神经调节素对大鼠脑缺血再灌注损伤AQP-4及GFAP表达的影响

应用线栓法经颈外-颈内动脉插线建立大脑中动脉闭塞再灌注(MCAO/R)大鼠动物模型,经颈内动脉单剂量注射1.5%神经调节素-1β(NRG-1β,0.3μg/kg)干预治疗。用干湿重法、免疫组化、免疫荧光双标记法和免疫印迹法观察NRG-1β对大鼠脑缺血再灌注损伤水通道蛋白(AQP-4)及胶质纤维酸性蛋白(GFAP)表达的影响。结果显示:随着缺血时间延长,对照组脑组织含水量逐渐增加,NRG-1β治疗能减少MCAO/R后脑组织含水量,与对照组相比,缺血1.5～2.0h组存在显著性差异(P<0.05)。脑缺血再灌注损伤可诱导脑组织AQP-4及GFAP表达,且随着缺血缺氧时间的延长,AQP-4及GFAP蛋白的表达逐渐增加。尽管它们均在胶质细胞中表达,但在脑组织中的分布略有不同。NRG-1β治疗可以增加二者在脑中的表达水平,与对照组相比具有显著性差异(P<0.01)。以上结果提示,NRG-1β可能通过激活内在的保护机制,增强胶质细胞的活性,从而抑制MCAO/R早期脑水肿的形成过程,改善神经元的生存环境,进而干扰脑缺血再灌注损伤的病理生理过程,对缺血性脑损伤具有积极的保护作用。     

Upregulation of erbB receptors in rat brain after middle cerebral arterial occlusion

We have previously demonstrated that neuregulin-1 (NRG-1) is upregulated and is neuroprotective in ischemic brain injury, however the expression and localization of its receptors during ischemia has not been investigated. Therefore, we used a rat middle cerebral artery occlusion (MCAO) model to examine the distribution of erbB receptors following ischemic stroke. Like neuregulin-1, we observed a dramatic induction of erbB4 in the peri-infarct regions of the ipsilateral cortex 24 h following MCAO. Using Fluoro-Jade B (FJB) staining as a marker of neurodegeneration, erbB4 was upregulated in FJB-positive cells, suggesting that erbB receptors are induced in injured neurons. The increase in erbB receptors was seen in neurons and a subpopulation of macrophages/microglia. There was no erbB co-localization with GFAP-positive astrocytes. These results demonstrate that erbB receptors are upregulated in neurons and macrophages/microglia following ischemic stroke and may be involved in neuroprotection and repair.     

Neuregulin-1beta modulates in vivo entorhinal-hippocampal synaptic transmission in adult rats

Neuregulin-1 (NRG-1) proteins and their erbB receptors are essential for neuronal development during embryogenesis and may contribute importantly to neuronal function in the adult brain. This study tests the hypothesis that NRG-1beta acts as a modulator of synaptic activity in the adult brain, specifically at hippocampal formation synapses. Adult, male Sprague-Dawley rats were anesthetized and a recording electrode with an attached stainless steel microinjector was stereotaxically positioned to record field potentials (fEPSP) in either the dentate gyrus or the cornu ammonis (CA) 1 field of the hippocampus. The entorhinal cortex was continuously stimulated via a paired stainless steel electrode. Microinjection of NRG-1beta significantly increased the slope of the fEPSP in the dentate gyrus in a dose-dependent manner. Compared with a low dose (20 nM), a high dose (100 nM) of NRG-1beta induced a shorter latency response that was of greater magnitude. Responses to NRG-1beta were abolished by pretreatment with a selective, reversible erbB tyrosine kinase inhibitor, PD158780 (100 microM). Further, PD158780 (100 microM) itself significantly decreased the entorhinal-dentate fESPS slope by about 15%. Neither equimolar (100 nM) nor hypermolar (100 microM) sucrose or heat-inactivated NRG-1beta (100 nM) significantly altered the entorhinal-dentate fEPSP slope. In contrast to its effect at the entorhinal-dentate synapse, NRG-1beta (100 nM) depressed, and PD158780 potentiated entorhinal-CA1 synaptic transmission. Thus, in adult rats NRG-1beta potentiates transmission at the entorhinal-dentate synapse but suppresses transmission at the entorhinal-CA1 synapse. These observations indicate that NRG-1 is not only a developmental growth factor, but also modifies synaptic transmission in adult rat brain.     

