RESUMEN

NEW FINDINGS: What is the central question of this study? We investigated the effects of intrathecal administration of a novel toxin, CTK 01512-2, in a mouse model of Huntington's disease. We asked whether spinal cord neurons can represent a therapeutic target, given that the spinal cord seems to be involved in motor symptoms of Huntington's disease. Pharmacological approaches focusing on the spinal cord and skeletal muscles might represent a more feasible strategy than a high-risk brain intervention. What is the main finding and its importance? We provided evidence of a novel, local, neuroprotective effect of CTK 01512-2, paving a path for the development of approaches to treat motor symptoms of Huntington's disease beyond the brain. ABSTRACT: Phα1ß is a neurotoxin from the venom of the Phoneutria nigriventer spider, available as CTK 01512-2, a recombinant peptide. Owing to its antinociceptive and analgesic properties, CTK 01512-2 has been described to alleviate neuroinflammatory responses. Despite the diverse actions of CTK 01512-2 on the nervous system, little is known regarding its neuroprotective effect, especially in neurodegenerative conditions such as Huntington's disease (HD), a genetic movement disorder without cure. Here, we investigated whether CTK 01512-2 has a neuroprotective effect in a mouse model of HD. We hypothesized that spinal cord neurons might represent a therapeutic target, because the spinal cord seems to be involved in the motor symptoms of HD (BACHD) mice. We treated BACHD mice with CTK 01512-2 by intrathecal injection and performed in vivo motor behavioural and morphological analyses in the CNS (brain and spinal cord) and muscles. Our data showed that intrathecal injection of CTK 01512-2 significantly improved motor performance in the open field task. CTK 01512-2 protected neurons in the spinal cord (but not in the brain) from death, suggesting a local effect. CTK 01512-2 exerted its neuroprotective effect by inhibiting BACHD neuronal apoptosis, as revealed by a reduction in caspase-3 in the spinal cord. CTK 01512-2 was also able to revert BACHD muscle atrophy. In conclusion, our data suggest a novel role for CTK 01512-2 acting directly in the spinal cord to ameliorate morphofunctional aspects of spinal cord neurons and muscles and improve the performance of BACHD mice in motor behavioural tests. Given that HD shares similar symptoms with many neurodegenerative conditions, the findings presented herein might also be applicable to other disorders.

Asunto(s)

Enfermedad de Huntington , Fármacos Neuroprotectores , Animales , Modelos Animales de Enfermedad , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/genética , Ratones , Ratones Transgénicos , Neuronas , Fármacos Neuroprotectores/farmacología , Médula Espinal

RESUMEN

Opioids are the first-line treatment for cancer pain. Incomplete pain relief and the high rate of adverse effects of these compounds bring a need to combine them with other drugs acting on different targets. AIMS: We here evaluate the antinociceptive interaction and adverse events of methadone combined with recombinant Phα1ß, an analgesic toxin from Phoneutria nigriventer. MAIN METHODS: Melanoma was produced by intraplantar inoculation of B16-F10 cells into the right paw. von Frey filaments measured the paw-withdrawal threshold after administration of methadone, Phα1ß, and their combination. The degree of interaction was evaluated using isobolographic analysis. Spontaneous performance and forced motor performance were assessed with the open-field and rotarod tests, respectively. Intestinal function was evaluated by the distance traveled by charcoal and opioid tolerance was induced by daily morphine injections. KEY FINDINGS: Co-administration of Phα1ß with methadone synergistically reverses the melanoma-induced mechanical hypersensitivity. No motor alterations were observed but mild alterations on intestinal function after treatment with the combination that was also capable of restoring morphine analgesia in the tail-flick test after an opioid-induced tolerance. SIGNIFICANCE: Combinatorial treatment with Phα1ß and methadone produces synergistic analgesic potentiation with potential implications to pain treatment even under opioid tolerance conditions.

Asunto(s)

Analgésicos/farmacología , Dolor en Cáncer/tratamiento farmacológico , Metadona/administración & dosificación , Manejo del Dolor/métodos , Venenos de Araña/administración & dosificación , Analgésicos Opioides/farmacología , Animales , Conducta Animal , Bloqueadores de los Canales de Calcio/farmacología , Sinergismo Farmacológico , Quimioterapia Combinada , Tolerancia a Medicamentos , Tracto Gastrointestinal/efectos de los fármacos , Masculino , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Neoplasias/complicaciones , Factores de Tiempo

