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. Author manuscript; available in PMC: 2024 Feb 1.
Published in final edited form as: Alcohol Clin Exp Res (Hoboken). 2022 Dec 30;47(2):382–394. doi: 10.1111/acer.14997

Effects of ethanol on mechanical allodynia and dynamic weight bearing in male and female mice with spared nerve injury

Mitchell A Nothem 1, Jason R Wickman 1,2, Laura L Giacometti 1, Jacqueline M Barker 1,*
PMCID: PMC9992011  NIHMSID: NIHMS1861604  PMID: 36521835

Abstract

Background:

Men and women with chronic pain report increased alcohol use and are more likely to be diagnosed with alcohol use disorder. The relationship between alcohol use and pain is bidirectional with alcohol used as an analgesic, but chronic intake increasing pain. Sex differences in the relationship between chronic pain and alcohol are reported in the clinical and preclinical literature, but due to this bidirectional relationship, it is challenging to investigate the mechanisms that contribute to these differences. Thus, animal models of chronic pain are needed to characterize the efficacy of ethanol as an analgesic in males and females. The current experiments tested the hypothesis that ethanol would differentially reduce pain behaviors in male and female mice in chronic neuropathic pain.

Methods:

The spared nerve injury (SNI) model was used to investigate the analgesic effects of multiple doses of ethanol (0.5, 1, 2, g/kg i.p.) male and female mice using von Frey and dynamic weight bearing (DWB) assays.

Results:

In both male and female mice, SNI led to robust allodynia and shifts in dynamic weight bearing. In male SNI mice, all three doses of ethanol fully reversed mechanical allodynia and shifts in DWB. In SNI females, only the highest dose (2.0g/kg) was fully antiallodynic in the von Frey assay, while shifts in weight bearing were reversed at the 1.0 and 2.0 g/kg doses. The differences between male and females were not due to decreased blood ethanol concentrations in female mice.

Conclusion:

These data indicate that while ethanol has antiallodynic and antinociceptive effects in males and female mice, the doses and time course of these effects are distinct. Studies investigating the relationship between pain and ethanol in mice should consider sex as a key variable. These data further inform reported sex differences in rodent models of chronic pain and in chronic pain patients.

Keywords: sex differences, ethanol, pain, spared nerve injury, mice

Introduction

Alcohol use disorder (AUD) and chronic pain are highly comorbid conditions associated with reduced quality-of-life (Andrew et al., 2014; Axley et al., 2019) and enormous social and economic costs. Over 50% of individuals with AUD also suffer from chronic pain, with higher comorbidity in women (Boissoneault et al., 2019). Individuals with high levels of pain are more likely to be dependent on alcohol, to meet the criteria for AUD, and to relapse after a period of abstinence compared to those without pain (Boissoneault et al., 2019; McDermott et al., 2018).

Alcohol’s ability to reduce pain sensitivity in humans (Mullin & Luckhardt, 1934) and utility as an analgesic are well-documented (Horn-Hofmann et al., 2015; Perrino et al., 2008). People with chronic pain are more likely to report using alcohol to deal with pain (Riley & King, 2009). However, the effectiveness of this strategy is short-lived as chronic alcohol intake can lead to worsened pain outcomes in the form of alcohol induced-polyneuropathy and, upon cessation, alcohol-induced hyperalgesia (Gatch, 2009; Sommer et al., 2018a). Thus, alcohol use and chronic pain interact in a positive feedback loop, whereby chronic pain increases excessive alcohol use, which in turn worsens pain outcomes (Cucinello-Ragland & Edwards, 2021; Ditre et al., 2018; Edwards et al., 2020; Egli et al., 2012; Pahng & Edwards, 2021). However, in clinical populations it is difficult to assess the directionality of this interaction and dissect the contributing mechanisms, thus necessitating the use of animal models to elucidate directional interactions between the effects of alcohol on pain and vice versa.

Ethanol is effective at reducing pain-related behaviors in animal models of both acute and chronic pain. In naïve animals, ethanol promotes hypoalgesic responses, reducing paw withdrawal responses to painful mechanical and thermal stimuli (Campbell et al., 2006; Neddenriep et al., 2019). Further, alcohol reduces mechanical and cold allodynia and reverses deficits in voluntary wheel running during chronic pain (Bilbao et al., 2019; Neddenriep et al., 2019).

The presence of ongoing chronic pain also drives ethanol consumption in animals. In a chronic inflammatory pain model, pain increased ethanol consumption and preference in a continuous access paradigm in male – but not female – mice (Yu et al., 2019). Together, these data suggest that ethanol is antinociceptive in animals and that animal models of chronic pain facilitate ethanol consumption, consistent with findings in humans.

The majority of the work investigating the effect of alcohol on chronic pain has used models of chronic inflammatory pain(Adrienne McGinn et al., 2020). Few have focused on chronic neuropathic pain (CNP), despite the large segment of the chronic pain population experiencing ongoing neuropathic pain(Sommer et al., 2018). Since the mechanisms contributing to CNP are vastly different than chronic inflammatory pain (Jensen & Finnerup, 2014; Shepherd et al., 2018; Vranken, 2011; Yezierski & Hansson, 2018), there is a pressing need for robust preclinical data on the effects of alcohol in animal models of CNP.

To close this gap, these experiments used the well-characterized spared nerve injury (SNI) model of CNP, in which severe mechanical hypersensitivity of the hindpaw ipsilateral to injury persists for months (Decosterd & Woolf, 2000; Erichsen & Blackburn-Munro, 2002; Guida et al., 2020). The effects of ethanol on mechanical hypersensitivity in SNI were assessed using von Frey and dynamic weight bearing (DWB) assays in male and female mice. Female mice have been reported to be insensitive to effects of voluntary ethanol drinking on pain-related behavior, despite drinking more than their male counterparts (Bilbao et al., 2019) while higher doses of experimenter-administered ethanol had similar effects on pain conditioned place avoidance in males and females. Therefore, it was hypothesized that female mice would require higher doses of ethanol to observe an antiallodynic effect.

