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Caspase-dependent and -independent pathways in neuronal apoptosis after spinal cord injury Kay L.H. Wu, Chin Hsu and Julie Y.H. Chan
Graduate Institute of Medicine, Kaohsiung Medical university, and Department o Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
Introduction
1. Spinal cord injury (SCI) results in irreversible neuronal damage leading to apoptotic cell death.2. Accumulated evidence indicates that mitochondria initiate apoptosis via cellular events that
include caspases-dependent and caspases-independent pathways.3. We study the hypotheses that mitochondrial respiratory impairment promotes neuronal apoptosis
and the impairment is induced via early stage of free radical production after SCI.
Aims of Study
1. To identify the temporal profiles of apoptosis in the spinal cord after injury.2. To delineate the relationship between mitochondrial respiratory dysfunction
and neuronal apoptosis after spinal cord injury.3. To investigate the involvement of free radical in mitochondrial dysfunction in
the spinal cord after injury.
Materials and Methods
1 52 3 4 7 10
Tissue fixation
Electromicroscopy
Genomic DNAProteins
Mitochondria
DNA LadderingWestern Blot
T8
14
Summary and ConclusionFree radical
NeuronNucleus
AIF
DNAfragmentation
Caspase-3activation
Apoptosis
PARPactivation
I
IIIII
IV
Caspase-9activation cyt. c
Figure 1. DNA fragmentation detected in the spinal cord after T8 transection (SCT) alone or with addition of coenzyme Q10 (CoQ 10) treatment. Values are mean SEM, n = 9-12 animals per group. *P<0.05 vs. baseline control (C) in the Dunnett multiple-range test, or #P<0.05 vs. SCT groups in the Scheffé multiple-range test.
M C 1 2 3 4 5 7 10 14
SCT only
SCT with CoQ10
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Figure 3. Time-course of changes in the mitochondrial respiratory complex I activity, ATP production in the spinal cord after SCT alone or with addition of CoQ10 treatment. Values are mean SEM, n = 9-12 animals per group. *P<0.05 vs. control (0) in the Dunnett multiple-range test, or #P<0.05 vs. SCT groups in the Scheffé multiple-range test. Also shown is the representative electron microscopic photomicrographs illustrating swelling of the mitochondrial cristae after SCT.
Normal control (50000 X) Post SCT 1 day (50000 X)Mitochondria complex I activityin the spinal cord after injury
ATP concentrationin the spinal cord after injury
Figure 5. Time-course changes of iNOS expression or NOx levels in the spinal cord after SCI alone or with addition of CoQ10 treatment. Values are mean SEM, n = 9-12 animals per group. *P<0.05 vs. control (0) in the Dunnett multiple-range test, or #P<0.05 vs. SCT groups in the Scheffé multiple-range test. Also shown is the representative photomicrographs illustrating the distribution of dihydroethidium or expression of NADPH oxidase subunits in the injured spinal cord.
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Cytosolic iNOS expressionin the spinal cord after injury
NOx concentrationin the spinal cord after injury
gp91phox
p47phox
p67phox
C 1 2 3 4 5 7 10 14Dihydroethidium stain
in 3rd day after SCI
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Nucleus AIF expression
(NeuN, AIF and TUNEL triple-labeled cells, 1000 X)
3rd day after SCI
NeuN AIF
TUNEL Merge
Figure 4. Time-course of changes in the expression of apoptosis inducing factor (AIF) in the spinal cord after SCT alone or with addition of CoQ10 treatment. Values are mean SEM, n = 9-12 animals per group. *P<0.05 vs. control (0) in the Dunnett multiple-range test, or #P<0.05 vs. SCT groups in the Scheffé multiple-range test. Also shown is the representative photomicrographs illustrating co-localization of AIF-immunoreactivity with NeuN-immunoreactivity in the TUNEL-positive cells.
0 1 2 3 4 5 7 10 14
Figure 2. Time-course of changes in the expression of caspase-dependent cytosolic proteins in the spinal cord after SCT alone or with addition of CoQ10 treatment. Values are mean SEM, n = 9-12 animals per group. *P<0.05 vs. control (0) in the Dunnett multiple-range test, or #P<0.05 vs. SCT groups in the Scheffé multiple-range test.
Cytosolic cleaved caspase-9 expression
Cytosolic cleaved caspase-3 expression
Cytosolic cytochrome c expression Cleaved PARP expression
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C 1 2 3 4 5 7 10 14 C 1 2 3 4 5 7 10 14C 1 2 3 4 5 7 10 14 C 1 2 3 4 5 7 10 14
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