RESUMEN

Abnormal calcium influx and glutamatergic excitotoxicity have been extensively associated with neuronal death in Huntington's disease (HD), a genetic movement disorder. Currently, there is no effective treatment for this fatal condition. The neurotoxin Phα1ß has demonstrated therapeutic effects as a calcium channel blocker, for example during pain control. However, little is known about its neuroprotective effect in HD. Herein, we investigated if Phα1ß is effective in inhibiting neuronal cell death in the BACHD mouse model for HD. We performed intrastriatal injection of Phα1ß in WT and BACHD mice. No side effects or unusual behaviors were observed upon Phα1ß administration. Using three different motor behavior tests, we observed that injection of the toxin in BACHD mice greatly improved the animals' motor-force as seen in the Wire-hang test, and also the locomotor performance, according to the Open field test. NeuN labeling for mature neuron detection revealed that Phα1ß toxin promoted neuronal preservation in the striatum and cortex, when injected locally. Intrastriatal injection of Phα1ß was not able to preserve neurons from the spinal cord and also not revert muscle atrophy in BACHD mice. Finally, we observed that Phα1ß might, at least in part, exert its protective effect by decreasing L-glutamate, measured in cerebrospinal fluid. Our data provide evidence of a novel neuroprotector effect of Phα1ß, paving a path for the development of new approaches to treat HD motor symptoms.

Asunto(s)

Enfermedad de Huntington/tratamiento farmacológico , Fármacos Neuroprotectores/administración & dosificación , Venenos de Araña/administración & dosificación , Animales , Modelos Animales de Enfermedad , Ácido Glutámico/metabolismo , Enfermedad de Huntington/patología , Ratones , Ratones Transgénicos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Neuronas/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Médula Espinal/patología

RESUMEN

PURPOSE: Abdominal hysterectomy is one of the most commonly performed gynecologic surgical procedures and is frequently associated with moderate to severe pain. The present study compared the effects of morphine and ketamine on postoperative analgesia, hemodynamic stability, and postoperative adverse effects in patients who underwent abdominal hysterectomy. DESIGN: This randomized controlled trial compares the effects of morphine plus adjuvants to those of ketamine plus adjuvants, administered as spinal anesthetic agents in patients who underwent abdominal hysterectomy. METHODS: Eighty patients were randomly assigned to two different groups: group M (morphine, 40 mcg) and group K (ketamine, 20 mg); the anesthetic agents were combined with equal quantities of other adjuvants. Postoperative analgesia was evaluated by means of a numeric pain rating scale; adverse effects (pruritus, nausea and vomiting, urinary retention, respiratory depression, and changes in bowel habits) at specific postoperative time intervals of T1 (4 hours), T2 (12 hours), and T3 (24 hours) were documented and compared. Hemodynamic stability was assessed intraoperatively. FINDINGS: Both groups displayed similar patient characteristics, comorbidities, paravertebral block level, and intraoperative hemodynamics. The present study observed a significant difference in postoperative analgesia between the two groups, 12 hours after the surgery, with group M exhibiting better results, compared with group K (P = .004). The pain scores obtained from group K were consistent with the amount of rescue medication (tramadol) administered to the subjects in the group, which showed a concomitant higher consumption of tramadol, compared with group M (42.5 and 71.8 mg in group M and group K, respectively, P = .011). Group M showed a higher incidence of pruritus, changes in bowel habits, and constipation compared with group K. CONCLUSIONS: Compared with ketamine, intrathecal morphine obtained better postoperative analgesia up to 12 hours after surgery, with a higher incidence of pruritus without any significant change in other variables.

Asunto(s)

Ketamina , Morfina/uso terapéutico , Dolor Postoperatorio/tratamiento farmacológico , Analgesia Controlada por el Paciente , Analgésicos Opioides , Método Doble Ciego , Femenino , Humanos , Histerectomía/efectos adversos , Ketamina/uso terapéutico

RESUMEN

Vascular dementia (VD) is a major cognitive disorder originated from a blood flow disruption in the brain. This process leads to chronic cerebral ischemia that deeply affects neuronal tissues and lipid homeostasis. The understanding of cerebral lipid dynamics during chronic ischemia can reveal biomarkers and novel pharmacological targets for the treatment of VD. In this study, we used the Desorption Electrospray Ionization - imaging mass spectrometry (DESI-IMS) technique to map lipids in the rat brain tissues after bilateral common carotid artery occlusion (BCCAO) rat model of chronic cerebral hypoperfusion. The brain imaging enabled the detection of differences in lipids from ischemic and non-ischemic brains. The analysis demonstrated that arachidonic acid (ARA), docosahexaenoic acid (DHA), dihomo-γ-linolenic acid, hydroxyeicosatetraenoic (HETE)-Ala and glycerophosphoethanolamine levels were significantly reduced in the hippocampus and cortex of animals submitted to BCCAO model when compared to control animals. Decanoic acid was increased after 30 days of BCCAO model. Partial least squares discriminant analysis (PLS-DA) could discriminate between BCCAO group and the control group, in which γ-linolenic acid (m/z 277) ion and stearic acid (m/z 283) had the highest discrimination potential. Taken together, these findings indicate that lipid dynamics are altered in chronic ischemia-induced by BCCAO in rats and indicate potential biomarkers and pharmacological targets for VD.

