The interaction between tumor necrosis factor-like weak inducer of apoptosis and its receptor fibroblast growth factor-inducible 14 promotes the recruitment of neutrophils into the ischemic brain

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are expressed in endothelial cells and perivascular astrocytes. Here, we show that TWEAK induces a dose-dependent increase in the expression of the chemokine monocyte chemoattractant protein-1 (MCP-1) in astrocytes, and that this effect is mediated by its interaction with Fn14 via nuclear factor-κB pathway activation. Exposure to oxygen-glucose deprivation (OGD) conditions increases TWEAK and Fn14 mRNA expression in wild-type (Wt) astrocytic cultures. Likewise, incubation under OGD conditions induces the expression of MCP-1 in Wt astrocytes but not in astrocytes deficient on either TWEAK (TWEAK^−/−) or Fn14 (Fn14^−/−). We also found that TWEAK induces the passage of neutrophils to the abluminal side of an in vitro model of the blood–brain barrier. Our earlier studies indicate that cerebral ischemia increases the expression of TWEAK and Fn14 in the endothelial cell-basement membrane-astrocyte interface. Here, we report that middle cerebral artery occlusion increases the expression of MCP-1 and the recruitment of neutrophils into the ischemic tissue in Wt but not in TWEAK^−/− or Fn14^−/− mice. These novel results indicate that during cerebral ischemia, the interaction between TWEAK and Fn14 leads to the recruitment of leukocytes into the ischemic tissue.     

Brain edema and tumor necrosis factor-like weak inducer of apoptosis in rats with cerebral ischemia

BACKGROUND： Recent studies have demonstrated that tumor necrosis factor-like weak inducer of apoptosis （TWEAK） participates in brain edema. However, it is unclear whether blood-brain barrier （BBB） disruption is associated with TWEAK during the process of brain edema OBJECTIVE： To investigate the effects of TWEAK on BBB permeability in brain edema. DESIGN, TIME AND SETTING： An immunohistochemical observation, randomized, controlled animal experiment was performed at the Laboratory of Neurosurgical Anatomy, Xiangya Medical College, Central South University ＆ Central Laboratory, Third Xiangya Hospital, Central South University between January 2006 and December 2007. MATERIALS： A total of 48 adult Wistar rats were randomly divided into three groups： normal control （n = 8）, sham-operated （n = 8）, and ischemia/reperfusion （n = 32）. Rats from the ischemia/reperfusion group were randomly assigned to four subgroups according to different time points, i.e., 2 hours of ischemia followed by 6 hours （n = 8）, 12 hours （n = 8）, 1 day （n = 8）, or 12 days （n = 8） of reperfusion. METHODS： Focal cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion （MCAO） using the suture method in rats from the ischemia/reperfusion group. Thread was introduced at a depth of 17-19 mm. Rats in the sham-operated group were subjected to experimental procedures similar to the ischemia/reperfusion group; however, the introducing depth of thread was 10 mm. The normal control group was not given any intervention. MAIN OUTCOME MEASURES： TWEAK expression was examined by immunohistochemistry; brain water content on the ischemic side was calculated as the ratio of dry to wet tissue weight; BBB permeability was measured by Evans blue extravasation. RESULTS： A total of eight rats died prior to and after surgery and an additional eight rats were randomly entered into the study. Thus 48 rats were included in the final analysis. In the ischemia/reperfusion group, TWEAK-positiv     

Cerebral mast cells regulate early ischemic brain swelling and neutrophil accumulation.

