitfalls of cell culture-based models of the BBB. In our opinion, none of these models yet fully expresses the G. A. Grant and D. Janigro are in the Department of Neuro- logical Surgery, University of Washington School of Medicine, unique features of the BBB in situ; it is thus impor- 325 9th Ave., Seattle, WA 98104, USA; N. J. Abbott is in the tant for physiological, pharmacological, and Biomedical Science Division, King’s College, London, UK. preclinical studies to compare, whenever possi- 0886-1714/98 5.00 @ 1998 Int. Union Physiol. Sci./Am.Physiol. Soc. News Physiol. Sci. • Volume 13 • December 1998 287 Table 1. Central nervous system disorders involving BBB dysfunction Neoplasia Brain tumors (histamine, tissue necrosis factor, interferons, interleukins, permeable tumor vessels) Meningiomas (vascular endothelial growth factor) Vascular Ischemia, hypoxia (glutamate, free radicals, vasodilatation, lactic acidosis, prostaglandins, glial dysfunction) Hypertension (mechanical damage to endothelium, free radicals, vasopressin, angiotensin) Subarachnoid hemorrhage (complement system-C3a, EC damage, vasospasm) Arteriovenous malformations (endothelial damage due to ischemia and high ﬂow state) Migraines (serotonin) X-irradiation (endothelial damage) Trauma Open and closed head injury (intracranial hypertension, endothelial disruption, vascular spasm and loss of cerebral autoregulation) Brain edema Vasogenic (EC damage, intracranial hypertension, arachidonic acid metabolites, histamine, oxygen free radicals, polyamines) Cytotoxic Metabolic Diabetes (hyperglycemia, ischemia) Toxins: lead, aluminum, mercury, dimethyl sulfoxide (endothelial damage) Epilepsy Seizures (glutamate, glial dysfunction following neuronal activation, hypertension) Inﬂammation Multiple sclerosis/experimental allergic encephalomyelitis Meningitis: bacterial, viral, fungal (bradykinin, ATP, histamine, serotonin, interleukins) Dementia: AIDS demential complex, Alzheimer’s (B-amyloid) BBB, blood-brain barrier; EC, endothelial cell. ble, results obtained in vitro with data collected of a luminal plasma membrane, the cytosol, and from intact animals. However, it should be the abluminal membrane of the endothelial cell. emphasized that, in addition to enormous techni- The capillary endothelium is closely invested by cal difﬁculties associated with in vivo studies of < endfeet of glia, which develop in proximity to the 2-µm-thick ECs lining the brain parenchymal ves- brain ECs (15). Studies both in vivo and in vitro sels, in vivo models have other technical provide evidence that astrocytes contribute at least disadvantages. These include the impossibility of in part to the induction and maintenance of BBB simultaneously sampling brain and plasma com- characteristics in the brain endothelium (3, 7, 13); partments with minimal damage to the BBB itself. thus it appears that the cerebral endothelium is Finally, the development of in vitro models holds under direct inﬂuence from neighboring (or signiﬁcant promise for the identiﬁcation of thera- “perivascular” glia) astrocytes and that chemical peutic strategies for the treatment of neurological signals released by or present on the membrane of “The capillary diseases. In this review, we will ﬁrst describe the these cells are in part responsible for the unique endothelium is BBB in vivo and in isolated vessels and then dis- characteristics of the mammalian BBB. closely invested by cuss several cell culture-based in vitro models of In addition to its functional barrier properties, endfeet of glia. . . .” the BBB and their application to the study of neu- the endothelium is capable of selective, bidirec- ropathological diseases. tional transcellular transfer. Many essential metabolic substances, such as glucose and some amino acids, are highly polar and have poor per- In vivo studies and isolated vessels meability via the membrane lipid barrier. Unlike peripheral endothelia, brain microvessel Therefore, these substances are transported ECs are characterized by the presence of a high across the BBB by saturable carrier systems to transendothelial electrical resistance (TER), inter- meet the high metabolic demand of the brain (5, cellular tight junctions, minimal pinocytotic 6). To achieve an effective regulation of activity, and the virtual absence of fenestrations energy/metabolic supply to the parenchyma, the (6). In vivo, the endothelial BBB actually consists BBB must be capable of responding to abluminal 288 News Physiol. Sci. • Volume 13 • December 1998 Table 2. Description of the main cellular and be used to obtain accurate cerebrovascular