RESUMEN
A response to environmental stress is critical to alleviate cellular injury and maintain cellular homeostasis. Eukaryotic initiation factor 2 (eIF2) is a key integrator of cellular stress responses and an important regulator of mRNA translation. Diverse stress signals lead to the phosphorylation of the α subunit of eIF2 (Ser51), resulting in inhibition of global protein synthesis while promoting expression of proteins that mediate cell adaptation to stress. Here we report that eIF2α is instrumental in the control of noxious heat sensation. Mice with decreased eIF2α phosphorylation (eIF2α+/S51A) exhibit reduced responses to noxious heat. Pharmacological attenuation of eIF2α phosphorylation decreases thermal, but not mechanical, pain sensitivity, whereas increasing eIF2α phosphorylation has the opposite effect on thermal nociception. The impact of eIF2α phosphorylation (p-eIF2α) on thermal thresholds is dependent on the transient receptor potential vanilloid 1. Moreover, we show that induction of eIF2α phosphorylation in primary sensory neurons in a chronic inflammation pain model contributes to thermal hypersensitivity. Our results demonstrate that the cellular stress response pathway, mediated via p-eIF2α, represents a mechanism that could be used to alleviate pathological heat sensation.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Nocicepción , Temperatura , Animales , Conducta Animal , Biomarcadores , Calcio/metabolismo , Células Cultivadas , Factor 2 Eucariótico de Iniciación/genética , Ganglios Espinales/metabolismo , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos , Imagen Molecular , Neuronas/metabolismo , Dolor/etiología , Dolor/metabolismo , Umbral del Dolor , Fosforilación , Transducción de Señal , Médula Espinal/metabolismo , Estrés Fisiológico , Canales Catiónicos TRPV/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
Although chronic pain is the most common symptom of arthritis, relatively little is known about the mechanisms driving it. Recently, a sprouting of autonomic sympathetic fibers into the upper dermis of the skin, an area that is normally devoid of them, was found in the skin following chronic inflammation of the rat hindpaw. While this sprouting only occurred when signs of joint and bone damage were present, it remained to be clarified whether it was a consequence of the chronic inflammation of the skin or of the arthritis and whether it also occurred in the joint. In the present study, we used a model of arthritis in which complete Freund's adjuvant (CFA) was injected into the rat ankle joint. At 4 weeks following CFA treatment, there was an increase in sympathetic and peptidergic fiber density in the ankle joint synovium. We also observed a sympathetic, but not peptidergic, fiber sprouting in the skin over the joint, which may be a consequence of the increased levels of mature nerve growth factor levels in skin, as revealed by Western blot analysis. The pharmacological suppression of sympathetic fiber function with systemic guanethidine significantly decreased the pain-related behavior associated with arthritis. Guanethidine completely suppressed the heat hyperalgesia and attenuated mechanical and cold hypersensitivity. These results suggest that transmitters released from the sprouted sympathetic fibers in the synovial membrane and upper dermis contribute to the pain-related behavior associated with arthritis. Blocking the sympathetic fiber sprouting may provide a novel therapeutic approach to alleviate pain in arthritis.
Asunto(s)
Fibras Adrenérgicas/patología , Articulación del Tobillo/fisiopatología , Artritis/complicaciones , Dermis/inervación , Dolor/etiología , Dolor/patología , Fibras Adrenérgicas/metabolismo , Análisis de Varianza , Animales , Articulación del Tobillo/inervación , Artritis/inducido químicamente , Péptido Relacionado con Gen de Calcitonina/metabolismo , Modelos Animales de Enfermedad , Adyuvante de Freund/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Guanetidina/administración & dosificación , Hiperalgesia/etiología , Hiperalgesia/fisiopatología , Masculino , Factor de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Dimensión del Dolor , Umbral del Dolor/fisiología , Precursores de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Simpaticolíticos/administración & dosificación , Factores de Tiempo , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
We report a novel model in which remote activation of peripheral nociceptive pathways in transgenic mice is achieved optogenetically, without any external noxious stimulus or injury. Taking advantage of a binary genetic approach, we selectively targeted Nav1.8(+) sensory neurons for conditional expression of channelrhodopsin-2 (ChR2) channels. Acute blue light illumination of the skin produced robust nocifensive behaviors, evoked by the remote stimulation of both peptidergic and nonpeptidergic nociceptive fibers as indicated by c-Fos labeling in laminae I and II of the dorsal horn of the spinal cord. A non-nociceptive component also contributes to the observed behaviors, as shown by c-Fos expression in lamina III of the dorsal horn and the expression of ChR2-EYFP in a subpopulation of large-diameter Nav1.8(+) dorsal root ganglion neurons. Selective activation of Nav1.8(+) afferents in vivo induced central sensitization and conditioned place aversion, thus providing a novel paradigm to investigate plasticity in the pain circuitry. Long-term potentiation was similarly evoked by light activation of the same afferents in isolated spinal cord preparations. These findings demonstrate, for the first time, the optical control of nociception and central sensitization in behaving mammals and enables selective activation of the same class of afferents in both in vivo and ex vivo preparations. Our results provide a proof-of-concept demonstration that optical dissection of the contribution of specific classes of afferents to central sensitization is possible. The high spatiotemporal precision offered by this non-invasive model will facilitate drug development and target validation for pain therapeutics.
