HDAC3 In Hippocampus Contributes To Neuropathic Pain Induced Memory Impairment

Background: Pain is a distressing experience associated with actualor potential tissue damage with sensory,emotional,cognitive,and social that can have a marked impact on both physiological and psychological state of an individual.Increasing clinical evidence has shown that patients with chronic pain commonly suffer from impaired cognitive function.A wide array of cognitive domains,including attention,learning memory,speed information processing and decision-making,were impaired in the patients with chronic pain.Previous study reported that two-thirds of the participants with chronic pain revealed clinically significant disruption of attention and impaired working memory processes.Recent preclinical studies also demonstrated memory deficits in the animal model of neuropathic pain.Despite the clinical and animal evidence of comorbidity of memory impairment under chronic pain conditions,molecular mechanism underlying this comorbidity remains poorly understood.Histone deacetylase(HDACs)plays an important role in chromosome structural modification and gene expression regulation.HDACs catalyze the removal of acetyl groups from histones to cause the chromatin to curl tightly,thereby inhibit gene transcription.HDACs were the targets for a variety of diseases ranging from pain to neurodegeneration.Previous studies revealed that the expression of HDACs increased under pain condiction and HDAC inhibitors can attenuate nociception in experimental models of neuropathic pain.Moreover,the hippocampal HDACs were increased in Alzheimer's disease and postoperative cognitive disorder and HDAC inhibitors can improve cognitive impairments.However,it is unclear whether HDACs are involved in chronic pain-associated memory impairment.HDAC3,one isoform of the class I HDACs,is highly expressed in the cortex and hippocampus,and negatively regulates learning and memory.Indeed,overexpression of HDAC3 promoted neuronal death and produced memory impairment and inhibition of HDAC3 significantly improved cognitive function in rodents.In the present study,we tested whether HDAC3 was specifically implicated in neuropathic pain and the associated memory impairment and the mechanisms of how HDAC3 participated in this process.Methods: First part: To explore the effect of neuropathic pain on the memory function.A neuropathic pain mouse model of chronic constriction injury(CCI)of the sciatic nerve was carried out.Pain behaviors,including paw withdrawal threshold(PWT),paw withdrawal latency(PWL)and conditioned place preference(CPP)and memory behaviors,including Y maze and novel object recognition(NOR)were tested at different time points.Mice were randomly divided into two groups: sham group and CCI group.The chromic gut was tied loosely around the exposed sciatic nerve in CCI group and the sciatic nerve was only exposed without ligation in sham group.The open field test(OFT),PWT and PWL were examined on day 1 before surgery and days 7,14 and 21 after surgery.The Y maze was performed on days 7,14 and 21 after surgery,whereas the NOR on day 21 after surgery.Second part: To observe the changes in the expression of HDACs.Mice were randomly divided into two groups: sham group and CCI group.The medial prefrontal cortex(m PFC),anterior cingulate area(ACC)and hippocampus were harvested 7,14 and 21 days after surgery.The protein and m RNA expression of HDAC1,HDAC2,HDAC3 and HDAC8 was measured using Western blot,qRT-PCR and immunofluorescence.Third part: To evaluate the effect of HDAC3 on nociceptive and pain induced memory deficit.(1)four groups of mice were used:sham + vehicle,sham + RGFP966,CCI + vehicle,and CCI + RGFP966.Selective HDAC3 inhibitor RGFP966 was intraperitoneally injected into the mice starting 2 h before surgery and then once every other day for 23 days.Mice were subjected to the OFT,Y maze,NOR,PWT,PWL and CPP on day 21 to day 23 after surgery.(2)To evaluate the effect of HDAC3 overexpression on nociceptive and memory behavior,two groups of mice were used: EYFP group(Control group)and HDAC3 group(HDAC3overexpression group).AAV-EYFP or AAV-HDAC3-EYFP was administered into the bilateral hippocampus.Mice were subjected to the OFT,Y maze,NOR,PWT,PWL and CPP on day 21 to day 23 after viral injection.Fourth part: To study the mechanism of HDAC3 involed in the memory impairment induced by neuropathic pain.Four groups of mice were used: sham + vehicle,sham + RGFP966,CCI + vehicle,and CCI +RGFP966.Selective HDAC3 inhibitor RGFP966 was intraperitoneally injected into the mice starting 2 h before surgery and then once every other day for 21 days.Hippocampus was harvest for the degree of acetylation of histones measurement,HDAC3 activation and dendrite spine density detection.Hippocampus slide was used for LTP recording.Results:(1)Compared with the sham group,the CCI group showed significant reductions in both PWT and PWL 7-21 days after surgery,indicating sciatic nerve injury leads to significant hyperalgesia.The spontaneous alternation in the Y maze test was significantly decreased 14 and 21 days after surgery as compared with that in the sham group,and discrimination index in the NOR test were significantly decreased 21 days after surgery as compared with that in the sham group,indicating CCI neuropathic pain can induce significant memory impairment.(2)The protein and m RNA level of HDAC3,but not HDAC1,HDAC2 and HDAC8,in the hippocampus from the CCI group was significantly higher than that in the sham group 21 days after surgery.The expression of HDAC3 at m RNA and protein levels in the hippocampus from the CCI group was significantly higher than that of the sham group at 14 and 21 days,but not at7 days,after CCI.These above results indicate that hippocampus HDAC3 may contribute to pain and pain induced memory impairment.(3)The HDAC3 inhibitor RGFP966 restored the memory deficit induced by CCI.RGFP966 had no effect on the CCI-induced reductions in PWT and PWL and on the CCI-induced increase in time spent on lidocaine-paired chamber.HDAC3 overexpression in the hippocampus led to memory deficit without affecting basal nociceptive responses.These results suggest that HDAC3 contributes to pain induced memory impairment but not pain.(4)Compared with the sham group,the CCI group showed significant increase in HDAC3 activity,reductions in acetylation levels of H3,H3K9,H4 and H4K12 and impairment in LTP and dendritic spines.These synaptic plastic impairments were significantly rescued in the CCI plus selective HDAC3 inhibitor RGFP966 group.Conclusion: CCI mice with long-lasting nociceptive threshold reduction exhibited the deficits of memory.Hippocampal HDAC3 contributes to neuropathic pain-associated memory impairment;HDAC3 contributes to neuropathic pain-induced memory impairment likely through disrupting synaptic plasticity.