Neuropathology of acute liver failure

Cerebral edema has been identified in all forms of liver disease and is closely related to the development of hepatic encephalopathy. Cerebral edema is most readily recognized in acute liver failure (ALF), while the main cause of death in patients with ALF is multi-organ failure; brain herniation as a result of intracranial hypertension does remain a major cause of mortality. The mechanisms responsible for cerebral edema in ALF suggest both cytotoxic and vasogenic injury. This article reviews the gross and ultrastructural changes associated with cerebral edema in ALF. The primary cause of cerebral edema is associated with astrocyte swelling, mainly perivascular edema and ammonia still remains the primary neurotoxin involved in its pathogenesis. The astrocytic changes were confined to the gray matter. The other organelles involved in the pathogenesis of ALF include mitochondria, basement membrane, pericytes, microglial cells, blood-brain barrier (BBB) etc. Discrete neuronal changes have recently been reported. Recent studies in animal and humans have demonstrated the microglial changes which have the potential to cause neuronal dysfunction in ALF. The alterations in BBB still remain unclear though few studies have showed disruption of tight junction proteins indicating the involvement of BBB in cellular swelling.     

Blood-brain barrier in acute liver failure

Brain edema remains a challenging obstacle in the management of acute liver failure (ALF). Cytotoxic mechanisms associated with brain edema have been well recognized, but evidence for vasogenic mechanisms in the pathogenesis of brain edema in ALF has been lacking. Recent reports have not only shown a role of matrix metalloproteinase-9 in the pathogenesis of brain edema in experimental ALF but have also found significant alterations in the tight junction elements including occludin and claudin-5, suggesting a vasogenic injury in the blood-brain barrier (BBB) integrity. This article reviews and explores the role of the paracellular tight junction proteins in the increased selective BBB permeability that leads to brain edema in ALF.     

Expression of Aquaporin 4 and Breakdown of the Blood-Brain Barrier after Hypoglycemia-Induced Brain Edema in Rats

Background

Hypoglycemia-induced brain edema is a severe clinical event that often results in death. The mechanisms by which hypoglycemia induces brain edema are unclear.

Methods

In a hypoglycemic injury model established in adult rats, brain edema was verified by measuring brain water content and visualizing water accumulation using hematoxylin and eosin staining. Temporal expression of aquaporin 4 (AQP4) and the integrity of the blood-brain barrier (BBB) were evaluated. We assessed the distribution and expression of AQP4 following glucose deprivation in astrocyte cultures.

Results

Brain edema was induced immediately after severe hypoglycemia but continued to progress even after recovery from hypoglycemia. Upregulation of AQP4 expression and moderate breakdown of the BBB were observed 24 h after recovery. In vitro, significant redistribution of AQP4 to the plasma membrane was induced following 6 h glucose deprivation.

Conclusion

Hypoglycemia-induced brain edema is caused by cytotoxic and vasogenic factors. Changes in AQP4 location and expression may play a protective role in edema resolution.     

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.     

Magnetic resonance analysis of the effects of acute ammonia intoxication on rat brain. Role of NMDA receptors

Acute ammonia intoxication leads to rapid death, which is prevented by blocking N -methyl- d -aspartate (NMDA) receptors. The subsequent mechanisms leading to death remain unclear. Brain edema seems an important step. The aim of this work was to study the effects of acute ammonia intoxication on different cerebral parameters in vivo using magnetic resonance and to assess which effects are mediated by NMDA receptors activation. To assess edema induction, we injected rats with ammonium acetate and measured apparent diffusion coefficient (ADC) in 16 brain areas. We also analyzed the effects on T1, T2, and T2* maps and whether these effects are prevented by blocking NMDA receptors. The effects of acute ammonia intoxication are different in different brain areas. T1 relaxation time is reduced in eight areas. T2 relaxation time is reduced only in ventral thalamus and globus pallidus. ADC values increased in hippocampus, caudate-putamen, substantia nigra and cerebellar cortex, reflecting vasogenic edema. ADC decreased in hypothalamus, reflecting cytotoxic edema. Myo-inositol increased in cerebellum and substantia nigra, reflecting vasogenic edema. N -acetyl-aspartate decreased in cerebellum, reflecting neuronal damage. Changes in N -acetyl-aspartate, T1 and T2 are prevented by blocking NMDA receptors with MK-801 while changes in ADC or myo-inositol (induction of edema) are not.     

