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. Author manuscript; available in PMC: 2025 Jun 1.
Published in final edited form as: Alcohol. 2024 Mar 11;117:1–9. doi: 10.1016/j.alcohol.2024.03.005

Surgical Incision Pain Induced an Increase in Alcohol Consumption in Mice

Sofia Ghani 1, Yasmin Alkhlaif 1, Jared Mann 1, Lauren Moncayo 1, Esad Ulker 1, Martial Caillaud 3, Mitali Barik 1, Joseph W Ditre 4, Michael F Miles 1, M Imad Damaj 1,2
PMCID: PMC11042971  NIHMSID: NIHMS1974082  PMID: 38479450

Abstract

Introduction.

Large population-based studies have suggested a link between increased alcohol use and reduced pain. In addition, these studies suggest that higher levels of pain intensity are associated with an increase in alcohol consumption and rates of hazardous drinking which potentiates the risk of developing alcohol use disorders (AUD). The mechanisms and determinants of the alcohol-pain interaction can be studied in preclinical studies.

Methods.

The overall goal of this study is to use animal models to explore the impact of acute postoperative pain on alcohol intake. To achieve this, we characterized the timeline and levels of alcohol intake and preference in mice after laparotomy in the 2-bottle choice paradigm.

Results.

Our results show that laparotomy surgery increased alcohol intake and preference in male mice but not females in the 2-bottle choice and 3-bottle choice assays. In addition, ketoprofen administration blocked the increase in alcohol consumption in male mice after laparotomy. We also found that changes in alcohol initial sensitivity and acute functional tolerance, using loss of righting reflex (LORR) response, occur after surgery in mice.

Conclusion:

Taken together, these findings suggests that sex, pain and alcohol sensitivity-related factors may modulate the relationship between alcohol consumption and pain.

Keywords: Laparotomy, Acute Pain, Mice, Alcohol

INTRODUCTION

There is a high comorbidity rate of alcohol use and pain and their co-occurrence is common. For example, population-based studies showed that patients diagnosed with alcohol use disorder (AUD) had higher risks of developing pain disorders (Hung et al., 2021). In addition, 27% of patients with chronic pain used alcohol as a pain-reliever at least “sometimes” as reported in an epidemiological study (Riley and King, 2009), whereas a prospective study conducted with NESARC data suggested that drinking cessation significantly predicted reduction in bodily pain (Imtiaz et al., 2018). In a primary care patients’ population, over 60% of patients diagnosed with AUD drink alcohol to alleviate pain (Alford et al., 2016). Comparatively, heavy drinkers report a higher occurrence of consuming alcohol to manage their pain (Brenna, Schutte, & Moos, 2005). In addition, there is a greater prevalence and intensity of pain among individuals who drink alcohol, relative to non-drinkers. Similarly, the prevalence of AUD was greater after bariatric surgery in patients (King et al., 2012). Although the acute analgesic effects of alcohol have been documented for centuries (Crocq, 2007; McGovern, Mirzoian, & Hall, 2009), chronic alcohol use has been associated with pain exacerbation and enhanced pain during abstinence (Zale et al., 2015; Ditre et al., 2019; Bergeson et al., 2016). Additionally, the hyperalgesia associated with alcohol withdrawal may serve as a potent negative reinforcer that contributes to drinking escalation and relapse during abstinence (Powers et al., 2019; Egli, Koob & Edwards, 2012; Zale, Maisto, & Ditre, 2015). Therefore, the positive feedback relationship between alcohol use and acute pain seems to contribute to the exacerbation of both conditions over time (Ditre et al., 2019; Edwards et al., 2021).

