Immediate or delayed mild hypothermia prevents focal cerebral infarction

The protective effect of mild hypothermia was studied in rodent models of both permanent and transient focal cerebral ischemia. In Expt. 1, Wistar rats were exposed to 6 h permanent ischemia by bilateral occlusion of both common carotid arteries and right middle cerebral artery. In Expt. 2, animals were exposed to 3 h transient ischemia followed by 21 h reperfusion, and in Expt. 3, 3 h transient ischemia was followed by 69 h of reperfusion. Expt. 4 used 3 h transient ischemia followed by 3 h reperfusion. In Expt. 1, animals maintained at 37 degrees C rectal (normothermia) suffered a mean infarct volume (+/- S.D.) of 142 +/- 44 mm3 (n = 6), which was reduced for those exposed to permanent hypothermic (32 degrees C) ischemia to 56 +/- 64 mm3 (n = 10) (P less than 0.05). In Expt. 2, normothermic ischemia and reperfusion resulted in an infarction of 211 +/- 35 mm3 (n = 6). Intra-ischemic hypothermia (32 degrees C) followed by 21 h of normothermic reperfusion resulted in 17 +/- 12 mm3 of infarction (n = 9) (P less than 0.001). Hypothermia for either the first or second 1.5 h of the 3 h ischemic insult reduced the infarct volume to 116 +/- 76 mm3 (n = 6) (P less than 0.05) or 108 +/- 73 mm3 (n = 7) (P less than 0.01), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

升压联合亚低温治疗对局灶性脑缺血再灌注的脑保护作用

目的　观察升压联合亚低温治疗对大鼠局灶性脑缺血再灌注的脑保护作用。方法　 32只大鼠随机分为对照组、升压组、亚低温组、升压 亚低温组 ,采用大鼠局灶性脑缺血再灌注模型 ,观察各组神经功能缺损评分和脑梗死体积。结果　升压组、亚低温组及升压 亚低温组神经功能缺损评分 (P <0 .0 5 )、脑梗死体积 (P <0 .0 1)均明显低于对照组 ;升压 亚低温组脑梗死体积明显低于升压组和亚低温组 (P <0 .0 5 )。结论　升压、亚低温对局灶性脑缺血再灌注损伤有明显脑保护作用 ,升压联合亚低温应用效果更佳     

Evolution of brain infarction after transient focal cerebral ischemia in mice.

The evolution of brain infarction after transient focal cerebral ischemia was studied in mice using multiparametric imaging techniques. One-hour focal cerebral ischemia was induced by occluding the middle cerebral artery using the intraluminal filament technique. Cerebral protein synthesis (CPS) and the regional tissue content of adenosine triphosphate (ATP) were measured after recirculation times from 0 hours to 3 days. The observed changes were correlated with the expression of the mRNAs of hsp-70, c-fos, and junB, as well as the distribution of DNA double-strand breaks, visualized by TUNEL. At the end of 1 hour of ischemia, protein synthesis was suppressed in a larger tissue volume than ATP in accordance with the biochemical differentiation between core and penumbra. Hsp70 mRNA was selectively expressed in the cortical penumbra, whereas c-fos and junB mRNAs were increased both in the lateral part of the penumbra and in the ipsilateral cingulate cortex with normal metabolism. During reperfusion after withdrawal of the intraluminal filament, suppression of CPS persisted except in the most peripheral parts of the middle cerebral artery territory, in which it recovered between 6 hours and 3 days. ATP, in contrast, returned to normal levels within 1 hour but secondarily deteriorated from 3 hours on until, between 1 and 3 days, the ATP-depleted area merged with that of suppressed protein synthesis leading to delayed brain infarction. Hsp70 mRNA, but not c-fos and junB, was strongly expressed during reperfusion, peaking at 3 hours after reperfusion. TUNEL-positive cells were detected from 3 hours on, mainly in areas with secondary ATP depletion. These results stress the importance of an early recovery of CPS for the prevention of ischemic injury and suggest that TUNEL is an unspecific response of delayed brain infarction.     