per- functional properties of the in situ BBB meability coefficients for poorly permeable BBB-speciﬁc markers Present compounds. A second methodological approach Inductive inﬂuence from glia Mandatory (e.g., intravenous administration, brain perfusion Tight junctions Present techniques) involves a prolonged uptake time TER >1,500 Ω•cm2 that depends on the permeability coefﬁcient of Psucrose Low (<10–7 cm/s) the solute and is therefore more sensitive than K+ permeability Low the ﬁrst approach. With the intravenous adminis- Exposure to ﬂow membrane Luminal tration technique, a solute is given parentally and Polarized transporters Ubiquitous (e.g., K+, the plasma concentration is monitored until a amino acids) speciﬁc time at which the brain content is deter- Stereoselective transport Glucose, amino acids mined. BBB permeability is calculated from the brain uptake of a solute using a kinetic model BBB, blood-brain barrier; TER, transendothelial resis- that describes solute exchange between the tance; P, permeability. plasma and CNS. It has been difﬁcult to quantify the amount of “Carriers mediating compound that traversed the brain endothelium speciﬁc efﬂux from the signals while controlling the rate of transport of alone in vivo, since numerous routes of clearance brain. . . .” metabolites and ions (e.g., K+, Na+, and H+). from the brain exist; thus the compound may enter The concept of the polarity of the brain endothe- the interstitium as well as the brain parenchyma lium emerged from functional transport studies (2). and become sequestered intracellularly and pro- For example, the Na+-dependent amino acid trans- tein bound or become metabolized. A porter (type A) is found only on the abluminal side determination of the kinetic characteristics of trans- and transports neutral amino acids from the brain port systems has proved difﬁcult in vivo because of to the blood against a concentration gradient. In the poor temporal and spatial resolution and poor contrast, the Na+-independent L system carries access to the brain side (abluminal) of the endothe- neutral amino acids and is expressed on both the lium. Powerful techniques have been developed to luminal and abluminal membranes (6). Carriers enable researchers to model the mammalian BBB mediating speciﬁc efﬂux from the brain have also in vitro. The development of in vitro models of the been described, including P-glycoprotein, which BBB, consisting of either isolated brain capillaries actively exports lipophylic molecules out of cells or cultured brain microvessel ECs, has enabled the and confers insensitivity to drugs used for cancer study of BBB transport phenomena at the cellular chemotherapy (multidrug resistance). level. The aim of such in vitro models is to func- Classically, in vivo experiments were used to tionally express as many as possible unique determine the permeability of compounds across characteristics of the BBB cerebral endothelium the brain endothelium (6). Such approaches described above in vivo. offered valuable information about the behavior of different classes of compounds and helped In vitro modeling of the BBB identify speciﬁc transport systems. A primary advantage of in vivo preparations is the preserva- Isolated brain microvessels. A better under- tion of the normal anatomic arrangement of cells standing of the physiology and pathophysiology at the blood-brain interface. In addition, regional of the BBB was gained with the development of differences within the brain can be studied. methods pioneered by the late Ferenc Joó to Most studies of BBB permeability in vivo use obtain functionally and morphologically intact one of two different methodological approaches. cerebral microvessels dissociated from surround- In the ﬁrst approach, a solute is injected as a ing neurons and glia. Several methods are now bolus into the carotid artery; brain uptake or available for the isolation and puriﬁcation of extraction is determined from a single pass of the brain microvessels, which involve separation of bolus through the brain capillaries. The brain brain capillaries from the brain by combinations uptake index technique was later introduced by of mechanical homogenization, enzymatic disso- Oldendorf in 1970 as an intracarotid injection ciation, ﬁltration, density gradient centrifugation, single-pass method to measure cerebrovascular and glass bead column ﬁltration. Capillaries, transport and permeability. In this method, the venules, and arterioles of 5- to 25-µm diameter brain uptake of a test tracer is normalized by the are typically isolated by these procedures. Corti- use of