大鼠干眼模型的建立及其角膜神经的改变

发布时间：2018-06-08 21:30

本文选题：干眼 + 动物模型　；参考：《眼科新进展》2014年05期

【摘要】：目的通过皮下注射氢溴酸东莨菪碱建立大鼠干眼模型,并对其眼表病理及角膜神经改变进行观察分析。方法 6周龄Wistar健康雌性大鼠32只,随机分为高(H)、中(M)、低(L)剂量组和对照(C)组,每组各8只,H、M、L组皮下注射东莨菪碱,剂量分别为6 mg·mL-1、4 mg·mL-1、2 mg·mL-1,C组皮下注射生理盐水。用药后0 d、7 d、14 d、21 d、28 d对各组大鼠行泪液分泌试验(Schirmer I test,SIt)、泪膜破裂时间(break-up time,BUT)、角膜上皮荧光素钠染色及泪液蕨类试验分析。用药后28 d时,处死大鼠取整个眼球,石蜡切片后行HE及PAS染色;其余眼取角膜行Real-time PCR及神经氯化金染色。结果 H组大鼠在用药后7 d即出现BUT缩短为(6.800±1.033)s,与C组(9.111±1.269)s比较差异有显著统计学意义(P0.01),H组较L组缩短(P0.05)。14 d时,H、M、L组的BUT分别为(5.200±0.632)s、(7.200±0.789)s和(9.000±0.816)s,短于C组(10.222±1.302)s,差异均有统计学意义(均为P0.05);H、L、M组两两比较,差异均有显著统计学意义(均为P0.05)。随着时间延长,H、M、L组的BUT进一步缩短。用药后21 d、28 d,除21 d时L组与C组、H组与M组差异无统计意义(均为P0.05)外,余组间差异均有显著统计学意义(均为P0.01)。用药后7 d,H组、M组出现少量角膜上皮细点状荧光素钠着色,并随实验进程角膜上皮染色分级逐渐加重。用药后14 d,H组、M组的泪液分泌开始减少,分别为(5.200±0.978)mm、(6.350±1.528)mm,分别与C组(7.944±1.130)mm比较,差异均有统计学意义(均为P0.05);21 d,各组的泪液分泌量略有增加;用药后28 d,H组、M组的泪液分泌量分别为(4.900±1.075)mm、(5.650±0.747)mm,较C组(7.600±1.600)mm显著减少(均为P0.01)。而L组的泪液分泌量在各时点均较C组无明显减少(均为P0.05)。泪液蕨类试验显示,H、M、L组泪液蕨类物结晶数量减少,分支减少,形态不完整,其中H组改变最明显。HE染色显示,H、M、L组角膜上皮增生,层数增加,角膜表面凹凸不平,基底细胞水肿、排列紊乱。其中,H组病变最明显,且可见空泡样变。用药后28 d,结膜杯状细胞除L组与C组差异无统计学意义外,余组间两两比较差异均有统计学意义(均为P0.05)。角膜神经氯化金染色及Real-time PCR结果显示,各组的角膜神经纤维密度和走形及神经生长因子的表达水平均无显著变化。结论皮下注射氢溴酸东莨菪碱可显著抑制大鼠泪液分泌,降低泪膜稳定性,引起眼表损伤,成功建立泪液缺乏为主的干眼模型,且干眼的进展呈时间和剂量依赖性,为进行干眼发病机制研究和干预治疗探索提供依据。
[Abstract]:Objective to establish the dry eye model of rats by subcutaneous injection of scopolamine hydrobromide, and to observe and analyze the ocular surface pathology and corneal nerve changes. Methods Thirty-two 6-week-old Wistar healthy female rats were randomly divided into three groups: the control group (n = 8) and the control group (n = 8). Each group (n = 8) was subcutaneously injected with scopolamine at a dose of 6 mg / ml ~ (4) mg / ml ~ (-1) and 2 mg / ml 路L ~ (-1) C, respectively, and normal saline was injected subcutaneously into the control group (n = 8). The tear secretion test (Schirmer I test), lacrimal film rupture time (DST), corneal epithelium fluorescein sodium staining and tear ferns test were performed at 0 d, 7 d, 14 d, 21 d and 28 d after administration. On the 28th day after administration, the rats were sacrificed to take out the whole eyeball, paraffin sections were stained with HE and pas, and the corneas were taken for real-time PCR and nerve gold chloride staining. Results on the 7th day after treatment, but in group H was reduced to 6.800 卤1.033 s, which was significantly lower than that in group C (9.111 卤1.269), and the BUT of group H was 5.200 卤0.632s (7.200 卤0.789) and 9.000 卤0.816 s, respectively, which was significantly shorter than that of group C (10.222 卤1.302s). (all P 0.05) and the comparison between two groups (P 0.05, P ~ (0.05) and (P ~ (0.05) (P ~ (0.05). The difference was statistically significant (P 0.05). With the time prolonging, the but was further shortened. After 21 days, there was no statistical difference between group L and group C (P 0.05), but there was significant difference between group C and group C (P 0.01). At 7 days after treatment, a small amount of sodium fluorescein staining of corneal epithelium was observed in group M, and the staining grading of corneal epithelium increased gradually with the process of the experiment. On the 14th day after treatment, the tear secretion began to decrease in group M (5.200 卤0.978mm), 6.350 卤1.528mm, respectively, which was significantly higher than that in group C (7.944 卤1.130)mm) (all P0.05 + 21 days), and the amount of tear secretion in each group was slightly increased. After 28 days of treatment, the tear secretion of group M was 4.900 卤1.075mm, 5.650 卤0.747mm, which was significantly lower than that of group C (7.600 卤1.600)mm) (P 0.01). The tear secretion in group L was not significantly decreased compared with that in group C at all time points (P 0.05). The tear fern test showed that the number of crystals, branches and morphology of tear ferns were decreased, and the most obvious changes were observed in the H group. He staining showed that the corneal epithelium proliferated, the number of layers increased, the corneal surface was uneven, and the basal cells were edema. The arrangement is disordered. In group H, the lesions were the most obvious and vacuolar changes were observed. On the 28th day after treatment, the conjunctival goblet cells showed significant differences (all P 0.05) except that there was no significant difference between L group and C group. The results of gold chloride staining and Real-time PCR showed that there were no significant changes in the density of corneal nerve fibers, the morphology of corneal nerve fibers and the expression level of nerve growth factor (NGF) in all groups. Conclusion Intradermal injection of scopolamine hydrobromide can significantly inhibit lacrimal secretion, decrease the stability of lacrimal film, cause ocular surface damage, and successfully establish dry eye model with lacrimal deficiency, and the progress of dry eye is in a time-and dose-dependent manner. To provide the basis for the study of the pathogenesis of dry eye and the exploration of intervention therapy.
【作者单位】：天津医科大学眼科医院;
【基金】：天津医科大学眼科医院院内基金(编号:20120404)~~
【分类号】：R777.34

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