A selective cysteine protease inhibitor is non-toxic and cerebroprotective in rats undergoing transient middle cerebral artery ischemia.

Ischemic neuronal injury mediated by cysteine proteases such as calpains and caspases has been demonstrated in various experimental models. Cathepsins B and L are also cysteine proteases which may contribute to neuronal death after ischemia. The authors measured in vitro and in vivo toxicity and post-ischemic cytoprotective effects of a cysteine protease inhibitor which does not block calpain or caspase but, rather, is relatively selective for cathepsins B and L. The compound belongs to the peptidyl-diazomethane family (cysteine protease inhibitor 1, termed CP-1). In vitro toxicity was measured using an assay of cell viability, and in vivo toxicity was measured by histological tissue analysis after infusion of CP-1 in rats. Two hours of middle cerebral artery (MCA) occlusion in rats was performed by the intravascular suture method. Immediately following reperfusion, intravenous infusion of CP-1 or vehicle was performed for 4 h at 0.9 ml/h. After a 7-day survival, the infarct volumes were measured. CP-1 was non-toxic to cultured glial cells to a local concentration of 200 microM, and relatively non-toxic to cultured endothelial cells at concentrations of 100-200 microM. No animal exhibited toxic effects at any of the doses used. Histologic comparisons revealed no signs of tissue toxicity. CP-1 significantly reduced hemispheric infarct volume compared to control (37+/-8.2%) at concentrations of 10, 50, and 250 microM [22+/-15%, P=0.008; 20+/-13%, P=0.002; 23+/-15%, P=0.022, respectively (mean+/-standard deviation; N=7-10 per group)]. CP-1, at the concentration of 50 microM, improved the functional score of the animals, but did not significantly alter cerebral blood flow. This study supports the hypothesis that the lysosomal cathepsins B and/or L contribute to cerebral injury after focal ischemia with reperfusion. Cysteine protease inhibitors which are relatively selective for cathepsins B and L, but not the calpains or caspases, are effective at reducing infarct volume after intravenous post-ischemic administration.

Early changes in concanavalin A-stimulated lymphocytes detected by the fluorescent probe N-phenyl-1-naphthylamine.

The hydrophobic fluorescent cell-membrane probe N-phenyl-1-naphthylamine (NPN) is a useful investigative tool for studies of early lymphocyte activation. NPN-labelled mouse thymus cells incubated with 5 micrograms/ml concanavalin A (Con A) for 30 min at 37 degrees C gave a reproducible increase in mean cell-fluorescence intensity measured by microfluorimetry on 100 single cells. The dose-response curve was similar to that obtained by 3H-thymidine assay. Increased fluorescence was not observed in the presence of 10 mM alpha-methyl mannoside, 5 mM sodium azide, 10(-5) M cytochalasin B, or Ca2+-free culture medium. However, incubation with 10(-5) M colchicine did not alter the probe response. Fluorescence change was also shown by spleen cells from a normal mouse but not from an athymic mouse, indicating T cell dependence of the response. Comparison with other lectins showed that increased fluorescence followed incubation with phytohaemagglutinin, and the non-mitogenic wheat germ lectin, but there was no change with succinyl-Con A, and decreased fluorescence with pokeweek mitogen. Use of fluorescent-labelled lectins showed that the NPN fluorescence change did not correlate with surface receptor patching and capping. Increased phospholipid-fatty acid turnover and subsequent increased membrane fluidity with alteration of molecular polarity are suggested as likely explanations of increased NPN fluorescence.