短暂局灶预缺血对脑梗死大鼠脑肝细胞生长因子表达的影响

目的:探讨短暂局灶缺血预处理对脑梗死大鼠脑肝细胞生长因子(HGF)表达的影响及其意义。方法:采取线栓法建立局灶脑缺血模型,预处理组大鼠经10分钟短暂预缺血处理.24h后二次处理造成大脑中动脉闭塞,脑梗死组只在第二次处理造成大脑中动脉闭塞。单纯短暂缺血组只在第一次处理时缺血10分钟。各组均在末次处理24小时后用RT-PCR法检测HGF的表达。结果:单纯短暂预缺血组脑缺血边缘区几乎无HGF的表达.预处理组脑梗死边缘区HGF的表达较单纯脑梗死组HGF的表达增强。结论:短暂局灶缺血预处理引起脑梗死边缘区HGF的高表达可能与缺血耐受的产生有一定的关系,对脑缺血损伤组织的保护及修复可能有重要意义。     

Mild postischemic hypothermia limits cerebral injury following transient focal ischemia in rat neocortex

Intraischemic mild hypothermia has been shown to attenuate cerebral infarction occurring after transient focal ischemia. In contrast, the capacity of mild hypothermia to provide a protective effect when administered postischemically has not been clearly defined for transient focal events such as occur in many types of stroke. The present study addressed this issue by investigating the influence of timing and duration of mild hypothermia on cerebral infarction in a rat model of reversible focal ischemia. Sprague-Dawley rats (n = 45) were subjected to 3 h of focal neocortical ischemia by occluding reversibly one middle cerebral artery and both carotid arteries. Mild hypothermia was established after reperfusion and maintained for brief (1 h) or prolonged (21 h) periods. Animals were sacrificed 24 or 48 h after ischemia. A significant reduction (32%) in the volume of infarction was obtained when hypothermia was established immediately after reperfusion and maintained for a prolonged (21 h) period. In contrast, immediate but brief (1 h) hypothermia did not reduce infarction volume. Delaying hypothermia until 30 min post reperfusion and maintaining it for 21 h reduced infarction volume by 22%; however, this effect did not achieve statistical significance. These findings demonstrate that mild postischemic hypothermia is capable of protecting against cerebral injury following transient focal ischemia but that prolonged hypothermia is required to achieve this effect. These findings are consistent with increasing evidence that the window of therapeutic opportunity after transient focal ischemia is rather brief and that critical mechanisms involved in this form of ischemic injury remain activated over a rather lengthy postischemic interval.     

Magnesium treatment and spontaneous mild hypothermia after transient focal cerebral ischemia in the rat

There is evidence from global cerebral ischemia experiments in the rat that the neuroprotection attributable to magnesium treatment depends on the concurrent presence of at least mild hypothermia. We set out to determine to what extent spontaneous hypothermia occurred after transient middle cerebral artery occlusion in the rat, and whether this hypothermia influenced the outcome of magnesium treatment. We found that rectal temperatures from 30 min to 3h after recovery from anaesthesia/surgery were 1 °C lower than in the period from 4 to 6h. Striatal infarcts were significantly reduced by 32% in animals treated with 360 μmol/kg MgSO(4) intravenously immediately prior to ischemia. A higher magnesium dose of 720 μmol/kg had not effect on infarct volume. Having previously established that these two doses of magnesium are ineffective in normothermic animals using this model, we conclude that the mild spontaneous hypothermia contributed to the observed neuroprotective effect of magnesium in this study, and that previous studies of magnesium in cerebral ischemia have likely been confounded in this way.     

A simple method to induce focal brain hypothermia in rats.

