Effect of Topical Losartan in the Treatment of Established Corneal Fibrosis in Rabbits

Abstract

Purpose: The purpose of this study was to evaluate the safety and efficacy of topical losartan in the therapeutic treatment of established corneal scaring fibrosis at 1 month after alkali burn in rabbits.

Methods: Standardized alkali burns were performed in 1 eye of 24 rabbits with 0.75N NaOH for 15 seconds. Corneas were allowed to heal and develop scaring of the cornea for 1 month. Twelve eyes per group were treated with 50 µL of topical 0.8 mg/mL losartan in balanced salt solution (BSS), pH 7.0, and 12 eyes were treated with vehicle BSS 6 times per day. Six corneas were analyzed at 1 week or 1 month in each group. Standardized slit lamp photographs were obtained at the end point for each cornea and opacity was quantitated using ImageJ. Corneoscleral rims were cryofixed in optimum cutting temperature (OCT) solution and combined duplex immunohistochemistry for myofibroblast marker alpha-smooth muscle actin (α-SMA), mesenchymal cell marker vimentin, and TUNEL assay for apoptosis was performed on all corneas.

Results: Topical losartan was effective in the treatment of established stromal fibrosis following alkali burn injury to the rabbit cornea. Stromal myofibroblast density was decreased and stromal cell apoptosis was increased (included both α-SMA-positive myofibroblasts and α-SMA-negative, vimentin-positive cells) at both 1 week and 1 month in the topical losartan-treated compared with vehicle-treated groups.

Conclusions: Topical losartan is effective in the treatment of established stromal fibrosis in rabbits. Most myofibroblasts disappear from the stroma within the first month of losartan treatment. Longer treatment with topical losartan is needed to allow time for corneal fibroblast regeneration of the epithelial basement membrane (in coordination with epithelial cells) and the removal of disordered extracellular matrix produced by myofibroblasts.

Figure 1.

Slit lamp images of each cornea 1 month after alkali burn prior (pre) to starting treatment with BSS vehicle or 0.8% losartan in BSS 6 times per day and after 1 week of treatment. The dotted yellow circle is the approximate area of ImageJ analysis of the opacity. One cornea in the losartan group (#4) developed a PED (arrows) during the interval between the alkali burn and beginning topical losartan treatment that persisted at the 1-week time point for euthanasia. It can be noted that the opacity tended to decrease in both the vehicle-treated and the losartan-treated corneas during the week of treatment. Magnification = 20×.

Figure 2.

Slit lamp images of each cornea 1 month after alkali burn prior (pre) to starting treatment with BSS vehicle or 0.8% losartan in BSS 6 times per day and after 1 month of treatment. The dotted yellow circle is the approximate area of ImageJ analysis of the opacity. One cornea in the losartan group (#1) developed a PED (arrows) during the interval between the alkali burn and beginning topical losartan treatment but the PED had resolved by the end of the month of treatment. A transient PED may have also developed in losartan cornea #6, but it was not present at the time of weekly screenings with fluorescein. It can be noted that the opacity tended to decrease in both the vehicle-treated and the losartan-treated corneas during the month of topical treatment. Magnification = 20×.

Figure 3.

ImageJ analysis of the changes in central corneal opacity in all eyes from 1 week or 1 month of treatment with either vehicle or losartan. Blue is post alkali burn opacity at 1 month and prior to topical treatment, and red is the final opacity just prior to euthanasia. Green dots are the values for one cornea with a PED. Note the overall trend toward lower opacity in the losartan treated corneas but these differences did not reach statistical significance over this time interval. As expected, the stromal opacity was greatest in one cornea in the 1-week losartan group that had developed a PED, and in this cornea also the opacity was higher pretreatment with losartan than after 1 week of treatment.

Figure 4.

Duplex immunohistochemistry combined with the TUNEL assay in the 1-week vehicle- or losartan-treated corneas. For each cornea, a composite is shown, in addition to IHC for myofibroblast marker α-SMA (arrowheads), TUNEL assay to detect apoptosis (arrowheads), and IHC for mesenchymal marker vimentin. The red dotted lined rectangles are examples of the approximate quantitation boxes for red α-SMA signal that were 120,000 pixels² in each cornea but the actual measurement was performed with the image in ImageJ so the area of the analysis could be controlled. The green dotted rectangles are representative 120,000 pixels² stromal quantitation boxes for green TUNEL-positive cells that were counted individually. Notice, in both groups, more of the myofibroblasts detected are in the posterior cornea, with only a few detected in the anterior half of the stroma in some corneas. Cells undergoing apoptosis were identified at the microscope as a combination of α-SMA-positive, vimentin-positive myofibroblasts and α-SMA-negative, vimentin-positive cells that were likely corneal fibroblast, fibrocytes, and the daughter cells of these precursors that have begun their transition into myofibroblasts. No epithelial cells undergoing apoptosis were counted because apoptosis is a normal part of epithelial maturation. The magnification bar shown is representative of all panels.

Figure 5.

Duplex immunohistochemistry with combined TUNEL assay in the 1-month vehicle- or losartan-treated corneas. For each cornea, a composite is shown, in addition to IHC for myofibroblast marker α-SMA (arrowheads), TUNEL assay to detect apoptosis (arrowheads), and IHC for the mesenchymal marker vimentin. The red dotted lined rectangles are representative quantitation boxes for red α-SMA signal that were 120,000 pixels² in each cornea but the actual measurement was performed with the image in ImageJ, so the area of the analysis could be controlled. The green dotted rectangles are examples of the 120,000 pixels² stromal quantitation boxes for green TUNEL-positive cells that were counted individually in each box. Notice, in both groups, more of the myofibroblasts detected are in the posterior cornea, with only a few detected in the anterior half of the stroma in some corneas. Cells undergoing apoptosis were a combination of α-SMA-positive, vimentin-positive myofibroblasts and α-SMA-negative, vimentin-positive cells that were likely corneal fibroblast, fibrocytes, and the daughter cells of these precursors that have begun their transition into myofibroblasts. No epithelial cells undergoing apoptosis were counted because apoptosis is a normal part of epithelial maturation. The magnification bar shown is representative of all panels.

Figure 6.

Myofibroblast and other stromal cell apoptosis after 1 week or 1 month of treatment with vehicle or losartan. (A and B) The stromal myofibroblast signal was significantly greater in the vehicle-treated groups than the losartan-treated groups after both 1 week and 1 month of treatment. (C and D) TUNEL-positive stromal cells were greater in the losartan-treated groups than the vehicle-treated groups at both the 1 week and 1 month of treatment time points.