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Chronic alcohol induced liver injury correlates with memory deficits: Role for neuroinflammation
Journal Pre-proof Chronic alcohol induced liver injury correlates with memory deficits: Role for neuroinflammation Jean A. King, Benjamin C. Nephew, Asmita Choudhury, Guillaume L. Poirier, Arlene Lim, Pranoti Mandrekar PII:
S0741-8329(19)30060-6
DOI:
https://doi.org/10.1016/j.alcohol.2019.07.005
Reference:
ALC 6930
To appear in:
Alcohol
Received Date: 15 March 2019 Revised Date:
19 July 2019
Accepted Date: 31 July 2019
Please cite this article as: King J.A, Nephew B.C., Choudhury A., Poirier G.L, Lim A. & Mandrekar P., Chronic alcohol induced liver injury correlates with memory deficits: Role for neuroinflammation, Alcohol (2019), doi: https://doi.org/10.1016/j.alcohol.2019.07.005. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Inc. All rights reserved.
Chronic alcohol induced liver injury correlates with memory deficits: Role for neuroinflammation
Jean A King 1,2, Benjamin C. Nephew1,2, Asmita Choudhury3, Guillaume L Poirier1, Arlene Lim3, Pranoti Mandrekar3
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Center for Comparative Neuroimaging, Department of Psychiatry, University of
Massachusetts Medical School, Worcester, MA, USA 2
Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester,
MA, USA 3
Department of Medicine, University of Massachusetts Medical School, Worcester, MA,
USA Corresponding authors: Pranoti Mandrekar, Ph.D. University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, United States. Tel.: +1 (508) 856 5391; fax: +1 (508) 856 4770. E-mail address: [email protected].
Jean King, Ph.D. University of Massachusetts Medical School, 55 Lake Avenue, Worcester, MA 01605, United States. Tel.: +1 (508) 856 4969 E-mail address: [email protected].
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Financial support statement This work was supported by NIAAA-NIH grants R01 AA17986-01A1 (to PM), R01 AA25289-01 (to PM), and S10OD018132 NIH (to JK) from the National Institute of Alcohol Abuse and Alcoholism, Bethesda, MD, USA.
Highlights •
Chronic alcohol consumption increased Tnfα, Il6, Mcp1, and Il1β mRNA in the hippocampus.
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Alcohol consumption increased Tnfα and Il1β mRNA in the prefrontal cortex.
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Memory and sensorimotor coordination were impaired by chronic alcohol.
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Hippocampal Il6 mRNA positively correlated with serum ALT, marker of liver injury.
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ALT and hippocampal Il6 mRNA were inversely correlated with memory.
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The liver-brain axis may mediate alcohol induced cognitive deficits.
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ABSTRACT Alcohol use disorder (AUD) affects over 15 million adults over age 18 in the United States, with estimated costs of 220 billion dollars annually—mainly due to poor quality of life and lost productivity, which in turn is intricately linked to cognitive dysfunction. AUD induced neuroinflammation in the brain, notably the hippocampus, is likely to contribute to cognitive impairments. The neuroinflammatory mechanisms mediating the impact of chronic alcohol on the central nervous system, specifically cognition, require further study. We hypothesized that chronic alcohol consumption impairs memory and increases the inflammatory cytokines TNFα, IL6, MCP1, and IL1β in the hippocampus and prefrontal cortex regions in the brain. Using the chronic-binge Gao-NIAAA alcohol mouse model of liver disease, representative of the drinking pattern common to human alcoholics, we investigated behavioral and neuroinflammatory parameters. Our data show that chronic alcohol intake elevated peripheral and brain alcohol levels, induced serum alanine aminotransferase (ALT), a marker of liver injury, impaired memory and sensorimotor coordination, increased inflammatory gene expression in the hippocampus and prefrontal cortex. Interestingly, serum ALT and hippocampal IL6 correlated with memory impairment, suggesting an intrinsic relationship between neuroinflammation, cognitive decline, and liver disease. Overall, our results point to a likely liver-brain functional partnership and suggest that future strategies to alleviate hepatic and/or neuroinflammatory impacts of chronic AUD may result in improved cognitive outcomes.
Keywords: Alcoholic liver disease, cognition, neuroinflammation, memory, IL6
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INTRODUCTION AUD is a consequence of chronic alcohol abuse affecting over 15 million adults over age 18 in the United States ("National Survey on Drug Use and Health (NSDUH). Substance Abuse and Mental Health Services Administration (SAMHSA)," 2015). Excessive drinking in the US costs over 220 billion dollars annually, including both healthcare costs and associated lost productivity which has been linked to poor quality of life and alcohol-induced cognitive impairments (Hingson & Rehm, 2013). In individuals with AUD, such cognitive alterations may persist up to at least after a year of abstinence (Parsons, 1998; Stavro, Pelletier, & Potvin, 2013), and the magnitude of the deficit is prognostic for relapse in abstinent individuals (Bowden-Jones, McPhillips, Rogers, Hutton, & Joyce, 2005). There remains a critical gap in knowledge regarding the underlying mechanisms promoting chronic alcohol-induced cognitive disturbances. Converging evidence points to plausible mechanistic links between alcohol-induced neuroinflammation and cognition and behavior (Crews et al., 2015; Kelley & Dantzer, 2011; M. Pascual, Baliño, Alfonso-Loeches, Aragón, & Guerri, 2011; Ureña-Peralta, Alfonso-Loeches, Cuesta-Diaz, García-García, & Guerri, 2018). However, much of the research on adverse effects of alcohol associated neuroinflammation has focused on early life exposures to developing brains (Zhang, Wang, Xu, Frank, & Luo, 2018). Taken together, a more comprehensive understanding of the role of neuroinflammatory factors in mediating behavioral phenotypes of AUD is urgently needed. One common adverse long-term consequence of AUD known to involve robust inflammatory mechanisms is alcoholic liver disease (ALD). Both innate and adaptive immune cell types have been implicated in ALD, and inflammatory signaling pathways
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play a critical role in AUD associated liver damage. A number of inflammatory cytokines and chemokines, including tumor necrosis factor alpha (TNFα), interleukin (IL) 6, monocyte chemoattractant protein 1 (MCP1), and IL1β, have been postulated to mediate both ALD pathogenesis and learning, neuroplasticity, and neurochemistry (Donzis & Tronson, 2014; Prieto & Cotman, 2017). To better understand the role of inflammation in cognitive impairments in AUD, we focus on the hippocampus and prefrontal cortex. The hippocampus plays a critical role in memory (Bird & Burgess, 2008), is implicated in alcohol consumption related neural changes (Huttunen & Myers, 1987; Martin-Garcia, Darbra, & Pallares, 2007), and is particularly vulnerable to the deleterious effects of AUD (Hermens et al., 2013; White, Matthews, & Best, 2000; Yaka, Phamluong, & Ron, 2003). Heavy alcohol drinking decreases hippocampal white matter, glial cells, and neurogenesis (Crews & Nixon, 2009; Harding, Wong, Svoboda, Kril, & Halliday, 1997; Korbo, 1999; Richardson et al., 2009), thus it is not surprising that memory is impaired as well (Hermens et al., 2013; White et al., 2000). The prefrontal cortex, critical to executive function and decision making, has also been implicated in the adverse cognitive effects of AUD (Staples & Mandyam, 2016). Structural abnormalities in the prefrontal cortex in individuals that report an AUD include reductions in white matter (Kril, Halliday, Svoboda, & Cartwright, 1997), decreased white matter integrity (Pfefferbaum et al., 1992; Zorlu et al., 2013), and reduced frontal cortex volume (Pfefferbaum et al., 1992). Damage to this brain region is predictive of impairments in executive function (Nakamura-Palacios et al., 2014). Exposure of adolescent rats to a binge paradigm induces long term cognitive
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deficits, increased TNFα and IL1β production, and changes in myelin proteins in the prefrontal cortex (Pascual, Pla, Miñarro, & Guerri, 2014). The objective of the current study was to use the clinically relevant Gao-NIAAA model of ALD, known to induce liver injury, to investigate the role of neuroinflammation in the adverse cognitive impacts of alcohol exposure. We hypothesized that chronic alcohol exposure induces cognitive deficits and neuroinflammation in the hippocampus and prefrontal cortex. We measured alcohol levels in blood and brain tissue, serum ALT, an indicator of alcoholic liver damage, assessed cognitive and sensorimotor effects, and documented the neuroinflammatory changes in the hippocampus and prefrontal cortex.
