轻、中、重度视神经挫伤的动物模型制作

发布时间：2018-01-22 06:18

本文关键词： 视神经损伤动物模型中度损伤　出处：《郑州大学》2007年硕士论文　论文类型：学位论文

【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／mm~2、1209.88g．s／mm~2、1549.74g．s／mm~2。 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P_2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P_2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P_2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P_2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，P_2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。
[Abstract]:objective
Traumatic optic neuropathy (Traumatic optic, neuropathy, TON) is the brain, eyes and facial trauma with serious complications, visual impairment after injury is serious, often left permanent visual impairment. For a long time, the treatment of TON has always been controversial. The study found that differences in the degree of damage is an important factor to determine the effect of treatment. To establish a standard and reliable, convenient and feasible, can quantify the degree of damage model repeated, can provide an important premise for the treatment of the in-depth study of the mechanism of TON.
The traumatic optic nerve injury model was only qualitative or semi quantitative damage damage quantification model and the lack of a unified standard. The application of three kinds of constant pressure vessel clip were made in rabbits, mild, severe optic nerve injury model, as the animal model of optic nerve injury treatment evaluation.
Materials and methods
1 animals and groups
A total of 48 adult healthy rabbits were used for both sexes. The weight was 2.0-2.5kg and the ocular examination was normal. 12 eyes were randomly selected, and the left and right eyes were randomly divided into normal control group and sham injury control group. The remaining 36 rats were randomly divided into injury group I, injury group II and injury group III.
Determination of 2 clamping force and injury intensity
The clamping force and the intensity of injury (average impulse) were accurately measured and calculated. The clamping force of three kinds of vascular clamps (small, medium and large) were 32g, 98g, 148g and the average impulse was 397.52g.s / mm~21209.88g.s / mm~21549.74g.s / mm~2., respectively.
3 animal model making
The use of small, large, three kinds of micro vascular clamp three animal side of optic nerve injury group 20s respectively, damage group, injury group II, group III damage of different degrees of injury model; sham injury group only exposed side of the optic nerve and does not impose holding; the other eye for the normal control group.
4 observation items and time points
3D after injury, injury and histopathological changes of partial optic nerve 1W and 2W observation of optic nerve Glee silver staining or Ponceau G brilliant green staining of optic nerve fibers and myelin integral optical density determination; and retinal morphology observation of retinal ganglion cells (Retinal Ganglion Cell, RGC) RGC apoptosis detection and counting. The apoptosis rate of the RGC calculation. After injured 1H, 6h, 1D, 3D, 1W F-VEP, to observe the latency and amplitude of P2 wave changes between the 5 groups.
5 statistical analysis
The data were statistically treated with single factor ANOVA and t test.
Result
Local pathological observation of 1 optic nerve injury
Local HE staining and histomorphological observation of 1.1 optic nerve injuries
The normal group and the sham injury group of optic nerve fibers arranged dense rules, uniform dyeing, has a small amount of glial cells. The damage group compared with normal group changed slightly. Injury group II 3D optic nerve edema, focal infarct were seen, glial cells arranged in disorder, with the passage of time, change the nerve edema obvious damage III increased. Group 3D, with multiple necrosis, with the passage of time, the rapid development of lesions, disappearance of nerve bundle structure of 2W. The time of injury group III changes compared with injury group II.
1.2 optic nerve fiber Glee immersion silver staining and integral light density measurement results
Each time point of the sham injury group and normal group of optic nerve morphology consistent; optic nerve fiber integral optical density compared with the normal group, no significant difference (P > 0.05). Each time I group of injury optic nerve morphology similar to the normal group; optic nerve fiber integral optical density lower than normal group, but no significant the difference (P > 0.05). Group II and III injury injury group 3D optic nerve fiber sparse distortions, over time, gradually changed obviously, each time point compared with injury group III injury II group changed significantly; the two groups at each time point of optic nerve fiber integral optical density were lower than the normal group, the difference is significant (P < 0.05), with the passage of time, the optic nerve fiber integral optical density was decreased. At the same time, between the injury group of optic nerve fiber integral optical density, there was significant difference (P < 0.01).
