Experimental evidence suggests that the massive release of
glutamate during experimental
brain ischemia both directly and indirectly regulates downstream mechanisms of cell suicide.
Cerebral ischemia was produced by distal, permanent 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 for 0-30 min, 1, 4, 12, 24, and 48 h, and 4 days. Additional animals were treated by local administration of
a 10 microM (in 10 microl) cocktail of
caspase inhibitors (
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 procaspase immunohistochemistry and in situ end-labeling of nuclear DNA fragmentation (TUNEL method). Both immunohistochemistry and double-labeling procaspase immunohistochemistry and TUNEL method were carried out on
formalin-fixed sections. For gel electrophoresis and Western blotting, we used
antibodies to
poly (ADP-ribose) polymerase (PARP),
lamin B, and PKC-delta, as specific cleavage substrates of
caspases. There was increased immunoreactivity ipsilaterally in the areas corresponding to the
infarct and surrounding penumbra with the peak of immunoreactivity between 12 and 24 h for most of the procaspases. Procaspases were present early in the infarcted tissue neurones and their dendrites and axons. Additional procaspase expression occurred in astrocytes and microglial cells at different times following
ischemia. Cells with positive in situ end-labeling of nuclear DNA fragmentation appeared 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 was no difference in the intensity of the bands corresponding to
lamin B or PKC-delta. Injection of procaspase inhibitors reduced the levels of major PARP products of 89 kDa and decreased the number of TUNEL-positive cells at 12 h post-MCAO. In conclusion, these results give support to further research on the use of
caspase inhibitors as add-on therapeutic agents for the treatment of
ischemia.