频闪光对豚鼠眼正视化影响的研究

发布时间：2018-06-07 07:38

本文选题：正视化 + 频闪　；参考：《复旦大学》2013年博士论文

【摘要】：出生后视觉环境的改变是诱发屈光发育的关键原因之一。迄今为止,任何一种人工照明均无法做到对自然光线的完全模拟,人工照明中的频闪对屈光发育的影响如何?本研究旨在探讨频闪光对屈光发育敏感期豚鼠的眼轴和屈光度的影响及其机理。主要方法是通过自动控制技术模拟不同频闪频率、亮度及节律的频闪光,利用频闪光诱导豚鼠近视模型,检测屈光度及眼轴长度改变,并观察离体眼球形状及后极部超微结构改变；通过视觉电生理的变化评估视网膜传导功能；通过分子生物学方法观察豚鼠视网膜视蛋白表达及巩膜胶原代谢的变化。主要结果：持续暴露于人工照明环境下特定频率的频闪光能刺激豚鼠眼球过度发育,并诱导近视性屈光改变；不同频率、亮度及节律的频闪光对豚鼠眼球发育的影响存在差异性；视网膜电图及超微结构改变表明豚鼠眼球视网膜的感光传导功能下降,并出现感光细胞形态异常；频闪组视蛋白表达增加,同时巩膜基质金属蛋白酶MMP-2表达增高。本文结论：频闪光这种异常光环境对豚鼠眼正视化存在影响,视觉感知过程中频闪光参数改变对视觉传导功能的影响、导致过度正视化最终导致近视的作用,及其中的机制值得进一步研究。具体分述如下：第一部分频闪光频率、亮度及节律对豚鼠眼屈光发育的影响目的：探讨持续暴露于不同刺激频率、亮度及节律的低频率频闪光对豚鼠屈光度及眼轴长度的影响以及频闪光不同参数间的差异性。对象与方法：实验对象为2w龄健康雄性豚鼠,使用频闪调光器进行频闪光刺激。本部分共分频率、亮度及节律ABC共三个实验组,A组72只豚鼠随机分为5个不同频率的频闪组和1个对照组(n=12),频闪组分别予5Hz、1Hz、0.5Hz、0.25Hz、0.1Hz的频率等时交替频闪(明暗周期50%),亮时光照度为6001ux,暗时照度为0,对照组为持续6001ux光照组。B组48只2w龄豚鼠随机分为3个不同亮度频闪组及1个对照组(n=12),频闪组均以0.5Hz(1s暗,1s亮)的频率以0-600lux、0-300lux及300-600lux的亮度等时交替频闪,对照组以6001ux持续亮度照明。AB两个研究组所有实验豚鼠均用同一时控开关控制照明时间为正常作息节律6：00-18：00。C组36只2w龄豚鼠随机分为2个不同节律的频闪组及1个对照组(n=12),频闪组以0.5Hz频率0-600lux照明亮度频闪,频闪暴露与停止频闪时间分别为12h/12h(正常作息节律6：00-18：00)和0.5h/0.5h两种节律,对照组以600lux持续照度照明,光照时间为6：00～18：00,与12h/12h频闪组相同。以上3个实验组均使用505nm波长发光二极管,应用照度测量仪测量即时照明亮度,各组实验豚鼠每2w记录屈光度、眼轴长度及角膜曲率半径,以首次测量日记录为0w,持续观察时间为12w。应用GRAPHPADPRISM Version5.0软件进行图表绘制,SPSS17.0进行统计分析,双眼间差异行配对t检验,每只豚鼠均取左眼参数进行进一步统计分析,组间差异采用单因素方差分析及多重比较,P0.05为差异有统计学意义。结果：实验前各组豚鼠生物学参数差异均无统计学意义(P0.05)。双眼间差异无统计学意义(P0.05)。随时间延长,ABC三个研究组的频闪光组与对照组相比均出现屈光度向近视偏移以及眼轴延长(P0.05),各组角膜曲率半径在整个实验过程中均逐渐增加,不同时间点各组间差异无统计学意义(P0.05)。12w时各组间近视屈光度及眼轴长度经统计学比较,A组里5种不同频率中0.5Hz频闪组近视程度及眼轴延长最明显,B组里不同亮度组中屈光度改变及眼轴延长0-6001ux0-3001ux300-6001ux对照组。C组中不同节律组屈光度改变及眼轴延长0.5h/0.5h12h/12h对照组。结论：(1)长期暴露于特定低频率的频闪光能刺激豚鼠眼球过度发育并诱导轴性近视形成,不同的刺激频率之间有差异性,0.5Hz频闪光的影响最为显著。(2)降低频闪光的刺激强度或明暗波动幅度有助于减缓屈光发展程度。(3)频闪暴露的节律会对眼球的过度发育造成影响。正常作息节律改变导致豚鼠眼球过度发育的现象提示中枢神经调节机制可能参与其中。第二部分0.5Hz频闪光环境对豚鼠眼球结构、功能的影响及机制目的：探讨持续0.5Hz频闪光暴露对豚鼠视觉传导功能及视网膜巩膜超微结构的影响及其相关机制。对象与方法：36只2w龄雄性豚鼠随机分为频闪组及对照组(n=18),频闪组予频闪调光器以0.5Hz频率(明暗周期50%,即1s亮、1s暗)等时交替频闪,实验前先通过照度测量仪测量笼内照明亮度,照度波动0-6001ux；对照组通过控制器旋钮调节笼内光源照度为持续6001ux。2组均使用505nm波长发光二极管,以上2组均由同一时控开关控制,控制光照时间为正常昼夜12h轮替,光照时间6：00-18：00。实验3周后两组豚鼠中各选取6只豚鼠麻醉后取出眼球分离视网膜及巩膜组织,Western-blot观察蛋白水平视网膜视蛋白及后部巩膜MMP-2表达的差异。其余24只豚鼠分别于实验前及第12w测量豚鼠的视网膜电图(F-ERG)及振荡电位(Ops),实验12w后所有豚鼠过量麻醉处死后取出眼球,测量离体眼球水平径、垂直径、前后径。病理学光镜及电镜下观察眼球后极部视网膜及巩膜超微结构,与对照组进行比较。应用GRAPHPADPRISM Version5.0软件进行图表绘制,SPSS17.0进行统计分析,组间差异采用t检验,P0.05为差异有统计学意义。