本文關(guān)鍵詞： 視神經(jīng)損傷 動(dòng)物模型 中度損傷　出處：《鄭州大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的 外傷性視神經(jīng)病變(Traumatic optic neuropathy，TON)是顱腦、眼眶和面部外傷嚴(yán)重的并發(fā)癥，傷后視力損害嚴(yán)重，常遺留永久性視力障礙。長(zhǎng)期以來(lái)，關(guān)于TON的治療一直存在著爭(zhēng)議。研究發(fā)現(xiàn)，損傷程度的差異是決定治療效果的重要因素。而建立規(guī)范可靠、方便易行、可重復(fù)的不同程度量化損傷模型，可為深入研究TON的治療機(jī)制提供重要前提。 以往的外傷性視神經(jīng)損傷模型僅是定性損傷或是半定量損傷，而缺乏統(tǒng)一標(biāo)準(zhǔn)的量化模型。本實(shí)驗(yàn)應(yīng)用三種壓力恒定的血管夾擬造成兔輕、中、重度視神經(jīng)損傷模型，作為視神經(jīng)損傷治療效果評(píng)價(jià)的動(dòng)物模型。 材料和方法 1動(dòng)物與分組 成年健康白兔48只，雌雄兼用，體重2.0-2.5kg，眼部檢查正常。隨機(jī)抽取12只，左右眼隨機(jī)分為正常對(duì)照組和假傷對(duì)照組，剩余36只隨機(jī)分為損傷Ⅰ組、損傷Ⅱ組、損傷Ⅲ組。 2夾持力和致傷強(qiáng)度測(cè)定 精確測(cè)定和計(jì)算血管夾的夾持力和致傷強(qiáng)度(平均沖量)。三種血管夾(小、中、大號(hào))夾持力分別為：32g、98g、148g，平均沖量分別為：397.52g．s／mm~2、1209.88g．s／mm~2、1549.74g．s／mm~2。 3動(dòng)物模型制作 使用小、中、大號(hào)三種顯微血管夾夾持三個(gè)損傷組動(dòng)物一側(cè)眼視神經(jīng)各20s，分別造成損傷Ⅰ組、損傷Ⅱ組、損傷Ⅲ組不同程度損傷模型；假傷組僅分離暴露一側(cè)眼視神經(jīng)而不施加夾持；另一眼作為正常對(duì)照。 4觀察項(xiàng)目和時(shí)間點(diǎn) 傷后3d、1w及2w觀察視神經(jīng)損傷局部的組織病理學(xué)改變、進(jìn)行視神經(jīng)Glee銀染或麗春紅G—亮綠染色后視神經(jīng)纖維及髓鞘積分光密度測(cè)定；以及視網(wǎng)膜的形態(tài)學(xué)觀察、視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(Retinal Ganglion Cell，RGC)計(jì)數(shù)、RGC凋亡檢測(cè)和RGC凋亡率計(jì)算。傷后1h、6h、1d、3d、1w行F-VEP檢查，觀察5組間P2波潛伏期和波幅變化。 5統(tǒng)計(jì)學(xué)分析 數(shù)據(jù)運(yùn)用單因素方差分析和t檢驗(yàn)進(jìn)行統(tǒng)計(jì)學(xué)處理。 結(jié)果 1視神經(jīng)損傷局部的病理學(xué)觀察結(jié)果 1．1視神經(jīng)損傷局部的HE染色和組織形態(tài)學(xué)觀察 正常組及假傷組視神經(jīng)纖維排列密集規(guī)則，染色均勻，有少量神經(jīng)膠質(zhì)細(xì)胞。損傷Ⅰ組與正常組相比改變輕微。損傷Ⅱ組3d時(shí)視神經(jīng)水腫，軸心區(qū)有梗死灶，膠質(zhì)細(xì)胞排列紊亂，隨時(shí)間推移，改變加重。損傷Ⅲ組3d時(shí)視神經(jīng)水腫明顯，有多處壞死，隨時(shí)間推移，病變迅速發(fā)展，2w時(shí)神經(jīng)束結(jié)構(gòu)消失。各時(shí)間點(diǎn)損傷Ⅲ組改變較損傷Ⅱ組嚴(yán)重。 1．2視神經(jīng)纖維Glee浸銀染色和積分光密度測(cè)定結(jié)果 各時(shí)間點(diǎn)假傷組視神經(jīng)形態(tài)與正常組基本一致；視神經(jīng)纖維積分光密度與正常組比較，無(wú)顯著性差異(P＞0.05)。各時(shí)間點(diǎn)損傷Ⅰ組視神經(jīng)形態(tài)與正常組相似；視神經(jīng)纖維積分光密度較正常組降低，但無(wú)顯著性差異(P＞0.05)。損傷Ⅱ組及損傷Ⅲ組3d時(shí)視神經(jīng)纖維稀疏扭曲，隨時(shí)間推移，改變漸明顯，各時(shí)間點(diǎn)損傷Ⅲ組較損傷Ⅱ組改變明顯；兩組在各時(shí)間點(diǎn)視神經(jīng)纖維積分光密度均較正常組降低，差異有顯著性(P＜0.05)，隨時(shí)間推移，視神經(jīng)纖維積分光密度進(jìn)行性降低。在同一時(shí)間點(diǎn)，各損傷組之間視神經(jīng)纖維積分光密度比較，差異有非常顯著性(P＜0.01)。 1．