Ischemia/hypoxia inhibits cardiomyocyte autophagy and promotes apoptosis via the Egr-1/Bim/Beclin-1 pathway

Background Myocardial injury caused by microvascular obstruction(MVO)is characterized by persistent ischemia/hypoxia(IH)of cardiomyocytes after microembolization.Autophagy and Egr-1 were closely associated with various cardiovascular diseases,including MVO.Bim and Beclin-1 are the important genes for autophagy and apoptosis.We aimed to explore whether the Egr-1/Bim/Beclin-1 pathway is involved in regulating autophagy and apoptosis in IH-exposed cardiomyocytes.Methods Neonatal rat cardiomyocytes exposed to the IH environment in vitro were transfected with lentivirus expressing Egr-1 or Egr-1 sh RNA,or further treated with 3-methyladenine(3-MA).The expressions of autophagy and apoptosis-associated genes were evaluated using RT-q PCR and Western blots assays.Autophagic vacuoles and autophagic flux were detected by transmission electron microscopy(TEM)and confocal microscope,respectively.Cell injury was assessed by lactate dehydrogenase(LDH)leakage,and apoptosis was determined by flow cytometry.Results IH exposure elevated Egr-1 and Bim expressions,and decreased Beclin-1 expression in rat cardiomyocytes.Egr-1 overexpression in IH-exposed cardiomyocytes significantly up-regulated the levels of Egr-1 and Bim,and down-regulated the level of Beclin-1.Egr-1 knockdown resulted in down-regulated expressions of Egr-1 and Bim,as well as up-regulated expression of Beclin-1.In addition,Egr-1 knockdown induced autophagy was suppressed by 3-MA treatments.TEM and autophagic flux experiments also confirmed that Egr-1 inhibited autophagy progression in IH-exposed cardiomyocytes.Egr-1 suppression protected cardiomyocytes from IH-induced injury,as evidenced by the positive correlations between Egr-1 expression and LDH leakage or apoptosis index in IH-exposed cardiomyocytes.Conclusions IH-induced cardiomyocyte autophagy and apoptosis are regulated by the Egr-1/Bim/Beclin-1 pathway,which is a potential target for treating cardiomyocyte injury caused by MVO in the IH environment.

Offspring of rats with cerebral hypoxia-ischemia manifest cognitive dysfunction in learning and memory abilities

Neonatal hypoxic-ischemic encephalopathy is a serious neurological disease,often resulting in long-term neurodevelopmental disorders among surviving children.However,whether these neurodevelopmental issues can be passed to offspring remains unclear.The right common carotid artery of 7-day-old parental-generation rats was subjected to permanent ligation using a vessel electrocoagulator.Neonatal hypoxic-ischemic rat models were established by subjecting the rats to 8%O2–92%N2 for 2 hours.The results showed that 24 hours after hypoxia and ischemia,pathological damage,cerebral atrophy,liquefaction,and impairment were found,and Zea-Longa scores were significantly increased.The parental-generation rats were propagated at 3 months old,and offspring were obtained.No changes in the overall brain structures of these offspring rats were identified by magnetic resonance imaging.However,the escape latency was longer and the number of platform crossings was reduced among these offspring compared with normal rats.These results indicated that the offspring of hypoxic-ischemic encephalopathy model rats displayed cognitive impairments in learning and memory.This study was approved by the Animal Care&Welfare Committee of Kunming Medical University,China in 2018(approval No.kmmu2019072).

Rosmarinic acid ameliorates hypoxia/ischemia induced cognitive deficits and promotes remyelination

Rosmarinic acid,a common ester extracted from Rosemary,Perilla frutescens,and Salvia miltiorrhiza Bunge,has been shown to have protective effects against various diseases.This is an investigation into whether rosmarinic acid can also affect the changes of white matter fibers and cognitive deficits caused by hypoxic injury.The right common carotid artery of 3-day-old rats was ligated for 2 hours.The rats were then prewarmed in a plastic container with holes in the lid,which was placed in 37°C water bath for 30 minutes.Afterwards,the rats were exposed to an atmosphere with 8% O2 and 92% N2 for 30 minutes to establish the perinatal hypoxia/ischemia injury models.The rat models were intraperitoneally injected with rosmarinic acid 20 mg/kg for 5 consecutive days.At 22 days after birth,rosmarinic acid was found to improve motor,anxiety,learning and spatial memory impairments induced by hypoxia/ischemia injury.Furthermore,rosmarinic acid promoted the proliferation of oligodendrocyte progenitor cells in the subventricular zone.After hypoxia/ischemia injury,rosmarinic acid reversed to some extent the downregulation of myelin basic protein and the loss of myelin sheath in the corpus callosum of white matter structure.Rosmarinic acid partially slowed down the expression of oligodendrocyte marker Olig2 and myelin basic protein and the increase of oligodendrocyte apoptosis marker inhibitors of DNA binding 2.These data indicate that rosmarinic acid ameliorated the cognitive dysfunction after perinatal hypoxia/ischemia injury by improving remyelination in corpus callosum.This study was approved by the Animal Experimental Ethics Committee of Xuzhou Medical University,China (approval No.20161636721) on September 16,2017.

