诱生型血红素氧合酶mRNA、诱生型一氧化氮合酶mRNA在脑缺血中的表达

目的观察诱生型血红素氧合酶(HO-1)mRNA、诱生型一氧化氮合酶(iNOS)mRNA在局灶性脑缺血中的表达及其不同作用。方法采用逆转录酶多聚酶链反应(RT-PCR)方法,测定HO-1mRNA、iNOSmRNA在局灶性缺血脑组织中不同时间点的表达变化。结果iNOSmRNA的表达在缺血后2 h出现,24 h达最高峰,以后逐渐下降。HO-1mRNA表达在缺血后2 h即出现,缺血后12 h达最高峰。结论脑缺血的病理生理过程中存在着一氧化氮(NO)及一氧化碳(CO)两种信使系统之间的相互作用。HO-1mRNA及iNOSmRNA的表达上调并具有时相性。缺血后期HO-1mRNA仍然维持在一定的水平,可能具有对抗后期iNOSmRNA增高所产生的NO毒性作用。     

大鼠局灶性脑缺血后再灌注期caspase—9 mRNA及Apaf—1mRNA表达的动态变化

目的探讨大鼠局灶性脑缺血后再灌注期caspase-9 mRNA及Apaf-1 mRNA表达的动态变化.方法采用线栓法制作大鼠局灶性脑缺血后再灌注模型,以逆转录聚合酶链式反应(RT-PCR)技术检测caspase-9 mRNA及Apf-1 mRNA的表达.结果缺血2 h后再灌注,缺血皮质中caspase-9 mRNA的表达在再灌注后24h达高峰,48h仍保持高水平,而Apaf-1 muRNA的表达无明显改变.结论局灶性脑缺血后再灌注48h内caspase-9 mRNA表达增强.     

Tumor necrosis factor alpha expression produces increased blood-brain barrier permeability following temporary focal cerebral ischemia in mice.

Alteration of blood-brain barrier (BBB) function occurs in both permanent and temporary cerebral ischemia. Studies in vivo and in vitro have shown that tumor necrosis factor-alpha (TNFalpha) is involved in changes of BBB permeability. However, the relationship between TNFalpha expression and BBB disruption during reperfusion is unclear. The aim of this study is to find the cell source of TNFalpha and to determine the relationship between TNFalpha expression and BBB disruption following temporary focal cerebral ischemia in mice. Adult CD-1 mice received 1 h middle cerebral artery occlusion (MCAO) followed by 2 h, 6 h, 12 h, 24 h, and 48 h of reperfusion. MCAO was achieved using an intraluminal suture technique and reperfusion was performed by the suture withdrawal. Neutralizing monoclonal anti-mouse TNFalpha antibody was administrated intraventricularly immediately after reperfusion. TNFalpha expression was determined by double labeling immunohistochemistry. BBB permeability was determined by albumin immunostaining. TNFalpha immunoreactivity (IR) was observed in the ipsilateral hemisphere from 1 h MCAO with 2 h reperfusion. TNFalpha positive cells included neurons, astrocytes, and ependymal cells. BBB disruption was detected beginning at 6 h reperfusion but was not present at 2 h of reperfusion. The areas of BBB disruption were significantly enlarged at 12 h reperfusion and plateaued at 24 h to 48 h reperfusion. BBB disruptions were significantly attenuated in the anti-TNFalpha antibody treated mice (p<0.05). Our results demonstrate that TNFalpha IR existed in neurons, astrocytes, and ependymal cells during reperfusion. TNFalpha IR following temporary focal cerebral ischemia precedes increased BBB permeability. Treatment with TNFalpha antibody reduces BBB disruption, suggesting TNFalpha may be an important mediator in altering BBB permeability during reperfusion.     

