Endothelial Cell Tight Junction Proteins Research Articles

Abstract TP307: oxLDL Preconditioning Intensifies Ischemic-Like Injury Mediated Alterations in Human Male Brain Endothelial Cell Tight Junction Protein Levels and Proinflammatory Mediators

Introduction: Acute ischemic stroke (AIS) triggers endothelial activation and induces cerebrovascular inflammation which can result in vascular function and integrity loss leading to worsened stroke outcome. A clinically correlated risk factor for stroke shown to mediate vascular dysfunction and injury is elevated levels of oxidized low density lipoprotein (oxLDL). We hypothesized that oxLDL would exacerbate acute ischemic injury mediated alteration of endothelial proinflammatory mediator and integral barrier protein levels. Methods: Primary adult male human brain microvascular endothelial cells (HBMEC) were preconditioned with human oxLDL (50 or 100ug/dL; 6, 12, 24, or 36h) or vehicle, using a serum dose reported in AIS patients. HBMECs were then exposed to normoxia (21% O 2 ) or ischemic-like injury, hypoxia plus glucose deprivation (HGD; 1% O 2 ), for 3 or 6h. HBMEC levels or expression of barrier markers, adhesion molecules, and inflammatory mediators were examined using qRT-PCR, in cell western, and FIJI mediated immunocytochemical analysis. Secondary analysis was performed on RNA sequencing of adult male aortic endothelial cells (HAoECs) exposed to oxLDL (50ug/mL; 3, 6, 12, 24h) obtained from GEO dataset GSE13139. Results: HGD injury concomitantly decreased HBMEC claudin-5, occludin, and ZO-1 as well as increased IL-1b, ICAM-1, VCAM-1, iNOS, and TNF-a. OxLDL exacerbated an HGD mediated decrease in HBMEC barrier levels and increase in proinflammatory levels in a dose and time dependent manner. HAoEC barrier, adhesion, and proinflammatory levels were not altered by oxLDL. Conclusion: OxLDL may preferentially alter brain endothelial barrier and proinflammatory levels, as opposed to aortic endothelial cells in dose and time dependent manner. Mitigating the effect of oxLDL on brain endothelial permeability and inflammation, possibly via receptor inhibition may be a novel, viable therapeutic target in the treatment of AIS.

Glucuronoxylomannan intranasal challenge prior to Cryptococcus neoformans pulmonary infection enhances cerebral cryptococcosis in rodents

The encapsulated fungus Cryptococcus neoformans is the most common cause of fungal meningitis, with the highest rate of disease in patients with AIDS or immunosuppression. This microbe enters the human body via inhalation of infectious particles. C. neoformans capsular polysaccharide, in which the major component is glucuronoxylomannan (GXM), extensively accumulates in tissues and compromises host immune responses. C. neoformans travels from the lungs to the bloodstream and crosses to the brain via transcytosis, paracytosis, or inside of phagocytes using a "Trojan horse" mechanism. The fungus causes life-threatening meningoencephalitis with high mortality rates. Hence, we investigated the impact of intranasal exogenous GXM administration on C. neoformans infection in C57BL/6 mice. GXM enhances cryptococcal pulmonary infection and facilitates fungal systemic dissemination and brain invasion. Pre-challenge of GXM results in detection of the polysaccharide in lungs, serum, and surprisingly brain, the latter likely reached through the nasal cavity. GXM significantly alters endothelial cell tight junction protein expression in vivo, suggesting significant implications for the C. neoformans mechanisms of brain invasion. Using a microtiter transwell system, we showed that GXM disrupts the trans-endothelial electrical resistance, weakening human brain endothelial cell monolayers co-cultured with pericytes, supportive cells of blood vessels/capillaries found in the blood-brain barrier (BBB) to promote C. neoformans BBB penetration. Our findings should be considered in the development of therapeutics to combat the devastating complications of cryptococcosis that results in an estimated ~200,000 deaths worldwide each year.

