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(211) Deep Pain Mechanisms in Sickle Cell Disease
S28 The Journal of Pain frequency-dependent [Ca2+]c regulation does, in fact, impact GCaMP6’s ability to encode neural activity. Here, we expanded upon this work to assess the relationship between neural activity and [Ca2+]c as well as the utility of GCaMP6s/m/f to detect activity changes in trigeminal sensory neuron subpopulations. To drive GCaMP6 expression in acutely dissociated rat trigeminal ganglion neurons, we used AAV9-CAG. Neurons were then stimulated using different protocols and frequencies. First, we measured infection efficiency of AAV9-CAG-GCAMP6(s/m/f). Then, comparing isoforms and neuronal subpopulations, we assessed the (i) ability to detect a single spike, (ii) ability to resolve single spikes in a spike train at different frequencies, (iii) dynamic range, and (iv) optimal firing frequency. Using Fura-2, we measured [Ca2+]c in GCaMP6-expressing neurons at rest and following stimulation. GCaMP6 expression was detected within 6 days, and results were collected 6-10d post-infection. Infection efficiency is >80% with all three isoforms in all neuron subpopulations. All three GCaMP6 isoforms detect a single spike in 84-98% of neurons in all subpopulations. However, GCaMP6s’s dynamic range is greater than 6m’s or 6f’s. We confirmed our initial observation that there is an optimal coding frequency that is isoform- and neuronal subpopulation-dependent, which is often below 30 Hz. These data suggest that care should be taken in selection, use, and interpretation of experiments that use GCaMP6 as a proxy for peripheral neurons activity.
(211) Deep Pain Mechanisms in Sickle Cell Disease A. Erb and C. Stucky; Medical College of Wisconsin, Milwaukee, WI Sickle Cell Disease (SCD), an autosomal recessive red blood cell disorder, affects more than 100,000 individuals in the US. Deep pain that appears to stem from muscle and bones is a major complaint of patients with SCD and very little is known about putative mechanisms that cause this deep pain. To assess this pain, we used the Townes HbSS (SS) transgenic mouse model of SCD to perform behavioral assays including evoked withdrawal responses to gastrocnemius muscle pinch and the inverted screen hang, a potential measure of muscle or bone pain. For the muscle pinch assay, SS mice exhibited lower withdrawal thresholds than Townes AA controls. Furthermore, there was a sex difference between SS males and SS females in their withdrawal thresholds where females had higher sensitivity and greater pain-like behaviors than males. For the inverted screen hang assay, SS mice had significantly lower total hang time and more time on 3 paws or less compared to AA controls. Together these data suggest that these mice may experience greater deep pain. Because the TRPV1 channel has been shown to contribute to mechanical hypersensitivity in SCD mice, we used the selective and potent TRPV1 inhibitor A-425619 in behavioral assays. Intraperitoneal injection of A-425619 (100mmol/kg) partially reversed the sensitized muscle pinch withdrawal thresholds in both sexes of SS SCD mice, with a greater effect on males than in females. To further assess mechanisms underlying the sensitization in SCD mice, calcium imaging of lumbar DRG sensory neurons was performed to begin to investigate whether there is increased sensitization of sensory neurons to endogenous metabolites that could act on TRPV1, ASIC3, and P2X channels. In conclusion, these data suggest that the Townes SS mouse model of SCD has increased deep pain sensitivity compared to controls and this sensitivity appears to be partly mediated by TRPV1.
