2结果
2.1缺血性视网膜组织学观察 正常组大鼠视网膜组织结构与人视网膜组织结构相似,内界膜清楚,神经纤维层较稀疏,水平排列较规整;RGCs呈单层排列,胞核较大,呈圆形或椭圆形,染色较淡,排列整齐;内丛状层较厚、疏松,呈较明显的网状结构;内核层由3~5层细胞构成,胞核较大,染色稍深;外丛状层明显薄于内丛状层;外核层较厚,由8~10层细胞组成,胞核较小,染色深,排列较紧密(图1A)。激光15~20s眼底后视乳头水肿,边界不清,动脉血管搏动,无血流中断现象。激光2min后眼底动脉无血流通过,静脉血流中断,似腊肠样,呈贫血状眼底,乳头周围苍白似棉絮状。激光3min后眼底:动脉闭塞,静脉呈串珠状。激光12h后:视网膜水肿减轻,内核层轻度变薄,神经纤维层扁平,RGC排列较稀疏。外核层变化不明显(图1B)。24h后:视网膜水肿基本消失,主要表现在RGCs数目减少,细胞变性,神经纤维层明显变薄,内核层排列紊乱,轻度变薄(图1C)。72h后视网膜水肿完全消退,内核层变薄,可见视网膜较长片段的 RGC消失。168h后RGC数目明显减少,神经纤维层变薄,内核层细胞排列紊乱,外核层变化不明显(图1D)。电镜下也有相应改变(图2A~H)。
2.2缺血性视神经病变定量观察 早期以水肿为主,晚期以细胞变性、萎缩及凋亡为主。正常大鼠视网膜内层平均厚度为102.4±4.2μm,缺血12h,视网膜内层厚度变为67.8±2.8μm,两者相比差异显著;正常大鼠视网膜神经节细胞密度为88.5±2.2个/mm,缺血12h后即变为43.2±3.2个/mm,两者相比差异显著。缺血24h上述变化最为明显,至缺血168h略有回升,但仍然显著低于正常组,实验组与对照组之间各时间点t检验结果P<0.05,差异具有显著性(表1)。
2.3视网膜打墨观察 正常大鼠视网膜血液供应。图3(A~B)视网膜缺血模型血液循环经孟加拉玫瑰红诱导激光30min后视乳头水肿,边界不清,动静脉血流中断,图3(C~F)。
2.4闪光VEP 观察实验眼较对照眼潜伏期均延迟,振幅降低(图4A~D)。
3讨论
视网膜缺血性改变是在眼科非常常见[1-4],如缺血性视神经病变、视网膜中央动、静脉阻塞、糖尿病视网膜病变、早产儿视网膜病变、青光眼等致盲性眼病,其组织学变化主要表现在视网膜厚度和神经节细胞数目的改变,早期以水肿为主,晚期以细胞变性、萎缩及凋亡为主[5-7];功能学改变包括视力下降,视网膜电图下降等改变。缺血性视神经病变的发病机制及临床治疗一直为广大眼科界所关注。视网膜缺血性改变发生机制比较复杂,目前比较公认的学说有:(1)刺激性氨基酸学说;(2)自由基损伤学说;(3)钙离子通道学说;(4)神经保护因子学说等,针对上诉学说,研究出缺血性视神经病变模型是目前亟待解决的课题[8-10]。
常用的视网膜缺血模型有高眼压模型、血管结扎模型、光化学阻断模型和游离视网膜模型等[11-13]。我们采用的孟加拉玫瑰红诱导的激光缺血模型[14-16],容易控制,可以通过激光时间的长短造成不同程度的视网膜缺血,且不受如眼压等其他因素影响,各项指标均相似缺血性视神经病变,通过在形态学及功能方面大量的研究已证明了此方法的可靠性和稳定性,经孟加拉玫瑰红诱导后,用激光的方法可以成功制作缺血性视神经病变模型,为进一步探讨视网膜缺血等疾病打下坚实基础。
【参考文献】
1 Hayreh SS. Anterior Ischemic optic neuropathy. I. Terminology and pathogenesis. Br J Ophthalmol,1974;58:955-963
2 Selles-Navarro I, Ellezam B, Fajardo R, Latour M, Mckerracher L. Retinal ganglion cell and non neuronal cell responses to a microcrush lesion of adult rat optic nerve. Exp Neurol ,2001;167:282-289
3 Krueger-Naug AM, Emsley JG, Myers TL, Currie RW, Clarke DB. Injury to retinal ganglion cells induces the expression of the small heat shock protein HSP27 in the rat visual system. Neuroscience ,2002 ;110:653-665
4 Johnson EC, Deppmeier LM, Wentzien SK, Hsu I, Morrison JC. Chronology of optic nerve head and retinal responses to elevated intraocular pressure. Invest Ophthalmol Vis Sci ,2000;41:431-442
5 Cioffi GA, Orgul S, Onda E, Bacon DR, Van Buskirk EM. An in-vivo model of chronic optic nerve ischemia: the dose-dependent effects of endothelin-1 on the optic nerve vasculature. Curr Eye Res ,1995;14:1147-1153
6 Rodgers MAJ. Light-induced generation of singlet oxygen in solutions of rose Bengal. Chem Phys Lett ,1981;78:509-514
7 Kikuchi S, Umemura K, Kondo K, Saniabadi AR, Nakashima M. Photochemically induced endothelial injury in the mouse as a screening model for inhibitors of vascular intimal thickening. Arterioscler Thromb Vasc Biol ,1998;18:1069-1078
8 Ocho S, Iwasaki S, Umemura K, Hoshino T. A new model for investigating hair cell degeneration in the guinea pig following damage of the stria vascularis using a photochemical reaction. Eur Arch Otorhinolaryngol ,2000; 257:182-187
9 Wilson CA, Hatchell DL. Photodynamic retinal vascular thrombosis. Invest Ophthalmol Vis Sci ,1991;32:2357-2365
10 Pooler JP, Valenzeno DP. Physicochemical determinants of the sensitizing effectiveness for photoxidation of nerve membranes by fluorescein derivatives. Photochem Photobiol ,1979;30:491-498
11 Luna MC, Wong S, Gomer CJ. Photodynamic therapy mediated induction of early response genes. Cancer Res ,1994;54:1374-1380
12 Grimm C, Wenzel A, Hafezi F, Reme CE. Gene expression in the mouse retina: the effect of damaging light. Mol Vis ,2000;6:252-260
13 Mosinger JL, Olney JW. Photothrombosis-induced ischemic neuronal degeneration in the rat retina. Exp Neurol ,1989;105:110-113
14 Bernstein SL, Guo Y, Kelman SE, Flower RW, Johnson MA. Functional and Cellular Responses in a Novel Rodent Model of Anterior Ischemic Optic Neuropathy . Invest Ophthalmol Vis Sci ,2003;44:4153-4162
15 Albert DM, Jakobiec FA. Principles and practice of Ophthalmology. Baltimore WB Saunders,1994:285-309
16 Levin LE. Apoptosis of retinal ganglion cells in anterior ischemic optic neuropathy. Arch Ophthalmol , 1996;114:488-491 上一页 [1] [2] |