leupeptins and Spinal-Cord-Injuries

Here, we investigated the effect of calpain inhibitors on apoptosis in organotypic adult spinal cord slices from mice. An increase in calpain I immunoreactivity was found in the nuclei of motor neurons from slices cultured for 30 min. After 4 h, the immunopositive motor neurons exhibited apoptotic changes including nuclear and chromatin condensation. Eight hours after excision, most motor neurons showed nuclear apoptotic features. Two calpain inhibitors, leupeptin and calpain inhibitor XI, inhibited apoptosis in the motor neurons while the caspase inhibitor Z-VAD.fmk had no effect. Leupeptin, but not calpain inhibitor XI and Z-VAD.fmk, also inhibited nucleosomal DNA fragmentation. These results suggest the involvement of calpain I in the induction of apoptosis in motor neurons of adult spinal cord and that apoptosis can be triggered independent of caspase activation.

Topics: Age Factors; Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Calpain; Caspases; Cysteine Proteinase Inhibitors; DNA Fragmentation; Female; Glycoproteins; Immunohistochemistry; Leupeptins; Mice; Motor Neurons; Nerve Degeneration; Organ Culture Techniques; Spinal Cord; Spinal Cord Injuries; Time Factors

Intraperitoneal administration of the neutral protease inhibitors leupeptin and E-64c substantially suppressed the degradation of neurofilament proteins (NFP) at the site of mechanical insult and secondary axonal degeneration, and facilitated the recovery of motor functions in acute spinal cord injury in rats. The drug effects were assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis of NFP fractions from the injured tissue and by morphometry of degenerating axons revealed by the Fink-Heimer method in distal spinal cord segments with the aid of an automated image analyzer. The role of calcium-activated neutral proteases in acute central nervous tissue damage and potential use of protease inhibitors as therapeutic modalities are discussed.

Topics: Animals; Electrophoresis, Polyacrylamide Gel; Intermediate Filament Proteins; Leucine; Leupeptins; Male; Nerve Tissue Proteins; Neurofilament Proteins; Oligopeptides; Protease Inhibitors; Rats; Rats, Inbred Strains; Spinal Cord; Spinal Cord Injuries

The effect of large doses of methylprednisolone sodium succinate (MPSS) and two protease inhibitors, leupeptin and bestatin, on experimental acute spinal cord injury was evaluated by morphometric analysis of degenerating axons with the aid of an automated image analyzer. Spinal cord injury was produced by epidural compression with a surgical clip on the T-11 segment in rats. The extent of axonal damage was assessed in Rexed's lamina VIII in the L-6 segment by measuring the amount of silver grains, representing degenerating axons and their terminals, using the Fink-Heimer method. The severity of axonal damage was expressed as the degeneration index: that is, the amount of silver grains in experimental animals/the amount of silver grains in cord-transected animals. When examined on the 7th postoperative day, axonal degeneration in MPSS-treated rats was significantly decreased, with an average degeneration index difference of 6 (p less than 0.05). Increased preservation of axons was seen in the leupeptin-treated rats sacrificed 7, 10, and 14 days after trauma. The difference in the degeneration index between the leupeptin-treated and untreated groups was 16 on Day 7 (p less than 0.001), 12 on Day 10 (p less than 0.001), and 13 on Day 14 (p less than 0.01). Bestatin had no beneficial effect. The implications for the use of calcium-activated neutral protease inhibitors in acute spinal cord injury are discussed.

Topics: Animals; Axons; Leucine; Leupeptins; Male; Methylprednisolone; Methylprednisolone Hemisuccinate; Rats; Rats, Inbred Strains; Spinal Cord Injuries

The degenerating axons and axon terminals developed in Rexed's lamina VIII in the anterior horn of the L6 segment after acute spinal cord compression at Th11 level in rats were visualized by the method of Fink-Heimer and the extent of axonal damage was quantitatively assayed with the aid of an automated image analyzer. Leupeptin, a potent protease inhibitor, substantially reduced the extent of the axonal damage (17% on average).

Topics: Animals; Leupeptins; Male; Oligopeptides; Rats; Rats, Inbred Strains; Spinal Cord; Spinal Cord Injuries; Wounds, Nonpenetrating