- Email: [email protected]
(256) Molecular mechanisms of stress exacerbated neuropathic pain in peripheral nerve injury
S40
Abstracts
The Journal of Pain
(256) Molecular mechanisms of stress exacerbated neuropathic pain in peripheral nerve injury J Lerch, N Hashi, A Zawerton, T Quach, and P Popovich; The Ohio State University, Columbus, OH Stress increases stimulus evoked pain after peripheral nerve injury but the mechanisms are unknown. Therefore to develop novel therapeutics to treat the pathophysiology of stress/pain, the molecular events that lead from stress to enhanced pain after injury need to be identified. Due to the ubiquitous nature of how stress can affect global physiology (e.g. stress elevates glucocorticoids (GCs), circulating steroid hormones involved in energy homeostasis, inflammation, and stress responses), identifying these molecular events is challenging. First, GCs bind to glucocorticoid receptors (GRs), transcription factors that are expressed in nearly every cell in the body. Second, GCs/GRs interactions can drive expression of thousands of downstream genes making identification of single, cell type specific targets difficult. To address this problem we used RNA-Seq specifically on sensory neurons to examine global gene expression after stress and spared nerve injury. By applying this approach specifically to sensory neurons we identified cell type specific transcriptional changes which are verified by in situ hybridization. We found expression changes in numerous regulatory (e.g. transcription factors, non-coding RNAs) and protein coding RNAs (e.g. voltage-gated ion channels). We tested the ability of these novel RNAs to either mitigate or increase stress/injury associated neuropathic pain after gain and loss of function in vivo using viral transduction in sensory neurons. In addition to defining many new molecular targets for the treatment of stress associated neuropathic pain, data from these studies has implications for how steroids, including exogenous GCs, are used clinically to treat disease and inflammation (e.g. multiple sclerosis, diabetes, arthritis, spinal cord injury).
(257) microRNA 320a is a potential mediator of chronic widespread hyperalgesia development after stress exposure S Linnstaedt, K Riker, J Nyland, E Zimny, C Lewandowski, P Hendry, K Damiron, C Pearson, M Velilla, J Jones, R Swor, R Domeier, and S McLean; University of North Carolina at Chapel Hill, Chapel Hill, NC In a well-explicated animal model, widespread hyperalgesia (WH) induced by unpredictable sound stress (USS) has been shown to be mediated by increased glucocorticoid and catecholamine levels acting at the level of the peripheral nerve.1 A recent study found that circulating levels of miRNA 320a are associated with fibromyalgia,2 and in silico analyses suggest that miR-320a targets multiple transcripts associated with glucocorticoid synthesis/regulation. Based on these data, we evaluated whether (1) circulating miR-320a levels predict chronic widespread pain (CWP) and fibromyalgia development after motor vehicle collision (MVC) stress exposure in humans, (2) miR-320a is expressed in tissues relevant to identified mechanisms mediating WH induced by induced by USS (3) miR-320a regulates glucocorticoid system-related transcripts in cell culture consistent with previous in silico analyses. We found that (1) among African Americans presenting to the ED after MVC and discharged home after evaluation (n = 69), reduced circulating miRNA-320a isoform levels in the ED predict both CWP (defined via ACR criteria) and fibromyalgia (defined via an annotated ACR criteria) at six months, (2) in animals miR-320a is expressed in tissues relevant to identified mechanisms mediating WH induced by USS, including the adrenal gland, dorsal root ganglion, and peripheral nerves (3) in neuroblastoma (SK-N-AS) cell lines, miR-320a expression affects glucocorticoid system-related transcripts that encode FK506 binding protein 5 (FKBP5), prostaglandin E receptor 3 (EP3), and adenylate cyclase activating polypeptide 1(PACAP). Together, these data suggest that miR-320a may help mediate the widespread changes in pain sensitivity which occur in some individuals after stress exposure. Further studies are needed to evaluate this hypothesis. If confirmed, these data could lead to new preventive/treatment interventions for high risk individuals that target miRNA-320a. Research Supported by The Mayday Fund and R01AR060852. (1. Khasar et al, J Neuroscience, 2008; 2. Bjersing et al, Rheumatology Int, 2014.)
