Effects of mild hypothermia on the expression of microtubule-associated protein 2 in neurons of the hippocampal dentate gyrus in a rat model of cerebral ischemia/reperfusion

BACKGROUND： It is widely accepted that mild hypothermia can protect against injury to cerebral ischemia/reperfusion. OBJECTIVE： To observe the effects of mild hypothermia on microtubule-associated protein 2 （MAP2） expression in the hippocampal dentate gyms in rats following cerebral ischemia/reperfusion. Also, to study neuronal ultrastmctural changes in the dentate gyms to investigate the mechanism of the protection against injury to cerebral ischemia/reperfusion conferred by mild hypothermia. DESIGN, TIME AND SETTING： This randomized grouping, neural cell morphology trial was performed at the Laboratory Animal Center of Yijishan Hospital between March and June 2007. MATERIALS： Eighty-five healthy male Sprague Dawley rats were randomly allocated to three groups： mild hypothermia （n = 40）, normothermia （n = 40）, and sham-operated （n = 5）. METHODS： Cerebral ischemia/reperfusion injury was induced by the suture method in the mild hypothermia and normothermia groups, with a threading depth of 180.5 mm. In the sham-operated group, the suture was inserted 15 mm, with no vascular ligafion, and was followed by reperfusion 2 hours later. In the sham-operated and normothermia groups, the rat rectal temperature was maintained at 36-37 ℃ ; in the mild hypothermia group, it was controlled at 32-33 ℃. MAIN OUTCOME MEASURES： The hippocampal dentate gyms was serially sectioned for hematoxylin-eosin staining and MAP2 immunohistochemistry. Ultrastructural changes and the MAP2 absorbance value of the hippocampal dentate gyms were examined by transmission electron microscopy. RESULTS： The sham-operated group exhibited approximately normal ultrastructure of neurons in the bilateral hippocampal dentate gyms. In the normothermia group, ischemic hippocampal dentate gyms neurons were found with markedly fewer normal mitochondria, greatly proliferated rough endoplasmic reticulum, and a swollen and dysmorphic Golgi. In the mild hypothermia group, at each corresponding time point, these abnormal changes w     

Brain monoamines in human cerebral infarcts

Summary Dopamine (DA), serotonin (5-HT), and 5-hydroxyindole acetic acid (5-HIAA) were assayed spectrofluorometrically in various brain regions of 8 human patients who died after acute and old cerebral infarction. In both recent and older infarcts a total depletion of DA and 5-HT was associated with slight reduction of DA and 5-HT levels in remote nonischemic areas and various nuclei of both the injured and contralateral hemispheres. 5-HIAA was significantly reduced in acute ischemic necrosis, while the perifocal edema zone showed considerable accumulation of both 5-HT and 5-HIAA. The degradation zone surrounding old infarcts showed a mild decrease of both 5-HT and 5-HIAA, indicating normalization of 5-HT metabolism and turnover after decrease of cerebral edema. These preliminary data which confirm previous findings in experimental cerebral ischemia and infarct indicate that disorders of brain monoamine metabolism are contributing to the development of postischemic brain damage and the complicating cerebral edena.     

病变侧亚低温对局部脑缺血再灌流损伤有关因素的影响

目的　研究病变侧脑亚低温对脑缺血再灌流损伤梗塞体积、 Ｎ Ｏ 的影响确定病变侧亚低温的疗效, 探讨机理。方法　应用可反馈控温半导体致冷块对大鼠局灶脑缺血模型病变侧降温至３２ ～３３ ℃研究持续缺血及再灌流损伤的保护作用及有关因素的影响。结果　持续缺血１０ 分钟低温组及缺血４０ 分钟再灌流并低温组梗塞体积均小于常温对照组。亚低温组 Ｎ Ｏ 含量明显低于常温对照组。结论　病变侧亚低温对脑缺血再灌流损伤在一定时间窗内有明显保护作用, 而亚低温使 Ｎ Ｏ 产生减少可能是其脑保护作用的部分机制。     

