The Roles And Mechanisms Of Resolvin D1 In Burn Injury Induced Pain In Rats

Background Resolvins are endogenous lipid mediators generated from the omega-3 polyunsaturated fatty acids, which have potent anti-inflammatory and pro-resolution actions in several animal models of inflammation.Recent findings also demonstrate that resolvin D1 can potently dampen inflammatory pain, postoperative pain and neuropathic pain by adjusting a variety of transient receptor potential channel activity and expression of proinflammatory cytokines and anti-inflammatory mediators in the formation of central sensitization. Spinal cord glial cells, p-p38 MAPK in microglia cells, brain-derived neurotrophic factor(BDNF) and its receptor tropomyosin-related kinase B(Trk B) play an important role in the development of neuropathic pain, and mechanisms like neuropathic pain are important components of burnpain, therefore, they may also be involved in the formation of burn pain; vivo studies indicate Rvs can inhibit microglia and astrocytes activation, inhibit p38 MAPK phosphorylationin microglia, reduce the secretion of inflammatory mediators, so we hypothesized that Rv D1 may attenuate burn pain via inhibiting spinal cord glial activation, the activation of p38 MAPKin microglia and BDNF/Trk B signaling in spinal dorsal horn.Objective In this study, we established burn injury model in rats and injected Rv D1 intraperitoneally in order to explore the roles and mechanisms of Rv D1 in burn injury induced pain in rats. After inhibiting p38 MAPK activation by intrathecal injection of SB203580, we observed the changes of pain behavior in burn injury rats and detected the expression of spinal dorsal horn Iba- 1, BDNF and Trk B, so as to explore the status of p38 MAPK activation in the mechanisms of Rv D1 in burn injury induced pain; After inhibiting BDNF / Trk B signal by intrathecal injection of Trk B-Fc, changes of pain behavior in burn injury rats was observed, and the expression level of the spinal dorsal horn Iba-1 and p-p38 MAPK was detected, so as to explore the status of BDNF / Trk B signal in the mechanism of Rv D1 in burn injury induced pain.Methods 1. Sprague-Dawley rats, weighing 140~150g, were randomly divided into 5 groups: sham group(group Sham + Veh1), burn group(group Burn + Veh1), small-dose Rv D1 treatment group(group Burn + R( S)), large-dose Rv D1 treatment group(group Burn + R(L)), group Sham + R(L). Anesthetized rats received burn injury on dorsal hind paw. Rv D1(100ng or 300 ng in 50 ul PBS) or control solvent Veh1 intraperitoneally, started at 30 minutes before burn injury, was administered for 7 days, twice a day. The rats were tested for mechanical paw withdrawal threshold(MWT) at 1 day before burn and 1?3?5?7 and 14 days postburn. Three rats in each group were sacrificed after measurement of MWT at 7 days postburn for detection of the expression of Iba-1(a specific marker of microglia), GFAP(a specific marker of astroglia),p-p38 MAPK, and BDNF/Trk B at the spinal dorsal horn by immunofluorescence. To examine the cellular populations within the spinal cord that expresses p-p38 MAPK, double labeling with either the microglial marker Iba-1, the astrocytic marker GFAP or the neuronal marker Neu N was performed. 2. Sprague-Dawley rats, weighing 140~150g, were randomly divided into 5 groups: group Sham+Veh2, group Burn+Veh2, group Burn+SB203580, group Sham+SB203580, group Burn+R(L). Anesthetized rats received burn injury on dorsal hind paw. SB203580 or control solvent Veh2 was given by intrathecal injection on day 7 postburn. Rats in group Burn+R(L) was given Rv D1 in the similar way with former. MWT was tested before intrathecal injection and 15 min, 30 min, 1h and 2h after intrathecal injection. Three rats in each group were sacrificed after measurement of MWT at 7 days postburn for detection of the expression of Iba-1 and BDNF/Trk B at the spinal dorsal horn by immunofluorescence. 3. Sprague-Dawley rats, weighing 140~150g, were randomly divided into 5 groups: group Sham+Veh3, group Burn+Veh3, group Burn+Trk B-Fc, group Sham+Trk B-Fc, group Burn+R(L). Anesthetized rats received burn injury on dorsal hind paw. Trk B-Fc or control solvent Veh3 was injected through the subarachnoid catheter. The injection started at 1 h before burn injury and once daily for 7 days after burn injury. Rats in group Burn+R(L) was given Rv D1 in the similar way with former. The rats were tested for MWT at 1 day before burn and 1?3?5?7 and 14 days postburn. Three rats in each group were sacrificed after measurement of MWT at 7 days postburn for detection of the expression of Iba-1 and p-p38 MAPK at the spinal dorsal horn by immunofluorescence.Results 1. Compared with group Sham + Veh1, MWT of group Burn+Veh1 was significantly decreased after burn(P<0.05); Compared with group Burn+Veh1, MWT of group Burn+R(S) and group Burn+R(L) was significantly increased after burn(P<0.05); Compared with group Burn+R(S), MWT of group Burn+R(L) was significantly increased after burn(P<0.05). Compared with group Sham+Veh1, immunofluorescence in group Burn+Veh1 showed the expression of Iba-1?GFAP?p-p38 MAPK?BDNF and Trk B were significantly increased(P<0.05); Compared with group Burn+Veh1, the expression of Iba-1?GFAP?p-p38 MAPK?BDNF and Trk B in group Burn+R(L) were decreased(P<0.05). Double-labeled immunohistochemical analysis showed that p-p38 MAPK was co-localized with Iba-1-positive microglia, but was not co-localized with GFAP-positive astrocytes and Neu N-positive neuron. 2. Compared with group Sham+Veh2, MWT of group Burn+Veh2 was decreased(P<0.05); Compared with group Burn+Veh2, MWT of group Burn+SB203580 was significantly increased(P<0.05); No significant difference was found between MWT of group Burn+R(L) andgroup Burn+SB203580 after intrathecal injection of SB203580(P>0.05). Compared with group Sham+Veh2, immunofluorescence in group Burn+Veh2 and group Burn+SB203580 showed the expression of Iba-1 ? BDNF and Trk B were significantly increased(P<0.05); Compared with group Burn+Veh2, the expression of Iba-1?BDNF and Trk B in group Burn+SB203580 were decreased(P<0.05). 3. Compared with group Sham+Veh3, MWT of group Burn+Veh3 was significantly decreased after burn(P<0.05); Compared with group Burn+Veh3, MWT of group Burn+Trk B-Fc was significantly increased(P<0.05); No significant difference was found between MWT ofgroup Burn+Trk B-Fc and group Burn+R(L)(P>0.05). Compared with group Sham+Veh3, immunofluorescence in group Burn+Veh3 and group Burn+Trk B-Fc showed the expression of Iba-1 and p-p38 MAPK were significantly increased(P<0.05); Compared with group Burn+Veh2, the expression of Iba-1 and p-p38 MAPK in group Burn+SB203580 were decreased(P<0.05).Conclusion Rv D1 may attenuate burn pain via inhibiting spinal cord glial activation, the activation of p38 MAPK in microglia and BDNF/Trk B signaling in spinal dorsal horn.