Electroacupuncture stimulation of the brachial plexus trunk on the healthy side promotes brain-derived neurotrophic factor mRNA expression in the ischemic cerebral cortex of a rat model of cerebral ischemia/reperfusion injury

A rat model of cerebral ischemia/reperfusion was established by suture occlusion of the left middle cerebral artery. In situ hybridization results showed that the number of brain-derived neurotrophic factor mRNA-positive cells in the ischemic rat cerebral cortex increased after cerebral ischemia/ reperfusion injury. Low frequency continuous wave electroacupuncture （frequency 2-6 Hz, current intensity 2 mA） stimulation of the brachial plexus trunk on the healthy （right） side increased the number of brain-derived neurotrophic factor mRNA-positive cells in the ischemic cerebral cortex 14 days after cerebral ischemia/reperfusion injury. At the same time, electroacupuncture stimulation of the healthy brachial plexus truck significantly decreased neurological function scores and alleviated neurological function deficits. These findings suggest that electroacupuncture stimulation of the brachial plexus trunk on the healthy （right） side can greatly increase brain-derived neurotrophic factor mRNA expression and improve neurological function.

Effect of Electroacupuncture on Expression of p53 Protein in Cerebral Cortex of Rats with Global Cerebral Ischemia/Reperfusion Injury

Objective: To observe the effect of electroacupuncture (EA) on expression of p53 protein in cerebral cortex of senile rats with global cerebral ischemia/reperfusion (IR) injury and to explore its mechanism. Methods: The cerebral IR injury rat model was established referring to Pulsinelli 4-vessel occlusion method. Thirty-six SD rats were randomly and evenly divided into the control group, the IR group and the IR plus EA (IR-EA) group. The animals in the control group were subjected to electrocauterization of vertebral arteries in bilateral flank orifice alone with the general carotid arteries unoccluded. To rats in the IR-EA group, immediately and 24h, 48h, 72h after cerebral IR, EA treatment on bilateral acupoint 'Zusanli' (ST36) was applied once a day, lasting for 60 minutes. After the final treatment, all the rats were sacrificed and their brains were taken to examine p53 protein expression by the immunohistochemical method. Results: Cells with positive p53 immunoreactivity in the cerebral cortex of rats in the IR group was significantly higher than that in the control group ( P<0. 05), while that in the IR-EA group was significantly lower than that in the IR group ( P<0. 05). Conclusion: EA could remarkably reduce expression of p53 protein in the cerebral cortex of senile rats with global cerebral IR injury, which might be one of the means for EA to inhibit neuronal ap-optosis after cerebral IR injury.

Effects of anisodamine on altered [Ca^(2+)]i and cerebral cortex ultrastructure following acute cerebral ischemia/reperfusion injury in rabbits

