Bumetanide inhibition of the blood-brain barrier Na-K-Cl cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke.

Increased transport of Na+ across an intact blood-brain barrier (BBB) participates in edema formation during the early hours of cerebral ischemia. In previous studies, the authors showed that the BBB Na-K-Cl cotransporter is stimulated by factors present during ischemia, suggesting that the cotransporter may contribute to the increased brain Na+ uptake in edema. The present study was conducted to determine (1) whether the Na-K-Cl cotransporter is located in the luminal membrane of the BBB, and (2) whether inhibition of the BBB cotransporter reduces brain edema formation. Perfusion-fixed rat brains were examined for cotransporter distribution by immunoelectron microscopy. Cerebral edema was evaluated in rats subjected to permanent middle cerebral artery occlusion (MCAO) by magnetic resonance diffusion-weighted imaging and calculation of apparent diffusion coefficients (ADC). The immunoelectron microscopy studies revealed a predominant (80%) luminal membrane distribution of the cotransporter. Magnetic resonance imaging studies showed ADC ratios (ipsilateral MCAO/contralateral control) ranging from 0.577 to 0.637 in cortex and striatum, indicating substantial edema formation. Intravenous bumetanide (7.6-30.4 mg/kg) given immediately before occlusion attenuated the decrease in ADC ratios for both cortex and striatum (by 40-67%), indicating reduced edema formation. Bumetanide also reduced infarct size, determined by TTC staining. These findings suggest that a luminal BBB Na-K-Cl cotransporter contributes to edema formation during cerebral ischemia.     

Tight junctions in the blood–brain barrier promote edema formation and infarct size in stroke – Ambivalent effects of sealing proteins

The outcome of stroke is greatly influenced by the state of the blood–brain barrier (BBB). The BBB endothelium is sealed paracellularly by tight junction (TJ) proteins, i.e., claudins (Cldns) and the redox regulator occludin. Functions of Cldn3 and occludin at the BBB are largely unknown, particularly after stroke. We address the effects of Cldn3 deficiency and stress factors on the BBB and its TJs. Cldn3 tightened the BBB for small molecules and ions, limited endothelial endocytosis, strengthened the TJ structure and controlled Cldn1 expression. After middle cerebral artery occlusion (MCAO) and 3-h reperfusion or hypoxia of isolated brain capillaries, Cldn1, Cldn3 and occludin were downregulated. In Cldn3 knockout mice (C3KO), the reduction in Cldn1 was even greater and TJ ultrastructure was impaired; 48 h after MCAO of wt mice, infarct volumes were enlarged and edema developed, but endothelial TJs were preserved. In contrast, junctional localization of Cldn5 and occludin, TJ density, swelling and infarction size were reduced in affected brain areas of C3KO. Taken together, Cldn3 and occludin protect TJs in stroke, and this keeps the BBB intact. However, functional Cldn3, Cldn3-regulated TJ proteins and occludin promote edema and infarction, which suggests that TJ modulation could improve the outcome of stroke.     

A Lipoxin A<Subscript>4</Subscript> Analog Ameliorates Blood–Brain Barrier Dysfunction and Reduces MMP-9 Expression in a Rat Model of Focal Cerebral Ischemia–Reperfusion Injury

LXA₄ methyl ester (LXA₄ME), a lipoxin A₄ analog, reduces ischemic insult in the rat models of transient or permanent cerebral ischemic injury. We investigated whether LXA₄ME could ameliorate blood–brain barrier (BBB) dysfunction after stroke by reducing matrix metalloproteinase (MMP)-9 expression. Adult male rats were subjected to 2-h middle cerebral artery occlusion (MCAO) followed by 24-h reperfusion. Brain infarctions were detected by triphenyltetrazolium chloride (TTC) staining. BBB dysfunction was determined by examining brain edema and Evans Blue extravasation. Temporal expression of MMP-9 was determined by zymography and Western blot. The presence of tissue inhibitors of metalloproteinase-1 (TIMP-1) was also determined by Western blot in tissue protein sample. Brain edema and Evans Blue leakage were significantly reduced after stroke in the LXA₄ME group and were associated with reduced brain infarct volumes. MMP-9 activity and expression were inhibited by LXA₄ME after stroke. In addition, LXA₄ME significantly increased TIMP-1 protein levels. Our results indicate that LXA₄ME reduces brain injury by improving BBB function in a rat model of MCAO, and that a relationship exists between BBB permeability and MMP-9 expression following ischemic insult. Furthermore, these results suggest that LXA₄ME-mediated reduction of MMP-9 following stroke are attributed to increased TIMP-1 expression.     

