Survivin-dependent angiogenesis in ischemic brain: molecular mechanisms of hypoxia-induced up-regulation

Approaches to regulating angiogenesis in the brain, which may diminish parenchymal damage after stroke, are lacking. Survivin, the inhibitor of apoptosis protein, is up-regulated in vitro in vascular endothelial cells by angiogenic factors, including vascular endothelial cell growth factor (VEGF). To evaluate the in vivo role of survivin in the brain in response to hypoxia/ischemia, we used a mouse model of stroke and show that 2 days after permanent middle cerebral artery occlusion, survivin is uniquely expressed by microvessels that form in the peri-infarct and infarct regions. The extent of vascularization of the infarct is dependent on expression of survivin, since vessel density is significantly reduced in mice with heterozygous deficiency of the survivin gene (survivin+/- mice), even though infarct sizes were not different. Hypoxia alone induces survivin expression in the brain, by cultured endothelial cells and by embryonic stem cells, but this response is at least partially independent of VEGF, hypoxia inducible factor 1alpha, or placental growth factor. Delineating the spatiotemporal pattern of expression of survivin after stroke, and the molecular mechanisms by which this is regulated, may provide novel approaches to therapeutically optimize angiogenesis in a variety of ischemic disorders.     

Long‐term survival and regeneration of neuronal and vasculature cells inside the core region after ischemic stroke in adult mice

Focal cerebral ischemia results in an ischemic core surrounded by the peri‐infarct region (penumbra). Most research attention has been focused on penumbra while the pattern of cell fates inside the ischemic core is poorly defined. In the present investigation, we tested the hypothesis that, inside the ischemic core, some neuronal and vascular cells could survive the initial ischemic insult while regenerative niches might exist many days after stroke in the adult brain. Adult mice were subjected to focal cerebral ischemia induced by permanent occlusion of distal branches of the middle cerebral artery (MCA) plus transient ligations of bilateral common carotid artery (CCA). The ischemic insult uniformly reduced the local cerebral blood flow (LCBF) by 90%. Massive cell death occurred due to multiple mechanisms and a significant infarction was cultivated in the ischemic cortex 24 h later. Nevertheless, normal or even higher levels of brain‐derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) persistently remained in the core tissue, some NeuN‐positive and Glut‐1/College IV‐positive cells with intact ultrastructural features resided in the core 7‐14 days post stroke. BrdU‐positive but TUNEL‐negative neuronal and endothelial cells were detected in the core where extensive extracellular matrix infrastructure developed. Meanwhile, GFAP‐positive astrocytes accumulated in the penumbra and Iba‐1‐positive microglial/macrophages invaded the core several days after stroke. The long term survival of neuronal and vascular cells inside the ischemic core was also seen after a severe ischemic stroke induced by permanent embolic occlusion of the MCA. We demonstrate that a therapeutic intervention of pharmacological hypothermia could save neurons/endothelial cells inside the core. These data suggest that the ischemic core is an actively regulated brain region with residual and newly formed viable neuronal and vascular cells acutely and chronically after at least some types of ischemic strokes.     