a permeant reference tracer of known cal microvessels isolated from adult mammalian uptake (11). However, because permeability is brain are enriched in the putative BBB markers, determined from a single pass through the brain, alkaline phosphatase and -glutamyl transpepti- this approach has limited sensitivity and cannot dase (GGTP), but are deﬁcient in choline News Physiol. Sci. • Volume 13 • December 1998 289 acetyltransferase, a protein exclusively expressed the monolayer, which is largely determined by its at the BBB (10). The expression of BBB markers impenetrability due to tight junctions. The TER in can be used to assess the purity of the microves- vivo measured ~1,500 Ω•cm2, whereas the meas- sel preparations but do not guarantee the integrity urements in endothelial monolayers cultured in or preservation of the normal BBB function. Iso- vitro have ranged from 20 to 1,400 Ω•cm2. Cyclic lated capillaries can be successfully used for nucleotides such as adenosine 3 ,5 -cyclic physiological, biochemical, and developmental monophosphate and cyclic guanosine monophos- studies of the BBB as well as transport studies. phate have been reported to modulate TER and Such preparations have also been used for the permeability across endothelial monolayers, but identiﬁcation of membrane receptors and trans- their mechanisms of action are poorly understood porters. The advantages of this isolated capillary (9, 13). system include the preservation of its three- Although primary cultures of brain endothelium dimensional structure, differentiation, availability, alone may form tight intercellular junctions, and ease of use. The disadvantages include the coculture with astrocytes resulted in the increased difﬁculties associated with the isolation proce- formation and complexity of endothelial tight dure, limited viability of the endothelium, junctions and induced the expression of speciﬁc possible metabolic deﬁciencies induced by the BBB markers, including GGTP, the glucose trans- isolation procedures, and the inability to study porter isotype (GLUT-1), OX-26 (mouse antibody transendothelial ﬂux. against human and rat transferrin receptor), and P- Given the unique interactions between EC and glycoprotein (1). In contrast, the ubiquitous “Cyclic nucleotides . . . glia, and owing to the difﬁculties associated with occurrence of ZO-1 (antibody against zonula modulate TER and the use of the capillary microvasculature in vitro, it occludens-associated protein) in the CNS makes permeability. . . .” is not surprising that among the most successful the application of this protein unreliable as a models of the BBB are actually isolated pial brain quantitative means to estimate tight junctional arterioles. Surface pial microvessels appear to permeability but can be used as a tool for the share many of the molecular, physiological, and direct detection of de novo tight junction assem- morphological properties of cortical parenchymal bly (8, 10). The advantages of cultured vessels, despite a lack of the perivascular astrocytic endothelium include the potential for using pure ensheathment. Although the ultrastructural fea- cell populations as well as their relative viability tures, permeability to tracers, electrical resistance, compared with isolated arterioles ex situ. The iso- and molecular properties are quite similar between lation procedure for primary cultures, however, is pial and cortical vessels, the distribution of the labor intensive and expensive, and it is difﬁcult to expression of BBB-speciﬁc antigens (e.g., endothe- avoid contamination by other cell types, chieﬂy lial barrier antigen) as well as the structure of tight pericytes, leptomeningeal cells, and smooth mus- junctions visualized by electron microscopy may cle. Therefore, developing immortalized cell lines differ (4). Because technical limitations have pre- that preserve a stable BBB phenotype is of great cluded the in vitro study of cortical parenchymal interest. Several cell lines have been established vessels, investigators have pursued cell culture- (e.g., RBE4). Interestingly, even nonbrain ECs such based in vitro models of the BBB. as bovine aortic ECs can be induced by glia to Cultured brain microvascular ECs. Highly puri- form complex tight junctions and express a barrier ﬁed populations of cultured microvascular cells phenotype (7, 14). Sophisticated systems have for the study of the developmental and patho- been developed to preserve the barrier-speciﬁc physiological processes of the BBB became functional polarity of the endothelium in culture, available in the early 1980s. With this