We previously observed degranulated mast cells (MC) in association with perivascular brain edema formation during focal cerebral ischemia. Brain MC are typically located perivascularly and contain potent fast-acting vasoactive and proteolytic substances. We examined in a rat model of transient middle cerebral artery occlusion (MCAO) whether, in the early phase of ischemia, MC regulate microcirculation, the blood-brain barrier (BBB) permeability, and edema formation. First, animals received MC inhibitor (cromoglycate), MC-degranulating drug (compound 48/80), or saline. Thereafter, we performed transient MCAO in gene-manipulated MC-deficient rats and their wild-type (WT) littermates, calculating brain swelling, visualizing BBB leakage by intravenously administered Evans blue albumin, and determining neutrophil infiltration with light microscopy. Cerebral blood flow, monitored by laser-Doppler flowmetry in separate experiments, was similar among pharmacological treatments. Ischemic swelling resulted in increased hemispheric volume of 13.4%+/-1.0% in controls, 8.1%+/-0.4% (39% reduction) after cromoglycate, and 25.2%+/-2.0% (89% increase) after compound 48/80 (P<0.05). Early ischemic BBB leakage was reduced by 51% after cromoglycate, and 50% enhanced by compound 48/80 (P<0.05). The cromoglycate group showed 37% less postischemic neutrophil infiltration than did controls (P<0.05). Furthermore, MC-deficient rats responded to focal ischemia with 58% less brain swelling (6.7%+/-1.2%) than did their WT littermates (15.8%+/-1.4%, P<0.05). Blood-brain barrier damage was 47% lower in MC-deficient rats than in the WT (P<0.05). Neutrophil infiltration after MCAO was decreased 47% in MC-deficient rats in comparison to WT (P<0.05). Pharmacological MC inhibition thus appears to deserve further investigation regarding reduction of brain swelling and inflammation early after stroke.     

Inhibition of integrin alphavbeta3 reduces blood-brain barrier breakdown in focal ischemia in rats

Ischemic stroke is a major cause of morbidity and mortality in industrialized nations. We tested the effect of postischemic treatment of cyclo-RGDfV (cRGDfV), a selective inhibitor of integrin alphavbeta3, in the middle cerebral artery occlusion (MCAO) model of ischemic stroke in rats. Rats were randomly divided into three groups: sham operation (n = 13), MCAO with no treatment (n = 18), and MCAO with cRGDfV treatment (n = 28). Focal ischemia was induced with the suture occlusion method for 2 hr, and treatment was given 1 hr after reperfusion (3 hr after ischemia). All animals were sacrificed 24 hr after reperfusion. Assessment included neurological scores, infarction volumes, brain water content, Evans blue exudation, IgG exudation, histology, immunohistochemistry, and Western blotting. Treatment with cRGDfV ameliorated neurological deficits, reduced brain edema, and reduced exudation of Evans blue dye and IgG, but failed to reduce infarction volumes. Western blotting showed a reduction in phosphorylation of one subset of vascular endothelial growth factor (VEGF) receptors in the cRGDfV treatment group. Western blotting also demonstrated a significant reduction of fibrinogen in the cRGDfV treatment group. We conclude that poststroke treatment with cRGDfV reduces blood-brain barrier breakdown in focal ischemia, possibly through inhibition of VEGF-mediated vascular breakdown.     

Ischemic brain edema and the osmotic gradient between blood and brain

The relationship of the osmotic pressure gradient between blood and brain, and the development of ischemic brain edema was studied. Focal cerebral ischemia was produced by left middle cerebral artery occlusion in rats. Brain osmolality was determined with a vapor pressure osmometer, brain water content by wet-dry weight, and tissue sodium and potassium contents by flame photometry. Permeability of the BBB was tested by Evans blue. Measurements were made from the ischemic cortex within 14 days of occlusion. Brain osmolality increased from 311 +/- 2 to 329 +/- 2 mOsm/kg by 6 h after occlusion. Serum osmolality did not change significantly. The osmotic gradient between blood and brain peaked at approximately 26 mOsm/kg. Brain osmolality then decreased to 310 +/- 2 mOsm/kg by 12 h after occlusion and remained at about that same level. Water content increased progressively within 1 day of occlusion, then gradually decreased by 14 days. Brain tissue sodium plus potassium content did not increase within 6 h of occlusion, and Evans blue extravasation was not seen within that time. These findings indicate that an osmotic pressure gradient contributes to the formation of edema only during the early stage of cerebral ischemia. Furthermore, the increase in brain osmolality is not related to tissue electrolyte change or BBB disruption to protein.     

