本文關鍵詞： 視神經損傷 動物模型 中度損傷　出處：《鄭州大學》2007年碩士論文　論文類型：學位論文

【摘要】： 目的 外傷性視神經病變(Traumatic optic neuropathy，TON)是顱腦、眼眶和面部外傷嚴重的并發(fā)癥，傷后視力損害嚴重，常遺留永久性視力障礙。長期以來，關于TON的治療一直存在著爭議。研究發(fā)現，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、可重復的不同程度量化損傷模型，可為深入研究TON的治療機制提供重要前提。 以往的外傷性視神經損傷模型僅是定性損傷或是半定量損傷，而缺乏統(tǒng)一標準的量化模型。本實驗應用三種壓力恒定的血管夾擬造成兔輕、中、重度視神經損傷模型，作為視神經損傷治療效果評價的動物模型。 材料和方法 1動物與分組 成年健康白兔48只，雌雄兼用，體重2.0-2.5kg，眼部檢查正常。隨機抽取12只，左右眼隨機分為正常對照組和假傷對照組，剩余36只隨機分為損傷Ⅰ組、損傷Ⅱ組、損傷Ⅲ組。 2夾持力和致傷強度測定 精確測定和計算血管夾的夾持力和致傷強度(平均沖量)。三種血管夾(小、中、大號)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動物模型制作 使用小、中、大號三種顯微血管夾夾持三個損傷組動物一側眼視神經各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眼視神經而不施加夾持；另一眼作為正常對照。 4觀察項目和時間點 傷后3d、1w及2w觀察視神經損傷局部的組織病理學改變、進行視神經Glee銀染或麗春紅G—亮綠染色后視神經纖維及髓鞘積分光密度測定；以及視網膜的形態(tài)學觀察、視網膜神經節(jié)細胞(Retinal Ganglion Cell，RGC)計數、RGC凋亡檢測和RGC凋亡率計算。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)計學分析 數據運用單因素方差分析和t檢驗進行統(tǒng)計學處理。 結果 1視神經損傷局部的病理學觀察結果 1．1視神經損傷局部的HE染色和組織形態(tài)學觀察 正常組及假傷組視神經纖維排列密集規(guī)則，染色均勻，有少量神經膠質細胞。損傷Ⅰ組與正常組相比改變輕微。損傷Ⅱ組3d時視神經水腫，軸心區(qū)有梗死灶，膠質細胞排列紊亂，隨時間推移，改變加重。損傷Ⅲ組3d時視神經水腫明顯，有多處壞死，隨時間推移，病變迅速發(fā)展，2w時神經束結構消失。各時間點損傷Ⅲ組改變較損傷Ⅱ組嚴重。 1．2視神經纖維Glee浸銀染色和積分光密度測定結果 各時間點假傷組視神經形態(tài)與正常組基本一致；視神經纖維積分光密度與正常組比較，無顯著性差異(P＞0.05)。各時間點損傷Ⅰ組視神經形態(tài)與正常組相似；視神經纖維積分光密度較正常組降低，但無顯著性差異(P＞0.05)。損傷Ⅱ組及損傷Ⅲ組3d時視神經纖維稀疏扭曲，隨時間推移，改變漸明顯，各時間點損傷Ⅲ組較損傷Ⅱ組改變明顯；兩組在各時間點視神經纖維積分光密度均較正常組降低，差異有顯著性(P＜0.05)，隨時間推移，視神經纖維積分光密度進行性降低。在同一時間點，各損傷組之間視神經纖維積分光密度比較，差異有非常顯著性(P＜0.01)。 1．3視神經切片麗春紅G—亮綠染色和髓鞘積分光密度測定結果 各時間點假傷組視神經形態(tài)與正常組基本一致；髓鞘積分光密度與正常組比較，無顯著性差異(P＞0.05)。各時間點損傷Ⅰ組視神經形態(tài)與正常組相似；髓鞘積分光密度較正常組降低，但無顯著性差異(P＞0.05)。損傷Ⅱ組及損傷Ⅲ組3d時有髓鞘脫失，隨時間推移，改變漸明顯，2w時損傷Ⅲ組大量髓鞘崩解，神經束結構基本消失，各時間點損傷Ⅲ組較損傷Ⅱ組改變明顯；兩組在各時間點髓鞘積分光密度均較正常組降低，差異有顯著性(P＜0.05)，隨時間推移，髓鞘積分光密度進行性降低。在同一時間點，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視網膜的病理學觀察結果 2．1視網膜HE染色組織形態(tài)學觀察 正常組及假傷組視網膜層次清晰，節(jié)細胞呈單層排列，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RGC出現核固縮，染色加深，數量減少；隨后視網膜各層變薄，病變隨時間進行性加重。損傷Ⅲ組3d時大量RGC出現核固縮，染色加深，RGC排列明顯稀疏，隨時間進行病變迅速加重。各時間點損傷Ⅲ組較Ⅱ組病變明顯。 2．2 RGC計數 各時間點假傷組RGC數與正常組比較，無顯著性差異(P＞0.05)。各時間點損傷Ⅰ組RGC數較正常組減少，，但無顯著性差異(P＞0.05)。各時間點損傷Ⅱ組及Ⅲ組RGC數與正常組相比，均明顯減少，差異有非常顯著性(P＜0.01)，RGC數隨時間進行性減少。在同一時間點，各損傷組之間RGC數比較，差異有顯著性(P＜0.05)。 2．3 RGC凋亡的檢測和平均凋亡率 正常組及假傷組視網膜切片未見凋亡細胞。各損傷組的凋亡細胞主要位于GCL。損傷Ⅰ組有少量凋亡細胞，各時間點RGC凋亡率之間無顯著性差異(P＞0.05)。損傷Ⅱ組凋亡細胞隨時間進行性增多，各時間點RGC凋亡率之間差異有顯著性(P＜0.05)。損傷Ⅲ組3d時出現大量凋亡細胞，RGC凋亡率隨時間進行性增高，各時間點RGC凋亡率之間差異有顯著性(P＜0.05)。在同一時間點，各損傷組之間RGC凋亡率有非常顯著性差異(P＜0.01)。 3 F-VEP檢測結果 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。各時間點假傷組與正常組的P_2波潛伏期和幅值比較，差異無顯著性(P＞0.05)。損傷后1h，各損傷組均出現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P_2波潛伏期及幅值基本恢復正常(P＞0.05)。損傷Ⅱ組及損傷Ⅲ組，隨時間推移，P_2波潛伏期進行性延遲，幅值進行性降低。 結論 1應用壓力為32g、98g、148g的顯微血管夾夾持兔視神經20s，可制作穩(wěn)定、可重復的輕、中、重度視神經損傷動物模型。 2輕度損傷組傷后視神經形態(tài)學改變輕微，無顯著病理改變，視神經傳導功能良好；中度損傷組傷后視神經形態(tài)學改變明顯，隨時間推移損傷進行性加重，但視神經有一定傳導功能；重度損傷組傷后視神經迅速出現不可逆性潰變，傷后視神經傳導功能完全喪失。 3中度視神經損傷模型可作為觀察外傷性視神經病變的治療方法和效果的的動物模型。
[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.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R-332

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