Another evidence that activated caspase-3 is not an exclusive apoptotic marker: a comprehensive study of activated caspase-3 population of cells in rat spinal cord

Abstract

Cell death is an essential process occurring during the development of the central nervous system. Despite the availability of wide range of commercially produced antibodies against various apoptotic markers, data regarding the apoptosis in intact spinal cord during postnatal development and adulthood are mostly missing. This study aimed to investigate the apoptosis in the rat spinal cord at different stages of ontogenesis (8, 29 and 90 postnatal days). For this purpose, we used immunofluorescent detection of two widely used apoptotic markers, activated caspase-3 (aC3) and cleaved PARP (cPARP). Surprisingly, we found significant discrepancy between the amounts of aC3+ cells and PARP+ cells, varying with ratio around 500:1–5,000:1 in the rat spinal cord in all postnatal time points. Majority of aC3+ cells were glial cells and did not exhibit apoptotic phenotype. In contrast with the results of in vivo study, in vitro analysis of primary cell culture derived from neonatal rat spinal cord, treated with apoptotic inductor staurosporine, revealed similar onset of occurrence of both markers in cells subjected to apoptosis. Gene expression analysis of spinal cord tissue revealed elevated expression of Birc4 (XIAP), Birc2 and Birc5 (Survivin) genes, which are known as potent inhibitors of apoptosis. Our data indicates that the activated caspase-3 is not an exclusive marker of apoptosis, especially in glial cells, due its possible presence in inhibited forms and/or its participation in other, non-apoptotic roles. Therefore, in the light of our recent results, cPARP appears to be more appropriate marker for detection of apoptosis.