亚低温及银杏叶提取物对大鼠缺血脑组织氨基酸水平的影响

目的 探讨亚低温及银杏叶提取物(GBE)对大鼠缺血脑组织谷氨酸(Glu)、天门冬氨酸(Asp)和γ-氨基丁酸(GABA)水平的影响.方法 48只Wistar大鼠随机分为假手术组、脑缺血对照组、亚低温组、轻度高温GBE组、常温GBE组及亚低温GBE组,每组8只.用线栓法制作大鼠脑缺血模型,GBE组在缺血前12 h～缺血后2 h 4次腹腔注射GBE 300 mg/kg.用高效液相色谱法测定脑组织Glu、Asp、GABA含量.结果 与假手术组比较,除亚低温GBE组Glu、Asp及亚低温组Asp外,其他各组缺血脑组织Glu、Asp、GABA水平均显著增高(P<0.05～0.001).与脑缺血对照组比较,常温GBE组、亚低温组及亚低温GBE组缺血脑组织Glu、Asp水平均显著降低(均P<0.001),GABA水平均显著增高(P<0.01～0.001).与常温GBE组比较,亚低温组及亚低温GBE组缺血脑组织Glu、Asp水平和轻度高温GBE组缺血脑组织GABA水平均显著降低(P<0.01～0.001);而亚低温组及亚低温GBE组缺血脑组织GABA水平和轻度高温GBE组缺血脑组织Glu、Asp水平均显著增高(P<0.01～0.001).亚低温组与亚低温GBE组缺血脑组织Glu、Asp、GABA水平差异无统计学意义.结论 亚低温和GBE都能降低缺血脑组织Glu、Asp水平,使GABA水平升高,两者联合应用效果更好.     

猴脑选择性超深低温断血流复苏对细胞因子影响的实验研究

目的观察猴脑选择性超深低温断血流复苏实验前后血清中IL-2,IL-10,TNF-α,IFN-γ的动态变化,了解脑缺血后选择性超深低温复苏对猴免疫功能的影响。方法健康恒河猴10只,随机分为两血管阻断冷灌注组(4只),四血管阻断冷灌注组(4只)和常温灌注组(2只)。应用ELISA法统一测定实验前后血清中IL-2,IL-10,TNF-α,IFN-γ的浓度。结果两血管组4只恒河猴术后安全复苏并长期存活,四种细胞因子在灌注结束后较术前均明显增高(P<0.05),24h后恢复至术前水平,72h测得值与术前无显著差异(P>0.05)。常温组2只恒河猴及四血管组4只恒河猴于灌注后均未能安全复苏,全部死亡。其中四血管组与两血管组比较在灌注结束后IL-10,TNF-α增高更明显(P<0.05),IL-2,IFN-γ增高与两血管阻断冷灌注组比较无显著差异(P>0.05)。结论猴脑选择性超深低温断血流复苏可引起抗炎细胞因子与促炎细胞因子一过性增高,由于促炎因子与抑炎因子之间互相抑制,互相作用,两类因子同时增高可使细胞因子网络维持动态平衡,有利于机体免疫功能的重新建立。     

选择性超深低温断血流复苏促进猴脑中神经生长因子和胶质细胞源性神经营养因子的表达

目的观察常温缺血10min后选择性超深低温断血流复苏后猴脑中神经生长因子（nerve growth factor,NGF）和胶质细胞源性神经营养因子（glial cell line-derived neurotrophicfactor,GDNF）表达的变化。方法等温组及超深低温组实验猴于灌注或复苏死亡后立即开颅取脑,用NGF和GDNF抗体进行免疫组化染色;对额叶恒定视野内NGF和GDNF的阳性细胞记数求阳性率,并统计学分析。结果等温组2只实验猴额叶NGF和GDNF有微量表达,超深低温组4只实验猴额叶NGF和GDNF表达明显上调,与等温组比较差异均极显著俨〈0．01）。结论猴脑选择性超深低温断血流复苏实验可引起NGF和GDNF表达上调,这可能是防止脑缺血的重要保护机制之一。     

