leupeptins and Ischemic-Attack--Transient

Transient forebrain ischemia induces activation of calpain and proteolysis of a neuronal cytoskeleton, fodrin, in gerbil hippocampus. This phenomenon precedes delayed neuronal death in hippocampal CA1 neurons. We examined effects of a calpain inhibitor on delayed neuronal death after transient forebrain ischemia. In gerbils, a selective calpain inhibitor entrapped in liposome was given transvenously and 30 min later, 5-min forebrain ischemia was produced by occlusion of both common carotid arteries. On day 7, CA1 neuronal damage was examined in the hippocampal slices stained with cresyl violet. Calpain-induced proteolysis of fodrin was also examined by immunohistochemistry and immunoblot. Additionally, to assure entrapment of the inhibitor by CA1 neurons, the inhibitor-liposome complex was labeled with FITC and given to gerbils. Fluorescence in the hippocampal slices was examined by confocal laser scanning microscope. Selective CA1 neuronal damage induced by forebrain ischemia was prevented by administration of the inhibitor in a dose-dependent manner. Calpain-induced proteolysis of fodrin was also extinguished by the calpain inhibitor in a dose-dependent manner. Bright fluorescence of the FITC-labeled inhibitor was observed in the CA1 neurons. The data show an important role of calpain in the development of the ischemic delayed neuronal death. Calpain seems to produce neuronal damage by degrading neuronal cytoskeleton. Our data also show a palliative effect of the calpain inhibitor on the neurotoxic damage, which offers a new and potent treatment of transient forebrain cerebral ischemia.

Topics: Animals; Blood-Brain Barrier; Blotting, Western; Calpain; Carrier Proteins; Cell Death; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Carriers; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gerbillinae; Hippocampus; Immunohistochemistry; Injections, Intravenous; Ischemic Attack, Transient; Leupeptins; Liposomes; Microfilament Proteins; Microscopy, Confocal; Neurons; Prosencephalon

The calpains are calcium-dependent intracellular proteases that are activated in a number of pathogenic conditions. We tested the capacities of protease inhibitors, calpain inhibitor I and leupeptin, to protect against the neuronal degeneration caused by cytotoxic hypoxia or transient global cerebral ischemia. Primary neuronal cultures were prepared from embryonic chick telencephalon, and cytotoxic hypoxia was induced by adding 1 mM NaCN to the culture medium for 30 min. Global ischemia was induced in rats by clamping both carotid arteries and lowering the arterial blood pressure to 40 mmHg for 10 min. Both calpain inhibitor I and leupeptin protected neurons against ischemic and hypoxic damage. Neuroprotection was indicated by increased cell viability and protein content in the cultures, and fewer damaged neurons in the hippocampal CA1-subfield. Thus, blockade of proteolysis can protect neurons against cytotoxic and ischemic damage.

Topics: Animals; Blood Pressure; Calpain; Cells, Cultured; Glycoproteins; Hypoxia, Brain; Ischemic Attack, Transient; Leupeptins; Male; Neurons; Prosencephalon; Rats; Rats, Wistar

Intense proteolysis of cytoskeletal proteins occurs in brain within minutes of transient ischemia, possibly because of the activation of calcium-sensitive proteases (calpains). This proteolytic event precedes overt signs of neuronal degeneration, is most pronounced in regions of selective neuronal vulnerability, and could have significant consequences for the integrity of cellular function. The present studies demonstrate that (i) the early phase of enhanced proteolysis is a direct response to hypoxia rather than other actions of ischemia, (ii) it is possible to pharmacologically inhibit the in vivo proteolytic response to ischemia, (iii) inhibition of proteolysis is associated with a marked reduction in the extent of neuronal death, and (iv) protected neurons exhibit normal-appearing electrophysiological responses and retain their capacity for expressing long-term potentiation, a form of physiological plasticity thought to be involved in memory function. These observations indicate that calcium-activated proteolysis is an important component of the post-ischemic neurodegenerative response and that targeting this response may be a viable therapeutic strategy for preserving both the structure and function of vulnerable neurons.

Topics: Animals; Calpain; Cerebral Ventricles; Electrophysiology; Endopeptidases; Evoked Potentials; Gerbillinae; Hippocampus; Hypoxia; Infusions, Parenteral; Ischemic Attack, Transient; Leupeptins; Neurons; Spectrin; Synapses