Asunto(s)

Isquemia Encefálica/diagnóstico por imagen , Corteza Cerebral/patología , Hipocampo/patología , Lípidos/análisis , Animales , Enfermedades de las Arterias Carótidas/patología , Enfermedad Crónica , Trastornos del Conocimiento/patología , Modelos Animales de Enfermedad , Masculino , Neuronas/patología , Ratas Wistar

RESUMEN

It is well-established that bacterial lipopolysaccharides (LPS) can promote neuroinflammation through receptor Toll-like 4 activation and induces sickness behavior in mice. This phenomenon triggers changes in membranes lipid dynamics to promote the intracellular cell signaling. Desorption electrospray ionization mass spectrometry (DESI-MS) is a powerful technique that can be used to image the distribution of lipids in the brain tissue directly. In this work, we characterize the LPS-induced neuroinflammation and the lipid dynamics in C57BL/6 mice at 3 and 24 h after LPS injection. We have observed that intraperitoneal administration of LPS (5 mg/kg body weight) induces sickness behavior and triggers a peripheral and cerebral increase of pro- and anti-inflammatory cytokine levels after 3 h, but only IL-10 was upregulated after 24 h. Morphological analysis of hypothalamus, cortex and hippocampus demonstrated that microglial activation was present after 24 h of LPS injection, but not at 3 h. DESI-MS revealed a total of 14 lipids significantly altered after 3 and 24 h and as well as their neuroanatomical distribution. Multivariate statistical analyzes have shown that ions associated with phosphatidylethanolamine [PE(38:4)] and docosatetraenoic acid [FA (22:4)] could be used as biomarkers to distinguish samples from the control or LPS treated groups. Finally, our data demonstrated that monitoring cerebral lipids dynamics and its neuroanatomical distribution can be helpful to understand sickness behavior and microglial activation after LPS administration.

Asunto(s)

Lípidos/inmunología , Inflamación Neurogénica/inmunología , Neuroinmunomodulación/inmunología , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/metabolismo , Citocinas/metabolismo , Hipocampo/diagnóstico por imagen , Hipocampo/metabolismo , Hipotálamo/diagnóstico por imagen , Hipotálamo/metabolismo , Conducta de Enfermedad/fisiología , Lipopolisacáridos/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/inmunología , Transducción de Señal , Espectrometría de Masa por Ionización de Electrospray/métodos

RESUMEN

Voltage-gated calcium channels (VGCCs) play a critical role in neuroinflammatory diseases, such as multiple sclerosis (MS). CTK 01512-2 is a recombinant version of the peptide Phα1ß derived from the spider Phoneutria nigriventer, which inhibits N-type VGCC/TRPA1-mediated calcium influx. We investigated the effects of this molecule in the mouse model of experimental autoimmune encephalomyelitis (EAE). The effects of CTK 01512-2 were compared to those displayed by ziconotide-a selective N-type VGCC blocker clinically used for chronic pain-and fingolimod-a drug employed for MS treatment. The intrathecal (i.t.) treatment with CTK 01512-2 displayed beneficial effects, by preventing nociception, body weight loss, splenomegaly, MS-like clinical and neurological scores, impaired motor coordination, and memory deficits, with an efficacy comparable to that observed for ziconotide and fingolimod. This molecule displayed a favorable profile on EAE-induced neuroinflammatory changes, including inflammatory infiltrate, demyelination, pro-inflammatory cytokine production, glial activation, and glucose metabolism in the brain and spinal cord. The recovery of spatial memory, besides a reduction of serum leptin levels, allied to central and peripheral elevation of the anti-inflammatory cytokine IL-10, was solely modulated by CTK 01512-2, dosed intrathecally. The intravenous (i.v.) administration of CTK 01512-2 also reduced the EAE-elicited MS-like symptoms, similarly to that seen in animals that received fingolimod orally. Ziconotide lacked any significant effect when dosed by i.v. route. Our results indicate that CTK 01512-2 greatly improved the neuroinflammatory responses in a mouse model of MS, with a higher efficacy when compared to ziconotide, pointing out this molecule as a promising adjuvant for MS management.

Asunto(s)

Bloqueadores de los Canales de Calcio/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Bloqueadores de los Canales de Calcio/farmacología , Quimiocinas/metabolismo , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/patología , Trastornos del Conocimiento/fisiopatología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/complicaciones , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Clorhidrato de Fingolimod/farmacología , Clorhidrato de Fingolimod/uso terapéutico , Hiperalgesia/complicaciones , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/patología , Inflamación/patología , Mediadores de Inflamación/metabolismo , Inyecciones Espinales , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Esclerosis Múltiple/complicaciones , Esclerosis Múltiple/patología , Esclerosis Múltiple/fisiopatología , Vaina de Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/metabolismo , Nocicepción/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , omega-Conotoxinas/farmacología , omega-Conotoxinas/uso terapéutico

