Effect And Mechanism Of Sevoflurane Postconditioning On Hippocampal Migration Abnormalities After Hypoxic-ischemic Brain Injury In Newborn Rats

Objective:Neonatal ischemic-hypoxic encephalopathy（HIE）refers to neonatal brain injury caused by perinatal asphyxia,with brain structure development injured and sequelae of cerebral palsy,epilepsy and behavioral abnormalities,which seriously threaten the health of newborn.Although the currently used methods such as mild hypothermia and oxygen therapy have a certain protective effect on neonatal ischemic hypoxic brain injury,the long-term treatment effect is still very optimistic.Seeking new treatments to reduce ischemic hypoxic brain injury and improve the long-term prognosis of children is still imminent.Many studies have gradually explained the underlying pathological mechanisms of neonatal hypoxic-ischemic encephalopathy.Ischemia and hypoxia mainly damage the cerebral cortex,hippocampus and neurons in the subventricular region.Damaged neurons went through internal environment homeostasis,abnormal glycolytic ATP production capacity leads to abnormal energy metabolism in newborn neurons and programmed molecular development of neural progenitors caused by excessive activation of molecular signals such as apoptosis,autophagy,necrosis,and inflammation The process was severely damaged or even killed.The hippocampus and the hippocampal loop,as well as cerebral cortex,play an important role in spatial memory,learning ability,and emotional regulation.Neonatal neurons in subgranular zone in dentate gyrus of the hippocampus undergo the processes of occurrence and migration,and finally integrate mature neurons into the pre-existed hippocampal neural circuit.It stores and organizes hippocampal dependent learning and memory functions,especially new information and transmits signals in the brain.Current research has found that there are two regions of the mammalian brain:the hippocampus and the subventricular zone（SVZ）along the wall.The lateral ventricle is considered to be an important part of neurogenesis.The progenitor cells of the hippocampal dentate gyrus（DG）are located in its subgranular zone（SGZ）.Neurogenesis and migration of immature hippocampal neurons gradually become an inseparable process during the development of hippocampal structures,and the entire migration process is extremely vulnerable to external environmental stimulation.Experimental studies have shown that a stroke in the whole brain can excessively induce nerve regeneration in the brain,and tracking these new neurons found that the hyperproliferative neurons eventually developed into functional cells,and the number of integrated into the hippocampus circuit was very small,and even differentiated into Glial cells form nerve scars,hinder the migration of normal neurons,and ultimately affect the formation of the normal structure of the hippocampus.Immature hippocampal neurons are extremely susceptible to external environmental stimuli.Many harmful stimuli such as hypoxia and restricted intrauterine development can cause fatal damage to hippocampal development and individual development.The hippocampal migration process,which is controlled by the integration of many internal mechanisms,is extremely susceptible to stimuli such as ischemia and hypoxia.It mainly involves neurodevelopmental regulators NeuroD1,BDNF,GDNF,CXCL12 and the extracellular matrix such as Reelin,as well as abnormal intercellular interactions.Other experiments have confirmed that the absence of migration regulation factor Reelin can cause diseases such as epilepsy and stunting in adulthood.The correct neural migration process regulated by the above-mentioned key neurodevelopmental regulators and the intrinsic molecular mechanisms of the neural molecules is crucial for the establishment of a hippocampal functional neural circuit.In recent years,researches on the development of Cajal-Retzius（CR）cells in the cerebral cortex and Reelin protein secreted by them have gradually revealed that the role of Reelin expression in hippocampal nerves migration,synapse formation and hippocampal dependent long-term learning during central nervous system development.After birth,Reelin protein is continuously secreted by GABAergic neurons,and its NPXY domain is further combined with cell membrane surface receptors ApoER2 and VLDLR.Dab1 is further activated by the activation of SFK family proteins Src/Fyn kinase,and finally the Gsk-3βfamily acted on downstream microtubule-associated protein（MAP）which affects the final dynamic migration process of neurons.Studies on the changes