BACKGROUND： Calcium ion （Ca^2＋） overload plays an important role in cerebral ischemia/reperfusion injury. Anisodamine, a type of alkaloid, can protect the myocardium from ischemia and reperfusion injury by inhibiting intracellular calcium [Ca^2＋]i overload. OBJECTIVE： To investigate effects of anisodamine on [Ca^2＋]i concentration and cortex ultrastructure following acute cerebral ischemia/reperfusion in rabbits. DESIGN, TIME AND SETTING： Randomized and controlled trial was performed at the Department of Emergency, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology from September to December 2006. MATERIALS： Forty healthy rabbits were used to establish models of acute cerebral ischemia/reperfusion. Anisodamine was provided by Lianyungang Dongfeng Pharmaceutical Factory; Fura-2 was purchased from Nanjing Jiancheng Bioengineering Institute; dual-wave length fluorescent spectrophotometry system and DM-300 software were provided by Bio-Rad, USA; OPTON-EM10C transmission electron microscope was product of Siemens, Germany. METHODS： Forty rabbits were randomly divided into the following groups： sham operation, ischemia, ischemia/reperfusion, and anisodamine, with ten rabbits in each group. Models of complete cerebral ischemia injury were established. In addition, blood was collected from the femoral artery of rats in the ischemia/reperfusion and anisodamine groups to induce hypotension and establish repeffusion injury models. The bilateral common carotid artery clamp was removed from the anisodamine group 20 minutes after ischemia, and anisodamine （10 mg/kg body mass） was injected via the femoral vein. Rabbits in the sham operation group underwent only venous cannulation. MAIN OUTCOME MEASURES： [Ca^2＋]i concentration was determined using a dual-wave length fluorescent spectrophotometry system, and cortical ultrastructure was observed following uranyl-lead citrate staining. RESULTS： The levels of [Ca^2＋]i in the ischemia and ischemia/reperfusion groups were significantly increased, compared with the sham operation group （P 〈 0.01）, and the levels of [Ca^2＋]i in the anisodamine group were remarkably less than the ischemia and ischemia/reperfusion groups （P 〈 0.01）. Ultrastructural damage to the cortex was greatly aggravated with increasing levels of [Ca^2＋]i. In the ischemia group, cortical neuronal membranes were fragmentally damaged, including the mitochoudria and endoplasmic reticulum, as well as neufite swelling, and slight chromatin margination. In the ischemia/reperfusion group, the cellular membrane was ruptured with aggravated mitochondrial swelling, increased chromatin margination, obscure neufite structure, and the disappearance of endoplasmic reticulum. However, in the anisodamine group, cellular damage was obviously alleviated. The appearance and structure of cortical neurons was relatively normal, with intact cells. There was slight swelling of the mitochondria and endoplasmic reticulum, as well as mild chromatin margination. CONCLUSION： Cerebral tissue injury was related to increased [Ca^2＋]i levels following ischemia/ reperfusion. Anisodamine exhibited a protective role on acute cerebral ischemia/reperfusion injury by inhibiting the increase in [Ca^2＋]i levels.

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.

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.

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.

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.

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.

Vav1 promotes inflammation and neuronal apoptosis in cerebral ischemia/reperfusion injury by upregulating microglial and NLRP3 inflammasome activation

Microglia,which are the resident macrophages of the central nervous system,are an important part of the inflammatory response that occurs after cerebral ischemia.Vav guanine nucleotide exchange factor 1(Vav1) is a guanine nucleotide exchange factor that is related to microglial activation.However,how Vav1 participates in the inflammato ry response after cerebral ischemia/reperfusion inj ury remains unclea r.In this study,we subjected rats to occlusion and repe rfusion of the middle cerebral artery and subjected the BV-2 mic roglia cell line to oxygen-glucose deprivatio n/reoxygenation to mimic cerebral ischemia/repe rfusion in vivo and in vitro,respectively.We found that Vav1 levels were increased in the brain tissue of rats subjected to occlusion and reperfusion of the middle cerebral arte ry and in BV-2 cells subjected to oxygen-glucose deprivation/reoxygenation.Silencing Vav1 reduced the cerebral infarct volume and brain water content,inhibited neuronal loss and apoptosis in the ischemic penumbra,and im p roved neurological function in rats subjected to occlusion and repe rfusion of the middle cerebral artery.Further analysis showed that Vav1 was almost exclusively localized to microglia and that Vav1 downregulation inhibited microglial activation and the NOD-like receptor pyrin 3(NLRP3) inflammasome in the ischemic penumbra,as well as the expression of inflammato ry facto rs.In addition,Vov1 knoc kdown decreased the inflammatory response exhibited by BV-2 cells after oxygen-glucose deprivation/reoxyge nation.Taken together,these findings show that silencing Vav1 attenuates inflammation and neuronal apoptosis in rats subjected to cerebral ischemia/repe rfusion through inhibiting the activation of mic roglia and NLRP3 inflammasome.

