Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJs) that are critical for maintaining brain homeostasis. The effects of initial reoxygenation after a hypoxic insult (H/R) on functional and molecular properties of the BBB and TJs remain unclear. In situ brain perfusion and Western blot analyses were performed to assess in vivo BBB integrity on reoxygenation after a hypoxic insult of 6% O2 for 1 h. Model conditions [blood pressure, blood gas chemistries, cerebral blood flow (CBF), and brain ATP concentration] were also assessed to ensure consistent levels and criteria for insult. In situ brain perfusion revealed that initial reoxygenation (10 min) significantly increased the uptake of [14C]sucrose into brain parenchyma. Capillary depletion and CBF analyses indicated the perturbations were due to increased paracellular permeability rather than vascular volume changes. Hypoxia with reoxygenation (10 min) produced an increase in BBB permeability with associated alterations in tight junctional protein expression. These results suggest that H/R leads to reorganization of TJs and increased paracellular diffusion at the BBB, which is not a result of increased CBF, vascular volume change, or endothelial uptake of marker. Additionally, the tight junctional protein occludin had a shift in bands that correlated with functional changes (i.e., increased permeability) without significant change in expression of claudin-3, zonula occludens-1, or actin. H/R-induced changes in the BBB may result in edema and/or associated pathological outcomes.     

Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders

The neurovascular unit (NVU) comprises brain endothelial cells, pericytes or vascular smooth muscle cells, glia and neurons. The NVU controls blood-brain barrier (BBB) permeability and cerebral blood flow, and maintains the chemical composition of the neuronal 'milieu', which is required for proper functioning of neuronal circuits. Recent evidence indicates that BBB dysfunction is associated with the accumulation of several vasculotoxic and neurotoxic molecules within brain parenchyma, a reduction in cerebral blood flow, and hypoxia. Together, these vascular-derived insults might initiate and/or contribute to neuronal degeneration. This article examines mechanisms of BBB dysfunction in neurodegenerative disorders, notably Alzheimer's disease, and highlights therapeutic opportunities relating to these neurovascular deficits.     

Human cerebral malaria and the blood-brain barrier

Malaria represents a continuing and major global health challenge and our understanding of how the Plasmodium parasite causes severe disease and death remains poor. One serious complication of the infection is cerebral malaria, a clinically complex syndrome of coma and potentially reversible encephalopathy, associated with a high mortality rate and increasingly recognised long-term sequelae in survivors. Research into the pathophysiology of cerebral malaria, using a combination of clinical and pathological studies, animal models and in vitro cell culture work, has focussed attention on the blood-brain barrier (BBB). This represents the key interface between the brain parenchyma and the parasite, which develops within an infected red cell but remains inside the vascular space. Studies of BBB function in cerebral malaria have provided some evidence for parasite-induced changes secondary to sequestration of parasitised red blood cells and host leukocytes within the cerebral microvasculature, such as redistribution of endothelial cell intercellular junction proteins and intracellular signaling. However, the evidence for a generalised increase in BBB permeability, leading to cerebral oedema, is conflicting. As well as direct cell adhesion-dependent effects, local adhesion-independent effects may activate and damage cerebral endothelial cells and perivascular cells, such as decreased blood flow, hypoxia or the effects of parasite toxins such as pigment. Finally, a number of systemic mechanisms could influence the BBB during malaria, such as the metabolic and inflammatory complications of severe disease acting 'at a distance'. This review will summarise evidence for these mechanisms from human studies of cerebral malaria and discuss the possible role for BBB dysfunction in this complex and challenging disease.     

Connexin Channels at the Glio-Vascular Interface: Gatekeepers of the Brain

Neuronal survival, electrical signaling and synaptic activity require a well-balanced micro-environment in the central nervous system. This is achieved by the blood–brain barrier (BBB), an endothelial barrier situated in the brain capillaries, that controls near-to-all passage in and out of the brain. The endothelial barrier function is highly dependent on signaling interactions with surrounding glial, neuronal and vascular cells, together forming the neuro-glio-vascular unit. Within this functional unit, connexin (Cx) channels are of utmost importance for intercellular communication between the different cellular compartments. Connexins are best known as the building blocks of gap junction (GJ) channels that enable direct cell–cell transfer of metabolic, biochemical and electric signals. In addition, beyond their role in direct intercellular communication, Cxs also form unapposed, non-junctional hemichannels in the plasma membrane that allow the passage of several paracrine messengers, complementing direct GJ communication. Within the NGVU, Cxs are expressed in vascular endothelial cells, including those that form the BBB, and are eminent in astrocytes, especially at their endfoot processes that wrap around cerebral vessels. However, despite the density of Cx channels at this so-called gliovascular interface, it remains unclear as to how Cx-based signaling between astrocytes and BBB endothelial cells may converge control over BBB permeability in health and disease. In this review we describe available evidence that supports a role for astroglial as well as endothelial Cxs in the regulation of BBB permeability during development as well as in disease states.     

