Animal Studies

Leptin is an adipocytokine that is primarily secreted by white adipose tissue, and it contributes to the pathogenesis of neuropathic pain in collaboration with N-methyl-D-aspartate receptors (NMDARs). Functional NMDARs are a heteromeric complex that primarily comprise two NR1 subunits and two NR2 subunits. NR2A is preferentially located at synaptic sites, and NR2B is enriched at extrasynaptic sites. The roles of synaptic and extrasynaptic NMDARs in the contribution of leptin to neuropathic pain are not clear. The present study examined whether the important role of leptin in neuropathic pain was related to synaptic or extrasynaptic NMDARs. We used a rat model of spared nerve injury (SNI) and demonstrated that the intrathecal administration of the NR2A-selective antagonist NVP-AAM077 and the NR2B-selective antagonist Ro25-6981 prevented and reversed mechanical allodynia following SNI. Administration of exogenous leptin mimicked SNI-induced behavioral allodynia, which was also prevented by NVP-AAM077 and Ro25-6981. Mechanistic studies showed that leptin enhanced NR2B- but not NR2A-mediated currents in spinal lamina II neurons of naïve rats. Leptin also upregulated the expression of NR2B, which was blocked by the NR2B-selective antagonist Ro25-6981, in cultured dorsal root ganglion (DRG) neurons. Leptin enhanced neuronal nitric oxide synthase (nNOS) expression, which was also blocked by Ro25-6981, in cultured DRG cells. However, leptin did not change NR2A expression, and the NR2A-selective antagonist NVP-AAM077 had no effect on leptin-enhanced nNOS expression. Our data suggest an important cellular link between the spinal effects of leptin and the extrasynaptic NMDAR-nNOS-mediated cellular mechanism of neuropathic pain.

One maladaptive consequence of inflammatory stimulation of the afferent somatosensory system is the manifestation of inflammatory pain. We established and characterized a neuroglial primary culture of the rat superficial dorsal horn (SDH) of the spinal cord to test responses of this structure to neurochemical, somatosensory, or inflammatory stimulation. Primary cultures of the rat SDH consist of neurons (43%), oligodendrocytes (35%), astrocytes (13%), and microglial cells (9%). Neurons of the SDH responded to cooling (7%), heating (18%), glutamate (80%), substance P (43%), prostaglandin E (8%), and KCl (100%) with transient increases in the intracellular calcium [Ca]. Short-term stimulation of SDH primary cultures with LPS (10 μg/ml, 2 h) caused increased expression of pro-inflammatory cytokines, inflammatory transcription factors, and inducible enzymes responsible for inflammatory prostaglandin E synthesis. At the protein level, increased concentrations of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) were measured in the supernatants of LPS-stimulated SDH cultures and enhanced TNFα and IL-6 immunoreactivity was observed specifically in microglial cells. LPS-exposed microglial cells further showed increased nuclear immunoreactivity for the inflammatory transcription factors NFκB, NF-IL6, and pCREB, indicative of their activation. The short-term exposure to LPS further caused a reduction in the strength of substance P as opposed to glutamate-evoked Ca-signals in SDH neurons. However, long-term stimulation with a low dose of LPS (0.01 μg/ml, 24 h) resulted in a significant enhancement of glutamate-induced Ca transients in SDH neurons, while substance P-evoked Ca signals were not influenced. Our data suggest a critical role for microglial cells in the initiation of inflammatory processes within the SDH of the spinal cord, which are accompanied by a modulation of neuronal responses.

Agonists to subtype C of the Mas-related G-protein-coupled receptors (MrgC) induce pain inhibition after intrathecal (i.t.) administration in rodent models of nerve injury. Here, we investigated whether tolerance develops after repeated MrgC agonist treatments and examined the underlying mechanisms. In animal behavior studies conducted in male rats at 4-5 weeks after an L5 spinal nerve ligation (SNL), the ability of dipeptide MrgC agonist JHU58 (0.1 mM, 10 μL, i.t.) to inhibit mechanical and heat hypersensitivity decreased after 3 days of treatment with a tolerance-inducing dose (0.5 mM, 10 μL, i.t., twice/day). In HEK293T cells, acute treatment with JHU58 or BAM8-22 (a large peptide MrgC agonist) led to MrgC endocytosis from the cell membrane, and later sorting to the membrane for reinsertion. However, chronic exposure to JHU58 increased the coupling of MrgC to β-arrestin-2 and led to the ubiquitination and degradation of MrgC. Importantly, pretreatment with TAK-243 (0.2 mM, 5 μL, i.t.), a small-molecule inhibitor of the ubiquitin activating enzyme, during tolerance induction attenuated the development of tolerance to JHU58-induced inhibition of mechanical and heat hypersensitivity in SNL rats. Interestingly, morphine analgesia was also decreased in SNL rats that had become tolerant to JHU58, suggesting a cross-tolerance. Furthermore, intrathecal pretreatment with TAK-243, which reduced JHU58 tolerance, also attenuated the cross-tolerance to morphine analgesia. These findings suggest that tolerance can develop to MrgC agonist-induced pain inhibition after repeated intrathecal administrations. This tolerance development to JHU58 may involve increased coupling of MrgC to β-arrestin-2 and ubiquitin-mediated receptor degradation.

Neuroinflammation is a critical feature of sensitisation of spinal nociceptive processing in chronic pain states. We hypothesised that the resolvin pathways, a unique endogenous control system, may ameliorate aberrant spinal processing of somatosensory inputs associated with chemotherapy-induced neuropathic pain (CINP).

Activated microglia involved in the development of orofacial pain hypersensitivity have two major polarization states. The aim of this study was to assess the involvement of the aging-related phenotypic conversion of medullary microglia in the enhancement of intraoral pain sensitivity using senescence-accelerated mice (SAM)-prone/8 (SAMP8) and SAM-resistant/1 (SAMR1) mice. Mechanical head-withdrawal threshold (MHWT) was measured for 21 days post palatal mucosal incision. The number of CD11c-immunoreactive (IR) cells [affective microglia (M1)] and CD163-IR cells [protective microglia (M2)], and tumor-necrosis-factor-α (TNF-α)-IR M1 and interleukin (IL)-10-IR M2 were analyzed via immunohistochemistry on days 3 and 11 following incision. The decrease in MHWT observed following incision was enhanced in SAMP8 mice. M1 levels and the number of TNF-α-IR M1 were increased on day 3 in SAMP8 mice compared with those in SAMR1 mice. On day 11, M1 and M2 activation was observed in both groups, whereas IL-10-IR M2 levels were attenuated in SAMP8 mice, and the number of TNF-α-IR M1 cells increased, compared to those in SAMR1 mice. These results suggest that the mechanical allodynia observed following intraoral injury is potentiated and sustained in SAMP8 mice due to enhancement of TNF-α signaling, M1 activation, and an attenuation of M2 activation accompanying IL-10 release.