Human eyes, collected within 30 hours of death from donors aged
between 10 and 88 years, were obtained from the Corneal Transplantation
Service Eye Bank at Bristol Eye Hospital (UK) after removal of corneas
for transplantation. Exclusion criteria included history of intraocular
surgery, human immunodeficiency virus (HIV), hepatitis, and systemic
diseases with ocular manifestations. Eyes were also excluded if any
signs of gross hemorrhage or tissue abnormality were observed. Each
sample, which consisted of vitreous dissected from both eyes of
individuals, was liquefied by centrifugation at 60,000 rpm for 1 hour
at 4(C (Optima TLX; Beckman, Berkeley CA) and the sedimented insoluble
material removed. Liquefied vitreous supernatants were homogenized by
mixing and diluted with equal volumes of 2 × electrophoresis sample
buffer (0.12 M Tris/HCl, [pH 6.8], containing 4% [wt/vol] sodium
dodecyl sulfate [SDS], 20% [vol/vol] glycerol, and 0.01%[
wt/vol] bromophenol blue).
By the methods to be described, samples were analyzed in two groups,
one of less than and one of more than 50 years of age. These groupings
were based on the assumption that increased levels of degradative
activity would be associated with the increased levels of vitreous
liquefaction observed in older people.
2 For all analyses,
values obtained were related to unit volumes of vitreous sample, as
opposed to vitreal protein concentrations, because the latter may be
subject to selective filtration of proteins on the basis of molecular
weight, if derived from extravitreal sources.