| Objective: Immunohistochemistry staining and quantitative analysis of stereological morphometry have been used in this research, to observe the changes of mechanical withdrawal threshold(MWT) and the proportion of GABA-immunoreactive neurons in spinal dorsal horn(SDH) after SNI(spared nerve injury) in rats, as well as the maintenance of those changes.Methods: Thirty-seven healthy male SD rats, weighing 286.12( ± 14.02) g, were randomly divided into two groups, sham-operated group(control group, n = 18) and SNI group(experimental group, n = 19). Then each group was divided into 7-day, 14-day and 28-day group by the time of modeling, with 6 rats in each subgroup(7 rats in SNI 28-day group). The left sciatic nerve trunk and three branches were exposed, two of which, known as tibial and the peroneal nerve, were ligated and cut off. And the sural nerve was preserved to build the SNI model. The left sciatic nerve trunk and three branches were only exposed. And the muscle and skin were sutured in sham-operated group. On 1st day before surgery, 3rd, 7th, 14 th and 28 th day after surgery, medial and lateral MWTs of left hind paw in rats were tested. On 14 th and 28 th day after surgery, rats in the two groups underwent cardiac perfusion of 4% paraformaldehyde, then the L5 spinal segments were dissected to prepare paraffin sections. GABA-immunoreactive neurons were labeled by immunohistochemical staining of GABA neurotransmitters in cytoplasm. The quantitative analysis of stereological morphometry were used to describe the proportions of GABA-immunoreactive neurons in both the operated and non-operated side of SDH. Values were expressed as means ± SD. Statistical comparison was made using T-test and one-way ANOVA with SPSS 18.0 software. P < 0.05 was considered statistically significant.Results: Firstly, the valgus deformity and contracture of left hind paw were noticed in SNI group rats. The spontaneous flinching reactions were observed in about 95 % rats without any stimulus situation, among which autophagic response occurred in a rat of 14-day group. No changes of shape and gait in left hind paw were observed in sham-operated group. Secondly, there is an obvious decrease of Von Frey MWTs on both lateral side(sural nerve area) and the medial side(saphenous nerve area) of the left hind paw in SNI group rats. Compared with the baseline on 1st day before surgery, the medial Von Frey MWTs on 1st, 7th, 14 th and 28 th day after SNI decreased obviously in SNI group(P < 0.05). The lateral MWTs on 14 th and 28 th day after surgery significantly decreased, compared with the baseline of SNI group(P <0.01). The MWTs on lateral side of the left hind paw dropped to a more significant level compared with those on medial side in rats(P < 0.05). The MWTs of left hind paw on both two sides decreased significantly compared with sham-operated group on 7th, 14 th and 28 th day after SNI(P < 0.05). There were no statistically significant differences of MWTs on both lateral side and the medial side of left hind paw between postoperative values and preoperative values in sham-operated group(P > 0.05). Thirdly, there weren’t statistically significant reductions in the proportions of GABAimmunoreactive neurons of left SDH on 7th and 28 th day after SNI, compared with right SDH(P > 0.05). The proportion of GABA-immunoreactive neurons in left SDH significantly decreased, compared with that in right side on 14 th day after SNI(Left side: 55.27±6.80 %, Right side: 64.68±9.77 %, P < 0.05). On the same way, the proportions of GABA-immunoreactive neurons on 7th, 14 th and 28 th day after surgery were not statistically different(P > 0.05) in sham-operated group. The proportions of GABA-immunoreactive neurons in left SDH decreased comparatively on 7th and 28 th day after SNI with those in sham-operated group, but the difference was not statistically significant(P > 0.05), while the proportion of GABA-immunoreactive neurons on 14 th day after SNI decreased significantly, compared with that of sham-operated group(SNI group: 55.27±6.80 %, Sham-operated group: 66.59±3.97 %, P < 0.01).Conclusion: First, SNI could cause the mechanical hyperalgesia of hind paw on nerve-injured side in rats, and the extent of lateral side was more serious than that of medial side. Second, SNI model could lead to the decrease of the proportions of GABA-immunoreactive neurons in SDH. And this change did not last long. It might have something to do with the transformation of neurons. |
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