Yanguo Hong

Activated microglia in the spinal cord underlies diabetic neuropathic pain.

Diabetes mellitus is an increasingly common chronic medical condition. Approximately 30% of diabetic patients develop neuropathic pain, manifested as spontaneous pain, hyperalgesia and allodynia. Hyperglycemia induces metabolic changes in peripheral tissues and enhances oxidative stress in nerve fibers. The damages and subsequent reactive inflammation affect structural properties of Schwann cells and axons leading to the release of neuropoietic mediators, such as pro-inflammatory cytokines and pro-nociceptive mediators. Therefore, diabetic neuropathic pain (DNP) shares some histological features and underlying mechanisms with traumatic neuropathy. DNP displays, however, other distinct features; for instance, sensory input to the spinal cord decreases rather than increasing in diabetic patients. Consequently, development of central sensitization in DNP involves mechanisms that are distinct from traumatic neuropathic pain. In DNP, the contribution of spinal cord microglia activation to central sensitization and pain processes is emerging as a new concept. Besides inflammation in the periphery, hyperglycemia and the resulting production of reactive oxygen species affect the local microenvironment in the spinal cord. All these alterations could trigger resting and sessile microglia to the activated phenotype. In turn, microglia synthesize and release pro-inflammatory cytokines and neuroactive molecules capable of inducing hyperactivity of spinal nociceptive neurons. Hence, it is imperative to elucidate glial mechanisms underlying DNP for the development of effective therapeutic agents. The present review highlights the recent developments regarding the contribution of spinal microglia as compelling target for the treatment of DNP.

The involvement of spinal bovine adrenal medulla 22-like peptide, the proenkephalin derivative, in modulation of nociceptive processing.

Bovine adrenal medulla 22 (BAM22), one of the cleavage products of proenkephalin A, possesses high affinity for opioid receptors and sensory neuron‐specific receptor (SNSR). The present study was designed to examine the expression of BAM22 in the spinal cord and dorsal root ganglion (DRG) of naive rats as well as in a model of inflammation. BAM22‐like immunoreactivity (BAM22‐IR) was expressed in fibers in the spinal cord, with high density seen in lamina I in naïve rats. The expression of BAM22‐IR in the superficial laminae was greatly reduced following dorsal rhizotomy. BAM22‐IR was also located in 19% of DRG cells, mainly in the small‐ and medium‐sized subpopulations. Following injection of complete Freunds adjuvant (CFA) in the hindpaw, the expression of BAM22‐IR in the superficial laminae of the spinal cord and small‐sized DRG neurons on the ipsilateral side was markedly increased. Double labeling showed that the Fos‐positive nucleus was surrounded by BAM22‐IR cytoplasm in the spinal dorsal horn neurons or closely associated with BAM22‐IR fibers in the superficial laminae. Furthermore, CFA‐induced mechanical allodynia in the inflamed paw was potentiated by intrathecal administration of anti‐BAM22 antibody. Together, these results demonstrate for the first time that BAM22‐like peptide is mainly located in the superficial laminae of the spinal cord and mostly originates from nociceptive DRG neurons. BAM22 could thus act as a ligand for presynaptic opioid receptors and SNSR. Our study also provides evidence suggesting that BAM22 plays a role in the modulation of nociceptive processing at the spinal level under normal and inflammatory conditions.

The contribution of peripheral 5-hydroxytryptamine2A receptor to carrageenan-evoked hyperalgesia, inflammation and spinal Fos protein expression in the rat

The present study was conducted to test the hypothesis that the peripheral 5-hydroxytryptamine (5-HT)2A receptor is involved in inflammatory hyperalgesia and production of noxious stimulus-induced neuronal activity at the level of the spinal cord dorsal horn. Intraplantar (i.pl.) injection of carrageenan dramatically reduced paw withdrawal latency to noxious heat (47 degrees C) and caused paw swelling. Pretreatment with ketanserin, a selective antagonist of 5-HT2A receptor, in the hindpaw produced dose-dependent inhibition of the hyperalgesia (0.5, 3 and 5 mug; i.pl.) with full relief at 5 mug. The drug also moderately reduced carrageenan-induced paw swelling in a dose-dependent manner. Carrageenan induced conspicuous expression of c-fos-like immunoreactivity (FLI) in the spinal dorsal horn of segments L4-5. Ketanserin (5 mug) markedly reduced carrageenan-induced FLI in all laminae of the dorsal horn. However, blockade of peripheral 5-HT1A receptors by (N-2-[4-(2-methoxyphenyl-1-piperazinyl] ethyl]-N-2-pyridinylcyclohexanecarboxamide at maximally effective doses (30 and 100 mug; i.pl.) did not alter carrageenan-induced hyperalgesia, edema or expression of FLI. The present study provided evidence at cellular level that the peripheral 5-HT2A receptor is preferentially involved in the development of thermal hyperalgesia in the carrageenan model of inflammation.

Topical ketanserin attenuates hyperalgesia and inflammation in arthritis in rats.

