- Email: [email protected]
(150) Development of a Murine Model of Acute Burn Injury and Chronic Pain
S14
Abstracts
The Journal of Pain
(149) CD163 in macrophages from diabetic patients and its role in the inflammatory response of THP-1 and primary human macrophages A Alvarado, R Grosick, and E Romero-Sandoval; Presbyterian College, Clinton, NC
Macrophages regulate inflammation, tissue repair, and pain by releasing cytokines and chemokines. A prolonged pro-inflammatory phenotype in macrophages has been associated with the development of chronic pain and impaired wound healing after major surgeries or in diabetic neuropathy. Macrophage CD163 expression has been proposed to contribute to the resolution of inflammation and tissue repair. We hypothesize that the specific blockade of CD163 induces a prolonged pro-inflammatory phenotype in stimulated THP-1 and primary human macrophages. We also hypothesize that macrophages from diabetic patients display lower levels of CD163. THP-1 and primary human macrophages were stimulated with lipopolysaccharide (LPS, 5 mg/ml). CD163 receptor was blocked using an anti-CD163 antibody (a control isotype was used) 48 hours after the addition of LPS. The concentration of tumor necrosis factor-alfa (TNF-a), interleukin (IL)-6, IL1b, IL-10, transforming growth factor-beta (TGF-b) and monocyte chemoattractant protein-1 were determined 24 hours after the addition of antibody using ELISA assays. Primary human monocytes were isolated from blood samples of healthy volunteers and from patients with diabetes or diabetic neuropathy, transformed into macrophages (IRB-PC-201523), and stimulated with LPS (5 mg/mL). After 48-hour incubation, cells were collected to determine the levels of CD163 using qPCR. The blockade of CD163 using the anti-CD163 antibody resulted in a decrease of TGF-b in THP-1 cells and an increase of TNF-a in THP-1 and in primary cells. No changes in the other studied cytokines were found. CD163 mRNA did not change in healthy macrophages, though it was downregulated in macrophages from diabetic patients after LPS stimulation. These results suggest that constitutive CD163 contributes to the resolution of pro-inflammatory processes by regulating cytokine release. The downregulation of CD163 in diabetic patients could explain the prolonged pro-inflammatory phenotype displayed in macrophages from diabetic patients. Supported by Rita Allen Foundation, APS-2011-Pain grant and NIH, NIGMS, R15GM109333.
(150) Development of a Murine Model of Acute Burn Injury and Chronic Pain A Nicol, J Ryals, M Winter, D Wright, and K McCarson; University of Kansas School of Medicine, Kansas City, KS
Acute and chronic pain is a prevalent and debilitating complication following burn injury. However, burn injury pain remains largely an untreatable condition and current strategies only address ongoing pain control. Little research has addressed this large clinical problem, with only a few studies using an animal model to explore molecular and cellular mechanisms underlying this form of acute and chronic pain. This paucity of information has hampered clinicians in developing effective treatment strategies for patients that suffer burn-related pain. Currently, there are a number of proposed mechanisms that lead to acute and chronic pain in burn injuries. Alterations in inflammation or neurotrophic factor expression are thought to modulate both acute and chronic pain. Our studies have shown that exercise intervention can modify each of these factors and exercise can correct alterations in pro-nociceptive peripheral nervous system adaptations. Together, our multidisciplinary team has developed a murine model of burn injury that leads to robust pain behaviors and pathological changes consistent with acute and chronic burn injury neuropathic pain. Furthermore, our preliminary studies show that mice with burn-injured hind paws voluntarily exercise on running wheels. Future studies will identify mechanisms of how exercise can modulate these acute and chronic neuropathic pain associated with burn injury. Importantly, our approaches are readily translatable to human studies and may be applicable to other forms of chronic pain. These studies will also aid future experiments to investigate predictive features regarding diet, lifestyle, and activity that will be important in treating chronic pain associated with burn injury.
