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Objective: Vitamin D deficiency (VDD) is emerging as a predictor of poor prognosis in various neurological conditions, where clinical outcomes are often worse in stroke patients with VDD. This study aimed to provide experimental evidence on whether and how pre-existing VDD would affect survival and neurofunctional outcomes in intracerebral haemorrhage (ICH), and to evaluate whether acute vitamin D (VD) supplementation would improve post-stroke outcomes., Methods: Experimental ICH models were induced in mice with and without VDD. Haematoma size was measured using T2*-weighted MRI and haemoglobin concentration. Post-ICH mortality, neurofunctional outcomes and the extent of blood-brain barrier (BBB) leakage were assessed to identify their correlations with VD status. Therapeutic benefits of acute VD administration were also evaluated., Results: Mice with VDD exhibited significantly higher acute mortality rates and more severe motor deficits than mice without VDD post-ICH. Marked haematoma expansion and increased Evans blue extravasation were observed in VDD mice, suggesting that VDD was associated outcomes with increased BBB disruption. Acute treatment with a loading dose of VD (calcitriol) significantly improved outcomes in VDD mice., Conclusion: This study provides novel insights into the pathophysiological mechanisms at play in ICH concomitant with VDD and a scientific rationale for acute treatment with VD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) is a pleiotropic peptide known to promote many beneficial processes following neural damage and cell death after stroke. Despite PACAP's known neurotrophic and anti-inflammatory properties, it has not realized its translational potential due to a poor pharmacokinetic profile (non-linear PK/PD), and limited Blood-Brain Barrier Penetration (BBB) permeability. We have previously shown that glycosylation of PACAP increases stability and enhances BBB penetration. In addition, our prior studies showed reduced neuronal cell death and neuroinflammation in models of Parkinson's disease and Traumatic Brain Injury (TBI). In this study we show that a PACAP (1-27) glucoside retains the known neurotrophic activity of native PACAP (1-27) in vitro and a 5-day daily treatment regimen (100 nM) leads to neurite-like extensions in PC12 cells. In addition, we show that intraperitoneal injection of a PACAP (1-27) lactoside (10 mg/kg) with improved BBB-penetration, given 1-hour after reperfusion in a Transient Middle Cerebral Artery Occlusion (tMCAO) mouse model, reduces the infarct size after the ischemic injury in males significantly by ∼ 36 %, and the data suggest a dose-dependency. In conclusion, our data support further development of PACAP glycopeptides as promising novel drug candidates for the treatment of stroke, an area with an urgent clinical need., Competing Interests: Declaration of Competing Interest MLH, TF, and RP hold patents related to the content. MLH, TF, and RP have equity in Teleport Pharmaceuticals, LLC, a UArizona biotech startup. This interest played no role in the design of the studies; in the collection, analyses, or interpretation of data; in the writing of the Paper, or in the decision to present the results. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

There is a critical need for safer and better-tolerated alternatives to address the current limitations of antidepressant treatments for major depressive disorder. Recently, drugs targeting the GABA system via α5-containing GABA A receptors (α5-GABA A R) as negative allosteric modulators (α5-NAMs) have shown promise in alleviating stress-related behaviors in preclinical studies, suggesting that α5-NAMs may have translational relevance as novel antidepressant medications. Here, we evaluated the efficacy of Basmisanil, an α5-NAM that has been evaluated in Phase 2 clinical studies as a cognitive enhancer, in a battery of behavioral tests relevant to coping strategies, motivation, and aversion in male mice, along with plasma and brain pharmacokinetic measurements. Our findings reveal that Basmisanil induces dose-dependent rapid antidepressant-like responses in the forced swim test and sucrose splash test without promoting locomotor stimulating effects. Furthermore, Basmisanil elicits sustained behavioral responses in the female urine sniffing test and sucrose splash test, observed 24 h and 48 h post-treatment, respectively. Bioanalysis of plasma and brain samples confirms effective blood-brain barrier penetration by Basmisanil and extrapolation to previously published data suggest that effects were observed at doses (10 and 30 mg/kg i.p.) corresponding to relatively modest levels of α5-GABA A R occupancy (40-65 %). These results suggest that Basmisanil exhibits a combination of rapid and sustained antidepressant-like effects highlighting the potential of α5-NAMs as a novel therapeutic strategy for depression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Intracerebral hemorrhage (ICH) is a severe neurological dysfunction and a medical emergency with a high mortality rate. Minocycline ameliorates deficits in rodent models of acute and chronic neurological diseases. However, the role of minocycline in ICH remains unclear. The extracellular matrix metalloproteinase inducer (EMMPRIN) is a key inflammatory mediator in some neurological diseases, triggering matrix metalloproteinases (MMPs) production. In this study, we aimed to use minocycline to inhibit EMMPRIN and thus the activity of MMPs. Male adult C57BL/6 mice were injected with collagenase type VII or saline into the right basal ganglia and euthanized at different time points. The minocycline was intraperitoneally injected once every 12 h for three days to block the expression of EMMPRIN from two hours after ICH. We found that breakdown of the BBB was most severe 3 days after ICH. The minocycline treatment significantly decreased EMMPRIN and MMP-9 expression, reduced zonula occludens-1 and occludin, and alleviated BBB disruption. Moreover, minocycline treatment displayed a lower brain water content, lesser neurological dysfunction, and smaller injury volume on day 3 than those of the vehicle-treated group. Minocycline also inhibited the activation of microglia/macrophages, infiltration of neutrophils, and production of inflammatory mediators, including tumor necrosis factor alpha and interleukin-1beta. The current study shows that minocycline exhibits protective roles in ICH by decreasing EMMPRIN and MMP-9 expression, alleviating BBB disruption, inhibiting neuroinflammation, areducing neuronal degeneration and death., (Copyright © 2021. Published by Elsevier B.V.)

Blood-brain barrier (BBB) disruption is one of the most important pathological changes following cerebral ischemia-reperfusion. We tested whether inhibition of the serum and glucocorticoid regulated kinase 1 (SGK1) would decrease BBB disruption and contribute to decreasing infarct size in the first few hours of cerebral ischemia-reperfusion within the thrombolysis therapy time window. After transient middle cerebral artery occlusion (MCAO), an SGK1 inhibitor GSK650394, or vehicle was administered into the lateral ventricle of rats. After one hour of MCAO and two hours of reperfusion, we determined BBB disruption using the transfer coefficient (K i ) of 14 C-α-aminoisobutyric acid, and also determined infarct size, phosphorylation of NDRG1, and MMP2 protein level. Ischemia-reperfusion increased (+34%, p < 0.05) and GSK650394 decreased (-25%, p < 0.05) the K i in the ischemic-reperfused cortex. GSK650394 decreased the percentage of cortical infarct (-31%, p < 0.001). At the same time GSK650394 reduced NDRG1 phosphorylation and MMP2 protein level in the ischemic-reperfused cortex suggesting that SGK1 was inhibited by GSK650394 and that lower MMP2 could be one of the mechanisms of decreased BBB disruption. Collectively our data suggest that GSK650394 could be neuroprotective and one of the mechanisms of the neuroprotection could be decreased BBB disruption. SGK1 inhibition within the thrombolysis therapy time window might reduce cerebral ischemia-reperfusion injury., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Extracellular RNAs (exRNAs) are present in all biofluids and incorporate many types of RNAs including miRNA. To enhance their stability outside of the cell, exRNAs are bound within ribonucleoprotein complexes or packaged into extracellular vesicles (EVs). The blood-brain barrier (BBB) is a dynamic interface between the systemic circulation and the CNS and is responsible for maintaining a stable extracellular environment for CNS cells. The intent of this study was to determine if EVs and their contents are transferred from the peripheral circulation to the CNS under conditions of an impaired BBB. The BBB of mice was disrupted by unilateral intracarotid artery infusion with hyperosmolar mannitol solution. To validate barrier opening, the uptake clearance of [ 13 C 12 ]-sucrose in the left forebrain (i.e. the ipsilateral, mannitol injected hemisphere) was quantified and revealed a 14-fold increase in the mannitol perfused hemisphere compared to sham treated mice. EVs were isolated from the extracellular spaces of the left forebrain following gentle tissue lysis and differential ultracentrifugation. EVs were confirmed using nanotracking analysis, electron microscopy and western blotting. qRT-PCR showed that the erythrocyte-enriched miR-451a in brain tissue EVs increased with mannitol treatment by 24-fold. Small RNA sequencing performed on the EVs isolated from the sham and mannitol treated mice showed that miR-9-5p was the most abundant miRNA contained within the brain EVs. qRT-PCR analysis of plasma EVs did not produce a statistically significant difference in the expression of the CNS-enriched miR-9-5p or miR-9-3p, suggesting that transfer of CNS EVs to the peripheral circulation did not occur under the conditions of our experiment. We demonstrate that EVs containing miR-451a, a highly abundant miRNA present within erythrocytes and erythrocyte EVs, are enhanced in the CNS upon BBB disruption., (Copyright © 2021. Published by Elsevier B.V.)

