脑缺血预处理的脑保护作用

脑缺血预处理(NIPC)可启动机体自身保护机制,提高脑组织对缺血、缺氧的耐受性,减少脑的缺血性损害,其保护作用是明确的。本文分析了脑缺血预处理现象,根据其形成、特点、时间过程和对重复缺血性损害的反应,结合功能恢复情况以及临床上短暂性脑缺血发作的影响,对于IPC的机制和其他的预处理方法进行了比较,试图找到一种安全有效、能够直接用于临床的预处理手段,通过“防病”的方式预防缺血性脑卒中的发生,减轻缺血性脑损伤。     

Topiramate reduces excitotoxic and ischemic injury in the rat retina

The effects of topiramate, a drug used clinically as an anti-epileptic, were investigated in excitotoxin-induced neurotoxicity models involving two different retinal primary cultures and in a rat model of retinal ischemic injury. For the in vitro studies, we used retinal-neuron cultures from rat embryos and purified retinal ganglion cells (RGCs) from newborn rats. In the retinal-neuron cultures, neurotoxicity was induced by a 10-min exposure to 1 mM glutamate or (+/-)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). In RGCs, neurotoxicity was induced by incubation for 3 days in a culture medium containing 25 microM glutamate. For the in vivo study, retinal ischemia was induced by elevating intraocular pressure to 130 mmHg for 45 min, and topiramate was administered intraperitoneally before and after the ischemia. Retinal damage was evaluated by measuring the number of cells in the ganglion cell layer (GCL) and the thickness of the inner plexiform layer (IPL), and by examining the a- and b-waves of the electroretinogram (ERG). Topiramate (> or =1 microM) markedly reduced the neuronal cell death induced by each of the excitotoxins in rat retinal-neuron cultures and in RGCs. Ischemia caused a decrease in GCL cells and in IPL thickness, and a diminution of the ERG waves. Histopathologic and functional analyses indicated that systemic treatment with topiramate prevented ischemia-induced damage in a dose-dependent manner. In conclusion, topiramate was protective against excitotoxic and ischemic retinal-neuron damage in vitro and in vivo, respectively. Therefore, it may be useful for treatment of the retina-related diseases such as central retinal artery occlusion, diabetic retinopathy, and glaucoma.     

Neuroprotective effects of ischemic preconditioning on hippocampal CA1 pyramidal neurons through maintaining calbindin D28k immunoreactivity following subsequent transient cerebral ischemia

Ischemic preconditioning elicited by a non-fatal brief occlusion of blood flow has been applied for an experimental therapeutic strategy against a subsequent fatal ischemic insult. In this study, we investi-gated the neuroprotective effects of ischemic preconditioning (2-minute transient cerebral ischemia) on calbindin D28k immunoreactivity in the gerbil hippocampal CA1 area following a subsequent fatal tran-sient ischemic insult (5-minute transient cerebral ischemia). A large number of pyramidal neurons in the hippocampal CA1 area died 4 days after 5-minute transient cerebral ischemia. Ischemic preconditioning reduced the death of pyramidal neurons in the hippocampal CA1 area. Calbindin D28k immunoreactivity was greatly attenuated at 2 days after 5-minute transient cerebral ischemia and it was hardly detected at 5 days post-ischemia. Ischemic preconditioning maintained calbindin D28k immunoreactivity after transient cerebral ischemia. These findings suggest that ischemic preconditioning can attenuate transient cerebral ischemia-caused damage to the pyramidal neurons in the hippocampal CA1 area through maintaining cal-bindin D28k immunoreactivity.     

脑缺血耐受机制的研究进展

2－3min的脑缺血对随后的严重脑缺血具有明显保护作用，即脑缺血耐受，目前发现蛋白合成、不同类型的离子通道的变化以及胶质细胞的支持参与了脑缺血耐受，研究缺血耐受机制能够为进一步研究脑损伤与保护机制提供新的视角。     

Modulation of valosin-containing protein by Kyoto University Substances (KUS) as a novel therapeutic strategy for ischemic neuronal diseases

