不同冻融条件对同种异体面神经许旺细胞活性的影响

Effects of different freeze-thaw condition on Schwann cell viability of the facial nerve in allotransplantation of rats

目的探索一种可简单准确地调控离体面神经许旺细胞(SchwannCells,SC)活性的方法并观察含不同活性SC的面神经在同种异体移植中的效果。方法(1)将动物分为新鲜神经组(F组)、冻融一次组(FTⅠ组)和冻融五次组(FTⅤ组),定量测定冻融法对面神经SC活性的影响,每组3条面神经用钙黄绿素-乙酰羟甲基酯(Calcein-AM)染色,以共聚焦显微镜测定荧光强度,反映SC活性。(2)每组9只大鼠接受单侧同种异体面神经移植,术后2、4、8周各取3只大鼠的面神经移植物中段行髓鞘锇酸染色,观察轴突再生情况。结果平均荧光强度:F组为140.93±17.55/μm2,FTⅠ组56.99±7.10/μm2,FTⅤ组28.46±4.11/μm2;术后第8周F组、FTⅠ组和FTⅤ组中段的轴突计数分别为225±41、357±76和303±51;术后第8周,FTⅠ组神经新生轴突分布较FTⅤ组和F组更为均匀,髓鞘厚度和轴突横截面积更大,髓鞘更为成熟。结论快速冻融法可以有效和准确地调控面神经SC的活性,冻融一次可以将神经SC活性降低到新鲜面神经的40%,而反复冻融五次可降低到20%;调控SC的数目可能影响同种异体面神经移植的效果。

Objectives To explore a simple and precise method to modulate viability of Schwann cells （SCs） of the facial nerve and evaluate regeneration of facial nerves with different SC viability which were allotransplanted to facial nerve-resected Sprague Dawley（SD） rats. Methods The effects of freeze-thaw method on SC viability of the facial nerve were measured. The allotransplanted SD rats were assigned to fresh nerve group（Group F）, one freeze-thaw cycle group（Group FTⅠ） and five freeze-thaw cycles group（Group FTⅤ）. Three nerves in each group were stained with Calcein-AM and the calcein fluorescence intensity was measured with a confocal microscope to reflect the viability of SC. Nine SD rats in each group received unilateral facial nerve allotransplantation. The middle grafts of 3 rats in each group were stained with osmic acid for histological observation and the regenerated axons were counted at 2, 4 and 8 weeks after transplantation. Results The mean fluorescent intensities were 140.93 ± 17.55/μm^2, 56.99 ± 7.10/μm^2 and 28.46 ± 4.11/μm^2 in Group F, Group FTⅠand Group FTⅤ, repectively. The axons counts of Group F, Group FTⅠ and Group FTⅤ at the 8th week after transplantation were 225 ± 41, 357 ± 76 and 303 ± 51, respectively. The distribution of regenerative axons in Group FTⅠ at the 8th week after transplantation was more even than that of Group FTⅤ and Group F, and the thickness of myelin and cross-section area of axons were larger and myelin was more mature in Group FTⅠas compared to Group FTⅤ and Group F. Conclusion The viability of SC can be effectively and precisely modulated by fast freeze-thaw method. It can be decreased to 40% of the fresh nerve by one freeze-thaw cycle, and even to 20% by five freeze-thaw cycles. It is suggested that modulating viability of SC in a certain range may be beneficial for facial nerve regeneration.