TSA Alleviate Neuropathic Pain Induced By RTX Through Regulating The Activity Of Neuron-Glial Cells

PART1:The changes of neuron-glial cell activity in neuropathic pain induced by RTXOBJECTIVETo evaluate the changes of neuron-glial cell activity in RTX induced neuropathic pain models(a PHN-mimic model).METHODS1.Male Sprague-Dawley rats,were randomly divided into 2 groups using a random number table:group Vehicle and group RTX.The RTX group was induced by a single intraperitoneal injection of RTX(210 ?g/kg),and Vehicle group was induced by the same dose of menstruum.2.Mechanical allodynia was quantified with von Frey filaments,and thermal sensitivity was tested with radiant heat stimulus.3.IB4/CGRP/TRPV1–expressing DRG neurons were shown by using immunofluorescence labeling at day 1(D1),7(D7),42(D42)after modeling,and GFAP–expressing was also shown by using immunofluorescence labeling in spinal cord.4.The expression of mRNA of IL-1?,TNF-?,IL-6,IL-10 and IL-17 a,BDNF in spinal cord were shown by qPCR(Quantitative polymerase chain reaction,PCR)at time of4 hours after RTX injection(H4),D1,D3,D7,D42;the expression of IL-1?,TNF-? and BDNF in DRG were also shown by qPCR at D3,D7,D42 after modeling.5.The expression of mRNA and protein of GFAP and CD11 b were shown by qPCR and western blotting at H4,D1,D3,and D7 and D42.1.Compared with group Vehicle,group RTX displayed a significantly decrease in mechanical allodynia,and thermal sensitivity was significantly increased on day 3 and persisted until day 42(P<0.05).Group Vehicle show no obviously change(P>0.05),RTX group and group Vehicle compared in the same time point,was statistically significant different(P<0.05).2.Compared with group Vehicle,IB4,CGRP and TRPV1 positive neurons in group RTX presented as long lasting,unrestorable decrease in RTX group(D3~D42)(P<0.05).3.Compared with group Vehicle,the expression of astrocyte marker product GFAP in group RTX was increased throughout the observation period(P<0.05),but the expression of CD11 b mRNA and its protein in spinal were up-regulated only after 4 hours of RTX injection(P<0.05).4.Compared with group Vehicle,in group RTX,the expression of IL-1? and TNF-?mRNA in DRG(D3,D7,D42)and SC(D1,D3,D7,D42)were significantly increased(P<0.05).And the expression of BDNF in the early stage of spinal cord(H4)was increased(P <0.05),and decreased at D3(P<0.05),and there was statistically significant difference between the two groups until D42(P <0.05).The expression of IL-10 and IL-17 a mRNA in the early stage(D1,D3)was higher than that in group Vehicle(P<0.05),and the expression of D7 and D42 were decreased(P <0.05).CONCLUSION1.The decrease of medium and small diameter DRG neurons such as IB4?CGRP?TRPV1 positive neurons may play an important role in RTX induced neuropathic pain.IB4 neurons may take part in the mechanical allodynia,while the deletion of CGRP and TRPV1 positive neurons fibers may be the reason of thermalgesia desensitization.2.Activated glia participate in the pathogenesis of RTX induced neuropathic pain and are likely to be the source of proinflammatory cytokines(such as IL-1?,TNF-?,IL-6,IL-17a).3.Anti-inflammatory factors IL-10 and BDNF expression imbalance play an important role in RTX-induced neuropathic pain.PART 2: TSA alleviate neuropathic pain induced by RTX throughregulating the activity of neuron-glial cellOBJECTIVETo evaluate the role of trichostatin A(TSA)in the regulation of the activity of neuron-glia in an RTX induced neuropathic pain(a PHN-mimic model).METHODS1.The neuropathic pain model was induced by a single intraperitoneal injection of RTX(210 ?g/kg)in rats.2.Male SD rats weighing 250-280 g were randomly divided into four groups :Vehicle group;RTX group;RTX +DMSO group;RTX +TSA group.TSA was dissolved in5% dimethyl sulfoxide(DMSO)in physiological saline.From 60 min before RTX injection(day 0)and once daily after modeling for 7 days.5% DMSO was injected intrathecally once a day in RTX+DMSO group and TSA(0.5 ?g/kg)was injected intrathecally once a day in RTX+TSA group.3.Mechanical allodynia was quantified with von Frey filaments;the measurement time after the modeling was 2 hours after intrathecal injection.4.The expression of GFAP,IL-1?,TNF-? and BDNF mRNA in SC and the expression of BDNF mRNA in DRG were measured by qRT-PCR after 2 hours of last time of TSA injection.RESULTS1.Compared with group Vehicle,group RTX and group RTX + DMSO displayed a significantly decrease in mechanical allodynia on 3th day(P <0.05),RTX + TSA group displayed on 5th day(P <0.05);Compared with group RTX and group RTX + DMSO,group RTX + TSA had significantly higher mechanical pain threshold(P<0.05).There was no significant statistical difference between group RTX and group RTX + DMSO(P>0.05).2.Compared with group Vehicle,in mRNA level,GFAP?IL-1??TNF-? wereincreased in group RTX,group RTX +DMSO,group RTX+TSA(P<0.05);Compared with group RTX and RTX+ DMSO group,IL-1?,TNF-? were decreased in m RNA level in group TSA+DMSO(P<0.05);.There was no significant statistical difference between group RTX+DMSO and group RTX(P> 0.05).3.Compared with group Vehicle,the expression of BDNF mRNA is down-regulated in the spinal cord and DRG(P<0.05)in group RTX and group RTX+DMSO;Compared with the group RTX and group RTX+DMSO,the expression of BDNF mRNA in the spinal cord was up-regulated(P<0.05)in group RTX+TSA.There was no significant statistical difference between group RTX+DMSO and group RTX(P> 0.05).CONCLUSIONIntrathecal TSA regulates neuronal-glial cell activity and relieves RTX-induced neuropathic pain by modulating the expression of BDNF and proinflammatory cytokines(IL-1?,TNF-?).