Penetration of verapamil across blood brain barrier following cerebral ischemia depending on both paracellular pathway and P-glycoprotein transportation

Background

Blood brain barrier (BBB) dysfunction is a common facet of cerebral ischemia, and the alteration of drug transporter, P-glycoprotein (P-gp), has been documented.

Aims

This study explores influence of damaged BBB and elevated P-gp on cerebral verapamil penetration after ischemia both in vivo and in vitro.

Methods

Middle cerebral artery occlusion (MCAO) induced ischemia/reperfusion (I/R) of rats, and Na₂S₂O₄ induced hypoxia/reoxygenation (H/R) damage of rat brain mirovessel endothelial cells (RBMECs) respectively, served as BBB breakdown model in vivo and in vitro. Evans-Blue (EB) extravagation and ¹²⁵I-albumin were used to quantify BBB dysfunction; UPLC–MS/MS analytical method was performed to determine accurately the concentration of verapamil in brain tissue and cell. Flow cytometry, immunohistochemistry and western blotting were applied to evaluate transport function and protein expression of P-gp.

Results

Overexpressed ICAM-1 and MMP-9 mediated BBB dysfunction after ischemia, which induced EB leakage and ¹²⁵I-albumin uptake increase. Enhanced accumulation of verapamil in brain tissue, but intracellular concentration reduced evidently after H/R injury. Transcellular transportation of verapamil elevated when P-gp function or expression was inhibited after H/R injury.

Conclusion

These data indicated that BBB penetration of verapamil under ischemia condition was not only depending on BBB breakdown, but also regulated by P-gp.     

Sufficient virus-neutralizing antibody in the central nerve system improves the survival of rabid rats

Background

Rabies is known to be lethal in human. Treatment with passive immunity for the rabies is effective only when the patients have not shown the central nerve system (CNS) signs. The blood–brain barrier (BBB) is a complex functional barrier that may compromise the therapeutic development in neurological diseases. The goal of this study is to determine the change of BBB integrity and to assess the therapeutic possibility of enhancing BBB permeability combined with passive immunity in the late stage of rabies virus infection.

Methods

The integrity of BBB permeability in rats was measured by quantitative ELISA for total IgG and albumin levels in the cerebrospinal fluid (CSF) and by exogenously applying Evans blue as a tracer. Western blotting of occludin and ZO-1, two tight junction proteins, was used to assess the molecular change of BBB structure.The breakdown of BBB with hypertonic arabinose, recombinant tumor necrosis factor-alpha (rTNF-γ), and focused ultrasound (FUS) were used to compare the extent of BBB disruption with rabies virus infection. Specific humoral immunity was analyzed by immunofluorescent assay and rapid fluorescent focus inhibition test. Virus-neutralizing monoclonal antibody (mAb) 8-10E was administered to rats with hypertonic breakdown of BBB as a passive immunotherapy to prevent the death from rabies.

Results

The BBB permeability was altered on day 7 post-infection. Increased BBB permeability induced by rabies virus infection was observed primarily in the cerebellum and spinal cord. Occludin was significantly decreased in both the cerebral cortex and cerebellum. The rabies virus-specific antibody was not strongly elicited even in the presence of clinical signs. Disruption of BBB had no direct association with the lethal outcome of rabies. Passive immunotherapy with virus-neutralizing mAb 8-10E with the hypertonic breakdown of BBB prolonged the survival of rabies virus-infected rats.

Conclusions

We demonstrated that the BBB permeability was altered in a rat model with rabies virus inoculation. Delivery of neutralizing mAb to the infected site in brain combined with effective breakdown of BBB could be an aggressive but feasible therapeutic mode in rabies when the CNS infection has been established.     

