BACKGROUND: In critically ill pediatric burn injury (BI) patients, repeated morphine doses not consistently alleviate basal and procedural-pain and can cause chronic neuropathic pain. Microglia activation exaggerates pain behaviors and meanwhile express a7 acetylcholine receptors (a7AChRs), which when agonist stimulated, exhibit anti-inflammatory properties. We tested the hypothesis that morphine via microglia activation aggravates BI-induced pain, which can be reversed by a7AChR selective agonist, GTS-21.

METHODS: Hind paw 1% BI was administered to very young rats and treated intraperitoneally with saline, morphine(10mg/kg) and/or GTS-21 (4mg/kg) daily for 14 days. Pain thresholds were tested every other day for 20 days prior to daily drug administration. Spinal microglia immunochemistry (IBa1-staining) and cytokine expression (western blot) were examined.

RESULTS: Morphine, started on day of BI, significantly increased both allodynia and hyperalgesia by day 3 after BI (0.019 ± 0.007 vs. 0.08 ± 0.020 gram; 6.564 ± 0.628 vs. 8.685 ± 0.283 seconds, P<0,05). The exaggerated nociceptive behaviors persisted even after termination of morphine at day 14.  The administration of GTS-21 to burn with morphine group increased the allodynia and hyperalgesia thresholds as early as day 1 and 3, respectively, to level of BI group with no morphine (8.895 ± 0.764sec vs. 6.564 ± 1.985secs, # P<0.05). That is, the morphine-induced exaggerated pain was completely reversed by GTS

Spinal cord samples were obtained at day 14. The ipsilateral dorsal side of burn injury site was separated, and the expression of inflammatory proteins result shows that: 1) BI animals treated with saline had significant and more profound increase in spinal inflammatory cytokine expression of TNF-α, IL-6 and IL-1β compared to sham BI rats. The superimposition of morphine to BI caused the highest expression of the inflammatory proteins. Compared to BI animals receiving morphine, the administration of GTS-21 to BI-morphine group significantly attenuated the increased inflammatory protein expression (P<0.05).

Lumber spinal cord was extracted at day 7 after burn injury and ipsilateral dorsal horn was stained with Iba1, a specific marker for microglia. Compared to sham-burn saline group, burn injury caused microgliosis to ~275% increase. Morphine to burn injury group had a significantly increased microgliosis to ~600% compared to non-burn group and was also had a significantly increased the microgliosis compared to burn alone group, which was ~275% increase (33.75 ± 2 .38/field vs. 4.8 ± 2/field vs. 18.01±4.11/field, respectively, *P<0.05). GTS-21 completely reversed the microgliosis to BI levels (P<0.05).

CONCLUSIONS: The administration of morphine to very young rats aggravates BI-induced nociception together with the increased spinal microgliosis and expression of inflammatory pain- transducers. The GTS-21-induced mitigation of exaggerated nociception by morphine and BI were paralleled by decreased spinal expression of inflammatory pain transducer proteins and microgliosis. These observations in very young 3-week-old rats indicate that the microglia innate immune responses contribute to the lowered pain threshold and GTS-21 can mitigate these changes and therefore shows promise as a therapeutic adjunct to treat Bi and/or morphine-induced pain.

Keywords: Morphine, α7AChR agonist, neuroinflammation, Allodynia, Hyperalgesia