During osmotic demyelination syndrome (ODS), myelin and oligodendrocyte are lost according to specific patterns in centro- or extra-pontine regions. In both experimental model of ODS and human cases, brain lesions are locally correlated with the disruption of the blood brain-barrier (BBB). The initiation, the degree and the duration of blood-brain barrier (BBB) opening as well as its contribution to brain damages are still a matter of debate. Using a panel of intravascular tracers from low- to high- molecular weight (from 0.45 kDa 150 kDa), we have assessed the BBB permeability at different timings of ODS induced experimentally in mice. ODS was mimicked according to a protocol of rapid correction of a chronic hyponatremia. We demonstrated that BBB leakage towards smallest tracers Lucifer Yellow (0.45 kDa) and Texas Red-dextran (3 kDa) was delayed by 36 h compared to the first clues of oligodendrocyte loss (occurring 12 h post-correction of hyponatremia). At 48 h post-correction and concomitantly to myelin loss, BBB was massively disrupted as attested by accumulation of Evans Blue (69 kDa) and IgG (150 kDa) in brain parenchyma. Analysis of BBB ultrastructure verified that brain endothelial cells had minimal alterations during chronic hyponatremia and at 12 h post-correction of hyponatremia. However, brain endothelium yielded worsened alterations at 48 h, such as enlarged vesicular to tubular-like cytoplasmic profiles of pinocytosis and/or transcytosis, local basal laminae abnormalities and sub-endothelial cavities. The protein expressions of occludin and claudin-1, involved in inter-endothelial tight junctions, were also downregulated at 48 h post-correction of hyponatremia. Our results revealed that functional BBB opening occured late in pre-established ODS lesions, and therefore was not a primary event initiating oligodendrocyte damages in the mouse model of ODS., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The blood-brain barrier (BBB) is the multicellular interface located between the peripheral circulation and the brain parenchyma. BBB dysfunction is reported in many CNS diseases, such cognitive impairment, depression, Alzheimer's disease (AD), and multiple sclerosis (MS). Emerging evidence indicates that liver-derived inflammatory mediators are upregulated in neurological diseases with BBB dysfunction. Serum amyloid A (SAA), an acute phase protein secreted by hepatocytes, could be a candidate inflammatory signaling molecule transmitted from the liver to the brain; however, its contribution to BBB dysfunction is poorly understood. The present study aimed to elucidate the involvement of SAA in BBB impairment in an in vitro BBB model using rat brain microvascular endothelial cells (RBECs). We demonstrated that Apo-SAA significantly decreased transendothelial electrical resistance (TEER) and increased sodium fluorescein (Na-F) permeability in RBEC monolayers. Apo-SAA also decreased claudin-5 expression levels in RBECs. Furthermore, the Apo-SAA-mediated impairment of the BBB with decreased claudin-5 expression was inhibited by the addition of a high-density lipoprotein (HDL) related to SAA in plasma. These findings suggest that HDL counteracts the effects of SAA on BBB function. Therefore, the functional imbalance between SAA and HDL may induce BBB impairment, thereby triggering development of neuroinflammation. SAA could be a significant endogenous mediator in the liver-to-brain inflammation axis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

White matter lesions are associated with impairment of the blood-brain barrier (BBB), an essential component of the cerebrovasculature. The BBB allows the brain to maintain its highly specialized microenvironment by restricting entry of blood-borne substances including molecules that induce myelin damage. Accumulating evidence suggests that interactions between brain endothelial cells and neighboring cells, including oligodendrocyte progenitor cells (OPCs), are required for the induction and maintenance of BBB function. Here, we compared the ability of OPCs and oligodendrocytes to modulate BBB integrity using co-cultures of rat brain endothelial cells with OPCs or oligodendrocytes. We found that OPCs lowered the brain endothelial permeability to sodium fluorescein, and this enhancement of BBB function was prevented by treatment with AG1296 (a PDGFRα inhibitor). Oligodendrocytes also enhanced BBB integrity. Pharmacological inhibition of PDGFRα did not affect the oligodendrocyte-induced BBB facilitation. These data indicate that oligodendrocytes enhance BBB integrity through pathways other than PDGF-BB/PDGFRα signaling triggered by the brain endothelial cell-derived PDGF-BB. Therefore, our findings suggest that oligodendrocytes constitutively support BBB integrity through soluble factors. Crosstalk between brain endothelial cells and oligodendrocytes could play a facilitatory role in maintaining BBB integrity in the white matter., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: In this study, the protective effects of 3,4-dihydroxyphenyl lactic acid (DLA) on blood-brain barrier (BBB) injury following subarachnoid hemorrhage (SAH) has been explored., Methods: Male Sprague-Dawley rats (weight 300-350 g) were used to establish the SAH model using the endovascular perforation method. The animals were randomly divided into four groups: sham (n = 40), SAH (n = 46), SAH + vehicle (n = 44), and SAH + DLA (n = 40) treatment groups. At 1 h after SAH, either DLA (10 mg/kg) or normal saline (vehicle) was administered by femoral vein injection. The effects of DLA on mortality, neurological function, brain water content, and BBB were observed. Additionally, immunohistochemistry and western blot techniques were applied to investigate the mechanism of action of DLA., Results: We found that the administration of DLA (10 mg/kg) following SAH could improve neurological functions, reduce brain water content, and maintain BBB integrity. The expression of pro-inflammatory and pro-apoptotic factors such as toll-like receptor 4 (TLR4), NF-κB (p-p65), tumor necrosis factor-α, p-p38 MAPK, p-p53, and caspase-3 were significantly increased after SAH. These same factors were markedly attenuated following treatment with DLA., Conclusions: These findings showed that DLA can alleviate BBB injury following SAH through its anti-inflammatory and anti-apoptotic effects via suppression of TLR4 and its downstream NF-κB and p38 MAPK pathways., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Postoperative neurocognitive disorder (PND) is a major complication in surgical patients, especially the elderly, leading to mild memory impairment after surgery. The underlying pathophysiology remains unknown, although neuroinflammation and blood-brain barrier (BBB) disruption have been increasingly implicated in PND. Emerging evidence suggests that neurokinin-1 receptor (NK-1R), the principal target of proinflammatory neuropeptide substance P (SP), plays a pivotal role in modulating neuroinflammation and BBB integrity. In this study, we used an established mouse model for PND to investigate the effects of a selective NK-1R antagonist L-733,060 on PND-like features after peripheral surgery. Hippocampal SP started to increase at 6 h, peaked at 1 day, and returned to baseline at 3 days after surgery. At 1 day after surgery, NK-1R expression was increased in the hippocampus. At this time point, NK-1R antagonist pretreatment attenuated microgliosis and prevented neutrophil infiltration after surgery. Similarly, proinflammatory cytokines interleukin-1 beta and interleukin-6 were reduced in the hippocampus in NK-1R antagonist-treated mice at 6 h after surgery. Furthermore, surgery-induced BBB disruption, assessed by albumin deposition and expression of tight junction protein claudin-5, was attenuated by NK-1R antagonism at postoperative day 1. Finally, trace fear conditioning test revealed NK-1R antagonism reversed surgery-induced cognitive impairment at 3 days after surgery. Our findings suggest that inhibition of NK-1R signaling protects hippocampus-dependent memory from surgical insult, probably through modulations of neuroinflammation and BBB integrity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