Hypothermia reduces cell death and promotes recovery in models of cerebral ischemia, intracerebral hemorrhage and trauma. Clinical studies report significant benefit for treating cardiac arrest and studies are investigating hypothermia for stroke and related conditions. Both local (head) and generalized hypothermia have been used. However, selective brain cooling has fewer side effects than systemic cooling. In this study, we developed a method to induce local (hemispheric) brain hypothermia in rats. The method involves using a small metal coil implanted between the Temporalis muscle and adjacent skull. This coil is then cooled by flushing it with cold water. In our first experiment, we tested whether this method induces focal brain hypothermia in anesthetized rats. Brain temperature was assessed in the ipsilateral cortex and striatum, and contralateral striatum, while body temperature was kept normothermic. Focal, ipsilateral cooling was successfully produced, while the other locations remained normothermic. In the second experiment, we implanted the coil, and brain and body temperature telemetry probes. The coil was connected via overhead swivel to a cold-water source. Brain hypothermia was produced for 24 h, while body temperature remained normothermic. A third experiment measured brain and body temperature along with heart rate and blood pressure. Brain cooling was produced for 24 h without significant alterations in pressure, heart rate or body temperature. In summary, our simple method allows for focal brain hypothermia to be safely induced in anesthetized or conscious rats, and is, therefore, ideally suited to stroke and trauma studies.     

Early changes of oxypurines in rat brain following focal cerebral ischemia

Cerebral hypoxanthine, xanthine, and uric acid levels were measured by high-performance liquid chromatography (HPLC) for up to 4 hours following focal cerebral ischemia in the rat. Fifty male Sprague-Dawley rats were subjected to occlusion of the left middle cerebral artery under halothane inhalation anesthesia. The animals were sacrificed with microwave at 30, 60, 120, and 240 minutes after surgery. The brains were removed and divided into right and left hemisphere. Each hemisphere was homogenized with perchloric acid and centrifuged. The supernates were filtrated with membrane filter. An aliquot of the filtrate was used for measurement of uric acid, xanthine, and hypoxanthine in both of the ischemic and contralateral hemisphere by a HPLC system. A HPLC with multiple ultraviolet spectroscopy was used for measuring hypoxanthine and xanthine. Identification of hypoxanthine and xanthine was made by parallel chromatography of standards, disappearance with xanthine oxidase, and the spectrum of UV absorption. Uric acid was measured by reversed-phase HPLC with electrochemical detection as reported previously. Hypoxanthine increased rapidly and arrived at a peak value at 60 minutes. Xanthine increased not so rapidly as hypoxanthine and showed the highest value at 120 minutes. Uric acid also increased significantly but very slowly and did not seem to reach the peak value during the observation period. Hypoxanthine is oxidized to xanthine and then xanthine is oxidized to uric acid at the terminal stage of purine degradation. The order of peak times of cerebral hypoxanthine, xanthine, and uric acid levels following cerebral ischemia corresponds to the order in purine metabolism. This result strongly suggests that hypoxanthine is degraded into uric acid in ischemic rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selenoprotein S expression in the rat brain following focal cerebral ischemia

Recent studies on cerebral ischemic stroke have demonstrated the importance of the inflammatory response. Ongoing inflammatory insults have been implicated as a secondary mechanism underlying neuronal injury induced by ischemia, and anti-inflammatory strategies have gained considerable interest. Selenoprotein S (SelS), which is an endoplasmic reticulum resident protein, is known to promote cell survival by regulating inflammation. Moreover, SelS has been shown to be responsive to ischemia in cultured astrocytes. A Finnish report revealed that a variation in the SelS gene locus is associated with a higher predisposition to ischemic stroke in humans, suggesting a crucial role for SelS in protection against brain ischemia. However, the time-course of SelS expression following cerebral ischemia in vivo remains unknown. In the present study, we show, for the first time, differential SelS expression from 3 h to 7 days after reperfusion in rats with transient focal cerebral ischemia induced by a 1-h middle cerebral artery occlusion. We found that the SelS protein level decreased in the ischemic core 3–7 days after reperfusion. Furthermore, SelS expression was upregulated in the ischemic penumbra adjacent to the ischemic core 3–7 days after reperfusion and is matched by reactive astrogliosis. Thus, we propose that the upregulation of Sels represents a reaction of astrocytes against inflammatory stimuli, and the findings of this study open a new chapter in the research of the interrelationships between SelS and cerebral ischemic stroke.     