Disruption of blood-brain barrier following bilateral carotid artery occlusion in spontaneously hypertensive rats. A quantitative study

The present study was designed to clarify the relationship of cerebral blood flow (CBF) to blood-brain barrier (BBB) in the ischemic brains with or without recirculation, which were produced by clipping of both common carotid arteries in spontaneously hypertensive rats. CBF was measured by the hydrogen clearance method and BBB function was evaluated by the permeability of 131I-albumin and Evans blue dye. Cortical CBF was reduced from 48.8 +/- 9.5 to 4.0 +/- 1.2 ml/100 gm/min during 1 hr ischemia and further to 2.6 +/- 0.3 ml/100 gm/min during 3 hrs ischemia, while thalamic CBF was reduced much less from 50.0 +/- 3.6 to 17.9 +/- 6.5 ml/100 gm/min and to 17.5 +/- 11.0 ml/100 gm/min, respectively. There was no increase in permeability to protein tracers observed in such 1 hr or 3 hrs ischemic brain. Both cortical and thalamic CBF were markedly increased 2.5 to 6 fold of resting values at 5 min after recirculation in the 1 hr ischemic brain. In the 3 hrs ischemic brain, however, both CBF were only slightly increased but never restored to the resting level even at 30 min after recirculation. In such reperfused brains, exudation to Evans blue dye was observed in none of 16 animals with 1 hr ischemia, but in 18 of 23 with 3 hrs ischemia. Disruption of BBB was twice more frequent in the cortex (77.8%) than in either thalamus (33.3%) or hippocampus (33.3%). Permeability index of 131I-albumin (brain albumin/blood albumin) was significantly higher in the ischemic areas stained with blue dye (2.07 +/- 0.45%) than in non-ischemic control brain (0.10 +/- 0.01%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of lipopolysaccharide priming on acute ischemic brain injury

BACKGROUND AND PURPOSE: Infection has been implicated as a stroke risk factor. Activation and infiltration of polymorphonuclear neutrophils (PMNs) after cerebral ischemia may contribute to ischemic brain injury. This study was conducted to investigate how enhanced postischemic PMN infiltration by lipopolysaccharide (LPS) altered the acute ischemic outcomes. METHODS: LPS (0.05 mg/kg SC) or vehicle was given to Long-Evans male rats 24 hours before ischemia. Focal cerebral ischemia was induced by temporary ligation of the right middle cerebral artery and both common carotid arteries for 45 minutes. Animals were killed 6 and 24 hours after reperfusion to determine the extent of PMN infiltration (myeloperoxidase assay), brain edema (wet-dry weight method), and vascular injury (fluorescein isothiocyanate-conjugated dextran extravasation). The infarct volumes were measured on the basis of TTC stain 24 hours after ischemia. RESULTS: LPS had little effect on body temperature or peripheral white count but substantially enhanced PMN infiltration into the ischemic right middle cerebral artery cortex on the basis of myeloperoxidase activity (6 hours: control, 0 U/g; LPS, 0.186+/-0. 025 U/g; 24 hours: control, 0.185+/-0.025 U/g; LPS, 0.290+/-0.040 U/g; P<0.001) and morphological studies. The extent of vascular injury defined by the extravasation of fluorescein isothiocyanate-conjugated dextran into the ischemic tissue (6 hours: control, 3.11+/-0.41 microliter/mg protein; LPS, 0.48+/-0.16 microliter/mg protein; 24 hours: control, 1.77+/-0.23 microliter/mg protein; LPS, 0. 90+/-0.19 microliter/mg protein; P<0.001) and brain edema determined by the brain water content (6 hours: control, 84.77+/-1.63%; LPS, 82. 09+/-1.25%; 24 hours: control, 89.40+/-0.43%; LPS, 87.88+/-0.58%; P<0.01) were paradoxically reduced by LPS priming. LPS-primed rats also had smaller infarct volumes (control, 135+/-5 mm(3); LPS, 108+/-12 mm(3); P<0.05). CONCLUSIONS: Enhanced postischemic PMN infiltration is anticipated to facilitate ischemic brain injury. Contrary to this expectation, results from the present study suggest that an increase in postischemic PMN infiltration after LPS priming was not detrimental. These findings challenge the notion that postischemic PMN infiltration is uniformly deleterious.     

