Animal Studies

Malfunctioning synaptic plasticity is one of the major mechanisms contributing to the development of chronic pain. We studied spike-timing dependent depression (tLTD) in the ACC of male mice, a brain region involved in processing emotional aspects of pain. tLTD onto layer 5 pyramidal neurons depended on postsynaptic calcium-influx through GluN2B-containing NMDARs and retrograde signaling via nitric oxide to reduce presynaptic release probability. After chronic constriction injury of the sciatic nerve, a model for neuropathic pain, tLTD was rapidly impaired; and this phenotype persisted even beyond the time of recovery from mechanical sensitization. Exclusion of GluN2B-containing NMDARs from the postsynaptic site specifically at projections from the anterior thalamus to the ACC caused the tLTD phenotype, whereas signaling downstream of nitric oxide synthesis remained intact. Thus, transient neuropathic pain can leave a permanent trace manifested in the disturbance of synaptic plasticity in a specific afferent pathway to the cortex.Synaptic plasticity is one of the main mechanisms that contributes to the development of chronic pain. Most studies have focused on potentiation of excitatory synaptic transmission, but very little is known about the reduction in synaptic strength. We have focused on the ACC, a brain region associated with the processing of emotional and affective components of pain. We studied spike-timing dependent LTD, which is a biologically plausible form of synaptic plasticity, which depends on the relative timing of presynaptic and postsynaptic activity. We found a long-lasting and pathway-specific suppression of the induction mechanism for spike-timing dependent LTD from the anterior thalamus to the ACC, suggesting that this pathology might be involved in altered emotional processing in pain.

Bone cancer pain (BCP) is the most frequently observed chronic cancer pain, and its development remains largely unexplored. Dysregulation of non-coding RNAs greatly contributes to the pathogenesis of BCP. In the present study, we found a new long noncoding RNA (lncRNA), NONRATT009773.2, and investigated its role in the spinal cord of BCP rats. Our results showed that NONRATT009773.2 was significantly up-regulated in BCP model rats, while depletion of NONRATT009773.2 attenuated BCP. In contrast, overexpression of NONRATT009773.2 triggered pain-like symptoms in normal animals. Moreover, NONRATT009773.2 functioned as a microRNA (miRNA) sponge to absorb miR-708-5p and up-regulated miRNA downstream target CXCL13, which plays fundamental roles in the initiation and maintenance of neuroinflammation and hyperalgesia. Collectively, our current findings indicated that NONRATT009773.2 could be employed as a new therapeutic target for BCP.

Central post stroke pain (CPSP) is an intractable neuropathic pain syndrome that occurs after the acute focal lesion of the central nervous system (CNS) due to a cerebrovascular cause. Epoxyeicosatrienoic acids (EETs) exert many pharmacological effects in vivo and in vitro, such as anti-apoptosis, anti-inflammatory, and anti-oxidative stress. Neuroinflammation and apoptosis are the potential pathophysiological mechanisms of neuropathic pain. This study aimed to investigate whether 14,15-EET has an antinociception effect on CPSP rats through its anti-inflammation and anti-apoptosis mechanisms. Rats were treated with type IV collagenase (CPSP group) or saline (Sham group) via injection with a Hamilton syringe into the ventral posterior lateral nucleus (VPL) according to the stereotaxic coordinates. We first tested the mechanical withdrawal threshold, as well as neuroinflammation- and apoptosis-related protein expressions in the per-lesion site of CPSP and Sham rats. Sprague-Dawley rats were randomly divided into five groups, as follows: vehicle; EET at 0.025, 0.05, and 0.1 μg; and EET (0.1 μg) + EEZE (3.25 ng). EET or and vehicle were administered into VPL nuclei three consecutive days after hemorrhagic stroke. Immunostaining, ELISA, and Western blot were performed to evaluate neuroinflammation and apoptosis. Hemorrhagic stroke induced mechanical allodynia, glial activation, neuroinflammation, and apoptosis-related protein upregulation. However, early treatment with 14,15-EET inhibited glial cell activation, decreased proinflammatory cytokines and apoptosis-related protein, and alleviated the pain behavior of CPSP rats. Our results provided strong evidence that antinociception produced by 14,15-EET is partly mediated by the inhibition of neuroinflammation and apoptosis.