The effects of trifluoperazine on brain edema,aquaporin-4 expression and metabolic markers during the acute phase of stroke using photothrombotic mouse model

Stroke is the second leading cause of death and the third leading cause of disability globally. Edema is a hallmark of stroke resulting from dysregulation of water homeostasis in the central nervous system (CNS) and plays the major role in stroke-associated morbidity and mortality. The overlap between cellular and vasogenic edema makes treating this condition complicated, and to date, there is no pathogenically oriented drug treatment for edema. Water balance in the brain is tightly regulated, primarily by aquaporin 4 (AQP4) channels, which are mainly expressed in perivascular astrocytic end-feet. Targeting AQP4 could be a useful therapeutic approach for treating brain edema; however, there is no approved drug for stroke treatment that can directly block AQP4. In this study, we demonstrate that the FDA-approved drug trifluoperazine (TFP) effectively reduces cerebral edema during the early acute phase in post-stroke mice using a photothrombotic stroke model. This effect was combined with an inhibition of AQP4 expression at gene and protein levels. Importantly, TFP does not appear to induce any deleterious changes on brain electrolytes or metabolic markers, including total protein or lipid levels. Our results support a possible role for TFP in providing a beneficial extra-osmotic effect on brain energy metabolism, as indicated by the increase of glycogen levels. We propose that targeting AQP4-mediated brain edema using TFP is a viable therapeutic strategy during the early and acute phase of stroke that can be further investigated during later stages to help in developing novel CNS edema therapies.     

脑水肿时紧密连接蛋白occludin及AQP4的表达变化

目的:采用枕大池内注入脂多糖(lipopolysaccharides,LPS)的方法建立大鼠脑水肿模型,观察脑组织病理形态学变化,脑组织含水量(brain water content,BWC),血脑屏障(blood brain barrier,BBB)的紧密连接蛋白Occludin和水通道蛋白-4(aquaporin 4,AQP4)表达水平的动态变化,研究AQP4及Occludin与脑水肿形成的关系,及其可能的作用机制,为临床脑水肿的治疗提供理论依据。方法:选用Wistar健康成年大鼠,随机分为正常对照组,生理盐水组和脂多糖组,后两组的观察时间点选定于造模后3 h、6h、12 h、24 h、72 h。采用经皮穿刺枕大池内注入脂多糖的方法制备脑水肿动物模型,正常对照组、生理盐水组及脂多糖组分别于各时间点进行开颅取脑,测定脑组织含水量,通过HE染色法观察脑组织的病理形态学变化,应用Western blot方法检测occludin的表达变化。应用RT-PCR技术测定脑组织内AQP4mRNA的表达变化。结果:生理盐水组各时间点中有少量AQP4mRNA及occludin蛋白的表达,与正常对照组之间无显著性差异;脂多糖组在造模后3 hAQP4的mRNA表达开始增加,6-12 h达高峰,此后明显下降,随后表达开始减弱,24-72 h表达显著低于生理盐水组;occludin蛋白表达下降出现于造模后3 h,12-24 h下降更明显,72 h表达开始升高。结论:枕大池内注入脂多糖(LPS)所建立脑水肿模型中,脑组织含水量及血脑屏障通透性增加,病理学特点是血管源性脑水肿出现早且持久,后期伴有细胞毒性脑水肿的改变。AQP4早期表达增强是胶质细胞的适应性反应,与血脑屏障的破坏有关,促进了血管源性脑水肿的发生。后期AQP4表达减弱是机体内在防御机制的表现,同时又促进细胞毒性脑水肿的形成。occludin在脑组织中表达量随脑水肿的加重而降低,即与脑水肿的程度呈负相关,目前认为这与脑水肿时内皮细胞通透性增加,血脑屏障的通透性改变,导致occludin的表达下调有关,促进了血管源性脑水肿的发生。针对以上特点,我们可以进一步研究调控AQP4及occludin表达的药物,从而减轻脑损伤后脑水肿的程度,为脑水肿的治疗提供新的临床策略。     