Methods

Animals

Adult male and female C57Bl/6J mice were used (Jackson Laboratories). Mice were 9 weeks old and between 18–23 g at the beginning of the experiments. All animals were singly housed in a temperature and humidity-controlled environment on a standard 12h:12h light dark cycle and provided food and water ad libitum for the duration of the experiment. All behavioral experiments occurred within the light cycle. Mice were handled and allowed to acclimate to the colony room for one week prior to beginning the baseline behavior experiments. All procedures were carried out under a protocol approved by the Institutional Animal Care and Use Committee of Drexel University.

Spared nerve injury (SNI) surgery

Mice underwent SNI surgery at approximately 11 weeks of age. Mice were anesthetized by inhaled isoflurane. An incision was made on the left leg and the sciatic nerve was exposed. The common peroneal and tibial nerves were ligated with 6–0 silk suture and cut distally from the ligation. The skin was closed using size 7 wound clips (Reflex). The sural nerve was left intact. Sham mice underwent a similar procedure in which the branches of the sciatic nerve were exposed but not ligated or cut. Animals recovered for 2 weeks for the development of hypersensitivity to occur and for changes in weight bearing to stabilize prior to behavioral testing (Mogil et al., 2010). Forty animals were used in these studies (Male Sham: 11; Male SNI: 14; Female Sham: 7; Female SNI: 8).

Behavioral testing: von Frey

To assess the effect of acute ethanol administration on mechanical hypersensitivity in the context of chronic pain, mice were stimulated with a set of von Frey fibers using the updown method beginning with a 0.16g fiber (Stoetling Touch Test, Chicago, IL) and with 2.56g being the upper limit (Chaplan et al., 1994; Dixon, 1980). The 50% paw withdrawal threshold was calculated per Chaplan & Dixon (1994); all withdrawal thresholds represent 50% paw withdrawal threshold. Animals were tested prior to surgery to establish baseline sensitivity. Two weeks after surgery, mice were tested before and after ethanol injections (saline, or 0.5, 1.0, or 2.0 g/kg of ethanol, i.p.). Repeated tests occurred at 5-,10-, 20-, and 60-minutes after injection. Ethanol doses were randomized and administered at least 48 hours apart to minimize the development of tolerance and withdrawal. Experimenter was blinded to the treatment. At the highest dose of ethanol some of the paw withdrawal thresholds in the male mice exceeded up the upper limit of the test (2.56g), thus, a value of 2.56g was used.

Behavioral testing: dynamic weight bearing (DWB)

To assess the effect of SNI surgery and acute ethanol administration on DWB, mice were recorded using a DWB apparatus (Dynamic Weight Bearing 2.0, Bioseb, France) prior to and 3 weeks after SNI surgery. At the post-test timepoint, mice received an injection of saline or 0.5,1.0, or 2.0 g/kg ethanol (i.p.) immediately prior to placement into the DWB apparatus. Mice were acclimated for 5 minutes prior to 5 min of recorded behavioral data. Throughout testing, mice were able to freely rear and explore the chamber, while a grid of floor force sensors measured the weight placed on each hindpaw and an overhead camera recorded the animal’s position and posture. Paw detection settings were defined with a central pixel sensitivity threshold of 0.8 g and an adjacent pixel threshold of 0.2 g, allowing for reliable detection of hindpaws. A minimum of 90 seconds of video was validated using a combination of Bioseb’s automatic validation (Level 1 and 2) and manual validation to ensure that paws were accurately identified.

Blood ethanol concentrations

After all behavioral studies, mice were injected with either 0.5 g/kg or 2.0 g/kg of ethanol in a counterbalanced fashion. Blood was collected by submandibular bleed either 5- or 20-minutes after injection. Blood ethanol concentrations (BECs) were measured using an AM1 Alcohol Analyzer (Analox Instruments).

Statistics

All statistics were conducted using Prism Graphpad. Analyses consisted of between-subjects or repeated measures (rm) ANOVA. In the case of missing values in repeated measures tests, a mixed-effects design was used. Significant interactions were followed by Sidak’s post hoc comparisons. For area under the curve (AUC) analysis, the AUC of the paw withdrawal threshold of the ipsilateral paw from 0 to 60 minutes for each animal was generated and averaged per sex for subsequent analysis.

Results

SNI induces mechanical allodynia and shifts in weight bearing in males and females

To confirm that male and female mice developed mechanical allodynia after SNI, basal paw withdrawal thresholds on the paw ipsilateral to surgery were assessed. A 2-way between-subjects ANOVA indicated a main effect of surgery [F(1,37) = 314.1, p < 0.0001] but no effect of sex [F(1,37) = 1.478, p = 0.2319] or sex x surgery interaction [F(1,37) = 2.987, p = 0.0923; Fig. 1A], indicating that SNI induced reductions in paw withdrawal thresholds that were similar in male and female mice.

Figure 1.

Figure 1.

