Objective: To investigate the spinal analgesic mechanism of minocycline in formalin-induced inflammatory pain. Methods: Behavioral test: Male Sprague-Dawley rats(3-5-week old) were randomly assigned into four groups: control, model, vehicle-controlled and minocycline group. Ten percent neutral formalin was injected subcutaneously into the right hind paw dorsum of the rats in model, vehicle-controlled and minocycline group. Normal saline was injected subcutaneously into the right hind paw dorsum of the rats in control group. Before 1 h of formalin injection, the rats in vehicle-controlled and minocycline group received intraperitoneal injection of saline and minocycline, respectively. Licking and lifting time was observed as the behavior results of inflammatory pain. Electrophysiologic experiment: In vitro spinal cord parasagittal slices were prepared from the same rats as above. The effect of minocycline on spontaneous inhibitory postsynaptic currents(sIPSCs) of substantia gelatinosa(SG) neurons was observed using whole-cell patch-clamp technique. Results: Compared with the control group, the licking and lifting time in the model group was significantly increased. Compared with the vehicle-controlled group, the licking and lifting time in the minocycline group was significantly decreased. Minocycline significantly increased the frequency(t=9.32, P<0.05)but not the amplitude(t=1.54, P>0.05) of sIPSCs of SG neurons, the frequency of sIPSCs of control and minocycline group were (2.5±0.3)Hz and (5.2±0.6)Hz, respectively. When calcium was removed from the extracellular solution, the frequency before and after minocycline perfusion were (0.9±0.1)Hz and (0.9±0.1)Hz, respectively, the amplitude before and after minocycline perfusion were (18.2±0.7)pA and (18.5± 0.6)pA, respectively, the difference of frequency or amplitude was not statistically significant(t=0.32, 0.82, all P>0.05). However, minocycline still increased the frequency of sIPSCs when glutamate receptor antagonists 6-Cyano-7-nitroquinoxaline-2, 3-dione(CNQX) and D-(-)-2-Amino-5-phosphonopentanoic acid(APV) were included in extracellular solution(t=13.51, P<0.05), the frequency of sIPSCs were (2.0±0.1)Hz and (4.3±0.4)Hz, respectively. Minocycline still increased the frequency of IPSCs when voltage-gated sodium channel blocker tetrodotoxin(TTX) were included in extracellular solution(t=8.67, P<0.05), the frequency of IPSCs were (2.2±0.2)Hz and (5.2±0.5)Hz. Conclusion: Minocycline can attenuate formalin-induced inflammatory pain which may be associated with its increase in the inhibitory synaptic transmission of SG neurons.
目的： 研究米诺环素缓解大鼠甲醛溶液炎性痛的脊髓机制。 方法： 行为学实验：3～5周龄雄性SD大鼠随机分为4组：对照组、模型组、溶媒对照组及米诺环素组。模型组、溶媒对照组及米诺环素组大鼠于右后足背皮下注射10%中性甲醛溶液，对照组大鼠右后足背皮下注射生理盐水，其中溶媒对照组和米诺环素组在甲醛溶液注射前1 h分别腹腔注射生理盐水和米诺环素，观察各组大鼠缩足和舔爪等炎性痛行为。电生理实验：选取同上SD大鼠，制作离体脊髓纵切片。采用全细胞膜片钳技术记录米诺环素对脊髓背角胶状质(SG)神经元的自发性抑制性突触后电流(sIPSCs)的作用。 结果： 与对照组相比，模型组缩足和舔爪时间显著增加；与溶媒对照组比较，米诺环素组缩足和舔爪时间显著减少。对照组和米诺环素组sIPSCs的频率分别为(2.5±0.3)Hz和(5.2±0.6)Hz，米诺环素可显著增加SG神经元sIPSCs的频率(t＝9.32, P<0.05)，而对其幅度无明显影响(t＝1.54, P>0.05)。去除细胞外液中的钙离子后，米诺环素用药前后sIPSCs的频率分别为(0.9± 0.1)Hz与(0.9± 0.1)Hz，振幅分别为(18.2±0.7)pA与(18.5± 0.6)pA，差异均无统计学意义(t＝0.32、0.82，均P>0.05)。在细胞外液中加入谷氨酸受体阻滞剂6-氰基-7-硝基喹喔啉-2，3-二酮(CNQX)和D-α-氨基磷酸基戊酸(APV)后，米诺环素仍可增加sIPSCs的频率，分别为(2.0±0.1)Hz与(4.3±0.4)Hz，差异有统计学意义(t＝13.51, P<0.05)。在细胞外液中加入电压门控钠通道阻滞剂河豚毒素(TTX)后，米诺环素仍可增加IPSCs的频率，分别为(2.2±0.2)Hz与(5.2±0.5)Hz，差异有统计学意义(t＝8.67, P<0.05)。 结论： 米诺环素可缓解甲醛溶液诱导的炎性痛，这一效应与其增加脊髓背角SG神经元的抑制性突触传递有关。.
Keywords: Inhibitory postsynaptic currents; Minocycline; Neurons; Pain measurement; Patch-clamp techniques.