Asunto(s)
Vías Aferentes/metabolismo , Optogenética , Umbral del Dolor/fisiología , Dolor/patología , Vigilia/fisiología , Vías Aferentes/patología , Animales , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Células Cultivadas , Channelrhodopsins , Femenino , Ganglios Espinales/citología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hiperalgesia/genética , Hiperalgesia/fisiopatología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfina/farmacología , Morfina/uso terapéutico , Canal de Sodio Activado por Voltaje NAV1.8/genética , Dolor/tratamiento farmacológico , Dolor/genética , Dolor/fisiopatología , Umbral del Dolor/efectos de los fármacos , Receptores Purinérgicos P2X3/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiología , Valina/análogos & derivados , Valina/farmacología , Vigilia/genética , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
BACKGROUND: We have previously shown a sprouting of sympathetic fibers into the upper dermis of the skin following subcutaneous injection of complete Freund's adjuvant (CFA) into the hindpaw. This sprouting correlated with an increase in pain-related sensitivity. We hypothesized that this sprouting and pain-related behavior were caused by an increase in nerve growth factor (NGF) levels. In this study, we investigated whether the inhibition of mature NGF degradation, using a matrix metalloproteinase 2 and 9 (MMP-2/9) inhibitor, was sufficient to reproduce a similar phenotype. RESULTS: Behavioral tests performed on male Sprague-Dawley rats at 1, 3, 7 and 14 days after intra-plantar MMP-2/9 inhibitor administration demonstrated that acute and chronic injections of the MMP-2/9 inhibitor induced sensitization, in a dose dependent manner, to mechanical, hot and cold stimuli as measured by von Frey filaments, Hargreaves and acetone tests, respectively. Moreover, the protein levels of mature NGF (mNGF) were increased, whereas the levels and enzymatic activity of matrix metalloproteinase 9 were reduced in the glabrous skin of the hind paw. MMP-2/9 inhibition also led to a robust sprouting of sympathetic fibers into the upper dermis but there were no changes in the density of peptidergic nociceptive afferents. CONCLUSIONS: These findings indicate that localized MMP-2/9 inhibition provokes a pattern of sensitization and fiber sprouting comparable to that previously obtained following CFA injection. Accordingly, the modulation of endogenous NGF levels should be considered as a potential therapeutic target for the management of inflammatory pain associated with arthritis.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Hiperalgesia/etiología , Hiperalgesia/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Animales , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratas , Ratas Sprague-Dawley , Piel/efectos de los fármacos , Piel/metabolismoRESUMEN
Pain is a major symptom associated with chronic inflammation. In previous work from our laboratory, we have shown that in animal models of neuropathic pain there is a sprouting of sympathetic fibers into the upper dermis, a territory normally devoid of them. However, it is not known whether such sympathetic sprouting, which is likely trophic factor mediated, also occurs in chronic inflammation and arthritis. In the present study, we used a rat model of chronic inflammation in which a small single dose of complete Freund's adjuvant (CFA) was injected subcutaneously, unilaterally, into the plantar surface of the hindpaw. This led to a localized long-term skin inflammation and arthritis in all joints of the hindpaw. Animals were perfused with histological fixatives at 1, 2, 3 or 4 weeks after the injection. Experimental animals treated with CFA were compared to saline-injected animals. We then investigated the changes in the pattern of peripheral innervation of the peptidergic nociceptors and sympathetic fibers in rat glabrous hindpaw skin. Antibodies directed towards calcitonin gene-related peptide (CGRP) and dopamine beta-hydroxylase (DBH) were used for the staining of peptidergic and sympathetic fibers, respectively. Immunofluorescence was then used to analyze the different nerve fiber populations of the upper dermis. At 4 weeks following CFA treatment, DBH-immunoreactive (IR) fibers were found to sprout into the upper dermis, in a pattern similar to the one we had observed in animals with a chronic constriction injury of the sciatic nerve in a previous publication. There was also a significant increase in the density of CGRP-IR fibers in the upper dermis in CFA treated animals at 2, 3 and 4 weeks post-injection. The increased peptidergic fiber innervation and the ectopic autonomic fibers found in the upper dermis may have a role in the pain-related behavior displayed by these animals.