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Experimental timeline and model characterization. (A) Adult male and female mice underwent SNI surgery following assessment of basal paw withdrawal thresholds using von Frey and weight bearing with the DWB assay. The effect of ethanol administration on mechanical allodynia and weight bearing were assessed two weeks following SNI surgery. Submandibular bleeds occurred after all behavior was completed. (B) SNI decreased paw withdrawal thresholds in male and female mice. Paw withdrawal thresholds did not change in mice that underwent sham surgery. (C) Following surgery, mice SNI shifted weight from the ipsilateral hindpaw to the contralateral hindpaw (reduced ipsilateral/contralateral weight bearing ratio) compared to mice that underwent sham surgery. All data expressed as mean +/− SEM. ****p<0.0001 SNI vs. Sham

SNI induced similar shifts in weight bearing in males and females. Ratios of weight on the ipsilateral versus contralateral paw were assessed before and after SNI surgery in male and female mice. A 3-way rmANOVA indicated a significant time x surgery interaction [F(1,29) = 109.6, p < 0.0001], but no main effect of sex [F(1,29) = 1.532, p = 0.2257] or sex interactions [overall interaction: F(1,29) = 0.6495, p = 0.4269; surgery x sex: F(1,29) = 0.2155, p = 0.646; time x sex: F(1,29) = 3.449, p = 0.0735; Fig. 1B]. Post hoc comparisons indicated a lower ipsilateral/contralateral ratio following surgery in the SNI mice (p < 0.0001) than in the sham mice (p = 0.0854).

Ethanol reverses mechanical allodynia in males

To assess the effect of acute ethanol administration on mechanical hypersensitivity in the context of chronic pain, mice with SNI underwent von Frey testing following acute intraperitoneal (i.p.) administration of ethanol.

At the 0.5g/kg dose of ethanol, a 3-way rmANOVA (surgery x paw x time) revealed a time-dependent effect of ethanol administration on paw withdrawal thresholds with significant interactions between time x paw [F(3.378, 77.70) = 3.498, p = 0.0156; Greenhouse-Geisser corrected] and surgery x paw [F(1, 23) = 22.72, p < 0.0001; Fig. 2A]. No 3-way interaction was observed [F(4, 92) = 1.024, p = 0.3992]. Significant 2-way interactions were deconstructed using post hoc comparisons. Prior to ethanol administration, the SNI mice had lower ipsilateral paw withdrawal thresholds (p = 0.0123). After ethanol administration, SNI ipsilateral paw withdrawal thresholds were increased at 10 and 20 minutes vs. baseline (p = 0.0253, p < 0.01, respectively). Consistent with this, ipsilateral paw withdrawal thresholds were significantly lower in SNI mice than shams at 5 minutes (p=.0059) and 60 minutes (p=.0229), but not at 10 or 20 minutes (p’s > 0.99). No effect was observed on the contralateral paw for SNI animals or on either paw for the sham-injured animals (all p’s > 0.9). Together these results indicated SNI male mice exhibited mechanical allodynia that was fully reversed from 10–20 minutes after injection of 0.5 g/kg ethanol.

Figure 2.

Figure 2.

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Ethanol effects on paw withdrawal thresholds in male and female mice with SNI. (A) In SNI males, administration of 0.5 g/kg ethanol (i.p) fully reversed mechanical allodynia at the 10- and 20-minute timepoints. (B) In SNI males, administration of 1.0 g/kg ethanol reversed mechanical allodynia at the 5-, 10-, and 20-minute timepoints. (C) In SNI males, administration of 2.0g/kg ethanol reversed allodynia at all time points. In sham males, 2.0g/kg ethanol had an antinociceptive effect at 5-, 10-, and 20-minute timepoints. (D) Administration of 0.5 g/kg ethanol did not alter SNI induced-mechanical allodynia in female mice. (E) Administration of 1.0 g/kg ethanol also did not alter allodynia in SNI females. (F). Administration of 2.0 g/kg ethanol reversed SNI-induced mechanical allodynia in females only at the 5-minute timepoint. There was no effect on sham females. All data expressed as mean +/− SEM. *p < 0.05, **p < 0.01 SNI vs sham ipsilateral paws. Green shading indicates p < 0.05 SNI ipsilateral paw post- vs pre-ethanol. ##p < 0.01 sham ipsilateral paw post- vs. pre-ethanol.

At the 1.0g/kg dose of ethanol, a 3-way rmANOVA (surgery x paw x time) revealed a time-dependent effect of ethanol administration on paw withdrawal thresholds with a significant 3-way interaction [F(4, 92) = 2.579, p = 0.0425; Greenhouse-Geisser corrected; Fig. 2B]. Additionally, there was a significant two-way interaction between surgery x paw [F(1, 23) = 11.46, p = 0.0025]. Sidak’s post hoc tests revealed lower withdrawal thresholds in the SNI ipsilateral paw compared to sham ipsilateral paw at baseline (p = 0.0002). Ethanol administration increased withdrawal thresholds for the SNI ipsilateral paw at 5 (p = 0.0197), 10 (p < 0.0001), and 20 minutes (p = 0.0173) versus baseline. No effect of time was observed on the contralateral paw or for the sham-injured animals (all p’s > 0.9) after ethanol administration. These results indicate that 1.0 g/kg injection of ethanol fully reversed mechanical allodynia between 5–20 minutes in the SNI males without affecting paw withdrawal thresholds in uninjured paws.

At the 2.0g/kg dose of ethanol, a 3-way rmANOVA (surgery x paw x time) revealed a time-dependent effect of ethanol administration on paw withdrawal thresholds with significant 2-way interactions between time x paw [F(3.443, 79.18) = 4.060, p = 0.0071; Greenhouse-Geisser corrected] and surgery x paw [F(1, 23) = 9.223, p = 0.0059; Fig. 2C]. The 3-way interaction was not significant [F(4, 92) = 0.1451, p = 0.9647]. Sidak’s post hoc comparisons indicated lower withdrawal thresholds in the SNI versus sham ipsilateral paw prior to ethanol administration (p = 0.0010), but not at any time point after ethanol administration (all p’s > 0.800) Consistent with this, paw withdrawal thresholds for the ipsilateral paw of SNI mice were increased compared to baseline at 5 (p = 0.0069), 10 (p = 0.0018), and 20 (p = 0.0072) minutes following injection. Additionally, the uninjured paws exhibited increased thresholds at several timepoints: The sham ipsilateral paw at 5 (p = 0.0005), 10, (p = 0.0001) and 20 minutes (p = 0.0040) and the SNI contralateral paw at 10 (p = 0.0048) and 20 minutes (p = 0.0040). These data indicate that ethanol fully reversed mechanical allodynia in SNI animals and had an antinociceptive effect in the uninjured paws.

