Neuroprotective effects of VEGF administration after focal cerebral ischemia/reperfusion: dose response and time window

Administration of vascular endothelial growth factor (VEGF) has been shown to increase cerebral blood flow and reduce neurological damage after experimental ischemic brain injury. The purpose of this study was to examine the optimal dose and time window for the neuroprotective effect of VEGF when administrated after focal ischemia/reperfusion injury in rabbits. Focal cerebral ischemia/reperfusion was induced by the middle cerebral artery occlusion (MCAO) method. In a dose response experiment, low (1.25 ng/μL), middle (2.5 ng/μL) and high (5.0 ng/μL) doses of VEGF were administered 2h after MCAO by the route of perifocal region. The VEGF at a dose of middle (2.5 ng/μL) displayed excellent effects on neuroprotective efficacy for focal cerebral ischemia/reperfusion injury. In another experiment, 2.5 ng/μL VEGF was administered at times varying from 2 to 8h after MCAO. Infarct volume, water content and neurological deficits were significantly reduced when VEGF was given at 2 and 3h after injury. The protective effect was less when the same dose was given at the later times. Thus, the present findings indicated that VEGF reduced ischemic neuronal danger with a therapeutic time window within the first 3h of transient MCAO and may be useful in the treatment of acute ischemic stroke in humans.     

TIPE2, a Novel Regulator of Immunity,Protects against Experimental Stroke

The inflammatory responses accompanying stroke are recognized to contribute to secondary ischemic injury. TIPE2 is a very recently identified negative regulator of inflammation that maintains immune homeostasis. However, it is unknown whether TIPE2 is expressed in the brain and contributes to the regulation of cerebral diseases. In this study, we explored the potential roles of TIPE2 in cerebral ischemia/reperfusion injury. TIPE2^−/− mice were used to assess whether TIPE2 provides neuroprotection following cerebral ischemia/reperfusion induced by middle cerebral artery occlusion (MCAO), and in vitro primary cerebral cell cultures were used to investigate the expression and regulation of TIPE2. Our results show that genetic ablation of the Tipe2 gene significantly increased the cerebral volume of infarction and neurological dysfunction in mice subjected to MCAO. Flow cytometric analysis revealed more infiltrating macrophages, neutrophils, and lymphocytes in the ischemic hemisphere of TIPE2^−/− mice. The responses to inflammatory cytokines and chemokines were significantly increased in TIPE2^−/− mouse brain after MCAO. We further observed that TIPE2 was highly induced in WT mice after cerebral ischemia and was expressed mainly in microglia/macrophages, but not in neurons and astrocytes. Finally, we found that regulation of TIPE2 expression was associated with NADPH oxidase activity. These findings demonstrate, for the first time, that TIPE2 is involved in the pathogenesis of stroke and suggest that TIPE2 plays an essential role in a signal transduction pathway that links the inflammatory immune response to specific conditions after cerebral ischemia. Targeting TIPE2 may be a new therapeutic strategy for stroke treatment.     

Atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in ischemic stroke

Statins have recently been shown to exert neuronal protection in ischemic stroke. Reactive oxygen species, specifically superoxide formed during the early phase of reperfusion, augment neuronal injury. NADPH oxidase is a key enzyme for superoxide production. The present study tested the hypothesis that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia. Transient focal ischemia was created in halothane-anesthetized adult male Sprague-Dawley rats (250-300 g) by middle cerebral artery occlusion (MCAO). Atorvastatin (Lipitor, 10 mg/kg sc) was administered three times before MCAO. Infarct volume was measured by triphenyltetrazolium chloride staining. NADPH oxidase enzymatic activity and superoxide levels were quantified in the ischemic core and penumbral regions by lucigenin (5 microM)-enhanced chemiluminescence. Expression of NADPH oxidase membrane subunit gp91(phox) and membrane-translocated subunit p47(phox) and small GTPase Rac-1 was analyzed by Western blot. NADPH oxidase activity and superoxide levels increased after reperfusion and peaked within 2 h of reperfusion in the penumbra, but not in the ischemic core, in MCAO rats. Atorvastatin pretreatment prevented these increases, blunted expression of membrane subunit gp91(phox), and prevented translocation of cytoplasmic subunit p47(phox) to the membrane in the penumbra 2 h after reperfusion. Consequently, cerebral infarct volume was significantly reduced in atorvastatin-treated compared with nontreated MCAO rats 24 h after reperfusion. These results indicate that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia.     

