【摘要】 目的：研究慢性高眼压大鼠视网膜髓鞘相关抑制蛋白NogoA表达的变化。 方法：成年雄性Wistar大鼠36只随机分为正常对照组6只和慢性高眼压组30只，应用免疫组织化学方法观察慢性高眼压大鼠视网膜不同时点NogoA表达的变化。结果：与正常对照组相比，慢性高眼压21d大鼠视网膜变薄，节细胞数量减少(P<0.05); NogoA表达增多，与形态学变化相一致(P<0.05)。结论：髓鞘相关抑制蛋白NogoA在慢性高眼压大鼠视网膜损伤过程中发挥了重要作用。
【关键词】 视网膜;慢性高眼压;NogoA;视网膜神经节细胞
Expression of NogoA on the retina in rat model with chronic ocular hypertension
QingZhu Nie, ZhiLi Liu, Qian Sha, DianWen Gao
Foundation item: Scientific and Technological Research Funded Projects in Liaoning Province, China (No.2009225021 )
1Department of Ophthalmology, Shengjing Hospital Affiliated to China Medical University, Shenyang 110004, Liaoning Province, China
2Heilongjiang Eye Hospital, Harbin 150001,
Heilongjiang Province, China
Department of Ophthalmology, Shengjing Hospital Affiliated to China Medical University, Shenyang 110004, Liaoning Province, China. [email protected]
AbstractAIM: To study the expressive variation of NogoA on rat retina in the process of chronic ocular hypertension.METHODS: Thirtysix healthy adult male Wistars were randomly divided into control group (6 rats) and chronic hypertension group (30 rats). Chronic hypertension was created by cauterizing the superficial scleral veins. Immunohistochemistry technique was used to evaluate the expressive varieties of NogoA at different time points during the course of chronic ocular hypertension.RESULTS: The success of the model was indicated by over 40% of increase in the IOP as compared with normal rats. Compared with control group, as time passed chronic hypertension group gradually had detectable morphology changes in the retina. At the 21st day of chronic ocular hypertension, retinas became thinner and the quantity of retinal ganglion cell (RGC) decreased (P<0.05). Assoicated with the morphological changes, the expression of NogoA was strongly increased (P<0.05).CONCLUSION: Myelin associated protein NogoA plays a part in the process of chronic ocular hypertension.
KEYWORDS: retina; chronic hypertension; NogoA; retinal ganglion cell
INTRODUCTION
The pathological mechanism of glaucomatous optic neuropathy is progressive death of retinal ganglion cells, which leads to irreversible damage. Regeneration of damaged central nervous system, including optic nerve, is difficult to achieve because of a number of reasons, such as inhibitors of axonal regeneration are present in myelin, lack of neurotrophic factors, and formation of the glia scar. It has been postulated that the regeneration of central nervous system is affected by myelin associated protein NogoA. NogoA, which is predominantly present in oligodendrocytes and myelin in the adult central nervous system (CNS), not only restricts neurite growth, plasticity, and axonal regeneration, but also limits the invasion and migration of cells and tumors in the CNS[1]. Therefore, our finding on the expressive variation of NogoA in rat retina indicates an important role of NogoA in the optic nerve injury in the process of chronic ocular hypertension and suggests a new approach to rehabilitate glaucomatous optic nerve.
MATERIALS AND METHODS
Materials Thirtysix male Wistar rats, weighing between 250300g were supplied by experiment animal department of China Medical University. The animals and experimental conditions were performed in accordance with laboratory animal regulations of State Science and Technology Commission. Animals were randomly divided into 2 groups, which were 6 in control group (12 eyes),and 30 in chronic hypertension group (60 eyes). Rabbit antirat NogoA (Wuhan Boster Biotechnology Co, Ltd), and SP kit (Fuzhou Maixin Biotechnology) were used.
Methods Rat model of chronic IOP elevation. Rats were anesthetized by intraperitoneal injection of 3.5mL/kg of chloral hydrate (100g/L). Bulbar conjunctiva was cut and two superficial venous tributaries were burnt. Signs of successful burn were shown as disappeared episclera venous blood flow on the distal end of the burnt point, and distension and darkness of the vessels near comeoscleral limbus. Bulbar conjunctiva was then reset with TobraDex drops and pasted with eyedrop. IOP was measured with Tonopen XL before the operation, half an hour after the operation, and at the 3rd day, the 7th day, the 14th day, the 21st day and the 28th day after operation. IOP that is 40% beyond preoperative value (916mmHg) indicates a success of the model.
After sampling fixation, dehydration and paraffin imbedding were performed according to the instruction of the kit. Positive cells were those with yellow or brownish yellow granules deposited in cytoplasm or nuclei. We selected 5 discontinued high power fields from each section to assess the expression intensity with metaMorph/BX51 microgram analytical system to determine the integrated A of positive cells.
Statistical Analysis Quantitative data were expressed as the mean±SEM, and were analyzed with oneway analysis of varianceFigure 1Expression of NogoA in retinas of rats with chronic ocular hypertension A: Control; B: 3 days; C: 7 days; D: 28 days.
(ANOVA) followed by Bonferroni test for multiple comparisons among experimental groups and control groups. Statistical analysis was performed using the SPSS 17.0 statistical software. The results were considered statistically different at P<0.05.
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