Effect of Phoneutria nigriventer Venom on the Expression of Junctional Protein and P-gp Efflux Pump Function in the Blood–Brain Barrier

Phoneutria nigriventer spider venom (PNV) contains Ca(2+), K(+) and Na(+) channel-acting peptides that affect neurotransmitter release and causes excitotoxicity in PNS and CNS. It has been demonstrated that PNV causes blood-brain barrier (BBB) breakdown of hippocampal microvessels time-dependently through enhanced microtubule-mediated vesicular transport. Herein, it is hypothesized that PNV can cause BBB breakdown in the hippocampus and cerebellum time-dependently through other molecular mechanisms. The BBB integrity was assessed through the analysis of expression of Poly-glycoprotein (P-gp) efflux transporter protein, laminin from basement membrane and endothelial tight junctional and adhesion junctional (TJ/AJ) proteins. Phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A) expression, which are known to have a role in the phosphorylation of junctional proteins and BBB opening, were also investigated. Astrocytes P-gp activity was determined by flow cytometry. The study demonstrated temporary decreased expression of laminin, TJ and AJ proteins (ZO1//occludin//claudin-5//beta-catenin) and P-gp (more prominently in hippocampus), which was completely or partially resolved between 2 and 5?h (and more quickly for cerebellum). PNV inhibited P-gp activity in astrocytes. PP2A phosphorylation, which inhibits the enzyme activity, was increased in both regions (15-45?min); however the phosphorylation level returned to baseline after 2?h. In conclusion, PNV disrupts paracellular transport in the BBB and possesses substrates for the active P-gp efflux transporter located in the BBB complex. Further studies into cellular mechanisms of astrocyte/endothelial interactions, using PNV as tool, may identify how astrocytes regulate the BBB, a characteristic that may be useful for the temporary opening of the BBB.     

The relationship between erythropoietin pretreatment with blood-brain barrier and lipid peroxidation after ischemia/reperfusion in rats

Blood-brain barrier (BBB) leakage plays a role in the pathogenesis of many pathological states of the brain including ischemia and some neurodegenerative disorders. In recent years, erythropoietin (EPO) has been shown to exert neuroprotection in many pathological conditions including ischemia in the brain. This study aimed to investigate the effects of EPO on BBB integrity, infarct size and lipid peroxidation following global brain ischemia/reperfusion in rats. Wistar male rats were divided into four groups (each group n=8); Group I; control group (sham-operated), Group II; ischemia/reperfusion group, Group III; EPO treated group (24 h before decapitation--000 U/kg r-Hu EPO i.p.), Group IV; EPO+ ischemia/reperfusion group (24 h before ischemia/reperfusion--3000 U/kg r-Hu EPO i.p.). Global brain ischemia was produced by the combination of bilateral common carotid arteries occlusion and hemorrhagic hypotension. Macroscopical and spectrophotometrical measurement of Evans Blue (EB) leakage was observed for BBB integrity. Infarct size was calculated based on 2,3,5-triphenyltetrazolium chloride (TTC) staining. Lipid peroxidation in the brain tissue was determined as the concentration of thiobarbituric acid-reactive substances (TBARS) for each group. Ischemic insult caused bilateral and regional BBB breakdown (hippocampus, cortex, corpus striatum, midbrain, brain stem and thalamus). EPO pretreatment reduced BBB disruption, infarct size and lipid peroxide levels in brain tissue with 20 min ischemia and 20 min reperfusion. These results suggest that EPO plays an important role in protecting against brain ischemia/reperfusion through inhibiting lipid peroxidation and decreasing BBB disruption.     

Blood-Brain Barrier Breakdown after Embolic Stroke in Rats Occurs without Ultrastructural Evidence for Disrupting Tight Junctions

