Biochemical evidence of apoptosis. The spinal cord was removed at 48 h post-SCI by being frozen in situ with liquid nitrogen and divided into three sections of equal length (1 cm each) with section 1 centered at the epicenter and sections 2 and 3 centered at 1 and 2 cm caudal to the epicenter for DNA laddering and ELISA as described in the Methods. A: The laddering of DNA fragments in the spinal cord tissue. Lane 1, standard DNA marker; lane 2, the tissue obtained from the epicenter; lanes 3 and 4, tissue centered 1 and 2 cm away from the epicenter, respectively; and lane 5, tissue from a sham-operated rat. The fragmented DNA shown in each lane was obtained from the corresponding sections of one rat (60 mg wet weight tissue). The results are representative of four separate experiments. B: Quantitative analysis of fragmented DNA by ELISA with 3 sections at epicenter, 1 and 2 cm caudal from the epicenter. Each sample for ELISA analysis equaled 2 mg wet weight tissue. Error bars: mean ± SEM. Both A and B show significantly decreased DNA fragmentation with an increase in distance from the epicenter. C: Caspase-3 activation analyzed at 4 h post-SCI. The injured cord was divided into two sections with section 1 centered at the injury epicenter and section 2 centered 1 cm from the epicenter. Error bars: mean ± SEM. The caspase-3-like protease activity was significantly higher at the injury epicenter compared to 1 cm from the epicenter and sham control.