benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Cerebral-Infarction

Energy requiring apoptosis and presumably unregulated necrosis are considered conceptually and morphologically distinct forms of cell death which have been initially identified as two exclusive pathways. However, several apoptotic characteristics have been observed in the necrotic core lesion in ischemia which led to the controversial theory that cell death advances via a number of hybrid pathways among a continuum between the two processes. ATP availability has been shown to influence the decision between apoptosis and necrosis. The aims of our study are 1) to determine if combined inhibitors administration of pan-caspase inhibitor Carbobenzoxy-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) and non-selective poly (ADP-ribose) polymerase (PARP) inhibitor 3-aminobenzamide (3-AB) can further reduce infarct volume compared to single modality of either inhibitor following ischemic insult, 2) to ascertain the pharmacological intervention up to 24 hour post-middle cerebral artery occlusion (MCAo), and 3) to correlate intracellular ATP level with infarct volume. Single modality treatment was optimised at 3 mg/kg z-VAD-fmk and 30 mg/kg 3-AB with infarct volume measured at 24.13%+/-3.89% and 26.98%+/-2.22% respectively, while untreated control group was determined at 45.97%+/-1.86%. Combined inhibitors treatment rendered further reduction in infarct volume, measuring 7.228%+/-1.988%, 21.02%+/-1.06%, 24.40%+/-2.12% at 30 min, 6 h, 24 h post-ischemia respectively. In conclusion, the combined inhibitors administration of both z-VAD-fmk and 3-AB show further increased in infarct volume reduction with our ischemic model up to the 24 hour post-MCAo. However, in our in vivo study, no correlation between intracellular ATP level and infarct size was established.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Benzamides; Caspases; Cerebral Infarction; Drug Therapy, Combination; Energy Metabolism; Enzyme Inhibitors; Male; Necrosis; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Stroke; Treatment Outcome

Recent studies have shown that release of mitochondrial cytochrome c is a critical step in the apoptosis process. We have reported that cytosolic redistribution of cytochrome c in vivo occurred after transient focal cerebral ischemia (FCI) in rats and preceded the peak of DNA fragmentation. Although the involvement of reactive oxygen species in the cytosolic redistribution of cytochrome c in vitro has been suggested, the detailed mechanism by which cytochrome c release is mediated in vivo has not yet been established. Also, the role of mitochondrial oxidative stress in cytochrome c release is unknown. These issues can be addressed using knock-out mutants that are deficient in the level of the mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD). In this study we examined the subcellular distribution of the cytochrome c protein in both wild-type mice and heterozygous knock-outs of the Mn-SOD gene (Sod2 -/+) after permanent FCI, in which apoptosis is assumed to participate. Cytosolic cytochrome c was detected as early as 1 hr after ischemia, and correspondingly, mitochondrial cytochrome c showed a significant reduction 2 hr after ischemia (p < 0.01). Cytosolic accumulation of cytochrome c was significantly higher in Sod2 -/+ mice compared with wild-type animals (p < 0.05). N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone (z-VAD.FMK), a nonselective caspase inhibitor, did not affect cytochrome c release after ischemia. A significant amount of DNA laddering was detected 24 hr after ischemia and increased in Sod2 -/+ mice. These data suggest that Mn-SOD blocks cytosolic release of cytochrome c and could thereby reduce apoptosis after permanent FCI.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blood Pressure; Brain; Brain Ischemia; Cardiomyopathy, Dilated; Cerebral Cortex; Cerebral Infarction; Cysteine Proteinase Inhibitors; Cytochrome c Group; Cytosol; DNA Fragmentation; Heterozygote; Ischemic Attack, Transient; Male; Mice; Mice, Knockout; Mitochondria; Oxidative Stress; Rats; Superoxide Dismutase; Superoxides

Inhibitors of apoptosis and of excitotoxic cell death reduce brain damage after transient and permanent middle cerebral artery occlusion. We compared the neuroprotective effects of two caspase family inhibitors with the N-methyl-D-aspartate receptor antagonist (+)-MK-801 hydrogen maleate (MK-801) in a newly characterized cycloheximide-sensitive murine model of transient middle cerebral artery occlusion (30 minutes) in which apoptotic cell death is prominent. Ischemic infarction, undetected by 2,3,5-triphenyltetrazolium chloride staining at 24-hour reperfusion, featured prominently in the striatum at 72 hours and 7 days on hematoxylin-eosin-stained sections. Markers of apoptosis, such as oligonucleosomal DNA damage (laddering) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-positive cells first appeared at 24 hours and increased significantly at 72 hours and 7 days after reperfusion. The TUNEL-labeled cells were mostly neurons and stained negative for glial (GFAP, glial fibrillary acid protein) and leukocyte specific markers (CD-45). The caspase inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.FMK; 120 ng intracerebroventricularly) or N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (z-DEVD.FMK; 480 ng intracerebroventricularly) decreased infarct size and neurologic deficits when administered 6 hours after reperfusion. The extent of protection was greater than in models of more prolonged ischemia or after permanent occlusion, and the therapeutic window was extended from 0 to 1 hours after 2-hour middle cerebral artery occlusion to at least 6 hours after brief ischemia. Also, z-VAD.FMK and z-DEVD.FMK treatment decreased oligonucleosomal DNA damage (DNA laddering) as assessed by quantitative autoradiography after gel electrophoresis. By contrast, MK-801 protected brain tissue only when given before ischemia (3 mg/kg intraperitoneally), but not at 3 or 6 hours after reperfusion. Despite a decrease in infarct size after MK-801 pretreatment, the amount of DNA laddering did not decrease 72 hours after reperfusion, thereby suggesting a mechanism distinct from inhibition of apoptosis. Hence, 30 minutes of reversible ischemia augments apoptotic cell death, which can be attenuated by delayed z-VAD.FMK and z-DEVD.FMK administration with preservation of neurologic function. By contrast, the therapeutic window for MK-801 does not extend beyond the time of occlusion, probably because its primary mecha

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Brain Ischemia; Caspase 1; Caspase 3; Caspases; Cell Death; Cerebral Infarction; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Injections, Intraventricular; Male; Mice; Neurons; Oligopeptides