N-acetylcysteine prevents ketamine-induced adverse effects on development, heart rate and monoaminergic neurons in zebrafish

Abstract

N-acetylcysteine, a precursor molecule of glutathione, is an antioxidant. Ketamine, a pediatric anesthetic, has been implicated in cardiotoxicity and neurotoxicity including modulation of monoaminergic systems in mammals and zebrafish. Here, we show that N-acetylcysteine prevents ketamine's adverse effects on development and monoaminergic neurons in zebrafish embryos. The effects of ketamine and N-acetylcysteine alone or in combination were measured on the heart rate, body length, brain serotonergic neurons and tyrosine hydroxylase-immunoreactive (TH-IR) neurons. In the absence of N-acetylcysteine, a concentration of ketamine that produces an internal embryo exposure level comparable to human anesthetic plasma concentrations significantly reduced heart rate and body length and those effects were prevented by N-acetylcysteine co-treatment. Ketamine also reduced the areas occupied by serotonergic neurons in the brain, whereas N-acetylcysteine co-exposure counteracted this effect. TH-IR neurons in the embryo brain and TH-IR cells in the trunk were significantly reduced with ketamine treatment, but not in the presence of N-acetylcysteine. In our continued search for compounds that can prevent ketamine toxicity, this study using specific endpoints of developmental toxicity, cardiotoxicity and neurotoxicity, demonstrates protective effects of N-acetylcysteine against ketamine's adverse effects. This is the first study that shows the protective effects of N-acetylcysteine on ketamine-induced developmental defects of monoaminergic neurons as observed in a whole organism.

Keywords: 5-HT; Developmental toxicity; Ketamine; N-acetyl cysteine; Tyrosine hydroxylase; Zebrafish.

Fig. 1

NAC prevents ketamine’s adverse effects on heart rate. Embryos (48 hpf) were exposed for 2 h to ketamine (2 mM) alone or in the presence of various concentrations of NAC and 1 mM NAC alone. Heart rate was measured as described in the Materials and methods section. Statistical significance (*) was set at P < 0.05.

Fig. 2

NAC prevents ketamine’s adverse effects on growth. Zebrafish embryos at 28 hpf (manually dechorionated) were exposed for 20 h to drugs. Linear body lengths of the untreated control (A) and embryos treated with 2 mM ketamine (B), 2 mM ketamine + 1 mM NAC (C) and 1.0 mM NAC alone, were compared as percentage change over control (E). Statistical significance is indicated (*).

Fig. 3

NAC prevents ketamine’s adverse effects on the 5-HT neurons. Zebrafish embryos at 28 hpf (manually dechorionated) were exposed for 20 h to drugs. Anti-5-HT-immunostained embryos at 48 hpf are shown. Anterior is upwards (A–D). Eyes (e) and yolk sac are indicated. Brain areas with 5-HT neurons in control (A); 20 h post-exposure to 2 mM ketamine (B), 2 mM ketamine + 1 mM NAC (C) and 1 mM NAC (D) are shown. Quantification of relative areas occupied by 5-HT neurons in the experimental groups are presented (E). Asterisk indicates significant difference.

Fig. 4

NAC counteracts ketamine-induced TH-IR neuron toxicity in the brain. Anti-TH antibody-stained whole mount immunohistochemistry of the anterior part of the embryos at 48 hpf are shown. Anterior is upwards (A–D). Eyes (e) and yolk sac are indicated. Specification of midbrain TH-IR neurons in the 48 hpf embryos, demarcated by dotted bracket lines in control (A); 20 h post-exposure to 2 mM ketamine (B), 2 mM ketamine + 1 mM NAC (C) and 1 mM NAC (D). Relative areas occupied by TH-IR neurons in each experimental group are presented (E). Asterisk indicates significant difference.

Fig. 5

NAC restores ketamine-induced reduction in TH-IR cells in the trunk. Anti-TH antibody-stained trunk regions (lateral view) of the 48 hpf embryo control (A), 20 h exposure at 28 hpf to 2 mM ketamine (B), 2 mM ketamine + 1 mM NAC (C) and 1 mM NAC alone (D). Anterior is to the left and posterior is to the right (A–D). Yolk extension (YE) and some TH-IR cells (arrows) are indicated. Quantification of relative numbers of TH-IR cells in each experimental group are presented (E). Asterisk indicates a significant difference.