保留后根的新型脊髓旁矢状面切片技术及其在脊髓突触传递研究中的应用

A novel parasagittal spinal slice model with dorsal root attached: application in the study of spinal synaptic transmission

目的:对保留脊神经后根的脊髓切片技术进行改良,增加所保留的脊神经后根纤维投射到脊髓后角浅层的完整性,以提高实验效率。方法:选用4～5周的SD大鼠,应用振动切片机对其脊髓腰骶膨大分别进行横断面或矢状面切片,在全细胞模式记录下,给予后根刺激观察两者中诱发出兴奋性突触后电流(EPSCs)的成功率,并对其进行比较;调整后根刺激参数分别刺激Aβ、Aδ和C纤维以诱发EPSCs,并对不同纤维诱发的EPSCs进行鉴别。结果:在保留后根的横断面和矢状面切片上诱发出EPSCs的成功率分别是38.43±9.97%和86.36±5.32%,具有显著的统计学差异(P<0.0001);与非保留后根的脊髓切片上诱发出的EPSCs相比,应用保留后根的脊髓横断面切片和矢状面切片所诱发的EPSCs均可通过刺激强度和潜伏期的差异,对不同纤维诱发的EPSCs进行有效的区分。结论:保留后根的脊髓矢状面切片刺激后根反应率显著高于横断面切片,且可对不同纤维诱发的EPSCs进行有效区分。因此,保留后根的脊髓矢状面切片比横断面切片更完整的保留了后根到脊髓后角浅层的投射,可提高实验效率,是研究脊髓中枢突触传递及其可塑性的可靠离体模型。

Objective： To improve the sectioning technique for the spinal slice preparation with dorsal root attached, and to retain the integrality of spinal nerve fibers projected to the superficial dorsal horn and to enhance the experimental efficacy. Methods： Transverse or parasagittal slices with a dorsal root attached were cut from the lumbosacral spinal cord of Sprague-Dawley rats （4 ~ 5 weeks old）. Whole-cell recordings were made from superficial dorsal horn neurons. Electric dorsal root stimulation through a suction electrode was used to evoke synaptic responses in the dorsal horn neurons. The success rate for inducing excitatory postsynaptic currents （eEPSCs） was compared in the two preparation groups. EPSCs evoked by AS, A[3 and C-fiber activation were also obtained by differential intensity of stimulation in dorsal root, and were further identified in association with the fiber type. Results： The proportion of neurons that eEPSCs can be elicited is 38.43 ± 9.97% in transverse slices and 86.36 ± 5.32% in parasagittal slices （P 〈 0.0001 ） , showing significant difference （P 〈 0.0001 ）. Compared with EPSCs evoked by local dorsal horn stimulation in the transverse slice without dorsal root, EPSCs evoked by stimulation of different fiber type, in transverse or parasagittal slices with attached dorsal root could be eventually characterized by stimulatily intensity and latency. Conclusion： eEPSCs occurrence rate is significantlyhigher in parasagittal slices than in transverse slices, and the fiber type for evoking EPSCs can be effectively distinguished. Thus, the parasagittal slice with dorsal root attached reserves more dorsal root fibers than transverse slices, and can be used to significantly enhance the experimental efficiency. The parasagittal slice preparation thus represents an ideal in vitro model for the study of spinal synaptic transmission and plasticity .