Ischemic accumulation of succinate induces Cdc42 succinylation and inhibits neural stem cell proliferation after cerebral ischemia/reperfusion

Ischemic accumulation of succinate causes cerebral damage by excess production of reactive oxygen species. However, it is unknown whether ischemic accumulation of succinate affects neural stem cell proliferation. In this study, we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery. We found that succinate levels increased in serum and brain tissue(cortex and hippocampus) after ischemia/reperfusion injury. Oxygen-glucose deprivation and reoxygenation stimulated primary neural stem cells to produce abundant succinate. Succinate can be converted into diethyl succinate in cells. Exogenous diethyl succinate inhibited the proliferation of mouse-derived C17.2 neural stem cells and increased the infarct volume in the rat model of cerebral ischemia/reperfusion injury. Exogenous diethyl succinate also increased the succinylation of the Rho family GTPase Cdc42 but repressed Cdc42 GTPase activity in C17.2 cells. Increasing Cdc42 succinylation by knockdown of the desuccinylase Sirt5 also inhibited Cdc42 GTPase activity in C17.2 cells. Our findings suggest that ischemic accumulation of succinate decreases Cdc42 GTPase activity by induction of Cdc42 succinylation, which inhibits the proliferation of neural stem cells and aggravates cerebral ischemia/reperfusion injury.

Upregulation of CDGSH iron sulfur domain 2 attenuates cerebral ischemia/reperfusion injury

CDGSH iron sulfur domain 2 can inhibit ferroptosis,which has been associated with cerebral ischemia/reperfusion,in individuals with head and neck cancer.Therefore,CDGSH iron sulfur domain 2 may be implicated in cerebral ischemia/reperfusion injury.To validate this hypothesis in the present study,we established mouse models of occlusion of the middle cerebral artery and HT22 cell models of oxygen-glucose deprivation and reoxygenation to mimic cerebral ischemia/reperfusion injury in vivo and in vitro,respectively.We found remarkably decreased CDGSH iron sulfur domain 2 expression in the mouse brain tissue and HT22 cells.When we used adeno-associated virus and plasmid to up-regulate CDGSH iron sulfur domain 2 expression in the brain tissue and HT22 cell models separately,mouse neurological dysfunction was greatly improved;the cerebral infarct volume was reduced;the survival rate of HT22 cells was increased;HT22 cell injury was alleviated;the expression of ferroptosis-related glutathione peroxidase 4,cystine-glutamate antiporter,and glutathione was increased;the levels of malondialdehyde,iron ions,and the expression of transferrin receptor 1 were decreased;and the expression of nuclear-factor E2-related factor 2/heme oxygenase 1 was increased.Inhibition of CDGSH iron sulfur domain 2 upregulation via the nuclear-factor E2-related factor 2 inhibitor ML385 in oxygen-glucose deprived and reoxygenated HT22 cells blocked the neuroprotective effects of CDGSH iron sulfur domain 2 up-regulation and the activation of the nuclear-factor E2-related factor 2/heme oxygenase 1 pathway.Our data indicate that the up-regulation of CDGSH iron sulfur domain 2 can attenuate cerebral ischemia/reperfusion injury,thus providing theoretical support from the perspectives of cytology and experimental zoology for the use of this protein as a therapeutic target in patients with cerebral ischemia/reperfusion injury.

A molecular probe carrying anti-tropomyosin 4 for early diagnosis of cerebral ischemia/reperfusion injury

In vivo imaging of cerebral ischemia/reperfusion injury remains an important challenge.We injected porous Ag/Au@SiO_(2) bimetallic hollow nanoshells carrying anti-tropomyosin 4 as a molecular probe into mice with cerebral ischemia/reperfusion injury and observed microvascular changes in the brain using photoacoustic imaging with ultrasonography.At each measured time point,the total photoacoustic signal was significantly higher on the affected side than on the healthy side.Twelve hours after reperfusion,cerebral perfusion on the affected side increased,cerebrovascular injury worsened,and anti-tropomyosin 4 expression increased.Twenty-four hours after reperfusion and later,perfusion on the affected side declined slowly and stabilized after 1 week;brain injury was also alleviated.Histopathological and immunohistochemical examinations confirmed the brain injury tissue changes.The nanoshell molecular probe carrying anti-tropomyosin 4 has potential for use in early diagnosis of cerebral ischemia/reperfusion injury and evaluating its progression.

DNA hypomethylation promotes learning and memory recovery in a rat model of cerebral ischemia/reperfusion injury

Cerebral ischemia/reperfusion injury impairs learning and memory in patients.Studies have shown that synaptic function is involved in the formation and development of memory,and that DNA methylation plays a key role in the regulation of learning and memory.To investigate the role of DNA hypomethylation in cerebral ischemia/reperfusion injury,in this study,we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery and then treated the rats with intraperitoneal 5-aza-2′-deoxycytidine,an inhibitor of DNA methylation.Our results showed that 5-aza-2′-deoxycytidine markedly improved the neurological function,and cognitive,social and spatial memory abilities,and dose-dependently increased the synaptic density and the expression of SYP and SHANK2 proteins in the hippocampus in a dose-dependent manner in rats with cerebral ischemia/reperfusion injury.The effects of 5-aza-2′-deoxycytidine were closely related to its reduction of genomic DNA methylation and DNA methylation at specific sites of the Syp and Shank2 genes in rats with cerebral ischemia/reperfusion injury.These findings suggest that inhibition of DNA methylation by 5-aza-2′-deoxycytidine promotes the recovery of learning and memory impairment in a rat model of cerebral ischemia/reperfusion injury.These results provide theoretical evidence for stroke treatment using epigenetic methods.

Edema and neuronal apoptosis in the hippocampus and cortex of elderly rats following transient cerebral ischemia/reperfusion injury

BACKGROUND： Previous studies of cerebral ischemia have used young animals, with an ischemic time greater than 5 minutes （safe time limit）. Despite an increased understanding of neuronal apoptosis, it remains uncertain whether brief cerebral ischemic events of 5 minutes or less damage brain tissue in elderly rodents. OBJECTIVE： To investigate the effects of transient cerebral ischemia （5 minutes）/reperfusion injury on brain cortical and hippocampal edema, aquaporin-4 （AQP-4） expression, and neuronal apoptosis in aged rats, and to compare ischemic sensitivity between cortex and hippocampus. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Institute of Cerebrovascular Disease, Qingdao University Medical School from April 2008 to March 2009. MATERIALS： Rabbit anti-AQP-4 polyclonal antibody, TUNEL kit, and SABC immunohistochemistry kit were purchased from Wuhan Boster Bioengineering, China. METHODS： A total of 160 healthy, male, aged 19-21 months, Wistar rats were randomly assigned to 4 groups： sham-surgery, and ischemia 1-, 3-, and 5-minute groups, with 40 rats in each group. The global cerebral ischemia model was established using the Pusinelli four-vessel occlusion, and the three cerebral ischemia groups were subdivided into reperfusion 12-hour, 1-, 2-, 3-, and 7-day subgroups, with 8 rats in each subgroup. The sham-surgery group was subjected to exposure of the first cervical bilateral alar foramina and bilateral common carotid arteries. MAIN OUTCOME MEASURES： The dry-wet weight assay was used to measure brain water content and histopathology of the cortex and hippocampus was observed following hematoxylin-eosin staining. In addition, cortical and hippocampal AQP-4 expression was detected by streptavidin-biotin complex immunohistochemistry, and neuronal apoptosis was detected by the TUNEL method. RESULTS： There was no significant difference in brain water content or AQP-4 expression in the cortex and hippocampus between ischemia 1- and 3-minute groups and the sham-surgery group or brain water content or AQP-4 expression in the cortex between ischemia 5-minute group and sham-surgery group （P 〉 0.05）. However, brain water content and AQP-4 expression in the hippocampus after 5 minutes of cerebral ischemia were significantly increased compared with the sham-surgery group （P 〈 0.05 or P 〈 0.01）. Several TUNEL-positive cells were observed in the cortex and hippocampus of the sham-surgery group and ischemia 1-minute group, as well as in the cortex of the ischemia 3-minute group. In addition, the number of apoptotic neurons in the hippocampus of ischemia 3-minute group and in the cortex and hippocampus of ischemia 5-minute group was significantly increased （P 〈 0.05 or P 〈 0.01 ）. Neuronal apoptosis was increased after 12 hours of ischemia/reperfusion, and it reached a peak by 2 days （P 〈 0.01）. CONCLUSION： Transient cerebral ischemia （5 minutes） resulted in increased hippocampal edema, AQP-4 expression, and neuronal apoptosis. Moreover, cerebral ischemia had a greater effect on neuronal apoptosis than brain edema or AQP-4 expression, and the hippocampus was more sensitive than the cortex.

Nerve growth factor downregulates c-jun mRNA and Caspase-3 in striate cortex of rats after transient global cerebral ischemia/reperfusion

BACKGROUND： Immediate early gene （lEG） c-jun is a sensitive marker for functional status of nerve cells. Caspase-3 is a cysteine protease, which is a critical regulator of apoptosis. The effect of exogenous nerve growth factor （NGF） on the expression of c-jun mRNA and Caspase-3 protein in striate cortex of rats with transient global cerebral ischemia/reperfusion （IR） is unclear. OBJECTIVE： To study the protective effect of exogenous NGF on the brain of rats with transient globa cerebral IR and its effecting pathway by observing the expression of c-jun mRNA and Caspase-3 protein. DESIGN： Randomized controlled animal trial SETTING： Department of Neural Anatomy, Institute of Brain, China Medical University MATERIALS：Eighteen healthy male SD rats of clean grade, aged 1 to 3 months, with body mass of 250 to 300 g, were involved in this study. NGF was provided by Dalian Svate Pharmaceutical Co.,Ltd. c-jun in situ hybridization detection kit, Caspase-3 antibody and SABC kit were purchased from Boster Biotechnology Co.. Ltd. METHODS： This trial was carried out in the Department of Neural Anatomy, Institute of Brain, China Medical University during September 2003 to April 2005. （1） Experimental animals were randomized into three groups with 6 in each： sham-operation group, IR group and NGF group.（2）After the rats were anesthetized, the bilateral common carotid arteries and right external carotid arteries of rats were bluntly dissected and bilateral common carotid arteries were clamped for 30 minutes with bulldog clamps. Reperfusion began after buldog clamps were removed. Normal saline of lmL and NGF （1×10^6 U/L） of 1 mL was injected into the common carotid artery of rats via right external carotid arteries in the IR group and NGF group respectively. The injection was conducted within 30 minutes, and then the right external carotid arteries were ligated. In the sham-operation group, occlusion of bilateral common carotid arteries and administration of drugs were omitted.GAll the rats were executed by decollation at 3 hours after modeling. The animals were fixed with phosphate buffer solution （PBS, 0.1 mol/L） containing 40 g/L polyformaldehyde, their brains were quickly removed. The coronal section tissue mass containing striate cortex about 3 mm before line between two ears was taken and made into successive frozen sections.（4）The expression of c-jun mRNA and Caspase-3 protein in striate cortex of global cerebral ischemia rats were detected with in situ hybridization, immunohistochemistry and microscope image analysis. （5）t test was used for comparing the difference of the measurement data. MAIN OUTCOME MEASURES：Comparison of the expression of lEG c-jun mRNA and Caspase-3 protein in striate cortex of brain of rats in each group. RESULTS：All the 18 SD rats were involved in the analysis of results. The c-jun mRNA and Caspase-3 protein positive reaction cells were found brown yellow in the striate cortex of rats, and most of them were in lamellas Ⅱ and Ⅲ, mainly presenting round or oval. The expression of c-jun mRNA and Caspase-3 protein in sham-operation group was weak or negative. The average gray value of c-jun mRNA and Caspase-3 protein in the IR group was significantly lower than that in the sham-operation group （49.52±4.13 vs. 95.48± 5.28; 74.73±4.29 vs. 162.38±9.16,P 〈 0.01）. The average gray value of c-jun mRNA and Caspase-3 protein in the NGF group was significantly higher than that in the IR group （63.96±4.25 vs.49.52±4.13; 83.98± 4.13 vs. 74.73±4.29, P〈 0.05）. CONCLUSION： NGF can protect ischemic neurons by down-regulating the expression of c-jun mRNA and Caspase-3 protein in striate cortex of global cerebral ischemia rats.

Changes in corticocerebral morphology in a rat model of focal cerebral ischemia/reperfusion injury following “Xingnao Kaiqiao” acupuncture

BACKGROUND： Cerebral ischemia/reperfusion injury has been shown to induce inflammatory reactions, including white blood cell activation and adhesion molecule expression. These reactions often lead to aggravated neuronal injury. OBJECTIVE： To observe corticocerebral pathology, as well as ultrastructural changes, in a rat model of focal cerebral ischemia/reperfusion injury through optical and electron microscopy, and to investigate interventional effects of ＂Xingnao Kaiqiao＂ acupuncture （a brain-activating and orifice-opening acupuncture method）. DESIGN, TIME AND SETTING： A randomized, controlled, neuropathology, animal experiment was performed at the Laboratory of Molecular Biology, First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine between April and June 2004. MATERIALS： A total of 50 healthy, male, Wistar rats were randomized into 5 groups, with 10 rats per group： control, sham-operated, model, non-acupoint, and ＂Xingnao Kaiqiao ＂. Transmission electron microscope （TEM 400ST） was provided by Philips, Netherlands. Electro-acupuncture treatment apparatus （KWD-8082） was provided by Changzhou Wujin Great Wall Medical Instrument, China. METHODS： Focal cerebral ischemia/reperfusion injury was induced by occlusion of the middle cerebral artery in the model, non-acupoint, and ＂Xingnao Kaiqiao＂ groups. Rats from the control group did not undergo any treatment. The sham-operated group received identical experimental procedures as the model group, except that the nylon suture was not inserted into the right internal carotid artery. At 1, 3, 6, and 12 hours following focal cerebral ischemia/reperfusion injury induction, rats from the Xingnao Kaiqiao group underwent 1-minute acupuncture at the bilateral ＂Neiguan＂ （PC 6） acupoint, using a reducing method of lifting-thrusting and twirling-rotating. Subsequently, the rats were subjected to acupuncture at the ＂Renzhong＂ （DU26） acupoint 10 times by a heavy bird-pecking method. The non-acupoint group received acupuncture administration at the bilateral costal region. MAIN OUTCOME MEASURES： After ischemia for 1 hour and reperfusion for 24 hours, corticocerebral morphology and ultrastructural changes were observed on the injured side through the use of optical and electron microscopy. RESULTS： Cerebral ischemia/reperfusion resulted in damage to neurons, glial cells, and capillary vessels in the rat brain. ＂Xingnao Kaiqiao＂ acupuncture produced superior curative effects when it was performed 3 hours after cerebral ischemia/reperfusion induction, resulting in slightly recovered neuronal structures, alleviated cellular interstitial edema, and more capillary vessels. At each corresponding time point, the ＂Xingnao Kaiqiao＂ group exhibited improved neuronal structure and cellular interstitial edema, compared with the non-acupoint group. CONCLUSION： ＂Xingnao Kaiqiao＂ acupuncture results in protective effects on corticocerebral neuronal morphology and ultrastructure in rats following focal cerebral ischemia/reperfusion.

Glucose Metabolic Alteration of Cerebral Cortical Subareas in Rats with Renal Ischemia/Reperfusion Based on Small-Animal Positron Emission Tomography

Objective:To investigate glucose metabolic alterations in cerebral cortical subareas using ^(18)F-labeled glucose derivative fluorodeoxyglucose(FDG)micro-positron emission tomography(PET)scanning in a rat renal ischemia/reperfusion(RIR)model.Methods:Small-animal PET imaging in vivo was performed with ^(18)F-labeled FDG as a PET tracer to identify glucose metabolic alterations in cerebral cortical subregions using a rat model of RIR.Results:We found that the average standardized uptake value(SUV_(average))of the cerebral cortical subareas in the RIR group was significantly increased compared to the sham group(P<0.05).We also found that glucose uptake in different cortical subregions including the left auditory cortex,right medial prefrontal cortex,right para cortex,left retrosplenial cortex,right retrosplenial cortex,and right visual cortex was significantly increased in the RIR group(P<0.05),but there was no significant difference in the SUV_(avcrage) of right auditory cortex,left medial prefrontal cortex,left para cortex,and left visual cortex between the two groups.Conclusion:The ^(18)F-FDG PET data suggests that RIR causes a profound shift in the metabolic machinery of cerebral cortex subregions.

Ligustrazine monomer against cerebral ischemia/reperfusion injury

Ligustrazine （2,3,5,6-tetramethylpyrazine） is a major active ingredient of the Szechwan lovage rhizome and is extensively used in treatment of ischemic cerebrovascular disease. The mecha- nism of action of ligustrazine use against ischemic cerebrovascular diseases remains unclear at present. This study summarizes its protective effect, the optimum time window of administra- tion, and the most effective mode of administration for clinical treatment of cerebral ischemia/ reperfusion injury. We examine the effects of ligustrazine on suppressing excitatory amino acid release, promoting migration, differentiation and proliferation of endogenous neural stem cells. We also looked at its effects on angiogenesis and how it inhibits thrombosis, the inflammatory response, and apoptosis after cerebral ischemia. We consider that ligustrazine gives noticeable protection from cerebral ischemia/reperfusion injury. The time window of ligustrazine admin- istration is limited. The protective effect and time window of a series of derivative monomers of ligustrazine such as 2-[（1,1-dimethylethyl）oxidoimino]methyl]-3,5,6-trimethylpyrazine, CXC137 and CXC 195 after cerebral ischemia were better than ligustrazine.

Neuronal apoptosis in cerebral ischemia/reperfusion area following electrical stimulation of fastigial nucleus

Previous studies have indicated that electrical stimulation of the cerebellar fastigial nucleus in rats may reduce brain infarct size, increase the expression of Ku70 in cerebral ischemia/ reperfusion area, and decrease the number of apoptotic neurons. However, the anti-apoptotic mechanism of Ku70 remains unclear. In this study, fastigial nucleus stimulation was given to rats 24, 48, and 72 hours before cerebral ischemia/reperfusion injury. Results from the electrical stim- ulation group revealed that rats exhibited a reduction in brain infarct size, a significant increase in the expression of KuT0 in cerebral ischemia/reperfusion regions, and a decreased number of terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL）-positive cells. Double immunofluorescence staining revealed no co-localization of Ku70 with TUNEL-positive cells. However, Ku70 partly co-localized with Bax protein in the cytoplasm of rats with cerebral ischemia/reperfusion injury. These findings suggest an involvement of Ku70 with Bax in the cy- toplasm of rats exposed to electrical stimulation of the cerebellar fastigial nucleus, and may thus provide an understanding into the anti-apoptotic activity of KuT0 in cerebral ischemia/reperfu- sion injury.