Contribution of astrocytic MMP9 toward progression of VCID pathology

AbstractBackgroundVascular contributions to cognitive impairment and dementia (VCID) is one of the leading causes of dementia. High levels of plasma homocysteine or hyperhomocysteinemia has been characterized as a risk factor for VCID however, the mechanism underlying the connection between hyperhomocysteinemia and development of VCID pathology remains elusive. I hypothesize that hyperhomocysteinemia initiates a pro‐inflammatory cascade that increases the activity of MMP9 causing both perivascular astrocytes to dissociate from their vessels and initiating the degradation of endothelial cell tight junction proteins, leading to blood brain barrier dysfunction and the progression toward VCID pathology.MethodFor in vivo studies, C57BL6 WT and MMP9KO mice were placed on a control diet or a diet deficient in folate, vitamins B6 and B12 and enriched in methionine to induce hyperhomocysteinemia for 4, 8, 12, and 16 weeks. For in vitro, experiments, primary astrocytes from WT and MMP9KO mice were treated with a 50μM homocysteine stimulus or an inflammatory stimulus (TNF‐a, IL‐1b and C1q) for 48 hours and the conditioned media was collected. This homocysteine or inflammatory astrocyte conditioned media was then used to treat WT endothelial cells. Immunohistochemistry and gene expression analysis were used to determine neuroinflammatory changes while histology was used to identify changes in astrocytic end‐feet proteins, tight junction proteins, vascular density and microhaemorrhages. Both gel and in situ zymography were used to assess proteinase activity of MMP9 and related gelatinases. Western blots were used to investigate substrates of MMP9 including claudin, occludin and β‐dystroglycan. Behaviour was assessed using rotarod, novel object recognition and spontaneous alternation testing. Transendothelial electrical resistance and sodium fluorescein assays were used to measure the integrity of endothelial cell tight junctions in vitro.ResultStudies are underway to examine changes in cognition, microhaemorrhages, neuroinflammation, astrocyte end‐foot integrity, tight junction protein integrity and activity of MMP9.ConclusionCollectively, our findings suggest that astrocytic MMP9 may play an integral role in the mechanism associating homocysteine induced neuroinflammation with vascular pathogenesis leading to VCID, highlighting this pathway as an important subject for future study.

Inhibiting P2Y12 receptor relieves LPS-induced inflammation and endothelial dysfunction.

BackgroundAcute lung injury (ALI) is characterized by abnormal inflammatory response without effective therapies. P2Y12 receptor (P2Y12R) plays a vital role in inflammatory response. This study intends to explore whether P2Y12R antagonists can inhibit LPS‐induced inflammatory injury of human pulmonary microvascular endothelial cells (HPMVECs) and endothelial cell dysfunction.MethodsUsing a cell model of ALI, the role of P2Y12R was investigated in LPS‐induced HPMVECs. The expression of P2Y12R was detected by RT‐qPCR and Western blot analysis assay and TNF‐α, IL‐1β, and IL‐6 levels were analyzed by RT‐qPCR. NO levels were also analyzed through NO kit. The levels of NF‐κB p65, P‐IκB‐α, and IκB‐α, as well as p‐AKT and eNOS levels were detected by Western blot analysis assay. Wound healing assay was performed to evaluate HPMVECs migration. FITC‐dextran was used to evaluate endothelial cell permeability, and the analysis of adherens junction protein VE‐cadherin and endothelial cell tight junction proteins ZO‐1, Claudin 5 and Occludin expression was performed by RT‐qPCR and Western blot analysis assay.ResultsIn vitro, LPS increased the expression levels of P2Y12R and pro‐inflammatory mediators (TNF‐α, IL‐1β, and IL‐6), followed by a decrease in HPMVECs migration. In addition, LPS led to an increase in endothelial cell permeability. P2Y12R antagonists Ticagrelor or clopidogrel treatment significantly reversed these effects of LPS.ConclusionThe inhibitor of P2Y12R was able to decrease inflammatory response, promote migration and improve endothelial cell function and permeability, suggesting a key role of P2Y12R in ALI.

Childhood social isolation causes anxiety-like behaviors via the damage of blood-brain barrier in amygdala in female mice.