Ornithine decarboxylase knockdown exacerbates transient focal cerebral ischemia-induced neuronal damage in rat brain.

Transient cerebral ischemia leads to increased expression of ornithine decarboxylase (ODC). Contradicting studies attributed neuroprotective and neurotoxic roles to ODC after ischemia. Using antisense oligonucleotides (ODNs), the current study evaluated the functional role of ODC in the process of neuronal damage after transient focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats. Transient MCAO significantly increased the ODC immunoreactive protein levels and catalytic activity in the ipsilateral cortex, which were completely prevented by the infusion of antisense ODN specific for ODC. Transient MCAO in rats infused with ODC antisense ODN increased the infarct volume, motor deficits, and mortality compared with the sense or random ODN-infused controls. Results of the current study support a neuroprotective or recovery role, or both, for ODC after transient focal ischemia.     

Quantitative proton magnetic resonance imaging in focal cerebral ischemia in rat brain

Proton magnetic resonance (MR) imaging has been recommended as a diagnostic tool for the detection of focal cerebral ischemia. We compared microscopic MR images of rat brains after focal cerebral ischemia with evidence of histological damage found on corresponding silver-impregnated or cresyl violet-stained brain sections. Ten male Wistar rats were subjected to permanent unilateral occlusions of the right middle cerebral and common carotid arteries under halothane anesthesia. Twenty-four hours later the area of injury on MR images amounted to 26% of the total slice area, whereas only 9% of the total slice area was necrotic on histological sections from the same animals. The infarcted areas on tissue sections were surrounded by regions of selective neuronal injury in the cerebral cortex and occasionally in the hippocampus. The area of injury on MR images was larger than the combined areas of infarction and selective neuronal injury on histological sections. Areas of increased T2 values on MR images extended medially into noninfarcted striatum and laterally and dorsally into noninfarcted cortex. The lateral and dorsal areas on MR images frequently coincided with cortical areas in which considerable selective neuronal injury was present in the upper cortical layers. We hypothesize that the abnormal areas on MR images above histologically normal brain tissue represent the ischemic penumbra. If true, this is the first demonstration of the ischemic penumbra by MR imaging and may reflect our use of Wistar rats, a new image analysis technique, and ultra-high resolution MR imaging.     

Quantitative assessment of early brain damage in a rat model of focal cerebral ischaemia.

A method for the volumetric assessment of early cerebral infarction, together with its statistical and biological validation, is described. In halothane anaesthetised rats the stem of the right middle cerebral artery was occluded and 3 hours later (with full monitoring of respiratory and cardiovascular status) the animals were killed by perfusion fixation. In normotensive normocapnic animals the volume of infarction was 52 +/- 4 mm3 in the cerebral cortex and 21 +/- 1 mm3 in the corpus striatum. The reproducibility of the volumetric assessment was found to be excellent (coefficient of correlation 0.995 on 18 replicate measurements). The minimum number of stereotactic levels which must be assessed to yield accurate volumetric measurements of infarction is 8. The method is sensitive at detecting alterations in the amount of infarction. For example, it can readily detect the increase in amount of structural damage in cerebral cortex following a transient episode of hypotension. This approach allows an objective assessment of drug therapy and management strategies in the treatment of cerebral infarction.     