结果：与对照组相比,频闪组各豚鼠眼球视网膜视蛋白及巩膜MMP-2表达均增加(P0.05)；第12w时与对照组相比,频闪组的F-ERG的a波潜伏期延长(P0.05)；a波振幅及b波潜伏期及振幅未见统计学差异(P0.05), Ops两组间差异未见统计学意义(P0.05)；眼球水平径、垂直径及前后径分别较对照组增加0.89±0.3mm,0.69±0.2m及0.96+0.3mm,差异有统计学意义(P0.05),组织学观察：光镜下频闪光组眼球巩膜纤维出现扩张,电镜下视网膜感光细胞层外段排列紊乱并可见较多变形及脱落的外节盘膜,对照组无明显异常。结论：1.频闪光诱导的豚鼠近视模型视蛋白表达增加,提示视锥细胞感受异常光觉信息有可能是眼轴变化的起始环节。2.频闪中巩膜MMP-2增加,与豚鼠眼轴的延长之间的关系值得进一步探讨。3.持续频闪光刺激诱导豚鼠眼球过度发育,可影响视网膜感光细胞的超微结构与传导功能。
[Abstract]:The changes in visual environment after birth are one of the key causes of refractive development. To date, any artificial lighting can not complete the complete simulation of natural light. How does the stroboscopic effect of artificial lighting affect the refractive development? This study aims to explore the eye axis and diopter of the frequency flashes in the flexion sensitive guinea pigs. The main method is to simulate frequency flashes of different stroboscopic frequencies, luminance and rhythm by automatic control technology. Using frequency flash to induce myopia model of guinea pig, detect the changes of refractive index and axial length, observe the shape of the eyeball and the ultrastructure of the posterior pole, and evaluate the retinal conduction work through the changes of visual electrophysiology. The changes in the retina protein expression and collagen metabolism in the retina of guinea pigs were observed by molecular biology methods. Main results: the frequency flash that sustained exposure to a specific frequency in the artificial lighting environment could stimulate the overdevelopment of the eyeball of guinea pigs and induce the myopic refractive changes; the frequency of frequency, brightness and rhythm of the guinea pig's eyeballs with different frequency, brightness and rhythm The effects of development were different. The change of electroretinogram and ultrastructure showed that the photosensitive conduction function of the retina of the guinea pig was decreased, and the morphologic abnormalities of the photoreceptor cells appeared; the expression of the stroboscopic protein expression increased and the expression of the scleral matrix metalloproteinase MMP-2 increased. In the process of visual perception, the effect of the change of frequency flashing parameters on the visual conduction function in the visual perception process leads to the effect of myopia, and the mechanism in it should be further studied.