不同脑温状态下地西泮对大鼠脑缺血组织氨基酸及自由基的影响

目的 探讨不同脑温状态下地西泮对大鼠脑缺血组织谷氨酸(Glu)、γ-氨基丁酸(GABA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)及丙二醛(MDA)的影响.方法 建立大鼠大脑中动脉缺血再灌注模型,诱导目标脑温,测定轻度高温、常温、亚低温状态下各组脑缺血组织Glu、GABA、SOD、GSH-Px及MDA含量.结果 (1)与常温假手术组比较,常温脑缺血对照组及常温地西泮组Glu、MDA水平显著增高(均P<0.001),SOD、GSH-Px水平显著降低(均P<0.001),GABA在常温脑缺血对照组差异无统计学意义(P>0.05),在常温地西泮组则显著增高(P<0.01).(2)与常温脑缺血对照组比较,常温地西泮组GSH-Px显著增高(P<0.001),MDA显著降低(P<0.001),而Glu、GABA、SOD差异均无统计学意义(均P>0.05).(3)与常温地西泮组比较,轻度高温地西泮组Glu、MDA显著增高(均P<0.001),SOD、GSH-Px显著降低(均P<0.001),GABA差异无统计学意义(P>0.05);亚低温地西泮组Glu、MDA显著降低(P<0.01～0.001),GABA、SOD、GSH-Px显著增高(均P<0.001).结论 亚低温状态下,地西泮显著上调GABA水平,有利于地西泮"抑制性保护"机制的建立,从而增强地西泮的神经保护作用.     

不同脑温状态地西泮对大鼠脑缺血组织氨基酸含量的影响

目的 探讨不同脑温状态下,地西泮对大鼠脑缺血组织谷氨酸(Glu)、天门冬氨酸(Asp)、甘氨酸(Gly)及γ-氨基丁酸(GABA)含量的影响.方法建立大鼠大脑中动脉缺血再灌注模型,诱导目标脑温,用HPLC荧光法检测轻度高温、常温、亚低温各组脑缺血组织Glu、Asp、Gly及GABA含量.结果 (1)与常温假手术组比较,常温脑缺血对照组及常温地西泮组Glu、Asp、Gly显著增高(P均<0.001),GABA在常温脑缺血对照组差异无统计学意义(P>0.05),在常温地西泮组则显著增高(P<0.01).(2)与常温脑缺血对照组比较,常温地西泮组Glu、Asp、Gly稍降低,GABA稍增高,但差异均无统计学意义(P均>0.05).(3)与常温地西泮组比较,轻度高温地西泮组Glu、Asp、Gly显著增高(P均<0.001),GABA差异无统计学意义(P>0.05);亚低温地西泮组Glu、Asp、Gly显著降低(P均<0.01),GABA显著增高(P<0.001).结论亚低温状态下,地西泮显著上调GABA水平,从而有利于地西泮"抑制性保护"机制的建立,可能使地西泮的神经保护作用得到增强.     

托吡酯对大鼠脑缺血再灌注损伤的神经保护作用

目的探讨托吡酯(TPM)对大鼠脑缺血再灌注损伤的神经保护作用及其机制。方法将健康30只雄性SD大鼠随机分为假手术组、缺血再灌注组和TPM干预组。用线栓法建立大鼠脑缺血再灌注模型,TPM干预组给予TPM80mg/kg腹腔注射,2次。缺血再灌注24h时进行神经功能评分、TTC染色法测量梗死体积、高效液相色谱分析法测定脑组织谷氨酸(Glu)及γ-氨基丁酸(GABA)的含量;免疫组化法检测GABAA受体阳性表达。结果(1)与缺血再灌注组比较,TPM干预组神经功能评分明显增高(P<0.01),脑梗死体积减少(P<0.05);(2)TPM干预组缺血侧脑皮质Glu含量显著低于缺血再灌注组(P<0.01),与假手术组比较差异无显著性;GABA含量显著高于假手术组和缺血再灌注组(均P<0.01);(3)TPM干预组缺血侧脑皮质GABAA受体阳性细胞数显著高于缺血再灌注组(P<0.01)。结论TPM对脑缺血再灌注损伤有神经保护作用,其机制可能为TPM降低兴奋性递质Glu水平、增加抑制性递质GABA的释放及GABAA受体的表达。     

