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. Author manuscript; available in PMC: 2025 Mar 1.
Published in final edited form as: Alcohol Clin Exp Res (Hoboken). 2024 Feb 20;48(3):478–487. doi: 10.1111/acer.15273

Histone deacetylase inhibitor decreases hyperalgesia in a mouse model of alcohol withdrawal-induced hyperalgesia

Jhoan Aguilar 1, Luana Martins De Carvalho 1, Hu Chen 1, Ryan Condon 1, Amy W Lasek 1,§, Amynah A Pradhan 1,§
PMCID: PMC10940188  NIHMSID: NIHMS1961146  PMID: 38378262

Abstract

Background:

Alcohol withdrawal induced hyperalgesia (AWH) is characterized as an increased pain sensitivity observed after cessation of chronic alcohol use. AWH can contribute to the negative affective state associated with abstinence and can increase susceptibility to relapse. The aim of this study was to characterize pain sensitivity in mice during withdrawal from two different models of alcohol exposure, chronic drinking in the dark (DID) and the Lieber-DeCarli liquid diet. We also investigated whether treatment with a histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), could ameliorate AWH in mice treated with the Lieber-DeCarli diet.

Methods:

Male and female C57BL/6J mice were used for these studies. In the DID model, mice received bottles of 20% ethanol or water during the dark cycle for 4 h per day for 4 consecutive days per week for six weeks. Peripheral mechanical sensitivity was measured weekly the morning of day five using von Frey filaments. In the Lieber-DeCarli model, mice received ethanol (5% v/v) or control liquid diet for 10 days, along with a single binge ethanol gavage (5 g/kg) or control gavage, respectively, on day 10. Peripheral mechanical sensitivity was measured during the liquid diet administration and at 24 and 72 hours into ethanol withdrawal. An independent group of mice that received the Lieber-DeCarli diet were administered SAHA (50 mg/kg, i.p.) during withdrawal.

Results:

Male mice exhibited mechanical hypersensitivity after consuming ethanol for 5 weeks in the DID procedure. In the Lieber-DeCarli model, ethanol withdrawal led to hyperalgesia in both sexes. SAHA treatment during withdrawal from the ethanol liquid diet alleviated AWH.

Conclusions:

These results demonstrate AWH in mice after chronic binge drinking in males and after Lieber-DeCarli liquid diet administration in both sexes. Like previous findings in rats, HDAC inhibition reduced AWH in mice, suggesting involvement of epigenetic mechanisms in AWH.

Keywords: allodynia, binge drinking, alcohol withdrawal, pain, epigenetic

Introduction

Alcohol use disorder (AUD) is highly prevalent and characterized by the inability to limit ethanol consumption despite adverse consequences (Cargiulo, 2007). In AUD patients, cessation of drinking induces acute physiological and psychological symptoms, as absence of the depressant effects of alcohol can cause a hyperactive state in the central nervous system (Heilig et al., 2010). Manifestations of alcohol withdrawal include seizures, nausea, tremors and mild pyrexia which subside 40–50 hours after the last alcohol consumption (Heilig et al., 2010). These physiological effects are often accompanied by negative emotional states such as increased anxiety and depressive-like symptoms that can continue long after the initial withdrawal period (Sullivan et al., 1989). Clinically, another feature of alcohol withdrawal is increased sensitivity to pain, also known as alcohol withdrawal-induced hyperalgesia (AWH), which can last for weeks into the abstinence period. The presence of AWH and other withdrawal symptoms contribute to a negative affective state that can ultimately lead to alcohol relapse. Further, persistent pain is also associated with AUD severity (Trafton et al., 2004, Caldeiro et al., 2008), and because alcohol can be analgesic it reinforces continued alcohol intake which can lead to alcohol dependence and associated neuroadaptations (Egli et al., 2012).

Similar to other neuropsychiatric conditions, preclinical AUD models are used to investigate a combination of behavioral, physiological, and neurobiological aspects of alcohol use and misuse (Goltseker et al., 2019). Preclinical work on AWH has been primarily performed in rodent models of alcohol exposure. An initial study on AWH involved the prolonged administration of a 6.5% ethanol solution to male Long-Evans rats over a period of 10 days. Hyperalgesia was observed during the initial 6 and 12 hours of withdrawal but was not present at the 36-hour mark (Gatch and Lal, 1999). Subsequent studies have utilized various alcohol intake protocols in rats, including two-bottle choice and ethanol vapor exposure, to induce hyperalgesia during withdrawal. These models have revealed molecular markers and neurological signaling mechanisms involved in the development of AWH, such as modulation of protein kinase C and regulation of the endogenous corticotropin releasing hormone system (Dina et al., 2000, Edwards et al., 2012). Withdrawal from chronic ethanol exposure can also induce epigenetic changes, altering the expression of genes that promote withdrawal symptoms (Pandey et al., 2008, Pandey et al., 2015, You et al., 2014). The Lieber-DeCarli liquid diet model has been used to investigate withdrawal symptomology in rats, where increased anxiety and depressive-like behaviors observed in withdrawal appear to be linked to aberrant acetylation dynamics in the brain (Chen et al., 2019). We also previously observed AWH in rats using the Lieber-DeCarli diet (Pradhan et al. 2019). In this procedure, increased mechanical hypersensitivity was observed at 24 and 72h of withdrawal. Furthermore, the pan-histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) was able to relieve acute hyperalgesia at these time points, revealing a novel role of epigenetic regulation in AWH pathogenesis (Pradhan et al., 2019).

