Calpain, a neutral protease activated by calcium, may promote microtub
ular proteolysis in ischemic brain. We tested this hypothesis in an an
imal model of focal cerebral ischemia without reperfusion. The earlies
t sign of tissue injury was observed after no more than 15 min of isch
emia, with coiling of apical dendrites immunolabeled to show microtubu
le-associated protein 2 (MAP2). After 6 h of ischemia, MAP2 immunoreac
tivity was markedly diminished in the infarct zone. Quantitative Weste
rn analysis demonstrated that MAP2 was almost unmeasurable after 24 h
of ischemia. An increase in calpain activity, shown by an antibody rec
ognizing calpain-cleaved spectrin fragments, paralleled the loss of MA
P2 immunostaining. Double-labeled immunofluorescent studies showed tha
t intraneuronal calpain activity preceded evidence of MAP2 proteolysis
. Perikaryal immunolabeling of tau protein became increasingly promine
nt between 1 and 6 h in neurons located within the transition zone bet
ween ischemic and unaffected tissue. Western blot experiments confirme
d that dephosphorylation of 7 protein occurred during 24 h of ischemia
, but was not associated with significant loss of 7 antigen. We conclu
de that focal cerebral ischemia is associated with early microtubular
proteolysis caused by calpain.