Ethanol only reverses mechanical allodynia in females at high doses

To determine the effect of acute ethanol administration on mechanical sensitivity during chronic pain, female mice received injections of 0.5, 1.0, and 2.0 g/kg ethanol immediately prior to testing in the von Frey assay. At the 0.5 g/kg dose, a 3-way rmANOVA (surgery x paw x time) revealed a time-dependent effect of ethanol administration on paw withdrawal thresholds with a significant 2-way interaction between surgery x paw [F(1, 13) = 50.95, p < 0.0001; Greenhouse-Geisser corrected; Fig. 2D]. The three-way interaction was not significant, [F(4, 52) = 0.5777 p=0.681]. A Sidak’s post hoc comparison between SNI and Sham ipsilateral paws at baseline revealed a significantly reduced withdrawal threshold in the SNI ipsilateral paw (p = 0.0307). After ethanol administration, paw withdrawal thresholds in the SNI ipsilateral paw were not increased at any time point compared to baseline (all p’s > 0.70). Consistent with this, paw withdrawal thresholds in uninjured paws were also not affected by 0.5 g/kg ethanol administration (all p’s > 0.90). These data indicate that 0.5g/kg ethanol did not have an antinociceptive or antiallodynic effect in female mice.

At the 1.0g/kg dose of ethanol, a 3-way rmANOVA (surgery x paw x time) revealed a time-dependent effect of ethanol administration on paw withdrawal thresholds with a significant 3-way interaction [F(4, 52) = 2.833, p = 0.0336; Greenhouse-Geisser corrected]. Additionally, there was a significant two-way interaction between surgery x paw [F(1, 13) = 6.564, p = 0.0237; Fig. 2E]. Sidak’s post hoc comparisons indicated lower withdrawal thresholds in the ipsilateral paw of SNI vs sham mice prior to ethanol administration (p = 0.0216). However, ethanol did not affect paw withdrawal thresholds for the SNI ipsilateral paw at any timepoint compared to baseline (all p’s < 0.70). Acute administration of 1.0g/kg ethanol also did not influence paw withdrawal thresholds in uninjured females (all p’s > 0.80), together indicating that 1.0 g/kg injection of ethanol did not affect paw withdrawal thresholds in female mice irrespective of pain status.

At the 2.0g/kg dose of ethanol, a 3-way rmANOVA (surgery x paw x time) revealed a time-dependent effect of ethanol administration on paw withdrawal thresholds with a significant surgery x paw interaction [F(1, 13) = 35.73 p < 0.0001; Greenhouse-Geisser corrected; Fig. 2F]. The 3-way interaction was not significant [F(4, 52) = 0.9945 p = 0.4189; Greenhouse-Geisser corrected]. Post hoc comparisons indicated a significant decrease in paw withdrawal thresholds at baseline between SNI ipsilateral and Sham ipsilateral hindpaws (p = 0.0039). Administration of 2.0g/kg ethanol increased paw withdrawal thresholds in the SNI ipsilateral paw as compared to baseline at 5 minutes (p = 0.0132). Consistent with this, a post hoc comparison between SNI and sham ipsilateral paws indicated no significant difference at 5 minutes (p > 0.99). Meanwhile, paw withdrawal thresholds in the uninjured paws were unaffected at all timepoints after injection (all p’s > 0.90). These results show that 2.0g/kg of ethanol fully reversed mechanical allodynia in SNI female mice 5 minutes after injection, but not at later timepoints or in sham mice.

Sex differences in antinociceptive and antiallodynic effects of ethanol

To further interrogate the emerging sex differences in the antiallodynic and antinociceptive effects of ethanol, responses in male and female mice were directly compared. The area under the curve (AUC) from –5 to 60 minutes after ethanol injection was calculated for each animal in the sham (Fig. 3A) and SNI (Fig. 3B) groups and compared between sexes. In sham animals, a 2-way ANOVA (sex x dose) revealed a significant interaction [F(3, 48) = 11.75 p < 0.0001; Greenhouse-Geisser corrected; Fig. 3A]. A Sidak’s post hoc comparison between sham males and females indicated significantly greater paw withdrawal thresholds in males at 1.0 (p = 0.0051) and 2.0 (p < 0.0001) g/kg ethanol, suggesting that females are less sensitive to the antinociceptive effects of ethanol at these doses.

Figure 3.

Figure 3.

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Sex differences in ethanol effects on paw withdrawal thresholds. (A) Sham male mice exhibited greater paw withdrawal thresholds at 1.0g/kg and 2.0g/kg doses of ethanol than sham female mice. (B) SNI male mice displayed greater paw withdrawal thresholds than SNI females at the 2.0g/kg dose. (C) In female mice, BECs were transiently elevated compared to males at 5-minutes, but not 20-minutes, after a 0.5 g/kg ethanol injection. (D) BECs in females were greater than in males following a 2.0g/kg ethanol injection. All data expressed as mean +/− SEM. **p < 0.01 ***, p < 0.001, **** p < 0.0001 males vs. females; ###p < 0.001, ####p < 0.0001, 20 minutes vs. 5 minutes.

