Hepatocyte growth factor reduces the infarct volume after transient focal cerebral ischemia in rats.

Hepatocyte growth factor (HGF) was originally discovered as a powerful mitogen for hepatocytes. HGF also has been reported to function as a neurotrophic factor as well as an angiogenetic factor. The present study examined the neuroprotective effect of HGF against transient focal cerebral ischemia in rats, in which an anti-apoptotic and an angiogenetic effect of HGF was assumed to contribute to the reduction of the infarct volume. The intraventricular administration of human recombinant HGF prevented neuronal death after 120 min of occlusion in the right middle cerebral artery and the bilateral common carotid arteries. HGF significantly reduced the infarct volume in a dose-dependent manner. In a separate series of experiments, we next histopathologically investigated both the anti-apoptotic effect on neurons and the angiogenetic effect of HGF. A large number of TUNEL positive neurons were observed in the inner boundary of the infarct area in both the control and the vehicle group whereas only a few TUNEL positive neurons were observed in the corresponding area in the HGF group. In the HGF group, Bcl-2 protein was obviously represented in surviving neurons subjected to ischemia. The number of the vascular lamina in HGF group were significantly higher than those in the vehicle group. These data suggest that HGF appears to have an ability to prevent apoptotic neuronal cell death while also possessing an angiogenetic effect in the central nervous system which was affected with transient focal cerebral ischemia.     

G-CSF reduces infarct volume and improves functional outcome after transient focal cerebral ischemia in mice.

Growth factors possess neuroprotective and neurotrophic properties in vitro, but few have been extensively studied in vivo after stroke. In the present study, we investigated the potential functional benefits of granulocyte colony-stimulating factor (G-CSF) administration after focal cerebral ischemia. Male mice underwent 60-minute middle cerebral artery occlusion (MCAO) and received G-CSF (50 microg/kg, subcutaneously) or vehicle (saline) at the onset of reperfusion. Granulocyte colony-stimulating factor-treated mice killed at 48 hours after MCAO revealed a >45% reduction (P<0.05) in lesion volume. In terms of body weight recovery, and in tests of motor (grid test and rotarod) and cognitive ability (water maze), MCAO significantly worsened the outcome in vehicle-treated mice as compared with shams (P<0.05). However, G-CSF treatment was beneficial as, compared with vehicle, this significantly improved weight recovery and motor ability. This effect was most apparent on the water maze where G-CSF-treated mice were indistinguishable from shams in terms of acquiring the task. These results indicate long-term beneficial effects of a single dose of G-CSF administered on reperfusion, and illustrate the need to further investigate the mechanisms of G-CSF action.     

Hyperbaric oxygen decreases infarct size and behavioral deficit after transient focal cerebral ischemia in rats

Cerebral hypoxia is a major component of immediate and secondary cell damage caused by ischemia. Hyperbaric oxygen (HBO) is a potent means to increase the amount of oxygen dissolved in blood plasma. The effectiveness of HBO in clinical and experimental cerebral ischemia, however, is controversial. We sought to determine whether treatment with HBO initiated early after focal cerebral ischemia-onset protects the brain when experimental conditions such as brain temperature are controlled. Male Wistar rats (n=57) underwent reversible filament occlusion of the right middle cerebral artery (MCA) for 75 min. Animals were awakened after filament introduction and assessed for presence of forelimb paresis. Rats then underwent a 60-min course of either 100% O(2) at 1.0 atmosphere absolute (ata; control group), HBO 1.5 ata, or HBO 2.5 ata in a customized HBO chamber allowing physiological monitoring and pericranial temperature control. The filament was then removed. Seven days after ischemia, rat behavior was scored from 3-18 (18=normal) and brains were removed for histological analysis of infarct volume. Rats treated with HBO 2.5 ata had better mean+/-standard deviation (S.D.) behavioral scores (14+/-2; p<0.05) than control (10+/-3) or HBO 1.5-ata-treated animals (11+/-3). Similarly, total infarct volumes (mean+/-S.D.) were smaller in animals receiving HBO at 2.5 ata (76+/-65 mm(3); p<0.05) compared to control (129+/-83 mm(3)) and HBO 1.5-ata (119+/-68 mm(3))-treated groups. Cortical infarction occurred less frequently in HBO 2. 5-ata-treated than in control animals (44% vs. 71%; p<0.05). We conclude that HBO can improve outcome after temporary focal ischemia when treatment is started early after ischemia-onset but HBO dose appears important. Potential mechanisms include enhanced oxygen supply to marginally perfused cells.     

