RESUMEN
Opioids are commonly used for the treatment of postoperative and post-traumatic pain; however, their therapeutic effectiveness is limited by undesirable and life-threatening side effects. Researchers have long attempted to develop opioid co-administration therapies that enhance analgesia, but the complexity of opioid analgesia and our incomplete mechanistic understanding has made this a daunting task. We discovered that subanalgesic morphine doses (100 ng/kg-10 µg/kg) augmented the acute analgesic effect of fentanyl (20 µg/kg) following subcutaneous drug co-administration to male rats. In addition, administration of equivalent drug ratios to naïve rat spinal cord membranes induced a twofold increase in G protein activation. The rate of GTP hydrolysis remained unchanged. We demonstrated that these behavioral and biochemical effects were mediated by the delta opioid receptor (DOP). Subanalgesic doses of the DOP-selective agonist SNC80 also augmented the acute analgesic effect of fentanyl. Furthermore, co-administration of the DOP antagonist naltrindole with both fentanyl-morphine and fentanyl-SNC80 combinations prevented augmentation of both analgesia and G protein activation. The mu opioid receptor (MOP) antagonist cyprodime did not block augmentation. Confocal microscopy of the substantia gelatinosa of rats treated with fentanyl, subanalgesic morphine, or this combination showed that changes in MOP internalization did not account for augmentation effects. Together, these findings suggest that augmentation of fentanyl analgesia by subanalgesic morphine is mediated by increased G protein activation resulting from a synergistic interaction between or heterodimerization of MOPs and DOPs. This finding is of great therapeutic significance because it suggests a strategy for the development of DOP-selective ligands that can enhance the therapeutic index of clinically used MOP drugs.
Asunto(s)
Analgesia , Morfina , Analgésicos Opioides/farmacología , Animales , Fentanilo/farmacología , Fentanilo/uso terapéutico , Masculino , Morfina/farmacología , Dolor , Ratas , Receptores Opioides delta , Receptores Opioides muRESUMEN
Mechanical sensitization is one of the most difficult clinical pain problems to treat. However, the molecular and genetic bases of mechanical nociception are unclear. Here we develop a Drosophila model of mechanical nociception to investigate the ion channels and signaling pathways that regulate mechanical nociception. We fabricated von Frey filaments that span the subthreshold to high noxious range for Drosophila larvae. Using these, we discovered that pressure (force/area), rather than force per se, is the main determinant of aversive rolling responses to noxious mechanical stimuli. We demonstrated that the RTK PDGF/VEGF receptor (Pvr) and its ligands (Pvfs 2 and 3) are required for mechanical nociception and normal dendritic branching. Pvr is expressed and functions in class IV sensory neurons, whereas Pvf2 and Pvf3 are produced by multiple tissues. Constitutive overexpression of Pvr and its ligands or inducible overexpression of Pvr led to mechanical hypersensitivity that could be partially separated from morphological effects. Genetic analyses revealed that the Piezo and Pain ion channels are required for mechanical hypersensitivity observed upon ectopic activation of Pvr signaling. PDGF, but not VEGF, peptides caused mechanical hypersensitivity in rats. Pharmacological inhibition of VEGF receptor Type 2 (VEGFR-2) signaling attenuated mechanical nociception in rats, suggesting a conserved role for PDGF and VEGFR-2 signaling in regulating mechanical nociception. VEGFR-2 inhibition also attenuated morphine analgesic tolerance in rats. Our results reveal that a conserved RTK signaling pathway regulates baseline mechanical nociception in flies and rats.SIGNIFICANCE STATEMENT Hypersensitivity to touch is poorly understood and extremely difficult to treat. Using a refined Drosophila model of mechanical nociception, we discovered a conserved VEGF-related receptor tyrosine kinase signaling pathway that regulates mechanical nociception in flies. Importantly, pharmacological inhibition of VEGF receptor Type 2 signaling in rats causes analgesia and blocks opioid tolerance. We have thus established a robust, genetically tractable system for the rapid identification and functional analysis of conserved genes underlying mechanical pain sensitivity.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Nocicepción/fisiología , Células Receptoras Sensoriales/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila melanogaster , Péptidos y Proteínas de Señalización Intercelular/genética , Larva , Masculino , Nocicepción/efectos de los fármacos , Estimulación Física/efectos adversos , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Especificidad de la Especie , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , VertebradosRESUMEN