VEGF expression in human brain tissue after acute ischemic stroke

Ischemic stroke is the third most common cause of death in humans, requiring further studies to elucidate its pathophysiological background. One potential mechanism to increase oxygen delivery to the affected tissue is induction of angiogenesis. The most potent proangiogenic factor is VEGF. For this reason, our study investigated immunohistochemically VEGF reactivity in different cellular brain compartments from 15 ischemic stroke patients, as well as from 2 age control cases. By enzymatic immunohistochemistry, we investigate VEGF expression in different brain cell compartments and then we quantified its signal intensity by assessing integrated optical densities (IOD). To establish the exact cellular brain topography of VEGF immunoreactivity we performed double fluorescent immunohistochemistry series (VEGF÷NeuN, GFAP, CD68, CD105). In control samples, VEGF reactivity was observed especially in neurons from the Brodmann cortical layers IV to VI and in protoplasmic astrocytes from the deeper layers of gray matter and in endothelial cells from normal blood vessels because of systemic hypoxia generated after death. In acute ischemic stroke samples, this reactivity was noticed in all brain cellular compartments but with different intensities. The most reactive compartment was the neurons, the intensity of VEGF reaction decreasing with the lesional age from the core infarct toward intact adjacent brain cortex. With a lower intensity, VEGF reaction was noticed in astrocytes compartments, especially in gemistocytic astrocytes adjacent to the liquefaction zone. We also noticed a weak reaction in activated non-phagocytic microglia from the periphery of liquefaction zones, and high VEGF-CD105 colocalization values at the level of microvessels that surround the infarcted brain area. In conclusion, this reactivity could suggest that VEGF might exhibit neuronal and glial protective effects and also a neoangiogenic property in acute ischemic stroke, facts that may have significant therapeutically impact on these patients.     

TRPM4 inhibition promotes angiogenesis after ischemic stroke

Transient receptor potential melastatin 4 (TRPM4) is a voltage-dependent, nonselective cation channel. Under pathological conditions, sustained activation of TRPM4 leads to oncotic cell death. Here, we report the upregulation of TRPM4 in vascular endothelium following hypoxia/ischemia in vitro and in vivo. In human umbilical vein endothelial cells, TRPM4 expression was increased at both the mRNA and protein levels following oxygen–glucose deprivation. Blocking TRPM4 with 9-phenanthrol greatly enhanced tube formation on Matrigel. In a rat permanent middle cerebral artery occlusion model, TRPM4 was upregulated in the vascular endothelium within the penumbra region after stroke. TRPM4 expression peaked 1 day post-occlusion and gradually decreased. In vivo siRNA-mediated TRPM4 silencing enhanced angiogenesis and improved capillary integrity. A twofold reduction in infarct volume and a substantial recovery of motor function were observed in animals receiving the siRNA treatment. Interestingly, the protective effect of TRPM4 suppression disappeared 5 days after stroke induction, indicating that TRPM4 upregulation is critical for cerebral damage during the acute phase of stroke. TRPM4 could be a potential therapeutic target for ischemic stroke.     

CT灌注成像在急性缺血性卒中患者诊断及预后评估中的应用

目的 探讨CT灌注成像在急性缺血性卒中（AIS）患者诊断及预后评估中的应用价值。方法 回顾性研究。纳入2020年2月—2021年7月河南省直第三人民医院收治的AIS患者104例,其中,男61例、女43例,年龄46~81（62.7±12.3）岁。患者均接受急诊脑CT灌注成像检查,获取核心梗死面积,以及脑血流量（CBF）、脑血容量（CBV）、对比剂平均通过时间（MTT）和峰值时间（TTP）等CT灌注成像血流动力学参数,对比分析患者脑梗死区和缺血半暗带脑组织间CBF、CBV、MTT、TTP值的差异。以《中国急性缺血性脑卒中诊疗指南2018》为金标准,分析CT灌注成像AIS患者的检出率。入院时进行美国国立卫生研究院卒中量表（NIHSS）评分评估患者神经功能状态。治疗后3个月采用改良Rankin量表（mRS）评分评估患者预后,并按照预后评估结果进行分组观察。采用logistic回归分析AIS预后的预测因素。结果 本组104例AIS患者中,经CT灌注成像检查显示梗死区及半暗带血流异常者共96例（检出率为92.31%）。CT灌注成像检出的96例中,mRS评分评估预后良好者69例、预后不良者27例。脑梗死区和缺血半暗带分别与健侧对应部位的正常脑组织相比,CBF、CBV值降低,MTT、TTP延长,差异均有统计学意义（P值均<0.05）。预后不良组核心梗死面积、发病至溶栓时间及高脂血症比例、NIHSS评分及缺血半暗带CT灌注参数MTT、TTP均高于预后良好组,CT灌注参数CBF、CBV均低于预后良好组,差异均有统计学意义（P值均<0.05）。logistic回归分析结果显示,核心梗死面积、发病至溶栓时间、高脂血症,NIHSS评分,以及缺血半暗带CT灌注成像血流动力学参数CBF、CBV、MTT、TTP等均是AIS患者预后的预测因素（P值均<0.05）。结论 CT灌注成像检查可快速反映脑部组织灌注损伤情况,对AIS患者的及时诊断具有重要意义;且CT灌注成像血流动力学参数可有效反映AIS患者溶栓治疗后受累血管再通情况,为其预后评估提供一定依据。     

