Transcriptomic characterization of the diabetic retinal response to chronic insulin treatment

The aim of this study was to identify alterations associated with diabetes-induced functional dysregulation of the retina and the effects of chronic insulin therapy. Transcriptional profiling through microarray analysis was analyzed to find mRNA targets of interest in diabetic samples, as well as in the insulin treated group. Verification of targets shown to be unrecovered in the insulin group was validated. Diabetes was induced in Sprague-Dawley male rats (Charles River Laboratories, Wilmington, MA) by intraperitoneal injection of 65 mg/kg streptozotocin (STZ)(Sigma-Aldrich, St. Louis, MO) in 10mM sodium citrate pH 4.5 vehicle. Control rats were injected with an equal dose of vehicle only. Rats had free access to food and water, and were maintained on a 12 hour light/dark cycle. Blood glucose level and body weight were measured 6 days post-STZ or vehicle injection, and biweekly throughout the experiment. Only rats with blood glucose levels >250 mg/dL at the time of the original test and throughout the experiment were included in the diabetic groups. The insulin treatment group received one 26mg subcutaneous pellet (LinShin Canada, Scarborough, Canada) delivered via trocar 6 weeks post-STZ injection. An additional 26 mg implant was introduced when body weight exceeded 300 g or when midday non-fasting blood glucose exceeded 250 mg/dL. At the time of retina harvest, rats were given a lethal dose of pentobarbital, 100 mg/kg, (Ovation Pharmaceuticals Inc., Deerfield, IL) by intraperitoneal injection and sacrificed by decapitation. Retinas were rapidly excised snap- frozen in liquid nitrogen for subsequent experimentation.

本研究旨在明确与糖尿病诱导的视网膜功能失调相关的病理改变，以及慢性胰岛素治疗的干预效应。通过微阵列分析（microarray analysis）开展转录组谱检测，以筛选糖尿病样本及胰岛素治疗组中具有研究价值的mRNA靶点，并针对胰岛素治疗组中未能恢复至正常水平的靶点进行了验证实验。本研究通过向雄性斯普拉-道来（Sprague-Dawley）大鼠（购自美国马萨诸塞州威尔明顿市查尔斯河实验室（Charles River Laboratories））腹腔注射溶于10mM pH 4.5柠檬酸钠缓冲液的65mg/kg链脲佐菌素（streptozotocin, STZ，购自美国密苏里州圣路易斯市西格玛-奥德里奇公司（Sigma-Aldrich, St. Louis, MO））构建糖尿病模型，对照组大鼠仅注射等量溶剂。大鼠自由进食饮水，饲养于12小时光照-12小时黑暗的循环环境中；于链脲佐菌素或溶剂注射后第6天，以及实验期间每两周检测一次大鼠血糖与体重，仅将造模初始检测及实验全程血糖均高于250mg/dL的大鼠纳入糖尿病组。胰岛素治疗组大鼠于链脲佐菌素注射后6周，通过套管针植入1枚26mg的皮下缓释制剂（购自加拿大斯卡伯勒市LinShin加拿大公司（LinShin Canada））；当大鼠体重超过300g或午间非禁食血糖高于250mg/dL时，追加植入1枚26mg的缓释制剂。视网膜取材时，大鼠经腹腔注射100mg/kg戊巴比妥钠（pentobarbital，购自美国伊利诺伊州迪尔菲尔德市Ovation制药公司（Ovation Pharmaceuticals Inc.））实施安乐死，随后通过断头法处死；快速摘取视网膜组织，置于液氮中快速冷冻以备后续实验使用。