Consistent with human laboratory findings (Ditre et al., 2023), results from animal models suggest that pain may increase voluntary alcohol consumption. In a mouse model of osteoarthritis, animals increased their alcohol intake in the 2-bottle choice (2BC) paradigm compared to their sham counterparts (Breese et al., 2017). In a partial sciatic nerve ligation mouse model, injured mice increased their alcohol intake and exhibited a persistent anxiety-like phenotype (Valverde et al., 2016). Furthermore, animal studies implicate sex as a determining factor in the relationship between pain and increased alcohol consumption. Exposure to complete Freund’s adjuvant (CFA) increased alcohol reinstatement drinking only in female rats (Lorente et al., 2021) but failed to increase alcohol consumption in male rats (Adrienne et al., 2020). In contrast, CFA-induced inflammatory pain was associated with significantly greater EtOH daily consumption in male mice exclusively compared to their female counterparts (Yu et al., 2019). This suggests an interesting sex-dependent relationship between pain and alcohol use. While chronic pain studies have showed a clear correlation between increased alcohol intake, few animal studies have investigated the impact of acute pain on drinking behaviors. Therefore, the present study characterized the impact of acute laparotomy pain on alcohol 2BC and 3BC drinking in both male and female C57BL/6J mice. The laparotomy surgical procedure is widely used as a model for acute surgical pain in rodents. Using this model, we also assessed sex differences related to alcohol intake, intoxication and metabolism.

MATERIAL AND METHODS

Animals

The experiments were performed on C57BL/6J adult female and male mice 8–10 weeks years old purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were housed four per cages with an enriched environment and maintained on a 12h light/dark cycle (lights on at 07:00 h), with a room temperature of 22°C, with ad libitum access to food (global 18% protein chow diet; Envigo Teklad, Indianapolis, IN, USA) and water. All experiments were performed during the light cycle. The study was approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. All studies were carried out following the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. All mice were observed daily for general well-being and their weight was measured daily.

Laparotomy Procedure

Laparotomy surgeries were performed as described by Oliver et al. (2018) with slight modification under sterile conditions. Mice were anesthetized with 5.0% isoflurane and maintained using a constant stream of 2.5% isoflurane in oxygen using a face mask and vaporizer. Abdominal hair was clipped with nose hair clippers and the surgical site was disinfected with beta-iodine. All surgeries were performed by the same surgeon to ensure consistency, and all surgeries performed between 08:00 AM and 11:00 AM. A 1.0- to 1.5-cm midline abdominal incision was made through the skin and then extended through the linea alba. To mimic visceral manipulation associated with various surgeries, a sterile cotton swab was inserted and moved throughout the abdomen for 30 seconds. The abdominal muscle layers and skin was closed by using 3 to 4 5-0 nylon sutures in a discontinuous pattern. The total duration of surgery was between 7 and 10 min. Once recovered from anesthesia, mice were housed back to their cages instead of new cages to avoid novelty-induced anxiety. Every surgical group’s corresponding sham group received the same duration of isoflurane at the same time and their hair was clipped and surgical site disinfected but no incisions were made.

Two bottle choice (2BC) drinking studies.

Testing protocol.

Mice were acclimated in the testing room for 5 days in individual cages with water 2BC before any intervention. Approximately 30 min after recovery from anesthesia, mice were evaluated for alcohol intake and preference in the 2BC test. For that they were housed individually and were given a choice of ethanol (20%, v/v) or water in a two-bottle choice paradigm for a period of 12-18 days. The bottle positions were alternated every day to control for the development of a side preference. Ethanol intake was reported as g/kg of body weight and ethanol preference was reported as the ratio between ethanol intake and the total fluid intake. Subjects were weighed daily to accurately calculate ethanol intake. Data was analyzed by day and also the average of all days’ results was determined. Laparotomy mice were evaluated in a separate testing room than the sham animals.

Study 1.

Impact of laparotomy on alcohol (20%, v/v) intake in male (n=10/group) and female (n=15/group) mice.

Study 2.

Impact of laparotomy on alcohol (10%, v/v) intake in female (n=8/group) mice.

Study 3.