(212) Gene Expression Profile of Nociceptor Hyper-Excitability in Painful Diabetic Neuropathy D. George, N. Jayaraj, D. Ren, R. Miller, and D. Menichella; Northwestern University, Chicago, IL Painful diabetic neuropathy (PDN) is an intractable complication of diabetes that affects 25% of patients. PDN is characterized by neuropathic pain and small-fiber degeneration, accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability and loss of their axons within the skin. The molecular mechanisms underlying DRG nociceptor hyperexcitability and small-fiber degeneration in PDN are unknown. To delineate the molecular mechanisms behind nociceptor hyperexcitability and small-fiber degeneration, it becomes important to uniquely identify changes in the nociceptor population from within the heterogeneous population of neurons in the DRG. To identify genes that are differentially expressed in PDN, we compared the RNA profile of mice fed a regular-diet (RD) or a highfat diet (HFD), a commonly used model of PDN. To capture the translational state of these nociceptive neurons, we used
Abstracts transgenic mice expressing tagged ribosomal subunits (RiboTag) in the nociceptor population expressing the sodium channel-Nav1.8. Analysis of the RiboTagged mRNA between HFD and RD mice at 10 weeks revealed 84 upregulated genes and 70 downregulated genes. We identified candidate genes like ApoD, known to increase excitatory signaling through CXCR4/CXCL12. Previously our work demonstrated that CXCR4/CXCR12 signaling is critical for the development of mechanical allodynia and small-fiber degeneration in PDN. Further analysis implicated changes in pathways relating to extracellular matrix organization, gliogenesis and, complex 1 biogenesis. In addition to the translational profiling, we plan to perform single-cell RNA sequencing of the Nav1.8-positive DRG neuron population using Nav1.8-Cre;Ai9 mice fed an RD or an HFD. Singlecell RNA sequencing will give us a better understanding of the transcriptional status of subpopulations within the Nav1.8-positive DRG neurons and generate candidate genes at a higher resolution. This study will provide insight into the genes that are involved in the pathogenesis of neuropathic pain and small-fiber degeneration and yield potential translational targets for disease-modifying treatments for PDN.
(213) IgG-Immune Complex Directly Activates Joint Sensory Neurons through Neuronal FcgRI to Induce Arthritis Pain L. Qu, L. Wang, X. Jiang, Q. Zheng, S. Jeon, Z. Dong, and M. Caterina; Johns Hopkins School of Medicine, Baltimore, MD Joint pain in rheumatoid arthritis (RA) represents a significant health burden. Although RA pain is conventionally thought to result from inflammation, it often persists even after control of inflammation with available therapies, suggesting the additional involvements of non-inflammatory mechanisms. Yet, such mechanisms remain largely unexplored. Our in situ hybridization assays suggested that the immune complex receptor FcgRI was expressed in a subpopulation of joint sensory neurons. Consistent with this finding, the proportion of joint sensory neurons exhibiting a Ca2+ increase in response to IgG-immune complex (IgG-IC), assayed both in vitro and in vivo, was lower in global FcgRI¡/¡ mice, compared with wildtype mice. Injection of IgG-IC but not monomeric IgG into one ankle of na€ıve mice evoked joint pain-related behaviors without concurrent joint swelling. This effect was diminished in global FcgRI¡/¡ mice. Ablation of T and B cells, mast cells or macrophages had no effect on IgG-IC evoked nocifensive behaviors. Under arthritic conditions, FcgRI mRNA expression and function were upregulated in dorsal root ganglion (DRG) in a mouse model of antigen-induced arthritis (AIA). In vivo extracellular electrophysiological recordings on intact DRG showed that genetic deletion of FcgRI reduced the incidence of abnormal activity and mechanical hypersensitivity of joint sensory neurons in the context of AIA. Acute blockade of FcgRI with neutralizing antibody and genetic knockout of FcgRI significantly attenuated pain-related behaviors in the AIA model. Conversely, no significant differences between treatments or genotypes were observed in the inflamed joint in the expression level of cytokines or in immune cell infiltration. These findings indicate that FcgRI may contribute to arthritis pain through a non-inflammatory mechanism that parallels inflammation and joint damage and point towards a promising therapeutic target to consider for RA pain that is resistant to current anti-inflammatory treatments. Supported by NIAMS 1R01AR072230 to L.Q.