The term blood-brain barrier (BBB) relates to the ability of cerebral vessels to hold back hydrophilic and large molecules from entering the brain, thereby crucially contributing to brain homeostasis. In fact, experimental opening of endothelial tight junctions causes a breakdown of the BBB evidenced as for instance by albumin leakage. This and similar observations led to the conclusion that BBB breakdown is predominantly mediated by damage to tight junction complexes, but evidentiary ultrastructural data are rare. Since functional deficits of the BBB contribute to an increased risk of hemorrhagic transformation and brain edema after stroke, which both critically impact on the clinical outcome, we studied the mechanism of BBB breakdown using an embolic model of focal cerebral ischemia in Wistar rats to closely mimic the essential human pathophysiology. Ischemia-induced BBB breakdown was detected using intravenous injection of FITC-albumin and tight junctions in areas of FITC-albumin extravasation were subsequently studied using fluorescence and electron microscopy. Against our expectation, 25 hours after ischemia induction the morphology of tight junction complexes (identified ultrastructurally and using antibodies against the transcellular proteins occludin and claudin-5) appeared to be regularly maintained in regions where FITC-albumin massively leaked into the neuropil. Furthermore, occludin signals along pan-laminin-labeled vessels in the affected hemisphere equaled the non-affected contralateral side (ratio: 0.966 vs. 0.963; P = 0.500). Additional ultrastructural analyses at 5 and 25 h after ischemia induction clearly indicated FITC-albumin extravasation around vessels with intact tight junctions, while the endothelium exhibited enhanced transendothelial vesicle trafficking and signs of degeneration. Thus, BBB breakdown and leakage of FITC-albumin cannot be correlated with staining patterns for common tight junction proteins alone. Understanding the mechanisms causing functional endothelial alterations and endothelial damage is likely to provide novel protective targets in stroke.     

Differential expression and cellular distribution of γ‐tubulin and βIII‐tubulin in medulloblastomas and human medulloblastoma cell lines

The breakdown of the blood–brain barrier (BBB) has been considered to be a key step in the disease process of a number of neurological disorders such as cerebral ischemia and Alzheimer's disease. Many in vitro BBB models derived from animal tissues have been established to elucidate the mechanism of BBB insufficiency. However, only a few human immortalized in vitro BBB models have been reported. In the present study, a temperature‐sensitive SV40‐T antigen was introduced to immortalize cells using a retrovirus to obtain a better human in vitro BBB model which sustains physiological properties. This endothelial cell (EC) line, termed TY08, showed a spindle‐shaped morphology. The cells expressed all key tight junctional proteins, such as occludin, claudin‐5, zonula occludens (ZO)‐1 and ZO‐2 at their cell‐to‐cell boundaries, and had low permeability to inulin across its monolayer. The cells also expressed various influx and efflux transporters and exhibited the functional expression of p‐glycoprotein. Furthermore, the TY08 cells grew and proliferated well under the permissive temperature and stopped growing under the non‐permissive temperature to serve as physiological ECs forming the BBB. Thus, conditionally immortalized TY08 cells retaining the in vivo BBB functions should facilitate analyses for determining the pathophysiology of various neurological diseases. J. Cell. Physiol. 225: 519–528, 2010. © 2010 Wiley‐Liss, Inc.     

Wnt/beta-catenin signaling controls development of the blood-brain barrier

The blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/β-catenin (β-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of β-cat in vivo enhances barrier maturation, whereas inactivation of β-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of β-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of β-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of β-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown.     

Matrix metalloproteinase-2 and -9 secreted by leukemic cells increase the permeability of blood-brain barrier by disrupting tight junction proteins

Central nervous system (CNS) involvement remains an important cause of morbidity and mortality in acute leukemia, the mechanisms of leukemic cell infiltration into the CNS have not yet been elucidated. The blood-brain barrier (BBB) makes CNS become a refugee to leukemic cells and serves as a resource of cells that seed extraneural sites. How can the leukemic cells disrupt this barrier and invasive the CNS, even if many of the currently available chemotherapies can not cross the BBB? Tight junction in endothelial cells occupies a central role in the function of the BBB. Except the well known role of degrading extracellular matrix in metastasis of cancer cells, here we show matrix metalloproteinase (MMP)-2 and -9, secreted by leukemic cells, mediate the BBB opening by disrupting tight junction proteins in the CNS leukemia. We demonstrated that leukemic cells impaired tight junction proteins ZO-1, claudin-5 and occludin resulting in increased permeability of the BBB. However, these alterations reduced when MMP-2 and -9 activities were inhibited by RNA interference strategy or by MMP inhibitor GM6001 in an in vitro BBB model. We also found that the disruption of the BBB in company with the down-regulation of ZO-1, claudin-5 and occludin and the up-regulation of MMP-2 and -9 in mouse brain tissues with leukemic cell infiltration by confocal imaging and the assay of in situ gelatin zymography. Besides, GM6001 protected all mice against CNS leukemia. Our findings suggest that the degradation of tight junction proteins ZO-1, claudin-5 and occludin by MMP-2 and -9 secreted by leukemic cells constitutes an important mechanism in the BBB breakdown which contributes to the invasion of leukemic cells to the CNS in acute leukemia.     