Role of arginine vasopressin V1 and V2 receptors for brain damage after transient focal cerebral ischemia.

Brain edema formation is one of the most important mechanisms responsible for brain damage after ischemic stroke. Despite considerable efforts, no specific therapy is available yet. Arginine vasopressin (AVP) regulates cerebral water homeostasis and has been involved in brain edema formation. In the current study, we investigated the role of AVP V1 and V2 receptors on brain damage, brain edema formation, and functional outcome after transient focal cerebral ischemia, a condition comparable with that of stroke patients undergoing thrombolysis. C57/BL6 mice were subjected to 60-min middle cerebral artery occlusion (MCAO) followed by 23 h of reperfusion. Five minutes after MCAO, 100 or 500 ng of [deamino-Pen(1), O-Me-Tyr(2), Arg(8)]-vasopressin (AVP V1 receptor antagonist) or [adamantaneacetyl(1), O-Et-D-Tyr(2), Val(4), Abu(6), Arg(8,9)]-vasopressin (AVP V2 receptor antagonist) were injected into the left ventricle. Inhibition of AVP V1 receptors reduced infarct volume in a dose-dependent manner by 54% and 70% (to 29+/-13 and 19+/-10 mm3 versus 63+/-17 mm3 in controls; P<0.001), brain edema formation by 67% (to 80.4%+/-1.0% versus 82.7%+/-1.2% in controls; P<0.001), blood-brain barrier disruption by 75% (P<0.001), and functional deficits 24 h after ischemia, while V2 receptor inhibition had no effect. The current findings indicate that AVP V1 but not V2 receptors are involved in the pathophysiology of secondary brain damage after focal cerebral ischemia. Although further studies are needed to clarify the mechanisms of neuroprotection, AVP V1 receptors seem to be promising targets for the treatment of ischemic stroke.     

Endothelin-1 overexpression leads to further water accumulation and brain edema after middle cerebral artery occlusion via aquaporin 4 expression in astrocytic end-feet.

Stroke patients have increased levels of endothelin-1 (ET-1), a strong vasoconstrictor, in their plasma or cerebrospinal fluid. Previously, we showed high level of ET-1 mRNA expression in astrocytes after hypoxia/ischemia. It is unclear whether the contribution of ET-1 induction in astrocytes is protective or destructive in cerebral ischemia. Here, we generated a transgenic mouse model that overexpress ET-1 in astrocytes (GET-1) using the glial fibrillary acidic protein promoter to examine the role of astrocytic ET-1 in ischemic stroke by challenging these mice with transient middle cerebral artery occlusion (MCAO). Under normal condition, GET-1 mice showed no abnormality in brain morphology, cerebrovasculature, absolute cerebral blood flow, blood-brain barrier (BBB) integrity, and mean arterial blood pressure. Yet, GET-1 mice subjected to transient MCAO showed more severe neurologic deficits and increased infarct, which were partially normalized by administration of ABT-627 (ET(A) antagonist) 5 mins after MCAO. In addition, GET-1 brains exhibited more Evans blue extravasation and showed decreased endothelial occludin expression after MCAO, correlating with higher brain water content and increased cerebral edema. Aquaporin 4 expression was also more pronounced in astrocytic end-feet on blood vessels in GET-1 ipsilateral brains. Our current data suggest that astrocytic ET-1 has deleterious effects on water homeostasis, cerebral edema and BBB integrity, which contribute to more severe ischemic brain injury.     