(258) CYP2C8 and CYP2C9 metabolic profiles and frequency of emergency department visits for sickle cell disease pain management C Jaja, L Bowman, L Wells, H Xu, M Lyon, R Gibson, and A Kutlar; University of Cincinnati, Cincinnati, OH The CYP2C8 and CYP2C9 variant alleles play a significant role in NSAIDs analgesic effect and toxicity. NSAIDs are used to treat vasoocclusive pain at the prodromal stage in sickle cell disease (SCD) patients. We describe CYP2C8 and CYP2C9 genotypes and phenotypes and frequency of emergency department (ED) visits in an African American SCD patient cohort. DNA from 165 unrelated SCD patients (82 males/83 females, aged from 16 to 61 years) was genotyped for seven CYP2C8 (*1, *2, *3, *4, *5, *7 & *8) alleles and 15 CYP2C9 alleles (*1, *2,*3, *4, *5, *6, *8, *9, *10, *11, *12, *13, *15, *25 & *27) using the iPLEXâ ADME PGx multiplexed panel. CYP2C8 *1(0.806),*2(0.164),*3(0.018), and *4 (0.012) alleles were identified. Genotype frequencies were distributed as homozygous wild-type (66.7%), heterozygous (27.8%), and homozygous variant/compound heterozygous (5.4%) respectively. CYP2C9 *1(0.824), *2 (0.027), *3 (0.012), *5 (0.009), *6 (0.009), *8 (0.042), *9 (0.061), and *11(0.015) were observed with extensive (68.5%), intermediate (18.1%) and poor predicted metabolizers (0.6%) respectively. Ten of twelve of the combined alleles of the CYP2C8 and CYP2C9 enzymes identified in our cohort contribute to deficient metabolic genotypes. Fifty-two subjects had at least one variant CYP2C9 allele associated with either the intermediate metabolizer, poor, or indeterminate phenotype. Fifty-five subjects had at least one CYP2C8 variant allele that contributes to impaired metabolic genotypes. Unlike the CYP2C9 (p= 0.0515), the distribution of CYP2C8 phenotypes was not significantly different in high and low ED users (p= 0.1668); but some CYP2C8 and CYP2C9 allelic combinations observed in 15.2% (25) of the cohort are associated with higher risks for analgesic failure or exacerbation of SCD related morbidity. Data on the association of NSAIDs treatment with analgesic failure are limited for SCD patients. CYP2C8 and CYP2C9 preemptive genotyping may facilitate early prediction of efficacy of NSAID treatment outcomes.
D05 Immune Mediators (259) Exercise attenuates LPS-induced musculoskeletal hyperalgesia in mice by preventing excessive macrophage activation R Abdelhamid, E Merriwether, S Kolker, L Allen, and K Sluka; The University of Iowa, Iowa City, IA Exercise is an effective method for alleviating pain symptoms in people with chronic musculoskeletal pain conditions. Macrophages are plastic mononuclear phagocytes that change their phenotype in response to the immunological microenvironment of the tissue, and release cytokines based on their phenotype. M1 macrophages release proinflammatory cytokines that promote tissue damage and hyperalgesia, whereas M2 macrophages release anti-inflammatory cytokines that promote wound healing and analgesia. Since the anti-inflammatory effects of exercise are established, we investigated the effects of exercise on LPS-induced hyperalgesia, inflammation and macrophage activation. We hypothesized that regular exercise produces analgesia by increasing M2 macrophage phenotype and decrease M1 macrophage phenotype. C57Bl/6 mice were divided into two groups: 1) exercise mice that were given free access to running wheels in their home cages (3-14 days), and 2) sedentary mice (no running wheels). Systemic inflammation was induced by i.p. injection of LPS (1mg/kg) to model widespread hyperalgesia. Muscle and cutaneous hyperalgesia were determined using muscle withdrawal thresholds of the gastrocnemius muscle, and hind paw withdrawal responses to repeated stimuli, respectively. Phenotypes of peritoneal macrophages were examined by measuring M1 and M2 markers (Ly-6C, CD206) by flow cytometry and by analysis of cytokine release profiles by multiplex. Our data show that regular exercise attenuated LPS-induced muscle and paw hyperalgesia. LPS also increased macrophage polarization towards an M1 phenotype, and enhanced inflammatory cytokine release. Effects of the time course of exercise on phenotype and cytokine release are presented. Taken together, these data indicate that activation of macrophages systemically can result in widespread cutaneous and muscle hyperalgesia, and increase inflammatory cytokines. The data also demonstrate that regular exercise on running wheels can prevent the development of this hyperalgesia. Supported by NIH R01 AR061371.
