Selective impairment of hippocampal neurogenesis by chronic alcoholism: protective effects of an antioxidant

Abstract

A major pathogenic mechanism of chronic alcoholism involves oxidative burden to liver and other cell types. We show that adult neurogenesis within the dentate gyrus of the hippocampus is selectively impaired in a rat model of alcoholism, and that it can be completely prevented by the antioxidant ebselen. Rats fed for 6 weeks with a liquid diet containing moderate doses of ethanol had a 66.3% decrease in the number of new neurons and a 227-279% increase in cell death in the dentate gyrus as compared with paired controls. Neurogenesis within the olfactory bulb was not affected by alcohol. Our studies indicate that alcohol abuse, even for a short duration, results in the death of newly formed neurons within the adult brain and that the underlying mechanism is related to oxidative or nitrosative stress. Moreover, these findings suggest that the impaired neurogenesis may be a mechanism mediating cognitive deficits observed in alcoholism.

Fig. 1.

BrdUrd administration protocols. (A) In protocol 1, rats received one daily BrdUrd injection over 10 consecutive days starting on the first day of full liquid diet (diet day 1) and then were allowed to survive for 6 weeks after the first BrdUrd injection. (B) Protocol 2 rats received seven BrdUrd injections spaced at 2-h intervals on 1 day only (day 10 of the full liquid diet) and then were allowed to survive for different time periods.

Fig. 2.

Ethanol selectively impairs adult neurogenesis in the hippocampus. (A and B) Immunofluorescence detection of BrdUrd-labeled cells in the DG. Photomicrograph of the DG from an animal that received control liquid diet (A) and ethanol-fed rat (B), both in BrdUrd protocol 1, 6-week survival; sgl, subgranular layer. (Bar = 150 μm.) (C) Merged confocal photomicrographs showing immunostaining for glial fibrillary acidic protein (green) and BrdUrd (red) with very few double-labeled cells in the DG. (Bar = 10 μm.) (D) Merged confocal photomicrographs of the DG showing immunostaining for BrdUrd (red) and the neuronal marker NeuN (green). Arrow points to a double-labeled cell in the DG; most BrdUrd-labeled cells in the DG were NeuN positive. (Bar = 10 μm.) (E) There was no statistical difference in the number of new cells either in the periglomerular or the granular cell layers of the OB between ethanol-fed or control rats. (F) There was an average 66.1% decrease in the number of BrdUrd-labeled cells in the DG of ethanol-fed rats as compared with controls. This effect was consistent throughout the DG. (G) Analysis at six different levels where BrdUrd-labeled cells were counted in the DG, BrdUrd protocol 1, 6-week survival. Distance of analyzed sections from bregma is expressed in mm.

Fig. 3.

Ethanol feeding decreases the number of newly formed neurons in the DG. (A and B) Photomicrographs of BrdUrd immunostaining in the DG of control (A) or ethanol-fed (B) rats. (Bar = 100 μm.) BrdUrd was administered in seven injections at 2-h intervals, and rats were perfused 1 h after the last injection (10 days into the liquid diet treatment, BrdUrd protocol 2). Labeling is observed in the basal layers only (Insets). (C) Counting of BrdUrd-positive cells in the DG shows no impairing effect of ethanol on proliferation. (D) Rats received seven injections of BrdUrd within a 12-h period (BrdUrd protocol 2). Then, animals exposed to ethanol or control diet were allowed to survive for different time periods. Rats exposed to ethanol showed a decrease of up to 70% in the number of BrdUrd-labeled cells in the DG after 14 days. Most of the decrease seemed to occur between 7 and 14 days after the uptake of BrdUrd. (E) The number of BrdUrd-positive cells in ethanol-exposed rats at 2 weeks was similar to that observed after a 6-week exposure period, suggesting that most of the cells died within the first 14 days after BrdUrd uptake and before establishing mature contacts. (F and G) Photomicrographs of PCNA immunostaining in the DG of control (F) or ethanol-fed (G) rats. (Bar = 75 μm.) Labeling is observed in the basal layers only (Insets). (H) Counting of PCNA-positive cells in the DG shows again no impairing effect of ethanol on proliferation.

Fig. 4.

Ethanol exposure increases cell death in the DG. (A) Photomicrograph of Nissl-stained coronal section of rat hippocampus. Arrow shows pyknotic nucleus in the basal granular layer where precursors are located (6-week survival). (B) Counting of pyknotic nuclei shows a marked increase in the DG of such figures in ethanol-fed rats (40.75 ± 2.9 pyknotic nuclei per DG section, n = 4) as compared with controls (14.67 ± 1.38, n = 4). P ≤ 0.01, Mann–Whitney test (6-week survival). (C) BrdUrd-labeled section of the DG counterstained with Nissl, 6-week survival. Dotted line indicates chosen division between basal layers (including subgranular cell layer) and the outer layers (OL) of the DG. Arrows point to BrdUrd-positive cells in the basal layers, where most of the newly born cells can be found at this survival point. The number of pyknotic cells per mm² (D) is clearly higher in the basal layers of the DG than in the outer layers (ol). (E) TUNEL was used to assess cell death in the DG of ethanol vs. control-fed rats. (Bar = 25 μm.) Positive labeling was observed predominantly in the basal layers (6-week survival). (F) There was a marked increase in the number of TUNEL-positive cells per section in ethanol-fed rats (6.5 ± 0.87, n = 4) as compared with controls (2.5 ± 0.87, n = 4). P ≤ 0.01, Mann–Whitney test (6-week survival). (G) BrdUrd-labeled pyknotic cells in the DG. BrdUrd was administered 3 days before perfusion (BrdUrd in red, nuclear staining in green with Cyquant 1:2,000; BrdUrd protocol 2). (Bar = 10 μm.) (H) Apoptotic nuclei were observed in the basal layers of the DG of ethanol-fed rats at the ultrastructural level (6-week survival). The arrow points to the typical fragmentation of the nuclear membranes in advanced stages of apoptosis. Also note dense chromatin within the periphery of the nucleus. (Bar = 5 μm.)

Fig. 5.

The antioxidant ebselen prevents ethanol effects on neurogenesis in the adult DG. (A–D) Photomicrographs of BrdUrd immunolabeling in coronal sections of the DG 6-week survival (BrdUrd protocol 1). (Bar = 100 μm). (A) Control rat. (B) Ethanol-fed rat. (C) Ebselen + control feedings. (D) Ebselen + ethanol feeding. (E) Ebselen prevented the deleterious effects of ethanol on newly born DG cells (ANOVA and Dunnett's post hoc analysis, P ≤ 0.01, n per group = 9) preventing cell death (F) (ANOVA and Dunnett's post hoc analysis, P ≤ 0.05, n per group = 4).