Endothelial Barrier Antigen Expression Research Articles

Exposure to Acute, High Dose Cadmium Significantly Downregulates Postnatal Expression of the Vascular Endothelial Barrier Antigen in the Rat Central Nervous System as Quantitated by Immunofluorescent Microscopy

Clinical and experimental studies have demonstrated the neurotoxic and behavioral effects of cadmium. The exact mechanism(s) of cadmium neurotoxicity on the human central nervous system is not completely understood. It has been suggested that the metal exerts its effect via oxidative stress‐induced morphological and functional disruption of blood‐brain barrier (BBB) structures. A sensitive and specific rat BBB marker, the endothelial barrier antigen (EBA), has been previously identified and characterized by lymphocyte hybridoma technology. Alterations in EBA expression have been reported to occur during infectious and non‐infectious central and peripheral nervous system (CNS and PNS) pathologies. We have previously demonstrated that in the rat, EBA first becomes detectable during the first week postnatally and is progressively expressed along with the biochemical and hemodynamic changes that accompany normal brain maturation and barriergenesis. We have also shown that this anti‐EBA IgG1 antibody exclusively recognizes barrier‐competent microvessels in rat central and peripheral nervous system (CNS and PNS). Endothelial cells of peripheral tissues such as the liver and kidney and brain circumventricular organs possessing fenestrated microvascular endothelia, do not display immunoreactivity for EBA.In the present study, a double, sequential immunofluorescent protocol was employed to colocalize EBA‐specific microvessels and astrocytes, using anti‐EBA and anti‐GFAP antibodies, respectively. Sections were examined with an Olympus BX63 Fluorescent microscope outfitted with a DP80 Camera and cellSense software. The EBA‐immunoreactive microvessels were quantitated by measuring the areal fraction of the microvessels per field using an algorithm that allowed for the microvessels to be segmented from the surrounding background within the region of interest (ROI) using a global threshold. The grand mean areal fractions of all microscopic fields captured from the normal control brain sections were compared for statistical significance with the areal fraction grand mean for the cadmium‐treated brain sections using a two‐sample t‐test at a 95% confidence interval. Positive staining was not detected in any of the tissue sections in which the primary antibodies were omitted. Also, EBA expression was not detected in sections from the kidney, known to be vascularized by fenestrated capillaries lacking a barrier function. Statistical analysis (two‐sample t‐test) of the Grand Mean Areal Fractions (μm2) for all microscopic fields evaluated for control and experimental groups yielded a p‐value of less than 0.001 (t = 5.8507, df = 1789, p‐value = 5.808e‐09; p < 0.001).This study indicates that acute exposure to a single, high dose of cadmium significantly downregulates the expression of EBA in the developing rat CNS. The results suggest that EBA is involved in the pathogenesis of cadmium neurotoxicity and could serve as a useful tool for studying the extent of cadmium‐induced impact on BBB structures of the central nervous system in the rat model system.Support or Funding InformationWe express our thanks and sincere appreciation of the research funding provided by the Alabama College of Osteopathic Medicine.

Hyper-oligodendrogenesis at the vascular niche and reduced blood–brain barrier integrity in the prefrontal cortex during protracted abstinence

Alcoholism is a relapsing disorder with limited treatment options, in part due to our limited understanding of the disease etiology. We have recently shown that increased ethanol-seeking in a behavioral model of relapse in a rat model of alcoholism was associated with increased oligodendrogenesis which was positively correlated with platelet/endothelial cell adhesion molecule (PECAM-1) expression in the medial prefrontal cortex (mPFC). The current study investigated whether newly born oligodendrocytes form close physical associations with endothelial cells expressing PECAM-1 and whether these changes were accompanied by altered blood–brain barrier (BBB) integrity. Colableling and confocal analysis demonstrate that newly born oligodendroglia were always located in close physical proximity to PECAM-1 in the mPFC of rats that were ethanol dependent and demonstrated high propensity for relapse. Notably, the endothelial proximity of new oligodendrocytes was associated with reduced expression of endothelial barrier antigen (SMI-71), a marker for BBB integrity. Furthermore, voluntary wheel running during abstinence enhanced SMI-71 expression in endothelial cells, indicating protection against abstinence-induced reduction in BBB integrity. Taken together, these results suggest that ethanol experience and abstinence disrupts homeostasis in the oligo-vascular niche in the mPFC. Reversing these mechanisms may hold the key to reducing propensity for relapse in individuals with moderate to severe alcohol use disorder.

