| Objective:Neonatal ischemic hypoxic encephalopathy(HIE)is a common disease in the neonatal period.It is caused by many causes such as intrauterine distress,neonatal asphyxia,hyaline membrane disease,etc.,and is the major factor responsible for infant death and neurological sequelae.In preterm infants,the incidence of HIE is as high as 60%.Among them,90%of the patients with chronic disease and have long-term nervous system dysfunction,including delayed development of the nervous system,epilepsy,learning cognitive dysfunction,and motor dysfunction.However,the treatment of HIE at this stage is mainly focused on symptomatic treatment in the acute phase,including mechanical ventilation,cooling,hyperbaric oxygen therapy,and rehabilitation after injury.The results are not satisfactory.We still need a treatment that can reverse or reduce long-term brain damage.With the widespread use of inhaled anesthetics in infant and children surgery,the brain protective effect of anesthesia has attracted attention.As a commonly used clinical drug with multiple pharmacological functions,inhalation anesthetics such as isoflurane and sevoflurane can improve the tolerance of ischemic brain tissue to hypoxia.Sevoflurane,as an inhalation anesthetic commonly used in neonatal clinical surgery,has the advantages of less respiratory irritation and easy adjustment of the depth of anesthesia.With intensive research into the protective effects of inhaled anesthetics,preconditioning and postconditioning of sevoflurane have been shown neuroprotective effects.However,due to the unpredictable onset of HIE,the clinical significance of post-treatment is relatively better.Previous studies have shown that sevoflurane post-treatment can reduce cerebral infarct volume,inhibit neuronal apoptosis,and promote hippocampal neuron regeneration.The previous experimental research of the research group showed that in the model of ischemia and hypoxia,sevoflurane can reduce the occurrence of autophagy and inhibit the apoptosis of nerve cells,thereby playing a neuroprotective role.The important functions of neural stem cells in the developing brain include proliferation,migration,and differentiation.These functions are performed simultaneously during development.The differentiation direction of neural stem cells includes neurons,astrocytes,and oligodendrocytes.Neurogenic brain tissues that persist in mammalian brains are the subventricular layer(SVZ)of the lateral ventricle and the subgranular layer(SGZ)of the hippocampal dentate gyrus(DG).There are a large number of neural stem cells in each brain area,and it is also the most vulnerable area during ischemia and hypoxia.Developmental brain damage can have severe effects on the cortex and hippocampus.In the acute phase after the onset of ischemia and hypoxia,neural stem cells received exogenous signals and differentiated in large numbers towards astrocytes.The differentiated astrocytes migrated to the damaged brain area for repair in the acute phase.Gliding scars.Earlier this was considered to be a stress-protecting nervous system self-protection.However,long-term observations have found that as astrocytes continue to proliferate and differentiate,the glial scars formed during the repair process will hinder and barrier neuronal synaptic connections after ischemia and hypoxia,affecting the arrangement of normal neurons.structure.Experiments have shown that in a stroke model,hypoxia-inducible factor(HIF-1α)is involved in regulating the direction of neural stem cell differentiation in the SVZ region of adult rats,promoting the differentiation of neural stem cells toward neurons and neuroblasts,and inhibiting astrocytes.Cell Differentiation.HIF-1α is an oxygen-sensitive transcription factor widely present in nature,which can control hundreds of important oxygen homeostasis molecules.Although HIF-1α is formed in neurons,its expression is very low under normoxic conditions.In most cell types,HIF-1α protein is stably expressed under hypoxia,but rapidly degrades under normoxic conditions.Recent studies have shown that post-translational modification plays a significant role in the regulation of HIF-1α expression level,especially its ubiquitination process can regulate its stability,thereby changing the expression level of HIF-1α.The ubiquitination of proteins involves ubiquitin activating enzyme E1,ubiquitin binding enzyme E2 and ubiquitin ligase E3.The specific E3 ligase of HIF-1α is pVHL.DJ-1(PARK7)is a neuroprotective protein under conditions of hypoxia and oxidative stress.It can reduce the ubiquitination level of HIF-1α by inhibiting the interaction between HIF-VHL and the oxidation of DJ-1 deficient mice.The expression of HIF-1α decreased in both the stress model and the stroke model.In the Parkinson’s disease model,an increase in DJ-1 expression levels was confirmed to suppress the expression of VHL activity.In summary,this topic first explores whether sevoflurane can reduce the glial scar caused by ischemic hypoxic brain injury by regulating the expression of HIF-1α,and then improve the arrangement of neurons.Second,it explores the expression of sevoflurane on HIF-1α The effect of the level is related to the ubiquitination process of HIF-1α.Finally,we explore whether sevoflurane can up-regulate the brain protective protein DJ-1,inhibit the level of ubiquitination of HIF-1α,and reduce the excessive proliferation of glial cells and glial scars.Formation,improve