(151) A novel murine neuroma model: sural ligation results in neuroma formation and the up-regulation of Sema3A J Lerch and K Madalena; The Ohio State University, Columbus, OH Uncontrolled proliferation of nerve tissue or neuroma is a painful and prevalent clinical problem. Surgical removal, the main treat-
ment option, is largely ineffective as neuromas often grow back. Available models show a high degree of variability and are often so traumatic they result in autonomy. Here we present a sural nerve ligation model of neuroma in the mouse, which produces no motor deficits or autonomy. We observed that a simple ligation of the sural branch of the sciatic nerve results in the appearance of a large neural mass reminiscent of a neuroma. Indeed, this mass expresses high amounts of the human neuroma marker semaphorin 3A, Sema3a, and is associated with strong behavioral changes in allodynia in female mice. To better understand neuroma formation we determined which sensory neuron subtypes contribute to the sural nerve using retrograde Fluoro-Gold (FG) labeling. We observed FG+ neuronal bodies in the L3, 4 and 5 dorsal root ganglia. Follow-up immunostaining revealed that 20-40% of these neurons are positive for neurofilament heavy, a marker of large diameter general sensory neurons. The remaining 60-80% of FG+ neurons were positive for isolectin B4 typical of small diameter nonpeptidergic nociceptive neurons. Continuing studies will investigate the effect of sural nerve ligation in male mice and will allow us to dissect the basic biology of neuroma.
NeuP: Animal (152) Characterization of chemotherapeutic-induced visceral neuropathy J Pineda, E Yilmaz, M Giraldo-Duque, J Loeza-Alcocer, M Dayal, V Nagarajan, Q Liu, and M Gold; University of Pittsburgh, Pittsburgh, PA
Chemotherapeutic-induced peripheral neuropathy (CIPN), characterized by numbness, tingling and ultimately pain in the hands and feet, remains the primary dose-limiting side effect of some of the most effective anti-cancer drugs. But while the primary focus of CIPN research has been on the somatic nervous system, clinical data suggest a variety of persistent visceral symptoms may have the most deleterious impact on the quality of life in cancer survivors. Both because of the nature of the persistent symptoms and our recent data suggesting that unique features of a subpopulation of somatic afferents make them particularly vulnerable to chemotherapeutics, we hypothesized that the persistent visceral symptoms reflect a chemotherapeutic-induced ‘‘visceral’’ neuropathy (CIVN). To test this hypothesis, we assessed changes in visceral sensory neurons (the vagus and nodose ganglia) and the enteric nervous system in rats a week after the last of six IV infusions of the combination of paclitaxel (2 mg/kg) and carboplatin (30 mg/kg) administered over three weeks. Changes in somatic afferents were used for comparison. Combination- but not vehicle-treated rats developed mechanical and cold sensitivity within the first week of drug administration that did not resolve. Combination-treatment was also associated with a significant (50%) reduction in the conduction velocity of A- and C-fibers in sciatic and vagal nerves. The chemokine MCP-1 was increase in a subpopulation of neurons in dorsal root ganglia (DRG) and nodose ganglia. There was also an increase in the mitochondria protein TOM-20 in a subpopulation of DRG and nodose neurons. Our results are consistent with our initial hypothesis. Whether the same mechanism(s) are responsible for the damage to visceral and somatic afferents remains to be determined. Identification of the mechanisms responsible for the damage to visceral neurons, may suggest novel treatments for patients suffering from these persistent symptoms. Supported by Grant R01 DK107966.
