Use this page as an execution guide, then fall back to the source paper whenever you need exact exclusions, dosing details, or assay-specific caveats.
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Protocol Steps
Start here. The step list is optimized for running the experiment, with direct vendor links available inline when you need to source a cited item.
Induce chronic constriction injury
Perform chronic constriction injury as one of three distinct partial peripheral nerve lesion models to induce transsynaptic apoptosis in the superficial dorsal horn
Note: This is one of three nerve lesion models tested; others include spared nerve injury and spinal nerve ligation
View evidence from paper
“transsynaptic apoptosis is induced in the superficial dorsal horn (laminas I-III) of the spinal cord by three distinct partial peripheral nerve lesions: spared nerve injury, chronic constriction, and spinal nerve ligation”
Allow time for apoptosis induction
Allow sufficient time for caspase-dependent degeneration of dorsal horn neurons to occur following nerve injury
Note: Apoptosis is slow in onset and persists for several weeks
View evidence from paper
“a caspase-dependent degeneration of dorsal horn neurons that is slow in onset and persists for several weeks”
Assess neuronal loss via stereological analysis
Determine cumulative loss of dorsal horn neurons using stereological analysis at four weeks post-injury
Note: Cumulative loss of dorsal horn neurons exceeds 20% at four weeks
View evidence from paper
“Four weeks after spared nerve injury, the cumulative loss of dorsal horn neurons, determined by stereological analysis, is >20%”
Measure inhibitory postsynaptic currents
Record inhibitory postsynaptic currents from lamina II neurons to assess GABAergic transmission changes
Note: Marked decrease in inhibitory postsynaptic currents coincides with apoptosis induction
View evidence from paper
“a marked decrease in inhibitory postsynaptic currents of lamina II neurons coincides with the induction of apoptosis”
Assess pain-like behavioral changes
Evaluate neuropathic pain-like behavioral changes characterized by hypersensitivity to tactile or cold stimuli
Note: Behavioral changes result from partial peripheral nerve injury
View evidence from paper
“Partial peripheral nerve injury results in pain-like behavioral changes characterized by hypersensitivity to tactile or cold stimuli”
Administer caspase inhibitor treatment
Treat animals with zVAD caspase inhibitor to block apoptosis
Note: zVAD has no intrinsic analgesic properties but prevents loss of GABAergic interneurons
View evidence from paper
“Treatment with zVAD, which has no intrinsic analgesic properties, attenuates this neuropathic pain-like syndrome”
Evaluate effect of apoptosis blockade on pain behavior
Assess whether blocking caspase activity reduces pain hypersensitivity and maintains inhibitory transmission
Note: Preventing apoptosis maintains inhibitory transmission in lamina II and reduces pain hypersensitivity
View evidence from paper
“Preventing nerve injury-induced apoptosis of dorsal horn neurons by blocking caspase activity maintains inhibitory transmission in lamina II and reduces pain hypersensitivity”