RESUMEN

Neurodegenerative diseases affect millions of individuals worldwide. So far, no disease-modifying drug is available to treat patients, making the search for effective drugs an urgent need. Neurodegeneration is triggered by the activation of several cellular processes, including oxidative stress, mitochondrial impairment, neuroinflammation, aging, aggregate formation, glutamatergic excitotoxicity, and apoptosis. Therefore, many research groups aim to identify drugs that may inhibit one or more of these events leading to neuronal cell death. Venoms are fruitful natural sources of new molecules, which have been relentlessly enhanced by evolution through natural selection. Several studies indicate that venom components can exhibit selectivity and affinity for a wide variety of targets in mammalian systems. For instance, an expressive number of natural peptides identified in venoms from animals, such as snakes, scorpions, bees, and spiders, were shown to lessen inflammation, regulate glutamate release, modify neurotransmitter levels, block ion channel activation, decrease the number of protein aggregates, and increase the levels of neuroprotective factors. Thus, these venom components hold potential as therapeutic tools to slow or even halt neurodegeneration. However, there are many technological issues to overcome, as venom peptides are hard to obtain and characterize and the amount obtained from natural sources is insufficient to perform all the necessary experiments and tests. Fortunately, technological improvements regarding heterologous protein expression, as well as peptide chemical synthesis will help to provide enough quantities and allow chemical and pharmacological enhancements of these natural occurring compounds. Thus, the main focus of this review is to highlight the most promising studies evaluating animal toxins as therapeutic tools to treat a wide variety of neurodegenerative conditions, including Alzheimer's disease, Parkinson's disease, brain ischemia, glaucoma, amyotrophic lateral sclerosis, and multiple sclerosis.

RESUMEN

Determining antinociceptive interaction between Phα1ß toxin (a voltage gated calcium channel blocker) and SB366791 (selective TRPV1 antagonist) may have both clinical and mechanistic implications for the pain management. This data in brief article is associated to the research paper "Synergistic antinociceptive effect of a calcium channel blocker and a TRPV1 blocker in an acute pain model in mice". This material supports the isobolographic analysis performed with the above drugs and shows: data of the dose response curves of the agents given as single drug or combination regimens. Mathematics and statistical processing of dose response curves, proportion of drugs dosage to be used in the combination, calculus of theoretical additive DE20 dose as well as experimentally obtained DE20 are provided. It is also presented details of statistical comparison between theoretical and experimentally obtained DE20.

RESUMEN

AIMS: Extensive evidence supports a role for voltage-gated calcium channels (VGCC) and TRPV1 receptors in pain transmission and modulation. We investigated the profile of analgesic interaction between Phα1ß toxin (a VGCC blocker) and SB366791 (selective TRPV1 antagonist) in a model of acute pain induced by capsaicin. Changes in body temperature induced by combination regimens were also evaluated. MAIN METHODS: Isobolographic approach with a fixed dose-ratio of combined drugs was used to determine whether antinociceptive interaction of Phα1ß and SB366791 are subadditive, additive or synergic. Body temperature was obtained by thermal infrared imaging. KEY FINDINGS: Phα1ß and SB366791 interact in a synergistic manner to cause antinociception. We found an interaction index (α) of 0.07 for Phα1ß and SB366791 when these drugs were injected together intraplantarly, which indicates that in vivo interaction between these drugs is greater than additive interaction. Synergism also occurred when intraplantar SB366791 was administered simultaneously with intrathecal Phα1ß (interaction index α=0.06) suggesting a 15 fold rise in potency on the analgesic effect of these drugs when they are added together. It was observed no significant alterations in body temperature of animals treated with this combination regimen. SIGNIFICANCE: Our data reveal that Phα1ß toxin potentiates in 15 fold the antinociceptive action of the TRPV1 blocker SB366791. Therefore, lower doses of these drugs are required to achieve antinociceptive effects when these agents are given in combination.

Asunto(s)

Dolor Agudo/tratamiento farmacológico , Analgésicos/farmacología , Anilidas/farmacología , Cinamatos/farmacología , Venenos de Araña/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidores , Analgésicos/administración & dosificación , Anilidas/administración & dosificación , Animales , Temperatura Corporal , Bloqueadores de los Canales de Calcio/administración & dosificación , Bloqueadores de los Canales de Calcio/farmacología , Capsaicina , Cinamatos/administración & dosificación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Ratones , Venenos de Araña/administración & dosificación

RESUMEN

The venom of the Brazilian armed spider Phoneutria nigriventer is a rich source of biologically active peptides that have potential as analgesic drugs. In this study, we investigated the analgesic and adverse effects of peptide 3-5 (Tx3-5), purified from P. nigriventer venom, in several mouse models of pain. Tx3-5 was administered by intrathecal injection to mice selected as models of postoperative (plantar incision), neuropathic (partial sciatic nerve ligation) and cancer-related pain (inoculation with melanoma cells) in animals that were either sensitive or tolerant to morphine. Intrathecal administration of Tx3-5 (3-300 fmol/site) in mice could either prevent or reverse postoperative nociception, with a 50 % inhibitory dose (ID50) of 16.6 (3.2-87.2) fmol/site and a maximum inhibition of 87 ± 10 % at a dose of 30 fmol/site. Its effect was prevented by the selective activator of L-type calcium channel Bay-K8644 (10 µg/site). Tx3-5 (30 fmol/site) also produced a partial antinociceptive effect in a neuropathic pain model (inhibition of 67 ± 10 %). Additionally, treatment with Tx3-5 (30 fmol/site) nearly abolished cancer-related nociception with similar efficacy in both morphine-sensitive and morphine-tolerant mice (96 ± 7 and 100 % inhibition, respectively). Notably, Tx3-5 did not produce visible adverse effects at doses that produced antinociception and presented a TD50 of 1125 (893-1418) fmol/site. Finally, Tx3-5 did not alter the normal mechanical or thermal sensitivity of the animals or cause immunogenicity. Our results suggest that Tx3-5 is a strong drug candidate for the treatment of painful conditions.