Characterization of neuronal cell death in normal and diabetic rats following exprimental focal cerebral ischemia.

We have studied the forms of cell death following ischemia/reperfusion, and the influence of diabetes mellitus (DM) as an additional factor. Based on the models of diabetes and middle cerebral artery occlusion (MCAO), characteristics of cell death after ischemia/reperfusion were evaluated synthetically by different methods: pathology, FCM, TUNEL and DNA agarose electrophoresis. The results showed that the occurrence of cerebral injury after ischemia/reperfusion was accompanied by cell necrosis and cell apoptosis. Cell apoptosis was mainly located in the ischemic penumbral (IP) zone around the densely ischemic focus. The ischemic core was characterized by cell necrosis. At the same time, the results showed that the process of ischemic cerebral injury worsened by DM was related to inducing cell apoptosis in IP and mid zone. In conclusion, there existed not only cell apoptosis but cell necrosis in brain damage following focal cerebral ischemia/reperfusion and showed a close, internal relationship between them. Brain damage following cerebral ischemia/reperfusion was worsened distinctly under diabetic conditions.     

CD38 exacerbates focal cytokine production, postischemic inflammation and brain injury after focal cerebral ischemia

Background

Converging evidence suggests that inflammatory processes significantly influence brain injury and clinical impairment in ischemic stroke. Although early studies suggested a key role of lymphocytes, recent data has emphasized the orchestrating function of innate immunity, i.e., macrophages and microglia. The bifunctional receptor and ectoenzyme CD38 synthesizes calcium-mobilizing second messengers (e.g., cyclic ADP-ribose), which have been shown to be necessary for activation and migration of myeloid immune cells. Therefore, we investigated the dynamics of CD38 in stroke and the impact of CD38-deficiency on cytokine production, inflammation and cerebral damage in a mouse model of cerebral ischemia-reperfusion.

Methodology/Principal Findings

We show that the local expression of the chemokine MCP-1 was attenuated in CD38-deficient mice compared with wildtype mice after focal cerebral ischemia and reperfusion. In contrast, no significant induction of MCP-1 expression was observed in peripheral blood after 6 hours. Flow cytometry analysis revealed less infiltrating macrophages and lymphocytes in the ischemic hemisphere of CD38-deficient mice, whereas the amount of resident microglia was unaltered. An up-regulation of CD38 expression was observed in macrophages and CD8⁺ cells after focal cerebral ischemia in wildtype mice, whereas CD38 expression was unchanged in microglia. Finally, we demonstrate that CD38-deficiency decreases the cerebral ischemic injury and the persistent neurological deficit after three days of reperfusion in this murine temporary middle cerebral artery occlusion (tMCAO) model.

Conclusion/Significance

CD38 is differentially regulated following stroke and its deficiency attenuates the postischemic chemokine production, the immune cell infiltration and the cerebral injury after temporary ischemia and reperfusion. Therefore CD38 might prove a therapeutic target in ischemic stroke.     

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.     

Upregulation of EMMPRIN after permanent focal cerebral ischemia

Elevated activities of matrix metalloproteinases (MMPs) following ischemic stroke have been shown to mediate ischemic injury as well as neurovascular remodeling. The extracellular MMP inducer (EMMPRIN) is a 58-kDa cell surface glycoprotein, which has been known to play a key regulatory role for MMP activities. The roles of EMMPRIN in stroke injury are not clearly understood. In this study, we investigated changes of EMMPRIN in a mouse model of permanent focal cerebral ischemia, and examined potential association between EMMPRIN and MMP-9 expression. Adult male CD-1 mice were subjected to permanent focal ischemia by intraluminal occlusion of the left middle cerebral artery (MCAO) under anesthesia. EMMPRIN expression was markedly upregulated in the peri-infarct area at 2-7 days after ischemia compared to the contralateral non-ischemic hemisphere by Western blot analysis. Immunofluorescent double staining demonstrated that EMMPRIN signals co-localized with vwF-positive endothelial cells and GFAP-positive peri-vascular astrocytes. In contrast, EMMPRIN signal did not co-localize with NeuN-positive neurons, or MPO-positive neutrophils. Dual fluorescent staining revealed that EMMPRIN co-localized with MMP-9. Our data also demonstrated that increased EMMPRIN expression correlated with increased MMP-9 levels in a temporal manner. In summary, we report for the first time that EMMPRIN expression was significantly increased in a mouse model of permanent focal cerebral ischemia. The spatial and temporal association between increased EMMPRIN expression and elevated MMP-9 levels suggest that EMMPRIN may modulate MMP-9 activity, and participate in neurovascular remodeling after ischemic stroke.     

