【作者】 张衡頔；

【导师】 王一；

【作者基本信息】 第三军医大学 ， 眼科学， 2011， 硕士

【摘要】 糖尿病视网膜病变(Diabetic retinopathy,DR)是一种严重的致盲性眼病,临床上缺乏有效的治疗方法。临床观察发现,双眼屈光参差(Anisometropia)的患者,高度近视(high myopia,HM)或弱视(amblyopia)的一眼相对另一眼DR程度轻,较少发展成为增殖性糖尿病视网膜病变(Proliferative diabetic retinopathy,PDR)。高度近视/弱视对DR影响的机制尚不清楚,既往的研究集中在视网膜血液流变学上。一些研究者认为,DR早期视网膜血流呈高灌注,而高度近视早期,视网膜血流减缓,高度近视可能通过减少视网膜高灌注性损伤而延缓DR发生发展;另一些研究者提出,高度近视患者眼轴增长,视网膜较正常眼明显变薄,对DR所致缺血缺氧耐受性提高,这可能是高度近视对DR的保护机制。临床上,一眼高度近视患者多合并有形觉剥夺性弱视。Murat等人的临床观察报告显示,弱视对DR也具有保护趋势[1]。弱视眼并无视网膜血流改变及形态学变化,提示其对DR的保护作用可能不仅是血流改变。既往多项研究证明,动物出生后在视觉形成关键期内进行形觉剥夺(Form deprivation,FD),可以造成形觉剥夺性近视(form deprivation myopia, FDM)和弱视(form deprivation amblyopia,FDA)。哺乳类动物因其眼球结构及眼生物化学特性与人类相似,在近视和弱视研究中受到重视。近年来用于建立的动物模型有小鼠[2]和豚鼠[3]模型。其中小鼠因其基因背景纯,且巩膜壁上具有人类所有的5种毒蕈碱样受体[4],并发现小鼠形觉剥夺性近视伴有玻璃体腔增长,类似于人类和其他非人类的灵长目动物[5]。因此,小鼠是一种具有研究前景的形觉剥夺性近视/弱视动物模型,能够较好的模拟人类病理性近视(pathological myopia)的形成过程,既往已有多项实验在小鼠上建立了形觉剥夺性近视[6,7,8]。然而,小鼠因其眼球直径短,球形晶体等,使得验光和眼轴长度较为困难。DR常用的实验动物有猴、猫、狗、猪、鼠等,因为其视网膜有精细复杂的血管网,可用于糖尿病视网膜病变实验研究。研究发现在糖尿病大鼠发病1个月内发生视网膜组织代谢异常,而其最早的组织病理损害(周细胞丧失)在6个月内出现(胡玉章等,1993年)。猴、狗等动物发生早期DR改变的时间则更长。而小鼠生长周期短,已有研究在C57/BL6小鼠上建立了糖尿病模型,并观察到周细胞大量凋亡、毛细血管基底膜增厚,血管渗漏等DR早期改变[9]。因此,小鼠是一种具有研究前景的高血糖合并形觉剥夺性近视/弱视实验动物模型。DR早期病理改变表现为血-视网膜内屏障(inner blood-retina barrier,IBB)损伤,其特征性改变是周细胞(pericytes)大量丢失,毛细血管基底膜增厚。继而失去周细胞接触性抑制的内皮细胞(endothelial cells,EC)大量增殖,肿胀,白细胞黏附,边集,堵塞管腔,形成无细胞结构血管,造成视网膜局部缺血缺氧。一般认为,周细胞的凋亡是由于持续高血糖,糖基化终末产物(advanced glycosylation end products,AGEs)堆积引起。高度近视/弱视是否能在DR早期起到保护血-视网膜内屏障的作用,目前尚未见文献报道。综合上述各项研究,本研究推测:形觉剥夺性近视/弱视可能能减轻DR早期的血-视网膜内屏障损伤从而延缓DR进展的关键。针对以上假设,本实验采用BALB/cJ小鼠,研究形觉剥夺性近视/弱视对小鼠高血糖视网膜损伤的影响。构建高血糖(hyperglycemia)模型和高血糖合并形觉剥夺性近视/弱视模型,分别测量其血糖、眼轴长度、眼屈光度,并观察视网膜血管网的形态学变化,结果采用单因素方差分析及独立样本T检验。本文的主要内容及研究结果如下:1、通过在BALB/cJ小鼠生后第10天缝合右眼睑,生后第30天起连续注射STZ造成高血糖合并形觉剥夺性近视/弱视模型。高血糖成模后第30天(即生后72-77天时)检影验光,测量眼轴长度,发现BALB/cJ小鼠连续注射STZ后5-7天血糖即达成模标准(>11.1mmol/L),此后均稳定在此水平以上。形觉剥夺的一侧眼在生后72-77天时验光,可获得-3.00D以上的近视化改变,眼轴测量发现,形觉剥夺眼眼轴明显长于对照眼。2、通过PAS染色、透射电镜观察发现,高血糖持续1月时,BALB/cJ小鼠视网膜微血管即可出现DR早期表现,周细胞大量丢失,毛细血管基底膜增厚,血-视网膜内屏障损伤。同时合并形觉剥夺组小鼠眼周细胞丢失及毛细血管基底膜增厚均较高血糖组轻。3、通过鬼笔环肽-FITC/DAPI免疫组化,染色消化铺片的视网膜血管网,行三维重建后计数内皮细胞/周细胞比值,可清晰的辨认周细胞核与血管的毗邻关系,易于区分形态不典型的周细胞核,以及计数因位于铺片底面,与内皮细胞核重叠的周细胞核。本方法弥补了传统的PAS染色技术的不足,可以更精确的计数周细胞核和内皮细胞核。本研究结论:1、BALB/cJ小鼠是一种成模快捷、稳定的高血糖合并形觉剥夺性近视/弱视动物模型。2、形觉剥夺性近视/弱视在高血糖极早期即有保护血-视网膜内屏障的作用。3、鬼笔环肽-FITC/DAPI染色后行三维重建,较传统的PAS染色能够更精确计数内皮细胞/周细胞比值。更多还原