高血糖和脑缺血对树鼩皮层不同区域VEGF表达的影响

目的：研究高血糖及局灶性脑缺血条件下,树鼩皮层不同区域VEGF表达的变化,探讨脑缺血、高血糖与VEGF之间的相互关系。方法：用链脲佐菌素复制树鼩高血糖模型,并建立光化学诱导皮层局灶性脑缺血,观察缺血4 h、24 h及72 h的病理形态学改变并计数海马神经元密度,用免疫组化法测定上述时间树鼩缺血中心区、半暗带、对侧皮层VEGF表达的动态变化。结果：形态学观察显示,光化学反应后4 h照射区皮层可见梗塞灶;24 h病损达高峰;72 h伴随胶质细胞增生等修复性反应。相应时点高血糖加缺血组的损伤大于缺血组,以缺血后24 h(P<0.01)和72 h(P<0.05)尤为显著。免疫组化染色表明,缺血后4 h皮层缺血半暗区可见VEGF表达增加, 24 h达高峰,72 h减弱;单纯高血糖也使VEGF表达上调;高血糖加缺血组VEGF表达强于单纯高血糖组(P<0.05),但高血糖加缺血组与缺血组的同期值比较,无显著差异。结论：(1)在低等灵长类动物树鼩体内注射链脲佐菌素,并结合血栓性局部脑缺血方法学的应用能成功复制出实验性高血糖及脑缺血模型;(2)实验证明高血糖对局灶性脑缺血有恶化加重作用;(3)脑缺血及高血糖均可分别作为独立因素诱导VEGF的表达;但缺血与高血糖相加对VEGF表达未显示出叠加效应。     

Transforming growth factor α induces angiogenesis and neurogenesis following stroke

The cytokine transforming growth factor α (TGFα) has proangiogenic and proneurogenic effects and can potentially reduce infarct volumes. Therefore, we administered TGFα or vehicle directly into the area surrounding the infarct in female mice that received gender-mismatched bone marrow transplants from green fluorescent protein (GFP)–expressing males prior to undergoing permanent middle cerebral artery occlusion. Newborn cells were tracked with bromodeoxyuridine (BrdU) labeling and immunohistochemistry at 90 days after stroke onset. We also studied the ingress of bone marrow–derived cells into the ischemic brain to determine whether such cells contribute to angiogenesis or neurogenesis. Infarct volumes were measured at 90 days poststroke. The results show that TGFα led to significant increments in the number of newborn neurons and glia in the ischemic hemisphere. TGFα also led to significant increments in the number of bone marrow–derived cells entering into the ischemic hemisphere. Most of these cells did not label with BrdU and represented endothelial cells that incorporated into blood vessels in the infarct border zone. Our results also show that infarct size was significantly reduced in animals treated with TGFα compared with controls. These results suggest that TGFα can induce angiogenesis, neurogenesis and neuroprotection after stroke. At least part of the pro-angiogenic effect appears to be secondary to the incorporation of bone marrow–derived endothelial cells into blood vessels in the infarct border zone.     

