Measuring depth of injury (DOI) in an isolated rabbit eye irritation test (IRE) using biomarkers of cell death and viability

Abstract

While DOI is a mechanistic correlate to the ocular irritation response, attempts to measure DOI in alternative tests have been limited to qualitative histopathologic assessment by veterinarian pathologists. The purpose of this study was to determine whether DOI could be measured objectively by fluorescent staining for biomarkers of cell death and viability using an ex vivo isolated rabbit eye (IRE) test. A panel of nine materials characterized by in vivo DOI were selected that caused slight (3% acetic acid and 5% SDS), mild (acetone, sodium hypochlorite and 10% acetic acid), moderate (cyclohexanol and parafluoroanaline) and severe (8% sodium hydroxide and 10% benzalkonium chloride) irritation. Materials were then tested using a modified IRE test with 3 h recovery and then processed for cyrosectioning and staining using a TUNEL assay to detect cell death, phalloidin to detect intracellular f-actin and DAPI staining to detect nuclei. Control eyes treated with water showed intense phalloidin staining of the corneal epithelium and stromal keratocytes but no TUNEL labeling. In general, eyes treated with mild, moderate and severe irritants showed regions of TUNEL labeled epithelial and keratocyte nuclei with no phalloidin stain overlying phalloidin stained, undamaged cells. DOI measurements showed that slight irritants damaged <40% of the epithelium, mild and moderate irritants damaged >50% of the epithelium, extending at times into the anterior stroma (<20%), and the severe irritant damaged >50% of the stroma. Regression analysis between ex vivo and in vivo DOI showed a significant ( p < 0.007) correlation ( r = 0.785). These data suggest that fluorescent staining of fixed and sectioned tissue using biomarkers can be used to objectively identify the depth of injury caused by ocular irritants.