Influence of hypoxia-inducible factor 1-alpha on neuronal apoptosis in a rat model of hypoxia-or hypoxia-ischemia-induced brain injury

BACKGROUND： In addition to neuroprotective genes, the targeted genes of hypoxia-inducible factor 1α （HIF-1α） include pro-apoptotic genes. However, the influence of HIF-1α on neuronal apoptosis in hypoxia-ischemia remains poorly understood. OBJECTIVE： To investigate the relationship between HIF-1α expression and neuronal apoptosis in hypoxia or hypoxia-ischemia brain injury and to determine the role of HIF-1α in regulating neuronal apoptosis. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed at the Laboratory of Children Neurology of Sichuan University between May 2006 and May 2007. MATERIALS： In situ cell death detected kit was provided by Roche, USA; rabbit anti-mouse HIF-1α polyclonal antibody was purchased from Santa Cruz Biotechnologies, USA; rabbit anti-mouse cleaved caspase-3 polyclonal antibody was purchased from Chemicon, USA. METHODS： A total of 36 Sprague Dawley rats aged 10 days were randomly assigned to 3 groups： sham-surgery, hypoxia, and hypoxia-ischemia, with 12 rats per group. The rats were treated at 3 time points： 4, 8, and 24 hours, with 4 rats per time point. In the hypoxia-ischemia group, the right common carotid artery was exposed and permanently ligated through a midline cervical incision. A 2.5-hour exposure to hypoxia （8% O2/92% N2） was used to induce hypoxia-ischemia injury. In the hypoxia group, rats were exposed to hypoxia without ligation of the common carotid artery. In the sham-surgery group, the common carotid artery was exposed without ligation or hypoxia. MAIN OUTCOME MEASURES： Histopathological changes, HIF-1α and activated caspase-3 protein expression, integrated optical density of positive cells, and apoptosis-positive cells. RESULTS： Hematoxylin and eosin staining showed that neuronal degeneration and edema was most prominent at 24 hours after hypoxia-ischemia. HIF-1α protein expression was significantly upregulated at 4 hours, peaked at 8 hours, and decreased at 24 hours after hypoxia or hypoxia-ischemia. HIF-1α protein expression was significant greater in the hypoxia and hypoxia-ischemia groups compared with the sham-surgery group （P 〈 0.01）. Activated caspase-3 protein expression began to increase at 4 and 8 hours following hypoxia or hypoxia-ischemia and was significantly upregulated at 24 hours. Activated caspase-3 protein expression remained at low levels in the sham controls compared with the hypoxia and hypoxia-ischemia groups （P〈 0.01）. TUNEL staining showed that the number of apoptotic cells significantly increased at 24 hours after hypoxia or hypoxia-ischemia. In addition, HIF-1α protein expression was greater in the hypoxia group compared with the hypoxia-ischemia group at the same time point （P 〈 0.05）. However, activated caspase-3 expression and the number of TUNEL-positive cells were less in the hypoxia group compared with the hypoxia-ischemia group at the same time point （P〈 0.05）. CONCLUSION： HIF-1α played a neuroprotective role following hypoxia-ischemia brain injury.

Changes of hypoxia-inducible factor-1 signaling and the effect of cilostazol in chronic cerebral ischemia

Hypoxiainducible factor1 and its specific target gene heme oxygenase1, are involved in acute cerebral ischemia. However, very few studies have examined in detail the changes in the hy poxiainducible factor1/heme oxygenase1 signaling pathway in chronic cerebral ischemia. In this study, a rat model of chronic cerebral ischemia was established by permanent bilateral common carotid artery occlusion, and these rats were treated with intragastric cilostazol （30 mg/kg） for 9 weeks. Morris water maze results showed that cognitive impairment gradually worsened as the cerebral ischemia proceeded. Immunohistochemistry, semiquantitative PCR and western blot analysis showed that hypoxiainducible factorla and heme oxygenase1 expression levels in creased after chronic cerebral ischemia, with hypoxiainducible factorla expression peaking at 3 weeks and heme oxygenase1 expression peaking at 6 weeks. These results suggest that the elevated levels of hypoxiainducible factorla may upregulate heine oxygenase1 expression fol lowing chronic cerebral ischemia and that the hypoxiainducible factor1/heme oxygenase1 sig naling pathway is involved in the development of cognitive impairment induced by chronic cerebral ischemia. Cilostazol treatment alleviated the cognitive impairment in rats with chronic cerebral ischemia, decreased hypoxiainducible factorla and heme oxygenase1 expression levels, and reduced apoptosis in the frontal cortex. These findings demonstrate that cilostazol can protect against cognitive impairment induced by chronic cerebral ischemic injury through an antiapoptotic mechanism.

Transfer of mitochondria from mesenchymal stem cells derived from induced pluripotent stem cells attenuates hypoxia-ischemia-induced mitochondrial dysfunction in PC12 cells

Mitochondrial dysfunction in neurons has been implicated in hypoxia-ischemia-induced brain injury.Although mesenchymal stem cell therapy has emerged as a novel treatment for this pathology,the mechanisms are not fully understood.To address this issue,we first co-cultured 1.5×10^5 PC12 cells with mesenchymal stem cells that were derived from induced pluripotent stem cells at a ratio of 1:1,and then intervened with cobalt chloride(CoCl2)for 24 hours.Reactive oxygen species in PC12 cells was measured by Mito-sox.Mitochondrial membrane potential(ΔΨm)in PC12 cells was determined by JC-1 staining.Apoptosis of PC12 cells was detected by terminal deoxynucleotidal transferase-mediated dUTP nick end-labeling staining.Mitochondrial morphology in PC12 cells was examined by transmission electron microscopy.Transfer of mitochondria from the mesenchymal stem cells derived from induced pluripotent stem cells to damaged PC12 cells was measured by flow cytometry.Mesenchymal stem cells were induced from pluripotent stem cells by lentivirus infection containing green fluorescent protein in mitochondria.Then they were co-cultured with PC12 cells in Transwell chambers and treated with CoCl2 for 24 hours to detect adenosine triphosphate level in PC12 cells.CoCl2-induced PC12 cell damage was dose-dependent.Co-culture with mesenchymal stem cells significantly reduced apoptosis and restoredΔΨm in the injured PC12 cells under CoCl2 challenge.Co-culture with mesenchymal stem cells ameliorated mitochondrial swelling,the disappearance of cristae,and chromatin margination in the injured PC12 cells.After direct co-culture,mitochondrial transfer from the mesenchymal stem cells stem cells to PC12 cells was detected via formed tunneling nanotubes between these two types of cells.The transfer efficiency was greatly enhanced in the presence of CoCl2.More importantly,inhibition of tunneling nanotubes partially abrogated the beneficial effects of mesenchymal stem cells on CoCl2-induced PC12 cell injury.Mesenchymal stem cells reduced CoCl2-induced PC12 cell injury and these effects were in part due to efficacious mitochondrial transfer.

Developmental changes of glutamate acid decarboxylase 67 in mouse brain after hypoxia ischemia

Objective To study the developmental changes of glutamic acid decarboxylase-67 （ GAD-67, a GABA synthetic enzyme） in normal and hypoxic ischemic （HI） brain. Methods C57/BL6 mice on postnatal day （P） 5, 9, 21 and 60, corresponding developmentally to premature, term, juvenile and adult human brain were investigated by using both Western blot and immunohistochemistry methods either in normal condition or after hypoxic ischemic insult. Results The immunoreactivity of GAD67 was up regulated with brain development and significant difference was seen between mature （P21, P60） and immature （P5, P9） brain. GAD67 immunoreactivity decreased in the ipsilateral hemisphere in all the ages after hypoxia ischemia （HI） insult, but, significant decrease was only seen in the immature brain. Double labeling of GAD67 and cell death marker, TUNEL, in the cortex at 8h post-HI in the P9 mice showed that （15.6±7.0）% TUNEL positive cells were GAD67 positive which was higher than that of P60 mice. Conclusion These data suggest that GABAergic neurons in immature brain were more vulnerable to HI insult than that of mature brain.

Neuroprotective effects of autophagy inhibition on hippocampal glutamate receptor subunits after hypoxia-ischemia-induced brain damage in newborn rats

Autophagy has been suggested to participate in the pathology of hypoxic-ischemic brain damage（HIBD）.However,its regulatory role in HIBD remains unclear and was thus examined here using a rat model.To induce HIBD,the left common carotid artery was ligated in neonatal rats,and the rats were subjected to hypoxia for 2 hours.Some of these rats were intraperitoneally pretreated with the autophagy inhibitor 3-methyladenine（10 m M in 10 μL） or the autophagy stimulator rapamycin（1 g/kg） 1 hour before artery ligation.Our findings demonstrated that hypoxia-ischemia-induced hippocampal injury in neonatal rats was accompanied by increased expression levels of the autophagy-related proteins light chain 3 and Beclin-1 as well as of the AMPA receptor subunit GluR 1,but by reduced expression of GluR 2.Pretreatment with the autophagy inhibitor 3-methyladenine blocked hypoxia-ischemia-induced hippocampal injury,whereas pretreatment with the autophagy stimulator rapamycin significantly augmented hippocampal injury.Additionally,3-methyladenine pretreatment blocked the hypoxia-ischemia-induced upregulation of Glu R1 and downregulation of GluR2 in the hippocampus.By contrast,rapamycin further elevated hippocampal Glu R1 levels and exacerbated decreased GluR2 expression levels in neonates with HIBD.Our results indicate that autophagy inhibition favors the prevention of HIBD in neonatal rats,at least in part,through normalizing Glu R1 and GluR2 expression.

Short Review of Ischemia- and Hypoxia-Protective Roles of “Big Potassium” (BK) Channels

There is accumulating evidence that the subfamily of large-conductance potassium (“big”, “BK”) channels are involved in diverse, and perhaps coordinated, protective or counteractive responses to local or generalized ischemia and hypoxia. Although widely distributed, the physiological differences among BK channels which results from posttranslational modification (alternative splicing) and co-assembly with auxiliary modulatory subunits (β1-4 and γ1-4), bestows localized differences in subunit composition, distribution, 2nd-messenger coupling, and pharmacologic properties. Due to the ubiquitous nature of BK channels and the multiplicity of subtypes, they have many potential therapeutic applications in the maintenance of oxygen homeostasis, cerebro- and cardio-protection, and stimulation of respiration in response to drug-induced respiratory depression. BK channels may also offer other potentially broad and underrecognized promising targets for novel pharmaceutical development.

Correlation of hypoxia-inducible factor-1 alpha and erythropoietin protein and mRNA to cerebral ischemic tolerance in a focal ischemia/reperfusion model using the twice suture method

BACKGROUND： Numerous studies have shown that transient ischemic preconditioning induces cerebral ischemic tolerance. However, the underlying mechanisms of endogenous protection following ischemic preconditioning remain unclear. OBJECTIVE： To dynamically measure erythropoietin and hypoxia-inducible factor-1α （HIF-1α） mRNA and protein expression at various times following preconditioning, and to investigate effects of erythropoietin and HIF-1α on cerebral ischemic tolerance in a model of focal ischemia/reperfusion established using the twice suture method. DESIGN, TIME AND SETTING： The randomized, controlled study was performed at the Institute of Anatomy, Medical College, Qingdao University, China from March 2006 to March 2007. MATERIALS： Rabbit anti-rat HIF-1α monoclonal antibody and biotinylated goat anti-rabbit IgG （Boster, China）, rabbit anti-rat erythropoietin monoclonal antibody （Santa Cruz Biotechnology, USA）, and one-step RT-PCR kit （Qiagen, Germany） were used in this study. METHODS： A total of 99 healthy, male, Wistar rats were randomly assigned to three groups： sham surgery （n = 9）, non-ischemic preconditioning （n = 45）, and ischemic preconditioning （n = 45）. In the ischemic preconditioning group, rat models of pre-ischemia-reperfusion-ischemia-reperfusion were established by occluding the left middle cerebral artery using the twice suture method. In the non-ischemic preconditioning group, pre-ischemia was replaced by sham surgery. Subsequently, the ischemic preconditioning and non-ischemic preconditioning groups were equally divided into five subgroups according to time of first reperfusion, including 1-, 3-, 7-, 14-, and 21-day subgroups. The sham surgery group received the sham surgery twice. MAIN OUTCOME MEASURES： HIF-la and erythropoietin protein expression was measured in the cerebral cortex, corpus striatum, and hippocampus of the ischemic hemisphere. HIF-1α and erythropoietin mRNA expression were determined in the frontal and parietal cortex of the ischemic hemisphere. RESULTS： （1） Intergroup comparison： compared with the non-ischemic preconditioning group, HIF-1α protein expression significantly increased in the rat cerebral cortex, corpus striatum, and hippocampus in the ischemic hemisphere at 1,3, and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05 or P 〈 0.01）. Erythropoietin protein expression significantly increased in the cerebral cortex, corpus striatum, and hippocampus, as well as HIF-1α and erythropoietin mRNA expression in the frontal and parietal cortex in the ischemic hemisphere, at 3 and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05）. （2） Temporal expression： HIF-1α protein expression in the rat cerebral cortex, corpus striatum, and hippocampus, as well as HIF-la mRNA expression in the frontal and parietal cortex, in the ischemic hemisphere increased at 3 days, and gradually decreased from 7 days following reperfusion in the ischemic preconditioning group. Temporal erythropoietin protein and mRNA expression was consistent with HIF-1α protein expression. （3） Correlation： erythropoietin mRNA expression positively correlated with HIF-1α mRNA expression （r= 0.737, P 〈 0.01）. CONCLUSION： Ischemic preconditioning induced cerebral ischemic tolerance. Pre-ischemiainduced increase in endogenous HIF-1αexpression, as well as its target gene erythropoietin, participated in the formation of cerebral ischemic tolerance.

Effects of exogenous ganglioside-1 on learning and memory in a neonatal rat model of hypoxia-ischemia brain injury

BACKGROUND： Exogenous ganglioside-1 （GM1） can cross the blood-brain barrier and play a protective role against hypoxia-ischemia-induced brain damage. OBJECTIVE： To examine the possible mechanisms of exogenous GM1 protection in hypoxia-ischemia-induced brain damage in a neonatal rat model by measuring changes in brain mass, pathological morphology, growth-associated protein-43 expression, and neurobehavioral manifestations. DESIGN, TIME AND SETTING： A randomized block-design study was performed at the Immunohistochemistry Laboratory of the Pediatric Research Institute, Children＇s Hospital of Chongqing Medical University from August 2005 to August 2006. MATERIALS： A total of 36 neonatal, 7-day-old, Sprague Dawley rats were used in this experiment. The hypoxia-ischemia-induced brain damage model was established by permanently occluding the right carotid artery, followed by oxygen inhalation at a low concentration （8% O2, 92% N2） for 2 hours, METHODS： All rats were randomly divided into the following groups： GMI, model, and sham operation, with 12 rats each group. Rats in the GM 1 and model groups received hypoxic/ischemic-induced brain damage. Rats in the GM1 group received injections of GM1 （i.p., 20 mg/kg） at 0, 24, 48, 72, 96, 120, and 144 hours following models established, and rats in the model group were administered （i.p.） the same amount of saline. The right carotid artery was separated, but not ligated, in the sham operation group rats. MAIN OUTCOME MEASURES： At 1 week after surgery, expression of growth-associated protein-43, a marker of neural development and plasticity, was detected in the hippocampal CA3 region by immunohistochemistry. Brain mass was measured, and the pathological morphology was observed. At 4 weeks after surgery, behavioral changes in the remaining rats were tested by Morris water maze, and growth-associated protein-43 expression was measured. RESULTS： （1） In the GMI and sham operation groups, growth-associated protein-43 expression was greater in the hippocampal CA3 region compared to the model group 1 week after surgery （P 〈 0.05）. In all three groups, brain weight of the right hemisphere was significantly less than the left hemisphere, in particular in the model group （P 〈 0.05）. In the GMI group, the weight difference between two hemispheres, as well as the extent of damage in the right hemisphere, was less than the model group （P 〈 0.01 ）. In the sham operation Uoup, brain tissue consisted of integrated structures and ordered cells. In the model group, the cerebral cortex layers of the right hemisphere were not defined, neurons were damaged, and neurons were disarranged in the hippocampal area. In the GM1 group, neurons were dense in the right cerebral cortex and hippocampal area, with no significant change in glial proliferation. （2） The average time of escape latency in the GM1 group was shortened 4 weeks alter surgery, and significantly less than the model group （P 〈 0.05）. In addition, the frequency platform passing in the GMI group was significantly greater than the model group （P 〈 0.01）. CONCLUSION： Exogenous GM1 may reduce brain injury and improve learning and memory in hypoxia-ischemia-induced brain damage rats. This protection may be associated with increased growth-associated protein-43 expression, which is involved in neuronal remodeling processes.

Hypoxia-ischemia in the immature rodent brain impairs serotonergic neuronal function in certain dorsal raphé nuclei

Neonatal hypoxia-ischemia(HI) results in losses of serotonergic neurons in specific dorsal raphé nuclei. However, not all serotonergic raphé neurons are lost and it is therefore important to assess the function of remaining neurons in order to understand their potential to contribute to neurological disorders in the HI-affected neonate. The main objective of this study was to determine how serotonergic neurons, remaining in the dorsal raphé nuclei after neonatal HI, respond to an external stimulus(restraint stress). On postnatal day 3(P3), male rat pups were randomly allocated to one of the following groups:(i) control + no restraint(n = 5),(ii) control + restraint(n = 6),(iii) P3 HI + no restraint(n = 5) or(iv) P3 HI + restraint(n = 7). In the two HI groups, rat pups underwent surgery to ligate the common carotid artery and were then exposed to 6% O2 for 30 minutes. Six weeks after P3 HI, on P45, rats were subjected to restraint stress for 30 minutes. Using dual immunolabeling for Fos protein, a marker for neuronal activity, and serotonin(5-hydroxytrypamine; 5-HT), numbers of Fos-positive 5-HT neurons were determined in five dorsal raphé nuclei. We found that restraint stress alone increased numbers of Fos-positive 5-HT neurons in all five dorsal raphé nuclei compared to control animals. However, following P3 HI, the number of stress-induced Fos-positive 5-HT neurons was decreased significantly in the dorsal raphé ventrolateral, interfascicular and ventral nuclei compared with control animals exposed to restraint stress. In contrast, numbers of stress-induced Fos-positive 5-HT neurons in the dorsal raphé dorsal and caudal nuclei were not affected by P3 HI. These data indicate that not only are dorsal raphé serotonergic neurons lost after neonatal HI, but also remaining dorsal raphé serotonergic neurons have reduced differential functional viability in response to an external stimulus. Procedures were approved by the University of Queensland Animal Ethics Committee(UQCCR958/08/NHMRC) on February 27, 2009.

Effects of Naotan Pill on repair of neural cells and cognitive disorders in juvenile rats following hypoxia and ischemia

BACKGROUND： Hypoxia and ischemia induce neuronal damage, decreased neuronal numbers and synaptophysin levels, and deficits in learning and memory functions. Previous studies have shown that lycium barbarum polysaccharide, the most effective component of barbary wolfberry fruit, has protective effects on neural cells in hypoxia-ischemia. OBJECTIVE： To investigate the effects of Naotan Pill on glutamate-treated neural cells and on cognitive function in juvenile rats following hypoxia-ischemia. DESIGN, TIME AND SETTING： The randomized, controlled, in vivo study was performed at the Cell Laboratory of Lanzhou University, Lanzhou Institute of Modern Physics of Chinese Academy of Sciences, and Department of Traditional Chinese Medicine of Gansu Provincial Rehabilitation Center Hospital, China from December 2005 to August 2006. The cellular neurobiology, in vitro experiment was conducted at the Institute of Human Anatomy, Histology, Embryology and Neuroscience, School of Basic Medical Sciences, Lanzhou University, and Department of Traditional Chinese Medicine of Gansu Provincial Rehabilitation Center Hospital, China from March 2007 to January 2008. MATERIALS： Naotan Pill, composed of barbary wolfberry fruit, danshen root, grassleaf sweetflag rhizome, and glossy privet fruit, was prepared by Gansu Provincial Rehabilitation Center, China. Rabbit anti-synaptophysin, choline acetyl transferase polyclonal antibody, streptavidin-biotin complex kit and diaminobenzidine kit （Boster, Wuhan, China）, as well as glutamate （Hualian, Shanghai, China） were used in this study. METHODS： Cortical neural cells were isolated from neonatal Wistar rats. Neural cell damage models were induced using glutamate, and administered Naotan Pill prior to and following damage. A total of 54 juvenile Wistar rats were equally and randomly assigned into model, Naotan Pill, and sham operation groups. The left common carotid artery was ligated, and then rat models of hypoxic-ischemic injury were assigned to the model and Naotan Pill groups. At 2 days following model induction, rats in the Naotan Pill group were administered Naotan Pillsuspension for 21 days. In the model and sham operation groups, rats received an equal volume of saline. MAIN OUTCOME MEASURES： Neural cell morphology was observed using an inverted phase contrast microscope. Survival rate of neural cells was measured by MTT assay. Synaptophysin and choline acetyl transferase expression was observed in the hippocampal CA1 region of juvenile rats using immunohistochemistry. Cognitive function was tested by the Morris water maze. RESULTS： Pathological changes were detected in glutamate-treated neural cells. Neural cell morphology remained normal after Naotan Pill intervention. Absorbance and survival rate of neural cells were significantly greater following Naotan Pill intervention, compared to glutamate-treated neural cells （P 〈 0.05）. Synaptophysin and choline acetyl transferase expression was lowest in the hippocampal CA1 region in the model group and highest in the sham operation group. Significant differences among groups were observed （P 〈 0.05）. Escape latency and swimming distance were significantly longer in the model group compared to the Naotan Pill group （P 〈 0.05）. CONCLUSION： Naotan Pill exhibited protective and repair effects on glutamate-treated neural cells. Naotan Pill upregulated synaptophysin and choline acetyl transferase expression in the hippocampus and improved cognitive function in rats following hypoxia-ischemia.

A new animal model for uterine torsion and uterine ischemia-reperfusion studies, but not fetal hypoxia studies

The aim of the present study was to develop a new animal model for use in uterine torsion, uterine ischemia-reperfusion, and fetal hypoxia studies in rats. A total of 14 pregnant rats on their 18 th-19 th gestational days were used. The animals were randomly divided into two groups: those undergoing the shame operation(group 1),and those in which a 360 uterine torsion was performed using a novel technique,which was corrected 6 hours later(group 2). Subsequently, seven female and seven male rat pups aged 1 month were separated from the mothers in each group. The female rats were monitored until puberty via measuring the vaginal apertures. The 1-month old male rats and the female rats on reaching puberty were decapitated and histopathological tests were performed on the dissected organs, including the cerebral, visceral and genital organs. At the end of the study, no differences were observed between the groups with regard to abortions, offspring death rates and congenital abnormalities. It was observed that the time to reach puberty in female rats born from mothers with uterine torsion was longer, but the difference was statistically insignificant. No microscopic lesions were detected in the cerebral, visceral or genital organs of the offspring. Accordingly, it was concluded that offspring of mothers with the uterine torsion were not affected, at least in the short term. It was generally concluded that this animal model is appropriate for use in uterine torsion and ischemia-reperfusion studies, but is not appropriate for fetal hypoxia studies.

Early expressions of hypoxia-inducible factor 1alpha and vascular endothelial growth factor increase the neuronal plasticity of activated endogenous neural stem cells after focal cerebral ischemia

Endogenous neural stem cells become ＂activated＂ after neuronal injury, but the activation sequence and fate of endogenous neural stem cells in focal cerebral ischemia model are little known. We evaluated the relationships between neural stem cells and hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a photothromobotic rat stroke model using immunohistochemistry and western blot analysis. We also evaluated the chronological changes of neural stem cells by 5-bromo-2′-deoxyuridine（BrdU） incorporation. Hypoxia-inducible factor-1α expression was initially increased from 1 hour after ischemic injury, followed by vascular endothelial growth factor expression. Hypoxia-inducible factor-1α immunoreactivity was detected in the ipsilateral cortical neurons of the infarct core and peri-infarct area. Vascular endothelial growth factor immunoreactivity was detected in bilateral cortex, but ipsilateral cortex staining intensity and numbers were greater than the contralateral cortex. Vascular endothelial growth factor immunoreactive cells were easily found along the peri-infarct area 12 hours after focal cerebral ischemia. The expression of nestin increased throughout the microvasculature in the ischemic core and the peri-infarct area in all experimental rats after 24 hours of ischemic injury. Nestin immunoreactivity increased in the subventricular zone during 12 hours to 3 days, and prominently increased in the ipsilateral cortex between 3–7 days. Nestin-labeled cells showed dual differentiation with microvessels near the infarct core and reactive astrocytes in the peri-infarct area. BrdU-labeled cells were increased gradually from day 1 in the ipsilateral subventricular zone and cortex, and numerous BrdU-labeled cells were observed in the peri-infarct area and non-lesioned cortex at 3 days. BrdU-labeled cells rather than neurons, were mainly co-labeled with nestin and GFAP. Early expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor after ischemia made up the microenvironment to increase the neuronal plasticity of activated endogenous neural stem cells. Moreover, neural precursor cells after large-scale cortical injury could be recruited from the cortex nearby infarct core and subventricular zone.

Adenovirus-mediated hypoxia-inducible factor-1 alpha gene transfer induces angiogenesis and neurogenesis following cerebral ischemia in rats

BACKGROUND： Hypoxia-inducible factor-1 （HIF-1） accumulates under conditions of hypoxia. HIF-1α target genes have pleiotropic effects on neurogenesis, neuroprotection and angiogenesis in the brain. OBJECTIVE： To investigate whether a recombinant adenovirus carrying HIF-1α can increase the expression of HIF-I a in vivo and thus promote angiogenesis and neurogenesis in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING： The randomized, controlled experiment was performed at the Department of Neurobiology, Third Military Medical University of Chinese PLA from September 2006 to October 2007. MATERIALS： 68 healthy adult male Sprague-Dawley （SD） rats, weighing 230-250 g, were used. HIF-I a antibody was purchased from Wuhan Boster Company. Vascular endothelial growth factor （VEGF） antibody was purchased from Santa Cruz Biotech Company. METHODS： All 68 rats were induced with a transient middle cerebral artery occlusion （MCAO）, according to the method of intra-luminal vascular occlusion. 54 rats, in which MCAO was successfully induced, were randomly divided into adenovirus （Ad） group and recombinant adenovirus with HIF-1α gene （Ad-HIF-1α ） group （27 rats for each group）. Rats were injected with 10 μL Ad （Ad group） or Ad-HIF-1α （Ad-HIF-1α group） into the lateral ventricle, 1 day after MCAO induction. MAIN OUTCOME MEASURES： Reverse transcription polymerase chain reaction was used to measure the expression of HIF-1α and of VEGF. Immunohistochemistry was used to detect the localization of HIF-1α, VEGF and factor Ⅷ in ischemic penumbra. Rat newborn nerve cells were labeled with 5-bromodeoxyuridine （BrdU） after ischemia. BrdU/neurofilament 200 （NF200） and BrdU/glial fibrillary acidic protein （GFAP） double labeled immunofluorescent histochemistry was used to identify the differentiation of newborn cells. Neurological function was evaluated using the modified neurological severity score （NSS）. RESULTS： Compared with Ad, Ad-HIF-1α enhanced the expression of HIF-1α and VEGF （P 〈 0.01）. The numbers of factor VIII, BrdU, BrdU/NF200 and BrdU/GFAP positive cells were increased significantly （P 〈 0.01） in the Ad-HIF-I a group compared to the Ad group. Levels of HIF-1α and VEGF mRNA in the Ad-HIF-1α group were enhanced compared with those in the Ad group. NSS scores of the Ad-HIF-1α group were superior to those of the Ad group at days 7, 14, 21, and 28 after MCAO （P 〈 0.05）. CONCLUSION： HIF-1α gene therapy can increase angiogenesis and neurogenesis, and thus improve neurological function following cerebral ischemia in rats.

复方人参对机体缺氧损伤的保护作用及其分子机制研究

探究复方人参(compound ginseng,CGS)的抗缺氧作用及其分子机制。构建小鼠密闭缺氧模型、亚硝酸钠中毒模型、急性脑缺血模型以及全心缺血缺氧模型,观察CGS对小鼠的存活时间、喘气时间、喘气次数、丙二醛(malondialdehyde,MDA)含量、超氧化物歧化酶(superoxide dismutase,SOD)活力、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性、乳酸脱氢酶(lactic dehydrogenase,LDH)活力和肌酸激酶(creatine kinase,CK)活力。利用网络药理学预测CGS的抗缺氧相关机制,并采用分子对接方法验证网络药理学筛选所得CGS活性成分与机体靶点之间的亲和性,检测小鼠体内缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)和血管内皮生长因子(vascular endothelial growth factor,VEGF)mRNA表达量。结果显示,CGS可以显著延长密闭缺氧实验、亚硝酸钠中毒实验和全心缺血缺氧实验小鼠的存活时间以及急性脑缺血实验的喘气时间和喘气次数;CGS能够降低小鼠血清中的MDA含量、LDH和CK活力,提升SOD、GSH-Px活性。采用网络药理学预测出226条CGS抗缺氧通路,其中关键通路有HIF-1α通路;密闭缺氧实验结果证实CGS可以显著提升HIF-1α表达,下调VEGF表达。综上,CGS具有耐缺氧作用,其作用可能与激活HIF-1α/VEGF通路,提高血液携氧能力和氧利用率,增强抗氧化活性有关。

缺氧缺血性脑损伤后乳酸通过GPR81抑制细胞凋亡

目的 观察缺氧缺血性脑损伤后乳酸(Lac)受体G蛋白偶联受体81 (GPR81)与细胞凋亡间的动态变化,明确Lac干预通过GPR81对细胞凋亡的作用。方法 选取30头新生大约克夏猪。将21头新生猪随机分为Sham组和缺血缺氧(HI)模型组,HI模型组细分为0~2 h、2~6 h、6~12 h、12~24 h、24~48 h、48~72 h HI组,每组3头,采用免疫组织化学和TUNEL染色观察基底节区GPR81和细胞凋亡变化。将9头新生猪随机分为Sham+生理盐水(NS)组、HI+NS组和HI+Lac组,每组3头。观察基底节区GPR81表达水平、凋亡细胞比例以及PI3K、AKT、Bax和Bcl-2表达水平。结果 HI脑损伤后细胞凋亡率逐渐上升,24~48 h达到峰值(P <0.05)。GPR81表达水平先升后降,2~6 h达到峰值(P <0.05)。与Sham+NS组相比,HI+NS组细胞凋亡率、Bax表达水平升高,p-AKT/AKT比值以及PI3K、Bcl-2表达水平降低(均P <0.05);与HI+NS组相比,HI+Lac组细胞凋亡率、Bax表达水平降低,p-AKT/AKT比值以及GPR81、PI3K、Bcl-2表达水平升高(均P <0.05)。结论 Lac干预能够通过GPR81介导PI3K/AKT信号通路活化,发挥抑制细胞凋亡的作用。

麝香通心滴丸调控HIF-1α/HO-1通路抑制铁死亡缓解心肌缺血再灌注损伤研究

目的:探究麝香通心滴丸(STDP)对心肌缺血再灌注损伤(MIRI)的保护作用及其潜在机制。方法:采用AnaeroPack方法构建H9c2细胞缺氧复氧损伤(H/R)模型以模拟MIRI。将细胞随机分为对照组、模型组和麝香通心滴丸组,分别在不含药或含药的培养液中培养24 h后收集细胞。采用细胞计数试剂盒法(CCK-8)检测细胞增殖与活性,酶联免疫吸附法(ELISA)测定细胞上清液乳酸脱氢酶(LDH)水平,荧光探针法检测活性氧(ROS)水平以评估细胞的氧化应激状态,透射电镜(TEM)观察线粒体超微结构变化,Western blot检测低氧诱导因子-1α(HIF-1α)、血红素加氧酶-1(HO-1)信号通路相关蛋白的表达水平。结果:麝香通心滴丸在H9c2心肌细胞中未表现出明显细胞毒性,同时在H/R条件下显著抑制了细胞损伤。麝香通心滴丸通过降低ROS生成和LDH释放,显著缓解了H/R诱导的氧化应激。TEM观察显示,与对照组相比,模型组的线粒体呈现明显的萎缩并伴随典型的铁死亡特征,而麝香通心滴丸组的线粒体形态显著改善,趋于正常。进一步通过Western blot检测发现,模型组HIF-1α和HO-1蛋白表达显著上调,而麝香通心滴丸组显著抑制了这些蛋白的过度表达(P<0.05)。结论:麝香通心滴丸可能通过抑制HIF-1α/HO-1信号通路介导的铁死亡,减轻心肌缺血再灌注损伤。

Effect of lentiviral vector-mediated overexpression of hypoxia-inducible factor 1 alpha delivered by pluronic F-127 hydrogel on brachial plexus avulsion in rats

Brachial plexus avulsion often results in massive motor neuron death and severe functional deficits of target muscles. However, no satisfactory treatment is currently available. Hypoxia-inducible factor 1α is a critical molecule targeting several genes associated with ischemia-hypoxia damage and angiogenesis. In this study, a rat model of brachial plexus avulsion-reimplantation was established, in which C5–7 ventral nerve roots were avulsed and only the C6 root reimplanted. Different implants were immediately injected using a microsyringe into the avulsion-reimplantation site of the C6 root post-brachial plexus avulsion. Rats were randomly divided into five groups: phosphate-buffered saline, negative control of lentivirus, hypoxia-inducible factor 1α(hypoxia-inducible factor 1α overexpression lentivirus), gel(pluronic F-127 hydrogel), and gel + hypoxia-inducible factor 1α(pluronic F-127 hydrogel + hypoxia-inducible factor 1α overexpression lentivirus). The Terzis grooming test was performed to assess recovery of motor function. Scores were higher in the hypoxia-inducible factor 1α and gel +hypoxia-inducible factor 1α groups(in particular the gel + hypoxia-inducible factor 1α group) compared with the phosphate-buffered saline group. Electrophysiology, fluorogold retrograde tracing, and immunofluorescent staining were further performed to investigate neural pathway reconstruction and changes of neurons, motor endplates, and angiogenesis. Compared with the phosphate-buffered saline group, action potential latency of musculocutaneous nerves was markedly shortened in the hypoxia-inducible factor 1α and gel + hypoxia-inducible factor1α groups. Meanwhile, the number of fluorogold-positive cells and ChAT-positive neurons, neovascular area(labeled by CD31 around av ulsed sites in ipsilateral spinal cord segments), and the number of motor endplates in biceps brachii(identified by α-bungarotoxin) were all visibly increased, as well as the morphology of motor endplate in biceps brachil was clear in the hypoxia-inducible factor 1α and gel + hypoxia-inducible factor 1α groups. Taken together, delivery of hypoxia-inducible factor 1α overexpression lentiviral vectors mediated by pluronic F-127 effectively promotes spinal root regeneration and functional recovery post-brachial plexus avulsion. All animal procedures were approved by the Institutional Animal Care and Use Committee of Guangdong Medical University, China.