活性氮自由基:脑缺血再灌注损伤过程中引起血脑屏障破坏及调节神经修复的双刃剑(英文)

在脑缺血再灌注损伤中,自由基发挥着重要作用。脑缺血及再灌注可产生大量的自由基,随着这些自由基的聚集,会引发一系列的分子级联反应,从而增加血脑屏障的通透性,诱发脑水肿、出血、炎症反应及细胞死亡。以一氧化氮(NO)及过氧亚硝基阴离子(ONOO-)为代表的活性氮(reactive nitrogen species,RNS),是自由基的重要组成部分,它们在脑缺血再灌注损伤中作用显著。一方面,活性氮能激活基质金属蛋白酶(MMPs),破坏血脑屏障。MMPs作为一大类含2价锌离子的水解酶,其激活可以降解脑血管及神经元细胞外基质。脑缺血再灌注损伤产生NO和ONOO-,它们均可以通过激活MMPs,降解紧密连接蛋白,从而破坏血脑屏障。另一方面,近期研究发现,活性氮也参与了脑缺血后神经再生及修复的调节过程。因此,了解这些活性小分子在血脑屏障破坏及神经再生中的复杂生物活性将很有意义。小窝蛋白1(Caveolin-1)就是活性氮自由基的重要靶分子,它是一种细胞表面的穴样内陷(caveolae)中的膜蛋白,可以通过抑制MMPs的激活保护血脑屏障的完整性。下调Caveolin-1的表达将引起血脑屏障的破坏。脑缺血所产生的NO能下调Caveolin-1的表达,而Caveolin-1的下调,能引起NO合酶的增加,促进生成更多的NO。活性氮与Caveolin-1互相作用,形成了一个反馈回路,通过激活MMPs而造成血脑屏障的不断破坏。此外,Caveolin-1通过调节不同的信号通路,抑制神经干细胞的增长及向神经元分化。因此,活性氮也很可能通过调节Caveolin-1及其他信号通路调控神经再生。在这篇文章中,我们对活性氮在血脑屏障及神经再生中的近期研究进展进行了综述。我们认为,活性氮可能在脑缺血再灌注中起双重作用,既是细胞毒性分子,亦可能是神经再生中的重要信号分子,其作用与其在神经元、内皮细胞及其微环境中产生的量有重要的关系。     

The Noncompetitive AMPAR Antagonist Perampanel Abrogates Brain Endothelial Cell Permeability in Response to Ischemia: Involvement of Claudin-5

The blood–brain barrier (BBB) is formed by brain endothelial cells, and decreased BBB integrity contributes to vasogenic cerebral edema and increased mortality after stroke. In the present study, we investigated the protective effect of perampanel, an orally active noncompetitive AMPA receptor antagonist, on BBB permeability in an in vitro ischemia model in murine brain endothelial cells (mBECs). The results showed that perampanel significantly attenuated oxygen glucose deprivation (OGD)-induced loss of cell viability, release of lactate dehydrogenase, and apoptotic cell death in a dose-dependent manner. Perampanel treatment did not alter the expression and surface distribution of various glutamate receptors. Furthermore, the results of calcium imaging showed that perampanel had no effect on OGD-induced increase in intracellular Ca²⁺ concentrations. Treatment with perampanel markedly reduced the paracellular permeability of mBECs after OGD in different time points, as measured by transepithelial electrical resistance assay. In addition, the expression of claudin-5 at protein level, but not at mRNA level, was increased by perampanel treatment after OGD. Knockdown of claudin-5 partially prevented perampanel-induced protection in cell viability and BBB integrity in OGD-injured mBECs. These data show that the noncompetitive AMPA receptor antagonist perampanel affords protection against ischemic stroke through caludin-5 mediated regulation of BBB permeability.     

