The Integrity of Mammalian Lenses in Organ Culture

Abstract

A method to quantitate protein leakage was developed to assess the integrity of mammalian lenses in organ culture. Protein leakage into the culture medium was found to be a strong indicator of lens integrity. Rat lens incubation medium that contained more than 14 μg protein per lens after 1 hr was indicative of lens damage. Damaged lenses almost always exhibited lens opacities within 24 hr. However, on occasion, a lens predicted by this method to be damaged would maintain clarity at the 24 hr timepoint. In these instances, damage was confirmed by the inability of the lens to accumulate [3H]choline and 86RbCl from the medium at levels equivalent to undamaged lenses. When the protein concentration of the incubation medium was greater than 14 μg per lens, there was a concomitant impairment of lens transport function. This indicates that quantitation of protein leakage is a better predictive measure of lens viability than is lens clarity.Rat lenses were oxidatively stressed by exposure to a bolus of hydrogen peroxide (final concentration, 1 mM). Within 24 hr, the lenses exhibited cortical opacities and the protein concentration of the medium was significantly increased over the controls. Western and slot blot analyses of the media revealed the presence of tens crystallins, βB2-crystallin was the major component leaking from damaged lenses. The incubation media from control lenses exhibited no protein leakage. Comparative studies with Rhesus monkey lenses yielded similar data.