Objective: Evaluation of dose-dependent antinociceptive and anti-hyperalgesic effects of intrathecal ketamine using mechanical withdrawal thresholds in rat pups at different postnatal ages
Materials & Equipment Checklist
8 items
Gather these items before starting the experiment. Check off items as you prepare.
Equipment1
Not specified • Not specified • Not specified • Not mentioned
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Protocol Steps
View Abstract
Background Systemic ketamine can trigger apoptosis in the brain of rodents and primates during susceptible developmental periods. Clinically, spinally administered ketamine may improve the duration or quality of analgesia in children. Ketamine-induced spinal cord toxicity has been reported in adult animals but has not been systematically studied in early development. Methods In anesthetized rat pups, intrathecal ketamine was administered by lumbar percutaneous injection. Changes in mechanical withdrawal threshold evaluated dose-dependent antinociceptive and carrageenan-induced antihyperalgesic effects in rat pups at postnatal day (P) 3 and 21. After intrathecal injection of ketamine at P3, 7, or 21, spinal cords were examined for apoptosis (Fluoro-Jade C and activated caspase-3), histopathologic change, and glial responses (ionized calcium-binding adapter molecule 1 and glial fibrillary acid protein). After maximal doses of ketamine or saline at P3 or P21, sensory thresholds and gait analysis were evaluated at P35. Results Intrathecal injection of 3 mg/kg ketamine at P3 and 15 mg/kg at P21 reverses carrageenan-induced hyperalgesia. Baseline neuronal apoptosis in the spinal cord was greater at P3 than P7, predominantly in the dorsal horn. Intrathecal injection of 3-10 mg/kg ketamine in P3 pups (but not 15 mg/kg at P21) acutely increased apoptosis and microglial activation in the spinal cord and altered spinal function (reduced mechanical withdrawal threshold and altered static gait parameters) at P35. Conclusions Because acute pathology and long-term behavioral change occurred in the same dose range as antihyperalgesic effects, the therapeutic ratio of intrathecal ketamine is less than one in the neonatal rat. This measure facilitates comparison of the relative safety of spinally administered analgesic agents.
1
Anesthetize rat pups
Anesthetize rat pups prior to intrathecal injection
Not specifiedNot specified
Note: Anesthesia type and dosage not specified
View evidence from paper
“In anesthetized rat pups, intrathecal ketamine was administered by lumbar percutaneous injection”
2
Administer intrathecal ketamine
Perform lumbar percutaneous injection of intrathecal ketamine at multiple doses to evaluate dose-dependent effects
Not specifiedNot specified
Note: Administered at P3, P7, or P21; multiple doses tested for dose-dependent evaluation
View evidence from paper
“intrathecal ketamine was administered by lumbar percutaneous injection. Changes in mechanical withdrawal threshold evaluated dose-dependent antinociceptive”
3
Evaluate mechanical withdrawal threshold
Measure mechanical withdrawal threshold to assess antinociceptive effects following ketamine administration
Not specifiedNot specified
Note: Evaluated at P3 and P21 timepoints
View evidence from paper
“Changes in mechanical withdrawal threshold evaluated dose-dependent antinociceptive and carrageenan-induced anti-hyperalgesic effects in postnatal day (P)3 and 21 rat pups”
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Induce hyperalgesia with carrageenan
Administer carrageenan to induce hyperalgesia for testing anti-hyperalgesic effects of ketamine
Not specifiedNot specified
Note: Used to evaluate anti-hyperalgesic effects at P3 and P21
View evidence from paper
“carrageenan-induced anti-hyperalgesic effects in postnatal day (P)3 and 21 rat pups”
5
Examine spinal cord for apoptosis
Following intrathecal ketamine administration at P3, P7, or P21, harvest and examine spinal cords for apoptotic markers
Not specifiedNot specified
Note: Use Fluoro-Jade C and activated caspase-3 staining
View evidence from paper
“Following intrathecal ketamine at P3, 7 or 21, spinal cords were examined for apoptosis (Fluoro-Jade C and activated caspase-3)”
6
Assess histopathological changes
Examine spinal cord tissue for histopathological changes following ketamine administration
Not specifiedNot specified
Note: Performed at P3, P7, or P21 timepoints
View evidence from paper
“spinal cords were examined for apoptosis (Fluoro-Jade C and activated caspase-3), histopathological change, and glial responses”
7
Evaluate glial responses
Examine spinal cord tissue for glial responses using Iba1 and GFAP markers
Not specifiedNot specified
Note: Performed at P3, P7, or P21 timepoints
View evidence from paper
“spinal cords were examined for apoptosis (Fluoro-Jade C and activated caspase-3), histopathological change, and glial responses (ionized calcium binding adapter molecule 1 and glial fibrillary acid protein)”
8
Evaluate sensory thresholds at P35
At P35, measure sensory thresholds in animals that received maximal doses of ketamine or saline at P3 or P21
Not specifiedNot specified
Note: Long-term follow-up assessment of sensory function
View evidence from paper
“Following maximal doses of ketamine or saline at P3 or P21, sensory thresholds and gait analysis were evaluated at P35”
9
Perform gait analysis at P35
At P35, conduct gait analysis in animals that received maximal doses of ketamine or saline at P3 or P21
Not specifiedNot specified
Note: Long-term follow-up assessment of motor function
View evidence from paper
“Following maximal doses of ketamine or saline at P3 or P21, sensory thresholds and gait analysis were evaluated at P35”
Subjects / Specimens
Species
rat
Strain
Not specified
Age
Postnatal day (P)3, P7, P21, and P35
Sex
unknown
Weight
Not specified
Pups tested at multiple developmental timepoints; sensory thresholds and gait analysis evaluated at P35 following maximal doses at P3 or P21