Neuroprotection against permanent focal cerebral ischemia by ginkgolides A and B is associated with obstruction of the mitochondrial apoptotic pathway via inhibition of c‐Jun N‐terminal kinase in rats

We have previously reported that ginkgolides containing ginkgolides A and B (GKAB) reduce infarct size in a rat model of focal ischemia. c‐Jun N‐terminal kinase (JNK), also known as stress‐activated kinase (SAPK), is a critical stress‐responsive kinase activated by various brain insults. Previous studies have demonstrated a brief increase in p‐SAPK/JNK levels after focal ischemic brain injuries. In this study, we sought to investigate whether the neuroprotective effects of GKAB in rat models of permanent focal cerebral ischemia are associated with the JNK signaling pathway. Sprague‐Dawley rats were subjected to permanent middle cerebral artery occlusion by intraluminal suture blockade. GKAB was injected intravenously immediately after ischemia onset. Here we demonstrate in rats that GKAB reduces neuronal apoptosis and blocks the increase of p‐SAPK/JNK levels and nuclear translocation after cerebral ischemia in a dose‐dependent manner. Furthermore, we report that cerebral ischemia increases ischemia‐induced induction of reactive oxygen species, and this effect was blocked by GKAB. In addition, we show that BimL is induced and attenuated by GKAB. GKAB also repressed the ischemia‐induced increase in the expression of Bax and reversed the decline in expression of Bcl‐2. Likewise, there was a reduction in the release or activation of several mitochondrial proapoptotic molecules, including cytochrome c, caspases 3 and 9, and PARP. Taken together, our findings strongly suggest that GKAB‐mediated neuroprotective effects against focal ischemia act through the inhibition of p‐SAPK/JNK activation, in which the obstruction of the mitochondrial apoptotic pathway via the JNK signaling pathway is a key downstream mechanism of GKAB. © 2013 Wiley Periodicals, Inc.     

Differential neuroprotective effects of a minocycline-based drug cocktail in transient and permanent focal cerebral ischemia

Considering that several pathways leading to cell death are activated in cerebral ischemia, we tested in mouse models of transient and permanent ischemia a drug cocktail aiming at distinct pharmacological targets during the evolution of ischemic injury. It consists of minocycline--an antibiotic with anti-inflammatory properties, riluzole--a glutamate antagonist, and nimodipine--a blocker of voltage-gated calcium channels. Administered 2 h after transient or permanent MCAO, it significantly decreased the size of infarction, by approximately 65% after transient and approximately 35% after permanent ischemia and markedly improve clinical recovery of mice. In both experimental models a three-drug cocktail achieved significantly more efficient neuroprotection than any of the components tested alone. However, some interesting observation emerged from the single-drug studies. Treatment with minocycline alone was efficient in both experimental models while treatment with glutamate antagonist riluzole conferred neuroprotection only after transient MCAO. Immunohistochemical analysis following three-drug treatment revealed reduced microglia/macrophages and caspase-3 activation as well as preserved GFAP immunoreactivity following transient ischemia. No detectable differences in the levels of Mac-2, GFAP and caspase-3 immunoreactivities were observed 72 h after permanent MCAO. These marked differences in the brain tissue responses to ischemic injury and to treatments suggest that different pathological mechanisms may be operating in transient and permanent ischemia. However, the three-drug cocktail exerted significant neuroprotection in both experimental models thus demonstrating that simultaneous targeting of several pathophysiological pathways involved in the evolution of ischemic injury may represent a rational therapeutic strategy for stroke.     

