S‐nitrosoglutathione prevents blood–brain barrier disruption associated with increased matrix metalloproteinase‐9 activity in experimental diabetes

Hyperglycemia is known to induce microvascular complications, thereby altering blood–brain barrier (BBB) permeability. This study investigated the role of matrix metalloproteinases (MMPs) and their endogenous inhibitors in increased BBB permeability and evaluated the protective effect of S‐nitrosoglutathione (GSNO) in diabetes. Diabetes was induced in mice by intraperitoneal injection of streptozotocin (40 mg/kg body weight) for 5 days and GSNO was administered orally (100 μg/kg body weight) daily for 8 weeks after the induction of diabetes. A significant decline in cognitive functions was observed in diabetic mice assessed by Morris water maze test. Increased permeability to different molecular size tracers accompanied by edema and ion imbalance was observed in cortex and hippocampus of diabetic mice. Furthermore, activity of both pro and active MMP‐9 was found to be significantly elevated in diabetic animals. Increased in situ gelatinase activity was observed in tissue sections and isolated microvessels from diabetic mice brain. The increase in activity of MMP‐9 was attributed to increased mRNA and protein expression in diabetic mice. In addition, a significant decrease in mRNA and protein expression of tissue inhibitor of matrix metalloproteinase‐1 was also observed in diabetic animals. However, GSNO supplementation to diabetic animals was able to abridge MMP‐9 activation as well as tissue inhibitor of matrix metalloproteinase‐1 levels, restoring BBB integrity and also improving learning and memory. Our findings clearly suggest that GSNO could prevent hyperglycemia‐induced disruption of BBB by suppressing MMP‐9 activity.

     

Valproate Administered after Traumatic Brain Injury Provides Neuroprotection and Improves Cognitive Function in Rats

Background

Traumatic brain injury (TBI) initiates a complex series of neurochemical and signaling changes that lead to pathological events including neuronal hyperactivity, excessive glutamate release, inflammation, increased blood-brain barrier (BBB) permeability and cerebral edema, altered gene expression, and neuronal dysfunction. It is believed that a drug combination, or a single drug acting on multiple targets, may be an effective strategy to treat TBI. Valproate, a widely used antiepileptic drug, has a number of targets including GABA transaminase, voltage-gated sodium channels, glycogen synthase kinase (GSK)-3, and histone deacetylases (HDACs), and therefore may attenuate a number of TBI-associated pathologies.

Methodology/Principal Findings

Using a rodent model of TBI, we tested if post-injury administration of valproate can decrease BBB permeability, reduce neural damage and improve cognitive outcome. Dose-response studies revealed that systemic administration of 400 mg/kg (i.p.), but not 15, 30, 60 or 100 mg/kg, increases histone H3 and H4 acetylation, and reduces GSK-3 activity, in the hippocampus. Thirty min post-injury administration of 400 mg/kg valproate improved BBB integrity as indicated by a reduction in Evans Blue dye extravasation. Consistent with its dose response to inhibit GSK-3 and HDACs, valproate at 400 mg/kg, but not 100 mg/kg, reduced TBI-associated hippocampal dendritic damage, lessened cortical contusion volume, and improved motor function and spatial memory. These behavioral improvements were not observed when SAHA (suberoylanilide hydroxamic acid), a selective HDAC inhibitor, was administered.

Conclusion/Significance

Our findings indicate that valproate given soon after TBI can be neuroprotective. As clinically proven interventions that can be used to minimize the damage following TBI are not currently available, the findings from this report support the further testing of valproate as an acute therapeutic strategy.     

Astrocyte mediated modulation of blood-brain barrier permeability does not correlate with a loss of tight junction proteins from the cellular contacts

In the central nervous system (CNS) complex endothelial tight junctions (TJs) form a restrictive paracellular diffusion barrier, the blood-brain barrier (BBB). Pathogenic changes within the CNS are frequently accompanied by the loss of BBB properties, resulting in brain edema. In order to investigate whether BBB leakiness can be monitored by a loss of TJ proteins from cellular borders, we used an in vitro BBB model where brain endothelial cells in co-culture with astrocytes form a tight permeability barrier for ³H-inulin and ¹⁴C-sucrose. Removal of astrocytes from the co-culture resulted in an increased permeability to small tracers across the brain endothelial cell monolayer and an opening of the TJs to horseradish peroxidase as detected by electron microscopy. Strikingly, opening of the endothelial TJs was not accompanied by any visible change in the molecular composition of endothelial TJs as junctional localization of the TJ-associated proteins claudin-3, claudin-5, occludin, ZO-1 or ZO-2 or the adherens junction-associated proteins -catenin or p120cas did not change. Thus, opening of BBB TJs is not readily accompanied by the complete loss of the junctional localization of TJ proteins.This work is dedicated to the memory of Werner Risau (died 13.12.1998), who initiated this collaboration     

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.     