Hypoxia-inducible factor 1 contributes to N-acetylcysteine’s protection in stroke

Stroke is a leading cause of adult morbidity and mortality with very limited treatment options. Evidence from preclinical models of ischemic stroke has demonstrated that the antioxidant N-acetylcysteine (NAC) effectively protects the brain from ischemic injury. Here, we evaluated a new pathway through which NAC exerted its neuroprotection in a transient cerebral ischemia animal model. Our results demonstrated that pretreatment with NAC increased protein levels of hypoxia-inducible factor-1α (HIF-1α), the regulatable subunit of HIF-1, and its target proteins erythropoietin (EPO) and glucose transporter (GLUT)-3, in the ipsilateral hemispheres of rodents subjected to 90 min middle cerebral artery occlusion (MCAO) and 24 h reperfusion. Interestingly, after NAC pretreatment and stroke, the contralateral hemisphere also demonstrated increased levels of HIF-1α, EPO, and GLUT-3, but to a lesser extent. Suppressing HIF-1 activity with two widely used pharmacological inhibitors, YC-1 and 2ME2, and specific knockout of neuronal HIF-1α abolished NAC’s neuroprotective effects. The results also showed that YC-1 and 2ME2 massively enlarged infarcts, indicating that their toxic effect was larger than just abolishing NAC's neuroprotective effects. Furthermore, we determined the mechanism of NAC-mediated HIF-1α induction. We observed that NAC pretreatment upregulated heat-shock protein 90 (Hsp90) expression and increased the interaction of Hsp90 with HIF-1α in ischemic brains. The enhanced association of Hsp90 with HIF-1α increased HIF-1α stability. Moreover, Hsp90 inhibition attenuated NAC-induced HIF-1α protein accumulation and diminished NAC-induced neuroprotection in the MCAO model. These results strongly indicate that HIF-1 plays an important role in NAC-mediated neuroprotection and provide a new molecular mechanism involved in the antioxidant’s neuroprotection in ischemic stroke.     

当归注射液对脑缺血/再灌注神经元代谢物的影响

目的：研究当归注射液对脑缺血／再灌注时神经元代谢物及血流速度的作用,阐明当归对脑缺血损伤神经修复过程的影响。方法：雄性SD大鼠69只,体重150～170g,随机分成假手术组(n=4)、缺血损伤组(n=30)和当归治疗组(n=35)。制作右大脑中动脉血供阻断(MCA0)模型。缺血2h后,当归治疗组立即腹腔注射当归注射液(5g／kgbw)。在再灌注后3～4h和5～6h,以磁共振成像(MRI)技术研究大脑T2加权成像(T2WI)和局域质子谱(^1H MRS)的变化,观察当归对成像和神经元代谢物N-乙酰天门冬氨酸(NAA)、肌酸／磷酸肌酸(Cr／PCr)和胆碱(Cho)的影响。激光多普勒血流仪观察当归注射液对血流速度的影响,测定脑表面血管密度。结果：与缺血损伤组比较,当归治疗组的高信号强度区信号减弱、体积小,NAA值大,Cr/NAA和Cho/NAA比值小,再灌注时的血流速度显著加快,单位面积内的血管长度增加。结论：当归注射液加快缺血脑组织的血液循环,改善神经元的代谢。     

Delayed activin A administration attenuates tissue death after transient focal cerebral ischemia and is associated with decreased stress-responsive kinase activation

Focal cerebral ischemia and reperfusion initiates complex cellular and molecular interactions that lead to either cell repair or destruction. In earlier work, we found that activin A is an early gene response to cerebral ischemia and supports cortical neuron survival in vitro. In this study, the ability of exogenous activin A to attenuate injury from transient middle cerebral artery occlusion was tested in adult mice. Intracerebroventricular administration of activin A prior to middle cerebral artery occlusion reduced infarct volume apparent 1 day after experimental stroke. A single activin A administration at 6 h following ischemia/reperfusion reduced lesion volumes at 1 and 3 days and led to improved neurobehavior. Moreover, activin A treatment spared neurons within the ischemic hemisphere and led to a concomitant reduction in microglial activation. Activation of the stress-responsive kinases p38 and c- jun N-terminal kinase implicated in neuronal apoptosis after stroke was reduced following activin A treatment. Together these findings suggest that activin A promotes tissue survival after focal cerebral ischemia/reperfusion with an extended therapeutic window.     

