Source Paper
Source Paper
Alexander G. Yakovlev, Susan M. Knoblach, Lei Fan, Gerard B. Fox, Randyll Goodnight et al.
Journal of Neuroscience • 1997
We examined the temporal profile of apoptosis after fluid percussion-induced traumatic brain injury (TBI) in rats and investigated the potential pathophysiological role of caspase-3-like proteases in this process. 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. 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 with high frequency in both traumatized rat cortex and hippocampus. Cytosolic extracts from injured cortex and hippocampus, but not from contralateral or control tissue, induced internucleosomal DNA fragmentation in isolated nuclei with temporal profiles consistent with those of DNA fragmentation observed in vivo . Caspase-3 mRNA levels, estimated by semiquantitative RT-PCR, were elevated fivefold in ipsilateral cortex and twofold in hippocampus by 24 hr after TBI. Caspase-1 mRNA content also was increased after trauma, but to a lesser extent in cortex. Increased caspase-3-like, but not caspase-1-like, enzymatic activity was found in cytosolic extracts from injured cortex. Intracerebroventricular administration of z-DEVD-fmk—a specific tetrapeptide inhibitor of caspase-3—before and after injury markedly reduced post-traumatic apoptosis, as demonstrated by DNA electrophoresis and TUNEL staining, and significantly improved neurological recovery. Together, these results implicate caspase-3-like proteases in neuronal apoptosis induced by TBI and suggest that the blockade of such caspases can reduce post-traumatic apoptosis and associated neurological dysfunction.
Objective: Examine the temporal profile of apoptosis after fluid percussion-induced traumatic brain injury (TBI) in rats and investigate the pathophysiological role of caspase-3-like proteases in post-traumatic neuronal apoptosis and neurological dysfunction
Gather these items before starting the experiment. Check off items as you prepare.
Not specified • Not specified • Not specified • Not specified
Not specified • Not specified
As an Amazon Associate, we earn from qualifying purchases. Product links help support this free resource.
Administer fluid percussion injury to rats
Note: Injury is the baseline event from which all subsequent measurements are timed
“fluid percussion-induced traumatic brain injury (TBI) in rats”
Intracerebroventricular administration of z-DEVD-fmk before and after injury
Note: Specific tetrapeptide inhibitor of caspase-3
“Intracerebroventricular administration of z-DEVD-fmk—a specific tetrapeptide inhibitor of caspase-3—before and after injury”
Harvest injured cortex, hippocampus, and contralateral tissue for DNA fragmentation analysis
Note: DNA fragmentation observed beginning at this timepoint
“DNA fragmentation was observed in samples from injured cortex and hippocampus, but not from contralateral tissue, beginning 4 hr after TBI”
Collect tissue samples at multiple timepoints up to 3 days after TBI
Note: DNA fragmentation continues for at least 3 days
“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”
Double label brain tissue with TUNEL and antibody to neuronal nuclear protein to identify apoptotic neurons
Note: Apoptotic neurons identified with high frequency in both traumatized cortex and hippocampus
“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 with high frequency”
Extract cytosolic fractions from injured cortex and hippocampus, contralateral tissue, and control tissue
Note: Used for in vitro DNA fragmentation assays
“Cytosolic extracts from injured cortex and hippocampus, but not from contralateral or control tissue, induced internucleosomal DNA fragmentation”
Incubate cytosolic extracts with isolated nuclei to assess internucleosomal DNA fragmentation
Note: Temporal profiles consistent with in vivo DNA fragmentation
“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 to quantify caspase-3 mRNA in ipsilateral cortex and hippocampus
Note: Elevated fivefold in ipsilateral cortex and twofold in hippocampus
“Caspase-3 mRNA levels, estimated by semiquantitative RT-PCR, were elevated fivefold in ipsilateral cortex and twofold in hippocampus by 24 hr after TBI”
Perform semiquantitative RT-PCR to quantify caspase-1 mRNA in injured tissue
Note: Increased after trauma but to lesser extent in cortex compared to caspase-3
“Caspase-1 mRNA content also was increased after trauma, but to a lesser extent in cortex”
Assess caspase-3-like and caspase-1-like enzymatic activity in cytosolic extracts from injured cortex
Note: Increased caspase-3-like activity found; caspase-1-like activity not increased
“Increased caspase-3-like, but not caspase-1-like, enzymatic activity was found in cytosolic extracts from injured cortex”
Analyze DNA fragmentation patterns from injured tissue
Note: Used to assess post-traumatic apoptosis reduction following caspase-3 inhibitor treatment
“markedly reduced post-traumatic apoptosis, as demonstrated by DNA electrophoresis and TUNEL staining”
Evaluate neurological function in animals treated with caspase-3 inhibitor versus controls
Note: Caspase-3 inhibitor treatment significantly improved neurological recovery
“significantly improved neurological recovery”
Both traumatized and contralateral tissue examined; control tissue used for comparison