本文選題：氨基胍 + 視神經(jīng)損傷　； 參考：《河北聯(lián)合大學》2011年碩士論文

【摘要】：目的 視神經(jīng)損傷是眼科常見疾病,多并發(fā)于顱腦外傷,預后不良,常致患者失明。由于視神經(jīng)損傷的發(fā)病機制尚未完全明了,所以迄今為止其治療仍是國內外眼科界的一大難題。本試驗通過建立兔眼視神經(jīng)夾傷的動物模型,及傷后早期應用氨基胍(Aminogunidine,AG)治療,動態(tài)觀察視神經(jīng)損傷后視網(wǎng)膜病理形態(tài)學改變,研究一氧化氮(NO)、誘導型一氧化氮合酶(iNOS)、丙二醛(MDA)、超氧化物歧化酶(SOD)含量變化及耳緣靜脈注射AG對其影響,力求探索一條治療視神經(jīng)損傷的新思路,為臨床治療提供實驗依據(jù),盡最大可能挽救患者視功能。 材料與方法 健康成年大耳白兔66只,3-4月齡,雌雄不限,體重2.5±0.2kg,檢查雙眼屈光間質清,瞳孔等大等圓,對光反射正常,眼底無異常。將實驗兔隨機分為正常對照組、損傷治療組、損傷對照組共3組。正常對照組(6只,12只眼):從66只兔中隨機抽取6只。損傷治療組:余60只兔用同一反向動脈夾于球后3mm處夾閉視神經(jīng)20s制成視神經(jīng)損傷的動物模型,然后隨機抽取30只給予2㳠AG80mg/kg耳緣靜脈注射,每日1次;損傷對照組:另外30只耳緣靜脈注射等量生理鹽水。按照視神經(jīng)損傷后1d、3d、7d、14d、21d又隨機分為5組,每組兔(6只,12只眼)。 造模成功后分別于傷后1d、3d、7d、14d、21d處死動物,取出眼球,其中一部分眼球固定、石蠟包埋常規(guī)做視網(wǎng)膜切片,進行HE染色及視網(wǎng)膜凋亡細胞(TUNEL)檢測,光鏡觀察視網(wǎng)膜形態(tài)改變。另一部分眼球于冰鹽水上小心剝下視網(wǎng)膜組織,勻漿,分光光度計檢測NO、iNOS、MDA、SOD的含量變化,結果進行圖像分析,所得數(shù)據(jù)資料均用spss13.0軟件包進行統(tǒng)計分析。 結果 1.視網(wǎng)膜HE染色:正常對照組視網(wǎng)膜內界膜平滑完整,三層細胞分界清楚,節(jié)細胞呈單層排列,按胞核大小分為兩類:一類大核淺染;另一類小核深染,核內染色質分布均勻。內、外核層細胞均呈多層排列,其厚度、染色均勻,細胞排列整齊緊密。 損傷對照組:傷后1d,可見視網(wǎng)膜組織輕度水腫,視網(wǎng)膜各層結構完整;傷后3d,視網(wǎng)膜水腫加重,偶見空泡變性;傷后7d、14d視網(wǎng)膜全層高度水腫,以神經(jīng)纖維層、神經(jīng)節(jié)細胞層與內叢狀層水腫最為明顯,各層細胞排列疏松,細胞間隙增大;傷后21d主要表現(xiàn)視網(wǎng)膜水腫減輕,RGCs數(shù)量減少,部分細胞空泡樣變,內、外核層細胞排列紊亂,細胞數(shù)量減少,視網(wǎng)膜內層明顯變薄。 損傷治療組:同一時間點損傷治療組視網(wǎng)膜的病理改變(細胞間隙,組織水腫,細胞減少),均較損傷對照組明顯減輕。 2.TUNEL染色凋亡細胞計數(shù)和定位:視網(wǎng)膜切片中顯示TUNEL陽性細胞(凋亡細胞)不僅見于視網(wǎng)膜神經(jīng)節(jié)細胞層,還見于內外核層。凋亡細胞胞核染色,呈棕黃色,部分可由于核固縮扭曲破裂而失去正常形態(tài),并可見凋亡小體。正常對照組視網(wǎng)膜切片極少見到凋亡細胞。本實驗僅計數(shù)節(jié)細胞層的凋亡情況進行比較研究。同一時間點損傷對照組和損傷治療組比較,差異具有統(tǒng)計學意義(P0.05)。 3.NO含量,iNOS活力測定:正常視網(wǎng)膜組織中很少表達iNOS,但含有少量NO,在損傷后二者含量逐漸增高,同一時間點損傷對照組和損傷治療組比較,NO含量和iNOS活性差異有統(tǒng)計學意義(P 0.05)。 4. MDA含量, SOD活力檢測:正常視網(wǎng)膜組織勻漿中含一定量的MDA和SOD。同一時間點損傷對照組和損傷治療組比較,MDA含量差異有統(tǒng)計學意義(P 0.05)。同一時間點損傷對照組和損傷治療組比較,SOD活性差異有統(tǒng)計學意義(P0.05)。 結論: 1.視神經(jīng)夾傷后,損傷治療組各時間點視網(wǎng)膜的病理改變,均較損傷對照組明顯減輕。提示傷后早期耳緣靜脈注射AG進行干預治療能夠及時挽救未受損的視網(wǎng)膜細胞,對維持視網(wǎng)膜的形態(tài)和功能起到了重要作用。 2.正常視網(wǎng)膜組織中很少表達iNOS,但含有少量NO,在損傷后二者含量逐漸增高,其結果與視神經(jīng)損傷后RGCs的凋亡趨勢相吻合,說明視神經(jīng)損傷后NO、iNOS的大量生成是引起RGCs凋亡的一個因素。 3.視神經(jīng)夾傷后視網(wǎng)膜組織中的MDA含量逐漸升高,隨著組織損傷的進一步加重,SOD逐漸減少,削弱了其對視神經(jīng)視網(wǎng)膜的保護作用。結果說明在視神經(jīng)損傷后視網(wǎng)膜組織內MDA不斷產(chǎn)生,SOD不斷被消耗,致使自由基不斷堆積,導致細胞出現(xiàn)不可逆損傷。 4.視神經(jīng)夾傷后早期耳緣靜脈注射AG進行干預治療能夠一定程度減少損傷后RGCs的凋亡。提示AG通過抑制iNOS合成減少NO的產(chǎn)生,同時也抑制自由基的生成,對視神經(jīng)損傷后的RGCs起到保護作用。
[Abstract]:objective
Optic nerve injury is a common disease in the ophthalmology, complicated with craniocerebral trauma and poor prognosis, and often leads to blindness. Because the pathogenesis of optic nerve injury is not completely clear, so far, the treatment is still a major problem in the field of Ophthalmology at home and abroad. This experiment has established an animal model of the rabbit eye nerve clamp injury and the early application of ammonia after injury. Aminogunidine (AG) was used to dynamically observe the pathological changes of retina after optic nerve injury, and study the changes of nitric oxide (NO), inducible nitric oxide synthase (iNOS), malondialdehyde (MDA), superoxide dismutase (SOD) and the influence of AG on the ear vein injection, and try to explore a new idea for the treatment of optic nerve injury. Treatment provides experimental evidence to maximize patient's visual function.
Materials and methods
66 healthy adult white rabbits, 3-4 months old, male and male, 2.5 + 0.2kg, checked the diopter interstitial clear, the pupil and so on, the light reflex was normal and the fundus had no abnormality. The experimental rabbits were randomly divided into normal control group, injury treatment group and the control group 3 groups. The normal control group (6, 12 eyes): 6 rabbits were randomly selected from 66 rabbits. The treatment group: the remaining 60 rabbits used the same reverse artery to clamp the optic nerve 20s into the optic nerve injury in the same reverse artery at 3mm after the ball, and then randomly selected 30 to give 2? AG80mg/kg ear vein injection, 1 times a day, and the injured control group: the other 30 ear veins were injected with the same amount of normal saline. After the optic nerve injury, 1D, 3D, 7d, 14d, 21d were random. There were 5 groups of rabbits in each group (6, 12 eyes).
After the success of the model, the animals were killed at 1D, 3D, 7d, 14d, and 21d respectively. The eyeballs were taken out. Some of them were fixed and paraffin embedded routine retinal section, HE staining and retinal apoptotic cells (TUNEL) detection, and the retinal morphological changes were observed by light microscope. The other part of the eyeball was carefully stripped off the retina tissue, homogenate, and light. Photometric detection of NO, iNOS, MDA, SOD content changes, the results of image analysis, data obtained by SPSS13.0 software package for statistical analysis.