of Reelin protein in the offspring of immature hippocampal neurons stimulated by hypoxia during pregnancy explain the potential link between Reelin-mediated neuronal migration disorder,hippocampal abnormalities and long-term behavioral cognitive impairments.In recent years,scholars have conducted a lot of research on the role of anesthetics in protecting immature damaged nerves.Sevoflurane is the most commonly used inhaled anesthetic in neonatal surgery.A large number of studies have confirmed that sevoflurane pre-treatment and post-treatment have significant protective effects on ischemic-hypoxic brain injury in rats.However,previous studies have focused on the effect of sevoflurane on reducing ischemic-hypoxic brain damage by inhibiting cerebral infarct volume,neuronal apoptosis and inhibiting secondary inflammatory responses after ischemia-reperfusion.There are few reports on its functions on regulating of hippocampal neural cell migration and the role of neuronal permutation under oxygen stimulation.This experiment aimed to investigate whether ischemia and hypoxia can cause neonatal hippocampal dentate gyrus migration disorder and long-term neurocognitive disorders.Moreover,we investigated whether sevoflurane post-treatment can protect neurological injury after hypoxia and hypoxia.Finally,we investigated whether the neuroprotective effect of sevoflurane on hypoxic-ischemic brain injury in neonatal rats can target the neural migration regulator Reelin and the downstream Reelin-Dab1 pathway to repair hippocampal migration impairments and improve hippocampus dependent learning and memory ability.Methods:Seven-day-old newborn rats of SPF grade,weighing 12-20g were provided by were provided by Benxi Experimental Animal Center,Shengjing Hospital,China Medical University.The temperature of the breeding environment is constant at 24±2℃,the relative humidity is 50±10%.Rats were free access to water.Day and night change 12h:12h.Seven-day-old neonatal SD rats were subjected to ligation of the left common carotid artery after continuous anesthesia with sevoflurane.After taking the supine position and disinfecting the skin with an iodine,a mid-incision was made to expose the left common carotid artery.The artery was ligated twice with a 7-0 silk suture and the skin incision was sutured.After the operation,rats were put back to their mother for 2h.Then the rats were put into a self-made hypoxic box（the bottom of the box was filled with soda lime to absorb carbon dioxide and moisture）and immersed in a 36.5±5℃constant temperature water bath.HIE model was prepared by continuously inputting a humidified mixed gas of 8%O₂-92%N₂ into the hypoxic tank at a flow rate of 2L/min for 2h.As for sevoflurane post-treatment,2.5%sevoflurane（1.0MAC）was inhaled for 30 min immediately after HIE.Newborn SD rats were randomly divided into groups.The first part of the experiment was divided into:The first part of the experiment was divided into:（1）Sham Group,（2）Ischemic-Hypoxic Group（HI Group）（n=20）.The second part of the experiment was divided into three groups:（1）Sham Group（Sham Group）（2）Ischemic-Hypoxic Group（HI Group）（3）Ischemia and hypoxia with 2.5%sevoflurane post-treatment group（HI+Sev Group）（n=25）.The third part of the experiment was divided into 4 groups:（1）Sham Group（Sham Group）（2）Ischemia and hypoxia group（HI Group）,（3）Ischemia and hypoxia with 2.5%sevoflurane post-treatment group（HI+Sev Group）（4）2.5%sevoflurane post-treatment plus Reelin degradation enzyme inhibitor group（HI+Sev+G Group）（n=40）.On the 14th and 21st day after birth,the neonatal rats in each group were tested for nerve migration with immunofluorescence.On the 8^th,14th and 21st day after birth,the neonatal rats in each group were tested for neurogenesis with immunofluorescence.On the 21st day after birth,the arrangement of neurons in DG area and the changes of neuron density were observed with Nissl stain.Reelin expression was detected by immunofluorescence on the 14^th,21^thh day after birth.The expression levels of Reelin,Dab1,P-Dab1,Gsk-3β,P-Gsk-3β,total Tau and P-Tau protein were detected by Western blot on the 14th day after birth.Open-field,water maze,and eight-arm maze experiments were performed at 28 to 35 days after birth to detect neurobehavioral changes in rats.Results:The first part of the experimental results:The immunofluorescence results at 14days and 21 days after birth showed that the migration of hippocampal dentate gyrus neonatal neurons was blocked in the granular layer of the dentate gyrus,and the migration process was significantly abnormal（p<0.05）.Immunofluorescence