Rationale
Abstinence-based approaches to treating alcohol use disorder (AUD) are highly prevalent, but abstinence from chronic drinking may exacerbate subsequent levels of alcohol intake in relapse.
...Objective
Use a non-human primate model that encompasses a range of chronic voluntary ethanol drinking to isolate biological responses to repeated cycles of imposed abstinence as a function of baseline voluntary alcohol drinking levels.
Methods
Over a 26-month protocol, young adult male rhesus macaques were first induced to drink alcohol and then given continuous access to 4% (
w
/
v
) ethanol (
n
= 8) or water (
n
= 4) for approximately 14 months, followed by three 28- to 35-day abstinence phases, with 3 months of ethanol access in between. Ethanol intake and blood ethanol concentration (BEC) were the primary dependent variables. Observational signs of physical dependence and circulating ACTH and cortisol were monitored.
Results
Prior to abstinence, stable, categorical, individual differences in voluntary ethanol intake under chronic access conditions were found. Following abstinence, categorical “non-heavy” drinking subjects increased drinking transiently (increased between 0.7 and 1.4 g/kg/day in first month after abstinence) but returned to baseline after 3 months. Categorical “heavy” drinkers, however, maintained drinking 1.0–2.6 g/kg above baseline for over 3 months following abstinence. Signs of physical dependence were rare, although huddling and social withdrawal increased in ethanol and control subjects. The most prominent effect on hormonal measures was heightened cortisol during abstinence that increased to a greater extent in ethanol subjects.
Conclusion
Involuntary abstinence increases drinking in the absence of overt physical withdrawal symptoms, and heavy drinkers are more robustly affected compared to non-heavy drinkers.
The Non-Human Primate (NHP) model for the study of Alcohol Use Disorders (AUD) as developed in our laboratories is critical to our understanding of the pathophysiology of voluntary, chronic, ethanol ...consumption. Previous work in this model established categories of ethanol consumption that parallel reported categories of human consumption across a spectrum spanning low drinking, binge drinking, heavy drinking, and very heavy drinking, albeit at generally higher daily intakes across categories than documented in people. Original categories assigned to ethanol consumption patterns were established using a limited cohort of rhesus macaques. This study revisits the validity of categorical drinking using an additional 28 monkeys. In addition to finding categorical representations consistent with the original 2014 report, our findings demonstrate that drinking categories remain stable across the observed 12 months of nearly consistent access to ethanol (22 h/day), termed "open access". Animals occupying the two ends of the spectrum, "low" and "very heavy" drinkers, exhibit the largest stability. The findings also indicate a slight escalatory drift over time, with very heavy drinking animals experiencing fatigue near the end of open access.
Background
Heavy alcohol drinking has aspects of inflexible behavior. This study addressed the consequences of chronic alcohol drinking on cognitive and sensory‐motor domains of behavioral ...flexibility in rhesus monkeys.
Methods
Behavioral flexibility was assessed in 12 monkeys (n = 9, ethanol EtOH drinkers) with a set‐shifting visual discrimination procedure before alcohol self‐administration and while maintaining consumption of 1.5 g/kg/d EtOH. Task performance was assessed in the morning after ~18 hours of drinking 1.5 g/kg, and 1 hour before the next day's drinking session began. The first 10 set‐shifting sessions had the original (preethanol) test parameters and were used to determine retention of preethanol performance. Then, an effect of sensory‐motor challenge (60% reduction in the size of the discriminative stimuli) on performance was assessed during 10 additional sessions.
Results
There were no average group‐dependent differences in the performance between control and EtOH groups at the preethanol time‐point. The daily consumption of 1.5 g/kg/d produced binge alcohol intakes in 7 of 9 monkeys (blood EtOH concentration BEC ≥ 80 mg/dl). Chronic daily intakes of 1.5 g/kg had no effect on retention of the task in the sober state. However, when challenged with a reduction in the size of the stimuli, daily 1.5 g/kg EtOH resulted in a decrement in performance due to an increase in the number of errors.
