Effect of dextromethorphan, a NMDA antagonist, on DNA repair in rat photochemical thrombotic cerebral ischemia

Photochemical thrombotic ischemia model was used to study the possible roles of excision repair cross-complementing group 6 (ERCC6), a DNA repair gene, in the neuroprotection of dextromethorphan (DM), a NMDA antagonist, in ischemic brain injury. The results showed that no obvious ERCC6 mRNA expression was found in the perifocal area of irradiated cerebral cortex before 24 h postischemia. Then, the number of ERCC6 mRNA positive cells gradually enhanced, and attained a peak value at 72 h after light irradiation, which followed a declined tendency at 7-day postlesion. These results suggest that DNA repair gene ERCC6 mRNA expression in the perifocal area may be involved in the pathophysiological processes following the photochemical thrombotic cerebral ischemia. By the administration of DM, we observed that it can significantly upregulate the expression of ERCC6 mRNA in the perifocal area at 48 h after ischemic event. The neuroprotective mechanisms of DM may be related to the upregulation of DNA repair gene ERCC6 mRNA.     

Bcl—2 antisense注射对脑缺血后DNA修复基因表达的影响

目的:研究Bcl-2对缺血性脑损伤保护作用的机制。方法:采用侧脑室微量注射、免疫组织化学法和焦油紫染色法在大鼠大脑中动脉栓塞模型上,观察Bcl-2反义寡脱氧核苷酸(antisense oligodeoxynucleotides,antisense ODNs)对DNA修复基因ERCC1(excisionrepair cross-complementing group 1)表达的影响。结果:antisense ODNs能抑制缺血周边Ⅰ区Bcl-2的表达(P<0.01),加重该区域脑缺血损伤。同时,ERCC1的表达也有下降的趋势(P=0.087),而且,在Bcl-2表达的区域,也有ERCC1的表达。结论:Bcl-2对缺血神经元的保护作用与调节脑细胞内源性的DNA修复有关。     

神经元外单胺递质载体在人脑肿瘤的表达及其在SarCNU化疗中的意义

目的：探讨神经元外单胺递质载体（EMT）在人脑肿瘤的表达与抗癌新药二氯乙基肌氨酰胺亚硝脲（SarCNU)敏感性之间的关系。方法：采用逆转录－聚合酶连锁反应（RT－PCR）测定EMT在人脑肿瘤细胞株和人脑肿瘤标本的表达，并与SarCNU细胞毒性试验结果进行相关性分析。结果：检测的15株人脑肿瘤细胞都有不同程度的EMT表达，30例人脑肿瘤标本中25例EMT阳性。虽然单因素分析，肿瘤细胞EMT表达与SarCNU细胞毒性结果之间的相关性不显，多元回归分析显示，SarCNU细胞毒性与EMT表达和六氧甲基鸟嘌呤NA甲基转移酶（MGMT），核苷酸切除修复系统的重要成员ERCC2表达显相关（r=0.983,P=0.0001)，结论：脑肿瘤EMT和DNA修复基因表达与SarCNU的敏感性都有关。EMT阳性肿瘤将对SarCNU敏感，而大多数脑肿瘤有EMT表面，因此，SarCNU可望是理想的脑肿瘤化疗效新药。     

Cell type specific upregulation of vascular endothelial growth factor in an MCA-occlusion model of cerebral infarct.

Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that has been implicated in hypoxia-mediated angiogenesis under physiological and pathological conditions. We used the middle cerebral artery occlusion model (MCAO) in the rat to investigate VEGF mRNA and protein localization, and VEGFR-1 mRNA and VEGFR-2 mRNA expression in cerebral ischemia. By nonradioactive in situ hybridization we observed upregulation of VEGF mRNA and VEGFR-1 mRNA, but not of VEGFR-2 mRNA in the hemisphere ipsilateral to MCA occlusion. VEGF mRNA was upregulated in the periphery of the ischemic area commencing 3 hours (h) after onset of MCAO, reached a peak after 24 h, and remained expressed at lower levels until 7 days (d) after MCAO. Double labelling experiments revealed that the majority of VEGF expressing cells in the penumbra and within the infarct were immunoreactive for Ox-42, Iba-1, and Ed1, but not for GFAP and neurofilament proteins, suggesting that microglial cells/macrophages are the major cell type expressing VEGE Since VEGF was also expressed in Ox-42 immunoreactive cells distant from the infarct (e.g. in the corpus callosum and hippocampus), activated microglial cells expressing VEGF may migrate towards the ischemic stimulus. VEGF protein was also detected on capillaries within the peri-ischemic area, suggesting that VEGF produced and secreted by microglial cells/macrophages binds to its receptors on nearby vascular endothelial cells and initiates an angiogenic response which counterbalances tissue hypoxia. Accordingly, apoptosis of neuroectodermal cells in the penumbra was highly depressed after the onset of angiogenesis. The spatial and temporal correlation between the induction of angiogenesis with VEGF and VEGFR-1 expression suggests that the ischemic upregulation of VEGF represents a physiological response of the brain to counterbalance hypoxia/ischemia in order to protect neuroectodermal tissue.     

Differential expression of vascular endothelial growth factor-A (VEGF-A) and VEGF-B after brain injury

Our previous study demonstrated that vascular endothelial growth factor (VEGF), now referred to as VEGF-A, plays a significant role in blood-brain barrier (BBB) breakdown and angiogenesis after brain injury. In this study, VEGF-A expression was compared with that of VEGF-B in the rat cortical cold injury model over a period of 6 hours to 6 days post-injury. VEGF-A and VEGF-B mRNA were detected by in situ hybridization and their protein was detected by immunohistochemistry. The presence of VEGF-A and VEGF-B proteins in endothelium of lesion vessels was related to BBB breakdown by double labeling for either of these growth factors and fibronectin, which was used as a marker of BBB breakdown. Significant induction of both VEGF-A and VEGF-B mRNA occurred at the lesion site during the period of maximal endothelial proliferation. VEGF-A mRNA levels peaked at 3 and 4 days post-injury and returned to basal expression by day 6, while VEGF-B mRNA levels remained elevated up to day 6. VEGF-B protein was constitutively expressed in endothelium of all cerebral vessels. After brain injury, there was increased immunoreactivity for VEGF-B at the lesion site, this protein being present in the endothelium and vascular smooth muscle cells of pial vessels, in inflammatory cells, and later in proliferating endothelial cells, endothelium of neovessels, and astrocytes. Lesion vessels showing BBB breakdown to fibronectin showed endothelial VEGF-A protein but not VEGF-B protein. Constitutive expression of VEGF-B in normal endothelium suggests that it may have a role in maintenance of the BBB in steady states, while its induction at both the gene and protein level post-injury indicates that it has an essential role in angiogenesis and the repair processes after brain injury.     

Recombinant tissue-type plasminogen activator transiently enhances blood–brain barrier permeability during cerebral ischemia through vascular endothelial growth factor-mediated endothelial endocytosis in mice

Recombinant tissue-type plasminogen activator (rt-PA) modulates cerebrovascular permeability and exacerbates brain injury in ischemic stroke, but its mechanisms remain unclear. We studied the involvement of vascular endothelial growth factor (VEGF)-mediated endocytosis in the increase of blood–brain barrier (BBB) permeability potentiated by rt-PA after ischemic stroke. The rt-PA treatment at 4 hours after middle cerebral artery occlusion induced a transient increase in BBB permeability after ischemic stroke in mice, which was suppressed by antagonists of either low-density lipoprotein receptor families (LDLRs) or VEGF receptor-2 (VEGFR-2). In immortalized bEnd.3 endothelial cells, rt-PA treatment upregulated VEGF expression and VEGFR-2 phosphorylation under ischemic conditions in an LDLR-dependent manner. In addition, rt-PA treatment increased endocytosis and transcellular transport in bEnd.3 monolayers under ischemic conditions, which were suppressed by the inhibition of LDLRs, VEGF, or VEGFR-2. The rt-PA treatment also increased the endocytosis of endothelial cells in the ischemic brain region after stroke in mice. These findings indicate that rt-PA increased BBB permeability via induction of VEGF, which at least partially mediates subsequent increase in endothelial endocytosis. Therefore, inhibition of VEGF induction may have beneficial effects after thrombolytic therapy with rt-PA treatment after stroke.     