The analgesic effect of opioids decreases over time due to the development of analgesic tolerance. We have shown that inhibition of the platelet-derived growth factor beta (PDGFR-ß) signaling eliminates morphine analgesic tolerance in rats. Although the PDGFR-ß and its ligand, the platelet-derived growth factor type B (PDGF-B), are expressed in the substantia gelatinosa of the spinal cord (SG) and in the dorsal root ganglia (DRG), their precise distribution within different cell types of these structures is unknown. Additionally, the impact of a tolerance-mediating chronic morphine treatment, on the expression and distribution of PDGF-B and PDGFR-ß has not yet been studied. Using immunohistochemistry (IHC), we found that in the spinal cord, PDGFR-ß and PDGF-B were expressed in neurons and oligodendrocytes and co-localized with the mu-opioid receptor (MOPr) in opioid naïve rats. PDGF-B was also found in microglia and astrocytes. Both PDGFR-ß and PDGF-B were detected in DRG neurons but not in spinal primary afferent terminals. Chronic morphine exposure did not change the cellular distribution of PDGFR-ß or PDGF-B. However, PDGFR-ß expression was downregulated in the SG and upregulated in the DRG. Consistent with our previous finding that morphine caused tolerance by inducing PDGF-B release, PDGF-B was upregulated in the spinal cord. We also found that chronic morphine exposure caused a spinal proliferation of oligodendrocytes. The changes in PDGFR-ß and PDGF-B expression induced by chronic morphine treatment suggest potential mechanistic substrates underlying opioid tolerance.
Asunto(s)
Analgésicos Opioides , Morfina , Ratas , Masculino , Animales , Morfina/farmacología , Analgésicos Opioides/farmacología , Analgésicos Opioides/metabolismo , Proteínas Proto-Oncogénicas c-sis/metabolismo , Proteínas Proto-Oncogénicas c-sis/farmacología , Ratas Sprague-Dawley , Ganglios Espinales/metabolismo , Tolerancia a Medicamentos/fisiología , Médula Espinal/metabolismoRESUMEN
Severe viral respiratory diseases, such as SARS-CoV-2, are transmitted through aerosol particles produced by coughing, talking, and breathing. Medical procedures including tracheal intubation, extubation, dental work, and any procedure involving close contact with a patient's airways can increase exposure to infectious aerosol particles. This presents a significant risk for viral exposure of nearby healthcare workers during and following patient care. Previous studies have examined the effectiveness of plastic enclosures for trapping aerosol particles and protecting health-care workers. However, many of these enclosures are expensive or are burdensome for healthcare workers to work with. In this study, a low-cost plastic enclosure was designed to reduce aerosol spread and viral transmission during medical procedures, while also alleviating issues found in the design and use of other medical enclosures to contain aerosols. This enclosure is fabricated from clear polycarbonate for maximum visibility. A large single-side cutout provides health care providers with ease of access to the patient with a separate cutout for equipment access. A survey of medical providers in a local hospital network demonstrated their approval of the enclosure's ease of use and design. The enclosure with appropriate plastic covers reduced total escaped particle number concentrations (diameter > 0.01 µm) by over 93% at 8 cm away from all openings. Concentration decay experiments indicated that the enclosure without active suction should be left on the patient for 15-20 minutes following a tracheal manipulation to allow sufficient time for >90% of aerosol particles to settle upon interior surfaces. This decreases to 5 minutes when 30 LPM suction is applied. This enclosure is an inexpensive, easily implemented additional layer of protection that can be used to help contain infectious or otherwise potentially hazardous aerosol particles while providing access into the enclosure.