Effects of ketoprofen on alcohol (20%, v/v) intake in male (n=10/group) laparotomy and sham mice. Mice were administered an injection of either vehicle (saline-10 ml/kg, s.c.) or ketorprofen (20 mg/kg, s.c.) 30 min before surgery and for 5 days after laparotomy (once/day). The dose of 20 mg/kg of ketorprofen was chosen because it was shown to induce an analgesic-like effects in the mouse laparotomy mouse model (Ulker et al., 2022).

Three bottle choice (3BC) drinking studies.

To assess the effect of surgery on alcohol intake with the availability of a nondrug reward, we evaluated the impact of laparotomy on alcohol and sucrose preference in male (n=12/group) and female (n=8/group) mice in a 3BC test. Approximately 30 min after recovery from anesthesia, mice were housed individually and were given a choice of ethanol (20%, v/v), sucrose (4%) or water in a three-bottle choice paradigm for a period of 7 days. The bottle positions were alternated every day to control for the development of a side preference. The concentration of sucrose was chosen based on pilot studies to achieve a preference between 40 to 60% in the 3BC test. We have not determined the respective preference of ethanol and sucrose concentrations to see if they are equally preferred over water or with each other in the absence of water in 2BC tests in mice. The goal of this experiment was to confirm that the increase we saw in the 2BC test extends to a 3-way choice between alcohol, a natural reward and water. Ethanol and sucrose preferences were reported as the ratio between ethanol or sucrose intake and the total fluid intake. Laparotomy mice were evaluated in a separate testing room than the sham animals.

Loss of Righting Reflex (LORR) studies.

The sedative-hypnotic effects of ethanol were measured using the loss of LORR. Male and female mice undergoing sham or laparotomy were injected four days after surgery with 3.5 g/kg ethanol (20%v/v, i.p.). Mice were placed in a supine position in a V-shaped trough and monitored for the onset of LORR, defined by the inability to right themselves 3 times in 30 seconds after being in the supine position. Time from ethanol injection to LORR was recorded as LORR onset. Mice taking longer than 6 min to experience LORR were eliminated from the study due to the possibility of misplacement. Immediately after the onset of LORR, a 20 μl blood sample was collected from the periorbital sinus. The time taken to recover from LORR, LORR duration, was defined as the time required for the subject to right itself three times within 30 seconds. A second 20 μl blood sample was collected from the periorbital sinus at this time. Care was taken to collect all blood samples within 10 seconds of each behavioral endpoint to minimally affect the true relationships between BEC and LORR onset or recovery. Finally, a third 20 μl blood sample was collected from the periorbital sinus 6 hr after alcohol injection.

A separate cohort of male and female mice undergoing sham or laparotomy were injected seven days after surgery with 3.5 g/kg ethanol (20%v/v, i.p.) and tested for LORR as described above. No BAC was determined for this time point.

We chose an early time point after the surgery where no significant increase in alcohol preference were seen in male mice (day 4) and a later time point (day 7) where male mice showed an increase in alcohol preference.

Blood Alcohol Concentration (BAC) Determination

Testing protocol.

Blood was collected via cheek bleed using 5 mm Lancets (Medipoint, Inc., Minenola, NY) or via the periorbital sinus. Blood was stored in heparinized BD microtainers and analyzed for ethanol content using gas chromatography similar to a previously described procedure (Gallaher et al. 1996). Animals were injected with saline (s.c) to avoid hypovolemia and received food/water ad libitum after blood collection.

Statistical Analysis

Data were analyzed using the GraphPad software, version 9.3 (GraphPad Software, Inc., La Jolla, CA), and are expressed as the mean ± S.E.M. Normality and equality of variances of all data sets except nesting were confirmed by using the Shapiro–Wilk and Levene tests, respectively. All other tests were analyzed with 3- or 2-way ANOVA [post hoc analysis (Sidak test)]. Data are expressed as the mean ± S.E.M. of mice/per sex/per group for all tests. P values less than 0.05 were considered significant.

