【作者】 王丽；

【导师】 朱豫；

【作者基本信息】 郑州大学 ， 眼科学， 2007， 硕士

【摘要】 目的外伤性视神经病变（Traumatic optic neuropathy，TON）是颅脑、眼眶和面部外伤严重的并发症，伤后视力损害严重，常遗留永久性视力障碍。长期以来，关于TON的治疗一直存在着争议。研究发现，损伤程度的差异是决定治疗效果的重要因素。而建立规范可靠、方便易行、可重复的不同程度量化损伤模型，可为深入研究TON的治疗机制提供重要前提。以往的外伤性视神经损伤模型仅是定性损伤或是半定量损伤，而缺乏统一标准的量化模型。本实验应用三种压力恒定的血管夹拟造成兔轻、中、重度视神经损伤模型，作为视神经损伤治疗效果评价的动物模型。材料和方法1动物与分组成年健康白兔48只，雌雄兼用，体重2.0-2.5kg，眼部检查正常。随机抽取12只，左右眼随机分为正常对照组和假伤对照组，剩余36只随机分为损伤Ⅰ组、损伤Ⅱ组、损伤Ⅲ组。2夹持力和致伤强度测定精确测定和计算血管夹的夹持力和致伤强度（平均冲量）。三种血管夹（小、中、大号）夹持力分别为：32g、98g、148g，平均冲量分别为：397.52g．s／mm²、1209.88g．s／mm²、1549.74g．s／mm²。3动物模型制作使用小、中、大号三种显微血管夹夹持三个损伤组动物一侧眼视神经各20s，分别造成损伤Ⅰ组、损伤Ⅱ组、损伤Ⅲ组不同程度损伤模型；假伤组仅分离暴露一侧眼视神经而不施加夹持；另一眼作为正常对照。4观察项目和时间点伤后3d、1w及2w观察视神经损伤局部的组织病理学改变、进行视神经Glee银染或丽春红G—亮绿染色后视神经纤维及髓鞘积分光密度测定；以及视网膜的形态学观察、视网膜神经节细胞（Retinal Ganglion Cell，RGC）计数、RGC凋亡检测和RGC凋亡率计算。伤后1h、6h、1d、3d、1w行F-VEP检查，观察5组间P2波潜伏期和波幅变化。5统计学分析数据运用单因素方差分析和t检验进行统计学处理。结果1视神经损伤局部的病理学观察结果1．1视神经损伤局部的HE染色和组织形态学观察正常组及假伤组视神经纤维排列密集规则，染色均匀，有少量神经胶质细胞。损伤Ⅰ组与正常组相比改变轻微。损伤Ⅱ组3d时视神经水肿，轴心区有梗死灶，胶质细胞排列紊乱，随时间推移，改变加重。损伤Ⅲ组3d时视神经水肿明显，有多处坏死，随时间推移，病变迅速发展，2w时神经束结构消失。各时间点损伤Ⅲ组改变较损伤Ⅱ组严重。1．2视神经纤维Glee浸银染色和积分光密度测定结果各时间点假伤组视神经形态与正常组基本一致；视神经纤维积分光密度与正常组比较，无显著性差异（P＞0.05）。各时间点损伤Ⅰ组视神经形态与正常组相似；视神经纤维积分光密度较正常组降低，但无显著性差异（P＞0.05）。损伤Ⅱ组及损伤Ⅲ组3d时视神经纤维稀疏扭曲，随时间推移，改变渐明显，各时间点损伤Ⅲ组较损伤Ⅱ组改变明显；两组在各时间点视神经纤维积分光密度均较正常组降低，差异有显著性（P＜0.05），随时间推移，视神经纤维积分光密度进行性降低。在同一时间点，各损伤组之间视神经纤维积分光密度比较，差异有非常显著性（P＜0.01）。1．3视神经切片丽春红G—亮绿染色和髓鞘积分光密度测定结果各时间点假伤组视神经形态与正常组基本一致；髓鞘积分光密度与正常组比较，无显著性差异（P＞0.05）。各时间点损伤Ⅰ组视神经形态与正常组相似；髓鞘积分光密度较正常组降低，但无显著性差异（P＞0.05）。损伤Ⅱ组及损伤Ⅲ组3d时有髓鞘脱失，随时间推移，改变渐明显，2w时损伤Ⅲ组大量髓鞘崩解，神经束结构基本消失，各时间点损伤Ⅲ组较损伤Ⅱ组改变明显；两组在各时间点髓鞘积分光密度均较正常组降低，差异有显著性（P＜0.05），随时间推移，髓鞘积分光密度进行性降低。在同一时间点，各损伤组之间髓鞘积分光密度比较，差异有非常显著性（P＜0.01）。2视网膜的病理学观察结果2．1视网膜HE染色组织形态学观察正常组及假伤组视网膜层次清晰，节细胞呈单层排列，整齐密集，胞核清楚，核膜光滑完整。损伤Ⅰ组与正常组相比改变轻微。损伤Ⅱ组3d时RGC出现核固缩，染色加深，数量减少；随后视网膜各层变薄，病变随时间进行性加重。损伤Ⅲ组3d时大量RGC出现核固缩，染色加深，RGC排列明显稀疏，随时间进行病变迅速加重。各时间点损伤Ⅲ组较Ⅱ组病变明显。2．2 RGC计数各时间点假伤组RGC数与正常组比较，无显著性差异（P＞0.05）。各时间点损伤Ⅰ组RGC数较正常组减少，但无显著性差异（P＞0.05）。各时间点损伤Ⅱ组及Ⅲ组RGC数与正常组相比，均明显减少，差异有非常显著性（P＜0.01），RGC数随时间进行性减少。在同一时间点，各损伤组之间RGC数比较，差异有显著性（P＜0.05）。2．3 RGC凋亡的检测和平均凋亡率正常组及假伤组视网膜切片未见凋亡细胞。各损伤组的凋亡细胞主要位于GCL。损伤Ⅰ组有少量凋亡细胞，各时间点RGC凋亡率之间无显著性差异（P＞0.05）。损伤Ⅱ组凋亡细胞随时间进行性增多，各时间点RGC凋亡率之间差异有显著性（P＜0.05）。损伤Ⅲ组3d时出现大量凋亡细胞，RGC凋亡率随时间进行性增高，各时间点RGC凋亡率之间差异有显著性（P＜0.05）。在同一时间点，各损伤组之间RGC凋亡率有非常显著性差异（P＜0.01）。3 F-VEP检测结果正常白兔P₂波潜伏期及波幅分别为（71.72±3.66）ms、（20.53±4.15）μv。各时间点假伤组与正常组的P₂波潜伏期和幅值比较，差异无显著性（P＞0.05）。损伤后1h，各损伤组均出现P₂波潜伏期延迟，幅值降低，与正常组相比差异均有显著性（P＜0.05）。损伤Ⅰ组1d时P₂波潜伏期及幅值基本恢复正常（P＞0.05）。损伤Ⅱ组及损伤Ⅲ组，随时间推移，P₂波潜伏期进行性延迟，幅值进行性降低。结论1应用压力为32g、98g、148g的显微血管夹夹持兔视神经20s，可制作稳定、可重复的轻、中、重度视神经损伤动物模型。2轻度损伤组伤后视神经形态学改变轻微，无显著病理改变，视神经传导功能良好；中度损伤组伤后视神经形态学改变明显，随时间推移损伤进行性加重，但视神经有一定传导功能；重度损伤组伤后视神经迅速出现不可逆性溃变，伤后视神经传导功能完全丧失。3中度视神经损伤模型可作为观察外伤性视神经病变的治疗方法和效果的的动物模型。更多还原

【Abstract】 ObjectiveTraumatic optic neuropathy（TON）, as a severe complication of brain, orbit and prosopo injury, often results in serious vision deterioration even permanent visual loss. For a long time, there have been arguments about the treatments of TON. The difference of injury degrees were recognized as the key for the reaction of treatment. Establishing a reliable, convenient and reproducible animal model with optic nerve injury which can be graded will be beneficial for the research of effects of different treating methods.Previous animal models of optic nerve injury were qualitative or semi-quantitative, there was not a unified standard calibrated model. In this experiment, mild, medium and severe optic nerve crush injury models were made in rabbits by using 3 kinds of vascular clamp of permanent pressure, as the animal model for the evaluation of effects of different treating methods.Materials and Methods1 Animal and grouping48 healthy adult albino rabbits without eye diseases were divided into 5 groups randomly: normal control group, false injury group, injury group I , injury group II, and injury group III. Among of them, 