Conclusions
Rhesus monkeys consuming 1.5 g/kg alcohol daily perform equally as could as control monkeys in retention of a well‐learned cognitive task. However, this pattern of daily alcohol intake robustly decreased the ability to flexibly adjust behavior when confronted with novel changes to perceptual stimuli.
Heavy alcohol drinking has aspects of inflexible behavior. This study addressed the consequences of chronic alcohol drinking on cognitive and sensory‐motor domains of behavioral flexibility in rhesus monkeys. Rhesus monkeys consuming approximately 6 alcohol drinks daily perform equally well as control monkeys in retention of a well‐learned cognitive task. However, this pattern of daily alcohol intake robustly decreased the ability to flexibly adjust behavior when confronted with novel changes to perceptual stimuli.
Background
Long‐term alcohol drinking is associated with numerous health complications including susceptibility to infection, cancer, and organ damage. However, due to the complex nature of human ...drinking behavior, it has been challenging to identify reliable biomarkers of alcohol drinking behavior prior to signs of overt organ damage. Recently, extracellular vesicle‐bound microRNAs (EV‐miRNAs) have been found to be consistent biomarkers of conditions that include cancer and liver disease.
Methods
In this study, we profiled the plasma EV‐miRNA content by miRNA‐Seq from 80 nonhuman primates after 12 months of voluntary alcohol drinking.
Results
We identified a list of up‐ and downregulated EV‐miRNA candidate biomarkers of heavy drinking and those positively correlated with ethanol dose. We overexpressed these candidate miRNAs in control primary peripheral immune cells to assess their potential functional mechanisms. We found that overexpression of miR‐155, miR‐154, miR‐34c, miR‐450a, and miR‐204 led to increased production of the inflammatory cytokines TNFα or IL‐6 in peripheral blood mononuclear cells after stimulation.
Conclusion
This exploratory study identified several EV‐miRNAs that could serve as biomarkers of long‐term alcohol drinking and provide a mechanism to explain alcohol‐induced peripheral inflammation.
Due to the complex nature of human drinking behavior, it has been challenging to identify reliable biomarkers of alcohol use that could be used to determine drinking behavior prior to signs of overt organ damage. This study profiles the plasma EV‐miRNA content of 80 non‐human primates after 12 months of voluntary ethanol drinking by miRNA‐Seq. We identified several EV‐miRNA that could serve as biomarkers of long‐term alcohol drinking as well as provided a mechanism for alcohol‐induced peripheral inflammation.
Chronic alcohol abuse is frequently considered a habitual or inflexible behavior; however, measures of pre-existing cognitive flexibility prior to initiation of alcohol use are usually not available. ...This study used rhesus monkeys and an attentional set-shifting task to investigate whether pre-existing cognitive flexibility would predict increased risk for heavy alcohol drinking. As previously reported, monkeys were given 30 daily set-shifting sessions prior to alcohol access. These sessions consisted of the same sequence of eight unique visual discriminations (sets) of two objects that varied on two dimensions (shapes and colors). The ratio of errors per trials, session duration, and maximum set reached were primary dependent variables from each session and were used to compose a session performance index (PI) that ranged from a low performance PI of 31 to an optimal performance PI of 247. Here, animals underwent an alcohol induction period followed by 22 weeks of daily (22-h) self-administration sessions with free access to water and alcohol. Based on average daily alcohol intake during 22 weeks of 22-h/day access, the monkeys were categorized as non-heavy (mean = 2.0 ± 0.3 g/kg/day; n = 3) and heavy (mean = 3.3 ± 0.5 g/kg/day; n = 6) drinkers. The two groups diverged in performance on the set-shifting task across the 30 pre-alcohol sessions, and at the end of the pre-alcohol testing, the group average PI was 216 ± 27 and 137 ± 71 for the future non-heavy and heavy drinkers, respectively. The data show that low cognitive flexibility assessed with a set-shifting procedure was predictive of future classification as a heavy alcohol drinker. The data highlight individual differences in both cognitive flexibility and in alcohol self-administration in this population of rhesus monkeys.