Effect of duration of osmotherapy on blood-brain barrier disruption and regional cerebral edema after experimental stroke.

Osmotherapy is the cornerstone of medical management for cerebral edema associated with large ischemic strokes. We determined the effect of duration of graded increases in serum osmolality with mannitol and hypertonic saline (HS) on blood-brain barrier (BBB) disruption and regional cerebral edema in a well-characterized rat model of large ischemic stroke. Halothane-anesthetized adult male Wistar rats were subjected to transient (2-h) middle cerebral artery occlusion (MCAO) by the intraluminal occlusion technique. Beginning at 6 h after MCAO, rats were treated with either no intravenous fluids or a continuous intravenous infusion (0.3 mL/h) of 0.9% saline, 20% mannitol, 3% HS, or 7.5% HS for 24, 48, 72, and 96 h. In the first series of experiments, BBB permeability was quantified by the Evans blue (EB) extravasation method. In the second series of experiments, water content was assessed by comparing wet-to-dry weight ratios in six predetermined brain regions. Blood-brain barrier disruption was maximal in rats treated with 0.9% saline for 48 h, but did not correlate with increases in serum osmolality or treatment duration with osmotic agents. Treatment with 7.5% HS attenuated water content in the periinfarct regions and all subregions of the contralateral nonischemic hemisphere to a greater extent than mannitol did with no adverse effect on survival rates. These data show that (1) BBB integrity is not affected by the duration and degree of serum osmolality with osmotic agents, and (2) attenuation of increases in brain water content with HS to target levels >350 mOsm/L may have therapeutic implications in the treatment of cerebral edema associated with ischemic stroke.     

Cerebral mast cells regulate early ischemic brain swelling and neutrophil accumulation.

We previously observed degranulated mast cells (MC) in association with perivascular brain edema formation during focal cerebral ischemia. Brain MC are typically located perivascularly and contain potent fast-acting vasoactive and proteolytic substances. We examined in a rat model of transient middle cerebral artery occlusion (MCAO) whether, in the early phase of ischemia, MC regulate microcirculation, the blood-brain barrier (BBB) permeability, and edema formation. First, animals received MC inhibitor (cromoglycate), MC-degranulating drug (compound 48/80), or saline. Thereafter, we performed transient MCAO in gene-manipulated MC-deficient rats and their wild-type (WT) littermates, calculating brain swelling, visualizing BBB leakage by intravenously administered Evans blue albumin, and determining neutrophil infiltration with light microscopy. Cerebral blood flow, monitored by laser-Doppler flowmetry in separate experiments, was similar among pharmacological treatments. Ischemic swelling resulted in increased hemispheric volume of 13.4%+/-1.0% in controls, 8.1%+/-0.4% (39% reduction) after cromoglycate, and 25.2%+/-2.0% (89% increase) after compound 48/80 (P<0.05). Early ischemic BBB leakage was reduced by 51% after cromoglycate, and 50% enhanced by compound 48/80 (P<0.05). The cromoglycate group showed 37% less postischemic neutrophil infiltration than did controls (P<0.05). Furthermore, MC-deficient rats responded to focal ischemia with 58% less brain swelling (6.7%+/-1.2%) than did their WT littermates (15.8%+/-1.4%, P<0.05). Blood-brain barrier damage was 47% lower in MC-deficient rats than in the WT (P<0.05). Neutrophil infiltration after MCAO was decreased 47% in MC-deficient rats in comparison to WT (P<0.05). Pharmacological MC inhibition thus appears to deserve further investigation regarding reduction of brain swelling and inflammation early after stroke.     

