Expression And Role Of HIF-1α In Hypoxia Tolerance Of Central Neurons Of Brandt’s Voles

BackgroundOxygen(O2)plays an important role in maintaining the survival of organism and hypoxia may lead to metabolic disorder and various degrees of tissue damage.Specially the central nervous system is very sensitive to hypoxia,it can cause irreversible damage to neurons.Some high-altitude mammals,diving mammals and subterranean mammals,which live in hypoxic environments for a long time,have developed effective strategies and physiological mechanisms.So,these animals can be used to provide a good model for hypoxia research.Brandt’s vole mainly live in underground caves with hardened soil and severe hypoxia.Based on comparative physiology,our lab found that Brandt’s voles possessed remarkable hypoxia tolerance through survival,cognitive behavior and hippocampal histomorphology experiments and thus,Brandt’s vole can be used as a type of natural hypoxia-tolerant animal model.However,the mechanisms of neuronal hypoxia tolerance in the brain of lasiopodomys brandtii are still unclear.Therefore,the exploration of the mechanisms of hypoxia tolerance in its central nervous system can not only provide theoretical basis for the study on mechanism of hypoxia tolerance in subterranean rodents,but also provide new ideas for the prevention of hypoxia-related diseases such as stroke.Hypoxia inducible factors(HIFs)are the most important regulatory factors to deal with hypoxic environment for mammals.HIFs are composed of functional subunit HIF-α and constitutive subunit HIF-β.HIF-α is the oxygen sensitive subunit and there are three isomers of HIF-α,namely HIF-1α,HIF-2α and HIF-3α.In this study,we hypothesized that central neurons of Brandt’s voles have high expression of HIFs isoforms and play a role in their hypoxia tolerance.Therefore,this study explored the expression and role of HIFs in the hypoxia tolerance of brain tissue in Brandt’s vole,and whether hypoxia-inducible factors(HIFs)played protective roles on neurons through EPO under hypoxia.Methods1.Animals were grouped and treated with hypoxiaThe tested animals were divided into four groups:Kunming mice and Brandt’s voles normoxia group(21%O2),Kunming mice and Brandt’s voles hypoxia group(7.5%O2 6 h).Brandt’s voles were divided into three groups in subsequent experiments:Brandt’s voles hypoxia group,DMSO+Brandt’s voles hypoxia group(Salt solution containing 1%DMSO),LW6+Brandt’s voles hypoxia group(LW6 was injected intraperitoneally,20 mg/kg/d,two weeks).Animals of hypoxic group were treated in a DS-Ⅱ hyperbaric cabin for animal experimentation.2.Explore the mechanism of hypoxia tolerance in central neurons of Brandt’s volesBy means of comparative physiology,the survival curves was used to evaluate the hypoxia tolerance of Brandt’s voles,HE and fluorescent immunohistochemical method were used to detect the morphological and structural changes,and loss of neurons in the cerebral cortex and hippocampus of Brandt’s voles,and verify its hypoxic tolerance.Western blot and fluorescent immunohistochemical method were used to detect the differential expression of HIFs in cerebral cortex and hippocampus of Brandt’s voles,LW6(A salt solution containing 1%DMSO,20 mg/kg/d),a HIF-1αinhibitor,was used to verify the role of HIF-1α in neuronal hypoxic tolerance of Brandt’s voles.Western blot and fluorescence immunohistochemistry were used to test the effect of HIF-1α on EPO expression.Results1.Neurons of the cerebral cortex and hippocampus have hypoxic tolerance in Brandt’s voles(1)Survival curve analysis showed that Brandt’s voles could survive for 48 h,while Kunming mice could only survive for 24 h(P<0.01).