Caspase‐3 Mediated Neuronal Death After Traumatic Brain Injury in Rats

Abstract

During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase-3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro-caspase-3 in cytosolic cellular fractions and an increase in caspase-3-like enzyme activity were seen in injured brain versus control. Cleavage of the caspase-3 substrates DNA-dependent protein kinase and inhibitor of caspase-activated deoxyribonuclease and co-localization of cytosolic caspase-3 in neurons with evidence of DNA fragmentation were also identified.

boc-Aspartyl(OMe)-Fluoromethylketone Attenuates Mitochondrial Release of Cytochrome c and Delays Brain Tissue Loss after Traumatic Brain Injury in Rats

“…Released cytochrome c engages with caspase-9 and apoptotic proteaseactivating factor-1 (Apaf-1) to form an 'apoptosome' that activates caspase-3 and results in cellular changes producing apoptotic cell death (Li et al, 1997;Zou et al, 1997). Several relatively selective caspase-3 inhibitors such as DEVD have been reported to be neuroprotective after experimental TBI, used primarily to target events downstream from cytochrome c release (Clark et al, 2000;Knoblach et al, 2004;Yakovlev et al, 1997). Fewer studies have tested the effects of less selective caspase inhibitors after TBI, which could be expected to have effects both upstream and downstream from cytochrome c release.…”

“…Brains were then cut into 5-mm coronal sections through the contusion, and mounted on glass slides. Sections were then processed for terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL) and counterstained with cresyl violet as described (Clark et al, 2000). Separation of Cellular Proteins: Separation of cytosolic, nuclear, and mitochondrial proteins was performed as previously described .…”

“…Washed membranes were incubated in the appropriate horseradish peroxidase-conjugated secondary antibody and immunoblotted proteins were detected using chemiluminescence (NEN, Boston, MA, USA). To assess efficiency of separation of cellular compartments and rule out nonspecific release of mitochondria-associated proteins, immunoblotting for cytochrome c oxidase (Clark et al, 2000) was also performed. Protein abundance was determined by measuring the relative optical density of the respective protein bands using a Kodak Image Station 440CF (Kodak, Rochester, NY, USA).…”

“…Neurons were counted in the entire anatomic CA1 and CA3 hippocampal regions by an observer masked to experimental group. Normal appearing neurons with a well-defined nucleus and cell body were counted in each hemisphere, and were discriminated from non-neuronal cells based on size and morphologic criteria as previously described (Clark et al, 1997(Clark et al, , 2000. Neuronal cell counts are expressed as the percentage of surviving neurons in the ipsilateral versus contralateral hemispheres.…”

Response of caspase-independent apoptotic factors to high salt diet-induced heart failure

“…The fractionation method described by Clark et al [15] was adopted with minor modifications to extract the mitochondrial and cytosolic S-100 fractions from frozen ventricular tissues. Briefly, ventricle muscle was homogenized on ice with 100 strokes of a glass homogenizer in ice-cold lysis buffer (10 mM NaCl, 1.5 mM MgCl 2 , 120 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and 250 mM sucrose) supplemented with a protease inhibitor cocktail.…”