Retinal ischemia causes several vision-threatening diseases, including diabetic retinopathy, retinal artery occlusion, and retinal vein occlusion. Intracellular adenosine triphosphate (ATP) depletion and sub-sequent induced endoplasmic reticulum (ER) stress are proposed to be the underlying mechanisms of ischemic retinal cell death. Recently, we found that a naphthalene derivative can inhibit ATPase activity of valosin-containing protein, universally expressed within various types of cells, including retinal neural cells, with strong cytoprotective activity. Based on the chemical structure, we developed novel valosin-con-taining protein modulators, Kyoto University Substances (KUSs), that not only inhibit intracellular ATP depletion, but also ameliorate ER stress. Suppressing ER stress by KUSs is associated with neural cell sur-vival in animal models of several neurodegenerative diseases, such as glaucoma and retinal degeneration. Given that a major pathology of ischemic retinal diseases, other than intracellular ATP depletion, is ER stress-induced cell death, KUSs may provide a novel strategy for cell protection in ischemic conditions. Hence, we investigated the efficacy of KUS121 in a rat model of retinal ischemic injury. Intravitreal injec-tions of KUS121, which is clinically preferable route of drug administration in retinal diseases, significantly suppressed inner retinal thinning and retinal cell death, and maintained visual functions. Valosin-contain-ing protein modulation by KUS is a promising novel therapeutic strategy for ischemic retinal diseases.     

Elevated erythropoietin in vitreous with ischemic retinal diseases

The aim of the current study was to measure the concentrations of erythropoietin in the vitreous fluid and analyze its association with vascular endothelial growth factor (VEGF) in ischemic vitreoretinal diseases. Vitreous fluid samples were collected from patients with proliferative diabetic retinopathy, branch retinal vein occlusion and idiopathic macular hole. Concentrations of erythropoietin and VEGF in vitreous fluid were significantly elevated in patients with proliferative diabetic retinopathy and branch retinal vein occlusion as compared to patients with macular hole. There were no differences in serum concentrations of erythropoietin and VEGF among patient groups. There was significant correlation between erythropoietin and VEGF concentrations in vitreous fluid. Erythropoietin was up-regulated in ischemic disorders and may act as an endogenous neuroprotective factor against ischemic retinal disorders.     

局灶性缺血预处理对脑梗死大鼠神经生长因子表达的影响

目的:研究局灶性缺血预处理对脑梗死大鼠神经生长因子(nerve growthfactor,NGF)表达的影响,探讨缺 血预处理诱导脑缺血耐受机制。方法:SD大鼠随机分为3组。预缺血组和假手术组在大脑中动脉缺血(MCAO)前3天 分别接受10min的预缺血或假手术,MCAO 2h后再灌注22h处死;对照组两次均为假手术,比较各组神经功能评分、梗 死体积及NGF的表达。结果:预缺血组神经功能评分、梗死体积较假手术组减少(P<0．05),NGF表达明显高于其余两 组(P<0．01)。结论:局灶性缺血预处理可诱导脑缺血耐受的产生,其作用机制可能与NGF的表达改变有关。     

Changes in neuronal response to ischemia in retinas with genetic alterations of somatostatin receptor expression

Ischemia is a primary cause of neuronal death in retinal diseases. The repertoire of expressed transmitter receptors would determine the neurons' responses to ischemic damage, and peptidergic receptors may be involved. With a new in vitro model of the ischemic mouse retina, we investigated whether an altered expression of somatostatin receptors could modulate retinal responses to ischemia. We used retinas of somatostatin receptor 1 (sst(1)) knock out (KO) mice, where sst(2) are over-expressed and over-functional, and of sst(2) KO mice. TUNEL analysis of ischemic retinas showed a marked reduction of cell death in sst(1) KO retinas, while there were no differences between wild-type (WT) and sst(2) KO retinas. In addition, caspase-3 mRNA expression was also reduced in sst(1) KO as compared to WT retinas. An immunohistochemical analysis demonstrated that different cell populations responded differently to the ischemic insult, and that the persistence of some immunohistochemical markers was greater in sst(1) KO than in WT or in sst(2) KO retinas. In particular, rod bipolar cell survival was markedly improved in sst(1) KO retinas, while it was dramatically decreased in sst(2) KO retinas. Furthermore, consistent with a role of glutamate excitotoxicity in ischemia-induced neuronal death, retinal glutamate release was observed to increase under ischemic conditions, but this increase was significantly reduced in sst(1) KO retinas. These observations demonstrate that an increased presence of functional sst(2) protects against retinal ischemia, thus implementing the background for the use of sst(2) analogs in therapies of retinal diseases such as glaucoma or diabetic retinopathy.     

