Sex-specific effects of ethanol consumption in older Fischer 344 rats on microglial dynamics and Aβ(1-42) accumulation

Abstract

Chronic alcohol consumption, Alzheimer's disease (AD), and vascular dementia are all associated with cognitive decline later in life, raising questions about whether their underlying neuropathology may share some common features. Indeed, recent evidence suggests that ethanol exposure during adolescence or intermittent drinking in young adulthood increased neuropathological markers of AD, including both tau phosphorylation and beta-amyloid (Aβ) accumulation. The goal of the present study was to determine whether alcohol consumption later in life, a time when microglia and other neuroimmune processes tend to become overactive, would influence microglial clearance of Aβ_(1-42), focusing specifically on microglia in close proximity to the neurovasculature. To do this, male and female Fischer 344 rats were exposed to a combination of voluntary and involuntary ethanol consumption from ∼10 months of age through ∼14 months of age. Immunofluorescence revealed profound sex differences in microglial co-localization, with Aβ_(1-42) showing that aged female rats with a history of ethanol consumption had a higher number of iba1+ cells and marginally reduced expression of Aβ_(1-42), suggesting greater phagocytic activity of Aβ_(1-42) among females after chronic ethanol consumption later in life. Interestingly, these effects were most prominent in Iba1+ cells near neurovasculature that was stained with tomato lectin. In contrast, no significant effects of ethanol consumption were observed on any markers in males. These findings are among the first reports of a sex-specific increase in microglia-mediated phagocytosis of Aβ_(1-42) by perivascular microglia in aged, ethanol-consuming rats, and may have important implications for understanding mechanisms of cognitive decline associated with chronic drinking.

Keywords: Aging; Amyloid beta; Chronic ethanol; Microglia; Sex differences.

Figure 1.. Timeline and behavioral characterization of drinking model.

(A) Body weight in rats across experimental timeline; no weight differences were observed as a result of ethanol consumption. (B) Females consumed more water in mL/kg than males (#), with data collapsed across sessions and exposure condition, and ethanol-exposed animals consumed more water than their water-exposed counterparts (&), with data collapsed across sex and session. Water consumption was highest (%) during session 3. (C) Females consumed more g/kg ethanol than males (#), with data collapsed across sessions, and the highest amounts of ethanol (see %) were consumed during sessions 2 and 3 (D) Preference for ethanol was highest during session 8 (%), but lowest during session 9. (E & F) A final, 30-minute acute drinking test was performed to determine whether ethanol preference had developed. No differences in water intake or ethanol intake were observed during the final drinking test. BECs reported in Table 1. All data expressed as mean ± SEM, alpha set to p = 0.05.

Figure 2.. Assessment of microglia and Aβ_(1–42) after chronic ethanol.

(A) Regions of interest denoted in the dorsal hippocampus (CA1, CA2, CA3, and DG) as well as amygdala (AMG), piriform cortex (Pir), and lateral entorhinal cortex (Lent) (B) Quantification of labeling in the dorsal hippocampus. Females with a history of ethanol had increased Iba-1+ positive cells, Iba-1 / Aβ_(1–42) co-localization, and Iba-1 / Aβ_(1–42) / tomato lectin triple localization. Asterisk (*) denotes a significant interaction (p < 0.05). (C) Representative images of immunofluorescence in the dorsal hippocampus. All representative images were collected at 40× for better clarity and include a 5-micron scale bar for reference.

Figure 3.. Immunofluorescence and 3D rendering of amyloid beta plaques.

Images to the left are immunofluorescent images taken with a confocal microscope, images to the right are rendered using IMARIS software for improved visual clarity. Arrows denote Aβ fragments and plaques. (A & B) Prodromal Aβ fragments, relatively small (<75 micron²), Aβ primarily found in vascular-bound microglia, captured at 40×. (C & D) Plaque development beginning, roughly the size of a microglial soma (350 micron²), captured at 40×. (E & F) Traditional Aβ plaque, larger than a microglial soma (519 micron²), with high density of Aβ internalized in surrounding microglia, captured at 40×. (G & H) Representative image of microglia-vasculature interaction, captured at 20×. Scale bar is included for size reference in each image.

Figure 4.. Quantification of immunofluorescence in subregions of the hippocampus.

(A) Females with a history of ethanol had increased Iba-1 / Aβ co-localization Iba-1 / Aβ / tomato lectin triple localization in the CA1 region of the dorsal hippocampus. (B) No differences were observed across groups in the CA2 region of the dorsal hippocampus. (C) No differences were observed across groups in the CA3 region of the dorsal hippocampus. (D) Ethanol history resulted in lowered Aβ_(1–42) in both males and females, while females with a history of ethanol had increased Iba-1 / Aβ co-localization Iba-1 / Aβ / tomato lectin triple localization in the dentate gyrus. Asterisk (*) denotes a significant interaction (p < 0.05). All data expressed as mean ± SEM.