Tetrandrine Attenuated Cerebral Ischemia/Reperfusion Injury and Induced Differential Proteomic Changes in a MCAO Mice Model Using 2-D DIGE

Ischemic stroke is the most common type of stroke and brings about a big disease burden because of high mortality and disability in China. Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the Chinese herb Radix Stephania tetrandra, has been demonstrated to possess anti-inflammatory and free radical scavenging effects and even regulate astrocyte activation, but the possible role of tetrandrine in ameliorating cerebral ischemia/reperfusion injury of ischemic stroke remains unknown. The aim of this study was to determine the effects of tetrandrine on neurological injury and differential proteomic changes induced by transient reversible middle cerebral artery occlusion (MCAO) in mice. Male Balb/c mice were divided into sham (n = 30), MCAO + saline as control (n = 30), and MCAO + Tet as tetrandrine-treated (n = 30) groups. Mice in the control and tetrandrine-treated groups underwent 120 min of MCAO following reperfusion. Immediately and 2 h after MCAO, the mice received either normal saline (sham operated and control groups) or tetrandrine (tetrandrine-treated group) intraperitoneally. Neurological defects, brain water content, and infarct volume at 24 h after stoke were used to evaluate neurological injury extent. Treatment with tetrandrine not only mitigated cerebral neurological deficits (P < 0.05) and infarct size (P < 0.01), but also decreased brian edema in the ischemic brain (P < 0.05). Then, fluorescence two-dimensional difference in gel electrophoresis was used to detect our systematic differential profiling of proteomic changes responding to tetrandrine administration. We validated that the expression of GRP78, DJ-1 and HYOU1 was associated with neuroprotective effect of tetrandrine in MCAO model by Western blotting. These findings indicate a potential neuroprotective role of tetrandrine for ischemic stroke and yield insights into cellular and molecular mechanisms of tetrandrine taking place in ischemic stroke.     

Down-Regulation of miRNA-30a Alleviates Cerebral Ischemic Injury Through Enhancing Beclin 1-Mediated Autophagy

The understanding of molecular mechanism underlying ischemia/reperfusion-induced neuronal death and neurological dysfunction may provide therapeutic targets for ischemic stroke. The up-regulated miRNA-30a among our previous identified 19 MicroRNAs (miRNAs) in mouse brain after 6 h middle cerebral artery occlusion (MCAO) could negatively regulate Beclin 1 messenger RNA (mRNA) resulting in decreased autophagic activity in tumor cells and cardiomyocytes, but its role in ischemic stroke is unclear. In this study, the effects of miRNA-30a on ischemic injury in N2A cells and cultured cortical neurons after oxygen glucose deprivation (OGD), and mouse brain with MCAO-induced ischemic stroke were evaluated. The results showed that miRNA-30a expression levels were up regulated in the brain of mice after 6 h MCAO without reperfusion, but significantly down regulated in the peri-infarct region of mice with 1 h MCAO/24 h reperfusion and in N2A cells after 1 h OGD/6–48 h reoxygenation. Both the conversion ratio of microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I and Beclin 1 protein level increased in N2A cells and cultured cortical neurons following 1 h OGD/24 h reoxygenation. The down-regulated miRNA-30a could attenuate 1 h OGD/24 h reoxygenation-induced ischemic injury in N2A cells and cultured cortical neurons through enhancing Beclin 1-mediated autophagy, as miRNA-30a recognized the 3′-untranslated region of beclin 1 mRNA to negatively regulate Beclin 1-protein level via promoting beclin 1 messenger RNA (mRNA) degradation, and Beclin 1 siRNA abolished anti-miR-30a-induced neuroprotection in 1 h OGD/24 h reoxygenation treated N2A cells. In addition, anti-miR-30a attenuated the neural cell loss and improved behavioral outcome of mice with ischemic stroke. These results suggested that down-regulation of miRNA-30a alleviates ischemic injury through enhancing beclin 1-mediated autophagy, providing a potential therapeutic target for ischemic stroke.     