局灶性脑缺血时血红素氧合酶-1 mRNA的表达

目的 观测永久性脑缺血后一氧化碳限速酶一血红素氧合酶-1(HO-1)mRNA表达的变化规律。方法 在建立MCAO局灶性脑缺血模型基础上，采用半定量RT-PCR技术观察并测定脑缺血后不同时相HO-1 mRNA的相对表达量。结果脑缺血后1h即有HO-1 mRNA的表达，随时间延长而逐渐升高，12h达最高，以后逐渐下降，至7d时仍有表达。结论 脑缺血后HO-1 mRNA表达变化是缺血脑组织损伤后重要的自身恢复机制之一。     

3-硝基丙酸预处理对大鼠局灶性脑缺血Bcl-2和Bax mRNA表达的影响

目的探讨3-硝基丙酸(3-NPA)预处理对大鼠局灶性脑缺血半暗带Bc l-2和Bax mRNA表达的影响。方法将大鼠腹腔注射3-NPA 20 mg/kg,3 d后制作局灶性脑缺血再灌注模型;采用逆转录聚合酶链反应,观察3-NPA预处理对脑缺血再灌注1 h、6 h、12 h、24 h及48 h额顶部皮质Bc l-2和Bax mRNA表达的影响,并与假手术组和缺血再灌注组比较。结果与假手术组比较,缺血再灌注组和3-NPA预处理组各时间点Bc l-2和Bax mRNA表达极显著增强(均P<0.01);与缺血再灌注组比较,3-NPA预处理组各时间点Bc l-2mRNA表达显著增强(均P<0.05),再灌注12~48 h Bax mRNA的表达显著降低(均P<0.05)。结论增强Bc l-2的表达、抑制Bax的表达,可能是3-NPA预处理抑制细胞凋亡、诱导脑缺血耐受的机制之一。     

Induction of inducible heme oxygenase (HO-1) in the central nervous system: is HO-1 helpful or harmful?

Heme oxygenase (HO) produced biliverdin and bilirubin, which are powerful antioxidants, therefore, it has been proposed as helpful against oxidative stress. In contrast, HO also produces iron, and it could increase oxidative stress if not handled properly. To clarify the effect of HO, i.e., helpful or harmful, we examined the expression, localization, and induction mechanism of HO-1 in the rat hippocampus after transient forebrain ischemia and injection of kainic acid (KA). Following ischemia, HO-1 expression was observed early but transiently in the CA1 pyramidal neurons and later but continuously in glial cells. In addition, HO-1 expressing pyramidal neurons were colocalized well with phosphorylated c-Jun, which is a critical step in neuronal apoptosis. After injection of KA, HO-1 expression was observed only in glial cells but not neurons, and HO-1 expression was observed in predominantly ameboid microglia, along with a few astrocytes. HO-1 expressing ameboid microglia expressed major histocompatibility complex class II antigen, suggesting strong activation. These results suggested that HO-1 may have double-edged effects, and its effects may depend on the cell type. This short review is intended to highlight on the effect of HO-1 in neurodegeneration.     

大鼠局灶性脑缺血再灌注后早期生长反应基因-1 mRNA的表达

目的探讨早期生长反应基因-1( early growth response gene-1,Egr-1)mRNA在大鼠局灶性脑缺血再灌注后的表达情况。方法取10只健康雄性SD大鼠,体重200 ～250 g,应用原位杂交和RT-PCR方法检测大鼠局灶性脑缺血再灌注后Egr-1 mRNA的表达情况。结果 (1)原位杂交结果:假手术组神经元及胶质细胞为轻度阳性表达。缺血2 h再灌注2 h后,缺血侧Egr-1 mRNA细胞阳性表达明显增强。再灌注4 h时Egr-1 mRNA细胞阳性表达最高,再灌注22 h Egr-1 mRNA细胞阳性表达下降,至166 h时下降更加明显,但仍显著高于假手术组。(2)RT-PCR结果:大鼠脑缺血再灌注后缺血侧Egr-1 mRNA的表达明显高于假手术组,P<0.01。动态观察发现,脑缺血2h再灌注2 h后,Egr-1 mRNA即高表达,缺血2 h再灌注4 h达高峰,再灌注46 h已明显下降,但仍高于假手术组,P<0.01,随缺血再灌注时间延长,Egr-1 mRNA表达又逐渐增多,至再灌注166 h其表达亦明显高于假手术组,P<0.01。结论缺血再灌注后Egr-1 mRNA有规律性表达。     

