Casein Zymogram Assessment of μ-Calpain and m-Calpain Activity After Traumatic Brain Injury in the Rat In Vivo

Abstract

A number of brain insults including traumatic brain injury (TBI) can result in excitotoxic consequences largely attributable to pathological increases in intracellular calcium (1,2). Loss of calcium homeostasis can result in activation of the calcium-dependent proteases, or calpains, that may be one of the principle causes of pathology after acute central nervous system (CNS) injury (3). A zymographic assay for calpains using nondenaturing casein-containing polyacrylamide gels was originally developed for in vitro studies (4). Recently, we have refined this approach to apply to in vivo studies of acute CNS injury (5,6). This technique allows for differential and concurrent measurement of the two major isoforms of calpain, m-calpain and μ-calpain. This technique also provides the opportunity for analyzing protease activity in cytosolic and total membrane fractions (with appropriate sample preparation), an important consideration because calpain translocation may be a determinant of its attack on membrane-bound cytoskeletal protein targets (7). The zymographic assay also assesses calpain activity independently of the effects of the endogenous calpain inhibitor, calpastatin. Acute CNS injury such as TBI can also importantly affect brain pH, and both μ-calpain and m-calpain in brain are sensitive to changes in pH (8,9).