In the SNI animals, a 2-way ANOVA (sex x dose) revealed a significant interaction [F(3, 60) = 10.59, p < 0.0001; Greenhouse-Geisser corrected; Fig. 3B]. A Sidak’s post hoc comparison between SNI males and females indicated a significant increase in withdrawal threshold AUC at 2.0g/kg ethanol, suggesting female mice were less sensitive to the antiallodynic effects of high dose ethanol administration (p < 0.0001).

Blood ethanol concentrations are sex-and time-dependent

To determine if the antinociceptive and antiallodynic effects of ethanol in male and female mice were associated with differing BECs, blood was collected after injection of 0.5 g/kg or 2.0 g/kg ethanol. Following administration of 0.5 g/kg of ethanol, a 3-way mixed-effects analysis (surgery x sex x time) revealed a significant main effect of time [F(1, 54) = 61.18, p < 0.0001] and sex [F(1, 54) = 5.101, p = 0.0280] on BEC. There was also a significant time x sex interaction [F(1, 54) = 8.684, p = 0.0047; Fig. 3C]. Since surgery did not impact BECs (all p’s > 0.58), data from SNI and sham data were consolidated, revealing a significant interaction [F(1, 58) = 8.277, p = 0.0056]. A Sidak’s post hoc comparison revealed that BECs were reduced in both males (p =0.0004) and females (p < 0.0001) at 20 minutes versus 5 minutes. BECs were significantly higher in females than males at 5 minutes (p = 0.0014), but not at 20 minutes (p = 0.8823). Following administration of 2.0g/kg ethanol, a 3-way mixed-effects analysis (surgery x sex x time) revealed a significant main effect of sex [F(1, 28) = 8.932, p = 0.0058] with greater BECs in females, but neither surgery nor time influenced BECs at this dose (Fig 3D).

SNI leads to changes in dynamic weight bearing in male and female mice

To test the hypothesis that SNI surgery would shift weight bearing from the ipsilateral to the contralateral paw, the ipsilateral/contralateral ratio of weight bearing was assessed where a ratio of 1.0 would indicate equal weight distribution on each hindpaw. In males, a 2-way mixed-effects analysis (surgery x dose) revealed a surgery-dependent effect of hindpaw weight bearing, with a significant 2-way interaction [F(4, 60) = 6.872, p = 0.0001; Greenhouse-Geisser corrected; Fig. 4A]. A Sidak’s post hoc comparison between SNI and sham revealed no significant difference prior to surgery (p = 0.9082). However, after surgery, the ipsilateral/contralateral ratio in SNI mice was significantly reduced compared to sham mice (p = 0.0010) indicating that male SNI mice shift their weight bearing towards the uninjured hind paw. Ethanol was administered to determine whether doses of ethanol that reduced mechanical allodynia in the von Frey assay were also able to reverse disrupted weight bearing after SNI surgery. Sidak’s post hoc comparison showed that administration of 0.5g/kg ethanol trended towards reversal and 1.0 and 2.0 g/kg ethanol fully reversed disrupted weight bearing when compared to sham controls (0.5g/kg: p = 0.046; 1.0g/kg ethanol: p = 0.0688; 2.0g/kg ethanol: p = 0.704)

Figure 4.

Figure 4.

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SNI-induced shifts in dynamic weight bearing are reversed by ethanol. (A) SNI male mice shifted weight to the contralateral paw (reduced ipsilateral/contralateral ratio) following SNI. This shift in DWB was reversed by administration of 1.0g/kg and 2.0g/kg of ethanol. (B) Female mice shifted weight to the contralateral paw following SNI. This shift was reversed by 1.0 and 2.0g/kg of ethanol. (C) In SNI male mice, paw weight as a % of total body weight increased on the contralateral paw following surgery. Weight on the contralateral paw was similar between SNI and sham mice following ethanol administration (D) There was a trend towards a decrease in % paw weight on the ipsilateral paw in female SNI mice following surgery. Following 1.0 and 2.0 g/kg ethanol administration, % paw weight on the SNI ipsilateral paw did not differ from sham animals. All data expressed as mean +/− SEM. **p < 0.001, ***p < 0.001 ipsilateral vs. contralateral. ##p < 0.01 ipsilateral paw post- vs. pre surgery.

In females, a 2-way mixed-effects analysis (surgery x dose) showed a significant 2-way interaction between surgery x dose [F(4, 47) = 5.495, p = 0.0010; Greenhouse-Geisser corrected; Fig. 4B]. As in males, comparison between SNI and sham females revealed no significant difference between ipsilateral/contralateral ratio prior to surgery (p = 0.999). However, after surgery, the ipsilateral/contralateral ratio was reduced in SNI mice compared to sham mice (p = 0.002), indicating that female SNI mice shift their weight bearing towards the uninjured hind paw. Next, the effect of doses of ethanol on disrupted weight bearing after SNI surgery were assessed. Post hoc comparisons showed that 0.5 g/kg ethanol was unable restore ipsilateral/contralateral ratio (p = 0.008 vs. sham), while at higher doses there was no difference between sham and SNI, suggesting these doses reversed deficits in ipsilateral/contralateral ratio of weight bearing (1.0g/kg ethanol: p = 0.291; 2.0g/kg ethanol: p = 0.244).

