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, 20 (8), 2825-34

Cytochrome C Release and Caspase Activation in Traumatic Axonal Injury

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Cytochrome C Release and Caspase Activation in Traumatic Axonal Injury

A Büki et al. J Neurosci.

Abstract

Axonal injury is a feature of traumatic brain injury (TBI) contributing to both morbidity and mortality. The traumatic axon injury (TAI) results from focal perturbations of the axolemma, allowing for calcium influx triggering local intraaxonal cytoskeletal and mitochondrial damage. This mitochondrial damage has been posited to cause local bioenergetic failure, leading to axonal failure and disconnection; however, this mitochondrial damage may also lead to the release of cytochrome c (cyto-c), which then activates caspases with significant adverse intraaxonal consequences. In the current communication, we examine this possibility. Rats were subjected to TBI, perfused with aldehydes at 15-360 min after injury, and processed for light microscopic (LM) and electron microscopic (EM) single-labeling immunohistochemistry to detect extramitochondrially localized cytochrome c (cyto-c) and the signature protein of caspase-3 activation (120 kDa breakdown product of alpha-spectrin) in TAI. Combinations of double-labeling fluorescent immunohistochemistry (D-FIHC) were also used to demonstrate colocalization of calpain activation with cyto-c release and caspase-3-induction. In foci of TAI qualitative-quantitative LM demonstrated a parallel, significant increase in cyto-c release and caspase-3 activation over time after injury. EM analysis demonstrated that cyto-c and caspase-3 immunoreactivity were associated with mitochondrial swelling-disruption in sites of TAI. Furthermore, D-IFHC revealed a colocalization of calpain activation, cyto-c release, and caspase-3 induction in these foci, which also revealed progressive TAI. The results demonstrate that cyto-c and caspase-3 participate in the terminal processes of TAI. This suggests that those factors that play a role in the apoptosis in the neuronal soma are also major contributors to the demise of the axonal appendage.

Figures

Fig. 1.
Fig. 1.
Axons displaying cyto-c-IR (A,B) and SBDP-120-IR (C,D) in rat medial longitudinal fasciculus (arrows). At 15 min after injury (A,C), the axonal segments appear swollen (A, C), and at 360 min after injury, both cyto-c- and SBDP-120-IR axonal profiles display evidence of imminent disconnection (B, D). Scale bar, 15 μm.
Fig. 2.
Fig. 2.
Bar charts representing the mean densities of immunopositive axons displaying cyto-c immunoreactivity in the CSpT (black) and the MLF (gray) at various postinjury survival times after impact acceleration injury. Error bars indicate SEM, and the asterisksindicate statistical significance (p < 0.05) between consecutive survival times. (Significant changes between non-neighboring survival times are not indicated.)
Fig. 3.
Fig. 3.
Bar chart of the mean densities of immunopositive axons displaying caspase-linked SBDP-120- immunoreactivity in the CSpT (black) and the MLF (gray) at various postinjury survival times after impact acceleration injury. Error bars indicate SEM, and the asterisks indicate statistical significance (p < 0.05) between consecutive survival times. (Significant changes between non-neighboring survival times are not indicated.)
Fig. 4.
Fig. 4.
Electron micrographs of axonal profiles demonstrating cyto-c-IR (A–D). Overview of a segment of the corticospinal tract 30 min after injury (A). The short white arrow marks an uninjured axonal segment illustrated in B in high-power magnification, and the long black arrowpoints to a portion of the traumatically altered axonal profile enlarged in C. Note the loosening of the myelin sheet around the injured axonal segment (M). Uninjured axonal profiles (B) display normal interfilament distance, unaltered mitochondrial structure (arrow), and the lack of DAB deposition. Traumatically injured scattered axonal segments from the same field (C) reveal neurofilament compaction and mitochondrial alteration with the electron-dense DAB chromogen partially covering the surface of the swollen mitochondria (arrows). Six hours after injury (D), the cytoskeletal alterations, such as neurofilament compaction and digestion, as well as the modificaton of the myelin sheet (M), is more obvious. Note the pooling of the mitochondria, many of which are disrupted or swollen (arrows). Also note the cyto-c immunoreactivity, which diffuses into the surrounding axoplasm (asterisks). Scale bar, 500 nm.
Fig. 5.
Fig. 5.
Electron micrograph of caspase-associated SBDP-120-immunoreactive axonal profiles. At 30 min after injury (A), the electron-dense DAB reaction product is primarily associated with cytoskeletal elements (arrows) and the perimitochondrial domain (double arrow). Note a damaged swollen mitochondrion with partially ruptured cristae approximating normal-appearing mitochondria. Also, note the loosening of the myelin sheet (M) and the reduction of interfilament distance (white asterisks). Three hours after injury (B, unstained section), the cytoskeletal damage (and the related SBDP-120-IR) is more obvious (white asterisk), as is the mitochondrial swelling (arrows). At 6 hr after injury (C), note the cytoskeletal alterations and the severe mitochondrial damage (arrows) reflected in their pooling, swelling, and rupture. Also note the myelin alterations (M) and the electron-dense DAB associated with damaged mitochondria and/or the disintegrated cytoskeletal elements (asterisk). Scale bar, 500 nm.
Fig. 6.
Fig. 6.
A–C, Images of Ab38 (A) and cyto-c (B) immunofluorescent damaged axonal foci and a digital overlay of the same fields (C) in rat medial longitudinal fasciculus at 60 min after injury. Note the segmental swelling and focal vacuolization and that cyto-c and CMSP immunoreactivity are clearly colocalized in this damaged axonal segment. D–F, Images demonstrating cyto-c (D) and caspase-associated SBDP-120 (E) immunofluorescence damaged axonal foci and a digital overlay of the same fields (F) in rat MLF at 3 hr after injury. The morphology of the damaged immunoreactive axonal segment is consistent with imminent axonal disconnection. Again, note the obvious colocalization of the immunohistochemical markers. G–I, One hour after injury, immunoreactivity representing the activated form of caspase-3 enzyme (G) and caspase-linked spectrin proteolysis-IR (SBDP-120) (H) are directly colocalized within the same vacuolated, swollen axonal profiles (I), providing direct evidence of the contribution of the caspase death cascade to the pathogenesis of traumatic axonal injury. J–L, Images demonstrating calpain-mediated spectrin proteolysis immunoreactivity (J) and caspase-linked SBDP-120-IR (K) 3 hr after injury. The digital overlay of the same fields (L) proves that both cysteine proteases are activated within the same, severely damaged axonal segments. Scale bar, 20 μm.

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