To date, the majority of AWH studies performed in mice have utilized a range of ethanol exposure models, such as chronic intermittent ethanol vapor (CIE), intermittent access two bottle choice (IA2BC), and drinking in the dark (DID) (Bergeson et al., 2016, Lopez et al., 2023, Quadir et al., 2021a, Quadir et al., 2021b, Smith et al., 2016, Smith et al., 2017, Okhuarobo et al., 2020, Morgan et al., 2022, Okhuarobo et al., 2023, Tonetto et al., 2023). However, to the best of our knowledge no one has reported AWH in mice using the Lieber-DeCarli model. One of the goals of this study was to determine if mice develop AWH after Lieber-DeCarli ethanol liquid diet and to compare it to chronic binge-like drinking in the DID procedure. In addition, we also used male and female mice, and tested the effect of SAHA in our model, to determine if sex differences and/or epigenetic mechanisms play a role in mouse AWH.

Materials and Methods

Subjects

Male and female C57BL/6J mice were used in this study and were purchased from the Jackson Laboratory at 8 weeks of age. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Illinois at Chicago and followed the NIH Guide for the Care and Use of Laboratory Animals. Mice were housed in a temperature-controlled room with a 12-hour light/dark cycle, with lights on at 6:00 AM and off at 6:00 PM. Mice utilized for the DID paradigm were housed in a reversed light cycle with lights on at 10 AM and off at 10 PM and acclimated for two weeks in the reversed light cycle room prior to DID. Mice were individually housed, except where specified below. Food and water were available ad libitum unless otherwise specified. Mechanical thresholds were tested between 10AM-1PM for the liquid diet model and 9–10AM in the drinking in the dark model.

DID

This procedure was used for the experiment described in Fig. 1. Beginning at 10 weeks of age, mice were given access to a single sipper tube of 20% ethanol (v/v in water) or water (as a control) for 4 h per day, 4 consecutive days per week (Monday through Thursday), starting 3 h into the dark cycle (Rhodes et al., 2005). The procedure was conducted for 6 weeks, with 4 days of DID and 3 days without ethanol access (Friday through Sunday) per week. Volumes of fluid consumed after each 4 h DID session were recorded, and amounts consumed were calculated as g ethanol consumed per kg body weight.

Figure 1.

Figure 1.

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Alcohol withdrawal-induced hyperalgesia (AWH) develops in male mice after 5 cycles of DID. A. Experimental design for DID experiment. Mice were given either water or 20% ethanol for 4 h per day for 4 days per week. On the 5th day, mice were tested for mechanical sensitivity using mechanical von Frey hair stimulation. This protocol was repeated every week for 6 weeks. B. Average weekly ethanol consumption during 4 h sessions (g/kg/4h) of male and female mice over six weeks of DID. Female mice consistently consumed more ethanol than males in weeks 5 and 6 (two-way ANOVA: effect of time, F(5, 108) = 18.89, p < 0.0001; effect of sex, F(1,22) = 11.52, p = 0.0026; time X sex interaction, F(5,108) = 2.506, p = 0.0346; **p<0.01, ***p<0.001 when comparing males and females by post-hoc Holm-Sidak multiple comparisons test). C. Average water consumption of control mice (in ml) over six weeks (two-way RM ANOVA: effect of time, F(5, 110) = 5.613, p = 0.0001; effect of sex, F (1, 22) = 0.1196, p = 0.7328; time x sex interaction F (5, 110) = 0.3773, p = 0.8638). D. Mechanical responses of ethanol- and water-treated female mice (n = 12 mice/ group) did not differ during the 6 cycles of DID (two-way mixed-effects ANOVA, effect of time, F (5, 57) = 0.5442, p = 0.7420; effect of treatment, F (1, 57) = 0.4162, p = 0.5214; time x treatment interaction, F (5, 57) = 0.9003, p = 0.4873). E. Mechanical responses of ethanol- and water-treated male mice (n = 12 mice/ group) did not differ during weeks 1– 4, but hyperalgesia was observed in weeks 5 and 6 of DID (two-way RM ANOVA: effect of time, F (5, 50) = 0.5170, p = 0.7622; effect of treatment F (1,10) = 3.133, p = 0.1072; time x treatment interaction, F (5, 50) = 3.431, p = 0.0096; *p = 0.0345 for control vs. ethanol in week 5, #p = 0.0598 for control vs. ethanol in week 6 by post-hoc Holm-Sidak multiple comparisons test).

Chronic Ethanol + Binge Exposure

This procedure was only used for the experiment described in Fig. 2. For the chronic ethanol + binge model, the NIAAA model (Bertola et al., 2013) was implemented to test for AWH. Briefly, adult C57BL/6J male and female mice were same-sex pair housed after being acclimated to the vivarium for three days. All mice were then given control liquid diet (Lieber-DeCarli Diet 82; Bio-Serv F1259SP, Frenchtown, NJ) for five days (“acclimation phase”). After acclimation, mice were separated into control and ethanol groups for the “control” or “ethanol” phase; the control group received the control diet for 10 days while the ethanol group was given 5% ethanol diet (Lieber-DeCarli Diet 82; Bio-Serv F1258SP, Frenchtown, NJ) for 10 days. At the end of the 10 days, ethanol-treated mice received a 5 g/kg oral gavage of ethanol, while control mice received an isocaloric maltodextrin gavage, as described in the NIAAA protocol (Bertola et al., 2013). At this point, ethanol-treated mice received control diet for the rest of the experiment to induce withdrawal. Fresh diet was supplied daily 3–4 hours before the beginning of the dark cycle.

Figure 2.

Figure 2.

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Withdrawal from chronic liquid diet + binge produces AWH. A. Experimental design. Mice were acclimated to control liquid diet for 5 days prior to being separated into control or ethanol diet groups. Following 10 days of control or ethanol diet, the mice were given a control maltodextrin gavage or 5 g/kg ethanol gavage respectively, at which time the ethanol diet was replaced with control diet. Both groups then received 10 days of control diet in the withdrawal phase. Mechanical sensitivity was evaluated periodically during liquid diet administration. B. Mechanical responses of control and ethanol groups did not differ throughout the control diet acclimation and control or ethanol liquid diet phase of the experiment (n=10 per group, two-way RM ANOVA: effect of time, F (5, 90) = 0.7838, p = 0.5648; effect of treatment, F (1,18) = 1.519, p = 0.2236; interaction, F (5,90) = 0.2071, p = 9587). C. Ethanol withdrawal produced peripheral hyperalgesia that was present at 24 and 72 hours into ethanol withdrawal compared to the control diet fed group (n=10 per group, two-way RM ANOVA: effect of time, F (1,18) = 0.2865, p = 0.5990; effect of treatment, F (1,18) = 22.82, p = 0.0002; interaction, F (1,18) = 0.0948, p = 0.0948). D. Blood alcohol concentration (BAC) of 6 male and 6 female mice in the ethanol liquid diet group taken from a separate batch of animals on day 7; t-test p=0.2246.