Neuroprotective potential of Piroxicam in cerebral ischemia: An in silico evaluation of the hypothesis to explore its therapeutic efficacy by inhibition of aquaporin-4 and acid sensing ion channel1a

Cerebral stroke is caused by acute interruption of the brain arterial blood supply and is one of the major health problems with no effective treatments so far apart from the thrombolytic recombinant tissue plasminogen activator (rt-PA), which must be administered within 3h of stroke onset. This emerges it as the third leading cause of mortality worldwide. Acidosis and brain edema are the prominent metabolic features of ischemic brain. The combined inhibition of aquaporin-4 (AQP4) and ASIC1a channels may offers a new neuroprotective approach in cerebral stroke management. Moreover, the combined inhibition of AQP4 and ASIC1a with NSAID remains unknown against neuroprotection in animal models of cerebral ischemia. NSAIDs are believed to act as a pharmacological molecule that reported to have an antioxidant and anti-inflammatory properties. Therefore, the target of the present in silico study was to determine the neuroprotective efficacy of Piroxicam, a NSAID in animal model of cerebral ischemia/reperfusion (I/R) injury and efforts were made to analyze its inhibitory effects on aquaporin-4 activation and ASIC1a channels mediated downstream survival/damage mechanisms. Thus we hypothesized that Piroxicam, a NSAID can act as a neuroprotective agent in animal model of cerebral ischemia/reperfusion (I/R) injury due to its inhibitory effects on aquaporin-4 channel and ASIC1a channels. It is indeed vital that we do not give up the fight to develop compounds to treat stroke despite the many years of setbacks. One of the mottos of our proposed hypothesis is to find out a successful modality of effective substantial treatment of brain stroke with other anti stroke therapeutics available till date.     

Protective effect of estrogen in endothelin-induced middle cerebral artery occlusion in female rats

Estrogen is a powerful endogenous and exogenous neuroprotective agent in animal models of brain injury, including focal cerebral ischemia. Although this protection has been demonstrated in several different treatment and injury paradigms, it has not been demonstrated in focal cerebral ischemia induced by intraparenchymal endothelin-1 injection, a model with many advantages over other models of experimental focal ischemia. Reproductively mature female Sprague–Dawley rats were ovariectomized and divided into placebo and estradiol-treated groups. Two weeks later, halothane-anesthetized rats underwent middle cerebral artery (MCA) occlusion by interparenchymal stereotactic injection of the potent vasoconstrictor endothelin 1 (180 pmoles/2 μl) near the middle cerebral artery. Laser-Doppler flowmetry (LDF) revealed similar reductions in cerebral blood flow in both groups. Animals were behaviorally evaluated before, and 2 days after, stroke induction, and infarct size was evaluated. In agreement with other models, estrogen treatment significantly reduced infarct size evaluated by both TTC and Fluoro-Jade staining and behavioral deficits associated with stroke. Stroke size was significantly correlated with LDF in both groups, suggesting that cranial perfusion measures can enhance success in this model.     

黑木耳多糖对抗大鼠慢性缺血性脑损伤

目的:观察黑木耳多糖(AAP)对大鼠慢性脑缺血损伤的保护作用,并探讨其相关机制。方法:雄性成年SD大鼠右侧永久性大脑中动脉栓塞(MCAO),建立慢性脑缺血模型,缺血后每天分别给予不同浓度的AAP灌胃4周。银杏叶提取物作为阳性对照。4周后采用Morris水迷宫检测大鼠学习记忆能力。取脑做冰冻切片进行Nissl染色,观察存活神经元数量,并测定脑组织丙二醛(MDA)水平和超氧化物歧化酶(SOD)活性。结果:AAP能明显改善脑缺血大鼠的学习记忆能力,增加海马神经元的存活数量,并且能够使脑组织长期MCAO诱导的MDA生成减少,使SOD活性显著升高。高剂量AAP(200mg/kg)的作用和银杏叶提取物相比更明显。结论:AAP明显减轻大鼠慢性脑缺血损伤,其作用与其对抗过氧化应激有关。     

Neuregulin attenuated cerebral ischemia-Creperfusion injury via inhibiting apoptosis and upregulating aquaporin-4

