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Ketamine is a commonly used clinical anesthetic and a popular recreational drug. However, with the exception of studies about the nervous system, studies about the effect of early ketamine exposure on embryos are rare. Xenopus laevis is a commonly used vertebrate model for assessing teratogenicity. Therefore, we treated Xenopus embryos with ketamine to evaluate its teratogenicity on embryos. Xenopus embryos were treated with ketamine from stages 8 to 21. Embryonic and cardiac morphology were analyzed using living embryo imaging and whole-mount RNA in situ hybridization (WMISH). Heart function was measured by heart rate and ventricular shortening fraction (VSF). The mRNA expression levels of several heart development-related genes were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The protein expression levels of XMLC2, phospho-histone H3 (pH3) and histone H3 were determined by western blot. Ketamine caused concentration-dependent increases in mortality and shortening of body length. At a dose of 0.5 mg/ml, ketamine exposure resulted in cardiac enlargement as the primary manifestation of several malformations: gut defects, a curved axis and shortened body length. Cardiac cells underwent increased proliferation. Moreover, the heart rate and ventricular shortening fraction were decreased, findings indicative of heart dysfunction. XMLC2 expression levels were down-regulated at stages 28, 32/33, 35/36 and 46. Ketamine exposure during early development has teratogenic effects on Xenopus embryos. The heart enlargement and decreased VSF may result from the down-regulation of XMLC2 mRNA and protein levels. These findings provide new insight into the potential fetal defects induced by ketamine exposure during early pregnancy.
Fig. 1. The effect of ketamine on embryos at stage 46. a Mortality rate of increasing concentrations of ketamine (0.1, 0.125, 0.25, 0.5, 1, 2, 5, 10, 25 mg/ml). b Malformations at three exposure concentrations of ketamine (0.125, 0.5, 2 mg/ml). Hearts are marked by white punctiform curves. The arrowhead indicates a curved axis. Scale barâ=â100 μm. c Comparison of body lengths at three exposure concentrations. a Pâ<â0.05 vs. control group; b Pâ<â0.05 vs. 0.125 mg/ml ketamine-treated group; c Pâ<â0.05 vs. 0.5 mg/ml ketamine-treated group. The data represent the meansâ±âSD
Fig. 2. The effect of ketamine on cardiac morphology. a-h Embryos at stage 46. a-b Live embryos. c-h Whole-mount RNA in situ hybridization (WMISH) analysis of the heart-specific gene XNkx2.5 and myocardium-specific genes XTnIc and XMLC2. i The dissected hearts from embryos at stage 46. oft, outflow tract; a, atria; v, ventricle. j-o WMISH analysis of XMLC2 at stage 28, 32/33 and 35/36. Scale bar = 20 μm
Fig. 3. The effect of ketamine on cardiac cell proliferation. a Phospho-histone H3 (pH3) and histone H3 protein levels. b Quantitation of the levels of pH3 and histone H3 protein shown in (a). The data are expressed as the meansâ±âSD
Fig. 4. The effect of ketamine on heart function. a Comparison of the heart rate (beats per minute) in the control and ketamine-treated embryos. n = 10. *P < 0.05. b Comparison of ventricular shortening fraction (VSF) in the control and ketamine-treated embryos. n = 6. *P < 0.05. The data are expressed as the means ± SD
Fig. 5. The effect of ketamine on heart development-related gene expression. a The mRNA levels of heart-specific genes XNkx2.5 and XTnIc in whole embryos, carcasses and dissected tissues at stage 46. *P < 0.05, #P < 0.05. b The mRNA levels of heart development-related genes, XNkx2.5, XTnIc, MHC-alpha, gata4, gata6b and XMLC2 in dissected tissues at stage 46. *P < 0.05. c XMLC2 mRNA levels at stage 28, 32/33 and 35/36. d XMLC2 protein levels at stage 28, 32/33, 35/36 and 46. e Quantitation of the levels of XMLC2 protein shown in D. The data are expressed as the means ± SD. Con :control, K: ketamine, st: stage.
Table 2. Characteristics of ketamine-treated embryos (0.5 mg/ml) at stage 46.
Table 1. The effect of ketamine at three concentrations on embryonic death and malformation.
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