Differential downregulation of vascular endothelial growth factor by dexamethasone in normoxic and hypoxic rat glioma cells

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a mitogen and chemotactic factor for endothelial cells in vitro and an angiogenesis and vascular permeability factor in vivo. Due to its properties, VEGF is a candidate for both angiogenesis and vascular permeability/oedema induction which typically occur in glioblastomas. In this study we test the hypothesis that the antioedema effect of dexamethasone is mediated by downregulation of VEGF or VEGF receptor expression. VEGF mRNA and protein levels of two rat glioma cells lines, C6 and GS-9L, were determined after incubation with dexamethasone under normoxic and hypoxic conditions. In normoxic C6 and GS9L cells, we observed 50-60% downregulation of VEGF mRNA by dexamethasone (P=0.015 and P=0. 01, respectively). This effect was dependent on glucocorticoid-receptor (GR) function. The inhibitory effect of dexamethasone on VEGF gene expression by tumour cells was markedly reduced by hypoxia which suggests that the upregulation of VEGF driven by hypoxia overcomes the effect of the dexamethasone. Dexamethasone did not alter VEGFR-2 mRNA levels in human umbilical endothelial cells. In a subcutaneous glioma tumour model, we observed only a 15% decrease in VEGF mRNA expression in dexamethasone treated animals (n = 12) compared with controls animals (P = 0.24). We conclude that dexamethasone may decrease brain tumour-associated oedema by reduction of VEGF expression in tumour cells. However, the highly reduced activity on hypoxic tumour cells suggests that dexamethasone efficacy may be limited by hypoxia in rapidly growing tumours.     

Up-regulation of neuropilin-1 in neovasculature after focal cerebral ischemia in the adult rat.

During development, neuropilin-1 is a receptor for semaphorin 3a-mediated axonal guidance and for vascular endothelial growth factor (VEGF) promotion of angiogenesis. The authors measured neuropilin-1 expression in the adult ischemic brain using Northern blot, in situ hybridization, and immunohistochemistry. Neuropilin-1 mRNA was significantly up-regulated as early as 2 hours and persisted at least 28 days after focal cerebral ischemia. Acute up-regulation of neuropilin-1 mRNA primarily localized to the ischemic neurons. A marked increase in both mRNA and protein of neuropilin-1 was detected in endothelial cells of cerebral blood vessels at the border and in the core of the ischemic lesion 7 days after ischemia, and neuropilin-1 gene expression persisted on these vessels for at least 28 days after ischemia. In these areas, neovascularization was detected using three-dimensional reconstructed images obtained from laser scanning confocal microscopy. Activated astrocytes also exhibited neuropilin-1 immunoreactivity during 7 to 28 days of ischemia. Double immunofluorescent staining showed colocalization of neuropilin-1 and VEGF to cerebral blood vessels and activated astrocytes. These data suggest that in addition to its role in axonal growth, up-regulation of neuropilin-1, in concert with VEGF and its receptors, may contribute to neovascular formation in the adult ischemic brain.     

Hypoxia-induced vascular endothelial growth factor expression in normal rat astrocyte cultures

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen, which also enhances vascular permeability. Because this angiogenic factor has been suggested to play a role in brain tumor biology, we have begun to investigate the regulation of VEGF expression in cultures of rat type I astrocytes. In this report, we have focused on the influence of hypoxia on VEGF expression. Under standard in vitro conditions (21% O₂) VEGF expression in astrocytes is barely detectable by northern analysis. However, after exposure to 0.2% O₂ for as little as 3 h VEGF mRNA levels are markedly increased reaching a maximum by approximately 8 h of exposure. Treatment of astrocytes with CoCl₂ or desferrioxamine results in a similar induction of VEGF, suggesting that the oxygen sensor regulating VEGF expression in astrocytes is a heme-containing molecule. Although acute treatment with TPA (6 h) induces VEGF expression, chronic exposure to TPA (24 h) to deplete PKC activity does not reduce the hypoxia-induced VEGF expression. These data indicate that VEGF induction in astrocytes can proceed through PKC-dependent and -independent pathways. Furthermore, chronic exposure to TPA or treatment with herbimycin A results in the enhancement of the hypoxia-mediated increase in VEGF mRNA levels. These results suggest that PKC and herbimycin-sensitive tyrosine kinase may serve as negative regulators of the hypoxia-activated signal transduction pathway that leads to the induction of VEGF expression. However, treatment of astrocytes with the nonspecific kinase inhibitors H7 and H8 reduced the level of VEGF induction by hypoxia, indicating that some type of kinase activity is required in this signaling pathway. © 1995 Wiley-Liss, Inc.     

