疏血通对局灶性脑缺血大鼠神经保护和hsp70表达水平的影响

目的 探讨疏血通对脑缺血-再灌注后神经保护作用的机制,拟为临床应用提供理论依据.方法 90只健康雄性SD大鼠,随机分为假手术组、缺血-再灌注组和疏血通治疗组,建立大脑中动脉闭塞模型.采用神经功能缺损评分对人鼠神经功能缺损程度进行评价,HE染色、TUNEL染色和免疫组织化学染色检测大鼠脑组织梗死灶体积、细胞凋亡及hsp70表达水平.结果 经疏血通治疗后,大鼠神经功能缺损程度明显改善,除再灌注后6h,其余各时间点疏血通治疗组评分均优于缺血-再灌注组(P<0.05).疏血通治疗组大鼠梗死灶体积明显缩小(P<0.01).再灌注后6 h,缺血半暗带区和海马区即可见凋亡细胞,分别于再灌注后48 h和72 h达峰值水平,疏血通治疗组表达高峰时间延迟,且各时间点凋亡细胞数目均少于缺血-再灌注组(P<0.05).再灌注后6 h,缺血半暗带区和大脑皮质即可见少量hsp70表达阳性细胞,两组均于再灌注后24 h达峰值水平,但疏血通治疗组各时间点hsp表达水平均高于缺血-再灌注组(P<0.05).结论 疏血通通过上调脑组织hsp70表达水平,减轻脑缺血-再灌注损伤,从而使梗死灶体积缩小、神经功能改善.     

丁苯酞对大鼠局灶性脑缺血再灌注损伤后脑程序性凋亡因子-5表达的影响

目的研究丁苯酞对大鼠局灶性脑缺血再灌注损伤后程序性细胞凋亡因子-5表达的影响。方法健康雄性SD大鼠132只,随机分为假手术组、缺血再灌注模型组、缺血再灌注治疗组,后两者再分别分为再灌注6h、24h、48h、3d、7d共5个亚组,每亚组12只。应用大脑中动脉线栓法制作大鼠局灶性脑缺血再灌注模型,治疗组造模成功后即刻给予丁苯酞80mg／（kg·d）灌胃,余各大鼠给予同剂量生理盐水灌胃。在相应时间点评定神经功能缺损评分,TTC染色法测定脑梗死体积,免疫组化法测定缺血脑组织PDCD-5的表达,TUNEL法测定凋亡细胞。结果假手术组未见梗死灶,偶有凋亡细胞,少见PDCD-5的表达;模型组及治疗组均可见梗死灶及凋亡细胞,有PDCD-5的表达;治疗组在对应时间点较模型组梗死灶小,凋亡细胞少,PDCD-5的表达少。结论丁苯酞对局灶性脑缺血再灌注损伤具有保护作用,其机制可能是通过减少PDCD-5的表达抑制细胞凋亡。     

大鼠脑缺血再灌注后梗死体积的动态变化

目的观察大鼠大脑中动脉缺血再灌注后梗死灶体积的变化规律。方法线栓法制作大鼠局灶性脑缺血再灌注模型,观察脑缺血2h再灌注3h、24h、3d、7d、14d及21d后的神经功能缺损评分及2％氯化三苯基四氮唑(TTC)标记的梗死体积。结果缺血2h再灌注3h组已经出现较明显的梗死灶(梗死体积占前脑体积14．4％),再灌注24h组梗死体积最大(24．3％),显著大于再灌注3h、7d、14d、21d组(P<0．05)。再灌注3d组梗死灶仍较大 (23．8％),再灌注7d组梗死体积缩小(5．0％),再灌注14d组梗死灶进一步缩小(1．2％),再灌注21d组梗死灶基本消失(0．2％)。大鼠神经功能缺损评分与梗死体积之间呈显著相关(r=0．61,P<0．01)。结论脑缺血再灌注后梗死体积于24h达最大,21d时基本消失。脑缺血再灌注后神经功能缺损评分与梗死体积之间显著相关。     

