Expression Of BRD4 In Spinal Cord Of Acute Inflammatory Pain Induced By Formalin In Mice

Objective:To detect the cellular localization and expression of BRD4 in the spinal dorsal horn of mice,and to investigate its role in acute inflammatory pain by establishing the formalin-induced pain model.Methods:Thirty-six mice were randomly divided into three groups:control group,formalin group and indomethacin plus formalin group.25μl1% formalin was injected into the right plantar to establish the acute pain model,and the control group was treated with the same amount of saline,while the indomethacin(20mg/kg)was injected intraperitoneally1 hour before formalin injection.All mice were recored for 1h by video to observe the spontous pain.Then,cell localization of BRD4 in the spinal cord of Normal mice was determined by immunofluorescence assasy.The expression of BRD4 in spinal cord was tested through immunohistochemistry(IHC)and Western blot.Results:(1)The expression and cell localization of BRD4 in spinal cord of mice.The results of immunohistochemical method showed that BRD4 positive signal was widely distributed in various regions of the spinal cord of the mice,mainly concentrated in the shallow layer of the posterior horn,and the expressions on the left and right sides were approximately the same.The co-expression of BRD4 with the neuron marker Neu N,the astrocyte marker GFAP,and the microglia cell marker Iba-1 was detected by immunofluorescence double-labeling.The results showed that BRD4 was mainly distributed and co-localized with neuron marker Neu N in the superficial layers of the mice spinal cord,but was less co-localized with the astrocyte marker GFAP and the microglia marker Iba-1.The results suggesting that BRD4 is located in neurons in the superficial layers of the mice spinal cord.(2)The expression changes of BRD4 in the spinal cord of formalin pain model mice.Behavioral results showed that compared with normal control group,the mice in the formalin group showed obvious biphasic pain behavior,and the first phase pain appeared in 0-5 minutes after injection and the second phase pain appeared in 10-40 minutes.After the treatment of Indomethacin,the formalin-induced secondary pain was reduced.Immunohistochemistry showed that the expression of BRD4 in the superficial layers of the spinal cord was significantly upregulated in the formalin group comparing with normal control group(P < 0.05).And the expression of BRD4 protein in Formalin + indomethacin group was significantly lower than the formalin group in the bilateral spinal dorsal horn of the mice(P < 0.05).Western blot showed that the expression of BRD4 protein of bilateral spinal cord was significantly upregulated in the formalin group comparing with normal control group(P < 0.05).And the expression of BRD4 in Formalin + indomethacin group was significantly lower than the formalin group in the bilateral spinal horn of the mice(P <0.05).Conclusion:The expression of BRD4 may be involved in the regulation of acute inflammatory pain in the superficial layer of spinal cord in mice.