technique, since the cells may rapidly dedifferentiate in the a viable and homogeneous population of brain absence of astrocytes with serial cell passage. The capillary ECs can be isolated for the establishment addition of ﬂow to the culture system has been of a tissue culture system. The ﬁrst endothelial shown to cause physiological shear stress and play monolayers were established using cerebral a critical role in the differentiation of ECs (12). microvessel ECs grown on culture dishes, micro- Comparison of in vitro models. Several in vitro carriers (e.g., dextran beads), and various kinds of models of the BBB are currently used to explore ﬁlters, including nylon mesh and polycarbonate. the inﬂuence of diseases on the dynamic barrier The sucrose permeability (Psucrose) of these mono- on cellular, biochemical, and molecular levels. layers ranged from 10–4 to 10–5 cm/s compared Each model attempts to mimic the complexity of with 10–6 to 10–8 cm/s in vivo. Despite the differ- the mammalian BBB, but each is characterized ences in Psucrose, the rank order for penetration of by different selective permeability to different test compounds was well maintained. TER is a compounds and may manifest a range of TER val- measure of the ionic conductance of the mono- ues. The term “blood-brain barrier” suggests that layer and is a useful measure of the “tightness” of brain capillaries are impermeable, but it is obvi- 290 News Physiol. Sci. • Volume 13 • December 1998 FIGURE 1. Schematics of in vitro models of the blood-brain barrier (BBB). A: Transwell system. Transwell system consists of cultured brain microvessel endothelial cell (EC) monolayers grown on microporous membranes. Cultured cortical astrocytes are compartmentalized below the endothelial monolayer and release soluble factors, which preserve the BBB properties. This sys- tem allows for study of bidirectional transport across the BBB. B: dynamic in vitro BBB model (DIV-BBB). ECs and cortical astrocytes are cocultured on hollow ﬁbers inside a sealed chamber accessible by ports. Left: schematic representation of a cross section of one hollow ﬁber. ECs are loaded intraluminally, and cortical astrocytes are loaded abluminally. Astrocytic foot processes grow toward ECs to induce and maintain their BBB phenotype. Cartridge-hollow ﬁber system consists of artiﬁcial cap- illaries connected by gas-permeable tubing to a source of growth medium, thus allowing exchange of O2 and CO2 and exposure to pulsatile ﬂow (right). C: isolated vessels. Pial surface and penetrating cortical arterioles have also been used for BBB studies. Left: schematic representation of a cross section of an end arteriole with one layer of endothelium surrounded by a single layer of vascular smooth muscle. Pia mater and its attached penetrating intracerebral arterioles are then separated from a section of cortical parenchyma as shown (right). ous that, although they are indeed impermeable or vertical diffusion system (Fig. 1A). Although to some plasma solutes, they must be freely per- this system lacks physiological ﬂow, a polarity is meable to others. A critical feature in any model inducible in the ECs when cocultured with glia system is the ability to discriminate between a or astrocytic-conditioned medium and affords an compound of high permeability through the lipid opportunity to study bidirectional transendothe- bilayer (transcellular) and a compound such as lial transport of solutes across the BBB. It is an sucrose or mannitol that traverses (albeit poorly) ideal system in which to establish Michaelis- via a paracellular pathway. A transcellular vesic- Menten kinetics of transport for different ular route may also need to be considered. physiological substances because of the ﬁxed The most commonly used tissue culture sub- volumes in each small compartment. strate used for EC culturing consists of a porous More recently, a dynamic in vitro culture sys- membrane support submerged in culture tem characterized by intraluminal flow medium (Transwell apparatus). The Transwell sys- (DIV-BBB) has been developed; the DIV-BBB tem is characterized by a horizontal side-by-side closely mimics the BBB phenotype in vivo (14). News Physiol. Sci. • Volume 13 • December 1998 291 In this system, cerebral or peripheral ECs are cul- fragmented basal lamina to a high degree, depend- tured intraluminally in the presence of astrocytes ing on tumor type and grade, which lead to a cultured abluminally using hollow ﬁber tubes considerable increase in permeability of the tumor inside a sealed chamber (Fig. 1B). The hollow vascular bed. Tumors also stimulate