Regulation of the BBB during viral encephalitis: roles of IL-12 and NOS.

Intranasal infection of mice by Vesicular Stomatitis Virus (VSV) often leads to breakdown of the blood-brain barrier (BBB). The role of Interleukin 12 (IL-12) and nitric oxide synthase (NOS) was examined here. Wild-type (WT), NOS-1 knockout (KO), and NOS-3 KO mice were infected with VSV and treated with either IL-12 or medium. IL-12 treatment of uninfected hosts did not result in pathology. In contrast with WT and NOS-1 KO mice, where extensive gross and ultrastructural correlation of BBB breakdown were evident following infection, in NOS-3 KO mice, integrity of the BBB was observed. Thus NOS-3 activity in astrocytes, endothelial cells, or ependymal cells may play an essential role in regulating the BBB.     

2-(2-Benzofuranyl)-2-Imidazoline Attenuates the Disruption of the Blood–Brain Barrier in EAE via NMDAR

Blood–brain barrier (BBB) disruption has been recognized as an early hallmark of multiple sclerosis (MS) pathology. Our previous studies have shown that 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) protected against experimental autoimmune encephalomyelitis (EAE), a classic animal model of MS. However, the potential effects of 2-BFI on BBB permeability have not yet been evaluated in the context of EAE. Herein, we aimed to investigate the effect of 2-BFI on BBB permeability in both an animal model and an in vitro BBB model using TNF-α to imitate the inflammatory damage to the BBB in MS. In the animal model, 2-BFI reduced neurological deficits and BBB permeability in EAE mice compared with saline treatment. The Western blot results indicated that 2-BFI not only alleviated the loss of the tight junction protein occludin caused by EAE but also inhibited the activation of the NR1-ERK signaling pathway. In an in vitro BBB model, 2-BFI (100 μM) alleviated the TNF-α-induced increase in permeability and reduction in expression of occludin in monolayer bEnd.3 cells. Similar protective effects were also observed after treatment with the NMDAR antagonist MK801. The Western blot results showed that the TNF-α-induced BBB breakdown and increase in NMDAR subunit 1 (NR1) levels and ERK phosphorylation could be blocked by pretreatment with 2-BFI or MK801. However, no additional effect was observed on BBB permeability or the expression of occludin and p-ERK after pretreatment with both 2-BFI and MK801. Our study indicates that 2-BFI alleviates the disruption of BBB in the context of inflammatory injury similar to that of MS by targeting NMDAR1, as well as by likely activating the subsequent ERK signaling pathway. These results provide further evidence for 2-BFI as a potential drug for the treatment of MS.

     

The blood-brain barrier and cerebral angiogenesis: lessons from the cold-injury model.

Brain injury is associated with an initial blood-brain barrier (BBB) breakdown, which can be life threatening. A second phase of BBB breakdown accompanies the angiogenesis occurring at the lesion margins. Studies of the molecular mechanisms involved in these processes are essential to determine targets for therapeutic intervention, as well as the time periods during which therapeutic intervention could ameliorate brain damage and thus improve the clinical outcome.     