亚低温持续不同时间对缺血脑组织中氨基酸和单胺类递质含量的影响

目的观察脑缺血后即时亚低温并维持不同时间对脑缺血损伤的影响,探讨亚低温脑保护作用的生物学机制. 方法脑缺血动物模型采用改良的Pulsinelli四动脉阻断法.采用氨基酸分析仪测定脑组织中谷氨酸(Glu)、天门冬氨酸(Asp)、甘氨酸(Gly)和γ-氨基丁酸(GABA)含量;采用荧光分光光度法测定多巴胺(DA)、去甲肾上腺素(NE)、5-羟色胺(5-HT)及其代谢产物5-羟吲哚乙酸(5-HIAA). 结果常温脑缺血组织中Glu、Asp、Gly和GABA的含量明显低于假手术组(P<0.01),亚低温持续30～240 min脑组织中Glu、Asp、Gly和GABA 含量明显高于常温脑缺血组(P<0.05 或P<0.01).常温脑缺血后组织中DA、NE和5-HT的含量明显低于假手术组(P<0.01),5-HIAA含量高于假手术组(P<0.01);亚低温持续30 min脑组织中5-HT含量明显高于常温脑缺血组(P<0.01),5-HIAA的含量低于常温脑缺血组(P<0.05);亚低温持续60～240 min脑组织中DA、NE和5-HT含量明显高于常温脑缺血组(P<0.05或P<0.01),5-HIAA含量低于常温脑缺血组(P<0.01). 结论脑缺血后即时亚低温明显减轻常温脑缺血时组织中氨基酸和单胺类神经递质的代谢紊乱,提示亚低温减轻脑缺血损伤作用最好是在脑缺血后立即实施,并持续达1 h以上效果更明显.     

脑超深低温下猴脑血流阻断时间窗的研究

目的 探明选择性脑超深低温技术对常温条件下猴脑血流阻断有效治疗时间窗.方法 10只恒河猴分为3组:常温组(n=3);深低温I组(n=4):脑血流阻断15min开始深低温治疗;深低温Ⅱ组(n=3):脑血流阻断20min开始深低温治疗.双侧颈动脉和颈静脉系统分离、脑血流阻断建立严重脑缺血缺氧模型.深低温组动物通过一侧颈内动脉灌注4~C林格氏液同侧颈内静脉回流,阻断其他颈动脉和颈静脉血管,脑温维持在(15.3±1.04)℃、中心体温(35.1±1.10)℃.维持深低温60min后恢复正常脑血流.常温组动物采用相同方法脑灌注37℃林格氏液.观察猴生存率、脑神经功能状况和病理形态.结果 4只脑血流阻断15min开始深低温治疗的猴死亡3只、1只长期存活;3只脑血流阻断20min开始深低温治疗和3只常温组猴全部死亡.死亡动物脑干出现神经元死亡.结论 常温条件下严重或完全脑缺血缺氧有效低温复苏时间为10min、15min内低温复苏成功率高,15min以上开始深低温复苏效果差.     