It is well established that tobacco smoking is associated with vascular endothelial dysfunction in a causative and dose dependent manner primarily related to the tobacco smoke (TS) content of reactive oxygen species (ROS), nicotine, and oxidative stress (OS) -driven inflammation. Preclinical studies have also shown that nicotine (the principal e-liquid's ingredient used in e-cigarettes (e-Cigs) can also cause OS, exacerbation of cerebral ischemia and secondary brain injury. Likewise, chronic e-Cig vaping could be prodromal to vascular endothelial dysfunctions. Herein, we provide direct evidence that similarly to TS, e-Cig promotes mitochondrial depolarization in primary brain vascular endothelial cells as well as the vascular endothelial cell line bEnd3. In addition, both TS and e-Cig exposure upregulated the transmembrane iron exporter Slc40a1 (crucial to maintain cellular iron and redox homeostasis) and that of porphyrin importer Abcb6 (linked to accelerated atherosclerosis). We then investigated in vivo whether gender plays a role in how chronic TS affect vascular endothelial functions. Our results clearly show chronic TS exposure differentially impacts the expression levels of Phase-II enzymes as well as the iron transporters previously investigated in vitro. Although the physiological implications of the gender-specific differential responses to TS are not fully clear, they do demonstrate that gender is a risk factor that needs to be investigated when assessing the potential impact of chronic smoking and perhaps e-Cig vaping., (Published by Elsevier B.V.)

Activation of Akt has been suggested to produce neuronal protection in cerebral ischemia. Decreasing blood-brain barrier (BBB) disruption has been associated with a better neuronal outcome in cerebral ischemia. We hypothesized that activation of Akt would decrease BBB disruption and contribute to decreasing the size of infarct in the early stage of cerebral ischemia-reperfusion within the therapeutic window. Transient middle cerebral artery occlusion (MCAO) was performed in rats under isoflurane anesthesia with controlled ventilation. Rats were treated with SC79 (a selective Akt activator which is cell and BBB permeable) 0.05 mg/kg × 3 i.p. or vehicle i.p. perioperatively. After one hour of MCAO and two hours of reperfusion, the transfer coefficient (K i ) of 14 C-α-aminoisobutyric acid ( 14 C-AIB, molecular weight 104 Da) and the volume of 3 H-dextran (molecular weight 70,000 Da) distribution were determined to measure the degree of BBB disruption. At the same time point, the size of infarction was determined using tetrazolium staining. In an additional group of rats, a higher dose of SC79 (0.5 mg/kg × 3) was administered to determine the size of infarct. Administration of SC79 increased the K i in the ischemic-reperfused cortex (IR-C, +32%, p < 0.05) as well as in the contralateral cortex (CC, +35%, p < 0.05) when compared with the untreated animals with MCAO/reperfusion. The volume of dextran distribution was not significantly changed by SC79. SC79 treatment significantly produced a decrease in the percentage of cortical infarct out of total cortical area (12.7 ± 1.7% vs 6.9 ± 0.9%, p < 0.001). Increasing the dose of SC79 by ten times did not significantly affect the size of cortical infarct. Contrary to our hypothesis, our data demonstrated that SC79 decreased the size of the infarct in the ischemic-reperfused cortex despite an increase in BBB disruption. Our data suggest the importance of activation of Akt for neuronal survival in the early stage of cerebral ischemia-reperfusion within the therapeutic window and that the mechanism of neuroprotection may not be related to the BBB effects of SC79., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: Cerebral venous sinus thrombosis (CVST) is a life-threatening disease with high misdiagnosis and mortality rates due to its complex etiology and unknown pathophysiology. The present study aimed to establish an animal model suitable for assessing the pathophysiology of CVST and develop treatment methods., Methods: 40% ferric chloride (FeCl3) was administered for 5min followed by thrombin injection to induce superior sagittal sinus thrombosis (SSST). Digital subtraction angiography (DSA) was performed to ensure thrombosis and evaluate the recanalization rate 7days post-CVST. Neurological evaluation, Evans blue injection, 2,3,5-Triphenyltetrazolium chloride (TTC), and hematoxylin-eosin (H&E) staining were used to assess thrombosis and the accompanying brain parenchyma., Results: SSST was detected in all model rabbits, with a thrombus recanalization rate of 10%. Brain infarction, hemorrhage, cell edema, and disruption of the blood-brain barrier (BBB) were also observed., Conclusion: The method of inducing cerebral venous sinus thrombosis by applying 40% FeCl3 and injecting thrombin is feasible and efficient. This experimental model mimics the pathogenesis and pathophysiology of actual CVST., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Diabetes causes functional and structural changes in blood-brain barrier (BBB). The mammalian target of rapamycin (mTOR) has been associated with glucose metabolism, diabetes, and altering BBB permeability. Since there is only a narrow therapeutic window (3h) for stroke victims, it is important to investigate BBB disruption in the early stage of cerebral ischemia. We compared the degree of BBB disruption in diabetic and in control rats at two hours of reperfusion after one hour of middle cerebral artery (MCA) occlusion with or without inhibition of mTOR. Two weeks after streptozotocin ip to induce diabetes, MCA occlusion was performed. In half of the rats, an mTOR inhibitor, rapamycin was given for 2days before MCA occlusion. After one hour of MCA occlusion and two hours of the reperfusion, the transfer coefficient (K i ) of 14 C-α-aminoisobutyric acid was determined to quantify degree of BBB disruption. Ischemia-reperfusion increased the K i in the control animals. Streptozotocin increased the K i in the ischemic-reperfused (IR-C, +22%) as well as in the contralateral cortex (CC, +40%). Rapamycin decreased the K i in the IR-C (-32%) as well as in the CC (-26%) in the control rats. However, rapamycin did not affect K i in the IR-C or in the CC in the diabetic rats. Our data demonstrated a greater BBB disruption in diabetes in the ischemic as well as non-ischemic cortex even in the early stage of cerebral ischemia-reperfusion and that acute administration of rapamycin did not significantly affect BBB permeability in diabetes. From our quantitative analysis of BBB disruption, the vulnerability of BBB in diabetes has been emphasized in the early stage of cerebral ischemia-reperfusion and a less important role of the mTOR pathway is suggested in altering BBB permeability in diabetes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Vasogenic edema induced by blood brain barrier disruption and neuronal loss play an important role in the epileptogenic process. 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) is a commonly used anion channel inhibitor that has been reported to exert an anticonvulsant effect in rat hippocampus in vitro. The present study aimed to investigate whether DIDS could prevent epileptogenic process in rat lithium-pilocarpine model of temporal lobe epilepsy. The tight junction proteins and serum extravasation were examined in the piriform cortex 3days after status epilepticus. The findings showed that status epilepticus induced vasogenic edema. Based on these findings, rats were intracerebroventricularly infused with saline and DIDS 1 week after surgery, DIDS reduced vasogenic edema and prevented neuronal loss following status epilepticus in the piriform cortex. Moreover, spontaneous recurrent seizures were recorded by continuous video monitoring. DIDS significantly reduced the frequency and duration of spontaneous recurrent seizures from day 28 to day 42 post status epilepticus. These findings demonstrated that DIDS attenuated vasogenic edema and neuronal apoptosis and might exert disease-modifying effect in animal model of temporal lobe epilepsy. These results explored a novel therapeutic strategy for treatment of epilepsy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate whether Cyclosporine A (CsA) attenuates early brain injury by alleviating matrix metalloproteinase 9 (MMP-9) associated blood-brain barrier (BBB) disruption after subarachnoid hemorrhage (SAH). A standard intravascular perforation model was used to produce the experimental SAH in C57B6J mice. Dosages of 5mg/kg, 10mg/kg and 15mg/kg CsA were evaluated for effects on neurological score, brain water content, Evans blue extravasation and fluorescence, P-p65, MMP-9 and BBB components' alterations after SAH. We found that CsA 15mg/kg is effective in attenuating BBB disruption, lowering edema, and improving neurological outcomes. In addition, Collagen IV, ZO-1, Occludin and Claudin 5 expressions in ipsilateral/left hemisphere were downregulated after SAH, but increased after CsA treatment. Our results suggest that CsA exert a neuroprotective role in SAH pathophysiology, possibly by alleviating MMP-9 associated BBB disruption., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Prolonged hydrocephalus is a major cause of severe disability and death of intraventricular hemorrhage (IVH) patients. However, the therapeutic options to minimize the detrimental effects of post-hemorrhagic hydrocephalus are limited. Curcumin has been reported to confer neuroprotective effects in numerous neurological diseases and injuries, but its role in IVH-induced hydrocephalus has not been determined. The aim of present study was to determine whether curcumin treatment ameliorates blood brain barrier (BBB) damage and reduces the incidence of post-hemorrhagic hydrocephalus in IVH rat model. Autologous blood intraventricular injection was used to establish the IVH model. Our results revealed that repeated intraperitoneal injection of curcumin ameliorated IVH-induced learning and memory deficits as determined by Morris water maze and reduced the incidence of post-hemorrhagic hydrocephalus in a dose-dependent manner at 28 d post-IVH induction. Further, the increased BBB permeability and brain edema induced by IVH were significantly reduced by curcumin administration. In summary, these findings highlighted the important role of curcumin in improving neurological function deficits and protecting against BBB disruption via promoting the neurovascular unit restoration, and thus it reduced the severity of post-hemorrhagic hydrocephalus in the long term. It is believed that curcumin might prove to be an effective therapeutic component in prevent the post-IVH hydrocephalus in the near future., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The study investigated the roles and mechanisms of Salvianolic acid B (Sal B) on permeability of rat brain microvascular endothelial cells (RBMECs) exposed to high glucose. The results demonstrated that Sal B greatly up-regulated the expression of tight junction (TJ) proteins and decreased the permeability of RBMECs compared with the control group. And the increase of reactive oxidative species (ROS) production, the upregulation of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) protein induced by high glucose were antagonized by Sal B. In addition, a great decrease of microRNA-200b (miR-200b) was observed in the RBMECs under high-glucose condition, which was significantly increased by Sal B pretreatment. And overexpression of miR-200b markedly attenuated the RBMECs permeability and inhibited the expression of VEGF protein by targeting with 3'-UTR of its mRNA. This led to the conclusion that Sal B-mediated improvement of blood-brain barrier dysfunction induced by high-glucose is related to the ROS/HIF-1α/VEGF and miR-200b/VEGF signaling pathways., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Designer drugs such as synthetic psychostimulants are indicative of a worldwide problem of drug abuse and addiction. In addition to methamphetamine (METH), these drugs include 3,4-methylenedioxy-methamphetamine (MDMA) and commercial preparations of synthetic cathinones including 3,4-methylenedioxypyrovalerone (MDPV), typically referred to as "bath salts." These psychostimulants exert neurotoxic effects by altering monoamine systems in the brain. Additionally, METH and MDMA adversely affect the integrity of the blood-brain barrier (BBB): there are no current reports on the effects of MDPV on the BBB. The aim of this study was to compare the effects of METH, MDMA and MDPV on bovine brain microvessel endothelial cells (bBMVECs), an accepted in vitro model of the BBB. Confluent bBMVEC monolayers were treated with METH, MDMA and MDPV (0.5mM-2.5mM) for 24h. METH and MDMA increased lactate dehydrogenase release only at the highest concentration (2.5mM), whereas MDPV induced cytotoxicity at all concentrations. MDMA and METH decreased cellular proliferation only at 2.5mM, with similar effects observed after MDPV exposures starting at 1mM. Only MDPV increased reactive oxygen species production at all concentrations tested whereas all 3 drugs increased nitric oxide production. Morphological analysis revealed different patterns of compound-induced cell damage. METH induced vacuole formation at 1mM and disruption of the monolayer at 2.5mM. MDMA induced disruption of the endothelial monolayer from 1mM without vacuolization. On the other hand, MDPV induced monolayer disruption at doses ≥0.5mM without vacuole formation; at 2.5mM, the few remaining cells lacked endothelial morphology. These data suggest that even though these synthetic psychostimulants alter monoaminergic systems, they each induce BBB toxicity by different mechanisms with MDPV being the most toxic., (Published by Elsevier Ireland Ltd.)