24 eyes of 12 rabbits were randomly assigned as the normal control group and the false injury group, another 36 rabbits were divided into 3 injury group, 12 rabbits in each group. Optic nerve crush injury was only made in unilateral eye of each animal.2 Measurement about pressure and injury intensity （average impulse） of vascular clampThe pressure power of the vascular clamp was measured by using the hang heavy tensile force, and the injury intensity （average impulse） was calculated according to the area of vascular clamp and the pressure power. The pressure power of small, moderate and big vascular clamp were respectively 32g, 98g and 148g, and the average impulse were 397.52g.s/mm², 1209.88g.s/ mm² and 1549.74 g.s/mm², respectively.3 Establishment of animal modelThe crush injury of optic nerve was made by using different vascular clamp in every group for twenty seconds, 32g for injury group I, 98g for injury group II and 148g for injury group III. The rabbits of false injury group only received optic nerve dissociation and exposure without optic nerve crush.4 Observation items and timePathologic changes of the optic nerve crush injury were examined at 3d, 1w and 2w after injury. The changes of tissue morphology of optic nerve were observed by routine HE stain, the integral light density of optic nerve fiber and myelin were measured by using Glee-sliver stain and ponceau red G-light green stain.At 3d, 1w and 2w after injury, pathologic changes of retina and the apoptosis of RGC were examined by routine HE stain and terminal deoxynucleotidyl transferase mediated DIG-dUTP nick end labeling method （TUNEL）. The number of RGC and the rate of apoptotic RGC were calculated.F-VEP was examined at 1h, 6h,1d, 3d and 1w after injury, the latency and amplitude of P₂ wave were analyzed.5 Statistical analysisAll the data were analyzed statistically by ANOVA and t test. Results1 Focal pathological changes of optic nerve crush injury1.1 HE stain and focal morphological observation of optic nerve crush injuryIn normal group and false injury group, the optic nerve fiber was intensive, regular and stained evenly.No obvious changes were found in injury group I at 3d, 1w and 2w after injury.Optic nerve edema and focal infarct were seen in injury group II at 3d after injury. These changes deteriorated gradually within 2w.Optic nerve edema and infarct in injury group III were more obvious than that in injury group III. At 2w after injury, the frame of optic nerve bundles disappeared. The pathological change of injury group III showed more serious than injury group II in any time.1.2 Glee-sliver stain and the examination of integral light density of optic nerve fiberNo significant difference of the integral light density of optic nerve fiber was found among the normal control group, the false injury group and injury group I （P >0.05）.Optic nerve fiber of injury group II and III were sparse. The value of integral light density was lower at 3d after injury. Pathological change deteriorated gradually within 2w. At each point, the change in injury group III was more obvious than that in injury group II. At each point, the integral light density of optic nerve fiber in injury group II and IIIwere lower than normal group（P<0.05）, and degraded gradually. At the same point, the difference of the integral light density of optic nerve fiber among these injury group was statistically significant（P<0.01）.1.3 Ponceau red G-light green stain and the examination of integral light density of myelinThe morphology of optic nerve of false injury group and injury group I were nearly identical with the normal group, and the difference of the integral light density of myelin showed no statistical significance, when we compared respectively false group and injury group I with normal group （P > 0.05）. At 3d after injury, demyelination was observed in injury group II and III. These changes deteriorated gradually with the lapse of time. At each point, the change in injury group III was more obvious than that in injury group II. At each point, the integral light density of myelin in injury group II and III were lower than normal group（P<0.05）, and degraded gradually. At the same point, the difference of the integral light density of myelin among these injury group was statistically significant（P< 0.01）. 