To expand upon the findings of shifted ipsilateral/contralateral hindpaw weight bearing, weight bearing per paw was calculated as a percent of total body weight. In males, a 3-way mixed-effects analysis (paw x surgery x dose) revealed a significant 3-way interaction [F(4, 50) = 3.040, p = 0.0255; Greenhouse-Geisser corrected; Fig. 4C]. Post hoc comparions between ipsilateral and contralateral paws at each dose revealed a significant difference between hindpaw weight bearing in SNI animals at the post surgery timepoint (p = 0.0001), but not prior to surgery or after 0.5, 1.0, 2.0 g/kg of ethanol (all p’s > 0.5). There were no differences in sham animals in weight bearing between the ipsilateral and contralateral paw (all p’s > 0.9). Since a significant difference in weight bearing after surgery was observed in the SNI animals, weight bearing on each paw was compared to the pre-surgery baseline, revealing a significant increase in weight bearing on the SNI contralateral hindpaw after SNI surgery (p = 0.0093). This was reversed by administration of 0.5, or 1.0g/kg or 2.0 g/kg of ethanol (all p’s > 0.4). No differences in weight bearing on the SNI ipsilateral paw compared to baseline were observed (all p’s > 0.84). These data suggest that decreases in the ipsilateral/contralateral ratio are primarily driven by increases in weight bearing on the contralateral paw and are reversed by ethanol.

In female mice, a 3-way mixed-effects analysis (paw x surgery x dose) revealed a significant 3-way interaction [F(4, 42) = 5.506, p < 0.0012; Greenhouse-Geisser corrected; Fig. 4D]. Post hoc comparisons between ipsilateral and contralateral paws at each dose revealed a significant difference between hindpaw weight bearing in SNI animals at the post surgery timepoint (p = 0.006), after 0.5g/kg (p = 0.0043), but not prior to surgery or after 1.0 or 2.0 g/kg of ethanol (all p’s > 0.4). No differences in weight bearing between paws were observed at any dose in sham mice (all p’s > 0.9). When weight bearing on each paw was compared to the pre-surgery baseline, a trend (p = 0.0512) towards a decrease in weight bearing on the ipsilateral paw was observed, but no change in the contralateral paw (p > 0.99) These data suggest that decreases in the ipsilateral/contralateral ratio that are reversed by high doses of ethanol are primarily driven by decreases in weight bearing on the ipsilateral paw in female mice.

Another strategy that the animals may use to avoid bearing weight on the injured hindlimb is by shifting weight towards the forelimbs (Clarke et al., 1997). Therefore, the forepaw/hindpaw ratio was calculated to see if SNI surgery or ethanol affected this behavior.

In male mice, a 2-way mixed effects analysis (surgery x dose) revealed a significant main effect of dose on forepaw/hindpaw ratio [F(1.305, 20.23) = 82.78 p < 0.0001; Greenhouse-Geisser corrected; Fig. 5A]. A post hoc comparison between the pre-surgery and post-surgery timepoint showed SNI surgery did not affect forepaw/hindpaw ratio in either SNI (p = 0.1985) or sham (p = 0.5989) males in the absence of ethanol. However, comparison between the saline and ethanol conditions revealed significant increases in forepaw/hindpaw ratio after 1.0g/kg ethanol and 2.0g/kg in SNI (p’s < 0.0041) and sham male mice (p’s < 0.006). These results suggest that the higher doses of ethanol increase forepaw/hindpaw ratio independent of surgery status.

Figure 5.

Figure 5.

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Changes in weight bearing and mobility following SNI. (A) Administration of 1.0 g/kg and 2.0 of ethanol increased the proportion of weight bearing on the forepaw vs hind paw (forepaw/hindpaw ratio) in male mice. (B) A similar pattern was observed in female mice. (C) Mobility time during the DWB assay was not affected by ethanol administration or surgery in male mice. (D) Female mice with a SNI spent a greater percentage of the DWB assay mobile than sham controls. Ethanol did not impact mobility time in female mice. All data expressed as mean +/− SEM. *p < 0.05 SNI vs. Sham; ##p < 0.01, ###p < 0.001, ####p < 0.0001 ethanol treated vs. baseline.

Female mice exhibited similar effects of ethanol on forepaw/hindpaw ratio. A 2-way mixed effect analysis (surgery x dose) revealed significant main effects of dose [F(4, 46) = 20.17 p<0.0001; Greenhouse-Geisser corrected] and surgery [F (1, 13) = 9.571 p = 0.0085; Greenhouse-Geisser corrected; Fig. 5B]. A Sidak’s post hoc comparison showed that SNI surgery did not affect forepaw/hindpaw ratio versus baseline in the SNI (p = 0.5099) or sham females (p > 0.9999). Ethanol administration at the 1.0g/kg and 2.0g/kg doses increased forepaw/hindpaw ratios in SNI (p’s < 0.0002) and sham mice (p = 0.0015) compared to baseline. Additionally, at 2.0g/kg of ethanol SNI mice have a significantly higher forepaw/hindpaw ratio versus shams (p = 0.0211). These results suggest that the higher doses of ethanol increase forepaw/hindpaw ratio in female mice, but to a greater extent in SNI animals.

Ethanol does not affect mobility time in male or female mice during DWB

To confirm that increased forepaw weight bearing after ethanol was not due to the sedative effects of ethanol, the % of total time that mice were mobile during DWB recordings was calculated. In male mice, a 2-way mixed effect analysis (surgery x dose) showed no significant main effects of surgery [F(2.866, 44.43) = 2.660; p = 0.062; Greenhouse-Geisser corrected] or dose [F(1, 16) = 0.3580; p = 0.558; Greenhouse-Geisser corrected; Fig. 5C]. In female mice, a 2-way mixed effect analysis (surgery x dose) revealed a main effect of surgery [F(1, 59) = 17.32, p < 0.0001; Greenhouse-Geisser corrected], but no effect of dose [F(2.815,41.52) = 1.0007, p = 0.3956; Greenhouse-Geisser corrected Fig. 5D]. These data show that ethanol did not affect mobility time in mice during DWB recordings.

