Initial Studies on Nitrite as a Topical Stiffening Agent for Corneo-Scleral Disorders

Abstract

Purpose:: Our group has previously shown that nitrite-bearing solutions can produce non-enzymatic collagen cross-linking and induce tissue stiffening. Recognizing the success of riboflavin/UVA therapy in the treatment of keratoconus through corneal collagen cross-linking, the present study was undertaken as an initial step in evaluating nitrite containing solutions as a topical stiffening agent.

Methods:: Adult pig eyes were obtained within 12 hrs of death and soaked in buffered solutions (pH 5-7.4) of NaNO₂ (0-200mM) and NaCl (0-200mM). Following a 30-80 hour incubation period at 37^oC, the corneo-scleral complexes were excised. In order to obtain a general assessment of the degree of collagen cross-linking induced, the samples were subjected to thermal shrinkage temperature (thermal denaturation) analysis. The intact cornea was heated in 2^oC increments every 5 min using a digitally controlled water bath. The maximal dimensions in the long and short axis were measured using a micrometer under visualization with an operating microscope. The percentage of tissue shrinkage was then determined by using 2 dimensional area calculations from the micrometer readings. In addition, penetration of nitrite through the intact cornea was assessed at 24hrs by sampling the aqueous humor and assaying for free nitrite using a modification of the Greiss colorimetric assay.

Results:: Dramatic contour changes were evident in both the cornea and sclera from cross-linked globes and were pH dependent, with the most significant changes seen under acidic conditions. The onset of tissue shrinkage was increased by 4^oC following 30 hrs of treatment and 8^oC following 80 hrs of treatment (200mM NaNO₂, pH 5, 37^oC), indicating a time-dependent increase in the formation of covalent collagen cross-links within the cornea. In addition, nitrite penetration through the intact cornea showed a concentration dependent effect. At 24 hrs, nitrite was detected in the anterior chamber at 35.5% of the incubation fluid (10mM NaNO₂) which increased to 62.8% at 100mM.

Conclusions:: Nitrite containing solutions can induce corneo-scleral crosslinking via nitrosation reactions. These preliminary studies suggest that nitrite may find a role as a pharmacologic topical stiffening agent for keratoconus and related disorders.

Keywords: keratoconus • cornea: stroma and keratocytes • nitric oxide