Changes in oxidative stress,iNOS activity and neutrophil infiltration in severe transient focal cerebral ischemia in rats

Oxidative stress, inducible nitric oxide synthase (iNOS) and neutrophils all contribute to post-ischemic brain damage. This study has determined the time courses of these three phenomena after ischemia in parallel with histological and functional outcomes. Ischemia was produced in rats by occluding the left middle cerebral artery and both common carotid arteries for 20 min. Regional cerebral blood flow (rCBF) rapidly decreased to 20% of its preischemic value during occlusion and stabilized at 60% following reperfusion. The striatal infarction was maximal 15 h after reperfusion (50+/-3 mm(3)), whereas the cortical infarction reached its maximum at 48 h (183+/-10 mm(3)). This drastic decrease in rCBF followed by incomplete reperfusion and massive infarction is, thus, extremely severe. The cortical infarction was strongly correlated with the neurologic deficit and loss of body weight. Oxidative stress, evaluated by the decrease in glutathione concentrations, appeared in the striatum at 6 h after reperfusion and in the cortex at 15 h. Calcium-independent NOS activity, considered as inducible NOS activity, was significantly enhanced at 24 h in the striatum and at 48 h in the cortex. Myeloperoxidase activity, a marker of neutrophil infiltration, was significantly increased at 48 h in both the striatum and cortex. These time courses show that the delayed iNOS activity and neutrophil infiltration that occur after the maturation of infarction in severe ischemia may not contribute to ischemic brain damage. By contrast, early oxidative stress may well be implicated in cerebral injury.     

大鼠局灶性缺血再灌注后大脑皮质ADM及其mRNA的表达

目的探讨肾上腺髓质素（ADM）与脑缺血再灌注损伤的关系。方法采用线栓法制成大鼠大脑中动脉缺血再灌注模型，阻断血流2h进行再灌注。应用免疫组织化学法和RT—PCR法检测不同时间段大鼠局灶性脑缺血再灌注后大脑皮质ADM及其mRNA的表达，并进行动态观察。结果正常大鼠大脑皮质有ADM及其mR—NA的表达，假手术组ADM及其mRNA表达略高于正常组，P〉0．05；大鼠脑缺血再灌注后大脑皮质ADM及其mRNA过表达，与正常对照组及假手术组相比差异显著，P〈0．05。动态观察发现，脑缺血2h再灌注2h，大脑皮质ADM及其mRNA即表达，再灌注22h达高峰，至1w仍明显多于正常对照组，P〈0．05。结论脑缺血再灌注后ADM及其mRNA呈现规律性过表达。     

白果内酯对高血糖大鼠脑缺血再灌注损伤的保护作用

目的 观察白果内酯对高血糖大鼠脑缺血再灌注损伤的保护作用及其可能机制.方法 采用50%的葡萄糖溶液腹腔注射(6 ml/kg)建立急性高血糖模型.采用线栓法建立大鼠脑缺血再灌注模型,按随机数字表方法将40只大鼠分为高血糖假手术组(假手术组),高血糖+缺血再灌注损伤组(模型组),高血糖+缺血再灌注损伤+白果内酯组(白果内酯组),白果内酯分三个剂量组(2.5,5,10 mg/kg),每组各8只.白果内酯组于术前3 d连续给予白果内酯腹腔注射,术前1 h再给予腹腔注射1次.脑缺血2 h,再灌注24 h后行神经功能缺损评分、脑梗死体积及脑含水量测定,同时测定脑组织中水通道蛋白-4(AQP4)mRNA的表达,超氧化物歧化酶(SOD)的活力,计算脑组织中丙二醛(MDA)的含量及去甲肾上腺素(NE)、多巴胺(DA)和5-羟色胺(5-HT)的表达.结果 白果内酯(5,10 mg/kg)组大鼠与模型组相比,神经功能缺损评分下降,脑梗死体积缩小,脑含水量降低,缺血侧脑组织中AQP4 mRNA的表达下调,SOD活力提高,MDA含量减低,NE、DA及5-HT的含量增加,差异均有统计学意义(P<0.05).结论 白果内酯对高血糖条件下的局灶性脑缺血再灌注损伤具有一定的保护作用.     