2 pathological observation of retina2.1 HE stain and morphological observation of retinaIn normal group and false injury group, the layers of retina were clear. Distribution of RGC was regular intensively, nucleus was distinct. The changes of injury group I were slight. At 3d after injury, nucleus pyknosis of RGC was seen in injury group II and injury group III, then RGC loss were seen within 2w, and the thickness of retina become thin. These pathological changes were more obvious in injury group III. With the lapse of time, these changes deteriorated quickly. At each point, the changes of injury group III were more serious than the injury group II.2.2 Count of RGCAt each point, the number of RGC in false injury group and injury group I were approximate to the normal control group（P>0.05）. The number of RGC both in injury group II and III were fewer than normal group（P<0.01）, and count of RGC decreased continuously. At the same point, the difference of count of RGC among each injury group showed statistically significant（P<0.05）.2.3 Examination of RGC apoptosis and rate of RGC apoptosisThere were few apoptotic RGC in normal control group and false injury group. In each injury group, apoptotic RGC distributed in ganglion cell layer.In injury group I , there was less apoptotic RGC at each point, but the difference of the rate of apoptotic RGC showed no statistically significant（P>0.05） at each time point.In injury group II, the rate of apoptotic RGC increased gradually within 3d～2w after injury, however, the rate of apoptotic RGC among different time points showed statistically significant（P<0.05）.In injury group III, there were more apoptotic RGC at 3d after injury, the number of apoptotic RGC increased gradually within 2w, and among different time points, the rate of apoptotic RGC showed statistically significant（P<0.05）.At the same point, the difference among different injury groups showed extremely statistical significance.3 The result of F-VEPIn normal group, the latency of P₂ wave was （71.72±3.66）ms, and the amplitude of P₂ wave was （20.53±4.15）μv. At each point, the difference of latency and amplitude between false injury group and normal group showed no statistical significance（P> 0.05）.The delay of P₂ wave latency was found in all three injury groups at 1h after injury compared with normal group （P>0.05）. and the amplitude was lower than normal group（P<0.05）.At 1d after injury, latency and amplitude of injury group I had recovered to normal（P>0.05）. But latency and amplitude of injury group II and III deteriorated gradually within 2w.Conclusions1 Stable and reproducible animal model of mild, medium and severe optic nerve crush was established by using the vascular clamp which pressure power was 32g, 98g and 148g, respectively.2 In mild injury group, changes of morphology were slight, and visual function was fine. In medium injury group, pathological changes of optic nerve were obvious, and the injury deteriorated gradually within 2w observing time, however, part visual function remained. In severe injury group, changes of morphology were irreversible sooner after injury, and the visual function lost completely3 The medium optic nerve crush model can be used to observe the therapy effect of different treatment methods of TON.更多还原