Increase of tumor necrosis factor‐α in the blood induces early activation of matrix metalloproteinase‐9 in the brain

Increases of cytokine in the blood play important roles in the pathogenesis of influenza‐associated encephalopathy. TNF‐α was administered intravenously to wild‐type mice, after which blood, CSF and brain tissue were obtained, and changes in BBB permeability, the amounts of MMP‐9 and TIMP‐1, and the localization of activated MMP were assessed. There was a significant increase in BBB permeability after 6 and 12 hr. MMP‐9 was increased after 3 hr in the brain and cerebrospinal fluid, which was earlier than in the serum. TIMP‐1 protein in the brain increased significantly after MMP‐9 had increased. Activation of MMP‐9 was observed in neurons in the cerebral cortex and hippocampus, and in vascular endothelial cells. These findings suggest that an increase in blood TNF‐α promotes activation of MMP‐9 in the brain, and may also induce an increase in permeability of the BBB. Early activation of MMP‐9 in the brain may contribute to an early onset of neurological disorders and brain edema prior to multiple organ failure in those inflammatory diseases associated with highly increased concentrations of TNF‐α in the blood, such as sepsis, burns, trauma and influenza‐associated encephalopathy.     

n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood–brain barrier

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood–brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (−23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 μM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [³H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood–brain barrier, possibly via changes in GLUT1 protein expression and activity.     

Role of VEGF in an experimental model of cortical micronecrosis

Vascular endothelial growth factor (VEGF) is a major mediator in angiogenesis and vascular permeability. In central nervous system (CNS) it plays a pivotal role as: 1. inductor of endothelial cell proliferation, migration and inhibition of apoptosis, and 2. mediator of vascular permeability and subsequently of brain edema. This ubiquitous epiphenomenon is a major complication in several CNS pathologies, including head trauma and stroke.After brain injury the expression of VEGF is increased contributing to disruption of the blood brain barrier (BBB). VEGF increase the permeability of BBB via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. The immunohistochemistry shows an increase of stained astrocytes and endothelial cells around cortical micronecrosis. VEGF immunopositivity distribution shows some correspondence with the blood brain barrier breakdown following a cortical micronecrosis.     

SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and low permeability. BBB maintenance is important in the central nervous system (CNS) because disruption of the BBB may contribute to many brain disorders, including Alzheimer disease and ischemic stroke. The molecular mechanisms of BBB development remain ill-defined, however. Here we report that src-suppressed C-kinase substrate (SSeCKS) decreases the expression of vascular endothelial growth factor (VEGF) through AP-1 reduction and stimulates expression of angiopoietin-1 (Ang-1), an antipermeability factor in astrocytes. Conditioned media from SSeCKS-overexpressing astrocytes (SSeCKS-CM) blocked angiogenesis in vivo and in vitro. Moreover, SSeCKS-CM increased tight junction proteins in endothelial cells, consequently decreasing [3H]sucrose permeability. Furthermore, immunoreactivity to SSeCKS gradually increased during the BBB maturation period, and SSeCKS-expressing astrocytes closely interacted with zonula occludens (ZO)-1-expressing blood vessels in vivo. Collectively, our results suggest that SSeCKS regulates BBB differentiation by modulating both brain angiogenesis and tight junction formation.     

Adrenomedullin Improves the Blood–Brain Barrier Function Through the Expression of Claudin-5

Summary 1. Aims: Brain vascular endothelial cells secret Adrenomedullin (AM) has multifunctional biological properties. AM affects cerebral blood flow and blood–brain barrier (BBB) function. We studied the role of AM on the permeability and tight junction proteins of brain microvascular endothelial cells (BMEC).2. Methods: BMEC were isolated from rats and a BBB in vitro model was generated. The barrier functions were studied by measuring the transendothelial electrical resistance (TEER) and the permeability of sodium fluorescein and Evans’ blue albumin. The expressions of tight junction proteins were analyzed using immunocytochemistry and immunoblotting.3. Results: AM increased TEER of BMEC monolayer dose-dependently. Immunocytochemistry revealed that AM enhanced the claudin-5 expression at a cell–cell contact site in a dose-dependent manner. Immunoblotting also showed an overexpression of claudin-5 in AM exposure.4.Conclusions: AM therefore inhibits the paracellular transport in a BBB in vitro model through claudin-5 overexpression.     

