Prenatal alcohol exposure promotes nerve injury-induced pathological pain following morphine treatment via NLRP3-mediated peripheral and central proinflammatory immune actions

Abstract

Adverse in-utero conditions may exert a lifelong impact on neuroimmune function. Our prior work showed that prenatal alcohol exposure (PAE) increases pathological pain sensitivity (allodynia) following peripheral sciatic nerve injury. While the immune mechanism(s) of PAE-induced immune dysfunction are poorly understood, prior studies implicated the involvement of Toll-like receptor 4 (TLR4) and the nucleotide-binding domain, leucine-rich repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasomes. Interestingly, emerging data suggest a surprising overlap of spinal glial proinflammatory activation via the TLR4-NLRP3-interleukin (IL)-1β axis due to opioid treatment in nerve-injured non-PAE rodents. Considering this preclinical evidence, we explored whether PAE poses a risk factor in creating proinflammatory immune bias consequent to opioid (morphine) exposure. We hypothesized that under nerve injury conditions, PAE may interact with morphine, promoting peripheral and CNS proinflammatory factors in a NLRP3-dependent manner. Using a minor nerve injury model in adult mice, we demonstrate that PAE prolongs the chronicity of ongoing allodynia in both sexes, with a more pronounced effect observed in male mice. Our study shows that PAE amplifies proinflammatory responses at the injury site and the spinal cord, driving morphine-prolonged allodynia through NLRP3 inflammasome activation. Furthermore, high mobility group box 1 (HMGB1), a well-established pain-promoting TLR4 agonist, is elevated in allodynic PAE mice. NLRP3 inhibitor, MCC950, effectively reverses morphine-induced allodynia and reduces Caspase-1 activity, IL-1β, and related proinflammatory factors. Although few sex-specific effects were observed, our data convincingly support that PAE and morphine interactions ultimately converge on NLRP3-driven mechanisms in both sexes. Together, this study suggests that PAE modulates later-life neuroimmune function and provides critical insights into immune regulators underlying PAE-induced biological vulnerability to pathological pain processing and adverse effects of opioids.

Keywords: MCC950; NLRP3 inflammasome; Prenatal alcohol exposure; allodynia; cytokines; glia; interleukin −1β; morphine; neuroimmune; peripheral immune.

Figure 1.. Morphine treatment prolongs the duration of allodynia only in minor nerve-injured PAE mice in both sexes.

Sac and PAE mice of both sexes were exposed to sham surgery or minor nerve injury (CCI), and the effects of morphine treatment on hind paw sensitivity were examined. Pre-surgery baseline (BL) values were similar in sac and PAE mice (F_1,54 = 0.48, p = 0.49). Ipsilateral hind paw sensitivity developed following minor CCI. (A & C) A significant interaction between PAE × time (F_3,150 = 7.06, p = 0.00002), PAE × surgery (F_1,50 = 104.71, p < 0.0001), and PAE × surgery × time (F_3,150 = 4.03, p = 0.009) was observed only on the ipsilateral side. In males and females, nerve-injured PAE mice displayed a significant increase in ipsilateral hind paw sensitivity by D7–D14 (p < 0.05). (B & D) In both sexes, no contralateral allodynia was observed post-minor nerve injury. Post-morphine hind paw responses were evaluated between the PAE morphine-treated and vehicle-treated mice. (A) Post-morphine treatment, there was a significant interaction of PAE × morphine (F_1,29 = 182.39, p < 0.0001). In males, morphine-injected PAE mice showed persistent ipsilateral hind paw sensitivity compared to vehicle-treated PAE mice up to D47 post-CCI (D23–D47; #p < 0.04). (C) Female PAE mice showed persistent ipsilateral hind paw sensitivity compared to vehicle-treated PAE mice up to D39 post-CCI (D19–D39; #p < 0.0001). (A & C) Morphine-injected PAE mice exhibited allodynia, in contrast to morphine-treated sac mice, which maintained baseline levels throughout the entire time course. (B & D) No significant changes were observed in the contralateral paw. Large effect sizes were observed for PAE exposure (η²_p = 0.870), morphine treatment (η²_p = 0.869), and their interaction (PAE × morphine; η²_p = 0.875), indicating that PAE mice treated with morphine developed significant allodynia compared to both sac morphine-treated and PAE vehicle-treated mice.

Figure 2.. Systemic treatment of NLRP3 inhibitor, MCC950, reverses morphine-mediated prolonged allodynia in PAE mice, regardless of sex.