Social interaction plays an essential role in species survival for socialized animals. Previous studies have shown that a lack of social interaction such as social isolation, especially in the early-life phase, increases the risk of developing mental diseases in adulthood. Chronic social stress alters blood-brain barrier (BBB) integrity and increases peripheral cytokines to infiltrate the brain, which is linked to the development of depressive-like behaviors in mice, suggesting that BBB function is crucial in environmental stimuli-driven mood disorders via increased neuroinflammation in the brain. However, the precise mechanisms of inflammation and BBB integrity underlying the behavioral profiles induced by social isolation remain poorly understood. Here we showed that chronic childhood social isolation from post-weaning for consecutive 8 weeks in female but not male C57BL/6J mice induces anxiety-like behaviors. The levels of peripheral inflammatory cytokines including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the plasma of socially isolated female mice were increased. Importantly, we found decreased expression of the endothelial cell tight junction protein Claudin-5, increased BBB breakdown and microglial activation in the amygdala of isolated but not group-housed female mice. Moreover, the neuronal activity in the amygdala was increased as evidenced by c-fos positive cells, and the levels of IL-1β in the amygdala, a critical brain region for regulating social processing and interaction, were also higher in female mice exposed to social isolation. Finally, down-regulation of Claudin-5 induced anxiety-like behaviors in group-housed females and overexpression of Claudin-5 with adeno-associated virus in the amygdala to restore BBB integrity decreased subsequent anxiety-like behaviors. Together, these findings suggest that chronic childhood social isolation impaired BBB permeability and caused neuroinflammation in the amygdala by recruiting peripheral cytokines into the brain and activating microglia, consequently triggering the development of anxiety-like behaviors in female mice.

Effect of plasma exosomes on endothelial cell tight junction proteins in SLE patients with immune thrombocytopenia.

To study the expression of exosomes in peripheral blood of systemic lupus erythematosus (SLE) immune thrombocytopenia patients with and without hemorrhage symptoms, respectively, and the effect of exosomes on endothelial cell tight junction proteins (TJs) in vitro, so as to investigate the related mechanisms involved in the occurrence of hemorrhage symptoms. Patients diagnosed with SLE and immune thrombocytopenia (<50x109/L) were divided into 2 groups according to the presence or absence of hemorrhage symptoms. Plasma exosomes were isolated, and observed by transmission electron microscopy. The exosomes were co-cultured with endothelial cells in vitro. The permeability of umbilical vein endothelial cells (HUVECs) was measured by transendothelial electrical resistance (TEER). The mRNA and protein expression of tight junctions (occludin and claudin-5) were detected by RT-PCR and Western blot, respectively. Plasma exosomes were increased in the group without hemorrhage symptoms. The TEER value of HUVECs after adding plasma exosomes of hemorrhage group in vitro was not significantly changed compared to the control while increased after adding exosomes of non-hemorrhage group. Plasma exosomes of the non-hemorrhage group could increase both the mRNA and protein expression of TJs in vitro, while exosomes of the hemorrhage group could decrease the expression, the difference was statistically significant (p < 0.05). Plasma exosomes may influence the hemorrhage symptoms of SLE patients with immune thrombocytopenia by regulating the expression of endothelial tight junction proteins. Key Points • The role of exosomes in SLE immune thrombocytopenia is first reported in this study. • We have explored the mechanism that exosomes may participate in hemorrhage, which will facilitate individualized treatment of SLE immune thrombocytopenia.

Angiogenesis and Blood-Brain Barrier Permeability in Vascular Remodeling after Stroke.

Angiogenesis, the growth of new blood vessels, is a natural defense mechanism helping to restore oxygen and nutrient supply to the affected brain tissue following an ischemic stroke. By stimulating vessel growth, angiogenesis may stabilize brain perfusion, thereby promoting neuronal survival, brain plasticity, and neurologic recovery. However, therapeutic angiogenesis after stroke faces challenges: new angiogenesis-induced vessels have a higher than normal permeability, and treatment to promote angiogenesis may exacerbate outcomes in stroke patients. The development of therapies requires elucidation of the precise cellular and molecular basis of the disease. Microenvironment homeostasis of the central nervous system is essential for its normal function and is maintained by the blood-brain barrier (BBB). Tight junction proteins (TJP) form the tight junction (TJ) between vascular endothelial cells (ECs) and play a key role in regulating the BBB permeability. We demonstrated that after stroke, new angiogenesis-induced vessels in peri-infarct areas have abnormally high BBB permeability due to a lack of major TJPs in ECs. Therefore, promoting TJ formation and BBB integrity in the new vessels coupled with speedy angiogenesis will provide a promising and safer treatment strategy for improving recovery from stroke. Pericyte is a central neurovascular unite component in vascular barriergenesis and are vital to BBB integrity. We found that pericytes also play a key role in stroke-induced angiogenesis and TJ formation in the newly formed vessels. Based on these findings, in this article, we focus on regulation aspects of the BBB functions and describe cellular and molecular special features of TJ formation with an emphasis on role of pericytes in BBB integrity during angiogenesis after stroke.

Neutralization of interleukin-9 ameliorates experimental stroke by repairing the blood-brain barrier via down-regulation of astrocyte-derived vascular endothelial growth factor-A.