The increased prevalence of temporomandibular joint osteoarthritis (TMJOA) in children and adolescents has drawn considerable attention as it may interfere with mandibular condyle growth, resulting in dento-maxillofacial deformities. However, treatments for osteoarthritis have been ineffective at restoring the damaged bone and cartilage structures due to poor understanding of the underlying degenerative mechanism. In this study, we demonstrate that Gli1 cells residing in the subchondral bone contribute to bone formation and homeostasis in the mandibular condyle, identifying them as osteogenic progenitors in vivo. Furthermore, we show that, in a TMJOA mouse model, derivatives of Gli1+ cells undergo excessive expansion along with increased but uneven distribution of osteogenic differentiation in the subchondral bone, which leads to abnormal subchondral bone remodeling via Hedgehog (Hh) signaling activation and to the development of TMJOA. The selective pharmacological inhibition and specific genetic inhibition of Hh signaling in Gli1 osteogenic progenitors result in improved subchondral bone microstructure, attenuated local immune inflammatory response in the subchondral bone, and reduced degeneration of the articular cartilage, providing in vivo functional evidence that targeting Hh signaling in Gli1 osteogenic progenitors can modulate bone homeostasis in osteoarthritis and provide a potential approach for treating TMJOA.

Neuropathic pain is a prevalent and debilitating chronic disease that is characterized by activation in glial cells in various pain-related regions within the central nervous system. Recent studies have suggested a sexually dimorphic role of microglia in the maintenance of neuropathic pain in rodents. Here, we utilized RNA sequencing analysis and in vitro primary cultures of microglia to identify whether there is a common neuropathic microglial signature and characterize the sex differences in microglia in pain-related regions in nerve injury and chemotherapy-induced peripheral neuropathy mouse models. While mechanical allodynia and behavioral changes were observed in all models, transcriptomic analysis of microglia revealed no common transcriptional changes in spinal and supraspinal regions and in the different neuropathic models. However, there was a substantial change in microglial gene expression within the ipsilateral lumbar spinal cord 7 days after chronic constriction injury (CCI) of the sciatic nerve. Both sexes upregulated genes associated with inflammation, phagosome, and lysosome activation, though males revealed a prominent global transcriptional shift not observed in female mice. Transcriptomic comparison between male spinal microglia after CCI and data from other nerve injury models and neurodegenerative microglia demonstrated a unique CCI-induced signature reflecting acute activation of microglia. Further, in vitro studies revealed that only male microglia from nerve-injured mice developed a reactive phenotype with increased phagocytotic activity. This study demonstrates a lack of a common neuropathic microglial signature and indicates distinct sex differences in spinal microglia, suggesting they contribute to the sex-specific pain processing following nerve injury.

Chronic pain is the most common neurological disorder of HIV patients. Multiple neuropathologies were identified in the pain pathway. Among them is the prominent astrocytic reaction (a.k.a. astrogliosis). However, the pathogenic role and mechanism of the astrogliosis are unclear. Here, we show that the astrogliosis is crucial for the pain development induced by a key neurotoxic HIV protein gp120 and that a neuron-to-astrocyte Wnt5a signal controls the astrogliosis. Ablation of astrogliosis blocked the development of gp120-induced mechanical hyperalgesia, and concomitantly the expression of neural circuit polarization (NCP) in the spinal dorsal horn (SDH). We demonstrated that conditional knockout (CKO) of either Wnt5a in neurons or its receptor ROR2 in astrocytes abolished not only gp120-induced astrogliosis but also hyperalgesia and NCP. Furthermore, we found that the astrogliosis promoted expression of hyperalgesia and NCP via IL-1β regulated by a Wnt5a-ROR2-MMP2 axis. Our results shed light on the role and mechanism of astrogliosis in the pathogenesis of HIV-associated pain.

Mesenchyme homeobox protein 2 (MEOX2) is a transcription factor involved in mesoderm differentiation, including development of bones, muscles, vasculature and dermatomes. We have previously identified dysregulation of MEOX2 in fibroblasts from Congenital Insensitivity to Pain (CIP) patients, and confirmed that btn, the Drosophila homologue of MEOX2, plays a role in nocifensive responses to noxious heat stimuli. To determine the importance of MEOX2 in the mammalian peripheral nervous system, we used a Meox2 heterozygous (Meox2 ) mouse model to characterize its function in the sensory nervous system, and more specifically, in nociception. MEOX2 is expressed in the mouse dorsal root ganglia (DRG) and spinal cord, and localizes in the nuclei of a subset of sensory neurons. Functional studies of the mouse model, including behavioral, cellular and electrophysiological analyses, showed altered nociception encompassing impaired action potential initiation upon depolarization. Mechanistically, we noted decreased expression of Scn9a and Scn11a genes encoding Na 1.7 and Na 1.9 voltage gated sodium channels, respectively, that are crucial in subthreshold amplification and action potential initiation in nociceptors. Further transcriptomic analyses of Meox2 DRG revealed downregulation of a specific subset of genes including those previously associated with pain perception, such as PENK and NPY. Based on these observations, we propose a novel role of MEOX2 in primary afferent nociceptor neurons for the maintenance of a transcriptional program required for proper perception of acute and inflammatory noxious stimuli.

The detailed pathological mechanism of orofacial neuropathic pain remains unknown. We aimed to examine the pannexin 1 (Panx1) signaling in the trigeminal ganglion (TG) involvement in infraorbital nerve injury (IONI)-induced orofacial neuropathic pain.