(214) NOD-Like Receptor Protein 3 Inflammasome Drives Postoperative Pain in a Sex-Dependent Manner A. Cowie, A. Menzel, C. O’Hara, M. Lawlor, and C. Stucky; Medical College of Wisconsin, Milwaukee, WI Inflammasomes are cytosolic receptors of the innate immune system that are responsible for activation and secretion of the proinflammatory cytokine interleukin-1b (IL-1b) where IL-1b is known to cause pain by directly acting on sensory neurons. Specifically, IL-1b is known to cause peripheral sensitization to mechanical stimuli in models of inflammatory and neuropathic pain. Of the multiple inflammasomes, the NOD-like receptor protein 3 (NLRP3) inflammasome is activated by endogenous molecules that are released as a result of tissue injury and thus is ideally positioned to produce the majority of postoperative IL-1b. Therefore, we aimed to uncover the mechanism by which NLRP3 modifies postoperative pain in males and females. Male mice lacking NLRP3 recovered from incisional surgery significantly faster at the behavioral and
(211) Deep Pain Mechanisms in Sickle Cell Disease A. Erb and C. Stucky; Medical College of Wisconsin, Milwaukee, WI Sickle Cell Disease (SCD), an autosomal recessive red blood cell disorder, affects more than 100,000 individuals in the US. Deep pain that appears to stem from muscle and bones is a major complaint of patients with SCD and very little is known about putative mechanisms that cause this deep pain. To assess this pain, we used the Townes HbSS (SS) transgenic mouse model of SCD to perform behavioral assays including evoked withdrawal responses to gastrocnemius muscle pinch and the inverted screen hang, a potential measure of muscle or bone pain. For the muscle pinch assay, SS mice exhibited lower withdrawal thresholds than Townes AA controls. Furthermore, there was a sex difference between SS males and SS females in their withdrawal thresholds where females had higher sensitivity and greater pain-like behaviors than males. For the inverted screen hang assay, SS mice had significantly lower total hang time and more time on 3 paws or less compared to AA controls. Together these data suggest that these mice may experience greater deep pain. Because the TRPV1 channel has been shown to contribute to mechanical hypersensitivity in SCD mice, we used the selective and potent TRPV1 inhibitor A-425619 in behavioral assays. Intraperitoneal injection of A-425619 (100mmol/kg) partially reversed the sensitized muscle pinch withdrawal thresholds in both sexes of SS SCD mice, with a greater effect on males than in females. To further assess mechanisms underlying the sensitization in SCD mice, calcium imaging of lumbar DRG sensory neurons was performed to begin to investigate whether there is increased sensitization of sensory neurons to endogenous metabolites that could act on TRPV1, ASIC3, and P2X channels. In conclusion, these data suggest that the Townes SS mouse model of SCD has increased deep pain sensitivity compared to controls and this sensitivity appears to be partly mediated by TRPV1.
(212) Gene Expression Profile of Nociceptor Hyper-Excitability in Painful Diabetic Neuropathy D. George, N. Jayaraj, D. Ren, R. Miller, and D. Menichella; Northwestern University, Chicago, IL Painful diabetic neuropathy (PDN) is an intractable complication of diabetes that affects 25% of patients. PDN is characterized by neuropathic pain and small-fiber degeneration, accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability and loss of their axons within the skin. The molecular mechanisms underlying DRG nociceptor hyperexcitability and small-fiber degeneration in PDN are unknown. To delineate the molecular mechanisms behind nociceptor hyperexcitability and small-fiber degeneration, it becomes important to uniquely identify changes in the nociceptor population from within the heterogeneous population of neurons in the DRG. To identify genes that are differentially expressed in PDN, we compared the RNA profile of mice fed a regular-diet (RD) or a highfat diet (HFD), a commonly used model of PDN. To capture the translational state of these nociceptive neurons, we used
Abstracts transgenic mice expressing tagged ribosomal subunits (RiboTag) in the nociceptor population expressing the sodium channel-Nav1.8. Analysis of the RiboTagged mRNA between HFD and RD mice at 10 weeks revealed 84 upregulated genes and 70 downregulated genes. We identified candidate genes like ApoD, known to increase excitatory signaling through CXCR4/CXCL12. Previously our work demonstrated that CXCR4/CXCR12 signaling is critical for the development of mechanical allodynia and small-fiber degeneration in PDN. Further analysis implicated changes in pathways relating to extracellular matrix organization, gliogenesis and, complex 1 biogenesis. In addition to the translational profiling, we plan to perform single-cell RNA sequencing of the Nav1.8-positive DRG neuron population using Nav1.8-Cre;Ai9 mice fed an RD or an HFD. Singlecell RNA sequencing will give us a better understanding of the transcriptional status of subpopulations within the Nav1.8-positive DRG neurons and generate candidate genes at a higher resolution. This study will provide insight into the genes that are involved in the pathogenesis of neuropathic pain and small-fiber degeneration and yield potential translational targets for disease-modifying treatments for PDN.