亚低温对大鼠局灶性脑缺血再灌注后MMP-9表达和细胞凋亡的影响

目的 通过研究亚低温对大鼠局灶性脑缺血再灌注后基质金属蛋白酶-9(MMP-9)表达和细胞凋亡的影响,探讨亚低温脑保护的可能机制.方法 将雄性SD大鼠39只分为假手术组、常温缺血组和缺血期亚低温组.制作大脑中动脉阻塞(MCAO)模型,缺血2h再灌注48h,HE染色观察各组大鼠脑组织形态学改变;采用TTC染色法观察梗死体积;TUNEL法检测细胞凋亡;免疫组化法检测MMP-9表达.结果 亚低温减轻脑缺血组织病理学损伤,明显缩小脑梗死体积(P<0.05).常温下缺血侧脑组织可见大量TUNEL阳性细胞和MMP-9免疫阳性细胞,主要位于皮质缺血半暗带区.亚低温减少脑缺血后TUNEL阳性细胞数目(P<0.05),明显下调MMP-9蛋白表达(P<0.05).结论 亚低温可能通过下调脑缺血再灌注后MMP-9表达,抑制细胞凋亡,从而发挥确实的脑保护作用.     

猴脑选择性深低温断血流复苏实验模型的建立

目的研究猴脑选择性深低温断血流复苏的可行性及最长断流时间;方法建立闭胸式脑局部体外循环,一侧颈内动脉冷灌注,同侧颈外静脉回流,60~80min后恢复脑血流,实验动物自然复苏。手术前后监测各主要脏器的血生化变化,术后行神经功能缺失评分;结果在7只实验猴中,2只未能建立实验模型,5只于术后安全复苏,其中4只术后长期存活;各主要脏器生化指标监测未见显著变化(P>0.05),术后神经功能评分无异常;结论猴脑选择性深低温断血流60~80min后可安全复苏,各主要脏器功能及神经功能均正常。     

Effect of mild hypothermia on ischemia-induced release of neurotransmitters and free fatty acids in rat brain

We have demonstrated previously that mild intraischemic hypothermia confers a marked protective effect on the final histopathological outcome. The present study was carried out to evaluate whether this protective effect involves changes in the degree of local cerebral blood flow reductions, tissue accumulation of free fatty acids, or alterations in the extracellular release of glutamate and dopamine. Rats whose intraischemic brain temperature was maintained at 36 degrees C, 33 degrees C, or 30 degrees C were subjected to 20 minutes of ischemia by four-vessel occlusion combined with systemic hypotension. Levels of local cerebral blood flow, as measured autoradiographically, were reduced uniformly in all experimental animals at the end of ischemia by gas chromatography after tissue extraction and separation by thin layer chromatography. A massive ischemia-induced accumulation of individual free fatty acids was observed in animal groups whose intraischemic brain temperature was maintained at either 36 degrees C or 30 degrees C. Extracellular neurotransmitter levels were measured by microdialysis; the perfusate was collected before, during, and after ischemia. In rats whose intraischemic brain temperature was maintained at 36 degrees C, dopamine and glutamate increased significantly during ischemia and the early period of recirculation (by 500-fold and sevenfold, respectively). In animals whose brain temperature was maintained at 33 degrees C and 30 degrees C, the release of glutamate was completely inhibited, and the release of dopamine was significantly attenuated (by 60%). These results suggest that mild intraischemic hypothermia does not affect the ischemia-induced local cerebral blood flow reduction or free fatty acid accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relationship between glutamate release and cerebral blood flow after focal cerebral ischaemia in the cat: effect of pretreatment with enadoline (a kappa receptor agonist)

The effect of the κ-opioid agonist enadoline (CI-977) upon the relationship between cerebral blood flow and glutamate release was simultaneously assessed (using microdialysis and hydrogen clearance techniques respectively) at the same anatomical locus in the cerebral cortex (suprasylvian gyrus) after permanent middle cerebral artery (MCA) occlusion in halothane-anaesthetised cats. During controlled graded ischaemia, pretreatment with enadoline (0.3 mg/kg i.v. followed by continuous infusion at 0.15 mg/kg/h), initiated 30 min prior to MCA occlusion, significantly attenuated the marked increases in extracellular glutamate, aspartate and GABA observed in the focal ischaemic penumbra. The present data are consistent with the hypothesis that the neuroprotective efficacy of enadoline in focal cerebral ischaemia is due to inhibition of glutamate release in the ischaemic penumbra.     