Retrovirus producer cells encoding antisense VEGF prolong survival of rats with intracranial GS9L gliomas

With increasing size tumors are continually dependent on a functional blood vessel system to guarantee the supply with oxygen and nutrients. Vascular endothelial growth factor (VEGF) is a key mediator not only of developmental but also of hypoxia-mediated and tumor-induced angiogenesis. Gene therapy using antisense VEGF with the aim to inhibit tumor angiogenesis may be a successful strategy for the treatment of highly vascular and invasive malignant gliomas. We investigated whether retrovirus producer cells encoding antisense VEGF can be used for in vivo gene transfer. The full length mouse VEGF164 cDNA was cloned in a sense and antisense direction into the retroviral expression vector pLEN. pLEN-VEGF (sense) and pLEN-FGEV (antisense) expression vectors were used to transfect the packaging cell line GP + E86 and to establish ecotropic virus producer cell lines. GP + E86:LEN-FGEV (#5) cells showed high expression of antisense VEGF mRNA, whereas GP+ E86:LEN-VEGF (#8) showed high expression of sense VEGF mRNA and active VEGF protein. Co-implantation of GS-9L cells with retrovirus producing cells containing the antisense VEGF construct into the brains of syngeneic rats showed a statistically significant inhibition of tumor growth and prolongation of survival time, while co-implantation of retrovirus producer cells containing the sense VEGF expression vector resulted in an increasing tumor growth and reduced survival time of the rats compared to control animals. Histological analysis of the tumors co-implanted with GP + E86:LEN-FGEV (#5) cells showed the suppression of angiogenesis, high degree of necrosis and no evidence of a significant immune response. Expression of antisense VEGF mRNA in these tumors was confirmed by in situ hybridization analysis. This is the first report demonstrating the potential utility of virus producer cells as in vivo gene transfer vehicles for antisense VEGF gene therapy of malignant gliomas.     

血管内皮生长因子基因对大鼠缺血脑组织的保护作用

目的　探讨血管内皮生长因子基因对大鼠缺血脑组织的保护作用 ,为临床治疗脑梗死提供实验依据。方法　用线拴法制成Wistar大鼠大脑中动脉永久性闭塞模型 ,将VEGF1 6 5真核表达质粒 (pUCCAGGS/hVEGF1 6 5)经颅骨注入到缺血区。术后 7d断头取脑 ,2 %红四氮唑 (TTC)染色测梗死体积、HE染色观察组织坏死情况、免疫组织化学检测VEGF1 6 5基因的表达。结果　治疗组VEGF表达高 ,脑组织坏死明显减轻 ,脑梗死体积明显缩小 (P <0 .0 1 )。结论　在质粒载体介导下 ,VEGF基因可转化到缺血脑组织中并表达VEGF ,进而保护神经细胞 ,治疗脑梗死     

Vascular endothelial growth factor upregulation in transient global ischemia induced by cardiac arrest and resuscitation in rat brain

This study examined vascular endothelial growth factor (VEGF) expression in rat brain after reversible global cerebral ischemia produced by cardiac arrest and resuscitation. Three alternative splicing forms, VEGF(188), VEGF(164) and VEGF(120), were observed in cortex, hippocampus and brainstem by RT-PCR analysis. After 24 h of recovery from cardiac arrest, mRNA levels corresponding to VEGF(188) and VEGF(164) were significantly increased by about double in all the regions analyzed. These mRNA levels remained elevated at 24 and 48 h of recovery but returned to basal expression after 7 days of recovery. Changes in VEGF(120) expression after cardiac arrest did not reach statistical significance. VEGF protein expression measured by Western blot was also increased by about double at 24 and 48 h of recovery but returned to control levels after 7 days of recovery. VEGF immunohistochemistry localized this increased expression mostly associated with astrocytes. Considering its biological activity, VEGF induction after cardiac arrest and resuscitation may be responsible for the increased vascular permeability and the resultant vasogenic edema, found 24-48 h after reversible global ischemia.     