血红素氧合酶-1及血红素氧合酶-2在大鼠局灶性脑缺血中的意义

目的　研究血红素氧合酶 1(HO 1)及血红素氧合酶 2 (HO 2 )在局灶性脑缺血中的作用。方法　采用大鼠大脑中动脉栓塞脑缺血模型 ,对 6 6只大鼠脑缺血后不同时间点进行HO 1、HO 2免疫组化染色及病理学研究 ,并用计算机图像分析技术计算两者表达水平。结果　栓塞后 30min大鼠皮质及海马即有HO 1阳性神经元及胶质细胞的表达 ,且随着时间推移HO 1的表达逐渐增强 ,到栓塞后 12h达峰值 (P <0 0 1) ,以后逐渐下降 ,栓塞后 1周仍有HO 1表达。HO 2在正常大鼠及梗死大鼠脑组织内均有表达。栓塞后不同时间段 ,HO 2阳性神经元的数量无明显变化 (P >0 0 5 ) ,但HO 2表达呈动态变化 ,2 4h时最高 (P <0 0 1) ,以后逐渐下降。结论　脑缺血时脑内HO 1、HO 2表达的不同变化 ,是脑组织对损伤恢复重要的机制之一。HO 1修复受损的神经元和胶质细胞 ,而HO 2在于维护正常细胞的稳定     

Acute administration of Ginkgo biloba extract (EGb 761) affords neuroprotection against permanent and transient focal cerebral ischemia in Sprague-Dawley rats

We examined the neuroprotective action of a standardized extract of Ginkgo biloba leaves (EGb 761) in permanent and transient middle cerebral artery (MCA) occlusion models in Sprague-Dawley rats. Forty-four animals were given either EGb 761 (50-200 mg/kg) or vehicle intraperitoneally, 1 hr before permanent MCA occlusion, to evaluate the dose-response effects. An additional 58 animals received EGb 761 (200 mg/kg) or vehicle, 0.5- 4 hr after permanent MCA occlusion, for establishing the therapeutic window. Delayed treatment was also employed in 110 animals treated with either EGb 761 (100-200 mg/kg) or vehicle at 2-3 hr following transient focal cerebral ischemia induced by MCA occlusion for 2 hr. Neurobehavioral scores were determined 22-24 hr after permanent MCA occlusion and either 3 or 7 days after transient MCA occlusion, and brain infarction volumes were measured upon sacrifice. Local cortical blood flow (LCBF) was serially measured in a subset of animals receiving EGb 761 (100-200 mg/kg) or vehicle, 0.5 hr and 2 hr after permanent and transient MCA occlusion, respectively. Relative to vehicle-treated controls, rats pretreated with EGb761 (100 and 200 mg/kg) had significantly reduced infarct volumes, by 36% and 49%, respectively, and improved sensory behavior (P < 0.05). Delayed treatment with EGb 761 also significantly reduced brain infarction, by 20-29% and 31%, when given up to 2 and 3 hr following transient and permanent MCA occlusion, respectively, whereas improved neurobehavioral scores were noted up to 2 hr after the onset of MCA occlusion (P < 0.05). LCBF was significantly improved in the ipsilateral cortex following the EGb 761 treatment, whereas a higher dose showed a more sustained effect. In conclusion, EGb 761 protected against transient and permanent focal cerebral ischemia and was effective after a prolonged reperfusion period even when therapy is delayed up to 2 hr. This neuroprotection may be at least partially attributed to the beneficial effects of selectively improved LCBF in the area at risk of infarction.     

L-NAME对大鼠局灶性脑缺血灌注损伤Fos蛋白表达的影响

目的观察一氧化氮含量的变化对缺血再灌注损伤后Fos蛋白表达的影响。方法采用线拴法制作大鼠局灶性脑缺血再灌注损伤模型,利用NADPH组化和Fos蛋白免疫组化双标技术研究NOS抑制剂L－NAME对大鼠局灶性脑缺血再灌注损伤脑皮层Fos蛋白表达的影响。结果缺血60min再灌注3h后损伤侧脑组织皮质一氧化氮合酶阳性神经元较正常增多并深染,Fos蛋白表达增加,L－NAME（3mg／kg）治疗组脑皮质神经元Fos蛋白的表达量较对照组减少,L－NAME（10mg/kg）治疗组脑皮质神经元Fos蛋白的表达量较对照组明显减少,同时也可见给予L－NAME后脑组织皮质内NOS阳性神经元无论在数量上还是在细胞着色、胞体突起均明显减少。结论c－fos基因表达也可能部分参与了NO的致神经细胞损伤过程。     

