TUNEL labeling in retinal explants treated with GJ blockers.

<p>Retinal explants were cultured for 4 hours with (A) PBS, (B) carbenoxolone (CBX), (C) quinine (Quin) and (D) CBX 100 µM + quinine 100 µM. Transverse sections of chick retina explants were submitted to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to characterize apoptotic spatial pattern. In each image it is possible to localize the focus of the lesion (arrows). (E–H) In order to determine whether GJ blockers caused changes in the distribution of apoptotic cells, we have counted the number of TUNEL- positive nuclei located as far as 150 µm away from the focus of the lesion. Values were plotted according to the distance of the focus, and were submitted to linear regression using the least square approach, generating mathematical parameters such as <i>R²</i>, <i>R</i> and also first order equation (<i>y = ax + b</i>). (I–L) The same procedure was undertaken using values from pixel analysis. <i>X–Y</i> axis bitmap analysis was used to view the pixel values in numeric format, where values correspond to the brightness of the pixels. (M) Considering the distribution of TUNEL-positive nuclei, the means of angular coefficient from first order equation (<i>a</i>) were calculated for each experimental condition (n = 3). When compared to the control (PBS), the means of <i>a</i> angular coefficient were higher for all evaluated conditions using GJ blockers (CBX, quinine and CBX + quinine). (N) Regarding values from bitmap pixel analysis, we observed that the means of <i>a</i> angular coefficient were higher for quinine and CBX + quinine conditions. *<i>P</i><0.01 and **<i>P</i><0.05 vs. PBS in Newman-Keuls pairwise comparisons after one-way ANOVA. Scale bar: 60 µm.