Facilitated angiogenesis induced by heme oxygenase-1 gene transfer in a rat model of hindlimb ischemia

Heme oxygenase-1 (HO-1) is an inducible form of heme oxygenase that catabolizes heme to carbon monoxide, biliverdin, and ferrous iron. We have investigated whether HO-1 can induce angiogenic effects in vivo. Rats were subjected to a bolus injection of either wild type adenovirus (ad-wt) or adenovirus encoding HO-1 (ad-HO-1) through the right femoral artery, which was then removed immediately. HO-1 gene transfer resulted in about a sixfold increase in HO-1 protein levels as compared to the non-treated animals. The increase in both blood flow and capillary density was significantly greater in the ischemic hindlimbs that had been injected with ad-HO-1 than in those injected with ad-wt. These angiogenic effects of ad-HO-1 infection could be completely abolished by treating the animals with the HO inhibitor, zinc protoporphyrin, indicating that they were specifically due to the expression of HO-1. Thus, HO-1 gene transfer improves the blood flow in ischemic hindlimb, at least in part, via angiogenesis facilitated by the induction of this molecule.     

双侧迷走神经和颈总动脉联合切断法建立急性脑缺血大鼠模型

目的建立一种操作简单的急性脑缺血动物模型。方法取雄性Wistar大鼠40只,体重200～230g,手术前禁食12 h,自由饮水,随机分为对照组A、B、C组及模型D组,共4组,每组10只。即A组:假手术组,仅切开颈部两侧皮肤,分离双侧颈总动脉和迷走神经,不切断,然后缝合;B组:仅切断双侧颈部迷走神经;C组:结扎并切断双侧颈总动脉(CCA);D组:联合组,即结扎并切断双侧颈总动脉,同时切断双侧颈部迷走神经。观察各组大鼠手术后的脑缺血症状,记录各组大鼠在8h内的死亡情况,超过8h死亡的动物按8h计,计算死亡率和死亡时间。结果 A组大鼠没有脑缺血症状,无死亡;B组大鼠无脑缺血症状,呼吸变慢变深,心率血压上升,但无死亡;C组大鼠部分出现脑缺血症状,眼睑下垂,活动能力低下,自发运动减少,有些大鼠术后自发运动增加,在8 h内无死亡;D组大鼠大多数出现较为明显的脑缺血症状,在8 h内全部死亡。结论采取同时结扎并切断大鼠双侧颈总动脉和双侧颈部迷走神经的方法,可以建立急性脑缺血大鼠动物模型,此方法具有手术简单,成功率高,术后动物缓慢死亡的特点。     

The effect of convulsions induced by flurothyl on ribonucleic acid synthesis in rat cerebral cortex during the recovery phase.

The effect of convulsions, induced by flurothyl, on RNA synthesis in purified unfractionated nuclei and the cytoplasm of rat cerebral cortex was studied by using a double-label technique involving injection of [3H]- and [14C]-orotate intracisternally. 2. Intact RNA was extracted in 80% yield by an enzymic method by using a proteinase in the presence of sodium dodecyl sulphate followed by deoxyribonuclease. Electrophoresis on 1.5% polyacrylamide-0.5% agarose gels revealed the presence of giant nuclear RNA of size up to approx. 300X 10(6) daltons and mRNA of maximal mol.wt. 9 X 10(6)-16 X 10(6). 3. Nuclear RNA synthesis was decreased to 27% in the first 15 min after convulsions but rapidly increased, so that at 1 1/2 h it was 124% of the control, and at 6 h 147%. 4. Labelling of cytoplasmic RNA was decreased to 15% at 15 min after convulsions but had not recovered to control values by 6 h. 5. Analysis of radioactive gel patterns and the 3H/14C ratio at six time-points (15 min-6h) showed that the major effect was inhibition of the processing of heterogeneous nuclear RNA resulting in a sharp decline in the export of newly synthesized RNA from the nucleus. 6. Cytoplasmic RNA patterns indicated that specific messengers were synthesized at different times during the recovery of the cell after convulsions.     