Astrocytes mediate the destruction of the blood-brain barrier (BBB) during ischemic stroke (IS). IL-9 is a pleiotropic cytokine that we previously found to be highly expressed in peripheral blood mononuclear cells from patients with IS, and the presence of IL-9 receptors on astrocytes has been reported in the literature. Here, we detected the effect of IL-9 on astrocytes using an anti-IL-9-neutralizing antibody to treat rats with experimental stroke. Supernatants from astrocytes treated with or without oxygen-glucose deprivation and/or IL-9 were incubated with bEnd.3 cell monolayers after blocking the IL-9 receptor on the endothelium. Immunofluorescence staining and Western blot analyses were conducted to observe the change in tight junction proteins (TJPs) in bEnd.3 cells as well as the level of VEGF-A and possible signal pathways in astrocytes. We also applied middle cerebral artery occlusion (MCAO) models to determine the effect of anti-IL-9-neutralizing antibodies on IS. As a result, astrocyte-conditioned medium treated with IL-9 aggravated the disruption of the BBB accomplished by the degradation of TJPs in endothelial cells. In addition, IL-9 increased the level of VEGF-A in astrocytes, and blocking the effect of VEGF-A reversed the breakdown of the BBB. In the MCAO model, anti-IL-9-neutralizing antibody reduced the infarct volume and BBB destruction. Mechanistically, the anti-IL-9-neutralizing antibody repaired the damaged TJPs (zonula occludens 1, occludin, and claudin-5) and induced a decrease in VEGF-A expression in ischemic lateral brain tissue. In contrast, a local injection of recombinant murine IL-9 to the brain resulted in a marked up-regulation of VEGF-A in the striatum. In conclusion, anti-IL-9-neutralizing antibody can reduce the severity of IS partially by alleviating the destruction of the BBB via down-regulation of astrocyte-derived VEGF-A. This finding suggests that targeting IL-9 or VEGF-A could provide a new direction for the treatment of IS.-Tan, S., Shan, Y., Lin, Y., Liao, S., Zhang, B., Zeng, Q., Wang, Y., Deng, Z., Chen, C., Hu, X., Peng, L., Qiu, W., Lu, Z. Neutralization of IL-9 ameliorates experimental stroke by repairing the blood-brain barrier via down-regulation of astrocyte-derived vascular endothelial growth factor-A.

Social stress induces neurovascular pathology promoting depression.

Studies suggest that heightened peripheral inflammation contributes to the pathogenesis of major depressive disorder. We investigated the effect of chronic social defeat stress, a mouse model of depression, on blood-brain barrier (BBB) permeability and infiltration of peripheral immune signals. We found reduced expression of endothelial cell tight junction protein claudin-5 (cldn5) and abnormal blood vessel morphology in nucleus accumbens (NAc) of stress-susceptible but not resilient mice. CLDN5 expression was also decreased in NAc of depressed patients. Cldn5 down-regulation was sufficient to induce depression-like behaviors following subthreshold social stress while chronic antidepressant treatment rescued cldn5 loss and promoted resilience. Reduced BBB integrity in NAc of stress-susceptible or AAV-shRNA-cldn5-injected mice caused infiltration of peripheral cytokine interleukin-6 (IL-6) into brain parenchyma and subsequent expression of depression-like behaviors. These findings suggest that chronic social stress alters BBB integrity through loss of tight junction protein cldn5, promoting peripheral IL-6 passage across the BBB and depression.

Lipid metabolites of the phospholipase A2 pathway and inflammatory cytokines are associated with brain volume in paediatric cerebral malaria.

BackgroundCerebral malaria (CM) remains a significant cause of morbidity and mortality in children in sub-Saharan Africa. CM mortality has been associated with increased brain volume, seen on neuroimaging studies.MethodsTo examine the potential role of blood metabolites and inflammatory mediators in increased brain volume in Malawian children with CM, an association study was performed between plasma metabolites, cytokine levels and phospholipase A2 (PLA2) activity with brain volume.ResultsThe metabolomics analysis demonstrated arachidonic acid and other lysophospholipids to be positively associated with brain swelling. These lipids are products of the PLA2 enzyme and an association of plasma PLA2 enzymatic activity with brain swelling was confirmed. TNFα, which can upregulate PLA2 activity, was associated with brain volume. In addition, CCL2 and IL-8 were also associated with brain volume. Some of these cytokines can alter endothelial cell tight junction proteins and increase blood brain barrier permeability.ConclusionsTaken together, paediatric CM brain volume was associated with products of the PLA2 pathway and inflammatory cytokines. Their role in causality is unknown. These molecules will need to undergo testing in vitro and in animal models to understand their role in processes of increased brain volume. These observations provide novel data on host physiology associated with paediatric CM brain swelling, and may both inform pathogenesis models and suggest adjunct therapies that could improve the morbidity and mortality associated with paediatric CM.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-1036-1) contains supplementary material, which is available to authorized users.

