小鼠真皮多能干细胞在缺血性损伤脑组织移植后的分布与分化

目的探讨小鼠真皮多能干细胞(SKP)经股静脉移植后在缺血性损伤脑组织中的分布及分化情况。方法分离培养绿色荧光蛋白转基因小鼠(C57BL/6-gfp)SKP,随机选取5只同基因型小鼠采用线栓法制作局灶性大脑中动脉栓塞(MCAO)模型,缺血2h后行再灌注,再灌注24h后将C57BL/6-gfp来源的SKP经小鼠股静脉输入动物模型体内,植入后第7天处死小鼠,作冷冻切片,采用免疫组织荧光染色法,检测SKP在脑缺血小鼠体内的分布及分化情况。结果移植SKP7d后,MCAO小鼠脑组织冷冻切片在荧光显微镜下可见移植的SKP主要分布在缺血灶周围,且这些细胞表达胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)。结论经股静脉移植的SKP主要分布在MCAO模型鼠脑缺血损伤区周围,并可向神经细胞分化。     

Microglial activation precedes and predominates over macrophage infiltration in transient focal cerebral ischemia: a study in green fluorescent protein transgenic bone marrow chimeric mice

Resident microglia and hematogenous macrophages play crucial roles in the pathogenetic cascade following cerebral ischemia but may functionally differ regarding neuroprotective and cytotoxic properties. Distinction between these cells has not been possible due to a lack of discriminating cellular markers. We generated bone marrow chimeric mice by transplanting bone marrow from green fluorescent protein (GFP) transgenic mice into irradiated wild-type recipients. Transient focal cerebral ischemia was induced by transient middle cerebral artery occlusion (MCAO) for 30 min. Resident microglia and infiltrating macrophages were identified by immunohistochemistry and GFP fluorescence after 1-28 days. The first blood-derived cells infiltrating the infarct area were seen on Day 1 and identified as granulocytes. Hematogenous GFP(+) macrophages were rarely observed on Day 2, reached peak numbers on Day 7, and decreased thereafter. In contrast, resident GFP(-) microglial cells rapidly became activated already on Day 1 after MCAO. Even on Days 4 and 7, most macrophage-like cells remained GFP(-), indicating their derivation from resident microglia. Hematogenous macrophages were able to acquire a ramified morphology indistinguishable from resident microglia while microglial cells could develop into a phagocytic phenotype indistinguishable from infiltrating macrophages. The vast majority of macrophages in the infarct area are derived from local microglia, revealing a remarkable predominance of local defense mechanisms over immune cells arriving from the blood. GFP bone marrow chimeric mice are a powerful tool to further differentiate the function of resident microglia and hematogenous macrophages following cerebral ischemia.     

Release of bradykinin and expression of kinin B2 receptors in the brain: role for cell death and brain edema formation after focal cerebral ischemia in mice.

Pharmacological studies using bradykinin B2 receptor antagonists suggest that bradykinin, an early mediator of inflammation and the main metabolite of the kallikrein-kinin system, is involved in secondary brain damage after cerebral ischemia. However, the time-course of bradykinin production and kinin receptor expression as well as the conclusive role of bradykinin B2 receptors for brain damage after experimental stroke have not been elucidated so far. C57/Bl6 mice were subjected to 45 mins of middle cerebral artery occlusion (MCAO) and 2, 4, 8, 24, and 48 h later brains were removed for the analysis of tissue bradykinin concentration and kinin B2 receptor mRNA and protein expression. Brain edema, infarct volume, functional outcome, and long-term survival were assessed in WT and B2-/- mice 24 h or 7 days after MCAO. Tissue bradykinin was maximally increased 12 h after ischemia (three-fold), while kinin B2 receptor mRNA upregulation peaked 24 to 48 h after MCAO (10- to 12-fold versus na?ve brain tissue). Immunohistochemistry revealed that kinin B2 receptors were constitutively and widely expressed in mouse brain, were upregulated 2 h after ischemia in cells showing signs of ischemic damage, and remained upregulated in the penumbra up to 24 h after ischemia. B2-/- mice had improved motor function (P<0.05), smaller infarct volumes (-38%; P<0.01), developed less brain edema (-87%; P<0.05), and survived longer (P<0.01) as compared with wild-type controls. The current results show that bradykinin is produced in the brain, kinin B2 receptors are upregulated on dying cells, and B2 receptors are involved in cell death and brain edema formation after experimental stroke.     

