Immunohistochemical Determination of Calcium—Calmodulin Binding Predicts Neuronal Damage after Global Ischemia

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Abstract
Since ionic Ca2+ binds with intracellular calmodulin (CaM) before activating proteases, kinases, and phospholipases, demonstration of persistent Ca2+ –CaM binding in neurons destined to show ischemic cellular injury would support the concept that elevated intracellular Ca2+ plays a causative role in ischemic neuronal damage. In order to characterize Ca2+ –CaM binding, we used a sheep anti-CaM antibody (CaM-Ab) which recognizes CaM that is not bound to Ca2+ or brain target proteins. Therefore, immunohistochemical staining of brain sections by labeled CaM-Ab represented only unbound CaM. Six normal rats were compared to 15 animals rendered ischemic for 30 min by a modification of the four-vessel occlusion model. Animals were killed immediately after ischemia, and after 2 and 24 h of reperfusion. Brain sections through hippocampus were incubated in CaM-Ab, and a diaminobenzadiene labeled anti-sheep secondary antibody was added to stain the CaM-Ab. Staining in the endal limb of dentate, dorsal CA1, lateral CA3, and parietal cortex was graded on a 4-point scale. All normal animals had grade 4 staining indicating the presence of unbound CaM in all four brain regions. Ischemic animals demonstrated reduced (grade 0 to 2) staining in the CA1 and CA3 regions immediately and 2 and 24 h after ischemia (p < 0.01 for both regions at all three time intervals) indicating persistent binding of CaM with Ca2+ and target proteins in these regions. Staining decreased in dentate and cortex up to 2 h after ischemia (p = 0.02 for both regions) but returned toward normal by 24 h. We conclude that while most brain regions demonstrate increased Ca2+ –CaM binding immediately after ischemia, this binding returns to normal in brain regions destined to recover, such as cortex and dentate, but persists beyond 24 h in selectively vulnerable CA1 and partially vulnerable CA3, which are destined to undergo irreversible damage. These findings support the hypothesis that calcium entry into neurons and consequent persistent activation of Ca2+ -dependent enzyme systems leads to irreversible cell damage.