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METHODS Animals All animals received proper care in agreement with animal protocols approved by the Institutional Animal Use and Care Committee of the University of Massachusetts Medical School. All animals received humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985). 12-week-old female C57BL/6 mice were obtained from Jackson laboratories. Two mice were housed per cage and were maintained in 12 light/12 dark cycle with free access to food and water. Females were used for the study as we have previously demonstrated sexual dimorphism in alcohol-induced inflammation and higher degree of inflammation in the female mice compared to the male mice (Fulham & Mandrekar, 2016).
Alcohol administration Our studies used the established Gao-NIAAA mouse model of ALD (Bertola, Mathews, Ki, Wang, & Gao, 2013) (Fig. 1). Based on this alcohol exposure regimen, female C57BL/6 mice were randomized into alcohol-fed and pair-fed groups and adapted to control Lieber DeCarli liquid diet (Bioserv, Frenchtown, NJ) for 5 days (Ambade et al., 2014). The alcohol group was allowed free access to alcohol-containing diet (36% calories) with increasing alcohol concentrations, 1%-5% for one day each and finally 5% for 10 days. Control mice were given pair-fed diets isocaloric substituted with maltose dextrin. On day 16, all mice were gavaged, either with a single dose of alcohol
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(5 g/kg body weight of 31.5% alcohol), or isocaloric dextrin maltose. Mice were behaviorally tested 7 hours following the gavage and euthanized two hours later for tissue collection.
Novel object discrimination task Rodents normally exhibit increased investigation of unfamiliar objects, and the discrimination with a familiar object serves as an index of memory. Memory was evaluated based on established memory test procedures for object identity and location (adapted from (Bello-Medina, Sanchez-Carrasco, Gonzalez-Ornelas, Jeffery, & Ramirez-Amaya, 2013; DeVito & Eichenbaum, 2010; Inostroza & Born, 2013)). Following three 10-min daily pre-exposure sessions to an open arena (60x50x30 cm), a 5-min familiarization phase was conducted by exposing a mouse to novel object quadruplicates. After an hour delay, the mouse was re-introduced to the arena, where two now-familiar objects were substituted by two novel objects. One of each pair of objects were positioned in a novel location to assess object memory, a) Familiar Identity in a Familiar Location, b) Familiar Identity in a New Location, c) New identity in a Familiar Location, and d) New Identity in a New Location. Blind video-scoring of the time spent investigating each object allowed for the calculation of overall preference ratios for object memory. The discrimination ratio for recognition of the distinct objects was calculated as the difference between the average exploration times for novel objects minus exploration times for familiar objects, divided by the sum of those times. This measure represents the animal's ability to differentiate between two groups of objects, relying on the natural tendency of mice to spend more time exploring more novel
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objects compared to less novel objects. Memory is indicated by a positive ratio, which reflects greater exploration time for novel objects compared to familiar objects.
Sensorimotor coordination Sensorimotor coordination was evaluated seven hours post binge using the static rod test, (Contet, 2012; Deacon, 2013) consisting of a sequential assay using five rods of diameters ranging from 1¼” to ¼”, from wide to narrow. Latencies for the mouse to re-orient 180o away from the edge and to traverse the length of the rod to safety after placement upon the overhanging extremity were recorded (120 s limit/trial), and the sum of the latencies was calculated for each animal.
Magnetic resonance spectroscopy methods to assess hippocampal ethanol levels Mice were anesthetized with 2% isoflurane and constantly monitored for vital signs during the entire time of imaging with a 4.7-T/40-cm horizontal magnet (Oxford) equipped with a Bruker Biospec/Avance console. Experiments for all other imaging were performed using the Oxford 4.7-T/40-cm horizontal magnet equipped with a Bruker Biospec/Avance III HD console. A 1H radiofrequency mouse head coil (Bruker) with inner diameter of 23 mm was used for the experiments. T1-weighted anatomical images were acquired using FLASH sequence with the following parameters: repetition time (TR) = 280.86 ms, echo time (TE) = 4.5 ms, matrix size = 384 × 384, field of view (FOV) = 18 × 18 mm2, slice number = 15, slice thickness = 0.5 mm, flip angle = 40°, and number of averages = 8. T2-weighted images were
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acquired using TurboRARE sequence with TR = 2,200 ms, TE = 36 ms, echo spacing = 12 ms, 8 averages, and rare factor = 8. 1H magnetic resonance spectra data were acquired using single-voxel point-resolved spectroscopy sequence (PRESS) (repetition time = 2,500 ms, echo time = 16 ms, number of averages = 512, voxel size = 3 × 3 × 3 mm). Proton spectra were fit using LC Model (Version 6.2-2B), which analyzed in vivo proton spectra as a linear combination of model in vitro spectra from individual metabolite solutions and generated data as absolute fits (in institutional units) and SD%. SD was used as a measure of the reliability of the fit.
Serum ALT Serum ALT levels were measured using the ALT reagent set (Teco Diagnostics, California, USA), as per the manufacturer’s protocol.
Blood alcohol level Blood alcohol content was measured in serum using the Alcohol Reagent and the AM1 Alcohol Analyzer (Analox Instruments Ltd., London, UK), as per the manufacturer’s protocol.
