Transmission Of Neonatal Surgery-induced Epigenetic Modifications And Behavioral Abnormality To Next Generation In Rats

Part 1 The effects of surgery on learning and memory function in adult rats and their offspringBackgroundLearning and memory dysfunction occurs in 20%of patients after surgery,which is associated with poor outcomes.The persistent characteristics of this learning and memory dysfunction that occurs after surgery and whether it affects the offspring are unclear.As health care continues to advance and the number of patients undergoing general anesthesia surgery increases each year,the above issues are of increasing concern to patients.ObjectiveTo investigate whether learning and memory dysfunction after surgery is persistent and whether there is intergenerational transmission on their offspring.MethodsTwo-month-old Sprague-Dawley rats had left carotid artery exposure under isoflurane anesthesia.Their learning and memory were evaluated by behavioral studies such as the Open field,Barnes maze and fear conditioning test from 5 days,60 days,or 120 days after surgery.Rats with or without surgery were mated when they were 4 or 6 months old.The learning and memory of the offspring were tested at an age of 2 months.ResultsMale rats exposed to surgery took a longer time to identify the target box after training sessions in Barnes maze and had less freezing behavior in context-related fear conditioning than control rats when the tests were started 5 days after surgery.Similarly,female rats had poorer performance than control rats in Barnes maze test given from 5 days after surgery.However,these poorer performances did not occur when the testes were administered 60 days or 120 days after surgery.The offspring of rats with surgery had a performance like that of the offspring of control rats.ConclusionOur results suggest that both male and female adult rats could develop short-term learning and memory dysfunction after surgery.The learning and memory dysfunction may not be persistent and does not have intergenerational transmission to affect the cognitive function of next generation.Part 2 The effects of surgery on learning and memory function in neonatal rats and their offspringBackgroundMillions of infants and children undergo surgery each year.Surgery with anesthesia in children may lead to learning,memory,and behavior deficits later in life.Clinical studies have shown that adolescences who had surgery at their early age have poor neurodevelopment and poor cognitive performance at school.Animal studies have also found similar results.Since childhood is a critical period for brain development,more and more people are concern about the effects of surgery on developing brains.Moreover,studies have confirmed that parental early life exposure to adverse factors could induce negative behavioral changes in the offspring.Epigenetic changes are proposed to be a mechanism for this transgenerational transmission.Whether parental early life exposure to surgery may affect their offspring through similar epigenetic modification mechanism needs to be further investigated.Glial cell line-derived neurotrophic factor(GDNF)and brain-derived neurotrophic factor(BDNF)play important roles in development and functional maintenance of the central nervous system.Whether neurotrophic factors are involved in the process of transgenerational transmission need to be further investigated.ObjectiveTo investigate whether parental early life exposure to surgery may induce learning and memory dysfunction on offspring whether there is epeginetic modifications on neurotrophic factors.MethodsSeven-day old Sprague-Dawley rats had left carotid artery exposure.They were subjected to Barnes maze,fear conditioning and open field tests from the age of 30 to 45 days and mated to produce the next generation when they were 65 days old.The behavior of the offspring was tested at the age of 30 to 45 days.Hippocampus was harvested at the age of 30 days and 7 days from the parent generation and offspring,respectively,for measuring the concentrations of GDNF and BDNF and the degree of methylation in the promoter regions of Gdnf and Bdnf genes.ResultsMale rats with surgery had poorer performance in Barnes maze and fear conditioning tests and reduced time in the center area of open field.This impairment of learning,memory and behavior occurred to the male offspring of surgery sires.The surgery male rats had decreased GDNF and BDNF in the hippocampus.Similarly,the male offspring of surgery rats had decreased GDNF.Surgery increased the methylation of gdnf and bdnf genes in the surgery male rats and male offspring of surgery sires.Although surgery female rats had impaired learning and memory,their BDNF and GDNF concentrations were not decreased and the methylation of Gdnf and Bdnf genes in these female rats was not altered.Only the female offspring whose both parents had surgery presented learning and memory impairment.None of the