CB2 receptor deletion on myeloid cells enhanced mechanical allodynia in a mouse model of neuropathic pain
Elisa Nent, Chihiro Nozaki, Anne-Caroline Schmöle, David Otte & Andreas Zimmer
www.nature.com/scientificreports, 2019, 9, 7468
Neuropathic pain can develop after nerve injury, leading to a chronic condition with spontaneous pain and hyperalgesia. Pain is typically restricted to the side of the injured nerve, but may occasionally spread to the contralateral side, a condition that is often referred to as mirror-image pain. Mechanisms leading to mirror-image pain are not completely understood, but cannabinoid CB2 receptors have been implicated. In this study, we use genetic mouse models to address the question if CB2 receptors on neurons or on microglia/macrophages are involved. First, we show that a GFP reporter protein under control of the CB2 promoter is induced upon partial sciatic nerve ligation in spinal cord, dorsal root ganglia, and highest in sciatic nerve macrophages, but not in neurons. Mice which lack CB2 receptors specifically on myeloid cells (microglia, macrophages) developed a mirror-image allodynia [treatment F1,48 = 45.69, p < 0.0001] similar to constitutive CB2 receptor knockout mice [treatment F1,70 = 92.41, p < 0.0001]. Such a phenotype was not observed after the deletion of CB2 from neurons [treatment F1,70 = 0.1315, p = 0.7180]. This behavioral pain phenotype was accompanied by an increased staining of microglia in the dorsal horn of the spinal cord, as evidenced by an enhanced Iba 1 expression [CB2KO, p = 0.0175; CB2-LysM, p = 0.0425]. Similarly, myeloid-selective knockouts showed an increased expression of the leptin receptor in the injured ipsilateral sciatic nerve, thus further supporting the notion that leptin signaling contributes to the increased neuropathic pain responses of CB2 receptor knockout mice. We conclude that CB2 receptors on microglia and macrophages, but not on neurons, modulate neuropathic pain responses.
Preparations of Cannabis sativa have been used for millennia to treat various pain conditions. One of the active ingredients of cannabis, Δ9-tetrahydrocannabinol (THC), can bind and activate the G-protein coupled cannabinoid receptor 1 (CB1)1 and cannabinoid receptor 2 (CB2)2, which are the two main receptors of the endocannabinoid system. Whereas CB1 is most abundantly expressed in the brain, CB2 is mainly found on immune cells, including macrophages and microglia3. CB2 expression in neurons is low under normal conditions4–6, but it is an important modulator of neuronal physiology and neuronal network activity7. Although neuronal expression of CB2 is low in healthy tissues, CB2 protein and mRNA levels are increased in DRG and spinal cord neurons under neuropathic pain conditions8–12. The induction of CB2 is probably part of the recuperative process, as activation of CB2 by natural and synthetic agonists reduced pain symptoms12–25. Genetically enhanced CB2 signaling in mice overexpressing CB2 receptors in microglia and neurons also reduced the manifestation of neuropathic pain symptoms26. Conversely, the deletion of CB2 resulted in an enhanced neuroinflammatory response after sciatic nerve injury and in mirror-image pain, as evidenced by the development of tactile allodynia on the contralateral side of the injured nerve26.
In most cases, neuropathic pain is restricted to the body region that is innervated by the affected nerve, but some animal models and human pain conditions with mirror-image pain have been described27,28. CB2 knockout (CB2KO) mice are one of very few genetic mouse models in which such a contralateral pain due to unilateral injury often develops, thus indicating that CB2 signaling is required to restrict the development of allodynia to the ipsilateral site. The mechanisms involved in this process nevertheless remain largely obscure, although peripheral and central inflammatory processes have been implicated 29,30. Previously, it was shown that leptin signaling promotes neuropathic pain via modulating CB2 signaling. Mice with a deletion of the CB2 receptor showed an increase in leptin receptor expression on the ipsi- and contralateral sciatic nerve. After perineural administration of a neutralizing anti-leptin antibody, increased leptin receptor expression and tactile hyperalgesia were reduced again on both sides, ipsi- and contralateral. A contribution of leptin in neuropathic pain development through CB2 was therefore concluded31.
Because CB2 receptors are expressed on neurons, as well as macrophages and microglia after sciatic nerve injury, it is not clear on which cells CB2 is acting during neuropathic pain. In this study, we use CB2-GFP reporter mice and mice with a cell-specific CB2 deletion on neurons or myeloid cells to address the question, which CB2- expressing cell type is important for the development of neuropathic pain.