the prolifera- ﬁber cartridge system consists of artiﬁcial capil- tion of abnormal capillaries by releasing laries that are exposed to luminal pulsatile ﬂow. angiogenic factors. In more malignant tumors, the This system is characterized by an extremely capillaries are fenestrated with increased pinocy- high TER estimated to be >1,000 Ω•cm2, resis- tosis and have an incomplete BBB. Tumors, tance that approximates that obtained in vivo. In therefore, permit contrast enhancement on radi- addition, there is a low permeability to sucrose ographic imaging studies (computerized (10–6–10–7 cm/s) and the functional expression of tomography or magnetic resonance imaging) and stereoselective transport (e.g., L- vs. D-aspartate) may exhibit marked vasogenic edema. More (14). However, because of the compartmental- recently, hyperosmolar solutions have been ization of ECs and glia in the hollow ﬁber system employed to improve the delivery of chemothera- in the presence of pulsatile ﬂow, the study of lin- peutic agents to the neoplastic cells. ear kinetics becomes more complex. Isolated Hyperosmolar agents cause an osmotically pial arterioles (surface and penetrating) have also induced shrinkage of brain microvessel ECs and a been used to study BBB function ex situ (Fig. 1C). reversible increase to BBB permeability. Future The induction and preservation of selective per- therapeutic strategies depend on an improved meability and transport mechanisms as well as understanding of the mechanisms responsible for expression of normally occurring ion channels the induction and maintenance of the barrier and responsible for the maintenance of brain home- modulation of the barrier under normal and patho- ostasis are necessary BBB phenotypic features. In logical conditions. Furthermore, by coculturing vitro models have proven valuable in the rigorous neuronal, glial, and brain ECs with tumor cell study of biochemical transport at the cellular level lines, we may obtain important information on the in the context of several disease states such as efﬁcacy of antineoplastic agents, while simultane- ischemia, neoplasia, and meningitis. Thus far, and ously monitoring passage of these agents across in addition to the aforementioned quasi-physio- the BBB, and on their potential neurotoxicity. logical TER, dynamic BBB models have There is increasing evidence that, in many dis- successfully replicated several morphological and eases of the CNS, the barrier dysfunction may be functional characteristics of the intact BBB. These brought about by the release or activation of a cas- include asymmetric K+ transport, stereoselective cade of mediator substances from damaged or transport of amino acids, a BBB-like glucose trans- activated cells. The study of conditions that porter, tight junctions, and negligible permeation increase BBB permeability has improved our by 14C-sucrose or inulin. Culture models have understanding of the mechanisms that maintain made it possible to investigate the site and mech- and modulate the barrier and our search for agents “. . .a group of. . . anism of action of toxic agents that affect the BBB, that can be used to open the barrier for therapeu- pathological including drugs and industrial toxins. The effects of tic purposes. The role of the BBB in the evolution conditions causes putative toxicants (e.g., lead, aluminum) and other of viral and bacterial CNS diseases remains early changes in pathogens on BBB viability have been studied incompletely deﬁned and is currently being BBB. . . .” using these models. Further studies will be chal- explored both in vivo and in vitro. Barrier dys- lenged by even more demanding tasks, such as function secondary to viral or bacterial pathogens mimicking chronic neurodegenerative processes may exacerbate the severity of the neurological (i.e., Alzheimer’s and AIDS dementia) in long-term injury, whereas an intact barrier may hinder recov- endothelial cell cultures. ery from disease by delaying the entry of immune complexes or therapeutic agents into the infected CNS. Seizures, whether induced by electrocon- Implications of the BBB in neurological disease vulsive shock or drugs, resulted in increased A group of toxic agents and pathological condi- permeability to intravascular markers. The induc- tions causes early changes in BBB function, which tion of hypertension also increased BBB are mediated via direct effects on the ECs, but is permeability; however, the increase was also asso- also usually associated with morphological ciated with increased pinocytosis. Hypercapnia changes in astrocytes. Tumors, for example, disrupt