Ionotropic glutamate receptor antagonists inhibit the proliferation of granule cell precursors in the adult brain after seizures induced by pentylenetrazol

Brief ischemia was reported to protect various cells against injury induced by subsequent ischemia-reperfusion, and this phenomenon is known as ischemic preconditioning. The aims of the present study were to clarify whether early ischemic preconditioning could be observed in the rat retina by histological examination. Male Sprague-Dawley rats were subjected to 60 min of retinal ischemia by raising intraocular pressure to 130 mm Hg. Ischemic preconditioning was achieved by applying 5 min of ischemia 5-60 min before 60 min of ischemia. Additional groups of rats received 10 mg/kg 8-phenyltheophiline and 4.5 mg/kg 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), adenosine A1 receptor antagonists, 5 mg/kg 5-hydroxydecanoate and 1 mg/kg glibenclamide, ATP-sensitive K+ channel blockers, or 2.5 mg/kg chelerythrine and 0.1 mg/kg bisindolylmaleimide I, protein kinase C inhibitors, 15 or 30 min before preconditioning. In the non-preconditioned group, cell loss in the ganglion cell layer and thinning of the inner plexiform and inner nuclear layer were observed 7 days after 60 min of ischemia. Five minutes of preconditioning ischemia 20-40 min before 60 min of sustained ischemia completely prevented the retinal tissue damage induced by the sustained ischemia. Treatment with 8-phenyltheophylline, DPCPX, 5-hydroxydecanoate, glibenclamide, chelerythrine and bisindolylmaleimide I almost completely reduced the protective effect of early ischemic preconditioning. The results in the present study indicated that early ischemic preconditioning was demonstrated in the rat retina. Stimulation of adenosine receptors, opening of ATP-sensitive K+ channels and activation of protein kinase C might be involved in the underlying protective mechanisms.     

Lycium barbarum polysaccharides protects retinal ganglion cells against oxidative stress injury

The accumulation of excessive reactive oxygen species can exacerbate any injury of retinal tissue because free radicals can trigger lipid peroxidation,protein damage and DNA fragmentation.Increased oxidative stress is associated with the common pathological process of many eye diseases,such as glaucoma,diabetic retinopathy and ischemic optic neuropathy.Many studies have demonstrated that Lycium barbarum polysaccharides(LBP)protects against oxidative injury in numerous cells and tissues.For the model of hypoxia we used cultured retinal ganglion cells and induced hypoxia by incubating with 200μM cobalt chloride(CoCl2)for 24 hours.To investigate the protective effect of LBP and its mechanism of action against oxidative stress injury,the retinal tissue was pretreated with 0.5 mg/mL LBP for 24 hours.The results of flow cytometric analysis showed LBP could effectively reduce the CoCl2-induced retinal ganglion cell apoptosis,inhibited the generation of reactive oxygen species and the reduction of mitochondrial membrane potential.These findings suggested that LBP could protect retinal ganglion cells from CoCl2-induced apoptosis by reducing mitochondrial membrane potential and reactive oxygen species.     

MicroRNAs as biomarkers of diabetic retinopathy and disease progression

Diabetes mellitus, together with its complications, has been increasing in prevalence worldwide. Its complications include cardiovascular disease(e.g., myocardial infarction, stroke), neuropathy, nephropathy, and eye complications(e.g., glaucoma, cataracts, retinopathy, and macular edema). In patients with either type 1 or type 2 diabetes mellitus, diabetic retinopathy is the leading cause of visual impairment or blindness. It is characterized by progressive changes in the retinal microvasculature. The progression from nonproliferative diabetic retinopathy to a more advanced stage of moderate to severe nonproliferative diabetic retinopathy and proliferative diabetic retinopathy occurs very quickly after diagnosis of mild nonproliferative diabetic retinopathy. The etiology of diabetic retinopathy is unclear, and present treatments have limited effectiveness. Currently diabetic retinopathy can only be diagnosed by a trained specialist, which reduces the population that can be examined. A screening biomarker of diabetic retinopathy with high sensitivity and specificity would aid considerably in identifying those individuals in need of clinical assessment and treatment. The majority of the studies reviewed identified specific microRNAs in blood serum/plasma able to distinguish diabetic patients with retinopathy from those without retinopathy and for the progresion of the disease from nonproliferative diabetic retinopathy to proliferative diabetic retinopathy. In addition,certain microRNAs in vitreous humor were dysregulated in proliferative diabetic retinopathy compared to controls. A very high percentage of patients with diabetic retinopathy develop Alzheimer's disease. Thus, identifying diabetic retinopathy by measurement of suitable biomarkers would also enable better screening and treatment of those individuals at risk of Alzheimer's disease.     

