Calpain activation and inhibition in organotypic rat hippocampal slice cultures deprived of oxygen and glucose

It has been suggested that, after ischaemia, activation of proteases such a s calpains could be involved in cytoskeletal degradation leading to neurona l cell death. In vivo, calpain inhibitors at high doses have been shown to reduce ischaemic damage and traumatic brain injury, however, the relationsh ip between calpain activation and cell death remains unclear. We have inves tigated the role of calpain activation in a model of ischaemia based on org anotypic hippocampal slice cultures using the appearance of spectrin breakd own products (BDPs) as a measure of calpain I activation. Calpain I activit y was detected on Western blot immediately after a 1-h exposure to ischaemi a. Up to 4 h post ischaemia, BDPs were found mainly in the CA1 region and a ppeared before uptake of the vital dye propidium iodide (PI). 24 h after th e insult, BDPs were detected extensively in CA1 and CA3 pyramidal cells, al l of which was PI-positive. However, there were many more PI-positive cells that did not have BDPs, indicating that the appearance of BDPs does not ne cessarily accompany ischaemic cell death. Inhibition of BDP formation by th e broad-spectrum protease inhibitor leupeptin was not accompanied by any ne uroprotective effects. The more specific and more cell-permeant calpain inh ibitor MDL 28170 had a clear neuroprotective effect when added after the is chaemic insult. In contrast, when MDL 28170 was present throughout the enti re pre- and post-incubation phases, PI labelling actually increased, indica ting a toxic effect. These results suggest that calpain activation is not a lways associated with cell death and that, while inhibition of calpains can be neuroprotective under some conditions, it may not always lead to benefi cial outcomes in ischaemia.