Asunto(s)
COVID-19 , Transmisión de Enfermedad Infecciosa de Paciente a Profesional , Partículas y Gotitas de Aerosol , COVID-19/prevención & control , Humanos , Transmisión de Enfermedad Infecciosa de Paciente a Profesional/prevención & control , Plásticos , Aerosoles y Gotitas Respiratorias , SARS-CoV-2RESUMEN
ABSTRACT: Pain experience can change the central processing of nociceptive inputs, resulting in persistent allodynia and hyperalgesia. However, the underlying circuit mechanisms remain underexplored. Here, we focus on pain-induced remodeling of the projection from the mediodorsal thalamus (MD) to the anterior cingulate cortex (ACC), a projection that relays spinal nociceptive input for central processing. Using optogenetics combined with slice electrophysiology, we detected in male mice that 7 days of chronic constriction injury (CCI; achieved by loose ligation of the sciatic nerve) generated AMPA receptor (AMPAR)-silent glutamatergic synapses within the contralateral MD-to-ACC projection. AMPAR-silent synapses are typically GluN2B-enriched nascent glutamatergic synapses that mediate the initial formation of neural circuits during early development. During development, some silent synapses mature and become "unsilenced" by recruiting and stabilizing AMPARs, consolidating and strengthening the newly formed circuits. Consistent with these synaptogenic features, pain-induced generation of silent synapses was accompanied by increased densities of immature dendritic spines in ACC neurons and increased synaptic weight of GluN2B-containing NMDA receptors (NMDARs) in the MD-to-ACC projection. After prolonged (â¼30 days) CCI, injury-generated silent synapses declined to low levels, which likely resulted from a synaptic maturation process that strengthens AMPAR-mediated MD-to-ACC transmission. Consistent with this hypothesis, viral-mediated knockdown of GluN2B in ACC neurons, which prevented pain-induced generation of silent synapses and silent synapse-mediated strengthening of MD-to-ACC projection after prolonged CCI, prevented the development of allodynia. Taken together, our results depict a silent synapse-mediated mechanism through which key supraspinal neural circuits that regulate pain sensitivity are remodeled to induce allodynia and hyperalgesia.
Asunto(s)
Giro del Cíngulo , Neuralgia , Animales , Giro del Cíngulo/metabolismo , Masculino , Ratones , Receptores AMPA/metabolismo , Sinapsis/metabolismo , TálamoRESUMEN
BACKGROUND CONTEXT: Psychological comorbidities are important prognostic factors for low back pain (LBP). To develop improved treatment paradigms, it is first necessary to characterize and determine current patterns of treatment in this population. PURPOSE: Identify how comorbid depression or anxiety in patients with LBP is related to use of healthcare resources. STUDY DESIGN/SETTING: Retrospective cohort study using electronic health records from outpatient offices at a large multisite academic medical center. PATIENT SAMPLE: Data from 513,088 unique patients seen between January 2010 and July 2020 (58.0% female, 52.6±19.5 years) with a diagnosis of LBP, indicated by predetermined ICD-9 and ICD-10 codes. OUTCOME MEASURES: Average self-reported pain scores, absolute differences and unadjusted risk ratios to compare opioid use, emergency department visits, hospitalizations, advanced imaging orders, spinal injections, and back surgeries between cohorts. METHODS: Clinical characteristics and data regarding use of healthcare resources were extracted from the electronic health record. Clinical features and patterns in healthcare utilization were determined for patients with depression or anxiety compared to those without. RESULTS: Depression or anxiety was coded for 21.4% of patients at first LBP visit. Those with depression or anxiety were more likely to be on opioids (unadjusted risk ratio: 1.22, CI: [1.22,1.23]), go to the emergency department (1.31 [1.30-1.33]), be hospitalized (1.15 [1.13, 1.17]), receive advanced imaging (1.09 [1.08, 1.11]), receive an epidural steroid injection (1.16 [1.15, 1.18]), and less likely to have back surgery (0.74 [0.72, 0.77]). Differences in pain scores for those with depression/anxiety compared to those without were not clinically significant. CONCLUSIONS: Depression/anxiety is associated with increased use of healthcare resources, and is not associated with clinically meaningful elevated pain scores. Limitations come from use of an aggregate data set and reliance on administrative coding.