Cerebrovascular Effects of Amyloid-ß Peptides: Mechanisms and Implications for Alzheimer's Dementia

1. The amyloid ß-peptide (Aß) is involved in the mechanisms of Alzheimer dementia. This paper reviews experimental evidence indicating that Aß exerts profound effects on the regulation of the cerebral circulation.2. Thus, Aß compromises the ability of cerebral endothelial cells to produce vascular relaxing factors, impairs the ability of cerebral blood vessels to maintain adequate flow during hypotension, and attenuates the increases in CBF evoked by enhanced brain activity.3. Studies in transgenic mice overexpressing the amyloid precursor protein suggest that these cerebrovascular alterations disrupt the delicate balance between the brain's energy requirements and cerebral blood supply, rendering the brain more vulnerable to ischemic injury.4. The findings support the recently emerged notion that vascular factors play a pathogenic role in the early stages of Alzheimer dementia.     

n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood-brain barrier

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood-brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (-23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 microM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [(3)H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood-brain barrier, possibly via changes in GLUT1 protein expression and activity.     

The blood-brain barrier: an alternative hypothesis

Brain blood vessels, unlike most vessels elsewhere in the body, exhibit a blood-brain barrier (BBB) to certain substances, e.g. trypan blue. Under some circumstances this barrier is no longer effective and the permeability of the vessels increases. Although capillarization is much less in the brain than in many other organs, e.g. heart muscle, total cerebral blood flow per minute is enormous. Consequently, to accommodate a large blood volume with a limited capillary bed, the velocity of blood through brain vessels must be extremely fast. The hypothesis presented in this paper is that this rapid flow results in a low or negative pressure on the endothelium, and plasma and trypan blue are prevented from passing through the wall. The tight junctions of cerebral endothelial cells may be able to withstand only a limited amount of pressure on their luminal surface. If the velocity of blood in brain capillaries decreases, pressure on the endothelium should increase, and brain vessels, like blood vessels elsewhere in the body, become permeable to vital dyes. Other conditions also increase capillary permeability, e.g. acute arterial hypertension or venous congestion. Although brain vessels can adapt to a moderate, gradual change in systemic pressure, when a significant rise in cerebral arterial pressure is abrupt, the compensatory changes in the postcapillary venous bed may be inadequate and consequently intracapillary pressure and vascular permeability are increased. Venous congestion increases intracapillary pressure by restricting capillary outflow as well as by reducing velocity through capillary beds. Under such conditions increased capillary permeability may be indicated by cerebral edema, and even, on occasion, by petechial hemorrhages. In short, if the flow is fast and unimpeded the BBB will be effective; if the velocity decreases, or intracapillary pressure increases for whatever reason, the permeability of the brain endothelium will be abnormally increased.     

Specific AHNAK expression in brain endothelial cells with barrier properties

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and low permeability. Because disruption of the BBB may contribute to many brain disorders, they are of considerable interests in the identification of the molecular mechanisms of BBB development and integrity. We here report that the giant protein AHNAK is expressed at the plasma membrane of endothelial cells (ECs) forming specific blood-tissue barriers, but is absent from the endothelium of capillaries characterized by extensive molecular exchanges between blood and extracellular fluid. In the brain, AHNAK is widely distributed in ECs with BBB properties, where it co-localizes with the tight junction protein ZO-1. AHNAK is absent from the permeable brain ECs of the choroid plexus and is down-regulated in permeable angiogenic ECs of brain tumors. In the choroid plexus, AHNAK accumulates at the tight junctions of the choroid epithelial cells that form the blood-cerebrospinal fluid (CSF) barrier. In EC cultures, the regulation of AHNAK expression and its localization corresponds to general criteria of a protein involved in barrier organization. AHNAK is up-regulated by angiopoietin-1 (Ang-1), a morphogenic factor that regulates brain EC permeability. In bovine cerebral ECs co-cultured with glial cells, AHNAK relocates from the cytosol to the plasma membrane when endothelial cells acquire BBB properties. Our results identify AHNAK as a protein marker of endothelial cells with barrier properties.     

n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood–brain barrier

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood–brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (−23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 μM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [³H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood–brain barrier, possibly via changes in GLUT1 protein expression and activity.     

