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. 2016 Feb:143:75-88.
doi: 10.1016/j.exer.2015.09.010. Epub 2015 Oct 9.

Development of a novel in vivo corneal fibrosis model in the dog

K M Gronkiewicz  1 , E A Giuliano  1 , K Kuroki  2 , F Bunyak  3 , A Sharma  1 , L B C Teixeira  4 , C W Hamm  5 , R R Mohan  6
Affiliations

Development of a novel in vivo corneal fibrosis model in the dog

K M Gronkiewicz et al. Exp Eye Res. 2016 Feb.

Abstract

The aim of this study was to develop a novel in vivo corneal model of fibrosis in dogs utilizing alkali burn and determine the ability of suberanilohydroxamic acid (SAHA) to inhibit corneal fibrosis using this large animal model. To accomplish this, we used seven research Beagle dogs. An axial corneal alkali burn in dogs was created using 1 N NaOH topically. Six dogs were randomly and equally assigned into 2 groups: A) vehicle (DMSO, 2 μL/mL); B) anti-fibrotic treatment (50 μM SAHA). The degree of corneal opacity, ocular health, and anti-fibrotic effects of SAHA were determined utilizing the Fantes grading scale, modified McDonald-Shadduck (mMS) scoring system, optical coherence tomography (OCT), corneal histopathology, immunohistochemistry (IHC), and transmission electron microscopy (TEM). The used alkali burn dose to produce corneal fibrosis was well tolerated as no significant difference in mMS scores between control and treatment groups (p = 0.89) were detected. The corneas of alkali burned dogs showed significantly greater levels of α-smooth muscle actin, the fibrotic marker, than the controls (p = 0.018). Total corneal thickness of all dogs post-burn was significantly greater than baseline OCT images irrespective of treatment (p = 0.004); TEM showed that alkali burned corneas had significantly greater minimum and maximum interfibrillar distances than the controls (p = 0.026, p = 0.018). The tested topical corneal alkali burn dose generated significant opacity and fibrosis in dog corneas without damaging the limbus as evidenced by histopathology, IHC, TEM, and OCT findings, and represents a viable large animal corneal fibrosis in vivo model. Additional in vivo SAHA dosing studies with larger sample size are warranted.

Keywords: Alkali burn; Corneal fibrosis; Dog; Suberanilohydroxamic acid; α-smooth muscle actin.

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Figures

Figure 1
Figure 1
Image analysis and quantification for TEM images of collagen fibril cross-sections. First row: original image at 25,000X magnification; second row: magnified region. A) Original TEM image. B) Collagen fibril segmentation results. D) Fibril centroids and fibril neighborhood graph. D) Nearest (red) and farthest (blue) immediate neighbors.
Figure 2
Figure 2
Clinical scores and corneal haze grades following the 20 second alkali burn in a representative dog. A) One day following alkali burn, the mMS score was 17.5 and corneal haze grade was 3. B) On day 5 the mMS score was 11 and corneal haze grade was 2. C) Ten days post-alkali burn the mMS score was 8.5 and corneal haze grade was 2. D) At the study’s endpoint (day 14), the mMS score was 6.5 and corneal haze grade was 2.
Figure 3
Figure 3
Representative OCT images of normal cornea at baseline and alkali-burned cornea 14 days after wounding. A) Normal cornea of a dog prior to wounding. The values in green represent epithelial and total corneal thickness. B) Alkali-burned cornea from dog in treatment group A. C) Alkali-burned cornea from dog in treatment group B. Note the variable epithelial thickness and the increased total corneal thickness in the alkali-burned cornea compared to the normal cornea at baseline.
Figure 4
Figure 4
Representative histological images of normal (A–D) and alkali-burned corneas (E–H). H&E stain demonstrated an increased cellularity of the anterior stroma in the alkali-burned corneas (E) compared to normal corneas (A). Masson’s trichrome stain of the alkali-burned cornea (F) confirmed presence of mesenchymal cells in the anterior stroma, which are absent in normal cornea (B). The formation of elastic fibers was demonstrated in the alkali-burned cornea with EVG (G). No elastic fibers were detected in normal cornea (C). PAS stained corneas showed increased basement membrane and fibrin deposition in the alkali-burned cornea (H) compared to normal cornea (D).
Figure 5
Figure 5
Representative Picrosirius red stained negative control (A) and alkali-burned (B) corneas. Note the increased amount of type III collagen fibers in the alkali-burned cornea compared to the negative control. This transition of type I to type III collagen fibers is typical of scar formation.
Figure 6
Figure 6
Representative immunohistochemistry of normal (A) and alkali-burned (B) corneas. Increased α-smooth muscle actin stain in the alkali-burned corneas confirmed the cell population in the anterior stroma as fibroblasts. There was only a mild-moderate increase in CD18 stain in the alkali-burned cornea indicating that the majority of the cellular infiltrate was not inflammatory in nature.
Figure 7
Figure 7
Representative transmission electron microscopy images of normal cornea (A) and alkali-burned (B) corneas, specifically measuring the minimum and maximum interfibrillar distances. Note the increased minimum (red) and maximum (blue) interfibrillar distances in the alkali-burned cornea compared to normal cornea.
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