It has been demonstrated that neuregulin-1beta (NRG-1beta) plays a neuroprotective role in cerebral ischemic injury, however, its defined mechanisms and the perfect treatment window are still elusive. Therefore, we established the animal model of MCAO/R to evaluate cerebral damage. As a result, neurological deficit scores were increased, and a small infarction focus could be seen in ischemic cortex in the control group at ischemic 0.5h/reperfusion 24h. With the duration of ischemia time, deficit scores and infarction sizes obviously elevated in the control group. A large number of positive-apoptotic cells were widespread in the ischemic cortex. Simultaneously, the expression of AQP-4 mRNA and its protein increased. NRG-1beta significantly improved neurological function, decreased the infarction volume, and elevated the expression levels of AQP-4 compared with that in the control group. The therapeutic effect of NRG-1beta was notable, especially at the ischemic 1.0h. These results demonstrate that NRG-1beta might play a neuroprotective effect on cerebral ischemia and reperfusion by inhibiting mitochondrial apoptosis pathway and regulating the activation of AQP-4. The perfect treatment window is at ischemic 1.0h after MCAO.     

Differential Expression of PTEN in Normal Adult Rat Brain and Upregulation of PTEN and p‐Akt in the Ischemic Cerebral Cortex

The tumor suppressor phosphatase and tensin homologue (PTEN) is a protein and lipid phosphatase. PTEN mutations have been associated with a large number of human cancers. To understand the physiological role of PTEN in the brain and its relationship to Akt in ischemic injury, we first investigated the localization of PTEN immunoreactivity in the brains of normal adult rats using immunohistochemistry. We then detected the modulation of PTEN and p‐Akt following transient global ischemia by Western blot and immunohistochemistry analyses. Our observation of normal brains showed that PTEN was heterogeneously distributed in the cytoplasm, nuclei, and processes in different regions. It was shown immunohistochemically that PTEN was distributed differentially in rat brain, with the highest levels in the anterior olfactory nucleus, cerebral cortex, amygdaloid nucleus, hippocampus, Purkinje's cells, and several nuclei in the basal ganglia, thalamus, midbrain, and pons. After global cerebral ischemia, PTEN and p‐Akt immunoreactivities were increased in the cerebral cortex. This was accompanied by the nuclear translocation of p‐Akt. Double‐labeling experiments revealed that PTEN and p‐Akt were most likely localized to neurons. These results suggest a role for PTEN in normal adult brain and that the PTEN/Akt pathway may be involved in neuronal survival or plasticity after ischemic injury. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

低氧/复氧刺激对体外培养的胎鼠脊髓OPCs内NRG-1表达的影响

为明确脊髓少突胶质前体细胞(oligodendrocyte progenitor cells,OPCs)内neuregulin-1(NRG-1)的表达情况,并探讨脊髓缺血后OPCs中NRG-1的表达变化,本实验采用胎鼠(E11～13)脊髓神经干细胞定向诱导分化所获得的OPCs为实验材料,用免疫组织化学和Western blot方法,比较观察了正常培养和低氧/复氧培养的OPCs中NRG-1的表达。结果显示:(1)正常培养的OPCs中有基础性NRG-1的表达;(2)经低氧/复氧培养后,OPCs中NRG-1的表达2h内骤降,4h时陡升,表达量显著高于其他各时点,此后较迅速地下降,24h后趋于消失。由此推测,脊髓损伤后,OPCs能在缺血缺氧的较早期高表达NRG-1,这可能对于损伤组织的保护和修复有重要意义。     

Midkine and retinoic acid reduce cerebral infarction induced by middle cerebral artery ligation in rats

The present study investigates the neuroprotective effects of midkine (MK) and retinoic acid (RA) against ischemia in the CNS. Primary cortical neurons, derived from rat E15 embryos (DIV9), were treated with 9-cis-RA (9cRA), all-trans-RA (atRA) or vehicle. Using quantitative PCR, the level of MK mRNA was significantly increased at 4h after 9cRA application. The protective effect of RA and MK was also investigated in adult Sprague-Dawley rats. 9cRA, atRA, MK, or vehicle was injected into the lateral ventricle prior to a 60-min-MCA ligation. Pretreatment with 9cRA or MK attenuated cerebral infarction in stroke animals. Application of a similar dose of atRA did not reduce the size of infarction. In conclusion, our data suggest that 9cRA has neuroprotective effects against ischemia-related brain injury which may involve upregulation of midkine.     

