Effects of kallikrein gene transfer on penumbral microvascular proliferation and on regional cerebral blood flow following cerebral ischemia/reperfusion injury

BACKGROUND： Recent findings have demonstrated that the kallikrein-kinin system （KKS） participates in the pathological process of cerebral ischemia/reperfusion injury. Kallikrein gene transfer exhibits neural protective effects following cerebral infarction. OBJECTIVE： To observe the effects of kallikrein gene transfer on vascular proliferation in the peripheral infarct focus and on regional cerebral blood flow （rCBF） following cerebral ischemia/reperfusion injury. DESIGN, TIME AND SETTING： The completely randomized, controlled experiment was performed at the Lin Baixin Laboratory Center, the Second Affiliated Hospital of Sun Yat-sun University between September 2007 and April 2008. MATERIALS： pUCI9-HTK plasmid was constructed and maintained in the Laboratory for Neurology, the Second Affiliated Hospital of Sun Yat-sen University, China. Mouse anti-human kallikrein 1 monoclonal antibody was purchased from R＆D Systems, USA. METHODS Ninety healthy, male, Sprague Dawley rats were used. Middle cerebral artery occlusion （MCAO） was established in all rats to induce cerebral ischemia/reperfusion injury. Following MCAO establishment, all rats were randomly divided into three groups （n = 30）： blank control, saline, and pAdCMV-HTK. The saline and pAdCMV-HTK groups were stereotactically micro-injected with 5μL of physiological saline or with pAdCMV-HTK [multiplicity of infection （MOI） = 20], respectively, into the ischemic penumbra. In the blank control group, only sham injection was performed. MAIN OUTCOME MEASURES： At 12, 24, and 72 hours after treatment, cerebral infarction volume was measured by 2, 3, 5-triphenyltetrazolium chloride （TTC） staining. Exogenous HTK expression, as well as regional vascular endothelial growth factor （VEGF） expression, was detected by immunohistochemistry. rCBF was examined by 14C-iodoantipyrine micro tracing. In addition, neurological severity score （NSS） was performed. Higher scores indicated more severe neurological deficits. RESULTS： NSS results demonstrated that compared with the saline and the blank control groups, the pAdCMV-HTK group exhibited lower NSSs 24 hours after pAdCMV-HTK injection （P 〈 0.05）. The NSSs were further decreased after 72 hours （P 〈 0.01）. Cerebral infarction volume at 24 hours, and in particular at 72 hours after treatment, was significantly reduced in the pAdCMV-HTK group compared with the blank control and saline groups （P 〈 0.05）. The rCBF in the area surrounding the infarction lesion was slightly decreased in all groups compared with the contralateral area. At 24 and 72 hours following treatment, the rCBF in the peripheral infarction lesion was significantly elevated in the pAdCMV-HTK group compared with the blank control and saline groups （P 〈 0.05）. Immunohistochemistry results revealed that VEGF-positive cells were primarily found in the cortex and in some white matter surrounding the cerebral infarction lesion. In addition, the expression of VEGF in the pAdCMV-HTK group was significantly higher compared with that in the blank control and saline groups at 12, 24, and 72 hours following treatment （P 〈 0.05）. CONCLUSION： Following cerebral ischemia/reperfusion, kallikrein gene transfer can promote vascular proliferation in the brain tissue surrounding the infarction lesion, improve rCBF, and reduce infarction volume, thereby exhibiting protective effects to attenuate neurological deficits.

Endoplasmic reticulum stress and autophagy in cerebral ischemia/reperfusion injury:PERK as a potential target for intervention

Several studies have shown that activation of unfolded protein response and endoplasmic reticulum(ER)stress plays a crucial role in severe cerebral ischemia/reperfusion injury.Autophagy occurs within hours after cerebral ischemia,but the relationship between ER stress and autophagy remains unclear.In this study,we established experimental models using oxygen-glucose deprivation/reoxygenation in PC12 cells and primary neurons to simulate cerebral ischemia/reperfusion injury.We found that prolongation of oxygen-glucose deprivation activated the ER stress pathway protein kinase-like endoplasmic reticulum kinase(PERK)/eukaryotic translation initiation factor 2 subunit alpha(e IF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP),increased neuronal apoptosis,and induced autophagy.Furthermore,inhibition of ER stress using inhibitors or by si RNA knockdown of the PERK gene significantly attenuated excessive autophagy and neuronal apoptosis,indicating an interaction between autophagy and ER stress and suggesting PERK as an essential target for regulating autophagy.Blocking autophagy with chloroquine exacerbated ER stress-induced apoptosis,indicating that normal levels of autophagy play a protective role in neuronal injury following cerebral ischemia/reperfusion injury.Findings from this study indicate that cerebral ischemia/reperfusion injury can trigger neuronal ER stress and promote autophagy,and suggest that PERK is a possible target for inhibiting excessive autophagy in cerebral ischemia/reperfusion injury.