The protective role of verbenalin in rat model of focal cerebral ischemia reperfusion

To investigate the protective mechanism of verbenalin on cerebral ischemia-reperfusion injury. Middle cerebral artery occlusion in the left hemisphere was induced in rats by filament insertion, and rat model of focal cerebral ischemia-reperfusion was established. The high, medium and low dose of verbenalin groups were injected in the tail vein of corresponding drugs 10?min before reperfusion, and submitted for 22?h of reperfusion after the operation. Mortality rate was then calculated, and neurological deficits of rats were scored. The serum of rats was got to determine the S-100β protein level, and the brain tissue was removed to determine the levels of Bax, Bcl-2, Caspase-3 and ATPase. TTC staining was performed on the brain tissue to calculate the percentage of cerebral infarct size. Changes in brain tissue morphology were observed. Rat model of focal cerebral ischemia-reperfusion was successfully replicated. In groups that have taken different doses of verbenalin, the mortality rate, neurological deficit score and the percentage of cerebral infarction size were significantly reduced, and the levels of Bax, Caspase-3, S-100β level of the serum in the brain tissue were also significantly reduced. Increases in the levels of Bcl-2 and ATPase in brain tissue and improvement of pathological damage of hippocampus and cortex were observed. Verbenalin can inhibit the expression of apoptosis genes, promote the expression of anti-apoptosis genes, improve brain microcirculation and energy metabolism, hence reducing cerebral ischemia-reperfusion injury.     

Diethylmaleate decreased ascorbic acid release induced by cerebral ischemia in cerebral cortex of the anesthetized rat

The effect of diethylmaleate administration on ascorbic acid release following cerebral ischemia was investigated in anesthetized rat brain cortex. Cerebral ischemia, induced by ligating bilateral common carotid arteries and unilateral middle cerebral artery, significantly increased the extracellular ascorbic acid levels. Diethylmaleate (4 mmoles/kg, i.p.), which has been shown in earlier studies to decrease the ischemia-induced glutamate release, significantly reduced the ischemia-induced ascorbic acid release. The ischemia-induced ascorbic acid release was unaffected by perfusing NMDA receptor antagonist MK 801 (75 microM). Additionally, elevated extracellular glutamate levels, achieved by either externally applied glutamate solutions or by perfusing L-trans-pyrrolidine-2,4-dicarboxylate (PDC) (31.4 mM and 15.7 mM) to inhibit the glutamate uptake transporter, also significantly increased the extracellular ascorbic acid levels. These results suggested that ascorbic acid release in cerebral ischemia might be related to the elevated extracellular glutamate levels, which occurs following cerebral ischemia.     

Effects of chronic L-NAME treatment on rat focal cerebral ischemia and cerebral vasoreactivity.

Nitric oxide has been shown to be involved in the regulation of cerebral blood flow and the consequences of cerebral ischemia. Short-term inhibition of its synthesis induces hypertension and increases the cortical infarct volume in focal ischemia. Our purpose was to investigate the influence of the long-term inhibition of nitric oxide synthase on infarct volume due to middle cerebral artery (MCA) occlusion and on the reactivity of cerebral arteries. Sprague Dawley rats were given N(omega)-nitro-L-arginine methyl ester (L-NAME) for 2 or 6 weeks and compared to untreated normotensive rats and untreated spontaneously hypertensive rats (SHRs). Brain nitric oxide synthase activity was measured by the 14C-L-arginine assay. Arterial blood pressure was measured in each group. Independently, the reactivity of MCA trees was studied in vitro by a perfusion technique. Cortical infarct volume was not significantly modified by either 2-week or 6-week L-NAME treatment, despite induced hypertension, whereas it was significantly higher in SHRs than in normotensive rats. The reactivity of the MCA tree was significantly affected by the treatment with a clearcut time-dependency. Compared to normotensive controls, contractility to noradrenaline and serotonin was reduced, more severely at 6 weeks, and while dilatation to acetylcholine and nitroprusside was moderately reduced at 6 weeks, dilatation to papaverine was then increased. A major difference of treated animals compared to SHRs was the decreased response to 5-hydroxytryptamine. We conclude that infarct expansion may be limited in treated animals by a progressive reduction in cerebral artery response to vasoconstrictory neurotransmitters, concomitant with augmented non-guanylate cyclase dilator responses (cf. papaverine) and some recovery of dilatation to acetylcholine.     

Involvement of free radicals in cerebral vascular reperfusion injury evaluated in a transient focal cerebral ischemia model of rat

Free radicals have been suggested to be largely involved in the genesis of ischemic brain damage, as shown in the protective effects of alpha-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic cerebral injury. In the present study, the effects of PBN as well as MCI-186, a newly-developed free radical scavenger, and oxypurinol, an inhibitor of xanthine oxidase, were evaluated in a rat transient middle cerebral aretery (MCA) occlusion model to clarify the possible role of free radicals in the reperfusion injury of brain. The volume of cerebral infarction, induced by 2-h occlusion and subsequent 2-h reperfusion of MCA in Fisher-344 rats, was evaluated. The administration of PBN (100 mg/kg) and MCI-186 (100 mg/kg) just before reperfusion of MCA significantly reduced the infarction volume. In contrast, oxypurinol (100 mg/kg) failed to show any preventive effect on the infarction. These results suggest that free radical formation is involved in the cerebral damage induced by ischemia-reperfusion of MCA, and that hydroxyl radical is responsible for the reperfusion injury after transient focal brain ischemia. It is also suggested that xanthine oxidase is not a major source of free radicals.     