Brain-Derived Neurotrophic Factor Improves Long-Term Potentiation and Cognitive Functions after Transient Forebrain Ischemia in the Rat

We investigated the effect of brain-derived neurotrophic factor (BDNF) on hippocampal long-term potentiation (LTP) and cognitive functions after global cerebral ischemia in the rat. After four-vessel occlusion, BDNF was administered via an osmotic minipump continuously over 14 days intracerebroventricularly. Electrophysiological experiments were performed 14 days after cerebral ischemia. Test stimuli and tetanization were delivered to the Schaffer collaterals of the hippocampus and field excitatory postsynaptic potentials (fEPSP) were recorded in the CA1 region. Cognitive impairment was analyzed repeatedly with a passive avoidance test, a hole-board test, and with an activity center on the same animal. In sham-operated animals, LTP was consistantly induced after delivering a tetanus (increase of initial slope of fEPSP to 173 +/- 12% of baseline; n = 6). After transient forebrain ischemia LTP could not be induced (117 +/- 4% of baseline; n = 7). In ischemic animals treated with BDNF, LTP could be induced (168 +/- 28% of baseline; n = 8). Transient forebrain ischemia resulted in a significant decrease in spatial discrimination performance but not of associative memory. The ratios for working memory (WM) and reference memory (RM) 15 days after ischemia were lower in the ischemic rats (n = 10) than in the sham-operated control animals (n = 10; WM: 22 +/- 6 vs 72 +/- 7; RM: 30 +/- 7 vs 72 +/- 5). Postischemic intracerebroventricular BDNF infusion increased both WM (63 +/- 4; n = 10) and RM (58 +/- 5; n = 10). The spontaneous locomotor activity did not differ significantly in the three groups. These data indicate a protective effect of BDNF for synaptic transmission and cognitive functions after transient forebrain ischemia.     

The angiotensin II type 1-receptor blocker candesartan increases cerebral blood flow, reduces infarct size, and improves neurologic outcome after transient cerebral ischemia in rats.

The goal of the present study was to test the impact of administration time of the angiotensin II type 1-receptor blocker candesartan on cerebral blood flow (CBF), infarct size, and neuroscore in transient cerebral ischemia. Therefore, 1-hour middle cerebral artery occlusion (MCAO) was followed by reperfusion. Rats received 0.5-mg/kg candesartan intravenously 2 hours before MCAO (pretreatment), 24 hours after MCAO, every 24 hours after MCAO, or 2 hours before and every 24 hours after MCAO. Infarct size (mm3) and a neuroscore at day 7 were compared with controls. CBF was quantified by radiolabeled microspheres and laser-Doppler flowmetry. Compared with controls (95 +/- 8), infarct size in candesartan-treated groups was smaller (59 +/- 5, 68 +/- 10, 28 +/- 3, and 15 +/- 3, respectively; P<0.05). Although there was no difference in neuroscore between pretreatment and controls (1.55 +/- 0.18, 1.80 +/- 0.13), other treatment regimens resulted in improved neuroscores (1.33 +/- 0.16, 1.11 +/- 0.11, 0.73 +/- 0.15; P<0.05). CBF in pretreated animals at 0.5 hours after MCAO was significantly higher than in controls (0.58 +/- 0.09 mL x g(-1) x min(-1) and 44% +/- 7% of baseline compared with 0.49 +/- 0.06 mL x g(-1) x min(-1) and 37% +/- 6%, microspheres and laser-Doppler flowmetry; P<0.05). Thus, candesartan reduces infarct size even if administered only during reperfusion. Apart from pretreatment, other treatment regimens result in significantly improved neuroscores. In the acute phase of cerebral ischemia, candesartan increases CBF.     

Endothelin-1 overexpression leads to further water accumulation and brain edema after middle cerebral artery occlusion via aquaporin 4 expression in astrocytic end-feet.

