Expression of caspases and their substrates in the rat model of focal cerebral ischemia

Experimental evidence suggests that the massive release of glutamate during experimental brain ischemia both directly and indirectly regulates downstr eam mechanisms of cell suicide. Cerebral ischemia was produced by distal, p ermanent occlusion of the middle cerebral artery (MCAO) in the rat. Sets of three animals and one sham-operated for each time-point were kept alive fo r 0-30 min, 1, 4, 12, 24, and 48 h, and 4 days. Additional animals were tre ated by local administration of a 10 mu M (in 10 pi) cocktail of caspase in hibitors (YVAD-cmk, DEVD-fmk, IETD). Immunohistochemistry was performed on free-floating tissue sections with goat polyclonal antibodies to procaspase -1, -2, -3, -6, and -8. Some sections were processed for double-labeling pr ocaspase immunohistochemistry and in situ end-labeling of nuclear DNA fragm entation (TUNEL method). Both immunohistochemistry and double-labeling proc aspase immunohistochemistry and TUNEL method were carried out on formalin-f ixed sections. For gel electrophoresis and Western blotting, we used antibo dies to poly (ADP-ribose) polymerase (PARP), lamin B, and PKC-delta, as spe cific cleavage substrates of caspases. There was increased immunoreactivity ipsilaterally in the areas corresponding to the infarct and surrounding pe numbra with the peak of immunoreactivity between 12 and 24 h for most of th e procaspases. Procaspases were present early in the infarcted tissue neuro nes and their dendrites and axons. Additional procaspase expression occurre d in astrocytes and microglial cells at different times following ischemia. Cells with positive in situ end-labeling of nuclear DNA fragmentation appe ared in high number predominantly in the infarcted areas and at the edge of the infarction and colocalized with enhanced procaspase expression. These findings suggest increased procaspase expression in dying cells at the edge of the infarction. A major product of PARP degradation of about 89 kDa was found in the samples taken from the infarcted and penumbra areas. There wa s no difference in the intensity of the bands corresponding to lamin B or P KC-delta. Injection of procaspase inhibitors reduced the levels of major PA RP products of 89 kDa and decreased the number of TUNEL-positive cells at 1 2 h post-MCAO. In conclusion, these results give support to further researc h on the use of caspase inhibitors as add-on therapeutic agents for the tre atment of ischemia. (C) 2000 Academic Press.