Vasoactive intestinal peptide in rats with focal cerebral ischemia enhances angiogenesis

We studied the effect of vasoactive intestinal peptide (VIP) on angiogenesis in the ischemic boundary area after focal cerebral ischemia. Adult male Sprague–Dawley rats underwent middle cerebral artery occlusion for 2 h. A single dose of VIP was given via i.c.v. injection at the beginning of reperfusion. Immunohistochemistry and Western blotting were performed to assay angiogenesis and brain levels of vascular endothelial growth factor (VEGF) protein, respectively. In addition, the expression of VEGF and its receptors (flt-1 and flk-1), as well as endothelial proliferation, was measured using rat brain microvascular endothelial cells. Immunohistochemical analyses revealed significant (P<0.05) increases in the numbers of bromodeoxyuridine (BrdU) positive endothelial cells and microvessels at the boundary of the ischemic lesion in rats treated with VIP compared with rats treated with saline. Western blotting analysis showed that treatment with VIP significantly (P<0.05) raised VEGF levels in the ischemic hemisphere. In addition, treatment with VIP increased flt-1 and flk-1 immunoreactivity in endothelial cells. In vitro, incubation with VIP significantly (P<0.01) increased the proliferation of endothelial cells and induced the expression of VEGF, flt-1 and flk-1 in endothelial cells. The stimulatory effect of VIP on the proliferation of endothelial cells was significantly (P<0.01) inhibited by SU5416, a selective inhibitor of VEGF receptor tyrosine kinase. Our data suggest that treatment with VIP enhances angiogenesis in the ischemic brain, and this effect may be mediated by increases in levels of VEGF and its receptors.     

MicroRNA-155对糖尿病大鼠脑缺血损伤血管再生的调控

目的:观察microRNA-155(miRNA-155)表达对糖尿病大鼠脑缺血损伤及CD31、血管内皮生长因子(VEGF)表达的影响,探讨miRNA-155对糖尿病加重脑缺血血管再生的调控作用。方法:SD大鼠腹腔内注射链脲佐菌素建立糖尿病大鼠模型,继而应用栓线法建立永久性局灶性脑缺血模型。随机分为5组:(1)假手术组(sham组);(2)脑缺血组(CI组);(3)糖尿病脑缺血组(DCI组);(4)糖尿病脑缺血+miRNA-155抑制物组(inhibitor组);(5)糖尿病脑缺血+阴性对照组(scramble组)。于缺血后24 h采用实时荧光定量聚合酶链反应检测miRNA-155的表达水平;参照Zea-Longa 5分制行神经功能缺损评分;2,3,5-氯化三苯基四氮唑(TTC)染色测梗死体积;Western blotting检测血小板内皮细胞黏附分子-1(PECAM-1/CD31)、VEGF的表达水平。结果:糖尿病脑缺血+miRNA-155抑制物组大鼠脑缺血侧皮质区的miRNA-155表达水平显著低于糖尿病脑缺血组(P0.05)。miRNA-155表达下调可显著减少糖尿病脑缺血大鼠的神经功能缺损评分及脑梗死体积,但显著增加CD31表达水平与VEGF表达水平(均P0.05)。结论:miRNA-155对糖尿病加重脑缺血损伤血管再生有重要的调控作用,miRNA-155表达下调可显著减轻糖尿病大鼠脑缺血损伤。     

一站式多模态平扫MR动态成像在急性脑卒中的应用及对缺血半暗带的早期干预效果

目的:探讨一站式多模态平扫MR动态成像在急性脑卒中的应用及对缺血半暗带的早期干预效果。方法:回顾性分析34例急性脑卒中患者影像资料。对比患者正常区域及病变区域的脑组织血流量（CBF）、造影剂平均峰值时间等参数。结果:弥散加权成像显示所有患者均有不同程度的高信号梗死灶,其中28例成像显示存在不同程度的狭窄与闭塞。在评价缺血半暗带方面,26例动态磁敏感对比增强（DSC）与磁共振动脉自旋标记（ASL）结果高度一致。8例患者两种检测结果不一致,ASL显示高灌注,而DSC显示正常灌注,DSC与ASL显示缺血半暗带无明显差异（P>0.05）。与正常对照区域各灌注成像参数相比,半暗带及梗死区域的中枢血容量（CBV）及CBF水平更低（P<0.05）,平均通过时间（MTT）水平更高（P<0.05）;与半暗带区域相比,梗死区域的CBF及CBV水平更低（P<0.05）,MTT水平更高（P<0.05）。结论:一站式平扫MR可快速准确评价急性缺血性脑卒中,为临床个性化治疗提供客观的影像学依据。     