N‐myc downstream‐regulated gene 2 protects blood–brain barrier integrity following cerebral ischemia

Disruption of the blood‐brain barrier (BBB) following cerebral ischemia is closely related to the infiltration of peripheral cells into the brain, progression of lesion formation, and clinical exacerbation. However, the mechanism that regulates BBB integrity, especially after permanent ischemia, remains unclear. Here, we present evidence that astrocytic N‐myc downstream‐regulated gene 2 (NDRG2), a differentiation‐ and stress‐associated molecule, may function as a modulator of BBB permeability following ischemic stroke, using a mouse model of permanent cerebral ischemia. Immunohistological analysis showed that the expression of NDRG2 increases dominantly in astrocytes following permanent middle cerebral artery occlusion (MCAO). Genetic deletion of Ndrg2 exhibited enhanced levels of infarct volume and accumulation of immune cells into the ipsilateral brain hemisphere following ischemia. Extravasation of serum proteins including fibrinogen and immunoglobulin, after MCAO, was enhanced at the ischemic core and perivascular region of the peri‐infarct area in the ipsilateral cortex of Ndrg2‐deficient mice. Furthermore, the expression of matrix metalloproteinases (MMPs) after MCAO markedly increased in Ndrg2^?/? mice. In culture, expression and secretion of MMP‐3 was increased in Ndrg2^?/? astrocytes, and this increase was reversed by adenovirus‐mediated re‐expression of NDRG2. These findings suggest that NDRG2, expressed in astrocytes, may play a critical role in the regulation of BBB permeability and immune cell infiltration through the modulation of MMP expression following cerebral ischemia.     

Clematichinenoside protects blood brain barrier against ischemic stroke superimposed on systemic inflammatory challenges through up-regulating A20

Suppression of excessive inflammation can ameliorate blood brain barrier (BBB) injury, which shows therapeutic potential for clinical treatment of brain injury induced by stroke superimposed on systemic inflammatory diseases. In this study, we investigated whether and how clematichinenoside (AR), an anti-inflammatory triterpene saponin, protects brain injury from stroke superimposed on systemic inflammation. Lipopolysaccharide (LPS) was intraperitoneally injected immediately after middle cerebral artery occlusion (MCAO) in rats. Rat microvessel endothelial cells (rBMECs) were exposed to hypoxia/reoxygenation (H/R) coexisting with LPS. The results revealed that AR suppressed the excessive inflammation, restored BBB dysfunction, alleviated brain edema, decreased neutrophil infiltration, lessened neurological dysfunction, and decreased infarct rate. Further study demonstrated that the expression of nucleus nuclear factor kappa B (NF-κB), inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α) and interlukin-1β (IL-1β) were suppressed by AR via zinc finger protein A20. Besides, AR increased in vitro BBB integrity through A20. In conclusion, AR alleviated cerebral inflammatory injury through A20-NF-κB signal pathway, offering an alternative medication for stroke associated with systemic inflammatory diseases.     

血脑屏障内皮细胞与细胞间粘附分子-1在鼠脑缺血性脑水肿发生机制中的作用

目的研究缺血性脑水肿病理过程中血脑屏障内皮细胞及其细胞间粘附分子-1(intercellularadhesionmolecule-1,ICAM-1)的表达与血脑屏障通透性的关系.方法用电镜技术和免疫组织化学方法观察脑缺血过程中大鼠血脑屏障内皮细胞超微结构及其ICAM-1的表达和对镧离子的通透性改变.结果脑缺血后1h,缺血区脑组织血脑屏障内皮细胞未见ICAM-1表达,但电镜下可见内皮细胞轻度肿胀.3h,ICAM-1开始表达,缺血区脑组织轻度水肿,内皮细胞肿胀.至12h,ICAM-1表达达到高峰,脑组织严重水肿,内皮细胞、神经细胞及胶质细胞明显肿胀;内皮细胞胞饮小泡包含有被吞噬的镧离子,有些镧离子进入紧密连接间隙.72h,ICAM-1表达已明显减少,脑组织水肿仍较重,内皮细胞严重破坏,大量镧离子漏出血管外,进入脑实质.结论脑缺血后血脑屏障的破坏与ICAM-1的表达密切相关,提示ICAM-1参与血脑屏障内皮细胞的破坏过程,而且这些改变在缺血性脑水肿的病理过程中起重要作用.     

