Ischemic preconditioning protects the mouse liver by inhibition of apoptosis through a caspase-dependent pathway

A short period of ischemia and reperfusion, called ischemic preconditioning , protects various tissues against subsequent sustained ischemic insults. W e previously showed that apoptosis of hepatocytes and sinusoidal endothelia l cells is a critical mechanism of injury in the ischemic liver. Because ca spases, calpains, and Bcl-2 have a pivotal role in the regulation of apopto sis, we hypothesized that ischemic preconditioning protects by inhibition o f apoptosis through down-regulation of caspase and calpain activities and u pregulation of Bcl-2. A preconditioning period of 10 minutes of ischemia fo llowed by 15 minutes of reperfusion maximally protected livers subjected to prolonged ischemia. After reperfusion, serum aspartate transaminase (AST) levels were reduced up to 3-fold in preconditioned animals. All animals sub jected to 75 minutes of ischemia died, whereas all those who received ische mic preconditioning survived. Apoptosis of hepatocytes and sinusoidal endot helial cells, assessed by in situ TUNEL assay and DNA fragmentation by gel electrophoresis, was dramatically reduced with preconditioning. Caspase act ivity, measured by poly (adenosine diphosphate ribose) polymerase (PARP) pr oteolysis and a specific caspase-3 fluorometric assay, was inhibited by isc hemic preconditioning. The antiapoptotic mechanism did not involve calpain- like activity or Bcl-2 expression because levels were similar in control an d preconditioned livers. In conclusion, ischemic preconditioning confers dr amatic protection against prolonged ischemia via inhibition of apoptosis th rough down-regulation of caspase 3 activity, independent of calpain-like ac tivity or Bcl-2 expression.