RESULTS

Impact of laparotomy on alcohol intake in the 2BC paradigm

Male mice consumed a significantly greater amount of ethanol per day (g/kg/24 h) after laparotomy than sham surgery, while females did not exhibit any consumption differences across treatments (Fig. 1 & 2). Indeed, a RM-ANOVA (Surgery x Time) analysis of alcohol intake in male mice revealed a main effect for Surgery [F(1,18) = 10.50, p=0.0045] and Time [F(17,306) = 8.735, p<0.0001] in males (Fig. 1A), and significant interaction for Surgery x Time [F (17, 306) = 2.098, p=0.0071]. Statistical analysis of the average drinking values of male mice across 18 days showed a significant increase in alcohol intake in the laparotomy group compared to sham mice (t=3.936, df=34, p= 0.0004) (Fig. 1D). Preference to alcohol in male mice exhibited similar results with laparotomy mice showing a significantly greater ethanol preference than their sham control mice with a main effect for Surgery [F(1,18) = 9.979, p=0.0054] and Time [F(17,306) = 4.934, p<0.0001] in males (Fig. 1B), but no significant interaction for Surgery x Time [F(17, 306) = 1.242, p=0.2303]. No statistical differences in total fluid intake were observed between the groups.

Figure 1. Alcohol intake in male mice.

Figure 1.

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Alcohol-drinking behavior (20% v/v) was measured amongst C57BL/6J male mice for 18 consecutive days after surgery. (A) alcohol intake (g/kg), (B) alcohol preference (%), (C) total fluid intake, and (D) average alcohol intake. Values are expressed as mean ± SEM of n = 18/group. (*p < 0.05 vs sham).

Figure 2. Alcohol intake in female mice.

Figure 2.

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Alcohol-drinking behavior (20% v/v) was measured amongst C57BL/6J female mice for 12 consecutive days after surgery. (A) alcohol intake (g/kg), (B) alcohol preference (%), (C) total fluid intake, and (D) average alcohol intake. Values are expressed as mean ± SEM of n = 12/group. (*p < 0.05 vs sham).

A RM-ANOVA (Surgery x Time) analysis of alcohol intake in female mice revealed a main effect for Time [F(15, 154) = 35.04, p<0.0001] but not for Surgery [F (1,28) = 0.3985, p=0.9501] (Fig. 2A), and statistical analysis of the average drinking values of these mice across 12 days showed no significant change in alcohol intake in the laparotomy group compared to sham mice (t=0.2692, df=22, p= 0.7903) (Figure 2D). Similarly, the preference to alcohol in female mice did not show significant difference between laparotomy and sham control mice with a main effect for Time [F(17, 306) = 14.60, p<0.0001] but not Surgery [F(1,18) = 0.6431, p=0.8027] (Fig. 2B). No statistical differences in total fluid intake were observed between the groups (Fig. 2C).

To elucidate a possible ceiling effect in female mice, we evaluated the effects of laparotomy on alcohol intake in female mice at a lower concentration (10%, v/v) of the drug in the 2BC test. As shown in Fig. 3, statistical analysis of alcohol intake in female mice did not show a main effect for Time [F(5,73) = 1.526, p=0.1897] or for Surgery [F(1,14) = 1.367, p= 0.2527] (Fig. 3A). In addition, average drinking values of these mice across 12 days showed no significant change in alcohol intake in the laparotomy group compared to sham mice (t=1.055, df=22, p= 0.3027) (Figure 3E). Similarly, preference to alcohol in female mice did not show significant difference between laparotomy and sham control mice for Time [F(5,73) = 14.60, p<0.0001] or Surgery [F (1, 14) = 1.894, p=0.1904] (Fig. 3B). No statistical differences in total fluid intake were observed between the groups.

Figure 3. Alcohol intake in female mice.

Figure 3.

