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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: Anesthetic agent not specified
View evidence from paper
“In anesthetized rat pups, intrathecal ketamine was administered by lumbar percutaneous injection”
2
Administer intrathecal injection at P3
Administer intrathecal ketamine or saline by lumbar percutaneous injection at postnatal day 3
not specifiednot specified
Note: Maximal doses used; exact doses not specified
View evidence from paper
“Following maximal doses of ketamine or saline at P3 or P21”
3
Administer intrathecal injection at P7
Administer intrathecal ketamine by lumbar percutaneous injection at postnatal day 7
not specifiednot specified
Note: For spinal cord examination only, not for sensory threshold testing
View evidence from paper
“Following intrathecal ketamine at P3, 7 or 21, spinal cords were examined”
4
Administer intrathecal injection at P21
Administer intrathecal ketamine or saline by lumbar percutaneous injection at postnatal day 21
not specifiednot specified
Note: Maximal doses used; exact doses not specified
View evidence from paper
“Following maximal doses of ketamine or saline at P3 or P21”
5
Evaluate mechanical withdrawal threshold at P3 and P21
Assess dose-dependent antinociceptive and carrageenan-induced anti-hyperalgesic effects using mechanical withdrawal threshold testing
not specifiednot specified
Note: Testing performed 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”
6
Examine spinal cords for apoptosis
Collect spinal cords and examine for apoptosis using Fluoro-Jade C and activated caspase-3 markers
not specifiednot specified
Note: Performed at P3, P7, and P21 timepoints
View evidence from paper
“spinal cords were examined for apoptosis (Fluoro-Jade C and activated caspase-3)”
7
Examine spinal cords for histopathological changes
Assess spinal cord tissue for histopathological changes
not specifiednot specified
Note: Performed at P3, P7, and P21 timepoints
View evidence from paper
“spinal cords were examined for apoptosis (Fluoro-Jade C and activated caspase-3), histopathological change, and glial responses”
8
Examine spinal cords for glial responses
Assess glial responses using ionized calcium binding adapter molecule 1 and glial fibrillary acid protein markers
not specifiednot specified
Note: Performed at P3, P7, and 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)”
9
Evaluate sensory thresholds at P35
Test sensory thresholds in rat pups at postnatal day 35 following early postnatal treatment
not specifiednot specified
Note: Performed in animals treated with maximal doses of ketamine or saline at P3 or P21
View evidence from paper
“Following maximal doses of ketamine or saline at P3 or P21, sensory thresholds and gait analysis were evaluated at P35”
10
Perform gait analysis at P35
Analyze gait in rat pups at postnatal day 35 following early postnatal treatment
not specifiednot specified
Note: Performed in animals treated with maximal doses of ketamine or saline at P3 or P21
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
P35 (postnatal day 35) at testing; P3, P7, P21 at treatment
Sex
unknown
Weight
not specified
Rat pups treated at P3 or P21 with maximal doses of ketamine or saline, then tested at P35