Administration of transforming growth factor-alpha reduces infarct volume after transient focal cerebral ischemia in the rat.

Growth factors promote cell growth and survival and protect the brain from developing injury after ischemia. In this article, the authors examined whether transforming growth factor-alpha (TGF-alpha) was protective in transient focal ischemia and whether alteration of cerebral circulation was involved. Rats received intraventricular TGF-alpha (50 ng, either split into 2 doses given 30 minutes before and 30 minutes after middle cerebral artery occlusion (MCAO), or 1 dose given 30 minutes after MCAO) or vehicle. Rats were subjected to 1-hour intraluminal MCAO and cerebral blood flow was recorded continuously by laser-Doppler flowmetry. Infarct volume was measured 1 and 4 days later. The effects of TGF-alpha on arterial tone were assessed in isolated rabbit basilar and common carotid arteries. Transforming growth factor-alpha before and after ischemia reduced infarct volume by 70% at 1 day and 50% at 4 days. Transforming growth factor-alpha given only after ischemia also did reduce infarct volume by 70% at 1 day and 80% at 4 days. The protective effect was more marked in cortex than in striatum. Transforming growth factor-alpha did not change cortical microvascular perfusion and did not modify arterial passive tone nor agonist-induced active tone. It can be concluded that TGF-alpha reduces infarct volume, even when the factor is exclusively administered at reperfusion, and that this effect is not mediated by changes in microvascular perfusion or cerebral arteries. It is therefore suggested that TGF-alpha has a protective effect against neuronal cell death after transient focal ischemia.     

Cycloheximide reduces infarct volume when administered up to 6 h after mild focal ischemia in rats

We have previously described a rodent model of brief (30 min) middle cerebral artery occlusion followed by reperfusion, in which infarction develops gradually, reaching completion more than 3 days after ischemia, accompanied by morphological, biochemical, and pharmacological evidence of apoptosis. In the present study, we tested the hypotheses that delayed administration of a protein synthesis inhibitor would be effective in reducing tissue injury in this slowly evolving ischemic infarction, and that efficacy of this treatment would wane with more prolonged ischemia. Focal cerebral ischemia was induced in Long-Evans rats by occlusion of the right middle cerebral artery. Infarction volume was analyzed using triphenyl tetrazolium chloride staining, and morphology was studied using hematoxylin and eosin stained sections. Following 30 min middle cerebral artery occlusion and reperfusion, the core ischemic region exhibited vacuolization in the neuropil by 36 h after ischemia, and infarction reached full size by 7 days after ischemia. Cycloheximide reduced infarct volume when given up to 6 h after ischemia. If the duration of ischemic insult was increased to 90 min, the therapeutic window for delayed cycloheximide was only 30 min. In permanent middle cerebral artery occlusion, cycloheximide was ineffective even when given prior to ischemia onset. After mild, but not severe, ischemic insults, cerebral infarction develops slowly and may be treatable with protein synthesis inhibitors, even when treatment is delayed for up to 6 h after the onset of ischemia.     

Treatment with dihydroethidium reduces infarct size after transient focal cerebral ischemia in mice

A transient focal ischemia model with C57Bl/6 mice was used to investigate whether dihydroethidium is neuroprotective. Different doses (25, 50, 100 mg/kg body weight) were used for pretreatment and the lowest effective dose was used for delayed treatment 1 and 2 h after reperfusion. Our results demonstrate that all the doses used for treatment reduced infarct volume. We conclude that dihydroethidium is neuroprotective by reducing superoxide in mice after stroke.     

Influence of EGF/bFGF treatment on proliferation, early neurogenesis and infarct volume after transient focal ischemia

The persistence of neurogenesis in the adult mammalian forebrain suggests that endogenous precursors may be a potential source for neuronal replacement after injury or neurodegeneration. On the other hand basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) can facilitate neural precursor proliferation in the adult rodent subventricular zone (SVZ) and dentate gyrus. As the application of EGF and bFGF was found to boost neurogenesis after global ischemia, in this study we investigated whether a combined intracerebroventricular (i.c.v.) EGF/bFGF treatment over a period of 2 weeks affects the proliferation of newly generated cells in the endothelin-1 model of transient focal ischemia in adult male Sprague-Dawley rats as well. As assessed by toluidine blue staining, EGF/bFGF substantially increased the infarct volume in ischemic animals. Chronic 5'-bromodeoxyuridine (BrdU) i.c.v. application revealed an EGF/bFGF-induced increase in cell proliferation in the lateral ventricle 14 days after surgery. Proliferation in the striatum increased after ischemia, whereas in the dentate gyrus and in the dorsal 3rd ventricle the number of cells decreased. Analysis of the neuronal fate of these cells by co-staining with a doublecortin (DCX) antibody showed that the growth factors concomitantly nearly doubled early neurogenesis in the ipsilateral striatum in ischemic animals but diminished it in the dentate gyrus. Because of the increased infarct volume and unclear long-term outcome further modifications of a chronic treatment schedule are needed before final conclusions concerning the perspectives of such an approach can be made.     