female offspring groups had altered GDNF and BDNF expression.Epigenetic modification on neurotrophic factor expression may contribute to this transmission in male rats.Highly methylated gdnf and bdnf gene can be passed to the male offspring rats.The male offspring from the surgery father showed learning and memory dysfunction.ConclusionTransgenerational transmission of the detrimental effects of surgery on learning,memory and behavior may have sex preference.Surgery induced increased methylation levels of Gdnf and Bdnf gene promoter regions,resulting in the down-expression of GDNF and BDNF protein levels in male rats.Highly methylated Gdnf and Bdnf gene can be passed to the offspring male rats.The male offspring from the surgery sires showed learning and memory dysfunction.Epigenetic modification on neurotrophic factor expression may contribute to this transmission in male rats.Part 3 The effects of PDTC on learning and memory function of neonatal rats with surgery and their offspringBackgroundNeuroinflammation is considered as a key pathological process for surgery induced learning and memory impairment.Studied have shown that neuroinflammation may be transmitted from systemic inflammation after a surgery via disrupted blood-brain barrier.Nuclear factor kappa-B(NF-κB)is an important transcription factor that regulates the expression of inflammatory cytokines.Surgery leads to elevated NF-κB expression,causing a decrease in downstream GDNF and BDNF expression and induce learning and memory dysfunction in animals.PDTC(Pyrrolidinedithiocarbamic)is a widely used NF-κB inhibitor.Studies have shown that PDTC improves surgery induced learning and memory dysfunction by inhibiting NF-κB pathway,which decreases the expression of pro-inflammatory cytokines IL-6 and IL-1β and increases the expression of GDNF and BDNF and synaptic proteins,improves ultrastructural synaptic plasticity.DNA methylation is regulated by DNA Methyltransferases(DNMTs).NF-κB is found to affect gene methylation by regulating DNMTs.Therefore,we hypothesized that PDTC inhibits the regulation of methylation of the promoter regions of Gdnf and Bdnf genes by DNMTs through inhibition of NF-κB,which in turn alleviates the reduced expression of GDNF and BDNF and improves learning and memory functions.ObjectiveTo investigate whether PDTC can ameliorates learning and memory dysfunction in neonatal surgery rats and their offspring by inhibiting methylation of the promoter regions of the Gdnf and Bdnf genes.MethodsSeven-day-old SD rats were randomly divided into control group,control+PDTC,anesthesia group,surgery group and surgery+PDTC group.The surgery group and surgery+PDTC group rats had left carotid artery exposure.In surgery+PDTC group,the learning and memory function of rats were evaluated,and IL-6 and IL-1β in blood and hippocampus were detected.The expression of GDNF and BDNF in hippocampus and the methylation degree of Gdnf and Bdnf gene promoter region were examined.To explore the role of PDTC in improving postoperative learning and memory impairment,7-day-old SD rats were randomly divided into control group,control+PDTC,anesthesia group,surgery group and surgery+PDTC group.At the age of 65 days,rats were randomly paired to produce next generation.Learning and memory were tested by open field,Barnes maze and fear conditioning,separate cohort of 7-day-old offspring was used to collect hippocampus to detect the expression of GDNF and BDNF.ResultsMale rats with surgery had poorer performance in Barnes maze and fear conditioning tests and reduced time in the center area of open field and reduced travelled distance in open field.PDTC can improve the learning and memory impairment after surgery.PDTC can improve memory and learning dysfunction in surgery rats.PDTC can alleviate the inflammatory reaction caused by surgery,reduce the decrease of GDNF and BDNF expression in hippocampus,and reduce the methylation level of GDNF and BDNF gene promoter.For the offspring male rats,all mothers were from control group.The offspring from the surgery father spend less time in the center of the open field,travelled less distance in the open field,showed short-term and long-term spatial memory impairment,showed context-related fear conditioning.Surgery reduced the hippocampus GDNF and BDNF expression.Compared with the male offspring from surgery father,the male offspring whose father from the surgery+PDTC group spent more time in the center of open field,didn’t showed short-term and long-term spatial memory impairment,their hippocampus GDNF and BDNF expression didn’t decrease.ConclusionPDTC reduce the methylation level of GDNF and BDNF Genes,reverse the decreased expression of GDNF and BDNF,and alleviate the learning and memory dysfunction after surgery.Post-operative PDTC treatment in the parental generation was able to prevent learning and memory dysfunction in their offspring.Therefore,the offspring do not show learning and memory dysfunction.