Treadmill Pre-Training Ameliorates Brain Edema in Ischemic Stroke via Down-Regulation of Aquaporin-4: An MRI Study in Rats

Objective

Treadmill pre-training can ameliorate blood brain barrier (BBB) dysfunction in ischemia-reperfusion injury, however, its role in ischemic brain edema remains unclear. This study assessed the neuroprotective effects induced by treadmill pre-training, particularly on brain edema in transient middle cerebral artery occluded model.

Methods

Transient middle cerebral artery occlusion to induce stroke was performed on rats after 2 weeks of treadmill pre-training. Magnetic resonance imaging (MRI) was used to evaluate the dynamic impairment of cerebral edema after ischemia-reperfusion injury. In addition, measurements of wet and dry brain weight, Evans Blue assay and Garcia scores were performed to investigate the cerebral water content, BBB permeability and neurologic deficit, respectively. Moreover, during ischemia-reperfusion injury, the expression of Aquaporin 4 (AQP4) was detected using immunofluorescence and Western bloting analyses.

Results

Treadmill pre-training improved the relative apparent diffusion coefficient (rADC) loss in the ipsilateral cortex and striatum at 1 hour and 2.5 hours after cerebral ischemia. In the treadmill pre-training group, T2W1 values of the ipsilateral cortex and striatum increased less at 7.5 hours, 1 day, and 2 days after stroke while the brain water content decreased at 2 days after ischemia. Regarding the BBB permeability, the semi-quantitative amount of contrast agent leakage of treadmill pre-training group significantly decreased. Less Evans Blue exudation was also observed in treadmill pre-training group at 2 days after stroke. In addition, treadmill pre-training mitigated the Garcia score deficits at 2 days after stroke. Immunofluorescence staining and Western blotting results showed a significant decrease in the expression of AQP4 after treadmill ischemia following pre-training.

Conclusions

Treadmill pre-training may reduce cerebral edema and BBB dysfunction during cerebral ischemia/reperfusion injury via the down-regulation of AQP4.     

Angiopoietin-1 Regulates Brain Endothelial Permeability through PTPN-2 Mediated Tyrosine Dephosphorylation of Occludin

Objective

Blood brain barrier (BBB) breakdown and increased endothelial permeability is a hallmark of neuro-vascular inflammation. Angiopoietin-1 (Ang-1), a Tie-2 receptor agonist ligand, is known to modulate barrier function of endothelial cells; however the molecular mechanisms related to Ang-1 mediated repair of Tight Junctions (TJs) in brain endothelium still remain elusive. In this study, we investigated a novel role of non-receptor protein tyrosine phosphatase N-2 (PTPN-2) in Ang-1 mediated stabilization of tight junction proteins.

Method and Result

To study the barrier protective mechanism of Ang-1, we challenged human brain microvascular endothelial cells in-vitro, with a potent inflammatory mediator thrombin. By using confocal microscopy and transwell permeability assay, we show that pretreatment of brain endothelial cells with Ang-1 diminish thrombin mediated disruption of TJs and increase in endothelial permeability. We also found that Ang-1 inhibits thrombin induced tyrosine phosphorylation of Occludin and promote Occludin interaction with Zona Occludens-1 (ZO-1) to stabilize TJs. Interestingly, our study revealed that depletion of PTPN-2 by siRNAs abolishes Ang-1 ability to promote tyrosine dephosphorylation of Occludin, resulting Occludin disassociation from ZO-1 and endothelial hyperpermeability.

Summary

Collectively, our findings suggest that in brain endothelial cells blocking PTPN-2 mediated tyrosine phosphorylation of Occludin is a novel mechanism to maintain BBB function, and may offer a key therapeutic strategy for neuro-inflammatory disorders associated with BBB disruption.     

Effects of radiofrequency radiation exposure on blood-brain barrier permeability in male and female rats

During the last several decades, numerous studies have been performed aiming at the question of whether or not exposure to radiofrequency radiation (RFR) influences the permeability of the blood-brain barrier (BBB). The objective of this study was to investigate the effect of RFR on the permeability of BBB in male and female Wistar albino rats. Right brain, left brain, cerebellum, and total brain were analyzed separately in the study. Rats were exposed to 0.9 and 1.8 GHz continuous-wave (CW) RFR for 20 min (at SARs of 4.26 mW/kg and 1.46 mW/kg, respectively) while under anesthesia. Control rats were sham-exposed. Disruption of BBB integrity was detected spectrophotometrically using the Evans-blue dye, which has been used as a BBB tracer and is known to be bound to serum albumin. Right brain, left brain, cerebellum, and total brain were evaluated for BBB permeability. In female rats, no albumin extravasation was found in in the brain after RFR exposure. A significant increase in albumin was found in the brains of the RF-exposed male rats when compared to sham-exposed male brains. These results suggest that exposure to 0.9 and 1.8 GHz CW RFR at levels below the international limits can affect the vascular permeability in the brain of male rats. The possible risk of RFR exposure in humans is a major concern for the society. Thus, this topic should be investigated more thoroughly in the future.     

