Transient Global Ischemia Model
Objective: To determine whether caspase-3 serves to regulate neuronal death after cerebral ischemia and study the effect of caspase-3 inhibition on cell survival and DNA fragmentation in the hippocampus
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Equipment4
Materials1
Not specified • Not specified • Not specified • Not specified
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Protocol Steps
Induction of transient global cerebral ischemia
Transient global cerebral ischemia was induced in rats using an unspecified method
Note: The specific ischemia induction method is not detailed in the provided text
View evidence from paper
“studied the effect of caspase-3 inhibition on cell survival and DNA fragmentation in the hippocampus in a rat model of transient global ischemia”
Examination of caspase-3 mRNA expression
caspase-3 mRNA expression in the brain was examined using in situ hybridization, Northern blot analyses
Note: Analysis performed at multiple timepoints post-ischemia
View evidence from paper
“At 8–72 hr after ischemia, caspase-3 mRNA and protein were induced in the hippocampus and caudate-putamen”
Examination of caspase-3 protein expression
caspase-3 protein expression in the brain was examined using Western blot analyses and double-labeled immunohistochemistry
Note: Protein predominantly increased in degenerating CA1 pyramidal neurons
View evidence from paper
“In the hippocampus, caspase-3 mRNA and protein were predominantly increased in degenerating CA1 pyramidal neurons”
Determination of caspase-3-like activity
Caspase-3-like activity was determined in brain cell extracts
Note: Proteolytic activation of caspase-3 precursor detected in hippocampus and caudate-putamen but not in cortex
View evidence from paper
“determined caspase-3-like activity in brain cell extracts; Proteolytic activation of the caspase-3 precursor was detected in hippocampus and CPu but not in cortex at 4–72 hr after ischemia”
Detection of DNA fragmentation
Double-label experiments were performed to detect DNA fragmentation in neurons overexpressing caspase-3
Note: DNA fragmentation detected in majority of CA1 neurons and selective caudate-putamen neurons
View evidence from paper
“Double-label experiments detected DNA fragmentation in the majority of CA1 neurons and selective CPu neurons that overexpressed caspase-3”
Ventricular infusion of caspase-3 inhibitor
Z-DEVD-FMK, a caspase-3 inhibitor, was administered via ventricular infusion to inhibit caspase-3 activity
Note: Inhibitor significantly reduced cell death and DNA fragmentation in CA1 sector
View evidence from paper
“ventricular infusion of Z-DEVD-FMK, a caspase-3 inhibitor, decreased caspase-3 activity in the hippocampus and significantly reduced cell death and DNA fragmentation in the CA1 sector up to 7 d after ischemia”