High susceptibility to cerebral ischemia in GFAP-null mice.

Astrocytes perform a variety of functions in the adult central nervous system (CNS) that contribute to the survival of neurons. Thus, it is likely that the activities of astrocytes affect the extent of brain damage after ischemic stroke. The authors tested this hypothesis by using a mouse ischemia model to compare the infarct volume produced in wild-type mice with that produced in mice lacking glial fibrillary acidic protein (GFAP), an astrocyte specific intermediate filament component. Astrocytes lacking GFAP have been shown to have defects in process formation, induction of the blood-brain barrier. and volume regulation; therefore, they might be compromised in their ability to protect the CNS after injury. The authors reported here that 48 hours after combined permanent middle cerebral artery occlusion (MCAO) and 15 minutes transient carotid artery occlusion (CAO) GFAP-null mice had a significantly (P < 0.001) larger cortical infarct volume (16.7 +/- 2.2 mm3) than their wild-type littermates (10.1 +/- 3.9 mm3). Laser-Doppler flowmetry revealed that the GFAP-null mice had a more extensive and profound decrease in cortical cerebral blood flow within 2 minutes after MCAO with CAO. These results indicated a high susceptibility to cerebral ischemia in GFAP-null mice and suggested an important role for astrocytes and GFAP in the progress of ischemic brain damage after focal cerebral ischemia with partial reperfusion.     

Obligatory role of inducible nitric oxide synthase in ischemic preconditioning.

Sublethal insults can induce a transient tolerance toward subsequent lethal ischemia, a phenomenon termed ischemic preconditioning (IPC). In the myocardium, nitric oxide derived from 'inducible' nitric oxide synthase (iNOS or NOS II) plays a critical role in the expression of IPC produced by sublethal ischemia. Here, we investigated whether iNOS is involved in IPC in brain. Ischemic preconditioning was produced in mice by three episodes of 1-min bilateral common carotid artery (BCCA) occlusion, each followed by 5 mins of reperfusion. After 24 h, mice underwent middle cerebral artery (MCA) occlusion for 20 mins. Intraischemic cerebral blood flow was monitored during both in BCCA and MCA occlusion (MCAO) by laser-Doppler flowmetry. Mice were killed 3 days after MCAO, and infarct volume was determined in thionine-stained sections. Infarct volume was significantly reduced 24 h after IPC (70%; P<0.05). Treatment with the iNOS inhibitor aminoguanidine (400 mg/kg), abolished the IPC-induced protection. Furthermore, IPC failed to induce ischemic tolerance in iNOS-null mice. In wild-type mice, IPC increased the resistance to Ca(2+)-mediated depolarization in isolated brain mitochondria. However, in iNOS-null mice IPC failed to induce such resistance. We conclude that iNOS is required for the full expression of IPC and that such effect is coupled to an increased resistance of mitochondria to injury. Thus, iNOS-derived nitric oxide, in addition to its deleterious effects on the late stages of ischemic brain damage, can also be beneficial by promoting ischemic tolerance through signaling, ultimately resulting in mitochondrial protection.     