Discussion

Together, findings from these experiments demonstrated that SNI led to similar levels of mechanical allodynia and shifts in DWB in male and female mice. Ethanol dose-dependently reversed mechanical allodynia and shifts in DWB, where female mice required higher doses of ethanol to reverse mechanical allodynia and shifts in weight bearing than males (Fig. 6). This difference was not explained by differences in BECs.

Figure 6.

Figure 6.

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Summary of dose- and sex-dependent effects of ethanol in the von Frey and dynamic weight bearing assays. (A) In sham female mice, paw withdrawal thresholds were unaffected by all doses of ethanol. Sham male mice exhibited antinociception at the highest dose of ethanol. Mechanical allodynia was present in both male and female SNI mice. All tested doses of ethanol reversed mechanical allodynia in male mice, while female mice only exhibited reversal of mechanical allodynia at the highest dose. (B) Hindpaw weight distribution in sham mice was not affected by all tested doses of ethanol. SNI male and female mice presented with decreased weightbearing on the ipsilateral hindpaw. All tested doses of ethanol restored hindpaw ratios in male SNI mice, while reversal was only achieved at the highest tested dose in female mice. Figure created with biorender.com.

SNI led to pronounced mechanical allodynia as indicated by robust reductions in paw withdrawal thresholds. No significant sex differences in basal or SNI-induced thresholds were observed, consistent with previous studies using the SNI model (Muralidharan et al., 2020; Sheehan et al., 2021; Shen et al., 2014) (Fig. 1A). Ethanol administration fully reversed mechanical allodynia in SNI mice in the von Frey assay, albeit at different doses and time points in males and females. In SNI males, mechanical allodynia was reversed at all doses of ethanol tested. This suggests that even low doses of ethanol reduced hypersensitivity in males in the context of chronic pain. At the 2.0 g/kg dose, there was also an antinociceptive effect of ethanol in the sham males. This is consistent with other reports in hotplate and tail flick assays where doses of 2.0 g/kg of ethanol or higher yield antinociceptive effects (Campbell et al., 2006; Neddenriep et al., 2019). These results suggest that the antiallodynic effects of ethanol are observed at lower doses than antinociceptive effects in males, which has been reported to occur in other chronic pain models (Neddenriep et al., 2019). This is consistent with findings that females are resistant to the effects of voluntary ethanol intake on cold and mechanical allodynia (Bilbao et al., 2019) at these lower doses, but exhibit reduced pain-induced conditioned place aversion at higher doses of ethanol. This may be one factor underlying increased alcohol drinking in chronic pain patients who may be more susceptible to using alcohol for pain relief, and act as an entry point into the feedback loop between chronic pain and alcohol use. Notably, male, but not female, mice were found not to escalate ethanol intake in response to chronic inflammatory pain (Yu et al., 2019). While this may reflect sex differences in the effect of chronic pain on ethanol intake, this may also result from higher basal ethanol intake in female mice (Barker et al., 2010; Middaugh et al., 1992, 1999; Yoneyama et al., 2008). Indeed a similar absence of escalation in intake is seen in female mice with higher baseline intake under chronic intermittent ethanol exposure parameters that escalate ethanol drinking in males (Jury et al., 2017).

In contrast to findings in males, low doses of ethanol did not impact paw withdrawal thresholds in females. While the highest dose of ethanol did cause a full reversal in females, this effect was only present at the 5-minute timepoint after injection. In contrast, males exhibited increased paw withdrawal thresholds for up to 60 minutes (Fig. 2 E-F). This suggests that in female mice with SNI, antiallodynic efficacy and duration of ethanol action is blunted compared to males. This conclusion is further supported by direct statistical comparisons of AUC between males and females at this dose (Fig. 3 A-B).

This sex difference contrasts with previous findings from Neddiereip and colleagues (2019) which report no sex differences in the antiallodynic and antinociceptive effects of ethanol administered via gavage to naïve or chronic constriction injury mice (CCI). The discrepancy between our results and previous reports may be due to differences in the chronic pain models or the route of administration of ethanol. For example, there are reports of sex differences in the development and of pain-related behavior in the CCI mode (Vacca et al., 2014), which may relate to distinct mechanisms underlying pain development and/or alleviation of pain in males and females (Fillingim et al., 2009; Racine et al., 2012)

Given the attenuated antiallodynic effect of ethanol in females, it was important to confirm that BECs were not lower in females compared to males. BECs were measured at 5 and 20-minutes after ethanol injection (Fig. 3C, D). After 0.5g/kg of ethanol injection females exhibited higher BECs than males, suggesting that the lack of antiallodynic effect in females is not due to lower BECs. At the 2.0g/kg dose, there was a main effect of sex in BECs with females displaying higher BECs than males. Additionally, BECs were stable between 5 minutes and 20 minutes at 2.0g/kg, suggesting that the loss of antiallodynic effect at 20 minutes in females was not due to elimination of ethanol. The loss of the effect of ethanol despite stable BECs may be attributed to acute functional tolerance (Neddenriep et al., 2019).

In addition to robust mechanical allodynia, SNI shifted weight bearing behavior in both males and females. Changes in weight bearing have been demonstrated in a variety of chronic pain models (Clarke et al., 1997; Griffioen et al., 2015; Mogil et al., 2010). Animals in chronic pain with paw hypersensitivity will compensate by shifting weight from the affected limb to other limbs, and this has been hypothesized to represent a guarding/avoidance behavior (Coulthard et al., 2003; Mogil et al., 2010). It was hypothesized that SNI would shift weight bearing away from the ipsilateral hindpaw onto either the contralateral hindpaw or the forepaws. While SNI did not lead to any increases in forepaw weight bearing, shifts in hindlimb weightbearing were altered to a similar degree in male and female SNI mice, but not sham controls (Fig. 4). This was congruent with the lack of sex differences after SNI surgery in mechanical allodynia in the von Frey assay (Fig. 1A).