A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

Vascular endothelial growth factor and its mimic peptide KLTWQELYQLKYKGI(QK)are widely used as the most potent angiogenic factors for the treatment of multiple ischemic diseases.However,conventional topical drug delivery often results in a burst release of the drug,leading to transient retention(inefficacy)and undesirable diffusion(toxicity)in vivo.Therefore,a drug delivery system that responds to changes in the microenvironment of tissue regeneration and controls vascular endothelial growth factor release is crucial to improve the treatment of ischemic stroke.Matrix metalloproteinase-2(MMP-2)is gradually upregulated after cerebral ischemia.Herein,vascular endothelial growth factor mimic peptide QK was self-assembled with MMP-2-cleaved peptide PLGLAG(TIMP)and customizable peptide amphiphilic(PA)molecules to construct nanofiber hydrogel PA-TIMP-QK.PA-TIMP-QK was found to control the delivery of QK by MMP-2 upregulation after cerebral ischemia/reperfusion and had a similar biological activity with vascular endothelial growth factor in vitro.The results indicated that PA-TIMP-QK promoted neuronal survival,restored local blood circulation,reduced blood-brain barrier permeability,and restored motor function.These findings suggest that the self-assembling nanofiber hydrogel PA-TIMP-QK may provide an intelligent drug delivery system that responds to the microenvironment and promotes regeneration and repair after cerebral ischemia/reperfusion injury.

Role of nitric oxide in cerebral ischemia/reperfusion injury:A biomolecular overview

Nitric oxide(NO)is a gaseous molecule produced by 3 different NO synthase(NOS)isoforms:Neural/brain NOS(nNOS/bNOS,type 1),endothelial NOS(eNOS,type 3)and inducible NOS(type 2).Type 1 and 3 NOS are constitutively expressed.NO can serve different purposes:As a vasoactive molecule,as a neurotransmitter or as an immunomodulator.It plays a key role in cerebral ischemia/reperfusion injury(CIRI).Hypoxic episodes simulate the production of oxygen free radicals,leading to mitochondrial and phospholipid damage.Upon reperfusion,increased levels of oxygen trigger oxide synthases;whose products are associated with neuronal damage by promoting lipid peroxidation,nitrosylation and excitotoxicity.Molecular pathways in CIRI can be altered by NOS.Neuroprotective effects are observed with eNOS activity.While nNOS interplay is prone to endothelial inflammation,oxidative stress and apoptosis.Therefore,nNOS appears to be detrimental.The interaction between NO and other free radicals develops peroxynitrite;which is a cytotoxic agent.It plays a main role in the likelihood of hemorrhagic events by tissue plasminogen activator(t-PA).Peroxynitrite scavengers are currently being studied as potential targets to prevent hemorrhagic transformation in CIRI.

Regional dynamic cerebral autoregulation across anterior and posterior circulatory territories:A detailed exploration and its clinical implications

Cerebral autoregulation(CA)is the mechanism that maintains stable cerebral blood flow(CBF)despite fluctuations in systemic blood pressure,crucial for brain homeostasis.Recent evidence highlights distinct regional variations in CA between the anterior(carotid)and posterior(vertebrobasilar)circulations.Noninvasive neuromonitoring techniques,such as transcranial Doppler,transfer function analysis,and near-infrared spectroscopy,facilitate the dynamic assessment of CBF and autoregulation.Studies indicate a robust autoregulatory capacity in the anterior circulation,characterized by rapid adjustments in vascular resistance.On the contrary,the posterior circulation,mainly supplied by the vertebral arteries,may have a lower autoregulatory capacity.in acute brain injuries such as intracerebral and subarachnoid hemorrhage,and traumatic brain injuries,dynamic CA can be significantly altered in the posterior circulation.Proposed physiological mechanisms of impaired CA in the posterior circulation include:(1)Decreased sympathetic innervation of the vasculature impairing compensatory vasoreactivity;(2)Endothelial dysfunction;(3)Increased cerebral metabolic rate of oxygen consumption within the visual cortex causing CBFmetabolism(i.e.,neurovascular)uncoupling;and(4)Impaired blood-brain barrier integrity leading to impaired astrocytic mediated release of vasoactive substances(e.g.nitric oxide,potassium,and calcium ions).Furthermore,more research is needed on the effects of collateral circulation,as well as the circle of Willis variants,such as the fetal-type posterior cerebral artery,on dynamic CA.Improving our understanding of these mechanisms is crucial to improving the diagnosis,prognosis,and management of various cerebrovascular disorders.