高压氧治疗时机对家兔实验性局部脑缺血治疗效果的影响及微透析监测研究

目的 观察高压氧(HBO)治疗时机对家兔实验性局部脑缺血治疗效果的影响.方法 75只家兔按随机数字表法平均分为3组,每组25只,即大脑巾动脉闭塞(MCAO)组:右侧大脑中动脉阻断.不行HBO治疗;MCAo+HBO组:右侧大脑巾动脉阻断后即予HBO治疗;MCAO+DHBO组:右侧大脑巾动脉阻断7d后行HBO治疗.动物放入HBO舱(100%O2,250 kPa,1 h/d)行HBO治疗30 d.MCAO后第1天、第3天、第10天、第30天观察各组动物的行为学变化、脑梗死体积,并采用微透析技术分析家兔缺血脑组织细胞间液中的葡萄糖、乳酸、丙酮酸、乳酸/丙酮酸比值以及谷氨酸水平的变化.结果 缺血后MCAO+HBO组行为学评分较其他组低;第3天至第30天,MCAO+HBO组脑梗死体积低于其他两组,差异有统计学意义(P<0.05).微透析分析能量代谢指标中乳酸丙酮酸比值变化最为明显.其中缺血后1 d和3 d右侧脑组织间液中MCAO+HBO组较其他两组降低,差异有统计学意义(P<0.05).缺血后家兔脑组织间液中谷氨酸浓度升高,3d时逐渐下降,缺血后1 d和3d右侧腩组织间液中MCAO+HBO组较其他两组降低,差异有统计学意义(P<0.05).结论 HBO治疗能通过改善神经组织的能量代谢,减轻兴奋性氨基酸毒性作用,改善缺血缺氧对神经的损害;而HBO治疗脑梗死应尽早进行.     

Therapeutic effect of post-ischemic hypothermia duration on cerebral ischemic injury

OBJECTIVE: To study the efficacy of post-ischemic mild brain hypothermia lasting for different time intervals on cerebral ischemic reperfusion injury. METHOD: Male Sprague-Dawley rats were divided into a sham-operated group, normothermia (37-38 degrees C) ischemia group and mild hypothermia (31-32 degrees C) group. The last group was subdivided into four groups: 30 minute hypothermia plus 210 minute normothermia, 60 minute hypothermia plus 180 minute nomothermia,120 minute hypothermia plus 120 minute normothermia, and 240 minute hypothermia (n=8). Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model. Brain tissue was collected following a 20 minute cerebral ischemia and 240 minute reperfusion, and was used to measure the levels of glutamate (Glu), aspartate (Asp), glycine (Gly), gamma-aminobutyric acid (GABA), dopamine (DA), norepinephrine (NE), serotonin(5-HT) and hydroxyindoleacetic acid (5-HIAA), nitrite (NO(2)), endothelin-1 (ET(1)), tumor necrosis factor alpha(TNFalpha) and interleukin-1beta (IL-1beta). Serum was collected to measure the levels of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), creatine kinase (CK) and its brain band isoenzyme (CK-BB). RESULTS: Hypothermia lasting for 60-240 minutes delayed the decrease in these amino acids, postponed the decrease in DA, NE and 5-HT and increase in hydroxyindoleacetic acid (5-HIAA), and decreased the levels of IL-1beta, TNFalpha, ET(1) and NO(2) in brain tissue. Hypothermia also decreased the levels of LDH, AST, CK and CK-BB in serum as compared to normothermia group (p<0.05 or p<0.01). Hypothermia lasting for 30 minutes delayed the decreases in these amino acids and 5-HT and increase in 5-HIAA in brain tissue (p<0.05), but failed to influence the levels of IL-1beta, TNFalpha, ET(1) and NO(2) in brain tissue and the amounts of LDH, AST, CK and CK-BB in serum as compared to normothermia ischemia group (p>0.05). CONCLUSIONS: Post-ischemic mild brain hypothermia can significantly suppress the excessive release of amino acids, monoamine neurotransmitters and inflammation response in ischemic tissue. It can also stabilize the function of the cell membrane, which is associated with the mechanism of cerebral protection by mild hypothermia. These results suggest that mild hypothermia should be applied immediately after ischemia and last for more than 60 minutes in order to obtain neuroprotective effects.     