Abstract We investigated effects of topical application of ketanserin, a 5‐HT2A receptor antagonist, on hyperalgesia and edema in the arthritic rat, a chronic pain model with inflammation. Unilateral, but not bilateral, arthritis was induced with intra‐articular injection of a mixture of kaolin and carrageenan in one side, as indicated by the shortened paw withdrawal latency and an increase in the circumference of the knee joint. Topical application of ketanserin onto skin over the arthritic joint delivered in a mixture of gelatin, glycerol and kaolin produced dose‐dependent attenuation of nociceptive and inflammatory effects resulting from intra‐articularly injected kaolin/carrageenan. One and 3% ketanserin produced significant or even complete anti‐hyperalgesia, as well as a remarkable anti‐inflammatory effect (50–70% reduction of edema) while 0.3% ketanserin and placebo failed to produce any effect. Moreover, the effects of ketanserin were maintained for 13 days without decline. In contrast, 3% ketanserin applied to skin of the knee joint on the non‐inflamed side for 2 weeks did not alter nociceptive thresholds of the paw and the size of the knee joint in both the inflamed and non‐inflamed limbs. These results indicate that 5‐HT2A receptors in the periphery play a significant role in the maintenance and/or development of inflammatory pain. The present study suggests that topical ketanserin is a promising direction for potential clinical exploration to relieve established hyperalgesia and inflammation in arthritis without adverse effects and tolerance.

Upregulation of adrenomedullin in the spinal cord and dorsal root ganglia in the early phase of CFA-induced inflammation in rats.

ABSTRACT Adrenomedullin (AM), a member of calcitonin gene‐related peptide (CGRP) family, has been demonstrated to be a pronociceptive mediator [28]. This study was undertaken to investigate the role of AM in a model of complete Freunds adjuvant (CFA)‐induced inflammatory pain. Injection of CFA, but not of saline, in the unilateral hindpaw produced an increase in the expression of AM‐like immunoreactivity (AM‐IR) in laminae I–II of the spinal cord as well as in small‐ and medium‐sized dorsal root ganglion (DRG) neurons at 48 h. The content of AM in DRG on the side ipsilateral to CFA injection started to increase at 4 h and remained at high levels at 24 and 48 h. The selective antagonist of AM receptors, AM22–52, administered intrathecally (i.t.) 24 h after CFA injection inhibited inflammation‐associated hyperalgesia in a dose‐dependent manner (2, 5 and 10 nmol). Impressively, this anti‐hyperalgesic effect lasted for at least 24 h. I.t. administration of AM22–52 (10 nmol) also reversed CFA‐induced increase in AM‐IR in the spinal dorsal horn and DRG. Furthermore, blockade of AM receptors abolished CFA‐induced changes in the expression and content of CGRP‐like immunoreactivity in these regions. Taken together, our results suggest that the upregulation of AM in DRG neurons contributes to the development of inflammatory pain, and this effect is mediated, at least in part, by enhancing the expression and release of CGRP. Blocking AM receptor downstream signaling effects using antagonists has the potential of relieving pain following the induction of inflammation.

Modulation of NMDA receptors by intrathecal administration of the sensory neuron-specific receptor agonist BAM8-22.

The sensory neuron-specific receptor (SNSR) is exclusively distributed in dorsal root ganglion (DRG) cells. We have demonstrated that intrathecal (i.t.) administration of SNSR agonists inhibits formalin-evoked responses and the development of morphine tolerance [Chen, T., Cai, Q., Hong, Y., 2006. Intrathecal sensory neuron-specific receptor agonists bovine adrenal medulla 8-22 and (tyr(6))-gamma2-msh-6-12 inhibit formalin-evoked nociception and neuronal fos-like immunoreactivity in the spinal cord of the rat. Neuroscience 141, 965-975]. The present study was undertaken to examine the possible impact of the activation of SNSR on NMDA receptors. I.t. administration of NMDA (6.8 nmol) induced nociceptive behaviors, including scratching, biting and lifting, followed by thermal hypoalgesia and hyperalgesia. These responses were associated with the expression of Fos-like immunoreactivity (FLI) throughout the spinal dorsal horn with highest effect seen in laminae I-II. I.t. NMDA also induced an increase in nitric oxide synthase (NOS) activity in superficial layers of the dorsal horn, but not around the central canal, as revealed by NADPH diaphorase histochemistry. Pretreatment with the SNSR agonist bovine adrenal medulla 8-22 (3, 10 and 30 nmol) dose-dependently diminished NMDA-evoked nocifensive behaviors and hyperalgesia. This agonist also reduced NMDA-evoked expression of FLI and NADPH reactivity in the spinal dorsal horn. Taken together, these data suggest that the activation of SNSR induces spinal analgesia by suppressing NMDA receptor-mediated activation of spinal dorsal horn neurons and an increase in NOS activity.

Effect of Mas-related gene (Mrg) receptors on hyperalgesia in rats with CFA-induced inflammation via direct and indirect mechanisms

Mas oncogene‐related gene (Mrg) receptors are exclusively distributed in small‐sized neurons in trigeminal and dorsal root ganglia (DRG). We investigated the effects of MrgC receptor activation on inflammatory hyperalgesia and its mechanisms.

