Itching is a pathological process in the somatosensory system at the levels of the primary sensory neurons, spinal cord, and brain. Although there has been distinct progress in the itching research field, the mechanisms underlying the sensation and transmission of itching, and in particular morphine-induced itch, are still unclear. Research over the past decade has increasingly shown that pain and itch are two clearly distinct sensations, and recent studies have identified separate neuronal pathways involved in each sensation.
One proposed mechanism for chronic itch involves an abnormal excitability of pruritic-specific neurons, such as the GRPR and NPPA neurons in the spinal dorsal horn[13, 22, 23]. However, although GRPR has been shown to be involved in MIS, an increasing body of evidence suggests that synaptic hyperexcitability in the spinal dorsal horn may not be a consequence simply of neuronal changes but may also be involved in multiple glial cell alterations.
The important role of microglia in itching sensations has also been verified in a series of studies[8, 24–26]. As CNS-dwelling macrophages, it is known that microglia play an important immunoregulatory role within the CNS . Moreover, it has been shown that microglia become activated in the spinal dorsal horn after peripheral nerve damage . Previous studies have also revealed the involvement of spinal microglia in pruritogen-induced itching, and recent research has shown that compound 48/80 and GNTI can elicit pronounced scratching behaviors in mice in conjunction with microglial activation in mouse spinal cords. However, it remains rather unclear exactly how microglia mediate MIS and how they affect the KOR activation process.
Previous evidence has suggested that PKCδ could play a specific role as an intracellular modulator of itch in sensory neurons. It is also known that KOR activation attenuates itch via PKCδ, although the broader role of PKCs in the development of acute itch has not yet been elucidated. We therefore set out to investigate PKC mechanisms, and we found that PKCδ affected the KOR-mediated inhibition of MIS. However, although PKCδ could still be activated by the administration of nalbuphine in mice treated with PKCδ siRNA, it had less effect on MIS. These observations are consistent with the hypothesis that PKCδ is activated in the KOR-mediated inhibition of MIS.
In addition, we also found that CD11b levels demonstrated an opposite trend to that of PKCδ. Since microglia were activated after treatment with PKCδ siRNA in the nalbuphine plus morphine group, it is clear that PKCδ had a negative feedback regulation of morphine, and this is also consistent with previous research. In contrast, mice treated with PKCβ siRNA demonstrated that PKCβ positively regulated microglia. The MIS model was therefore associated with increased PKCβ expression, which was in turn inhibited by nalbuphine. The expressions of both PKCδ and PKCβ were also both associated with microglial activation. However, whether PKCδ activates microglia directly via phosphorylation or indirectly via other kinases requires additional research. We were also unable to identify any role for PKCα in acute itching.
Our research used knockdown PKC isoforms to observe the role of microglia in MIS and to explore the relationship between microglial activation and the KOR/PKC pathways in nalbuphine’s antipruritic effect. Using a series of pharmacological, behavioral, immunochemical, and molecular biology experiments, we first found that the KOR agonist nalbuphine inhibited MIS in a dose-dependent manner and that this inhibitory effect was PKCδ-dependent. Second, we demonstrated that MIS could induce microglial activation and that this activation was PKCβ dependent. Third and finally, we found that nalbuphine inhibited MIS via the retroegulation of microglial activation by the KOR-PKCδ pathway. Our research has thus identified a new mechanism underlying nalbuphine’s antipruritic effect and provides novel insights about MIS that may lead to drug targets.
Moreover, previous research has shown that KOR agonists are effective at suppressing scratching in mice treated with different pruritogens. Nalbuphine is a nonscheduled KOR agonist and mu opioid receptor antagonist that has been approved by the FDA since the 1980s for clinical treatment of moderate to severe pain, including in postoperative pain management; more recently, it has also been studied in clinical trials as a treatment for chronic itch, or prurigo- nodularis. Although the use of nalbuphine as a treatment for the pruritus associated with neuraxial opioid use has not been approved by the FDA, it has nevertheless been used in this capacity. In fact, one study found that nalbuphine was a better treatment for MIS than either a placebo, a control, diphenhydramine, naloxone, or propofol in patients receiving neuraxial opioids for acute pain related to surgery or childbirth . However, the mechanism underlying nalbuphine’s antipruritic effects is still unclear.
There has been relatively little research on this topic to demonstrated that spinal KOR activation reduced itch transmission by inhibiting the function of GRPR. However, this is the only study to date that has investigated the role of a KOR agonist in MIS, and it investigated the role of neurons rather than microglia. The present research demonstrated that microglia’s immunoregulatory roles may be involved in MIS and that nalbuphine may affect microglial functioning in some manner. As expected, we observed significantly increased microglial activation after morphine i.t. injections; moreover, this activation was related to PKCβ function. Since this is the first time that this phenomenon has been observed in MIS, we used siRNA knockdowns to confirm that it was PKC isoform–specific; we observed that, in knockdown PKCβ mice, microglial activation was dramatically inhibited following decreases in MIS, thus demonstrating PKCβ’s importance in MIS.
Two recent photon in vivo imaging studies found that microglial processes are highly dynamic in the brain and spinal cord: stimulated microglia rapidly move toward the site of an injury and directly appose synaptic regions, in response to neuronal activity, thereafter facilitating contact with highly active neurons. Microglial activation had not been previously reported in MIS. Moreover, KOR activation was thought to attenuate the expression of microglia via some other mechanism than the G-protein signaling pathway. However, we found that microglia were activated in the spinal dorsal horns. Microglia are known to dramatically alter gene expressions at a molecular level, including via Iba1, CD11, and p-p38, and our results demonstrate that microglia play an important role in physical condition monitoring, such as itching, regardless of the severity of an injury.
Research has also shown that various microglia signaling molecules, including cell-surface receptors, are increased in the spinal dorsal horn once microglia are activated following a nerve injury. Following a nerve injury, the phosphorylation of p38 MAPK increases, although this is highly restricted to spinal microglia. In vitro studies have furthermore shown that activated p38 MAPK is present in spinal microglia and participates in the signaling pathways that mediate nociception at the spinal level . Microglia and p-p38 have also been shown to have a role in chronic itch.
Since we knew the potential roles of PKCs and microglia, we therefore hypothesized that p38 MAPK was also involved. An examination of p-p38 expression showed increased levels of p-p38 after treatment with morphine that were furthermore inhibited by nalbuphine. Iba1 cells were also co-expressed with p-p38 in the spinal cord, confirming our western blot results. These results suggest that microglial activation may occur by phosphorylating p38 to maintain an acute MIS signal. Our previous research has also suggested that the microglial inhibitor minocycline inhibited the p-p38 MAPK pathway in our MIS model. Overall, our present results suggest that, as the regular downstream microglial pathway, p38 MAPK precisely regulated microglia during the KOR-mediated inhibition of MIS. Although significant prior evidence existed that microglia play a role in chronic itch and pain, the field has lacked a deep understanding of microglia’s role in pruritus-induced itching and in neuraxial opioid-induced itching. Although our research observed an increased expression of microglia and the involvement of the p-p38 pathway, the exact microglial mechanisms underlying MIS still require further research.
Taken together, our acute itch mouse experiments have suggested a new regulation mechanism involving KORs and microglia. Moreover, the signaling pathway that we identified here as a mediator of acute MIS is similar to the one involved in pain, which also hinges on microglia and p-p38 activation. Ultimately, our findings about PKC isoform involvement and the downstream effects of p38 MAPK and KOR activation suggest new ways to treat MIS.