Result
1. retinal HE staining: the retinal inner boundary membrane in the normal control group was smooth and complete, the three layers of cells separated clearly and the ganglion cells were arranged in single layer. The nucleus of the nucleus was divided into two types according to the nucleus size of the nucleus: a kind of large nucleus light staining; the other kind of nuclei deep dyed, and the chromatin in the nucleus was evenly distributed. The outer nucleus layer cells were arranged in multilayer, the thickness, the coloring uniformity and the orderly and tight arrangement of the cells.
Injury control group: 1D after injury, the retina tissue was mild edema, and the structure of retina was complete. After injury, 3D, retinal edema aggravated, and occasionally vacuolated degeneration, 7d, 14d retinal edema after injury, the most obvious edema of the retinal layer, ganglion cell layer and inner plexiform layer, the cells in each layer were loosely arranged and the cell space increased; 2 after injury. The main manifestations of 1D were the reduction of retinal edema, the decrease of the number of RGCs, the vacuolation of some cells, the disorder of the outer nuclear layer cells, the decrease of the number of cells, and the obvious thinning of the inner retina.
Injury treatment group: at the same time point, the pathological changes of the retina in the injury treatment group (cell gap, tissue edema, cell reduction) were significantly reduced compared with the injury control group.
2.TUNEL staining apoptotic cells count and locate: the retinal slices showed that TUNEL positive cells (apoptotic cells) were not only seen in the retinal ganglion cell layer, but also in the inner and outer nuclear layers. The nuclei of the apoptotic cells were stained brown and yellow, and the apoptotic bodies could be lost in part due to the distortion and rupture of the nucleus. The apoptotic cells were rarely seen in the membrane slices. The apoptosis of the ganglion cell layer was compared in this experiment. The difference was statistically significant (P0.05) compared with the injury control group and the injury treatment group at the same time point.
The determination of 3.NO content and iNOS activity: iNOS was rarely expressed in normal retina, but a small amount of NO was contained, and the content of the two was increased gradually after injury. Compared with the injury control group and the injury treatment group, the difference of NO content and iNOS activity was statistically significant (P 0.05) at the same time point.
4. MDA content, SOD activity detection: normal retinal tissue homogenate containing a certain amount of MDA and SOD. in the same time point damage control group compared with the injury treatment group, MDA content difference was statistically significant (P 0.05). Compared with the same time point injury control group and the injury treatment group, SOD survival difference was statistically significant (P0.05).
Conclusion:
After 1. optic nerve clamp injury, the pathological changes of retina at all time points in the injury treatment group were significantly lower than that of the damage control group. It was suggested that the early ear vein injection of AG after injury could save the undamaged retinal cells in time and play an important role in maintaining the morphology and function of the retina.
2. in normal retina, iNOS is rarely expressed, but a small amount of NO is contained, and the content of the two is increased gradually after the injury. The result is consistent with the apoptosis trend of RGCs after the optic nerve injury, indicating that after the optic nerve injury, the formation of NO, iNOS is a factor that causes the apoptosis of RGCs.
3. the content of MDA in the retinal tissue was gradually increased after the clipping of the optic nerve. With the further aggravation of the tissue damage, the SOD decreased gradually and weakened its protective effect on the optic retina. The results showed that MDA produced in the retinal tissue after the optic nerve injury, and the SOD was constantly dissipated, resulting in the continuous accumulation of free radicals, resulting in the appearance of cells. Irreversible damage.
4. the early auricular vein injection of AG after the clipping of the optic nerve can reduce the apoptosis of RGCs after injury to a certain extent. It suggests that AG reduces the production of NO by inhibiting the iNOS synthesis and also inhibits the formation of free radicals, and protects the RGCs after the optic nerve injury.