CD8 T cell mediated disruption of tight junctions is associated with vascular permeability in vital brain regions during experimental cerebral malaria (MPF7P.708)

Abstract One of the most severe complications of Plasmodium falciparum infection in humans is cerebral malaria. Cerebral malaria causes coma, neurological deficits, and potentially death. The disease is characterized by blood-brain barrier (BBB) disruption leading to vascular permeability. In this study, we investigated cerebral endothelial cell tight junction protein alterations and BBB breakdown in the Plasmodium berghei ANKA (PbA) model of experimental cerebral malaria. PbA infected C57BL/6 mice displayed significant areas of vascular leakage within the central nervous system at six days post infection. Regions of permeability co-localized with disruption of the BBB tight junction proteins claudin-5 and occludin on microvasculature as shown using confocal microscopy. Additionally, 3D MRI analysis revealed that PbA infected mice exhibited vascular leakage specific to the hypothalamus, thalamus, and brain stem. In contrast, PbA infected perforin deficient mice retained tight junction integrity and displayed significantly reduced vascular leakage. We propose that CD8 T cells initiate vascular permeability through disruption of cerebral endothelial cell tight junctions. In turn, the resulting imbalance of homeostasis in vital brain regions likely contributes to the pathology and morbidity associated with PbA infection.

CD8 T Cell-Initiated Vascular Endothelial Growth Factor Expression Promotes Central Nervous System Vascular Permeability under Neuroinflammatory Conditions

Dysregulation of the blood-brain barrier (BBB) is a hallmark feature of numerous neurologic disorders as diverse as multiple sclerosis, stroke, epilepsy, viral hemorrhagic fevers, cerebral malaria, and acute hemorrhagic leukoencephalitis. CD8 T cells are one immune cell type that have been implicated in promoting vascular permeability in these conditions. Our laboratory has created a murine model of CD8 T cell-mediated CNS vascular permeability using a variation of the Theiler's murine encephalomyelitis virus system traditionally used to study multiple sclerosis. Previously, we demonstrated that CD8 T cells have the capacity to initiate astrocyte activation, cerebral endothelial cell tight junction protein alterations and CNS vascular permeability through a perforin-dependent process. To address the downstream mechanism by which CD8 T cells promote BBB dysregulation, in this study, we assess the role of vascular endothelial growth factor (VEGF) expression in this model. We demonstrate that neuronal expression of VEGF is significantly upregulated prior to, and coinciding with, CNS vascular permeability. Phosphorylation of fetal liver kinase-1 is significantly increased early in this process indicating activation of this receptor. Specific inhibition of neuropilin-1 significantly reduced CNS vascular permeability and fetal liver kinase-1 activation, and preserved levels of the cerebral endothelial cell tight junction protein occludin. Our data demonstrate that CD8 T cells initiate neuronal expression of VEGF in the CNS under neuroinflammatory conditions, and that VEGF may be a viable therapeutic target in neurologic disease characterized by inflammation-induced BBB disruption.

Fibrinogen induces alterations of endothelial cell tight junction proteins

We previously showed that an elevated content of fibrinogen (Fg) increased formation of filamentous actin and enhanced endothelial layer permeability. In the present work we tested the hypothesis that Fg binding to endothelial cells (ECs) alters expression of actin-associated endothelial tight junction proteins (TJP). Rat cardiac microvascular ECs were grown in gold plated chambers of an electrical cell-substrate impedance system, 8-well chambered, or in 12-well plates. Confluent ECs were treated with Fg (2 or 4 mg/ml), Fg (4 mg/ml) with mitogen-activated protein kinase (MEK) kinase inhibitors (PD98059 or U0126), Fg (4 mg/ml) with anti-ICAM-1 antibody or BQ788 (endothelin type B receptor blocker), endothelin-1, endothelin-1 with BQ788, or medium alone for 24 h. Fg induced a dose-dependent decrease in EC junction integrity as determined by transendothelial electrical resistance (TEER). Western blot analysis and RT-PCR data showed that the higher dose of Fg decreased the contents of TJPs, occludin, zona occluden-1 (ZO-1), and zona occluden-2 (ZO-2) in ECs. Fg-induced decreases in contents of the TJPs were blocked by PD98059, U0126, or anti-ICAM-1 antibody. While BQ788 inhibited endothelin-1-induced decrease in TEER, it did not affect Fg-induced decrease in TEER. These data suggest that Fg increases EC layer permeability via the MEK kinase signaling pathway by affecting occludin, ZO-1, and ZO-2, TJPs, which are bound to actin filaments. Therefore, increased binding of Fg to its major EC receptor, ICAM-1, during cardiovascular diseases may increase microvascular permeability by altering the content and possibly subcellular localization of endothelial TJPs.