Adipocytokine apelin-13 is a peptide which could reportedly protect the brain against ischemic reperfusion injury and traumatic brain injury (TBI). Whether apelin-13 has any roles to play in intracerebral hemorrhage (ICH) has not been clarified. We aimed to investigate the roles of apelin-13 in ICH and effects on ICH-induced apoptosis. Firstly, CD-1 mice were subjected to infusion of Type IV collagenase (to induce ICH) or saline (for shams) into the left striatum. ICH animals received intracerebroventricular administration of vehicle, apelin-13 (50μg dissolved in 5μl saline) immediately after ICH. The motor function and the cerebral water content (CWC) as well as blood brain barrier (BBB) disruption were measured, coupled with determination of ICH-induced neural cell death by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL). The apoptosis-associated proteins caspase-3 and Bcl-2 as well as the brain edema-associated proteins aquaporin-4 (AQP4) and MMP-9 were all assessed with western blotting. The results showed that apelin-13 decreased CWC and reduced Evans blue leakage into injured hemispheres, with the motor function significantly improved. Additionally, apelin-13 also acutely decreased the number of ICH-induced TUNEL-positive (TUNEL(+)) cells at 48h after ICH. The expressions of AQP4, MMP-9, caspse-3 and Bcl-2 were all downregulated by apelin-13 at 24h and 48h after ICH. All these results revealed that apelin-13 attenuated brain edema and reduced cellular death by suppressing apoptosis after ICH in mice., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The mammalian target of rapamycin (mTOR) pathway is essential in neuronal survival and repair in cerebral ischemia. Decreases in blood-brain barrier (BBB) disruption are associated with a decrease in neuronal damage in cerebral ischemia. This study was performed to investigate how pre-inhibition of the mTOR pathway with rapamycin would affect BBB disruption and the size of the infarcted cortical area in the early stage of focal cerebral ischemia-reperfusion using quantitative analysis of BBB disruption. Rats were treated with 20mg/kg of rapamycin i.p. once a day for 2days (Rapamycin Group) or vehicle (Control Group) before transient middle cerebral artery (MCA) occlusion. After one hour of MCA occlusion and two hours of reperfusion, the transfer coefficient (Ki) of (14)C-α-aminoisobutyric acid ((14)C-AIB) to measure the degree of BBB disruption and the size of the cortical infarct were determined. Ischemia-reperfusion increased the Ki in the Rapamycin treated (+15%) as well as in the untreated control group (+13%). However, rapamycin pretreatment moderately decreased Ki in the contralateral (-30%) as well as in the ischemic-reperfused (-29%) cortex when compared with the untreated control group. Rapamycin pretreatment substantially increased the percentage of cortical infarct compared with the control group (+56%). Our data suggest that activation of mTOR pathway is necessary for neuronal survival in the early stage of cerebral ischemia-perfusion and that the reason for the enlarged cortical infarct by rapamycin pretreatment may be related to its non-BBB effects on the mTOR pathway., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Early brain injury, which is associated with brain cell death, blood-brain barrier disruption, brain edema, and other pathophysiological events, is thought to be the main target in the prevention of poor outcomes after subarachnoid hemorrhage (SAH). Emerging evidences indicates that complement system, especially complement C3 is detrimental to neurological outcomes of SAH patients. Recently, Ephedra sinica extract was extracted and purified, which exhibits ability to block the activity of the classical and alternative pathways of complement, and improve neurological outcomes after spinal cord injury and ischemic brain injury. However, it is still unclear whether Ephedra sinica extract could attenuate early brain injury after SAH. In the present study, a standard endovascular perforation model was used to produce the experimental SAH in Sprague-Dawley rats. Ephedra sinica extract (15mg/kg) was orally administrated daily and evaluated for effects on modified Garcia score, brain water content, Evans blue extravasation and fluorescence, cortex cell death by TUNEL staining, and the expressions of complement C3/C3b, activated C3, sonic hedgehog, osteopontin and matrix metalloproteinase-9 by western bolt and immunofluorescence staining. We founded that the Ephedra sinica extract alleviated the blood-brain barrier disruption and brain edema, eventually improved neurological functions after SAH in rats. These neuroprotective effects was associated with the inhibition of complement C3, possibly via upregulating sonic hedgehog and osteopontin signal, and reducing the expressions of matrix metalloproteinase-9. Taking together, these observations suggested complement C3 inhibition by the Ephedra sinica extract may be a protective factor against early brain injury after SAH., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The underlying mechanism of isoflurane-induced cognitive dysfunction in older individuals is unknown. In this study, the effects of isoflurane exposure on the hippocampal blood-brain barrier (BBB) in aged rats were investigated because it was previously shown that BBB disruption involves in cognitive dysfunction. Twenty-month-old rats randomly received 1.5% isoflurane or vehicle gas as control. Hippocampal BBB ultrastructure was analyzed by transmission electron microscopy and expression of tight junction proteins was measured by western blot analysis. BBB permeability was detected with sodium fluorescein extravasation and further confirmed by immunoglobulin G immunohistochemistry. Spatial learning and memory were assessed by the Morris water maze test. Isoflurane anesthesia resulted in reversible time-dependent BBB ultrastructure morphological damage and significant decreases in expression of the tight junction proteins occludin, which contributed to sodium fluorescein and IgG leakage. Rats with isoflurane exposure also showed significant cognitive deficits in the Morris water maze test. This in vivo data indicate that occludin down-regulation may be one of the mediators of isoflurane-induced hippocampus BBB disruption, and may contribute to hippocampus-dependent cognitive impairment after isoflurane exposure in aged rats., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Ursolic acid (UA), a well-known anti-oxidative reagent, has been reported to protect the brain against ischemic stoke. However, the potential role of UA in protecting against early brain injury (EBI) after subarachnoid hemorrhage (SAH) remains unclear. The present study aimed to examine the effect of UA against EBI following SAH, and to demonstrate whether the effect is associated with its powerful antioxidant property. Male SD rats were divided into vehicle-treated sham, vehicle-treated SAH, and UA-treated SAH groups. The endovascular puncture model was used to induce SAH and all the rats were subsequently sacrificed at 48h after SAH. The results show that UA administration could significantly attenuate EBI (including brain edema, blood-brain barrier disruption, neural cell apoptosis, and neurological deficient) after SAH in rats and up-regulate the antioxidative levels in the rat cerebral cortex, suggesting that administration of UA in experimental SAH rats could alleviate brain injury symptom, potentially through its powerful antioxidant property. Hence, we concluded that UA might be a novel therapeutic agent for EBI following SAH., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Matrix metalloproteinase-9 (MMP-9) plays key roles in the brain pathophysiology, especially in blood-brain barrier (BBB) breakdown. Therefore, inhibiting MMP-9 activity may be a promising therapy for protecting brains in cerebrovascular diseases. Here we show that in a mouse prolonged cerebral hypoperfusion model, a clinically proven radical scavenger edaravone suppressed MMP-9 and reduced BBB damage in cerebral white matter. Prolonged cerebral hypoperfusion was induced by bilateral common carotid artery stenosis in male adult C57BL/6J mice (10 weeks old). After 7 days of cerebral hypoperfusion, white matter region (e.g. corpus callosum) exhibited significant BBB leakage, assessed by IgG staining. Correspondingly, immunostaining and western blotting showed that MMP-9 was upregulated in the white matter. Edaravone treatment (3mg/kg, i.p. at days 0 and 3) inhibited both BBB leakage and MMP-9 