Animal Studies

Identification of genetic variants that influence susceptibility to pain is key to identifying molecular mechanisms and targets for effective and safe therapeutic alternatives to opioids. To identify genes and variants associated with persistent pain, we measured late-phase response to formalin injection in 275 male and female Diversity Outbred mice genotyped for over 70,000 single nucleotide polymorphisms. One quantitative trait locus reached genome-wide significance on chromosome 1 with a support interval of 3.1 Mb. This locus, Nociq4 (nociceptive sensitivity quantitative trait locus 4; MGI: 5661503), harbors the well-known pain gene Trpa1 (transient receptor potential cation channel, subfamily A, member 1). Trpa1 is a cation channel known to play an important role in acute and chronic pain in both humans and mice. Analysis of Diversity Outbred founder strain allele effects revealed a significant effect of the CAST/EiJ allele at Trpa1, with CAST/EiJ carrier mice showing an early, but not late, response to formalin relative to carriers of the 7 other inbred founder alleles (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ). We characterized possible functional consequences of sequence variants in Trpa1 by assessing channel conductance, TRPA1-TRPV1 interactions, and isoform expression. The phenotypic differences observed in CAST/EiJ relative to C57BL/6J carriers were best explained by Trpa1 isoform expression differences, implicating a splice junction variant as the causal functional variant. This study demonstrates the utility of advanced, high-precision genetic mapping populations in resolving specific molecular mechanisms of variation in pain sensitivity.

Synovitis contributes to temporomandibular joint（TMJ）pain, nevertheless, the detailed nociceptive mechanism remains unclear. In this study, a rat model of TMJ synovitis was induced by intra-articular injection with complete Freund's adjuvant (CFA). After CFA-induced synovitis, pain behaviors were observed. Then, TMJ, trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC) tissues were collected, and immunohistochemistry (IHC) was used to detect the expression of substance P (SP) and protein gene product 9.5 (PGP9.5) in the synovium tissue. Furthermore, the gene expression level of SP and PGP9.5 in synovium were detected by reverse transcription-polymerase chain reaction (RT-PCR). Afterwards, the expression of SP in the TG and TNC and c-fos in the TNC was detected by IHC. Compared with the control group, the expression of SP and PGP9.5 nerve fibers density and gene levels of them in the synovium tissue were significantly increased in CFA-induced TMJ synovitis rats. Similarly, SP expression in the TG and TNC, and c-fos expression in the TNC were also obviously increased in CFA-induced TMJ synovitis rats. Collectively, CFA-induced rat TMJ synovitis resulted in obvious pain. This nociceptive reaction could be attributed to the augmented quantity of SP and PGP9.5 positive-stained nerve fibers distributed in the inflammatory synovium, as well as enhanced SP expression in the TG and TNC tissue. C-fos expression in the rat TNC illustrates CFA-induced TMJ synovitis can evoke the acute pain.

Ephrin-B2/EphB receptor signaling contributes to persistent pain states such as postinflammatory and neuropathic pain. Visceral hypersensitivity (VHS) is a major mechanism underlying abdominal pain in patients with irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBD) in remission, but the underlying pathophysiology remains unclear. Here, we evaluated the spinal ephrin-B2/EphB pathway in VHS in 2 murine models of VHS, that is, postinflammatory TNBS colitis and maternal separation (MS).

The loss of sensory input following a spinal deafferentation injury can be debilitating, and this is especially true in primates when the hand is involved. While significant recovery of function occurs, little is currently understood about the reorganization of the neuronal circuitry, particularly within the dorsal horn. This region receives primary afferent input from the periphery, and cortical input via the somatosensory subcomponent of the corticospinal tract (S1 CST), and is critically important in modulating sensory transmission, both in normal and lesioned states. To determine how dorsal horn circuitry alters to facilitate recovery post-injury, we used an established deafferentation lesion model (DRL/DCL – dorsal root/dorsal column) in male monkeys to remove sensory input from just the opposing digits (D1-D3) of one hand. This results in a deficit in fine dexterity that recovers over several months. Electrophysiological mapping, tract tracing, and immunolabeling techniques were combined to delineate specific changes to dorsal horn input circuitry. Our main findings show that (1) there is complementary sprouting of the primary afferent and S1 CST populations into an overlapping region of the reorganizing dorsal horn, (2) S1 CST and primary afferent inputs connect in different ways within this region to facilitate sensory integration (3) there is a loss of larger S1 CST terminal boutons in the affected dorsal horn, but no change in the size profile of the spared/sprouted primary afferent terminal boutons post-lesion. Understanding such changes helps to inform new and targeted therapies that best promote recovery.Spinal injuries that remove sensation from the hand, can be debilitating, though functional recovery does occur. We examined changes to the neuronal circuitry of the dorsal horn in monkeys following a lesion that deafferented three digits of one hand. Little is understood about dorsal horn circuitry, despite the fact that this region loses most of its normal input after such an injury, and is clearly a major focus of reorganization. We found that both the spared primary afferents and somatosensory corticospinal efferents sprouted in an overlapping region of the dorsal horn after injury, and that larger (presumably faster) corticospinal terminals are lost, suggesting a significantly altered cortical modulation of primary afferents. Understanding this changing circuitry is important for designing targeted therapies.