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Alcohol-drinking behavior (10% v/v) was measured amongst C57BL/6J female mice for 12 consecutive days after surgery. (A) alcohol intake (g/kg), (B) alcohol preference (%), (C) total fluid intake, and (D) average alcohol intake. Values are expressed as mean ± SEM of n = 12/group. (*p < 0.05 vs sham).

Finally, a two-way ANOVA analysis of average drinking values after surgery in male and female mice revealed a main effect for Surgery [F(1,52) = 5.097, p=0.0282] and for Sex [F(1,52) = 4.051, p=0.0493]. A similar change was seen when the cumulative intake was calculated (Sham: 252.40 ± 23.21 g/kg vs Surgery: 346.62 ± 17.50 g/kg*).

Ketoprofen reduces increase of alcohol intake in male mice after of laparotomy in the 2BC assay

To investigate if increased pain severity mediates the escalation of alcohol intake after laparotomy in male mice, animals were treated with repeated ketoprofen before and after surgery. As shown in Fig.4, ketoprofen reduced laparotomy-induced increase in alcohol consumption without altering its intake in sham-treated mice. Indeed a RM-ANOVA (Surgery x Ketoprofen x Time) analysis of alcohol intake in male mice revealed a main effect for Surgery [F(1,126) = 7.250, p=0.0081], Ketoprofen [F(1,126) = 14.37, p=0.0002] and Time [F(13,126) = 10.20, p<0.0001] (Fig. 4A), and significant interaction for Surgery x Ketoprofen [F(1,126) = 27.86, p<0.0001]. Statistical analysis of the average drinking values of male mice across 14 days [F(3,52) = 5.567, p=0.0022] showed the significant increase in alcohol intake in the laparotomy group compared to sham mice (p= 0.0022) was completely blocked by ketoprofen treatment (p= 0.0136 (Figure 4D). A similar change was seen when the cumulative intake was calculated (Veh/Sham: 172.2 ± 9.9 g/kg; Keto/Sham: 184.4 ± 14.8; Veh/SUR : 236.0 ± 15.2 g/kg; Keto/SUR: 163.1 ± 6.4 g/kg).

Figure 4. Alcohol intake in male mice after treatment with ketoprofen.

Figure 4.

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Alcohol drinking behavior (20% v/v) was measured amongst C57BL/6J male mice for 14 consecutive days after surgery and treatment of ketoprofen 20 mg/kg, s.c.) 30 min before surgery and for 5 days after laparotomy (once/day). (A) alcohol intake (g/kg), (B) alcohol preference (%), (C) total fluid intake, and (D) average alcohol intake. Values are expressed as mean ± SEM of n = 10/group

Similarly, preference to alcohol in male mice did show significant difference between laparotomy and sham control mice for Time [F(13,126) = 13.05, p<0.0001], Surgery [F(1,126) = 34.05, p<0.0001] and Ketoprofen [F(1,126) = 13.63, p=0.0003] and significant interaction for Surgery x Ketoprofen [F (1, 126) = 19.82, p<0.0001] (Fig. 4B). No statistical differences in total fluid intake were observed between the groups.

Impact of laparotomy on alcohol intake in the 3BC paradigm

In this experiment we evaluated if mice undergoing laparotomy will increase their alcohol consumption at the expense of a high-value alternative, a sucrose solution, in a 3BC procedure. As seen in Figure 5A, laparotomy surgery induced a shift in the preference ration from sucrose to alcohol in male mice compared to sham-treated controls. Indeed, a RM-ANOVA (Surgery x Sucrose x Time) analysis of alcohol preference in male mice revealed a main effect for Surgery [F(1,333) = 414.2, p<0.0001], Sucrose [F(1, 333) = 4.479, p=0.0351] (Fig. 5A), and significant interaction for Surgery x Sucrose [F(1, 333) = 35.82, p<0.0001] and Time x Surgery x Sucrose [F (8, 333) = 5.433, p<0.0001].

Figure 5. Alcohol preference in male and female mice in the 3-BC assay.