Inducible nitric oxide synthase expression in human cerebral infarcts

The inducible or “immunological” isoform of nitric oxide synthase (iNOS) is induced in many cell types by inflammatory stimuli and synthesizes toxic amounts of NO. In rodent models of focal cerebral ischemia, iNOS is expressed in neutrophils invading the injured brain and in local blood vessels. Studies with iNOS inhibitors and iNOS null mice indicate that NO produced by iNOS contributes to ischemic brain injury. In the present study, we sought to determine whether iNOS is also expressed in the human brain after ischemic stroke. Studies were conducted using immunohistochemistry on autopsy brains with neuropathological evidence of acute cerebral infarction. iNOS immunoreactivity was observed in neutrophils infiltrating the ischemic brain and in blood vessels within the ischemic territory. iNOS-positive cells also were immunoreactive for nitrotyrosine, reflecting protein nitration by NO-derived peroxynitrite and nitrites. iNOS or nitrotyrosine immunoreactivity was not detected outside the region of the infarct. These observations provide evidence that iNOS is expressed in the human brain after ischemic infarction and support the hypothesis that iNOS inhibitors may be useful in the treatment of ischemic stroke in humans. Received: 8 June 1998 / Revised, accepted: 23 September 1998     

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.     

Inhibiting cell cycle progression reduces reactive astrogliosis initiated by scratch injury in vitro and by cerebral ischemia in vivo

Astrogliosis occurs in a variety of neuropathological disorders and injuries, and excessive astrogliosis can be devastating to the recovery of neuronal function. In this study, we asked whether reactive astrogliosis can be suppressed in the lesion area by cell cycle inhibition and thus have therapeutic benefits. Reactive astrogliosis induced in either cultured astrocytes by hypoxia or scratch injury, or in a middle cerebral artery occlusion (MCAO) ischemia model were combined to address this issue. In the cultured astrocytes, hypoxia induced a cell cycle activation that was associated with upregulation of the proliferating cell nuclear marker (PCNA). Significantly, the cell cycle inhibitor, olomoucine, inhibited hypoxia-induced cell cycle activation by arresting the cells at G1/S and G2/M in a dose-dependent manner and also reversed hypoxia-induced upregulation of PCNA. Also in the cultured astrocytes, scratch injury induced reactive astrogliosis, such as hypertrophy and an increase in BrdU(+) astrocytes, both of which were ameliorated by olomoucine. In the MCAO ischemia mouse model, dense reactive glial fibrillary acidic protein and PCNA immunoreactivity were evident at the boundary zone of focal cerebral ischemia at days 7 and 30 after MCAO. We found that intraperitoneal olomoucine administration significantly inhibited these astrogliosis-associated changes. To demonstrate further that cell cycle regulation impacts on astrogliosis, cyclin D1 gene knockout mice (cyclin D1(-/-)) were subjected to ischemia, and we found that the percentage of Ki67-positive astrocytes in these mice was markedly reduced in the boundary zone. The number of apoptotic neurons and the lesion volume in cyclin D1(-/-) mice also decreased as compared to cyclin D1(+/+) and cyclin D1(+/-) mice at days 3, 7, and 30 after local cerebral ischemia. Together, these in vitro and in vivo results strongly suggest that astrogliosis can be significantly affected by cell cycle inhibition, which therefore emerges as a promising intervention to attenuate reactive glia-related damage to neuronal function in brain pathology.     

ADAMTS13 gene deletion enhances plasma high-mobility group box1 elevation and neuroinflammation in brain ischemia–reperfusion injury

Highly adhesive glycoprotein von Willebrand factor (VWF) multimer induces platelet aggregation and leukocyte tethering or extravasation on the injured vascular wall, contributing to microvascular plugging and inflammation in brain ischemia–reperfusion. A disintegrin and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) cleaves the VWF multimer strand and reduces its prothrombotic and proinflammatory functions. Although ADAMTS13 deficiency is known to amplify post-ischemic cerebral hypoperfusion, there is no report available on the effect of ADAMTS13 on inflammation after brain ischemia. We investigated if ADAMTS13 deficiency intensifies the increase of extracellular HMGB1, a hallmark of post-stroke inflammation, and exacerbates brain injury after ischemia–reperfusion. ADAMTS13 gene knockout (KO) and wild-type (WT) mice were subjected to 30-min middle cerebral artery occlusion (MCAO) and 23.5-h reperfusion under continuous monitoring of regional cerebral blood flow (rCBF). The infarct volume, plasma high-mobility group box1 (HMGB1) level, and immunoreactivity of the ischemic cerebral cortical tissue (double immunofluorescent labeling) against HMGB1/NeuN (neuron-specific nuclear protein) or HMGB1/MPO (myeloperoxidase) were estimated 24 h after MCAO. ADAMTS13KO mice had larger brain infarcts compared with WT 24 h after MCAO (p < 0.05). The rCBF during reperfusion decreased more in ADAMTS13KO mice. The plasma HMGB1 increased more in ADAMTS13KO mice than in WT after ischemia–reperfusion (p < 0.05). Brain ischemia induced more prominent activation of inflammatory cells co-expressing HMGB1 and MPO and more marked neuronal death in the cortical ischemic penumbra of ADAMTS13KO mice. ADAMTS13 deficiency may enhance systemic and brain inflammation associated with HMGB1 neurotoxicity, and aggravate brain damage in mice after brief focal ischemia. We hypothesize that ADAMTS13 protects brain from ischemia–reperfusion injury by regulating VWF-dependent inflammation as well as microvascular plugging.     