Endothelial Transient Receptor Potential Conical Channel (TRPC)-3 Activation Induces Vasogenic Edema Formation in the Rat Piriform Cortex Following Status Epilepticus

Transient receptor potential canonical channel (TRPC) is a nonselective cation channel permeable to Ca²⁺, which express in many cell types, including neurons. However the alterations in TRPC receptor expressions in response to status epilepticus (SE) have not been explored. Therefore, the present study was designated to elucidate the roles of TRPC3 in neuronal death and vasogenic edema within the rat piriform cortex (PC) following SE. In non-SE animals, TRPC3 immunoreactivity was abundantly detected in the PC. Following SE, TRPC3 immunoreactivity was increased in neurons. Furthermore, TRPC3 expression was detected in endothelial cells that did not contain it in non-SE animals. Loss of SMI-71 (a blood–brain barrier antigen) immunoreactivity was also observed in TRPC3 positive endothelial cells. In addition, FJB positive neurons and vasogenic edema were noticeably detected in the PC. To directly determine whether TRPC3 activation is correlated to SE-induced vasogenic edema formation and neuronal damages in the PC, the effect of Pyr-3 (a TRPC3 antagonist) on SE-induced insults were investigated. Pyr-3 infusion effectively attenuated vasogenic edema in the PC as compared to the vehicle. Therefore, our findings indicate that TRPC3 activation/overexpression induced by SE may involve BBB disruption and neuronal damages in the rat PC following SE. Therefore, the present study was TRPC3 may play an important role in SE-induced vasogenic edema formation through BBB disruptions in the rat PC.     

Intracerebral VEGF injection highly upregulates AQP4 mRNA and protein in the perivascular space and glia limitans externa

Stroke is the third leading cause of death and the leading cause of adult disability in the industrialized nations. One of the consequences of stroke is blood-brain barrier (BBB) leakage and subsequent edema, which is one of the causes of mortality in this pathology. Aquaporin-4 (AQP4) is the most abundant water channel in the brain. Studies in AQP4 knock-out mice have shown a prominent role of this water channel in edema development and resolution after ischemia. Here we have studied changes in AQP4 mRNA and protein expression in response to vascular endothelial growth factor (VEGF), a potent angiogenic factor. VEGF administration highly upregulated AQP4 mRNA and protein in the ventral midbrain. Perfusion of the animals with FITC-albumin prior to sacrifice demonstrated localization of AQP4 protein in close proximity to the VEGF-induced new blood vessels. Expression levels of AQP4 mRNA were maximum 7 days after VEGF injection whereas our previous report showed that BBB leakage is resolved at this time point. Therefore, we speculate a positive role of AQP4 in edema resolution, which may partially explain the previously reported beneficial effects of delayed VEGF administration in ischemic rats. Our results provide new insights into the molecular changes in the edematous brain and may help in future therapeutical directions.     

Effect of Baicalin on Matrix Metalloproteinase-9 Expression and Blood–Brain Barrier Permeability Following Focal Cerebral Ischemia in Rats