3視神經(jīng)切片麗春紅G—亮綠染色和髓鞘積分光密度測(cè)定結(jié)果 各時(shí)間點(diǎn)假傷組視神經(jīng)形態(tài)與正常組基本一致；髓鞘積分光密度與正常組比較，無(wú)顯著性差異(P＞0.05)。各時(shí)間點(diǎn)損傷Ⅰ組視神經(jīng)形態(tài)與正常組相似；髓鞘積分光密度較正常組降低，但無(wú)顯著性差異(P＞0.05)。損傷Ⅱ組及損傷Ⅲ組3d時(shí)有髓鞘脫失，隨時(shí)間推移，改變漸明顯，2w時(shí)損傷Ⅲ組大量髓鞘崩解，神經(jīng)束結(jié)構(gòu)基本消失，各時(shí)間點(diǎn)損傷Ⅲ組較損傷Ⅱ組改變明顯；兩組在各時(shí)間點(diǎn)髓鞘積分光密度均較正常組降低，差異有顯著性(P＜0.05)，隨時(shí)間推移，髓鞘積分光密度進(jìn)行性降低。在同一時(shí)間點(diǎn)，各損傷組之間髓鞘積分光密度比較，差異有非常顯著性(P＜0.01)。 2視網(wǎng)膜的病理學(xué)觀察結(jié)果 2．1視網(wǎng)膜HE染色組織形態(tài)學(xué)觀察 正常組及假傷組視網(wǎng)膜層次清晰，節(jié)細(xì)胞呈單層排列，整齊密集，胞核清楚，核膜光滑完整。損傷Ⅰ組與正常組相比改變輕微。損傷Ⅱ組3d時(shí)RGC出現(xiàn)核固縮，染色加深，數(shù)量減少；隨后視網(wǎng)膜各層變薄，病變隨時(shí)間進(jìn)行性加重。損傷Ⅲ組3d時(shí)大量RGC出現(xiàn)核固縮，染色加深，RGC排列明顯稀疏，隨時(shí)間進(jìn)行病變迅速加重。各時(shí)間點(diǎn)損傷Ⅲ組較Ⅱ組病變明顯。 2．2 RGC計(jì)數(shù) 各時(shí)間點(diǎn)假傷組RGC數(shù)與正常組比較，無(wú)顯著性差異(P＞0.05)。各時(shí)間點(diǎn)損傷Ⅰ組RGC數(shù)較正常組減少，，但無(wú)顯著性差異(P＞0.05)。各時(shí)間點(diǎn)損傷Ⅱ組及Ⅲ組RGC數(shù)與正常組相比，均明顯減少，差異有非常顯著性(P＜0.01)，RGC數(shù)隨時(shí)間進(jìn)行性減少。在同一時(shí)間點(diǎn)，各損傷組之間RGC數(shù)比較，差異有顯著性(P＜0.05)。 2．3 RGC凋亡的檢測(cè)和平均凋亡率 正常組及假傷組視網(wǎng)膜切片未見(jiàn)凋亡細(xì)胞。各損傷組的凋亡細(xì)胞主要位于GCL。損傷Ⅰ組有少量凋亡細(xì)胞，各時(shí)間點(diǎn)RGC凋亡率之間無(wú)顯著性差異(P＞0.05)。損傷Ⅱ組凋亡細(xì)胞隨時(shí)間進(jìn)行性增多，各時(shí)間點(diǎn)RGC凋亡率之間差異有顯著性(P＜0.05)。損傷Ⅲ組3d時(shí)出現(xiàn)大量凋亡細(xì)胞，RGC凋亡率隨時(shí)間進(jìn)行性增高，各時(shí)間點(diǎn)RGC凋亡率之間差異有顯著性(P＜0.05)。在同一時(shí)間點(diǎn)，各損傷組之間RGC凋亡率有非常顯著性差異(P＜0.01)。 3 F-VEP檢測(cè)結(jié)果 正常白兔P_2波潛伏期及波幅分別為(71.72±3.66)ms、(20.53±4.15)μv。各時(shí)間點(diǎn)假傷組與正常組的P_2波潛伏期和幅值比較，差異無(wú)顯著性(P＞0.05)。損傷后1h，各損傷組均出現(xiàn)P_2波潛伏期延遲，幅值降低，與正常組相比差異均有顯著性(P＜0.05)。損傷Ⅰ組1d時(shí)P_2波潛伏期及幅值基本恢復(fù)正常(P＞0.05)。損傷Ⅱ組及損傷Ⅲ組，隨時(shí)間推移，P_2波潛伏期進(jìn)行性延遲，幅值進(jìn)行性降低。 結(jié)論 1應(yīng)用壓力為32g、98g、148g的顯微血管夾夾持兔視神經(jīng)20s，可制作穩(wěn)定、可重復(fù)的輕、中、重度視神經(jīng)損傷動(dòng)物模型。 2輕度損傷組傷后視神經(jīng)形態(tài)學(xué)改變輕微，無(wú)顯著病理改變，視神經(jīng)傳導(dǎo)功能良好；中度損傷組傷后視神經(jīng)形態(tài)學(xué)改變明顯，隨時(shí)間推移損傷進(jìn)行性加重，但視神經(jīng)有一定傳導(dǎo)功能；重度損傷組傷后視神經(jīng)迅速出現(xiàn)不可逆性潰變，傷后視神經(jīng)傳導(dǎo)功能完全喪失。 3中度視神經(jīng)損傷模型可作為觀察外傷性視神經(jīng)病變的治療方法和效果的的動(dòng)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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R-332

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