Genome Editing with AAV-BR1-CRISPR in Postnatal Mouse Brain Endothelial Cells

Brain endothelial cells (ECs) are an important component of the blood-brain barrier (BBB) and play key roles in restricting entrance of possible toxic components and pathogens into the brain. However, identifying endothelial genes that regulate BBB homeostasis remains a time-consuming process. Although somatic genome editing has emerged as a powerful tool for discovery of essential genes regulating tissue homeostasis, its application in brain ECs is yet to be demonstrated in vivo. Here, we used an adeno-associated virus targeting brain endothelium (AAV-BR1) combined with the CRISPR/Cas9 system (AAV-BR1-CRISPR) to specifically knock out genes of interest in brain ECs of adult mice. We first generated a mouse model expressing Cas9 in ECs (Tie2^Cas9). We selected endothelial β-catenin (Ctnnb1) gene, which is essential for maintaining adult BBB integrity, as the target gene. After intravenous injection of AAV-BR1-sgCtnnb1-tdTomato in 4-week-old Tie2^Cas9 transgenic mice resulted in mutation of 36.1% of the Ctnnb1 alleles, thereby leading to a dramatic decrease in the level of CTNNB1 in brain ECs. Consequently, Ctnnb1 gene editing in brain ECs resulted in BBB breakdown. Taken together, these results demonstrate that the AAV-BR1-CRISPR system is a useful tool for rapid identification of endothelial genes that regulate BBB integrity in vivo.     

Oxygen-glucose deprivation increases the enzymatic activity and the microvesicle-mediated release of ectonucleotidases in the cells composing the blood-brain barrier

The blood-brain barrier (BBB), the dynamic interface between the nervous tissue and the blood, is composed by endothelial cells, pericytes and astrocytes. Extracellular nucleotides and nucleosides and their receptors (the purinergic system) constitute a widely diffused signaling system involved in many pathophysiological processes. However, the role of this system in controlling BBB functions is still largely unknown. By using cultures of these three cell types grown separately and a BBB in vitro model consisting of triple co-cultures, we studied for the first time the expression and distribution of the ecto-enzymes nucleoside triphosphate diphosphohydrolases (NTPDases, the enzymes which hydrolyze extracellular nucleotides) under control and ischemic (oxygen-glucose deprivation in vitro; OGD) conditions. NTPDase1 was detected in all three cell types, whereas NTPDase2 was expressed by astrocytes and pericytes and, to a lesser extent, by endothelial cells. Endothelial cells were extremely susceptible to cell death when OGD was applied to mimic in vitro the cytotoxicity induced by ischemia, whereas astrocytes and pericytes were more resistant. A semi-quantitative assay highlighted markedly increased e-ATPase activity following exposure to OGD in all three cell types, either when grown separately or when co-cultured together to resemble the composition of the BBB. Moreover, electron microscopy analysis showed that both endothelial cells and astrocytes shed microvesicles containing NTPDases from their membrane, which may suggest a novel mechanism to increase the breakdown of ATP released to toxic levels by damaged BBB cells. We hypothesize that this phenomenon could have a protective and/or modulatory effect for brain parenchymal cells. This in vitro model is therefore useful to study the role of extracellular nucleotides in modulating BBB responses to ischemic events, and to develop new effective purinergic-based approaches for brain ischemia.     

Annexin A1 restores Aβ1‐42‐induced blood–brain barrier disruption through the inhibition of RhoA‐ROCK signaling pathway