Chronic Ethanol Exposure

This protocol was used in Fig. 3. To assess the necessity of the oral gavage ethanol “binge” to induce hyperalgesia in withdrawal, a separate experiment was performed where singly housed animals underwent the same chronic liquid diet as described above. In this experiment, however, at the end of the control/ethanol phase, the ethanol group was divided into two cohorts and one group received an ethanol oral gavage (5 g/kg) while the other received isocaloric maltodextrin oral gavage. The control group also received the isocaloric maltodextrin gavage. Following gavage, all groups received control diet for the rest of the experiment. In subsequent experiments (Fig. 4), mice were singly housed and did not receive gavage, but were otherwise treated as described above.

Figure 3.

Figure 3.

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Withdrawal from chronic liquid diet alone produces AWH. A. Experimental design. Mice were acclimated to the liquid diet for 5 days prior to separation into control or ethanol diet groups. To assess the necessity of the ethanol gavage to induce AWH, following 10 days of control or ethanol diet, the ethanol mice were separated into two groups; one that received control maltodextrin gavage and the other group was given a 5 g/kg ethanol gavage. The control diet group was given a control maltodextrin gavage. All groups then received 10 days of control diet during the withdrawal phase. Mechanical sensitivity was evaluated periodically during the liquid diet administration. B. Blood alcohol concentration (BAC) of 6 male and 5 female mice in the ethanol liquid diet group was assessed on day 7. C. Mechanical responses of control and both ethanol groups did not differ throughout the control diet acclimation and control or ethanol liquid diet phases of the experiment (n=10 mice per group, two-way RM ANOVA: effect of time, F (6, 63) = 0.6900, p = 0.5615; effect of time, F (2, 21) = 0.07493, p = 0.9281; interaction, F (6, 63) = 0.6750, p = 0.6702). D. Ethanol withdrawal produced peripheral hyperalgesia that was present at 24 and 72 hours into ethanol withdrawal compared to the control diet group independent of gavage treatment (n=8 mice per group, two-way RM ANOVA: effect of time, F (1.974, 41.46) = 3.070, p = 0.0577; effect of treatment, F (2, 21) = 7.342, p = 0.0038; interaction F 4,42) = 0.6072, p = 0.6597; **p<0.01 significant effect of treatment.

Figure 4.

Figure 4.

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SAHA, a pan-histone deacetylase (HDAC) inhibitor, alleviates acute AWH. A. Experimental design. Mice were acclimated to the liquid diet for 5 days prior to being separated into control or ethanol diet groups. After 10 days of control or ethanol diet, the ethanol liquid diet was replaced with control liquid diet. At 24 (day 16), 48 (day 17), and 72 h (day 18) into withdrawal, mice were given a SAHA injection (50 mg/kg, i.p.). B. Depiction of times when mechanical testing was performed at 24 h and 72 h into withdrawal. Mice were first tested for baseline mechanical sensitivity prior to SAHA injection. Two hours after SAHA injection, mice were again tested for mechanical sensitivity. C. A single dose of SAHA (50 mg/kg IP) 24 h into withdrawal was sufficient to increase the mechanical thresholds of mice administered ethanol liquid diet that were experiencing hyperalgesia (n=6 males and 6 females per group, three-way RM ANOVA: effect of time, F (1, 44) = 8.124, p = 0.0066; effect of liquid diet, F(1, 44) = 168.5, p < 0.0001; effect of drug treatment, F(1, 44) = 7.434, p = 0.0092; time x liquid diet, F(1,44) = 4.144, p = 0.0478; time x drug, F(1, 44) = 10.92, p = 0.0019; liquid diet x drug, F(1, 44) = 14.12, p = 0.0005; time x liquid diet x drug interaction, F (1, 44) = 8.634, p = 0.0052; ****p < 0.0001, when comparing ethanol-SAHA vs. ethanol-vehicle by post hoc Holm-Sidak multiple comparisons testing. D. Two prior doses of SAHA sustained the increase in mechanical thresholds at 72 h into withdrawal; an additional dose maintained this effect (n=6 males and 6 females per group for control-vehicle, control-SAHA, ethanol-vehicle groups. Two mice died before the third day of SAHA administration, leaving an n= 6 males and 4 females for the ethanol -SAHA group; three-way RM ANOVA: effect of time, F (1, 42) = 0.0052, p = 0.9426; effect of liquid diet, F(1, 42) = 5.189, p = 0.0279; effect of drug, F(1, 42) = 4.182, p = 0.0472; time x liquid diet, F(1, 42) = 0.6771, p = 0.4152; time x drug, F(1, 42) = 0.8213, p = 0.3700; liquid diet x drug, F(1, 42) = 11.00, p = 0.0019; time x liquid diet x drug interaction, F (1, 42) = 0.4198, p = 0.5206; *p<0.05, ethanol-vehicle vs. ethanol-SAHA treated animals at baseline and post-SAHA timepoints.

Blood Alcohol Concentration (BAC) Measurements

Seven days into the control/ethanol diet phase of the Lieber DeCarli model, 20 μL of blood was collected within 1 hour of lights on (~7:00 am) from the lateral tail vein of a subgroup of ethanol-treated mice with heparinized capillary tubes. The serum was extracted with 3.4% perchloric acid, centrifuged to remove precipitates, and then the supernatant was assayed for ethanol concentration using an alcohol dehydrogenase (ADH) assay and measuring conversion of β-NAD to NADH at 340 nm in a spectrophotometer (Zapata et al., 2006). BACs were determined using a calibration curve.

