经瞳孔温热疗法诱导大鼠热休克蛋白60在视盘中的表达

目的探讨经瞳孔温热疗法（TTT）阈下刺激诱导大鼠视盘中热休克蛋白（HSP）60的表达,探讨TTT对视盘组织超微结构的影响。方法采用810nm二极管激光对12周龄的BN大鼠56只右眼视盘进行阈值下TTT照射,选择激光参数为光斑直径0.5mm、持续时间60s、能量60mW;另设8只正常大鼠为对照。实验鼠分别于TTT照射后24h、72h和1周过量麻醉处死,实验眼的视盘组织切片制备后应用免疫组织化学染色法鉴别BN大鼠视盘TTT照射后HSP60的表达;Western blot法对TTT干预后HSP60在视盘组织中的表达进行半定量分析,并以吸光度（A）值表示。透射电镜下观察TTT照射后1周实验眼视盘组织的超微结构变化。结果免疫组织化学染色显示,HSP60在正常大鼠视盘局部呈弱阳性表达,TTT干预后24h、72h及1周HSP60的阳性染色明显增强,72h最强;Western blot蛋白质表达半定量分析显示,HSP60在正常大鼠视盘中的表达量为21458.13±156.32,在TTT干预后24h、72h及1周表达量分别为46907.24±10099.20、61848.02±2714.49、40738.01±5670.12,较正常大鼠视盘中的表达量明显上调,差异均有统计学意义（t=0.002、t=0.000、t=0.001,P〈0.01）,72h表达最强。阈下TTT干预后1周透射电镜下可见视盘神经纤维轴索部分髓鞘板层离散,个别轴索神经微丝、微管有溶解现象,有轻度轴膜下水肿,但神经纤维密度无明显减少。结论 TTT阈下刺激可诱导大鼠视盘中HSP60的表达,并对正常组织的超微结构产生轻度影响。

Expression of HSP60 in optic nerve head following transpupillary thermotherapy

Background The latest study demonstrated that the subthreshold transpupillary thermotherapy（TTT）laser irradiation on optical never head can protect retinal ganglion cells（RGCs）after optic nerve crushing.The underlying mechanism may be related to TTT-induced heat shock protein（HSP）expression in the RGCs.ObjectiveThis study was to investigate the expression of HSP60 in optic nerve head and ultrastructural change of optic nerve head after subthreshold response of TTT.MethodsTTT was performed on the right eyes of 56 BN rats with the diode laser of 810nm.The laser was excited targeting on the center of optic nerve head with a spot size of 0.5mm,power of 60mW and exposure duration of 60 seconds.The experimental rats were sacrificed at 24 hours,72 hours and 1 week after laser irradiation and the eyeballs were enucleated respectively.Immunohistochemical staining and Western blot assay were used for the identification and semiquantitation of HSP60 expression in optic nerve head.1 week after subthreshold response of TTT,ultrastructure of optic nerve head was evaluated under the transmission electron microscopy.The usage of animals followed the Statement of ARVO.ResultsThe immunohistochemistry revealed that HSP60 was weakly expressed in the cytoplasm of optic nerve tissue in normal control rats.However,24 hours,72 hours and 1 week after TTT,HSP60 was positively expressed in the cytoplasm of optic nerve tissue.Western blot assay showed that the expression level of HSP60（A value）in optic nerve tissue was 21458.13±156.32 in normal control rats and 46907.24±10099.20,61848.02±2714.49,40738.01±5670.12 respectively at 24 hours,72 hours and 1week after TTT,indicating a significant enhance in comparison with control rats（t=0.002,t=0.000,t=0.001,P0.01）with the peak value at 72 hours after TTT.Under the transmission electron microscope,redundancy of the myelin sheath and dissociation of the lamellae were found in the axons of the optic nerve,and some of the axons showed lysis of the microfilaments and microtubes.A few axons showed the sub-axolemma hydrops.But no obvious difference was found in the axon density between TTT group and normal control group.ConclusionSubthreshold TTT laser irradiation to the optic nerve head induces the upregulation of HSP60 expression.TTT can cause slight ultrastructure change in optic nerve tissue.