Abstracts
The Journal of Pain
(256) Molecular mechanisms of stress exacerbated neuropathic pain in peripheral nerve injury J Lerch, N Hashi, A Zawerton, T Quach, and P Popovich; The Ohio State University, Columbus, OH Stress increases stimulus evoked pain after peripheral nerve injury but the mechanisms are unknown. Therefore to develop novel therapeutics to treat the pathophysiology of stress/pain, the molecular events that lead from stress to enhanced pain after injury need to be identified. Due to the ubiquitous nature of how stress can affect global physiology (e.g. stress elevates glucocorticoids (GCs), circulating steroid hormones involved in energy homeostasis, inflammation, and stress responses), identifying these molecular events is challenging. First, GCs bind to glucocorticoid receptors (GRs), transcription factors that are expressed in nearly every cell in the body. Second, GCs/GRs interactions can drive expression of thousands of downstream genes making identification of single, cell type specific targets difficult. To address this problem we used RNA-Seq specifically on sensory neurons to examine global gene expression after stress and spared nerve injury. By applying this approach specifically to sensory neurons we identified cell type specific transcriptional changes which are verified by in situ hybridization. We found expression changes in numerous regulatory (e.g. transcription factors, non-coding RNAs) and protein coding RNAs (e.g. voltage-gated ion channels). We tested the ability of these novel RNAs to either mitigate or increase stress/injury associated neuropathic pain after gain and loss of function in vivo using viral transduction in sensory neurons. In addition to defining many new molecular targets for the treatment of stress associated neuropathic pain, data from these studies has implications for how steroids, including exogenous GCs, are used clinically to treat disease and inflammation (e.g. multiple sclerosis, diabetes, arthritis, spinal cord injury).
(257) microRNA 320a is a potential mediator of chronic widespread hyperalgesia development after stress exposure S Linnstaedt, K Riker, J Nyland, E Zimny, C Lewandowski, P Hendry, K Damiron, C Pearson, M Velilla, J Jones, R Swor, R Domeier, and S McLean; University of North Carolina at Chapel Hill, Chapel Hill, NC In a well-explicated animal model, widespread hyperalgesia (WH) induced by unpredictable sound stress (USS) has been shown to be mediated by increased glucocorticoid and catecholamine levels acting at the level of the peripheral nerve.1 A recent study found that circulating levels of miRNA 320a are associated with fibromyalgia,2 and in silico analyses suggest that miR-320a targets multiple transcripts associated with glucocorticoid synthesis/regulation. Based on these data, we evaluated whether (1) circulating miR-320a levels predict chronic widespread pain (CWP) and fibromyalgia development after motor vehicle collision (MVC) stress exposure in humans, (2) miR-320a is expressed in tissues relevant to identified mechanisms mediating WH induced by induced by USS (3) miR-320a regulates glucocorticoid system-related transcripts in cell culture consistent with previous in silico analyses. We found that (1) among African Americans presenting to the ED after MVC and discharged home after evaluation (n = 69), reduced circulating miRNA-320a isoform levels in the ED predict both CWP (defined via ACR criteria) and fibromyalgia (defined via an annotated ACR criteria) at six months, (2) in animals miR-320a is expressed in tissues relevant to identified mechanisms mediating WH induced by USS, including the adrenal gland, dorsal root ganglion, and peripheral nerves (3) in neuroblastoma (SK-N-AS) cell lines, miR-320a expression affects glucocorticoid system-related transcripts that encode FK506 binding protein 5 (FKBP5), prostaglandin E receptor 3 (EP3), and adenylate cyclase activating polypeptide 1(PACAP). Together, these data suggest that miR-320a may help mediate the widespread changes in pain sensitivity which occur in some individuals after stress exposure. Further studies are needed to evaluate this hypothesis. If confirmed, these data could lead to new preventive/treatment interventions for high risk individuals that target miRNA-320a. Research Supported by The Mayday Fund and R01AR060852. (1. Khasar et al, J Neuroscience, 2008; 2. Bjersing et al, Rheumatology Int, 2014.)