Brain tissue collection Brains were rapidly extracted, immediately snap frozen using -40oC isopentane (2methylbutane), and then stored at -80oC until further processing for RT-PCR. Tissue micropunches (0.5mm diameter) of the dorsal hippocampus and prefrontal cortex were
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obtained from slices (0.5mm) cut on a Leica cryostat (CM3050 S), using visible anatomical landmarks.
qRT-PCR methods Dorsal hippocampus and prefrontal cortex samples were homogenized in QIAzol (Qiagen GmbH, Hilden, Germany) and subjected to a chloroform extraction. Total RNA was extracted using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany), according to manufacturer’s instructions. Yield of RNA was measured using Nano Drop 2000 (Thermo Scientific, Wilmington, DE). cDNA was synthesized using the Reverse Transcription System (Promega, Madison, WI). mRNA levels of genes were quantified using iTAQ Universal SYBR Green Supermix and CFX Connect Real-Time PCR Detection System (Biorad Laboratories, Hercules, CA). 18s ribosomal RNA served as the housekeeping control. Relative gene expression was analyzed by the 2–∆∆Ct method. Primers were synthesized by IDT, Inc. (Coralville, IA). Primer sequences used for this study are enlisted in table 1.
Statistics Data are represented as mean ± SEM. Statistical significance was determined by the Student t-test or Pearson’s Correlation, using Graphpad Prism 7.04, and denoted as p≤0.05.
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RESULTS Alcohol in serum and hippocampal tissue The Gao-NIAAA model of alcohol administration resulted in significantly elevated blood alcohol levels (BAC) in serum (82.6 ± 4.6 (ethanol-fed) vs. 1.5 ± 0.4 (pair-fed), t=15.83, p<0.001, df=9) and hippocampal tissue (12.5 ± 1.6 (ethanol-fed) vs. 0 (pairfed), t=7.813, p<0.001, df=8), as well as high serum ALT (158.24 ± 35 (ethanol-fed) vs. 77.2 ± 0.78 (pair-fed), t=1.96, p<0.01, df=5), a marker of liver injury confirming chronic alcohol exposure.
Object Memory and Sensorimotor Coordination Chronic ethanol exposure impaired memory in the ethanol-fed group compared to the pair-fed group (p<0.05, t=3.28, df=8, Fig. 2). Although there was no difference in completion of the static rod test between the groups, ethanol-fed mice had longer total latencies to traverse the rods (336.3 ± 141.8 (ethanol-fed) vs. 22.1 ± 4.3 (pair-fed) seconds, t=2.89, p<0.01, df=8), indicating impaired sensorimotor functioning.
Neuroinflammation To determine whether pro-inflammatory cytokine alterations were associated with cognitive impairments, cytokine gene expression was measured in the dorsal hippocampus and prefrontal cortex. Levels of Tnfα, Il1β, Mcp1, and Il6 mRNA were elevated in the dorsal hippocampus of ethanol-fed mice compared to pair-fed controls (all p’s <0.05, Fig. 3A-D), and levels of Tnfα and Il1β were also elevated in the prefrontal
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cortex (all p’s < 0.05 Fig. 3A, B) indicating differential inflammation in these two brain regions.
Correlation Analysis Next, we analyzed whether alterations in memory correlated with any cytokine gene expression and/or liver injury. Interestingly, memory is negatively correlated with serum ALT in both groups combined (r = -0.8254 p=0.0033, Table II). It is also negatively correlated with Il6 levels in the hippocampus in both groups combined (r = 0.9021, p = 0.0022, Table II). Furthermore, Il6 and ALT are positively correlated (r= 0.9759, p<0.0001, Table II). There were no other significant memory-cytokine mRNA correlations, and no correlations between ALT or cytokines and sensorimotor coordination.
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Discussion Using the Gao-NIAAA binge model of alcohol administration, which effectively increased serum and hippocampal ethanol levels and serum ALT, we report memory deficits, sensorimotor impairment, and increased neuroinflammation in ethanol-fed mice compared to pair-fed controls. Expression levels of several ALD-associated inflammatory factors were increased in both the hippocampus and the prefrontal cortex, regions critical for memory (Preston & Eichenbaum, 2013) and implicated in sensorimotor control (Bast & Feldon, 2003; Hyder et al., 1997; Siegel, Buschman, & Miller, 2015). Memory was negatively associated with both peripheral ALT levels and hippocampal Il6 mRNA, and these factors were positively correlated. Taken together, the results indicate that the Gao-NIAAA binge model is a valuable tool to investigate relationships between the adverse hepatic, neuroinflammatory, cognitive, and sensorimotor impacts of chronic alcohol, topics of growing concern in efforts to comprehend the role of the brain-liver axis in alcohol mediated cognitive disease etiology. Previous alcohol-induced studies of neural effects have primarily used models of repeated binge exposures (Majchrowicz 4-day binge paradigm) (Faingold, 2008) or alcohol binges followed by abstinence (Ward et al., 2009). The current study augments this prior work with the use of the clinically relevant chronic Gao-NIAAA binge model which effectively and substantially increased alcohol levels in serum and brain tissue and elevated ALT, a primary marker of liver damage. The present investigation focused on memory to align with a major clinical presentation of AUD and to address the paucity of data linking alcohol-induced hippocampal alterations and cognitive deficits (Obernier, White, Swartzwelder, & Crews,
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2002; Vedder, Hall, Jabrouin, & Savage, 2015). Chronic administration studies in mice report that an 8 week consumption paradigm produces long-term memory deficits (Farr, Scherrer, Banks, Flood, & Morley, 2005). Long-term moderate oral self-administration of alcohol in rats adversely affects memory, and it is postulated that this accelerates aging induced cognitive decline (Baird et al., 2006). Memory is also impaired with acute treatment, and similarities between alcohol induced deficits and those induced by hippocampal lesions supports a central role of the hippocampus in alcohol-associated cognitive impairments (Hoffmann & Matthews, 2006; Van Skike, Goodlett, & Matthews, 2019). In our study using the Gao-NIAAA model, cognitive and neuroinflammatory parameters are linked to liver injury and indicate that the brain liver axis and may be crucial in the etiology of cognitive decline and/or could serve as a biomarker of increased risk. Elevated ALT may indicate increased likelihood of adverse neuroinflammatory changes, such as the reported increases in mRNA levels of inflammatory factors in the hippocampus and prefrontal cortex. While the specific literature on neuroinflammation in ALD is limited, it is known that liver failure in general is strongly associated with neuroinflammation (Butterworth, 2013, 2015). Several of the cytokines altered in the current study are implicated in acute liver failure and induce neuroinflammation (TNFα, IL6, IL1β) (Butterworth, 2013). Peripheral inflammation is associated with cognitive and motor deficits with hepatic encephalopathy, and this is postulated to be mediated by neuroinflammation (Felipo et al., 2012; Shawcross, Davies, Williams, & Jalan, 2004). The strong negative associations between memory, serum ALT, and Il6 expression in the hippocampus, combined with the positive association between ALT and
