Measuring depth of injury (DOI) in an isolated rabbit eye irritation test (IRE) using biomarkers of cell death and viability
| Autor: | Joseph Ling, James V. Jester, John W. Harbell |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Pathology
medicine.medical_specialty Cell Survival Phalloidine Phalloidin Eye Toxicology medicine.disease_cause Stain Cornea chemistry.chemical_compound Organ Culture Techniques In vivo Toxicity Tests In Situ Nick-End Labeling medicine Animals DAPI Corneal epithelium Cell Death Staining and Labeling General Medicine Staining medicine.anatomical_structure chemistry Irritants Rabbits Irritation Biomarkers Ex vivo |
| Zdroj: | Toxicology in Vitro. 24:597-604 |
| ISSN: | 0887-2333 |
| Popis: | 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 3h 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 damaged40% of the epithelium, mild and moderate irritants damaged50% of the epithelium, extending at times into the anterior stroma (20%), and the severe irritant damaged50% of the stroma. Regression analysis between ex vivo and in vivo DOI showed a significant (p0.007) 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. |
| Databáze: | OpenAIRE |
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