| Hypoxic stress is a pathological stimulus for most mammals.Large amounts of glutamate can be released from presynaptic membrane under hypoxia.The overrealeased glutamate accumulates in the synaptic cleft and overactivites ionotropic glutamate receptor N-methyl-D-aspartate(NMDA)receptor.Once the NMDA receptor is activated,it triggers a large permanent influx of intracellular Ca2+ and calcium overload,which can result in an over-depolarization of the postsynaptic neuron,and ultimately neuronal death and brain damage through excitotoxicity.Glutamate is the principal excitatory neurotransmitter in the brain.Its ionotropic receptor,NMDA receptor is a heterotetramer composed of the structural subunit NR1 and the functional subunit NR2.The abnormalities in glutamate and NMDA receptor alter neurological function and have been linked to many diseases.However,rodents of underground life can survive for long periods in low oxygen tensions,which forms a hypoxic adaptation.Gansu zokor(Eospalax cansus)is a typical subterranean rodent which lives in underground tunnels with low oxygen and high carbon dioxide through all its life.It leads to the evolution of a series of hypoxic adaptation mechanisms.It is an ideal animal model to study hypoxic tolerance.At present,some studies on subterranean rodents have shown that NMDA receptor plays an important role in hypoxic brain protection.But it is limited to expression of NMDA receptor subunits at the transcription level,thus,the NMDA receptor and its pathway is not studied fully.In this experiment,the hypoxic tolerance subterranean rodent Gansu zokor was used as the research object,and oxygen sensitive rodent SD rat(Rattus norvegicus)was used as the control.Three groups were established,such as normoxia group:21%O2;hypoxia I group:10.5%O2 for up to 44 h;hypoxia ? group:6.5%O2 for 6 h.To explore the protective effects of glutamate and its NMDA receptor and to reveal the brain protective mechanism in the brain of Gansu zokor under hypoxic stress from the morphological and molecular levels,the damage degree of hippocampal neurons in Gansu zokor was detected by Nissl staining,and the density of hippocampus dendritic spines was measured by Golgi staining.In addition,the glutamate contents of hippocampus was detected by Enzyme-Linked Immunosorbent(ELISA).The expressions of NMDA receptor subunits were identified by real-time quantitative PCR and Western Blot.The experimental results are as follows:1.Effects of hypoxic stress on the number of pyramidal neurons in hippocampal CA1 region of Gansu zokor and SD rat.Under hypoxic stress treatments,the hippocampal pyramidal neurons of Gansu zokor had complete structure,clear nucleus and no loss of neurons.The pyramidal neurons in the hippocampus of SD rat were arranged loosely,and the density of neurons decreased and neurons loss occurred.Compared with normoxia group,the number of neurons in Gansu zokor was significantly increased of normoxia group in hypoxia I group,but not significant change in hypoxia ? group.In the both hypoxia groups,the neurons number of SD rat were extremely significant decreased.Compared with SD rat,the number of neurons in Gansu zokor was significantly lower than that in SD rat.The results indicated that hypoxic stress did not damage hippocampal neurons in Gansu zokor,but reduces the neurons number of hippocampal and damages neurons in SD rat.It showed that there was a hypoxia tolerance in hippocampal neurons of Gansu zokor.2.Effects of hypoxic stress on the density of dendritic spines in hippocampus of Gansu zokor and SD rat.There was no significant difference in the density of total dendritic spines and mushroom dendritic spines in two hypoxic treatment groups compared with the normoxia group.The density of thin dendritic spines of Gansu zokor was significantly decreased in hypoxia ? group,while the density of stubby dendritic spines was markedly increased in hypoxia ? group.The density of total dendritic spines of SD rat in both hypoxia treatment groups were significantly decreased.The density of mushroom dendritic spines was significantly decreased in hypoxia ? group.The density of thin dendritic spines was significantly decreased in hypoxia ? group,and the density of stubby dendritic spines were significantly decreased in both hypoxia groups.Compared with SD rat,the total dendritic spines density of Gansu zokor in three groups were significantly higher than that in SD rat,and the mushroom dendritic spines density in both hypoxia groups were significantly higher than that in SD rat.The results indicated that the density of total dendritic spines and mushroom spines in the Gansu zokor hippocampus did not change under hypoxic stress.Nonetheless,the density of total dendritic spines and mushroom spines in oxygen-sensitive SD rat hippocampus was decreased.The results showed that the number and shape of dendritic spines in the Gansu zokor hippocampal neurons were not affected by hypoxic stress,and they were tolerant