国产降纤酶对大鼠局灶性脑缺血/再灌注后行为学和梗死体积的影响

目的：探讨国产降纤酶对大鼠局灶性脑缺血／再灌注动物模型的神经行为学和脑梗死体积的影响，以证明其是否有脑保护作用。方法：采用线检法制作大鼠大脑中动脉闭塞模型，用Zea Longa 5分制评分和TTC染色法评价神经行为学和脑梗死体积。由吉林大学第一医院和复旦大学华山医院共同进行实验研究，每部分实验动物各做一半。动物随机分为缺血3h及再灌注3h,6h,24h和72h；缺血6h及再灌注3h,6h,24h;缺血24h等共10组。降纤酶采用8U／kg腹腔注射给药。结果：行为学结果：在10组中仅于缺血6h再灌注6h组Zea Longa评分有明显改善（P＜0．05），其余各组与盐水对照组比较无显著性差异。梗死体积测定结果：在缺血3h各组中，只有缺血3h再灌注72h这个时间点比盐水组无明显缩小（P＞0．05），其余各时间点梗死体积均明显缩小（P分别＜0．05、0．01和0．001）。在缺血6h和24h组中，各时间点与盐水组比较均明显缩小（分别为P＜0．01和P＜0．05）。结论：国产降纤酶对大鼠局灶性脑缺血及再灌注损伤有一定的保护作用。     

Delayed post-ischemic albumin treatment neither improves nor worsens the outcome of transient focal cerebral ischemia in rats

Human albumin therapy within the first 4 h is highly neuroprotective in focal ischemia, but it is unknown whether delayed albumin therapy is deleterious. Rats received 2 h middle cerebral artery suture-occlusion. Human albumin (25%, 2.5 mg/kg; n=12) or vehicle (0.9% saline, 5 ml/kg; n=9) were administered at 19 h. Neurological status was evaluated daily, and histopathology and brain swelling were quantified at 3 days. Delayed albumin treatment, while ineffective, failed to show adverse effects.     

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.     

Delayed treatment with nicotinamide (Vitamin B(3)) improves neurological outcome and reduces infarct volume after transient focal cerebral ischemia in Wistar rats

BACKGROUND AND PURPOSE: We have previously shown that nicotinamide (NAm) acutely reduces brain infarction induced by permanent middle cerebral artery occlusion (MCAo) in rats. In this study, we investigate whether NAm may protect against ischemia/reperfusion injury by improving sensory and motor behavior as well as brain infarction volumes in a model of transient focal cerebral ischemia. METHODS: Forty-eight male Wistar rats were used, and transient focal cerebral ischemia was induced by MCAo for 2 hours, followed by reperfusion for either 3 or 7 days. Animals were treated with either intraperitoneal saline or NAm (500 mg/kg) 2 hours after the onset of MCAo (ie, on reperfusion). Sensory and motor behavior scores and body weight were obtained daily, and brain infarction volumes were measured on euthanasia. RESULTS: Relative to treatment with saline, treatment with NAm (500 mg/kg IP) 2 hours after the onset of transient focal cerebral ischemia in Wistar rats significantly improved sensory (38%, P<0.005) and motor (42%, P<0.05) neurological behavior and weight gain (7%, P<0.05) up to 7 days after MCAo. The cerebral infarct volumes were also reduced 46% (P<0.05) at 3 days and 35% (P=0.09) at 7 days after MCAo. CONCLUSIONS: NAm is a robust neuroprotective agent against ischemia/reperfusion-induced brain injury in rats, even when administered up to 2 hours after the onset of stroke. Delayed NAm treatment improved both anatomic and functional indices of brain damage. Further studies are needed to clarify whether multiple doses of NAm will improve the extent and duration of this neuroprotective effect and to determine the mechanism(s) of action underlying the neuroprotection observed. Because NAm is already used clinically in large doses and has few side effects, these results are encouraging for the further examination of the possible use of NAm as a therapeutic neuroprotective agent in the clinical treatment of acute ischemic stroke.     