A Role for Protein Kinase C-Dependent Upregulation of Adrenomedullin in the Development of Morphine Tolerance in Male Rats

Adrenomedullin (AM) belongs to calcitonin gene-related peptide (CGRP) family and is a pronociceptive mediator. This study investigated whether AM plays a role in the development of tolerance to morphine-induced analgesia. Repetitive intrathecal injection of morphine increased the expression of AM-like immunoreactivity (AM-IR) in the spinal dorsal horn and dorsal root ganglion (DRG) neurons. Ganglion explant culture study showed that this upregulation of AM-IR was μ-opioid receptor dependent through the use of another agonist, fentanyl, and a selective antagonist, CTAP (d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2). The coadministration of the selective AM receptor antagonist AM22-52 markedly attenuated the development of morphine tolerance, associated thermal hyperalgesia, and increase in AM-IR. A likely autocrine mechanism is supported by the finding that AM-IR is colocalized with AM receptor components in DRG neurons. Furthermore, opiate-induced increase in AM content was blocked by protein kinase C (PKC) inhibitors, whereas a PKC activator increased AM synthesis and release. A treatment with AM22-52 also inhibited increases in the expression of CGRP-IR in the spinal cord and DRGs as well as in culture ganglion explants, whereas exposure to CGRP failed to alter AM content. Together, these results reveal that a sustained opiate treatment induces an upregulation of AM through the activation of μ-opioid receptors and the PKC signaling pathway. This phenomenon contributes to the development of tolerance to the antinociceptive effects of opiates at least partially via the upregulation of CGRP. Targeting AM and its receptors should be considered as a novel approach to preserve the analgesic potency of opiates during their chronic use.

Topical and systemic administrations of ketanserin attenuate hypersensitivity and expression of CGRP in rats with spinal nerve ligation

The aim of this study was to examine the involvement of 5-HT and 5-HT(2A) receptors in neuropathic pain and their possible cellular mechanism. To evaluate a potential therapy the 5-HT(2A) receptor antagonist ketanserin was administered topically or subcutaneously in rats with L5 nerve ligation. Unilateral spinal nerve ligation induced hypersensitivity to thermal and mechanical stimuli in the ipsilateral hindpaw and an increase in calcitonin gene-related peptide (CGRP) immunoreactivity (CGRP-IR) in small and medium diameter neurons in dorsal root ganglia (DRG) at L4 and L6, but not at L5. Topical application of ketanserin (3%) delivered in a mixture of gelatin, glycerol and kaolin onto skin over the hindpaw produced significant elevation of nociceptive threshold in the paw. Daily injection of ketanserin inhibited the thermal hyperalgesia in a dose dependent manner (0.1 and 0.3mg/kg, s.c.). The inhibition occurred at 1-3 days after the injection started and persisted for at least 2 weeks without decline. Injection of ketanserin (0.3mg/kg) also suppressed tactile allodynia. Moreover, ketanserin (0.3mg/kg) reversed the increase in CGRP-IR expression in L4 and L6 DRG neurons. These results support the hypothesis that adaptive change in CGRP expression that occurred in the DRG adjacent to the DRG containing injured neurons underlies the nerve ligation-induced hypersensitivity. This study suggests that 5-HT and 5-HT(2A) receptors contribute to the maintenance of neuropathic pain by up-regulating the expression of CGRP-IR, and that topical and systemic administrations of ketanserin are promising therapies for relieving neuropathic pain without tolerance.

Blockade of adrenomedullin receptors reverses morphine tolerance and its neurochemical mechanisms

Adrenomedullin (AM) has been demonstrated to be involved in the development of opioid tolerance. The present study further investigated the role of AM in the maintenance of morphine tolerance, morphine-associated hyperalgesia and its cellular mechanisms. Intrathecal (i.t.) injection of morphine for 6 days induced a decline of its analgesic effect and hyperalgesia. Acute administration of the AM receptor antagonist AM(22-52) resumed the potency of morphine in a dose-dependent manner (12, 35.8 and 71.5 μg, i.t.). The AM(22-52) treatment also suppressed morphine tolerance-associated hyperalgesia. Furthermore, i.t. administration of AM(22-52) at a dose of 35.8 μg reversed the morphine induced-enhancement of nNOS (neuronal nitric oxide synthase) and CGRP immunoreactivity in the spinal dorsal horn and/or dorsal root ganglia (DRG). Interestingly, chronic administration of morphine reduced the expression of the endogenous opioid peptide bovine adrenal medulla 22 (BAM22) in small- and medium-sized neurons in DRG and this reduction was partially reversed by the administration of AM(22-52) (35.8 μg). These results suggest that the activation of AM receptors was involved in the maintenance of morphine tolerance mediating by not only upregulation of the pronociceptive mediators, nNOS and CGRP but also the down-regulation of pain-inhibiting molecule BAM22. Our data support the hypothesis that the level of both pronociceptive mediators and endogenous pain-inhibiting molecules has an impact on the potency of morphine analgesia. Targeting AM receptors is a promising approach to maintain the potency of morphine analgesia during chronic use of this drug.