Caspase-3 is activated following axotomy of neonatal facial motoneurons and caspase-3 gene deletion delays axotomy-induced cell death in rodents

In this report, we examined the possible functions of the cell death protea se, caspase-3, in the axotomy-induced apoptosis of facial motoneurons in ne wborn rodents. Using in situ hybridization and Western blot, we found highe r levels of caspase-3 mRNA and pro-caspase-3 protein expression in motoneur ons of neonatal and 2-week-old rats than adult rats. Following facial moton euron axotomy, caspase-3 mRNA and protein expression increased in motoneuro ns of both neonatal and adult rats. However, using an antibody directed to the activated form of the caspase-3 protease, we found that catalytically a ctive caspase-3 was present only in axotomized neonatal motoneurons. As mot oneurons in neonatal but not adult rodents are susceptible to axotomy-induc ed apoptosis, we hypothesized that caspase-3 may play a role in their demis e. To determine the necessity of caspase-3 activation in axotomy-induced ap optosis, we counted the number of surviving motoneurons at 4 and 7 days fol lowing axotomy in wild type mice and caspase-3 gene-deleted mice. There wer e nearly three times more surviving motoneurons in caspase-3 gene-deleted m ice than in wild type mice at both 4 days (mean 1074 vs. 464, P < 0.005) an d 7 days (mean 469 vs. 190, P < 0.005) following injury, indicating a slowe r rate of death. Examination of the dying motoneurons using TUNEL staining (for fragmented DNA) and bisbenzimide staining (for nuclear morphology) rev ealed incomplete nuclear condensation in caspase-3-deficient motoneurons. T hese results demonstrate that caspase-3 activation plays important roles in the rapid demise of axotomized neonatal motoneurons.