Inhibition of Na/H exchanger reduces infarct volume of focal cerebral ischemia in rats

Activation of Na⁺/H⁺ exchanger (NHE) may have an important role in ischemic cell death by means of intracellular overload of Na⁺ and Ca²⁺. Recent evidence has suggested that inhibitors of NHE have protective effects on myocardial ischemia both in vivo and in vitro. In this study, we tested the hypothesis that FR183998, an inhibitor of NHE, reduces infarct volume produced by focal cerebral ischemia in rats. We used 20 male spontaneously hypertensive rats. Either FR183998 (1 mg/kg; n=10), or vehicle (n=10) was given intravenously to the rats and the distal middle cerebral artery of each animal was occluded using a photothrombotic technique. We measured regional cerebral blood flow using laser-Doppler flowmetry throughout the experiments. After 3 days, infarct volume was measured in each animal group. To estimate the brain edema, we also calculated the cortical volume in both hemispheres. The infarct volume in the FR183998-treated group (82±8 mm³, mean±S.E.M.) was significantly smaller than that in the control group (115±12 mm³) (P=0.034). The cortical volume of the occluded side in the FR183998-treated group (359±7 mm³) tended to be smaller than that in the control group (378±9 mm³) (P=0.116). The regional cerebral blood flow and physiological variables during ischemia were not significantly different between the two groups throughout the experiments. These results suggest that inhibition of NHE by FR183998 may have beneficial effects in reducing infarct volume and brain edema during cerebral ischemia. Thus, NHE may play an important role in the development of neuronal damage during acute cerebral ischemia.     

Dietary phytoestrogens improve stroke outcome after transient focal cerebral ischemia in rats

As phytoestrogens are postulated as being neuroprotectants, we assessed the hypothesis that dietary isoflavone-type phytoestrogens are neuroprotective against ischemic stroke. Transient focal cerebral ischemia (90 min) was induced by middle cerebral artery occlusion (MCAO) following the intraluminal thread technique, both in rats fed with soy-based diet and in rats fed with isoflavone-free diet. Cerebro-cortical laser-Doppler flow (cortical perfusion, CP), arterial blood pressure, core temperature, PaO2, PaCO2, pH and glycemia were measured before, during and after MCAO. Neurological examination and infarct volume measurements were carried out 3 days after the ischemic insult. Dietary isoflavones (both glycosides and aglycones) were measured by high-performance liquid chromatography. Neither pre-ischemic, intra-ischemic nor post-ischemic CP values were significantly different between the soy-based diet and the isoflavone-free diet groups. Animals fed with the soy-based diet showed an infarct volume of 122 +/- 20.2 mm3 (19 +/- 3.3% of the whole ipsilateral hemisphere volume). In animals fed with the isoflavone-free diet the mean infarct volume was significantly higher, 191 +/- 26.7 mm3 (28 +/- 4.1%, P < 0.05). Neurological examination revealed significantly higher impairment in the isoflavone-free diet group compared with the soy-based diet group (3.3 +/- 0.5 vs. 1.9 +/- 0.5, P < 0.05). These results demonstrate that dietary isoflavones improve stroke outcome after transient focal cerebral ischemia in such a way that a higher dietary isoflavone content results in a lower infarct volume and a better neurological status.     

Intracerebral adenosine infusion improves neurological outcome after transient focal ischemia in rats

Second Institute of New Drug Research, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan In order to elucidate the role of adenosine in brain ischemia, the possible protective effects of adenosine on ischemic brain injury were investigated in a rat model of brain ischemia both in vitro and in vivo. Exogenous adenosine dose-dependently rescued cortical neuronal cells from injury after glucose deprivation in vitro. Adenosine (1 mM) also significantly reduced hypoglycemia/hypoxia-induced glutamate release from the hippocampal slice. In a rat model of transient middle cerebral artery occlusion (MCAO), extracellular adenosine concentration was increased immediately after occlusion, and then returned to the baseline by 30 min after reperfusion. Adenosine infusion through a microdialysis probe into the ipsilateral striatum (1 mM adenosine, 2 microl min(-1), total 4.5 h from the occlusion to 3 h after reperfusion) showed a significant improvement in the neurological outcome, and about 25% reduction of infarct volume, although the effect did not reach statistical significance, compared with the vehicle-treated group at 20 h after 90 min of MCAO. These results demonstrated the neuroprotective effect of adenosine against ischemic brain injury both in vitro and in vivo, suggesting the possible therapeutic application of adenosine regulating agents, which inhibit adenosine uptake or metabolism to enhance or maintain extracellular endogenous adenosine levels, for stroke treatment.     