Treatment with β-sitosterol ameliorates the effects of cerebral ischemia/reperfusion injury by suppressing cholesterol overload, endoplasmic reticulum stress, and apoptosis

β-Sitosterol is a type of phytosterol that occurs naturally in plants.Previous studies have shown that it has anti-oxidant,anti-hyperlipidemic,anti-inflammatory,immunomodulatory,and anti-tumor effects,but it is unknown whetherβ-sitosterol treatment reduces the effects of ischemic stroke.Here we found that,in a mouse model of ischemic stroke induced by middle cerebral artery occlusion,β-sitosterol reduced the volume of cerebral infarction and brain edema,reduced neuronal apoptosis in brain tissue,and alleviated neurological dysfunction;moreover,β-sitosterol increased the activity of oxygen-and glucose-deprived cerebral cortex neurons and reduced apoptosis.Further investigation showed that the neuroprotective effects ofβ-sitosterol may be related to inhibition of endoplasmic reticulum stress caused by intracellular cholesterol accumulation after ischemic stroke.In addition,β-sitosterol showed high affinity for NPC1L1,a key transporter of cholesterol,and antagonized its activity.In conclusion,β-sitosterol may help treat ischemic stroke by inhibiting neuronal intracellular cholesterol overload/endoplasmic reticulum stress/apoptosis signaling pathways.

The action mechanism by which C1q/tumor necrosis factor-related protein-6 alleviates cerebral ischemia/reperfusion injury in diabetic mice

Studies have shown that C1q/tumor necrosis factor-related protein-6 (CTRP6) can alleviate renal ischemia/reperfusion injury in mice. However, its role in the brain remains poorly understood. To investigate the role of CTRP6 in cerebral ischemia/reperfusion injury associated with diabetes mellitus, a diabetes mellitus mouse model of cerebral ischemia/reperfusion injury was established by occlusion of the middle cerebral artery. To overexpress CTRP6 in the brain, an adeno-associated virus carrying CTRP6 was injected into the lateral ventricle. The result was that oxygen injury and inflammation in brain tissue were clearly attenuated, and the number of neurons was greatly reduced. In vitro experiments showed that CTRP6 knockout exacerbated oxidative damage, inflammatory reaction, and apoptosis in cerebral cortical neurons in high glucose hypoxia-simulated diabetic cerebral ischemia/reperfusion injury. CTRP6 overexpression enhanced the sirtuin-1 signaling pathway in diabetic brains after ischemia/reperfusion injury. To investigate the mechanism underlying these effects, we examined mice with depletion of brain tissue-specific sirtuin-1. CTRP6-like protection was achieved by activating the sirtuin-1 signaling pathway. Taken together, these results indicate that CTRP6 likely attenuates cerebral ischemia/reperfusion injury through activation of the sirtuin-1 signaling pathway.

Cav3.2 channel regulates cerebral ischemia/reperfusion injury:a promising target for intervention

Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type calcium channels.T-type calcium channel blockers,such as pimozide and mibefradil,have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury.However,the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear.Here,in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons.The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons.We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury.Cav3.2 knockout markedly reduced infarct volume and brain water content,and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury.Additionally,Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress,inflammatory response,and neuronal apoptosis.In the hippocampus of Cav3.2-knockout mice,calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury.These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling.Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.