Attenuation of ischemic brain edema and cerebrovascular injury after ischemic preconditioning in the rat.

Ischemic preconditioning (IPC) induces neuroprotection to subsequent severe ischemia, but its effect on the cerebrovasculature has not been studied extensively. This study evaluated the effects of IPC on brain edema formation and endothelial cell damage that follows subsequent permanent focal cerebral ischemia in the rat. Transient (15 minute) middle cerebral artery occlusion (MCAO) was used for IPC. Three days after IPC or a sham operation, permanent MCAO was induced. Twenty-four hours after permanent MCAO, neurologic deficit, infarction volume, and water and ion content were evaluated. Six hours post-ischemia, blood-brain barrier (BBB) permeability was examined using [3H]-inulin. Water, ion contents, and BBB permeability were assessed in three zones (core, intermediate, and outer) depending on their relation to the MCA territory. Heat shock protein 70 (HSP70) was also examined as a potential marker of vascular injury. The model of IPC significantly reduced brain infarction and neurologic deficit. Compared with a sham operation, IPC also significantly attenuated brain edema formation in the intermediate (sham and IPC water contents: 5.99+/-0.65 vs. 4.99+/-0.81 g/g dry weight; P < 0.01) and outer zones (5.02+/-0.48 vs. 4.37+/-0.42 g/g dry weight; P < 0.01) of the ipsilateral hemisphere but not in the core zone. Blood-brain barrier disruption assessed by [3H]-inulin was significantly attenuated in the IPC group and the number of blood vessels that displayed HSP70 immunoreactivity was also reduced. Thus, IPC significantly attenuates ischemic brain edema formation, BBB disruption, and, as assessed by HSP70, vascular injury. Understanding the mechanisms involved in IPC may provide insight into methods for preserving cerebrovascular function during ischemia.     

Cerebralcare Granule® attenuates blood-brain barrier disruption after middle cerebral artery occlusion in rats

Disruption of blood–brain barrier (BBB) and subsequent edema are major contributors to the pathogenesis of ischemic stroke, for which the current clinical therapy remains unsatisfied. Cerebralcare Granule® (CG) is a compound Chinese medicine widely used in China for treatment of cerebrovascular diseases. CG has been demonstrated efficacy in attenuating the cerebral microcirculatory disturbance and hippocampal neuron injury following global cerebral ischemia. However, the effects of CG on BBB disruption following cerebral ischemia have not been investigated. In this study, we examined the therapeutic effect of CG on the BBB disruption in a focal cerebral ischemia/reperfusion (I/R) rat model. Male Sprague–Dawley rats (250 to 300 g) were subjected to 1 h middle cerebral artery occlusion (MCAO). CG (0.4 g/kg or 0.8 g/kg) was administrated orally 3 h after reperfusion for the first time and then once daily up to 6 days. The results showed that Evans blue extravasation, brain water content, albumin leakage, infarction volume and neurological deficits increased in MCAO model rats, and were attenuated significantly by CG treatment. T2-weighted MRI and electron microscopy further confirmed the brain edema reduction in CG-treated rats. Treatment with CG improved cerebral blood flow (CBF). Western blot analysis and confocal microscopy showed that the tight junction proteins claudin-5, JAM-1, occludin and zonula occluden-1 between endothelial cells were significantly degradated, but the protein expression of caveolin-1, the principal marker of caveolae in endothelial cells, increased after ischemia, all of which were alleviated by CG treatment. In conclusion, the post-treatment with CG significantly reduced BBB permeability and brain edema, which were correlated with preventing the degradation of the tight junction proteins and inhibiting the expression of caveolin-1 in the endothelial cells. These findings provide a novel approach to the treatment of ischemic stroke.     

