Purpose

To develop a stromal wound healing model and a reliable scar classification score system that correlates photographic evaluation with changes in the structure and organization of the extracellular matrix.

Materials and methods

We tested three stromal injury techniques in adult C57BL/6 mice. Technique 1, a lineal partial thickness keratotomy in the horizontal axis. Technique 2, corneal epithelial and stromal debridement using a diamond burr in the horizontal axis, and technique 3, a combination of techniques 1 and 2. To assess intra-observer and inter-observer agreement between two examiners evaluating formed stromal scars, stereo microscopic photographs of anterior segment were scored by two masked examiners at around 1-month. Depending on the severity of opacification and the area of involvement, scars were classified on a scale from 0 to 3 based on a modified Fantes haze scale. Extracellular matrix composition as well as matrix organization, macrophage infiltration and neovascularization were evaluated with immunofluorescence and second harmonic generation (SHG) microscopy.

Results

Technique 1 created mild scars, with a score of 0.5 ± 0.43, while techniques 2 (score 2.1 ± 0.45) and 3 (score 2 ± 0.66), created dense scars with a higher score. A significant difference in scar severity score was noted between the 3 techniques (one way ANOVA, p < 0.0001). Masked graders demonstrated excellent agreement (intraclass correlation = 0.927 [95% confidence interval: 0.87–0.96]). The severity of scars noted at stereo microscopy correlated with the severity of changes in extracellular matrix in the stroma as demonstrated by the expression of collagens I, IV and fibronectin and evaluation of matrix hierarchical organization. In contrast to mild scarring, moderate and severe scars had increased expression of CD31 and CD68, markers of vascular endothelial cells and macrophages, respectively.

Conclusion

Mouse models of stromal scarring using simple surgical techniques are described. Corneal scars can be consistently classified by two observers. Grading of scar severity positively correlates with changes in extracellular matrix composition, disorganization and cell infiltration.

中文翻译：

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小鼠模型实验性角膜瘢痕的产生和分级

目的

开发基质伤口愈合模型和可靠的疤痕分类评分系统，将摄影评估与细胞外基质结构和组织的变化相关联。

材料和方法

我们在成年 C57BL/6 小鼠中测试了三种基质损伤技术。技术1，在水平轴上进行线性部分厚度角膜切开术。技术 2，在水平轴上使用金刚石毛刺进行角膜上皮和间质清创术，以及技术 3，技术 1 和 2 的组合。为了评估两个检查者之间的观察者内部和观察者之间的一致性，评估形成的基质疤痕，立体显微照片由两名蒙面检查员在大约 1 个月时对眼前节进行评分。根据混浊的严重程度和受累区域，疤痕根据修改后的 Fantes 雾度等级按 0 到 3 的等级分类。细胞外基质组成以及基质组织，

结果

技术 1 产生轻度疤痕，评分为 0.5 ± 0.43，而技术 2（评分 2.1 ± 0.45）和技术 3（评分 2 ± 0.66）产生的致密疤痕评分较高。注意到 3 种技术之间疤痕严重程度评分的显着差异（单向方差分析，p < 0.0001）。蒙面评分者表现出极好的一致性（类内相关性 = 0.927 [95% 置信区间：0.87–0.96]）。立体显微镜下观察到的疤痕严重程度与基质中细胞外基质变化的严重程度相关，如胶原蛋白 I、IV 和纤连蛋白的表达以及基质分层组织的评估所证明的。与轻度瘢痕形成相反，中度和重度瘢痕的 CD31 和 CD68 表达增加，分别是血管内皮细胞和巨噬细胞的标志物。

结论

描述了使用简单手术技术的基质疤痕小鼠模型。角膜疤痕可以由两个观察者一致地分类。瘢痕严重程度的分级与细胞外基质组成、解体和细胞浸润的变化呈正相关。