Intraischemic hypothermia during pretreatment with sublethal ischemia reduces the induction of ischemic tolerance in the gerbil hippocampus

We examined whether mild brain hypothermia during pretreatment with sublethal 2-min ischemia affected the tolerance to subsequent lethal 5-min ischemia. The neuronal densities in the hippocampal CA1 sector of gerbils preconditioned at mild brain hypothermia (32% of normal) were significantly lower than those in gerbils preconditioned at brain normothermia (70% of normal). 72-kDa heat-shock protein immunoreactivity in the CA1 sector preconditioned at mild hypothermia was reduced. These results suggest that mild brain hypothermia during pretreatment with sublethal ischemia reduces the tolerance to subsequent lethal ischemia.     

Mild hypothermia ameliorates ubiquitin synthesis and prevents delayed neuronal death in the gerbil hippocampus.

BACKGROUND AND PURPOSE: The purpose of the present study is to determine the effect of mild hypothermia on the synthesis of ubiquitin, an important protein for maintenance of cell viability, in the hippocampal neurons following transient cerebral ischemia. METHODS: Transient ischemia was induced by occluding both common carotid arteries for 5 minutes. In experiment 1, the animals were divided into four groups according to the rectal and scalp temperatures during ischemia: the normothermia group and the graded hypothermia A, B, and C groups (n = 9 per group). CA1 neuronal density was assessed at 7 days after ischemia. In experiment 2, the animals were divided into two groups designated the normothermia and the hypothermia groups (n = 6 per group). The presence of ubiquitin was examined by immunohistochemistry at 6, 24, and 48 hours after transient ischemia in various regions of the hippocampus. RESULTS: In experiment 1, the mean +/- SEM neuronal density per millimeter was 12 +/- 1 in the normothermia group and 126 +/- 25, 225 +/- 10, and 214 +/- 9 in hypothermia groups A, B, and C, respectively. Mild hypothermia in groups B and C, in which the brain temperature was below 33 degrees C, ameliorated markedly the extent of ischemic neuronal damage in the CA1 sector (p less than 0.01). In experiment 2, ubiquitin immunoreactivity had disappeared in all regions of the hippocampus at 6 hours after ischemia and showed no subsequent recovery in the CA1 pyramidal neurons under normothermic conditions. Under hypothermic conditions, however, it had recovered significantly in the CA1 pyramidal neurons at 24 and 48 hours after ischemia (p less than 0.01). CONCLUSIONS: We conclude that mild hypothermia, in which the brain temperature is below 33 degrees C, markedly improves the ischemic delayed neuronal damage in the CA1 sector, and that increased ubiquitin synthesis and protein ubiquitination could be one essential part of the protective mechanism afforded by mild hypothermia against delayed neuronal death.     

Mild hypothermia inhibits systemic and cerebral complement activation in a swine model of cardiac arrest

Complement activation has been implicated in ischemia/reperfusion injury. This study aimed to determine whether mild hypothermia (HT) inhibits systemic and cerebral complement activation after resuscitation from cardiac arrest. Sixteen minipigs resuscitated from 8 minutes of untreated ventricular fibrillation were randomized into two groups: HT group (n=8), treated with HT (33°C) for 12 hours; and normothermia group (n=8), treated similarly as HT group except for cooling. Blood samples were collected at baseline and 0.5, 6, 12, and 24 hours after return of spontaneous circulation (ROSC). The brain cortex was harvested 24 hours after ROSC. Complement and pro-inflammatory markers were detected using enzyme-linked immunosorbent assay. Neurologic deficit scores were evaluated 24 hours after ROSC. C1q, Bb, mannose-binding lectin (MBL), C3b, C3a, C5a, interleukin-6, and tumor necrosis factor-α levels were significantly increased under normothermia within 24 hours after ROSC. However, these increases were significantly reduced by HT. Hypothermia decreased brain C1q, MBL, C3b, and C5a contents 24 hours after ROSC. Hypothermic pigs had a better neurologic outcome than normothermic pigs. In conclusion, complement is activated through classic, alternative, and MBL pathways after ROSC. Hypothermia inhibits systemic and cerebral complement activation, which may provide an additional mechanism of cerebral protection.     

