Effect Of C8-ceramide On Cognitivefunction After Surgery In Mice

Background:Postoperative cognitive dysfunction(POCD)is a common clinical complication in patients after surgery,which lasts for several weeks and even more than one year in serious patients.POCD always reduce the life quality of patients and bringing heavy economic burden to families and society.Although the pathogenesis of POCD have no definite understanding,neuroinflammation is the most important independent risk factor.Aseptic operation stimulates the peripheral immune system and produces excess proinflammatory cytokine,which can into the brain in different ways.Proinflammatory cytokine will activate microglia,the immunocompetent cells of the central nervous system,ultimately causes inflammation in the nervous system.The excessive neuroinflammation will induce neuronal death and damage synaptic plasticity,leading to cognitive disorder.Brain derived neurotrophic factor(BDNF),which enhance neuron survival and neurogenesis.What’ more,BDNF can increase synaptic plasticity and benefits the long-term potentiation.In our previous study,we found that microglia-derived BDNF can reduce the levels of TNF-α by inhibiting the PKC patyway,improving the postoperative learning and memory dysfunction.There have studies proven that an analogue of ceramide,C8-ceramide,can inhibit the relase of proinflammational factors.What’ more,C8-ceramide can enhance the expression of BDNF from microglia.So,this study intends to explore the relationship between the effects of C8-ceramide on microglia-derived BDNF and the postoperative cognitive dysfunction.Objective:The study is to investigate the association between the effects of exogenous C8-ceramide on the synthesis and secretion of BDNF from microglia and the postoperative cognitive function,and explore the possible mechanism.Methods:1.The effects of C8-ceramide on the expression of BDNF in pimary microglia.The method to isolate microglial cells from C57BL/6 newborn mouse in vitro culture.The purified primary microglia were randomly divided into four groups: sham group,LPS treatmented group(LPS),LPS plus C8-ceramide treatmented group(C8+LPS)and C8-ceramide treatmented group(C8).The C8+LPS and C8 group recives 25 uM C8-ceramide before LPS challenge.The total RNA from microglia were collected by Trizol and then evaluate the mRNA levels of BDNF by RT-PCR.2.Explore the effect of C8-ceramide on POCD and the possible mechanismThe animal model was established by a right carotid artery exposureoperation and anesthetized with 3% sevoflurane.The C57BL/6 mice were divided randomly into four groups: Sham group(n=43),suegery group(n=43),C8-ceramide + surgery group(C8 + surgery,n=43)and C8-ceramide group(C8,n=43).The micetreatmented with C8-ceramide(10 mg/kg)via intraperitoneal injection daily for 4 days or the same dosage of saline.Their spatial learning and memory abilities were evaluated by morris water maze and fear conditioning test at 7d after surgery.The expression of IL-6,TNF-α in hippocampus and serum was assessed at 6h,24 h by ELISA.Theconcentration of BDNF and the p-TrkBwas detected by western blotting method at day 3,7.The phosphorylation of PKCδ and NF-κBp65 was assessed at third day after surgery.The activation of microglia at hippocampus was detected by immunofluorescent staining assay at 24 h after surgery.Results:1.The RT-PCR results shown that: LPS significantly reduces the the mRNA level of BDNF(p<0.01),but C8-ceramide completely blocked this reduction.what’s more,C8-ceramide will significantly increase the mRNA level than sham group(p<0.05).2.The morris water maze results shown that: the escape latency gradually shortened as the increase in the number oftraining,and the escape latency of surgery group is significantly elongatedthan normal in day 2,3(p<0.05).The test results shown that the times across platform and time percent during platform quadrant of surgery group was significantly reduced than sham group.There has no significant difference between C8 plus surgery group and sham group.3.The fear conditioning test results shown that: the context-related freezing time of suegery group was significantly decreased,but the results of C8-treatmented mice have no different with sham group.There have no significant difference between the four groups in tone-related conditioning test.4.The ELISA results shown that: the levels of TNF-α and IL-6 were significantly increased in serum and hippocampusat at 6h,24 h after surgery(p<0.01,p<0.05).However,C8-ceramide blocked the increase of IL-6 and TNF-α.5.The western blotting results shown that: the levels of BDNF and p-TrkB were significantly reduced BDNF at day 3(p<0.05),but they will return to baseline at day 7.There have no significant difference between the sham group and C8+surgey group at day 3,7.The phosphorylation of PKCδ and NF-κBp65 are significantly incerased in surgery group at day 3(p<0.05).The levels of p-PKCδ and p-P65 of the C8+surgery group are significantly lower than surgery group(p<0.05).6.The immunofluorescent staining results shown that: the number of iNOS immunoreactivity microglial cells in surgery group was significantly increased than sham group and C8+surgery group(p<0.01).The number of Arg-1 positive microglial cells in C8+surgery groupand C8 group were significantly increased than mice in sham group(p<0.01).Conclusion:Pre-treatment with C8-ceramide can prevent the reduction of BDNF induced by aseptic operation or LPS.C8-ceramide can enhance the activation of BDNF/TrkB patyway and alleviate postoperation cognitive dysfunction by suppressing the production of proinflammational cytokines in the periphery and the central nervous system,inhibiting the PKC/NF-κB pathway.