RESUMO
OBJECTIVES: Spinal cord ischemia secondary to trauma or a vascular occlusive event is a threatening phenomenon. The neuroprotective properties of minocycline have been shown in several models of central nervous system diseases and after spinal cord ischemia; however, the benefit of using the drug requires additional confirmation in different animal models. Astrocytes are essential as regulators of neuronal functions and for providing nutrients. The authors hypothesized that astrocytes in the spinal cord may be an important target for minocycline action after ischemia and thus in the prevention of secondary spreading damage. DESIGN: A prospective, randomized animal study. SETTING: University research laboratory, single institution. PARTICIPANTS: Adult male Sprague Dawley rats, weighing between 400 and 450 g. INTERVENTIONS: A model of spinal cord ischemia in the rat was used for this study to determine whether a single, high-dose (10 mg/kg) of minocycline protects against damage to the neuronal cytoskeleton, both in the white and gray matter, and whether it reduces glial fibrillary acidic protein levels, which is an index for prevention of astrocyte activation during ischemia. Thirty minutes before thoracic aorta occlusion, minocycline was administered for 18 minutes using a 2 F Fogarty catheter. MEASUREMENTS AND MAIN RESULTS: Minocycline given prophylactically significantly mitigated severe hindlimb motor impairment and reduced glial fibrillary acidic protein plus astrocytosis in both the white and gray matter of the spinal cord, caudal to the occlusion. Neuronal histologic cytoarchitecture, which was severely and significantly compromised in control animals, was preserved in the minocycline-treated animals. CONCLUSIONS: This study's data imply that minocycline may attenuate reactive astrocytosis in response to injury with better neurologic outcome in a model of spinal cord ischemia in rats. The data suggest that future use of minocycline, clinically, might be advantageous in surgeries with a potential risk for paraplegia due to spinal cord ischemia.
Assuntos
Arteriopatias Oclusivas/prevenção & controle , Gliose/tratamento farmacológico , Membro Posterior/irrigação sanguínea , Minociclina/administração & dosagem , Paraplegia/tratamento farmacológico , Isquemia do Cordão Espinal/tratamento farmacológico , Animais , Arteriopatias Oclusivas/patologia , Gliose/patologia , Membro Posterior/efeitos dos fármacos , Membro Posterior/patologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/patologia , Paraplegia/patologia , Profilaxia Pré-Exposição/métodos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Isquemia do Cordão Espinal/patologiaRESUMO
BACKGROUND: Volatile anesthetics decrease Ca²âº entry through voltage-dependent Ca²âº channels. Ca influences neurotransmitter release and neuronal excitability. Because volatile anesthetics act specifically on the spinal cord to produce immobility, we examined the effect of isoflurane and the nonimmobilizers F6 (1, 2-dichlorohexafluorocyclobutane) and F8 (2, 3-dichlorooctafluorobutane) on CaV1 and CaV2 Ca²âº channels in spinal cord motor neurons and dorsal root ganglion neurons. METHODS: Using patch clamping, we compared the effects of isoflurane with those of F6 and F8 on CaV1 and CaV2 channels in isolated, cultured adult rat spinal cord motor neurons and on CaV1 and CaV2 channels in adult rat dorsal root ganglion sensory neurons. RESULTS: In spinal cord motor neurons, isoflurane, but not F6 or F8, inhibited currents through CaV1 channels. Isoflurane and at least one of the nonimmobilizers inhibited currents through CaV1 and CaV2 channels in dorsal root ganglion neurons and CaV2 in spinal cord motor neurons. CONCLUSIONS: The findings that isoflurane, but not nonimmobilizers, inhibited CaV1 Ca²âº channels in spinal cord motor neurons are consistent with the notion that spinal cord motor neurons might mediate isoflurane-induced immobility. Additional studies are required to examine whether inhibition of CaV1 calcium currents in spinal cord motor neurons is sufficient or whether actions on other channels/proteins contribute to isoflurane-induced immobility.
