Developmental neurotoxicity of ketamine: morphometric confirmation, exposure parameters, and multiple fluorescent labeling of apoptotic neurons.

Ketamine is a widely used pediatric anesthetic recently reported (C. Ikonomidou et al., 1999, Science 283, 70-74) to enhance neuronal death in neonatal rats. To confirm and extend these results, we treated four groups of PND 7 rats with seven sc doses, one every 90 min, of either saline, 10 mg/kg ketamine, 20 mg/kg ketamine, or a single dose of 20 mg/kg ketamine. The repeated doses of 20 mg/kg ketamine increased the number of silver-positive (degenerating) neurons in the dorsolateral thalamus to a degree comparable to previous results (Ikonomidou et al., 1999, Science 283, 70-74), i.e., 28-fold vs. 31-fold respectively. However, blood levels of ketamine immediately after the repeated 20 mg/kg doses were about 14 micrograms/ml, about seven-fold greater than anesthetic blood levels in humans (J. M. Malinovsky et al., 1996, Br. J. Anaesth. 77, 203-207; R. A. Mueller and R. Hunt, 1998, Pharmacol. Biochem. Behav. 60, 15-22). Levels of ketamine in blood following exposure to the multiple 10 mg/kg doses of ketamine or to a single 20 mg/kg dose ranged around 2-5 micrograms/ml; although these blood levels are close to an anesthetic level in humans, they failed to produce neurodegeneration. To investigate the mode of ketamine-induced neuronal death, coronal sections were stained with both Fluoro-Jade B (a green fluorescent stain selective for neurodegeneration) and DAPI (a blue DNA stain), as well as for caspase-3 (using an antisera labeled red with rhodamine). These histochemical results confirmed the developmental neurotoxicity of ketamine, demonstrated that Fluoro-Jade B (FJ-B), like silver methods, successfully stained degenerating neurons in neonatal rats, and indicated that ketamine acts by increasing the rate of neuronal apoptosis.