| Objective:To observe the effects of botulinum toxin A (BoNT-A) on pain behaviors and calcitonin gene related peptide (CGRP) expression in the spinal motor neurons by L5, L6 spinal nerve ligation model (SNL model) in rats.Methods:The left L5, L6 spinal nerve ligation models (SNL model) were established by Wistar rats and were then randomly allocated into observation group and control group (n= 8). The sham-operated group was set by the following method:left L5, L6 spinal nerves were only exposed without ligation. After all the SNL models were finished, BoNT-A 30 U/kg was subcutaneously injected into the ipsilateral plantar metapede in the observation group, while the subcutaneous injection of the same volume of saline was offered for the rats in the control group. All the rats were tested for mechanical withdrawal threshold (MWT, in units of g) and thermal withdrawal latency (TWL, in unit of s) at three days preoperatively and 1,3,5,7,14,21 d after administration. Four rats were randomly selected for immunohistochemical staining from each group. After total anesthetization,120 ml 4℃ saline and 200 ml 4% paraformaldehyde (pH 7.4) were injected through the ascending aorta. The L4-6 spinal marrow cord was then exposed and cut out. The CGRP positive expression in motor neurons was tested and counted by immunohistochemical staining and image analysis technique after a several steps of L4-6 spinal marrow cord.Results:There was no accident death of rats and the wound healed in primary time without infection. Compared with the preoperative baseline, there were no significant differences of MWT and TWL of contralateral leg in the rats from sham-operated group. The MWT and TWL of contralateral leg in the rats from both observation and control group decreased because of the ligation of nerve, however, they maintained at the similar level without significant difference after injection of BoNT-A or saline through the study. On the contrary, MWT increased significantly of ipsilateral leg in the observation group from (4.2±1.5) preoperatively to (13.8±4.8) at 5 days after injection of BoNT-A and decreased to similar level of first day postoperatively. Similar change happened of TWL from (13.9±0.3) to (22.1±2.0) and decreased latter to the level of first day postoperatively. The results of immunohistochemical staining and image analysis showed that the CGRP positive expression located in the spinal motor neurons with light yellow color. It was darker in the control group compared with sham-operated group and lighter in the observation group. There was no significant change of CGRP positive expression in the sham-operated group. It increased form 154.7±2.9 to 178.2±3.6 at the second week postoperatively and decreased to 162.1 ±2.9 at forth week postoperatively in the control group. CGRP positive expression of observation group decreased form 152.3±3.0 to 120.4±3.1 at the second week postoperatively and increased to 131.6±3.4 at forth week postoperatively.Conclusions:Plantar subcutaneous injection of BoNT-A can show analgesic effect with improving MWT and TWL in SNL rat model of ipsilateral limb. The mechanism may be the suppression effect of the CGRP in the spinal motor neurons. |
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