increase. Under the early phase of cerebral hypoperfusion conditions, oligodendrocyte precursor cells (OPCs) mainly contribute to the MMP-9 increase, but our immunostaining data showed that very little OPCs expressed MMP-9 in the edaravone-treated animals at day 7. Therefore, in vitro studies with primary rat OPCs were conducted to examine whether edaravone would directly suppressed MMP-9 expressions in OPCs. OPC cultures were exposed to sub-lethal CoCl2 for 7 days to induce prolonged chemical hypoxic stress. Prolonged chemical hypoxic stress increased MMP-9 expression in OPCs, and radical scavenging with edaravone (10μM for 7 days) ameliorated the increase. Taken together, our proof-of-concept study demonstrates that radical scavengers may provide a potential therapeutic approach for white matter injury by suppressing BBB damage., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The blood-brain barrier (BBB) is composed of a network of tight junctions (TJ) which interconnect cerebral endothelial cells (EC). Alterations in the TJ proteins are common in inflammatory diseases of the central nervous system (CNS) like multiple sclerosis (MS). Modulation of the BBB could thus represent a therapeutic mechanism. One pathway to modulate BBB integrity could be the induction of nuclear-factor (erythroid derived 2) related factor-2 (Nrf2) mediated oxidative stress responses which are targeted by fumaric acid esters (FAE). Here we analyze effects of FAE on the expression of TJ proteins in the human cerebral endothelial cell line hCMEC/D3 and experimental autoimmune encephalomyelitis (EAE). We show that dimethylfumarate (DMF) and its primary metabolite monomethylfumarate (MMF) induce the expression of the Nrf2/NQO1 pathway in endothelial cells. Neither MMF nor DMF had a consistent modulatory effect on the expression of TJ molecules in hCMEC/D3 cells. Tumor necrosis factor (TNFα)-induced downregulation of TJ proteins was at least partially reversed by treatment with FAE. However, DMF had no effect on claudin-5 expression in EAE, despite its effect on the clinical score and infiltration of immune cells. These data suggest that the modulation of the BBB is not a major mechanism of action of FAE in inflammatory demyelinating diseases of the CNS., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Carvacrol is a natural compound extracted from many plants of the family Lamiaceae. Previous studies have demonstrated that carvacrol has potential neuroprotective effects in central nervous system diseases such as Alzheimer's disease and cerebral ischemia. In this study, we investigated the preclinical effect of carvacrol on cerebral edema after intracerebral hemorrhage (ICH) using a bacterial collagenase-induced ICH mouse model. Mice were randomly divided into sham (n=43), vehicle-treated (n=51), and carvacrol-treated groups (n=101). In carvacrol-treated group, carvacrol was administrated to mice at 0h, 1h, or 3h after ICH induction. Carvacrol was injected intraperitoneally with single doses of 10, 25, 50, or 100mg/kg. Neurologic dysfunctions, brain water content, aquaporins (AQPs) mRNAs level and AQP4 protein expression in the perihematomal area were evaluated post ICH. Our results showed that carvacrol administration improved neurological deficits after day 3 following ICH (p<0.05). Carvacrol reduced cerebral edema and Evans Blue leakage at day 3 (p<0.05). We also found that carvacrol treatment decreased AQP4 mRNA in a dose-dependent manner at 24h. Furthermore, AQP4 protein expression in the perihematomal area was reduced by carvacrol significantly at day 3 after ICH (p<0.05). Our findings suggest that carvacrol may exert its protective effect on ICH injury by ameliorating AQP4-mediated cerebral edema., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Previous studies have demonstrated that the sonic hedgehog (Shh) pathway plays a neuro-protective role. However, whether the Shh pathway is induced by subarachnoid hemorrhage (SAH) has not been investigated. We sought to investigate Shh activation in the cortex in the early stage of SAH, and assessed the effect of cyclopamine (a specific inhibitor of the Shh pathway) on Shh pathway regulation and evaluated the impact of cyclopamine on SAH. We found that the Shh pathway was up-regulated in the cortex after SAH, and that blocking the Shh pathway increased cell apoptosis. Early brain damages, including brain edema, blood-brain barrier impairment, and cortical apoptosis were significantly aggravated following with cyclopamine treatment compared with vehicle treatment. Our results suggest that the Shh pathway should be activated in the brain after SAH, and plays a beneficial role in SAH development, possibly by inhibiting cerebral oxidative stress through induction of antioxidant and detoxifying enzymes., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Because α-synuclein (Snca) is involved in neuroinflammatory response, we determined if its expression altered blood-brain barrier (BBB) permeability. To induce increased BBB permeability, Snca gene-ablated (KO) and wild-type (WT) mice were injected (i.p.) with lipopolysaccharide (LPS). To assess changes in BBB permeability, Evans blue was injected (i.p.) and extravasation into the brain assessed using fluorescence spectroscopy. WT mice had a significant increase in BBB permeability at 1, 3, and 6h post-injection of LPS relative to untreated mice. Contrary to WT mice, LPS did not induce a time-dependent change in BBB permeability in KO mice. Although brain edema is associated with increased BBB permeability, no significant difference in edema was found between groups. These results show that Snca expression is associated with increased reactive opening of the BBB in response to LPS., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background and Purpose: Although the neuroprotective effects of progesterone against early brain injury (EBI) after trauma have been demonstrated in several studies, whether progesterone reduces EBI after subarachnoid hemorrhage (SAH) remains unknown. In this study, we explored the effect of progesterone on cell apoptosis, stability of the blood-brain barrier (BBB), brain edema, and mortality in male Sprague-Dawley rats subjected to subarachnoid hemorrhage-induced EBI by endovascular perforation., Method: Rats (n=66) were randomly assigned to sham, SAH+vehicle, and SAH+progesterone groups. Progesterone (16 mg/kg) or an equal volume of vehicle was administered at 1h, 6h and 12h after SAH. Mortality within 24h, neurological scores, brain edema, Evans blue dye extravasation, cell apoptosis, and the expression of caspase-3 and matrix metalloproteinase (MMP)-9 were assayed after 24h of SAH., Result: Progesterone treatment significantly reduced mortality, brain edema, Evans blue dye extravasation, cell apoptosis, expression of caspase-3 and MMP-9, and improved neurological scores compared with the vehicle group., Conclusion: Progesterone may reduce EBI after SAH by inhibiting cell apoptosis and stabilizing the BBB., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Clavulanic acid is a CNS-modulating compound with exceptional blood-brain barrier permeability and safety profile. Clavulanic acid has been proposed to have anti-depressant activity and is currently entering Phase IIb clinical trials for the treatment of Major Depressive Disorder (MDD). Studies have also shown that clavulanic acid suppresses anxiety and enhances sexual functions in rodent and primate models by a mechanism involving central nervous system (CNS) modulation, although its detailed mechanism of action has yet to be elucidated. To further examine its potential as a CNS modulating agent as well as its mechanism of action, we investigated the effects of clavulanic acid in neuronal cells. Our results indicate that clavulanic acid enhances dopamine release in PC12 and SH-SY5Y cells without affecting dopamine synthesis. Furthermore, using affinity chromatography we were able to identify two proteins, Munc18-1 and Rab4 that potentially bind to clavulanic acid and play a critical role in neurosecretion and the vesicle trafficking process. Consistent with this result, an increase in the translocation of Munc18-1 and Rab4 from the cytoplasm to the plasma membrane was observed in clavulanic acid treated cells. Overall, these data suggest that clavulanic acid enhances dopamine release in a mechanism involving Munc18-1 and Rab4 modulation and warrants further investigation of its therapeutic use in CNS disorders, such as depression., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