Focal cerebral ischemia results in an increased expression of matrix metalloproteinase-9 (MMP-9), which induces vasogenic brain edema via disrupting the blood–brain barrier (BBB) integrity. Recent studies from our laboratory showed that baicalin reduces ischemic brain damage by inhibiting inflammatory reaction and neuronal apoptosis in a rat model of focal cerebral ischemia. In the present study, we first explored the effect of baicalin on the neuronal damage, brain edema and BBB permeability, then further investigated its potential mechanisms. Sprague–Dawley rats underwent permanent middle cerebral artery occlusion (MCAO). Baicalin was administrated by intraperitoneally injected twice at 2 and 12 h after the onset of MCAO. Neuronal damage, brain edema and BBB permeability were measured 24 h following MCAO. Expression of MMP-9 protein and mRNA were determined by western blot and RT–PCR, respectively. Expression of tight junction protein (TJP) occludin was detected by western blot. Neuronal damage, brain edema and BBB permeability were significantly reduced by baicalin administration following focal cerebral ischemia. Elevated expression of MMP-9 protein and mRNA were significantly down-regulated by baicalin administration. In addition, MCAO caused the decreased expression of occludin, which was significantly up-regulated by baicalin administration. Our study suggested that baicalin reduces MCAO-induced neuronal damage, brain edema and BBB permeability, which might be associated with the inhibition of MMP-9 expression and MMP-9-mediated occludin degradation.     

The m.3243A>G mtDNA mutation is pathogenic in an in vitro model of the human blood brain barrier

MELAS is a common mitochondrial disease frequently associated with the m.3243A>G point mutation in the tRNA^Leu(UUR) of mitochondrial DNA and characterized by stroke-like episodes with vasogenic edema and lactic acidosis. The pathogenic mechanism of stroke and brain edema is not known. Alterations in the blood brain barrier (BBB) caused by respiratory chain defects in the cortical microvessels could explain the pathogenesis. To test this hypothesis we developed a tissue culture model of the human BBB. The MELAS mutation was introduced into immortalized brain capillary endothelial cells and astrocytes. Respiratory chain activity and transendothelial electrical resistance, TEER was measured. Severe defects of respiratory chain complex I and IV activities, and a moderate deficiency of complex II activity in cells harboring the MELAS mutation were associated with low TEER, indicating that the integrity of the BBB was compromised. These data support our hypothesis that respiratory chain defects in the components of the BBB cause changes in permeability.     

Sepsis Associated Encephalopathy Studied by MRI and Cerebral Spinal Fluid S100B Measurement

The pathogenesis of sepsis associated encephalopathy (SAE) is not yet clear: the blood–brain barrier (BBB) disruption has been indicated among the possible causative mechanisms. S100B, a calcium binding protein, originates in the central nervous system but it can be also produced by extra-cerebral sources; it is passively released from damaged glial cells and neurons; it has limited passage through the BBB. We aimed to demonstrate BBB damage as part of the pathogenesis of SAE by cerebral spinal fluid (CSF) and serum S100B measurements and by magnetic resonance imaging (MRI). This paper describes four septic patients in whom SAE was clinically evident, who underwent MRI and S100B measurement. We have not found any evidence of CSF-S100B increase. Serum S100B increase was found in three out of four patients. MRI did not identify images attributable to BBB disruption but vasogenic edema, probably caused by an alteration of autoregulation, was diagnosed. S100B does not increase in CSF of septic patients; S100B increase in serum may be due to extracerebral sources and does not prove any injury of BBB. MRI can exclude other cerebral pathologies causing brain dysfunction but is not specific of SAE. BBB damage may be numbered among the contributors of SAE, which aetiology is certainly multifactorial: an interplay between the toxic mediators involved in sepsis and the indirect effects of hyperthermia, hypossia and hypoperfusion.     

Leukotriene D4 induces brain edema and enhances CysLT2 receptor-mediated aquaporin 4 expression

Cysteinyl leukotrienes (including LTC(4), LTD(4), and LTE(4)), potent inflammatory mediators, can induce brain-blood barrier (BBB) disruption and brain edema. These reactions are mediated by their receptors, CysLT(1) and CysLT(2) receptors. On the other hand, aquaporin 4 (AQP4) primarily modulates brain water homeostasis and edema after various injuries. Here, we aimed to determine whether AQP4 is involved in LTD(4)-induced brain edema. LTD(4) (1ng in 0.5mul PBS) microinjection into the cortex increased endogenous IgG exudation (BBB disruption) and water content (brain edema), and enhanced AQP4 expression in mouse brain. The selective CysLT(1) receptor antagonist pranlukast inhibited the IgG exudation, but not the increased water content and AQP4 expression induced by LTD(4). In the cultured rat astrocytes, LTD(4) (10(-9)-10(-7)M, for 24h) similarly enhanced AQP4 expression. The enhanced AQP4 expression was inhibited by Bay u9773, a non-selective CysLT(1)/CysLT(2) receptor antagonist, but not by pranlukast. LTD(4) (10(-9)-10(-7)M) also induced the mRNA expression of CysLT(2) (not CysLT(1)) receptor in astrocytes. These results indicate that LTD(4) modulates brain edema; CysLT(1) receptor mediates vasogenic edema while CysLT(2) receptor may mediate cytotoxic edema via up-regulating AQP4 expression.     

