神经减压缓解糖尿病大鼠触诱发痛的机制研究

目的探讨周围神经减压术缓解糖尿病大鼠触诱发痛的机制。方法将健康成年雄性SD大鼠按随机数字表法分为5组,分别为Ⅰ组(正常对照,n=10)、Ⅱ组(糖尿病模型,n=20)、Ⅲ组(糖尿病模型+乳胶管置入,n=10)、Ⅳ组(糖尿病模型+乳胶管置入+神经减压,n=10)及Ⅴ组(糖尿病模型+乳胶管置入+单纯术区显露,n=10)。糖尿病模型采用链脲佐菌素(STZ)腹腔注射,神经减压法即去除坐骨神经乳胶管。建模后3 d,采用up-down法检测各组大鼠的缩足阈值,保留Ⅱ~Ⅴ组中出现触诱发痛的大鼠。采用透射电镜观察5组大鼠坐骨神经的形态,分别对有髓和无髓神经纤维进行测量。进一步采用蛋白质免疫印迹(WB)和免疫荧光实验对5组大鼠脊髓后角γ-氨基丁酸B型(GABAB)受体的表达进行定量和定位检测。结果术后3周,STZ注射结合乳胶管置入(Ⅲ、Ⅳ、Ⅴ组)较单纯性STZ注射(Ⅱ组)大鼠触诱发痛的发生率高分别为86.7%(26/30)、55.0%(11/20),χ^2=6.254,P=0.012]。Ⅱ、Ⅲ、Ⅳ、Ⅴ组的缩足阈值分别为(4.06±1.28)g、(3.09±1.43)g、(4.02±1.96)g、(4.15±1.87)g]均低于Ⅰ组(13.41±1.88)g,均P<0.05];术后5周,Ⅳ组的缩足阈值高于Ⅱ、Ⅲ、Ⅴ组(均P<0.05)。电镜观察结果显示,Ⅱ、Ⅲ、Ⅳ、Ⅴ组有髓和无髓神经纤维的面积、密度均较Ⅰ组减小(均P<0.05),有髓神经纤维的g比例较Ⅰ组增加(均P<0.05);Ⅳ组有髓纤维的面积和密度均大于Ⅱ、Ⅴ组(均P<0.05)、g比例低于Ⅱ、Ⅴ组(均P<0.05)。WB结果显示,神经减压术后3周,Ⅱ、Ⅲ、Ⅳ、Ⅴ组GABAB受体的表达量均较Ⅰ组下降(均P<0.05),而Ⅳ组高于Ⅴ组(P<0.05)。免疫荧光结果显示,Ⅱ、Ⅲ、Ⅳ、Ⅴ组中GABAB受体在脊髓背角内神经丝蛋白(NF)-200+区和NF-200+神经元的表达均较Ⅰ组下调(均P<0.05)。结论周围神经减压术可缓解糖尿病大鼠触诱发痛,主要通过去除有髓神经纤维的压迫、解除GABAB受体下调介导的中枢敏化,从而恢复脊髓兴奋性升高的病理状态。

Research on the pathological mechanisms of nerve decompression in relieving tactile allodynia in diabetic rats

Objectives To explore the pathological mechanism of peripheral nerve decompression in relieving tactile allodynia in diabetic rats.Methods Healthy adult male Sprague Dawley rats were randomly divided into 5 groups for different interventions:groupⅠ(healthy control,n=10),groupⅡ(diabetes model,n=20),groupⅢ(diabetes model with latex tube placement,n=10),groupⅣ(diabetes model with latex tube placement and nerve decompression,n=10),group V(diabetes model with latex placement and merely operational area exposure,n=10).The diabetes model was induced by intraperitoneal injection of streptozotocin(STZ).Nerve decompression was performed by removal of latex tube encircling the sciatic nerve at 3 weeks post model establishment.The paw withdrawal threshold was tested 3 days after modeling with the use of"up-down"method.Diabetic rats with tactile allodynia in 4 experimental groups(groupsⅡ-Ⅳ)were preserved.Morphometric analysis of myelinated and non-myelinated nerve fibers was performed with the use of projection electron microscope.Western blot and immunofluorescence were used to localized and determined the expression of GABAB receptor protein in spinal dorsal horn.Results Three weeks after operation,the incidence of tactile allodynia in STZ-induced rats55.0%(11/20)]was lower than that in diabetic rats with latex tube placement(groupsⅢ,ⅣandⅤ)86.7%(26/30),χ^2=6.254,P=0.012].The paw withdrawal threshold in groupⅠ(13.41±1.88 g)was higher than those in groupsⅡ-Ⅳ(4.06±1.28 g,3.09±1.43 g,4.02±1.96 g,4.15±1.87 g respectively,P<0.05).At 5 weeks post operation,the paw withdrawal threshold in groupⅣwas higher than those of groupⅡ,ⅢandⅤ(all P<0.05).When compared with groupⅠ,smaller myelinated fibers area and density,as well as higher g-ratio were revealed by electron microscope in each experimental group(all P<0.05).Larger myelinated fiber area and density,as well as lower g-ratio were noted in groupⅣwhen compared with those in groupⅡandⅤ(all P<0.05).The results of western blot showed that lower expression of GABAB receptor was noted in experimental groups(Ⅱ,Ⅲ,ⅣandⅤ)when compared with groupⅠ(all P<0.05),and higher expression of GABAB receptor in both NF-200+areas and neurons of spinal dorsal horn was noted in groupⅣwhen compared with group V(P<0.05)at 3 weeks post nerve decompression.The results of immunofluorescence showed that lower expression of GABAB receptor in both NF-200+areas and neurons of spinal dorsal horn was noted in experimental groups(Ⅱ,Ⅲ,ⅣandⅤ)when compared with groupⅠ(all P<0.05).Conclusions Peripheral nerve decompression can relieve the tactile allodynia of diabetic rats mainly by removing the compression damage of myelinated nerve fibers,lifting the GABAB receptor downregulation-mediated central sensitivity,thereby relieving the pathological state of elevated spinal excitability.