Figure 5.

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Alcohol preference was measured in male and female C57BL/6J mice in the 3-BC paradigm between alcohol (20% v/v), sucrose (4%) or water for 7 consecutive days after surgery. Alcohol preference (%) values are expressed as mean ± SEM of n = 8/group/sex; *p<0.05 vs surgery alcohol group.

In contrast to male mice, laparotomy surgery did not induce a shift in the preference ratio from sucrose to alcohol in female mice compared to sham-treated controls. A RM-ANOVA (Surgery x Sucrose x Time) analysis of alcohol preference in female mice showed no effect for Surgery [F(8,63) = 0.6453, p= 0.7367], Sucrose [F(1,63) = 0.1487, p=0.7011] (Fig. 5B), and no significant interaction for Surgery x Sucrose [F (1,63) = 0.1198, p=0.7304] and Time x Surgery x Sucrose [F (8, 63) = 0.7181, p=0.6748].

A similar changes in alcohol intake was seen in male and female mice in the 3-BC test (Supplementary Figure 1).

Impact of laparotomy on alcohol-induced LORR and blood alcohol concentration

In this experiment we evaluated the effect of laparotomy on the sedative/hypnotic effects of alcohol. Laparotomy and sham control male and female mice were injected with 3.5 g/kg ethanol and onset latency and duration of LORR were recorded 4 and 7 days after surgery in different cohorts of animals. In male mice, laparotomy induced a significant decrease in the onset time at day 4 but that effect was lost on day 7 (Fig. 6A). In addition, a significant decrease in the duration of LORR was observed in surgery male mice compared to control sham animals on both days 4 and 7 (Fig. 6B). In female mice, laparotomy induced a significant decrease in the onset of LORR on both days 4 and 7 (Fig. 6C) but a decrease in the duration of LORR on day 4 only (Fig. 6D). Importantly, BAC on day 4 after laparotomy was similar between treatment groups at the onset of LORR, loss of function or 6 hr after injection in male (Surgery [F(1,10) = 0.5642, p= 0.4699]) (Fig. 7A) and female mice (Surgery [F(1,11) = 0.9894, p= 0.3413]) (Fig. 7B).

Figure 6. LORR after alcohol injection in laparotomy and sham male and female mice.

Figure 6.

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(A) Onset of LORR in min in C57BL/6J male mice after i.p. injection of 3.5 g/kg alcohol, (B) duration of LORR in min in C57BL/6J male mice, (C) onset of LORR in C57BL/6J female C57BL/6J mice and (D) duration of LORR in C57BL/6J female mice. Values are expressed as mean ± SEM of n = 8/group/sex; *p<0.05 vs sham.

Figure 7. Laparotomy does not affect alcohol blood levels in C57BL/6J male and female mice.

Figure 7.

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Blood samples were taken at onset, end of duration of sleep and 6 hr post-alcohol injection at 4 days after surgery. Values are expressed as mean ± SEM of n = 6-7/group/sex

DISCUSSION

In this study, we describe sex differences in alcohol drinking following laparotomy surgery. Male incised mice consumed more alcohol and exhibited higher preference for the drug than sham controls, while incised female mice and sham controls consumed a similar amount of alcohol in the 2BC model. A similar pattern was observed when mice were given access to sucrose while given continuous free access to alcohol and water. The increase in alcohol consumption following surgery in male mice was prevented by the administration of ketoprofen. These findings suggest that acute surgical pain increases alcohol drinking in male mice, and that this effect can be attenuated via administration of a nonsteroidal anti-inflammatory drug (NSAID).

This is the first study reporting an increase in voluntary ethanol consumption after acute surgical injury in male mice. It is consistent with some recent preclinical research showing an increase in voluntary ethanol consumption following chronic inflammation and neuropathy in male mice (Valverde et al., 2016; Breese et al., 2017; Yu et al., 2019). The time-course of alcohol intake increase in the 2-BC assay after laparotomy in male mice is parallel to the time-course of mechanical hypersensitivity but not spontaneous pain-like behaviors after surgery (Ulker et al., 2022). In addition, the effectiveness of the NSAID ketoprofen to prevent surgical injury-induced escalation of alcohol consumption provide evidence for an important role for pain and inflammation.

