Reduction of infarct volume by magnesium after middle cerebral artery occlusion in rats

The effects of magnesium, an endogenous inhibitor of calcium entry into neurons, upon ischemic brain damage were investigated using a well-characterized model of focal cerebral ischemia in rats. Infarct volumes were determined by 2,3,5-triphenyltetrazolium chloride transcardiac perfusion 48 h after middle cerebral artery (MCA) occlusion. The area of ischemic damage was quantified by image analysis in coronal sections taken every 0.5 mm. MgCl2 (1 mmol/kg) was injected intraperitoneally just after MCA occlusion and again 1 h later. Posttreatment with MgCl2 (16 control and 16 treated rats) significantly reduced the cortical infarct volume. Compensation for the hyperglycemic effect of MgCl2 with insulin (17 rats) further reduced the infarct volume in the neocortex. No systemic effects of either treatment could account for the observed neuroprotection.     

Lack of effect of PN 200-110 on neuronal injury and neurological outcome in middle cerebral artery-occluded rats.

We evaluated the influence of a continuous intravenous infusion of 0.24 mg/kg PN 200-110 started 20 minutes before the induction of ischemia and continued for 2 hours on infarct size, histopathology, and neurological outcome in middle cerebral artery-occluded rats treated with PN 200-110 (n = 8), placebo (n = 7), or saline (n = 8). Neurological examination was performed 24 hours after occlusion. We quantified infarct size by 2,3,5-triphenyl-tetrazolium chloride, hematoxylin and eosin, and Nissl staining and by computerized analysis of tracings of the infarcted areas and evaluated neuronal injury at the infarct periphery. The different types of ischemic cell damage were quantified by direct visual counting. We found no differences among saline-, placebo-, and PN 200-110-treated rats regarding infarct size, amount of neuronal alteration, and neurological outcome. Our results indicate the lack of a significant protective effect of this drug in experimental focal ischemia.     

Liposome-entrapped superoxide dismutase reduces cerebral infarction in cerebral ischemia in rats

We studied the role of superoxide radicals in the pathogenesis of ischemic brain injury using a model of focal cerebral ischemia in 102 rats and liposome-entrapped CuZn-superoxide dismutase, which can penetrate the blood-brain barrier and cell membranes efficiently. The bolus intravenous administration of 25,000 units of liposome-entrapped CuZn-superoxide dismutase elevated superoxide dismutase activities in the blood and brain 1, 2, 8, and 24 hours later as well as in the ischemic hemisphere and contralateral cortex. Determined 24 hours after right middle cerebral and bilateral common carotid artery occlusion by the lack of staining for mitochondrial dehydrogenase activity with 2,3,5-triphenyltetrazolium chloride, infarct sizes were reduced by 33%, 25%, and 18% in the anterior, middle, and posterior brain slices, respectively, by treatment with liposome-entrapped CuZn-superoxide dismutase. Our data demonstrate that superoxide radicals are important determinants of infarct size following focal cerebral ischemia and that liposome-entrapped CuZn-superoxide dismutase may have pharmacologic value for the treatment of focal cerebral ischemic injury.     

大鼠大脑中动脉缺血/再灌注模型中Caspase-3的表达

目的 研究Caspase-3在缺血性脑损伤中的作用，进一步探讨缺血性脑血管病的分子机制。方法 用Belayev改良的Longa线栓法制备大鼠局灶性大脑中动脉(MCA)缺血／再灌注模型，TTC染色观察梗死灶的形成，分别用原位杂交及免疫组化技术检测鼠脑中Caspase-3 mRNA与活性蛋白的表达。结果 缺血2小时再灌注24小时，TTC染色见明显的梗死灶形成，正常脑组织、假手术组及MCAO缺血对侧脑中有少量的Caspase-3 mRNA表达，但活性蛋白几无表达；再灌注24小时后，缺血侧脑中Caspase-3 mRNA表达明显增强，蛋白质活化增多，再灌注48小时进一步增加。结论 细胞凋亡机制参与了缺血后迟发性神经元死亡，Caspase-3在其中起重要作用。     

Diffusion-weighted magnetic resonance imaging: rapid and quantitative detection of focal brain ischemia.