Annexin A2 Plus Low-Dose Tissue Plasminogen Activator Combination Attenuates Cerebrovascular Dysfunction After Focal Embolic Stroke of Rats.

Previous studies showed recombinant annexin A2 (rA2) in combination with low-dose tissue-type plasminogen activator (tPA) improved thrombolytic efficacy and long-term neurological outcomes after embolic focal ischemia in rats. The objective of this study was to investigate the effects and mechanisms of the combination in early BBB integrity and cerebrovascular patency in the rat focal embolic stroke model. Ischemic brain infarct volume and hemorrhagic transformation were quantified at 24h after stroke. At an earlier time point, 16h after stroke, BBB integrity was evaluated by IgG extravasation, and the involved mechanisms were assessed for tight junction ZO-1 and adhesion junction ve-cadherin protein expression, matrix metalloproteinase activation, extracellular matrix collagen IV and endothelial barrier antigen expression, and activation of microglia/macrophages and astrocytes. While at the same time point, cerebrovascular patency was assessed by intravascular fibrin and platelet depositions. At 24h after stroke, the combination showed significant reduction in brain infarction and intracerebral hemorrhage. At 16h after stroke onset, the combination therapy significantly reduced BBB disruption, and improved preservation of the junction proteins ZO-1 and ve-cadherin, decreased activation of matrix metalloproteinase, inhibited degradation of extracellular matrix collagen IV and endothelial barrier antigen, and reduced microglia/macrophage and astrocytes activations. Meanwhile, the combination also significantly improved cerebrovascular patency by reducing intravascular fibrin and platelet depositions in the peri-infarct brain tissues. These results suggest the beneficial effects of the rA2 plus low-dose tPA combination may be mediated in part by the amelioration of BBB disruption and improvement of cerebrovascular patency.

The correlationship between MMP-9 expression and the blood-spinal cord barrier disruption in chronic compressive cervical myelopathy

Objective To investigate the correlation between the expression of MMP-9 and the disruption of the blood spinal cord barrier (BSCB) in chronic cervical cord compressive myelopathy rat model. Methods 48 adult rats were randomly divided into group A (sham surgical group, n=24) and group B (spinal cord compressive group, n=24). A water-absorbing polymer sheet was implanted into the C5 epidural space on the posterolateral side to induce a chronic spinal cord compression model. BBB scores and SEP were collected 4 weeks after surgery. Expression of MMP-9 and endothelial barrier antigen (EBA) were detected immunohistochemically in different spinal cord areas of 12 rats in each group. The other 12 rats were performed with Evans blue (EB) perfusion to observe the permeability of BSCB. The results and the correlation between MMP-9 and EBA expression were analyzed. Results BBB score was lower in spinal cord compressive group than that of sham surgical group. Latency was delayed and amplitude was reduced significantly in spinal cord compressive group. Expression level of MMP-9 in spinal cord compressive group was significant higher than that in both grey matter and white matter in sham surgical group. In the contrary, EBA expression was obviously more in grey matter of sham surgical group than that in spinal cord compressive group, while it showed no significant change in white matter between the 2 groups. EB perfusion in sham surgical group was less than that in spinal cord compressive group. MMP-9 expression was correlated with EBA staining significantly. Conclusion Neurological function of spinal cord in the compression group was abnormal, with a marked decline 4 weeks after model made. The increased permeability and structural failure of BSCB in chronic spinal cord compression myelopathy, which was characterized and correlated with increment of MMP-9 expression and decrement of EBA expression concurrently. Disruption of BSCB may be correlated with distinct increasing expression of MMP-9. Key words: Spinal cord compression; Rats; Matrix metalloproteinase 9

The expression of endothelial barrier antigen (EBA) and S100B in the rat parietal cortex following brain irradiation

Objective: To visualize the dynamic expression of endothelial barrier antigen (EBA) and S100B in the rat parietal cortex at the acute phase of radiation-induced brain injury using computed tomography (CT). Methods: A rat model of brain injury was established by CT scanning. The expression of EBA and S100B in the parietal cortex was analyzed at different time points by immunohistochemistry (IHC) and western blotting. Results: Significantly increased EBA expression was detected in the animals in the control group compared with the animals receiving CT radiation, which exhibited significantly reduced EBA levels within the vessel walls (F=33.29, p<0.05), particularly at day 3 after radiation. Both immunohistochemical staining and western blot analysis indicated that the positive expression levels of S100B among radiation groups were increased compared with the control group (IHC, F=28.05, p<0.05; WB, F=175.3, p<0.05). The expression of S100B peaked at day 3 (IHC, 102718±8710; WB, 2320±0.129), and subsequently decreased. Conclusion: CT radiation can induce altered EBA and S100B protein expression. Decreased EBA expression levels indicated that the integrity of the blood–brain barrier (BBB) was affected by radiation. The destruction of the BBB and the expression of S100B might play important roles in the incidence and repair of the early radiation-induced brain injury, and radiation represents a cause of mental disorders.

Effect of intracortical vascular endothelial growth factor infusion and blockade during the critical period in the rat visual cortex

VEGF is the major angiogenic and vascular permeability factor in health and disease. Vascular development depends on function, and in sensory areas is experience-dependent. Our aim was to investigate, qualitatively and quantitatively, the effects of intracortical infusion and neutralisation of VEGF during the first days of the critical visual period, when peak levels of endogenous VEGF secretion are reached.VEGF was intracortically delivered into middle cortical layers of P18 Long–Evans rats. Another cohort received anti-VEGF. Vehicle (PBS)-infused and non-operated animals were used as controls. Various immunopathological analyses were performed: Endothelial Barrier Antigen (EBA) for the BBB integrity and GFAP for astroglial response. Vascular density was measured by Butyryl Cholinesterase Histochemistry, neuronal density by NeuN immunohistochemistry and apoptosis by TUNEL staining. VEGF levels were measured by Western Blot.Decreased vascular permeability was evoked in VEGF-infused rats whilst EBA expression remained constant, suggesting a preserved BBB function. When VEGF was blocked, tissue showed a higher degree of extravasation and a decreased number of EBA-positive vessels surrounding the injury. Lesion induced by cannula implantation annulled the normal increase in vascular density and the decrease in neuronal density during this time. VEGF rescued in part the vascular increase, and also prevented physiological and pathological neuronal death. VEGF blockade induced a higher amount of neural loss and lower astrocytic reaction.Our results support the role of VEGF as extending beyond vascularization, preventing physiological and pathological neuronal death, not only in the injured hemisphere but also in the intact one suggesting a process of transhemispheric diaschisis.

Effects of rhEPO on endothelial barrier antigen expression and blood-brain barrier permeability after brain ischemia reperfusion injury in rats

Effects of rhEPO on endothelial barrier antigen expression and blood-brain barrier permeability after brain ischemia reperfusion injury in rats

Effects of ischemia/reperfusion injury on endothelial barrier antigen expression and blood-brain barrier permeability in rats

Effects of ischemia/reperfusion injury on endothelial barrier antigen expression and blood-brain barrier permeability in rats

Role of the blood-spinal cord barrier in posttraumatic syringomyelia

Posttraumatic syringomyelia produces a significant burden of pain and neurological deficits in patients with spinal cord injury. The mechanism of syrinx formation is unknown and treatment is often ineffective. A possible explanation for syrinx formation is fluid leakage from the microcirculation in the presence of a compromised blood-spinal cord barrier (BSCB). The aim of this study was to investigate the structural and functional integrity of the BSCB in a model of posttraumatic syringomyelia. The excitotoxic amino acid and arachnoiditis model of syringomyelia was used in 27 Sprague-Dawley rats. Structural integrity of the BSCB was assessed using immunoreactivity to endothelial barrier antigen (EBA), and loss of functional integrity was assessed by extravasation of intravascular horseradish peroxidase. Animals were studied after 3 days, or at 1, 3, 6, or 12 weeks after surgery. There were laminectomy-only and saline injection control animals for comparison at each time point. Syrinxes formed in 16 of the 17 animals injected with excitotoxic amino acid. Loss of structural and functional integrity of the BSCB in syrinx animals was noted at all time points. Disruption of the BSCB was most dramatic in tissue adjacent to the syrinx, and in the central and dorsal gray matter. Changes in EBA expression generally corresponded with altered vascular permeability, although in the acute stages, widespread vascular permeability occurred without a corresponding decrease in EBA expression. At the later time points (3-12 weeks) EBA expression was often absent, although no vascular leakage was observed. This study demonstrated a prolonged structural and functional disruption of the BSCB in this model of posttraumatic syringomyelia. Loss of functional integrity of the BSCB, with fluid entering the interstitial space of the spinal cord, may contribute to initial cyst formation after spinal cord injury and subsequent enlargement of the cyst, to produce posttraumatic syringomyelia.

Effects of acute hypoxia and hyperthermia on the permeability of the blood-brain barrier in adult rats

Acute mountain sickness (AMS) develops within a few hours after arrival at high altitude and includes headache, anorexia, nausea, vomiting, and malaise. This afflicts 15-25% of the general tourist population at moderate altitudes. High-altitude cerebral edema (HACE) is considered to be the end stage of severe AMS and has been suggested to be a vasogenic edema, raising the possibility that acute hypoxia may increase blood-brain barrier (BBB) permeability. At present, there are no good small-animal models to study this syndrome. We hypothesize 1) that acute hypoxia can damage the BBB and 2) that rat can be used as a model to study hypoxia-induced changes in BBB permeability, especially if hypoxia-induced hypothermia could be minimized with high ambient temperature (HAT). Male Wistar rats were exposed to 1, 2, and 7 days of hypobaric hypoxia (equivalent to 0.5 atm), and changes in the temperature and BBB permeability were studied. The extravasation of endogenous immunoglobulin G, a large molecule, did not increase during room temperature hypoxia but did increase when hypoxia was combined with HAT. Hypoxia caused a significant increase in the leakage of sodium fluorescein (mol wt 376 Da). The expression of endothelial barrier antigen (EBA), a protein associated with the BBB, was reduced to 50% between 24 and 48 h after exposure to hypoxia, and the loss was exacerbated by HAT. The values almost returned to control levels by 7 days, showing adaptation to hypoxia. Hypoxic rats exhibited sodium fluorescein leakage mainly in focal areas in the brain parenchyma. In conclusion, it is possible to have transient BBB damage through exposure to acute hypoxia, and this damage is exacerbated by increasing body temperature to more of a normothermic value.

Characteristics of blood-optic nerve barrier: experiment with rats

To investigate the difference of endothelial cells in ultrastructure, marker expression, and permeability between blood-brain barrier and blood-optic nerve barrier. The optic nerve, including the prelaminar region, lamina cribrosa, retro-laminar region, intraorbital portion, intracanalicular portion, and intracranial portion, and frontal cortex of 20 male SD rats were obtained to undergo electron microscopy and immunohistochemistry was used to detect the expression of endothelial barrier antigen (EBA) and the extravasation of fibrinogen around the microvessels. Electron microscopy showed tight junction among the endothelial cells in the microvessels of all portions of optic nerve and cerebral cortex. The number of plasmalemmal vesicles of the prelaminar region was (108.0 +/- 12.0)/microm2, significantly greater than that of the cerebral cortex [(31.8 +/- 2.9)/microm2, P < 0.05]. However, there was no significantly difference in the number of plasmalemmal vesicle among the other portion of optic nerve and cerebral cortex (all P > 0.05). Immunohistochemistry showed that EBA was negative in the endothelial cells in the microvessels of prelaminar region, but strongly positive in the microvessels in the other portions of the optic nerve and in the cerebral cortex. Fibrinogen was present around the microvessels in the prelaminar region of optic nerve in a small amount, however, not present in the other portions of optic nerve and cerebral cortex. There is significant differences in the number of plasmalemmal vesicle, EBA expression, and permeability between the prelaminar region of optic nerve and cerebral cortex, which demonstrates that this region lacks the characteristics of typical blood-brain barrier. However, the other parts of optic nerve possess the properties of classical blood-brain barrier.

Lack of experience-mediated differences in the immunohistochemical expression of blood–brain barrier markers (EBA and GluT-1) during the postnatal development of the rat visual cortex

The development of the cortical vascular tree depends on functional development. External inputs are an essential requirement in the modeling of the visual cortex, mainly during the critical period, when congruous blood supply is needed. The blood–brain barrier (BBB) function regulates the passage of substances between the blood and the brain parenchyma, which is one of the main differential features of central nervous system (CNS) microvessels. The endothelial barrier antigen (EBA) has been reported as a specific marker for the BBB physiological function in rats. We studied the postnatal development of EBA expression in the visual cortex of rats reared under opposite paradigms of visual experience, e.g., standard laboratory conditions, dark rearing, and enriched environment at 14, 21, 28, 35, 42, 49, 56, and 63 days postnatal (dpn). Parallel sections were immunohistochemically processed for endothelial barrier antigen (EBA) and glucose transporter-1 (GluT-1). Total vasculature was quantified by Lycopersicon esculentum (LEA) lectin histochemistry. No differences in EBA expression were found between groups, although quantitative differences were recorded paralleling differences in vascular density. Paradoxically, there was no expression in certain cortical vessels which were GluT-1 immunopositive and positivity was consistent in non-barrier areas such as the pineal gland. These findings were completely independent of age or experimental conditions. Therefore, the role of the EBA antigen in the BBB remains unclear: it has been undeniably linked to vascular permeability, but its presence in non-barrier vessels suggests another vascular function. Although visual experience modifies vascular density in the visual cortex, it has not been shown to have an influence on the maturation of the BBB function.

Disruption of the blood–brain barrier during TNBS colitis

Well-documented central nervous system changes during colitis suggest possible alterations of blood-brain barrier (BBB) permeability, yet the integrity of the BBB has not been fully evaluated in experimental colitis. Our aim was to investigate whether trinitrobenzene sulphonic acid (TNBS) colitis was associated with an increase in the permeability of the BBB. Sprague-Dawley rats were given an intracolonic injection of saline or TNBS and studied 1, 2, 3, 7 and 21 days after treatment. The extravasation of endogenous immunoglobulin G, a large molecule, was not altered at any time after TNBS treatment. In contrast, significant increases in the BBB leakage of sodium fluorescein, a much smaller molecule, were observed 1 and 2 days after the induction of colitis, in and around the circumventricular organs; the organum vasculosum of the lamina terminalis, subfornical organ and median eminence of the hypothalamus. TNBS-treated rats also exhibited sodium fluorescein leakage in focal areas in the brain parenchyma. The expression of endothelial barrier antigen, a protein associated with the BBB, was reduced about 60% 48 h after the induction of colitis. This returned to control values by 3 weeks, when colitis had largely subsided. In conclusion, experimental colitis transiently increased permeability of the brain to small molecules through a mild disruption of the BBB.

305. Effects of moderate spinal cord injury on the expression of a barrier marker in endothelial cells of the testis and in the prostate of rats

It was recently shown that the endothelial barrier antigen (EBA), previously thought to be specific to endothelial cells in the central nervous system, was also present in endothelial cells in the testis and in epithelial cells in the dorsolateral prostate of adult rats.1 In the present study, we examined the effect of moderate spinal cord injury (SCI), produced by compression for 5 min of the cord at T 10/11. There was a slight reduction in EBA in the testis and prostate 24 h after SCI, and this became more obvious after 3days. EBA was completely absent from the prostate and testis at 1 week. By 2 and 4 weeks some expression of EBA returned, and at these times EBA was also detected in the ventral prostate. Brain endothelial cells remained positive throughout. We cannot yet say whether these changes are due directly to interference with the nerve supply, or involve changes in androgen status. (1)Ghabriel MN, Lu JJ, Hermanis G, Zhu C, Setchell BP (2002) Reproduction 123, 389–397.

Hemorrhagic transformation is related to the duration of occlusion and treatment with tissue plasminogen activator in a nonembolic stroke model

The availability of reperfusion therapy for acute ischemic stroke patients has made the causes and significance of hemorrhagic transformation an area of intense interest and controversy. Ninety-two male Wistar rats underwent transient middle cerebral artery occlusion (MCAO) of between 1 and 6 h. Forty animals received 10 mg kg-1 of recombinant tissue plasminogen activator (rtPA), infused over 20 min, starting 5 min before reperfusion. At 18-24 h, the animals were sacrificed. The presence of hemorrhagic transformation (HT) on stained sections was recorded and total ischemic lesion area was quantified using image analysis software. Seventeen animals (11 with HT) were subjected to immunohistochemical analysis for detection of endothelial barrier antigen (EBA), quantified in three sections, in eight different fields per section. Chi-squared analysis and logistic regression were used to assess the contribution of rtPA and duration of occlusion to HT development. Nested, repeated measures analyses of variance were performed to assess the changes in EBA caused by ischemia and associated with HT. Fifty-nine animals developed HT that was significantly associated with occlusion duration (p < 0.0001) and ischemic lesion size (p = 0.0007). The presence of rtPA accelerated HT development. Statistically significant side-to-side differences in the presence of EBA were found in the striatum (core of the infarct) of animals with HT (p < 0.001) and without HT (p < 0.001), but only in animals with durations of occlusion of 2 h or more. Duration of occlusion is an important predictor of HT in transient MCAO in the rat and is closely associated with EBA expression.

Acute exposure to sarin increases blood brain barrier permeability and induces neuropathological changes in the rat brain: dose–response relationships

We hypothesize that a single exposure to an LD 50 dose of sarin induces widespread early neuropathological changes in the adult brain. In this study, we evaluated the early changes in the adult brain after a single exposure to different doses of sarin. Adult male rats were exposed to sarin by a single intramuscular injection at doses of 1, 0.5, 0.1 and 0.01×LD 50. Twenty-four hours after the treatment, both sarin-treated and vehicle-treated (controls) animals were analyzed for: (i) plasma butyrylcholinesterase (BChE) activity; (ii) brain acetylcholinesterase (AChE) activity, (iii) m2 muscarinic acetylcholine receptor (m2 mAChR) ligand binding; (iv) blood brain barrier (BBB) permeability using [H 3]hexamethonium iodide uptake assay and immunostaining for endothelial barrier antigen (EBA); and (v) histopathological changes in the brain using H&E staining, and microtubule-associated protein (MAP-2) and glial fibrillary acidic protein immunostaining. In animals treated with 1×LD 50 sarin, the significant changes include a decreased plasma BChE, a decreased AChE in the cerebrum, brainstem, midbrain and the cerebellum, a decreased m2 mAChR ligand binding in the cerebrum, an increased BBB permeability in the cerebrum, brainstem, midbrain and the cerebellum associated with a decreased EBA expression, a diffuse neuronal cell death and a decreased MAP-2 expression in the cerebral cortex and the hippocampus, and degeneration of Purkinje neurons in the cerebellum. Animals treated with 0.5×LD 50 sarin however exhibited only a few alterations, which include decreased plasma BChE, an increased BBB permeability in the midbrain and the brain stem but without a decrease in EBA expression, and degeneration of Purkinje neurons in the cerebellum. In contrast, animals treated with 0.1 and 0.01×LD 50 did not exhibit any of the above changes. However, m2 mAChR ligand binding in the brainstem was increased after exposure to all doses of the sarin. Collectively, the above results indicate that, the early brain damage after acute exposure to sarin is clearly dose-dependent, and that exposure to 1×LD 50 sarin induces detrimental changes in many regions of the adult rat brain as early as 24 hours after the exposure. The early neuropathological changes observed after a single dose of 1×LD 50 sarin could lead to a profound long-term neurodegenerative changes in many regions of the brain, and resulting behavioral abnormalities.

Electron microscope study of blood–brain barrier opening induced by immunological targeting of the endothelial barrier antigen

Barrier vessels in the central nervous system are lined with endothelial cells which constitute the blood–brain barrier (BBB) and show selective expression of certain biochemical markers. One of these, the endothelial barrier antigen (EBA), is specific to the rat. The exact role of EBA in the BBB is not known, although several studies have shown a correlation between the reduction in EBA expression in endothelial cells and the opening of the BBB. However, in these studies it was not possible to determine if EBA reduction was a primary event or was secondary to opening of the BBB. A recent light microscope study demonstrated that immunological targeting of EBA in vivo, by intravenous injection of a monoclonal antibody (anti-EBA), leads to acute and widespread opening of the BBB. In the current study we have employed this model together with tracer application and immunoperoxidase electron microscopy to determine the site of binding of the injected antibody and the route of opening of the BBB. The results showed that (a) the anti-EBA injected in vivo became bound to brain endothelial cells, principally to luminal membranes. (b) Endothelial cells showed widened intercellular junctions and increased cytoplasmic vesicles and vacuoles. (c) Many perivascular astrocytic processes were swollen. (d) The macromolecular tracer HRP was present in vesicles, vacuoles, widened paracellular clefts, the perivascular space and brain parenchyma. In conclusion, the in vivo targeting of EBA leads to opening of the BBB apparently via paracellular and transcellular routes. This model is useful for the study of vascular permeability in the CNS and experimental manipulation of the BBB. It may have a potential application in experimental studies on drug delivery throughout the CNS.