•A novel set-shifting task was used to explore poor cognitive flexibility as a risk for developing heavy alcohol drinking.•A non-human primate model of alcohol self-administration was used.•Cognitive flexibility assessed with the set-shifting procedure was lower in a group of future heavy alcohol drinkers.•Pre-existing low cognitive flexibility predicts increased risk for classification as a heavy drinker in non-human primates.
The worldwide incidence of hepatocellular carcinoma (HCC) continues to rise, in part due to poor diet, limited exercise, and alcohol abuse. Numerous studies have suggested that the loss or mutation ...of PTEN plays a critical role in HCC tumorigenesis through the activation of the PI3K/Akt signaling axis. The homozygous knockout of PTEN in the livers of mice results in the accumulation of fat (steatosis), inflammation, fibrosis, and eventually progression to HCC. This phenotype bears a striking similarity to non-alcoholic steatohepatitis (NASH) which is thought to occupy an intermediate stage between non-alcoholic fatty liver disease (NAFLD), fibrosis, and HCC. The molecular and physiological phenotypes that manifest during the transition to HCC suggest that molecular imaging could provide a non-invasive screening platform to identify the hallmarks of HCC initiation prior to the presentation of clinical disease. We have carried out longitudinal imaging studies on the liver-specific PTEN knockout mouse model using CT, MRI, and multi-tracer PET to interrogate liver size, steatosis, inflammation, and apoptosis. In male PTEN knockout mice, significant steatosis was observed as early as 3 months using both magnetic resonance spectroscopy (MRS) and computed tomography (CT). Enhanced uptake of the apoptosis tracer
F-TBD was also observed in the livers of male PTEN homozygous knockout mice between 3 and 4 months of age relative to heterozygous knockout controls. Liver uptake of the inflammation tracer
F4FN remained relatively low and constant over 7 months in male PTEN homozygous knockout mice, suggesting the suppression of high-energy ROS/RNS with PTEN deletion relative to heterozygous males where the
F4FN liver uptake was elevated at early and late time points. All male PTEN homozygous mice developed HCC lesions by month 10. In contrast to the male cohort, only 20% (2 out of 10) of female PTEN homozygous knockout mice developed HCC lesions by month 10. Steatosis was significantly less pronounced in the female PTEN homozygous knockout mice relative to males and could not accurately predict the eventual occurrence of HCC. As with the males, the
F4FN uptake in female PTEN homozygous knockout mice was low and constant throughout the time course. The liver uptake of
F-TBD at 3 and 4.5 months was higher in the two female PTEN knockout mice that would eventually develop HCC and was the most predictive imaging biomarker for HCC in the female cohort. These studies demonstrate the diagnostic and prognostic role of multi-modal imaging in HCC mouse models and provide compelling evidence that disease progression in the PTEN knockout model is highly dependent on gender.
Chronic heavy alcohol consumption is a risk factor for low trauma bone fracture. Using a non-human primate model of voluntary alcohol consumption, we investigated the effects of 6 months of ethanol ...intake on cortical bone in cynomolgus macaques (Macaca fascicularis). Young adult (6.4 ± 0.1 years old, mean ± SE) male cynomolgus macaques (n = 17) were subjected to a 4-month graded ethanol induction period, followed by voluntary self-administration of water or ethanol (4 % w/v) for 22 h/d, 7 d/wk. for 6 months. Control animals (n = 6) consumed an isocaloric maltose-dextrin solution. Tibial response was evaluated using densitometry, microcomputed tomography, histomorphometry, biomechanical testing, and Raman spectroscopy. Global bone response was evaluated using biochemical markers of bone turnover. Monkeys in the ethanol group consumed an average of 2.3 ± 0.2 g/kg/d ethanol resulting in a blood ethanol concentration of 90 ± 12 mg/dl in longitudinal samples taken 7 h after the daily session began. Ethanol consumption had no effect on tibia length, mass, density, mechanical properties, or mineralization (p > 0.642). However, compared to controls, ethanol intake resulted in a dose-dependent reduction in intracortical bone porosity (Spearman rank correlation = −0.770; p < 0.0001) and compared to baseline, a strong tendency (p = 0.058) for lower plasma CTX, a biochemical marker of global bone resorption. These findings are important because suppressed cortical bone remodeling can result in a decrease in bone quality. In conclusion, intracortical bone porosity was reduced to subnormal values 6 months following initiation of voluntary ethanol consumption but other measures of tibia architecture, mineralization, or mechanics were not altered.