The blood–brain barrier (BBB) is composed of brain capillary endothelial cells and has an important role in maintaining homeostasis of the brain separating the blood from the parenchyma of the central nervous system (CNS). It is widely known that disruption of the BBB occurs in various neurodegenerative diseases, including Alzheimer's disease (AD). Annexin A1 (ANXA1), an anti‐inflammatory messenger, is expressed in brain endothelial cells and regulates the BBB integrity. However, its role and mechanism for protecting BBB in AD have not been identified. We found that β‐Amyloid 1‐42 (Aβ42)‐induced BBB disruption was rescued by human recombinant ANXA1 (hrANXA1) in the murine brain endothelial cell line bEnd.3. Also, ANXA1 was decreased in the bEnd.3 cells, the capillaries of 5XFAD mice, and the human serum of patients with AD. To find out the mechanism by which ANXA1 recovers the BBB integrity in AD, the RhoA‐ROCK signaling pathway was examined in both Aβ42‐treated bEnd.3 cells and the capillaries of 5XFAD mice as RhoA was activated in both cases. RhoA inhibitors alleviated Aβ42‐induced BBB disruption and constitutively overexpressed RhoA‐GTP (active form of RhoA) attenuated the protective effect of ANXA1. When pericytes were cocultured with bEnd.3 cells, Aβ42‐induced RhoA activation of bEnd.3 cells was inhibited by the secretion of ANXA1 from pericytes. Taken together, our results suggest that ANXA1 restores Aβ42‐induced BBB disruption through inhibition of RhoA‐ROCK signaling pathway and we propose ANXA1 as a therapeutic reagent, protecting against the breakdown of the BBB in AD.     

Blood-brain barrier damage induces release of alpha2-macroglobulin

Blood-brain barrier (BBB) failure occurs in many neurological diseases and is caused in part by activation of proinflammatory factors including matrix metalloproteinases. Counterbalancing, "BBB protective" cascades have recently been described, including NO-mediated interleukin 6 release by glia. Interleukin 6 has been shown to trigger production of matrix metalloproteinase inhibitors such as alpha2-macroglobulin (alpha2M). We hypothesized that BBB failure may result in increased alpha(2)M release by perivascular astrocytes. This was initially tested in patients undergoing iatrogenic BBB disruption by hyperosmotic mannitol for intra-arterial chemotherapy of brain tumors. Serum samples revealed significantly increased levels of alpha2M at 4 h after BBB disruption by hyperosmotic mannitol. In parallel in vitro experiments, we observed a similar increase of alpha2M release by astrocytes under conditions mimicking BBB failure and perivascular edema. For both experiments, protein analysis was initially performed by bidimensional gel electrophoresis and mass spectrometry followed by Western blotting immunodetection. We conclude that, in addition to proinflammatory changes, BBB failure may also trigger protective release of alpha2M by perivascular astrocytes as well as peripheral source.     

Breakdown of the blood-brain barrier during tick-borne encephalitis in mice is not dependent on CD8+ T-cells

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. The disease is characterized by fever and debilitating encephalitis that can progress to chronic illness or fatal infection. In this study, changes in permeability of the blood-brain barrier (BBB) in two susceptible animal models (BALB/c, and C57Bl/6 mice) infected with TBE virus were investigated at various days after infection by measuring fluorescence in brain homogenates after intraperitoneal injection of sodium fluorescein, a compound that is normally excluded from the central nervous system. We demonstrate here that TBE virus infection, in addition to causing fatal encephalitis in mice, induces considerable breakdown of the BBB. The permeability of the BBB increased at later stages of TBE infection when high virus load was present in the brain (i.e., BBB breakdown was not necessary for TBE virus entry into the brain), and at the onset of the first severe clinical symptoms of the disease, which included neurological signs associated with sharp declines in body weight and temperature. The increased BBB permeability was in association with dramatic upregulation of proinflammatory cytokine/chemokine mRNA expression in the brain. Breakdown of the BBB was also observed in mice deficient in CD8(+) T-cells, indicating that these cells are not necessary for the increase in BBB permeability that occurs during TBE. These novel findings are highly relevant to the development of future therapies designed to control this important human infectious disease.     