Assuntos
Anestésicos Inalatórios/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Clorofluorcarbonetos/farmacologia , Ciclobutanos/farmacologia , Isoflurano/farmacologia , Neurônios Motores/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Animais , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Técnicas de Patch-Clamp , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacos , Medula Espinal/citologiaRESUMO
BACKGROUND: Neuraxial local anesthetics may have neurological complications thought to be due to neurotoxicity. A primary site of action of local anesthetics is the dorsal root ganglia (DRG) neuron. Physiologic differences have been noted between young and adult DRG neurons; hence, the authors examined whether there were any differences in lidocaine-induced changes in calcium and lidocaine toxicity in neonatal and adult rat DRG neurons. METHODS: DRG neurons were cultured from postnatal day 7 (P7) and adult rats. Lidocaine-induced changes in cytosolic calcium were examined with the calcium indicator Fluo-4. Cells were incubated with varying concentrations of lidocaine and examined for viability using calcein AM and ethidium homodimer-1 staining. Live imaging of caspase-3/7 activation was performed after incubation with lidocaine. RESULTS: The mean KCl-induced calcium transient was greater in P7 neurons (P < 0.05), and lidocaine significantly inhibited KCl-induced calcium responses in both ages (P < 0.05). Frequency distribution histograms of KCl-evoked calcium increases were more heterogeneous in P7 than in adult neurons. With lidocaine, KCl-induced calcium transients in both ages became more homogeneous but remained different between the groups. Interestingly, cell viability was decreased by lidocaine in a dose-dependent manner similarly in both ages. Lidocaine treatment also activated caspase-3/7 in a dose- and time-dependent manner similarly in both ages. CONCLUSIONS: Despite physiological differences in P7 and adult DRG neurons, lidocaine cytotoxicity is similar in P7 and adult DRG neurons in vitro. Differences in lidocaine- and KCl-evoked calcium responses suggest the similarity in lidocaine cytotoxicity involves other actions in addition to lidocaine-evoked effects on cytosolic calcium responses.
Assuntos
Envelhecimento/fisiologia , Anestésicos Locais/toxicidade , Cálcio/metabolismo , Citosol/metabolismo , Gânglios Espinais/patologia , Lidocaína/toxicidade , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Microscopia de Fluorescência , Cloreto de Potássio/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
A novel role of melatonin was unveiled, using immortalized human keratinocyte cells (HaCaT) as a model system. Within a time window compatible with its circadian rhythm, melatonin at nanomolar concentration raised both the expression level of the neuronal nitric oxide synthase mRNA and the nitric oxide oxidation products, nitrite and nitrate. On the same time scale, a depression of the mitochondrial membrane potential was detected together with a decrease of the oxidative phosphorylation efficiency, compensated by glycolysis as testified by an increased production of lactate. The melatonin concentration, â¼ nmolar, inducing the bioenergetic effects and their time dependence, both suggest that the observed nitric oxide-induced mitochondrial changes might play a role in the metabolic pathways characterizing the circadian melatonin chemistry.
Assuntos
Antioxidantes/farmacologia , Metabolismo Energético/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Queratinócitos/enzimologia , Melatonina/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Trifosfato de Adenosina/metabolismo , Western Blotting , Células Cultivadas , Humanos , Queratinócitos/citologia , Ácido Láctico/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Nitritos/metabolismo , Oxirredução , Fosforilação Oxidativa , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Anesthetics are widely used to induce unconsciousness, pain relief, and immobility during surgery. It remains unclear whether the use of anesthetics has significant and long-lasting effects on synapse development and plasticity in the brain. To address this question, the authors examined the formation and elimination of dendritic spines, postsynaptic sites of excitatory synapses, in the developing mouse cortex during and after anesthetics exposure. METHODS: Transgenic mice expressing yellow fluorescence protein in layer 5 pyramidal neurons were used in this study. Mice at 1 month of age underwent ketamine-xylazine and isoflurane anesthesia over a period of hours. The elimination and formation rates of dendritic spines and filopodia, the precursors of spines, were followed over hours to days in the primary somatosensory cortex using transcranial two-photon microscopy. Four to five animals were examined under each experimental condition. Student t test and Mann-Whitney U test were used to analyze the data. RESULTS: Administration of either ketamine-xylazine or isoflurane rapidly altered dendritic filopodial dynamics but had no significant effects on spine dynamics. Ketamine-xylazine increased filopodial formation whereas isoflurane decreased filopodial elimination during 4 h of anesthesia. Both effects were transient and disappeared within a day after the animals woke up. CONCLUSION: Studies suggest that exposure to anesthetics transiently affects the dynamics of dendritic filopodia but has no significant effect on dendritic spine development and plasticity in the cortex of 1-month-old mice.