【Abstract】 Diabetic retinopathy(DR) is a severe disease that eventually causes blindness, with actually no effective therapy. Clinical researcheres has found that patients suffered simultaneously from anisometropia and diabetic, their high myopic or amblyopia eyes do not have proliferative diabetic retinopathy. The mechanism of how high myopia and amblyopia affected DR has not been clarified, and previous studies had focused on the hemorheology of the retina.Some researchers found that during the early stage of DR, the retina had more blood perfusion, which was contrary to the situation in early stage of high myopia, during which the retinal blood perfusion was decreased. It suggested that high myopia prevented the development of DR through decreasing of hypertransfusion damage in retina. Others found that in high myopia,the increase of axial length of the eye ball resulted in the decrease of the thickness of retina which improve the tolerance of ischemia and anoxia caused by DR, this might be the possible mechanism of the protective effect of HM during diabetic retinopathy. However, amblyopia eyes do not have morphological or hemorheological changes as formerly mentioned. In one of their clinical surveys , Murat et al. reported that amblyopia could also prevent the progression of DR[1]. These findings indicate that there may be other unknown mechanisms for the protective effect of HM and amblyopia against DR.It was reported that form deprivation(FD) in critical period of visual maturity could cause form deprivation myopia(FDM) and amblyopia. And mammals were the ideal animal models for FDM researches due to their similarities of eye configuration and biochemical properties. Recently, FDM/FDA mice models and guinea pigs models were established [2,3]. Mice are even better models than guinea pigs because they have pure gene backgrounds, and all the 5 kinds of muscarine receptor on sclera as human beings[4], and FDM in mice can cause elongation of vitrous chamber,which is the same in human beings and nonhuman primates[5]。Therefore mice model can imitate human pathological myopia, many studies has established FDM in mice[6,7,8]. However, there are difficulties on the measuring of eye diopter and axial length on mice due to their small eye size and spherophakia. Animal models commonly used in DR researches are monkeys, cats, dogs, pigs, rats and mice, because they have complicate retina vascular net. Some researches found retinal metabolic abnormality occurred in 1 month after diabetic mellitus(DM) had started, but the earliest morphological changes could only be observed after 6 months of DM. Morphological changes requires even longer pathogenesis in dogs and monkeys. Mice again shows superiority in DR study because they have short growth cycle and therefore requires less time to develop DR morphological changes. Researchers have established DM model on C57/BL6 mice, and they found early DR retina damages as loss of pericytes, thickening of microvascular membrane, vascular leackage in C57/BL6 mice model, thus proved mice can be a potential DR model.Early DR pathological damages are mainly damages of inner blood-retina barrier, the typical changes are loss of pericytes, thickening of microvascular membrane. Then endothelial cells proliferate because of the absent of pericyte contact inhibition, lumen of blood vessels were blocked because of endothelial cells swelling, leucocytes adhesion, finally causes retina ischemia and anoxia. It is reported that pericytes apoptosis are caused by enduring hyperglycemia and accumulation of advanced glycosylation end products(AGEs). It is yet unclear wether high myopia/amblyopia affects the development of DR through protecting inner blood-retina barrier, and our research is to study the protective effect of form deprivation myopia/amblyopia on inner blood-retina barrier during hyperglycemia retinal disease.This experiment established hyperglycemia model and hyperglycemia and FDM/FDA (Form deprivation amblyopia) model on BALB/cJ mice, observed the effect of form deprivation myopia/amblyopia on inner blood-retina barrier during hyperglycemia retinal disease, through measuring the blood glucose concentration, axial length of mice eyes, eye diopters and retinal vascular morphology changes. Our results are as follow.1. Through lid suturing of the right eye of BALB/cJ mice at P10, injecting of STZ at P30, we established FDM/FDA (Form deprivation amblyopia) model on BALB/cJ mice. Retinoscopy and eye axial length measuring at the 30th day (P72-77) after hyperglycemia was formed reveals that BALB/cJ mice can develop a -3.00D of FM at that time point, and the axial length also grows obviously longer than control group. 7-10 days after STZ injection, the blood glucose concentration of BALB/cJ mice raised over 11.1mmol/L, and kept elevating through out the experiment.2. Through PAS staining and transmission electron microscope we found that after 1month of hyperglucemia, BALB/cJ mice showed early DR damages (damage of inner blood-retina barrier) such as pericytes loss, thickening of microvacular membrane, hyperglycemia combined with form deprivation myopia/amblyopia group showed less pericytes loss and thickening of microvacular membrane than hyperglycemia group.3. Through phalloidine-FITC/DAPI immunohistochemisty staining the microvacular and 3D reconstruction, counting the endothelial cells/ pericytes, we could obtain the accurate number of endothelial cells/ pericytes as this method could identify non-typical pericyte nuclear and pericytes that are covered or overlapped by endothelial cells.Conclusions:1. BALB/cJ mice can be used as a stable, easily established animal model of hyperglycemia and form deprivation myopia/amblyopia.2. Form deprivation myopia/amblyopia showed protective effect on inner blood-retina barrier during early stage of hyperglycemia.3. Phalloidine-FITC/DAPI immunohistochemisty staining and 3D reconstruction provide a new way to count the endothelial cells/ pericytes, which can discern non-typical pericyte from endothelial cells, and is thus a more precise way to count the pericytes and ECs.更多还原