频谱照射对受损视网膜节细胞的早期保护作用

Early protective effect of frequency spectrum treatment on injured retinal ganglion cells

目的:观察频谱照射对受损中枢神经元自我保护及修复机制的激发和增强作用,使受损的神经元在早期启动自我保护及修复机能,为进一步促进受损中枢神经元存活和纤维再生创造有利的条件。方法:实验于2004-09/2005-03在北京大学医学部神经解剖实验室完成。选取成年雄性SD大鼠36只,实验中动物随机分成2组:①正常对照组(n=4):动物不造模,取正常视网膜。②实验组(n=32):再随机分成2组,手术损伤组,即实施视神经轴索切断术(n=16);照射治疗组(n=16),建立中枢神经损伤模型(视神经轴索切断术),外加频谱治疗仪照射(术后30min,将实验动物置于周林频谱下,保持照射源与动物之间的距离在30cm,照射30min,连续照射3d)。于实验开始后的7,14,21,28d,取视网膜,制作冰冻切片,Nissl染色观察视网膜节细胞形态和存活数量的变化,用免疫组织化学染色的方法观察节细胞层中热休克蛋白27的表达程度。结果:实验组32只SD大鼠进入结果分析。①视网膜节细胞层Nissl染色的形态学改变:与手术损伤组相比,照射治疗组细胞排列的较整齐,发生中晚期溃变的细胞比例较小。②视网膜节细胞计数:照射治疗组各时间点细胞计数均高于手术损伤组[7d:(600±25.40,480±22.65);14d:(468±12.73,324±12.19);21d:(236±9.15,192±8.23);28d:(120±9.57,104±5.62),P<0.01]。③热休克蛋白27在视网膜节细胞层的分布:各时间点照射治疗组热休克蛋白27的表达(用平均吸光度表示)均强于手术损伤组[7d:(127.179±8.127,93.799±4.80);14d:(117.291±9.575,80.157±2.904);21d:(100.589±8.275,70.847±5.052);28d:(102.078±31.283,63.669±2.523),P<0.01],并且能观察到清晰的树突野。热休克蛋白27在节细胞层分布的变化趋势与视网膜节细胞存活率的变化趋势近似。结论:形态学结果表明频谱照射可使具有保护作用的热休克蛋白27表达上调,受损细胞存活数量增加,激发并增强了受损中枢神经的自我保护及修复作用,将为进一步治疗做准备。

AIM： To observe the effect of frequency spectrum treatment on the provocation and enhancement of self-protection and self：repair of injured central neuron, which promote the early initiation of self-protective and self-repaired mechanism of injured central neuron, in order to create beneficial condition to the survival of injured central neuron and fiber regeneration. METHODS： The experiment was carried out in the Neuroanatomical Laboratory of Peking University Health Science Center between September 2004 and March 2005. A total of 36 male SD rats were randomly divided into two groups： ①normal control group （n=4） to remove normal retinas without modeling; ②experimental group （n=32）： This group was randomly subdivided into surgery group （n=16） with axonal amputation of optic nerve and irradiation group （n=16） with the spectrum treatment after axonal amputation of the optic nerve （the experimental animals were posed under the Zhoulin spetrum equipment for 30 minutes of irradiation with a distance of 30 em continuously for 3 days at the 30 minutes after the amputation）. On the 7^th, 14^th, 21^st and 28^th days after experiment, the retinas were taken to prepare the frozen section, and the changes of morphous and survival number of the retinal ganglion cells （RGCs） were observed and measured with Nissl staining. The expression of heat shock protein 27 （HSP27） in the ganglion cell layers was observed with the immunohistechemical staining method. RESULTS： All the 32 rats in the experimental group were involved in the result analysis, ①Morphological change of the RGC with Nissl staining： Compared with the surgery group, the cells in the irradiation group were arranged in order and had a lower proportion of occurrence of the intermediate and late degeneration. ②Count of the RGCs with Nissl staining： The number in the irradiation group was more than that in the surgery group at every time point [the 7^th day：（600±25.40, 480±22.65） ;the 14^th day：（468±12.73, 324±12.19）;the 21^st day：（236±9.15, 192 ±8.23）;the 28^th day： （120±9.57, 104±5.62）, P 〈 0.01]. ③The expression of HSP27 in the retinal ganglion cells： At every time point, it was stronger in the irradiation group than in the surgery group [A value： the 7^th day： （127.179±8.127, 93.799±4.800）;the 14^th day：（117.291±9.575, 80.157±2.904） ;the 21^st day： （ 100.589 ±8.275, 70.847 ±5.0520） ; the 28^th day： （ 102.078 ±31.283, 63.669±2.523）, P 〈 0.01], and the dendritic field could be observed clearly. The change tendency of HSP27 distributed in the ganglion cell layers was similar to the change tendency of the survival rate of the RGCs. CONCLUSION： The morphological results indicate that frequency spectrum treatment can increase the expression of HSP27 and the survival number of injured cells, so as to arouse the self-protective and self-repaired mechanism of the injured central nerve, which are prepared for further treatment.