脂质体介导的VEGF质粒对成年大鼠脑缺血再灌注后大脑皮质内P-MeCP2表达的影响

目的观察脂质体介导的VEGF质粒对成年大鼠脑缺血再灌注后大脑神经元phosphorylation of methyl-CpG binding protein 2 (P-MeCP2)表达的影响,探讨VEGF促进缺血损伤后神经元新生的可能机制,进一步为VEGF治疗缺血性脑中风提供实验和理论依据。方法 30只SD大鼠,随机分为假手术组(sham组)、质粒组(VEGF组)和对照质粒组(vehicle组),采用左侧大脑中动脉线栓(MCAO)模型,侧脑室给药,免疫印迹、免疫荧光三标染色及激光共聚焦扫描技术等方法检测P-MeCP2的表达。结果再灌2周VEGF质粒组P-MeCP2、BrdU和NeuN同时表达于缺血侧大脑皮质神经元,而缺血对侧没有找到,VEGF质粒组皮质内P-MeCP2的表达较对照组、假手术组显著增高(p<0.01)。结论脂质体介导的VEGF质粒能促进成年大鼠脑缺血神经元的新生,可能与上调P-MeCP2的表达有关。     

BMP7 reduces synergistic injury induced by methamphetamine and ischemia in mouse brain

Previous studies have indicated that methamphetamine (MA) potentiates neurodegeneration induced by ischemia in brain. We, and others, have reported that bone morphogenetic protein 7 (BMP7) is protective against MA and ischemic brain injury. The purpose of this study is to examine whether BMP7 reduces synergistic injury induced by both MA and cerebral ischemia. Adult CD-1 mice were treated with MA (4x 10mg/kg, each dose 2h apart) or saline. Using the quantitative real time polymerase chain reaction, we found that MA suppressed the expression of BMP7 mRNA in the cerebral cortex 1 day after injection. Ischemic and reperfusional injuries were introduced by ligation of the right middle cerebral artery for 90min after MA injection. Animals were sacrificed for caspase-3/7 activity assay and tri-phenyl-tetrazolium chloride staining at 1h and 2 days after reperfusion, respectively. Cerebral infarction and caspase-3/7 activity were enhanced in the stroke animals pretreated with MA; both responses were attenuated by pretreatment with BMP7. In conclusion, our data suggest that MA facilitates cerebral infarction after ischemia possibly mediated, in part, through the suppression of BMP7.     

Rapamycin alleviates brain edema after focal cerebral ischemia reperfusion in rats

Brain edema is a major consequence of cerebral ischemia reperfusion. However, few effective therapeutic options are available for retarding the brain edema progression after cerebral ischemia. Recently, rapamycin has been shown to produce neuroprotective effects in rats after cerebral ischemia reperfusion. Whether rapamycin could alleviate this brain edema injury is still unclear. In this study, the rat stroke model was induced by a 1-h left transient middle cerebral artery occlusion using an intraluminal filament, followed by 48?h of reperfusion. The effects of rapamycin (250?μg/kg body weight, intraperitoneal; i.p.) on brain edema progression were evaluated. The results showed that rapamycin treatment significantly reduced the infarct volume, the water content of the brain tissue and the Evans blue extravasation through the blood–brain barrier (BBB). Rapamycin treatment could improve histological appearance of the brain tissue, increased the capillary lumen space and maintain the integrity of BBB. Rapamycin also inhibited matrix metalloproteinase 9 (MMP9) and aquaporin 4 (AQP4) expression. These data imply that rapamycin could improve brain edema progression after reperfusion injury through maintaining BBB integrity and inhibiting MMP9 and AQP4 expression. The data of this study provide a new possible approach for improving brain edema after cerebral ischemia reperfusion by administration of rapamycin.     

