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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:

34卷

期数:

2014年5期

页码:

422-427

栏目:

实验研究

出版日期:

2014-05-05

文章信息/Info

Title:

Establishment of rat dry eye model and changes of corneal nerve

作者:

刘会娟; 黄悦; 张琰; 李斐; 赵少贞

３００３８４　天津市，天津医科大学眼科医院

Author(s):

LIU Hui-Juan ;  HUANG Yue ;  ZHANG Yan ; Ll Fei ;  ZHAO Shao-Zhen

关键词:

干眼; 动物模型; 氢溴酸东莨菪碱; 皮下注射; 角膜神经

Keywords:

dry eye ;  rat model;  scopolanune hydrobromide;  subcutaneous injection ;  corneal nerve

DOI:

10.13389/j.cnki.rao.2014.0116

文献标志码:

A

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６ｍｇ?ｍＬ－１、４ｍｇ?ｍＬ－１、２ｍｇ?ｍＬ－１，Ｃ组皮下注射生理盐水。用药后０ｄ、７ｄ、１４ｄ、２１ｄ、２８ｄ对各组大鼠行泪液分泌试验（ＳｃｈｉｒｍｅｒＩｔｅｓｔ，ＳＩｔ）、泪膜破裂时间（ｂｒｅａｋ-ｕｐｔｉｍｅ，ＢＵＴ）、角膜上皮荧光素钠染色及泪液蕨类试验分析。用药后２８ｄ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-ｔｉｍｅＰＣＲ及神经氯化金染色。结果　Ｈ组大鼠在用药后７ｄ即出现ＢＵＴ缩短为（６．８００±１．０３３）ｓ，与Ｃ组（９�保保保薄溃豹保玻叮梗�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�保埃担�。１４ｄ时，Ｈ、Ｍ、Ｌ组的ＢＵＴ分别为（５．２００±０�保叮常玻�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�保埃担�。随着时间延长，Ｈ、Ｍ、Ｌ组的ＢＵＴ进一步缩短。用药后２１ｄ、２８ｄ，除２１ｄ时Ｌ组与Ｃ组、Ｈ组与Ｍ组差异无统计意义（均为Ｐ＞ ０�保埃担┩猓�余组间差异均有显著统计学意义（均为Ｐ＜０．０１）。用药后７ｄ，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１４ｄ，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）；２１ｄ，各组的泪液分泌量略有增加；用药后２８ｄ，Ｈ组、Ｍ组的泪液分泌量分别为（４．９００±１．０７５）ｍｍ、（５�保叮担啊溃蔼保罚矗罚�ｍｍ，较Ｃ组（７�保叮埃啊溃豹保叮埃埃�ｍｍ显著减少（均为Ｐ＜０�保埃保�。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２８ｄ，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-ｔｉｍｅＰＣＲ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 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 -l and 2 mg . mL ") . and normal 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 paraffin 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. 269 ) 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 C were (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 statistical differences among group H, L and M ( all P < 0. 05 ) . There were statistical differences 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 of fine 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. 650 + 0. 747 ) mm , respectively , compared with group C (7. 600 + 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, increased levels , surface uneven , basal cell edema and disarrangement in experimental group. Group H changed more obviously 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 injection of scopolanune hydrobronude can sigruficantly inhibit lacrimal secretion , reduce tear film 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 expe-imental platform for the research of pathogenesis of dry eye and clinical treatment.

备注/Memo

备注/Memo:

天津医科大学眼科医院院内基金（编号：２０１２０４０４）

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更新日期/Last Update: 2014-05-04