We examined serial changes of diffusion- (DWI) and T2-weighted (T2WI) magnetic resonance images 30 minutes to 3 hours after intraluminal suture occlusion of the middle cerebral artery (MCA) in eight rats and after sham occlusion in four. We correlated the abnormal areas on DWI and T2WI with postmortem areas of infarction determined by 2,3,5-triphenyltetrazolium chloride (TTC), 24 hours after the operation. The 30-minute DWI in each MCA-occluded rat demonstrated increased signal intensity in the ipsilateral MCA territory, while T2WI showed no changes. At 3 hours, the ipsilateral DWI signal intensity increased further and the area of abnormality slightly increased. In some animals, the 3-hour T2WI disclosed an area of hyperintensity significantly smaller than that seen on the 30-minute DWI. TTC staining demonstrated an extensive MCA infarction in all rats with permanent MCA occlusion, confirmed by hematoxylin and eosin staining. The percent infarcted area of coronal brain sections, as determined by TTC staining, correlated significantly with areas on similar DWI sections at both 30 minutes and 3 hours. Sham-occluded control animals did not display any changes on DWI, T2WI, or TTC staining. The present study suggests that DWI is a very sensitive modality for detecting early ischemic brain injury, being highly correlated with post-mortem area of infarction, and may be useful to assess pharmacologic intervention.     

Postischemic intraventricular administration of FGF-2 expressing adenoviral vectors improves neurologic outcome and reduces infarct volume after transient focal cerebral ischemia in rats.

Fibroblast growth factor (FGF)-2 is a potent neurotrophic and angiogenic peptide. To examine possible protective effects of FGF-2 gene expression against transient focal cerebral ischemia in rats, a replication defective, recombinant adenovirus vector expressing FGF-2, was injected intraventricularly 2 hours after middle cerebral artery occlusion (MCAO). The treatment group showed significant recovery compared with the vehicle-treated groups in terms of serial neurologic severity scores over the 35 days after MCAO. Further, 2,3,5-triphenyltetrazolium chloride staining showed that FGF-2 gene transfer decreased infarct volume by 44% as compared with that in the vehicle-treated groups at 2 days after MCAO. The same tendency of gene transfer effects on infarct volume was confirmed at 35 days after MCAO with hematoxylin/eosin staining. Enzyme-linked immunosorbent assay revealed that FGF-2 concentration was increased significantly at 2 days after MCAO, not only in cerebrospinal fluid but also in cerebral substance in the lesioned and treated animals. These results suggested that FGF-2 gene transfer using these adenoviral vectors might be a useful modality for the treatment of occlusive cerebrovascular disease even after the onset of stroke.     

莱菔硫烷对大鼠局灶性脑缺血再灌注损伤的保护作用及机制研究

目的 探讨莱菔硫烷对大鼠局灶性脑缺血再灌注损伤的保护作用及机制.方法 采用线栓法制备大鼠大脑中动脉阻断局灶性脑缺血模型,分别于MCAO后1h腹腔注射莱菔硫烷2.5mg/kg、5mg/kg、10mg/kg.于缺血2h再灌注24h时进行神经行为缺损评分,TTC染色评价脑梗死体积,测定脑组织中超氧化物歧化酶(SOD)活力和丙二醛(MDA)含量.免疫荧光组织化学染色法检测黄核蛋白NQ01和脂质过氧化酶Prx6的表达.结果 莱菔硫烷给药组与对照组相比均能改善大鼠脑缺血再灌注后神经行为缺损评分,减少脑梗死体积.其中5mg/kg组能显著改善大鼠脑缺血再灌注后神经行为缺损评分,减少脑梗死体积,增强SOD活性,降低MDA含量.免疫荧光组织化学染色法提示NQ01和Prx6的表达明显增强.结论 莱菔硫烷对大鼠局灶性脑缺血再灌注损伤有神经保护作用,其机制可能与上调内源性抗氧化蛋白NQ01和Prx6的表达有关.     

Cerebroprotective effect of a non-N-methyl-D-aspartate antagonist, GYKI 52466, after focal ischemia in the rat.