兔大脑中动脉阻断所致局灶性脑缺血脑组织内皮抑素蛋白及mRNA表达增加(英文)

目的内皮抑素(endostatin)是强烈的抗血管再生因子。本文探讨大脑中动脉闭塞 (middle cerebral artery occlusion, MCAO)致局灶性脑缺血后脑组织内皮抑素蛋白及mRNA基因表达的变化,同时检测缺血脑组织血管内皮生长因子(vascular endothelial growth factor,VEGF)的含量。方法24只新西兰白兔随机分为正常对照组(n=5)、假手术组(n=4)、缺血2小时组(n=5)、缺血24小时组 (n=5)及缺血48小时组(n=5)共5组。酶联免疫吸附试验测定VEGF含量,免疫组化分析内皮抑素蛋白变化,原位杂交检测内皮抑素mRNA表达。结果与对照组相比,MCAO局灶性脑缺血后内皮抑素蛋白和mRNA表达均明显增加,至少分别增加了50%(P<0.01)和70%(P<0.05),同时缺血脑组织VEGF含量也明显增加,至少增加了270%。结论缺血导致脑组织内皮抑素表达增加,且内皮抑素的增加与缺血后脑组织VEGF变化无相关性,但可能抑制脑缺血后的血管再生,从而加重脑缺血损伤。     

C—fos反义寡脱氧核苷酸部分阻断MCAO大鼠缺血侧皮质内BDNF的表达

用c fos反义寡脱氧核苷酸侧脑室微量注射和细胞免疫化学等技术和方法 ,探讨大鼠局灶性脑缺血(MCAO)模型中 ,即早反应基因c fos表达与脑源性神经营养因子 (BDNF)表达的关系。结果表明 ,局灶性脑缺血再灌注可引起c fos和BDNF在缺血侧皮质的大量表达。侧脑室微量注射c fos反义寡脱氧核苷酸后 ,脑内BDNF的部分表达明显被阻断 ,脑缺血损伤加重。提示脑缺血损伤后 ,脑内BDNF的表达对脑缺血再灌注损伤起一定的保护作用 ;脑缺血后BDNF的表达可能部分通过c fos调控。     

脑膜瘤组织VEGF／VPF、KDR的表达及意义

目的研究脑膜瘤血管生成及瘤周脑水肿与VEGF/VPF及其受体KDR的关系。方法采用Northern印迹分子杂交和免疫组化技术检测47例脑膜瘤组织VEGF/VPFmRNA、VEGF/VPF和KDR蛋白表达,分析其与微血管密度(MVD)和瘤周脑水肿度DPTBE的关系。结果47例脑膜瘤均表达4.2Kb的VEGF/VPFmRNA片段,VEGF/VPF蛋白表达与其mRNA的表达一致,80.9%的肿瘤KDR蛋白表达阳性,VEGF/VPF和KDR分别定位于脑膜瘤瘤细胞和血管内皮细胞胞浆。VEGF/VPFmRNA表达与脑膜瘤MVD和DPTBE呈正相关r=0.67,P<0.01r=0.54,P<0.01;KDR阳性组脑膜瘤VEGF/VPFmRNA表达水平、MVD、DPTBE均显著高于KDR阴性组(P<0.01)。结论脑膜瘤细胞可产生VEGF/VPF,通过KDR介导促肿瘤血管生成和增加血管通透性,在脑膜瘤瘤周脑水肿的发生发展过程中起重要作用。     