Differential effects of HIF-1 inhibition by YC-1 on the overall outcome and blood-brain barrier damage in a rat model of ischemic stroke

Hypoxia-inducible factor 1 (HIF-1) is a master regulator of cellular adaptation to hypoxia and has been suggested as a potent therapeutic target in cerebral ischemia. Here we show in an ischemic stroke model of rats that inhibiting HIF-1 and its downstream genes by 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) significantly increases mortality and enlarges infarct volume evaluated by MRI and histological staining. Interestingly, the HIF-1 inhibition remarkably ameliorates ischemia-induced blood-brain barrier (BBB) disruption determined by Evans blue leakage although it does not affect brain edema. The result demonstrates that HIF-1 inhibition has differential effects on ischemic outcomes and BBB permeability. It indicates that HIF-1 may have different functions in different brain cells. Further analyses show that ischemia upregulates HIF-1 and its downstream genes erythropoietin (EPO), vascular endothelial growth factor (VEGF), and glucose transporter (Glut) in neurons and brain endothelial cells and that YC-1 inhibits their expression. We postulate that HIF-1-induced VEGF increases BBB permeability while certain other proteins coded by HIF-1's downstream genes such as epo and glut provide neuroprotection in an ischemic brain. The results indicate that YC-1 lacks the potential as a cerebral ischemic treatment although it confers certain protection to the cerebral vascular system.     

Combined treatment of sodium ferulate,n‐butylidenephthalide,and ADSCs rehabilitates neurovascular unit in rats after photothrombotic stroke

The remodelling of structural and functional neurovascular unit (NVU) becomes a central therapeutic strategy after cerebral ischaemic stroke. In the present study, we investigated the effect of combined therapy of sodium ferulate (SF), n‐butylidenephthalide (BP) and adipose‐derived stromal cells (ADSCs) to ameliorate the injured NVU in the photochemically induced thrombotic stroke in rats. After solely or combined treatment, the neovascularization, activation of astrocytes, neurogenesis, expressions of vascular endothelial growth factor (VEGF) and claudin‐5 were assessed by immunohistochemical or immunofluorescence staining. In order to uncover the underlying mechanism of therapeutic effect, signalling of protein kinase B/mammalian target of rapamycin (AKT/mTOR), extracellular signal‐regulated kinase 1/2 (ERK1/2), and Notch1 in infarct zone were analysed by western blot. ¹⁸F‐2‐deoxy‐glucose/positron emission tomography, magnetic resonance imaging, Evans blue staining were employed to evaluate the glucose metabolism, cerebral blood flow (CBF), and brain‐blood barrier (BBB) permeability, respectively. The results showed that combined treatment increased the neovascularization, neurogenesis, and VEGF secretion, modulated the astrocyte activation, enhanced the regional CBF, and glucose metabolism, as well as reduced BBB permeability and promoted claudin‐5 expression, indicating the restoration of structure and function of NVU. The activation of ERK1/2 and Notch1 pathways and inhibition of AKT/mTOR pathway might be involved in the therapeutic mechanism. In summary, we have demonstrated that combined ADSCs with SF and BP, targeting the NVU remodelling, is a potential treatment for ischaemic stroke. These results may provide valuable information for developing future combined cellular and pharmacological therapeutic strategy for ischaemic stroke.     

How can microbial interactions with the blood–brain barrier modulate astroglial and neuronal function?

The vascular endothelium of the blood-brain barrier (BBB) is regarded as a part of the neurovascular unit (NVU). This emerging NVU concept emphasizes the need for homeostatic signalling among the neuronal, glial and vascular endothelial cellular compartments in maintaining normal brain function. Conversely, dysfunction in any component of the NVU affects another, thus contributing to disease. Brain endothelial activation and dysfunction is observed in various neurological diseases, such as (ischemic) stroke, seizure, brain inflammation and infectious diseases and likely contributes to or exacerbates neurological conditions. The role and impact of brain endothelial factors on astroglial and neuronal activation is unclear. Similarly, it is not clear which stages of BBB endothelial activation can be considered beneficial versus detrimental. Although the BBB plays an important role in context of encephalopathies caused by neurotropic microbes that must first penetrate into the brain, a crucial role of the BBB in contributing to neurological dysfunction may be seen in cerebral malaria (CM), where the Plasmodium parasite remains sequestered in the brain vasculature, does not enter the brain parenchyma, and yet causes coma and seizures. In this minireview some of the scenarios and factors that may play a role in BBB as a relay station to modulate astroneuronal functioning are discussed.     

