Experimental Study Of NF-κB And Its Downstream Related Factors Expressions In Spinal Dorsal Horn In A Rat Model Of Neuropathic Pain

Objective:To investigate the expressions of NF-κB, TNF-a and iNOS in lumbar spinal cord in a rat model of chronic constrictive injury (CCI).Methods:A total of 104 male SD rats weighing 220-280 g were randomly divided into 2 groups (n=52 each):the CCI group which received the chronic constriction injury by placing 4 ligatures on the right sciatic nerve at lmm intervals with 4-0 chromic catgut and the sham group which received the sham operation as control. The mechanical and thermal nociceptive thresholds were assessed with paw withdrawal latency (PWL) to von Frey filaments and radiant heat at different time points. Eight animals were killed at each time point for Real time polymerase chain reaction (Real time-PCR) (n=4) and Western blot (n=4), another eight animals were killed at 7d after operation for double-immunofluorescence histochemical staining. Lumbar segments of spinal cord were removed. The protein expressions of NF-κB in spinal cord were examined by Western blot. The expressions of NF-κB, TNF-a and iNOS were examined by Real time-PCR and double-immunofluorescence histochemical technique.Results:1. Behavioral studies:CCI, and not sham surgery, produced significant mechanical allodynia and thermal hyperalgesia. The thermal threshold and the mechanical threshold both began to decrease at 1d after the operation(P<0.05), peaked at 4d and 7d separately (P<0.01), then lasted throughout the experiment (P<0.05)2. Western blot of NF-κB protein expression:CCI induced a significant upregulation of NF-κB in the spinal cord ipsilateral but not contraleteral to CCI, which occurred at 4-7 days after CCI. There was no significant difference in NF-κB expression between the contralateral side of CCI rats and the ipsilateral side of sham-operated rats.3.Real-time PCR examined NF-κB,TNF-α,iNOS mRNA expressions:The expression of NF-κB and TNF-a mRNA began to increase at 4 day (P<0.05) after operation and reached a peak at 7 day (P<0.01), then TNF-a began to decrease, while NF-κB maintained at a high level throughout the experiment (P<0.05). The expressions of NF-κB and TNF-a mRNA increased significantly in the ipsilateral spinal cord, compared with the contralateral side (P<0.05) and the sham group (P< 0.05), while no such alterations on iNOS expression could be seen in the ipsilateral spinal cord.4.Double-immunofluorescence histochemical examined NF-κB,TNF-α,iNOS protein expressions:At 7 day after operation, within the superficial laminae of ipsilateral spinal cord dorsal horn, there was a pattern of colocalization between the expressions of NF-κB and TNF-a, a majority of NF-κB and TNF-a positive cells could be seen, compared with sham-operatived spinal cord dorsal horn, while no iNOS positive cells could be seen in the superficial laminae of ipsilateral spinal cord dorsal horn in both CCI group and sham-operated group.Conclusions:The activation of NF-κB may be involved in the modulation of neuropathic pain arised from chronic constriction injury to the sciatic nerve. NF-κB activation may be upregulate TNF-a expression. But the activation of iNOS was not observed in the dorsal horn of the CCI rats in our studies.