Mechanical Sensitivity Assessment

Mechanical sensitivity was determined using manual von Frey hair stimulation of the hindpaw. For all behavioral experiments, mice were counterbalanced into groups following the first basal test for mechanical sensitivity. The experimenter was blinded to the drug/condition being tested. No adverse effects were observed in any of the experiments. All mice were tested in a separate behavior room with low-light (~35–50 lux) and low-noise conditions, between 09:00 and 16:00. For all behavioral tests, mice were habituated to the testing rack for 2 days prior to the first test day, and on each test day for 30 minutes prior to the first measurement. During the habituation days, a medium force von Frey filament was used to accustom the mice to the filaments. To assess mechanical sensitivity, the threshold for responses to punctate mechanical stimuli (mechanical allodynia) was tested according to the up-and-down method. Although allodynia was measured we refer to the mechanical hypersensitivity as hyperalgesia to stay consistent with the AWH terminology. The plantar surface of the left hindpaw was stimulated with a series of eight von Frey filaments (bending force ranging from 0.001g to 2g). A response was defined as a lifting, shaking, or licking of the hind paw. The first filament tested was 0.4g. In the absence of a response, a heavier filament (up) was tried, and in the presence of a response, a lighter filament (down) was tested. This pattern was followed for a maximum of four filaments following the first response. For the DID paradigm, mechanical sensitivity was tested each week on the morning of day 5 (Friday) one hour prior to the beginning of the dark cycle (16 hours after the DID session). For the Lieber-DeCarli model, mechanical assessments were performed periodically 4–7 hours into the light cycle during the diet acclimation and control/ethanol diet phase to investigate if ethanol or liquid diet alters mechanical thresholds. Mice were also evaluated at multiple time points following withdrawal, including 24h, 72h, and up to 7 days (see Fig 2 and 3 for exact days).

SAHA Treatment

To determine if HDAC inhibition can influence the development of AWH, SAHA (50 mg/kg, Selleck Chemicals, Houston, TX) or vehicle (2% dimethyl sulfoxide, 40% polyethylene glycol-300, 5% propylene glycol, 1% polysorbate 80 in 0.9% NaCl) was administered by intraperitoneal (i.p.) injection at 24-, 48- and 72-hours post-withdrawal. Mice were separated into four groups: control diet with vehicle injection, control diet with SAHA injection, ethanol diet in withdrawal with vehicle injection, and ethanol diet in withdrawal with SAHA injection. During the days in which mechanical thresholds were evaluated, 24- and 72-hours post-withdrawal, baseline thresholds were calculated the morning before SAHA injections. After SAHA injections, the mice were returned to their cages for 2 hours, at which point post-injection thresholds were taken to assess the acute effect of SAHA on mechanical responses.

Statistical Analysis

The data are reported as mean ± SEM. Statistical analyses were conducted using GraphPad Prism software 9 (GraphPad, San Diego, CA). Data in Figs. 1B & D were analyzed by two-way mixed effects ANOVA. Mixed effects ANOVA was used because one female ethanol-drinking mouse died during week 5. Behavioral data in Figures 1, 2 and 3 were analyzed with two-way ANOVA, while behavioral data in Figure 4 was analyzed by three-way ANOVA. Post hoc Holm-Sidak multiple comparisons testing was performed if there was a significant interaction by ANOVA. BAC data were analyzed by student’s t-test. The level of significance (α) for all tests was set to p < 0.05.

Results

DID induces AWH in male mice

We utilized DID, an established binge-like ethanol drinking model, to determine if acute AWH would develop in a drinking paradigm that does not induce physical symptoms of alcohol withdrawal. Twelve male and 12 female mice were given access to a sipper tube of 20% ethanol or water for 4 hours a day for 4 consecutive days per week (Monday through Thursday) for 6 weeks (Fig. 1A). Mice consumed increasing amounts of ethanol over the 6 weeks. Female mice drank significantly more ethanol than male mice in weeks 5 and 6 (Fig. 1B). Control mice that were only given water in sipper tubes during the DID procedure consumed increasing amounts of water each week, but there were no sex differences (Fig 1C). Mechanical responses were determined using von Frey hair stimulation of the hindpaw every Friday morning, 16 hours after the end of the Thursday drinking session, over the course of 6 weeks. There were no significant differences in mechanical responses observed between female mice treated with ethanol or water (Fig 1D). In contrast, male mice consuming ethanol showed a significant decrease in mechanical responses (hyperalgesia) on weeks 5 and 6 compared to water-consuming controls (Fig 1E). These findings indicate that AWH is observed in a sex specific manner after prolonged bouts of binge-like ethanol consumption.

Development of AWH during withdrawal from chronic ethanol plus binge exposure (NIAAA model)

We employed the NIAAA model, referred to as the chronic ethanol + binge procedure (Bertola et al., 2013), to investigate the development of hyperalgesia during withdrawal from chronic ethanol exposure in a group of 6 male and 4 female mice that received control liquid diet and 4 male and 6 female mice that received ethanol liquid diet (Fig. 2A). For this experiment mice were housed 2 same sex animals per cage. Naïve mechanical responses were assessed on day 1 prior to initiating the ethanol diet. Throughout the acclimation and control or ethanol diet phases, mechanical responses were similar to the initial/naïve levels (Fig. 2B). On day 15, mice were administered a single gavage dose of maltodextrin or 5% ethanol, after which all mice received the control diet (withdrawal phase). During the withdrawal period, there was a significant reduction in mechanical thresholds in mice fed the ethanol versus control diet, observed at 24- and 72-hours withdrawal (Fig. 2C). No sex differences were observed in alcohol withdrawal-induced hyperalgesia (AWH), so the data were combined across sexes. To ensure that animals were drinking sufficient and relevant quantities of ethanol, in a separate group of mice, the average blood alcohol concentration (BAC) was measured. Female BACs were 193.2 ± 26.72 mg/dL and males BACs were 120.5 ± 49.35 mg/dL. There was no significant difference between the sexes (Fig. 2D, Student’s t-test, p=0.2246). These findings show that the chronic ethanol + binge procedure can induce AWH in mice.