(213) IgG-Immune Complex Directly Activates Joint Sensory Neurons through Neuronal FcgRI to Induce Arthritis Pain L. Qu, L. Wang, X. Jiang, Q. Zheng, S. Jeon, Z. Dong, and M. Caterina; Johns Hopkins School of Medicine, Baltimore, MD Joint pain in rheumatoid arthritis (RA) represents a significant health burden. Although RA pain is conventionally thought to result from inflammation, it often persists even after control of inflammation with available therapies, suggesting the additional involvements of non-inflammatory mechanisms. Yet, such mechanisms remain largely unexplored. Our in situ hybridization assays suggested that the immune complex receptor FcgRI was expressed in a subpopulation of joint sensory neurons. Consistent with this finding, the proportion of joint sensory neurons exhibiting a Ca2+ increase in response to IgG-immune complex (IgG-IC), assayed both in vitro and in vivo, was lower in global FcgRI¡/¡ mice, compared with wildtype mice. Injection of IgG-IC but not monomeric IgG into one ankle of na€ıve mice evoked joint pain-related behaviors without concurrent joint swelling. This effect was diminished in global FcgRI¡/¡ mice. Ablation of T and B cells, mast cells or macrophages had no effect on IgG-IC evoked nocifensive behaviors. Under arthritic conditions, FcgRI mRNA expression and function were upregulated in dorsal root ganglion (DRG) in a mouse model of antigen-induced arthritis (AIA). In vivo extracellular electrophysiological recordings on intact DRG showed that genetic deletion of FcgRI reduced the incidence of abnormal activity and mechanical hypersensitivity of joint sensory neurons in the context of AIA. Acute blockade of FcgRI with neutralizing antibody and genetic knockout of FcgRI significantly attenuated pain-related behaviors in the AIA model. Conversely, no significant differences between treatments or genotypes were observed in the inflamed joint in the expression level of cytokines or in immune cell infiltration. These findings indicate that FcgRI may contribute to arthritis pain through a non-inflammatory mechanism that parallels inflammation and joint damage and point towards a promising therapeutic target to consider for RA pain that is resistant to current anti-inflammatory treatments. Supported by NIAMS 1R01AR072230 to L.Q.
(214) NOD-Like Receptor Protein 3 Inflammasome Drives Postoperative Pain in a Sex-Dependent Manner A. Cowie, A. Menzel, C. O’Hara, M. Lawlor, and C. Stucky; Medical College of Wisconsin, Milwaukee, WI Inflammasomes are cytosolic receptors of the innate immune system that are responsible for activation and secretion of the proinflammatory cytokine interleukin-1b (IL-1b) where IL-1b is known to cause pain by directly acting on sensory neurons. Specifically, IL-1b is known to cause peripheral sensitization to mechanical stimuli in models of inflammatory and neuropathic pain. Of the multiple inflammasomes, the NOD-like receptor protein 3 (NLRP3) inflammasome is activated by endogenous molecules that are released as a result of tissue injury and thus is ideally positioned to produce the majority of postoperative IL-1b. Therefore, we aimed to uncover the mechanism by which NLRP3 modifies postoperative pain in males and females. Male mice lacking NLRP3 recovered from incisional surgery significantly faster at the behavioral and