(258) CYP2C8 and CYP2C9 metabolic profiles and frequency of emergency department visits for sickle cell disease pain management C Jaja, L Bowman, L Wells, H Xu, M Lyon, R Gibson, and A Kutlar; University of Cincinnati, Cincinnati, OH The CYP2C8 and CYP2C9 variant alleles play a significant role in NSAIDs analgesic effect and toxicity. NSAIDs are used to treat vasoocclusive pain at the prodromal stage in sickle cell disease (SCD) patients. We describe CYP2C8 and CYP2C9 genotypes and phenotypes and frequency of emergency department (ED) visits in an African American SCD patient cohort. DNA from 165 unrelated SCD patients (82 males/83 females, aged from 16 to 61 years) was genotyped for seven CYP2C8 (*1, *2, *3, *4, *5, *7 & *8) alleles and 15 CYP2C9 alleles (*1, *2,*3, *4, *5, *6, *8, *9, *10, *11, *12, *13, *15, *25 & *27) using the iPLEXâ ADME PGx multiplexed panel. CYP2C8 *1(0.806),*2(0.164),*3(0.018), and *4 (0.012) alleles were identified. Genotype frequencies were distributed as homozygous wild-type (66.7%), heterozygous (27.8%), and homozygous variant/compound heterozygous (5.4%) respectively. CYP2C9 *1(0.824), *2 (0.027), *3 (0.012), *5 (0.009), *6 (0.009), *8 (0.042), *9 (0.061), and *11(0.015) were observed with extensive (68.5%), intermediate (18.1%) and poor predicted metabolizers (0.6%) respectively. Ten of twelve of the combined alleles of the CYP2C8 and CYP2C9 enzymes identified in our cohort contribute to deficient metabolic genotypes. Fifty-two subjects had at least one variant CYP2C9 allele associated with either the intermediate metabolizer, poor, or indeterminate phenotype. Fifty-five subjects had at least one CYP2C8 variant allele that contributes to impaired metabolic genotypes. Unlike the CYP2C9 (p= 0.0515), the distribution of CYP2C8 phenotypes was not significantly different in high and low ED users (p= 0.1668); but some CYP2C8 and CYP2C9 allelic combinations observed in 15.2% (25) of the cohort are associated with higher risks for analgesic failure or exacerbation of SCD related morbidity. Data on the association of NSAIDs treatment with analgesic failure are limited for SCD patients. CYP2C8 and CYP2C9 preemptive genotyping may facilitate early prediction of efficacy of NSAID treatment outcomes.
D05 Immune Mediators (259) Exercise attenuates LPS-induced musculoskeletal hyperalgesia in mice by preventing excessive macrophage activation R Abdelhamid, E Merriwether, S Kolker, L Allen, and K Sluka; The University of Iowa, Iowa City, IA Exercise is an effective method for alleviating pain symptoms in people with chronic musculoskeletal pain conditions. Macrophages are plastic mononuclear phagocytes that change their phenotype in response to the immunological microenvironment of the tissue, and release cytokines based on their phenotype. M1 macrophages release proinflammatory cytokines that promote tissue damage and hyperalgesia, whereas M2 macrophages release anti-inflammatory cytokines that promote wound healing and analgesia. Since the anti-inflammatory effects of exercise are established, we investigated the effects of exercise on LPS-induced hyperalgesia, inflammation and macrophage activation. We hypothesized that regular exercise produces analgesia by increasing M2 macrophage phenotype and decrease M1 macrophage phenotype. C57Bl/6 mice were divided into two groups: 1) exercise mice that were given free access to running wheels in their home cages (3-14 days), and 2) sedentary mice (no running wheels). Systemic inflammation was induced by i.p. injection of LPS (1mg/kg) to model widespread hyperalgesia. Muscle and cutaneous hyperalgesia were determined using muscle withdrawal thresholds of the gastrocnemius muscle, and hind paw withdrawal responses to repeated stimuli, respectively. Phenotypes of peritoneal macrophages were examined by measuring M1 and M2 markers (Ly-6C, CD206) by flow cytometry and by analysis of cytokine release profiles by multiplex. Our data show that regular exercise attenuated LPS-induced muscle and paw hyperalgesia. LPS also increased macrophage polarization towards an M1 phenotype, and enhanced inflammatory cytokine release. Effects of the time course of exercise on phenotype and cytokine release are presented. Taken together, these data indicate that activation of macrophages systemically can result in widespread cutaneous and muscle hyperalgesia, and increase inflammatory cytokines. The data also demonstrate that regular exercise on running wheels can prevent the development of this hyperalgesia. Supported by NIH R01 AR061371.