bcl-xl over-expression in transgenic mice reduces cerebral ischemia/reperfusion injury*☆

BACKGROUND： Basal cell lymphoma-extra large （bcl-xl） can inhibit neuronal apoptosis by stabilizing the mitochondrial membrane and suppressing cytochrome C release into the cytoplasm. OBJECTIVE： This study aimed to further investigate the cascade reaction pathway of cellular apoptosis. We established an ischemia/repcrfusion model by middle cerebral artery occlusion （MCAO） in transgenic and wild-type mice, and observed changes in the number and distribution of apoptotic neural cells, differences in cerebral infarct volume, in neurological function score, and in cytochrome C expression in the ischemic cerebral cortex, at different time points, DESIGN AND SETTING： The present gene engineering and cell biology experiment was performed at the Laboratory of Biology, Hubei Academy of Agricultural Sciences and at the Laboratory of Immunology, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS： Male bcl-xl over-expression Kunming mice aged 8 weeks and age-matched male wild-type mice were used for this study. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling （TUNEL） kits were purchased from Boliman, France. Cytochrome C antibody and Bcl-x immunohistochemical kit were purchased from PharMingen, USA and Santa Cruz Biotechnology, USA, respectively. METHODS： Following MCAO and reperfusion, apoptosis in the ischemic cerebral cortex was detected by the TUNEL assay. Prior to MCAO and 3 hours after reperfusion, the Bcl-xl protein level in the ischemic cerebral cortex was measured by immunohistochemistry. At 3, 6, 12 and 24 hours after reperfusion, the level of cytochrome C in the ischemic cerebral cortex was examined by western blot analysis. Subsequent to MCAO, cerebral infarct volume measurement and neurological examination were performed. MAIN OUTCOME MEASURES： Neural cell apoptosis and cytochrome C expression in the ischemic cerebral cortex; cerebral infarct volume and neurological function score. RESULTS： Twenty-four hours after reperfusion, cerebral inf     

Transgenic overexpression of adenosine kinase aggravates cell death in ischemia.

Adenosine is an endogenous neuromodulator with anticonvulsive and neuroprotective activity. Adenosine levels are normally kept in the range of 20 to 200 nmol/L by low basal expression of its main metabolic enzyme, adenosine kinase (ADK). Dysfunction of the adenosinergic system has been demonstrated to contribute to epileptogenesis. To investigate whether upregulation of ADK may render the brain more susceptible to ischemic cell death, mutant mice overexpressing an Adk transgene in brain were subjected to middle cerebral artery occlusion (MCAO). One day after either 15 or 60 mins of MCAO, wild-type (WT) animals had infarct areas encompassing about 5% and 50% of their ischemic hemisphere, respectively. In marked contrast, the volume of the infarcts increased three-fold in Adk transgenic mutants after 15 mins of MCAO, and after 60 mins of MCAO all mutants died within 24 h. Pretreatment of the mutants with the ADK inhibitor 5-iodotubercidin led to lesions similar to those in WT mice. Thus, low levels of ADK are essential to maintain adenosine-mediated neuroprotection. We conclude that pathologic overexpression of ADK as in epilepsy may also render the brain more susceptible to injury from ischemia. Consequently, ADK emerges as a rational therapeutic target to enhance neuroprotection.     