Neurovascular protection conferred by 2-BFI treatment during rat cerebral ischemia

Stroke is caused by vascular dysfunction and currently there are no effective therapeutics to stroke induced brain damage. In contrast to an intense emphasis on neuroprotection, relatively few studies have addressed means of vascular protection in cerebral ischemia. Here we discovered that the ligand to immidazolin receptor, 2-BFI, not only provided potent neuroprotection during middle cerebral artery occlusion in rat, which confirmed our previous reports, but also protected the integrity of the cerebral vasculature. Treatment with 2-BFI twice daily after the occlusion of the middle cerebral artery for 14d significantly improved the neurological deficits, reduced brain infarction, and importantly, protected the cerebral vasculature as evidenced by the increased expression of an endothelial marker, von Willebrand factor, and better preservation of the cerebral vasculature, as viewed under a confocal microscope on rat brain perfused with FITC-labeled dextran. These results indicated that 2-BFI contributes to protection of neurovasculature. Understanding the molecular mechanisms could eventually lead to development of more effective therapies for stroke.     

线栓法制备不同体重KM小鼠局灶性脑缺血/再灌注模型的建立及评价

目的线栓法制备小鼠脑缺血/再灌注模型,并探讨影响该模型稳定性的因素。方法 KM小鼠100只,雌雄各半,体重20～39 g,分别将雌雄KM小鼠随机分为假手术组(n=20)和不同体重实验组(n=80)。其中实验组按小鼠体重分为以下8组:A组(♂,20～24 g)、B组(♂,25～29 g)、C组(♂,30～34 g)、D组(♂,35～39g),E组(♀,20～24 g)、F组(♀,25～29 g)、G组(♀,30～34 g)、H组(♀,35～39 g),每组10只。线栓栓塞法制备局灶性脑缺血/再灌注模型,选用单丝尼龙线(直径0.128～0.180 mm),液体石蜡处理浸泡后,从右侧颈总动脉进线,阻塞大脑中动脉血流,再灌注时拔出线栓,并对模型进行评价,采用的主要观察指标是神经功能损伤评分和2,3,5-三苯基氯化四氮唑(TTC)染色。结果同假手术相比,不同体重实验组小鼠脑缺血再灌注后,出现神经行为功能异常,TTC染色脑组织显示出明显的梗塞灶。与C、D组相比,A、B两组行为学评分以及梗死灶体积显著性增加(P<0.01);与G、H组相比,E、F两组的行为学评分以及梗死灶体积也显著性增加(P<0.01,P<0.05)。雄性KM小鼠与雌性相比,成模率较高且死亡率相对较低。此外,A组与E组相比,其行为学评分显著增加(P<0.01);B组与F组相比,小鼠行为学评分以及脑梗塞体积显著增加(P<0.01)。结论体重在25 g左右的雄性KM小鼠制备模型成功率较高,死亡率较低,适合建立小鼠局灶性脑缺血/再灌注模型。     

Neuroprotective effect of pioglitazone on acute phase changes induced by partial global cerebral ischemia in mice

Present study was carried out to investigate the possible neuroprotective effect of pioglitazone, an antidiabetic agent, peroxisome proliferator-activated receptor gamma (PPARgamma) agonist on acute phase changes in mice model of cerebral ischemia induced by Bilateral Common Carotid artery Occlusion (BCCAO). BCCAO model was used to induce partial global cerebral ischemia. BCCAO induced significant brain infarct size and edema in saline treated control group along with high increase in oxidative stress showed by increase lipid peroxidation and decreased levels of antioxidants like superoxide superoxide dismutage, catalase, glutathione peroxidase. Pioglitazone (20 mg/kg, orally) administration showed neuroprotective effects by reducing cerebral infarct size significantly as compared to control group. Postischemic seizure susceptibility was also reduced as number of positive responders decreased to a significant number. Brain edema was subsided to a significant level. Pioglitazone reduced the plasma TNF-alpha levels as compared to ischemia group significantly. Pioglitazone treatment also improved all the antioxidants levels showing activity against oxidative stress induced by BCCAO. Pioglitazone showed neuroprotection against ischemic insult suggesting the role of PPARgamma agonist in neuroprotective agents.     