Asunto(s)
Dolor de la Región Lumbar , Ansiedad/diagnóstico , Ansiedad/epidemiología , Depresión/diagnóstico , Depresión/epidemiología , Femenino , Humanos , Dolor de la Región Lumbar/diagnóstico , Dolor de la Región Lumbar/epidemiología , Dolor de la Región Lumbar/terapia , Masculino , Aceptación de la Atención de Salud , Estudios RetrospectivosRESUMEN
Spot detection and quantification for 2-DE are challenging and important tasks to fully extract the proteomic information from these data. Traditional analytical methods have significant weaknesses, including spot mismatching and missing data, which require time-consuming manual editing to correct, dramatically decreasing throughput and compromising the objectivity and reproducibility of the analysis. To address this issue, we developed Pinnacle, a novel, quick, automatic, noncommercial method that borrows strength across gels in spot detection and has been shown to yield more precise spot quantifications than traditional methods. New commercial software, notably SameSpots, has also recently been developed as an improvement over traditional workflows. In this paper, we briefly describe Pinnacle and compare its performance to SameSpots in spot detection, spot quantification precision, and differential expression. Our analysis is performed in a rigorous fashion that, unlike other comparisons in the literature, summarizes performance across all spots detected on the gels, and we manually optimize SameSpots results while simply running Pinnacle with standard settings and no manual editing. While both methods showed marked improvement over a commercially available traditional method PG240, Pinnacle consistently yielded spot quantifications with greater validity and reliability, avoided spot delineation problems, and detected more differentially expressed proteins than SameSpots, and represents a significant noncommercial alternative for 2-DE processing.
Asunto(s)
Algoritmos , Electroforesis en Gel Bidimensional/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Proteínas/análisis , Proteómica/métodos , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: Opioids have relieved more human suffering than any other medication, but their use is still fraught with significant concerns of misuse, abuse, and addiction. This theoretical article explores the hypothesis that opioid misuse in the context of pain management produces a hypersensitivity to emotional distress, termed hyperkatifeia. RESULTS: In the misuse of opioids, neural substrates that mediate positive emotional states (brain reward systems) are compromised, and substrates mediating negative emotional states (brain stress systems) are enhanced. A reflection and early marker of such a nonhomeostatic state may be the development of opioid-induced hyperkatifeia, defined as the increased intensity of the constellation of negative emotional/motivational symptoms and signs observed during withdrawal from drugs of abuse (derived from the Greek "katifeia" for dejection or negative emotional state) and is most likely to occur in subjects in whom the opioid produces a break with homeostasis and less likely to occur when the opioid is restoring homeostasis, such as in effective pain treatment. When the opioid appropriately relieves pain, opponent processes are not engaged. However, if the opioid is administered in excess of need because of overdose, pharmacokinetic variables, or treating an individual without pain, then the body will react to that perturbation by engaging opponent processes in the domains of both pain (hyperalgesia) and negative emotional states (hyperkatifeia). CONCLUSIONS: Repeated engagement of opponent processes without time for the brain's emotional systems to reestablish homeostasis will further drive changes in emotional processes that may produce opioid abuse or addiction, particularly in individuals with genetic or environmental vulnerability.