Propofol Protects Against Focal Cerebral Ischemia via Inhibition of Microglia-Mediated Proinflammatory Cytokines in a Rat Model of Experimental Stroke

Ischemic stroke induces microglial activation and release of proinflammatory cytokines, contributing to the expansion of brain injury and poor clinical outcome. Propofol has been shown to ameliorate neuronal injury in a number of experimental studies, but the precise mechanisms involved in its neuroprotective effects remain unclear. We tested the hypothesis that propofol confers neuroprotection against focal ischemia by inhibiting microglia-mediated inflammatory response in a rat model of ischemic stroke. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by 24 h of reperfusion. Propofol (50 mg/kg/h) or vehicle was infused intravenously at the onset of reperfusion for 30 minutes. In vehicle-treated rats, MCAO resulted in significant cerebral infarction, higher neurological deficit scores and decreased time on the rotarod compared with sham-operated rats. Propofol treatment reduced infarct volume and improved the neurological functions. In addition, molecular studies demonstrated that mRNA expression of microglial marker Cd68 and Emr1 was significantly increased, and mRNA and protein expressions of proinflammatory cytokines tumor necrosis factor-α, interleukin-1β and interleukin-6 were augmented in the peri-infarct cortical regions of vehicle-treated rats 24 h after MCAO. Immunohistochemical study revealed that number of total microglia and proportion of activated microglia in the peri-infarct cortical regions were markedly elevated. All of these findings were ameliorated in propofol-treated rats. Furthermore, vehicle-treated rats had higher plasma levels of interleukin-6 and C-reactive protein 24 h after MCAO, which were decreased after treatment with propofol. These results suggest that propofol protects against focal cerebral ischemia via inhibition of microglia-mediated proinflammatory cytokines. Propofol may be a promising therapeutic agent for the treatment of ischemic stroke and other neurodegenerative diseases associated with microglial activation.     

Parathyroid hormone-related protein induction in focal stroke: a neuroprotective vascular peptide

Parathyroid hormone-related protein (PTHrP) is a multifunctional peptide that enhances blood flow in non-central nervous system (CNS) vascular beds by causing vasodilation. PTHrP expression is induced in non-CNS organs in response to ischemia. Experiments were therefore undertaken to determine whether PTHrP can be induced in brain in response to ischemic injury and whether PTHrP can act locally as a vasodilator in the cerebral vasculature, an effect that could be neuroprotective in the setting of stroke. PTHrP expression was examined by Northern analysis and immunohistochemical staining in male Sprague-Dawley rats subjected to permanent middle cerebral artery occlusion (MCAO). Vasodilatory effects of superfused PTHrP(1-34) on pial arterioles were determined by intravital fluorescence microscopy. Effects of PTHrP(1-34) peptide administration on MCAO infarction size reduction were assessed. PTHrP expression was induced in the ischemic hemisphere as early as 4 h after MCAO and remained elevated for up to 24 h. Increased immunoreactive PTHrP at sites of ischemic tissue injury was located in the cerebral microvessels. Superfusion with PTHrP(1-34) peptide for up to 25 min increased pial arteriolar diameter by 30% in normal animals. In animals with permanent MCAO, PTHrP(1-34) peptide treatment significantly decreased cortical infarct size (-47%). In summary, PTHrP expression increases at sites of ischemic brain injury in the cerebrovasculature. This local increase in PTHrP could be an adaptive response that enhances blood flow to the ischemic brain, thus limiting cell injury.     

Possible Involvement of PTEN Signaling Pathway in the Anti-apoptotic Effect of Electroacupuncture Following Ischemic Stroke in Rats