(2)After normoxia and 7.5%O2 treatment respectively,the cerebral cortex and hippocampus of Brandt’s voles expressed a clear structure,normal cell morphology,neat arrangement,complete and clear nuclei,and uniform staining.No significant difference was observed in the expression of NeuN in the cerebral cortex and hippocampal CA3 region under two conditions.The cerebral cortex and hippocampus tissue of Kunming mice were blurred and some nerve cells showed punctate degeneration,necrosis,nuclear pyknosis,and the numbers of neurons in the cerebral cortex and hippocampus were significantly decreased after treatment with 7.5%oxygen(P<0.01).The expression of NeuN in the cerebral cortex and hippocampal CA3 region of Brandt’s voles was higher than that of Kunming mice under hypoxia(P<0.05).The above results indicated that Brandt’s voles had better hypoxia tolerance than Kunming mice.Hypoxia exerted no significant effect on the brain structure and neuron loss,which also proved that the central neurons of Brandt’s voles possessed hypoxia tolerance.2.High expression of HIF-1α in cortex and hippocampus of microtus brucei is associated with the neuronal hypoxic tolerance(1)Under normoxic and hypoxic conditions,the expression of HIF-1α in the cerebral cortex and hippocampus of Brandt’s voles was significantly higher than that in Kunming mice(P<0.01);The expression of HIF-1α in the brain tissue of Brandt’s voles was significantly higher under hypoxia than that in normoxia group(P<0.05).No significant difference was observed in the expression of HIF-1α in the cerebral cortex and hippocampus between normoxia group and hypoxia group for Kunming mice.These results suggested that the high expression of HIF-1α in cerebral cortex and hippocampus might be related to hypoxia tolerance.(2)For both Brandt’s voles and Kunming mice,the expression of HIF-2α and HIF-3α of cerebral cortex and hippocampus was no statistical significance.These results suggested that HIF-2α or HIF-3α may play a limited role in hypoxia tolerance in cerebral cortex and hippocampus of Brandt’s voles.(3)Under normoxic and hypoxic conditions,the co-localized expression of HIF1α and neurons was detected in the cerebral cortex and hippocampal CA3 region both species.The co-expression of HIF-1α and neurons increased when HIF-1α was highly expressed.These results suggested that the high expression of HIF-1α in neurons increased hypoxia tolerance and may have a protective effect on neurons.3.Inhibition of HIF-la affects the structure of cerebral cortex and hippocampus and the number of neuronsAfter treatment with the inhibitor for two weeks and subsequent 7.5%O2 treatment,Brandt’s voles injected with LW6 expressed decreased survival rate,reduced expression of NeuN and diminished the co-localization of HIF-lα with neurons in the cerebral cortex and hippocampal neuron compared with DMSO+B-H group.The cerebral cortex and hippocampus tissue in Brandt’s voles after treated with LW6 were blurred and some nerve cells showed punctate degeneration,necrosis,and the expression of NeuN in the cerebral cortex and hippocampal neurons was significantly lost(P<0.05).The results showed that the high expression of HIF-1α in cerebral cortex and hippocampus might play a protective role in the neuronal hypoxic tolerance.4.HIF-1α protects neurons by EPO in the cortex and hippocampus of Brandt’s volesUnder both normoxic and hypoxic conditions,the expression level of EPO in the brain of Brandt’s voles was significantly higher than that in Kunming mice(P<0.05 and P<0.01).For Brandt’s voles,the expression of EPO in the cerebral cortex or hippocampus in hypoxia group was significantly higher compared with normoxia group(P<0.05).No significant difference in the expression of EPO was observed in the cerebral cortex and hippocampus between normoxia group and hypoxia group for Kunming mice.Compared with DMSO+B-H group,the expression of EPO was decreased in cerebral cortex and hippocampus and the co-localization of EPO with neurons was reduced in LW6 group of Brandt’s voles.These results suggested that HIF-1α may play a protective role in the cerebral cortex and hippocampal neurons by regulating the expression of EPO,and be responsible for hypoxia tolerance.ConclusionThe expression of HIF-1α in cerebral cortex and hippocampus of Brandt’s voles is significantly higher than that of Kunming mice.The high expression of HIF-1α in neurons may enhances the hypoxia tolerance of central neurons from Brandt’s voles through the cytoprotective effect of EPO.