The Influence Of Parental Neuropathic Pain On Emotion-related Behavior In Offspring And The Association With DNA Methylation Of Amygdale In Rats

Objectives:To investigate the influences of neuropathic pain in parental rats to basic pain threshold, anxiety and depression of F1generation, to reveal the relationship between maternal care and such influences with cross feeding method, and to explore the association between transgenerational influences of neuropathic pain and global DNA methylation in amygdale.Methods:1、12male and12female rats were randomly divided into4groups: group C1(CCI male, n=6), group C2(CCI female, n=6), group S1(sham male, n=12) and group S2(sham female, n=6). Group C1and C2rats were undergone chronic constriction injury (CCI), and group S1and S2were undergone sham operation.10days after operation, mechanical and thermal pain threshold were tested, as well as anxity and depression behavior with elevated-plus maze test and open-field test, then male and female rats were mated. After the the birth of F1generation rats, the F1generation rats were divided into4groups according to their parents: group FC1C2, groupFC1S2, groupFS1C2and group FS1S2.They were fed normally to the21st day after birth before being separated with their mother,30days after birth the test of mechanical and thermal pain threshold, anxity and depression behavior, and formalin test were performed in all the F1rats, after that, F1rats were euthanized and blood samples were taken from heart, and the CRH and COR in serum were test with ELISA method.2、24male and24female rats were randomly divided into4groups: group C1(CCI male, n=12), group C2(CCI female, n=12), group S1(sham male, n=12) and group S2(sham female, n=12). All the rats were undergone the same operation as section1,10days after operation, male and female rats were mated, and after the birtha of F1generation rats, the F1generation rats were also divided into4groups according to their mothers:group FC2(offspring of C2), group FC2’(offspring of C2) group FS2’(offspring of S2), and groupFS2(offspring of S2), group FS2’ and FC2’rats were cross fed by females rats in group C2and S2respectively, group FS2and FC2rats were fed by their own mothers. Then the maternal care of all the mother rats were investigated with gather and lick behaviors.21days after birth, F1generation rats were isolated.30days after birth, the mechanical and thermal pain threshold, anxiety and depression behavior were performed in all the F1rats. And after behavior tests, the F1rats were euthanized and the oxytocin expression in supraoptic nucleus of hypothalamus were measured by immunohistochemistry.3、24male and24female rats were grouped, operated and mated following the same protocols in section2, the F1generation rats were grouped and feded as those in section2.30days after birth, each F1group was divided randomly into2subgroups, one subgroup rats were perfused with4%paraformaldehyde for immunohistochemistry of DNMT1, DNMT3a/b in amygdale, the other subgroup rats were sacrificed for assessment of DNMT1, DNMT3a/b expression in amygdale with western blot, as well as the global DNA methylation with global methylation kit.Results1> For parental rats, the mechanical and thermal pain threshold, the rate of time spent in open arms (central zone)/time spent in closed arms (peripheral zone) in group C1and C2rats were significantly lower than that of rats in group S1and S2. For F1generation rats, the mechanical and thermal pain threshold in all4groups were similar; however, the rate of time spent in open arms (central zone)/time spent in closed arms (peripheral zone) in group FC1C2and FS1C2were lower than that of group FC1S2and FS2S2; in Formalin test, no difference in pain score were detected in phase I between the4groups, but the pain score in phase II in group FC1C2FS1C2were much higher than that of group FC1S2、FS1S2the serum concentration of CRH and COR of group FC1C2and FS1C2rats were higher than that of group FC1S2and FS1S2rats.2、The latency of gathering and licking, as well the total time spent in gathering in group C2and C2’were longer compared with that in group S2and S2’, and the total time spent in licking in group C2and C2’ were shorter compared with that in group S2and S2’ The mechanical and thermal pain threshold in all4offspring groups. The offspring fed by female rats with neuropathic pain (group FC2and FS2’) showed a reduced rate of time spent in open arms (central zone)/time spent in closed arms (peripheral zone) and lowere expression of oxytocin in supraoptic nucleus. than that in group FC2’ and FS2.3、the expression of DNMT1in amygdale in group FC1C2and FS2’ were higher than that in group FC2’ and FS2detected by immunohistochemistry and western blot, however, the expression of DNMT3a/b were similar in all the4groups; the level total DNA methylation in amygdale in group FC2and FS2’ were also higher than that in group FC2’ and FS2-All these indexes were similar between gourp FC2and Fs2’, as well as between group FC2’ and FS2Conclusions1、Neuropathic pain occurred in mother rather than in father induces negative influence such as anxiety and depression in offspring. Although neuropathic pain in mothers has no influence on the basic pain threshold in offspring, it may enhance the pain severity cognition when tissue injury occurs. Also it activates the HPA axis in offspring.2、The negative influence of neuropathic pain in mothers rats on offspring may be caused by the reduced quality of maternal care, and such deficit may be maintained in generations.3、The negative influence of neuropathic pain in mother mats on offspring may be associated with DNA methylation in amygdale, and this effect may be mediated by DNMT1rarher than DNMT3a/b.