Hyperbilirubinemia protects against focal ischemia in rats

Heme oxygenase-1 (HO1) catalyzes oxidation of the heme molecule in concert with NADPH-cytochrome P450 reductase following the specific cleavage of heme into carbon monoxide, iron, and biliverdin, which is rapidly metabolized to bilirubin. HO1 is a stress-inducible protein that protects cells against oxidative injury, but its protective mechanism is not fully understood. The Eizai hyperbilirubinemic rat (EHBR), a mutant strain derived from the Sprague-Dawley rat (SDR), has a mutation in the gene for the canalicular multispecific organic anion transporter, which results in a phenotype of hyperbilirubinemia, and thus is a model of Dubin-Johnson syndrome in humans. In this study, we compared EHBR and SDR with regard to neuronal death induced by 2 hr of occlusion of the middle cerebral artery and reperfusion. In EHBR, the area that was immunoreactive for microtubule-associated protein-2 was significantly reduced, and the HO1-immunoreactive area was smaller than that in SDR. These results suggest that bilirubin has essentially a neuroprotective effect against focal ischemia and may participate in HO1-induced neuroprotection.     

Rho-kinase activation in endothelial cells contributes to expansion of infarction after focal cerebral ischemia

Microcirculatory disturbances contribute to the expansion of infarct lesions after focal cerebral ischemia. Recently, it was shown that Rho-kinase involves in endothelial dysfunction via down-regulation of endothelial nitric oxide synthase function in a rodent stroke model. However, it is not clear whether endothelial Rho-kinase is activated in vivo or Rho-kinase activation contributes to microcirculatory disturbances after cerebral ischemia. In this study, we assessed the temporal and spatial profiles of Rho-kianse activity and the effect of the Rho-kinase inhibitor fasudil on microcirculatory disturbances in the focal brain ischemia. Rho-kinase activation was evaluated by analyzing the phosphorylation of adducin, a substrate of Rho-kinase, by immunohistochemistry. Staining for p-adducin was found in endothelia in the ischemic area 6 hr after induction of ischemia. Microcirculatory disturbances and increased endothelial cell staining for von Willebrand factor (vWF) were observed in the same area. Postischemic treatment with fasudil suppressed endothelial Rho-kinase activation, preserved microcirculation, and inhibited endothelial cell vWF staining. These effects resulted in inhibition of infarct expansion and improvement of neurologic deficits. These findings indicate that Rho-kinase is activated in the endothelial cells and contributes to microcirculatory disturbances in cerebral ischemia. The vascular protective effect of Rho-kinase inhibitors may be useful in the treatment of the acute phase of ischemic stroke.     

c—fos反义寡聚核苷酸对脑缺血性损伤和电针抗损伤作用的影响

用c-fos反义寡聚核苷酸脑内微量注射、TTC染色、c-Fos免疫组织化学和电针等技术和方法,探讨即早反应基因c-fos在大鼠局灶性脑缺血（MCAO）模型的脑损伤中和电针抗局灶性脑缺血脑损伤中所起的作用。实验结果表明,局灶性脑缺血可引起c-fos在缺血侧皮质的大量表达,电针能部分抑制这种表达,使脑缺血梗死灶体积减小。在缺血中心区注射c-fos反义寡聚核苷酸后,脑内c-fos的表达基本上被完全阻断,导致脑梗死灶的体积明显增大,电针抗脑缺血脑损伤的作用也被取消,提示脑缺血后,脑内的c-fos适度表达可能对脑损伤有一定的保护作用。电针可能部分抑制了脑内c-fos表达,调整了缺血后的c-fos表达的程度,对脑缺血损伤起一定的保护作用。     

