Creation and grading of experimental corneal scars in mice models

Abstract

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.

Keywords: Fibroblasts; Macrophages; Scar; Stroma; Wound.

Fig. 1.

A custom-made diamond blade with 70 μm guarded depth to avoid stromal perforation was used in these experiments.

Fig. 2.

Photographs illustrate different surgical techniques used to induce scar formation. A horizontal cut, technique 1 (A), horizontal abrasion into the stroma, technique 2 (B), and central cut and abrasion on both sides, technique 3 (C). Dash lines symbolize abrasion to the stroma with an AlgerBrush II burr. Arrowheads show keratotomy. Scar formation in the three surgical injury models studied. Different examples of scar tissue obtained by a horizontal cut into the stroma, technique 1 (D and G), horizontal abrasion technique 2 (E and H), and central cut and abrasion on both sides, technique 3 (F and I).

Fig. 3.

Plot shows differences in scar formation between the three different techniques. T1: partial keratotomy, T2: horizontal abrasion only, T3: central cut and abrasion on both sides.

Fig. 4.

Comparison of stromal matrix organization, neovascularization and macrophage infiltration in a normal uninjured adult eye.

Fig. 5.

Comparison of stromal matrix organization, neovascularization and macrophage infiltration in an eye classified by masked examiner as mild scar, grade 0.

Fig. 6.

Differences in stromal matrix organization, neovascularization and macrophage infiltration become more notorious between an eye classified as grade 1.

Fig. 7.

Poor stromal matrix organization, significant neovascularization and macrophage infiltration in an eye classified by masked examiner as severe scar, (Grades 2 and 3).

Fig. 8.

Changes in stromal organization in scars of different severity. Normal well-organized matrix and keratocytes (control normal stroma). More prominent matrix disorganization mostly localized to the anterior stroma noted in mild and moderate scars (Grades 0 and 1). Progressive worsening in matrix organization, cellular infiltration and neovascularization in more dense scars (Grades 2 and 3). Arrows show lamellae organization and asterisks presumed neovascularization.