Monocyte chemoattractant protein-1 deficiency is protective in a murine stroke model.

Inflammatory processes have been implicated in the pathogenesis of brain damage after stroke. In rodent stroke models, focal ischemia induces several proinflammatory chemokines, including monocyte chemoattractant protein-1 (MCP-1). The individual contribution to ischemic tissue damage, however, is largely unknown. To address this question, the authors subjected MCP-1-deficient mice (MCP-1-/-) to permanent middle cerebral artery occlusion (MCAO). Measurement of basal blood pressure, cerebral blood flow, and blood volume revealed no differences between wild-type (wt) and MCP-1-/- mice. MCAO led to similar cerebral perfusion deficits in wt and MCP-1-/- mice, excluding differences in the MCA supply territory and collaterals. However, compared with wt mice, the mean infarct volume was 29% smaller in MCP-1-/- mice 24 hours after MCAO (P = 0.022). Immunostaining showed a reduction of phagocytic macrophage accumulation within infarcts and the infarct border in MCP-1-/- mice 2 weeks after MCAO. At the same time point, the authors found an attenuation of astrocytic hypertrophy in the infarct border and thalamus in MCP-1-/- mice. However, these effects on macrophages and astrocytes in MCP-1-/- mice occurred too late to suggest a protective role in acute infarct growth. Of note: at 6 hours after MCAO, MCP-1-/- mice produced significantly less interleukin-1beta in ischemic tissue; this might be related to tissue protection. The results of this study indicate that inhibition of MCP-1 signaling could be a new acute treatment approach to limit infarct size after stroke.     

L-Arginine increases ischemic injury in wild-type mice but not in iNOS-deficient mice

Delayed administration of the nitric oxide precursor L-arginine increases brain injury in models of focal cerebral ischemia. We tested the hypothesis that L-arginine worsens the damage by acting as a substrate for inducible nitric oxide synthase (iNOS) and increasing the output of this enzyme. iNOS-null mice or wild-type littermates were treated with L-arginine (300 mg/kg; i.p, three times/day) starting 12 h after occlusion of the middle cerebral artery. Infarct volume was determined 96 h after ischemia. We found that L-arginine enlarges infarct volume in wild-type mice (+28+/-5% in neocortex) but not in iNOS-null mice. Thus, the worsening of ischemic damage produced by L-arginine depends on iNOS. The findings support the hypothesis that L-arginine worsens ischemic injury by increasing the catalytic output of iNOS and suggest that administration of L-arginine should be avoided in patients with acute stroke.     

The cyclooxygenase-2 inhibitor NS-398 ameliorates ischemic brain injury in wild-type mice but not in mice with deletion of the inducible nitric oxide synthase gene.

The authors investigated the role of the prostaglandin-synthesizing enzyme cyclooxygenase-2 (COX-2) in the mechanisms of focal cerebral ischemia and its interaction with inducible nitric oxide synthase (iNOS). Focal cerebral ischemia was produced by permanent occlusion of the middle cerebral artery (MCA) in mice. Infarct volume was measured 96 hours later by computer-assisted planimetry in thionin-stained brain sections. The highly selective COX-2 inhibitor NS398 (20 mg/kg; intraperitoneally), administered twice a day starting 6 hours after MCA occlusion, reduced total infarct volume in C57BL/6 (-23%) and 129/SVeV mice (-21%), and ameliorated the motor deficits produced by MCA occlusion (P < .05). However, NS398 did not influence infarct volume in mice with deletion of the iNOS gene (P > .05). In contrast, the neuronal NOS inhibitor 7-NI (50 mg/kg; intraperitoneally), administered once 5 minutes after MCA occlusion, reduced neocortical infarct volume by 20% in iNOS -/- mice (P < .05). NS398 did not affect arterial pressure, resting CBF or the CBF reactivity to hypercapnia in anesthetized iNOS null mice (P > .05). The data suggest that COX-2 reaction products, in mouse as in rat, contribute to ischemic brain injury. However, the failure of NS398 to reduce infarct volume in iNOS null mice suggests that iNOS-derived NO is required for the deleterious effects of COX-2 to occur. Thus, COX-2 reaction products may be another mechanism by which iNOS-derived NO contributes to ischemic brain injury.     