Conjugation of brain-derived neurotrophic factor to a blood-brain barrier drug targeting system enables neuroprotection in regional brain ischemia following intravenous injection of the neurotrophin

Neurotrophins such as brain-derived neurotrophic factor (BDNF) are potential neuroprotective agents that could be used in the treatment of acute stroke, should these proteins be made transportable through the blood-brain barrier (BBB) in vivo. One approach to the BBB problem is to attach the nontransportable peptide to a brain targeting vector, which is a peptide or peptidomimetic monoclonal antibody (MAb), that is transported into brain from blood via an endogenous BBB transport system. The present studies describe a conjugate of BDNF and the OX26 monoclonal antibody (MAb) to the transferrin receptor. Avidin-biotin technology is used to link the BDNF and the MAb. The surface of the BDNF is conjugated with 2000 Da polyethylene glycol at carboxyl residues to optimize the plasma pharmacokinetics of the neurotrophin. Adult rats subjected to 24 h of permanent middle cerebral artery occlusion (MCAO) were treated intravenously with either unconjugated BDNF, unconjugated MAb, or the BDNF-OX26 conjugate at a dose of 1, 5 and 50 microg/rat of the BDNF. These doses decreased the infarct volume by 6% (not significant), 43% (P<0.01), and 65% (P<0.01), respectively. Significant reduction in stroke volume was still observed if the administration of the BDNF conjugate was delayed for 1-2 h after MCAO, although the pharmacological effect was progressively diminished in proportion to the time delay between MCAO and treatment. In conclusion, these studies demonstrate that large reductions in stroke volume can be achieved with the noninvasive intravenous administration of neurotrophins such as BDNF, providing the peptide is conjugated to a BBB drug targeting system.     

Pre-treatment of adrenomedullin suppresses cerebral edema caused by transient focal cerebral ischemia in rats detected by magnetic resonance imaging

Recent studies suggest the protective effects of adrenomedullin (AM) on ischemic brain damage. The present study was aimed at investigating the effects of AM and its receptor antagonist, AM_22-52, on ischemia-induced cerebral edema and brain swelling in rats using magnetic resonance imaging. Rats were subjected to 60 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. Intravenous injection of AM (1.0 μg/kg), AM_22-52 (1.0 μg/kg), or saline was made before MCAO. Effects of AM injection just after reperfusion were also investigated. One day after ischemia, increases in T₂-weighted signals in the brain were clearly observed. Total edema volume, as well as brain swelling, was greatly and significantly reduced by pre-treatment of AM (reduced by 53%). Extent of brain swelling was significantly correlated with the volume of cerebral edema. The protective effect of AM against edema was more clearly observed in the cerebral cortex (reduced by 63%) than the striatum (reduced by 31%). Increased T₂ relaxation time in the cortex was recovered partially by pre-treatment of AM. Post-treatment of AM had no effects. Pre-treatment of AM_22-52 tended to exacerbate the edema. In another line of experiment, cocktail administration of AM with melatonin, a pineal product having neuroprotective potential as a free radical scavenger, failed to enhance the protective effects of AM alone. The present study clearly suggests the prophylactic effects of AM against cerebral edema, especially the cortical edema, in a rat stroke model.     

Gain-of-function of progesterone receptor membrane component 2 ameliorates ischemic brain injury

Objective

Progesterone receptor membrane component 2 (PGRMC2) belongs to the membrane-associated progesterone receptor family, which regulates multiple pathophysiological processes. However, the role of PGRMC2 in ischemic stroke remains unexplored. The present study sought to determine the regulatory role of PGRMC2 in ischemic stroke.