(153) The interleukin 27 (IL-27) protects mice from neuropathic pain development through up-regulation of anti-inflammatory cytokine IL-10 M Mendes Fonseca, F Santa-Cecilia, R Kusuda, D Ferreira, F Bezerra, M Ferreira, R Guimar~ aes, F Cunha, and T Cunha; Universidade de S~ao Paulo, S~ao Paulo, Brazil
During some inflammatory conditions in the central nervous system (CNS), interleukin IL-27 (p28/EBI3 subunits)1 is produced and promotes the protection of the CNS by regulating the exacerbated inflammatory response2 being an interesting target for neuropathic pain resolution. Based on that, we aimed to evaluate the protective role of IL-27 in spinal cord (SC) and dorsal root ganglia (DRG) during the genesis and maintenance of chronic neuropathic pain. The
Abstracts
The Journal of Pain
(149) CD163 in macrophages from diabetic patients and its role in the inflammatory response of THP-1 and primary human macrophages A Alvarado, R Grosick, and E Romero-Sandoval; Presbyterian College, Clinton, NC
Macrophages regulate inflammation, tissue repair, and pain by releasing cytokines and chemokines. A prolonged pro-inflammatory phenotype in macrophages has been associated with the development of chronic pain and impaired wound healing after major surgeries or in diabetic neuropathy. Macrophage CD163 expression has been proposed to contribute to the resolution of inflammation and tissue repair. We hypothesize that the specific blockade of CD163 induces a prolonged pro-inflammatory phenotype in stimulated THP-1 and primary human macrophages. We also hypothesize that macrophages from diabetic patients display lower levels of CD163. THP-1 and primary human macrophages were stimulated with lipopolysaccharide (LPS, 5 mg/ml). CD163 receptor was blocked using an anti-CD163 antibody (a control isotype was used) 48 hours after the addition of LPS. The concentration of tumor necrosis factor-alfa (TNF-a), interleukin (IL)-6, IL1b, IL-10, transforming growth factor-beta (TGF-b) and monocyte chemoattractant protein-1 were determined 24 hours after the addition of antibody using ELISA assays. Primary human monocytes were isolated from blood samples of healthy volunteers and from patients with diabetes or diabetic neuropathy, transformed into macrophages (IRB-PC-201523), and stimulated with LPS (5 mg/mL). After 48-hour incubation, cells were collected to determine the levels of CD163 using qPCR. The blockade of CD163 using the anti-CD163 antibody resulted in a decrease of TGF-b in THP-1 cells and an increase of TNF-a in THP-1 and in primary cells. No changes in the other studied cytokines were found. CD163 mRNA did not change in healthy macrophages, though it was downregulated in macrophages from diabetic patients after LPS stimulation. These results suggest that constitutive CD163 contributes to the resolution of pro-inflammatory processes by regulating cytokine release. The downregulation of CD163 in diabetic patients could explain the prolonged pro-inflammatory phenotype displayed in macrophages from diabetic patients. Supported by Rita Allen Foundation, APS-2011-Pain grant and NIH, NIGMS, R15GM109333.
(150) Development of a Murine Model of Acute Burn Injury and Chronic Pain A Nicol, J Ryals, M Winter, D Wright, and K McCarson; University of Kansas School of Medicine, Kansas City, KS
Acute and chronic pain is a prevalent and debilitating complication following burn injury. However, burn injury pain remains largely an untreatable condition and current strategies only address ongoing pain control. Little research has addressed this large clinical problem, with only a few studies using an animal model to explore molecular and cellular mechanisms underlying this form of acute and chronic pain. This paucity of information has hampered clinicians in developing effective treatment strategies for patients that suffer burn-related pain. Currently, there are a number of proposed mechanisms that lead to acute and chronic pain in burn injuries. Alterations in inflammation or neurotrophic factor expression are thought to modulate both acute and chronic pain. Our studies have shown that exercise intervention can modify each of these factors and exercise can correct alterations in pro-nociceptive peripheral nervous system adaptations. Together, our multidisciplinary team has developed a murine model of burn injury that leads to robust pain behaviors and pathological changes consistent with acute and chronic burn injury neuropathic pain. Furthermore, our preliminary studies show that mice with burn-injured hind paws voluntarily exercise on running wheels. Future studies will identify mechanisms of how exercise can modulate these acute and chronic neuropathic pain associated with burn injury. Importantly, our approaches are readily translatable to human studies and may be applicable to other forms of chronic pain. These studies will also aid future experiments to investigate predictive features regarding diet, lifestyle, and activity that will be important in treating chronic pain associated with burn injury.