Alteration of sphingolipid metabolism as a putative mechanism underlying LPS‐induced BBB disruption

Septic encephalopathy with confusion and agitation occurs early during sepsis and contributes to the severity of the disease. A decrease in the sphingosine‐1‐phosphate (S1P) blood levels has been shown in patients and in animal models of sepsis. The lipid mediator S1P is known to be involved in endothelial barrier function in a context‐dependent manner. We utilized lipopolysaccharide (LPS )‐injected mice as a model for septic encephalopathy and first performed tracer permeability assays to assess the blood–brain barrier (BBB ) breakdown in vivo. At time points corresponding to the BBB breakdown post LPS injection, we aimed to characterize the regulation of the sphingolipid signaling pathway at the BBB during sepsis. We measured sphingolipid concentrations in blood, in mouse brain microvessels (MBMV s), and brain tissue. We also analyzed the expression of S1P receptors, transporters, and metabolizing enzymes in MBMV s and brain tissue. Primary mouse brain microvascular endothelial cells (MBMEC s) were isolated to evaluate the effects of LPS on transendothelial electrical resistance (TEER ) as a measure of permeability in vitro . We observed a relevant decrease in S1P levels after LPS injection in all three compartments (blood, MBMV s, brain tissue) that was accompanied by an increased expression of the S1P receptor type 1 and of sphingosine kinase 1 on one hand and of the S1P degrading enzymes lipid phosphate phosphatase 1 (LPP 1) and S1P phosphatase 1 on the other hand, as well as a down‐regulation of sphingosine kinase 2. Application of LPS to a monolayer of primary MBMEC s did not alter TEER , but serum from LPS ‐treated mice lead to a breakdown of the barrier compared to serum from vehicle‐treated mice. We observed profound alterations of the sphingolipid metabolism at the BBB after LPS injection that point toward a therapeutic potential of drugs interfering with this pathway as novel approach for the detrimental overwhelming immune response in sepsis.

Read the Editorial Highlight for this article on page 115 . Cover Image for this Issue: doi. 10.1111/jnc.14161 .

     

Involvement of ROS in BBB dysfunction

The blood–brain barrier (BBB) forms a protective barrier around the brain, with the important function of maintaining brain homeostasis. Pathways thought to initiate BBB dysfunction include the kinin system, excitotoxicity, neutrophil recruitment, mitochondrial alterations and macrophage/microglial activation, all of which converge on the same point—reactive oxygen species (ROS). Interestingly, ROS also provide a common trigger for many downstream pathways that directly mediate BBB compromise such as oxidative damage, tight junction (TJ) modification and matrix metalloproteinases (MMP) activation. These observations suggest that ROS are key mediators of BBB breakdown and implicate antioxidants as potential neuroprotectants in conditions like stroke and traumatic brain injury (TBI). This review explores some of the pathways both upstream and downstream of ROS that have been implicated in increased BBB permeability and discusses the role of ROS and antioxidants in neuropathology.     

eNOS gene deletion restores blood-brain barrier integrity and attenuates neurodegeneration in the thiamine-deficient mouse brain

Wernicke's encephalopathy is a cerebral disorder caused by thiamine (vitamin B₁) deficiency (TD). Neuropathologic consequences of TD include region-selective neuronal cell loss and blood-brain barrier (BBB) breakdown. Early increased expression of the endothelial isoform of nitric oxide synthase (eNOS) occurs selectively in vulnerable brain regions in TD. We hypothesize that region-selective eNOS induction in TD leads to altered expression of tight junction proteins and BBB breakdown. In order to address this issue, TD was induced in C57BL/6 wild-type (WT) and eNOS^−/− mice by feeding a thiamine-deficient diet and treatment with the thiamine antagonist pyrithiamine. Pair-fed control mice were fed the same diet with additional thiamine. In medial thalamus of TD-WT mice (vulnerable area), increased heme oxygenase-1 and S -nitrosocysteine immunostaining was observed in vessel walls, compared to pair-fed control-WT mice. Concomitant increases in IgG extravasation, decreases in expression of the tight junction proteins occludin, zona occludens-1 and zona occludens-2, and up-regulation of matrix metalloproteinase-9 in endothelial cells were observed in the medial thalamus of TD-WT mice. eNOS gene deletion restored these BBB alterations, suggesting that eNOS-derived nitric oxide is a major factor leading to cerebrovascular alterations in TD. However, eNOS gene deletion only partially attenuated TD-related neuronal cell loss, suggesting the presence of mechanisms additional to BBB disruption in the pathogenesis of these changes.