经瞳孔温热疗法对大鼠视神经钳夹视网膜神经节细胞的保护作用

The protective effects of transpupillary thermotherapy on retinal ganglion cells in rat with optic nerve crush

目的探讨经瞳孔温热疗法(TTT)阈下反应对BN大鼠视神经钳夹后视网膜神经节细胞(RGCs)的保护作用。方法采用阈下TTT对BN大鼠视网膜进行照射后3d,通过逆行标记RGCs的方法,对TTT+视神经钳夹组(A组)、TTT+假手术组(B组)、单纯视神经钳夹组(C组)和空白对照组(D组)在视神经钳夹后1、2、4周进行RGCs计数并比较;检测视网膜TTT阈下反应的热休克蛋白70(HSP70)表达;观察TTT阈下反应对视网膜的影响。结果视神经钳夹后4周,A组RGCs数显著高于C组(P=0.006),而1周和2周时2组之间差异无统计学意义(P>0.05);各时间点B组和D组的RGCs数差异无统计学意义(P>0.05)。视网膜经阈下TTT干预后,HSP70表达高于对照眼。阈下TTT照射能引起视网膜组织形态上的改变。结论阈下TTT可显著提高视神经钳夹4周后RGCs的存活数量;其保护机制可能与诱导视网膜内源性HSP70表达、启动内源性保护机制有关。

Objective This study was designed to explore the protective effect of subthreshold transpupillary themotherapy （TTT） on retinal ganglion ceils（RGCs） in the rat with partial optic nerve crush. Methods 810 nm diode laser was used to irradiate the retina surrounding optic nerve（5 spots） for 30 seconds in the left eyes of BN rats. Three days later,the partial optic nerve crush models were created in the rats by clamping the retrobulbar optic nerve for 60 seconds. 102 female BN rats were randomly divided into TTT with optic nerve crush（ TTT ＋ ONC） group, optic nerve crush（ONC） group,TTT with sham operation （TTT） group and normal control group. RGCs were detected by the retrograde label of fluorogold at 1,2,4 weeks after optic nerve crush. HSP70 expression was assessed using immunohistochemistry and Western blot in 12 rats underwent TTT irradiation. The histological examination of rats retina with TTT irradiation was performed under the light and transmission electron microscope. Results The RGCs density in ONC group was（863 ±87） cells/mm^2 at 1 week and decreased to （614±124） at 2 weeks and （424±112 ） cells/mm^2 at 4 weeks after irradiation. In TTT ＋ ONC group, the density of RGCs was （819 ± 93 ） cells/mm^2 at 1 week and decreased to （623±123） at 2 weeks and （615±112） cells/mm^2 at 4 weeks. The RGCs density at 4 weeks in ONC group was significantly lower than in TTT ＋ ONC group （ P = 0. 006）. The expression of HSP70 in the retina underwent TTT irradiation was higher than the fellow eye. The thinning of retina and degranulation of retinal pigment epithelial cells were found under the light microscope, and shrinking of nuclear membrane and pyknosis of chromoplasm were seen in the 7th day after TTT under the transmission electron microscope. Conclusion TTT has a protective effect on RGCs in BN rats with partial optic nerve crush, and TTT can initiate the endogenous protective mechanism by increasing the expression of HSP70 in retina.