Ketamine-induced neuronal damage and altered N-methyl-D-aspartate receptor function in rat primary forebrain culture

Abstract

Ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, is frequently used in pediatric general anesthesia. Accumulating evidence from animal experiments has demonstrated that ketamine causes neuronal cell death during the brain growth spurt. To elucidate the underlying mechanisms associated with ketamine-induced neuronal toxicity and search for approaches or agents to prevent ketamine's adverse effects on the developing brain, a primary nerve cell culture system was utilized. Neurons harvested from the forebrain of newborn rats were maintained under normal control conditions or exposed to either ketamine (10 µM) or ketamine plus L-carnitine (an antioxidant; 1-100 µM) for 24h, followed by a 24-h withdrawal period. Ketamine exposure resulted in elevated NMDA receptor (NR1) expression, increased generation of reactive oxygen species (ROS) as indicated by higher levels of 8-oxoguanine production, and enhanced neuronal damage. Coadministration of L-carnitine significantly diminished ROS generation and provided near complete protection of neurons from ketamine-induced cell death. NMDA receptors regulate channels that are highly permeable to calcium, and calcium imaging data demonstrated that neurons exposed to ketamine had a significantly elevated amplitude of calcium influx and higher intracellular free calcium concentrations ([Ca(2+)]i) evoked by NMDA (50 µM), compared with control neurons. These findings suggest that prolonged ketamine exposure produces an increase in NMDA receptor expression (compensatory upregulation), which allows for a higher/toxic influx of calcium into neurons once ketamine is removed from the system, leading to elevated ROS generation and neuronal cell death. L-Carnitine appears to be a promising agent in preventing or reversing ketamine's toxic effects on neurons at an early developmental stage.

Figures

FIG. 1.

Immunostained micrographs from primary forebrain cultures. Cells from a control culture (A) and a ketamine-exposed culture (B) were double-immunostained with a mouse monoclonal antibody to NMDA receptor NR1 subunit protein (green) and a rabbit polyclonal antibody to GFAP (red; astrocytes). NR1 immunoreactivity (green) was localized specifically on neurons and the fluorescent density was remarkably upregulated in ketamine-exposed cultures. Scale bar = 50 µm. The NMDA receptor NR1 protein levels were also evaluated by Western blot analysis. A major protein band at about 130kDa was observed in both control and ketamine-exposed cultures (D). Ketamine administration produced a marked upregulation of the NR1 protein compared with controls. No significant difference was detected in GFAP expression levels (astrocytes) between control and ketamine-treated cultures. Densitometry measurements from three independent experiments were used to calculate a ratio of NMDA receptor NR1 protein to β-actin (C). The data are shown as means ± S.D. *p < 0.05 was considered significant compared with control.

FIG. 2.

Dynamic changes in intracellular calcium concentrations [Ca2+]i of a control neuron (A) and a ketamine-exposed neuron (C). Application of NMDA (50µM) or glutamate (25µM) caused an immediate elevation in intracellular free Ca2+ for both control (B) and ketamine-exposed (D) neurons. No NMDA-evoked [Ca2+]i rise was observed when the extracellular Ca2+ was chelated and, thus, unavailable for intracellular transport (50µM NMDA + 200µM EGTA in the perfusion buffer). A significant increase in intracellular free calcium [Ca2+]i was detected in ketamine-exposed neurons (D and E) compared with control neurons (B and E) after NMDA (50µM) stimulation. Each condition was assessed at least in triplicate and experiments were repeated independently three times. Data are presented as means ± SD.

FIG. 3.

Immunohistochemical staining of oxidized DNA with 8-oxoguanine (green) and nuclear staining with DAPI (blue). A 24-h exposure to ketamine (10µM) markedly increased oxidative DNA damage as evidenced by increased 8-oxoguanine formation in ketamine-exposed cultures (B) compared with controls (A). Scale bar = 50 µm. An 8-oxo-dG ELISA (C) showed that coadministration of l-carnitine (30 or 100µM) effectively blocked the 8-oxo-dG increase induced by ketamine. No significant effects were observed when l-carnitine was administered alone. Each condition was assessed at least in triplicate and experiments were repeated independently three times. Data are presented as means ± SD. *p < 0.05 was considered significant compared with control.

FIG. 4.

Effect of l-carnitine on ketamine-induced apoptotic cell death. Ketamine (10µM) exposure for 24h resulted in a significant increase in cell death as indicated by an ELISA for histone-associated DNA fragmentation. Coadministration of l-carnitine (30 or 100µM) effectively blocked the cell death induced by ketamine. No significant protective effect of l-carnitine was observed at a concentration of 1µM, and no significant neurotoxic effects were observed when l-carnitine was administered alone. Each condition was assessed at least in triplicate and the experiments were repeated independently three times. Data are presented as means ± SD. *p < 0.05 was considered significant compared with control.

FIG. 5.

Single-cell gel electrophoresis (Comet) assay for rat primary neuronal cells. A marked increase in the number of cells with DNA strand breaks (tails) was apparent in the cultures exposed to ketamine (B) compared with control cultures (A). Quantitative analysis of the percent of cells exhibiting DNA strand breaks indicated a significant increase in DNA damage in ketamine-exposed neurons, but not in ketamine-exposed cells that were also treated with l-carnitine or in cells treated with l-carnitine alone. Experiments were repeated independently three times. Data are presented as means ± SD. *p < 0.05 was considered significant.

FIG. 6.

Effect of ketamine on levels of PSA-NCAM (a neuron-specific marker). PSA-NCAM immunoreactivity was intense in control cultures (A) and diminished in ketamine-treated cultures (B). Scale bar = 50 µm.