A forebrain ischemic preconditioning model established in C57Black/Crj6 mice

Although many kinds of rat and gerbil cerebral ischemic preconditioning models are available, only a focal ischemic preconditioning model in mice has been reported. As most genetic alterations have been performed in mice, it is urgent to develop mouse ischemic preconditioning models for investigating the molecular mechanisms of ischemic preconditioning in transgenic mice. In the present study, we developed a forebrain ischemic preconditioning model in C57Black/Crj6 (C57BL/6) mice. Forebrain ischemia was induced in C57BL/6 mice (8-10 weeks old) by bilateral common carotid artery occlusion (BCCAO) for 18 min. The conditioning ischemic insult lasting for 6 min was carried out 48 h before the 18-min BCCAO. On the seventh day after BCCAO, neuronal damage was visualized by microtubule-associated protein-2 immunohistochemistry and quantified by cresyl violet staining. Terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) was performed 72 h after reperfusion to detect DNA fragmentation. Ischemia for 18 min resulted in injury to the striatum, cortex and hippocampus. In comparison to the hippocampus, striatal neuronal injury was more severe and reproducible. Although the conditioning ischemia itself caused neither noticeable striatal neuronal damage nor DNA fragmentation, it significantly reduced striatal neuronal damage and DNA fragmentation caused by the subsequent 18-min ischemia. These results indicate that striatal neuronal injury after transient BCCAO can be strongly reduced by a sublethal ischemic episode in C57BL/6 mice. As many kinds of gene-altered C57BL/6 mice are available, this preconditioning model may be useful for investigating the molecular mechanisms of ischemic preconditioning in transgenic mice.     

Molecular Mechanisms Mediating Diabetic Retinal Neurodegeneration: Potential Research Avenues and Therapeutic Targets

Diabetic retinopathy (DR) is a devastating complication of diabetes with a prevalence rate of 35%, and no effective treatment options. Since the most visible clinical features of DR are microvascular irregularities, therapeutic interventions often attempt to reduce microvascular injury, but only after permanent retinal damage has ensued. However, recent data suggests that diabetes initially affects retinal neurons, leading to neurodegeneration as an early occurrence in DR, before onset of the more noticeable vascular abnormalities. In this review, we delineate the sequence of initiating events leading to retinal degeneration in DR, considering neuronal dysfunction as a primary event. Key molecular mechanisms and potential biomarkers associated with retinal neuronal degeneration in diabetes are discussed. In addition to glial reactivity and inflammation in the diabetic retina, the contribution of neurotrophic factors, cell adhesion molecules, apoptosis markers, and G protein signaling to neurodegenerative pathways warrants further investigation. These studies could complement recent developments in innovative treatment strategies for diabetic retinopathy, such as targeting retinal neuroprotection, promoting neuronal regeneration, and attempts to re-program other retinal cell types into functional neurons. Indeed, several ongoing clinical trials are currently attempting treatment of retinal neurodegeneration by means of such novel therapeutic avenues. The aim of this article is to highlight the crucial role of neurodegeneration in early retinopathy progression, and to review the molecular basis of neuronal dysfunction as a first step toward developing early therapeutic interventions that can prevent permanent retinal damage in diabetes. ClinicalTrials.gov: NCT02471651, NCT01492400     

miR-30a-5p inhibition promotes interaction of Fas+ endothelial cells and FasL+ microglia to decrease pathological neovascularization and promote physiological angiogenesis