The effects of systemic MCC950 treatment were examined during morphine-prolonged allodynia in nerve-injured PAE mice. A two-way ANOVA revealed no significant differences in pre-surgery (BL) values (F_1,91 = 2.9, p = 0.09). Following CCI, only PAE mice developed morphine-prolonged allodynia, as indicated by a significant PAE interaction (F_1,364 = 11.17, p = 0.0009). Male (A) and female (C) PAE mice developed allodynia in comparison to sac mice post-CCI at D7–D14 (p < 0.003). (A & C) Post-morphine treatment, there was a significant interaction of PAE × morphine (F_1,86 = 132.77, p < 0.0001), as well as a sex × time interaction (F_2,172 = 4.17, p = 0.0170). Morphine-treated PAE mice exhibited prolonged allodynia on D19–D23 (#p < 0.0001) compared to PAE vehicle-treated mice for both males and females. Additionally, regardless of sex, significant differences in allodynia were observed between morphine-treated PAE mice and morphine-treated sac mice at D19–D23 (p < 0.04). (B & D) Morphine treatment did not result in any changes in the contralateral hind paw. (A & C) There was a significant sex effect (F_1,60 = 240.3, p < 0.0001) when evaluating the effects of MCC950. In both males and females, MCC950 treatment decreased sensitivity in morphine-treated PAE mice compared to vehicle-treated morphine-treated PAE mice, beginning as early as 90 minutes post-injection, with reversal continuing 24 hours post-injection (#p < 0.0001), at which time tissues were collected. (B & D) Regardless of sex, MCC950 did not alter hind paw responses in the absence of injury on the contralateral side.

Figure 3.. Effects of PAE and morphine immune interactions, and MCC950 treatment on Caspase-1, IL-1β, NLRP3, and TNF-α levels at the injured nerve.

Ipsilateral sciatic nerves were collected from behaviorally verified mice, as represented in Figure 2. Prenatal exposure (+), morphine (+), and MCC950 (−) in purple denote the allodynic group; all other groups are non-allodynic or allodynia-reversed at this post-CCI time point. (A) Despite all mice being exposed to minor nerve injury, morphine treatment significantly increased caspase-1 activity regardless of sex in PAE mice compared to the two key control groups: sac male mice ($p < 0.0001, PAE vs. sac, morphine-treated mice) and PAE mice without morphine treatment (##p < 0.0001, PAE, morphine vs. vehicle). Post hoc comparisons revealed that male PAE morphine-vehicle mice exhibited significantly higher values compared to their female counterparts (#p < 0.0001). In both males and females, MCC950 treatment reduced caspase-1 activity in morphine-treated PAE mice that displayed allodynia reversal (*p < 0.0001). (B) Despite all mice being exposed to minor nerve injury, morphine treatment significantly increased IL-1β protein regardless of sex in PAE mice compared to the two key control groups: sac male mice ($p < 0.0009, PAE vs. sac, morphine-treated mice) and PAE mice without morphine treatment (##p < 0.004, PAE, morphine vs. vehicle). In males, MCC950 treatment reduced IL-1β protein in morphine-treated PAE mice that displayed allodynia reversal (*p < 0.04). (C) In females, morphine-prolonged allodynia coincided with a significant increase in IL-1β mRNA in PAE females compared to sac female mice ($p < 0.0001, PAE vs. sac, morphine-treated mice) and PAE mice without morphine treatment (##p < 0.0001, PAE, morphine vs. vehicle). MCC950 treatment reduced IL-1β mRNA in morphine-treated PAE females that displayed allodynia reversal (*p = 0.01). (D) In males, morphine-prolonged allodynia resulted in a significant increase in TNF-α mRNA levels exclusively in PAE mice treated with morphine compared to sac mice treated with morphine (p = 0.003, unpaired t-test). Morphine-prolonged allodynia resulted in a significant increase in TNF-α mRNA levels in PAE female mice treated with morphine compared to PAE females treated with vehicle (##p = 0.04, PAE, morphine vs. vehicle). MCC950 treatment reduced TNF-α mRNA in morphine-treated male PAE mice that displayed allodynia reversal (*p = 0.03). We observed significant sex differences in TNF-α mRNA levels, with females exhibiting higher levels than males in allodynic, morphine-treated PAE mice (#p = 0.04). (E) In male mice, morphine treatment significantly increased NLRP3 mRNA in PAE mice compared to the two key control groups: sac male mice ($p = 0.05, PAE vs. sac, morphine-treated mice) and PAE mice without morphine treatment (##p = 0.002, PAE, morphine vs. vehicle). NLRP3 mRNA was significantly higher in allodynic male morphine-treated PAE mice compared to their female counterparts (#p < 0.0001). MCC950 significantly reduced NLRP3 mRNA only in morphine-treated male PAE mice that displayed allodynia reversal (*p = 0.007).

