Twelve months of voluntary heavy alcohol consumption in male rhesus macaques suppresses intracortical bone remodeling

Chronic heavy alcohol consumption is a risk factor for cortical bone fractures in males. The increase in fracture risk may be due, in part, to reduced bone quality. Intracortical (osteonal) bone remodeling is the principle mechanism for maintaining cortical bone quality. However, it is not clear how alcohol abuse impacts intracortical bone remodeling. This study investigated the effects of long-duration heavy alcohol consumption on intracortical bone remodeling in a non-human primate model. Following a 4-month induction period, male rhesus macaques (Macaca mulatta, n = 21) were allowed to voluntarily self-administer water or alcohol (4% ethanol w/v) for 22 h/d, 7 d/wk for 12 months. Control monkeys (n = 13) received water and an isocaloric maltose-dextrin solution. Tetracycline hydrochloride was administered orally 17 and 3 days prior to sacrifice for determination of active mineralization sites. Animals in the alcohol group consumed 2.7 ± 0.2 g alcohol/kg/d (mean ± SE) during the 12 months of self-administration, resulting in a mean daily blood alcohol concentration of 77 ± 9 mg/dl from samples taken at 7 h after the start of a daily session. However, blood alcohol concentration varied widely from day to day, with peak levels exceeding 250 mg/dl, modeling a binge-drinking pattern of alcohol consumption. The skeletal response to alcohol was determined by densitometry, microcomputed tomography and histomorphometry. Significant differences in tibial bone mineral content, bone mineral density, and cortical bone architecture (cross-sectional volume, cortical volume, marrow volume, cortical thickness, and polar moment of inertia) in the tibial diaphysis were not detected with treatment. However, cortical porosity was lower (1.8 ± 0.5 % versus 0.6 ± 0.1 %, p = 0.021) and labeled osteon density was lower (0.41 ± 0.2/mm² versus 0.04 ± 0.01/mm², p < 0.003) in alcohol-consuming monkeys compared to controls, indicating a reduced rate of intracortical bone remodeling. In concordance, plasma CTx was lower (2.5 ± 0.3 ng/ml versus 1.7 ± 0.1 ng/ml, p = 0.028) in the alcohol group. These results suggest that chronic heavy alcohol consumption may negatively impact bone health, in part, by suppressing intracortical bone remodeling.Keywords: Ethanol, Histomorphometry, Non-human primate, Haversian remodeling, Microcomputed tomograph

Video Conference Technology as a Tool for Pair Introduction in Rhesus Macaques

Pair housing is known to promote welfare for macaques in captivity. However, finding compatible partners can be challenging, particularly when animals are not located near one another. Because macaques show interest in videos of conspecifics, we examined the use of video conference technology (Zoom) as a potential tool to assess compatibility in 84 rhesus macaques (2&ndash;22 years old) prior to pair introduction. Monkeys involved in the pairs (12 female&ndash;female, 21 male&ndash;male, 9 female&ndash;male) were unfamiliar with each other. We set up a 10 min Zoom session between potential partners (on an iPad in front of the cage). We scored attention to the screen, anxiety, and prosocial behaviors and examined whether these behaviors predicted future pair success. Monkeys spent relatively little time attending to the tablet (median = 13.3%), and attention did not predict pair success (B = &minus;0.06, NS). However, pairs in which attention was primarily shown by one animal had a higher chance of success than those in which both individuals showed similar levels (B = &minus;4.66. p = 0.03). Neither prosocial (B = 0.89, NS) nor anxiety (B = &minus;1.95, p = 0.07) behavior correlated with pair success. While preliminary, our data suggest that video conferencing technology may be useful as a tool for introducing unfamiliar partners prior to a socialization attempt

Transcriptome Profiling Reveals Disruption of Innate Immunity in Chronic Heavy Ethanol Consuming Female Rhesus Macaques