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hippocampal Il6, suggests that ALT may be a valuable biomarker for ALD induced neuroinflammation and associated cognitive impairments. Similar to the current results, Tnfα mRNA levels are increased in the hippocampus of rats exposed to a 10-week alcohol regimen that adversely affects learning and memory (Tiwari, Kuhad, & Chopra, 2009). While inhibition of TNF synthesis can reverse hippocampal-dependent learning in a rodent model of chronic inflammation (Belarbi et al., 2012), intermittent ethanol exposure (simulated binge drinking) acutely increases hippocampal levels of both TNFα and IL1β and adversely impacts learning and memory (Zhao et al., 2013). Exogenous IL1β also inhibits the acquisition of learning, where the neutralization of this cytokine prevents Legionella infection induced cognitive impairment (Gibertini, Newton, Friedman, & Klein, 1995). Hippocampal IL1β, IL6, along with TNF, have been implicated in metabolic syndrome induced impairments in memory (Dinel et al., 2011), and MCP1 levels are elevated in the brain tissue of mice exposed to a high fat diet (Pistell et al., 2010), supporting the hypothesis that the adverse cognitive effects of metabolic changes and chronic alcohol exposure are both mediated by similar neuroinflammatory mechanisms. Given the specific association between memory and hippocampal Il6 mRNA in the current study, this cytokine could play a particularly potent role in the adverse cognitive effects of chronic alcohol exposure. This cytokine has been specifically implicated in neuroinflammatory mediated neurodegenerative diseases, including Alzheimer’s disease (Hull, Strauss, Berger, Volk, & Bauer, 1996), where β-amyloid peptide potentiates IL6 production from cells in culture (Sondag, Dhawan, & Combs, 2009). Further in vitro work indicates that IL6 potentiates β-amyloid induced
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neurotoxicity (Qiu & Gruol, 2003), and it may have common neurodegenerative actions in a range of pathological states, including AUD. Taken in conjunction with the present findings, it appears that a variety of insults impair cognition through inflammatory effects in the hippocampus, and research on AUD associated cognitive impairment can benefit from leveraging this literature to accelerate the identification and development of novel hippocampus immune-targeted preventative measures and treatments. The evidence for a role of the prefrontal cortex in inflammation mediated cognitive impairment is not as robust when compared to the findings from the hippocampus. In accordance with this observation, there were no significant cognition/memory cytokine correlations in the prefrontal cortex in the current study. Although others have shown that adult alcohol exposure in rats impairs recognition memory and increases IL6 levels in prefrontal cortex, (Terasaki & Schwarz, 2017) prefrontal cortex IL6 expression was not elevated in the present study. This could be, due to species differences in the response to alcohol and/or differences in alcohol exposure duration. Intermittent binge paradigms in rats induce learning, sensorimotor, and recognition deficits and increase cell death in the neocortex (M. Pascual, Blanco, Cauli, Minarro, & Guerri, 2007), but this work was in an adolescent rodent model, as are many of the animal studies of alcohol exposure. Changes in prefrontal cortex related neural connectivity have also been implicated in inflammatory pain related deficits in working memory (Cardoso-Cruz, Sousa, Vieira, Lima, & Galhardo, 2013), which is consistent with earlier studies of memory related functions in this region (Jung, Qin, McNaughton, & Barnes, 1998; Wang & Cai, 2006). The potential role of the prefrontal cortex in the
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present cognitive deficit is likely indirect and dependent on its structural and functional connectivity to the hippocampus. The cytokine results herein support additional investigation of the roles of prefrontal cortex TNFα and IL1β in AUD related cognitive deficits. While the NIAAA alcohol feeding paradigm did impair sensorimotor coordination along with memory, the dexterity required for the sensorimotor static rod task was greater than that necessary for the memory task in an open field, therefore it is unlikely that it contributed to the adverse effects on memory in a meaningful way. It is possible that the present effects of alcohol in the sensorimotor task may involve anxiety or ataxia related mechanisms (Gallate, Morley, Ambermoon, & McGregor, 2003), however, since we did not specifically assess either, we cannot be certain which of these mechanisms is at play. Progressive alcohol intake in humans impairs cognitive and motor processes, and it is postulated that central (such as neuroinflammatory) and peripheral mechanisms are involved in these effects (De Wilde, Dom, Hulstijn, & Sabbe, 2007). Although it is possible that we would have observed cognitive deficits without sensorimotor impairments with an extended period of abstinence, gait and behavior deficits are present following one year of abstinence in humans (Fein & Greenstein, 2013; Smith & Fein, 2011). Furthermore, both cognitive and sensorimotor deficits have been documented in detoxified alcoholic men, and the specific deficits implicate the prefrontal cortex in some of these impairments (Sullivan, Rosenbloom, & Pfefferbaum, 2000). Chronic ethanol exposure induces motor impairments in other rodent models, and these effects are also associated with cortical damage (da Silva et al., 2018; Teixeira et al., 2014), further supporting the use of animal models in studying AUD.
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Given the substantial increased risk of falling due to alcohol intake, especially with higher intake levels (Chen & Yoon, 2017), research aimed at understanding mechanisms to target for reversing alcohol use induced motor impairments, along with cognitive deficits, is warranted. The present data support our hypothesis that that the Gao-NIAAA model of ALD is a valuable tool to investigate the adverse cognitive and neuroinflammatory effects of AUD. Increased focus on the contributions of liver damage to the progression of alcohol associated cognitive deficits may generate novel etiological findings. An enhanced understanding of the role of the liver and the timing of the inflammatory/neurotrophic cascade induced by AUD, with a specific focus on plasma ALT and hippocampal IL6, will provide a strong foundation for the development of effective preventative measures and treatments that are likely to be pertinent to both AUD associated cognitive deficits and ALD etiology.
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Acknowledgements The authors would like to thank the UMass Medical School Department of Animal Medicine for outstanding animal care and cooperative support of the rodent experiments.
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Legends Figure 1: Study design. Mice drank 5% alcohol for 10 days after habituation to liquid diet and 5 days of progression to the final concentration. A 5g/kg binge gavage was administered on day 16. Figure 2: Impaired memory in ethanol fed mice. Mean ± SEM memory of ethanoland pair-fed mice, t=3.28, p<0.05, n=6. Figure 3: Neuroinflammation in ethanol fed mice. Mean ± SEM mRNA levels of Tnfα (t=4.914, df=6; dorsal hippocampus, t=3.53, df=8; prefrontal cortex) (A), Il1β (t=3.431, df=6; dorsal hippocampus, t=3.782, df=7; prefrontal cortex) (B), Mcp1 (t=2.864, df=8; dorsal hippocampus) (C), and Il6 (t=4.47, df=8; dorsal hippocampus) (D) in the dorsal hippocampus and prefrontal cortex of ethanol and pair-fed rats. * denotes p<0.05, n=6, ** denotes p<0.01, n=6.
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Table 1: Primer sequences used for real time PCR Gene
Forward
Reverse
18s
GTA ACC CGT TGA ACC CCA TT
CCA TCC AAT CGG TAG TAG CG
Il-1β
CAG GCA GGC AGT ATC ACT CA
TGT CCT CAT CCT GGA AGG TC
Il-6
ACA ACC ACG GCC TTC CCT ACT T
CAC GAT TTC CCA GAG AAC ATG TG
Mcp-1 CAG GTC CCT GTC ATG CTT CT
TCT GGA CCC ATT CCT TCT TG
Tnf-α
GTG AGG GTC TGG GCC ATA GA
GAA GTT CCC AAA TGG CCT CC
Table 2: Correlation analysis
Parameter I Parameter II
r
R2
p value
ALT
Novel Spatial preference
-0.8254
0.6813
0.0033
HPC Il-6
Novel Spatial preference
-0.9021
0.8137
0.0022
HPC Il-6
ALT
0.9759
0.9523
<0.0001
S0741-8329(19)30060-6
DOI:
https://doi.org/10.1016/j.alcohol.2019.07.005
Reference:
ALC 6930
To appear in:
Alcohol
Received Date: 15 March 2019 Revised Date:
19 July 2019
Accepted Date: 31 July 2019
Please cite this article as: King J.A, Nephew B.C., Choudhury A., Poirier G.L, Lim A. & Mandrekar P., Chronic alcohol induced liver injury correlates with memory deficits: Role for neuroinflammation, Alcohol (2019), doi: https://doi.org/10.1016/j.alcohol.2019.07.005. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Inc. All rights reserved.