【學位授予單位】：河北聯(lián)合大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R779.1

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5 曾勇;視神經(jīng)損傷大鼠視網(wǎng)膜神經(jīng)節(jié)細胞軸突再生與保護研究[D];昆明醫(yī)學院;2009年

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8 喬彤;Slit_2-siRNA玻璃體腔注射對急慢性高眼壓大鼠模型視網(wǎng)膜神經(jīng)節(jié)細胞凋亡的影響[D];華中科技大學;2009年

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10 陳芝清;鈣蛋白酶在高眼壓誘導的視網(wǎng)膜缺血再灌注損傷發(fā)生機制中的作用[D];浙江大學;2008年

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1 孫文婷;黃體酮對大鼠視神經(jīng)損傷修復的影響[D];河北醫(yī)科大學;2012年

2 張俊琦;氨基胍在兔視神經(jīng)損傷后對視網(wǎng)膜神經(jīng)節(jié)細胞的保護性作用研究[D];河北聯(lián)合大學;2011年

3 馬曉蕾;視神經(jīng)損傷后視網(wǎng)膜Müller細胞未折疊蛋白反應及與GLAST的關系[D];第四軍醫(yī)大學;2011年

4 王瑞嘉;視神經(jīng)損傷后視網(wǎng)膜神經(jīng)節(jié)細胞中腫瘤壞死因子在視神經(jīng)管減壓術后和藥物干預的免疫活性研究[D];新疆醫(yī)科大學;2012年

5 徐e，

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