Asunto(s)
Sistema Nervioso Autónomo/patología , Inflamación/patología , Fibras Nerviosas/patología , Piel/inervación , Piel/patología , Animales , Sistema Nervioso Autónomo/efectos de los fármacos , Enfermedad Crónica , Dermis/efectos de los fármacos , Dermis/inervación , Dermis/patología , Adyuvante de Freund/administración & dosificación , Adyuvante de Freund/farmacología , Microscopía Confocal , Fibras Nerviosas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Piel/efectos de los fármacos , Cloruro de Sodio/administración & dosificación , Cloruro de Sodio/farmacologíaRESUMEN
Functional changes in hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels have been shown to contribute to medial prefrontal (mPFC) hyperexcitability after peripheral nerve injury. A reduction in the open probability of these neuronal channels might be relevant since this can enhance membrane input resistance and synaptic summation. However, the molecular mechanisms underlying neuropathy-associated alterations in HCN channel activity remain elusive. Using the spared nerve injury model of neuropathic pain in Long-Evans rats, we first discovered a significant increase in noradrenergic innervation within the mPFC of nerve-injured compared to control animals. Patch-clamp recordings in layer II/III pyramidal neurons of the mPFC revealed that adrenoceptors, primarily the α2 subtype, can modulate the voltage-dependent activation of HCN channels and the abnormal prefrontal excitability following peripheral neuropathy. Additionally, microinfusions of the α2 adrenoceptor agonist clonidine in the mPFC of neuropathic rats provided analgesic effects, indicating the behavioral significance for this noradrenergic pathway in manifestations of the chronic pain state. Taken together, our results provide insights into the role of cortical catecholaminergic neuromodulation in neuropathic pain and suggest that altered noradrenergic transduction may play a major role in the HCN channel dysfunction and pyramidal hyperactivity observed in several chronic pain conditions.
Asunto(s)
Fibras Nerviosas/fisiología , Neuralgia/patología , Norepinefrina/metabolismo , Percepción del Dolor/fisiología , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Adrenérgicos/farmacología , Animales , Compuestos de Bario/farmacología , Cloruros/farmacología , Modelos Animales de Enfermedad , Dopamina beta-Hidroxilasa/metabolismo , Estimulación Eléctrica , Hiperalgesia/fisiopatología , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Fibras Nerviosas/efectos de los fármacos , Neuralgia/tratamiento farmacológico , Neuronas , Percepción del Dolor/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Ratas Long-Evans , Corteza Visual/patologíaRESUMEN
Recent studies have suggested that in humans and animals with significant skeletal pain, changes in the mechanical hypersensitivity of the skin can be detected. However, whether measuring changes in skin hypersensitivity can be a reliable surrogate for measuring skeletal pain itself remains unclear. To explore this question, we generated skeletal pain by injecting and confining GFP-transfected NCTC 2472 osteosarcoma cells unilaterally to the femur of C3H male mice. Beginning at day 7 post-tumor injection, animals were administered vehicle, an antibody to the P2X3 receptor (anti-P2X3) or anti-NGF antibody. Pain and analgesic efficacy were then measured on days 21, 28, and 35 post-tumor injection using a battery of skeletal pain-related behaviors and von Frey assessment of mechanical hypersensitivity on the plantar surface of the hind paw. Animals with bone cancer pain treated with anti-P2X3 showed a reduction in skin hypersensitivity but no attenuation of skeletal pain behaviors, whereas animals with bone cancer pain treated with anti-NGF showed a reduction in both skin hypersensitivity and skeletal pain behaviors. These results suggest that although bone cancer can induce significant skeletal pain-related behaviors and hypersensitivity of the skin, relief of hypersensitivity of the skin is not always accompanied by attenuation of skeletal pain. Understanding the relationship between skeletal and skin pain may provide insight into how pain is processed and integrated and help define the preclinical measures of skeletal pain that are predictive end points for clinical trials.
