大鼠干眼模型的建立及其角膜神经的改变

Establishment of rat dry eye model and changes of corneal nerve

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(P<0.01),H组较L组缩短(P<0.05)。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P<0.05);H、L、M组两两比较,差异均有显著统计学意义(均为P<0.05)。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P>0.05)外,余组间差异均有显著统计学意义(均为P<0.01)。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P<0.05);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P<0.01)。而L组的泪液分泌量在各时点均较C组无明显减少(均为P>0.05)。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P<0.05)。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。

Objective To evaluate the pathology and corneal nerve changes of rat dry eye model induced by subcutaneous injection of scopolamine hydrobromide. Methods Thirty-two female Wistar rats about six weeks old were randomly divided into high （H） ,medium （ M）, low （L） dose group and control group （ C ）, 8 cases in each group. Group H, M, and L were respectively injected with different concentrations of scopolamine hydrobromide （6 mg·mL-1, 4 mg · mL -1 and 2 mg · mL- 1 ）, and nor- mal sodium was injected in group C. The clinical signs of dry eye such as tear volume （Schirmer I test）, tear break-up time （BUT）, corneal epithelial fluorescein staining and tear ferns experiment were evaluated on day 0,7,14,21, and 28. On the 28th day, five eyes in each group were enucleated and processed for paraffm sections for HE and PAS staining;And the corneas of the other eyes were used for Real-time PCR and nervous gold chloride staining. Results BUT of group C and group H on day 7 were （ 9.111± 1. 259） s and （ 6.800 ± 1. 033 ） s, respectively, there was significant difference （P 〈 0.01 ）, and group H was shorter than group L （ P 〈 0.05 ）. On day 14, BUT of group H, M, L and Cwere （5.200 ±0.632）s,（7.200±0.789）s,（9.000±0. 816）s and （10.222± 1.302）s, respectively ,group H,M,L was shorter than group C （ all P 〈0.05 ） ,there were statisti- cal differences among group H, L and M （ all P 〈 0.05 ）. There were statistical differ- ences in BUT on day 28 between group L and C,group H and M （all P 〈0.01 ） ,but no statistical difference on day 21 （ all P 〉 0.05 ）. Group H and M showed a small amount offine punctate corneal epithelial fluorescein staining on day 7, and the score of corneal epithelial staining gradually increased with the time prolong. The tear volume of group H and M decreased on day 14, which were （ 5. 200 ± 0. 978 ） mm and （6.350 ± 1. 528） mm, respectively, compared with group C （7.944± 1. 130） mm,there were statistical differences （ all P 〈 0. 05 ） ; The tear volume of group H and M on day 28 were （4. 900± 1. 075 ） mm and （ 5. 550 ± 0. 747 ） mm, respectively, com- pared with group C （7.500± 1. 600） mm, there were statistical differences （ all P 〈 0.01 ）, there was no statistical difference between group L and C at different time points （ all P 〉 0.05 ）. Tear ferns test showed crystallization of the graphics and branches of the experimental group reduced, especially in group H. HE staining showed corneal epithelial hyperplasia, in- creased levels, surface uneven, basal cell edema and disarrangement in experimental group. Group H changed more obvious- ly with vacuolations. There was no statistical difference in conjunctival goblet cells between group L and group C （P 〉 0. 05 ）, but there were statistical differences among other groups （ all P 〈 0.05 ）. Nerve gold chloride staining and Real-time PCR results showed no change in corneal nerve fiber and nerve growth factor mRNA. Conclusion Subcutaneous injec- tion of scopolamine hydrobromide can significantly inhibit lacrimal secretion, reduce tear from stability and cause damage to ocular surface ,successfully establishing a rat dry eye model in a time-and dose-dependent manner to provide an ideal exper- imentalplatform for the research of pathogenesis of dry eye and clinical treatment.