颈交感神经干离断对局灶性脑缺血再灌注后大鼠海马iNOS表达的影响

目的采用颈交感干离断（TCST）模拟星状神经节阻滞，观察其对局灶性脑缺血再灌注损伤（CIRI）大鼠脑梗死容积及海马诱导型一氧化氮合酶（iNOS）表达等的影响，并探讨其脑保护作用的机制。方法将大鼠随机分成实验组（A组）、对照组（B组）和假手术组（C组）；采用线栓法行大脑中动脉栓塞（MCAO）制作大鼠局灶性CIRI模型，A组于TCST后即行MCAO，2h后再恢复灌注；B组为单纯CIRI组；C组仅完成与A组相似的手术步骤但不造成MCAO、不行TCST；再灌注24h后观察各组大鼠神经行为学评分、脑梗死容积及海马iNOs的表达变化。结果A组大鼠脑梗死容积和神经行为学评分均低于B组（P〈0．05）；与A组、C组相比，B组大鼠海马iNOS的表达增加（P〈0．05），而A组与C组间无显著差异（P〉0．05）。结论TCST可通过下调大鼠海马iNOS的表达而对局灶性CIRI发挥脑保护作用。     

Transplantation of human mesenchymal stem cells promotes functional improvement and increased expression of neurotrophic factors in a rat focal cerebral ischemia model

Previous studies have suggested that intravenous transplantation of mesenchymal stem cells (MSCs) in rat ischemia models reduces ischemia‐induced brain damage. Here, we analyzed the expression of neurotrophic factors in transplanted human MSCs and host brain tissue in rat middle cerebral artery occlusion (MCAO) ischemia model. At 1 day after transient MCAO, 3 × 10⁶ immortalized human MSC line (B10) cells or PBS was intravenously transplanted. Behavioral tests, infarction volume, and B10 cell migration were investigated at 1, 3, 7, and 14 days after MCAO. The expression of endogenous (rat origin) and exogenous (human origin) neurotorphic factors and cytokines was evaluated by quantitative real‐time RT‐PCR and Western blot analysis. Compared with PBS controls, rats receiving MSC transplantation showed improved functional recovery and reduced brain infarction volume at 7 and 14 days after MCAO. In MSC‐transplanted brain, among many neurotrofic factors, only human insulin‐like growth factor 1 (IGF‐1) was detected in the core and ischemic border zone at 3 days after MCAO, whereas host cells expressed markedly higher neurotrophic factors (rat origin) than control rats, especially vascular endothelial growth factor (VEGF) at 3 days and epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) at 7 days after MCAO. Intravenously transplanted human MSCs induced functional improvement, reduced infarct volume, and neuroprotection in ischemic rats, possibly by providing IGF‐1 and inducing VEGF, EGF, and bFGF neurotrophic factors in host brain. © 2009 Wiley‐Liss, Inc.     

Comparison of ischemia-directed migration of neural precursor cells after intrastriatal, intraventricular, or intravenous transplantation in the rat

Cell replacement therapy may have the potential to promote brain repair and recovery after stroke. To compare how focal cerebral ischemia affects the entry, migration, and phenotypic features of neural precursor cells transplanted by different routes, we administered neuronal precursors from embryonic cerebral cortex of green fluorescent protein (GFP)-expressing transgenic mice to rats that had undergone middle cerebral artery occlusion (MCAO) by the intrastriatal, intraventricular, and intravenous routes. MCAO increased the entry of GFP-immunoreactive cells, most of which expressed neuroepithelial (nestin) or neuronal (doublecortin) markers, from the ventricles and bloodstream into the brain, and enhanced their migration when delivered by any of these routes. Transplanted neural precursors migrated into the ischemic striatum and cerebral cortex. Thus, transplantation of neural precursors by a variety of routes can deliver cells with the potential to replace injured neurons to ischemic brain regions.     