<div><p>It is well established that heavy ethanol consumption interferes with the immune system and inflammatory processes, resulting in increased risk for infectious and chronic diseases. However, these processes have yet to be systematically studied in a dose and sex-dependent manner. In this study, we investigated the impact of chronic heavy ethanol consumption on gene expression using RNA-seq in peripheral blood mononuclear cells isolated from female rhesus macaques with daily consumption of 4% ethanol available 22hr/day for 12 months resulting in average ethanol consumption of 4.3 g/kg/day (considered heavy drinking). Differential gene expression analysis was performed using edgeR and gene enrichment analysis using MetaCore™. We identified 1106 differentially expressed genes, meeting the criterion of ≥ two-fold change and p-value ≤ 0.05 in expression (445 up- and 661 down-regulated). Pathway analysis of the 879 genes with characterized identifiers showed that the most enriched gene ontology processes were “response to wounding”, “blood coagulation”, “immune system process”, and “regulation of signaling”. Changes in gene expression were seen despite the lack of differences in the frequency of any major immune cell subtype between ethanol and controls, suggesting that heavy ethanol consumption modulates gene expression at the cellular level rather than altering the distribution of peripheral blood mononuclear cells. Collectively, these observations provide mechanisms to explain the higher incidence of infection, delay in wound healing, and increase in cardiovascular disease seen in subjects with Alcohol use disorder.</p></div

Chronic heavy ethanol consumption results in dysregulation of genes involved in innate immunity and immune system development.

<p>(A) Heatmap of DEGs that map to “Defense Response/Innate Immune Response” (B) Heatmap of DEGs that map to “Immune System Development”/“Myeloid Cell differentiation”.</p

Summary of down-regulated microRNAs and their up-regulated targets in drinkers.

<p>Summary of down-regulated microRNAs and their up-regulated targets in drinkers.</p

Chronic heavy ethanol consumption results in up-regulation of genes involved in blood coagulation and wound-healing.

<p>(A) Network of DEGs with direct interactions that mapped to “Wound healing”. (B) Heatmap of the 40 DEGs with a fold-change ≥ four-fold (30 up-regulated and 10 down-regulated) involved in “Blood Coagulation”. (C) Heatmap of the 18 DEGs that mapped to “Wound healing” but did not map to “Blood Coagulation”.</p

Chronic heavy ethanol consumption changes the expression of genes involved in heart diseases and cancer.

<p>(A) Bar graph depicting 8 disease terms enriched among the up-regulated genes. The line graph in both figures represents negative log (FDR) of the enriched term. (B) Heatmap of up-regulated genes involved in cardiovascular diseases. (C) Bar graph depicting 8 disease terms enriched among the down-regulated genes. (D) Heatmap of the down-regulated genes involved in cancer.</p

Chronic heavy ethanol consumption results in changes in expression of epigenetic regulators.

<p>(A) Functional profiles of the 128 down-regulated genes mapping to ‘Regulation of Gene Expression’ (B) Bar graph of expression levels (RPKM) of genes involved in chromatin remodeling (**—FDR of 5% and *—FDR of 10%). (C) Bar graph of 5 most significantly up- and down-regulated transcription factor networks. Green bars indicate up-regulated network and blue bars indicate down-regulated networks. Each bar is linked to a group of target genes (orange–up-regulated and grey down-regulated) that are differentially expressed in heavy drinkers.</p

Chronic heavy ethanol consumption results in robust changes in gene expression within PBMC.

<p>(A) Volcano plot of global gene expression changes with red specks denoting genes with significant fold changes in gene expression, with gene names annotated for those with fold change ≥ 32. (B) Bar graph depicting the 8 most significant Gene Ontology (GO) terms enriched among all differentially expressed genes (DEGs), (C) Venn diagram depicting the overlap of genes enriched for four major GO terms—Signaling, Blood Coagulation, Wounding and Immune System Process. (D) Heatmap of the 27 differentially expressed that belong to all four GO processes—red depicts higher expression and grey, lower expression.</p