Chronic alcohol induced liver injury correlates with memory deficits: Role for neuroinflammation
Jean A King 1,2, Benjamin C. Nephew1,2, Asmita Choudhury3, Guillaume L Poirier1, Arlene Lim3, Pranoti Mandrekar3
1
Center for Comparative Neuroimaging, Department of Psychiatry, University of
Massachusetts Medical School, Worcester, MA, USA 2
Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester,
MA, USA 3
Department of Medicine, University of Massachusetts Medical School, Worcester, MA,
USA Corresponding authors: Pranoti Mandrekar, Ph.D. University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, United States. Tel.: +1 (508) 856 5391; fax: +1 (508) 856 4770. E-mail address: [email protected].
Jean King, Ph.D. University of Massachusetts Medical School, 55 Lake Avenue, Worcester, MA 01605, United States. Tel.: +1 (508) 856 4969 E-mail address: [email protected].
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Financial support statement This work was supported by NIAAA-NIH grants R01 AA17986-01A1 (to PM), R01 AA25289-01 (to PM), and S10OD018132 NIH (to JK) from the National Institute of Alcohol Abuse and Alcoholism, Bethesda, MD, USA.
Highlights •
Chronic alcohol consumption increased Tnfα, Il6, Mcp1, and Il1β mRNA in the hippocampus.
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Alcohol consumption increased Tnfα and Il1β mRNA in the prefrontal cortex.
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Memory and sensorimotor coordination were impaired by chronic alcohol.
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Hippocampal Il6 mRNA positively correlated with serum ALT, marker of liver injury.
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ALT and hippocampal Il6 mRNA were inversely correlated with memory.
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The liver-brain axis may mediate alcohol induced cognitive deficits.
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ABSTRACT Alcohol use disorder (AUD) affects over 15 million adults over age 18 in the United States, with estimated costs of 220 billion dollars annually—mainly due to poor quality of life and lost productivity, which in turn is intricately linked to cognitive dysfunction. AUD induced neuroinflammation in the brain, notably the hippocampus, is likely to contribute to cognitive impairments. The neuroinflammatory mechanisms mediating the impact of chronic alcohol on the central nervous system, specifically cognition, require further study. We hypothesized that chronic alcohol consumption impairs memory and increases the inflammatory cytokines TNFα, IL6, MCP1, and IL1β in the hippocampus and prefrontal cortex regions in the brain. Using the chronic-binge Gao-NIAAA alcohol mouse model of liver disease, representative of the drinking pattern common to human alcoholics, we investigated behavioral and neuroinflammatory parameters. Our data show that chronic alcohol intake elevated peripheral and brain alcohol levels, induced serum alanine aminotransferase (ALT), a marker of liver injury, impaired memory and sensorimotor coordination, increased inflammatory gene expression in the hippocampus and prefrontal cortex. Interestingly, serum ALT and hippocampal IL6 correlated with memory impairment, suggesting an intrinsic relationship between neuroinflammation, cognitive decline, and liver disease. Overall, our results point to a likely liver-brain functional partnership and suggest that future strategies to alleviate hepatic and/or neuroinflammatory impacts of chronic AUD may result in improved cognitive outcomes.
Keywords: Alcoholic liver disease, cognition, neuroinflammation, memory, IL6
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INTRODUCTION AUD is a consequence of chronic alcohol abuse affecting over 15 million adults over age 18 in the United States ("National Survey on Drug Use and Health (NSDUH). Substance Abuse and Mental Health Services Administration (SAMHSA)," 2015). Excessive drinking in the US costs over 220 billion dollars annually, including both healthcare costs and associated lost productivity which has been linked to poor quality of life and alcohol-induced cognitive impairments (Hingson & Rehm, 2013). In individuals with AUD, such cognitive alterations may persist up to at least after a year of abstinence (Parsons, 1998; Stavro, Pelletier, & Potvin, 2013), and the magnitude of the deficit is prognostic for relapse in abstinent individuals (Bowden-Jones, McPhillips, Rogers, Hutton, & Joyce, 2005). There remains a critical gap in knowledge regarding the underlying mechanisms promoting chronic alcohol-induced cognitive disturbances. Converging evidence points to plausible mechanistic links between alcohol-induced neuroinflammation and cognition and behavior (Crews et al., 2015; Kelley & Dantzer, 2011; M. Pascual, Baliño, Alfonso-Loeches, Aragón, & Guerri, 2011; Ureña-Peralta, Alfonso-Loeches, Cuesta-Diaz, García-García, & Guerri, 2018). However, much of the research on adverse effects of alcohol associated neuroinflammation has focused on early life exposures to developing brains (Zhang, Wang, Xu, Frank, & Luo, 2018). Taken together, a more comprehensive understanding of the role of neuroinflammatory factors in mediating behavioral phenotypes of AUD is urgently needed. One common adverse long-term consequence of AUD known to involve robust inflammatory mechanisms is alcoholic liver disease (ALD). Both innate and adaptive immune cell types have been implicated in ALD, and inflammatory signaling pathways
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play a critical role in AUD associated liver damage. A number of inflammatory cytokines and chemokines, including tumor necrosis factor alpha (TNFα), interleukin (IL) 6, monocyte chemoattractant protein 1 (MCP1), and IL1β, have been postulated to mediate both ALD pathogenesis and learning, neuroplasticity, and neurochemistry (Donzis & Tronson, 2014; Prieto & Cotman, 2017). To better understand the role of inflammation in cognitive impairments in AUD, we focus on the hippocampus and prefrontal cortex. The hippocampus plays a critical role in memory (Bird & Burgess, 2008), is implicated in alcohol consumption related neural changes (Huttunen & Myers, 1987; Martin-Garcia, Darbra, & Pallares, 2007), and is particularly vulnerable to the deleterious effects of AUD (Hermens et al., 2013; White, Matthews, & Best, 2000; Yaka, Phamluong, & Ron, 2003). Heavy alcohol drinking decreases hippocampal white matter, glial cells, and neurogenesis (Crews & Nixon, 2009; Harding, Wong, Svoboda, Kril, & Halliday, 1997; Korbo, 1999; Richardson et al., 2009), thus it is not surprising that memory is impaired as well (Hermens et al., 2013; White et al., 2000). The prefrontal cortex, critical to executive function and decision making, has also been implicated in the adverse cognitive effects of AUD (Staples & Mandyam, 2016). Structural abnormalities in the prefrontal cortex in individuals that report an AUD include reductions in white matter (Kril, Halliday, Svoboda, & Cartwright, 1997), decreased white matter integrity (Pfefferbaum et al., 1992; Zorlu et al., 2013), and reduced frontal cortex volume (Pfefferbaum et al., 1992). Damage to this brain region is predictive of impairments in executive function (Nakamura-Palacios et al., 2014). Exposure of adolescent rats to a binge paradigm induces long term cognitive
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deficits, increased TNFα and IL1β production, and changes in myelin proteins in the prefrontal cortex (Pascual, Pla, Miñarro, & Guerri, 2014). The objective of the current study was to use the clinically relevant Gao-NIAAA model of ALD, known to induce liver injury, to investigate the role of neuroinflammation in the adverse cognitive impacts of alcohol exposure. We hypothesized that chronic alcohol exposure induces cognitive deficits and neuroinflammation in the hippocampus and prefrontal cortex. We measured alcohol levels in blood and brain tissue, serum ALT, an indicator of alcoholic liver damage, assessed cognitive and sensorimotor effects, and documented the neuroinflammatory changes in the hippocampus and prefrontal cortex.