Asunto(s)

Analgésicos/uso terapéutico , Dolor en Cáncer/tratamiento farmacológico , Neuralgia/tratamiento farmacológico , Neuropéptidos/uso terapéutico , Neurotoxinas/uso terapéutico , Venenos de Araña/uso terapéutico , Analgésicos/efectos adversos , Analgésicos/farmacología , Animales , Agonistas de los Canales de Calcio/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuropéptidos/efectos adversos , Neuropéptidos/farmacología , Neurotoxinas/efectos adversos , Neurotoxinas/farmacología , Nocicepción/efectos de los fármacos , Venenos de Araña/efectos adversos , Venenos de Araña/farmacología

RESUMEN

Spider toxins are recognized as useful sources of bioactive substances, showing a wide range of pharmacological effects on neurotransmission. Several spider toxins have been identified biochemically and some of them are specific glutamate receptors antagonists. Previous data indicate that PnTx4-5-5, a toxin isolated from the spider Phoneutria nigriventer, inhibits the N-methyl-d-aspartate receptor (NMDAR), with little or no effect on AMPA, kainate or GABA receptors. In agreement with these results, our findings in this study show that PnTx4-5-5 reduces the amplitude of NMDAR-mediated EPSCs in hippocampal slices. It is well established that glutamate-mediated excitotoxic neuronal cell death occurs mainly via NMDAR activation. Thus, we decided to investigate whether PnTx4-5-5 would protect against various cell death insults. For that, we used primary-cultured corticostriatal neurons from wild type (WT) mice, as well as from a mouse model of Huntington's disease, BACHD. Our results showed that PnTx4-5-5 promotes neuroprotection of WT and BACHD neurons under the insult of high levels of glutamate. Moreover, the toxin is also able to protect WT neurons against amyloid ß (Aß) peptide toxicity. These results indicate that the toxin PnTx4-5-5 is a potential neuroprotective drug.

Asunto(s)

Péptidos beta-Amiloides/antagonistas & inhibidores , Proteínas de Artrópodos/farmacología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Venenos de Araña/farmacología , Péptidos beta-Amiloides/toxicidad , Animales , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Cuerpo Estriado/citología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Embrión de Mamíferos/citología , Embrión de Mamíferos/patología , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Técnicas In Vitro , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Neuronas/patología , Técnicas de Placa-Clamp , Células Piramidales/citología , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo

RESUMEN

The mechanical properties of the local microenvironment may have important influence on the fate and function of adult tissue progenitor cells, altering the regenerative process. This is particularly critical following a myocardial infarction, in which the normal, compliant myocardial tissue is replaced with fibrotic, stiff scar tissue. In this study, we examined the effects of matrix stiffness on adult cardiac side population (CSP) progenitor cell behavior. Ovine and murine CSP cells were isolated and cultured on polydimethylsiloxane substrates, replicating the elastic moduli of normal and fibrotic myocardium. Proliferation capacity and cell cycling were increased in CSP cells cultured on the stiff substrate with an associated reduction in cardiomyogeneic differentiation and accelerated cell ageing. In addition, culture on stiff substrate stimulated upregulation of extracellular matrix and adhesion proteins gene expression in CSP cells. Collectively, we demonstrate that microenvironment properties, including matrix stiffness, play a critical role in regulating progenitor cell functions of endogenous resident CSP cells. Understanding the effects of the tissue microenvironment on resident cardiac progenitor cells is a critical step toward achieving functional cardiac regeneration.

Asunto(s)

Células Madre Adultas/fisiología , Dimetilpolisiloxanos/química , Mecanotransducción Celular , Miocitos Cardíacos/fisiología , Células de Población Lateral/fisiología , Nicho de Células Madre , Células Madre Adultas/metabolismo , Animales , Adhesión Celular , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Senescencia Celular , Técnicas de Cocultivo , Módulo de Elasticidad , Masculino , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Fenotipo , Ovinos , Células de Población Lateral/metabolismo , Factores de Tiempo

