氨基胍在兔视神经损伤后对视网膜神经节细胞的保护性作用研究

发布时间：2018-05-11 04:00

本文选题：氨基胍 + 视神经损伤　；参考：《河北联合大学》2011年硕士论文

【摘要】：目的视神经损伤是眼科常见疾病,多并发于颅脑外伤,预后不良,常致患者失明。由于视神经损伤的发病机制尚未完全明了,所以迄今为止其治疗仍是国内外眼科界的一大难题。本试验通过建立兔眼视神经夹伤的动物模型,及伤后早期应用氨基胍(Aminogunidine,AG)治疗,动态观察视神经损伤后视网膜病理形态学改变,研究一氧化氮(NO)、诱导型一氧化氮合酶(iNOS)、丙二醛(MDA)、超氧化物歧化酶(SOD)含量变化及耳缘静脉注射AG对其影响,力求探索一条治疗视神经损伤的新思路,为临床治疗提供实验依据,尽最大可能挽救患者视功能。材料与方法健康成年大耳白兔66只,3-4月龄,雌雄不限,体重2.5±0.2kg,检查双眼屈光间质清,瞳孔等大等圆,对光反射正常,眼底无异常。将实验兔随机分为正常对照组、损伤治疗组、损伤对照组共3组。正常对照组(6只,12只眼):从66只兔中随机抽取6只。损伤治疗组:余60只兔用同一反向动脉夹于球后3mm处夹闭视神经20s制成视神经损伤的动物模型,然后随机抽取30只给予2㳠AG80mg/kg耳缘静脉注射,每日1次;损伤对照组:另外30只耳缘静脉注射等量生理盐水。按照视神经损伤后1d、3d、7d、14d、21d又随机分为5组,每组兔(6只,12只眼)。造模成功后分别于伤后1d、3d、7d、14d、21d处死动物,取出眼球,其中一部分眼球固定、石蜡包埋常规做视网膜切片,进行HE染色及视网膜凋亡细胞(TUNEL)检测,光镜观察视网膜形态改变。另一部分眼球于冰盐水上小心剥下视网膜组织,匀浆,分光光度计检测NO、iNOS、MDA、SOD的含量变化,结果进行图像分析,所得数据资料均用spss13.0软件包进行统计分析。结果 1.视网膜HE染色:正常对照组视网膜内界膜平滑完整,三层细胞分界清楚,节细胞呈单层排列,按胞核大小分为两类:一类大核浅染;另一类小核深染,核内染色质分布均匀。内、外核层细胞均呈多层排列,其厚度、染色均匀,细胞排列整齐紧密。损伤对照组:伤后1d,可见视网膜组织轻度水肿,视网膜各层结构完整;伤后3d,视网膜水肿加重,偶见空泡变性;伤后7d、14d视网膜全层高度水肿,以神经纤维层、神经节细胞层与内丛状层水肿最为明显,各层细胞排列疏松,细胞间隙增大;伤后21d主要表现视网膜水肿减轻,RGCs数量减少,部分细胞空泡样变,内、外核层细胞排列紊乱,细胞数量减少,视网膜内层明显变薄。损伤治疗组:同一时间点损伤治疗组视网膜的病理改变(细胞间隙,组织水肿,细胞减少),均较损伤对照组明显减轻。 2.TUNEL染色凋亡细胞计数和定位:视网膜切片中显示TUNEL阳性细胞(凋亡细胞)不仅见于视网膜神经节细胞层,还见于内外核层。凋亡细胞胞核染色,呈棕黄色,部分可由于核固缩扭曲破裂而失去正常形态,并可见凋亡小体。正常对照组视网膜切片极少见到凋亡细胞。本实验仅计数节细胞层的凋亡情况进行比较研究。同一时间点损伤对照组和损伤治疗组比较,差异具有统计学意义(P0.05)。 3.NO含量,iNOS活力测定:正常视网膜组织中很少表达iNOS,但含有少量NO,在损伤后二者含量逐渐增高,同一时间点损伤对照组和损伤治疗组比较,NO含量和iNOS活性差异有统计学意义(P 0.05)。 4. MDA含量, SOD活力检测:正常视网膜组织匀浆中含一定量的MDA和SOD。同一时间点损伤对照组和损伤治疗组比较,MDA含量差异有统计学意义(P 0.05)。同一时间点损伤对照组和损伤治疗组比较,SOD活性差异有统计学意义(P0.05)。结论: 1.视神经夹伤后,损伤治疗组各时间点视网膜的病理改变,均较损伤对照组明显减轻。提示伤后早期耳缘静脉注射AG进行干预治疗能够及时挽救未受损的视网膜细胞,对维持视网膜的形态和功能起到了重要作用。 2.正常视网膜组织中很少表达iNOS,但含有少量NO,在损伤后二者含量逐渐增高,其结果与视神经损伤后RGCs的凋亡趋势相吻合,说明视神经损伤后NO、iNOS的大量生成是引起RGCs凋亡的一个因素。 3.视神经夹伤后视网膜组织中的MDA含量逐渐升高,随着组织损伤的进一步加重,SOD逐渐减少,削弱了其对视神经视网膜的保护作用。结果说明在视神经损伤后视网膜组织内MDA不断产生,SOD不断被消耗,致使自由基不断堆积,导致细胞出现不可逆损伤。 4.视神经夹伤后早期耳缘静脉注射AG进行干预治疗能够一定程度减少损伤后RGCs的凋亡。提示AG通过抑制iNOS合成减少NO的产生,同时也抑制自由基的生成,对视神经损伤后的RGCs起到保护作用。
[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.

【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R779.1

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