Minocycline inhibits 5-lipoxygenase expression and accelerates functional recovery in chronic phase of focal cerebral ischemia in rats

AimsWe previously reported that minocycline attenuates acute brain injury and inflammation after focal cerebral ischemia, and this is partly mediated by inhibition of 5-lipoxygenase (5-LOX) expression. Here, we determined the protective effect of minocycline on chronic ischemic brain injury and its relation with the inhibition of 5-LOX expression after focal cerebral ischemia.Main methodsFocal cerebral ischemia was induced by 90 min of middle cerebral artery occlusion followed by reperfusion for 36 days. Minocycline (45 mg/kg) was administered intraperitoneally 2 h and 12 h after ischemia and then every 12 h for 5 days. Sensorimotor function was evaluated 1–28 days after ischemia and cognitive function was determined 30–35 days after ischemia. Thereafter, infarct volume, neuron density, astrogliosis, and 5-LOX expression in the brain were determined.Key findingsMinocycline accelerated the recovery of sensorimotor and cognitive functions, attenuated the loss of neuron density, and inhibited astrogliosis in the boundary zone around the ischemic core, but did not affect infarct volume. Minocycline significantly inhibited the increased 5-LOX expression in the proliferated astrocytes in the boundary zone, and in the macrophages/microglia in the ischemic core.SignificanceMinocycline accelerates functional recovery in the chronic phase of focal cerebral ischemia, which may be partly associated with the reduction of 5-LOX expression.     

Ischemic post-conditioning protects brain and reduces inflammation in a rat model of focal cerebral ischemia/reperfusion

Ischemic post-conditioning (Post-cond) is a phenomenon in which intermittent interruptions of blood flow in the early phase of reperfusion can protect organ from ischemia/reperfusion (I/R) injury. Recent studies demonstrated ischemic Post-cond reduced infarct size in cerebral I/R injury. However, the molecular mechanisms underlying this phenomenon are not completely understood. As inflammation is known to be detrimental to the neurological outcome during the acute phase after stroke, we investigated whether ischemic Post-cond played its protective role in preventing post-ischemic inflammation in the rat middle cerebral artery occlusion model. Rats were treated with ischemic Post-cond after 60 min of occlusion (beginning of reperfusion). The infarct volume and myeloperoxidase activity were assessed at 24 h. The lipid peroxidation levels was evaluated by malondialdehyde assay and the expressions of interleukin-1β, tumor necrosis factor-α, and intercellular adhesion molecule 1 were studied by RT-PCR or western blotting. Ischemic Post-cond decreased myeloperoxidase activity and expressions of interleukin-1β, tumor necrosis factor-α, and intercellular adhesion molecule 1. Ischemic Post-cond also reduced infarct volume and lipid peroxidation levels. These findings indicated that ischemic Post-cond may be a promising neuroprotective approach for focal cerebral I/R injury and it is achieved, at least in part, by the inhibition of inflammation.     

Propofol post-conditioning induced long-term neuroprotection and reduced internalization of AMPAR GluR2 subunit in a rat model of focal cerebral ischemia/reperfusion

We previously reported that propofol (20 mg/kg/h) post-conditioning provided acute (up to 24 h) neuroprotection in rats with transient middle cerebral artery occlusion. In this study, we extend these data by examining long-term protection and exploring underlying mechanisms involving AMPA receptor GluR2 subunit internalization. Rats were treated with propofol 20 mg/kg/h after 60 min of occlusion (beginning of reperfusion for 4 h). Propofol post-conditioning reduced infarct volume and improved spatial memory deficiencies (up to 28 days) induced by ischemia/reperfusion injury. Additionally, Propofol post-conditioning promoted neurogenesis in the dentate gyrus of hippocampus, as measured by bromodeoxyuridine and neuron-specific nuclear protein immunofluorescence-double staining at day 28 after reperfusion. Finally, propofol post-conditioning increased the surface expression of AMPA receptor GluR2 subunit, thus inhibited the internalization of this part until 28 days after stroke. In conclusion, our data suggest that propofol post-conditioning provides long-term protection against focal cerebral ischemia/reperfusion injury in rats. Furthermore, we found that the inhibition of AMPA receptor GluR2 subunit internalization may contributed to this long-term neuroprotection.     