RESUMEN

GABA is an inhibitory neurotransmitter that appears to be associated with the action of volatile anesthetics. These anesthetics potentiate GABA-induced postsynaptic currents by synaptic GABAA receptors, although recent evidence suggests that these agents also significantly affect extrasynaptic GABA receptors. However, the effect of volatile anesthetics on the extracellular concentration of GABA in the central nervous system has not been fully established. In the present study, rat brain cortical slices loaded with [(3)H]GABA were used to investigate the effect of halothane and sevoflurane on the extracellular accumulation of this neurotransmitter. The accumulation of [(3)H]GABA was significantly increased by sevoflurane (0.058, 0.11, 0.23, 0.46, and 0.93 mM) and halothane (0.006, 0.012, 0.024, 0.048, 0072, and 0.096 mM) with an EC50 of 0.26 mM and 35 µM, respectively. TTX (blocker of voltage-dependent Na(+) channels), EGTA (an extracellular Ca(2+) chelator) and BAPTA-AM (an intracellular Ca(2+) chelator) did not interfere with the accumulation of [(3)H]GABA induced by 0.23 mM sevoflurane and 0.048 mM halothane. SKF 89976A, a GABA transporter type 1 (GAT-1) inhibitor, reduced the sevoflurane- and halothane-induced increase in the accumulation of GABA by 57 and 63 %, respectively. Incubation of brain cortical slices at low temperature (17 °C), a condition that inhibits GAT function and reduces GABA release through reverse transport, reduced the sevoflurane- and halothane-induced increase in the accumulation of [(3)H]GABA by 82 and 75 %, respectively, relative to that at normal temperature (37 °C). Ouabain, a Na(+)/K(+) ATPase pump inhibitor, which is known to induce GABA release through reverse transport, abolished the sevoflurane and halothane effects on the accumulation of [(3)H]GABA. The effect of sevoflurane and halothane did not involve glial transporters because ß-alanine, a blocker of GAT-2 and GAT-3, did not inhibit the effect of the anesthetics. In conclusion, the present study suggests that sevoflurane and halothane increase the accumulation of GABA by inducing the reverse transport of this neurotransmitter. Therefore, volatile anesthetics could interfere with neuronal excitability by increasing the action of GABA on synaptic and extrasynaptic GABA receptors.

Asunto(s)

Anestésicos/farmacología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Anestésicos/administración & dosificación , Animales , Calcio/farmacología , Frío , Halotano/administración & dosificación , Halotano/farmacología , Éteres Metílicos/administración & dosificación , Éteres Metílicos/farmacología , Ácidos Nipecóticos/farmacología , Ouabaína/farmacología , Ratas , Sevoflurano , Tetrodotoxina/toxicidad , Tritio/metabolismo , Volatilización , beta-Alanina/farmacología

RESUMEN

The treatment with the chemotherapeutic agent paclitaxel produces a painful peripheral neuropathy, and is associated with an acute pain syndrome in a clinically significant number of patients. However, no standard therapy has been established to manage the acute pain or the chronic neuropathic pain related to paclitaxel. In the present study, we evaluated the analgesic potential of two N-type voltage-gated calcium channel (VGCC) blockers, ω-conotoxin MVIIA and Phα1ß, on acute and chronic pain induced by paclitaxel. Adult male rats were treated with four intraperitoneal injections of paclitaxel (1+1+1+1mg/kg, in alternate days) and the development of mechanical hyperalgesia was evaluated 24h (acute painful stage) or 15days (chronic painful stage) after the first paclitaxel injection. Not all animals showed mechanical hyperalgesia 24h after the first paclitaxel injection, but those that showed developed a more intense mechanical hyperalgesia at the chronic painful stage. Intrathecal administration (i.t.) of ω-conotoxin MVIIA (3-300pmol/site) or Phα1ß (10-300pmol/site) reduced the mechanical hyperalgesia either at the acute or at the chronic painful stage induced by paclitaxel. When administered at the acute painful stage, ω-conotoxin MVIIA (300pmol/site, i.t.) and Phα1ß (300pmol/site, i.t.) prevented the worsening of chronic mechanical hyperalgesia. Furthermore, Phα1ß (30-300pmol/site, i.t.) elicited less adverse effects than ω-conotoxin MVIIA (10-300 pmol/site, i.t.). Taken together, our data evidence the involvement of N-type VGCC in pain sensitization induced by paclitaxel and point out the potential of Phα1ß as a safer alternative than ω-conotoxin MVIIA to treat the pain related to paclitaxel.

Asunto(s)

Analgésicos/uso terapéutico , Paclitaxel/efectos adversos , Dolor/tratamiento farmacológico , Venenos de Araña/uso terapéutico , omega-Conotoxinas/uso terapéutico , Enfermedad Aguda , Analgésicos/farmacología , Animales , Conducta Animal/efectos de los fármacos , Enfermedad Crónica , Masculino , Ratas , Ratas Wistar , Venenos de Araña/farmacología , omega-Conotoxinas/farmacología