Asunto(s)
Analgésicos Opioides/uso terapéutico , Encéfalo/fisiología , Hiperalgesia , Trastornos Relacionados con Opioides , Dolor , Estrés Psicológico , Encéfalo/anatomía & histología , Emociones , Homeostasis , Humanos , Hiperalgesia/fisiopatología , Hiperalgesia/psicología , Trastornos Relacionados con Opioides/fisiopatología , Trastornos Relacionados con Opioides/psicología , Dolor/tratamiento farmacológico , Dolor/psicología , Estrés Psicológico/fisiopatología , Estrés Psicológico/psicologíaRESUMEN
The safety and efficacy of opioids are compromised as analgesic tolerance develops. Opioids are also ineffective against neuropathic pain. Recent reports have suggested that inhibitors of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), may have analgesic effects in cancer patients suffering from neuropathic pain. It has been shown that the platelet-derived growth factor receptor-ß (PDGFR-ß), an RTK that has been shown to interact with the EGFR, mediates opioid tolerance but does not induce analgesia. Therefore, we sought to determine whether EGFR signaling was involved in opioid tolerance and whether EGFR and PDGFR signaling could induce pain in rats. We found that gefitinib, an EGFR antagonist, eliminated morphine tolerance. In addition, repeated EGF administration rendered animals unresponsive to subsequent analgesic doses of morphine, a phenomenon we call "pre-tolerance." Using a nerve injury model, we found that gefitinib alone was not analgesic. Rather, it reversed insensitivity to morphine analgesia (pre-tolerance) caused by the release of EGF by injured nerves. We also showed that repeated, but not acute EGF or PDGF-BB administration induced mechanical hypersensitivity in rats. EGFR and PDGFR-ß signaling interacted to produce this sensitization. EGFR was widely expressed in primary sensory afferent cell bodies, demonstrating a neuroanatomical substrate for our findings. Taken together, our results suggest a direct mechanistic link between opioid tolerance and mechanical sensitization. EGFR antagonism could eventually play an important clinical role in the treatment of opioid tolerance and neuropathic pain that is refractory to opioid treatment.
Asunto(s)
Analgésicos Opioides , Morfina , Analgésicos/farmacología , Analgésicos Opioides/farmacología , Animales , Tolerancia a Medicamentos , Receptores ErbB , Humanos , Morfina/farmacología , RatasRESUMEN
MOTIVATION: One of the key limitations for proteomic studies using 2-dimensional gel electrophoresis (2DE) is the lack of rapid, robust and reproducible methods for detecting, matching and quantifying protein spots. The most commonly used approaches involve first detecting spots and drawing spot boundaries on individual gels, then matching spots across gels and finally quantifying each spot by calculating normalized spot volumes. This approach is time consuming, error-prone and frequently requires extensive manual editing, which can unintentionally introduce bias into the results. RESULTS: We introduce a new method for spot detection and quantification called Pinnacle that is automatic, quick, sensitive and specific and yields spot quantifications that are reliable and precise. This method incorporates a spot definition that is based on simple, straightforward criteria rather than complex arbitrary definitions, and results in no missing data. Using dilution series for validation, we demonstrate Pinnacle outperformed two well-established 2DE analysis packages, proving to be more accurate and yielding smaller coefficiant of variations (CVs). More accurate quantifications may lead to increased power for detecting differentially expressed spots, an idea supported by the results of our group comparison experiment. Our fast, automatic analysis method makes it feasible to conduct very large 2DE-based proteomic studies that are adequately powered to find important protein expression differences. AVAILABILITY: Matlab code to implement Pinnacle is available from the authors upon request for non-commercial use.