门冬氨酸钾对局灶性脑缺血再灌注大鼠的保护作用研究

目的 观察门冬氨酸钾对大鼠局灶性脑缺血再灌注后神经细胞凋亡的保护作用。 方法 采用雄性SD大鼠右侧大脑中动脉闭塞模型,缺血2 h,再灌注22 h。大鼠随机分为5组,每组10 只,在缺血后1 h经腹腔注射给予生理盐水（1 ml/kg）或不同剂量门冬氨酸钾（10 mg/kg、25 mg/kg、 62.5 mg/kg和125 mg/kg）,观察不同剂量门冬氨酸钾对大鼠脑缺血再灌注后神经功能缺损和梗死 体积的影响。另取32只大鼠随机分为溶剂对照组和门冬氨酸钾组,在缺血后1 h腹腔注射生理盐水 （1 ml/kg）或门冬氨酸钾（62.5 mg/kg）,同时设立假手术组16只,检测三组大鼠脑组织三磷酸腺苷 （adenosine triphosphate,ATP）和乳酸水平（每组10只）,以及凋亡性细胞情况（每组6只）。 结果 与溶剂对照组比较,62.5 mg/kg剂量的门冬氨酸钾能显著改善神经功能缺损（P <0.001）, 降低梗死体积（P =0.011）;与溶剂对照组比较,25 mg/kg剂量的门冬氨酸钾能减少梗死体积 （P =0.040）,但神经功能评分无差异;10 mg/kg和125 mg/kg剂量的门冬氨酸钾组神经功能评分和 梗死体积与溶剂对照组均无差异。与溶剂对照组比较,门冬氨酸钾（62.5 mg/kg）能减少ATP的下降 （P =0.036）和细胞凋亡（P <0.001）。 结论 门冬氨酸钾对大鼠局灶性脑缺血再灌注后的细胞凋亡有保护作用。     

亚低温对局灶性脑缺血-再灌注大鼠脑皮质脑源性神经营养因子表达及神经元凋亡的影响

目的 观察局灶性脑缺血-再灌注后亚低温干预对大鼠脑源性神经营养因子表达及神经元凋亡的影响，并探讨脑源性神经营养因子在亚低温脑保护机制中的作用。方法 采用线栓法制备成年雄性SD大鼠左侧大脑中动脉闭塞局灶性脑缺血-再灌注改良模型，缺血时间2h。随机分为常温缺血组和亚低温缺血组。常温时大鼠脑温控制于36．5℃～37．5℃，肛温为35．9℃～36．9℃；亚低温时脑温维持于32．5℃～33．5℃，肛温为32．2℃～33．1℃。两组大鼠分别于脑缺血一再灌注及亚低温干预后2、6、24和72h进行神经功能缺损评分，并同时行三苯基氯化四唑（1TC）染色、HE染色、TUNEL染色、免疫组化染色及免疫组化与TUNEL双重染色，从而评估大鼠神经功能缺损状况；检测脑梗死体积及脑源性神经营养因子表达水平；观察组织病理学变化和神经元凋亡数量。结果 与常温缺血组相比，亚低温缺血组大鼠神经功能缺损评分低（P〈0．01），脑梗死体积小（P〈0．01），缺血灶周围脑皮质中的脑源性神经营养因子表达水平增高（P〈0．01），而且神经元凋亡数量少（P〈0．01）。在脑源性神经营养因子免疫组化染色呈阳性反应的神经元细胞核中，未发现TUNEL染色阳性者。结论 亚低温干预治疗可促进缺血灶周围的脑皮质对脑源性神经营养因子的表达，从而抑制神经元凋亡，减少大鼠脑梗死体积，改善神经功能缺损体征。     