Assuntos
Anestésicos/farmacologia , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Espinhas Dendríticas/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Anestésicos Dissociativos/farmacologia , Anestésicos Inalatórios/farmacologia , Animais , Córtex Cerebral/crescimento & desenvolvimento , Espinhas Dendríticas/ultraestrutura , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Isoflurano/farmacologia , Ketamina/farmacologia , Camundongos , Camundongos Transgênicos , Pseudópodes/ultraestrutura , Células Piramidais/efeitos dos fármacos , Células Piramidais/ultraestrutura , Córtex Somatossensorial/citologia , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/crescimento & desenvolvimento , Xilazina/farmacologiaRESUMO
BACKGROUND: Chronic pain is associated with depression. In rodents, pain is often assessed by sensory hypersensitivity, which does not sufficiently measure affective responses. Low-dose ketamine has been used to treat both pain and depression, but it is not clear whether ketamine can relieve depression associated with chronic pain and whether this antidepressant effect depends on its antinociceptive properties. METHODS: The authors examined whether the spared nerve injury model of neuropathic pain induces depressive behavior in rats, using sucrose preference test and forced swim test, and tested whether a subanesthetic dose of ketamine treats spared nerve injury-induced depression. RESULTS: Spared nerve injury-treated rats, compared with control rats, showed decreased sucrose preference (0.719 ± 0.068 (mean ± SEM) vs. 0.946 ± 0.010) and enhanced immobility in the forced swim test (107.3 ± 14.6s vs. 56.2 ± 12.5s). Further, sham-operated rats demonstrated depressive behaviors in the acute postoperative period (0.790 ± 0.062 on postoperative day 2). A single subanesthetic dose of ketamine (10 mg/kg) did not alter spared nerve injury-induced hypersensitivity; however, it treated spared nerve injury-associated depression-like behaviors (0.896 ± 0.020 for ketamine vs. 0.663 ± 0.080 for control rats 1 day after administration; 0.858 ± 0.017 for ketamine vs. 0.683 ± 0.077 for control rats 5 days after administration). CONCLUSIONS: Chronic neuropathic pain leads to depression-like behaviors. The postoperative period also confers vulnerability to depression, possibly due to acute pain. Sucrose preference test and forced swim test may be used to compliment sensory tests for assessment of pain in animal studies. Low-dose ketamine can treat depression-like behaviors induced by chronic neuropathic pain.
Assuntos
Anestésicos Dissociativos/farmacologia , Antidepressivos , Depressão/etiologia , Depressão/psicologia , Ketamina/farmacologia , Neuralgia/tratamento farmacológico , Neuralgia/psicologia , Animais , Comportamento Animal/efeitos dos fármacos , Temperatura Baixa , Corticosterona/sangue , Relação Dose-Resposta a Droga , Hiperalgesia/psicologia , Masculino , Neuralgia/complicações , Medição da Dor/efeitos dos fármacos , Estimulação Física , Ratos , Ratos Sprague-Dawley , Sacarose , Natação/psicologia , Paladar/efeitos dos fármacosRESUMO
Ataxia Telangiectasia (AT) patients are particularly sensitive to oxidative-nitrosative stress. Nitric oxide (NO) controls mitochondrial respiration via the reversible inhibition of complex IV. The mitochondrial response to NO of AT lymphoblastoid cells was investigated. Cells isolated from three patients and three intrafamilial healthy controls were selected showing within each group a normal diploid karyotype and homogeneous telomere length. Different complex IV NO-inhibition patterns were induced by varying the electron flux through the respiratory chain, using exogenous cell membrane permeable electron donors. Under conditions of high electron flux the mitochondrial NO inhibition of respiration was greater in AT than in control cells (P< or =0.05). This property appears peculiar to AT, and correlates well to the higher concentration of cytochrome c detected in the AT cells. This finding is discussed on the basis of the proposed mechanism of reaction of NO with complex IV. It is suggested that the peculiar response of AT mitochondria to NO stress may be relevant to the mitochondrial metabolism of AT patients.
Assuntos
Ataxia Telangiectasia/metabolismo , Respiração Celular , Linfócitos/metabolismo , Óxido Nítrico/fisiologia , Trifosfato de Adenosina/metabolismo , Células Cultivadas , Complexo IV da Cadeia de Transporte de Elétrons/fisiologia , Glicólise , Humanos , Mitocôndrias/metabolismo , Fosforilação OxidativaRESUMO
