Figure 1.
No difference in acute morphine-induced antinociception between in (A) C3H/HeJ TLR4 mutant or (C) B10ScNJ TLR4 null mice compared to their appropriate genotype/strain control.
Animals were administered morphine (3 mg/kg, i.p.) and thermal nociceptive responses were measured every 15 minutes post-injection. No difference in antinociception was observed between genotypes at any time point (p>0.05). The mean area under the curve was not significantly different between (B) control and TLR4 mutant mice (p>0.05) or (D) control and TLR4 null mice (p>0.05). Data represent means for n = 10 per group. Statistical analyses were performed using a two-way ANOVA followed by Bonferroni post hoc test or unpaired t-test.
Figure 2.
TLR4 mutant (C3H/HeJ) and null (B10ScNJ) mice are insensitive to lipopolysaccharide (LPS)-induced mechanical hyperalgesia.
Mechanical thresholds in control (C3H/HeOuJ, B10ScSNJ) or TLR4 mutant/null mice were measured prior to and 24 h after LPS (1 mg/kg, i.p.) injection. No difference between baseline withdrawal thresholds was observed between genotypes. The number of paw withdrawals significantly increased in control mice 24 h post-LPS injection. No difference in mechanical sensitivity was observed between baseline and 24 h post-injection responses in TLR4 mutant mice. Data represent means for n = 10 per group. Statistical analyses were performed using a two-way ANOVA followed by Bonferroni post hoc test. The asterisk denotes a significant difference from baseline control response. ***p<0.001.
Figure 3.
Ultra-low dose naloxone stereoselectively attenuated the loss in morphine-induced antinociception following chronic administration independent of TLR4.
(A,B) Control (C3H/HeOuJ) and (A,C) TLR4 mutant (C3H/HeJ) mice were administered morphine (MS; 10 mg/kg i.p.), or MS and (−) or (+)naloxone (NLX; 1 ng/kg i.p.) once daily. Thermal nociception was measured by tail flick test 30 minutes post-injection on days 1 and 5. (A) MS-induced antinociception did not differ between TLR4 mutant or control mice over time. MS-induced antinociception was significantly reduced in both control (A,B) and TLR4 mutant (A,C) mice following chronic MS treatment. Co-treatment with (−)NLX significantly attenuated the loss in antinociception compared to MS-only treated mice in both genotypes. (+)NLX did not significantly reduce the loss of MS-induced antinociception on Day 5 compared to mice treated with MS alone in either genotype. Data represent means of n = 4–6 per group, with each n-value comprising 3–5 animals. Statistical analyses were performed using a two-way ANOVA followed by Bonferroni post hoc test. The asterisk denotes a significant difference from morphine-treated mice. *p<0.05.
Table 1.
Mean thermal antinociceptive responses to chronic administration of morphine with or without ULD naloxone.
Figure 4.
Ultra-low dose naloxone non-stereoselectively inhibited morphine-induced hyperalgesia in control and TLR4 mutant mice.
(A,C) Mice were administered treatments (i.p.) twice daily, with mechanical thresholds measured daily prior to the morning injection. Number of paw withdrawals in (A) control (C3H/HeOuJ) and TLR4 mutant (C3H/HeJ) mice or (C) control (B10ScSNJ) and TLR4 null (B10ScNJ) mice treated with morphine, morphine and (+)naloxone (NLX), or vehicle (saline). A small but significant increase was observed with saline treatment over time within control and TLR4 mutant mice (p<0.01 and p<0.05, respectively) (A). (B,D) The mean number of paw withdrawals per treatment group on Day 7 in (B) TLR4 mutant (C3H/HeJ) and (D) null (B10ScNJ) mice vs respective control strains. Morphine significantly increased mechanical hyperalgesia in all genotypes. Co-administration of (−) or (+)naloxone (NLX) blocked morphine-induced hyperalgesia in control and TLR4 mutant and null mice. Data represent means for n = 7–11 per group. Statistical analyses were performed using a two-way ANOVA followed by Bonferroni post hoc test. The asterisk denotes a significant difference from saline-treated mice. *** = p<0.001. The ψ denotes a significant difference between genotypes receiving saline treatment. ψ ψ ψ = p<0.001.