丁苯酞预处理对大鼠脑缺血再灌注损伤的神经保护作用

目的研究丁苯酞预处理对大鼠局灶性脑缺血再灌注损伤的神经保护作用。方法健康成年SD雄性大鼠48只,随机分为假手术组、缺血再灌注组、丁苯酞预处理组,每组各16只。各组均灌胃5d后,采用线栓法制作大鼠局灶性脑缺血再灌注(MCAO)模型,缺血2h、再灌注24h,进行神经功能缺损评分,TTC染色及图像分析观察脑梗死体积,免疫组化法检测脑组织caspase-3、bcl-2表达的变化。结果与缺血再灌注组相比,丁苯酞预处理组神经缺损程度改善,梗死灶体积减少,caspase-3阳性细胞数量减少,bcl-2表达上调。结论丁苯酞可减轻缺血性脑血管病的发作,具有一定的神经保护作用。     

大鼠脑缺血再灌注模型粘附分子VCAM-1表达的研究

目的 探讨血管内皮细胞粘附分子（vascular cell molecule-1,VCAM-1)在脑缺血再灌注损伤中的作用和机制。方法 制作大脑中动脉梗死模型。分别于再灌注后6h,24h,36h,48h和7d5个时相点用免疫组化方法观察大鼠模型脑缺血区域VCAM-1的表达，并与永久性脑梗死，假手术组和正常大鼠组比较。结果 （1）正常对照组无VCAM-1的表达，假手术组在6h,24h,36h和48h均无VCAM-1的表达；（2）永久性脑梗死组6h后，在梗死周边区大脑微血管内皮细胞上和神经元上有VCAM-1的明显表达，24h表达达高峰，36-48h开始减弱，7d后完全消失；（3）缺血再灌注组6h后，在缺血周边区大脑神经元上和微血管内皮细胞上有较弱表达，24-48h达高峰，7d后在内皮细胞上仍有VCAM-1的持续表达。结论 缺血再灌注模型脑缺血区域有VCAM-1的表达，VCAM-1可能是缺血再灌注损伤的机制之一。     

大鼠脑缺血再灌注半暗带β淀粉样前蛋白mRNA表达与梗死体积的关系

为了探讨大鼠局灶性脑缺血再灌注缺血半暗带β淀粉样前蛋白(APP)转录水平与缺血时间及梗死体积的相互关系，用插线法建立大鼠局灶性脑缺血再灌注模型，剥取缺血半暗带皮质组织，采用半定量逆转录－聚合酶链式反应（RT－PCR），测定永久性缺血48h和不同缺血时间再灌注48h后，APPmRNA水平的变化。结果显示，梗死体积随再灌注前缺血时间的延长而增大，皮质半暗带缺血30min再灌注48h APPmRNA表达升高；缺血60min和缺血120min再灌注48h APPmRNA升高明显；缺血180min再灌注48h和永久性缺血48h APPmRNA达到高峰。提示缺血半暗带APPmRNA的表达随再灌注前缺血时间延长而增加并与梗死体积有一定的相关性，早期再灌注可减少其表达。（（GFDA1））。     

丁苯酞注射液预处理通过PI3K/Akt通路对大鼠脑缺血再灌注损伤的神经保护作用

目的研究丁苯酞预处理对大鼠脑缺血再灌注损伤的神经保护作用,并初步探讨PI3K/Akt信号通路在该过程中的作用。方法健康成年SD雄性大鼠随机原则分为假手术组、缺血再灌注组、丁苯酞预处理组(丁苯酞氯化钠注射液预处理后脑缺血再灌注组)。缺血2h再灌注24h后对各组大鼠进行神经功能缺损评分,并分别行TTC染色观察脑梗死体积,HE染色观察大鼠脑组织的病理形态,免疫组织化学检测方法观察caspase-3、p-Akt表达的变化。结果与假手术组相比缺血再灌注组有明显神经功能缺损,出现脑组织梗死,梗死区细胞受损,caspase-3、p-Akt阳性细胞表达增加;丁苯酞预处理组与缺血再灌注组相比神经功能缺损程度减轻,梗死灶体积减小,梗死区细胞损伤减轻,caspase-3阳性细胞表达减少,而p-Akt阳性细胞表达增加。结论丁苯酞预处理可以通过上调PI3K/Akt信号通路中p-Akt的表达,降低caspase-3的表达而起到神经保护作用。     

