benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Stroke

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

In primary neuronal-astrocyte cultures from mouse brain, ischemic conditions were simulated by combined oxygen-glucose deprival (OGD) for 2 hrs. This treatment resulted in near complete neuronal damage 24 hrs. later and was accompanied by DNA degradation and apoptotic nuclear morphology. Since caspases are key enzymes in the propagation and execution of programmed cell death, we evaluated the effect of the caspase inhibitor z-VAD-fmk. Damage following 2 hrs. OGD could be reduced by up to 56% with z-VAD-fmk (p<0.05). DNA-fragmentation and caspase activation has been also reported in an in vivo model of cerebral ischemia imitating human stroke. In this model the middle cerebral artery (MCA) is permanently occluded resulting in focal cerebral ischemia and subsequent infarction. Since z-VAD.fmk does not penetrate the blood-brain barrier it was applied intraventricularly as a bolus injection given 30 min. before MCA occlusion which was followed by 24 hrs. of infusion. Infarct volume was determined 48 hrs. after MCA occlusion by means of in vivo magnetic resonance imaging. Z-VAD.fmk dose dependently reduced infarct volume reaching a significant decrease of the cortical infarct by 45% when given as a 120 ng bolus followed by 40 ng/hr. infusion (p<0.05). In summary, our study supports the concept that caspase inhibitors are beneficial in brain ischemia.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Astrocytes; Brain; Brain Ischemia; Caspase Inhibitors; Dizocilpine Maleate; Enzyme Inhibitors; Glucose; Immunohistochemistry; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Magnetic Resonance Imaging; Mice; Neuroprotective Agents; Rats; Rats, Inbred F344; Stroke