to hypoxic stress.It provided the structural basis for synaptic function in hippocampus of Gansu zokor under hypoxic stress.3.Effects of hypoxic stress on glutamate content in hippocampus of Gansu zokor and SD rat.Compared with normaxia group,glutamate content in the hippocampus of Gansu zokor in both hypoxia groups were significantly decreased.While in SD rat the two hypoxia groups were significantly increased.Compared with SD rat,the content of glutamate in Gansu zokor normaxia group was significanytly higher than that in SD rat,and the content in both hypoxia groups of Gansu zokor were significantly lower than that in SD rat.The results indicated that the glutamate content in the hippocampus of Gansu zokor decreased and that in SD rat increased under hypoxic stress.The result showed that the reducing glutamate content of the hippocampus in Gansu zokor can resist neuron injury caused by excitatory toxicity which is the result of excessive glutamate release.4.Effects of hypoxic stress on NMDA receptor in hippocampus of Gansu zokor and SD rat.(1)Changes in mRNA relative expression levels of NMDA receptor subunits under hypoxic stress.NR1 subunit of Gansu zokor were considerably down-regulated in both hypoxia groups compared with normaxia group.That in both hypoxia groups were significantly up-regulated in SD rat,respectively.NR2A subunit of Gansu zokor and SD rat in the hypoxia ? group were significantly down-regulated,but there was no significant difference in the hypoxia ? group.NR2B subunit of Gansu zokor in the two hypoxia groups were markedly down-regulated,while in SD rat there were significantly upregulated in the two hypoxia groups.NR2C subunit of Gansu zokor in the hypoxia I group was extremely significantly down-regulated,but there was no significant difference in the hypoxia ? group.NR2C subunit in the both hypoxia groups were significantly down-regulated.NR2D subunit of Gansu zokor in the two hypoxia groups were extremely significantly down-regulated.NR2C subunit in SD rat were markedly down-regulated in both hypoxia groups.Compared with SD rat,NR1 subunit in three groups were dramatically higher than those in SD rat,while NR2A,NR2B and NR2C were significantly lower than those in SD rat.NR2D subunit in hypoxia I group was considerably higher than that in SD rat,but there was no significant difference in normaxia group and hypoxia? group.(2)Changes in protein expression levels of NMD A receptor subunits under hypoxic stress.Compared with normoxia group,NR2A subunit of Gansu zokor had no significant change in two hypoxia groups.It was considerably down-regulated in SD rat in the hypoxia ? group,but not significantly changed in the hypoxia ? group.NR2B subunit of Gansu zokor were dramatically decreased in both hypoxia groups.In the hypoxia ? group,NR2B subunit in SD rat was decreased,but there was no significant difference in the hypoxia ? group.NR2C subunit of Gansu zokor was significantly up-regulated in hypoxia ? group,but there was no significant difference in hypoxia ? group but upregulated.NR2C subunit of SD rat were markedly down-regulated in both hypoxia groups.There were no significant difference in NR2D subunit of the two hypoxia groups in both Gansu zokor and SD rat.Compared with SD rat,NR2A,NR2B and NR2C subunits of Gansu zokor were markedly lower in three groups than those of SD rat.NR2D subunit in normaxia group and hypoxia ? group were significantly higher than that in SD rat,but there was no significant difference in hypoxia ? group.These results indicated that under hypoxic stress,NR1 subunit in the hippocampus of Gansu Zokor is down-regulated to reduce the number of NMDA receptor and avoid the binding of glutamate to NMDA receptors in large amounts.Down-regulation of NR2B subunit,up-regulation of NR2C subunit,maintenance of NR2A and NR2D subunits,can reduce the channel opening of NMDA receptor,prevent the over-activation of NMDA receptor,reduce the influx of Ca2+ play the normal function of NMDA receptor,and prevent the occurrence of neuronal excitotoxicity.Therefore,NMDA receptor subunits play an important role in preventing brain damage caused by hypoxic stress in the hippocampus of Gansu zokor.In conclusion,the density of dendritic spines and the number of neuron in Gansu zokor hippocampus did not change under hypoxic condition.Gansu zokor can reduce the glutamate content in the hippocampus,stabilize NR2A subunit and NR2D subunit of normal expression,down-regulate NR1 and NR2B subunits,up-regulate NR2D subunit,which against neuronal excitability toxicity and neuronal dysfunction caused by hypoxic stress,and activate brain protection mechanism.It can be seen that under hypoxic stress,the hippocampus of Gansu zokor is not damaged by neuronal excitotoxicity,and its synaptic function is normal.It has evolved a unique tolerance mechanism that is different from oxygen-sensitive SD rat in terms of hypoxic brain protection. |
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