BACKGROUND: In addition to inhibiting the excitation conduction process in peripheral nerves, local anesthetics (LAs) cause toxic effects on the central nervous system, cardiovascular system, neuromuscular junction, and cell metabolism. Different postoperative neurological complications are ascribed to the cytotoxicity of LAs, but the underlying mechanisms remain unclear. Because the clinical concentrations of LAs far exceed their EC(50) for inhibiting ion channel activity, ion channel block alone might not be sufficient to explain LA-induced cell death. However, it may contribute to cell death in combination with other actions. In this study, we compared the cytotoxicity of six frequently used LAs and will discuss the possible mechanism(s) underlying their toxicity. METHODS: In human SH-SY5Y neuroblastoma cells, viability upon exposure to six LAs (bupivacaine, ropivacaine, mepivacaine, lidocaine, procaine, and chloroprocaine) was quantitatively determined by the MTT-(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-odium bromide) colorimetry assay and qualitatively confirmed by fluorescence imaging, using the LIVE/DEAD assay reagents (calcein/AM and ethidium homodimer-1). In addition, apoptotic activity was assessed by measuring the activation of caspase-3/-7 by imaging using a fluorescent caspase inhibitor (FLICA). Furthermore, LA effects on depolarization- and carbachol-stimulated intracellular Ca(2+)-responses were also evaluated. RESULTS: 1) After a 10-min treatment, all six LAs decreased cell viability in a concentration-dependent fashion. Their killing potency was procaine < or = mepivacaine < lidocaine < chloroprocaine < ropivacaine < bupivacaine (based on LD(50), the concentration at which 50% of cells were dead). Among these six LAs, only bupivacaine and lidocaine killed all cells with increasing concentration. 2) Both bupivacaine and lidocaine activated caspase-3/-7. Caspase activation required higher levels of lidocaine than bupivacaine. Moreover, the caspase activation by bupivacaine was slower than by lidocaine. Lidocaine at high concentrations caused an immediate caspase activation, but did not cause significant caspase activation at concentrations lower than 10 mM. 3) Procaine and chloroprocaine concentration-dependently inhibited the cytosolic Ca(2+)-response evoked by depolarization or receptor-activation in a similar manner as a previous observation made with bupivacaine, ropivacaine, mepivacaine, and lidocaine. None of the LAs caused a significant increase in the basal and Ca(2+)-evoked cytosolic Ca(2+)-level. CONCLUSION: LAs can cause rapid cell death, which is primarily due to necrosis. Lidocaine and bupivacaine can trigger apoptosis with either increased time of exposure or increased concentration. These effects might be related to postoperative neurologic injury. Lidocaine, linked to the highest incidence of transient neurological symptoms, was not the most toxic LA, whereas bupivacaine, a drug causing a very low incidence of transient neurological symptoms, was the most toxic LA in our cell model. This suggests that cytotoxicity-induced nerve injury might have different mechanisms for different LAs and different target(s) other than neurons.
Assuntos
Anestésicos Locais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Carbacol/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Colorimetria , Ativação Enzimática/efeitos dos fármacos , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Agonistas Muscarínicos/farmacologia , Neurônios/ultraestrutura , Bloqueadores dos Canais de Potássio/farmacologia , Tetraetilamônio/farmacologia , Sais de Tetrazólio , Tetrodotoxina/farmacologia , TiazóisRESUMO
BACKGROUND: Calmodulin (CaM) activation by Ca(2+), its translocation to the nucleus, and stimulation of phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) (P-CREB) are necessary for new gene expression and have been linked to long-term potentiation, a process important in memory formation. Because isoflurane affects memory, we tested whether isoflurane interfered with the translocation of CaM to the neuronal cell nucleus and attenuated the formation P-CREB. METHODS: SH-SY5Y cells, a human neuroblastoma cell line, were cultured. Cells were depolarized with KCl and the phosphorylation of CREB examined by Western blotting, enzyme-linked immunosorbant assay, and immunocytochemistry. The translocation of CaM from the cytosol to the nucleus was also examined after depolarization. Cells were depolarized and lysed and fractionated by centrifugation to determine the amount of CaM translocated to the nucleus. CaM was localized by immunocytochemistry and quantitated by Western blotting and imaging. Before and during KCl depolarization, cells were exposed to isoflurane, isoflurane plus Bay K 8644, nitrendipine, and omega-conotoxin GVIa, respectively. RESULTS: P-CREB increased after KCl depolarization. The increase of P-CREB peaked at depolarization duration of 30 s. The increase in P-CREB formation was inhibited by nitrendipine, but not omega-conotoxin, and by isoflurane in a concentration-dependent fashion. Pretreatment with the L-type Ca(2+) channel agonist, Bay K 8644, attenuated the inhibition of P-CREB formation by isoflurane. CaM presence in the nucleus occurred after KCl depolarization. CaM translocation was inhibited by nitrendipine and attenuated by isoflurane. Bay K 8644 pretreatment decreased the isoflurane inhibition of CaM translocation to the nucleus. CONCLUSIONS: Our data demonstrate that isoflurane inhibits CaM translocation and P-CREB formation. This most likely occurs through isoflurane inhibition of Ca(2+)entry through L-type Ca(2+) channels.