内源性一氧化碳对局灶性脑缺血大鼠神经功能、梗死灶体积、脑含水量的影响

目的研究内源性CO浓度变化对局灶性脑缺血大鼠神经功能及脑组织含水量、梗死灶体积的影响.方法将48只S.D.大鼠随机分为2组(n=24), 一组为梗死灶体积组, 一组为脑含水量组.每一组又分为3小组, 分别为HO诱导剂、 HO抑制剂、生理盐水组(n=8).使用HO诱导剂、 HO抑制剂腹腔注射, 等量生理盐水腹腔注射作为对照组, 1 h后制成MCAO模型.栓塞后24 h观察局灶性脑缺血大鼠神经功能改变, 同时检测CO浓度、梗死灶体积、脑含水量.结果与生理盐水组相比, HO诱导剂组CO浓度明显升高(P＜0.01), 大鼠神经功能明显改善, 梗死灶体积、脑含水量明显降低, 各为(P＜0.01、 P＜0.01、 P＜0.05), 而HO抑制剂组CO浓度明显降低(P＜0.01), 大鼠神经功能缺失加剧, 梗死灶体积、脑含水量明显升高, 各为(P＜0.01、 P＜0.05、 P＜0.05).结论内源性CO是一种信使分子, 浓度升高对局灶性缺血的脑组织具有保护作用.     

Mannitol infusion immediately after reperfusion suppresses the development of focal cortical infarction after temporary cerebral ischemia in gerbils

Previously we found that, after temporary cerebral ischemia, microvasculogenic secondary focal cerebral cortical ischemia occurred, caused by microvascular obstruction due to compression by swollen astrocytic end‐feet, resulting in focal infarction. Herein, we examined whether mannitol infusion immediately after restoration of blood flow could protect the cerebral cortex against the development of such an infarction. If so, the infusion of mannitol might improve the results of vascular reperfusion therapy. We selected stroke‐positive animals during the first 10 min after left carotid occlusion performed twice with a 5‐h interval, and allocated them into four groups: sham‐operated control, no‐treatment, mannitol‐infusion, and saline‐infusion groups. Light‐ and electron‐microscopic studies were performed on cerebral cortices of coronal sections prepared at the chiasmatic level, where the focal infarction develops abruptly in the area where disseminated selective neuronal necrosis is maturing. Measurements were performed to determine the following: (A) infarct size in HE‐stained specimens from all groups at 72 and 120 h after return of blood flow; (B) number of carbon‐black‐suspension‐perfused microvessels in the control and at 0.5, 3, 5, 8, 12 and 24 h in the no‐treatment and mannitol‐infusion groups; (C) area of astrocytic end‐feet; and (D) number of mitochondria in the astrocytic end‐feet in electron microscopic pictures taken at 5 h. The average decimal fraction area ratio of infarct size in the mannitol group was significantly reduced at 72 and 120 h, associated with an increased decimal fraction number ratio of carbon‐black‐suspension‐perfused microvessels at 3, 5 and 8 h, and a marked reduction in the size of the end‐feet at 5 h. Mannitol infusion performed immediately after restitution of blood flow following temporary cerebral ischemia remarkably reduced the size of the cerebral cortical focal infarction by decreasing the swelling of the end‐feet, thus preventing the microvascular compression and stasis and thereby microvasculogenic secondary focal cerebral ischemia.     

TET3 regulates DNA hydroxymethylation of neuroprotective genes following focal ischemia

The 5-hydroxymethylcytosine (5hmC) epigenetic modification is highly enriched in the CNS and a critical modulator of neuronal function and development. We found that cortical 5hmC was enhanced from 5 min to three days of reperfusion following focal ischemia in adult mice. Blockade of the 5hmC-producing enzyme ten-eleven translocase 3 (TET3) increased edema, infarct volume, and motor function impairments. To determine the mechanism by which TET3 provides ischemic neuroprotection, we assessed the genomic regions where TET3 modulates 5hmC. Genome-wide sequencing analysis of differentially hydroxymethylated regions (DhMRs) revealed that focal ischemia robustly increased 5hmC at the promoters of thousands of genes in a TET3-dependent manner. TET3 inhibition reduced 5hmC at the promoters of neuroprotective genes involved in cell survival, angiogenesis, neurogenesis, antioxidant defense, DNA repair, and metabolism demonstrating a role for TET3 in endogenous protection against stroke. The mRNA expression of several genes with known involvement in ischemic neuroprotection were also reduced with TET3 knockdown in both male and female mice, establishing a correlation between decreased promoter 5hmC levels and decreased gene expression. Collectively, our results indicate that TET3 globally increases 5hmC at regulatory regions and overwhelmingly modulates 5hmC in several neuroprotective pathways that may improve outcome after ischemic injury.     