局灶缺血诱导大鼠脑肝细胞生长因子表达的动态观察

目的:探讨肝细胞生长因子(HGF)在局灶脑缺血边缘区表达的动态变化及其意义。方法:采取线栓法建立局灶脑缺血模型,根据缺血时间分为1h、3h、12h、24h组,RT-PCR法检测HGF的表达及其动态变化。结果:缺血1h脑缺血边缘区即有HGF的表达,且随着缺血时间的延长其表达逐渐增强,24h尤为明显。结论:HGF在局灶脑缺血诱导的高表达可能在脑缺血损伤组织的保护及修复过程中起重要作用。     

亚低温治疗重症脑梗死的疗效观察

目的 :探讨亚低温技术治疗重症脑梗死的临床效果。方法 :30例住院的重症脑梗死患者 ,分为治疗和对照两组 ,在给予抗脑梗死治疗的同时 ,治疗组加用亚低温技术 ,治疗后 7天、2 1天分别进行神经功能缺损评分 ,和病死率等一起进行分析。结果 :治疗 7天与 2 1天后 ,治疗和对照两组的评分分别为 30 .4 5± 5 .86 ,34.1 4± 5 .37( P=0 .0 5 ) ;和 2 1 .92± 2 .4 6 ,2 6 .82± 1 .99( P<0 .0 5 ) ;病死率分别为 2 0 %和 2 6 % ( P>0 .0 5 )。结论 :亚低温技术可以明显改善重症脑梗死患者的神经功能和预后。     

Normobaric hyperoxia retards the evolution of ischemic brain tissue toward infarction in a rat model of transient focal cerebral ischemia

Objectives: Oxygen therapy has been long considered a logical therapy for ischemic stroke. Our previous studies showed that normobaric hyperoxia (normobaric hyperoxia (NBO), 95% O₂ with 5% CO₂) treatment during ischemia reduced ischemic neuronal death and cerebromicrovascular injury in animal stroke models. In this study, we studied the effects of NBO on the evolution of ischemic brain tissue to infarction in a rat model of transient focal cerebral ischemia.

Methods: Male Sprague-Dawley rats were given NBO (95% O₂) or normoxia (21% O₂) during 90-min filament occlusion of the middle cerebral artery (MCAO), followed by 3 or 22.5 h of reperfusion. 2,3,5-triphenyltetrazolium chloride (TTC) staining was used to evaluate the longitudinal evolution of tissue infarction.

Results： In normoxic rats, MCA-supplied cortical and striatal tissue was infarcted after 90-min MCAO with 22.5 h of reperfusion. NBO-treated rats showed a 61.4% reduction in infarct size and tissue infarction mainly occurred in the ischemic striatum. When infarction was assessed at an earlier time point, i.e. at 3 h of reperfusion, normoxic rats showed significantly smaller but mature infarction (no TTC staining, white color), with the infarction mainly occurring in the striatum. Unexpectedly, NBO-treated rats only showed immature lesion (partially stained by TTC, light white color) in the ischemic striatum, indicating that NBO treatment also retarded the process of neuronal death in the ischemic core. Of note, NBO-preserved striatal tissue underwent infarction after prolonged reperfusion.

Conclusions： Our results demonstrate that NBO treatment given during cerebral ischemia retards the evolution of ischemic brain tissue toward infarction and NBO-preserved cortical tissue survives better than NBO-preserved striatal tissue during the phase of reperfusion.     