Chronic ethanol administration, without the binge, is sufficient to induce AWH

We next wanted to determine the necessity of binge ethanol treatment at the conclusion of the chronic ethanol diet to induce AWH. A separate experiment was conducted in which 5 single-housed males and 5 single-housed females per group were given either ethanol or control liquid diet. Before ethanol withdrawal, one group received an ethanol gavage while the other groups received an isocaloric gavage of maltodextrin (Fig. 3A). The average BACs during the ethanol liquid diet phase were 187.1 ± 15.51 mg/dL for females and 185.7 ± 16.88 mg/dL for males (Fig. 3B). Similar to the previous experiment, no significant differences in mechanical thresholds were observed between the control and ethanol diet groups during the acclimation and liquid diet periods (Fig. 3C). During withdrawal, there was a significant effect of treatment (p=0.0038), and both groups that received ethanol appeared to exhibit AWH, regardless of whether they received a bolus ethanol gavage before withdrawal (Fig. 3D). There was a trend towards a significant effect of time (p=0.0577) suggesting that AWH was transient, and by day 7 all groups showed similar mechanical thresholds (Fig. 3D, day 7). These findings show that in a chronic ethanol consumption model that is similar to the rat Lieber-DeCarli model (Pradhan et al., 2019), we also observe AWH in mice.

Treatment with a pan-HDAC inhibitor alleviates AWH

Our lab had previously shown in a rat Lieber-DeCarli model of chronic ethanol exposure that AWH was decreased by SAHA, a pan-HDAC inhibitor (Pradhan et al., 2019). Male and female mice were treated with control or ethanol diet (no binge), and 50 mg/kg SAHA was administered at 22, 46 and 70 h into withdrawal (Fig. 4A, B). Basal mechanical thresholds taken prior to the first dose of SAHA demonstrated mechanical hypersensitivity in the ethanol-treated mice compared to the control mice. One dose of SAHA was sufficient to alleviate the mechanical hyperalgesia observed at 24 h of alcohol withdrawal (Fig. 4C). Additionally, basal thresholds taken during day 3 of SAHA administration revealed that 2 doses of SAHA at 24 and 48 h into withdrawal were sufficient to sustain the pain-relieving effect of the HDAC inhibitor (Fig. 4D, baseline). Lastly, an additional dose of SAHA at 72h withdrawal had no further effect on mechanical sensitivity (Fig 4D, post-SAHA). These data demonstrate that SAHA alleviates AWH, suggesting that this increased pain sensitivity during withdrawal may be regulated by epigenetic mechanisms.

Discussion

In this study we demonstrate that AWH develops after chronic binge-like drinking in the DID procedure in male mice and after Lieber-DeCarli liquid diet administration in male and female mice. We also found that SAHA treatment alleviates the development of AWH in the liquid diet model, further corroborating previous data in rats (Pradhan et al., 2019). AUD is characterized by a progressive increase in alcohol consumption over time, and the emergence of negative emotional states (such as depression and anxiety) during withdrawal can contribute to the cycle of addiction. Alcohol withdrawal induced hyperalgesia (AWH) is a phenomenon observed in individuals with AUD during acute and protracted withdrawal and could contribute to this “dark side” of addiction (Jochum et al., 2010). Previous studies conducted in rats have explored the development of AWH, revealing that exposure to a 10-day ethanol liquid diet (6.5%) is sufficient to induce hyperalgesia at 6 and 12 hours of withdrawal (Gatch and Lal, 1999). Similarly, we have also shown that in rats, 15 days of ethanol diet (9%) produces AWH at 24 and 72h withdrawal (Pradhan et al., 2019). Apart from the liquid diet approach, voluntary ethanol consumption through a two-bottle choice method and chronic intermittent ethanol (CIE) vapor exposure in rats and mice have also been shown to increase pain sensitivity during withdrawal (Kang et al., 2019, Alongkronrusmee et al., 2016, Brandner et al., 2023, Quadir et al., 2021a, Quadir et al., 2021b, Okhuarobo et al., 2023, Okhuarobo et al., 2020).

We used two different models of alcohol exposure to test for the development of AWH: the DID procedure and Lieber-DeCarli liquid diet. The DID protocol is a less severe exposure model than the Lieber-DeCarli liquid diet and mimics binge-like ethanol consumption. AWH was observed with DID, but it took at least 5 weeks to establish AWH, and mechanical thresholds were only decreased by 40–50% in ethanol vs. control groups. Furthermore, AWH was only observed in males in this model, despite higher ethanol consumption (when normalized by body weight) by females. Our results differ from those of Bergeson et al (2016), who found decreased mechanical thresholds after 4 days of DID in male and female mice that returned to baseline by the 2nd (males) or 3rd (females) day after the last drinking session, with a greater effect in females (Bergeson et al., 2016). The reason for this discrepancy is not clear even though C57BL/6J mice were used in both studies and the DID procedure was similar (20% ethanol for 4 h per day).