Figure 4.. Effects of PAE and morphine immune interactions, and MCC950 treatment on Caspase-1 and IL-1β protein in the spinal cord from minor nerve-injured mice.

Spinal cords were collected from behaviorally verified mice, as represented in Figure 2. Prenatal exposure (+), morphine (+), and MCC950 (−) in purple denote the allodynic group; all other groups are non-allodynic or allodynia-reversed at this post-CCI time point. (A) Despite all female mice being exposed to minor nerve injury, morphine treatment significantly increased spinal Caspase-1 activity in PAE compared to sac mice ($ p < 0.0001, PAE vs. sac, morphine-treated mice) and compared to PAE mice without morphine treatment (## p < 0.0001, PAE morphine vs. vehicle). MCC950 treatment reduced spinal Caspase-1 activity in morphine-treated PAE female mice that displayed allodynia reversal (*p = 0.002). (B) In males, Caspase-1 activity was elevated between allodynic morphine-treated PAE mice and morphine-treated sac mice (p = 0.004, unpaired t-test). MCC950 treatment reduced spinal Caspase-1 activity in morphine-treated PAE male mice that displayed allodynia reversal (p = 0.04, unpaired t-test). (C) Despite all mice being exposed to minor nerve injury, morphine treatment significantly increased IL-1β protein levels in PAE female and male mice compared to sac mice ($ p < 0.005, PAE vs. Sac, morphine-treated mice) and PAE mice without morphine treatment (## p < 0.003, PAE morphine vs. PAE vehicle). MCC950 treatment reduced spinal IL-1β protein in morphine-treated PAE mice that displayed allodynia reversal (*p < 0.05). We observed significant sex differences in IL-1β protein, with females exhibiting higher levels compared to males in allodynic minor nerve-injured morphine-treated PAE mice (#p = 0.0001).

Figure 5.. Effects of PAE, morphine interactions, and MCC950 treatment on IL-1β, NLRP3, and TNF-α mRNA levels in the spinal cord from minor-nerve injured mice.

Spinal cords were collected from behaviorally verified mice, as represented in Figure 2. Prenatal exposure (+), morphine (+), and MCC950 (−) in purple denote the allodynic group; all other groups are non-allodynic or allodynia-reversed at this post-CCI time point. (A) Despite all mice being exposed to minor nerve injury, morphine treatment significantly increased IL-1β mRNA in both sexes in PAE mice compared to sac mice ($p < 0.0008, PAE vs. sac, morphine-treated mice) and PAE mice without morphine treatment (##p < 0.03, PAE, morphine vs. vehicle). Regardless of sex, MCC950 treatment reduced IL-1β mRNA in morphine-treated PAE mice that displayed allodynia reversal (*p < 0.004). (B) In males, morphine-prolonged allodynia coincided with a significant increase in TNF-α mRNA in PAE mice compared to sac mice (p = 0.03, unpaired t-test, PAE vs. sac, morphine-treated mice). In females, morphine-prolonged allodynia coincided with a significant increase in TNF-α mRNA in PAE mice compared to sac mice ($p = 0.0005, PAE vs. sac, morphine-treated mice) and PAE mice without morphine treatment (##p = 0.006, PAE, morphine vs. vehicle). Regardless of sex, MCC950 treatment significantly reduced TNF-α mRNA levels in morphine-treated PAE mice that displayed allodynia reversal (female: *p = 0.009; male: p = 0.03, unpaired t-test). (C) In males, morphine-prolonged allodynia coincided with a significant increase in NLRP3 mRNA in PAE mice compared to sac mice (p = 0.02, unpaired t-test, PAE vs. sac, morphine-treated mice) and PAE mice without morphine treatment (p = 0.05, unpaired t-test, morphine vs. vehicle). In females, regardless of minor nerve injury, morphine treatment significantly increased NLRP3 mRNA levels in morphine-treated PAE mice compared to vehicle-treated PAE mice (##p = 0.004). (D) In males and females, regardless of minor nerve injury, morphine treatment significantly increased IL-18 mRNA levels in PAE mice compared to morphine-treated sac male mice ($p < 0.03) and vehicle-treated PAE mice (female: ##p = 0.0002; male: p = 0.01, unpaired t-test). Regardless of sex, MCC950 treatment significantly reduced IL-18 mRNA levels in morphine-treated PAE mice that displayed allodynia reversal (*p = 0.03).

Figure 6.. Effects of PAE, morphine and MCC950 treatment on TLR4 and IκBα mRNA levels in the spinal cord.