亚低温治疗大鼠缺血性脑水肿研究

目的研究亚低温对延迟时间窗再灌注的局灶脑缺血大鼠缺血性脑水肿的治疗作用。方法 SD雄性大鼠96只,线栓法制作大脑中动脉闭塞模型后随机分为缺血3 h组、缺血6 h组、缺血9 h组(每组各30只),分别在造模3 h、6 h和9 h后拔出线栓,使大脑中动脉再灌注。各缺血组按照再灌注后是否给予亚低温治疗及亚低温持续时间分为常温、亚低温3 h和亚低温5 h三个亚组,每个亚组有10只大鼠。另设假手术组6只。缺血组大鼠在再灌注24 h后处死取脑,假手术组在术后24 h处死取脑,干-湿重法测定各组缺血侧脑组织含水量并进行比较。结果与假手术组比较,缺血组缺血侧脑组织含水量明显增高。缺血3 h组中3 h亚低温和5 h亚低温亚组的缺血侧脑组织含水量与缺血3 h常温组比较,差异有统计学意义(79.39%±2.44%vs82.16%±1.50%,P0.05;79.20%±1.55%vs 82.16%±1.50%,P0.05)。其余各缺血组中经过亚低温治疗的大鼠与常温亚组的脑组织含水量无统计学差异。结论亚低温可减轻缺血早期(3 h)再灌注的脑组织水肿,保护缺血脑组织,而对晚期(6 h和9 h)再灌注的缺血性脑水肿无论亚低温时间长短均无明显保护作用。     

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。     

Effects of graded hypothermia on outcome from brain ischemia

Abstract

The effects of mild (34° C) and moderate (31° C) hypothermia on the electroencephalogrami, cerebral blood flow and outcome from incomplete brain ischemia were compared to normothermia (37°C) in the rat Rats were anesthetized with fentanyl (25 fig kg^–1 h^–1) and 70% nitrous oxide in oxygen. Ischemia was produced by right carotid ligation combined with hemorrhagic hypotension to 25 mmHg for 30 min. CBF was measured in the right parietal cortex by laser Doppler. Neurologic outcome was measured daily for 3 days after ischemia and histopathology determined at the end of the study. Before ischemia, neither EEC nor CBF were changed by hypothermia compared to normothermic controls. Total EEG and beta EEG (12.5- 32 Hz) were abolished and CBF decreased by 70% during ischemia. During a 2.5 h recovery period total EEG and beta EEG activity recovered better with moderate hypothermia compared to normothermia. Recovery of cerebral blood flow was not significantly different between the treatment groups. Neurologic and histopathologic outcome were improved in rats receiving moderate hypothermia compared to normothermic controls. These results show that during severe incomplete ischemia\, hypothermia has a graded effect on outcome which is consistent with its effects on brain metabolism. [Neurol Res 1996; 18: 185–189]     

病变侧亚低温对大鼠局脑缺血再灌注后Akt、Survivin表达的影响

目的 通过检测Akt及Survivin蛋白的表达,探讨亚低温对局脑缺血再灌注后神经元存活的影响.方法 用线拴法制作大鼠大脑中动脉闭塞（MCAO）局脑缺血再灌注模型,将44只SD大鼠随机分成假手术组、常温缺血组和亚低温缺血组,缺血组分别缺血2,6 h再灌注4,24,72 h,1周,2周后处死,亚低温组于缺血后13～14 min实施病灶侧亚低温持续4 h.免疫组织化学法检测Akt、Survivin 蛋白的表达.结果 相同缺血再灌注时间点,亚低温组比常温组缺血侧Akt、Survivin、表达水平显著增高（P＜0.05）.结论 病灶侧亚低温通过促进缺血半暗带脑组织Akt、Survivin表达的核内移,从而抑制神经元凋亡,促进脑缺血后神经功能恢复.     