With regard to sex differences in AWH in other alcohol exposure models, Brandner et al (2023) found that withdrawal from CIE vapor exposure produced significant mechanical hypersensitivity in male mice at 48 h of withdrawal beginning after the first week of CIE, but in females, mechanical hypersensitivity at 48 h of withdrawal did not develop until after the fourth week of CIE (Brandner et al., 2023). However, Morgan et al found that female mice developed mechanical hypersensitivity at 72 h of withdrawal from 21 days of continuous two-bottle choice 10% ethanol consumption. Morgan et al (2022), and Okhuarobo et al (2020 & 2023) found mechanical hypersensitivity in both female and male mice in the tail pressure test at 32 h of withdrawal from 4 days of CIE vapor (Morgan et al., 2022, Okhuarobo et al., 2023, Okhuarobo et al., 2020). Sex differences in AWH in these procedures is likely dependent on the method of alcohol exposure (voluntary vs. forced; continuous vs. intermittent), dose of alcohol, duration of exposure, and/or timepoints during which testing is conducted during withdrawal. Previous literature shows that male mice are more susceptible to the antinociceptive effects of alcohol than females (White et al., 2023, Bilbao et al., 2019). Sex differences have also been observed in pain processing (Mogil, 2020), and our observation of increased mechanical sensitivity after DID in males may be related to these differences. Finally, in the CIE vapor model, male mice exhibited greater sensitivity to mechanical stimuli compared to female mice; whereas female mice experienced increased thermal sensitivity relative to males (Brandner et al., 2023), suggesting different sex-specific mechanisms involved in the pain modality tested during alcohol withdrawal.

In comparison to the DID procedure, the chronic ethanol exposure liquid diet model more reliably produced AWH. In this case, both males and females developed AWH, and the effect size was >50%. In this model, pain hypersensitivity was associated specifically with ethanol withdrawal, as there was no change in mechanical thresholds while animals were maintained on the ethanol diet. Further, chronic ethanol diet was sufficient to produce AWH, as the same effect size was observed with or without the large binge ethanol dose that is used in the NIAAA model. One factor that might account for the difference in AWH between the Lieber-DeCarli liquid diet and DID is blood alcohol levels. In the Lieber-DeCarli liquid diet, mice achieved BACs of ~180–190 mg/dL. In our previous studies using DID, female mice attained BACs of ~180 mg/dL, consistent with their higher levels of ethanol intake, while males achieved BACs of ~130–140 mg/dL (Hamada et al., 2021, Chen et al., 2015). Given that female mice have roughly comparable BACs between the DID and Lieber-DeCarli alcohol exposure models, but only developed AWH during withdrawal from Lieber-DeCarli liquid diet, suggests that high BACs are not the only contributor to AWH. However, this conclusion is limited by the fact that we only measured BACs at one time point during the liquid diet administration, and it is not clear how long high BACs persist over the course of the day during liquid diet administration, or even the maximal BACs that are achieved. Related to this point, treatment of mice during ethanol vapor exposure with pyrazole, an inhibitor of alcohol dehydrogenase, to maintain high BACs, resulted in increased AWH (Brandner et al., 2023), suggesting that persistently high BACs are important for this behavioral phenomenon.

Similar to findings in the rat Lieber-DeCarli liquid diet paradigm (Pradhan et al., 2019), AWH in mice lasted for at least 72h post-ethanol withdrawal. AWH in mice was not permanent and decreased mechanical responses were nearly completely resolved by day 7 of ethanol withdrawal. In a human study, individuals with AUD were shown to have increased pain sensitivity upon admission to a rehabilitation program, and at 2 weeks abstinence there was a trend towards normalized pain tolerance in the same subjects (Jochum et al., 2010). An interesting question is whether re-exposure to ethanol inhibits AWH, and future studies will test this hypothesis in the liquid diet and DID protocol.

Further investigations of AWH in mice have utilized established paradigms of ethanol administration to investigate the underlying mechanisms of this phenomenon. Genetics may contribute to AWH, as two mouse strains (C57BL/6JRj and C3H/HeNRj) showed differences in thermal hyperalgesia when females (males were not tested) were tested after withdrawal from three cycles of 6.7% ethanol liquid diet administration plus 2 days of abstinence (Tonetto et al., 2023). Administration of ethanol through an intermittent two bottle choice voluntary ethanol consumption procedure elicited the development of AWH in male mice (females were not tested), an effect that was attenuated with the antagonism and modulation of sigma—1 and 2 receptors, proteins that serve as chaperones and calcium sensors on the mitochondrial endoplasmic reticulum (Quadir et al., 2021a, Quadir et al., 2021b). The endocannabinoid system is also involved in AWH in mice, as elevating levels of the endocannabinoid 2-arachidonoylglycerol alleviated AWH, and inhibiting endocannabinoid receptors exacerbated AWH, following continuous two-bottle choice ethanol administration (Morgan et al., 2022). Additionally, AWH was also observed in a modified mouse two bottle choice model and appeared to be inhibited by delta opioid receptor activation (Alongkronrusmee et al., 2016), which suggests a role for the endogenous opioid system.

Epigenetic mechanisms may also play a role in AWH. We found that the pan-HDAC inhibitor, SAHA, significantly reversed AWH in the chronic ethanol exposure model. A single dose of SAHA was sufficient to ameliorate AWH in mice (Fig. 4), and this pain-relieving effect was sustained with repeated SAHA administration. These findings are very similar to that observed in rats (Pradhan et al. 2019) and suggests that in this model of AWH, mechanisms may be conserved across species. We did not test SAHA for its ability to reduce mechanical sensitivity in the DID model, but future studies should determine if SAHA has the same effect in this model of ethanol exposure. Previous work has also shown that SAHA can also inhibit other behaviors associated with alcohol withdrawal, including anxiety- and depression-like behaviors (Chen et al., 2019). The histone deacetylase family is made up of class I, II and III HDACs (Park and Kim, 2020). Class I HDACs, including HDAC2, are enzymes that deacetylate histones resulting in chromatin condensation and repression of gene expression. In contrast, Class II HDACs, such as HDAC6, are primarily localized in the cytosol and influence the cytoarchitecture of the cell by deacetylating α-tubulin of microtubules and promoting destabilization of the cytoskeleton (Park and Kim, 2020). As a pan-HDAC inhibitor, SAHA affects both epigenetic and cytoskeletal aspects of HDAC function.