The disturbed blood-brain barrier in human glioblastoma

The aim of this article is to describe alterations of the blood-brain barrier (BBB) in gliomas. The main clinical problem of human gliomas is the edematous swelling and the dramatic increase of intracerebral pressure, also compromising healthy areas of the brain. According to our concept, one of the main reasons on the cellular level for these clinical problems is the loss or reduction of astroglial polarity. Astroglial polarity means the specific accumulation of potassium and water channels in the superficial and perivascular astroglial endfeet membranes. The most important water channel in the CNS is the astroglial water channel protein aquaporin-4 (AQP4) which is arranged in a morphologically spectacular way, the so-called orthogonal arrays of particles (OAPs) to be observed in freeze-fracture replicas. In brain tumors, but also under conditions of trauma or inflammation, these OAPs are redistributed to membrane domains apart from endfeet areas. Probably, this dislocation might be due to the degradation of the proteoglycan agrin by the matrix metalloproteinase 3 (MMP3). Agrin binds to the dystrophin-dystroglycan-complex (DDC), which in turn is connected to AQP4. As a consequence, agrin loss may lead to a redistribution of AQP4 and a compromised directionality of water transport out of the cell, finally to cytotoxic edema. This in turn is hypothesized to lead to a breakdown of the BBB characterized by disturbed tight junctions, and thus to the development of vasogenic edema. However, the mechanism how the loss of polarity is related to the disturbance of microvascular tight junctions is completely unknown so far.     

microRNAs in stroke pathogenesis

Stroke is one of the leading causes of death and disability worldwide. There are two major types of stroke: cerebral ischemia caused by obstruction of blood vessels in the brain and haemorrhagic stroke that is triggered by the disruption of blood vessels. Thrombolytic therapy involving recombinant tissue plasminogen activator (rtPA) has been shown to be beneficial only when used within 4.5 hours of onset of acute ischemic stroke. rtPA treatment beyond this time window has been found to be unsuitable and usually resulting in haemorrhagic transformation. Stroke is a multifactorial disease that forms a possible end state for majority of patients suffering from diabetes, atherosclerosis and hypertension which are known risk factors. Although the biochemistry of stroke and related diseases is quite well understood, the knowledge on the molecular mechanisms underlying these diseases is still at its infancy. microRNAs that form a unique class of endogenous riboregulators of gene function, offer tremendous potential in unraveling the mechanisms underlying stroke pathogenesis. microRNA expression also reflects the response of individuals to drugs and therapy. Several microRNAs and their target genes, known to be involved in endothelial dysfunction, dysregulation of neurovascular integrity, edema formation, pro-apoptosis, inflammation and extra-cellular matrix remodeling contribute to the critical processes in the pathogenesis of stroke. In this review, we will also be discussing the role of microRNAs as possible diagnostic and prognostic biomarkers as well as potential therapeutic targets in stroke pathogenesis.     