results at 8,14,and 21 days after birth showed that the hippocampal dentate gyrus neurogenesis was significantly increased on PND 8 compared with the Sham group（p<0.05）.However,we found that there was no significant difference between the HI group and the Sham group.Nissl staining on 21 days showed that the neurons were arranged disorderly and the density was significantly reduced（p<0.05）.The results of long-term learning and memory function tests showed that compared with the Sham group,the HI group had a significantly longer evasion latency from the second day of training（p<0.05）,and the sixth day crossing platform quadrant results showed that the HI group crossed the platform quadrants significantly reduced（p<0.001）.The second part experimental results:The immunofluorescence results performed at 14days and 21 days after birth showed that compared with the Sham group,the block of hippocampal dentate gyrus neuron migration in rats was significantly relieved（p<0.05）.Immunofluorescence results at 8,14 and 21 days after birth showed that compared with the HI group,the occurrence of hippocampal dentate gyrus in the HI+Sev group was significantly reduced on the 8th day（p<0.05）.There was no significant difference in neurogenesis ability between the groups at day and day 21 detection time points.The survival rate and the basic structure of neurons were restored（p<0.05）.Water maze results showed that sevoflurane post-treatment significantly shortened the latency and increased the number of crossing the platform quadrant（p<0.05）.Immunofluorescence test results showed that the Reelin protein in the hippocampal dentate gyrus of the HI group continued to be highly expressed on 14^th,21^thh day after birth（p<0.05）.Post-treatment with sevoflurane significantly reduced the persistently high expression of Reelin protein（p<0.05）.The third part of the experimental results:The immunofluorescence results performed at14 and 21 days after birth showed that hippocampal dentate gyrus nerve migration was blocked,the proportion of neuron survival and the basic structure of neuron arrangement was disorder compared with the Sham group（p<0.05）.Immunofluorescence results performed at 8,14 and 21 days after birth showed that compared with the Sham group,the occurrence of hippocampal dentate gyrus in the HI group was significantly increased on day 8（p<0.05）.Significantly reduce hyperproliferative neonatal neurons（p<0.05）.The application of Piceatannol has no obvious effect on neurogenesis.There was no significant difference in neurogenesis ability between the groups on the 14th and 21st detection time points.Compared with the HI group,sevoflurane post-treatment could recover the injuries above（p<0.05）.In the HI+Sev+G group,the effect of sevoflurane was reversed（p<0.05）.Western-blot results showed that compared with the Sham group,levels of Reelin,activated Dab1（P-Dab1）,activated GSK-3β（P-GSK-3β）,and phosphorylated microtubule-associated protein Tau in the HI group were significantly increased（p<0.05）.The sevoflurane-treated group could significantly inhibit the Reelin/Dab1/GSK-3β/Tau pathway activated by HI（p<0.05）.Compared with the HI+Sev group,the HI+Sev+G group can block the effect of sevoflurane post-treatment（p<0.05）.The results of the water maze experiment showed that compared with the Sham group,the evasion latency was significantly longer（p<0.05）from the second day of training,and the number of crossing the platform quadrant was significantly reduced（p<0.05）.Sevoflurane post-treatment can significantly shorten the latency and increase the number of times to cross the platform quadrant（p<0.05）.Piceatannol can reverse the effect of sevoflurane.The eight-arm maze results showed that the total exercise distance and reference memory errors in the HI group increased significantly compared with the Sham group（p<0.05）.Sevoflurane post-treatment significantly reduced the total exercise distance and reference memory in the HI group（p<0.05）.The effect of sevoflurane was reversed after application of Piceatannol.Conclusion:After HIE,neural migration in the hippocampal dentate gyrus of neonatal rats is blocked,neurons are arranged in disorder and long-term spatial memory learning ability is impaired.Reactive neurogenesis was occured in 24h.Sevoflurane post-treatment targeted Reelin regulation,inhibited the Reelin/Dab1 signaling pathway activated by ischemia and hypoxia stimulation,improved hippocampal dentate gyrus neuron migration block phenomenon to restore damaged hippocampal structure and relieve long-term spatial learning and memory impairment in rats.