Pharmacologic management of brain edema

Cerebral edema is an intrinsic response to a variety of structural and metabolic insults. It is a major contributing factor in the development of intracranial hypertension and brain herniation, underscoring the need for early identification through an integration of clinical and neuroimaging findings, followed by timely institution of measures to reduce brain edema and intracranial hypertension. The management of cerebral edema requires a comprehensive approach in which pharmacologic treatments play a central role. These include glucocorticoids, hyperosmolar agents, diuretics, and sedative-anesthetic agents. Basic, translational, and clinical studies are needed to further unravel mechanisms underlying brain edema, with the goal of identifying new treatment strategies. Promising targets include modulators of endothelial cell tight junction proteins and of aquaporin channel expression within the blood-brain barrier.

Endothelial Cell Barrier Impairment Induced by Glioblastomas and Transforming Growth Factor β2Involves Matrix Metalloproteinases and Tight Junction Proteins

Gliomas, particularly glioblastoma multiforme, perturb the blood-brain barrier and cause brain edema that contributes to morbidity and mortality. The mechanisms underlying this vasogenic edema are poorly understood. We examined the effects of cocultured primary cultured human glioblastoma cells and glioma-derived growth factors on the endothelial cell tight junction proteins claudin 1, claudin 5, occludin, and zonula occludens 1 of brain-derived microvascular endothelial cells and a human umbilical vein endothelial cell line. Cocultured glioblastoma cells and glioma-derived factors (e.g. transforming growth factor beta2) enhanced the paracellular flux of endothelial cell monolayers in conjunction with downregulation of the tight junction proteins. Neutralizing anti-transforming growth factor beta2 antibodies partially restored the barrier properties in this in vitro blood-brain barrier model. The involvement of endothelial cell-derived matrix metalloproteinases (MMPs) was demonstrated by quantitative reverse-transcriptase-polymerase chain reaction analysis and by the determination of MMP activities via zymography and fluorometry in the presence or absence of the MMP inhibitor GM6001. Occludin, claudin 1, and claudin 5 were expressed in microvascular endothelial cells in nonneoplastic brain samples but were significantly reduced in anaplastic astrocytoma and glioblastoma samples. Taken together, these in vitro and in vivo results indicate that glioma-derived factors may induce MMPs and downregulate endothelial tight junction protein and, thus, play a key role in glioma-induced impairment of the blood-brain barrier.

Changes of tight junction protein and GFAP in the retina of experimental diabetic rats and their relationship with blood aqueous barrier

To investigate the changes of vascular endothelial cell tight junction protein (occludin) and glial cell morphology as well as their relationship with blood-retinal barrier (BRB) in the retina of diabetic rats. The distribution of occludin and GFAP were explored by immunofluorescence histochemical studies in the retina of streptozotocin (STZ)-diabetic rats and age-matched control rats. Evans blue was used to evaluate the impairment of BRB. GFAP immunoreactivity was limited to ganglion cell layer and nerve fiber layer in the control retina. GFAP immunoreactivity was significantly increased in ganglion cell layer and nerve fiber layer in one month diabetic rats. GFAP positive Müller cells were increased in three months and six months diabetic rats. Occludin immunoreactivity progressively decreased in three months and six months diabetic rats but not in the one month diabetic rats. Evans blue injection showed a progressive impairment of BRB. Astrocytes activation in the early stage of diabetes plays an important role in the maintaining of the BRB function. But the activation of Müller cells in the later stage destroyed the BRB eventually. These changes are consistent with the concept that BRB changes caused by altered glial-endothelial cell interactions contributes to the occurrence of diabetic retinopathy.