It has been previously shown that peroxisome proliferators-activated receptor gamma (PPAR-γ) is beneficial for nervous system injury. In present study, we examined the effect of rosiglitazone, a PPAR-γ agonist, on spinal cord injury (SCI) in rats. SCI was induced by dropping a 10g weight rod at a height of 25mm. The animals were randomly divided into vehicle group, rosiglitazone treated group, and G3335 treated group. Locomotor function recovery was evaluated by the Basso-Beattie-Bresnahan locomotor rating scale (BBB scale), NF-κB expression and endogenous neural progenitor cells (NPCs) proliferation and differentiation was assessed by flow cytometry and immunohistochemistry. Compared with the vehicle groups, we found that the rosiglitazone could significantly ameliorate locomotor recovery, reduce NF-κB expression, and increase the proliferation of endogenous NPCs. when the PPAR-γ antagonist was use, these effects were abolished. However, neurons differentiating from endogenous NPCs were inhibited when PPAR-γ was activated. Our results suggest that the activation of PPAR-γ may be a potential alternative treatment for spinal cord injury., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

This study was performed to investigate the mechanism of the blood-brain tumor-barrier (BTB) permeability increase, which was induced by NS1619, a selective K(Ca) channel activator. Using a rat brain glioma (C6) model, we exam the expression of ZO-1 and occludin in mRNA and protein level at different time point after intracarotid infusion of NS1619 (30 μg/kg/min) to tumor sites via RT-PCR and Western blot analysis. The mRNA and protein expression of ZO-1 and occludin had no great change before infusion and began to decrease significantly after 2 h NS1619 infusion, which was significantly attenuated by reactive oxygen species (ROS) scavenger (N-2-mercaptopropionyl glycine, MPG). In addition, MPG also significantly inhibited the increase of BTB permeability and malonaldehyde (MDA) level induced by NS1619. This led to the conclusion that NS1619 could time-dependently increase the BTB permeability by down-regulating the expression of tight junction protein, and this effect could be reversed by ROS., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Brain glioma is a malignant tumor which needs surgery followed by chemotherapy. Low-frequency ultrasound (LFU) and Optison could open blood-tumor barrier (BTB) selectively and noninvasively and thus increase the permeability of BTB. Endothelial monocyte-activating polypeptide II (EMAP-II) induces cytoskeletal remodeling in endothelial cells. In this study, we asked whether LFU, Optison, and/or EMAP-II used in combination have additive effects on increasing the permeability of BTB by tight junction (TJ)-associated protein-dependent manner and thus help understand the possible mechanisms for TJ-based drug delivery to the central nervous system through BTB. Evans Blue assay was used to measure the permeability of BTB in rat model of C6 glioma. The mRNA and protein levels of TJ-associated proteins, claudin-5, occludin, and ZO-1, were determined. Results showed that Evans blue content significantly increased and the mRNA and protein levels of claudin-5, occludin, and ZO-1 significantly reduced after the treatment in groups treated with EMAP-II and LFU combined with or without Optison (LFU+EMAP-II and LFU+Optison+EMAP-II groups) and in the group treated with LFU and Optison (LFU+Optison group). In conclusion, LFU and EMAP-II used in combination have additive effects on increasing the permeability of BTB and remodeling of TJ-associated proteins., (2010 Elsevier Ireland Ltd. All rights reserved.)

The blood-brain barrier (BBB) maintains the homeostasis between the central nervous system and the blood circulation. One of the main efflux transporter proteins at the BBB is P-glycoprotein (P-gP) also known as ABCB1 or MDR1. Due to the important role of P-gP for the transport barrier function of the BBB, the presence and functionality of P-gP was investigated in porcine cell line PBMEC/C1-2. Presence of P-gP was confirmed on the protein level by western blotting and immunofluorescence microscopy as well as on the mRNA level by qPCR. Functional assessment was accomplished by an established 96-well uptake assay using Rhodamine 123 and Doxorubicin as P-gP substrates and Verapamil as moderate P-gP inhibitor. In this regard, fluorescence microscopy confirmed a significant higher uptake of Rhodamine 123 into PBMEC/C1-2 cells when preincubated with Verapamil. Finally, knock-down of P-gP by antisense oligonucleotides revealed an increase of Rhodamine 123 uptake indicating decreased P-gP functionality. In summary, the presence and functionality of P-gP in the immortalised cell line PBMEC/C1-2 was proven with several techniques and assays. Thus, this cell line could be used for P-gP studies in the context of BBB relevant issues., ((c) 2009 Elsevier Ireland Ltd. All rights reserved.)