Effects of hyperthermia on infarct volume in focal embolic model of cerebral ischemia in rats

Hyperthermia worsens outcome of stroke in patients and also in animal models. In the present study, we tested the effects of hyperthermia in a focal embolic model of cerebral ischemia in rats. Focal ischemic injury was induced by embolizing a preformed clot into the middle cerebral artery. Hyperthermia significantly increased the brain infarct volume as compared to the normothermic controls measured at 48 h following the ischemic insult. Neurological deficits were also significantly elevated in the animals undergoing hyperthermic treatment. In addition, hyperthermia also significantly increased mortality. Our data thus show that hyperthermia exacerbates neuronal injury in the focal embolic model of cerebral ischemia in rats.     

Angiotensin type 1 receptor blockage improves ischemic injury following transient focal cerebral ischemia

Following cerebral ischemia, i.v. infusion of angiotensin II increases cerebral edema and mortality. Angiotensin type 1 receptor blockage should therefore improve acute cerebral ischemia. Left middle cerebral artery occlusion (120 min) followed by reperfusion was performed with the thread method under halothane anesthesia in Sprague-Dawley rats. Olmesartan (angiotensin type 1 receptor blocker; 0.01 or 0.1mumol/kg/h) was infused i.p. for 7 days following middle cerebral artery occlusion followed by reperfusion. Stroke index score, infarct volume, specific gravity, and brain angiotensin II and matrix metalloproteinases were quantified in the ischemic and non-ischemic hemispheres. Olmesartan treatment improved stroke index score, infarct volume, and cerebral edema in our cerebral ischemia model. In particular, stroke index score, infarct volume, and cerebral edema were reduced even with a low dose of olmesartan that did not decrease blood pressure. Paralleling these effects on cerebral ischemia, olmesartan treatment also reduced the reactive upregulation in brain angiotensin II, matrix metalloproteinase-2, matrix metalloproteinase-9, and membrane type 1-matrix metalloproteinase in the ischemic area. Angiotensin type 1 receptor stimulation may be one of the important factors that cause cerebral edema following cerebral ischemia, and that its inhibition may be of therapeutic advantage in cerebral ischemia.     

Expression of interleukin-17 in ischemic brain tissue

Ischemic brain injury is acute local inflammation, leading to accumulation of pro-inflammatory cytokines. Cytokines influence the recruitment of leucocytes and play a key role in the inflammatory injury processes. Recently, a number of studies have demonstrated a close relationship between brain ischemia and cytokines. Interleukin-17 (IL-17) is a newly identified T-cell-specific cytokine. In this study, we evaluated the source and the action of IL-17 over the course of cerebral ischemia in rats (Sprague-Dawley) and humans. The levels of IL-17 in the ischemic hemisphere of the human brain, which was removed at necropsy, were assayed immunohistochemically. In rats, permanent middle cerebral artery occlusion (pMCAO) was obtained by inserting nylon monofilament into the right external carotid artery, occluding the right middle cerebral artery. The expression of IL-17 mRNA in rat was assayed using oligoprobe in situ hybridization. IL-17 production by neuroglial cells was assayed by double-staining using antibody glial fibrillary acidic protein (GFAP) and antibody IL-17. Levels of IL-17 were elevated in the ischemic hemispheres of human brain compared with the opposite normal hemispheres and peaked at days 3-5 after brain ischemia. The IL-17-positive cells were found in the ischemic lesion region. IL-17 mRNA was also elevated in ischemic hemispheres of pMCAO-operated rats, which were slightly elevated after 1 h and peaked at 6 days. IL-17 and GFAP double-stained were extensive in rat ischemic hemisphere. The ischemia-induced IL-17 expression in human brain reported here for the first time was very similar to that in rat model except that the peak was slightly earlier. We found for the first time that IL-17 was involved in an intense inflammatory reaction of brain ischemic injury in human. In pMCAO-operated rats, our findings suggest that IL-17 is produced by the neuroglial cells in the brain region undergoing ischemic insult. We suggest that in additional to T cells the neuroglial cell may be another cellular origin of IL-17 in later progression of brain ischemia.     