Intravenously delivered neural stem cells migrate into ischemic brain,differentiate and improve functional recovery after transient ischemic stroke in adult rats

Stem cell transplantation may provide an alternative therapy to promote functional recovery after various neurological disorders including cerebral infarct. Due to the minimal immunogenicity and neuronal differentiation potential of neural stem cells (NSCs), we tested whether intravenous administration of mice-derived C17.2 NSCs could improve neurological function deficit and cerebral infarction volume after ischemic stroke in rats. Additionally, we evaluated the survival, migration, proliferation, and differentiation capacity of transplanted NSCs in the rat brain. Intravenous infusion of NSCs after middle cerebral artery occlusion (MCAO) showed better performance in neurobiological severity scores after MCAO compared to control. However, the volume of cerebral infarction was not different at 7 days after MCAO compared with control. Transplanted NSCs were detected in the ischemic region but not in the contralateral hemisphere. NSCs differentiated into neurons or astrocytes after MCAO. These data suggest that intravenously transplanted NSCs can migrate, proliferate, and differentiate into neurons and astrocytes in the rat brain with focal ischemia and improve functional recovery.     

Changes in extracellular amino acid neurotransmitters produced by focal cerebral ischemia

Excitatory amino acids (EAAs) have been implicated in the pathophysiology of cellular injury after brain ischemia. Changes in extracellular levels of amino acids in rat cerebral cortex after permanent proximal middle cerebral artery (MCA) occlusion were examined using microdialysis. Significant increases were found in dialysate concentrations of glutamate, aspartate and gamma-aminobutyric acid (GABA) from the ischemic cortex during the first 90 min after MCA occlusion compared to pre-ischemic concentrations and contralateral hemispheric controls. Total tissue levels of these amino acids in the infarcted hemisphere 90 min after onset of ischemia were not different from the contralateral hemisphere. These results are consistent with the hypothesis that the release of EAAs may contribute to tissue damage in focal cerebral ischemia.     

A soluble Fn14-Fc decoy receptor reduces infarct volume in a murine model of cerebral ischemia

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily. TWEAK acts on responsive cells via binding to a small cell surface receptor named Fn14. Recent studies have demonstrated that TWEAK can stimulate numerous cellular responses including cell proliferation, migration, and proinflammatory molecule production, but the role of this cytokine in cardiovascular disease and stroke has not been established. The present study investigated whether TWEAK or Fn14 expression was regulated in a murine model of cerebral ischemia and whether TWEAK played a role in ischemia-mediated cell death. We found that TWEAK and Fn14 were expressed by primary mouse cerebral cortex-derived astrocytes and neurons cultured in vitro. Also, both the TWEAK and Fn14 proteins were present at elevated levels in the ischemic penumbra region after middle cerebral artery occlusion. Finally, we report that intracerebroventricular injection of a soluble Fn14-Fc decoy receptor immediately after middle cerebral artery occlusion significantly reduced infarct volume and the extent of microglial cell activation and apoptotic cell death in the ischemic penumbra. We conclude that the cytokine TWEAK may play an important role in ischemia-induced brain injury and that inhibition of TWEAK expression or function in the brain may represent a novel neuroprotective strategy to treat ischemic stroke.     

Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat

Vascular endothelial growth factor (VEGF), a key regulator of vasculogenesis and embryonic angiogenesis, was recently found to be up-regulated in an animal model of stroke. Unlike VEGF, angiopoietin (Ang)-1 and -2, their receptor tie-2, and the associated receptor tie-1 exert their functions at later stages of vascular development, i.e., during vascular remodeling and maturation. To assess the role of the angiopoietin/tie family in ischemia-triggered angiogenesis we analyzed their temporal and spatial expression pattern after middle cerebral artery occlusion (MCAO) using in situ hybridization and immunohistochemistry. Ang-1 mRNA was constitutively expressed in a subset of glial and neuronal cells with no apparent change in expression after MCAO. Ang-2 mRNA was up-regulated 6 hours after MCAO and was mainly observed in endothelial cell (EC) cord tips in the peri-infarct and infarct area. Up-regulation of both Ang-2 and VEGF coincided with EC proliferation. Interestingly, EC proliferation was preceded by a transient period of EC apoptosis, correlating with a change in VEGF/Ang-2 balance. Our observation of specific stages of vascular regression and growth after MCAO are in agreement with recent findings suggesting a dual role of Ang-2 in blood vessel formation, depending on the availability of VEGF.     

短暂性脑缺血再灌注对大鼠脑PGRN表达的影响

目的检测短暂性脑缺血再灌注早期,大鼠的额颞叶皮质、海马及纹状体中颗粒蛋白前体(progranulin,PGRN)的表达变化,为进一步探讨PGRN对脑缺血的治疗提供实验基础。方法 SPF级成年SD大鼠随机分为2组,假手术组(shamoperation group)和实验组(MCAO group)。用线栓法制作右侧大脑中动脉阻塞(MCAO)1 h,然后再灌注30 min、2 h、12 h、24 h模型,以缺血侧为实验组(ipsilateral group),其对侧为对照组(contralateral group)。假手术组为处理对照。用TTC染色法观察缺血梗死体积。用免疫荧光组织化学法和Western-blot分别检测PGRN的细胞定位及其表达变化。结果 TTC染色表明,脑缺血区域呈现白色,而对照无缺血区为红色。免疫组织化学结果提示,PGRN在神经元中大量表达,在小胶质细胞中有少量表达,而在星形胶质细胞中几乎无表达。Western blot结果提示,与对照组相比,缺血再灌注后2 h大鼠脑额颞叶皮质的缺血侧与非缺血侧PGRN含量均显著上调。在海马区缺血再灌注能瞬间降低PGRN水平,随着时间延长PGRN表达水平在24 h逐步恢复到正常水平。与皮质和海马相比,纹状体在短时间再灌注后PGRN具有较高的表达,特别是在缺血再灌注24 h后。结论短暂性脑缺血再灌注能显著影响PGRN在大鼠脑缺血区及半暗带区的水平,提示PGRN可能参与缺血后脑的调节。     

Post-treatment of a BiP inducer prevents cell death after middle cerebral artery occlusion in mice

We previously reported the effect of a selective inducer of BiP (a BiP inducer X; BIX) after permanent middle cerebral artery occlusion (MCAO) in mice. However, in acute stroke, almost all drugs have been used clinically after the onset of events. We evaluated the effect of post-treatment of BIX after permanent MCAO in mice, and examined its neuroprotective properties in in vivo mechanism. BIX (intracerebroventricular injection at 20 μg) administered either at 5 min or 3 h after occlusion reduced both infarct volume and brain swelling, but at 6 h after occlusion there was no reduction. BIX protected against the decrease in a dose-dependent manner. Furthermore, BIX reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells induced by the ischemia in ischemic penumbra. These findings indicate that post-treatment with BIX after ischemia has neuroprotective effects against acute ischemic neuronal damage in mice even when given up to 3 h after MCAO. BIX may therefore be a potential drug for stroke.     