RESUMEN

Potassium channels regulate many neuronal functions, including neuronal excitability and synaptic plasticity, contributing, by these means, to mnemonic processes. In particular, A-type K(+) currents (IA) play a key role in hippocampal synaptic plasticity. Therefore, we evaluated the effect of the peptidic toxin Tx3-1, a selective blocker of IA currents, extracted from the venom of the spider Phoneutria nigriventer, on memory of mice. Administration of Tx3-1 (i.c.v., 300 pmol/site) enhanced both short- and long-term memory consolidation of mice tested in the novel object recognition task. In comparison, 4-aminopyridine (4-AP; i.c.v., 30-300 pmol/site), a non-selective K(+) channel blocker did not alter long-term memory and caused toxic side effects such as circling, freezing and tonic-clonic seizures. Moreover, Tx3-1 (i.c.v., 10-100 pmol/site) restored memory of Aß25-35-injected mice, and exhibited a higher potency to improve memory of Aß25-35-injected mice when compared to control group. These results show the effect of the selective blocker of IA currents Tx3-1 in both short- and long-term memory retention and in memory impairment caused by Aß25-35, reinforcing the role of IA in physiological and pathological memory processes.

Asunto(s)

Memoria/efectos de los fármacos , Neuropéptidos/farmacología , Neurotoxinas/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Venenos de Araña/química , 4-Aminopiridina/efectos adversos , 4-Aminopiridina/farmacología , Animales , Masculino , Ratones , Neuropéptidos/aislamiento & purificación , Neurotoxinas/aislamiento & purificación , Bloqueadores de los Canales de Potasio/aislamiento & purificación , Canales de Potasio , Reconocimiento en Psicología , Arañas/química

RESUMEN

We investigated the effects of cholesterol removal on spontaneous and KCl-evoked synaptic vesicle recycling at the frog neuromuscular junction. Cholesterol removal by methyl-ß-cyclodextrin (MßCD) induced an increase in the frequency of miniature end-plate potentials (MEPPs) and spontaneous destaining of synaptic vesicles labeled with the styryl dye FM1-43. Treatment with MßCD also increased the size of MEPPs without causing significant changes in nicotinic receptor clustering. At the ultrastructural level, synaptic vesicles from nerve terminals treated with MßCD were larger than those from control. In addition, treatment with MßCD reduced the fusion of synaptic vesicles that are mobilized during KCl-evoked stimulation, but induced recycling of those vesicles that fuse spontaneously. We therefore suggest that MßCD might favor the release of vesicles that belong to a pool that is different from that involved in the KCl-evoked release. These results reveal fundamental differences in the synaptic vesicle cycle for spontaneous and evoked release, and suggest that deregulation of cholesterol affects synaptic vesicle biogenesis and increases transmitter packing.

Asunto(s)

Membrana Celular/fisiología , Colesterol/metabolismo , Unión Neuromuscular/fisiología , Vesículas Sinápticas/fisiología , Animales , Membrana Celular/efectos de los fármacos , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Microelectrodos , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Potenciales Postsinápticos Miniatura/fisiología , Fármacos Neuromusculares/farmacología , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/ultraestructura , Cloruro de Potasio/farmacología , Compuestos de Piridinio , Compuestos de Amonio Cuaternario , Rana catesbeiana , Receptores Nicotínicos/metabolismo , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/ultraestructura , Técnicas de Cultivo de Tejidos , beta-Ciclodextrinas/farmacología

RESUMEN

BACKGROUND: The frequency of accidental spider bites in Brazil is growing, and poisoning due to bites from the spider genus Phoneutria nigriventer is the second most frequent source of such accidents. Intense local pain is the major symptom reported after bites of P. nigriventer, although the mechanisms involved are still poorly understood. Therefore, the aim of this study was to identify the mechanisms involved in nociception triggered by the venom of Phoneutria nigriventer (PNV). METHODOLOGY/PRINCIPAL FINDINGS: Twenty microliters of PNV or PBS was injected into the mouse paw (intraplantar, i.pl.). The time spent licking the injected paw was considered indicative of the level of nociception. I.pl. injection of PNV produced spontaneous nociception, which was reduced by arachnid antivenin (ArAv), local anaesthetics, opioids, acetaminophen and dipyrone, but not indomethacin. Boiling or dialysing the venom reduced the nociception induced by the venom. PNV-induced nociception is not dependent on glutamate or histamine receptors or on mast cell degranulation, but it is mediated by the stimulation of sensory fibres that contain serotonin 4 (5-HT4) and vanilloid receptors (TRPV1). We detected a kallikrein-like kinin-generating enzyme activity in tissue treated with PNV, which also contributes to nociception. Inhibition of enzymatic activity or administration of a receptor antagonist for kinin B2 was able to inhibit the nociception induced by PNV. PNV nociception was also reduced by the blockade of tetrodotoxin-sensitive Na(+) channels, acid-sensitive ion channels (ASIC) and TRPV1 receptors. CONCLUSION/SIGNIFICANCE: Results suggest that both low- and high-molecular-weight toxins of PNV produce spontaneous nociception through direct or indirect action of kinin B2, TRPV1, 5-HT4 or ASIC receptors and voltage-dependent sodium channels present in sensory neurons but not in mast cells. Understanding the mechanisms involved in nociception caused by PNV are of interest not only for better treating poisoning by P. nigriventer but also appreciating the diversity of targets triggered by PNV toxins.

