Submitted to: Journal of Experimental Neurology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/9/2003
Publication Date: 9/1/2003
Citation: Zhao, C., Tao, Y., Tall, J.M., Donovan, D.M., Meyer, R.A., Raja, S.N. Role of u-opioid receptors in formalin-induced pain behavior in mice. Journal of Experimental Neurology. 2003. 184(2):839-45.
Interpretive Summary: The Mu Opioid Receptor (MOR) is one of four known Opioid receptors in the mammalian body. One function of the opioid receptors is to modulate pain perception. However, pain responses are very complex, and the role of each member of the Opioid receptor family is unknown for each type of pain or in the various stages of a pain response. Formalin injection into one footpad of the mouse is a standardized assay of pain, is very mild for the mouse, and involves both the central and peripheral nervous system. The level of pain can be assayed by applying increasing amounts of pressure against the injected foot pad, and measurement of the flinch response that results. The animal can easily walk on the three unaffected feet, until the formalin injected foot can heal. In order to study the role of the MOR in foot pad pain, both wild type and MOR Knockout (MORKO) mice were examined. In this study, measurements of pain are taken every five minutes for sixty minutes post injection. The MORKO mice demonstrated greater pain response in Phase II than the wild type animals. Phase II is the second period that occurs 10-60 minutes after the formalin injection, and follows immediately after the acute pain (Phase I). Overall, the MORKO mice demonstrated a level of pain response that was similar to wild type mice that had received naloxone. Naloxone is an opioid antagonist that can block the opioid receptor's pain reducing effect and thus block the action of endogenous opioids or morphine. These data suggest that endogenous opioid peptides interacting with u-opioid receptors exert a significant antinociceptive role during the second phase of the formalin test in wild type mice. C-Fos is an immediate early transcription factor that is known to be induced in pain responses. Lumbar vertebrae 4 and 5 are the locations where the foot pad peripheral nerves join with the central nervous system, and is one region where the c-Fos induction occurs. Some of the experimental mice had this region of the spinal cord harvested and levels of c-Fos measured via immunohistochemistry. The MORKO mice showed a response identical to wild type in the levels of c-Fos induced in the spinal cord. Although this study showed that endogenous u-opioid systems may inhibit the formalin-induced flinching response, we did not observe a significant change in the expression of Fos, thus providing further evidence for the dissociation between "pain" and Fos expression. The reason for this dissociation is not clear. These results indicate that the MOR system plays an important modulatory role in tonic pain states, and that the first phase of the nociceptive response to formalin stimulation in u-opioid receptor knockout mice is not different than wild type mice. These finding suggests that the endogenous u-opioid receptor system is probably not tonically active under normal circumstances, but is activated by formalin injection and produces its antinociceptive effects during tonic phase.
Technical Abstract: Opioid receptors exert different influences on the perception of stimuli from distinct sensory modalities. Intraplantar formalin injection is widely used as an experimental model of tonic pain. We investigated the role of endogenous '-opioid receptor mechanisms in formalin-induced nocifensive behavior in mice. The flinching response induced by formalin (2%, 20'l) was studied in mice with normal (wild type, n=8) and absent (homozygous '-opioid receptor knockout, n=8) '-opioid receptor levels. The flinch responses were counted every 5 min for 60 min post formalin injection. Lumbar spinal cord (L4, 5) was harvested 2 h post formalin injection to examine c-Fos expression using immunohistochemistry. The effects of naloxone (5 mg/kg, s.c.) administered 30 min prior to the intraplantar formalin injection on the flinching response of wild type mice (n=7) were also recorded. The second phase formalin response (10-60 min after formalin) was higher in homozygous '-opioid receptor knockout mice compared to the wild type mice (p<0.01). Naloxone administration in wild type mice prior to formalin injection resulted in pain behavior similar to that observed in homozygous '-opioid receptor mice (p>0.05). The c-Fos expression induced by formalin injection in '-opioid receptor knockout mice was not different from that observed in wild type mice. Our results suggested that the endogenous '-opioid system is activated by intraplantar formalin injection and exerts a tonic inhibitory effect on the pain behavior. These results suggest an important modulatory role of endogenous '-opioid receptor mechanisms in tonic pain states.