| Objectives:1.To examine the effects of intrathecal nuclear factor-kappaB(NF-κB) inhibitor,pyrrolidine dithiocarbamate(PDTC),on the development of neuropathic pain,spinal microglial activation and spinal NF-κBp-p65,CX3CR1,p-p38MAPK,tumor necrosis factor(TNF)αexpression induced by sciatic chronic constriction injury(CCI) in rats. 2.To examine the effects of NF-κB inhibitor PDTC on TNFα-induced CX3CR1 expression in BV-2 microglial cells.3.To examine the effects of intrathecal resveratrol(Res) on the development of neuropathic pain, spinal microglial activation and spinal NF-κBp-p65,CX3CR1,TNFαexpression induced by sciatic CCI in rats,and investigate the therapeutic potential of Res in neuropathic pain.Methods 1.288 male Sprague-Dawley(SD) rats(250～300 g) fitted with intrathecal(i.t.) catheters,underwent surgery.Randomly,rats were divided into 8 groups (n=36):normal control group:rats didn't undergo any surgery;sham group:rats underwent surgery of exposing sciatic nerve without ligation; sham+PDTC group:rats undergoing sham surgery were intrathecally administrated PDTC(1000pmol/d);CCI group:rats underwent sciatic CCI surgery;CCI+PDTC group 1:Rats were divided into 2 subgroups, and intrathecal PDTC(100pmol/d) was infused 1 day before and 3 days after sciatic CCI respectively;CCI+PDTC group 2:Rats were divided into 2 subgroups,and intrathecal PDTC(1000pmol/d) was infused 1 day before and 3 days after sciatic CCI respectively;sham+saline group: Intrathecal normal saline(NS) was infused 1 day before and 3 days after sham surgery respectively;CCI+saline group:Intrathecal NS was infused 1 day before and 3 days after sciatic CCI,respectively.5 days after intrathecal catheters implantation,rats were administrated various intrathecal medicine.And 1 day after administration,rats experiented CCI and sham surgery,respectively.The rat hind paw withdrawal threshold(WT) to mechanical stimuli and withdrawal latency(WL) to radiant heat were determined before surgery and from day 1 to 7 following CCI.4 days after administration,the spinal cord around lumbar enlargement was removed.Spinal microglial activation was evaluated with OX-42 immunoreactivity and spinal NF-κBp-p65,TNFαexpression were determined by immunohistochemisty,spinal CX3CR1,p-p38MAPK expression were assessed by western blotting,spinal CX3CR1 mRNA expression was assessed by reverse transcriptase polymerase chain reation(RT-PCR),and spinal cord sections were also stained with hematoxylin and eosin(HE).2.BV-2 microglial cells were cultured in vitro.For experiments,cells were treated with TNFα(20ng/ml) as the experimental group,TNFα(20ng/ml) and PDTC(100μmol/l) as TNFα+PDTC group,PDTC(100μmol/l) as PDTC group,and serum-free medium as the control group.At the time point of 10min,20min,40min, 60min,2h,3h,4h,6h,8h after treatment,we used MTT,the inverted microscope,RT-PCR,immunocytochemistry and western blotting to assess cell viability,morphology changes,NF-κBp-p65 protein expression,CX3CR1 protein and mRNA expression.3.252 male Sprague-Dawley(SD) rats(250～300 g) fitted with intrathecal catheters, underwent surgery.Randomly,rats were divided into 7 groups(n=36): normal control group:rats didn't undergo any surgery;sham group:rats underwent surgery of exposing sciatic nerve without ligation;sham+Res group:rats with sham surgery were intrathecally administrated Res (500ug/d);CCI group:rats underwent sciatic CCI surgery;CCI+Res group:Rats were divided into 2 subgroups and intrathecal Res(500ug/d) was infused 1 day before and 3 days after sciatic CCI respectively; sham+saline group:Intrathecal normal saline(NS) was infused 1 day before and 3 days after sham surgery,respectively;CCI+saline group: Rats were divided into 2 subgroups and intrathecal NS was infused 1 day before and 3 days after sciatic CCI respectively.5 days after intrathecal catheters implantation,rats were administrated various intrathecal medicine.And 1 day after administration,rats experiented sciatic CCI and sham surgery according to groups arrangement.The rat hind paw withdrawal threshold to mechanical stimuli and withdrawal latency to radiant heat were determined before surgery and from day 1 to 7 following CCI.4 days after administration,the spinal cord around lumbar enlargement was removed.Spinal microglial activation was evaluated with OX-42 immunoreactivity and spinal NF-κBp-p65,TNFαexpression were determined by immunohistochemisty,spinal CX3CR1 expression was assessed by western blotting and spinal cord sections were also stained with hematoxylin and eosin.Results 1.Compared with sham group,a significant decrease in WT and WL was observed after surgery in CCI group,and the lowest point was 3 days after surgery(P＜0.05), spinal CX3CR1,p-p38MAPK,p-p65,OX42,TNFαexpression were significantly increased(P＜0.05).The WTs and WLs of PDTC+CCI group were significantly increased compared with CCI group,spinal CX3CR1,p-p38MAPK,p-p65,OX42,TNFαexpression were significantly decreased(P＜0.05).HE staining didn't show obvious spinal histopathologic change in sham+Res group compard with sham+NS group.2.TNFα-stimulated BV-2 cells showed a thicker process,and a significant increase of NF-κBp-p65 expression in cell nuclear within 20 min(P＜0.05),peaked at 1 h.TNFα-induced CX3CR1 protein and mRNA expression increased significantly,peaked at 4h and 2h after treatment, and PDTC(100μmol/l) suppressed TNFα-induced NF-κBp-p65 expression,significantly downregulated TNFα-induced CX3CR1 protein and mRNA expression.3.Compared with sham group,a significant decrease in WT and WL was observed after surgery in CCI group,and the lowest point was 3 days after surgery(P＜0.05),spinal CX3CR1,p-p65,OX42,TNFαexpression were significantly increased(P＜0.05).The WTs and WLs of Res+CCI group were significantly increased compared with CCI group,spinal CX3CR1,p-p65,OX42,TNFαexpression were significantly decreased(P＜0.05).HE staining didn't show obvious spinal histopathologic change in sham+Res group compard with sham+NS group.Conclusions:1.Intrathecal PDTC attenuated the CCI-induced neuropathic pain in rats,and suppressed spinal CX3CR1,p-p38MAPK,p-p65,TNFαexpression and microglial activation.And the activation of NF-κB pathway may contribute to spinal CX3CR1,p-p38MAPK,TNFαupregulation and microglial activation in neuropathic pain.2.TNFαrapidly activated the NF-κB pathway,changed the morphology of cells and upregulated CX3CR1 protein and mRNA expression in BV-2 microglial cells.And the morphological changes and upregulation of CX3CR1 expression may be mediated by NF-κB pathway.3.Inthathecal Res attenuated the CCI-induced neuropathic pain in rats,and suppressed spinal CX3CR1,p-p65,TNFαexpression and microglial activation.The regulation of spinal neuroinflammation may contribute to therapeutic potential of Res in neuropathic pain.
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