局灶性脑缺血/再灌注大鼠白细胞介素-1β蛋白和mRNA的表达

目的 :观察脑缺血对白细胞介素 - 1β(IL- 1β)表达的影响 ,及脑缺血后 IL- 1β的细胞来源。方法 :采用线栓法制备大鼠局灶性大脑中动脉栓塞 (MCAO)模型 ,应用原位杂交观察脑缺血再灌注对 IL- 1β m RNA表达的影响 ;应用荧光双标检测 IL- 1β的表达细胞。结果 :(1)正常和假手术大鼠大脑皮层 IL- 1β m RNA阳性细胞表达较少 ,缺血再灌注后缺血侧皮层 IL- 1β m RNA阳性细胞表达明显增加 ,再灌注后 2 h IL- 1β m RNA表达显著增加 ,再灌注后 2 4h逐渐降至正常水平。 (2 )缺血后表达 IL- 1β m RNA的主要细胞为神经元、星形胶质细胞和小胶质细胞。缺血再灌注后12 h IL- 1β蛋白主要表达于星形胶质细胞和小胶质细胞 ,神经元未见 IL- 1β的表达。结论 :脑缺血后 IL- 1β m RNA表达增加 ,IL- 1β可能在缺血性脑损伤中起重要作用。缺血再灌注后 IL- 1β主要来源于星形胶质细胞和小胶质细胞     

Heme-oxygenase-1 induction in glia throughout rat brain following experimental subarachnoid hemorrhage

The heme released following subarachnoid hemorrhage is metabolized by heme-oxygenase (HO) to biliverdin and carbon monoxide (CO) with the release of iron. The HO reaction is important since heme may contribute to vasospasm and increase oxidative stress in cells. HO is comprised of at least two isozymes, HO-2 and HO-1. HO-1, also known as heat shock protein HSP32, is inducible by many factors including heme and heat shock. HO-2 does not respond to these stresses. To begin to examine HO activity following subarachnoid hemorrhage (SAH), the expression of HO-1 and HO-2 was investigated after experimental SAH in adult rats. Immunocytochemistry for HO-1, HO-2 and HSP70 proteins was performed at 1, 2, 3 and 4 days after injections of lysed blood, whole blood, oxyhemoglobin and saline into the cisterna magna. A large increase in HO-1 immunoreactivity was seen in cells throughout brain following injections of lysed blood, whole blood, and oxyhemoglobin but not saline. Lysed blood, whole blood and oxyhemoglobin induced HO-1 in all of the cortex, hippocampus, striatum, thalamus, forebrain white matter and in cerebellar cortex. HO-1 immunoreactivity was greatest in those regions adjacent to the basal subarachnoid cisterns where blood and oxyhemoglobin concentrations were likely highest. Double immunofluorescence studies showed the HO-1 positive cells to be predominately microglia, though HO-1 was induced in some astrocytes. HO-1 expression resolved by 48 h. HO-2 immunoreactivity was abundant but did not change following injections of blood. A generalized induction of HSP70 heat shock protein was not observed following injections of lysed blood, whole blood, oxyhemoglobin, or saline. These results suggest that HO-1 is induced in microglia throughout rat brain as a general, parenchymal response to the presence of oxyhemoglobin in the subarachnoid space and not as a stress response. This microglial HO-1 response could be protective against the lipid peroxidation and vasospasm induced by hemoglobin, by increasing heme clearance and iron sequestration, and enhancing the production of the antioxidant bilirubin.     