Effects of radiofrequency radiation exposure on blood-brain barrier permeability in male and female rats

During the last several decades, numerous studies have been performed aiming at the question of whether or not exposure to radiofrequency radiation (RFR) influences the permeability of the blood-brain barrier (BBB). The objective of this study was to investigate the effect of RFR on the permeability of BBB in male and female Wistar albino rats. Right brain, left brain, cerebellum, and total brain were analyzed separately in the study. Rats were exposed to 0.9 and 1.8?GHz continuous-wave (CW) RFR for 20?min (at SARs of 4.26?mW/kg and 1.46?mW/kg, respectively) while under anesthesia. Control rats were sham-exposed. Disruption of BBB integrity was detected spectrophotometrically using the Evans-blue dye, which has been used as a BBB tracer and is known to be bound to serum albumin. Right brain, left brain, cerebellum, and total brain were evaluated for BBB permeability. In female rats, no albumin extravasation was found in in the brain after RFR exposure. A significant increase in albumin was found in the brains of the RF-exposed male rats when compared to sham-exposed male brains. These results suggest that exposure to 0.9 and 1.8?GHz CW RFR at levels below the international limits can affect the vascular permeability in the brain of male rats. The possible risk of RFR exposure in humans is a major concern for the society. Thus, this topic should be investigated more thoroughly in the future.     

Phospholipid transfer protein (PLTP) deficiency impaired blood–brain barrier integrity by increasing cerebrovascular oxidative stress

Phospholipid transfer protein (PLTP) regulates lipid metabolism and plays an important role in oxidative stress. PLTP is highly expressed in blood–brain barrier (BBB), but the role of PLTP in BBB integrity is not clear. In this study, BBB permeability was detected with in vivo multiphoton imaging and Evans blue assay. We found that PLTP deficient mice exhibited increased BBB permeability, as well as decreased expression of tight junction proteins occludin, zona occludens-1 (ZO-1) and claudin-5 in brain vessels. Cerebrovascular oxidative stress increased in PLTP deficient mice, including increased levels of reactive oxygen species (ROS) and lipid peroxidation marker 4-hydroxy-2-nonenal (HNE) and reduced superoxide dismutase (SOD) activity. Dietary supplementation of antioxidant vitamin E increased BBB integrity and tight junction proteins expression via reducing cerebrovascular oxidative stress. These findings indicated an essential role of PLTP in maintaining BBB integrity, possibly through its ability to transfer vitamin E, and modulate cerebrovascular oxidative stress.     

The protective effect of alpha lipoic acid against traumatic brain injury in rats

Traumatic brain injury (TBI) was induced by a weight-drop device using 300 g–1 m weight-height impact. The study groups were: control, alpha-lipoic acid (LA) (100 mg/kg, po), TBI, and TBI + LA (100 mg/kg, po). Forty-eight hours after the injury, neurological scores were measured and brain samples were taken for histological examination or determination of thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) levels, myeloperoxidase (MPO) and Na⁺-K⁺ ATPase activities, whereas cytokines (TNF-α, IL-1β) were determined in blood. Brain oedema was evaluated by wet–dry weight method and blood–brain barrier (BBB) permeability was evaluated by Evans Blue (EB) extravasation. As a result, neurological scores mildly increased in trauma groups. Moreover, TBI caused a significant decrease in brain GSH and Na⁺-K⁺ ATPase activity, which was accompanied with significant increases in TBARS level, MPO activity and plasma proinflammatory cytokines. LA treatment reversed all these biochemical indices as well as histopathological alterations. TBI also caused a significant increase in brain water content and EB extravasation which were partially reversed by LA treatment. These findings suggest that LA exerts neuroprotection by preserving BBB permeability and by reducing brain oedema probably by its anti-inflammatory and antioxidant properties in the TBI model.     