Estimation of time-dependent microRNA expression patterns in brain tissue,leukocytes, and blood plasma of rats under photochemically induced focal cerebral ischemia

miRNA expression over different time periods (24 and 48 h) using the quantitative RT-PCR and deep sequencing has been evaluated in a model of photochemically induced thrombosis. A combination of two approaches allowed us to determine the miRNA expression patterns caused by ischemia. Nine miRNAs, including let-7f-5p, miR-221-3p, miR-21-5p, miR-30c-5p, miR-30a-3p, miR-223-3p, miR-23a-3p, miR-22-5p, and miR-99a-5p, were differentially expressed in brain tissue and leukocytes of rats 48 h after onset of ischemia. In addition, six miRNAs were differentially expressed in the brain tissue and blood plasma of rats 24 h after exposure, among which miR-145-3p and miR-375-3p were downregulated and miR-19a-3p, miR-92a-3p, miR-188-5p, and miR-532-5p were upregulated. In our opinion, miR-188-5p and miR-532-5p may be considered to be new potential markers of ischemic injury. The level of miRNA expression tended to increase 48 h after the onset of ischemia in brain tissue and leukocytes, which reflects not only the local response in brain tissue due to inflammation, vascular endothelial dysfunction, and disorders of the permeability of the blood–brain barrier, but also the systemic response of the organism to multifactor molecular processes induced by ischemic injury.     

Neuroprotective effect of curcumin in middle cerebral artery occlusion induced focal cerebral ischemia in rats

Free radical induced neuronal damage is implicated in cerebral ischemia reperfusion (IR) injury and antioxidants are reported to have neuroprotective activity. Several in vitro and in vivo studies have proved the antioxidant potential of curcumin and its metabolites. Hence, in the present study the neuroprotective potential of curcumin was investigated in middle cerebral artery occlusion (MCAO) induced focal cerebral IR injury. 2 h of MCAO and 22 h of reperfusion resulted in the infarct volume of 210.39 +/- 31.25 mm3. Administration of curcumin 100 and 300 mg/kg, i.p. 30 min. after MCAO produced 37.23 +/- 5.10% and 46.39 +/- 10.23% (p < 0.05) reduction in infarct volume, respectively. Ischemia induced cerebral edema was reduced in a dose dependent manner. Curcumin at 300 mg/kg, i.p. produced 50.96 +/- 6.04% reduction in edema (p < 0.05) volume. Increase in lipid peroxidation after MCAO in ipsilateral and contralateral hemisphere of brain was observed, which was reduced by curcumin (300 mg/kg, i.p.)-treatment. Decrease in superoxide dismutase and glutathione peroxidase activity was observed in ipsilateral hemisphere of MCAO animal. Curcumin-treatment (300 mg/kg, i.p.) prevented IR injury mediated fall in glutathione peroxide activity. Peroxynitrite measured using rhodamine123 fluorescence and anti-nitrotyrosine immunofluorescence indicated increased peroxynitrite formation after IR insult. Curcumin-treatment reduced peroxynitrite formation and hence the extent of tyrosine nitration in the cytosolic proteins. These results suggest the neuroprotective potential of curcumin in cerebral ischemia and is mediated through its antioxidant activity.     

Nitric oxide down-regulates caveolin-1 expression in rat brains during focal cerebral ischemia and reperfusion injury

As a signalling molecule of the integral membrane protein family, caveolin participates in cellular signal transduction via interaction with other signalling molecules. The nature of interaction between nitric oxide (NO) and caveolin in the brain, however, remains largely unknown. In this study we investigated the role(s) of NO in regulating caveolin-1 expression in rat ischemic brains with middle cerebral artery occlusion (MCAO). Exposure to 1 h ischemia induced the increases in neuronal nitric oxide synthase (nNOS) and NO concentration with concurrent down-regulation of caveolin-1 expression in the ischemic core of rat brains. Subsequent 24 h or more reperfusion time led to an increase in inducible NOS (iNOS) expression and NO production, as well as a decline of caveolin-1 protein at the core and penumbra of the ischemic brain. Afterwards, NOS inhibitors and an NO donor were utilized to clarify the link between NO production and caveolin-1 expression in the rats with 1 h ischemia plus 24 h reperfusion. N(G)-nitro-l-arginine methyl ester (L-NAME, a non-selective NOS inhibitor), N(6)-(1-iminoethyl)-lysine (NIL, an iNOS inhibitor), and 7-nitroindazole (7-NI, a nNOS inhibitor) prevented the loss of caveolin-1 in the core and penumbra of the ischemic brain, whereas l-N(5)-(1-iminoethyl)-ornithine (L-NIO, an endothelial NOS inhibitor) showed less effect than the other NOS inhibitors. S-Nitroso-N-acetylpenicillamine (SNAP, a NO donor) down-regulated the expression of caveolin-1 protein in normal and ischemic brains. These results, when taken together, suggest that NO modulates the expression of caveolin-1 in the brain and that the loss of caveolin-1 is associated with NO production in the ischemic brain.