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METHODS Animals All animals received proper care in agreement with animal protocols approved by the Institutional Animal Use and Care Committee of the University of Massachusetts Medical School. All animals received humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985). 12-week-old female C57BL/6 mice were obtained from Jackson laboratories. Two mice were housed per cage and were maintained in 12 light/12 dark cycle with free access to food and water. Females were used for the study as we have previously demonstrated sexual dimorphism in alcohol-induced inflammation and higher degree of inflammation in the female mice compared to the male mice (Fulham & Mandrekar, 2016).
Alcohol administration Our studies used the established Gao-NIAAA mouse model of ALD (Bertola, Mathews, Ki, Wang, & Gao, 2013) (Fig. 1). Based on this alcohol exposure regimen, female C57BL/6 mice were randomized into alcohol-fed and pair-fed groups and adapted to control Lieber DeCarli liquid diet (Bioserv, Frenchtown, NJ) for 5 days (Ambade et al., 2014). The alcohol group was allowed free access to alcohol-containing diet (36% calories) with increasing alcohol concentrations, 1%-5% for one day each and finally 5% for 10 days. Control mice were given pair-fed diets isocaloric substituted with maltose dextrin. On day 16, all mice were gavaged, either with a single dose of alcohol
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(5 g/kg body weight of 31.5% alcohol), or isocaloric dextrin maltose. Mice were behaviorally tested 7 hours following the gavage and euthanized two hours later for tissue collection.
Novel object discrimination task Rodents normally exhibit increased investigation of unfamiliar objects, and the discrimination with a familiar object serves as an index of memory. Memory was evaluated based on established memory test procedures for object identity and location (adapted from (Bello-Medina, Sanchez-Carrasco, Gonzalez-Ornelas, Jeffery, & Ramirez-Amaya, 2013; DeVito & Eichenbaum, 2010; Inostroza & Born, 2013)). Following three 10-min daily pre-exposure sessions to an open arena (60x50x30 cm), a 5-min familiarization phase was conducted by exposing a mouse to novel object quadruplicates. After an hour delay, the mouse was re-introduced to the arena, where two now-familiar objects were substituted by two novel objects. One of each pair of objects were positioned in a novel location to assess object memory, a) Familiar Identity in a Familiar Location, b) Familiar Identity in a New Location, c) New identity in a Familiar Location, and d) New Identity in a New Location. Blind video-scoring of the time spent investigating each object allowed for the calculation of overall preference ratios for object memory. The discrimination ratio for recognition of the distinct objects was calculated as the difference between the average exploration times for novel objects minus exploration times for familiar objects, divided by the sum of those times. This measure represents the animal's ability to differentiate between two groups of objects, relying on the natural tendency of mice to spend more time exploring more novel
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objects compared to less novel objects. Memory is indicated by a positive ratio, which reflects greater exploration time for novel objects compared to familiar objects.
Sensorimotor coordination Sensorimotor coordination was evaluated seven hours post binge using the static rod test, (Contet, 2012; Deacon, 2013) consisting of a sequential assay using five rods of diameters ranging from 1¼” to ¼”, from wide to narrow. Latencies for the mouse to re-orient 180o away from the edge and to traverse the length of the rod to safety after placement upon the overhanging extremity were recorded (120 s limit/trial), and the sum of the latencies was calculated for each animal.
Magnetic resonance spectroscopy methods to assess hippocampal ethanol levels Mice were anesthetized with 2% isoflurane and constantly monitored for vital signs during the entire time of imaging with a 4.7-T/40-cm horizontal magnet (Oxford) equipped with a Bruker Biospec/Avance console. Experiments for all other imaging were performed using the Oxford 4.7-T/40-cm horizontal magnet equipped with a Bruker Biospec/Avance III HD console. A 1H radiofrequency mouse head coil (Bruker) with inner diameter of 23 mm was used for the experiments. T1-weighted anatomical images were acquired using FLASH sequence with the following parameters: repetition time (TR) = 280.86 ms, echo time (TE) = 4.5 ms, matrix size = 384 × 384, field of view (FOV) = 18 × 18 mm2, slice number = 15, slice thickness = 0.5 mm, flip angle = 40°, and number of averages = 8. T2-weighted images were
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acquired using TurboRARE sequence with TR = 2,200 ms, TE = 36 ms, echo spacing = 12 ms, 8 averages, and rare factor = 8. 1H magnetic resonance spectra data were acquired using single-voxel point-resolved spectroscopy sequence (PRESS) (repetition time = 2,500 ms, echo time = 16 ms, number of averages = 512, voxel size = 3 × 3 × 3 mm). Proton spectra were fit using LC Model (Version 6.2-2B), which analyzed in vivo proton spectra as a linear combination of model in vitro spectra from individual metabolite solutions and generated data as absolute fits (in institutional units) and SD%. SD was used as a measure of the reliability of the fit.
Serum ALT Serum ALT levels were measured using the ALT reagent set (Teco Diagnostics, California, USA), as per the manufacturer’s protocol.
Blood alcohol level Blood alcohol content was measured in serum using the Alcohol Reagent and the AM1 Alcohol Analyzer (Analox Instruments Ltd., London, UK), as per the manufacturer’s protocol.
Brain tissue collection Brains were rapidly extracted, immediately snap frozen using -40oC isopentane (2methylbutane), and then stored at -80oC until further processing for RT-PCR. Tissue micropunches (0.5mm diameter) of the dorsal hippocampus and prefrontal cortex were
10
obtained from slices (0.5mm) cut on a Leica cryostat (CM3050 S), using visible anatomical landmarks.
qRT-PCR methods Dorsal hippocampus and prefrontal cortex samples were homogenized in QIAzol (Qiagen GmbH, Hilden, Germany) and subjected to a chloroform extraction. Total RNA was extracted using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany), according to manufacturer’s instructions. Yield of RNA was measured using Nano Drop 2000 (Thermo Scientific, Wilmington, DE). cDNA was synthesized using the Reverse Transcription System (Promega, Madison, WI). mRNA levels of genes were quantified using iTAQ Universal SYBR Green Supermix and CFX Connect Real-Time PCR Detection System (Biorad Laboratories, Hercules, CA). 18s ribosomal RNA served as the housekeeping control. Relative gene expression was analyzed by the 2–∆∆Ct method. Primers were synthesized by IDT, Inc. (Coralville, IA). Primer sequences used for this study are enlisted in table 1.
