Caspase-3 Inhibitor Administration
Objective: To investigate the temporal profile of apoptosis after traumatic brain injury and assess the effects of caspase-3 inhibitor administration on post-traumatic apoptosis and neurological recovery
Gather these items before starting the experiment. Check off items as you prepare.
Equipment1
Not specified • Not specified • Not specified • Not specified
Materials1
Not specified • Not specified • Not specified • Not specified
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Protocol Steps
Induce traumatic brain injury
Rats undergo fluid percussion-induced traumatic brain injury
Note: This is the baseline injury model
View evidence from paper
“fluid percussion-induced traumatic brain injury (TBI) in rats”
Administer z-DEVD-fmk before injury
Intracerebroventricular administration of z-DEVD-fmk caspase-3 inhibitor prior to traumatic brain injury induction
Note: Pre-injury administration timepoint
View evidence from paper
“Intracerebroventricular administration of z-DEVD-fmk—a specific tetrapeptide inhibitor of caspase-3—before and after injury”
Administer z-DEVD-fmk after injury
Intracerebroventricular administration of z-DEVD-fmk caspase-3 inhibitor following traumatic brain injury induction
Note: Post-injury administration timepoint
View evidence from paper
“Intracerebroventricular administration of z-DEVD-fmk—a specific tetrapeptide inhibitor of caspase-3—before and after injury”
Collect tissue samples
Harvest cortex and hippocampus tissue from injured and contralateral hemispheres at specified timepoints
Note: Samples collected from both ipsilateral injured tissue and contralateral control tissue
View evidence from paper
“DNA fragmentation was observed in samples from injured cortex and hippocampus, but not from contralateral tissue, beginning 4 hr after TBI and continuing for at least 3 d”
Perform DNA electrophoresis
Analyze DNA fragmentation patterns in tissue samples to assess apoptosis
Note: Used to demonstrate reduction in post-traumatic apoptosis with z-DEVD-fmk treatment
View evidence from paper
“markedly reduced post-traumatic apoptosis, as demonstrated by DNA electrophoresis and TUNEL staining”
Perform TUNEL staining
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling to identify apoptotic cells
Note: Double labeling with antibody to neuronal nuclear protein to identify apoptotic neurons
View evidence from paper
“Double labeling of brain with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and an antibody directed to neuronal nuclear protein identified apoptotic neurons”
Prepare cytosolic extracts
Extract cytosolic proteins from injured and control tissue for enzymatic assays
Note: Extracts from injured cortex and hippocampus used for DNA fragmentation assays
View evidence from paper
“Cytosolic extracts from injured cortex and hippocampus, but not from contralateral or control tissue, induced internucleosomal DNA fragmentation in isolated nuclei”
Perform semiquantitative RT-PCR
Measure caspase-3 and caspase-1 mRNA levels in tissue samples
Note: Samples collected at 24 hours after TBI
View evidence from paper
“Caspase-3 mRNA levels, estimated by semiquantitative RT-PCR, were elevated fivefold in ipsilateral cortex and twofold in hippocampus by 24 hr after TBI”
Assess neurological recovery
Evaluate neurological function in treated and control animals
Note: z-DEVD-fmk treatment significantly improved neurological recovery
View evidence from paper
“significantly improved neurological recovery”