Asunto(s)
Biología Computacional/métodos , Proteínas/análisis , Programas Informáticos , Animales , Línea Celular Tumoral , Electroforesis en Gel Bidimensional , Humanos , Masculino , Morfina/farmacología , Proteínas de Neoplasias/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
Chemically induced nociception has not yet been studied intensively in genetically tractable models. Hence, our goal was to establish a Drosophila assay that can be used to study the cellular and molecular/genetic bases of chemically induced nociception. Drosophila larvae exposed to increasing concentrations of hydrochloric acid (HCl) produced an increasingly intense aversive rolling response. HCl (0.5%) was subthreshold and provoked no response. All classes of peripheral multidendritic (md) sensory neurons (classes I-IV) are required for full responsiveness to acid, with class IV making the largest contribution. At the cellular level, classes IV, III and I showed increases in calcium following acid exposure. In the central nervous system, Basin-4 second-order neurons are the key regulators of chemically induced nociception, with a slight contribution from other types. Finally, chemical nociception can be sensitized by tissue damage. Subthreshold HCl provoked chemical allodynia in larvae 4 h after physical puncture wounding. Pinch wounding and UV irradiation, which do not compromise the cuticle, did not cause chemical allodynia. In sum, we developed a novel assay to study chemically induced nociception in Drosophila larvae. This assay, combined with the high genetic resolving power of Drosophila, should improve our basic understanding of fundamental mechanisms of chemical nociception. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.
Asunto(s)
Drosophila/fisiología , Etología/métodos , Nocicepción/fisiología , Células Receptoras Sensoriales/fisiología , Animales , Escala de Evaluación de la Conducta , Drosophila/crecimiento & desarrollo , Larva/fisiología , Nocicepción/efectos de los fármacosRESUMEN
Endogenous inflammatory mediators contribute to the pathogenesis of pain by acting on nociceptors, specialized sensory neurons that detect noxious stimuli. Here, we describe a new factor mediating inflammatory pain. We show that platelet-derived growth factor (PDGF)-BB applied in vitro causes repetitive firing of dissociated nociceptor-like rat dorsal root ganglion neurons and decreased their threshold for action potential generation. Injection of PDGF-BB into the paw produced nocifensive behavior in rats and led to thermal and mechanical pain hypersensitivity. We further detailed the biophysical mechanisms of these PDGF-BB effects and show that PDGF receptor-induced inhibition of nociceptive M-current underlies PDGF-BB-mediated nociceptive hyperexcitability. Moreover, in vivo sequestration of PDGF or inhibition of the PDGF receptor attenuates acute formalin-induced inflammatory pain. Our discovery of a new pain-facilitating proinflammatory mediator, which by inhibiting M-current activates nociceptive neurons and thus contributes to inflammatory pain, improves our understanding of inflammatory pain pathophysiology and may have important clinical implications for pain treatment.
Asunto(s)
Inflamación/tratamiento farmacológico , Nociceptores/fisiología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células Receptoras Sensoriales/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Ganglios Espinales/metabolismo , Ganglios Espinales/fisiopatología , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Masculino , Nociceptores/efectos de los fármacos , Dolor/metabolismo , Dolor/fisiopatología , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Ratas Sprague-Dawley , Células Receptoras Sensoriales/efectos de los fármacosRESUMEN
Many software packages have been developed to process and analyze 2-D gel images. Some programs have been touted as automated, high-throughput solutions. We tested five commercially available programs using 18 replicate gels of a rat brain protein extract. We determined computer processing time, approximate spot editing time, time required to correct spot mismatches, as well as total processing time. We also determined the number of spots automatically detected, number of spots kept after manual editing, and the percentage of automatically generated correct matches. We also determined the effect of increasing the number of replicate gels on spot matching efficiency for two of the programs. We found that for all programs tested, less than 3% of the total processing time was automated. The remainder of the time was spent in manual, subjective editing of detected spots and computer generated matches. Total processing time for 18 gels varied from 22 to 84 h. The percentage of correct matches generated automatically varied from 1 to 62%. Increasing the number of gels in an experiment dramatically reduced the percentage of automatically generated correct matches. Our results demonstrate that these 2-D gel analysis programs are not automatic or rapid, and also suggest that matching accuracy decreases as experiment size increases.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
The application of MS to imaging, or MS imaging (MSI), allows for the direct investigation of tissue sections to identify biological compounds and determine their spatial distribution. We present an approach to MSI that combines secondary ion MS (SIMS) and MALDI MS for the imaging and analysis of rat spinal cord sections, thereby enhancing the chemical coverage obtained from an MSI experiment. The spinal cord is organized into discrete, anatomically defined areas that include motor and sensory networks composed of chemically diverse cells. The MSI data presented here reveal the spatial distribution of multiple phospholipids, proteins, and neuropeptides obtained within single, 20 mum sections of rat spinal cord. Analyte identities are initially determined by primary mass match and confirmed in follow-up experiments using LC MS/MS from extracts of adjacent spinal cord sections. Additionally, a regional analysis of differentially localized signals serves to rapidly screen compounds of varying intensities across multiple spinal regions. These MSI analyses reveal new insights into the chemical architecture of the spinal cord and set the stage for future imaging studies of the chemical changes induced by pain, anesthesia, and drug tolerance.