Stroke patients have increased levels of endothelin-1 (ET-1), a strong vasoconstrictor, in their plasma or cerebrospinal fluid. Previously, we showed high level of ET-1 mRNA expression in astrocytes after hypoxia/ischemia. It is unclear whether the contribution of ET-1 induction in astrocytes is protective or destructive in cerebral ischemia. Here, we generated a transgenic mouse model that overexpress ET-1 in astrocytes (GET-1) using the glial fibrillary acidic protein promoter to examine the role of astrocytic ET-1 in ischemic stroke by challenging these mice with transient middle cerebral artery occlusion (MCAO). Under normal condition, GET-1 mice showed no abnormality in brain morphology, cerebrovasculature, absolute cerebral blood flow, blood-brain barrier (BBB) integrity, and mean arterial blood pressure. Yet, GET-1 mice subjected to transient MCAO showed more severe neurologic deficits and increased infarct, which were partially normalized by administration of ABT-627 (ET(A) antagonist) 5 mins after MCAO. In addition, GET-1 brains exhibited more Evans blue extravasation and showed decreased endothelial occludin expression after MCAO, correlating with higher brain water content and increased cerebral edema. Aquaporin 4 expression was also more pronounced in astrocytic end-feet on blood vessels in GET-1 ipsilateral brains. Our current data suggest that astrocytic ET-1 has deleterious effects on water homeostasis, cerebral edema and BBB integrity, which contribute to more severe ischemic brain injury.     

Microvascular basal lamina injury after experimental focal cerebral ischemia and reperfusion in the rat.

To define the location and extent of microvascular damage of the basal lamina after cerebral ischemia and reperfusion in rats, the authors subjected animals (n = 16) to 3 hours of focal cerebral ischemia and 24 hours of reperfusion using the suture middle cerebral artery occlusion model; sham-operated animals served as controls (n = 6). The Western blot technique was used to define the collagen type IV protein content in various brain regions, whereas immunohistochemistry identified microvascular basal lamina loss (anticollagen type IV staining). The extent of damage was quantified by automatic morphometric video-imaging analysis. Statistical analysis was based on the Mann-Whitney test and the paired Student's t-test. The ischemic hemisphere showed a reduction of the collagen type IV protein content after ischemia and reperfusion in the Western blot (reduction compared with the nonischemic side: total hemisphere, 33% +/- 6%; basal ganglia, 25% +/- 7%; cortex 49% +/- 4%; P < 0.01) [corrected]. There was also a decrease in the number of cerebral microvessels between the ischemic and nonischemic hemispheres (20% +/- 2%), cortical (8% +/- 3%), and basal ganglia areas (31% +/- 3%) (P < 0.001). Besides a reduction of the vessel number, there was also a loss in basal lamina antigen-positive stained area in ischemic areas (hemisphere, 16% +/- 3%; cortex, 14% +/- 3%; basal ganglia, 21% +/- 4%; P < 0.01) [corrected]. Cortical areas had a less pronounced basal lamina loss than basal ganglia (P < 0.05). For the first time, microvascular basal lamina damage, indicated by collagen type IV loss, is proven in rats by biochemical and morphometric analysis. These changes are comparable with those found in nonhuman primates. The authors report novel data regarding microvascular ischemic changes in the cortex. These data provide a basis for future experiments to determine the mechanisms of ischemic microvascular damage and to devise new therapeutic strategies.     

NXY-059, a novel free radical trapping compound, reduces cortical infarction after permanent focal cerebral ischemia in the rat