(A) Morphine treatment increased TLR4 mRNA in the spinal cord only in allodynic PAE females compared to sac mice ($p = 0.01, PAE vs. sac, morphine-treated mice) as well as vehicle-treated PAE mice (##p = 0.03). MCC950 treatment reduced TLR4 mRNA in the spinal cord in female PAE mice that displayed allodynia reversal (*p = 0.05). No changes were observed in males. We observed significant sex differences in TLR4 mRNA, with females exhibiting higher levels compared to males in allodynic, minor nerve-injured, morphine-treated PAE mice (#p = 0.002). (B) Morphine treatment increased IκBα mRNA in the spinal cord only in allodynic PAE females compared to sac mice ($p = 0.03, PAE vs. sac, morphine-treated mice). MCC950 treatment reduced IκBα mRNA in the spinal cord in morphine-treated female PAE mice that displayed allodynia reversal (*p = 0.02). IκBα mRNA levels were significantly higher in allodynic female, minor nerve-injured, morphine-treated PAE mice than in their male counterparts (#p = 0.006). (C) Despite all mice being exposed to minor nerve injury, morphine treatment significantly increased GFAP mRNA in the spinal cord of allodynic PAE mice compared to sac mice regardless of sex ($p < 0.04, PAE vs. sac, morphine-treated mice) and PAE mice without morphine treatment (##p < 0.03, PAE, morphine vs. vehicle). In males, MCC950 treatment reduced GFAP mRNA in morphine-treated male PAE mice that displayed allodynia reversal (*p = 0.005). (D) Despite all mice being exposed to minor nerve injury, morphine treatment significantly increased Iba-1 mRNA in the spinal cord of allodynic PAE mice compared to sac mice regardless of sex (female: $p < 0.04; male: p = 0.02, unpaired t-test, PAE vs. sac, morphine-treated mice) and PAE mice without morphine treatment (##p < 0.03, PAE, morphine vs. vehicle). In males, MCC950 treatment reduced Iba-1 mRNA in morphine-treated male PAE mice that displayed allodynia reversal (p = 0.04, unpaired t-test).

Figure 7.. Effects of PAE and morphine and MCC950 treatment on HMGB1 mRNA levels in the injured nerve and spinal cord.

(A) Despite all mice being exposed to minor nerve injury, and in both sexes, morphine treatment significantly increased HMGB1 mRNA in the sciatic nerve of allodynic PAE mice compared to sac mice ($p < 0.03, PAE vs. sac, morphine-treated mice) and PAE mice without morphine treatment (##p < 0.03, PAE, morphine vs. vehicle). MCC950 treatment reduced HMGB1 mRNA in morphine-treated male PAE mice that displayed allodynia reversal (male: *p < 0.002; female: p = 0.004, unpaired t-test). We observed that HMGB1 mRNA was significantly higher in allodynic male, minor nerve-injured, morphine-treated PAE mice compared to their female counterparts (#p = 0.001). (B) Independent of minor nerve injury, morphine treatment significantly increased spinal HMGB1 mRNA in both sexes in PAE morphine-treated mice compared to sac morphine-treated mice ($p < 0.0001, PAE vs. sac, morphine-treated mice) and PAE mice without morphine treatment (##p < 0.0003, PAE, morphine vs. vehicle). MCC950 treatment reduced HMGB1 mRNA in allodynia-reversed mice (*p < 0.0004).

Figure 8.. Effects of PAE and morphine immune interactions and MCC950 treatment on μ-opioid receptor mRNA in the spinal cord and midbrain in minor-nerve injured mice.

(A) Regardless of sex, morphine-treated PAE mice exhibited significantly higher μ-opioid receptor mRNA levels compared to morphine-treated saccharin controls (female: $p = 0.03; male: p = 0.02, unpaired t-test). μ-Opioid receptor mRNA was significantly higher in allodynic female, minor nerve-injured, morphine-treated PAE mice compared to their male counterparts (#p = 0.0007). MCC950 treatment reduced HMGB1 mRNA in allodynia-reversed mice (*p < 0.01) (B) No change was observed in the midbrain.

Figure 9.. Effects of PAE and morphine and MCC950 on NLRP3 and TNF-α mRNA in the midbrain and anterior cingulate cortex.

(A) Morphine treatment significantly increased NLRP3 mRNA in the midbrain of female PAE mice treated with morphine compared to PAE mice without morphine treatment (##p = 0.02). (B) Morphine treatment significantly increased TNF-α mRNA in the anterior cingulate cortex of female PAE mice treated with morphine compared to PAE mice without morphine treatment (p = 0.003, unpaired t-test). (C & D) No changes were observed in IL-1β or TNF-α mRNA in the midbrain regardless of sex.

Figure 10. Schematic diagram of the experimental paradigm and summary of key findings of this study.

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