had the same effect as hypertension, although the glial sheath that envelops the ECs and are asso- vasodilatation and the stretch of tight junctions are ciated with an increased capillary permeability. critical (1, 6, 10). Classical vasoactive mediators of The capillary endothelium of tumor vessels is inﬂammation (e.g., histamine, serotonin, ATP) highly abnormal and expresses a varied degree of cause a rapid and dramatic increase in permeabil- fenestrated regions, vesicles, open junctions, and ity. Experiments performed on brain microvascular 292 News Physiol. Sci. • Volume 13 • December 1998 endothelium showed that tight junction openings 2. Betz, A. L., and G. W. Goldstein. Polarity of the blood- caused by agents such as ATP and bradykinin may brain barrier: neutral amino acid transport into isolated brain capillaries. Science 202: 225–227, 1978. be mediated via a rise in cytosolic Ca2+ and 3. Brightman, M. Implication of astroglia in the blood-brain endothelial contraction. Oxygen free radicals barrier. Ann. NY Acad. Sci. 633: 343–347, 1991. released following ischemia or hypoxia caused an 4. Cassella, J. P., J. G. Lawrenson, L. Lawrence, and J. A. even more dramatic opening of the barrier, which Firth. Differential distribution of an endothelial barrier was Ca2+ independent. The breakdown of the BBB antigen between the pial and cortical microvessels of the rat. Brain Res. 744: 335–338, 1997. following an ischemic episode has been well doc- 5. Crone, C. Facilitated transfer of glucose from blood to umented; however, the extent and duration of the brain tissue. J. Physiol. (Lond.) 181: 103–113, 1965. opening largely depends on the duration of the 6. Davson, H. and M. B. Segal (Eds.). The blood-brain bar- “. . .opening of the ischemia and degree of reperfusion. The temporal rier. In: Physiology of the CSF and of the Blood-Brain BBB is thought to course of the BBB opening following ischemic Barrier. New York: CRC, 1995, p. 49–91. contribute to the 7. Dehouck, M. P., S. Meresse, P. Delorme, J. Fruchart, and reperfusion injury and even traumatic brain injury development of R. Cecchelli. An easier, reproducible, and mass-produc- appears biphasic. The early opening of the BBB is tion method to study the blood-brain barrier in vitro. J. cerebral edema. . ..” thought to contribute to the development of cere- Neurochem. 54: 1798–1801, 1990. bral edema formation both in tumors and 8. Dermietzel, R., and D. Krause. Molecular anatomy of the following ischemia, although the pathogenesis of blood-brain barrier as deﬁned by immunocytochemistry. Int. Rev. Cytol. 125: 57–109, 1991. the disruption has not been clearly elucidated. 9. Joó, F. The role of second messenger molecules in the Novel dynamic cell culture systems offer valuable regulation of permeability in the cerebral endothelial tools to study the effect of ischemia and hypoxia cells. Adv. Exp. Med. Biol. 331: 155–164, 1993. on barrier function. 10. Laterra, J., and G. W. Goldstein. The Blood-Brain Barrier: Cellular and Molecular Biology, edited by W. M. Pardridge. New York: Raven, 1993. Conclusion 11. Oldendorf, W. H. Measurement of brain uptake of radio- labeled substances using a tritiated water internal In vitro models of the mammalian BBB are now standard. Brain Res. 24: 372–376, 1970. able to mimic key features of the in situ BBB and, 12. Ott, M. J., J. L. Olson, and B. J. Ballermann. Chronic in moreover, can provide detailed information vitro ﬂow promotes ultrastructural differentiation of endothelial cells. Endothelium 3: 21–30, 1995. about the cellular and molecular mechanisms of 13. Rubin, L. L., D. E. Hall, S. Porter, K. Barbu, C. Cannon, dynamic endothelial function. Study of in vitro H. C. Horner, M. Janatpour, C. W. Liaw, K. Manning, J. preparations for application of modern tech- Morales, L. I. Tanner, K. J. Tomaselli, and F. Bard. A cell niques of molecular and cellular biology and culture model of the blood-brain barrier. J. Cell Biol. 115: physiology at the single cell level is critical for the 1725–1735, 1991. 14. Stanness, K. A., L. E. Westrum, P. Mascagni, E. Fornaciari, study of the BBB. Further developments in vitro J. A. Nelson, S. G. Stenglein, and D. Janigro. Morpholog- now will allow sophisticated genetic manipula- ical and functional characterization of an in vitro tions to explore novel clinical applications. blood-brain barrier model. Brain Res. 771: 329–342, 1997. References 15. Stewart, P. A., and M. J. Wiley. Developing nervous sys- tem induces formation of blood-brain barrier 1. Abbott, N. J., and P. A. Revest. 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