(151) A novel murine neuroma model: sural ligation results in neuroma formation and the up-regulation of Sema3A J Lerch and K Madalena; The Ohio State University, Columbus, OH Uncontrolled proliferation of nerve tissue or neuroma is a painful and prevalent clinical problem. Surgical removal, the main treat-
ment option, is largely ineffective as neuromas often grow back. Available models show a high degree of variability and are often so traumatic they result in autonomy. Here we present a sural nerve ligation model of neuroma in the mouse, which produces no motor deficits or autonomy. We observed that a simple ligation of the sural branch of the sciatic nerve results in the appearance of a large neural mass reminiscent of a neuroma. Indeed, this mass expresses high amounts of the human neuroma marker semaphorin 3A, Sema3a, and is associated with strong behavioral changes in allodynia in female mice. To better understand neuroma formation we determined which sensory neuron subtypes contribute to the sural nerve using retrograde Fluoro-Gold (FG) labeling. We observed FG+ neuronal bodies in the L3, 4 and 5 dorsal root ganglia. Follow-up immunostaining revealed that 20-40% of these neurons are positive for neurofilament heavy, a marker of large diameter general sensory neurons. The remaining 60-80% of FG+ neurons were positive for isolectin B4 typical of small diameter nonpeptidergic nociceptive neurons. Continuing studies will investigate the effect of sural nerve ligation in male mice and will allow us to dissect the basic biology of neuroma.
NeuP: Animal (152) Characterization of chemotherapeutic-induced visceral neuropathy J Pineda, E Yilmaz, M Giraldo-Duque, J Loeza-Alcocer, M Dayal, V Nagarajan, Q Liu, and M Gold; University of Pittsburgh, Pittsburgh, PA
Chemotherapeutic-induced peripheral neuropathy (CIPN), characterized by numbness, tingling and ultimately pain in the hands and feet, remains the primary dose-limiting side effect of some of the most effective anti-cancer drugs. But while the primary focus of CIPN research has been on the somatic nervous system, clinical data suggest a variety of persistent visceral symptoms may have the most deleterious impact on the quality of life in cancer survivors. Both because of the nature of the persistent symptoms and our recent data suggesting that unique features of a subpopulation of somatic afferents make them particularly vulnerable to chemotherapeutics, we hypothesized that the persistent visceral symptoms reflect a chemotherapeutic-induced ‘‘visceral’’ neuropathy (CIVN). To test this hypothesis, we assessed changes in visceral sensory neurons (the vagus and nodose ganglia) and the enteric nervous system in rats a week after the last of six IV infusions of the combination of paclitaxel (2 mg/kg) and carboplatin (30 mg/kg) administered over three weeks. Changes in somatic afferents were used for comparison. Combination- but not vehicle-treated rats developed mechanical and cold sensitivity within the first week of drug administration that did not resolve. Combination-treatment was also associated with a significant (50%) reduction in the conduction velocity of A- and C-fibers in sciatic and vagal nerves. The chemokine MCP-1 was increase in a subpopulation of neurons in dorsal root ganglia (DRG) and nodose ganglia. There was also an increase in the mitochondria protein TOM-20 in a subpopulation of DRG and nodose neurons. Our results are consistent with our initial hypothesis. Whether the same mechanism(s) are responsible for the damage to visceral and somatic afferents remains to be determined. Identification of the mechanisms responsible for the damage to visceral neurons, may suggest novel treatments for patients suffering from these persistent symptoms. Supported by Grant R01 DK107966.
(153) The interleukin 27 (IL-27) protects mice from neuropathic pain development through up-regulation of anti-inflammatory cytokine IL-10 M Mendes Fonseca, F Santa-Cecilia, R Kusuda, D Ferreira, F Bezerra, M Ferreira, R Guimar~ aes, F Cunha, and T Cunha; Universidade de S~ao Paulo, S~ao Paulo, Brazil
During some inflammatory conditions in the central nervous system (CNS), interleukin IL-27 (p28/EBI3 subunits)1 is produced and promotes the protection of the CNS by regulating the exacerbated inflammatory response2 being an interesting target for neuropathic pain resolution. Based on that, we aimed to evaluate the protective role of IL-27 in spinal cord (SC) and dorsal root ganglia (DRG) during the genesis and maintenance of chronic neuropathic pain. The