局灶预缺血诱导脑缺血耐受的动物模型

目的　建立一种简便可靠的 SD大鼠局灶性脑缺血预处理模型。方法　将大鼠随机分为 3组 ,分别给予 10 m in大脑中动脉缺血 ( MCAO)预处理 ( PC) ;10 min PC后 2 h MCAO( PC MCAO)及假手术 ( SS)后 2 hMCAO( SS MCAO) ,再灌注 2 2 h后处死 ,观察各组神经功能缺损、梗死体积及脑含水量变化。结果　 PC MCAO组神经功能评分、梗死体积及含水量均明显低于 SS MCAO组 ,PC组无神经功能缺损及梗死灶形成。结论　 2次线栓法建立的大鼠局灶脑缺血预处理模型 ,能有效减轻 MCAO所致的神经损伤 ,操作简便 ,稳定性好 ,是一种研究局灶脑缺血耐受的有用工具。     

视频脑电图在小儿癫痫诊断中的应用

目的评价视频脑电图(video-EEG)在小儿癫诊断中的应用价值。方法对126例具有发作性症状的患儿进行连续8h的包括清醒、睡眠、诱发试验及必要的认知测验的视频脑电图监测。结果经发作期视频脑电图证实,39例初诊为癫性发作的患儿中14例(35%)为非癫性发作;15例其他症状发作中13例(86%)为非癫性发作。64例样放电患儿中51例(80%)确定发作类型,22例(34%)确定癫类型。视频脑电图可发现短暂轻微的癫发作及样放电引起的一过性认知损伤。结论视频脑电图在排除非癫性发作、确定癫性发作的类型、评价脑电-临床关系方面可提供准确可靠的证据,进一步提高癫的临床诊断水平。     

Storage of lipofuscin in neurons in mucopolysaccharidosis

Summary A histochemical and ultrastructural study was made on the brain of a 23-year-old man with Sanfilippo's syndrome. In accordance with previous reports the cortical nerve cells contained a PAS-positive lipid storage substance. This showed intense autofluorescence in UV-light and was positive with various stains for lipofuscin. The storage material appeared ultrastructurally as inclusion bodies composed of short lamellated membranes, granular material, and vacuoles. In addition, concentrically and transversely lamellated membranous cytoplasmic bodies were observed in the nerve cells. It is concluded that the PAS-positive lipid storage material in the neurons was composed partly of lipofuscin in addition to other lipids presumably glycosphingolipids.Supported by a grant from the Expressen Prenatal Research Foundation     

Role of glutathione in repair of free radical damage in hippocampus in vitro.

Depletion of glutathione (GSH), an intrinsic antioxidant, increases vulnerability to free radical damage in a number of cell systems. This study investigates the role of GSH in limiting electrophysiological damage and/or recovery from free radical exposure in slices of guinea pig hippocampus. Synaptic potentials (PSPs) and population spikes (PSs) were recorded from field CA1. Free radicals were generated from 0.006% peroxide through the Fenton reaction. Analysis of the input-output curves showed that peroxide treatment decreased PSPs and impaired ability of the PSPs to generate PSs as previously reported. Recovery was nearly total within a half hour. Treatment with 5 mM buthionine sulfoximine (BSO) for 2 h depleted hippocampal GSH to 79.2% of control values. The extent of free radical damage was not increased. Recovery, however, was only partial. GSH was further depleted by oxidation with diamide or covalent bonding with dimethyl fumarate (DMF) immediately before and during the peroxide treatment. Neither diamide nor DMF treatment in BSO-incubated tissue enhanced peroxide-induced electrophysiological deficits. Following these treatments, however, tissue showed little recovery from free radical damage. We conclude that glutathione is essential for repair processes in hippocampal neurons exposed to oxidative damage.     