Similar to the 2-BC assay, we saw an increase in alcohol consumption and preference in the 3-BC paradigm in male mice only. However, the time-course of the increase in alcohol intake differs between the two tests after laparotomy. The onset of the increase in drinking behavior in the 3-BC assay after laparotomy was faster and its duration was shorter compared to the 2-BC assay. While the time-course in the 2-BC assay was parallel to the time-course of the evoked abdominal mechanical hypersensitivity, the time-course of alcohol intake after incision in the 3-BC test onset was closely related to that of spontaneous behavior changes after laparotomy (Ulker et al., 2022).

The lack of postoperative pain-induced increase in alcohol drinking in female mice is consistent with a recent preclinical study with chronic inflammation in mice (Yu et al., 2019) and several human studies wherein male chronic pain patients more commonly endorsed pain-induced alcohol consumption and self-medicating pain with alcohol (Riley & King, 2009; Wilsnack et al., 2009; Brennan et al., 2011). More recently, results of a human laboratory study showed that experimental pain (capsaicin + thermal heat paradigm) induction increased ad-lib alcohol consumption among moderate-to-heavy drinkers without chronic pain, and that this effect was most pronounced among male participants (Ditre et al., 2023). Collectively, these findings underscore the importance of investigating sex as a factor in pain-alcohol relations, especially considering that females are disproportionally affected by many pain syndromes (Edwards et al., 2020).

This raises the possibility of sex-dependent differences in pain responses/intensity or alcohol metabolism, or both after laparotomy. Firstly, our pain-evoked and non-evoked behavioral recently published results (Ulker et al., 2020) showed no significant sex differences after injury. Next, our female drinking data corresponds with previous studies showing a higher baseline alcohol consumption than males (Yu et al., 2019). It has been suggested that the lack of an increase in alcohol in the females could be due to a ceiling effect, but our data with female mice did not show a significant increase in alcohol intake at a lower concentration of the drug (10%). Therefore, sex differences regarding their ability to escalate alcohol consumption is not likely prevented by pain-related or sex-related drinking ceiling effects for females. Instead, differences in alcohol analgesia may provide a better explanation for sex-dependent differences in alcohol consumption. Previous literature has shown female rodents being less prone to attenuation of inflammation by alcohol (Alfonso-Loeches et al., 2013). Therefore, it is possible that the laparotomy-treated females are less susceptible to increasing their alcohol intake and preference than their sham counterparts because alcohol fails to provide more analgesia upon further drinking. We recently reported that compared to male C57BL/6J mice, alcohol did not produce antinociception in same-strain female mice in an acute thermal nociceptive pain test (White et al. 2023). Additionally, the stimulatory and depressive locomotor effects of alcohol were greater in male mice than in females. These results may explain the lack of increase in alcohol consumption amongst the females after incision.

It is also possible that sex hormones can be an underlying factor in the difference in postoperative alcohol drinking between the sexes. Estrogen has been reported to have an influence on reward-seeking behaviors (Quigley et al., 2021). However, ovariectomized mice did not consume a significantly different amount of alcohol compared to their sham counterparts (White et al., 2023).

Generally, it has been proven that having high initial sensitivity to the effects of alcohol could decrease alcohol drinking (Palmer et al., 2004). Given the alcohol drinking data after surgery, we hypothesized that if an animal has a high initial sensitivity to alcohol, then that may inhibit alcohol consumption. In addition, if they developed a high acute functional tolerance (AFT) to the drug, then that may enhance alcohol-drinking. We used the loss of righting reflex (LORR) assay to assess the mice’s initial sensitivity to alcohol’s ataxic and sedative effects by measuring the onset of LORR and assessing their AFT through their total duration of LORR. Our results show that female mice exhibited an increase in sensitivity to alcohol as expressed by a significant and longer decrease in their onset of LORR on postoperative days 4 and 7 compared to day 4 only in male mice after laparotomy. These findings suggest that the sustained and longer increase in alcohol initial sensitivity in female mice may decrease alcohol drinking after laparotomy compared to male mice.

