Effects of Intrathecal Ketamine in the Neonatal Rat: Evaluation of Apoptosis and Long-term Functional Outcome methods
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Intrathecal ketamine and neuronal apoptosis
In P3 animals, apoptotic cells were also identified in haematoxylin and eosin sections of the spinal cord, and qualitative differences were not apparent between treatment groups. No necrosis, gliosis or inflammation was identified. One P21 animal (ketamine 15 mg/kg, 24-h survival) had focal subpial inflammation. No other histopathological changes were seen in the P21 animals.
Intrathecal ketamine and glial activation
Iba1 immunoreactivity in the spinal cord was increased 7 days following injection of intrathecal ketamine 3 or 10 mg/kg in P3 pups ( ). No change was apparent following ketamine injection in P21 pups.
Changes in spinal cord function
The degree of apoptosis in different brain regions varies following systemic ketamine. Repeated doses of 20 mg/kg ketamine (6 doses at 2-h intervals) increased activated caspase-3 positive cells by a factor of 10 in the frontal cortex and 2.5 fold in the hippocampus, whereas single doses of ketamine increased cortical apoptosis by a factor of two following 20 mg/kg and by greater than four after 40 mg/kg. In the spinal cord, the number of apoptotic profiles was similarly increased by a factor of 2-3 following intrathecal ketamine, and while statistically significant, the importance of the magnitude of such changes is evidently greater if associated with a persistent functional deficit. For example, increased apoptosis in the hippocampus following NMDA antagonists and gamma-amino butyric acid agonists has been associated with long-term deficits in learning and memory. Sensory...
Dose-response of intrathecal ketamine in rat pups
P21 pups received 15 mg/kg (mean body weight 60 g giving nominal injection volume of 30 mcl of 30 mg/ml ketamine and average total dose per animal of 900 mcg).
Dose-response of intrathecal ketamine in rat pups
In adult rodents, antihyperalgesic effects of intrathecal ketamine have been demonstrated in tissue injury models. Therefore, hindpaw inflammation was induced in P3 or P21 pups, and changes in mechanical withdrawal threshold were used to evaluate acute antihyperalgesic effects of intrathecal ketamine. Pups were lightly restrained on a flat bench surface and calibrated von Frey hairs (Stoelting, Wood Dale, IL) that deliver increasing mechanical stimuli (0.4 to 60 g) were applied to the dorsal surface of the hindpaw five times at 1-s intervals. The number of evoked flexion withdrawals was recorded, and the maximum force applied was that which evoked five withdrawal responses. Animals were anesthetized with isoflurane (3-5%) in oxygen and air, and 1 mcl/g of 2% lambda carrageenan (Sigma-Aldrich) was injected into the mid-plantar surface of the left hindpaw. Mechanical withdrawal t...
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 anti-hyperalgesic effects in postnatal day (P)3 and 21 rat pups. Following intrathecal ketamine at P3, 7 or 21, 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). Following maximal doses of ketamine or saline at P3 or P21, sensory thresholds and gait analysis were evaluated at P35.
Intrathecal ketamine and neuronal apoptosis
Consistent results were seen using activated caspase-3 to identify apoptotic neurons ( ). The number of apoptotic cell profiles was significantly increased 24 h after ketamine 3 mg/kg, and 6 and 24 h after ketamine 10 mg/kg in P3 pups ( P < 0.05, one-way ANOVA with Bonferroni post hoc comparisons). In P7 animals, increases following ketamine were not statistically significant.
Materials and Methods
All experiments were carried out according to protocols approved by the Institutional Animal Care and Use Committee of the University of California San Diego, La Jolla, California (under the Guide for Care and Use of Laboratory Animals, National Institutes of Health publication 85-23, Bethesda, MD). Timed pregnant Holtzman rats were obtained (Sprague-Dawley, Harlan, Indianapolis, IN) and housed in a 12-h light-dark cycle with free access to food and water. On postnatal (P) day 3 or 7, pups were randomly assigned to treatment groups containing equal numbers of males and females, and if necessary litters were culled to a maximum of 12 pups. Pups were kept under radiant heat during treatment to maintain body temperature. The duration of maternal separation was minimized and was the same for control and treatment animals. Pups were weaned into same sex cages at P21. Separate groups of mal...
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Evidence quotes (8)
In P3 animals, apoptotic cells were also identified in haematoxylin and eosin sections of the spinal cord, and qualitative differences were not apparent between treatment groups. No necrosis, gliosis or inflammation was identified. One P21 animal (ketamine 15 mg/kg, 24-h survival) had focal subpial inflammation. No other histopathological changes were seen in the P21 animals.
