老年Fischer 344大鼠耳蜗损伤外毛细胞的定量观察

Quantitative analysis of damaging hair cells in cochleae of aging Fischer 344 rats

目的通过定量观察老年Fischer344大鼠耳蜗损伤的外毛细胞,揭示年龄相关的大鼠耳蜗基底膜外毛细胞损伤的发展趋势和外毛细胞死亡的主要途径。方法 32只Fischer344大鼠分为两大组:青年组(13只,3～4月龄)和老年组(19只,20～27月龄),再将老年组大鼠进一步分为20～23月龄组(12只)和24～27月龄组(7只)。应用电位反应测听仪检测不同频率短纯音(5、10、20和40kHz)诱发的青年和老年大鼠双侧听性脑干诱发电位反应阈值(ABR)。听觉功能测试后,将动物断头,解剖取出双耳听泡,固定耳蜗组织,分离耳蜗基底膜。分别用细胞核DNA染料碘化丙锭(PI)和原位末端标记(TUNEL)染色不同月龄大鼠耳蜗基底膜细胞核,鉴别凋亡、坏死的毛细胞。异硫氰酸荧光素标记的鬼笔环肽对存在于毛细胞表皮板和纤毛的丝状肌动蛋白进行染色,确认缺失的耳蜗毛细胞。荧光显微镜下自耳蜗顶部到底部计数损伤(核缺失、核固缩和核肿胀)的外毛细胞,绘制耳蜗图。结果老年Fischer344大鼠的不同频率ABR阈值均高于青年组(P<0.05)。明显的TUNEL染色阳性反应不仅出现于不同程度固缩的外毛细胞核,而且见于细胞核型不变、PI染色略有加深的老年大鼠耳蜗外毛细胞核。定量观察发现,老年大鼠耳蜗外毛细胞损伤率(核缺失、核固缩和核肿胀)平均为32.75%±11.80%,其中24～27月龄组耳蜗外毛细胞损伤率(37.85%±9.00%)明显高于20～23月龄组(23.75%±7.65%,P=0.012)。与20～23月龄组大鼠比较,24～27月龄组耳蜗底回外毛细胞损伤增加率(32.20%±11.78%)明显高于顶回(12.80%±11.41%,P=0.022)。20～27月龄大鼠耳蜗基底膜核固缩外毛细胞数目(16.74±7.16)明显多于核肿胀外毛细胞(2.31±3.20,P=0.0001)。结论老年大鼠凋亡外毛细胞DNA特征性变化早于细胞核形态学的改变,耳蜗基底膜底回为老年耳蜗退行性变发展的主要部位,凋亡为老年大鼠耳蜗外毛细胞死亡的主要途径。

Objective To make a quantitative analysis of damaging hair cells,and investigate the progression pattern of cochlear pathology and the primary death pathway of hair cells in cochleae of aging Fischer 344 rats.Methods Thirty-two Fischer 344 rats were used in this experiment.The animals were assigned to two groups：rats in young group （n=13） were 3-4 months old,and in aging group （n=19） were 20-27 months old.Rats in aging group were further divided into two subgroups based on the animals＇ age：20-23-month subgroup （n=13） and 24-27-month subgroup （n=7）.The auditory brainstem response （ABR） thresholds of both ears elicited with tone bursts at 5,10,20 and 40 kHz were measured in both young,and aging Fischer 344 rats.Upon completion of the auditory test,animals were decapitated,and both left and right bullae were exposed.Following fixation,whole specimens comprising the basilar membrane with Corti organ were separated from the modiolus.Propidium iodide （PI）,a popular DNA intercalating fluorescent probe was used to trace the morphological changes in hair cell nuclei,and a terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay was used to detect nuclear DNA fragmentation in the aging cochleae.Filamentous actin （F-actin）,an important structural protein of outer hair cells （OHCs）,was stained with FITC-labeled phalloidin to illustrate the morphologic viability of cuticular plate and the stereocilia for affirming the missed OHCs.Each Corti organ was thoroughly inspected from the apical to the basal turns of the cochlea under a fluorescence microscope.The numbers of damaging OHCs including missed nuclei,condensed nuclei and swollen nuclei were documented,and a cochleogram was drawn.Results There were significant thresholds at all tested frequencies between the young and aging rats （P0.05）.A strong TUNEL fluorescence exhibited not only in condensed nuclei of OHCs,but also in certain OHCs of aging rats with a relatively normal nuclear morphology and slightly increased PI staining.The average percentage of damaging OHCs （the sum of the missed OHCs and the OHCs with condensed and swollen nuclei） in aging rats was 32.75%±11.80%,and in 24-27-month-old rats was 37.85%±9.00%,significantly greater than in 20-23-month-old rats （23.75%±7.65%,P=0.012）.From 20-23 months to 24-27 months,the basal lesion （an increase of 32.20%±11.78%） grew faster than did the apical lesion （an increase of 12.80%±11.41%,P=0.022） of the cochleae.The average number of condensed OHC nuclei was significantly greater than that of swollen OHC nuclei in 20-27-month-old rats （16.74±7.16 vs 2.31±3.2,P=0.0001）.Conclusions During the process of HC degeneration,the onset of DNA fragmentation precedes the onset of nuclear condensation.The basal end of the cochleae exhibits a great expansion of the OHC lesion with the increase in age.The action mode of age-related pathogenesis of OHC lesion proceeds primarily through apoptosis in the cochleae of Fischer 344 rats.