Ischemia-induced angiogenesis contributes to various neuronal and retinal diseases, and often results in neurodegeneration and visual impairment. Current treatments involve the use of anti-VEGF agents but are not successful in all cases. In this study we determined that miR-30a-5p is another important mediator of retinal angiogenesis. Using a rodent model of ischemic retinopathy, we show that inhibiting miR-30a-5p reduces neovascularization and promotes tissue repair, through modulation of microglial and endothelial cell cross-talk. miR-30a-5p inhibition results in increased expression of the death receptor Fas and CCL2, to decrease endothelial cell survival and promote microglial migration and phagocytic function in focal regions of ischemic injury. Our data suggest that miR-30a-5p inhibition accelerates tissue repair by enhancing FasL–Fas crosstalk between microglia and endothelial cells, to promote endothelial cell apoptosis and removal of dead endothelial cells. Finally, we found that miR-30a levels were increased in the vitreous of patients with proliferative diabetic retinopathy. Our study identifies a role for miR-30a in the pathogenesis of neovascular retinal disease by modulating microglial and endothelial cell function, and suggests it may be a therapeutic target to treat ischemia-mediated conditions.     

Poly-IC preconditioning protects against cerebral and renal ischemia-reperfusion injury

Preconditioning induces ischemic tolerance, which confers robust protection against ischemic damage. We show marked protection with polyinosinic polycytidylic acid (poly-IC) preconditioning in three models of murine ischemia-reperfusion injury. Poly-IC preconditioning induced protection against ischemia modeled in vitro in brain cortical cells and in vivo in models of brain ischemia and renal ischemia. Further, unlike other Toll-like receptor (TLR) ligands, which generally induce significant inflammatory responses, poly-IC elicits only modest systemic inflammation. Results show that poly-IC is a new powerful prophylactic treatment that offers promise as a clinical therapeutic strategy to minimize damage in patient populations at risk of ischemic injury.     

无创伤双后肢缺血后处理对移植胰腺缺血再灌注损伤的远隔保护

背景：缺血预处理及缺血后处理是近年来提出减轻缺血再灌注损伤有效方法。 目的：探讨无创伤双后肢缺血后处理对移植胰腺缺血再灌注损伤的影响及机制。 方法：18只糖尿病SD大鼠数字表法随机分为3组,对照组仅行开腹术;缺血再灌注组仅行胰腺移植;缺血后处理组,移植前行非创伤性双后肢缺血后处理。 结果与结论：缺血再灌注组血糖和胰腺组织中丙二醛水平均高于缺血后处理组(P < 0.01)、而超氧化物歧化酶活性低于缺血后处理组(P < 0.01);与缺血后处理组比较,缺血再灌注组胰腺组织凋亡指数明显增高(P < 0.01)。结果提示,无创伤双后肢缺血后处理对大鼠移植胰的缺血再灌注损伤具有保护作用,机制可能与可通过减少超氧化物歧化酶失活,从而清除氧自由基以及减少胰腺细胞凋亡等有关。     

核因子—κB(NF—κB)在大鼠局灶性脑缺血耐受产生中的意义

目的:通过观察局灶脑缺血预处理对核因子κB(nuclear factor—κB,NF—κB)表达的影响初步探讨其在脑缺血耐受中的意义。方法:SD大鼠随机分为三组,对照组给予两次相前3天的假手术,其余两组分别在2小时大脑中动脉缺血(MCAO)及22小时再灌注前3天给予10分钟的预缺血或假手术,比较各组梗死体积、组织病理变化及NF—κB的表达。结果:预缺血组梗死体积较假手术组减小53.15％(p〈0.01),神经元损伤、坏死轻于后者,NF—κB表达低于假手术组及对照组(p〈0.01)。结论:10分钟大脑中动脉预缺血可有效诱导缺血耐受,减小梗死体积,减轻缺血性神经损伤,抑制NF—κB表达。NF—κB表达下调可能是局灶性脑缺血耐受产生的分子机制之一。     

ERK1 plays a critical protective role against N-methyl-D-aspartate-induced retinal injury

