Chronic alcohol drinking delays recovery from capsaicin- and nerve injury-induced hypersensitivity in mice

Abstract

Chronic pain and alcohol use disorder (AUD) are major health concerns that significantly impair quality of life. Persistent pain is common in individuals with alcohol dependence, and alcohol is commonly used by chronic pain patients for self-medication. The neural mechanisms linking these conditions remain unclear. We hypothesized that chronic alcohol exposure induces hypersensitivity in multiple modalities and increases the duration of recovery in acute and persistent pain models. Using the two-bottle free-choice alcohol consumption paradigm in adult mice, alcohol-induced hypersensitivity (AIH), indicated by reduced mechanical withdrawal thresholds, developed after 4-6 weeks of alcohol consumption compared to age- and sex-matched water-consuming controls. Alcohol-consuming female mice, but not male mice, developed cold hypersensitivity after AIH emerged. To assess the impact of chronic alcohol consumption on acute and persistent pain, we used intraplantar capsaicin and sciatic nerve crush models, respectively. In the capsaicin model, water-treated, but not alcohol-treated, mice recovered from hypersensitivity by 24 hours post-injection. In the sciatic nerve crush model, alcohol-consuming mice exhibited slower recovery of mechanical withdrawal thresholds compared to water-consuming controls. While mechanical hypersensitivity in water-consuming mice returned to pre-surgical thresholds by 3-4 weeks post-surgery, recovery in alcohol-consuming mice was both delayed and partial. Surgical intervention did not impact alcohol intake. Overall, our results suggest that chronic voluntary alcohol consumption facilitates the transition to chronic pain by prolonging hypersensitivity and delaying recovery from injuries.

Keywords: Alcohol; chronic pain; hypersensitivity; injury; mice; recovery.

Figure 1.. Diagram of the timeline of voluntary alcohol consumption using the intermittent access two-bottle choice model.

Mechanical thresholds were measured on Tuesdays or Thursdays (water only days) using von Frey filaments and computed using the up down method. In some cases, cold sensitivity was assessed on the same days immediately following von Frey testing. Sciatic nerve crush was performed immediately after von Frey testing during week 6, and capsaicin injections were performed immediately after week 8 testing. Figure created with BioRender.com.

Figure 2.. Alcohol consumption is greater in C57BL/6J females.

Female mice voluntarily drink more alcohol than male mice. Average alcohol consumed per day measured relative to (A) body weight and (B) % preference for alcohol for males (n=15) and females (n=13). RM ANOVA, main effect of sex **p=0.002, ****p<0.0001.

Figure 3.. Chronic alcohol consumption produces alcohol-induced mechanical hypersensitivity (AIH).

(A) No difference in magnitude of mechanical AIH between males and females (n=13–14/group/sex, Sidak’s multiple comparisons test p>0.05, males vs. females weeks 4–6), determined by changes in paw withdrawal thresholds (PWT). Significant reductions in PWT (relative to week 0 baseline) appeared from 4–6 weeks of alcohol consumption (Sidak’s post hoc test, ⁺⁺p<0.01, ⁺⁺⁺p<0.001, ⁺⁺⁺⁺p<0.0001). (B) Alcohol-consuming mice developed mechanical hypersensitivity 4–6 weeks after initiation of alcohol exposure (n=13–14/group/sex; Tukey’s multiple comparisons test **p<0.01, ****p<0.0001 relative to same-sex water-drinking mice). Hind paw thresholds were averaged, since there was no significant difference between left/right thresholds within any group during weeks 1–6 (Tukey’s multiple comparisons test p>0.05). (C- E) Simple linear regression analyses of paw withdrawal threshold vs amount of alcohol consumed in male (n=32) and female (n=26) mice following 2-, 4- and 5-weeks of alcohol consumption (C, D and E, respectively) indicates paw withdrawal thresholds are not directly proportional to alcohol consumption (p>0.05). (F) No relationship was observed between body weight and paw withdrawal threshold at 5 weeks.

Figure 4.. Chronic alcohol consumption produces alcohol-induced cold hypersensitivity in females only.

Cold sensitivity was observed in female, but not male, mice after 8 weeks of drinking, Sidak’s multiple comparisons test between groups Week 8 **p<0.01, ****p<0.0001. Males n=6–7/group, females n=8–9/group.

Figure 5.. Chronic alcohol consumption delays resolution of mechanical hypersensitivity induced by capsaicin.

Mechanical thresholds following hind paw intradermal capsaicin (30μg) injection in male (n=7–8/group) and female mice (n=6–7/group). (A) Red arrows indicate capsaicin or vehicle hind paw injection, which occurred after 8 weeks of CIA. Capsaicin significantly lowered tactile thresholds in the ipsilateral paw of (B) males and (C) females regardless of drinking group assignment (Sidak’s multiple comparisons test *p<0.05,**p<0.01,****p<0.0001 compared with −2hr within alcohol- or water-drinking groups). While water-drinking male and female mice had recovered to normal thresholds by 24 hours, hypersensitivity persisted in alcohol drinking mice (Sidak’s multiple comparisons test ++p<0.01 and +++p<0.001 compared with +2hr within alcohol- or water-drinking groups). Intradermal capsaicin had no significant effect on the non-injected paw in alcohol- or water-drinking group in (D) males or (E) females (p>0.05, Sidak’s multiple comparisons test).

Figure 6.. Chronic intermittent alcohol delays resolution of mechanical hypersensitivity induced by transient nerve injury.

(A) Red arrows indicate sciatic crush/sham surgery was performed after 6 weeks of CIA, with CIA continuing for a total of 9 weeks. Mechanical thresholds in (B, C) ipsilateral injured and (D, E) contralateral uninjured hind paws across all weeks of the experiment (*p<0.05,**p<0.01,****p<0.0001 compared within group to pre-surgery week 0, ++p<0.01 and ++++p<0.0001 compared within group to post-surgery week 1, Sidak’s multiple comparisons test). Male and female data had no significant differences between thresholds of the same treatment group.

Figure 7.. Female mice voluntarily drink more alcohol than male mice regardless of injury.

(A) Average alcohol consumed per day measured relative to body weight and (B) % preference for the alcohol bottle for males (n=8–9/group) and females (n=6–7/group). There was no effect of injury on alcohol consumption (3-way RM ANOVA p>0.05). (C) Alcohol consumption and (D) preference shown with the sham and crush groups combined within sex. Female mice consumed more alcohol (g/kg/24h) and had a higher preference for the alcohol bottle compared with male mice (Sidak’s multiple comparisons test between male sham + crush vs. female sham + crush at each time point, *p<0.05, ***p<0.001, ****p<0.0001).