Intraluminal increase of superoxide anion following transient focal cerebral ischemia in rats

Using a modification of Karnovsky's Mn²⁺/diaminobenzizine (DAB) technique, we examined the production of superoxide anion (O⁻₂) in the vascular lumen following transient occlusion and reperfusion of the left middle cerebral artery (MCA) in Sprague–Dawley rats. The MCA was occluded for 2 h using an intraluminal suture method. Zero, 15, 30, and 60 min after reperfusion, animals were perfused transcardially with buffer containing Mn²⁺ and DAB, and brain samples were prepared for light and electron microscopic examination. The amber reaction deposits of O⁻₂ were observable to the naked eye along the major cerebral vessels of the ischemic hemisphere after each reperfusion period. Upon microscopic examination the deposits were revealed to be within arterial, capillary, and venular lumen. The amount of reaction deposits in the ischemic hemisphere corresponded to the duration of reperfusion. The formation of O⁻₂ was suppressed when the perfusate contained superoxide dismutase and when either Mn²⁺ or DAB was omitted, confirming that the reaction products produced are due to the enhanced production of O⁻₂. These results show that there is a progressive increase in intraluminal O⁻₂ during reperfusion following an ischemic insult which may participate in the aggravation of cerebral damage.     

黄芩苷、栀子苷及其配伍治疗局灶性脑缺血的海马基因表达：一项基因网络功能分析

The compound traditional Chinese medicine Qingkailing,which is an ingredient used to treat cerebral ischemia,has been limited to studies concerning single genes or single pathways.Interactions and pharmacological mechanisms of the compound ingredients(baicalin and jasminoidin) remain poorly understood.In the present study,baicalin and jasminoidin,as well as the combination,were used to separately treat mouse models of cerebral ischemia.cDNA microarray analyses of 374 cerebral ischemia-related genes were utilized to determine changes in gene-expression profiles.Arraytrack 3.40 and Ingenuity Pathway Analysis(IPA) databases were utilized to analyze changes in gene molecular functions and network path functions.Baicalin or jasminoidin alone effectively reduced infarct area,and the combination resulted in significantly better outcomes.IPA showed inhibited cell apoptosis in the baicalin group and Ca2+ channel regulation in the jasminoidin group.The combination of baicalin and jasminoidin activated HTR3A and F5 expression,regulated Ca2+ channels,activated kappa light polypeptide gene enhancer inhibitor IKBKG in B cells to control IkB kinase/nuclear factor-kB cascade,suppressed activation of inflammatory cytokine interleukin-6 receptors and activated transduction of guanine-nucleotide-binding protein(G protein) signal.Results suggested that the combination of baicalin and jasminoidin resulted in similar molecular mechanisms to baicalin and jasminoidin alone.However,novel pharmacological actions of compatibility were detected,demonstrating significant protection against cerebral ischemia.     

Therapeutic window of Qingkailing injection for focal cerebral ischemia/reperfusion injury

The time window in which a drug is effective varies between drugs. The present study investigated the therapeutic window of Qingkailing injection for focal cerebral ischemia/reperfusion in mice. Animals underwent middle cerebral artery occlusion and were injected with Qingkailing (1.5, 3, 6 mL/kg). Infarct volume and neurological function were assessed after 24 hours of ischemia. In addition, to establish the therapeutic time window, mice were injected with 3 mL/kg Qingkailing at 0, 1, 3, 4, 6, 9 and 12 hours after occlusion. Results revealed that Qingkailing injection significantly reduced infarct volume and improved neurological function in model mice after cerebral infarction for up to 9 hours, demonstrating that the therapeutic window of Qingkailing injection can extend to 9 hours for cerebral ischemia/reperfusion in mice.     