The selective agonist of serotonin 5-HT(3) receptor 1-(3-chlorophenyl)biguanide hydrochloride (m-CPBG) administered intracerebroventricularly (40, 80 or 160 nmol) produced long-lasting dose-dependent hypothermic response in AKR/2J mice. m-CPBG (160 nmol i.c.v.) induced profound hypothermia (delta t=-4 degrees C) that lasted up to 7 h. m-CPBG (40 nmol i.c.v.)-induced hypothermia was attenuated by 5-HT(3) receptor antagonist ondansetron pretreatment. At the same time, intraperitoneal administration of m-CPBG in a wide range of doses (0.5, 1.0, 5.0 or 10.0 mg/kg) did not affect the body temperature. These findings indicate: (1) the implication of central, rather than peripheral 5-HT(3) receptor in the thermoregulation; (2) the inability of m-CPBG to cross blood-brain barrier in mice. The comparison of brain 5-HT(3)-induced hypothermic reaction in six inbred mouse strains (DBA/2J, CBA/Lac, C57BL/6, BALB/c, ICR, AKR/J) was performed and two highly sensitive to m-CPBG strains (CBA/Lac and C57BL/6) were found. In the same six mouse strains the functional activity of 5-HT(1A) receptor was studied. The comparison of hypothermic reactions produced by 5-HT(1A) receptor agonist 8-OH-DPAT (1.0 mg/kg i.p.) and m-CPBG revealed significant correlation between 5-HT(3) and 5-HT(1A)-induced hypothermia in five out of six investigated mouse strains. 5-HT(1A) receptor antagonist p-MPPI pretreatment (1 mg/kg i.p.) diminished hypothermia produced by centrally administered m-CPBG (40 nmol i.c.v.). The data suggest the cross-talk between 5-HT(1A) and 5-HT(3) receptors in the mechanism of 5-HT-related hypothermia.

This study was performed to determine whether dexamethasone (DEX) had an effect on calcium-activated potassium channels (K(Ca) channels) and Occludin protein in blood-brain tumor barrier (BTB). Using a rat brain glioma model, we found that the expression of K(Ca) channels protein and Occludin protein was significantly increased in brain tumor tissue after DEX treatment for 3 days. Compared with non-DEX-treated animals, Evans Blue levels were greatly attenuated in DEX-treated animals. These effects were significantly reversed by the glucocorticoid receptor antagonist RU38486. In addition, DEX treatment enhanced the density of I(KCa) in the rat brain microvascular endothelial cells (RBMECs) in vitro BTB. All of these results strongly suggest that DEX could be involved in the regulation of both transcellular and paracellular pathway.

Bradykinin has been shown to increase the permeability of blood-tumor barrier (BTB) selectively. This study was performed to determine whether tumor necrosis factor-alpha (TNF-alpha) was involved in the regulation of this biological process. We found that the levels of TNF-alpha mRNA and heat shock factor-1 (HSF1) protein in C6 cells were markedly up-regulated by bradykinin via real-time RT-PCR and Western blot methods. And the most obvious increase of HSF1 protein and TNF-alpha mRNA in C6 cells were observed at 5 min and 10 min of bradykinin perfusion, respectively. In addition, the radioactivity of TNF-alpha in C6 cells' culture fluid also mostly increased at 15 min of bradykinin perfusion. And the Evans blue content of brain tumor tissues in rats and the concentration of TNF-alpha reached the maximum at 15 min of bradykinin perfusion. Our results suggested that the bradykinin-mediated BTB permeability increase is due to accelerated release of TNF-alpha, which could cause the increase of BTB permeability by promoting to the release HSF1 from neurospongioma cells.

The aim of the present study was to examine the signaling pathways of hypoxia followed by reoxygenation (H/R)-induced disruption of the blood-brain-barrier (BBB) in a co-culture of astrocytes and brain endothelial cells (BEC) in vitro. We analyzed the possible stabilizing effect of MK801, a highly selective N-methyl-d-aspartate receptor (NMDAR) antagonist, on BBB integrity. Levels of reactive oxygen species (ROS), glutamate (Glut) release and monocyte adhesion were measured under normoxia and H/R. BBB integrity was monitored measuring the trans-endothelial electrical resistance (TEER). TEER values dropped under H/R conditions which was abolished by MK801. Glut release from astrocytes, but not from endothelial cells was significantly increased under H/R, as were ROS levels and monocyte adhesion. The oxidative stress was blocked by MK801 and the NAD(P)H-oxidase inhibitor apocynin. We observed that calcium (Ca(2+)) signaling plays a crucial role during ROS generation and monocyte adhesion under H/R. ROS levels were decreased by applying ryanodine, a blocker of Ca(2+) release from the endoplasmic reticulum (ER) and by lowering the extracellular Ca(2+) concentration. Xestospongin C, which blocks IP(3) mediated Ca(2+) release from the ER did not alter ROS production under H/R conditions. These findings indicate that both extracellular Ca(2+) influx and ryanodine-mediated intracellular Ca(2+) release from the ER during H/R contribute to ROS formation at the BBB. Blocking ROS or Ca(2+) signaling prevented H/R-induced monocyte adhesion to BEC. We conclude, that the activation of NMDAR under H/R by Glut increases intracellular Ca(2+) levels, contributes to BBB disruption, ROS generation and monocyte adhesion.

The aim of the present study was to assess the potential of delivering VEGF directly into the central nervous system (CNS) following intranasal administration. Adult Sprague-Dawley rats were randomized into two groups, given [(125)I]-VEGF intranasally or intravenously. VEGF was intranasally administered in both nares alternately, the single dose is 10 microl with time interval of 2 min for about 18.5 min. The intravenous (IV) group was treated with 100 microl [(125)I]-VEGF intravenously. Thirty minutes after administration, rats were killed following blood sample collections, then the brains were removed, and olfactory bulb, striatum corpora, cortex, thalamus, pons, cerebella, medulla, hippocampus, cervical cord and other tissues were collected, weighted, under auto gamma counting and autoradiography analysis. Cisternal sampling of cerebrospinal fluid (CSF) was performed in an additional group of animals. Both gamma counting and high resolution phosphor imaging of tissue sections showed that intranasal administration of [(125)I]-VEGF resulted in substantial delivery throughout the CNS. The highest CNS tissue concentration following IN delivery was found in the trigeminal nerve, followed by the optic nerve, olfactory bulbs, olfactory tubercle, striatum, medulla, frontal cortex, midbrain, pons, appendix cerebri, thalamus, hippocampus, cerebellum. Intranasal administration of [(125)I]-VEGF also targeted the deep cervical lymph nodes. CSF did not contain [(125)I]-VEGF following intranasal administration. Intravenous [(125)I]-VEGF resulted in blood and peripheral tissue exposure higher concentrations than that intranasal administration, but CNS concentrations were significantly lower. The results suggest intranasally delivered VEGF can bypass the blood-brain barrier via olfactory- and trigeminal-associated extracellular pathways to directly entry into the CNS. Intranasal administration of VEGF may provide an effective way for the treatments of CNS diseases.

The study investigated the effect of aminoguanidine (AG) on surgical brain injury (SBI) in rat. AG (75, 150 and 300 mg/kg, i.p.) was administered immediately following surgical resection. Using a SBI model, we found that AG (150 mg/kg) significantly reduced cerebral edema, while AG at the doses of 75 and 300 mg/kg had no effect. And AG (150 mg/kg) significantly reduced Evans Blue extravasation into brain tissue and improved the neurological outcome compared to control group. Moreover, the expression of TNF-alpha and nuclear factor-kappaB (NF-kappaB) mRNA and protein in brain tissue at the edge of the resection site increased at 24h after SBI, which could be significantly attenuated by the treatment with AG via RT-PCR and Western blots methods. Our results demonstrated that SBI causes increased brain edema, BBB disruption and inflammation along the periphery of the site of surgical resection, which could be significantly improved by the treatment of AG.

Up to now no standard cell culture model of the blood-brain barrier is available. However, several models based on primary cells or continuous cell lines have been characterized and described in respect of different applications. One of the most important characteristics of the blood-brain barrier is the restriction of paracellular transport, respectively its tightness. Human cell line ECV304 is one of the promising continuous cell lines for blood-brain barrier modelling due to two reasons: on the one hand the cells are able to form significant tighter layers than most of the other cell lines used and on the other hand several properties of the blood-brain barrier are inducible by using glioma-conditioned medium. Claudins are transmembranal proteins which form the backbone of the tight junctions at the blood-brain barrier. We have investigated the presence and inducibility of the expression of Claudin-1, Claudin-3 and Claudin-5 using immunofluorescence microscopy. For the first time this study proves the presence of Claudin-1, Claudin-3 and Claudin-5 in ECV304 (obtained from ECACC) cell layers and the inducibility of their expression by glioma-conditioned media.

