Morphine analgesic tolerance in 129P3/J and 129S6/SvEv mice

Abstract

Morphine analgesic tolerance is heritable in both humans and rodents, with some individuals and strains exhibiting little and others exhibiting robust tolerance. 129S6/SvEv and 129P3/J mice reportedly do not demonstrate tolerance to morphine analgesia. Using our laboratory's standard morphine tolerance regimen and a between-subjects design, tolerance developed in the hot plate and tail withdrawal assays as indicated by a change in analgesic efficacy following a morphine challenge dose. Furthermore, the non-competitive NMDA receptor antagonist MK-801 (dizocilipine) blocked morphine tolerance in 129S6/SvEv and CD-1 mice in the hot plate assay. As previously reported, when a within-subjects design and cumulative dosing was employed, no tolerance was observed in the 129P3/J strain. However, using the same morphine regimen and a between-subjects design, comparable tolerance developed between 129P3/J and C57BL/6J strains following a single challenge dose of morphine. Spontaneous hyperalgesia was observed in the tail withdrawal assay following chronic morphine in C57BL/6J, but not 129P3/J mice. Additionally, morphine-tolerant C57BL/6J mice, but not 129P3/J mice, exhibited a large increase in the frequency of tail flicks during the first second following the baseline nociceptive response which may facilitate detection of the response during the tolerant state. We conclude that the method of tolerance assessment affects the ability to detect tolerance and thus may affect the degree and pattern of heritability of this trait and this could have implications for gene mapping studies.

FIGURE 1. Morphine tolerance in 129S6/SvEv and 129P3/J strains in the hot plate and tail withdrawal assays following a between-subjects design and a once daily regimen of escalating morphine doses (Bryant et al., 2006)

Male 129S6/SvEv (N=8) or 129P3/J mice (N=8) were treated once daily for 6 days with either saline (10 ml/kg, s.c.) or morphine (10–40 mg/kg, s.c.). On day 7, following baseline measurements, separate mice for each pain assay were tested for morphine analgesia (7.5 mg/kg, s.c.) from 30–120 min on either the 52.5°C hot plate or the 49.0°C tail withdrawal. A and B). Tolerance develops in 129S6/SvEv and 129P3/J strains in the hot plate assay. C and D). Tolerance develops in 129S6/SvEv and 129P3/J strains in the hot plate assay as indicated by area under the curve (AUC). E and F). Tolerance develops in 129S6/SvEv and 129P3/J strains in the tail withdrawal assay. G and H). Tolerance develops in 129S6/SvEv and 129P3/J strains in the tail withdrawal assay as indicated AUC. Close circles = chronic saline treatment. Open circles = chronic morphine treatment. Data are presented as the mean %MPE ± S.E.M. A p value of 0.05 was considered significant (*).

FIGURE 2. Morphine tolerance to both the first and second nociceptive response in the tail withdrawal assay in 129P3/J mice following a between-subjects design and a once daily regimen of escalating morphine doses (Bryant et al., 2006)

Male 129P3/J mice (N=16) were treated once daily for 6 days with either saline (10 ml/kg, s.c.) or morphine (10–40 mg/kg, s.c.). On day 7, following baseline measurements, mice were administered a challenge dose of morphine (5 mg/kg, s.c.) and tested for analgesia, measuring both the first and second analgesic response from 30–120 min in the 49.0°C tail withdrawal assay. A and B). Tolerance develops to the first and second analgesic response in 129P3/J mice. C and D). Tolerance develops to the first and second nociceptive response as indicated by AUC. Data are presented as the mean %MPE ± S.E.M. A p-value of 0.05 was considered significant ("*").