Assuntos
Anestésicos Inalatórios/farmacologia , Calmodulina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Isoflurano/farmacologia , Neurônios/efeitos dos fármacos , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação para Baixo , Humanos , Potenciais da Membrana , Neuroblastoma/metabolismo , Neurônios/metabolismo , Nitrendipino/farmacologia , Fosforilação , Cloreto de Potássio/farmacologia , Fatores de Tempo , ômega-Conotoxina GVIA/farmacologiaRESUMO
In SH-SY5Y cells we have shown that stimulation with high extracellular K+ ([K+]e) evokes a transient increase in cytoplasmic Ca2+ ([Ca2+]cyt) (K+on) that is triggered by the opening of voltage-dependent Ca2+ channels and followed by Ca2+ -induced Ca2+ release from the endoplasmic reticulum (Xu, F., Zhang, J., Recio-Pinto, E. and Blanck, T.J., Halothane and isoflurane augment depolarization-induced cytosolic CA2+ transients and attenuate carbachol-stimulated CA2+ transients, Anesthesiology, 92 (2000) 1746-56). The removal of high-[K+]e results in a second transient increase in [Ca2+]cyt (K+off) that is independent of extracellular Ca2+ (Corrales, A., Montoya, G.J., Sutachan, J.J., Cornillez-Ty, G., Garavito-Aguilar, Z., Xu, F., Blanck, T.J. and Recio-Pinto, E., Transient increases in extracellular K+ produce two pharmacological distinct cytosolic Ca2+ transients, Brain Res., 1031 (2005) 174-184). In this study we further characterize the properties of K+off. We found that K+off was detectable at near physiological temperatures (34-36 degrees C) but, depending on the level of [K+]e, it was undetectable or highly diminished at room temperature. In contrast, K+on was increased by lowering the temperature. Extracellular Na+ -replacement with K+ did not affect K+off, indicating that K+off was not generated by osmolarity changes. Replacement of extracellular Na+ with choline+ did not affect K+off, indicating that K+off did not result from activity changes of the plasma membrane Na+/Ca2+ exchanger. Caffeine decreased K+on but not K+off. CCCP (carbonyl cyanide m-chlorophenyl), a protonophore uncoupler that decreases mitochondrial Ca2+ uptake, decreased K+on but not K+off. CGP37157, an inhibitor of the mitochondria Na+/Ca2+ exchanger, decreased K+off when added alone but not when added simultaneously with CCCP. Clonazepam had similar effects as CGP37157. These findings indicate that the generation of K+off is strongly temperature-dependent and its pharmacology is distinct from the [Ca2+]cyt changes observed previously at room temperature.
Assuntos
Cálcio/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Citosol/metabolismo , Líquido Extracelular/efeitos dos fármacos , Ionóforos/farmacologia , Cloreto de Potássio/farmacologia , Temperatura , Anticonvulsivantes/farmacologia , Cafeína/farmacologia , Linhagem Celular Tumoral , Clonazepam/análogos & derivados , Clonazepam/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Humanos , Neuroblastoma/patologia , Nitrilas , Inibidores de Fosfodiesterase/farmacologia , Tiazepinas/farmacologia , Fatores de TempoRESUMO
In DRG an increase in miR-133b-3p, miR-143-3p, and miR-1-3p correlates with the lack of development of neuropathic pain following a peripheral nerve injury. Using lentiviral (LV) vectors we found that a single injection of LV-miR-133b-3p or LV-miR-143-3p immediately after a peripheral nerve injury prevented the development of sustained mechanical and cold allodynia. Injection of LV-miR-133b-3p or LV-miR-143-3p by themselves or in combination, on day 3 post-injury produced a partial and transient reduction in mechanical allodynia and a sustained decrease in cold allodynia. Injection of LV-miR-1-3p has no effect. Co-injection of LV-miR-1a with miR-133b-3p or miR-143-3p on day 3 post-injury produced a sustained decrease in mechanical and cold allodynia. In DRG cultures, miR-133b-3p and miR-143-3p but not miR-1-3p, enhanced the depolarization-evoked cytoplasmic calcium increase. Using 3'UTR target clones containing a Gaussian luciferase reporter gene we found that with the 3'UTR-Scn2b, miR-133-3p and miR-143-3p reduced the expression while miR-1-3p enhanced the expression of the reporter gene. With the 3'UTR-TRPM8, miR-133-3p and miR-143-3p reduced the expression and miR-1-3p had no effect. With the 3'UTR-Piezo2, miR-133-3p increased the expression while miR-143-3p and miR-1-3p had no effect. LV-miR133b-3p, LV-miR-143-3p and LV-miR1a-3p reduced Scn2b-mRNA and Piezo2-mRNA. LV-miR133b-3p and LV-miR-143-3p reduced TRPM8-mRNA. LV-miR-133b-3p and LV-miR-143-3p prevent the development of chronic pain when injected immediately after the injury, but are only partially effective when injected at later times. LV-miR-1a-3p had no effect on pain, but complemented the actions of LV-miR-133b-3p or LV-miR-143-3p resulting in a sustained reversal of pain when co-injected 3â¯days following nerve injury.