Chaperonin GroEL and its mutant D87K protect from ischemia in vivo and in vitro

Protein aggregation and misfolding are central mechanisms of both acute and chronic neurodegeneration. Overexpression of chaperone Hsp70 protects from stroke in animal and cell culture models. Although it is accepted that chaperones protect cells, the mechanism of protection by chaperones in ischemic injury is poorly understood. In particular, the relative importance of preventing protein aggregation compared to facilitating correct protein folding during ischemia and recovery is not known. To test the importance of protein folding and minimize interaction with co-chaperones we studied the bacterial chaperonin GroEL (HSPD1) and a folding-deficient mutant D87K. Both molecules protected cells from ischemia-like injury, and reduced infarct volume and improved neurological outcome after middle cerebral artery occlusion in rats. Protection was associated with reduced protein aggregation, assessed by ubiquitin immunohistochemistry. Marked neuroprotection by the folding-deficient chaperonin demonstrates that inhibition of aggregation is sufficient to protect the brain from ischemia. This suggests that strategies to maintain protein solubility and inhibit aggregation in the face of acute insults such as stroke may be a useful protective strategy.     

Atorvastatin protects rat brains against permanent focal ischemia and downregulates HMGB1, HMGB1 receptors (RAGE and TLR4), NF-κB expression

Inflammatory processes play a key, mainly detrimental role in the pathophysiology of ischemic stroke. Currently, HMGB1-induced NF-κB activation pathway has been recognized as a key contributor to the proinflammatory response. It has been proved that chronic administration and pre-treatment with statins could protect brain tissue against ischemic injury. However, little is known about the effects of statins in the acute phase after cerebral ischemia. Thus, this study investigated the atorvastatin's protective role and the underlying mechanisms in cerebral ischemia. After middle cerebral artery occlusion (MCAO), atorvastatin was administered immediately. We found that atorvastatin dramatically improved neurological deficits, reduced brain water contents and infarct sizes at 24 h after stroke. Moreover, the over-expression of HMGB1, RAGE, TLR4 and NF-κB induced by ischemia was significantly attenuated by atorvastatin.     

中枢神经系统损伤过程中Nogo-A降解片段的检测

目的观察大鼠脊髓横断、脑缺血和海人藻酸(KA)注射所致的脑损伤模型Nogo-A的降解情况，以推测Nogo-A降解片段产生的机制和可能的作用。方法制作大鼠脊髓横断、脑缺血和KA注射所致的脑损伤模型，Western免疫印迹方法检测Nogo-A降解片段。结果大鼠脊髓横断和脑缺血模型均检测到Nogo-A降解片段，而KA注射所致的脑损伤模型未发现Nogo-A降解片段。结论CNS机械性和缺血性损伤可能通过某些机制导致Nogo-A发生降解。     

Iptakalim protects against hypoxic brain injury through multiple pathways associated with ATP-sensitive potassium channels

The rapid and irreversible brain injury produced by anoxia when stroke occurs is well known. Cumulative evidence suggests that the activation of neuronal ATP-sensitive potassium (KATP) channels may have inherent protective effects during cerebral hypoxia, yet little information regarding the therapeutic effects of KATP channel openers is available. We hypothesized that pretreatment with a KATP channel opener might protect against brain injury induced by cerebral hypoxia. In this study, adult Wistar rats were treated with iptakalim, a new KATP channel opener, which is selective for SUR2 type KATP channels, by intragastric administration at doses of 2, 4, or 8 mg/kg/day for 7 days before being exposed to simulated high altitude equivalent to 8000 m in a decompression chamber for 8 h leading to hypoxic brain injury. By light and electron microscopic images, we observed that hypobaric hypoxia-induced brain injury could be prevented by pretreatment with iptakalim. It was also observed that the permeability of the blood-brain barrier, water content, Na+ and Ca2+ concentration, and activities of Na+,K+-ATPase, Ca2+-ATPase and Mg2+-ATPase in rat cerebral cortex were increased and the gene expression of the occludin or aquaporin-4 was down- or upregulated respectively, which could also be prevented by the pretreatment with iptakalim at doses of 2, 4, or 8 mg/kg in a dose-dependent manner. Furthermore, we found that in an oxygen-and-glucose-deprived model in ECV304 cells and rat cortical astrocytes, pretreatment with iptakalim significantly increased survived cell rates and decreased lactate dehydrogenate release, which were significantly antagonized by glibenclamide, a K(ATP) channel blocker. We conclude that iptakalim is a promising drug that may protect against brain injury induced by acute hypobaric hypoxia through multiple pathways associated with SUR2-type K(ATP) channels, suggesting a new therapeutic strategy for stroke treatment.