不同低温对鼠永久性大脑中动脉阻塞后NF-κB表达及脑梗死体积的影响

目的:观察不同低温对大鼠永久性大脑中动脉阻塞后NF-κB表达及梗死体积的影响。方法:120只雄性SD大鼠随机分为三组:常温组(37℃),轻度低温组(34℃),中度低温组(32℃),每组分缺血2,4,6,12小时共四个亚组。采用栓线法制作永久性大脑中动脉阻塞(MCAO)模型,各组大鼠分别于缺血2,4,6,12小时后置于常温操作台和冰毯机上,分别保持肛温为37±0．5℃,34±0．5℃和32±0．5℃12小时。在各自规定时间点处死大鼠,其中三只作TTC染色,剩余作免疫组织化学染色。结果:与常温组相比,在缺血的各个时间点,除轻度低温组缺血6h外,两低温组的脑梗死体积均显著减小(P<0．05或P<0．01)。随缺血时间的延续,常温组NF-κB蛋白表达逐渐升高,在缺血6小时达高峰,并持续升高:除轻度低温组缺血4h外,在其余各时间点,两低温组的NF-κB蛋白水平较常温组显著下降(P<0．01),且低温治疗使NF-κB蛋白表达高峰前移。结论:低温治疗组与常温组比较,脑梗死体积显著减小(P<0．05或P<0．01);低温治疗还显著抑制NF-κB的蛋白表达(P<0．01),使NF-κB蛋白表达高峰前移。低温的脑保护作用可能与抑制NF-κB的表达有关。     

大鼠颅脑损伤后亚低温治疗对Calpain及作用底物MAP-2表达的影响

目的探讨亚低温治疗对颅脑损伤后Calpain、MAP-2基因和蛋白表达的影响。方法将54只SD大鼠随机分为假手术组（n=6）、常温脑损伤组（n=24）和亚低温脑损伤组（n=24）。亚低温脑损伤组在液压打击伤后即接受持续4h的亚低温治疗。伤后6h、12h、24h和72h4个时间点分别处死3只常温脑损伤组和亚低温脑损伤组大鼠。荧光PCR、Western blot半定量检测皮质Calpain及MAP-2基因转录和蛋白的表达。结果颅脑损伤后12h及24h亚低温使Calpain mRNA表达增加（P〈0．05）,伤后6h、12h、24h和72h亚低温均可减少Calpain蛋白的升高,伤后12h及72h尤其显著（P〈0．05）。与假手术组比较,常温脑损伤组和亚低温脑损伤组MAP-2基因转录均减少（P〈0．05）;与常温脑损伤组比较,伤后6h、12h和24h亚低温可抑制MAP-2基因转录的下调,但亚低温脑损伤组MAP-2蛋白的表达均比同时间点常温组低（P〈0.05）。结论颅脑损伤后亚低温治疗的脑保护机制可能与调节Calpain蛋白的表达有关,而亚低温与MAP-2的关系还有待进一步研究。     

亚低温对局灶性脑缺血大鼠模型C3表达的影响

目的观察亚低温（32～35℃）对局灶性脑缺血后大鼠脑组织C3表达的影响。方法 100只大鼠随机分成假手术组、常温组和亚低温组,各组根据观察时间点分为术后6h、1d、2d、3d、7d五个亚组。建立大鼠左侧大脑中动脉闭塞（MCAO）模型,应用冰袋降温的方法使亚低温组大鼠肛温10min内降至（33℃±1℃）,维持低温6h后复温。假手术组和常温组大鼠保持肛温（37℃±0.5℃）。在各时间点采用神经缺陷评分对各大鼠进行神经功能评分后断头取脑,行苏木素伊红染色观察脑缺血病理学改变,免疫组织化学染色观察不同时间点C3表达情况。结果假手术组大鼠清醒后无神经功能障碍,常温组及亚低温组大鼠清醒后均出现左侧Horner征及不同程度右侧前肢为重的偏瘫,两组大鼠神经功能缺损3d时最明显,7d时均有所减轻。除6h外各时间点亚低温组大鼠神经功能缺损评分均较常温组低（P〈0.05）,各时间点亚低温组大鼠脑组织病理学损伤较常温组轻。在各时间点假手术组大鼠脑组织中C3可见少许表达,各组间比较差异无统计学意义（P〉0.05）。常温组和亚低温组大鼠缺血侧脑组织于缺血后6h补体C3阳性细胞数均开始增加,至3d均达到高峰,到7d时C3阳性细胞数明显减少,与常温组相比,亚低温组各时间点缺血侧脑组织C3表达明显减少（P〈0.05）。结论脑缺血时,缺血侧脑组织C3有表达,亚低温可使C3表达减少。亚低温可能通过降低C3的表达减轻补体级联反应起脑保护作用。     