Iba1 immunoreactivity in the spinal cord was increased 7 days following injection of intrathecal ketamine 3 or 10 mg/kg in P3 pups ( ). No change was apparent following ketamine injection in P21 pups.
The degree of apoptosis in different brain regions varies following systemic ketamine. Repeated doses of 20 mg/kg ketamine (6 doses at 2-h intervals) increased activated caspase-3 positive cells by a factor of 10 in the frontal cortex and 2.5 fold in the hippocampus, whereas single doses of ketamine increased cortical apoptosis by a factor of two following 20 mg/kg and by greater than four after 40 mg/kg. In the spinal cord, the number of apoptotic profiles was similarly increased by a factor of 2-3 following intrathecal ketamine, and while statistically significant, the importance of the magnitude of such changes is evidently greater if associated with a persistent functional deficit. For example, increased apoptosis in the hippocampus following NMDA antagonists and gamma-amino butyric acid agonists has been associated with long-term deficits in learning and memory. Sensory thresholds and motor function can be used to evaluate long-term changes in spinal cord function. Following prolonged general anesthesia at P7, increased apoptosis in the cord was reported. The sum of positive cells in four sections was reported, but if converted to mean values, numbers are similar...
P21 pups received 15 mg/kg (mean body weight 60 g giving nominal injection volume of 30 mcl of 30 mg/ml ketamine and average total dose per animal of 900 mcg).
In adult rodents, antihyperalgesic effects of intrathecal ketamine have been demonstrated in tissue injury models. Therefore, hindpaw inflammation was induced in P3 or P21 pups, and changes in mechanical withdrawal threshold were used to evaluate acute antihyperalgesic effects of intrathecal ketamine. Pups were lightly restrained on a flat bench surface and calibrated von Frey hairs (Stoelting, Wood Dale, IL) that deliver increasing mechanical stimuli (0.4 to 60 g) were applied to the dorsal surface of the hindpaw five times at 1-s intervals. The number of evoked flexion withdrawals was recorded, and the maximum force applied was that which evoked five withdrawal responses. Animals were anesthetized with isoflurane (3-5%) in oxygen and air, and 1 mcl/g of 2% lambda carrageenan (Sigma-Aldrich) was injected into the mid-plantar surface of the left hindpaw. Mechanical withdrawal thresholds were again determined 3 h after carrageenan. Then, under brief anesthesia, 0.5 mcl/g intrathecal saline or ketamine (P3: 0.03, 0.3, or 3 mg/kg; P21: 1.5, 5, or 15 mg/kg; n = 4-6 all groups) was administered via percutaneous injection. Mechanical withdrawal thresholds were measured 30...
In anesthetized rat pups, intrathecal ketamine was administered by lumbar percutaneous injection. Changes in mechanical withdrawal threshold evaluated dose-dependent antinociceptive and carrageenan-induced anti-hyperalgesic effects in postnatal day (P)3 and 21 rat pups. Following intrathecal ketamine at P3, 7 or 21, 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). Following maximal doses of ketamine or saline at P3 or P21, sensory thresholds and gait analysis were evaluated at P35.
Consistent results were seen using activated caspase-3 to identify apoptotic neurons ( ). The number of apoptotic cell profiles was significantly increased 24 h after ketamine 3 mg/kg, and 6 and 24 h after ketamine 10 mg/kg in P3 pups ( P < 0.05, one-way ANOVA with Bonferroni post hoc comparisons). In P7 animals, increases following ketamine were not statistically significant.
All experiments were carried out according to protocols approved by the Institutional Animal Care and Use Committee of the University of California San Diego, La Jolla, California (under the Guide for Care and Use of Laboratory Animals, National Institutes of Health publication 85-23, Bethesda, MD). Timed pregnant Holtzman rats were obtained (Sprague-Dawley, Harlan, Indianapolis, IN) and housed in a 12-h light-dark cycle with free access to food and water. On postnatal (P) day 3 or 7, pups were randomly assigned to treatment groups containing equal numbers of males and females, and if necessary litters were culled to a maximum of 12 pups. Pups were kept under radiant heat during treatment to maintain body temperature. The duration of maternal separation was minimized and was the same for control and treatment animals. Pups were weaned into same sex cages at P21. Separate groups of male and female P17-19 Holtzman rats were assigned to treatment groups at P21. Animals were regularly monitored and maintained until further testing at 5 weeks of age.
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