Epigenetic mechanisms are demonstrated to play an important role in alcohol withdrawal. In a liquid diet paradigm using male rats, withdrawal induced a decrease in the expression of the activity regulated cytoskeleton associated (Arc) protein and brain-derived neurotrophic factor (BDNF) protein, which influence synaptic plasticity. This decrease in protein expression was correlated with a decrease in dendritic spine density. The anxiety -like behaviors observed during withdrawal were associated with increased HDAC activity and decreased acetylation of histone H3 and H4 (Pandey et al., 2008a). Additionally, administration of trichostatin a (TSA), an HDAC inhibitor, rescued the aberrant acetylation of histone H3 and H4 and alleviated anxiety-like behaviors observed during withdrawal (Pandey et al., 2008, You et al., 2014). Cytoarchitectural changes, through HDAC6, have also been shown to play an important role in the development of pain sensitivity, including in models of migraine, inflammatory pain, and neuropathic pain (Bertels et al., 2021, Sakloth et al., 2020, Bertels et al., 2022). In our model, we cannot definitively conclude whether the pain-relieving effects of SAHA are mediated by epigenetic or cytostructural changes. For this reason, future studies will focus on investigating how inhibition of different classes of HDACs affect AWH, and whether this hyperalgesia is mediated through epigenetic, cytoskeletal, or both mechanisms. Ultimately, this model recapitulates previous findings in rat, and serves as a useful tool to investigate AWH and the underlying biology that modulates this pain hypersensitivity.

ACKNOWLEDGEMENTS

This work was supported by the National Institutes of Health (grants U01 AA020912 and R01 AA027231 to AWL and R01 NS130882 and UG3 DA053094 to AAP). We would like to thank Dr. Subhash Pandey for helpful discussions on implementing the Lieber-DeCarli procedure in mice. The authors declare no conflicts of interest.