As a traditional therapeutic method, electroacupuncture (EA) has been adopted as an alternative therapy for stroke recovery. Here, we aimed to evaluate whether EA therapy at points of Quchi (LI11) and Zusanli (ST36) alleviated neuronal apoptosis by PTEN signaling pathway after ischemic stroke. A total of 72 male Sprague–Dawley rats were randomized into three groups, including sham group, MCAO group, and EA group. EA was initiated after 24 h of reperfusion for 3 consecutive days. At 72 h following ischemia/reperfusion, neurological deficits, infarct volumes, and TUNEL staining were evaluated and the PTEN pathway-related proteins together with apoptosis-related proteins were detected. The results indicated that EA treatment significantly decreased cerebral infarct volume, neurological deficits and alleviated proportion of apoptotic cells in cerebral ischemic rats. Furthermore, EA significantly up-regulated the phosphorylation levels of PDK1, Akt(Thr308), GSK-3β, and down-regulated the phosphorylation levels of PTEN, Akt(Ser473) in the peri-infarct cortex. EA treatment significantly reduced the up-regulation of caspase-3, cleaved-caspase-3, Bim, and reversed the reduction of Bcl-2 induced by the ischemic stroke. These findings suggest that EA treatment at points of Quchi (LI11)- and Zusanli (ST36)-induced neuroprotection might involve inhibition of apoptosis via PTEN pathway.     

Bone morphogenetic protein-7 (BMP-7) mediates ischemic preconditioning-induced ischemic tolerance via attenuating apoptosis in rat brain

A mild cerebral ischemic insult, also known as ischemic preconditioning (IPC), confers transient tolerance to a subsequent ischemic challenge in the brain. This study was conducted to investigate whether bone morphogenetic protein-7 (BMP-7) is involved in neuroprotection elicited by IPC in a rat model of ischemia. Ischemic tolerance was induced in rats by IPC (15 min middle cerebral artery occlusion, MCAO) at 48 h before lethal ischemia (2 h MCAO). The present data showed that IPC increased BMP-7 mRNA and protein expression after 24 h reperfusion following ischemia in the brain. In rats of ischemia, IPC-induced reduction of cerebral infarct volume and improvement of neuronal morphology were attenuated when BMP-7 was inhibited either by antagonist noggin or short interfering RNA (siRNA) pre-treatment. Besides, cerebral IPC-induced up-regulation of B-cell lymphoma 2 (Bcl-2) and down-regulation of cleaved caspase-3 at 24 h after ischemia/reperfusion (I/R) injury were reversed via inhibition of BMP-7. These findings indicate that BMP-7 mediates IPC-induced tolerance to cerebral I/R, probably through inhibition of apoptosis.     

δ-Opioid Receptor Activation Rescues the Functional TrkB Receptor and Protects the Brain from Ischemia-Reperfusion Injury in the Rat

Objectives

δ-opioid receptor (DOR) activation reduced brain ischemic infarction and attenuated neurological deficits, while DOR inhibition aggravated the ischemic damage. The underlying mechanisms are, however, not well understood yet. In this work, we asked if DOR activation protects the brain against ischemic injury through a brain-derived neurotrophic factor (BDNF) -TrkB pathway.

Methods

We exposed adult male Sprague-Dawley rats to focal cerebral ischemia, which was induced by middle cerebral artery occlusion (MCAO). DOR agonist TAN-67 (60 nmol), antagonist Naltrindole (100 nmol) or artificial cerebral spinal fluid was injected into the lateral cerebroventricle 30 min before MCAO. Besides the detection of ischemic injury, the expression of BDNF, full-length and truncated TrkB, total CREB, p-CREB, p-ATF and CD11b was detected by Western blot and fluorescence immunostaining.

Results

DOR activation with TAN-67 significantly reduced the ischemic volume and largely reversed the decrease in full-length TrkB protein expression in the ischemic cortex and striatum without any appreciable change in cerebral blood flow, while the DOR antagonist Naltrindole aggregated the ischemic injury. However, the level of BDNF remained unchanged in the cortex, striatum and hippocampus at 24 hours after MCAO and did not change in response to DOR activation or inhibition. MCAO decreased both total CREB and pCREB in the striatum, but not in the cortex, while DOR inhibition promoted a further decrease in total and phosphorylated CREB in the striatum and decreased pATF-1 expression in the cortex. In addition, MCAO increased C11b expression in the cortex, striatum and hippocampus, and DOR activation specifically attenuated the ischemic increase in the cortex but not in the striatum and hippocampus.

Conclusions

DOR activation rescues TrkB signaling by reversing ischemia/reperfusion induced decrease in the full-length TrkB receptor and reduces brain injury in ischemia/reperfusion     

Role of AT1 receptors and NAD(P)H oxidase in diabetes-aggravated ischemic brain injury

The objective of the present study was to examine the role of the angiotensin II type 1 receptor (AT(1)-R) in the diabetes-aggravated oxidative stress and brain injury observed in a rat model of combined diabetes and focal cerebral ischemia. Diabetes was induced by an injection of streptozotoxin (STZ; 55 mg/kg iv) at 8 wk of age. Two weeks after the induction of diabetes, some animals received continuous subcutaneous infusion of the AT(1)-R antagonist candesartan (0.5 mg.kg(-1).day(-1)) for 14 days. Focal cerebral ischemia, induced by middle cerebral artery occlusion/reperfusion (MCAO), was conducted at 4 wk after STZ injection. Male Sprague-Dawley rats (n = 189) were divided into five groups: normal control, diabetes, MCAO, diabetes + MCAO, and diabetes + MCAO + candesartan. The major observations were that 1) MCAO produced typical cerebral infarction and neurological deficits at 24 h that were accompanied by elevation of NAD(P)H oxidase gp91(phox) and p22(phox) mRNAs, and lipid hydroperoxide production in the ipsilateral hemisphere; 2) diabetes enhanced NAD(P)H oxidase gp91(phox) and p22(phox) mRNA expression, potentiated lipid peroxidation, aggravated neurological deficits, and enlarged cerebral infarction; and 3) candesartan reduced the expression of gp91(phox) and p22(phox), decreased lipid peroxidation, lessened cerebral infarction, and improved the neurological outcome. We conclude that diabetes exaggerates the oxidative stress, NAD(P)H oxidase induction, and brain injury induced by focal cerebral ischemia. The diabetes-aggravated brain injury involves AT(1)-Rs. We have shown for the first time that candesartan reduces brain injury in a combined model of diabetes and cerebral ischemia.     

松龄血脉康胶囊对局灶性缺血损伤后大鼠脑组织TNF-α表达的影响

目的：探讨松龄血脉康预处理对大鼠急性局灶性脑缺血再灌注损伤脑组织TNF-α表达的影响。方法：36只雄性SD大鼠,随机分为松龄血脉康（SL-xmk）预处理组、假手术组、对照组,SL-xmk预处理组采用SL-xmk（937.5mg/kg）悬浮液对大鼠进行4w预防性灌胃处理,假手术组、对照组采用等容量生理盐水预防性灌胃处理,在预处理终点采用线栓法制作大鼠大脑中动脉阻塞（middlecerebralarteryocclusion,MCAO）模型。观察SL-xmk预处理后MCAO大鼠脑组织含水量、脑梗死体积变化的影响,运用免疫组织化学法检测大鼠缺血脑组织TNF-α免疫反应阳性细胞表达。结果：SL-xmk预处理后脑组织TNF-α表达显著下降,缺血脑组织含水量和脑梗死体积明显降低。结论：松龄血脉康可抑制急性局灶性脑缺血再灌注损伤后脑组织TNF-α的表达。     

Mechanism of Protection by Soluble Epoxide Hydrolase Inhibition in Type 2 Diabetic Stroke

Inhibition of soluble epoxide hydrolase (sEH) is a potential target of therapy for ischemic injury. sEH metabolizes neuroprotective epoxyeicosatrienoic acids (EETs). We recently demonstrated that sEH inhibition reduces infarct size after middle cerebral artery occlusion (MCAO) in type 1 diabetic mice. We hypothesized that inhibition of sEH would protect against ischemic injury in type 2 diabetic mice. Type 2 diabetes was produced by combined high-fat diet, nicotinamide and streptozotocin in male mice. Diabetic and control mice were treated with vehicle or the sEH inhibitor t-AUCB then subjected to 60-min MCAO. Compared to chow-fed mice, high fat diet-fed mice exhibited an upregulation of sEH mRNA and protein in brain, but no differences in brain EETs levels were observed between groups. Type 2 diabetic mice had increased blood glucose levels at baseline and throughout ischemia, decreased laser-Doppler perfusion of the MCA territory after reperfusion, and sustained larger cortical infarcts compared to control mice. t-AUCB decreased fasting glucose levels at baseline and throughout ischemia, improved cortical perfusion after MCAO and significantly reduced infarct size in diabetic mice. We conclude that sEH inhibition, as a preventative treatment, improves glycemic status, post-ischemic reperfusion in the ischemic territory, and stroke outcome in type 2 diabetic mice.