FIGURE 3. Disruption of morphine tolerance by MK-801 in 129S6/SvEv and CD-1 mice in the hot plate assay following a between-subjects design and a once daily regimen of escalating morphine doses (Bryant et al., 2006)

Male mice were treated once daily for 6 days with the non-competitive NMDA receptor antagonist MK-801 (1 mg/kg, i.p.) and a simultaneous dose of morphine (10–40 mg/kg, s.c.) followed 2 h later by an additional injection of antagonist (1 mg/kg, i.p.). On day 7, 30 min following measurement of baseline hot plate latencies, mice were then administered a morphine challenge (5 mg/kg, s.c. for 129S6/SvEv mice; N=8; 10 mg/kg, s.c. for CD-1 mice; N=12–14) and tested for analgesia every 30 min for 120 min. A and B). In both strains, mice previously receiving chronic administration of MK-801 plus morphine (“MK+mor”) showed significantly greater analgesia than mice previously receiving morphine alone (“*”; “morphine”) at 30 min, indicating an attenuation of tolerance. C and D). In analysis of AUC, significant tolerance developed in CD-1, but not 129S6/SvEv mice, which was attenuated by MK+mor treatment ("*"). Data are presented as the mean %MPE ± S.E.M. A p value of 0.05 was considered significant.

FIGURE 4. Morphine tolerance under a separate morphine regimen (Kest et al., 2002) following a within-subjects design and cumulative dosing or a between-subjects design and a single challenge dose

Mice (N=8) were assayed for baseline latencies on day 1, and immediately administered morphine (s.c.). Thirty min later, immediately following post-injection latency assessment mice, were administered a subsequent dose of morphine. This was repeated every 30 min. Doses of 1, 2, 3.6, 6.5, 11.7, and 21.0 mg/kg were administered. In the case of extra injections, 21.0 mg/kg was administered (Kest et al. 2002). All mice received the same number of injections and doses of morphine on day 1, although if they reached cut-off, they were not subjected to further pain testing (Kest et al. 2002). The x-axis represents the cumulative morphine dose. A and B). No tolerance developed in 129P3/J mice in the tail withdrawal assay as indicated by a lack of shift in the dose-response curve. Comparable ED₅₀ values were obtained between days 1 and 4 (see Results). ED₅₀ estimates for C57BL/6J mice were not possible, due to the lack of comparable maximum possible effects that could be reached, even with subsequent dosing on day 4 (data not shown). C and D). Tolerance developed in 129P3/J and C57BL/6J mice in the tail withdrawal assay following a between-subjects design, the regimen of Kest et al. (2002), and a single challenge dose of morphine on test day (129P3/J = 7.5 mg/kg, s.c.; C57BL/6J = 25 mg/kg, s.c.) . E and F). Tolerance as indicated by AUC in 129P3/J and C57BL/6J mice. Data are presented as the mean ± S.E.M. A p value of 0.05 was considered significant ("*").

FIGURE 5. Increase in the frequency of the baseline nociceptive response following chronic morphine administration in C57BL/6J mice, but not 129P3/J mice

Using a between-subjects design, mice (N=4) were administered escalating doses of morphine over 3 days (Kest et al. 2002). On day 4, following the first baseline tail flick, the tails were left in the waterbath for 1 s and the total number of flicks was recorded on a digital video. A). Morphine-tolerant C57BL/6J mice, but not morphine-tolerant 129P3/J mice, exhibited a large, significant increase in the number of baseline flicks following the first nociceptive response. Following a subsequent morphine challenge, regardless of strain, tolerant mice did not exhibit any flicking during the 1 s following the first flick (data not shown). B). To confirm tolerance in both strains, we used a lower challenge dose of morphine in C57BL/6J mice (15 mg/kg, s.c.) as compared to Figure 4 (25 mg/kg, s.c.) and the same challenge dose in 129P3/J mice (7.5 mg/kg, s.c.). Comparable acute analgesia and tolerance was observed in both strains at 30 min post-injection. "sal" = chronic saline treatment. "mor" = chronic morphine treatment. "BL" = baseline. "TW" = tail withdrawal. Data are presented as the mean ± S.E.M. A p value of 0.05 was considered significant.