A novel method for generating corneal haze in anterior stroma of the mouse eye with the excimer laser
A novel method for generating corneal haze in anterior stroma of the mouse eye with the excimer laser
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DOI：
10.1016/j.exer.2007.10.014
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发表时间：
2008-02-01
影响因子：
3.4
通讯作者：
Wilson, Steven E.
中科院分区：
文献类型：
--
作者：
Mohan, Rajiv R.;Stapleton, W. Michael;Wilson, Steven E.
屈光手术是一种流行的方法，用于减少或消除对眼镜和隐形眼镜的依赖。角膜雾状混浊是准分子激光屈光性角膜切削术（PRK）后常见的并发症之一。本研究的目的是开发一种体内小鼠模型，该模型在准分子激光治疗后在小鼠角膜的前基质中持续产生中度至重度角膜混浊，以在遗传定义的模型中研究肌成纤维细胞生物学和角膜伤口愈合。用Summit Apex准分子激光器（Alcon，Ft. Worth，TX）。在准分子激光消融之前刮擦上皮后，在中央角膜上发射不同数量的激光脉冲（45;消融深度类似于10 μ m）。通过在发射50%的脉冲后在激光路径中放置细网筛来产生不规则性。在手术后1、2、3或4周收集眼睛。用裂隙灯生物显微镜测量雾度形成。使用对肌成纤维细胞标志物α-平滑肌肌动蛋白（SMA）具有特异性的抗体，采用免疫细胞化学法测定小鼠角膜中肌成纤维细胞的数量。通过在基质内计数确定SMA阳性细胞/400 x显微镜的数量。使用方差分析（AVOVA）进行统计分析，并对重复测量进行Bonferonni-Dunn调整。在小鼠眼睛中进行具有上皮刮擦的规则PTK（组3）和具有上皮刮擦的不规则PTK（组4）以产生角膜混浊。在上皮刮除后常规或不规则PTK后4周收集的眼睛在前角膜中显示22 6.6（组3）或34 7.9（组4）SMA阳性细胞。各组间检测到的SMA阳性细胞的差异具有统计学意义（p < 0.01）。在常规PTK（组3）或不规则PTK（组4）或对照1、2或3周后收集的小鼠眼组织切片中检测到少于4个SMA阳性细胞。（第1组和第2组）。在这项研究中开发的优化PTK准分子激光条件下产生混浊选择性在上皮基底膜下的小鼠角膜前基质。发现在上皮刮除后通过施加45个激光脉冲进行的不规则PTK是产生肌成纤维细胞的最有效方法。这种用于在体内诱导小鼠角膜混浊的PTK技术为研究转基因小鼠中的伤口愈合和肌成纤维细胞生物学提供了有用的模型。(c)2007爱思唯尔有限公司出版
Refractive surgery is a popular method used to reduce or eliminate dependence on glasses and contact lenses. Corneal haze is one of the common complications observed after photorefractive keratectmomy (PRK). The objective of this study was to develop an in vivo mouse model that consistently produces moderate to severe corneal haze in the anterior stroma of the mouse cornea after excimer laser treatment to study myofibroblast biology and corneal wound healing in a genetically defined model. Regular- or irregular-phototherapeutic keratectomy (PTK) was performed on black C57BL/6 mice with the Summit Apex excimer laser (Alcon, Ft. Worth, TX). Different numbers of laser pulses (45; ablation depth similar to 10 mu m) were fired on the central cornea, after scraping the epithelium prior to excimer laser ablation. Irregularity was generated by positioning a fine mesh screen in the path of laser after firing 50% of the pulses. Eyes were collected 1, 2, 3 or 4 weeks after the procedure. Haze formation was gauged with slit lamp biomicroscopy. Immunocytochemistry was used to determine number of myofibroblasts in the mouse cornea using antibodies specific for the myofibroblast marker alpha-smooth muscle actin (SMA). The numbers of SMA-positive cells/400x microscopic were determined by counting within the stroma. Statistical analysis was performed using analysis of variance (AVOVA) with the Bonferonni-Dunn adjustment for repeated measures. Regular-PTK with epithelial scrape (group 3) and irregular-PTK with epithelial scrape (group 4) in the mouse eyes were performed to produce corneal haze. Eyes collected 4 weeks after regular- or irregular-PTK after epithelial scrape showed 22 6.6 (group 3) or 34 7.9 (group 4) SMA-positive cells in the anterior cornea. The difference in the SMA-positive cells detected among the groups was statistically significant (p < 0.01). Less than 4 SMA-positive cells were detected in the tissue sections of the mouse eyes collected after 1, 2 or 3 weeks of regular (group 3) or irregular PTK (group 4) or controls. (groups 1 and 2). The optimized PTK excimer laser conditions developed in this study produces haze selectively in anterior stroma of the mouse cornea immediately beneath the epithelial basement membrane. Irregular PTK performed after epithelial scrape by applying 45 laser pulses was found to be the most effective method to generate myofibroblasts. This PTK technique for inducing haze in mouse cornea in vivo provides a useful model for studying wound healing and myofibroblast biology in transgenic mice. (c) 2007 Published by Elsevier Ltd.