Apoptosis in normal lymphoid organs from healthy normal, conventional pigs at different ages detected by TUNEL and cleaved caspase-3 immunohistochemistry in paraffin-embedded tissues

Abstract

The frequency and the distribution of apoptotic cells were investigated in formalin-fixed paraffin-embedded lymphoid tissues from healthy conventional pigs at four different ages (6 days, 2 months, 3.5 months and 5 months). Samples of tonsil, mesenteric lymph node, spleen, thymus and Peyer's patches were histologically processed and apoptosis evaluated with the TUNEL reaction and cleaved caspase-3 immunohistochemistry. In each technique, quantification of positive labelling was done for each particular lymphoid tissue area. The labelling pattern and distribution were similar for TUNEL and cleaved caspase-3. TUNEL stained mainly apoptotic bodies inside macrophages, but signal was also seen in free apoptotic bodies and in the nuclei of lymphocyte-like cells. The anti-cleaved caspase-3 antibody labelled mainly nuclei of lymphocyte-like cells. All tissues presented a similar distribution pattern of apoptosis, except for the 6-day-old group. In this group, a scattered distribution of positive cells was detected in tonsil, lymph node and spleen. In the tonsil and mesenteric lymph nodes from the older pigs, follicular areas presented higher amounts of positive cells than interfollicular areas. Moreover, the splenic white pulp showed more positive reaction than the red pulp, especially when they included germinal centres. In all groups, the follicular areas of ileal Peyer's patches presented more labelled cells than the dome and the lamina propria. In the thymus, the higher apoptotic rates were found in the cortex. In general, TUNEL yielded higher rates of positive cells than cleaved caspase-3 immunolabelling. A good correlation between the two techniques was found for thymus, tonsil and mesenteric lymph node, but not for Peyer's patches and spleen. This study describes a detailed histochemical characterization of apoptosis in pig lymphoid tissues using TUNEL and a cleaved caspase-3 immunolabelling at different ages. Moreover, our results indicate that TUNEL and cleaved caspase-3 techniques can be equivalent only when tissues have a high or low levels of apoptosis, since a considerable discrepancy was found in intermediate situations. Data from this study should be useful for future comparative studies under disease conditions.