Effects of the first frequency, brightness and rhythm on the refractive development of guinea pigs
Objective: To investigate the effects of low frequency frequency flashes on the refractive index and axial length of guinea pigs and the difference between different parameters of frequency flash.
Subjects and methods: the experimental subjects were 2W aged healthy male guinea pigs, using stroboscopic light to stimulate frequency flash stimulation. The total frequency division rate, brightness and rhythm of ABC were three experimental groups, and 72 guinea pigs in group A were randomly divided into 5 different frequency stroboscopic groups and 1 control groups (n=12). The frequency group was given the frequency of 5Hz, 1Hz, 0.5Hz, 0.25Hz, 0.1Hz, respectively. For stroboscopic (50%), bright light time was 6001ux, dark time was 0, and 48 2W old guinea pigs in group.B of the control group were randomly divided into 3 different luminance stroboscopic groups and 1 control groups (n=12). The frequency of stroboscopic group was 0.5Hz (1s dark, 1s bright) frequency with 0-600lux, 0-300lux and 300-600lux brightness alternately stroboscopic, and the control group was 6. 001ux continuous luminance lighting.AB two study groups all experimental guinea pigs were used the same time control switch to control the lighting time as normal resting rhythm 6:00-18:00.C group 36 2W old guinea pigs randomly divided into 2 different rhythm stroboscopic groups and 1 control groups (n=12). The stroboscopic group was flicker with 0.5Hz frequency 0-600lux illumination, stroboscopic exposure and stop stroboscopic The time was 12h/12h (normal resting rhythm 6:00-18:00) and 0.5h/0.5h two kinds of rhythms respectively. The control group was illuminated with 600lux continuous illumination, the light time was from 6:00 to 18:00, and the same as the 12h/12h stroboscopic group. The above 3 experimental groups all used 505nm wavelength light emitting diodes, and the illumination measuring instrument was used to measure instant illumination brightness, each group of guinea pigs of each group was 2 W recorded the diopter, the length of the eye axis and the radius of the curvature of the cornea. The record was 0W for the first day of measurement, and the continuous observation time was drawn by the GRAPHPADPRISM Version5.0 software for 12w., and the SPSS17.0 was statistically analyzed. The double binocular difference was paired t test. The left eye parameters of each guinea pig were further analyzed, and the difference between the groups was adopted. Factor analysis of variance and multiple comparisons showed that P0.05 was statistically significant.
