Preprotachykinin-A gene disruption attenuates nociceptive sensitivity after opioid administration and incision by peripheral and spinal mechanisms in mice

Abstract

The preprotachykinin A gene (ppt-A) codes for Substance P (SP), supports nociceptive sensitization, and modulates inflammatory responses after incision. Repeated opioid use produces paradoxical pain sensitization-termed opioid-induced hyperalgesia (OIH) -which can exacerbate pain after incision. Here the contribution of SP to peri-incisional nociceptive sensitization and nociceptive mediator production after opioid treatment was examined utilizing ppt-A knockout (-/-) mice and the neurokinin (NK1) receptor antagonist LY303870. Less mechanical allodynia was observed in ppt-A(-/-) mice compared to wild types (wt) after morphine treatment both before and after incision. Moreover, LY303870 administered with morphine reduced incisional hyperalgesia in wt mice. Incision after saline or escalating morphine treatment upregulated skin IL-1β, IL-6, G-CSF and MIP-1α levels in ppt-A(-/-) and wt mice similarly. However, chronic morphine treatment greatly exacerbated increases in skin nerve growth factor levels after incision, an effect entirely dependent upon intact SP signaling. Additionally, SP dependent upregulation of prodynorphin, NMDA1 and NK1 receptor expression in spinal cord was seen after morphine treatment and incision. A similar pattern was seen for 5-HT3 receptor expression in tissue from dorsal root ganglia. Therefore, SP may work at both central and peripheral sites to enhance nociceptive sensitization after morphine treatment and incision.

Perspective: These studies show that SP signaling modulates enhanced nerve growth factor production and changes in neuronal gene expression seen after incision in mice previously exposed to morphine.

Figure 1

Assessment of mechanical allodynia after escalating morphine treatment and/or hind paw incision. Mechanical allodynia was measured in the wild type (WT) and ppt-A^−/− mice using calibrated von Frey filaments before and at different time points after morphine treatment (A), incision (B) and morphine treatment followed by hind paw incision (C). Data are presented as Mean ± S.E.M and were analyzed by two way ANOVA with post hoc Bonferroni tests comparing strains (n=8 each) at corresponding time points. * p<0.05; **p<0.01; ***p<0.001 indicate difference from baseline for each strain and ^# p<0.05; ^## p<0.01; ^###p<0.001 represents strain differences at corresponding time points.

Figure 2

Assessment of selective NK-1 receptor antagonist Ly303870 effects on escalating morphine and/or incision induced mechanical allodynia. Mechanical hypersensitivity was measured in four treatment groups of wild type mice: vehicle/vehicle, vehicle/morphine, LY303870/vehicle and LY303870/morphine (A). The effect of NK-1 receptor blockade was also measured in hindpaw incision alone (B) and in hindpaw incision after morphine treatment (C). LY303870 (30 mg/kg. i.p.) and morphine treatments (Escalating dose, s.c.) were carried out for 4 days (n=8 each group). Mean ± S.E.M values of each group were analyzed by a two way ANOVA with post hoc Bonferroni tests comparing treatment groups at each time point. * p<0.05; **p<0.01; ***p<0.001 indicate difference from baseline for each individual group and ^# p<0.05; ^## p<0.01; ^###p<0.001 represents group differences at corresponding time points.

Figure 3

Effects of escalating morphine treatment and/or hind paw incision on local cytokines levels. The levels of cytokines were measured at 24 hours time point after incision. The 24 hour time point was selected as incisional and opioid mediated sensitizations were maximal at this point (Figure 1). Different groups of mice of both wild type and ppt-A^−/− were used (n=6 per group). Data are presented as Mean ± S.E.M and were analyzed by two way ANOVA with post hoc Bonferroni tests comparing strains at corresponding time points. **p<0.01; ***p<0.001 indicate difference from baseline for WT and ^## p<0.01; ^###p<0.001 represents difference from baseline for ppt-A^−/− mice. ⁺ p<0.05 represents strain differences.

Figure 4

Effects of escalating morphine treatment and/or hind paw incision on NGF levels. NGF levels were measured at 24 hours time points after incision. Different groups of mice of both wild type and ppt-A(−/−) were used for assessment (A). **p<0.01; ***p<0.001 indicate difference from baseline for WT and ^# p<0.05; ^###p<0.001 represents difference from baseline for ppt-A^−/− mice. ⁺ p<0.05 represents strain differences. Assessment of selective NK-1 receptor antagonist Ly303870 effects on NGF levels after morphine and/or incision in WT mice (B). * p<0.05; **p<0.01; ***p<0.001 indicate differences between incised and non incised in each treatment group and ^###p<0.001 represents differences in relation to both other treatment groups. Data are presented (n=6 per group) as Mean ± S.E.M and were analyzed by two way ANOVA with post hoc Bonferroni tests comparing strains at corresponding time points.

Figure 5

Effects of hind paw incision on spinal cord ppt-A gene (tac1) expression after escalating morphine treatment in WT mice. The ppt-A mRNA levels in DRG were measured by real time PCR in different treatment groups (n=6 each). Data are presented as Mean ± S.E.M and were analyzed by one way ANOVA with post hoc Bonferroni tests comparing treatment groups. **p<0.01 and ***p<0.001 represents significant difference from saline/No Incision group; ^## p<0.01 represents difference from saline/Incision group and ⁺⁺⁺ p<0.05 represents differences between Morphine/No Incision vs. Morphine/Incision groups.

Figure 6

Effects of hind paw incision on DRG and Spinal Cord gene Expression after escalating morphine treatment in the wild type and ppt-A^−/− mice. The mRNA levels of pdyn (prodynorphin), grin1 (NMDA1 receptor) and tacr1 (NK1 receptor) genes (spinal cord) and htr3a (5-HT3 receptor) gene (DRG) were measured by real time PCR in different treatment groups(n=4–6). Data are presented as Mean ± S.E.M and were analyzed by a two way ANOVA with post hoc Bonferroni tests. * p<0.05; **p<0.01; ***p<0.001 indicate difference from baseline for each strain and ^# p<0.05; ^## p<0.01; ^###p<0.001 represents strain differences for corresponding treatments.

Figure 7

Assessment of mechanical allodynia after recovery from escalating morphine treatment induced hyperalgesia followed by hind paw incision. Mechanical allodynia was measured in the wild type (WT) mice using calibrated von Frey filaments before and at different time points after morphine treatment. Data are presented as Mean ± S.E.M and were analyzed by two way ANOVA with post hoc Bonferroni tests comparing saline to morphine treated groups (n=8 each) at corresponding time points. * p<0.05; **p<0.01; ***p<0.001 indicate difference treatment group differences at corresponding time points.