Effects of tetrahydrobiopterin on cerebral infarction after transient focal ischemia in rats

Abstract

Objectives: The present study investigated the effects of tetrahydrobiopterin (BH4) on cerebral infarction after transient focal ischemia in rats.

Methods: Focal ischemia (1·5 hours) was created in male Sprague-Dawley rats (250-280 g) by middle cerebral artery occlusion. Some rats were treated with 20 mg/kg tetrahydrobiopterin by intraperitoneal injection 30 minutes before reperfusion. At 2, 6, and 12 hours of reperfusion, the brains were harvested for the nitric oxide synthase (NOS) activity and nitric oxide (NO) level assays. At 12 hours of reperfusion, the brains were harvested for infarct size measurement.

Results: NOS activity and NO level were all augmented after reperfusion. BH4 treatment significantly further increased NOS activity and NO level. Cerebral infarct size was significantly bigger in BH4 treatment group compared to that in no treatment group.

Conclusions: The data indicate that BH4 enhances cerebral infarction after transient focal ischemia in rats, through NOS and NO pathway.     

Reduction of infarct size by the NO donors sodium nitroprusside and spermine/NO after transient focal cerebral ischemia in rats

Nitric oxide (NO) plays a dual role (neuroprotection and neurotoxicity) in cerebral ischemia. NO promoting strategies may be beneficial shortly after ischemia. Therefore, we have studied the hemodynamic and possible neuroprotective effects of two NO donors, the classical nitrovasodilator sodium nitroprusside (SNP) and the NONOate spermine/NO, after transient focal cerebral ischemia in rats. Parietal cortical perfusion was measured by laser-Doppler flowmetry. The effects of increasing intravenous doses (10-300 microgram) of sodium nitroprusside and spermine/NO on cortical perfusion and arterial blood pressure were assessed. Transient (2 h) focal cerebral ischemia was carried out by the intraluminal thread method. The effects of intraischemic intravenous infusion of SNP (0.11, 1.1 mg/kg) and spermine/NO (0.36, 3.6 mg/kg) on hemodynamic parameters and infarct size developed after 1 week reperfusion were assessed. In control conditions, SNP and, to a lesser extent, spermine/NO induced dose-dependent hypotension and concomitant reduction in cortical perfusion. In focal cerebral ischemia, infusion of SNP (0.11 mg/kg) and spermine/NO (0.36, 3.6 mg/kg) reduced the infarct size. In the case of spermine/NO, cortical perfusion was maintained above the control levels during the ischemic insult. No significant hypotension was elicited by NO donors at the dose-ratios infused. In conclusion, brain damage induced by transient focal ischemia is reduced by intravenous NO donors. Neuroprotective effects of spermine/NO are due at least in part to improvement of brain perfusion, while sodium nitroprusside must provide direct cytoprotection. These results give further support to the protective effect of NO in the early stages of cerebral ischemia and point to the therapeutic potential of NONOates in the management of brain ischemic damage.     

Hyperbaric oxygen reduces basal lamina degradation after transient focal cerebral ischemia in rats

Hyperbaric oxygen (HBO) has been shown to preserve the integrity of the blood-brain barrier after cerebral ischemia. However, the underlying molecular mechanisms are currently unknown. We examined the effect of HBO on postischemic expression of the basal laminar component laminin-5 and on plasma matrix metalloproteinase-9 (MMP) levels. Wistar rats underwent occlusion of the middle cerebral artery (MCAO) for 2 h. With a delay of 45 min after filament introduction, animals breathed either 100% O2 at 1.0 atmosphere absolute (ata; NBO) or at 3.0 ata (HBO) for 1 h in an HBO chamber. Laminin-5 expression was quantified on immunohistochemical sections after 24 h of reperfusion. Plasma MMP-9 levels were measured using gelatin zymography before MCAO as well as 0, 6 and 24 h after reperfusion. Immunohistochemistry 24 h after ischemia revealed a decrease of vascular laminin-5 staining in the ischemic striatum to 43 +/- 26% of the contralateral hemisphere in the NBO group which was significantly attenuated to 73 +/- 31% in the HBO group. Densitometric analysis of zymography bands yielded significantly larger plasma MMP-9 levels in the NBO group compared to the HBO group 24 h after ischemia. In conclusion, HBO therapy attenuates ischemic degradation of cerebral microvascular laminin-5 and blocks postischemic plasma MMP-9 upregulation.