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。     

Hyperlipidemia affects neuronal nitric oxide synthase expression in brains of focal cerebral ischemia rat model★

BACKGROUND: Hyperlipidemia, a risk factor for ischemic cerebrovascular disease, may mediate production of neuronal nitric oxide synthase (nNOS) to induce increased nitric oxide levels, resulting in brain neuronal injury. OBJECTIVE: To investigate effects of hyperlipidemia on brain nNOS expression, and to verify changes in infarct volume and pathology during reperfusion, as well as neuronal injury following ischemia/reperfusion in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING: Complete, randomized grouping experiment was performed at the Laboratory of Physiology, Shanxi Medical University from March 2005 to March 2006. MATERIALS: A total of 144 eight-week-old, male, Wistar rats, weighing 160-180 g, were selected. A rat model of middle cerebral artery occlusion was established by suture method after 4 weeks of formulated diet. Nitric oxide kit and rabbit anti-rat nNOS kit were respectively purchased from Nanjing Jiancheng Bioengineering Institute, China and Wuhan Boster Biological Technology, Ltd., China. METHODS: The rats were equally and randomly divided into high-fat diet and a normal diet groups. Rats in the high-fat diet group were fed a high-fat diet, consisting of 10% egg yolk powder, 5% pork fat, and 0.5% pig bile salt combined with standard chow to create hyperlipidemia. Rats in the normal diet group were fed a standard rat chow. A total of 72 rats in both groups were randomly divided into 6 subgroups: sham-operated, 4-hour ischemia, 4-hour ischemia/2-hour reperfusion, 4-hour ischemia/4-hour reperfusion, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion, with 12 rats in each subgroup. MAIN OUTCOME MEASURES: nNOS expression was measured by immunohistochemistry, and pathomorphology changes were detected by hematoxylin-eosin staining. Infarct volume and nitric oxide levels were respectively measured using 2, 3, 5-triphenyltetrazolium chloride (TTC) and immunohistochemistry. RESULTS: In the ischemic region, pathology changes were significant in the 4-hour ischemia/4-hour, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion subgroups fed on a high-fat diet compared to the same groups fed on a normal diet. In each ischemia subgroup, nNOS expression in brain tissues was higher than in the sham-operated subgroups fed on either the high-fat diet or normal diet (P< 0.01). At each ischemia/reperfusion time point, rats fed on a high-fat diet expressed higher levels of nNOS compared to rats fed on the normal diet (P<0.05). When tissue was stained with TTC, a white infarction area was detected in the ischemic hemisphere, demonstrating that the infarct volume gradually increased with prolonged reperfusion time in each ischemia subgroup. At each ischemia/reperfusion time point, the infarct volume was larger in rats fed on a high-fat diet compared to those fed on a normal diet. CONCLUSION: nNOS expression was greater in hyperlipidemia rats following ischemia/reperfusion. Cerebral ischemia/reperfusion injury is aggravated with prolonged reperfusion time.     

Spatiotemporal expression of Nogo-66 receptor after focal cerebral ischemia

NgR, the receptor for the neurite outgrowth inhibitor Nogo-66, plays a critical role in the plasticity and regeneration of the nervous system after injury such as ischemic stroke. In the present study, we used immunohistochemistry to investigate the regional expression of NgR in rat brain following middle cerebral artery occlusion (MCAO). NgR protein expression was not observed in the center of the lesion, but was elevated in the marginal zone compared with control and sham-operated rats. The cerebral cortex and hippocampus (CA1, CA2, and CA3) showed the greatest expression of NgR. Furthermore, NgR expression was higher in the ipsilesional hemisphere than on the control side in the same coronal section. Although time-dependent changes in NgR expression across brain regions had their own characteristics, the overall trend complied with the following rules: NgR expression changes with time showed two peaks and one trough; the ifrst peak in expression appeared between 1 and 3 days after MCAO; expression declined at 5 days; and the second peak occurred at 28 days.     

