Desferoxamine preconditioning protects against cerebral ischemia in rats by inducing expressions of hypoxia inducible factor 1α and erythropoietin

Objective To investigate whether desferoxamine （DFO） preconditioning can induce tolerance against cerebral ischemia and its effect on the expression of hypoxia inducible factor 1 α （HIF- 1α） and erythropoietin （EPO） in vivo and in vitro. Methods Rat model of cerebral ischemia was established by middle cerebral artery occlusion with or without DFO administration. Infarct size was examined by TTC staining, and the neurological severity score was evaluated according to published method. Cortical neurons were cultured under ischemia stress which was mimicked by oxygen-glucose deprivation （OGD）, and the neuron damage was assessed by MTT assay. Immunofluorescent staining was employed to detect the expressions of HIF-1 and EPO. Results The protective effect induced by DFO （decreasing the infarction volume and ameliorating the neurological function） appeared at 2 d after administration ofDFO （post-DFO）, lasted until 7 d and disappeared at 14 d （P 〈 0.05）; the most effective action was observed at 3 d post-DFO. DFO induced tolerance of cultured neurons against OGD： neuronal viability was increased 23%, 34%, 40%, 48% and 56% at 8 h, 12 h, 24 h, 36 h, and 48 h, respectively, post-DFO （P 〈 0.05）. Immunofluorescent staining found that HIF-1 α and EPO were upregulated in the neurons of rat brain at 3 d and 7 d post-DFO; increase of HIF-1 α and EPO appeared in cultured cortex neurons at 36 h and 48 h post-DFO. Conclusion DFO induced tolerance against focal cerebral ischemia in rats, and exerted protective effect on OGD cultured cortical neurons. DFO significant induced the expression of HIF- 1 α and EPO both in vivo and in vitro. DFO preconditioning can protect against cerebral ischemia, which may be associated with the synthesis of HIF- 1 α and EPO.

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.

Is longer sevoflurane preconditioning neuroprotective in permanent focal cerebral ischemia?

Sevoflurane preconditioning has neuroprotective effects in the cerebral ischemia/reperfusion model. However, its influence on permanent cerebral ischemia remains unclear. In the present study, the rats were exposed to sevoflurane for 15, 30, 60, and 120 minutes, followed by induction of perma- nent cerebral ischemia. Results demonstrated that 30- and 60-minute sevoflurane preconditioning significantly reduced the infarct volume at 24 hours after cerebral ischemia, and 60-minute se- voflurane preconditioning additionally reduced the number of TUNEL- and caspase-3-positive cells in the ischemic penumbra. However, 120-minute sevoflurane preconditioning did not show evident neuroprotective effects. Moreover, 60-minute sevoflurane preconditioning significantly attenuated neurological deficits and infarct volume in rats at 4 days after cerebral ischemia. These findings in- dicated that 60-minute sevoflurane preconditioning can induce the best neuroprotective effects in rats with permanent cerebral ischemia through the inhibition of apoptosis.

Ischemic preconditioning protects against ischemic brain injury

In this study, we hypothesized that an increase in integrin αβand its co-activator vascular endothelial growth factor play important neuroprotective roles in ischemic injury. We performed ischemic preconditioning with bilateral common carotid artery occlusion for 5 minutes in C57BL/6J mice. This was followed by ischemic injury with bilateral common carotid artery occlusion for 30 minutes. The time interval between ischemic preconditioning and lethal ischemia was 48 hours. Histopathological analysis showed that ischemic preconditioning substantially diminished damage to neurons in the hippocampus 7 days after ischemia. Evans Blue dye assay showed that ischemic preconditioning reduced damage to the blood-brain barrier 24 hours after ischemia. This demonstrates the neuroprotective effect of ischemic preconditioning. Western blot assay revealed a significant reduction in protein levels of integrin αβ, vascular endothelial growth factor and its receptor in mice given ischemic preconditioning compared with mice not given ischemic preconditioning 24 hours after ischemia. These findings suggest that the neuroprotective effect of ischemic preconditioning is associated with lower integrin αβand vascular endothelial growth factor levels in the brain following ischemia.

Acupuncture preconditioning protects hippocampal neurons from transient ischemia/reperfusion injury

The present study established a model of brain ischemia in aged rats using four-vessel occlusion.We observed hippocampal CA1 neuronal apoptosis and apoptosis-mediated protease caspase-3 expression following preconditioning of electroacupuncture at Baihui（GV 20）.Our results showed that the number of hippocampal CA1 normal neurons was decreased,and degenerated neurons were increased 12 hours to 3 days following cerebral ischemia/reperfusion.The number of hippocampal CA1 apoptotic neurons and caspase-3-positive neurons in rats with cerebral ischemia/reperfusion injury was significantly decreased following acupuncture preconditioning.Acupuncture preconditioning protects aged rats against ischemia/reperfusion injury by regulating caspase-3 protein expression.