The strategy used to redistribute weight was distinct for males and females. Changes in weight bearing on each hindpaw were individually calculated as a percent of total body weight, and the data indicate that shifts in hindpaw weight bearing after SNI were driven by distinct changes in behavior in male and female mice. Male mice primarily increased weight bearing on the contralateral hindpaw after surgery, similar to previous reports (Blaszczyk et al., 2018; Sheehan et al., 2021). In contrast, female mice decreased weight bearing on the ipsilateral paw. This also highlights the importance of measuring the contribution of individual paws and not relying solely on measures of paw/paw weight distribution in the DWB assay.

Ethanol shifted weight distribution in a dose- and time-dependent manner in both males and females. Similar to the pattern observed in the von Frey assay, ethanol effects on behavior were more pronounced in males than in females. This suggests that there may be a relationship between hindpaw sensitivity generated by the SNI model and shifts in hindpaw weight bearing measured with DWB. It was also observed that ethanol increased forepaw weight bearing independent of sex or surgery status at the 2.0g/kg dose. However, mobility time was not affected by any of the doses tested. This suggests that increased forepaw weight bearing is not due to the sedative effects of ethanol at these doses. However, this does not preclude the possibility that higher doses of ethanol produce motor impairment. Indeed, impaired performance in the rotarod test occurs after administration of the higher doses of ethanol used in this study (Pandey, 2012; Smoker et al., 2019; Tornberg et al., 2007) which may contribute to the increased paw withdraw thresholds observed in both SNI and sham control animals following administration of the higher doses of ethanol. One challenge in interpreting DWB data in nerve injury models, is determining whether the changes in weight bearing are observed due to the pain and hypersensitivity caused by the injury or other aspects of the injury, such as motor deficits (Sik Naa et al., 1996). SNI animals have large portions of their hindpaw denervated, so they may learn to avoid contact by walking on the denervated portion to avoid allodynia. Many groups have reported inflammatory models of chronic pain alter weight bearing, and changes in weight bearing are reversed by appropriate anti-inflammatory compounds (Ängeby Möller et al., 2018; Clarke et al., 1997; Coulthard et al., 2003). However, it has been argued that changes in weight bearing are unrelated to hypersensitivity and pain in the context of neuropathic pain due to lack of analgesic efficacy of opioids and gabapentinoids in the catwalk assay (Mogil et al., 2010). In the context of chronic pain, weight bearing in freely moving animals has primarily been measured via the catwalk, where animals walk across a smooth surface and the intensity of paw prints is a correlate of weight bearing (Tétreault et al., 2011). It is important to consider the differences between the catwalk and the method used in this study because animals walking on a catwalk are engaged in a behavioral task that is motivated by social reward while our animals are freely moving. Additionally, the animals in this study are walking on a surface that may provide stimulation necessary to trigger dynamic allodynia. Despite both methods being interpreted as “weight bearing” these differences may explain discrepancies between the current findings and studies using the catwalk platform. Alternatively, the differences may be attributed to the broad pharmacological effects of ethanol.

Repeated ethanol administration can induce tolerance. In order to avoid the effect of tolerance on behavioral outcomes, ethanol doses were randomized and each test was separated by at least 48 hours. Others have reported that tolerance to the antiallodynic and antinociceptive effects of ethanol does not occur following four doses and can take up to 7–10 days of chronic administration (Kotagale et al., 2022; Neddenriep et al., 2019). For these reasons, it is not expected that the observed sex differences reflect tolerance to the effects of ethanol.

These results are consistent with studies that have used the same dynamic weight bearing apparatus in other nerve injury models. In the spinal nerve ligation model, decreases in ipsilateral hindpaw and increases in contralateral hindpaw weight were reported (Blaszczyk et al., 2018). In a model of sciatic nerve cuff injury model which induces hyperalgesia that later resolves, shifts in weight bearing were temporally correlated with hyperalgesia (Sheehan et al., 2021), potentially consistent with the current findings of reversal of mechanical hypersensitivity altered weightbearing by ethanol in the SNI model. While it may be difficult to disentangle changes in DWB due to injury and changes in DWB due to hypersensitivity caused by injury, DWB captures aspects of the pain experience that are not captured with von Frey. While von Frey can measure evoked allodynia, suffering in chronic pain extends well beyond hypersensitivity. Since measures of weight bearing are non-evoked, they may better model clinical and translational outcomes for chronic pain, which involves non-evoked pain. This highlights the importance and value of integrating multiple pain assays in interpreting findings.

Together, these findings describe similar outcomes of SNI in male and female mice, with distinct patterns of deficit reversal by ethanol in the von Frey and DWB assays. These findings parallel the broader literature and emphasize the importance of understanding the analgesic properties of ethanol. Furthermore, the doses of ethanol used are consistent with doses that have been reported to reduce both pain threshold and intensity in healthy human subjects (Thompson et al., 2017). While women are more likely than men to have both chronic pain and AUD, men are more likely to report using alcohol to deal with chronic pain (Egli et al., 2012; Riley & King, 2009). It remains an open question whether ethanol antiallodynia or analgesia are attenuated in women with chronic pain as compared to men, due to the unavailability of data. Our current findings characterize profound sex differences in response to ethanol in the SNI model and inform our understanding of factors placing chronic pain patients at higher risk for increased alcohol use and development of AUD.

Supplementary Material

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Acknowledgements

This research was supported by the National Institute on Alcohol Abuse and Alcoholism under Award Number R21AA027629 (JMB) and by the Mary DeWitt Pettit, MD Foundation Fellowship (JMB). The authors thank Dr. Seena Ajit and her laboratory for training in and use of dynamic weight bearing equipment.

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