Assuntos
Temperatura Baixa/efeitos adversos , Hiperalgesia/prevenção & controle , MicroRNAs/administração & dosagem , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Tato/fisiologia , Animais , Células HEK293 , Humanos , Hiperalgesia/metabolismo , Injeções Espinhais , Masculino , MicroRNAs/biossíntese , Traumatismos dos Nervos Periféricos/metabolismo , Ratos , Ratos Sprague-Dawley , Tato/efeitos dos fármacosRESUMO
Neuropathic pain involves long-lasting modifications of pain pathways that result in abnormal cortical activity. How cortical circuits are altered and contribute to the intense sensation associated with allodynia is unclear. Here we report a persistent elevation of layer V pyramidal neuron activity in the somatosensory cortex of a mouse model of neuropathic pain. This enhanced pyramidal neuron activity was caused in part by increases of synaptic activity and NMDA-receptor-dependent calcium spikes in apical tuft dendrites. Furthermore, local inhibitory interneuron networks shifted their activity in favor of pyramidal neuron hyperactivity: somatostatin-expressing and parvalbumin-expressing inhibitory neurons reduced their activity, whereas vasoactive intestinal polypeptide-expressing interneurons increased their activity. Pharmacogenetic activation of somatostatin-expressing cells reduced pyramidal neuron hyperactivity and reversed mechanical allodynia. These findings reveal cortical circuit changes that arise during the development of neuropathic pain and identify the activation of specific cortical interneurons as therapeutic targets for chronic pain treatment.
Assuntos
Interneurônios/fisiologia , Rede Nervosa/fisiopatologia , Neuralgia/fisiopatologia , Células Piramidais/fisiologia , Córtex Somatossensorial/fisiopatologia , Somatostatina/metabolismo , Potenciais de Ação/fisiologia , Animais , Dendritos/metabolismo , Camundongos Transgênicos , Neuralgia/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Córtex Somatossensorial/fisiologia , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
Fura-2 is one of the most widely used cytoplasmic Ca2+ ([Ca2+]cyt) sensors. In studies using isolated dorsal root ganglion (DRG) neurons, the loading of Fura-2 AM is often facilitated by the use of pluronic F-127. In preliminary studies, we detected that the use of pluronic F-127 appeared to be affecting the depolarization-evoked [Ca2+]cyt transient in DRG neurons. To determine whether this was the case, we conducted a systematic study. Adult rat DRG neurons were cultured, and their response to 50 mM KCl was measured in sister cultured cells (isolated on the same day) that were loaded with 5 microM Fura-2AM in the absence or in the presence of 0.02% pluronic F-127. In the absence of pluronic F-127, the KCl-evoked [Ca2+]cyt transient changed with time, being wider on day 1 than on day 2 after plating. On day 2, the KCl-evoked [Ca2+]cyt transient was wider in neurons Fura-2 loaded in the presence of pluronic F-127. These results indicate that pluronic F-127 significantly alters depolarization-evoked [Ca2+]cyt transients, which may reflect alteration in regulation of [Ca2+]cyt in neuronal cells.
Assuntos
Cálcio/metabolismo , Citoplasma/metabolismo , Neurônios/metabolismo , Poloxâmero/farmacologia , Tensoativos/farmacologia , Animais , Adesão Celular , Células Cultivadas , Citoplasma/efeitos dos fármacos , Corantes Fluorescentes , Fura-2 , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , TemperaturaRESUMO
STUDY OBJECTIVE: The objective of this study was to determine whether lavender fleur oil (LFO) aromatherapy would reduce anxiety when administered to women before undergoing breast surgery. DESIGN: This was a single-site, randomized study comparing the effect of LFO to unscented oil (UO). SETTING: The study was conducted in the preoperative holding area of the ambulatory surgery department of NYU Langone Medical Center. PATIENTS: Ninety three women, 18 years and older, scheduled for breast surgery. Women meeting inclusion/exclusion criteria were randomized to either LFO or UO aromatherapy and were blind to their assigned treatment. OUTCOME MEASURES: Subjects completed a Speilberger State Anxiety Inventory for Adults (STAI) before and after aromatherapy. Vital signs were recorded before and after aromatherapy. RESULTS: STAI-State questions were divided into positive and negative emotions for analysis. Before aromatherapy, there was no significant difference between groups by individual questions or overall average answer of either positive or negative questions. The use of both LFO and UO increased the positive STAI score totals, with the LFO group having a slightly, but statistically significant, greater increase. Both resulted in a statistically significant decrease in the negative score totals after treatment. There were no differences in vital signs between groups for either treatment. Following the conclusion of the trial LFO was analyzed and found to contain a very low content of the 2 major Lavandula angustifolia constituents. CONCLUSIONS: Both LFO and UO aromatherapy treatments lowered anxiety before surgery despite no significant changes in vital signs. LFO treatment generated a slight but statistically significant increase in positive feelings compared with UO treatment. It is probable that the beneficial effect observed was due to both aromatherapy with LFO and a placebo effect related to the added attention given to the patients.