Table 2.
Effect of chronic morphine with or without ULD naloxone enantiomers on mechanical withdrawal thresholds.
Figure 5.
Naloxone-precipitated withdrawal syndrome in control and TLR4 mutant and null mice.
(A) Withdrawal cumulative score and (B) Total number of jumps were significantly higher in both morphine-treated groups as compared with the saline controls, but no differences were detected between C3H/HeJ or B10ScNJ and their respective control strain. Data represent means for n = 5–6 per group. Statistical analyses were performed using a two-way ANOVA followed by Bonferroni post hoc test. p>0.05 for genotype.
Figure 6.
c-Fos immunohistochemical labeling in the dorsal spinal cord following naloxone precipitated withdrawal in morphine treated control and TLR null mice.
Mice underwent cardiac perfusions with 4% PFA 2 h following naloxone precipitated withdrawal and spinal cords were processed for immunohistochemistry of c-fos labeling. Significant differences in labeling intensity were produced by morphine in both control (B10SCSNJ) and mutant (B10ScNJ) mice. Data represent means for n = 5–6 per group. Statistical analyses were performed using a two-way ANOVA followed by Bonferroni post hoc test. p>0.05 for genotype.
Figure 7.
Effect of ultra-low dose naloxone and morphine treatment on CD11b and glial fibrillary acidic protein (GFAP) mRNA expression in mouse spinal cord.
Wild type (C3H/HeOuJ) and TLR4 mutant (C3H/HeJ) mice were treated (i.p.) with vehicle (saline; SAL), morphine (MS; 10 mg/kg), MS and (−)naloxone (NLX; 1 ng/kg), or MS and (+)NLX (1 ng/kg), once daily for five days. Total RNA was extracted from the dorsal lumbar spinal cord, converted to cDNA templates and the expression levels of CD11b and GFAP were then assessed by quantitative real-time polymerase chain reactions as described. Values for each target gene were measured in triplicate (n = 4–5), and expressed as mean percentage expression ± s.e.m. relative to respective vehicle-treated controls (A–D) or vehicle-treated control mice (E,F). CD11b (A) and GFAP (C) mRNA expression were significantly up-regulated in MS-treated control mice; this increase was non-stereoselectively blocked by naloxone. No difference was observed between MS and (−) or (+)NLX and saline treated controls. Morphine did not significantly induce CD11b (B) or GFAP (D) mRNA up-regulation in TLR4 mutant mice, nor was there an effect of naloxone; no difference was observed between treatment groups. Significantly increased CD11b (E) and GFAP (F) mRNA expression was observed in saline treated TLR4 mutant mice compared to control mice. Data represent means for n = 5 per group. Statistical analyses were performed using a two-way ANOVA followed by Bonferroni’s post hoc test. The asterisk denotes a significant difference from morphine-treated mice. *P<0.05, **p<0.01, ***p<0.001. The ψ denotes a significant difference between genotypes receiving saline treatment. ψ ψ ψ = p<0.001.
Figure 8.
Morphine induces microglial activation in control and TLR4 null mice.
Control (B10ScSNJ) and TLR4 null (B10ScNJ) mice were treated with escalating doses of morphine over 5 days. Mice were underwent cardiac perfusions with 4% PFA 2 h following naloxone precipitated withdrawal and processed for immunohistochemistry of Iba-1 labeling. Significant differences in labeling intensity were produced by morphine in both groups of mice. Additionally, a genotype effect was identified where more microglial activation was identified in the TLR4 null mouse compared to control strain. Data represent means for n = 5 per group. The asterisk denotes a significant difference from saline-treated control response. **p<0.01, ***p<0.001. The ψ denotes a significant difference between genotypes receiving saline treatment.