Statistics Data are represented as mean ± SEM. Statistical significance was determined by the Student t-test or Pearson’s Correlation, using Graphpad Prism 7.04, and denoted as p≤0.05.
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RESULTS Alcohol in serum and hippocampal tissue The Gao-NIAAA model of alcohol administration resulted in significantly elevated blood alcohol levels (BAC) in serum (82.6 ± 4.6 (ethanol-fed) vs. 1.5 ± 0.4 (pair-fed), t=15.83, p<0.001, df=9) and hippocampal tissue (12.5 ± 1.6 (ethanol-fed) vs. 0 (pairfed), t=7.813, p<0.001, df=8), as well as high serum ALT (158.24 ± 35 (ethanol-fed) vs. 77.2 ± 0.78 (pair-fed), t=1.96, p<0.01, df=5), a marker of liver injury confirming chronic alcohol exposure.
Object Memory and Sensorimotor Coordination Chronic ethanol exposure impaired memory in the ethanol-fed group compared to the pair-fed group (p<0.05, t=3.28, df=8, Fig. 2). Although there was no difference in completion of the static rod test between the groups, ethanol-fed mice had longer total latencies to traverse the rods (336.3 ± 141.8 (ethanol-fed) vs. 22.1 ± 4.3 (pair-fed) seconds, t=2.89, p<0.01, df=8), indicating impaired sensorimotor functioning.
Neuroinflammation To determine whether pro-inflammatory cytokine alterations were associated with cognitive impairments, cytokine gene expression was measured in the dorsal hippocampus and prefrontal cortex. Levels of Tnfα, Il1β, Mcp1, and Il6 mRNA were elevated in the dorsal hippocampus of ethanol-fed mice compared to pair-fed controls (all p’s <0.05, Fig. 3A-D), and levels of Tnfα and Il1β were also elevated in the prefrontal
12
cortex (all p’s < 0.05 Fig. 3A, B) indicating differential inflammation in these two brain regions.
Correlation Analysis Next, we analyzed whether alterations in memory correlated with any cytokine gene expression and/or liver injury. Interestingly, memory is negatively correlated with serum ALT in both groups combined (r = -0.8254 p=0.0033, Table II). It is also negatively correlated with Il6 levels in the hippocampus in both groups combined (r = 0.9021, p = 0.0022, Table II). Furthermore, Il6 and ALT are positively correlated (r= 0.9759, p<0.0001, Table II). There were no other significant memory-cytokine mRNA correlations, and no correlations between ALT or cytokines and sensorimotor coordination.
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Discussion Using the Gao-NIAAA binge model of alcohol administration, which effectively increased serum and hippocampal ethanol levels and serum ALT, we report memory deficits, sensorimotor impairment, and increased neuroinflammation in ethanol-fed mice compared to pair-fed controls. Expression levels of several ALD-associated inflammatory factors were increased in both the hippocampus and the prefrontal cortex, regions critical for memory (Preston & Eichenbaum, 2013) and implicated in sensorimotor control (Bast & Feldon, 2003; Hyder et al., 1997; Siegel, Buschman, & Miller, 2015). Memory was negatively associated with both peripheral ALT levels and hippocampal Il6 mRNA, and these factors were positively correlated. Taken together, the results indicate that the Gao-NIAAA binge model is a valuable tool to investigate relationships between the adverse hepatic, neuroinflammatory, cognitive, and sensorimotor impacts of chronic alcohol, topics of growing concern in efforts to comprehend the role of the brain-liver axis in alcohol mediated cognitive disease etiology. Previous alcohol-induced studies of neural effects have primarily used models of repeated binge exposures (Majchrowicz 4-day binge paradigm) (Faingold, 2008) or alcohol binges followed by abstinence (Ward et al., 2009). The current study augments this prior work with the use of the clinically relevant chronic Gao-NIAAA binge model which effectively and substantially increased alcohol levels in serum and brain tissue and elevated ALT, a primary marker of liver damage. The present investigation focused on memory to align with a major clinical presentation of AUD and to address the paucity of data linking alcohol-induced hippocampal alterations and cognitive deficits (Obernier, White, Swartzwelder, & Crews,
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2002; Vedder, Hall, Jabrouin, & Savage, 2015). Chronic administration studies in mice report that an 8 week consumption paradigm produces long-term memory deficits (Farr, Scherrer, Banks, Flood, & Morley, 2005). Long-term moderate oral self-administration of alcohol in rats adversely affects memory, and it is postulated that this accelerates aging induced cognitive decline (Baird et al., 2006). Memory is also impaired with acute treatment, and similarities between alcohol induced deficits and those induced by hippocampal lesions supports a central role of the hippocampus in alcohol-associated cognitive impairments (Hoffmann & Matthews, 2006; Van Skike, Goodlett, & Matthews, 2019). In our study using the Gao-NIAAA model, cognitive and neuroinflammatory parameters are linked to liver injury and indicate that the brain liver axis and may be crucial in the etiology of cognitive decline and/or could serve as a biomarker of increased risk. Elevated ALT may indicate increased likelihood of adverse neuroinflammatory changes, such as the reported increases in mRNA levels of inflammatory factors in the hippocampus and prefrontal cortex. While the specific literature on neuroinflammation in ALD is limited, it is known that liver failure in general is strongly associated with neuroinflammation (Butterworth, 2013, 2015). Several of the cytokines altered in the current study are implicated in acute liver failure and induce neuroinflammation (TNFα, IL6, IL1β) (Butterworth, 2013). Peripheral inflammation is associated with cognitive and motor deficits with hepatic encephalopathy, and this is postulated to be mediated by neuroinflammation (Felipo et al., 2012; Shawcross, Davies, Williams, & Jalan, 2004). The strong negative associations between memory, serum ALT, and Il6 expression in the hippocampus, combined with the positive association between ALT and
15
hippocampal Il6, suggests that ALT may be a valuable biomarker for ALD induced neuroinflammation and associated cognitive impairments. Similar to the current results, Tnfα mRNA levels are increased in the hippocampus of rats exposed to a 10-week alcohol regimen that adversely affects learning and memory (Tiwari, Kuhad, & Chopra, 2009). While inhibition of TNF synthesis can reverse hippocampal-dependent learning in a rodent model of chronic inflammation (Belarbi et al., 2012), intermittent ethanol exposure (simulated binge drinking) acutely increases hippocampal levels of both TNFα and IL1β and adversely impacts learning and memory (Zhao et al., 2013). Exogenous IL1β also inhibits the acquisition of learning, where the neutralization of this cytokine prevents Legionella infection induced cognitive impairment (Gibertini, Newton, Friedman, & Klein, 1995). Hippocampal IL1β, IL6, along with TNF, have been implicated in metabolic syndrome induced impairments in memory (Dinel et al., 2011), and MCP1 levels are elevated in the brain tissue of mice exposed to a high fat diet (Pistell et al., 2010), supporting the hypothesis that the adverse cognitive effects of metabolic changes and chronic alcohol exposure are both mediated by similar neuroinflammatory mechanisms. Given the specific association between memory and hippocampal Il6 mRNA in the current study, this cytokine could play a particularly potent role in the adverse cognitive effects of chronic alcohol exposure. This cytokine has been specifically implicated in neuroinflammatory mediated neurodegenerative diseases, including Alzheimer’s disease (Hull, Strauss, Berger, Volk, & Bauer, 1996), where β-amyloid peptide potentiates IL6 production from cells in culture (Sondag, Dhawan, & Combs, 2009). Further in vitro work indicates that IL6 potentiates β-amyloid induced