亚低温对大鼠脑缺血再灌注后MMP-9表达和脑水肿的影响

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）表达和脑水肿的影响，探讨亚低温脑保护的可能机制。方法雄性SD大鼠72只，随机分为假手术组、亚低温组、常温组，线栓法制备大脑中动脉闭塞（MCAO）再灌注48h模型，缺血时间2h。采用比色法测定脑组织中伊文思蓝（EB）含量反映血脑屏障（BBB）通透性；干-湿重法检测脑组织含水量；免疫组化法检测MMP-9表达。结果与假手术组相比，缺血鼠缺血侧脑组织的EB含量及脑含水量明显增高，可见大量MMP-9免疫阳性细胞（P〈0．05）。亚低温组和常温组大鼠脑EB含量分别为（22．42±1．86）μg/g脑重和（38．67±2．94）μg／g脑重，脑含水量分别为80．07％±0．56％和82．49％±0．97％，皮质缺血区MMP-9阳性细胞IOD值分别为380．33±40．87和695．16±67．67，纹状体区MMP-9阳性细胞IOD值分别为294．19±33．47和451．87±5．77，差异均有显著性意义（P〈0．05）。亚低温明显减轻缺血脑组织病理学损伤。结论推测亚低温可通过抑制MMP-9表达，减轻BBB的破坏，继而减轻脑水肿，从而发挥确实的脑保护作用。     

大鼠局灶性脑缺血脑内 bFGF基因表达的实验研究

目的　了解局灶性脑缺血病灶周围组织碱性成纤维细胞生长因子 (bFGF)mRNA表达水平 ,探讨bFGF在脑缺血病理过程中的作用机制。方法　采用“线栓法”建立SD大鼠大脑中动脉局灶性脑缺血模型 ,运用NorthernBlot杂交技术并结合图像处理对杂交信号密度扫描以反映bFGFmRNA相对水平。结果　缺血 6小时病灶周期组织即出现bFGFmRNA表达增强 ,1天后达到高峰 (P <0 .0 1)。 1周左右降至对照组水平 ,2周后再次呈现表达增高趋势 ,第 3周时与对照组相比增加约 5 0 % (P <0 .0 5 )。结论　局灶性脑缺血可诱发脑内bFGF基因表达 ,它在保护病灶周围神经元、促进细胞增殖和组织修复中可能发挥着重要作用。     

Allopurinol inhibits uric acid accumulation in the rat brain following focal cerebral ischemia

Uric acid (UA) in the rat brain was measured by HPLC with an electrochemical detector following focal ischemia. At 24 h after the operation, the UA level in the ischemic center was 105.47 +/- 8.39 nmol/g tissue, whereas it was 8.36 +/- 1.86 in the sham-operated group. Allopurinol, xanthine oxidase inhibitor, almost completely inhibited this UA accumulation. These data demonstrate that the UA increase in the ischemic brain is due to the xanthine oxidase reaction.     

3-Aminobenzamide reduces brain infarction and neutrophil infiltration after transient focal cerebral ischemia in mice

Poly(ADP-ribose) polymerase (PARP) was shown to be detrimental in cerebral ischemia but the mechanisms whereby PARP is deleterious have yet to be determined. They may include a role in neutrophil infiltration known to aggravate ischemic damage. In this context, we investigated the effect of 3-aminobenzamide (3-AB), a PARP inhibitor, on brain damage and neutrophil infiltration after transient focal cerebral ischemia in mice. Ischemia was induced in male Swiss mice, anaesthetized with chloral hydrate (400 mg/kg, i.p.), by a 15-min-occlusion of the left middle cerebral artery using an intraluminal suture. Treatments with 3-AB were first administered intraperitoneally 15 min before reperfusion and endpoints measured at 24 h. Among the range of dosages studied (20-320 mg/kg), 40 mg/kg gave the maximal neuroprotection with a 30% decrease in the infarct volume and tended to improve the neurological score evaluated by a grip test. The same dosage was, however, devoid of effect when injection was delayed 2 or 6 h after reperfusion. Myeloperoxidase (MPO) activity used as an index of neutrophil infiltration showed that infiltration peaked 48 h after reperfusion in our model. At this time point, 3-AB (40 mg/kg given 15 min before reperfusion) markedly reduced the neutrophil infiltration, as evidenced by a 72%-decrease in MPO activity, and was still neuroprotective. Our results confirm that 3-AB reduces brain damage. Moreover, for the first time, a quantitative study shows that 3-AB decreases neutrophil infiltration elicited by cerebral ischemia.     

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。