Pericytes from Brain Microvessels Strengthen the Barrier Integrity in Primary Cultures of Rat Brain Endothelial Cells

(1) The blood–brain barrier (BBB) characteristics of cerebral endothelial cells are induced by organ-specific local signals. Brain endothelial cells lose their phenotype in cultures without cross-talk with neighboring cells. (2) In contrast to astrocytes, pericytes, another neighboring cell of endothelial cells in brain capillaries, are rarely used in BBB co-culture systems. (3) Seven different types of BBB models, mono-culture, double and triple co-cultures, were constructed from primary rat brain endothelial cells, astrocytes and pericytes on culture inserts. The barrier integrity of the models were compared by measurement of transendothelial electrical resistance and permeability for the small molecular weight marker fluorescein. (4) We could confirm that brain endothelial monolayers in mono-culture do not form tight barrier. Pericytes induced higher electrical resistance and lower permeability for fluorescein than type I astrocytes in co-culture conditions. In triple co-culture models the tightest barrier was observed when endothelial cells and pericytes were positioned on the two sides of the porous filter membrane of the inserts and astrocytes at the bottom of the culture dish. (5) For the first time a rat primary culture based syngeneic triple co-culture BBB model has been constructed using brain pericytes beside brain endothelial cells and astrocytes. This model, mimicking closely the anatomical position of the cells at the BBB in vivo, was superior to the other BBB models tested. (6) The influence of pericytes on the BBB properties of brain endothelial cells may be as important as that of astrocytes and could be exploited in the construction of better BBB models.     

Blood vessel formation in cerebral organoids formed from human embryonic stem cells

Current cerebral organoid technology provides excellent in vitro models mimicking the structure and function of the developing human brain, which enables studies on normal and pathological brain; however, further improvements are necessary to overcome the problems of immaturity and dearth of non-parenchymal cells. Vascularization is one of the major challenges for recapitulating processes in the developing human brain. Here, we examined the formation of blood vessel-like structures in cerebral organoids induced by vascular endothelial growth factor (VEGF) in vitro. The results indicated that VEGF enhanced differentiation of vascular endothelial cells (ECs) without reducing neuronal markers in the embryonic bodies (EBs), which then successfully developed into cerebral organoids with open-circle vascular structures expressing an EC marker, CD31, and a tight junction marker, claudin-5, characteristic of the blood-brain barrier (BBB). Further treatment with VEGF and Wnt7a promoted the formation of the outer lining consisting of pericyte-like cells, which surrounded the vascular tubes. RNA sequencing revealed that VEGF upregulated genes associated with tube formation, vasculogenesis, and the BBB; it also changed the expression of genes involved in brain embryogenesis, suggesting a role of VEGF in neuronal development. These results indicate that VEGF treatment can be used to generate vessel-like structures with mature BBB characteristics in cerebral organoids in vitro.     

Volatile Anesthetics Influence Blood-Brain Barrier Integrity by Modulation of Tight Junction Protein Expression in Traumatic Brain Injury

Disruption of the blood-brain barrier (BBB) results in cerebral edema formation, which is a major cause for high mortality after traumatic brain injury (TBI). As anesthetic care is mandatory in patients suffering from severe TBI it may be important to elucidate the effect of different anesthetics on cerebral edema formation. Tight junction proteins (TJ) such as zonula occludens-1 (ZO-1) and claudin-5 (cl5) play a central role for BBB stability. First, the influence of the volatile anesthetics sevoflurane and isoflurane on in-vitro BBB integrity was investigated by quantification of the electrical resistance (TEER) in murine brain endothelial monolayers and neurovascular co-cultures of the BBB. Secondly brain edema and TJ expression of ZO-1 and cl5 were measured in-vivo after exposure towards volatile anesthetics in native mice and after controlled cortical impact (CCI). In in-vitro endothelial monocultures, both anesthetics significantly reduced TEER within 24 hours after exposure. In BBB co-cultures mimicking the neurovascular unit (NVU) volatile anesthetics had no impact on TEER. In healthy mice, anesthesia did not influence brain water content and TJ expression, while 24 hours after CCI brain water content increased significantly stronger with isoflurane compared to sevoflurane. In line with the brain edema data, ZO-1 expression was significantly higher in sevoflurane compared to isoflurane exposed CCI animals. Immunohistochemical analyses revealed disruption of ZO-1 at the cerebrovascular level, while cl5 was less affected in the pericontusional area. The study demonstrates that anesthetics influence brain edema formation after experimental TBI. This effect may be attributed to modulation of BBB permeability by differential TJ protein expression. Therefore, selection of anesthetics may influence the barrier function and introduce a strong bias in experimental research on pathophysiology of BBB dysfunction. Future research is required to investigate adverse or beneficial effects of volatile anesthetics on patients at risk for cerebral edema.