Purchase this article with an account.
Nooshin TalebiZadeh, Zhaohua Yu, Martin Kronschlager, Per Soderberg; Caspase-3 expression in rat lens epithelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):483.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Caspase-3 is one of the most important caspases in apoptosis required for tissue growth control and development. Knowledge about caspase-3 activity in normal lens epithelial cells is essential to understand the turnover of lens epithelial cells, physiologically as well as after toxic events. The present study aimed to determine the distribution and localization of active caspase-3 in normal rat lens epithelium by immunohistochemistry.
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 at the first nucleus visible in one nuclear bow and ended in the last nucleus visible in the opposite nuclear bow. The nuclei labelled with active caspase-3 and their relative position was identified three times, counts, in each section. The middle cell in each count was identified and the epithelium was split into two opposite halves. Then, each half was divided into fifteen segments and the fraction of active caspase-3 labelled nuclei in each segment was calculated.
The variance for animals, sections and countings was estimated to 11, 3 and 83 %′2, respectively. Active caspase-3 was expressed more often in the center than in the periphery. In total, an average of 38 % active caspase-3 labelled cells was found close to the anterior pole. Fitting the fraction of labelling normalized to 1 as a function of cell number starting from the equatorial end of the epithelium to a double exponential model, assuming an exponential increase from the periphery, an inflection point, and an exponential decrease towards the anterior pole, resulted in a 95 % confidence interval for increase rate (1/k) = 48 ± 5 cells and inflection point = 124 ±12 cells.
In our hands, quantitative analysis of immunoflorescence of active caspase-3 requires at least 3 sections per lens and further, the number of countings of labelled nuclei is the limiting factor for the precision in an estimate of fraction of labelling. Even in the normal lens, there is an abundance of active caspase-3 with a maximum activity around the anterior pole that tapers off towards the periphery.
This PDF is available to Subscribers Only