Results: there was no significant difference in biological parameters between the guinea pigs before the experiment (P0.05). There was no significant difference between the two groups (P0.05). As time extended, the frequency of flash groups in the three groups of the three study groups were all offset to myopia and eye axis extension (P0.05) compared with the control group. The radius of curvature of the cornea of each group was all in the whole experimental process. There was no statistical difference between each group (P0.05) at different time points (P0.05).12w. The myopia diopter and eye axis length of each group were compared statistically. The myopia and ocular axis of 0.5Hz stroboscopic group in group A were the most obvious, and the diopter changes in different luminance groups in B group and eye axis lengthening 0-6001ux0-3001ux300-6001ux control In group.C, the change of flexion and axial length of 0.5h/0.5h12h/12h in different rhythm groups were compared.
Conclusions: (1) long exposure to a specific low frequency flash can stimulate the overdevelopment of the eyeball and induce the formation of axial myopia in guinea pigs. There is a difference between the different stimuli frequency and the most significant effect of 0.5Hz frequency flash. (2) the intensity of low frequency flash or the amplitude of light and shade helps to reduce the degree of refractive development. (3) stroboscopic exposure Rhythms may affect the overdevelopment of the eyeball. The phenomenon of abnormal development of the eyeball in guinea pigs suggests that the central nervous regulation mechanism may be involved.
The second part is the effect and mechanism of 0.5Hz flash on guinea pig eyeball structure and function.
Objective: To investigate the effects of sustained 0.5Hz frequency flash exposure on visual transmission function and ultrastructure of retinal sclera in guinea pigs.
Objects and methods: 36 male 2W old male guinea pigs were randomly divided into stroboscopic and control group (n=18). Stroboscopic light flicker was given at the frequency of 0.5Hz (50%, 1s light, 1s dark). The illumination of the cage was measured by the illuminance measuring instrument and the illumination fluctuation was 0-6001ux before the experiment, and the control group adjusted the cage light through the controller knob. 505nm wavelength light emitting diode was used in the continuous 6001ux.2 group. The above 2 groups were controlled by the same control switch. The control light time was normal day and night 12h rotation. After 3 weeks of 6:00-18:00. experiment, 6 guinea pigs of two guinea pigs were selected to separate the retina and sclera tissue, and the Western-blot observation protein was observed. The difference in the expression of the horizontal retina and posterior sclera MMP-2. The other 24 guinea pigs were measured by the electroretinogram (F-ERG) and oscillatory potential (Ops) in the guinea pig before and after the experiment. After the experimental 12W, all guinea pigs were killed and the eyeball was taken out to measure the diameter of the eye water, the vertical diameter and the front and back diameter. The pathological light and the electron microscope view were measured. The ultrastructure of retina and sclera in the posterior polar part of the eyeball was compared with the control group. The GRAPHPADPRISM Version5.0 software was used to chart, and the SPSS17.0 was statistically analyzed. The difference between the groups was determined by t test, and the difference of P0.05 was statistically significant.
Results: compared with the control group, the expression of retina retina and MMP-2 in the retina of each guinea pig increased (P0.05). At 12W, the a wave latency of F-ERG in stroboscopic group was prolonged (P0.05) compared with the control group, and there was no statistical difference between the amplitude of a wave and the latency and amplitude of B wave (P0.05), and there was no statistical significance (P0.05) between the two groups of Ops. The horizontal diameter, vertical diameter and anterior and posterior diameter of the eyeball were 0.89 + 0.3mm, 0.69 + 0.2m and 0.96+0.3mm respectively. The difference was statistically significant (P0.05). The histological observation: the scleral fiber of the light microscope flash group appeared dilatation, the outer segments of the retinal photoreceptor layer were arranged in disorder under electron microscope, and more deformed and exfoliated outer segment membranes were visible. There was no obvious abnormality in the control group.
Conclusion: the expression of visual protein in the 1. frequency flashing induced guinea pig myopia model suggests that the abnormal light perception information of the cone cells may be the increase of the scleral MMP-2 in the starting link of the ocular axis.2. stroboscopic. The relationship between the eye axis of the guinea pig and the eye axis of the guinea pig is worthy of further exploring the overdevelopment of the eyeball induced by.3. continuous frequency flash stimulation. Affect the ultrastructure and conduction function of retinal photoreceptors.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R778.1

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