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neurotoxicity (Qiu & Gruol, 2003), and it may have common neurodegenerative actions in a range of pathological states, including AUD. Taken in conjunction with the present findings, it appears that a variety of insults impair cognition through inflammatory effects in the hippocampus, and research on AUD associated cognitive impairment can benefit from leveraging this literature to accelerate the identification and development of novel hippocampus immune-targeted preventative measures and treatments. The evidence for a role of the prefrontal cortex in inflammation mediated cognitive impairment is not as robust when compared to the findings from the hippocampus. In accordance with this observation, there were no significant cognition/memory cytokine correlations in the prefrontal cortex in the current study. Although others have shown that adult alcohol exposure in rats impairs recognition memory and increases IL6 levels in prefrontal cortex, (Terasaki & Schwarz, 2017) prefrontal cortex IL6 expression was not elevated in the present study. This could be, due to species differences in the response to alcohol and/or differences in alcohol exposure duration. Intermittent binge paradigms in rats induce learning, sensorimotor, and recognition deficits and increase cell death in the neocortex (M. Pascual, Blanco, Cauli, Minarro, & Guerri, 2007), but this work was in an adolescent rodent model, as are many of the animal studies of alcohol exposure. Changes in prefrontal cortex related neural connectivity have also been implicated in inflammatory pain related deficits in working memory (Cardoso-Cruz, Sousa, Vieira, Lima, & Galhardo, 2013), which is consistent with earlier studies of memory related functions in this region (Jung, Qin, McNaughton, & Barnes, 1998; Wang & Cai, 2006). The potential role of the prefrontal cortex in the
17
present cognitive deficit is likely indirect and dependent on its structural and functional connectivity to the hippocampus. The cytokine results herein support additional investigation of the roles of prefrontal cortex TNFα and IL1β in AUD related cognitive deficits. While the NIAAA alcohol feeding paradigm did impair sensorimotor coordination along with memory, the dexterity required for the sensorimotor static rod task was greater than that necessary for the memory task in an open field, therefore it is unlikely that it contributed to the adverse effects on memory in a meaningful way. It is possible that the present effects of alcohol in the sensorimotor task may involve anxiety or ataxia related mechanisms (Gallate, Morley, Ambermoon, & McGregor, 2003), however, since we did not specifically assess either, we cannot be certain which of these mechanisms is at play. Progressive alcohol intake in humans impairs cognitive and motor processes, and it is postulated that central (such as neuroinflammatory) and peripheral mechanisms are involved in these effects (De Wilde, Dom, Hulstijn, & Sabbe, 2007). Although it is possible that we would have observed cognitive deficits without sensorimotor impairments with an extended period of abstinence, gait and behavior deficits are present following one year of abstinence in humans (Fein & Greenstein, 2013; Smith & Fein, 2011). Furthermore, both cognitive and sensorimotor deficits have been documented in detoxified alcoholic men, and the specific deficits implicate the prefrontal cortex in some of these impairments (Sullivan, Rosenbloom, & Pfefferbaum, 2000). Chronic ethanol exposure induces motor impairments in other rodent models, and these effects are also associated with cortical damage (da Silva et al., 2018; Teixeira et al., 2014), further supporting the use of animal models in studying AUD.
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Given the substantial increased risk of falling due to alcohol intake, especially with higher intake levels (Chen & Yoon, 2017), research aimed at understanding mechanisms to target for reversing alcohol use induced motor impairments, along with cognitive deficits, is warranted. The present data support our hypothesis that that the Gao-NIAAA model of ALD is a valuable tool to investigate the adverse cognitive and neuroinflammatory effects of AUD. Increased focus on the contributions of liver damage to the progression of alcohol associated cognitive deficits may generate novel etiological findings. An enhanced understanding of the role of the liver and the timing of the inflammatory/neurotrophic cascade induced by AUD, with a specific focus on plasma ALT and hippocampal IL6, will provide a strong foundation for the development of effective preventative measures and treatments that are likely to be pertinent to both AUD associated cognitive deficits and ALD etiology.
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Acknowledgements The authors would like to thank the UMass Medical School Department of Animal Medicine for outstanding animal care and cooperative support of the rodent experiments.
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Legends Figure 1: Study design. Mice drank 5% alcohol for 10 days after habituation to liquid diet and 5 days of progression to the final concentration. A 5g/kg binge gavage was administered on day 16. Figure 2: Impaired memory in ethanol fed mice. Mean ± SEM memory of ethanoland pair-fed mice, t=3.28, p<0.05, n=6. Figure 3: Neuroinflammation in ethanol fed mice. Mean ± SEM mRNA levels of Tnfα (t=4.914, df=6; dorsal hippocampus, t=3.53, df=8; prefrontal cortex) (A), Il1β (t=3.431, df=6; dorsal hippocampus, t=3.782, df=7; prefrontal cortex) (B), Mcp1 (t=2.864, df=8; dorsal hippocampus) (C), and Il6 (t=4.47, df=8; dorsal hippocampus) (D) in the dorsal hippocampus and prefrontal cortex of ethanol and pair-fed rats. * denotes p<0.05, n=6, ** denotes p<0.01, n=6.
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Table 1: Primer sequences used for real time PCR Gene
Forward
Reverse
18s
GTA ACC CGT TGA ACC CCA TT
CCA TCC AAT CGG TAG TAG CG
Il-1β
CAG GCA GGC AGT ATC ACT CA
TGT CCT CAT CCT GGA AGG TC
Il-6
ACA ACC ACG GCC TTC CCT ACT T
CAC GAT TTC CCA GAG AAC ATG TG
Mcp-1 CAG GTC CCT GTC ATG CTT CT
TCT GGA CCC ATT CCT TCT TG
Tnf-α
GTG AGG GTC TGG GCC ATA GA
GAA GTT CCC AAA TGG CCT CC
Table 2: Correlation analysis
Parameter I Parameter II
r
R2
p value
ALT
Novel Spatial preference
-0.8254
0.6813
0.0033
HPC Il-6
Novel Spatial preference
-0.9021
0.8137
0.0022
HPC Il-6
ALT
0.9759
0.9523
<0.0001