Asunto(s)

Nocicepción/fisiología , Dolor/metabolismo , Venenos de Araña/toxicidad , Animales , Masculino , Ratones , Dolor/inducido químicamente

RESUMEN

The marine snail peptide ziconotide (ω-conotoxin MVIIA) is used as an analgesic in cancer patients refractory to opioids, but may induce severe adverse effects. Animal venoms represent a rich source of novel drugs, so we investigated the analgesic effects and the side-effects of spider peptide Phα1ß in a model of cancer pain in mice with or without tolerance to morphine analgesia. Cancer pain was induced by the inoculation of melanoma B16-F10 cells into the hind paw of C57BL/6 mice. After 14 days, painful hypersensitivity was detected and Phα1ß or ω-conotoxin MVIIA (10-100 pmol/site) was intrathecally injected to evaluate the development of antinociception and side-effects in control and morphine-tolerant mice. The treatment with Phα1ß or ω-conotoxin MVIIA fully reversed cancer-related painful hypersensitivity, with long-lasting results, at effective doses 50% of 48 (32-72) or 33 (21-53) pmol/site, respectively. Phα1ß produced only mild adverse effects, whereas ω-conotoxin MVIIA induced dose-related side-effects in mice at analgesic doses (estimated toxic dose 50% of 30 pmol/site). In addition, we observed that Phα1ß was capable of controlling cancer-related pain even in mice tolerant to morphine antinociception (100% of inhibition) and was able to partially restore morphine analgesia in such animals (56 ± 5% of inhibition). In this study, Phα1ß was as efficacious as ω-conotoxin MVIIA in inducing analgesia in a model of cancer pain without producing severe adverse effects or losing efficacy in opioid-tolerant mice, indicating that Phα1ß has a good profile for the treatment of cancer pain in patients.

Asunto(s)

Analgésicos/farmacología , Melanoma Experimental/tratamiento farmacológico , Dolor/tratamiento farmacológico , Péptidos/farmacología , Venenos de Araña/farmacología , Arañas/metabolismo , Analgésicos/efectos adversos , Animales , Línea Celular Tumoral , Tolerancia a Medicamentos , Masculino , Melanoma Experimental/complicaciones , Ratones , Ratones Endogámicos C57BL , Morfina/efectos adversos , Morfina/farmacología , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Péptidos/efectos adversos , omega-Conotoxinas/efectos adversos , omega-Conotoxinas/farmacología

RESUMEN

Phα1ß toxin is a peptide purified from the venom of the armed spider Phoneutria nigriventer, with markedly antinociceptive action in models of acute and persistent pain in rats. Similarly to ziconotide, its analgesic action is related to inhibition of high voltage activated calcium channels with more selectivity for N-type. In this study we evaluated the effect of Phα1ß when injected peripherally or intrathecally in a rat model of spontaneous pain induced by capsaicin. We also investigated the effect of Phα1ß on Ca²âº transients in cultured dorsal root ganglia (DRG) neurons and HEK293 cells expressing the TRPV1 receptor. Intraplantar or intrathecal administered Phα1ß reduced both nocifensive behavior and mechanical hypersensitivity induced by capsaicin similarly to that observed with SB366791, a specific TRPV1 antagonist. Peripheral nifedipine and mibefradil did also decrease nociceptive behavior induced by intraplantar capsaicin. In contrast, ω-conotoxin MVIIA (a selective N-type Ca²âº channel blocker) was effective only when administered intrathecally. Phα1ß, MVIIA and SB366791 inhibited, with similar potency, the capsaicin-induced Ca²âº transients in DRG neurons. The simultaneous administration of Phα1ß and SB366791 inhibited the capsaicin-induced Ca²âº transients that were additive suggesting that they act through different targets. Moreover, Phα1ß did not inhibit capsaicin-activated currents in patch-clamp recordings of HEK293 cells that expressed TRPV1 receptors. Our results show that Phα1ß may be effective as a therapeutic strategy for pain and this effect is not related to the inhibition of TRPV1 receptors.

Asunto(s)

Analgésicos no Narcóticos/uso terapéutico , Modelos Animales de Enfermedad , Ganglios Espinales/efectos de los fármacos , Moduladores del Transporte de Membrana/uso terapéutico , Neuralgia/tratamiento farmacológico , Neuronas/efectos de los fármacos , Venenos de Araña/uso terapéutico , Analgésicos no Narcóticos/farmacología , Animales , Conducta Animal/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Capsaicina , Células Cultivadas , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Células HEK293 , Humanos , Proteínas de Insectos/farmacología , Proteínas de Insectos/uso terapéutico , Masculino , Moduladores del Transporte de Membrana/farmacología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuralgia/metabolismo , Neuralgia/patología , Neuronas/citología , Neuronas/metabolismo , Neuronas/patología , Péptidos/farmacología , Péptidos/uso terapéutico , Ratas , Ratas Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Venenos de Araña/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo