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This study investigates the effect of beta-hydroxybutyrate (BHB) on blood-brain barrier (BBB) integrity during traumatic brain injury (TBI) in rats. Evans blue (EB) and horseradish peroxidase (HRP) were used as determinants of BBB permeability. Glutathione (GSH) and malondialdehyde (MDA) levels were estimated in the right (injury side) cerebral cortex of animals. The gene expression levels for occludin, glucose transporter (Glut)-1, aquaporin4 (AQP4) and nuclear factor-kappaB (NF-κB) were performed, and Glut-1 and NF-κB activities were analyzed. BHB treatment decreased GSH and MDA levels in intact animals and in those exposed to TBI (P<0.05). Glut-1 protein levels decreased in sham, BHB and TBI plus BHB groups (P<0.05). NF-κB protein levels increased in animals treated with BHB and/or exposed to TBI (P<0.05). The expression levels of occludin and AQP4 did not significantly change among experimental groups. Glut-1 expression levels increased in BHB treated and untreated animals exposed to TBI (P<0.05). While NF-κB expression levels increased in animals in TBI (P<0.01), a decrease was noticed in these animals upon BHB treatment (P<0.01). In animals exposed to TBI, EB extravasation was observed in the ipsilateral cortex regardless of BHB treatment. Ultrastructurally, BHB attenuated but did not prevent the presence of HRP in brain capillary endothelial cells of animals with TBI; moreover, the drug also led to the observation of the tracer when used in intact rats (P<0.01). Altogether, these results showed that BHB not only failed to provide overall protective effects on BBB in TBI but also led to BBB disruption in healthy animals., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We investigated the effects of a cell-permeable superoxide dismutase mimetic, manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) on blood-brain barrier (BBB) integrity following pentylenetetrazole (PTZ)-induced seizures in experimental preeclampsia symptoms induced by N(omega)-nitro-l-arginine methyl ester (l-NAME) in pregnant rats. To show the functional and morphological alterations in BBB integrity, quantitative analysis of sodium fluorescein (NaFlu) extravasation, immunohistochemistry and electron microscopic assessment of horseradish peroxidase (HRP) permeability were performed. Varying degrees of proteinuria were seen and arterial blood pressure increased in l-NAME-treated pregnant rats (p<0.01). MnTMPyP pretreatment and convulsive PTZ challenge significantly decreased the immunoreactivity of occludin in hippocampal capillaries in l-NAME-treated pregnant rats (p<0.01). BBB permeability to NaFlu significantly increased in pregnant rats treated with l-NAME plus PTZ (p<0.01), but MnTMPyP pretreatment did not significantly decrease NaFlu penetration into the brain parenchyma in these animals. Ultrastructurally, frequent vesicles containing HRP reaction products were observed in the capillary endothelial cells in the cerebral cortex and hippocampus of pregnant rats treated with l-NAME and l-NAME plus PTZ with the abundance being more in the latter group. MnTMPyP pretreatment caused a marked reduction in the frequency of HRP reaction product containing vesicles in both experimental settings. In conclusion, the results of the present study provide evidence that MnTMPyP plays an important role in limiting the enhanced vesicle-mediated transcellular transport in BBB endothelium in a rat model of preeclampsia and the differences in the way of transports of NaFlu and HRP might be responsible for the different effects of MnTMPyP on the BBB permeability to these two tracers., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Hyperbaric oxygen (HBO) treatment yields conflicting results on blood-brain barrier (BBB) integrity under various pathological conditions and the effects of HBO on healthy brain is poorly understood. In this experimental study, the effects of HBO on BBB integrity were investigated in comparison with hyperbaric air (HBA) in intact rats. Four sessions of HBA or HBO were applied to intact rats in 24h. BBB integrity was functionally and structurally evaluated by determining extravasation of Evans blue (EB) dye and horseradish peroxidase (HRP) tracers. In immunohistochemical evaluation, relative staining intensity for occludin, a tight junction (TJ) protein, and aquaporin 4 (AQP4), a water-channel protein, was detected in the barrier type of microvessels of brain by image analysis. BBB permeability to EB dye significantly increased in animals in HBO treatment group compared to those in HBA and control groups (p<0.05). The immunoreactivity of occludin, a tight junction protein, remained essentially unaltered in capillaries of hippocampus in all groups. In animals exposed to HBO, AQP4 immunoreactivity significantly increased in parietal cortex compared to those in HBA and control groups (p<0.01). Ultrastructurally, frequent vesicles containing HRP reaction products were observed in capillary endothelial cells in cerebral cortex and hippocampus of rats subjected to both HBA and HBO. Our results indicate that the HBO administration to intact rats increased BBB permeability to both EB and HRP while HBA increased only HRP extravasation in these animals. The results of this study suggest that HBA also impairs the BBB integrity in intact rats as well as HBO., (© 2013 Elsevier B.V. All rights reserved.)

We investigated the effects of topiramate (TPM), a novel broad spectrum anticonvulsant, on seizure severity, survival rate and blood-brain barrier (BBB) integrity during hyperthermic seizures in rats with cortical dysplasia (CD). Offsprings of irradiated mothers were used in this study. To show the functional and morphological alterations in BBB integrity, quantitative analysis of Evans blue (EB) extravasation, immunohistochemistry and electron microscopic assessment of horseradish peroxidase (HRP) permeability were performed. Rats with CD exposed to hyperthermia exhibited seizures with mean Racine's scores of 3.92 ± 1.2. Among the rats with CD pretreated with TPM, 21 of 24 rats showed no sign of seizure activity upon exposure to hyperthermia (p<0.01). The immunoreactivity of occludin, a tight junction protein, remained essentially unaltered in capillaries of hippocampus in all groups. In animals with CD exposed to hyperthermia, the significantly increased p-glycoprotein immunoreactivity in hippocampus (p<0.01) was slightly decreased by TPM pretreatment. Hyperthermic seizures increased BBB permeability to EB in animals with CD, but TPM pretreatment decreased the penetration of the tracer into the brain in these animals (p<0.01). Ultrastructurally frequent vesicles containing HRP reaction products were observed in capillary endothelial cells in cerebral cortex and hippocampus of rats with CD subjected to hyperthermia-induced seizures, and TPM pretreatment prevented the development of HRP reaction products in these animals. The results of this study suggest that TPM inhibits seizure activity and maintains BBB integrity in the course of febrile seizures in the setting of CD., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The mechanisms underlying the changes in blood-brain barrier (BBB) integrity in septic encephalopathy are poorly understood. The present study was designed to examine whether hyperbaric oxygen therapy (HBOT) influences the response of BBB to sepsis induced by cecal ligation and puncture (CLP) in rats. Cerebral cortical and hippocampal tissue levels of tumor necrosis factor-alpha (TNF-α), malondialdehyde (MDA) and glutathione (GSH) levels were measured. BBB permeability was functionally and structurally evaluated by determining extravasation of Evans blue (EB) and horseradish peroxidase (HRP) tracers, respectively. Immunohistochemistry and western blotting for occludin were performed. HBOT did not alter TNF-α levels in CLP operated rats while a significant decrease was noted when the therapy was subjected to intact rats. MDA levels in animals subjected to CLP plus HBOT were significantly decreased. In septic rats, the decreased GSH levels were significantly increased by HBOT. While HBOT attenuated the increased BBB permeability to EB in rats subjected to CLP (P<0.01), no macroscopic alteration was observed in the enhanced HRP extravasation. An increase in HRP extravasation was also observed by HBOT in intact animals. Occludin immunoreactivity and expression remained essentially unchanged in the brain capillaries of animals in all groups. Ultrastructurally, frequent vesicles containing HRP reaction products were observed in brain capillary endothelial cells of animals treated with CLP and/or HBOT. In conclusion, our results revealed that HBOT did not provide overall protective effects on the BBB integrity in septic conditions and even led to BBB disruption in intact animals., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This study investigates the effects of levetiracetam (LEV) on the functional and structural properties of blood-brain barrier (BBB) in pentylenetetrazole (PTZ)-kindled rats with cortical dysplasia (CD). Pregnant rats were exposed to 145 cGy of gamma-irradiation on embryonic day 17. In offsprings, kindling was induced by giving subconvulsive doses of PTZ three times per week for 45 days. While all kindled rats with CD died during epileptic seizures evoked by the administration of a convulsive dose of PTZ in 15 to 25 min, one week LEV (80 mg/kg) pretreatment decreased the mortality to 38% in the same setting. LEV caused a remarkable decrease (p<0.01) in extravasation of sodium fluorescein dye into the brain tissue of kindled animals with CD treated with convulsive dose of PTZ. Occludin immunoreactivity and expression remained essentially unchanged in all groups. Immunoreactivity for glial fibrillary acidic protein (GFAP) was observed to be slightly increased by acute convulsive challenge in kindled rats with CD while LEV pretreatment led to GFAP immunoreactivity comparable to that of controls. An increased c-fos immunoreactivity in kindled rats with CD exposed to convulsive PTZ challenge was also observed with LEV pretreatment. Tight junctions were ultrastructurally intact, whereas LEV decreased the increased pinocytotic activity in brain endothelium of kindled rats with CD treated with convulsive dose of PTZ. The present study showed that LEV decreased the increased BBB permeability considerably by diminishing vesicular transport in epileptic seizures induced by convulsive PTZ challenge in kindled animals with CD.

Cortical dysplasia (CD) is one of the major causes contributing to epileptogenesis associated with blood-brain-barrier (BBB) disturbances. The current study investigated the functional and ultrastructural changes of BBB in pentylenetetrazole (PTZ)-kindled rats with CD. Pregnant rats on E17 were exposed to 145 cGy of gamma-irradiation and offspring were used for experiments. The rats were given PTZ three times per week to induce kindling. The permeability of BBB was determined by using sodium fluorescein (NaFlu). Immunohistochemistry for occludin, GFAP and c-fos, western-blot analysis for occludin and electron microscopy for the ultrastructural alterations in BBB were performed. The brain level of NaFlu did not increase in rats with CD and/or kindling. Following administration of a convulsive dose of PTZ, a significant increase in BBB permeability was observed in kindled rats with CD. Occludin immunoreactivity and expression remained essentially unchanged in all groups. Slightly enhanced immunoreactivity for GFAP was observed in all groups except control. c-fos immunoreactivity in brain sections of kindled rats with CD displayed a striking increase by convulsive PTZ challenge. Tight junctions were ultrastructurally intact, whereas markedly increased number of pinocytotic vesicles was noted in brain endothelium of kindled rats with CD by convulsive dose of PTZ. The present study showed that epileptic seizures induced by convulsive PTZ challenge during kindling-mediated epileptogenesis in the presence of CD changed both functional and ultrastructural properties of the BBB and considerably enhanced transendothelial vesicular transport, while paracellular pathway was apparently not involved in this setting.

Recent studies suggest that 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, statins, can have direct effects on blood vessels beyond their cholesterol-lowering effects. We investigated the effects of atorvastatin on the functional and structural properties of blood-brain barrier (BBB) and the activity of astrocytes during the N(omega)-nitro-L-arginine methyl ester (L-NAME) hypertension followed by angiotensin (ANG) II. We found that decreases in concentration of serum catalase and plasma nitric oxide (NO) induced by L-NAME were significantly ameliorated by atorvastatin, whereas L-NAME-induced serum malondialdehyde and cholesterol concentration increases were significantly reduced by atorvastatin. The content of Evans blue (EB) dye significantly increased in cerebellum, left cerebral cortex and diencephalon regions but atorvastatin markedly reduced the increased BBB permeability to EB in the brain regions of animals treated with L-NAME and L-NAME plus ANG II. Brain vessels of L-NAME-treated animals showed a considerable loss of immunoreactivity of tight junction proteins, zonula occludens (ZO)-1 and occludin. Immunoreactivity for ZO-1 and occludin increased in animals treated with atorvastatin and L-NAME plus atorvastatin. Glial fibrillary acidic protein (GFAP) immunoreactivity was seen in few astrocytes in the brain sections of L-NAME, but immunoreactivity for GFAP increased in L-NAME plus atorvastatin-treated animals. We suggest that long-term L-NAME treatment may affect BBB permeability through disruption of tight junction proteins, at least partly, via decreased NO concentration and increased oxidant capacity; the improvement of BBB integrity and astrocytic activity would be more closely associated with the action of atorvastatin favoring the increase in anti-oxidant capacity and expression of tight junction proteins and GFAP.

The use of radiation to improve the efficacy of chemotherapy on malignant brain tumors is also known to cause side effects on vascular endothelial cells and astrocytes in normal parts of the brain. We investigated the effects of lipopolysaccharide (LPS) on the functional and structural properties of blood-brain barrier (BBB) and the activity of astrocytes during whole-brain irradiation in rats. The permeability of the BBB to Evans blue (EB) dye significantly increased in the cerebral cortex, diencephalon and cerebellum regions of rats exposed to irradiation (P<0.01). In contrast, the BBB permeability in irradiated rats was significantly reduced by LPS (P<0.05). Tumor necrosis factor-alpha (TNF-alpha) levels were increased following LPS, irradiation and irradiation plus LPS (P<0.05, P<0.01). Irradiated brain vessels showed a considerable loss of staining intensity of tight junction proteins Zonula occludens-1 (ZO-1) and occludin. Staining for Zonula occludens-1 and occludin was intensive in animals treated with LPS and irradiation plus LPS. Glial fibrillary acidic protein (GFAP) immunoreactivity was seen in very few astrocytes of irradiated brains. However, this staining showed an increased positive intensity in the brain sections of LPS-treated as well as of irradiation plus LPS-treated animals. These results indicate that LPS reduces the passage of exogenous vascular tracer EB-binding albumin into the brain, at least partly, by increasing the expression of tight junction proteins and GFAP, following the irradiation. We suggest that irradiation may affect paracellular permeability through disruption of tight junction proteins, Zonula occludens-1 and occludin, and LPS could provide beneficial effects on the BBB integrity and the astrocytes against irradiation damage.