Asunto(s)
Médula Espinal/metabolismo , Animales , Ratas , Ratas Long-Evans , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masa de Ion Secundario , Espectrometría de Masas en TándemRESUMEN
Proteomics holds the promise of evaluating global changes in protein expression and post-translational modification in response to environmental stimuli. However, difficulties in achieving cellular anatomic resolution and extracting specific types of proteins from cells have limited the efficacy of these techniques. Laser capture microdissection has provided a solution to the problem of anatomical resolution in tissues. New extraction methodologies have expanded the range of proteins identified in subsequent analyses. This review will examine the application of laser capture microdissection to proteomic tissue sampling, and subsequent extraction of these samples for differential expression analysis. Statistical and other quantitative issues important for the analysis of the highly complex datasets generated are also reviewed.
Asunto(s)
Rayos Láser , Proteómica/métodos , Animales , Humanos , Microdisección , Proteínas/aislamiento & purificación , Proteómica/estadística & datos numéricosRESUMEN
The clinical use of opioids is limited by the development of tolerance and physical dependence. Opioid tolerance and dependence are believed to result from complex adaptations in the CNS, representing a form of neural plasticity. Extracellular signal-regulated kinases (ERKs) are involved in many forms of neural plasticity, and therefore could also be involved in the development of opioid tolerance and dependence. In this study, we investigated the effect of a systemically bioavailable MEK (ERK kinase) inhibitor, SL327, upon the development and the expression of tolerance to and dependence on morphine in mice. In tolerance and dependence development studies, two strains of mice were treated daily for 8 or 9 days with 5mg/kg morphine s.c. Tolerance development was assessed by tail flick latency. Withdrawal was then precipitated by subcutaneous injection of 2mg/kg naloxone s.c. and signs recorded. Co-administration of 50mg/kg SL327 i.p. prior to morphine administration had no effect on the development of tolerance or withdrawal signs. To study possible effects of ERK inhibition on the expression of tolerance and dependence, mice were implanted with 75mg morphine pellets s.c. Tolerance and dependence were assessed as previously described. An acute i.p. injection of 50mg/kg SL327 after 4 days of morphine exposure had no effect on the expression of either morphine tolerance or physical dependence. To verify that this dose of SL327 inhibited morphine-induced ERK modulation, mice received an acute i.p. injection of 50mg/kg SL327 prior to morphine administration, and sacrificed 30min later. Western blots demonstrated that SL327 did inhibit morphine-induced ERK modulation. Taken together, these data suggest that unlike many other observed forms of neural plasticity, the ERK signaling cascade is not involved in the development or expression of opioid tolerance and dependence.