BACKGROUND AND PURPOSE: Cerebroprotection after the administration of N-methyl-D-aspartate antagonists has been well documented. The present study sought to determine whether a cerebroprotective effect could be achieved with the administration of a non-N-methyl-D-aspartate antagonist, GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylene-dioxy-5H-2,3-benzodiazep ine hydrochloride; molecular weight, 330) after middle cerebral artery occlusion in the rat. METHODS: Neurological deficit and infarct volume 24 hours after permanent left middle cerebral artery occlusion in Fischer rats (n = 7-13 per group per dose) were studied. Cerebral infarcts was visualized by the lack of reduction of 2,3,5-triphenyltetrazolium chloride. RESULTS: GYKI 52466 (10 mg.kg-1 i.p. at 0, 2, 4 hours) after middle cerebral artery occlusion had no effect on infarct volume. GYKI 52466 (10 mg.kg-1 i.v. for 5 minutes followed by 15 mg.kg-1.hr-1 i.v. for 2 hours immediately after middle cerebral artery occlusion reduced cortical infarct volume by 68% (from 69 mm3 in vehicle-treated to 22 mm3 in GYKI 52466-treated animals; p less than 0.05). A 1-hour delay before initiation of drug infusion resulted in a 48% reduction in cortical infarct volume (from 60 mm3 vehicle-treated rats to 31 mm3 in GYKI 52466-treated rats; p less than 0.05). A 2-hour delay before initiation of drug infusion had no effect on cortical infarct volume. Neurological deficits (with blinded assessment after 24 hours) were improved after immediate treatment and after delayed treatment (1 or 2 hours). CONCLUSIONS: The cerebroprotective effect of GYKI 52466 in the rat suggests a possible therapeutic role for non-N-methyl-D-aspartate antagonists given shortly after the onset of stroke.     

Effect of sevoflurane preconditioning on astrocytic dynamics and neural network formation after cerebral ischemia and reperfusion in rats

Astrocytes, the major component of blood-brain barriers, have presented paradoxical profiles after cerebral ischemia and reperfusion in vivo and in vitro. Our previous study showed that sevoflurane preconditioning improved the integrity of blood-brain barriers after ischemia and reperfusion injury in rats. This led us to investigate the effects of sevoflurane preconditioning on the astrocytic dynamics in ischemia and reperfusion rats, in order to explore astrocytic cell-based mechanisms of sevoflurane preconditioning. In the present study, 2,3,5-triphenyltetrazolium chloride staining and Garcia behavioral scores were utilized to evaluate cerebral infarction and neurological outcome from day 1 to day 3 after transient middle cerebral artery occlusion surgery. Using immunofluorescent staining, we found that sevoflurane preconditioning substantially promoted the astrocytic activation and migration from the penumbra to the infarct with microglial activation from day 3 after middle cerebral artery occlusion. The formation of astrocytic scaffolds facilitated neuroblasts migrating from the subventricular zone to the lesion sites on day 14 after injury. Neural networks increased in the infarct of sevoflurane preconditioned rats, consistent with decreased infarct volume and improved neurological scores after ischemia and reperfusion injury. These findings demonstrate that sevoflurane preconditioning confers neuroprotection, not only by accelerating astrocytic spatial and temporal dynamics, but also providing astrocytic scaffolds for neuroblasts migration to ischemic regions, which facilitates neural reconstruction after brain ischemia.     

Broad-spectrum cation channel inhibition by LOE 908 MS reduces infarct volume in vivo and postmortem in focal cerebral ischemia in the rat

