The Effect of Direct Application of Mesenchymal Stem Cells on the Corneal Wound Healing in a Rat Limbal Deficiency Model

Abstract

Purpose:: To evaluate the effect of direct application of mesenchymal stem cells (MSC) on the corneal wound healing in a rat limbal deficiency model .

Methods:: This study was approved by the Institutional Review Board and Institutional Animal Care and Use Committee of the Seoul National University Hospital, and also performed according to ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.Corneal epithelial cells including limbal epithelium were totally removed using a blade after instillation of 100% alcohol for 30 seconds in right eye of six rats. Rat bone marrow-derived MSC (2X10⁶ cells) with PKH labeling were directly applied on damaged cornea using 3 mm diameter of column for two hours immediately after the injury (n=21), and PBS was applied in the control group (n=2). Corneal opacity, neovascularization, and epithelial defect were observed by slit lamp and graded weekly. The harvested corneas were stained with H&E and PKH. Immunofluorescent staining for CD4 and CD8 were performed. Inflammatory or stained cells were counted in 5 consecutive high power fields (HPF). The expression of IL-2 and IL-10 in the cornea was analyzed using ELISA.

Results:: MSC-transplanted cornea showed rapid reepithelialization, and reduced neovascularization and opacity, compared to PBS-applied control. PKH was positive in MSC-applied cornea. Inflammatory cells were more severely infiltrated in control (48.7±12.7/HPF) than in MSC applied eyes (13.6±12.1/HPF; p=0.004), but the mean number of CD8+ T cells showed no differences in both groups. Level of IL-2 increased in both MSC- and PBS-applied cornea, showing no significant differences, while level of IL-10 was significantly higher in MSC group (2.89 ± 0.89 ng/ml) than that in control (1.49 ± 0.94 ng/ml; p=0.016).

Conclusions:: The application of MSC transplantation in a rat limbal deficiency model reduces inflammation, neovascularization, and corneal opacity possibly by immune-modulatory action associated with increased IL-10.

Keywords: cornea: basic science • inflammation • immunomodulation/immunoregulation