Aluminum and zinc are two important trace elements in an organism. Although several studies have demonstrated their impacts on the intelligence, very little was known about their effects on the integrity of blood-brain barrier (BBB). To study the effects of aluminum and zinc on the permeability of BBB, different doses of aluminum and appropriate zinc were administered to rats. Evans blue was detected in brain to determine the permeability of BBB. The ultrastructure of BBB was observed under the transmission electron microscope. Immunohistochemistry and Western blot method were used to detect the expression of skeleton protein F-actin and tight junction protein occludin in brain capillary endothelium. The data indicated that compared with the control group, Evans blue in brains increased (P < 0.01), the ultrastructure of BBB changed and the expression of F-actin and occludin decreased (P < 0.01) in the aluminum-toxic group. Compared with the aluminum-toxic groups, the permeability of BBB to Evans blue decreased (P < 0.01), the damage of the BBB ultrastructure was attenuated and the expression of F-actin and occludin increased (P < 0.05) in the aluminum-zinc group. Our present studies suggest that aluminum increases the permeability of BBB by changing its ultrastructure and the expression of occludin and F-actin. Zinc can protect the integrity of BBB in juvenile rats that are exposed to aluminum and inhibit the decrease of tight junction protein occludin and F-actin expression in BBB.

Parkinson's disease is a neurodegenerative disorder that requires treatment by dopaminergic agonists, which may be responsible for central side effects. We hypothesized that the efflux transporter ABCB1/P-glycoprotein played a role in brain disposition of antiparkinsonian drugs and could control central toxicity. We aimed to evaluate antiparkinsonian drugs as ABCB1 substrates and/or inhibitors in rat brain endothelial cells GPNT, in order to predict potential clinical drug-drug interactions. Among the antiparkinsonian drugs tested, levodopa, bromocriptine, pergolide and pramipexole were ABCB1 substrates. However, only bromocriptine could inhibit ABCB1 functionality with an IC(50) of 6.71 microM on Rhodamine 123 uptake and an IC(50) of 1.71 microM on digoxine uptake. Thus, bromocriptine at 100 microM is responsible for an increase of levodopa intracellular transport of about 2.05-fold versus control. Therefore, we can conclude that bromocriptine is a potent drug for medicinal interactions in vitro. Hence, in patients with Parkinson's disease, these results may be considered to optimise treatments individually.

To investigate whether long-term exposure to four typical antiepileptic drugs (AEDs): phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ) and valproic acid (VPA) can increase P-glycoprotein (P-gp) level and function in primary cultured rat brain microvascular endothelial cells (rBMECs) in vitro, the rBMECs were incubated in culture medium containing indicated drugs (PB, PHT, CBZ, VPA and rifampin) for 60 days in a gradient concentration manner. Age-matched cells were incubated in normal culture medium. After a 60-day exposure to the indicated drugs, P-gp function and level in cells were measured using rhodamine 123 (Rho123) accumulations and Western blot analysis, respectively. Lower Rho123 accumulation in drug-treated cells was found than that in age-matched cells. Cyclosporin A (CsA) and verapamil (Ver) increased Rho123 accumulation both in drug-treated cells and age-matched cells. The magnitude of increased Rho123 accumulation in drug-treated cells was larger than that in age-matched cells. Higher P-gp levels were found to be consistent with decrease of Rho123 accumulation in drug-treated cells. The results verified the hypothesis that long-term exposure to the four antiepileptic drugs can induce P-gp function and level in rBMECs.

The scorpion envenoming syndrome is a serious public health matter in Brazil. The most severe cases occur during childhood and elderly. Previous results from our laboratory suggest that the effects of scorpion toxins on the central nervous system play a major role on the lethality induced by scorpion envenoming. The aim of this work is to evaluate the therapeutic potential of carbamazepine (CBZ) injected i.p. 90 min before s.c. tityustoxin (TsTX) injection in weanling rats. Rats were divided into six experimental groups according to s.c. injection (saline or TsTX) and i.p. treatment (vehicle or CBZ 12, 50 and 100 mg/kg): Sal/Veh group (n=4); Sal/CBZ100 (n=4); TsTX/CBZ12 (n=6); TsTX/CBZ50 (n=8); TsTX/CBZ100 (n=8) and, at last, TsTX/Veh (n=8). The dose of TsTX was the same for all groups: 6.0mg/kg, twice the DL50 for weanling rats. Video images were recorded until death or for a maximum period of 240 min. Lungs were excised and weighed to evaluate edema. The results showed that CBZ (12, 50 and 100mg/kg) was able to increase the survival rate and latency-to-death of the rats. Only the group treated with 100mg/kg of CBZ had a decrease in the pulmonary edema. The known effect of CBZ reducing neuronal excitability most likely protected the neural substrates targeted by TsTX. Although treatment was performed before TsTX inoculation, the results are promising regarding CBZ as a therapeutic coadjuvant in the treatment of scorpion poisoning. The pharmacokinetics of CBZ can be very much improved by either changing the form of administration or encapsulating the drug in order to enhance solubility.

P-glycoprotein (P-gp) is an important factor at the blood-brain barrier preventing passage of a wide variety of substances into the brain. Several studies have provided evidence that some drugs and certain steroid hormones are substrates or inhibitors of P-gp. However, the situation is unclear with regard to gonadal steroids, which have considerable central nervous effects. In vitro, experiments on the relationship between estradiol and P-gp are equivocal. We used abcb1ab knock-out mice and wild-type mice to determine the uptake of [3H]-17-beta-estradiol and [3H]-testosterone into the cerebrum and other organs after subcutaneous administration. The organ/plasma quotients showed no significant group effects. We concluded that P-gp does not influence the penetration of testosterone and estradiol to a biologically significant extent.

Erythropoietin (EPO) has recently been shown to have a neuroprotective effect in animal models of traumatic brain injury (TBI). However, the precise mechanisms remain unclear. Cerebral inflammation plays an important role in the pathogenesis of secondary brain injury after TBI. We, therefore, tried to analyze how recombinant human erythropoietin (rhEPO) might effect the inflammation-related factors common to TBI: nuclear factor kappa B (NF-kappaB), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) in a rat TBI model. Male rats were given 0 or 5000 units/kg injections of rhEPO 1h post-injury and on days 1, 2 and 3 after surgery. Brain samples were extracted at 3 days after trauma. We measured NF-kappaB by electrophoretic mobility shift assay (EMSA); IL-1beta, TNF-alpha and IL-6 by enzyme-linked immunosorbent assay (ELISA); ICAM-1 by immunohistochemistry; brain edema by wet/dry method; blood-brain barrier (BBB) permeability by Evans blue extravasation and cortical apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. We found that NF-kappaB, pro-inflammatory cytokines and ICAM-1 were increased in all injured animals. In animals given rhEPO post-TBI, NF-kappaB, IL-1beta, TNF-alpha and ICAM-1 were decreased in comparison to vehicle-treated animals. Measures of IL-6 showed no change after rhEPO treatment. Administration of rhEPO reduced brain edema, BBB permeability and apoptotic cells in the injured brain. In conclusion, post-TBI rhEPO administration may attenuate inflammatory response in the injured rat brain, and this may be one mechanism by which rhEPO improves outcome following TBI.

The systemic administration of nitroglycerine, regarded as a migraine model, was previously observed to result in an increased number of c-fos immunoreactive secondary sensory neurons in the caudal trigeminal nucleus, which forward nociceptive impulses to the thalamus. The present investigation tested the hypothesis of whether kynurenine in combination with systemically administered probenecid protects second-order trigeminal neurons against stimulation arriving via central processes of trigeminal ganglion cells. Electrical stimulation of the trigeminal ganglion, one of the experimental migraine models, is known to induce an increase in the number of c-fos immunoreactive second-order nerve cells projecting to the thalamus. Since the synapses between first- and second-order trigeminal neurons are presumed to be mediated by excitatory amino acids, postsynaptic NMDA receptors should be inhibited by kynurenic acid, an endogenous NMDA receptor antagonist. Kynurenic acid, however, does not cross the blood-brain barrier, and its use as a neuroprotective agent is therefore not feasible. In contrast, kynurenine, from which kynurenic acid is formed on the action of kynurenine aminotransferase, passes the blood-brain barrier without difficulty. After the i.p. injection of kynurenine combined with probenecid it was found that the stimulation-induced increase in the c-fos immunoreactivity of the secondary sensory neurons does not occur.