Cation channels conduct calcium, sodium and potassium, cations that are likely deleterious in the evolution of focal ischemic injury. We studied the effects of a novel, broad-spectrum inhibitor of several cation channels, LOE 908 MS, on acute ischemic lesion development with diffusion-weighted magnetic resonance imaging (DWI) and on cerebral perfusion with perfusion imaging (PI) in vivo and on cerebral infarct size using 2,3,5-triphenyltetrazolium chloride (TTC) staining postmortem. A total of 18 male Sprague-Dawley rats underwent 90 min of middle cerebral artery occlusion (MCAO) and were randomly and blindly assigned to either LOE 908 MS or vehicle starting 30 min after inducing focal ischemia and continuing for 4 h. Whole-brain DWI and multislice PI were done before initiation of treatment and repeated frequently for the next 3.5 h. DWI-derived lesion volume at 4 h showed a significant difference in favor of the drug treated group (P=0.03), whereas PI-derived perfusion deficit volumes did not significantly differ between the groups. The postmortem infarct volume at 24 h was significantly attenuated in the treated group in comparison to controls (P=0.0001) and neurological score was significantly better in the treated group (P<0.02). Blocking several distinct cation channels with LOE 908 MS significantly reduced infarct size and improved neurological outcome without observable adverse effects in this focal ischemia model.     

Neuroprotective effect of baicalin on focal cerebral ischemia in rats

Baicalin, a flavonoid compound from the root of the herb Scutellaria baicalensis Georgi, has been widely used to treat patients with inflammatory disease. The aim of this study was to assess the efficacy of baicalin in a rat model of focal cerebral ischemia. Adult male Sprague-Dawley rat models of cerebral artery occlusion were established and then randomly and equally divided into three groups: ischemia(cerebral ischemia and reperfusion), valproic acid(cerebral ischemia and reperfusion + three intraperitoneal injections of valproic acid; positive control), and baicalin(cerebral ischemia and reperfusion + intraperitoneal injection of baicalin for 21 days). Neurological deficits were assessed using the postural reflex test and forelimb placing test at 3, 7, 14, and 21 days after ischemia. Rat cerebral infarct volume was measured using 2,3,5-triphenyltetrazolium chloride(TTC) staining method. Pathological change of ischemic brain tissue was assessed using hematoxylin-eosin staining. In the baicalin group, rat neurological function was obviously improved, cerebral infarct volume was obviously reduced, and the pathological impairment of ischemic brain tissue was obviously alleviated compared to the ischemia group. Cerebral infarct volume was similar in the valproic acid and baicalin groups. These findings suggest that baicalin has a neuroprotective effect on cerebral ischemia.     

Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats

We have evaluated the use of 2,3,5-triphenyltetrazolium chloride (TTC) as an histopathologic stain for identification of infarcted rat brain tissue. The middle cerebral artery (MCA) of 35 normal adult rats was occluded surgically. At various times after surgical occlusion, rats were sacrificed and brain slices were obtained and stained with TTC or hematoxolin and eosin (H & E); the size of the area of infarcted tissue stained by each method was quantified. In rats sacrificed 24 hr after occlusion of the MCA, the size of the area of infarction was 21 +/- 2% of the coronal section for TTC, and 21 +/- 2% for H & E (mean +/- S.D., N = 13). The size of areas of infarction determined by either staining method was not significantly different in area by the paired test, and a significant correlation between sizes determined by each method was found by linear regression analysis (r = 0.91, slope = 0.89, and the y intercept = 4.4%). Staining with TTC is a rapid, convenient, inexpensive, and reliable method for the detection and quantification of cerebral infarction in rats 24 hr after the onset of ischemia.     

大鼠短暂局灶性大脑中动脉缺血后calpain的表达

目的：研究calpain在缺血性脑损伤中的作用，进一步探讨缺血性脑血管病的分子机制，为治疗研发提供理论依据。方法：用Belayev改良的Langa线栓法制备大鼠局灶性大脑中动脉(MCA)缺血／再灌注模型，TTC染色观察梗死灶的形成，分别用原位杂交及免疫组化技术检测鼠脑中calpain mRNA与活性蛋白的表达。结果：缺血2h再灌注24h,TTC染色见明显的梗死灶形成，正常脑组织、假手术组及：MCAO缺血对侧脑中有少量的calpain mRNA表达，但活性蛋白几无表达；缺血脑组织calpain mRNA表达及蛋白质活化均显著增加，呈双峰式，MCA缺血2h增加，再灌注4h减少，至24h更明显增高，而48h又有所下降。结论：Calpain参与了缺血性脑损伤过程，尤其在迟发性神经元死亡中起重要作用。     