1.3 optic nerve sections with Ponceau G brilliant green staining and myelin integral optical density determination results
Each time point of the sham injury group and normal group of optic nerve morphology consistent; myelin integral optical density compared with the normal group, no significant difference (P > 0.05). Each time I group of injury optic nerve morphology similar to the normal group; myelin integral optical density lower than normal group, but no significant difference (P > 0.05). Injury group II and group III 3D injury have demyelination, over time, gradually changed significantly, a large number of myelin disintegration injury group III 2W, nerve bundle structure disappeared, each time point compared with injury group III injury II group changed significantly; the two group at each time point medullary sheath integral optical density were lower than the normal group, there was significant difference (P < 0.05), with the passage of time, myelin integral optical density was decreased. At the same time, compare the integral optical density between the myelin injury group, there was significant difference (P < 0.01).
2 Pathological Observation of retina
Histomorphological observation of 2.1 retinal HE staining
The normal group and the sham injury group of retinal ganglion cells showed a clear hierarchy, monolayer, and dense, clear nucleus, nuclear membrane was smooth and complete. Injury group compared with normal group. RGC change slightly injury group II 3D nuclear condensation and deep staining, reducing the number of; then each retinal layer becomes thinner, changes with time progressive. The emergence of a large number of RGC injury group III 3D nuclear condensation and deep staining, RGC arrangement was sparse, with the time of disease rapidly worse. At each time point compared with the injury group III group II lesions.
2.2 RGC count
Each time point of the sham injury group RGC compared with the normal group, no significant difference (P > 0.05). Each time I damage the number of RGC was less than that of normal group, but no significant difference (P > 0.05). Each time point in group II and III injury, the number of RGC was compared with the normal group, were decreased, there was significant difference (P < 0.01), the number of RGC were decreased with time. At the same time, the number of RGC between the injury group, there was significant difference (P < 0.05).
2.3 RGC apoptosis detection and average apoptosis rate
The normal group and the sham injury group showed no retinal slice apoptosis. Apoptotic cells mainly located in the injury group, GCL. injury group the apoptotic cells, no significant difference between each time point, apoptosis rate of RGC (P > 0.05). Injury group II cell apoptosis with time increasing, there was significant different between RGC apoptosis rate difference (P < 0.05). A large number of apoptotic cells in group III 3D damage was increased with time, the apoptosis rate of RGC, there were significant differences between each time point, apoptosis rate of RGC (P < 0.05). At the same time, the apoptosis rate of RGC injury group was very significant the difference (P < 0.01).
3 F-VEP detection results
The normal rabbit P_2 latency and amplitude were (71.72 + 3.66) ms, (20.53 + 4.15) P_2 wave latency and amplitude of V. at each time point of the sham injury group and normal group, no significant difference (P > 0.05). 1h after injury, the injury group showed P_2 wave latency. The amplitude decreased obviously compared with normal group (P < 0.05). Injury group I 1D P_2 wave amplitude and latency recovered (P > 0.05). Group II and III injury injury group, with the passage of time, the latency of P_2 was delayed, the amplitude was decreased.
conclusion
1 the microvascular clips of 32g, 98g and 148g were sandwiched with the rabbit optic nerve 20s, which could be used to produce stable, repeatable animal models of light, moderate, and severe optic nerve injury.
2 mild injury group after optic nerve morphology changes slightly, no significant pathological changes of optic nerve conduction function; moderate injury group after optic nerve morphology changed obviously, progressive damage over time, but there are certain optic nerve conduction function; severe injury group rapid irreversible optic nerve degeneration, conduction function optic nerve injury is completely lost.
3 the model of moderate optic nerve injury can be used as an animal model to observe the therapeutic methods and effects of traumatic optic neuropathy.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R-332

【参考文献】