REFERENCES

  1. Alongkronrusmee D, Chiang T, van Rijn RM (2016) Involvement of delta opioid receptors in alcohol withdrawal-induced mechanical allodynia in male C57BL/6 mice. Drug Alcohol Depend. 167:190–198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bergeson SE, Blanton H, Martinez JM, Curtis DC, Sherfey C, Seegmiller B, Marquardt PC, Groot JA, Allison CL, Bezboruah C, Guindon J (2016) Binge Ethanol Consumption Increases Inflammatory Pain Responses and Mechanical and Cold Sensitivity: Tigecycline Treatment Efficacy Shows Sex Differences. Alcohol Clin Exp Res 40:2506–2515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bertels Z, Mangutov E, Conway C, Siegersma K, Asif S, Shah P, Huck N, Tawfik VL, Pradhan AA (2022) Migraine and peripheral pain models show differential alterations in neuronal complexity. Headache 62:780–791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bertels Z, Singh H, Dripps I, Siegersma K, Tipton AF, Witkowski WD, Sheets Z, Shah P, Conway C, Mangutov E, Ao M, Petukhova V, Karumudi B, Petukhov PA, Baca SM, Rasenick MM, Pradhan AA (2021) Neuronal complexity is attenuated in preclinical models of migraine and restored by HDAC6 inhibition. eLife 10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bertola A, Mathews S, Ki SH, Wang H, Gao B (2013) Mouse model of chronic and binge ethanol feeding (the NIAAA model). Nat. Protoc. 8:627–637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bilbao A, Leixner S, Wei S, Cantacorps L, Valverde O, Spanagel R (2019) Reduced sensitivity to ethanol and excessive drinking in a mouse model of neuropathic pain. Addict Biol 24:1008–1018. [DOI] [PubMed] [Google Scholar]
  7. Brandner AJ, Baratta AM, Rathod RS, Ferguson C, Taylor BK, Farris SP (2023) Mechanical and Heat Hyperalgesia upon Withdrawal From Chronic Intermittent Ethanol Vapor Depends on Sex, Exposure Duration, and Blood Alcohol Concentration in Mice. J Pain. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Caldeiro RM, Malte CA, Calsyn DA, Baer JS, Nichol P, Kivlahan DR, Saxon AJ (2008) The association of persistent pain with out-patient addiction treatment outcomes and service utilization. Addiction 103:1996–2005. [DOI] [PubMed] [Google Scholar]
  9. Cargiulo T (2007) Understanding the health impact of alcohol dependence. Am J Health Syst Pharm 64:S5–11. [DOI] [PubMed] [Google Scholar]
  10. Chen H, He D, Lasek AW (2015) Repeated Binge Drinking Increases Perineuronal Nets in the Insular Cortex. Alcohol Clin Exp Res 39:1930–1938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen WY, Zhang H, Gatta E, Glover EJ, Pandey SC, Lasek AW (2019) The histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) alleviates depression-like behavior and normalizes epigenetic changes in the hippocampus during ethanol withdrawal. Alcohol 78:79–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dina OA, Barletta J, Chen X, Mutero A, Martin A, Messing RO, Levine JD (2000) Key role for the epsilon isoform of protein kinase C in painful alcoholic neuropathy in the rat. J. Neurosci. 20:8614–8619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Edwards S, Vendruscolo LF, Schlosburg JE, Misra KK, Wee S, Park PE, Schulteis G, Koob GF (2012) Development of mechanical hypersensitivity in rats during heroin and ethanol dependence: alleviation by CRF(1) receptor antagonism. Neuropharmacology 62:1142–1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Egli M, Koob GF, Edwards S (2012) Alcohol dependence as a chronic pain disorder. Neurosci. Biobehav. Rev. 36:2179–2192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goltseker K, Hopf FW, Barak S (2019) Advances in behavioral animal models of alcohol use disorder. Alcohol 74:73–82. [DOI] [PubMed] [Google Scholar]
  16. Hamada K, Ferguson LB, Mayfield RD, Krishnan HR, Maienschein-Cline M, Lasek AW (2021) Binge-like ethanol drinking activates anaplastic lymphoma kinase signaling and increases the expression of STAT3 target genes in the mouse hippocampus and prefrontal cortex. Genes Brain Behav:e12729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Heilig M, Egli M, Crabbe JC, Becker HC (2010) Acute withdrawal, protracted abstinence and negative affect in alcoholism: are they linked? Addict Biol 15:169–184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jochum T, Boettger MK, Burkhardt C, Juckel G, Bar KJ (2010) Increased pain sensitivity in alcohol withdrawal syndrome. Eur. J. Pain 14:713–718. [DOI] [PubMed] [Google Scholar]
  19. Kang S, Li J, Zuo W, Chen P, Gregor D, Fu R, Han X, Bekker A, Ye JH (2019) Downregulation of M-channels in lateral habenula mediates hyperalgesia during alcohol withdrawal in rats. Sci Rep 9:2714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lopez MF, Davis EC, Cucinello-Ragland JA, Regunathan S, Edwards S, Becker HC (2023) Agmatine reduces alcohol drinking and produces antinociceptive effects in rodent models of alcohol use disorder. Alcohol 109:23–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mogil JS (2020) Qualitative sex differences in pain processing: emerging evidence of a biased literature. Nat. Rev. Neurosci. 21:353–365. [DOI] [PubMed] [Google Scholar]
  22. Morgan A, Adank D, Johnson K, Butler E, Patel S (2022) 2-Arachidonoylglycerol-mediated endocannabinoid signaling modulates mechanical hypersensitivity associated with alcohol withdrawal in mice. Alcohol Clin Exp Res 46:2010–2024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Okhuarobo A, Angelo M, Bolton JL, Lopez C, Igbe I, Baram TZ, Contet C (2023) Influence of early-life adversity on responses to acute and chronic ethanol in female mice. Alcohol Clin Exp Res 47:336–347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Okhuarobo A, Bolton JL, Igbe I, Zorrilla EP, Baram TZ, Contet C (2020) A novel mouse model for vulnerability to alcohol dependence induced by early-life adversity. Neurobiol Stress 13:100269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pandey SC, Sakharkar AJ, Tang L, Zhang H (2015) Potential role of adolescent alcohol exposure-induced amygdaloid histone modifications in anxiety and alcohol intake during adulthood. Neurobiol Dis 82:607–619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pandey SC, Ugale R, Zhang H, Tang L, Prakash A (2008) Brain chromatin remodeling: a novel mechanism of alcoholism. J. Neurosci. 28:3729–3737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Park SY, Kim JS (2020) A short guide to histone deacetylases including recent progress on class II enzymes. Exp Mol Med 52:204–212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pradhan AA, Tipton AF, Zhang H, Akbari A, Pandey SC (2019) Effect of Histone Deacetylase Inhibitor on Ethanol Withdrawal-Induced Hyperalgesia in Rats. Int. J. Neuropsychopharmacol. 22:523–527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Quadir SG, Tanino SM, Rohl CD, Sahn JJ, Yao EJ, Cruz LDR, Cottone P, Martin SF, Sabino V (2021a) The Sigma-2 receptor / transmembrane protein 97 (sigma2R/TMEM97) modulator JVW-1034 reduces heavy alcohol drinking and associated pain states in male mice. Neuropharmacology 184:108409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Quadir SG, Tanino SM, Sami YN, Minnig MA, Iyer MR, Rice KC, Cottone P, Sabino V (2021b) Antagonism of Sigma-1 receptor blocks heavy alcohol drinking and associated hyperalgesia in male mice. Alcohol Clin Exp Res 45:1398–1407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rhodes JS, Best K, Belknap JK, Finn DA, Crabbe JC (2005) Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiol Behav 84:53–63. [DOI] [PubMed] [Google Scholar]
  32. Sakloth F, Manouras L, Avrampou K, Mitsi V, Serafini RA, Pryce KD, Cogliani V, Berton O, Jarpe M, Zachariou V (2020) HDAC6-selective inhibitors decrease nerve-injury and inflammation-associated mechanical hypersensitivity in mice. Psychopharmacology (Berl.) 237:2139–2149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Smith ML, Hostetler CM, Heinricher MM, Ryabinin AE (2016) Social transfer of pain in mice. Sci Adv 2:e1600855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Smith ML, Walcott AT, Heinricher MM, Ryabinin AE (2017) Anterior Cingulate Cortex Contributes to Alcohol Withdrawal- Induced and Socially Transferred Hyperalgesia. eNeuro 4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sullivan JT, Sykora K, Schneiderman J, Naranjo CA, Sellers EM (1989) Assessment of alcohol withdrawal: the revised clinical institute withdrawal assessment for alcohol scale (CIWA-Ar). Br J Addict 84:1353–1357. [DOI] [PubMed] [Google Scholar]
  36. Tonetto S, Weikop P, Brudek T, Thomsen M (2023) Behavioral and biochemical effects of alcohol withdrawal in female C3H/HeNRj and C57BL/6JRj mice. Front Behav Neurosci 17:1143720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Trafton JA, Oliva EM, Horst DA, Minkel JD, Humphreys K (2004) Treatment needs associated with pain in substance use disorder patients: implications for concurrent treatment. Drug Alcohol Depend 73:23–31. [DOI] [PubMed] [Google Scholar]
  38. White A, Caillaud M, Carper M, Poklis J, Miles MF, Damaj MI (2023) Thermal antinociceptive responses to alcohol in DBA/2J and C57BL/6J inbred male and female mouse strains. Behav Brain Res 436:114087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. You C, Zhang H, Sakharkar AJ, Teppen T, Pandey SC (2014) Reversal of deficits in dendritic spines, BDNF and Arc expression in the amygdala during alcohol dependence by HDAC inhibitor treatment. Int. J. Neuropsychopharmacol. 17:313–322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zapata A, Gonzales RA, Shippenberg TS (2006) Repeated ethanol intoxication induces behavioral sensitization in the absence of a sensitized accumbens dopamine response in C57BL/6J and DBA/2J mice. Neuropsychopharmacology 31:396–405. [DOI] [PMC free article] [PubMed] [Google Scholar]

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