MMP-9 Inhibition: a Therapeutic Strategy in Ischemic Stroke

Ischemic stroke is a leading cause of disability worldwide. In cerebral ischemia there is an enhanced expression of matrix metallo-proteinase-9 (MMP-9), which has been associated with various complications including excitotoxicity, neuronal damage, apoptosis, blood–brain barrier (BBB) opening leading to cerebral edema, and hemorrhagic transformation. Moreover, the tissue plasminogen activator (tPA), which is the only US-FDA approved treatment of ischemic stroke, has a brief 3 to 4 h time window and it has been proposed that detrimental effects of tPA beyond the 3 h since the onset of stroke are derived from its ability to activate MMP-9 that in turn contributes to the breakdown of BBB. Therefore, the available literature suggests that MMP-9 inhibition can be of therapeutic importance in ischemic stroke. Hence, combination therapies of MMP-9 inhibitor along with tPA can be beneficial in ischemic stroke. In this review we will discuss the current status of various strategies which have shown neuroprotection and extension of thrombolytic window by directly or indirectly inhibiting MMP-9 activity. In the introductory part of the review, we briefly provide an overview on ischemic stroke, commonly used models of ischemic stroke and a role of MMP-9 in ischemia. In next part, the literature is organized as various approaches which have proven neuroprotective effects through direct or indirect decrease in MMP-9 activity, namely, using biotherapeutics, involving MMP-9 gene inhibition using viral vectors; using endogenous inhibitor of MMP-9, repurposing of old drugs such as minocycline, new chemical entities like DP-b99, and finally other approaches like therapeutic hypothermia.     

Novel insights into the development and maintenance of the blood–brain barrier

The blood–brain barrier (BBB) is essential for maintaining homeostasis within the central nervous system (CNS) and is a prerequisite for proper neuronal function. The BBB is localized to microvascular endothelial cells that strictly control the passage of metabolites into and out of the CNS. Complex and continuous tight junctions and lack of fenestrae combined with low pinocytotic activity make the BBB endothelium a tight barrier for water soluble moleucles. In combination with its expression of specific enzymes and transport molecules, the BBB endothelium is unique and distinguishable from all other endothelial cells in the body. During embryonic development, the CNS is vascularized by angiogenic sprouting from vascular networks originating outside of the CNS in a precise spatio-temporal manner. The particular barrier characteristics of BBB endothelial cells are induced during CNS angiogenesis by cross-talk with cellular and acellular elements within the developing CNS. In this review, we summarize the currently known cellular and molecular mechanisms mediating brain angiogenesis and introduce more recently discovered CNS-specific pathways (Wnt/β?catenin, Norrin/Frizzled4 and hedgehog) and molecules (GPR124) that are crucial in BBB differentiation and maturation. Finally, based on observations that BBB dysfunction is associated with many human diseases such as multiple sclerosis, stroke and brain tumors, we discuss recent insights into the molecular mechanisms involved in maintaining barrier characteristics in the mature BBB endothelium.     

Simultaneous activation of several second messengers in hypoxia-induced hyperpermeability of brain derived endothelial cells

In vivo, ischemia is known to damage the blood-brain barrier (BBB) leading to the development of vasogenic brain edema. Hypoxia-induced vascular endothelial growth factor (VEGF) has been shown to be a key regulator of these permeability changes. However, the signaling pathways that underlie VEGF-induced hyperpermeability are incompletely understood. In this study, we demonstrate that hypoxia- and VEGF-induced permeability changes depend on activation of phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), and protein kinase G (PKG). Inhibition of mitogen-activated protein kinases (MAPK) and of the protein kinase C (PKC) did not affect permeability at all. Paralleling hypoxia- and VEGF-induced permeability changes, localization of the tight junction proteins occludin, zonula occludens-1 (ZO-1), and ZO-2 along the cell membrane changed from a continuous to a more discontinuous expression pattern during hypoxia. In particular, localization of ZO-1 and ZO-2 expression moved from the cell membrane to the cytoplasm and nucleus whereas occludin expression remained at the cell membrane. Inhibition of PLCgamma, PI3-kinase, and PKG abolished these hypoxia-induced changes. These findings demonstrate that hypoxia and VEGF induce permeability through rearrangement of endothelial junctional proteins which involves activation of the PLCgamma and PI3-K/AKT pathway leading to the activation of PKG.