Comparison of 12, 24 and 48 h of systemic hypothermia on outcome after permanent focal ischemia in rat

Mild hypothermia reduces injury in models of global and focal cerebral ischemia even when initiated after the insult. Neuroprotection depends critically upon the duration of hypothermia with longer treatments often being more efficacious. However, the ideal treatment duration is not known for most insults and this knowledge would facilitate clinical studies. Thus, we compared 12, 24 and 48 h of systemic hypothermia (33 °C vs. normothermia) initiated 1 h after permanent middle cerebral artery occlusion (pMCAO), which was produced by permanent occlusion of the carotid arteries and cauterization of the distal MCA in rat. Behavioral recovery and lesion volume were determined 7 days after pMCAO. All three treatments significantly and equally attenuated neurological deficits (e.g., forelimb placing response). Conversely, stepping error rate in the horizontal ladder test was significantly reduced only by the 24-h (18.7%) and 48-h treatments (11.7%) compared to normothermic rats (34.4%), and the 48-h treatment was significantly better than the 12-h treatment (28.8%). Similarly, brain injury was significantly reduced by 24-h (78.8 mm³ lesion volume) and 48-h (66.8 mm³) treatments compared to normothermia (142.6 mm³), and the 48-h treatment was significantly better than the 12-h duration (114.6 mm³). In separate experiments cerebral edema was measured via wet–dry weight measurements and significantly reduced by hypothermia (e.g., from 83.7% water in the injured cortex of normothermic rats to 81.4% in rats cooled for one day), but for this there were no significant duration effects. In summary, prolonged hypothermia treatment provides superior protection overall, but this is not explained by reductions in edema.     

The neuroprotective effect of glial cell line-derived neurotrophic factor in fibrin glue against chronic focal cerebral ischemia in conscious rats

Glial cell line-derived neurotrophic factor (GDNF) is a transforming growth factor-beta which has shown beneficial effects in rats after acute focal cerebral ischemia (FCI). To study the effects of GDNF on chronic FCI injury in conscious rats, we used fibrin glue (GDNF-fibrin glue) and fibrin glue free (GDNF-only)-GDNF topically applied to the ischemic brain after right middle cerebral artery (MCA) ligation. Infarct brain volume and functional motor deficits were measured before and after FCI injury. After FCI injury induced by right MCA ligation, rats were randomly assigned to one of four treatment groups: (a) sham, (b) control, (c) topically applied GDNF (1 mug)-only, and (d) topically applied GDNF (1 mug)-fibrin glue. The degree of ischemic brain injury was estimated by infarct volume of right MCA territory at 4 weeks after occlusion. The functional motor deficits were quantified with rotarod test and grasping power test once a week. Topically applied GDNF-fibrin glue at infarct brain tissue after 4 weeks FCI injury significantly reduced the total infarct volume by 44.3% and 36%, respectively, compared to that of control group and GDNF-only group. The mean latencies for rats to stay on the rotarod were 55.0%, 50.3%, and 92.2% (P < 0.05 vs. control group and GDNF-only group) of baseline, respectively, in the control, GDNF-only, and GDNF-fibrin glue groups at the end of the 1st week after FCI injury but 75.3%, 67.3%, and 106.6% (P < 0.05 vs. control group and GDNF-only group) of baseline at the end of the 4th week after FCI injury. The mean values of grasping power were 78.7%, 71.7%, and 101.2% (P < 0.05 vs. control group and GDNF-only group) of baseline, respectively, in the control, GDNF-only, and GDNF-fibrin glue groups at the end of 1st week after FCI injury but 89.6%, 97.6%, and 120.7% (P < 0.05 vs. control group) of baseline at the end of 4th week after FCI injury. These results indicate that GDNF-fibrin glue not only reduced the total infarct volume after FCI injury but can also improve motor deficits after FCI injury. We concluded GDNF-fibrin glue could facilitate delivery of GDNF to the damaged brain tissue with subsequent reduction of ischemic brain injury accompanied by enhancing functional recovery in rats with chronic FCI injury.