Assuntos
Ansiedade/prevenção & controle , Aromaterapia/métodos , Mama/cirurgia , Óleos Voláteis/uso terapêutico , Óleos de Plantas/uso terapêutico , Cuidados Pré-Operatórios/métodos , Adulto , Idoso , Ansiedade/psicologia , Feminino , Humanos , Lavandula/química , Pessoa de Meia-Idade , Testes NeuropsicológicosRESUMO
Following injury, primary sensory neurons undergo changes that drive central sensitization and contribute to the maintenance of persistent hypersensitivity. NR2B expression in the dorsal root ganglia (DRG) has not been previously examined in neuropathic pain models. Here, we investigated if changes in NR2B expression within the DRG are associated with hypersensitivities that result from peripheral nerve injuries. This was done by comparing the NR2B expression in the DRG derived from two modalities of the spared nerve injury (SNI) model, since each variant produces different neuropathic pain phenotypes. Using the electronic von Frey to stimulate the spared and non-spared regions of the hindpaws, we demonstrated that sural-SNI animals develop sustained neuropathic pain in both regions while the tibial-SNI animals recover. NR2B expression was measured at Day 23 and Day 86 post-injury. At Day 23 and 86 post-injury, sural-SNI animals display strong hypersensitivity, whereas tibial-SNI animals display 50 and 100% recovery from post-injury-induced hypersensitivity, respectively. In tibial-SNI at Day 86, but not at Day 23 the perinuclear region of the neuronal somata displayed an increase in NR2B protein. This retention of NR2B protein within the perinuclear region, which will render them non-functional, correlates with the recovery observed in tibial-SNI. In sural-SNI at Day 86, DRG displayed an increase in NR2B mRNA which correlates with the development of sustained hypersensitivity in this model. The increase in NR2B mRNA was not associated with an increase in NR2B protein within the neuronal somata. The latter may result from a decrease in kinesin Kif17, since Kif17 mediates NR2B transport to the soma's plasma membrane. In both SNIs, microglia/macrophages showed a transient increase in NR2B protein detected at Day 23 but not at Day 86, which correlates with the initial post-injury induced hypersensitivity in both SNIs. In tibial-SNI at Day 86, but not at Day 23, satellite glia cells (SGCs) displayed an increase in NR2B protein. This study is the first to characterize of cell-specific changes in NR2B expression within the DRG following peripheral nerve injury. We discuss how the observed NR2B changes in DRG can contribute to the different neuropathic pain phenotypes displayed by each SNI variant.
RESUMO
OBJECTIVES: We sought to identify characteristic lipid abnormalities in patients with Barth syndrome (BTHS) and to correlate the lipid profile to phenotype and genotype. BACKGROUND: Barth syndrome typically includes cardiomyopathy, skeletal myopathy, neutropenia, growth retardation, and 3-methylglutaconic aciduria, and it is commonly associated with mutations in the tafazzin (TAZ) gene, whose products are homologous to phospholipid acyltransferases. However, clinical features of BTHS have also been found in patients with normal TAZ gene. METHODS: We analyzed molecular species of phospholipids in left and right ventricle, skeletal muscle, platelets, lymphoblasts, and fibroblasts from 19 children with BTHS (positive TAZ mutation), 6 children with BTHS-like syndromes (wild-type TAZ), 4 children with isolated cardiomyopathy (wild-type TAZ), and various controls. RESULTS: Cardiolipin, the specific lipid found only in mitochondria, was decreased in all tissues from BTHS patients, whereas concentrations of other phospholipids were normal. The molecular composition of cardiolipin was altered in all tissues from BTHS patients. The molecular compositions of phosphatidylcholine and phosphatidylethanolamine were altered in the heart. Cardiolipin abnormalities were only found in children with true BTHS, not in children with BTHS-like disease or with isolated cardiomyopathy. The degree of cardiolipin deficiency was tissue-specific but did not correlate with severity or specific phenotypic expression of BTHS. CONCLUSIONS: Abnormal cardiolipin is a specific diagnostic marker of cardiomyopathies caused by TAZ mutations. These mutations lead to alterations in the fatty acid composition of several phospholipids, supporting the idea that TAZ encodes a human acyltransferase.