亚低温对大鼠脑缺血再灌注后炎性反应的影响

目的：观察亚低温的不同时程对大鼠脑缺血再灌注后炎性反应的影响。方法：24只SD大鼠随机分为4组：常温组，亚低温1／2h组，亚低温1h组和亚低温3h组。每组各6只大鼠。参照Zea Longa的方法建立脑缺血再灌注动物模型，于再灌注24h断头取脑，行HE染色，观察脑组织中自细胞浸润及神经细胞的变化情况。结果：常温组在缺血梗死灶周围区可见白细胞浸润明显及深染受损的神经元；随亚低温时间的延长，白细胞浸润逐渐减少，神经元亦表现为淡染的轻度损伤。结论：亚低温可抑制脑缺血再灌注后的白细胞浸润，具有神经保护作用。     

Cerebral blood flow does not mediate the effect of brain temperature on recovery of extracellular potassium ion activity after transient focal ischemia in the rat

Temperature plays an important role in determining outcome following both global and focal brain ischemia. After focal ischemia, the degree of infarction decreases with mild hypothermia and increases with mild hyperthermia. In this study, brain extracellular potassium ion activity and local cerebral blood flow were measured in cerebral cortex during 60 min of middle cerebral artery occlusion and 60 min of re-perfusion. Brain temperature was maintained at 32-34 degrees C (mild hypothermia), 35.5-36.5 degrees C (normothermia), or 37.5-38.5 degrees C (mild hyperthermia) throughout ischemia and re-perfusion. In normothermic animals and to a greater degree in hyperthermic animals, extracellular potassium ion activity showed delayed secondary elevation above pre-ischemia values within 40-60 min after re-perfusion. No secondary elevation of extracellular potassium ion activity was observed in hypothermic animals. There was no difference in cortical blood flow among groups with varying brain temperature, indicating that delayed deterioration of brain potassium ion homeostasis was not caused by temperature dependent alteration of cerebral blood flow. The data suggest that loss of potassium ion homeostasis during re-perfusion after focal cerebral ischemia is caused by cellular rather than vascular dysfunction and may reflect secondary inhibition of energy metabolism.     

Mild hypothermia decreases the incidence of transient ADC reduction detected with diffusion MRI and expression of c-fos and hsp70 mRNA during acute focal ischemia in rats

The effects of mild hypothermia on the apparent diffusion coefficient of water (ADC) and expression of c-fos and hsp70 mRNA were examined during acute focal cerebral ischemia. Young adult rats were subjected to 60-min middle cerebral artery occlusion under either normothermia (37.5°C) or hypothermia (33°C). Diffusion-weighted echo-planar magnetic resonance imaging was used to monitor changes in ADC throughout the ischemic period. Perfusion MRI with dysprosium contrast was used at the end of the ischemic period to verify that the occlusion was successful. C-fos and hsp70 mRNA expression were examined with in situ hybridization at the end of the ischemic period. The results indicate that the size of the region that exhibited reduced ADC was smaller during hypothermia than during normothermia. Hypothermia also decreased the frequency of occurrence of transient ADC reductions, especially in dorsal aspects of cortex. Expression of both c-fos and hsp70 mRNA were markedly reduced by hypothermia. Transient ADC reduction and c-fos expression are associated with spreading depression, which is believed to contribute to lesion expansion during acute focal ischemia. The results suggest that part of the neuroprotective effect of hypothermia may be due to a reduced incidence of spreading depression.     

