benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Spinal-Cord-Injuries

The aim of this study was to investigate the effects of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketones (z-VAD-fmk) on the degree of experimental spinal cord trauma induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, neutrophil infiltration, production of a range of inflammatory mediators, tissue damage, and apoptosis. Treatment of the mice with z-VAD-fmk, a potent broad specific caspase inhibitor, significantly reduced the degree of (1) spinal cord inflammation and tissue injury (histological score), (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation, and (4) apoptosis (TUNEL staining and Bax and Bcl-2 expression). In a separate set of experiments, z-VAD-fmk significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with z-VAD-fmk reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Humans; In Situ Nick-End Labeling; Inflammation; Male; Mice; Neuroprotective Agents; Neutrophils; Peroxidase; Proto-Oncogene Proteins c-bcl-2; Spinal Cord Injuries

Apoptosis-modulating therapeutics using active-site mimetic peptide ketones (z-VAD-fluoromethylketone (fmk)) have been reported to be efficacious in delaying the apoptotic response in central nervous system lesions. The purpose of the present study was to examine whether the caspase inhibitor z-VAD fmk prevents apoptosis and improves neurological deficit and tissue damage. One-hundred twenty female Sprague-Dawley rats were randomized into groups that were administered 25 microg of z-VAD-fmk or vehicle 30 min and 24 h after moderate spinal cord contusion (NYU impactor, 12.5 mm at T10). Several routes of administration were tested: (1) via Gelfoam placed on the spinal cord, (2) into the cisterna magna via a subarachnoidal catheter, (3) intravenously via the external jugular vein, or (4) intraperitoneally. Another group was injected with 50 microg of zVAD-fmk or vehicle intraperitoneally 30 min, 24, 48, and 72 h after injury. Animals were evaluated for locomotor function (BBB score) at weekly intervals for 6 weeks after injury and treatment. Spinal cords were then processed for histological analysis to determine whether zVAD-fmk treatment decreased contusion volume. Other spinal cord samples were harvested 24 h after injury and examined for cleavage of XIAP by immunoblot analysis. There were no significant differences in the BBB scores, contusion volumes, and XIAP cleavage between animals receiving the broad specific caspase inhibitor by the various routes and animals receiving vehicle alone. These findings raise critical questions about the use of peptide ketone apoptotic inhibitors in improving functional and histopathological outcomes following spinal cord injury.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Enzyme Inhibitors; Female; Proteins; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; X-Linked Inhibitor of Apoptosis Protein

Evidence indicates that both necrotic and apoptotic cell death contribute to tissue injury and neurological dysfunction following spinal cord injury. Caspases have been implicated as important mediators of apoptosis following acute central nervous system insults. We investigated whether caspase-1 and caspase-3 are involved in spinal cord injury-mediated cell death, and whether caspase inhibition may reduce tissue damage and improve outcome following spinal cord injury. We demonstrate a 17-fold increase in caspase-1 activity in traumatized spinal cord samples when compared with samples from sham-operated mice. Caspase-1 and caspase-3 activation were also detected by western blot following spinal cord injury, which was significantly inhibited by the broad caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. By immunofluorescence or in situ fluorogenic substrate assay, caspase-1 and caspase-3 expression were detected in neuronal and non-neuronal cells following spinal cord injury. N-Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone treated mice, and transgenic mice expressing a caspase-1 dominant negative mutant, demonstrated a significant improvement of motor function and a reduction of lesion size compared with vehicle-treated mice. Our results demonstrate for the first time that both caspase-1 and caspase-3 are activated in neurons following spinal cord injury, and that caspase inhibition reduces post-traumatic lesion size and improves motor performance. Caspase inhibitors may be one of the agents to be used for the treatment of spinal cord injury.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 1; Caspase 3; Caspase Inhibitors; Caspases; Enzyme Activation; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Mice, Transgenic; Motor Activity; Neurons; Neuroprotective Agents; Spinal Cord Injuries