•Chronic heavy alcohol consumption is a risk factor for low trauma bone fractures.•We assessed effects of 6 mo of voluntary ethanol intake on cortical bone in monkeys.•Ethanol had no effect on tibia density, mechanical properties, or mineralization.•Ethanol resulted in a dose-dependent reduction in intracortical porosity.•Long-term suppressed intracortical remodeling may result in decreased bone quality.
Rationale
Consumption of alcohol begins during late adolescence in a majority of humans, and the greatest drinking occurs at 18–25 years then decreases with age.
Objectives
The present study measured ...the differences in ethanol intake in relation to age at the onset of ethanol access among nonhuman primates to control for self-selection in humans and isolate age effects on heavy drinking.
Methods
Male rhesus macaques were assigned first access to ethanol during late adolescence (
n
= 8), young adulthood (
n
= 8), or early middle age (
n
= 11). The monkeys were induced to drink ethanol (4 %
w
/
v
in water) in increasing doses (water then 0.5, 1.0, 1.5 g/kg ethanol) using a fixed-time (FT) 300-s schedule of food delivery, followed by 22 h/day concurrent access to ethanol and water for 12 months. Age-matched controls consumed isocaloric maltose–dextrin solution yoked to the late adolescents expected to be rapidly maturing (
n
= 4).
Results
Young adult monkeys had the greatest daily ethanol intake and blood-ethanol concentration (BEC). Only late adolescents escalated their intake (ethanol, not water) during the second compared to the first 6 months of access. On average, plasma testosterone level was consistent with age differences in maturation and tended to increase throughout the experiment more for control than ethanol-drinking adolescent monkeys.
Conclusions
Young adulthood in nonhuman primates strongly disposes toward heavy drinking, which is independent of sociocultural factors present in humans. Ethanol drinking to intoxication during the critical period of late adolescence is associated with escalation to heavy drinking.
Alcohol consumption suppressed bone turnover in male non-human primates; however, it is unclear the extent to which this effect depends upon biological variables. Using archived plasma samples, we ...investigated whether sex, age of onset of alcohol intake, and species influence the effects of graded increases in alcohol consumption on bone turnover markers.
91 male and female macaques (rhesus and cynomolgus), ranging in age from 4 years (adolescent) to 10 years (adult) were required to increase their consumption of ethanol in 30-day increments: 0 g/kg/day, followed by 0.5 g/kg/day, 1.0 g/kg/day, and, finally, 1.5 g/kg/day. Plasma osteocalcin (formation), plasma CTX (resorption) and osteocalcin to CTX ratio (turnover balance) were measured during these intervals to assess the dose-response effects of alcohol.
We detected no relationship between dose and osteocalcin when all monkeys were combined, but there was a significant effect of sex (lower levels in females) and interactions between alcohol dose and sex (osteocalcin levels increased with dose in rhesus females). In contrast, we detected a negative linear dose-response relationship for ethanol and CTX. We did not detect a relationship between dose and osteocalcin to CTX ratio overall, but there was a significant positive relationship detected in females (no change in males). Increased age predicted lower biomarker levels for both osteocalcin and CTX. Species was a significant predictor for osteocalcin and the osteocalcin to CTX ratio in these models.
These findings indicate that age, sex, and species influence bone turnover and support the concept that factors beyond quantity of alcohol affect skeletal response to alcohol consumption.
•Age, sex, and species influenced markers of bone turnover in non-human primates.•Ethanol consumption resulted in a dose-dependent reduction in CTX.•Ethanol consumption resulted in increased osteocalcin in rhesus females.
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