The effects of L-carnitine on the combination of, inhalation anesthetic-induced developmental, neuronal apoptosis in the rat frontal cortex.

The anesthetic gas nitrous oxide (N2O) and the volatile anesthetic isoflurane (ISO) are commonly used in surgical procedures for human infants and in veterinary and laboratory animal practice to produce loss of consciousness and analgesia. Recent reports indicate that exposure of the developing brain to general anesthetics that block N-methyl-D-aspartate (NMDA) glutamate receptors or potentiate GABA(A) receptors can trigger widespread apoptotic neurodegeneration. In the present study, the question arises whether a relatively low dose of ISO alone or its combination with N2O entails significant risk of inducing enhanced apoptosis. In addition, the role of L-carnitine to attenuate these effects was also examined. Postnatal day 7 (PND-7) rat pups were exposed to N2O (75%) or a low dose of ISO (0.55%) alone, or N2O plus ISO for 2, 4, 6 or 8 h with or without L-carnitine. The neurotoxic effects were evaluated 6 h after completion of anesthetic administration. No significant neurotoxic effects were observed for the animals exposed to N2O or ISO alone. However, enhanced apoptotic cell death was apparent when N2O was combined with ISO at exposure durations of 6 h or more. Co-administration of L-carnitine (300 or 500 mg/kg, i.p.) effectively protected neurons from the anesthetic-induced damage. These data indicate that 6 h or more of inhaled anesthetic exposure consisting of a combination of N2O and ISO results in enhanced neuronal apoptosis, and L-carnitine effectively blocks the neuronal apoptosis caused by inhalation anesthetics in the developing rat brain.

The utility of the K6/ODC transgenic mouse as an alternative short term dermal model for carcinogenicity testing of pharmaceuticals.

The use of transgenic rodents may overcome many limitations of traditional cancer studies. Regulatory perspectives continue to evolve as new models are developed and validated. The transgenic mouse, K6/ODC, develops epidermal tumors when exposed to genotoxic carcinogens. In this study, K6/ODC mice were evaluated for model fitness and health robustness in a 36-week study to determine oncogenic risk of residual DNA in vaccines from neoplastic cell substrates. K6/ODC and C57BL/6 mice were treated with T24-H-ras expression plasmid, carrier vector DNA, or saline topically or by subcutaneous injection. One group of K6/ODC mice received 7,12-dimethylbenz-[a]anthracene [DMBA] dermally. Only DMBA-treated mice developed papillomas by six weeks, increasing in incidence to 25 weeks. By week 11, many K6/ODC mice showed severe dehydration and dermal eczema. By week 32, (6/8) surviving K6/ODC mice showed loss of mobility and balance. Microscopic evaluation of tissues revealed dermal/sebaceous gland hyperplasia, follicular dystrophy, splenic atrophy, and amyloid deposition/neutrophilic infiltration within liver, heart, and spleen, in all K6/ODC mice. Pathology was not detected in C57BL/6 mice. Progressive adverse health, decreased survival, and failure to develop papillomas to the H-ras plasmid suggest that K6/ODC mice may be an inappropriate alternative model for detection of oncogenic DNA and pharmaceutical carcinogenicity testing.

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.

Interaction of hexachlorophene and other compounds with spin-labeled brain membranes.

Experiments reported here demonstrate that hexachlorophene influences oxidation-reduction events inside the brain membrane, possibly via a free radical mechanism. This was shown by nitroxide spin label quenching inside the rat cerebellum membrane bilayer due to the interaction between hexachlorophene and peroxidase-hydrogen peroxide system. Prior addition of antioxidants, e.g., vitamin E or butylated hydroxytoluene, prevented such membrane-bound fatty acid spin label reduction, presumably due to their free radical scavenging abilities. The 5-doxyl stearic acid spin probe attached to the brain membranes did not exhibit any detectable changes in their ESR spectra nor, consequently, in the microviscosity of the membranes when exposed to up to 40 mM hexachlorophene.