Endocannabinoids are showing great promise as effective mediators for controlling joint inflammation and pain. One strategy that could be harnessed to promote endogenous cannabinoid function is to inhibit the enzymatic break down of endocannabinoids locally in the joint. KML29 is an inhibitor of monoacylglycerol lipase (MAGL) activity which has been shown to promote increased 2-arachodonylglycerol (2-AG) levels in the circulation and in peripheral tissues. It is also known that 2-AG can be metabolised via the cyclo-oxygenase-2 (COX-2) pathway leading to the production of pro-inflammatory prostaglandins, which may counteract the effects of 2-AG. Therefore, this study examined the effect of KML29 alone as well as in combination with low-dose celecoxib (CXB) on joint pain and inflammation in the monoiodoacetate (MIA) model of osteoarthritis (OA) pain.

Some individuals recover from the pain of nerve trauma within 12 months or less whereas others experience life-long intractable pain. This transition between reversible pain and the establishment of chronic neuropathic pain is poorly understood. We examined the role of persistent inflammation in the dorsal root ganglia (DRG) in the long-term maintenance of mechanical allodynia; an index of neuropathic pain. Male Sprague-Dawley rats underwent chronic constriction injury (CCI), spared nerve injury (SNI) or sham surgery. Both CCI and SNI animals displayed robust mechanical allodynia in the ipsilateral paw at 7d post-surgery; however, only SNI animals maintained mechanical allodynia at 42d post-surgery. DRGs were extracted at 7d or 42d post-surgery to assess inflammation via rt-qPCR or immunohistochemistry to measure colony stimulating factor 1 (CSF1) expression, satellite glial cell (SGC) activation, presence of Iba1 positive macrophages and interleukin1 β (IL-1β) mRNA levels. Whereas DRGs from SNI animals continued to display inflammatory markers at 42d, those from CCI animals did not. Moreover, the level of allodynia displayed by each individual animal correlated with the extent of DRG inflammation. These data support the hypothesis that the amount of CSF1 immunoreactivity and the persistence of inflammation in ipsilateral DRGs contribute to the difference between transient and persistent mechanical allodynia observed in the CCI and SNI models. We also suggest that feedback loops involving cytokines and neurotransmitters may contribute to increased DRG activity in chronic neuropathic pain. Consequently, targeting persistent CSF1 production and peripheral neuroinflammation may be an effective approach to the management of chronic neuropathic pain.

Although microglia activation plays an important role in the development of nerve injury-induced neuropathic pain, the molecular mechanisms of spinal cord microglia activation in nerve injury are not completely understood. Recently, two injured sensory neuron-derived molecules, colony stimulating factor-1 (CSF-1) and GT1b, were proposed to trigger spinal cord microglia activation, yet their relationship and relative contribution to microglia activation have not been addressed. In the present study, the role of GT1b and CSF-1 in microglia activation and proliferation was characterized. GT1b stimulation upregulated proinflammatory mediators such as IL-1β, TNF-α, and NADPH oxidase 2 (Nox2), without microglia proliferation. Conversely, CSF-1 stimulation induced microglia proliferation with minimal proinflammatory gene induction. Notably, neither GT1b nor CSF-1 induced mechanical hypersensitivity in female mice; however, they induced similar microglial proliferation in both male and female mice. Taken together, our data indicate that injured sensory neuron-derived GT1b and CSF-1 activate spinal cord microglia in concert through distinct activation pathways.