Conjunctival epitheliopathy induced by topical exposure to bacterial peptidoglycan, muramyl dipeptide

Abstract

Noninfectious exudative conjunctivitis can be experimentally produced in rabbits by application of the apoptogenic bacterial cell wall peptidoglycan, muramyl dipeptide (MDP) to the ocular surface. The purpose of this study was to investigate the acute conjunctival cytopathology induced by unilateral ocular surface exposure to MDP. Hematoxylin and eosin staining assessed bilateral tear cytopathology and conjunctival histopathology. The caspases levels in conjunctival tissue and tears were measured in standard assays utilizing p-nitroanaline tagged caspase-specific substrates. Immunofluorescent antibody identified intracellular caspase-3, nuclear factor-κβ (NF-κβ), and oxidative DNA damage (8-OHdG; 8-oxo-2′-deoxyguanosine) in tear and conjunctiva cells. DNA extracted from conjunctival tissues and pooled tear fluids were visualized by ethydium bromide agarose gel electrophoresis. Onset of ipsilateral conjunctivitis was due to an epitheliopathy characterized by loss of conjunctival epithelial cell adherence, exuviation of conjunctival epithelial cells, and neutrophil infiltration. Caspase-3 levels were significantly higher in exuviated cells in ipsilateral than contralateral tear (p's ≤ 0.001) collected at 3–5 h post MDP. Significantly higher caspase-2, -3, -6, -8 and -9 (p's ≤ 0.03) levels were detected in ipsilateral than contralateral conjunctival tissue at 5 h. Polymeric DNA was detected in ipsilateral but not contralateral conjunctival tissue and tears. Caspase-3, NF-κβ, and 8-OHdG positive neutrophils were detected in bilateral conjunctiva and tear. The caspase-3/NF-κβ epithelial cells and polymeric DNA in conjunctival tissue and shedding of caspase positive cells and polymeric DNA into ipsilateral tears support MDP induction of acute programmed cell death in vivo. The results suggest that ipsilateral exudative conjunctivitis is due to acute caspase-mediated conjunctival epitheliopathy induced by topical exposure to the bacterial peptidoglycan MDP.