Asunto(s)
Conducta Animal/fisiología , Neoplasias Óseas/fisiopatología , Dolor en Cáncer/fisiopatología , Osteosarcoma/fisiopatología , Piel/fisiopatología , Animales , Neoplasias Óseas/psicología , Dolor en Cáncer/psicología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Hiperalgesia/fisiopatología , Hiperalgesia/psicología , Masculino , Ratones , Ratones Endogámicos C3H , Trasplante de Neoplasias , Osteosarcoma/psicologíaRESUMEN
The number of patients suffering from postoperative pain due to orthopedic surgery and bone fracture is projected to dramatically increase because the human life span, weight, and involvement in high-activity sports continue to rise worldwide. Joint replacement or bone fracture frequently results in skeletal pain that needs to be adequately controlled for the patient to fully participate in needed physical rehabilitation. Currently, the 2 major therapies used to control skeletal pain are nonsteroidal anti-inflammatory drugs and opiates, both of which have significant unwanted side effects. To assess the efficacy of novel therapies, mouse models of orthopedic and fracture pain were developed and evaluated here. These models, orthopedic surgery pain and bone fracture pain, resulted in skeletal pain-related behaviors that lasted 3 weeks and 8 to 10 weeks, respectively. These skeletal pain behaviors included spontaneous and palpation-induced nocifensive behaviors, dynamic weight bearing, limb use, and voluntary mechanical loading of the injured hind limb. Administration of anti-nerve growth factor before orthopedic surgery or after bone fracture attenuated skeletal pain behaviors by 40% to 70% depending on the end point being assessed. These data suggest that nerve growth factor is involved in driving pain due to orthopedic surgery or bone fracture. These animal models may be useful in developing an understanding of the mechanisms that drive postoperative orthopedic and bone fracture pain and the development of novel therapies to treat these skeletal pains.
Asunto(s)
Fémur/lesiones , Fracturas Óseas/tratamiento farmacológico , Factor de Crecimiento Nervioso/antagonistas & inhibidores , Procedimientos Ortopédicos/efectos adversos , Dolor Postoperatorio/tratamiento farmacológico , Dolor/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Fémur/diagnóstico por imagen , Fracturas Óseas/diagnóstico por imagen , Masculino , Ratones , Ratones Endogámicos C3H , Dolor/diagnóstico por imagen , Dolor Postoperatorio/diagnóstico por imagen , Radiografía , Factores de TiempoRESUMEN
Skeletal injury is a leading cause of chronic pain and long-term disability worldwide. While most acute skeletal pain can be effectively managed with nonsteroidal anti-inflammatory drugs and opiates, chronic skeletal pain is more difficult to control using these same therapy regimens. One possibility as to why chronic skeletal pain is more difficult to manage over time is that there may be nerve sprouting in nonhealed areas of the skeleton that normally receive little (mineralized bone) to no (articular cartilage) innervation. If such ectopic sprouting did occur, it could result in normally nonnoxious loading of the skeleton being perceived as noxious and/or the generation of a neuropathic pain state. To explore this possibility, a mouse model of skeletal pain was generated by inducing a closed fracture of the femur. Examined animals had comminuted fractures and did not fully heal even at 90+days post fracture. In all mice with nonhealed fractures, exuberant sensory and sympathetic nerve sprouting, an increase in the density of nerve fibers, and the formation of neuroma-like structures near the fracture site were observed. Additionally, all of these animals exhibited significant pain behaviors upon palpation of the nonhealed fracture site. In contrast, sprouting of sensory and sympathetic nerve fibers or significant palpation-induced pain behaviors was never observed in naïve animals. Understanding what drives this ectopic nerve sprouting and the role it plays in skeletal pain may allow a better understanding and treatment of this currently difficult-to-control pain state.
Asunto(s)
Fibras Adrenérgicas/patología , Fracturas Óseas/complicaciones , Dolor Musculoesquelético/etiología , Dolor Musculoesquelético/patología , Fibras Adrenérgicas/fisiología , Animales , Calcificación Fisiológica/fisiología , Péptido Relacionado con Gen de Calcitonina/metabolismo , Dolor Crónico , Modelos Animales de Enfermedad , Fracturas Óseas/patología , Proteína GAP-43/metabolismo , Imagenología Tridimensional , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Neurofilamentos/metabolismo , Neuroma/etiología , Neuroma/patología , Dimensión del Dolor , Palpación/efectos adversos , Estadísticas no Paramétricas , Rayos XRESUMEN
Studies in animals and humans show that blockade of nerve growth factor (NGF) attenuates both malignant and nonmalignant skeletal pain. While reduction of pain is important, a largely unanswered question is what other benefits NGF blockade might confer in patients with bone cancer. Using a mouse graft model of bone sarcoma, we demonstrate that early treatment with an NGF antibody reduced tumor-induced bone destruction, delayed time to bone fracture, and increased the use of the tumor-bearing limb. Consistent with animal studies in osteoarthritis and head and neck cancer, early blockade of NGF reduced weight loss in mice with bone sarcoma. In terms of the extent and time course of pain relief, NGF blockade also reduced pain 40% to 70%, depending on the metric assessed. Importantly, this analgesic effect was maintained even in animals with late-stage disease. Our results suggest that NGF blockade immediately upon detection of tumor metastasis to bone may help preserve the integrity and use, delay the time to tumor-induced bone fracture, and maintain body weight.