In contrast, male laparotomy mice had a significant and longer reduction in their duration of LORR (days 4 and 7 post-surgery) compared to female mice (day 4 only). The male surgery mice’s significantly shorter duration suggests that they developed a more sustained (day 4 vs day 7) acute functional tolerance (AFT) to alcohol’s sedative effects than female animals. This can provide further explanation as to why male laparotomy mice consumed a significantly greater amount of alcohol over time especially at later days after surgery. Although the results displayed significant differences in the onset and duration of LORR between the sexes, there were no differences in BAC as time progressed after LORR began in either sex. These results suggest that postoperative pain does not alter the metabolism of alcohol in rodents of either sex.

While our results have demonstrated that acute postoperative pain can induce an increase in alcohol consumption driven by pain-related mechanisms, it is important to address some limitations of our study.

First, we used naïve mice with no history of alcohol consumption, which may best relate to a population of patients who endure postoperative pain and would use alcohol for analgesic purposes. It will be important to examine alcohol consumption after laparotomy in mice with a history of alcohol exposure, as this will better reflect patients with a history of drinking who may turn to alcohol for analgesic or pain-coping effects (e.g., LaRowe et al., 2021; Ferguson et al., 2022). For example, the results of a recent study show that CFA-induced inflammatory pain does not alter total alcohol intake in male or female rats with a history of alcohol exposure (intermittent access for 4 weeks) before the insult (Campos-Jurado and Morón, 2023).

Secondly, pain and alcohol have been reported to have a bidirectional relationship (Ditre et al., 2019; Powers, Zvolensky, and Ditre, 2019); however, our study only investigated how pain impacts alcohol consumption. Therefore, a limitation of this study is that we did not consider if or how alcohol alters pain-like behaviors in mice. Ultimately, this is an important relationship to investigate and understand because it has been reported that individuals who drink alcohol report greater intensity and prevalence of pain (Boissoneault et al., 2019). However, our studies here provide important new information as to the mechanisms and sex-dependent nature of pain-induced changes in alcohol consumption.

Supplementary Material

1

Supplementary Figure 1. Alcohol intake in male and female mice in the 3-BC assay. Alcohol intake was measured in male and female C57BL/6J mice in the 3-BC paradigm between alcohol (20% v/v), sucrose (4%) or water for 7 consecutive days after surgery. Alcohol intake values are expressed as mean ± SEM of n = 8/group/sex; *p<0.05 surgery factor.

  • Acute laparotomy postoperative pain increases alcohol intake in male but not female mice

  • ketoprofen administration blocked the increase in alcohol consumption in mice after laparotomy.

  • Alcohol initial sensitivity and acute functional tolerance occur after surgery in mice.

Acknowledgements.

This work was funded by RO1AA027175 (NIH) to MID and MFM and P30DA033934 (NIH) to William Dewey.

Footnotes

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Declarations of interest: none.

On behalf of all authors, the corresponding author states that there is no conflict of interest.

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Associated Data

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Supplementary Materials

1

Supplementary Figure 1. Alcohol intake in male and female mice in the 3-BC assay. Alcohol intake was measured in male and female C57BL/6J mice in the 3-BC paradigm between alcohol (20% v/v), sucrose (4%) or water for 7 consecutive days after surgery. Alcohol intake values are expressed as mean ± SEM of n = 8/group/sex; *p<0.05 surgery factor.

NIHMS1974082-supplement-1.jpg (378.7KB, jpg)

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