Nicotinamide protects against ethanol-induced apoptotic neurodegeneration in the developing mouse brain

Abstract

Background: Exposure to alcohol during brain development may cause a neurological syndrome called fetal alcohol syndrome (FAS). Ethanol induces apoptotic neuronal death at specific developmental stages, particularly during the brain-growth spurt, which occurs from the beginning of third trimester of gestation and continues for several years after birth in humans, whilst occurring in the first two postnatal weeks in mice. Administration of a single dose of ethanol in 7-d postnatal (P7) mice triggers activation of caspase-3 and widespread apoptotic neuronal death in the forebrain, providing a possible explanation for the microencephaly observed in human FAS. The present study was aimed at determining whether nicotinamide may prevent ethanol-induced neurodegeneration.

Methods and findings: P7 mice were treated with a single dose of ethanol (5 g/kg), and nicotinamide was administered from 0 h to 8 h after ethanol exposure. The effects of nicotinamide on ethanol-induced activation of caspase-3 and release of cytochrome-c from the mitochondria were analyzed by Western blot (n = 4-7/group). Density of Fluoro-Jade B-positive cells and NeuN-positive cells was determined in the cingulated cortex, CA1 region of the hippocampus, and lateral dorsal nucleus of the thalamus (n = 5-6/group). Open field, plus maze, and fear conditioning tests were used to study the behavior in adult mice (n = 31-34/group). Nicotinamide reduced the activation of caspase-3 (85.14 +/- 4.1%) and the release of cytochrome-c (80.78 +/- 4.39%) in postnatal mouse forebrain, too. Nicotinamide prevented also the ethanol-induced increase of apoptosis. We demonstrated that ethanol-exposed mice showed impaired performance in the fear conditioning test and increased activity in the open field and in the plus maze. Administration of nicotinamide prevented all these behavioral abnormalities in ethanol-exposed mice.

Conclusions: Our findings indicate that nicotinamide can prevent some of the deleterious effects of ethanol on the developing mouse brain when given shortly after ethanol exposure. These results suggest that nicotinamide, which has been used in humans for the treatment of diabetes and bullous pemphigoid, may hold promise as a preventive therapy of FAS.

Figure 1. Nicotinamide Inhibits Ethanol-Induced Caspase-3 Activation

(A–D) P7 mice were injected with ethanol and received nicotinamide 2 h later. (A and B) Level of cleaved caspase-3 was analyzed by Western blot at 12 h (A) and 24 h (B) after ethanol administration with different doses of nicotinamide. (C and D) Densitometric quantification of cleaved caspase-3 at 12 h (ANOVA F _5,27 = 10.989; p < 0.0001; n = 5–6 each treatment group) (C) and 24 h (ANOVA F _3,18 = 8.851; p = 0.0008; n = 6 each treatment group) (D). (E and G) Nicotinamide was administered to P7 mice at different time points after ethanol exposure. Western blot analysis of caspase-3 activation 12 h after ethanol injection (E) and densitometric quantification (ANOVA F _5,33 = 15.061; p < 0.0001; n = 6–7 each treatment group) (G). (F and H) Different doses of ethanol were administered to P7 mice and nicotinamide was injected 2 h later. Western blot analysis of caspase-3 activation 12 h after ethanol injection (F) and densitometric quantification (ANOVA F _6,28 = 18.183; p < 0.0001; n = 5 each treatment group) (H). Values are shown as mean ± SD. Bonferroni correction for multiple comparisons revealed a significant difference between the ethanol treatment group and all other groups. ∗ p < 0.0001.

Figure 2. Nicotinamide Blocks Release of Cytochrome-C from Mitochondria

(A) Western blot analysis of cytochrome-c release in the cytosolic fraction 12 h after ethanol administration. (C) Densitometric quantification of cytochrome-c release (ANOVA F _3,20 = 43.546 p < 0.0001; n = 6 each treatment group). Values are shown as mean ± SD. Bonferroni correction for multiple comparison revealed a significant difference between ethanol treatment group and all other groups. ∗ p < 0.0001. (B and D) Nicotinamide (1mg/g) does not alter ethanol absorption or excretion. Ethanol levels in blood (B) and brain (D) were measured at different time points after 5g/kg of ethanol treatment in P7 mice. Nicotinamide was administered 2 h after ethanol exposure. No significant differences were seen between the different treatments ( n = 4 each treatment group).

Figure 3. Nicotinamide Inhibits Ethanol-Induced Neurodegeneration

(A) Fluoro-Jade B staining 24 h after ethanol treatments. (B) Quantification of Fluoro-Jade–B positive cells in the cingulate cortex (ANOVA F _2,13 = 25.541; p < 0.0001; n = 5–6 each treatment group), in the total CA1 region of hippocampus (ANOVA F _2,13 = 9.988; p = 0.0024; n = 5–6 each treatment group), and in the LDN of thalamus (ANOVA F _2,13 = 20.785; p < 0.0001; n = 5–6 each treatment group). (C) Total number of NeuN positive cells were counted 2 wk after ethanol administration in the cingulate cortex (ANOVA F _2,12 = 9.894; p = 0.0029; n = 5 each treatment group), in the CA1 region of hippocampus (ANOVA F _2,12 = 7.289; p = 0.0085; n = 5 each treatment group) and in the LDN of thalamus (ANOVA F _2,12 = 5.551; p = 0.0196; n = 5 each treatment group). Values are shown as mean ± SD. Bonferroni correction for multiple comparison revealed a significant difference between ethanol treatment group and all other groups. S, saline; E, ethanol; Etoh+Nic, ethanol + nicotinamide. Scale bars 200μm.