Animal Studies

Back pain is the leading cause of disability worldwide and contributes to significant socioeconomic impacts worldwide. It has been hypothesized that the degenerative intervertebral disc (IVD) contributes to back pain by sensitizing nociceptive neurons innervating the IVD to stimuli that would not be painful to healthy patients; however, the inflammatory signaling networks mediating this sensitization remain poorly understood. A better understanding of the underlying mechanisms of degenerative IVD induced changes in nociception are required to improve our understanding and treatment of back pain. Towards these ends, we developed a novel in vitro model to investigate degenerative IVD induced changes in dorsal root ganglion (DRG) neuron activation by measuring DRG neuron activity following neuron seeding on human degenerative IVD tissue collected from patients undergoing surgical treatment for back pain. Lentiviral CRISPR epigenome editing vectors were built to down-regulate the inflammatory receptors TNFR1, IL1R1, and IL6st in DRG neurons in single-plex and multi-plex. Multiplex CRISPR epigenome editing of inflammatory receptors demonstrated that degenerative IVD tissue drives thermal sensitization through the simultaneous and redundant signaling of IL-6, TNF-α, and IL-1β. This work elucidates redundant signaling pathways in neuron interactions with the degenerative IVD and suggests the need for multiplex targeting of IL-6, TNF-α, and IL-1β for pain modulation in the degenerative IVD.

Dysfunction of inhibitory circuits in the rostral anterior cingulate cortex underlies the affective (aversive), but not the sensory-discriminative features (hypersensitivity) of the pain experience. To restore inhibitory controls, we transplanted inhibitory interneuron progenitor cells into the rostral anterior cingulate cortex in a chemotherapy-induced neuropathic pain model. The transplants integrated, exerted a GABA-A mediated inhibition of host pyramidal cells and blocked gabapentin preference (i.e. relieved ongoing pain) in a conditioned place preference paradigm. Surprisingly, pain aversiveness persisted when the transplants populated both the rostral and posterior anterior cingulate cortex. We conclude that selective and long lasting inhibition of the rostral anterior cingulate cortex, in the mouse, has a profound pain relieving effect against nerve injury-induced neuropathic pain. However, the interplay between the rostral and posterior anterior cingulate cortices must be considered when examining circuits that influence ongoing pain and pain aversiveness.

Emerging evidence implicates the upregulation of matrix metalloproteinase (MMP)-9/2 in the dorsal root ganglion (DRG) and spinal cord as a contributor to the pathogenesis of chronic pain. In the current study, the expression of MMP-9/2 in wounded tissue, ipsilateral DRG, and the spinal dorsal horn as well as its role in the development of postoperative pain were examined following plantar incision in rats. Our results showed that plantar incision resulted in increased expression of MMP-9/2 in wounded tissue and ipsilateral L4/5 DRGs. Although gelatin zymography detected an increased activity of MMP-9, only MMP-2 protein was increased in the spinal cord. Results of double immunofluorescence staining showed MMP-2 expression in DRG neurons and satellite glial cells, but MMP-9 was found only in neurons. In the spinal cord, MMP-2 was expressed in neurons and astrocytes, and MMP-9 was expressed in neurons and somewhat in microglial cells. Planter incision also elicited increased expression of p-Erk, p-p38, and IL-1β in wounded tissue, ipsilateral L4/5 DRGs, and dorsal horn. Prior intraplantar or intrathecal injection of MMP-9- and MMP-2-specific inhibitors partially prevented reductions of paw withdrawal threshold and paw withdrawal latency following plantar incision. The maturation of IL-1β was also inhibited by the treatment. Moreover, MMP-9 inhibition suppressed p38, and MMP-2 inhibitor reduced the Erk phosphorylation in wounded tissue, DRGs, and dorsal horn. Immunofluorescence staining detected colocalization of MMP-9 with p38 and MMP-2 with Erk in DRG and spinal cord. Together, the above results reveal that upregulated MMP-9 via p38/IL-1β and MMP-2 via Erk/IL-1β signaling in the wounded tissue, ipsilateral DRG, and dorsal horn contribute to the development of postoperative pain.

AbstarctPreviously, distinct sex differences were observed in the pronociceptive role of spinal immune cells in neuropathic and inflammatory mouse pain models. Both peripheral and central innate and adaptive immune changes contribute to sensitization in the tibia fracture rodent model of complex regional pain syndrome (CRPS), and the current study evaluated sex differences in the development of pronociceptive immune responses after fracture. At 4 and 7 weeks post fracture the analgesic effects of a microglia inhibitor were tested in male and female mice and PCR was used to measure inflammatory mediator expression in skin and spinal cord. The temporal progression of CRPS-like changes in male and female wild-type and muMT fracture mice lacking B cells and antibodies were evaluated and IgM antibody deposition measured. Pronociceptive effects of injecting wild-type fracture mouse serum into muMT fracture mice were also tested in both sexes and the role of sex hormones was evaluated in the post fracture development of pronociceptive immune responses. Long lasting immune changes developed in the fracture limb and corresponding spinal cord of both male and female mice, including upregulated neuropeptide and cytokine signaling, microglial activation, and pronociceptive autoimmunity. These complex post fracture immune responses were sexually dichotomous and interacted in temporally evolving patterns that generated post traumatic nociceptive sensitization in both sexes lasting for up to 5 months. Unfortunately, the redundancy and plasticity of these chronic post traumatic immune responses suggest that clinical interventions focusing on any single specific pronociceptive immune change are likely to be ineffectual.

The mechanisms underlying chronic and neuropathic pain pathology involve peripheral and central sensitisation. The medial prefrontal cortex (mPFC) seems to participate in pain chronification, and glutamatergic neurotransmission may be involved in this process. Thus, the aim of the present work was to investigate the participation of the prelimbic (PrL) area of the mPFC in neuropathic pain as well as the role of N-methyl D-aspartate (NMDA) glutamate receptors in neuropathic pain induced by a modified sciatic nerve chronic constriction injury (CCI) protocol in Wistar rats. Neural inputs to the PrL cortex were inactivated by intracortical treatment with the synapse blocker cobalt chloride (CoCl, 1.0 mM/200 nL) 7, 14, 21, or 28 days after the CCI or sham procedure. The glutamatergic agonist NMDA (0.25, 1 or 4 nmol) or the selective NMDA receptor antagonist LY235959 (2, 4 or 8 nmol) was microinjected into the PrL cortex 21 days after surgery. CoCl administration in the PrL cortex decreased allodynia 21 and 28 days after CCI. NMDA at 1 and 4 nmol increased allodynia, whereas LY235959 decreased mechanical allodynia at the highest dose (8 nmol) microinjected into the PrL cortex. These findings suggest that NMDA receptors in the PrL cortex participate in enhancing the late phase of mechanical allodynia after NMDA-induced increases and LY235959-induced decreases in allodynia 21 days after CCI. The glutamatergic system potentiates chronic neuropathic pain by NMDA receptor activation in the PrL cortex. Mechanism of neuropathic pain. The infusion of CoCl, a synapse activity blocker, into the prelimbic (PrL) division of the medial prefrontal cortex (mPFC) decreased the severity of mechanical allodynia, showing the late participation of the limbic cortex. The glutamatergic system potentiates chronic neuropathic pain via NMDA receptor activation in the PrL cortex.