A New Method for Evaluating Regional Cerebral Blood Flow Changes:Laser Speckle Contrast Imaging in a C57BL/6J Mouse Model of Photothrombotic Ischemia

The present study aimed to improve the processing of data acquired from laser speckle contrast imaging（LSCI） to provide a standardization method to explore changes in regional cerebral blood flow（r CBF） and to determine the correlations among r CBF, cerebral ischemic lesion volume and microvascular density over time in a focal ischemic region. C57BL/6J mice were subjected to focal photothrombotic（PT） ischemia. r CBF was measured using LSCI at different time points before and after PT ischemia through an intact skull. Standardized r CBF（Sr CBF）, defined as the ratio of r CBF measured in the ipsilateral region of interest（ROI） to that in the corresponding contralateral region, was calculated to evaluate potential changes. In addition, the volume of the ischemic lesion and the microvascular density were determined using Nissl staining and immunofluorescence, respectively. The relationships among the ischemic lesion volume, microvascular density and Sr CBF were analyzed over time. The results showed that the cortical r CBF measured using LSCI following PT ischemia in the C57BL/6J mice gradually increased. Changes in the cerebral ischemic lesion volume were negatively correlated with Sr CBF in the ischemic region. Changes in the microvascular density were similar to those observed in Sr CBF. Our findings indicate that LSCI is a practical technique for observing changes in murine cortical r CBF without skull opening and for analyzing the relationships among the ischemic lesion volume, microvascular density and Sr CBF following focal cerebral ischemia. Preliminary results also suggest that the use of LSCI to observe the formation of collateral circulation is feasible.

Formononetin Enhances Autophagy Flux in The Penumbra of Cerebral Ischemia and Improves Nerve Damage

Objective Formononetin(FOR),a traditional Chinese medicine,has been widely used for nerve protection and nerve function rehabilitation after cerebral stroke.However,the role of FOR in autophagic lysosome function in cerebral ischemiareperfusion damage has not been investigated.This study aimed to explore whether the therapeutic benefits of FOR were influenced by the regulation of autophagy flux.Methods Male Sprague-Dawley rats were separated into sham,model,and MCAO+FOR(30 mg/kg)groups after undergoing middle cerebral artery occlusion(MCAO)and ischemia-reperfusion(I/R).Then,the brain tissues in the ischemic penumbra were obtained to detect the proteins in autophagic/lysosomal pathway with antibodies of Beclin-1,LC3,SQSTM1/P62,Ubiquitin,LAMP-2,Cathepsin B(CTSB)and Cathepsin D(CTSD)by Western blot and immunofluorescence,respectively.Meanwhile,the therapeutic effectiveness was evaluated by measuring infarct volume,neurological impairments,and neuronal necrosis.Results The findings of this study demonstrate that FOR treatment exhibits a dual effect by enhancing the autophagic activities of Beclin-1 and LC3 in neurons,while simultaneously improving the autophagic clearance function,as evidenced by reinforced lysosomal activities of LAMP-2,CTSB,and CTSD,as well as reduced autophagic accumulation of Ubiquitin and P62 in the MCAO+FOR group compared to the MCAO group.Additionally,7 d of FOR treatment dramatically reduced neurological deficits,infarct volume,and neuronal death caused by cerebral ischemia.Conclusion These findings suggest that the neuroprotective mechanism of FOR therapy in accelerating recovery from ischemic stroke may involve the increase of autophagy flux in the penumbra.

Lactiplantibacillus plantarum AR113 alleviates microbiota dysbiosis of tongue coating and cerebral ischemia/reperfusion injury in rat

Stroke is one of the leading causes of death and disability worldwide.However,information on stroke-related tongue coating microbiome(TCM)is limited,and whether TCM modulation could benefit for stroke prevention and rehabilitation is unknown.Here,TCM from stroke patients(SP)was characterized using molecular techniques.The occurrence of stroke resulted in TCM dysbiosis with significantly reduced species richness and diversity.The abundance of Prevotella,Leptotrichia,Actinomyces,Alloprevotella,Haemophilus,and TM7_[G-1]were greatly reduced,but common infection Streptococcus and Pseudomonas were remarkably increased.Furthermore,an antioxidative probiotic Lactiplantibacillus plantarum AR113 was used for TCM intervention in stroke rats with cerebral ischemia/reperfusion(I/R).AR113 partly restored I/R induced change of TCM and gut microbiota with significantly improved neurological deficit,relieved histopathologic change,increased activities of antioxidant enzymes,and decreased contents of oxidative stress biomarkers.Moreover,the gene expression of antioxidant-related proteins and apoptosis-related factors heme oxygenase-1(HO-1),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),nuclear factor erythroid 2-related factor 2(Nrf2),NAD(P)H:quinone oxidoreductase-1(NQO-1),and Bcl-2 was significantly increased,but cytochrome C,cleaved caspase-3,and Bax were markedly decreased in the brain by AR113 treatment.The results suggested that AR113 could ameliorate cerebral I/R injury through antioxidation and anti-apoptosis pathways,and AR113 intervention of TCM may have the application potential for stroke prevention and control.

Investigation multi-target synergistic mechanism of Choerospondias axillaris in the treat of cerebral ischemia based on systems pharmacology and experimental verification

Background:Choerospondias axillaris(CA)is a traditional Mongolian medicine that has been proven to have a good therapeutic effect on cerebrovascular disease.Cerebral Ischemia(CI)is a severe and life-threatening cerebrovascular disease.However,the specific mechanism of action of CA in the treatment of CI is still unclear.Methods:In this study,the related targets and pathways of CA in the treatment of CI were first predicted by system pharmacology and then verified by relevant experiments.Results:The results showed that 12 active ingredients and 208 targets were selected.Further validation through protein-protein interaction(PPI)network analysis and active ingredients-target-pathway(A-T-P)network analysis has confirmed the pivotal roles of the main bioactive constituents,including quercetin,kaempferol,naringin,β-sitosterol,and gallic acid.These components exert their anti-ischemic effects by modulating key targets such as IL6,TNF,MAPK3,and CASP3,thereby regulating the PI3K-Akt,HIF-1,and MAPK signaling pathways,which are integral to processes like inflammation,apoptosis,and oxidative stress.More importantly,through experimental verification,this study confirmed our prediction that CAE significantly reduced neurological function scores,infarct volume,and the percentage of apoptosis neurons.Conclusion:This indicates that CA acts on CI through multi-target synergistic mechanism,and this study provides theoretical basis for the clinical application of CA.

EFFECTS OF ELECTROACUPUNCTURE ON EXPRESSION OF INTERCELLULAR ADHESION MOLECULE-1 IN THE RAT OF LOCAL CEREBRAL ISCHEMIA-REPERFUSION

Objective To investigate effects of electroacupuncture （EA） on expression of intercellular adhesion molecule-1 （ICAM-1） in the rat of local cerebral ischemia-reperfusion. Methods Eighty SD rats were randomly divided into a normal control group, a sham operation group, a model group and an EA treatment group, 20 rats in each group. The thread-obstruction method was used for preparation of ischemia-reperfusion model. Zea-Longa rating criteria were used for evaluation of nervous function disorder; Immunohistochemical SABC method was used for detection of ICAM-1 expression in the microvascular endothelial cell of the ischemic brain region, and ELISA method for the soluble ICAM-1 （slCAM-1） content in peripheral blood. Re. suits After cerebral ischemia-reperfusion, both ICAM-1 expression level in the microvascular endethelium cell of the ischemic brain region and slCAM-1 content in the peripheral blood significantly increased in the model group as compared with the normal group and the sham operation group （P〈0.01）; After EA treatment, the ICAM-1 expression level in the microvascular endothelial cell of the ischemic brain region and slCAM-1 content in the peripheral blood were significantly down-regulated in the EA treatment group as com- pared with the model group （P〈 0.05）. Conclusion After cerebral ischemia-reperfusion, the microvascular endothelial cell of the ischemic brain region releases ICAM-1, which induces inflammatory injury of cerebral tissues; EA treatment can decease the expression of ICAM-1, so as to prevent the brain from the injury.

Pinacidil reduces neuronal apoptosis following cerebral ischemia-reperfusion in rats through both mitochondrial and death-receptor signal pathways

Objective To investigate effect of pinacidil, an ATP sensitive potassium channel （KATP） opener, on the neuronal apoptosis and its signaling transduction mechanism following focal cerebral ischemia-reperfusion in rats. Methods One hundred male Wistar rats were randomly divided into four groups： A, sham-operated group; B, ischemia-reperfusion group; C, KATe opener treatment group; and D, KATe opener and blocker treatment group. The middle cerebral artery occlusion （MCAO） model was established by using the intraluminal suture occlusion method, neuronal apoptosis was determined by TUNEL staining, and expressions of caspase-8, caspase-9 and caspase-3 mRNA were detected by in situ hybridization. Results （1） The numbers of apoptotic neurons at 12 h, 24 h, 48 h, and 72 h were significantly less in group C than in groups B and D （P 〈 0.01 or P 〈 0.05）; and there was no difference between groups B and D at all time points （P 〉 0.05）. （2） The expressions of caspase-3 mRNA and caspase-8 mRNA at all times and the expressions of caspase-9 mRNA at 12 h, 24 h, 48 h, 72 h were significantly lower in group C than in groups B and D （P 〈 0.01 or P 〈 0.05）; and there were no differences between groups B and D at all time points （P 〉 0.05）. Conclusions KATP opener can significantly decrease the neuronal apoptosis and the expressions of caspase-3, caspase-8 and caspase-9 mRNAs following cerebral ischemiareperfusion. The neuronal apoptosis may be decreased by the inhibition of both mitochondrial and death-receptor signal pathways.

N-methyl-D-aspartate receptors mediate diphosphorylation of extracellular signal-regulated kinases through Src family tyrosine kinases and Ca^2＋/calmodulin-dependent protein kinase Ⅱ in rat hippocampus after cerebral ischemia

Objective： Extracellular signal-regulated kinases （ERKs） can be activated by calcium signals. In this study, we investigated whether calcium-dependent kinases were involved in ERKs cascade activation after global cerebral ischemia. Methods Cerebral ischemia was induced by four-vessel occlusion, and the calcium-dependent proteins were detected by immunoblot. Results Lethal-simulated ischemia significantly resulted in ERKs activation in N-methyl-D-aspartate （NMDA） receptor-dependent manner, accompanying with differential upregulation of Src kinase and Ca^2＋/calmodulin-dependent protein kinase Ⅱ （CaMKⅡ） activities. With the inhibition of Src family tyrosine kinases or CaMKⅡ by administration of PP2 or KN62, the phosphorylation of ERKs was impaired dramatically during post-ischemia recovery. However, ischemic challenge also repressed ERKs activity when Src kinase was excessively activated. Conclusions Src family tyrosine kinases and CaMKⅡ might be involved in the activation of ERKs mediated by NMDA receptor in response to acute ischemic stimuli in vivo, but the intense activation of Src kinase resulted from ischemia may play a reverse role in the ERKs cascade.

Expression profiles of microRNAs after focal cerebral ischemia/reperfusion injury in rats

Rat models of focal cerebral ischemia/reperfusion injury were established by occlusion of the middle cerebral artery. Microarray analysis showed that 24 hours after cerebral ischemia, there were nine up-regulated and 27 down-regulated microRNA genes in cortical tissue. Bioinformatic analysis showed that bcl-2 was the target gene of microRNA-384-5p and microRNA-494, and caspase-3 was the target gene of microRNA-129, microRNA-320 and microRNA-326. Real-time PCR and western blot analyses showed that 24 hours after cerebral ischemia, bcl-2 mRNA and protein levels in brain tissue were significantly decreased, while caspase-3 mRNA and protein levels were significantly increased. This suggests that following cerebral ischemia, differentially expressed microRNA-384-5p, microRNA-494, microRNA-320, microRNA-129 and microRNA-326 can regulate bcl-2 and caspase-3 expression in brain tissue.

A feasible strategy for focal cerebral ischemiareperfusion injury: remote ischemic postconditioning

It is difficult to control the degree of ischemic postconditioning in the brain and other isch- emia-sensitive organs. Remote ischemic postconditioning could protect some ischemia-sensitive organs through measures on terminal organs. In this study, a focal cerebral ischemia-reperftlsion injury model was established using three cycles of remote ischernic postconditioning, each cycle consisted of 10-minute occlusion of the femoral artery and 10-minute opening. The results showed that, remote ischemic postconditioning significantly decreased the percentage of the in- farct area and attenuated brain edema. In addition, inflammatory nuclear factor-KB expression was significantly lower, while anti-apoptotic Bcl-2 expression was significantly elevated in the ce- rebral cortex on the ischemic side. Our findings indicate that remote ischemic postconditioning attenuates focal cerebral ischemia/reperfusion injury, and that the neuroprotective mechanism is mediated by an anti-apoptotic effect and reduction of the inflammatory response.

Electroacupuncture preconditioning protects against focal cerebral ischemia/reperfusion injury via suppression of dynamin-related protein 1

Electroacupuncture preconditioning at acupoint Baihui （GV20） can reduce focal cerebral ischemia/reperfusion injury. However, the precise protective mechanism remains unknown. Mitochondrial fission mediated by dynamin-related protein 1 （Drp1） can trigger neuronal apoptosis following cerebral ischemia/reperfusion injury. Herein, we examined the hypothesis that electroacupuncture pretreatment can regulate Drp1, and thus inhibit mitochondrial fission to provide cerebral protection. Rat models of focal cerebral ischemia/reperfusion injury were established by middle cerebral artery occlusion at 24 hours after 5 consecutive days of preconditioning with electroacupuncture at GV20 （depth 2 mm, intensity 1 mA, frequency 2/15 Hz, for 30 minutes, once a day）. Neurological function was assessed using the Longa neurological deficit score. Pathological changes in the ischemic penumbra on the injury side were assessed by hematoxylin-eosin staining. Cellular apoptosis in the ischemic penumbra on the injury side was assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling staining. Mitochondrial ultrastructure in the ischemic penumbra on the injury side was assessed by transmission electron microscopy. Drp1 and cytochrome c expression in the ischemic penumbra on the injury side were assessed by western blot assay. Results showed that electroacupuncture preconditioning decreased expression of total and mitochondrial Drp1, decreased expression of total and cytosolic cytochrome c, maintained mitochondrial morphology and reduced the proportion of apoptotic cells in the ischemic penumbra on the injury side, with associated improvements in neurological function. These data suggest that electroacupuncture preconditioning-induced neuronal protection involves inhibition of the expression and translocation of Drp1.

The role of Rho/Rho-kinase pathway and the neuroprotective effects of fasudil in chronic cerebral ischemia

The Rho/Rho-kinase signaling pathway plays an important role in cerebral ischemia/reperfusion injury. However, very few studies have examined in detail the changes in the Rho/Rho-kinase signaling pathway in chronic cerebral ischemia. In this study, rat models of chronic cerebral ischemia were established by permanent bilateral common carotid artery occlusion and intra- gastrically administered 9 mg/kg fasudil, a powerful ROCK inhibitor, for 9 weeks. Morris water maze results showed that cognitive impairment progressively worsened as the cerebral ischemia proceeded. Immunohistochemistry, semi-quantitative RT-PCR and western blot analysis showed that the expression levels of Rho-kinase, its substrate myosin-binding subunit, and its relat- ed protein alpha smooth muscle actin, significantly increased after chronic cerebral ischemia. TUNEL staining showed that chronic cerebral ischemia could lead to an increase in neuronal apoptosis, as well as the expression level of caspase-3 in the frontal cortex of rats subjected to chronic cerebral ischemia. Fasudil treatment alleviated the cognitive impairment in rats with chronic cerebral ischemia, and decreased the expression level of Rho-kinase, myosin-binding subunit and alpha smooth muscle actin. Furthermore, fasudil could regulate cerebral injury by reducing cell apoptosis and decreasing caspase-3 expression in the frontal cortex. These findings demonstrate that fasudil can protect against cognitive impairment induced by chronic cerebral ischemia via the Rho/Rho-kinase signaling pathway and anti-apoptosis mechanism.

Effects of Fujian tablet on Nogo-A mRNA expression and plasticity of the corticospinal tract in a rat model of focal cerebral ischemia

The present study investigated the effects of Fujian tablet, a Chinese medicine compound that can nourish liver and kidney, on corticospinal tract plasticity and cervical cord microenvironment in rats with focal cerebral ischemia. Results showed that motor function of rats with right proximal middle cerebral artery occlusion was significantly improved following treatment with Fujian tablet, 9 g crude drug/kg. Anterograde tracing revealed significantly increased biotinylated dextran amine expression in the denervated （left） side of the cervical cord （C4-6） following Fujian tablet treatment, and significantly decreased Nogo-A mRNA expression was detected in the denervated side of the cervical cord （C4-6） using in situ hybridization. Pearson＇s correlation analysis showed a negative correlation between biotinylated dextran amine and Nogo-A mRNA expression （r = -0.943, P 〈 0.01）. Results demonstrated that Fujian tablet can promote corticospinal tract plasticity possibly through the inhibitory effect on Nogo-A mRNA expression in the cervical spinal cord, thereby improving motor dysfunction.