电刺激对大鼠缺血性脑卒中的保护作用

目的探讨皮层电刺激对大鼠永久性缺血性脑卒中的保护作用。方法雄性sD大鼠随机分成对照组和实验组。实验组根据刺激频率不同分成2Hz组,20Hz组,100Hz组,100／2Hz组。建立永久性梗塞模型。梗塞3h后,电极植入右侧大脑缺血区皮层进行刺激。分别在梗塞5h和24h进行行为学评分。在梗塞24h行2％氯化三苯基四氮唑溶液染色测量脑梗死比。于刺激结束1h后取脑组织Westernblot分析脑源性神经营养因子（BDNF）和血管内皮生长因子（VEGF）表达量的变化。结果2Hz电刺激组在梗死比的减少及行为学评分的改善方面最显著（均P〈0．05）。VEGF的表达量下降,而BDNF表达水平较其他各组无显著性差异。结论对于脑缺血溶栓未通患者,皮层电刺激对脑缺血可能有保护作用。     

Effects of transection of cervical sympathetic trunk on cerebral infarct volume and oxygen free radical levels in rats with focal cerebral ischemia/reperfusion injury

BACKGROUND: Stellate ganglion block (SGB) plays a protective role on the brain, but the precise mechanism of action is not clear.OBJECTIVE: To simulate SGB by transection of the cervical sympathetic trunk (TCST) and to investigate the TCST effects on changes in cerebral infarct volume and oxygen free radical levels in rats with focal cerebral ischemia/reperfusion injury.DESIGN, TIME AND SETTING: A complete randomized control animal experiment was performed at the Institute of Neurological Diseases of Taihe Hospital, Yunyang Medical College from February to December 2005.MATERIALS: A total of 101 healthy Wistar rats, weighing 280-320g, of both genders, aged 17-18 weeks, were used in this study. 2,3,5-triphenyltetrazolium chloride (TTC) was purchased from Changsha Hongyuan Biological Company. Superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide (NO) assay kits were provided by Nanjing Jiancheng Bioengineering Institute.METHODS: Rats were randomly divided into a TCST group, a model group and a sham operation group. Successful models were included in the final analysis, with at least 20 rats in each group. After TCST, rat models of focal cerebral ischemia/reperfusion injury were established in the TCST group by receiving middle cerebral artery occlusion (MCAO) by the intraluminal suture method for 2 hours, followed by 24 hours of reperfusion. Rat models of focal cerebral ischemia/reperfusion injury were made in the model group. Rats in the sham operation group underwent experimental procedures as for the model group, threading depth of 10mm, and middle cerebral artery was not ligated.MAIN OUTCOME MEASURES: Brain tissue sections of ten rats from each group were used to measure cerebral infarct volume by TTC staining. Brain tissue homogenate of another ten rats from each group was used to detect SOD activities, MDA contents and NO levels. Rat neurological function was assessed by neurobehavioral measures.RESULTS: Cerebral infarct volume was bigger in the model group than in the TCST group (P<0.05). Twenty four hours after cerebral ischemia/reperfusion, SOD activities were lower, whereas MDA contents and NO levels were higher in the TCST and model groups, compared with the sham operation group (P<0.05 or P<0.01). Compared with the model group, SOD activities were higher, whereas MDA contents and NO levels were lower in the TCST group (P<0.05).CONCLUSION: After TCST, cerebral infarct volume is reduced, SOD activities are increased, and MDA contents and NO levels are decreased compared to the model group in rats with focal cerebral ischemia/reperfusion injury. These changes may be associated with TCST.     

Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries

This article describes a 3-year experience with focal neocortical ischemia in three rat strains. Multiple groups of adult Wistar (n = 50), Fisher 344 (n = 31), and spontaneously hypertensive (n = 72) rats were subjected to permanent occlusion of the distal middle cerebral (MCA) and ipsilateral common carotid arteries (CCA). Twenty-four hours later the animals were killed, and frozen brain sections were stained with hematoxylin and eosin to demarcate infarcted tissue. The infarct volume for each section was quantified with an image analyzer, and the total infarct volume was calculated with an iterative program that summed all interval volumes. Neocortical infarct volume was the largest and most reproducible in the spontaneously hypertensive rats (SHR). Statistical power analysis to project the numbers of animals necessary to detect a 25 or 50% change in infarct volume with alpha = 0.05 and beta = 0.2 revealed that only the SHR model was practical in terms of requisite animals: i.e., less than 10 animals per group. Tandem occlusion of the distal MCA and ipsilateral CCA in the SHR strain provides a surgically simple method for causing large neocortical infarcts with reproducible topography and volume. The interanimal variability in infarct volume that occurs even in the SHR strain dictates that randomized, concomitant controls are necessary in each study to ensure the accurate assessment of experimental manipulations or pharmacologic therapies.     

Differential recovery of multimodal MRI and behavior after transient focal cerebral ischemia in rats.

The association between recovery of brain function and behavior after transient cerebral ischemia in animals and humans is incompletely characterized. Quantitative diffusion- (DWI), perfusion- (PWI), T(2)-weighted (T(2)WI), and functional magnetic resonance imaging (fMRI) were performed before, during, and up to 1 day after 20-mins transient middle cerebral artery occlusion (tMCAO; n=6) or sham operation (n=6) in male Sprague-Dawley rats. Viability thresholds were employed to calculate diffusion, perfusion, and T(2) lesion volumes. Region of interest analysis was used to evaluate structural and functional MR signal changes within the sensorimotor network, which were then related to corresponding behavioral measures. Post-mortem 2,3,5-triphenyltetrazolium chloride (TTC) staining was performed 24 h after ischemia. Transient middle cerebral artery occlusion produced lesions on DWI and PWI, which fully recovered by 30 mins after reperfusion. Ipsilesional fMRI responses to hypercapnia and forepaw stimulation were significantly impaired after ischemia and did not fully normalize until 3 and 24 h after tMCAO, respectively. No abnormalities were observed on imaging or TTC at 24 h despite significant behavioral dysfunctions including contralesional forelimb impairment and ipsilesional neglect. No MRI, behavioral, or TTC anomalies were observed in sham-operated rats. There were no significant correlations between MRI parameters, behavior, and TTC in either group. Together, these results suggest that normal findings on diffusion, perfusion, and T(2) imaging shortly after transient ischemia may not indicate normal tissue status as indicated by fMRI and behavior, which may help explain the persistence of neurologic deficits in patients with normal conventional MRI after cerebral ischemia.     

Effect of propentofylline (HWA 285) on focal ischemia in rats: effect of treatment and posttreatment duration on infarct size

BACKGROUND AND PUROSEe: in this study we tested the potentially neuroprotective properties of propentofylline in a model of focal ischemia with long-term, repeated treatment. METHODS: 37 male Wistar rats (280-300 g) underwent permanent occlusion of the middle cerebral artery (MCA). Infusion was started 30 min after occlusion of the MCA over a period of 2 h with a dosage of 0.01 mg/kg body weight. Immediately after the termination of infusion repetitive intraperitoneal injections were started. Animals were assigned to four groups: continuous treatment for a period of 12 h with 24-h survival (group A, n=9) or 48-h survival (group B, n=10), continuous treatment for a period of 48 h with 48-h survival (group C, n=9) and placebo (group D, n=9). Infarct size was calculated from brain slices stained with 2,3,5-triphenyltetrazolium chloride. RESULTS: the infarct size was significantly reduced in group C (treatment for 48 h) (163.9+/-30.5 mm(3)) compared to the placebo group (297.4+/-17. 7 mm(3)). No effect on infarct size was observed in group A (196. 8+/-37.3 mm(3)) and group B (239.6+/-42.9 mm(3)) compared to placebo. CONCLUSION: continuous i.p. injections of propentofylline over a period of 48 h significantly reduces infarct size in an animal model of focal cerebral ischemia. With shorter periods of continuous administration of the drug and delayed postmortem analysis, reductions in the infarct size did not reach a level of significance. These data show the importance of continuous long-term administration after ischemic stroke in clinical trials to achieve the beneficial effects of neuroprotection by propentofylline.