亚低温对局灶性脑缺血再灌注大鼠脑皮质神经元凋亡及存活素、脑源性神经营养因子表达的影响

目的观察亚低温干预对局灶性脑缺血再灌注大鼠脑皮质神经元凋亡及存活累(Survivin)、脑源性神经营养因子(BDNF)表达的影响,探讨Survivin、BDNF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大鼠大脑中动脉闭塞(MCAO)局灶性脑缺血再灌注改良模型,将90只大鼠随机分为假手术组、常温缺血组和亚低温缺血组,缺血组分别于缺血3h再灌注3h、6h、12h、24h、48h、72h、7d处死,亚低温缺血组于缺血后10min实施全身亚低温持续3h。进行TUNEL染色及免疫组化染色,检测梗死灶周围皮质神经元凋亡数量和Sur-vivin、BDNF的表达水平。结果 (1)亚低温缺血组和常温缺血组于再灌注6h皮质区均出现TUNEL染色阳性细胞,72h达高峰,随后逐渐减少,两组内相邻时间点比较差异均有统计学意义(P<0.05);在相同时间点亚低温缺血组凋亡细胞数明显少于常温缺血组,两组间比较差异有统计学意义(P<0.05)。(2)亚低温缺血组于再灌注3hSurvivin、BDNF表达有所增加,BDNF于24h达高峰,Survivin于48h达高峰,随后表达逐渐降低,但7d时仍高于假手术组,常温缺血组表达趋势与之相似,两组各时间点Survivin、BDNF表达均高于假手术组,差异有统计学意义(P<0.05);除再灌注3h Survivin表达在亚低温缺血组与常温缺血组间无明显差异外,其余各时间点亚低温缺血组Sur-vivin、BDNF表达均高于常温缺血组,差异有统计学意义(P<0.05)。结论亚低温干预可抑制梗死灶周围脑皮质神经细胞凋亡,促进存活素及脑源性神经营养因子的表达,发挥脑保护作用。     

亚低温对局灶性脑缺血大鼠白细胞介素-6和细胞间黏附分子-1表达的影响

目的 探讨亚低温对局灶性脑缺血大鼠缺血局部细胞间黏附分子-1（ICAM-1）表达和血清白细胞介素-6（IL-6）含量的影响。方法 将30只体重在250～300&#8197;g的雌性SD大鼠随机分为实验组（n=15）和对照组（n=15），采用线栓法阻断大鼠一侧大脑中动脉制作局灶性脑缺血模型.制模成功后，实验组和对照组大鼠分别给予亚低温、常温处理，使其肛温保持在（33±1）℃和（37±0.5）℃。12&#8197;h后，自左室取血，断头取脑，检测缺血区ICAM-1阳性血管数目（免疫组化方法）和血清IL-6含量（免疫放射测定法）。结果 实验组缺血局部ICAM-1的表达明显低于对照组[（2.45±1.56）vs（18.27±2.45）个/HP，P<0.01］；对照组血清IL-6含量、神经功能缺陷评分明显高于实验组[（184±38）vs（124±41）ng/L），P<0.01；（2.13±0.35）vs（1.48±0.25），P<0.01]。结论 亚低温对脑缺血性损害的神经保护作用可能与降低IL-6含量和减少ICAM-1的表达有关。     

Hypothermia attenuates the activation of protein kinase C in focal ischemic rat brain: dual autoradiographic study of [H]phorbol 12,13-dibutyrate and iodo[C]antipyrine

Using phorbol 12,13-dibutyrate (PDBu) autoradiography, we investigated the effect of hypothermia on protein kinase C (PKC) activation in rat brain 2 h after focal ischemia. In normothermia, a significant increase of PDBu binding was observed in ischemic brain. Hypothermia suppressed the increase of PDBu binding in degree and extent. These observations suggest that intraischemic hypothermia attenuates the activation of PKC, and this may in part be participate in the protective effect of hypothermia.