大鼠脑缺血再灌注损伤后GAP-43及IGF-1在神经系统中的表达

目的探讨大鼠脑缺血再灌注损伤后生长相关蛋白-43（GAP-43）和胰岛素样生长因子-1（IGF-1）的表达。方法成年健康雄性Wistar大鼠72只，随机分为GAP-43组36只和IGF-1组36只，每组再分为假手术组和缺血1h再灌注2h、6h、12h、24h、48h、3d、7d、14d组，每组4只（n=4）。应用线栓法制备大鼠脑缺血再灌注动物模型，免疫组织化学方法检测GAP-43与IGF-1在神经元中的表达。结果GAP-43组：缺血再灌注2h，皮质区、海马及纹状体区神经元GAP-43呈基础表达，6h后表达逐渐增高，7d达高峰，14d开始降低，较假手术组高（P〈0．05）。IGF-1组：缺血再灌2h IGF-1表达明显增高，24h达高峰，48h恒定表达，14d仍维持高表达，较假手术组高（P〈0．05）。结论GAP-43和IGF-1可能参与促进神经元轴突的再生。     

大鼠局灶性脑缺血再灌注后FADD mRNA及其蛋白的表达变化

目的观察大鼠脑缺血再灌注后缺血半暗带皮质内Fas死亡结构域相关蛋白（FADD）mRNA及蛋白的表达变化。方法用半定量的逆转录PCR（RT-PCR）法检测缺血2h再灌注不同时间点缺血半暗带皮质内FADD mRNA的表达,Western blot检测FADD蛋白表达的变化。结果缺血半暗带脑皮质内FADD mRNA及其蛋白的表达于缺血灌注后3h明显升高,再灌注后12h达高峰（P＜0．01）,至再灌注后24h明显下降。结论脑缺血再灌注后缺血半暗带皮质内FADD mRNA及蛋白表达均明显增加,提示FADD可能在脑缺血再灌注损伤中发挥重要作用。     

亚低温通过抑制两种凋亡通路对大鼠脑缺血再灌注损伤发挥保护作用

目的探讨亚低温对大鼠局灶性脑缺血再灌注后胱冬酶(caspase)依赖性及非依赖性两种凋亡通路的影响。方法线栓法建立大鼠大脑中动脉阻塞(M CAO)及再通模型,分为假手术组、常温及亚低温脑缺血再灌注组,应用RT-PCR技术检测再灌注后不同时相缺血侧皮层凋亡诱导因子(A IF)及caspase-3 mRNA的表达。结果脑缺血2h再灌注2~4h,A IF及caspase-3 mRNA表达开始增加,随着再灌注时间的延长表达逐渐增强,至再灌注24h达高峰。每一再灌注时间点亚低温组与常温组A IF及caspase-3 mRNA表达均有显著差异,亚低温组mRNA表达均低于相应常温组。结论亚低温不仅降低caspase依赖性通路中的关键蛋白酶—caspase-3的mRNA的表达,而且降低caspase非依赖性通路中的关键蛋白—A IF的mRNA的表达,亚低温通过抑制两种凋亡通路对大鼠脑缺血再灌注损伤发挥保护作用。     

Expression of the glial cell line-derived neurotrophic factor gene in rat brain after transient MCA occlusion

Change of the glial cell line-derived neurotrophic factor (GDNF) gene expression in rat brain was examined after transient middle cerebral artery (MCA) occlusion of adult rats. Northern blot analysis showed that the mRNA began to be induced in the occluded MCA from 1 h of reperfusion with a peak at 3 h, and almost diminished by 1 day of reperfusion. Immunohistochemical analysis with brain sections showed an expression of GDNF-like immunoreactivity in neurons of the cerebral cortex and caudate after 90 min of ischemia in a similar way to the mRNA, but the staining was more disseminated and stronger in the cerebral cortex than the caudate. No glial cell was stained in the brain sections. The present results indicate that the GDNF gene was expressed in an early stage of reperfusion in neuronal cells of the MCA territory, but that the staining property was different between in the cerebral cortex and caudate.