Free radicals have gained wide acceptance as mediators of cerebral ischemic injury. It has previously been reported that a spin trap nitrone, alpha-phenyl-N-tert-butyl nitrone (PBN), can reduce infarct volumes in rats subjected to either permanent or transient focal cerebral ischemia. A recent study has demonstrated that NXY-059, a novel free radical trapping nitrone compound, has a neuroprotective effect against transient focal cerebral ischemia. This study was designed to determine the effect of NXY-059 in a rodent model of permanent focal cerebral ischemia. Male spontaneously hypertensive rats were subjected to permanent middle cerebral artery occlusion (MCAO) by placement of a microaneurysm clip on the middle cerebral artery (MCA). Animals were divided into three groups: (1) physiological saline given as a 1 ml/kg i.v. bolus administered 5 min post MCAO followed immediately by a continuous i.v. infusion of 0.5 ml/h of physiological saline for 24 h (n=10); (2) 30 mg/kg, 1 ml/kg, i.v. bolus of NXY-059 dissolved in physiological saline administered 5 min post MCAO followed immediately by a continuous i.v. infusion of 30 mg/kg/h, 0.5 ml/h, of NXY-059 for 24 h (n=9); (3) 60 mg/kg, 1 ml/kg, i.v. bolus of NXY-059 dissolved in physiological saline administered 5 min post MCAO followed immediately by a continuous i.v. infusion of 60 mg/kg/h, 0.5 ml/h, of NXY-059 for 24 h (n=12). Infarction was quantified after a survival period of 24 h. Differences in infarct volume were examined with one-way ANOVA following Dunnet's multiple comparison test. The percentage of cortical infarction in the saline control group was 22.6 +/- 6.8% (mean+/-S.D.) of contra-lateral hemisphere, and in the 30 mg/kg/h NXY-059-treated group was 17.4% +/- 6.8% (NS). Plasma concentration (microM/l) of NXY-059 in the 30 mg/kg/h group was 80.2 +/- 52.2 (n=9), while in the 60 mg/kg/h group plasma concentration (microM/l) of NXY-059 was 391.0 +/- 207.0 (n=10). Infarction in the 60 mg/kg/h NXY-059-treated group was significantly reduced (P=0.009) to 14.5 +/- 5%. Our preliminary data demonstrate that administration of NXY-059 (60 mg/kg/h for 24 h) ameliorates cortical infarction in rats subjected to permanent focal cerebral ischemia with 24 h survival.     

Progesterone exacerbates striatal stroke injury in progesterone-deficient female animals

BACKGROUND AND PURPOSE: We have previously shown that female animals experience substantial protection from brain injury after reversible middle cerebral artery occlusion (MCAO) compared with their male or ovariectomized female counterparts. The reproductive steroid estrogen has been shown to provide neuroprotection from a variety of experimental insults, but the importance of progesterone as an anti-ischemic treatment has not been well explored. We evaluated histological outcomes after MCAO in ovariectomized female rats with or without acute or chronic progesterone replacement therapy. METHODS: Age-matched, adult female Wistar rats were ovariectomized and treated with 0, 30, or 60 mg/kg progesterone IP 30 minutes before ischemia (n=12 to 14 per group) or with 30 mg/kg progesterone IP daily for 7 to 10 days before ischemia (n=16). Each animal subsequently underwent 2 hours of MCAO with the intraluminal filament technique, followed by 22 hours of reperfusion. Ipsilateral parietal cortex perfusion was monitored with laser Doppler flowmetry throughout ischemia. Cortical, caudate-putamen, and hemispheric infarction volumes were determined with 2,3,5-triphenyltetrazolium chloride staining and digital image analysis. RESULTS: Intraischemic plasma progesterone levels were 5+/-3, 102+/-20,* 181+/-28,* and 133+/-25* ng/mL in the 0, 30, and 60 mg/kg acute progesterone group and the 30 mg/kg chronic progesterone group, respectively (*P<0.05 compared with 0 mg/kg). Caudate-putamen infarction volume (percent contralateral structure) was significantly increased by chronic progesterone treatment: 45.6+/-5.1%* in the 30 mg/kg chronic progesterone group and 29.2+/-5.3%, 35.8+/-5.1%, and 42.0+/-5.0% in the 0, 30, and 60 mg/kg acute progesterone groups, respectively (*P<0.05 compared with 0 mg/kg). Cortical and total hemispheric infarction volumes (percent contralateral structure) were unchanged by progesterone treatment. CONCLUSIONS: Exogenous progesterone therapy does not ameliorate histological injury after MCAO in previously ovariectomized, adult female rats. Furthermore, chronic progesterone administration can exacerbate infarction in subcortical regions.     