Indomethacin modulates ischemia-evoked release of glutamate and adenosine from the rat cerebral cortex

The effects of indomethacin (10 mg/kg) on the release of the transmitter amino acids, glutamate, aspartate, GABA, and of the purines, adenosine and inosine, from the cerebral cortex was studied in a four-vessel occlusion rat model of cerebral ischemia/reperfusion. In comparison with the control group, indomethacin significantly attenuated the ischemia-evoked release of glutamate and aspartate, but not of GABA. Adenosine levels in the cortical superfusates were significantly elevated following indomethacin administration. As indomethacin is a potent inhibitor of adenosine uptake, these results suggest that, by blocking adenosine uptake, indomethacin could elevate extracellular adenosine levels and depress glutamate and asparte efflux as a consequence of the activation of adenosine A₁ receptors.     

Alpha-MSH decreases core and brain temperature during global cerebral ischemia in rats

A key pathological event during cerebral ischemia is the excitotoxic release of glutamate. We have shown previously that alpha-melanocyte-stimulating hormone (alpha-MSH) enhances the hypothermia induced by kainic acid. We have investigated the effects of systemic administration of alpha-MSH on four-vessel occlusion forebrain ischemia on core temperature (CT) and brain temperature (BT), respectively. After 10 min cerebral ischemia, BT was lower in alpha-MSH- than in saline-injected animals. After 10 min reperfusion, both CT and BT were lower than the corresponding pre-ischemic levels after injection of alpha-MSH. alpha-MSH did not influence CT or BT in sham-operated rats. The alpha-MSH-induced hypothermia and its potentiation of reduction in BT during global cerebral ischemia, may contribute to neuroprotective effects of alpha-MSH.     

Hypothermia and brain-derived neurotrophic factor reduce glutamate synergistically in acute stroke

Moderate hypothermia and application of brain-derived neurotrophic factor (BDNF) have separately been identified as neuroprotective strategies in experimental cerebral ischemia. To assess their separate and combined effects on striatal glutamate release in the hyperacute phase of stroke, we inserted microdialysis probes into the striatum of rats 2 h before permanent middle cerebral artery occlusion (MCAO). The animals (N = 28) were randomly assigned to one of four treatment strategies commencing 30 min after MCAO: (1) hypothermia at 33 degrees C (n = 7); (2) intravenous BDNF infusion [300 microg/(kg/h) for 2 h, n = 7]; (3) combination of hypothermia and BDNF (n = 7); (4) control group (saline, n = 7). Infarct size at 5 h after MCAO was assessed with the silver-staining method. Total infarct volume was significantly reduced in the hypothermia (202.7 +/- 3.5 mm(3), P = 0.0002) and BDNF group (206.5 +/- 6.9 mm(3), P = 0.0006) as compared to control group (254.4 +/- 9.3 mm(3)). In the combination group, infarct size was further reduced with overall significance in post hoc tests (157.3 +/- 6.2 mm(3), P < 0.0001). Postischemic glutamate concentrations in the control group constantly remained significantly higher than in all other treatment groups. At 255 and 270 min after MCAO, striatal glutamate in the combination group decreased significantly more than in animals treated with hypothermia or BDNF alone.Combining hypothermia and BDNF therapy in the acute stage of ischemia has a synergistic effect in attenuating striatal glutamate release and reducing early infarct size.