Gliotoxin penetrates and impairs the integrity of the human blood-brain barrier in vitro

Cerebral fungal infections represent an important public health concern, where a key element of pathophysiology is the ability of the fungi to cross the blood-brain barrier (BBB). Yet the mechanism used by micro-organisms to cross such a barrier and invade the brain parenchyma remains unclear. This study investigated the effects of gliotoxin (GTX), a mycotoxin secreted by Aspergillus fumigatus, on the BBB using brain microvascular endothelial cells (BMECs) derived from induced pluripotent stem cells (iPSCs). We observed that both acute (2 h) and prolonged (24 h) exposure to GTX at the level of 1 μM or higher compromised BMECs monolayer integrity. Notably, acute exposure was sufficient to disrupt the barrier function in iPSC-derived BMECs, resulting in decreased transendothelial electrical resistance (TEER) and increased fluorescein permeability. Further, our data suggest that such disruption occurred without affecting tight junction complexes, via alteration of cell-matrix interactions, alterations in F-actin distribution, through a protein kinase C-independent signaling. In addition to its effect on the barrier function, we have observed a low permeability of GTX across the BBB. This fact can be partially explained by possible interactions of GTX with membrane proteins. Taken together, this study suggests that GTX may contribute in cerebral invasion processes of Aspergillus fumigatus by altering the blood-brain barrier integrity without disrupting tight junction complexes.     

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.     

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.     

Blood-brain barrier impairment in an animal model of MPS III B

Background

Sanfilippo syndrome type B (MPS III B) is caused by a deficiency of α-N-acetylglucosaminidase enzyme, leading to accumulation of heparan sulfate within lysosomes and eventual progressive cerebral and systemic multiple organ abnormalities. However, little is known about the competence of the blood-brain barrier (BBB) in MPS III B. BBB dysfunction in this devastating disorder could contribute to neuropathological disease manifestations.

Methodology/Principal Findings

In the present study, we investigated structural (electron microscope) and functional (vascular leakage) integrity of the BBB in a mouse model of MPS III B at different stages of disease, focusing on brain structures known to experience neuropathological changes. Major findings of our study were: (1) endothelial cell damage in capillary ultrastructure, compromising the BBB and resulting in vascular leakage, (2) formation of numerous large vacuoles in endothelial cells and perivascular cells (pericytes and perivascular macrophages) in the large majority of vessels, (3) edematous space around microvessels, (4) microaneurysm adjacent to a ruptured endothelium, (6) Evans Blue and albumin microvascular leakage in various brain structures, (7) GM3 ganglioside accumulation in endothelium of the brain microvasculature.

Conclusions/Significance

These new findings of BBB structural and function impairment in MPS III B mice even at early disease stage may have implications for disease pathogenesis and should be considered in current and future development of treatments for MPS III B.     

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.     

In vivo monitoring blood‐brain barrier permeability using spectral imaging through optical clearing skull window

The blood‐brain barrier (BBB) plays a key role in the health of the central nervous system. Opening the BBB is very important for drug delivery to brain tissues to enhance the therapeutic effect on brain diseases. It is necessary to in vivo monitor the BBB permeability for assessing drug release with high resolution; however, an effective method is lacking. In this work, we developed a new method that combined spectral imaging with an optical clearing skull window to in vivo dynamically monitor BBB opening caused by 5‐aminolevulinic acid (5‐ALA)‐mediated photodynamic therapy (PDT), in which the Evans blue dye (EBd) acted as an indicator of the BBB permeability. Using this method, we effectively monitored the cerebrovascular EBd leakage process. Moreover, the analysis of changes in the vascular and extravascular EBd concentrations demonstrated that the PDT‐induced BBB opening exhibited spatiotemporal differences in the cortex. This spectral imaging method based on the optical clearing skull window provides a low‐cost and simply operated tool for in vivo monitoring BBB opening process. This has a high potential for the visualization of drug delivery to the central nervous system. Thus, it is of tremendous significance in brain disease therapy. Monitoring the changes in PDT‐induced BBB permeability by evaluating the EBd concentration using an optical clearing skull window. (A) Entire brains and coronal sections following treatment of PDT with/without an optical clearing skull window after injection of EBd. (B) Typical EBd distribution maps before and after laser irradiation captured by the spectral imaging method. (Colorbar represents the EBd concentration).      

Pericytes Contribute to the Disruption of the Cerebral Endothelial Barrier via Increasing VEGF Expression: Implications for Stroke

Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progression of stroke. Although it has been well documented that brain microvascular endothelial cells and astrocytes play a critical role in the maintenance of BBB integrity, pericytes, sandwiched between endothelial cells and astrocytes, remain poorly studied in the pathogenesis of stroke. Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus. Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody. The in vivo relevance of these findings was further corroborated in the stroke model of mice wherein the mice, demonstrated disruption of the BBB integrity and concomitant increase in the expression of VEGF in the brain tissue as well as in the isolated microvessel. These findings thus suggest the role of pericyte-derived VEGF in modulating increased permeability of BBB during stroke. Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease.