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...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.展开更多
Extracellular vesicles(EVs)from mesenchymal stromal cells(MSCs)have previously been shown to protect against brain injury caused by hypoxia-ischemia(HI).The neuroprotective effects have been found to relate to the ant...Extracellular vesicles(EVs)from mesenchymal stromal cells(MSCs)have previously been shown to protect against brain injury caused by hypoxia-ischemia(HI).The neuroprotective effects have been found to relate to the anti-inflammatory effects of EVs.However,the underlying mechanisms have not previously been determined.In this study,we induced oxygen-glucose deprivation in BV-2 cells(a microglia cell line),which mimics HI in vitro,and found that treatment with MSCs-EVs increased the cell viability.The treatment was also found to reduce the expression of pro-inflammatory cytokines,induce the polarization of microglia towards the M2 phenotype,and suppress the phosphorylation of selective signal transducer and activator of transcription 3(STAT3)in the microglia.These results were also obtained in vivo using neonatal mice with induced HI.We investigated the potential role of miR-21a-5p in mediating these effects,as it is the most highly expressed miRNA in MSCs-EVs and interacts with the STAT3 pathway.We found that treatment with MSCs-EVs increased the levels of miR-21a-5p in BV-2 cells,which had been lowered following oxygen-glucose deprivation.When the level of miR-21a-5p in the MSCs-EVs was reduced,the effects on microglial polarization and STAT3 phosphorylation were reduced,for both the in vitro and in vivo HI models.These results indicate that MSCs-EVs attenuate HI brain injury in neonatal mice by shuttling miR-21a-5p,which induces microglial M2 polarization by targeting STAT3.展开更多
Neonatal hypoxia-ischemia(HI)results in losses of serotonergic neurons in specific dorsal raphe nuclei.However,not all serotonergic raphe neurons are lost and it is therefore important to assess the function of remain...Neonatal hypoxia-ischemia(HI)results in losses of serotonergic neurons in specific dorsal raphe nuclei.However,not all serotonergic raphe 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 raphe 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 raphe nuclei.We found that restraint stress alone increased numbers of Fos-positive 5-HT neurons in all five dorsal raphe 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 raphe 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 raphe dorsal and caudal nuclei were not affected by P3 HI.These data indicate that not only are dorsal raphe serotonergic neurons lost after neonatal HI,but also remaining dorsal raphe 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.展开更多
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 l...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.展开更多
BACKGROUND: In addition to neuroprotective genes, the targeted genes of hypoxia-induciblefactor 1α (HIF-1α) include pro-apoptotic genes. However, the influence of HIF-1α on neuronalapoptosis in hypoxia-ischemia rem...BACKGROUND: In addition to neuroprotective genes, the targeted genes of hypoxia-induciblefactor 1α (HIF-1α) include pro-apoptotic genes. However, the influence of HIF-1α on neuronalapoptosis in hypoxia-ischemia remains poorly understood.OBJECTIVE: To investigate the relationship between HIF-1α expression and neuronal apoptosis inhypoxia or hypoxia-ischemia brain injury and to determine the role of HIF-1α in regulating neuronalapoptosis.DESIGN, TIME AND SETTING: A randomized, controlled animal experiment was performed at theLaboratory 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-1αpolyclonal antibody was purchased from Santa Cruz Biotechnologies, USA; rabbit anti-mousecleaved 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 3time points: 4, 8, and 24 hours, with 4 rats per time point. In the hypoxia-ischemia group, the rightcommon carotid artery was exposed and permanently ligated through a midline cervical incision. A2.5-hour exposure to hypoxia (8% O_2/92% N_2) was used to induce hypoxia-ischemia injury. In thehypoxia group, rats were exposed to hypoxia without ligation of the common carotid artery. In thesham-surgery group, the common carotid artery was exposed without ligation or hypoxia.MAIN OUTCOME MEASURES: Histopathological changes, HIF-1α and activated caspase-3 proteinexpression, integrated optical density of positive cells, and apoptosis-positive cells.RESULTS: Hematoxylin and eosin staining showed that neuronal degeneration and edema wasmost prominent at 24 hours after hypoxia-ischemia. HIF-1α protein expression was significantlyupregulated at 4 hours, peaked at 8 hours, and decreased at 24 hours after hypoxia orhypoxia-ischemia. HIF-1α protein expression was significant greater in the hypoxia andhypoxia-ischemia groups compared with the sham-surgery group (P < 0.01). Activated caspase-3protein expression began to increase at 4 and 8 hours following hypoxia or hypoxia-ischemia andwas significantly upregulated at 24 hours. Activated caspase-3 protein expression remained at lowlevels 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 afterhypoxia or hypoxia-ischemia. In addition, HIF-1α protein expression was greater in the hypoxiagroup 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 hypoxiagroup 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.展开更多
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 pass...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).展开更多
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 protecti...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:GM1,model,and sham operation,with 12 rats each group.Rats in the GM1 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 GM1 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 GM1 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 group,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 after surgery,and significantly less than the model group(P < 0.05).In addition,the frequency platform passing in the GM1 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.展开更多
目的:基于缺血缺氧脑瘫大鼠神经功能评分(Zea-Longa评分)、脑组织肉眼观和大脑海马区胱天蛋白酶-9(Caspase-9)、胱天蛋白酶-3(Caspase-3)的表达水平变化,探讨缺血缺氧模型脑瘫大鼠的有效时长。方法:选取3周龄斯泼累格·多雷(SD)健...目的:基于缺血缺氧脑瘫大鼠神经功能评分(Zea-Longa评分)、脑组织肉眼观和大脑海马区胱天蛋白酶-9(Caspase-9)、胱天蛋白酶-3(Caspase-3)的表达水平变化,探讨缺血缺氧模型脑瘫大鼠的有效时长。方法:选取3周龄斯泼累格·多雷(SD)健康大鼠,随机分为正常组和模型组,采用改良的Rice-Vannucci方法建立脑瘫模型,造模后第1、7、14、21天,观察各组大鼠的一般情况并进行神经功能评分,在第7、14、21天分批处死大鼠并取脑组织,观察各组大鼠左侧脑组织,检测海马区Caspase-9、Caspase-3的表达水平。结果:一般情况:造模后第1天,与正常组比较,模型组大鼠左侧瞳孔缩小、姿势异常、自发或夹尾左旋、自主活动减少、兴奋性降低、肌肉颤动、头颤,抽搐,抓取时抵抗反应明显,随着时间延长,以上异常行为逐渐消失,造模后21 d基本消失不见,但左侧瞳孔一直小于对侧;Zea-Longa评分:与正常组比较,模型组造模后7、14 d Zea-Longa评分较高,差异有统计学意义(P<0.05);脑组织肉眼观:与正常组比较,模型组造模后7、14及21 d大鼠左侧脑组织有不同程度的萎缩和坏死;免疫组化结果:与正常组比较,模型组造模后7 d、14 d Caspase-9、Caspase-3的表达水平均显著升高,差异有统计学意义(P<0.05)。结论:3周龄缺血缺氧脑瘫模型大鼠的有效时长为14~21 d,可干预14 d。展开更多
基金supported by the National Natural Science Foundation of China,No.81671882,81471832the Natural Science Foundation of Guangdong Province of China,No.2016A030311039+1 种基金the Science and Technology Foundation of Guangdong Province of China,No.2015A020212012,2017A020224012the Science and Technology Foundation of Guangzhou City of China,No.201707010373(all to XL)
文摘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.
基金supported by the National Natural Science Foundation of China,Nos.81873768,82072535,81671213(to ZW),81770436(to WQC)the National Key Project of Chronic Non-Communicable Disease of China,No.2016YFC1300403(to WQC).
文摘Extracellular vesicles(EVs)from mesenchymal stromal cells(MSCs)have previously been shown to protect against brain injury caused by hypoxia-ischemia(HI).The neuroprotective effects have been found to relate to the anti-inflammatory effects of EVs.However,the underlying mechanisms have not previously been determined.In this study,we induced oxygen-glucose deprivation in BV-2 cells(a microglia cell line),which mimics HI in vitro,and found that treatment with MSCs-EVs increased the cell viability.The treatment was also found to reduce the expression of pro-inflammatory cytokines,induce the polarization of microglia towards the M2 phenotype,and suppress the phosphorylation of selective signal transducer and activator of transcription 3(STAT3)in the microglia.These results were also obtained in vivo using neonatal mice with induced HI.We investigated the potential role of miR-21a-5p in mediating these effects,as it is the most highly expressed miRNA in MSCs-EVs and interacts with the STAT3 pathway.We found that treatment with MSCs-EVs increased the levels of miR-21a-5p in BV-2 cells,which had been lowered following oxygen-glucose deprivation.When the level of miR-21a-5p in the MSCs-EVs was reduced,the effects on microglial polarization and STAT3 phosphorylation were reduced,for both the in vitro and in vivo HI models.These results indicate that MSCs-EVs attenuate HI brain injury in neonatal mice by shuttling miR-21a-5p,which induces microglial M2 polarization by targeting STAT3.
基金funded by the National Health and Medical Research Council of Australia(to KMB)HER was supported by a University of Queensland International Research Tuition Award and University of Queensland Research Scholarship.JAW was supported by an Australian Postgraduate Award
文摘Neonatal hypoxia-ischemia(HI)results in losses of serotonergic neurons in specific dorsal raphe nuclei.However,not all serotonergic raphe 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 raphe 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 raphe nuclei.We found that restraint stress alone increased numbers of Fos-positive 5-HT neurons in all five dorsal raphe 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 raphe 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 raphe dorsal and caudal nuclei were not affected by P3 HI.These data indicate that not only are dorsal raphe serotonergic neurons lost after neonatal HI,but also remaining dorsal raphe 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.
基金supported by the National Natural Science Foundation of China,No.81471488,81271378,81502157,and 81501291the Key Medical Subjects of Jiangsu Province of China,No.XK201120+3 种基金the Jiangsu Province Key Research and Development of Special Funds in China,No.BE2015644the Science and Technology Project of Suzhou City of China,No.SYSD2013105,SYS201446,SYS201441the Public Health Technology Project of Suzhou City of China,No.SS201536the Department of Pediatrics Clinical Center of Suzhou City of China,No.Szzx201504
文摘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.
基金Supported by: the National Natural Science Foundation of China, No. 30825039, 30973236, 30770748Outstanding Young Scientist Foundation of Sichuan Province, China, No. 08ZQ026-069
文摘BACKGROUND: In addition to neuroprotective genes, the targeted genes of hypoxia-induciblefactor 1α (HIF-1α) include pro-apoptotic genes. However, the influence of HIF-1α on neuronalapoptosis in hypoxia-ischemia remains poorly understood.OBJECTIVE: To investigate the relationship between HIF-1α expression and neuronal apoptosis inhypoxia or hypoxia-ischemia brain injury and to determine the role of HIF-1α in regulating neuronalapoptosis.DESIGN, TIME AND SETTING: A randomized, controlled animal experiment was performed at theLaboratory 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-1αpolyclonal antibody was purchased from Santa Cruz Biotechnologies, USA; rabbit anti-mousecleaved 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 3time points: 4, 8, and 24 hours, with 4 rats per time point. In the hypoxia-ischemia group, the rightcommon carotid artery was exposed and permanently ligated through a midline cervical incision. A2.5-hour exposure to hypoxia (8% O_2/92% N_2) was used to induce hypoxia-ischemia injury. In thehypoxia group, rats were exposed to hypoxia without ligation of the common carotid artery. In thesham-surgery group, the common carotid artery was exposed without ligation or hypoxia.MAIN OUTCOME MEASURES: Histopathological changes, HIF-1α and activated caspase-3 proteinexpression, integrated optical density of positive cells, and apoptosis-positive cells.RESULTS: Hematoxylin and eosin staining showed that neuronal degeneration and edema wasmost prominent at 24 hours after hypoxia-ischemia. HIF-1α protein expression was significantlyupregulated at 4 hours, peaked at 8 hours, and decreased at 24 hours after hypoxia orhypoxia-ischemia. HIF-1α protein expression was significant greater in the hypoxia andhypoxia-ischemia groups compared with the sham-surgery group (P < 0.01). Activated caspase-3protein expression began to increase at 4 and 8 hours following hypoxia or hypoxia-ischemia andwas significantly upregulated at 24 hours. Activated caspase-3 protein expression remained at lowlevels 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 afterhypoxia or hypoxia-ischemia. In addition, HIF-1α protein expression was greater in the hypoxiagroup 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 hypoxiagroup 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.
基金supported by the National Natural Science Foundation of China,No.81560215(FW)the Innovative Research Team Program of Science and Technology in Yunnan Province of China,No.2017HC007
文摘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).
基金supported by the Chongqing Municipal Health Bureau "Effect of ephedrine on neuronal plasticity of hypoxic-ischemic brain damage in neonatal rats" (Grant No. [Yu health science and education (2007) NO.1 (07-2-153)]).
文摘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:GM1,model,and sham operation,with 12 rats each group.Rats in the GM1 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 GM1 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 GM1 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 group,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 after surgery,and significantly less than the model group(P < 0.05).In addition,the frequency platform passing in the GM1 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.
文摘目的:基于缺血缺氧脑瘫大鼠神经功能评分(Zea-Longa评分)、脑组织肉眼观和大脑海马区胱天蛋白酶-9(Caspase-9)、胱天蛋白酶-3(Caspase-3)的表达水平变化,探讨缺血缺氧模型脑瘫大鼠的有效时长。方法:选取3周龄斯泼累格·多雷(SD)健康大鼠,随机分为正常组和模型组,采用改良的Rice-Vannucci方法建立脑瘫模型,造模后第1、7、14、21天,观察各组大鼠的一般情况并进行神经功能评分,在第7、14、21天分批处死大鼠并取脑组织,观察各组大鼠左侧脑组织,检测海马区Caspase-9、Caspase-3的表达水平。结果:一般情况:造模后第1天,与正常组比较,模型组大鼠左侧瞳孔缩小、姿势异常、自发或夹尾左旋、自主活动减少、兴奋性降低、肌肉颤动、头颤,抽搐,抓取时抵抗反应明显,随着时间延长,以上异常行为逐渐消失,造模后21 d基本消失不见,但左侧瞳孔一直小于对侧;Zea-Longa评分:与正常组比较,模型组造模后7、14 d Zea-Longa评分较高,差异有统计学意义(P<0.05);脑组织肉眼观:与正常组比较,模型组造模后7、14及21 d大鼠左侧脑组织有不同程度的萎缩和坏死;免疫组化结果:与正常组比较,模型组造模后7 d、14 d Caspase-9、Caspase-3的表达水平均显著升高,差异有统计学意义(P<0.05)。结论:3周龄缺血缺氧脑瘫模型大鼠的有效时长为14~21 d,可干预14 d。