Excitotoxicity has been implicated in several ischemic diseases of the retina, including retinal vessel occlusion and diabetic retinopathy. Glutamate signaling mediated through the N-methyl-D-aspartate (NMDA) receptor contributes to ischemic cell death. The NMDA receptor antagonists MK-801 and memantine have substantial neuroprotective effects in experimental retinal disease models, but the mechanisms by which NMDA receptor activity leads to cell death is not clear. Here we describe a previously unknown role for retinal glial cells in NMDA-induced retinal injury that involves the activation of ERK1/2. Within 1 hr after injecting NMDA intravitreally, activation of ERK1/2 and c-Fos induction were observed in retinal Müller cells. The roles of activated ERK1/2 in neuronal damage were examined using ERK1 gene deficient mice (homozygous ERK1(-/-) mice). NMDA-induced ERK1/2 activation in retina was significantly suppressed in ERK1(-/-) mice, and these mice had significantly higher numbers of TUNEL-positive retinal cells than wild-type mice 24 hr after NMDA injection. These data suggest that, during NMDA injury, Müller cells are activated and play a protective role against NMDA-induced retinal cell death. ERK1 appears to play a major role in this process. These new findings on retinal glial cell response during NMDA injury offer an important new therapeutic target for preventing many retinal disorders associated with excitotoxicity.     

Blood microRNAs as potential diagnostic and prognostic markers in cerebral ischemic injury

MicroRNAs are a family of small, genome-encoded endogenous RNAs that are transcribed but are not translated into proteins. They serve essential roles in virtually every aspect of brain function, including neurogenesis, neural development, and cellular responses leading to changes in synaptic plasticity. They are also implicated in neurodegeneration and neurological disorders, in responses to hypoxia and ischemia, and in ischemic tolerance induced by ischemic preconditioning. In recent developments, miRNA expres-sion proifling has been examined in stroke, and these studies indicate that miRNAs have emerged as key mediators in ischemic stroke biology. Both increased and decreased miRNA levels may be needed either as prevention or treatment of stroke. Novel approaches are being developed to get miRNA related therapeu-tics into the brain across an intact blood-brain barrier, including chemical modiifcation, use of targeting molecules and methods to disrupt the blood-brain barrier.     

Reduction of Glut1 in the Neural Retina But Not the RPE Alleviates Polyol Accumulation and Normalizes Early Characteristics of Diabetic Retinopathy

Hyperglycemia is a key determinant for development of diabetic retinopathy (DR). Inadequate glycemic control exacerbates retinopathy, while normalization of glucose levels delays its progression. In hyperglycemia, hexokinase is saturated and excess glucose is metabolized to sorbitol by aldose reductase via the polyol pathway. Therapies to reduce retinal polyol accumulation for the prevention of DR have been elusive because of low sorbitol dehydrogenase levels in the retina and inadequate inhibition of aldose reductase. Using systemic and conditional genetic inactivation, we targeted the primary facilitative glucose transporter in the retina, Glut1, as a preventative therapeutic in diabetic male and female mice. Unlike WT diabetics, diabetic Glut1^+/− mice did not display elevated Glut1 levels in the retina. Furthermore, diabetic Glut1^+/− mice exhibited ameliorated ERG defects, inflammation, and oxidative stress, which was correlated with a significant reduction in retinal sorbitol accumulation. Retinal pigment epithelium-specific reduction of Glut1 did not prevent an increase in retinal sorbitol content or early hallmarks of DR. However, like diabetic Glut1^+/− mice, reduction of Glut1 specifically in the retina mitigated polyol accumulation and diminished retinal dysfunction and the elevation of markers for oxidative stress and inflammation associated with diabetes. These results suggest that modulation of retinal polyol accumulation via Glut1 in photoreceptors can circumvent the difficulties in regulating systemic glucose metabolism and be exploited to prevent DR.SIGNIFICANCE STATEMENT Diabetic retinopathy affects one-third of diabetic patients and is the primary cause of vision loss in adults 20-74 years of age. While anti-VEGF and photocoagulation treatments for the late-stage vision threatening complications can prevent vision loss, a significant proportion of patients do not respond to anti-VEGF therapies, and mechanisms to stop progression of early-stage symptoms remain elusive. Glut1 is the primary facilitative glucose transporter for the retina. We determined that a moderate reduction in Glut1 levels, specifically in the retina, but not the retinal pigment epithelium, was sufficient to prevent retinal polyol accumulation and the earliest functional defects to be identified in the diabetic retina. Our study defines modulation of Glut1 in retinal neurons as a targetable molecule for prevention of diabetic retinopathy.