VEGF在实验性心肌梗塞中促血管生成的实验研究

目的探讨VEGF在实验性心肌梗塞中促血管生成的形态学依据和作用机理。方法结扎30只家兔左冠状动脉的前降支,造成实验性心肌梗塞。15只作为实验组,在梗塞区内注射VEGF;其余15只作为对照组,仅做前降支单纯性结扎。两组动物分别于结扎术后1、2、4周处死,利用组织切片染色法观察梗塞区血管的生成与重建及相关的病理学变化。结果实验组梗塞区内,可见许多呈岛状存活的心肌组织,对照组则极为少见。术后2周为血管形成的高峰期,实验组梗塞区内新生血管的数量和密度明显高于对照组,两组具有极其显著性差异,血管密度实验组为(24.19±0.77)条/4×10倍,对照组为(1.01±0.21)条/4×10倍。结论VEGF对急性实验性心肌梗塞后的血管形成及心肌组织存活具有明显的促进作用。     

Fibroblast Growth Factor Type 1 (FGF1)-Overexpressed Adipose-Derived Mesenchaymal Stem Cells (AD-MSC<Superscript>FGF1</Superscript>) Induce Neuroprotection and Functional Recovery in a Rat Stroke Model

Stroke, as the second most common cause of death, imposes a great financial burden on both the individual and society. Mesenchymal stem cells from rodents have demonstrated efficacy in experimental animal models of stroke due to enhanced neurological recovery. Since FGF1 (fibroblast growth factor 1) displays neuroprotective properties, for the first time, we investigated the effect of acute intravenous administration of FGF1 gene transfected adipose-derived mesenchymal stem cell (AD-MSC^FGF1) on transient experimental ischemic stroke in rats. Stroke induction was made by transient middle cerebral artery occlusion (tMCAO). 2?×?10⁶ AD-MSC^FGF1 was administrated intravenously 30 min after carotid reperfusion. The ability of technetium^99m-hexamethyl propylene amine oxime (^99mTc-HMPAO)-labeled AD-MSC^FGF1 to enter into ischemic brain was evaluated 2 h post injection. 24 h post operation, the neurological recovery (rotarod and Roger’s tests), the infarct volume (2, 3, 5-triphenyltetrazolium chloride, TTC assay), apoptosis rate (TUNEL assay), and the expression of FGF1 protein (western blotting) in the ischemic hemisphere were assessed. The ^99mTc-HMPAO-labeled AD-MSC^FGF1 could enter into the ischemic brain. Ischemic hemisphere activity was significantly higher than that observed in the contralateral hemisphere (p?=?0.002). The administration of AD-MSC^FGF1 resulted in significant improvement of neurological function tests and increased density of FGF1 protein in the peri-infarct area, while the infarct volume and the apoptotic index were significantly decreased, in comparison to the other treated groups. In conclusion, acute intravenous administration of AD-MSC^FGF1 can be a novel and promising candidate approach for the treatment of ischemic stroke.     

大鼠侧脑室注射VEGF重组质粒预防脑缺血再灌注损伤及其对PI3K／AKT通路的影响

目的：探讨血管内皮生长因子（VEGF）重组质粒预先导入鼠脑能否有效预防缺血性脑梗塞并初探其可能的作用机制。方法将pIERS2-EGFP/VEGF重组质粒预先注入质粒组大鼠侧脑室,通过大脑中动脉（MCA）线栓法制备大鼠局灶性缺血2h/再灌注24h模型,采用TTC染色法测量梗塞灶大小;用电镜观察鼠脑神经元超微结构的变化;并通过Western印迹方法检测各组鼠脑中P-AKT蛋白的表达。结果①VEGF质粒组右脑梗死体积较缺血组明显减小（P＜0．01）。②与缺血组相比,质粒组神经元损伤和水肿程度明显降低。③与正常组相比,缺血组与质粒组的P-AKT表达量均显著增加（P＜0．01）,且质粒组比缺血组增加显著（P＜0．01）。结论VEGF重组质粒的导入能有效预防缺血性脑梗塞,推测该机制可能是VEGF蛋白的表达能进一步活化PI3K/AKT信号通路。该研究为缺血性脑梗塞的二级预防提供一条新思路。