bcl-xl over-expression in transgenic mice reduces cerebral ischemia/reperfusion injury

BACKGROUND:Basal cell lymphoma-extra large (bcl-xl) can inhibit neuronal apoptosis by stabilizing the mitochondrial membrane and suppressing cytochrome C release into the cytoplasm. OBJECTIVE: This study aimed to further investigate the cascade reaction pathway of cellular apoptosis. We established an ischemia/reperfusion model by middle cerebral artery occlusion (MCAO) in transgenic and wild-type mice,and observed changes in the number and distribution of apoptotic neural cells,differences in cerebral infa...     

Agmatine reduces infarct area in a mouse model of transient focal cerebral ischemia and protects cultured neurons from ischemia-like injury

Agmatine is a primary amine formed by the decarboxylation of L-arginine synthesized in mammalian brain. In this study, we investigated the neuroprotective effect of agmatine on ischemic and ischemia-like insults. Primary cortical neuronal cultures were subjected to oxygen-glucose deprivation (OGD), a model of ischemia-like injury, and treated with agmatine before or at the start of OGD, or upon reperfusion. Neuronal death was reduced when agmatine was present during OGD, and this protection was associated with a reduction of nitric oxide (NO) and neuronal nitric oxide synthase (nNOS), but not inducible NOS (iNOS). Protection by agmatine was also studied at the in vivo level using a model of middle cerebral artery occlusion (MCAO) in mice. Mice were subjected to 2 h MCAO. Agmatine was administered either 30 min before ischemia, at the start of MCAO, at the start of reperfusion, or 2 or 5 h into reperfusion. Agmatine markedly reduced infarct area in all treatment groups except when treatment was delayed 5 h. The number of nNOS immunopositive cells was correlated with neuroprotection. Interestingly, immunoreactivity for iNOS was reduced only when agmatine was administered before and at the onset of MCAO. Our study suggests that agmatine may be a novel therapeutic strategy to reduce cerebral ischemic injury, and may act by inhibiting the detrimental effects of nNOS.     

Early NFkappaB activation is inhibited during focal cerebral ischemia in interleukin-1beta-converting enzyme deficient mice

Our previous study demonstrated that the inhibition of interleukin-1beta (IL-1beta) reduces ischemic brain injury; however, the molecular mechanism of the action of IL-1 in cerebral ischemia is unclear. We are investigating currently the role of NFkappaB during focal cerebral ischemia, using mutant mice deficient in the interleukin-1 converting enzyme gene (ICE KO) in a middle cerebral artery occlusion (MCAO) model. Adult male ICE KO and wild-type mice (n = 120) underwent up to 24 hr of permanent MCAO. Cytoplasmic phospho-NFkappaB/p65 expression in ischemic brain was examined using Western blot analysis and immunohistochemistry. NFkappaB DNA-binding activity was detected using electrophoretic mobility shift assay (EMSA). Furthermore, ICAM-1 expression was examined in both the ICE KO and wild-type mice (WT). Western blot analysis and immunostaining showed that the level of cytosolic phosphorylated NFkappaB/p65 increased after 2 and 4 hr of MCAO in WT mice; however, NFkappaB/p65 was significantly reduced after MCAO in the ICE KO mice (P < 0.05). EMSA showed that NFkappaB DNA-binding activity increased after MCAO in WT mice; but this effect was reduced in the ICE KO mice. The number of ICAM-1-positive vessels in the ischemic hemisphere was greatly attenuated in the ICE KO mice (P < 0.05), which paralleled the results of immunohistochemistry. Our results demonstrate that NFkappaB phosphorylation is reduced in ICE KO mice, suggesting that ICE or IL-1 are involved in early NFkappaB phosphorylation. Because cerebral ischemia induced infarction is significantly reduced in ICE KO mice, we conclude that early NFkappaB phosphorylation plays a disruptive role in the ischemic process.