Methods

Male C57BL/6J mice were subjected to middle cerebral artery occlusion (MCAO). The protein expression level and localization of PGRMC2 were examined by western blotting and immunofluorescence staining. The gain-of-function ligand of PGRMC2 (CPAG-1, 45 mg/kg) was intraperitoneally injected into sham/MCAO mice, and brain infarction, blood–brain barrier (BBB) leakage, and sensorimotor functions were evaluated by magnetic resonance imaging, brain water content, Evans blue extravasation, immunofluorescence staining, and neurobehavioral tests. The astrocyte and microglial activation, neuronal functions, and gene expression profiles were revealed by RNA sequencing, qPCR, western blotting, and immunofluorescence staining after surgery and CPAG-1 treatment.

Results

Progesterone receptor membrane component 2 was elevated in different brain cells after ischemic stroke. Intraperitoneal delivery of CPAG-1 reduced infarct size, brain edema, BBB leakage, astrocyte and microglial activation, and neuronal death, and improved sensorimotor deficits after ischemic stroke.

Conclusion

CPAG-1 acts as a novel neuroprotective compound that could reduce neuropathologic damage and improve functional recovery after ischemic stroke.     

Expression of Na–K–Cl cotransporter and edema formation are age dependent after ischemic stroke

Age is the most important independent risk factor for stroke; however aging animals are rarely used in stroke studies. Previous work demonstrated that young male mice had more edema formation after an induced stroke than aging animals. An important contributor to cerebral edema formation is the Na–K–Cl cotransporter (NKCC). We examined the expression of NKCC in young (10–12 weeks) and aging (15–16 months) C57BL6 male mice after middle cerebral artery occlusion (MCAO) and investigated the effect of pharmacological inhibition of NKCC with Bumetanide on cerebral edema formation. Both immunofluorescent staining and Western blotting analysis showed that NKCC expression was significantly higher in the ischemic penumbra of young compared to aging mice after stroke. Edema formation was significantly more robust in young mice and was reduced with Bumetanide. Bumetanide had no effect on cerebral edema in aging mice after MCAO. This suggests that NKCC expression and edema formation are age dependent after ischemic stroke.     

Deficiency of PAR4 attenuates cerebral ischemia/reperfusion injury in mice

Stroke is the third leading cause of death in the USA. Antithrombotic therapy targeting platelet activation is one of the treatments for ischemic stroke. Here we investigate the role of one of the thrombin receptors, protease-activated receptor 4 (PAR4), in a mouse transient middle cerebral artery occlusion (MCAO) model. After a 60 min MCAO and 23 h reperfusion, leukocyte and platelet rolling and adhesion on cerebral venules, blood–brain barrier (BBB) permeability, and cerebral edema were compared in PAR4-deficient mice and wild-type mice. Cerebral infarction volume and neuronal death were also measured. PAR4−/− mice had more than an 80% reduction of infarct volume and significantly improved neurologic and motor function compared with wild-type mice after MCAO. Furthermore, deficiency of PAR4 significantly inhibits the rolling and adhesion of both platelets and leukocytes after MCAO. BBB disruption and cerebral edema were also attenuated in PAR4−/− mice compared with wild-type animals. The results of this investigation indicate that deficiency of PAR4 protects mice from cerebral ischemia/reperfusion (I/R) injury, partially through inhibition of platelet activation and attenuation of microvascular inflammation.     

Spatiotemporal evolution of blood brain barrier damage and tissue infarction within the first 3h after ischemia onset