Protective effect of the 20-HETE inhibitor HET0016 on brain damage after temporary focal ischemia.

Cytochrome P450 metabolism of arachidonic acid produces the potent vasoconstrictive metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE). Recent studies have implicated 20-HETE as a vasoconstrictive mediator in hemorrhagic stroke. The purpose of this study was to determine the effect of the 20-HETE inhibitor, HET0016, on lesion volume and cerebral blood flow (CBF) after temporary middle cerebral artery occlusion (MCAO) in rats. Plasma pharmacokinetics and tissue concentrations of HET0016 were determined after a 10 mg/kg intraperitoneal dose. Separate rats were treated with HET0016 or vehicle before 90 mins of MCAO. Lesion volume was assessed by 2,3,5-triphenyl-tetrazolium-chloride and cerebral flow was determined using laser Doppler flow. The effect of MCAO on in vitro microsomal formation of mono-oxygenated arachidonic acid metabolites was also determined. Results show that HET0016 has a short biologic half-life, distributes into the brain, and is associated with a 79.6% reduction in 20-HETE concentration in the cortex. Lesion volume was greatly reduced in HET0016-treated (9.1%+/-4.9%) versus vehicle-treated (57.4%+/-9.8%; n=6; P<0.001) rats. An attenuation of the observed decrease in CBF was observed in HET0016-treated (180 mins 89.2%+/-6.2%; 240 mins 88.1%+/-5.7% of baseline flow) versus vehicle control (180 mins 57.6%+/-19.0%; 240 mins 53.8%+/-20.0% of baseline flow; n=6; P<0.05). Brain cortical microsomal formation rate of 20-HETE was also reduced at 24 h in the ipsilateral hemisphere after MCAO. These data support a significant role for 20-HETE in the pathogenesis of ischemic stroke.     

兔大脑中动脉阻断所致局灶性脑缺血脑组织内皮抑素蛋白及mRNA表达增加(英文)

目的内皮抑素(endostatin)是强烈的抗血管再生因子。本文探讨大脑中动脉闭塞 (middle cerebral artery occlusion, MCAO)致局灶性脑缺血后脑组织内皮抑素蛋白及mRNA基因表达的变化,同时检测缺血脑组织血管内皮生长因子(vascular endothelial growth factor,VEGF)的含量。方法24只新西兰白兔随机分为正常对照组(n=5)、假手术组(n=4)、缺血2小时组(n=5)、缺血24小时组 (n=5)及缺血48小时组(n=5)共5组。酶联免疫吸附试验测定VEGF含量,免疫组化分析内皮抑素蛋白变化,原位杂交检测内皮抑素mRNA表达。结果与对照组相比,MCAO局灶性脑缺血后内皮抑素蛋白和mRNA表达均明显增加,至少分别增加了50%(P<0.01)和70%(P<0.05),同时缺血脑组织VEGF含量也明显增加,至少增加了270%。结论缺血导致脑组织内皮抑素表达增加,且内皮抑素的增加与缺血后脑组织VEGF变化无相关性,但可能抑制脑缺血后的血管再生,从而加重脑缺血损伤。     

Extended therapeutic window and functional recovery after intraarterial administration of neuregulin-1 after focal ischemic stroke.

We have previously shown that neuregulin-1 (NRG-1) protects neurons from ischemic brain injury if administered before focal stroke. Here, we examined the therapeutic window and functional recovery after NRG-1 treatment in rats subjected to 90 mins of middle cerebral artery occlusion (MCAO) and 24 h of reperfusion. Neuregulin-1 (2.5 ng/kg bolus, 1.25 ng/kg/min infusion) reduced infarct volume by 89.2%+/-41.9% (mean+/-s.d.; n=8; P<0.01) if administered immediately after the onset of reperfusion. Neuroprotection was also evident if NRG-1 was administered 4 h (66.4%+/-52.6%; n=7; P<0.01) and 12 h (57.0%+/-20.8%; n=8; P<0.01) after reperfusion. Neuregulin-1 administration also resulted in a significant improvement of functional neurologic outcome compared with vehicle-treated animals (32.1%+/-5.7%; n=9; P<0.01). The neuroprotective effect of the single administration of NRG-1 was seen as long as 2 weeks after treatment. Neurons labeled with the neurodegeneration marker dye Fluoro-JadeB were observed after MCAO in the cortex, but the numbers were significantly reduced after NRG-1 treatment. These results indicate that NRG-1 is a potent neuroprotective compound with an extended therapeutic window that has practical therapeutic potential in treating individuals after ischemic brain injury.     