Expression of a blood-brain barrier-specific antigen in the reproductive tract of the male rat.

The endothelial barrier antigen (EBA) is a protein expressed specifically by the endothelial cells of the rat brain barrier vessels. This antigen has been described as a 'barrier protein' and is used as a marker for the competent blood-brain barrier. A blood-testis barrier has also been described. However, unlike the blood-brain barrier, which is formed by endothelial cells, the blood-testis barrier is formed mainly by the Sertoli cells, which provide an isolated environment for spermatogenic cells within the seminiferous tubules. Testicular blood vessels express the erythroid glucose transporter protein and other markers, which are strongly expressed in brain blood vessels, and may contribute to the blood-testis barrier. This study was carried out to determine whether Sertoli cells or testicular blood vessels express EBA. Tissues of other organs were used as controls for EBA expression. EBA was expressed by the endothelial cells in most microvessels of the testis, and in a few vessels of the epididymis, seminal vesicle, prostate gland, vas deferens and bladder-neck region. Furthermore, EBA was strongly and consistently detected in epithelial cells of the rete testis and dorsolateral prostate gland, and in a few epithelial cells of the ventral prostate gland, the seminal vesicle and the coagulating gland. However, Sertoli cells, which are the main site of the blood-testis barrier, were negative for EBA. In conclusion, EBA may have a wider role in rat tissues than has been previously appreciated.

Clostridium perfringens Prototoxin-Induced Alteration of Endothelial Barrier Antigen (EBA) Immunoreactivity at the Blood–Brain Barrier (BBB)

It has been reported that the severe cerebral edema produced in experimental animals by Clostridium perfringens (Cl p) type D ε toxin can be prevented by prior treatment with its precursor prototoxin due to competitive binding to endothelial cells (ECs) at the blood–brain barrier (BBB). In this study we investigate the effects of the prototoxin on the BBB, without added toxin. The integrity of the BBB was assessed by its ability to prevent leakage of endogenous albumin. ECs at the BBB were studied by immunocytochemistry for any alteration in the endothelial barrier antigen (EBA), a molecular marker for the intact BBB. Immunocytochemistry showed rapid but mild opening of the BBB to endogenous albumin. Light and electron immunocytochemistry showed qualitative and quantitative reduction in EBA immunoreactivity, with a spectrum of changes at time intervals from 1 h to 14 days post-prototoxin injection. Some vessels with ultrastructural changes and widening of the perivascular space retained EBA immunoreactivity. Many vessels showed partial or complete loss of EBA staining with minimal widening of the perivascular space and edema. Recovery of EBA expression was still incomplete at 14 days postinjection. This is the first report to show endothelial cell damage, mild reversible cerebral edema, and alteration in BBB markers following administration of Cl p prototoxin. This model of mild brain edema may be useful for BBB studies.

Immunological targeting of the endothelial barrier antigen (EBA) in vivo leads to opening of the blood–brain barrier

The role of the endothelial barrier antigen (EBA) in the blood–brain barrier (BBB) of the rat is not fully understood. Pathological conditions which show BBB disruption and leakage of plasma proteins are associated with reduced EBA expression in brain endothelial cells (ECs). However, it is not known if the reduction in EBA is the primary event or is secondary to protein extravasation. We hypothesized that immunological targeting of EBA in vivo would lead to opening of the BBB. To test this hypothesis, a monoclonal antibody (anti-EBA) was intravenously injected in anaesthetized experimental rats. Control animals received intravenous injections of phosphate-buffered saline (PBS) or non-specific antibodies (anti-human cytokeratin, anti-Salmonella bacterial antigen, or anti-pan endothelial cell antigen). Two groups of rats were used, each included experimental and control animals. The first group was used for immunocytochemical detection of EBA in brain ECs and rat albumin in brain parenchyma. In the second group, the permeability of the BBB to horseradish peroxidase (HRP) was tested. Experimental animals, injected with anti-EBA antibody, showed extensive leakage of HRP and albumin in the grey and white matter of the brain. Immunocytochemistry of experimental brains showed that the intravenously injected anti-EBA became bound to ECs and was detected in tissue sections. Control animals did not show leakage of HRP or albumin, and EBA distribution was normal. This study demonstrated for the first time, that immunological ‘neutralisation’ of EBA leads to opening of the BBB, and provided direct evidence for the importance of EBA in maintaining the integrity of the BBB in the rat.