睡眠剥夺对豚鼠离焦性近视及视网膜多巴胺代谢的影响

Contributions of Sleep Deprivation to the Development of Lens-Induced Myopia and Retinal Dopamine Metabolism in Guinea Pigs

目的:探究睡眠剥夺(SD)对豚鼠光学离焦性近视(LIM)形成及视网膜近视相关信号因子多巴胺(DA)代谢的影响。方法:实验研究。2022年3月采用随机数表法将30只21日龄三色豚鼠分为LIM组和LIM+SD组,予以右眼离焦处理,左眼为自身对照,所有实验动物予以14 d正常睡眠环境或每天SD处理20 h。每组进行角膜曲率半径、屈光度和眼轴长度等生物学参数测量。采用高效液相色谱电化学法测定视网膜DA、3,4-二羟基苯乙酸(DOPAC)含量,计算DOPAC/DA比值;通过免疫组织化学染色法及Western blot检测视网膜酪氨酸羟化酶(TH)分布及表达。豚鼠左右眼屈光参数及神经递质含量比较采用配对样本t检验,LIM组与LIM+SD组间屈光参数及神经递质含量比较采用独立样本t检验。离焦眼视网膜DA含量分别与眼球屈光度、眼轴长度进行Pearson相关分析。结果:LIM组对照眼与LIM+SD组对照眼屈光度、眼轴长度比较差异均无统计学意义(均P>0.05);与LIM组离焦眼相比,LIM+SD组离焦眼的近视程度更深、眼轴延长更长(t=5.63,P<0.001;t=-3.87,P=0.001)。2组豚鼠离焦眼视网膜DA、DOPAC含量均低于自身对照眼(LIM组:t=-8.64,P<0.001;t=-13.03,P<0.001;LIM+SD组:t=-11.72,P<0.001;t=-16.32,P<0.001),其中LIM+SD组离焦眼视网膜DA、DOPAC含量均较LIM组更低(t=5.35,P<0.001;t=3.80,P=0.002),而DOPAC/DA比值升高且组间差异具有统计学意义(t=-3.60,P=0.003)。豚鼠离焦眼视网膜DA含量与屈光度、眼轴长度均有相关性(r=0.93,P<0.001;r=-0.77,P=0.001)。免疫组织化学染色结果显示,TH阳性反应细胞主要分布在视网膜神经纤维层、神经节细胞层、内丛状层和内核层,外丛状层可见少量着色;与自身对照眼相比,豚鼠离焦眼视网膜染色均减弱。Western blot检测显示,离焦眼视网膜TH蛋白表达均明显低于自身对照眼(LIM组:t=-40.25,P<0.001;LIM+SD组:t=-17.64,P<0.001),但LIM组与LIM+SD组离焦眼视网膜TH蛋白表达差异无统计学意义(P>0.05)。结论:SD对正常屈光发育豚鼠的屈光发育无明显作用,但能促进豚鼠LIM进展,可能通过加快DA代谢而不影响DA合成水平,降低DA含量,促进近视进展。

Objective:To investigate the effects of sleep deprivation(SD)on the development of lens-induced myopia(LIM)and the dopamine(DA)metabolism in guinea pigs.Methods:This was an animal experimental study.A total of 30 three-week-old guinea pigs supplied were randomly divided into two groups:LIM group and LIM+SD group,raised either in a normal sleep environment or subjected to sleep deprivation for 20 hours every day.Refraction,corneal curvature radius,and ocular axial components were measured.The content of DA and 3,4-dihydroxyphenylacetic acid(DOPAC)in the retina was assessed by the highperformance liquid chromatography electrochemical method,and the DOPAC/DA ratio was calculated.The expression of retinal tyrosine hydroxylase(TH)protein was evaluated by immunohistochemical staining and Western blot.Paired t-test was used to compare the refraction and DA turnover between the left and right eyes of guinea pigs.Independent sample t-test was used for comparison between the LIM group and the LIM+SD group.The refractive error and axial length of the guinea pig eyes in the defocus eyes underwent a correlation analysis with DA content by using Pearson correlation analysis.Results:Normal refractive development was not affected by SD in guinea pigs.However,SD significantly deepened myopia and prolonged the ocular axis of defocus eyes(t=5.63,P<0.001;t=-3.87,P=0.001).The content of retinal DA and DOPAC in all defocus eyes was lower than that in their control eyes(LIM group:t=-8.64,P<0.001;t=-13.03,P<0.001;LIM+SD group:t=-11.72,P<0.001;t=-16.32,P<0.001).Compared with LIM group,the defocus eyes of LIM+SD group had lower DA and DOPAC obviously(t=5.35,P<0.001;t=3.80,P=0.002),while the DOPAC/DA ratio increased and the difference was statistically significant(t=-3.60,P=0.003).The content of retinal DA in defocus eyes was correlated with refractive error(r=0.93,P<0.001)and eye axis length(r=-0.77,P=0.001).Retinal immunohistochemical staining showed that TH positive cells were mainly distributed in the nerve fiber layer,ganglion cell layer,inner plexiform layer,and inner nuclear layer,and had a minor portion in outer plexiform layer.The expression of TH protein in defocus eyes was reduced compared with control eyes.Western blot showed that the expression of TH protein in defocus eyes was significantly lower than control eyes(LIM group:t=-40.25,P<0.001;LIM+SD group:t=-17.64,P<0.001).But the retinal TH protein expression had no significant difference in the defocus eyes between the LIM and LIM+SD groups(P>0.05).Conclusions:SD had no significant effects on normal refractive development.However,in a lens-induced environment,SD promotes the development of LIM in guinea pigs probably by accelerating DA catabolism without affecting DA anabolism.