long-term learning and memory function of neonatal rats after ischemia and hypoxia,and play a protective role in the brain.Materials and methods:1.Experimental animals:7-day-old SPF freshmen(Sprague-Dawley)SD rats weighing 12 to 16 g were provided by Benxi Medical Research and Education Development Base,Shengjing Hospital,China Medical University.This experiment was performed in strict accordance with international ethical guidelines and guidelines for laboratory animal care and use of the National Institutes of Health,and was reviewed and approved by the Medical Ethics Committee of Shengjing Hospital,China Medical University(2017PSO20K).2.Preparation of neonatal rat ischemic hypoxic brain injury model(HIBI):left carotid artery ligation of 7-day-old neonatal SD rats under anesthesia,return to the mother and rest for 2 hours after breastfeeding,then put in 37℃ constant temperature Water bath,8%O2,92%N2,and mixed gas for 2 hours.Thirty minutes before HIBI model surgery,the left ventricle of SD neonatal rats was injected with HIF-1α inhibitor for 7 days,and the remaining groups were injected with the same amount of blank solvent.3.The expression levels of hypoxia-inducible factor HIF-1α,ubiquitinated E3 ligase VHL,and brain protective protein DJ-1 in the left hippocampus of newborn rats were detected by Western blot after 12,24,and 48 hours after modeling.Realtime PCR was used to detect the mRNA expression of HIF-1α in the left hippocampus of newborn rats.After 21 days of modeling,the open field test,Morris Water Maze test(MWM),and suspension test were used to test the long-term motor activity,emotional changes,and spatial learning and memory ability of newborn rats.4.After the behavioral test was completed,the number of neurons in the left hippocampal DG,CA1 and CA3 areas was counted with Nissl staining.Western blot was used to detect the changes of the left hippocampal astrocyte marker protein GFAP,neuron marker NeuN and glial scar marker Neurocan,and the expression of synapse formation related proteins PSD95 and GAP43.Golgi staining was used to observe the dendritic spine density of neurons.The immunofluorescence double staining method was used to detect the changes of GFAP/Neun and GFAP/Neurocan expression in the hippocampus DG,CA1 and CA3 areas of the left side of the rat.5.Statistical analysis:All raw data are statistically analyzed using SPSS,expressed as mean plus or minus standard error.The Shapiro-Wilk method was used to test whether the data conformed to the normal distribution.One-way-anova analysis was used and then Tukey’s post hoc test was performed.Avoidance latency is compared using a two-way-anova analysis method.p<0.05 was considered statistically significant.Result:1.In the first part of the experiment:the results of Nissl staining showed that compared with the sham group,the neurons in the hippocampal CA1,CA3,and DG areas of the HI group were abnormally arranged,and the number of cells was significantly reduced(p<0.05);Golgi staining results showed that dendritic spine densities in hippocampal CA1 area of the HI group were reduced.Western Blot results showed that synapse formation-related proteins were reduced in the HI group.Immunofluorescence staining results showed that the glial cells in the hippocampal area of the HI group were hypertrophic and glial.The neuronal arrangement structure is abnormal.After sevoflurane treatment,the learning and memory function of the rats is improved(p<0.05 compared with the HI group),the number of neurons is increased,the dendritic spine density is increased,synaptic protein is increased,Reduced mass scars and improved neuronal alignment.2.In the second part of the experiment:21-25 days after the HI modeling of newborn rats,the water maze test results show that compared with the Sham group,the model building starts from the second day of training and the escape latency is significantly longer(p<0.05);the long-term behavioral and group chemistry results of the sevoflurane treatment group were significantly improved.After the HIF-1α inhibitor YC-1 was given,the protective effect of sevoflurane was reversed.The expression of hypoxia-inducible factor HIF-1α gradually decreased in neonatal rats 12 hours,24 hours,and 48 hours after HI treatment;sevoflurane post-treatment stabilized the sustained expression of HIF-1α in the brain.3.In the third part of the experiment:real-time PCR results showed that compared with the HI group,the mRNA expression of HIF-1α in the sevoflurane post-treatment group had no significant change.In newborn rats at 12,24,and 48 hours after HI treatment,DJ-1 expression decreased and VHL expression level increased in the HI group.After sevoflurane treatment,DJ-1 expression increased,VHL expression was suppressed,and HIF-1α Expression is stable.Conclusions:1.After HI,astrocytes in the hippocampal region of neonatal rats are hyperproliferated,forming glial scars,damaged neuronal arrangement,and inhibiting synapses.Post-treatment with sevoflurane reduces astrocytes hyperproliferation.And the formation of glial scars,play a protective role in the brain.2.Sevoflurane reduces glial scars by regulating HIF-a levels,protects the structure of neurons,promotes synapses,and improves long-term learning and memory in rats.3.Sevoflurane up-regulates DJ-1,reduces VHL expression,inhibits HIF-1α ubiquitination,and then affects the ratio of astrocytes and neurons,and exerts brain protective effects. |