Asunto(s)
Aminoacetonitrilo/análogos & derivados , Inhibidores Enzimáticos/uso terapéutico , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Dependencia de Morfina/prevención & control , Aminoacetonitrilo/uso terapéutico , Animales , Western Blotting , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Dependencia de Morfina/psicología , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Plasticidad Neuronal/efectos de los fármacos , Dimensión del Dolor/efectos de los fármacos , Tiempo de Reacción/efectos de los fármacos , Síndrome de Abstinencia a Sustancias/psicologíaRESUMEN
Opioids are the gold-standard treatment for severe pain. However, potentially life-threatening side effects decrease the safety and effectiveness of these compounds. The addiction liability of these drugs has led to the current epidemic of opioid abuse in the US. Extensive research efforts have focused on trying to dissociate the analgesic properties of opioids from their undesirable side effects. Splice variants of the mu opioid receptor (MOR), which mediates opioid actions, have unique pharmacological properties and anatomic distributions that make them attractive candidates for therapeutic pain relief. In this issue of the JCI, Xu et al. show that specific C-terminal regions of the MOR can modulate side effects without altering analgesia. This discovery greatly improves our understanding of opioid side effects and suggests intriguing therapeutic approaches that could improve both the safety and long-term effectiveness of opioids.
Asunto(s)
Empalme Alternativo , Trastornos Relacionados con Opioides/metabolismo , Receptores Opioides mu/metabolismo , Humanos , Trastornos Relacionados con Opioides/epidemiología , Trastornos Relacionados con Opioides/genética , Receptores Opioides mu/genética , Estados Unidos/epidemiologíaRESUMEN
Spinal subarachnoid opioid administration in rats has been a very important method for studying the pharmacological effects of opioids, including analgesia and tolerance. Intrathecal catheterization, either through the cervical or lumbar approach, has been the predominant method used to deliver opioids spinally. However, these methods have potential undesirable complications. To help mitigate these problems, we have developed a method of intermittent lumbar puncture in rats to study the effects of chronic spinal opioid administration. This method avoids catheter-associated morbidity. We demonstrate that this method can be readily used to induce spinal opioid tolerance without causing morbidity. Intermittent lumbar puncture should prove to be a useful technique for investigating mechanisms of spinal opioid analgesia and opioid tolerance development.
Asunto(s)
Analgésicos Opioides/farmacología , Inyecciones Espinales/métodos , Narcóticos/farmacología , Punción Espinal , Animales , Tolerancia a Medicamentos , Inflamación , Masculino , Morfina/farmacología , Dolor/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Médula Espinal/patología , Espacio Subaracnoideo/patologíaRESUMEN
Accurate spot detection and quantification is a challenging task that must be performed effectively in order to properly extract the proteomic information from two-dimensional (2-D) gel electrophoresis images. In Morris et al., Bioinformatics 24:529-536, 2008, we introduced Pinnacle, an automatic, fast, effective noncommercial package for spot detection and quantification for 2-D gel images, and subsequently we have developed a freely available gui-based interface for applying the method to a set of gels. In this chapter, we overview Pinnacle, and in a step-by-step manner we describe how to use the software to obtain spot lists and quantifications, to be used for comparative proteomic analysis.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Proteínas/aislamiento & purificación , Proteómica/métodos , Programas Informáticos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
While enkephalin and dynorphin peptides have been well characterized in the spinal cord, the cellular localization of beta-endorphin (beta E) and the processing of pro-opiomelanocortin (POMC) to beta E and other non-opioid peptides in the cord have not been extensively investigated. Other investigators have characterized the various beta E forms present in rat spinal cord regions. Previous studies have also suggested that spinal POMC content is entirely derived from supraspinal sources. However, high proportions of beta E precursors present in spinal cord sieving profiles led us to suspect the presence of POMC cell bodies intrinsic to the cord. In this study, we performed thoracic spinal cord lesions on a group of animals and demonstrated the persistence of about one-third of control levels of beta E immunoreactivity (beta E-IR) below the level of the lesions. We also characterized POMC processing in various regions of the spinal cord both before and after lesioning. These data suggested that there may be intrinsic POMC/endorphinergic neuronal systems in the spinal cord.