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.

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.

Ischemic preconditioning partially suppresses and postpones integrin α_Vβ_3 mRNA expression following transient global cerebral ischemia in C57BL/6 mice

Previous studies of integrin αvβ3 have focused on ischemic brain damage, although the role of integrin αvβ3 in ischemic preconditioning （IP） has rarely been reported. The present study analyzed the effects of IP on integrin αvβ3 mRNA expression following cerebral ischemia through the use of hematoxylin-eosin staining and real-time quantitative polymerase chain reaction techniques. Integrin avid3 mRNA expression in the ischemia group peaked at 24 hours after ischemia-reperfusion. In the IP ＋ ischemia group, integrin αvβ3 mRNA expression increased after 24 hours, but remained significantly less than the ischemia group, and expression continued to increase until 7 days after ischemiaJreperfusion. These results demonstrate that IP effectively attenuated upregulation of integrin αvβ3 mRNA expression at 24 hours after ischemia.

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 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.

Molecular mechanisms of liver preconditioning

Ischemia/reperfusion (I/R) injury still represents an important cause of morbidity following hepatic surgery and limits the use of marginal livers in hepatic transplantation. Transient blood flow interruption followed by reperfusion protects tissues against damage induced by subsequent I/R. This process known as ischemic pre-conditioning (IP) depends upon intrinsic cytoprotective systems whose activation can inhibit the progression of irreversible tissue damage. Compared to other organs,liver IP has additional features as it reduces inflammation and promotes hepatic regeneration. Our present understanding of the molecular mechanisms involved in liver IP is still largely incomplete. Experimental studies have shown that the protective effects of liver IP are triggered by the release of adenosine and nitric oxide and the subsequent activation of signal networks involving protein kinases such as phosphatidylinositol 3-kinase,protein kinase C δ/ε and p38 MAP kinase,and transcription factors such as signal transducer and activator of transcription 3,nuclear factor-κB and hypoxia-inducible factor 1. This article offers an overview of the molecular events underlying the preconditioning effects in the liver and points to the possibility of developing pharmacological approaches aimed at activating the intrinsic protective systems in patients undergoing liver surgery.

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).

Neuroprotective effect of ischemic preconditioning in focal cerebral infarction: relationship with upregulation of vascular endothelial growth factor

Neuroprotection by ischemic preconditioning has been confirmed by many studies, but the precise mechanism remains unclear. In the present study, we performed cerebral ischemic pre- conditioning in rats by simulating a transient ischemic attack twice （each a 20-minute occlusion of the middle cerebral artery） before inducing focal cerebral infarction （2 hour occlusion-reper- fusion in the same artery）. We also explored the mechanism underlying the neuroprotective effect of ischemic preconditioning. Seven days after ocdusion-reperfusion, tetrazolium chloride staining and immunohistochemistry revealed that the infarct volume was significantly smaller in the group that underwent preconditioning than in the model group. Furthermore, vascular endothelial growth factor immunoreactivity was considerably greater in the hippocampal CA3 region of preconditioned rats than model rats. Our results suggest that the protective effects of ischemic preconditioning on focal cerebral infarction are associated with upregulation of vascu- lar endothelial growth factor.

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.

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.

Hypoxic preconditioning：effect,mechanism and clinical implication(Part 1)

Hypoxic preconditioning(HPC) refers to exposure of organisms,systems,organs,tissues or cells to moderate hypoxia/ischemia that is able to result in a resistance to subsequent severe hypoxia/ischemia in tissues and cells.The effects exerted by HPC are well documented.The original local in situ(LiHPC) is now broadened to remote ectopic organs-tissues(ReHPC) and extended crossly to cross pluripotential HPC(CpHPC) induced by a variety of stresses other than hypoxia/ischemia,including cancer,for example.We developed a unique animal model of repetitive autohypoxia in adult mice,and studied systematically on the effects and mechanisms of HPC on the model in our laboratory since the early 1960 s.The tolerances to hypoxia and protection from injury increased significantly in this model.The adult mice behave like hypoxia-intolerant mammalian newborns and hypoxia-tolerant adult animals during their exposure to repetitive autohypoxia.The overall energy supply and demand decreased,the microorganization of the brain maintained and the spacial learning and memory ability improved but not impaired,the detrimental neurochemicals such as free radicals down-regulated and the beneficial neurochemicals such as adenosine(ADO) and antihypoxic gene(s)/factor(s)(AHGs/AHFs) up-regulated.Accordingly,we hypothesize that mechanisms for the tolerance / protective effects of HPC are fundamentally depending on energy saving and brain plasticity in particular.It is thought that these two major mechanisms are triggered by exposure to hypoxia/ischemia via oxygen sensing-transduction pathways and HIF-1 initiation cascades.We suggest that HPC is an intrinsic mechanism developed in biological evolution and is a novel potential strategy for fighting against hypoxia-ischemia and other stresses.Motivation of endogenous antihypoxic potential,activation of oxygen sensing- signal transduction systems and supplement of exogenous antihypoxic substances as well as development of HPC appliances and HPC medicines such as AHFs are encouraged based on our basic research on HPC.HPC may result in therapeutic augmentation of the endogenous cytoprotection in hypoxic-ischemic or suffering from other diseases' patients.Evolutionary consideration of HPC and clinical implications of HPC are both discussed to guide future research.The product of AHF is expected to be one of the most effective first aid medicines to rescue patients in critical condition.HPC is beginning to be used in surgery and is expected to be developed into a feasible adaptive medicine in the near future.