Minocycline promotes axonal regeneration through suppression of RGMa in rat MCAO/reperfusion model

Minocycline has been recently implicated in protection against focal cerebral ischemia reperfusion (I/R), but the protective effects on neurobehavioral abnormalities remains contradictory. In the present study, we investigate whether minocycline improves axonal regeneration and neurological function recovery by inhibiting the expression of the repulsive guidance molecular A (RGMa) after focal cerebral ischemia reperfusion. Male Sprague‐Dawley (SD) rats were subjected to occlusion of the right middle cerebral artery (MCAO) for 2 h and 3 mg kg^?1 minocycline was injected intravenously immediately after reperfusion twice a day for 14 days. The staircase test and modified neurological severity score (mNSS) were performed to evaluate functional outcome and blood‐brain barrier (BBB) permeability was assessed by Evan's blue dye extravasation (EB) at the expected time point. The expression of RGMa in ischemic cortex was measured by immunohistochemical staining and Western blot 2 weeks after MCAO. Neurofilament protein 200 (NF‐200) immunohistochemical staining was used to assess axonal damage. Treatment with minocycline at a dose of 3 mg kg^?1 via the caudal vein significantly reduced the extravasation of EB, elevated mNSS and improved forelimb motor function as assessed by the staircase test when compared to the I/R group (P < 0.05). Moreover, axonal regrowth was enhanced in the minocycline treatment group when compared to the I/R group (P < 0.05). In addition, minocycline significantly reduced the expression of RGMa protein 2 weeks after MCAO as assessed by both immunostaining and Western blot. Our studies suggest that early minocycline treatment promotes neurological functional recovery and axonal regeneration in rats after MCAO, which might be mediated by down‐regulating RGMa expression. Synapse, 2013. © 2012 Wiley Periodicals, Inc.     

Effect of mild hypothermia on focal cerebral ischemia. Review of experimental studies

The purposes of this review are to clarify the effect of hypothermia therapy on focal cerebral ischemia in rats, and to consider the relevancy of its application to human focal cerebral ischemia. Since 1990, 26 reports confirming the brain-protecting effect of hypothermia in rat focal cerebral ischemia models have been published. Seventy-four experimental groups in these 26 reports were classified as having transient middle cerebral arterial occlusion (MCAO) with mild hypothermia (group A; 43 groups), permanent MCAO with mild hypothermia (group B; 14 groups), permanent MCAO with deep hypothermia (group C; 8 groups) and transient or permanent MCAO with mild hyperthermia (group D; 9 groups). The results were evaluated as the % infarct volume change caused by hypothermia or hyperthermia compared with the infarct volume in normothermic animals. The effectiveness was confirmed in 36 (83%) of the 43 groups in group A, 10 (71%) of the 14 in group B, and six (75%) of the eight in group C. The infarct volume of eight of the nine groups in group D was markedly aggravated. The percent infarct volume change was 55.3% +/- 27.1% in group A, 57.6% +/- 24.7% in group B, 60.8% +/- 45.5% in group C, and 189.7% +/- 89.4% in group D. For effective reduction of the infarct volume, hypothermia should be started during ischemia or within 1 h, at latest, after the beginning of reperfusion in the rat transient MCAO model. However, it is not clear whether this neuroprotective effect of hypothermia can also be observed in the chronic stage, such as several months later. Keeping the body temperature normothermic in order to avoid mild hyperthermia seems to be rather important for not aggravating cerebral infarction. Clinical randomized studies on the efficacy of mild hypothermia for focal cerebral ischemia and sophisticated mild hypothermia therapy techniques are mandatory.