兔大脑中动脉阻断所致局灶性脑缺血脑组织内皮抑素蛋白及mRNA表达增加

目的内皮抑素（endostatin）是强烈的抗血管再生因子。本文探讨大脑中动脉闭塞（middle cerebral artery occlusion,MCAO）致局灶性脑缺血后脑组织内皮抑素蛋白及mRNA基因表达的变化,同时检测缺血脑组织血管内皮生长因子（vascular endothelial growth factor,VEGV0的含量。方法24只新西兰白兔随机分为正常对照组（n=5）、假手术组（n=4）、缺血2小时组（n=5）、缺血24小时组（n=5）及缺血48小时组（n=5）共5组。酶联免疫吸附试验测定VEGF含量,免疫组化分析内皮抑素蛋白变化,原位杂交检测内皮抑素mRNA表达。结果与对照组相比,MCAO局灶性脑缺血后内皮抑素蛋白和mRNA表达均明显增加,至少分别增加了50％（P〈0．01）和70％（P〈0．05）,同时缺血脑组织VEGF含量也明显增加,至少增加了270％。结论缺血导致脑组织内皮抑素表达增加,且内皮抑素的增加与缺血后脑组织VEGF变化无相关性,但可能抑制脑缺血后的血管再生,从而加重脑缺血损伤。     

纤维蛋白对大鼠脑血管内皮细胞血管内皮生长因子表达的影响

目的观察纤维蛋白对大鼠脑血管内皮细胞血管内皮生长因子(VEGF)转录及蛋白水平表达的影响。方法大鼠脑血管内皮细胞分离后培养,加入不同浓度的纤维蛋白,通过Real-time PCR检测VEGF转录水平,应用酶联免疫方法(ELISA)定量检测培养基和细胞裂解液中的VEGF水平。结果纤维蛋白可以特异性诱导大鼠脑血管内皮细胞表达VEGF;加入不同浓度的纤维蛋白(0.03mg/ml、0.1mg/ml、0.3mg/ml和1.0mg/ml),24h后,1.0mg/ml纤维蛋白组的培养基VEGF水平显著增高(P<0.001);1.0mg/ml纤维蛋白与大鼠脑血管内皮细胞分别培养0、2、4、8、24、48h,VEGF浓度在共培养2h已经升高,8h时显著升高,在24h时仍然保持在显著升高表达水平(P<0.005),48h有所下降;Real-time PCR结果提示,大鼠脑血管内皮细胞中VEGF mRNA的上调呈现出剂量和时间依赖性增加。结论纤维蛋白可以上调大鼠血管内皮细胞中的VEGF。     

Correlation of VEGF and angiopoietin expression with disruption of blood-brain barrier and angiogenesis after focal cerebral ischemia.

In an effort to elucidate the molecular mechanisms underlying cerebral vascular alteration after stroke, the authors measured the spatial and temporal profiles of blood-brain barrier (BBB) leakage, angiogenesis, vascular endothelial growth factor (VEGF), associated receptors, and angiopoietins and receptors after embolic stroke in the rat. Two to four hours after onset of ischemia, VEGF mRNA increased, whereas angiopoietin 1 (Ang 1) mRNA decreased. Three-dimensional immunofluorescent analysis revealed spatial coincidence between increases of VEGF immunoreactivity and BBB leakage in the ischemic core. Two to 28 days after the onset of stroke, increased expression of VEGF/VEGF receptors and Ang/Tie2 was detected at the boundary of the ischemic lesion. Concurrently, enlarged and thin-walled vessels were detected at the boundary of the ischemic lesion, and these vessels developed into smaller vessels via sprouting and intussusception. Three-dimensional quantitative analysis of cerebral vessels at the boundary zone 14 days after ischemia revealed a significant (P < 0.05) increase in numbers of vessels (n = 365) compared with numbers (n = 66) in the homologous tissue of the contralateral hemisphere. Furthermore, capillaries in the penumbra had a significantly smaller diameter (4.8 +/- 2.0 microm) than capillaries (5.4 +/- 1.5 microm) in the homologous regions of the contralateral hemisphere. Together, these data suggest that acute alteration of VEGF and Ang 1 in the ischemic core may mediate BBB leakage, whereas upregulation of VEGF/VEGF receptors and Ang/Tie2 at the boundary zone may regulate neovascularization in ischemic brain.