To determine the distribution and localization of active caspase-3 in normal rat lens epithelium by immunohistochemistry.Methods
Alltogether, 40 Sprague Dawley rats were sacrificed and eye lenses were removed. One eye from each animal was fixed and then stored at -80°C. Three mid-sagittal sections of each lens were processed for immunohistochemistry. Lens epithelial cell position was identified and specified as its consecutive position in relation to the first nucleus counted. The counting started from one nuclear bow and ended in the opposite side nuclear bow. The nuclei labelled with active caspase-3 and their relative position was identified three times, counts, in each section. Then, each lens was divided into fifteen segments and active caspase-3 labelling was calculated in each segment.Results
Active caspase-3 was more expressed in the anterior pole of the lens with the of 37 % active caspase-3 labelled cells. The variance for animals, sections and countings was estimated to 11, 3 and 83 %, respectively. Fitting the fraction of labelling to a double exponential model, as a function of segment number starting from the anterior pole, assuming an exponential decrease from the anterior pole toward periphery, resulted in a 95 % confidence interval for decrease rate (1/k) = -1.5±0.2 segments and inflection point = 7.7±0.2 segmentsConclusion
Active caspase-3 is present in the normal lens, with a maximum activity around the anterior pole that tapers off towards the periphery. Quantitative analysis of immunoflorescence of active caspase-3 requires at least 3 sections per lens. The number of countings of labelled nuclei is the limiting factor for the precision in an estimate of fraction of labelling.