Blood brain barrier (BBB) damage that occurs within the thrombolytic time window is increasingly appreciated to negatively impact the safety and efficacy profiles of thrombolytic therapy for ischemic stroke. However, the spatiotemporal evolution of BBB damage in this early stroke stage and the underlying mechanisms remain unclear. Here, we investigated the topographical distribution of BBB damage and its association with tissue injury within the first 3h after ischemia onset and the roles of matrix metalloproteinase (MMP)-2/9 in this process. Rats were subjected to 1, 2, or 3h of middle cerebral artery occlusion (MCAO) followed by 10min reperfusion with fluorescence-labeled dextran as BBB permeability marker. Acute tissue infarction was evidenced by staining defect with triphenyltetrazolium chloride (TTC). Cerebral blood flow (CBF) was measured by magnetic resonance imaging. MMP-2/9 were assessed by gel and in situ zymography. After 2-h MCAO, dextran leakage was seen in the ischemic ventromedial striatum and the preoptic area which showed ~70% CBF reduction, and expanded to other MCA regions including the cortex after 3-h MCAO. Interestingly, high (2000kDa) and low (70kDa) molecular weight dextrans displayed almost identical leakage patterns. Different from BBB damage, tissue infarction was first seen in the ischemic dorsal striatum and the parietal/insular cortex which experienced ~90% CBF reduction. Increased gelatinolytic activity colocalized with dextran leakage, and MMP-2 was found to be the major enzymatic source on gelatin zymograms. Pretreatment with MMP inhibitor GM6001 significantly reduced dextran leakage induced by 2-h and 3-h MCAO. Taken together, our findings reveal substantial differences in the topographic distribution of BBB damage and tissue infarction within the first 3h after MCAO onset. Unlike ischemic neuronal damage, BBB damage appears to develop faster in brain regions with moderately severe ischemia, and MMP-2 contributes to this early ischemic BBB damage.     

Conjugation of brain-derived neurotrophic factor to a blood–brain barrier drug targeting system enables neuroprotection in regional brain ischemia following intravenous injection of the neurotrophin

Neurotrophins such as brain-derived neurotrophic factor (BDNF) are potential neuroprotective agents that could be used in the treatment of acute stroke, should these proteins be made transportable through the blood–brain barrier (BBB) in vivo. One approach to the BBB problem is to attach the nontransportable peptide to a brain targeting vector, which is a peptide or peptidomimetic monoclonal antibody (MAb), that is transported into brain from blood via an endogenous BBB transport system. The present studies describe a conjugate of BDNF and the OX26 monoclonal antibody (MAb) to the transferrin receptor. Avidin–biotin technology is used to link the BDNF and the MAb. The surface of the BDNF is conjugated with 2000 Da polyethylene glycol at carboxyl residues to optimize the plasma pharmacokinetics of the neurotrophin. Adult rats subjected to 24 h of permanent middle cerebral artery occlusion (MCAO) were treated intravenously with either unconjugated BDNF, unconjugated MAb, or the BDNF–OX26 conjugate at a dose of 1, 5 and 50 μg/rat of the BDNF. These doses decreased the infarct volume by 6% (not significant), 43% (P<0.01), and 65% (P<0.01), respectively. Significant reduction in stroke volume was still observed if the administration of the BDNF conjugate was delayed for 1–2 h after MCAO, although the pharmacological effect was progressively diminished in proportion to the time delay between MCAO and treatment. In conclusion, these studies demonstrate that large reductions in stroke volume can be achieved with the noninvasive intravenous administration of neurotrophins such as BDNF, providing the peptide is conjugated to a BBB drug targeting system.     

Agmatine attenuates brain edema through reducing the expression of aquaporin-1 after cerebral ischemia

Brain edema is frequently shown after cerebral ischemia. It is an expansion of brain volume because of increasing water content in brain. It causes to increase mortality after stroke. Agmatine, formed by the decarboxylation of -arginine by arginine decarboxylase, has been shown to be neuroprotective in trauma and ischemia models. The purpose of this study was to investigate the effect of agmatine for brain edema in ischemic brain damage and to evaluate the expression of aquaporins (AQPs). Results showed that agmatine significantly reduced brain swelling volume 22 h after 2 h middle cerebral artery occlusion in mice. Water content in brain tissue was clearly decreased 24 h after ischemic injury by agmatine treatment. Blood–brain barrier (BBB) disruption was diminished with agmatine than without. The expressions of AQPs-1 and -9 were well correlated with brain edema as water channels, were significantly decreased by agmatine treatment. It can thus be suggested that agmatine could attenuate brain edema by limitting BBB disruption and blocking the accumulation of brain water content through lessening the expression of AQP-1 after cerebral ischemia.