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.

β淀粉样蛋白对缺氧缺血性脑损伤新生鼠神经元凋亡影响的实验研究

目的:通过建立新生大鼠缺氧缺血性脑损伤(HIBD)模型,以β淀粉样蛋白(Aβ)为切入点,研究其在模型的表达以及与神经元凋亡的关系,探讨Aβ在新生鼠缺氧缺血性脑损伤模型中对神经元的作用及机制。方法:建立10日龄新生大鼠缺血性脑损伤模型,模型后2、4、8、24 h心脏灌注,分别检测脑组织中Aβ、脑内淀粉样前体蛋白(APP)、β-分泌酶(BACE1)、Caspase-3、Cleaved caspase-3、B淋巴细胞瘤-2(Bcl-2)的蛋白表达,BACE1的mRNA表达。使用BACE1抑制剂干预,实验分为三组,缺氧缺血组、抑制剂组和溶剂组,抑制剂组在缺氧缺血后即给予BACE1抑制剂AZD3293处理24 h后再次检测以上指标。结果:APP、Aβ的蛋白表达、BACE1的蛋白表达和mRNA水平在建模后呈时间依赖的上升,24 h达到高峰。同时,促凋亡蛋白Cleaved caspase-3在建模后也呈时间依赖的上升,24 h达到高峰。而在缺氧缺血2 h后,凋亡抑制蛋白Bcl-2的蛋白水平显著升高(P<0.05)。之后逐渐降低,24 h最低。当使用BACE1抑制剂后,Aβ及BACE1在脑组织中的表达显著下降(均P<0.05),而BACE1mRNA的表达没有变化(P>0.05)。同时促凋亡蛋白Cleaved caspase-3的表达明显下降(P<0.05),同时,Bcl-2蛋白的表达也显著升高(P<0.05)。结论:在新生鼠HIBD时Aβ产生增多,应用BACE1抑制剂可降低Aβ的表达,增加Bcl-2的表达,减轻神经元凋亡。

miR-582-5p靶向调控FOXO1对新生大鼠缺血缺氧性脑病神经元损伤的影响

目的 探讨微小RNA-582-5p(miR-582-5p)靶向调控叉头框转录因子1(FOXO1)对新生大鼠缺血缺氧性脑病(HIE)神经元损伤的影响。方法 90只新生大鼠按照随机数字表法均分为对照(NC)组、模型(HIE)组、miRNA对照(LV-miRNA-NC)组、miR-582-5p过表达(LV-miR-582-5p)组、miR-582-5p过表达+mRNA对照(LV-miR-582-5p+LV-NC)组、miR-582-5p过表达+FOXO1过表达(LV-miR-582-5p+LV-FOXO1)组。除NC组外的各组大鼠建立HIE模型,对大鼠进行神经功能缺损评分,TTC染色测定脑梗死体积,Real-time PCR检测miR-582-5p和FOXO1表达,双萤光素酶报告基因实验检测miR-582-5p和FOXO1靶向关系,HE染色观察海马组织病理变化,TUNEL和Neu N荧光双标共定位检测海马组织神经元凋亡,免疫组化染色检测FOXO1、胱天蛋白酶3(Caspase-3)蛋白表达。结果miR-582-5p和FOXO1具有靶向关系,与NC组比较,HIE组大鼠神经功能缺损评分、脑梗死体积、FOXO1表达、神经元凋亡率、FOXO1、Caspase-3蛋白表达增加,miR-582-5p表达降低,海马组织出现病理损伤(P<0.05);与LVmiRNA-NC组比较,LV-miR-582-5p组大鼠神经功能缺损评分、脑梗死体积、FOXO1表达、神经元凋亡率、FOXO1、Caspase-3蛋白表达降低,miR-582-5p表达增加,海马组织病理损伤好转(P<0.05);LV-FOXO1可以逆转LV-miR-582-5p对于HIE大鼠神经元损伤的保护作用。结论 miR-582-5p可以直接靶向负调控FOXO1表达,减少HIE新生大鼠神经元凋亡,对神经损伤具有保护作用。

缺血缺氧脑瘫大鼠的时效研究

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。