Neuroprotective properties of memantine in different in vitro and in vivo models of excitotoxicity

The pathogenesis of stroke, trauma and chronic degenerative diseases, such as Alzheimer's disease (AD), has been linked to excitotoxic processes due to inappropriate stimulation of the N-methyl-D-aspartate receptor (NMDA-R). Attempts to use potent competitive NMDA-R antagonists as neuroprotectants have shown serious side-effects in patients. As an alternative approach, we were interested in the anti-excitotoxic properties of memantine, a well-tolerated low affinity uncompetitive NMDA-R antagonist presently used as an anti-dementia agent. We explored in a series of models of increasing complexity, whether this voltage-dependent channel blocker had neuroprotective properties at clinically relevant concentrations. As expected, memantine protected neurons in organotypic hippocampal slices or dissociated cultures from direct NMDA-induced excitotoxicity. However, low concentrations of memantine were also effective in neuronal (cortical neurons and cerebellar granule cells) stress models dependent on endogenous glutamate stimulation and mitochondrial stress, i.e. exposure to hypoxia, the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+) or a nitric oxide (NO) donor. Furthermore, memantine reduced lethality and brain damage in vivo in a model of neonatal hypoxia-ischemia (HI). Finally, we investigated functional rescue (neuronal capacity to migrate along radial glia) by memantine in cerebellar microexplant cultures exposed to the indirect excitotoxin 3-nitropropionic acid (3-NP). Potent NMDA-R antagonists, such as (+)MK-801, are known to block neuronal migration in microexplant cultures. Interestingly, memantine significantly restored the number of neurons able to migrate out of the stressed microexplants. These findings suggest that inhibition of the NMDA-R by memantine is sufficient to block excitotoxicity, while still allowing some degree of signalling.     

Role of vincristine in the inhibition of angiogenesis in glioblastoma

Objective: Vincristine, a microtubule-destabilizing drug, was found to exhibit anti-angiogenic effects and anti-tumoral activity. However, the precise mechanism by which vincristine inhibits angiogenesis in glioblastomas is not well understood. Our aim was to investigate whether vincristine affects vascular endothelial growth factor (VEGF) expression in glioblastoma cells and determine whether it is mediated by the downregulation of hypoxia-inducible factor-1α (HIF-1α).

Methods: We investigated the expression of HIF-1α in glioblastoma tissues resected from patients and in human glioblastoma cell lines using immunohistochemistry, Western blot analysis, and immunocytochemistry. In addition to an MTT assay assessing the effect of vincristine on cell proliferation and viability, the effects of vincristine on VEGF mRNA expression and HIF-1α protein were examined using real-time RT-PCR and Western blot analysis under 1% O₂ (hypoxia).

Results: HIF-1α was expressed in the majority of glioblastoma tissues and was detected mainly in the nucleus. Strong immunoreactivity for HIF- 1 α was found often in the hypercellular zones. Under hypoxic conditions, HIF-1α protein levels in the glioblastoma cell lines increased, primarily localizing into the nucleus similar to glioblastoma tissues. Exposure of glioblastoma cells to vincristine resulted in enrichment of the G₂-M fraction of the cell cycle, which suggests that vincristine-mediated growth inhibition of glioblastoma is correlated with mitotic inhibition. Using doses lower than those found to reduce the viability and proliferation of cells by 50% (IC₅₀), vincristine decreased both the expression of VEGF mRNA and the level of HIF-1α protein in hypoxic glioblastoma cells. In addition, following exposure to vincristine, the expression of VEGF mRNA was correlated with HIF-1α protein levels.

Conclusions: Our results suggest that the mechanism by which vincristine elicits an anti-angiogenic effect in glioblastomas under hypoxic conditions might be mediated, in part, by HIF-1α inhibition.