Assuntos
Cardiomiopatias/metabolismo , Doenças Musculares/metabolismo , Fosfolipídeos/análise , Fatores de Transcrição , Aciltransferases , Plaquetas/química , Cardiolipinas/análise , Fibroblastos/química , Glutaratos/urina , Transtornos do Crescimento/metabolismo , Ventrículos do Coração/metabolismo , Linfócitos/química , Músculo Esquelético/metabolismo , Mutação , Miocárdio/química , Neutropenia/metabolismo , Proteínas/genética , SíndromeRESUMO
Transient increases in extracellular K+ are observed under various conditions, including repetitive neuronal firing, anoxia, ischemia and hypoglycemic coma. We studied changes in cytoplasmic Ca2+ ([Ca2+]cyt) evoked by pulses of KCl in human neuroblastoma SH-SY5Y cells and rat dorsal root ganglia (DRG) neurons at 37 degrees C. A "pulse" of KCl evoked two transient increases in [Ca2+]cyt, one upon addition of KCl (K+on) and the other upon removal of KCl (K+off). The K+on transient has been described in many cell types and is initiated by the activation of voltage-dependent Ca2+ channels followed by Ca2+-evoked Ca2+ release from intracellular Ca2+ stores. The level of KCl necessary to evoke the K+off transient depends on the type of neuron, in SH-SY5Y cells it required 100 mM KCl, in most (but not all) of dorsal root ganglia neurons it could be detected with 100-200 mM KCl and in a very few dorsal root ganglia neurons it was detectable at 20-50 mM KCl. In SH-SY5Y cells, reduction of extracellular Ca2+ inhibited the K+on more strongly than the K+off and slowed the decay of K+off. Isoflurane (1 mM) reduced the K+on)- but not the K+off-peak. However, isoflurane slowed the decay of K+off. The nonspecific cationic channel blocker La3+ (100 microM) had an effect similar to that of isoflurane. Treatment with thapsigargin (TG) at a concentration known to only deplete IP3-sensitive Ca2+ stores did not affect K+on or K+off, suggesting that Ca2+ release from the IP3-sensitive Ca2+ stores does not contribute to K+on and K+off transients and that the thapsigargin-sensitive Ca2+ ATPases do not contribute significantly to the rise or decay rates of these transients. These findings indicate that a pulse of extracellular K+ produces two distinct transient increases in [Ca2+]cyt.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Citosol/metabolismo , Neurônios/metabolismo , Potássio/metabolismo , Transdução de Sinais/fisiologia , Animais , Canais de Cálcio/metabolismo , Líquido Extracelular/química , Gânglios Espinais/citologia , Humanos , Potenciais da Membrana/fisiologia , Neuroblastoma , Neurônios/citologia , Potássio/análise , Ratos , Células Tumorais CultivadasRESUMO
BACKGROUND: Failing hearts can exhibit elements of structural and molecular "reverse remodeling" after support with a left ventricular assist device (LVAD). The present study examined LVAD-induced remodeling of cardiac mitochondria. METHODS: Left ventricular tissue from 20 failing and 21 LVAD-supported hearts, catagorized as ischemic (ICM) or dilated (DCM) cardiomyopathy and four nonfailing hearts were studied. Myocyte mitochondrial ultrastructure was assessed by high-performance liquid chromatography determination of cardiolipin, a specific lipid component of the inner membrane, and its three major molecular species: L4, L3O, and L2O2. RESULTS: Both failing and LVAD-supported hearts exhibited a reduction in cardiolipin content that was independent of the type of cardiomyopathy. However, in failing/ICM hearts, there was a 25% increase in the L4/L3O ratio and a 70% increase in the L4/L2O2 ratio, indicating a change in cardiolipin composition. These alterations were normalized by LVAD support. In sharp contrast, molecular species ratios in DCM hearts were the same as those in nonfailing hearts regardless of whether LVAD support had been used or not. CONCLUSIONS: These data demonstrate LVAD-induced reverse remodeling of myocyte cardiolipin composition in ICM but not DCM hearts.
Assuntos
Cardiolipinas/análise , Coração Auxiliar , Mitocôndrias Cardíacas/metabolismo , Adulto , Cardiolipinas/química , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Cardiomiopatias/terapia , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/terapia , Tamanho Celular , Cromatografia Líquida de Alta Pressão , Feminino , Ventrículos do Coração/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias Cardíacas/patologia , Isquemia Miocárdica/complicaçõesRESUMO
In human SH-SY5Y neuroblastoma cells, two distinct intracellular Ca2+ stores, a KCl-/caffeine-sensitive and a carbachol-/IP3-sensitive store, were demonstrated previously. In this study, responses of these two intracellular Ca2+ stores to thapsigargin were characterized. Ca2+-release from these stores was evoked either by high K+ (100 mM KCl) or by 1 mM carbachol, and changes in the intracellular Ca2+ level were monitored using Fura-2 fluorimetry. A sequential stimulation protocol (KCl-->carbachol or vice versa) allowed evaluation of the individual contribution of different Ca2+ stores to the evoked intracellular Ca2+ ([Ca2+]i)-transients and the dynamic interaction between them. Thapsigargin (0.05 nM - 20 microM) alone induced a [Ca2+]i-transient. Both the carbachol- and the KCl-evoked [Ca2+]i-transients were inhibited by thapsigargin, but with very different sensitivities. Thapsigargin inhibited the carbachol-evoked [Ca2+]i-transients with (IC50 = 0.353 nM) or without (IC50 = 0.448 nM) a KCl-prestimulation, but an additional small component, with a much lower sensitivity (IC50=4814 nM), was observed in the absence of a KCl-prestimulation. In contrast, the KCl-evoked [Ca2+]i-transients displayed only one component with a very low sensitivity to thapsigargin in both absence (IC50=3343 nM) and presence (IC50=6858 nM) of a carbachol-prestimulation. These findings suggest that the sarco-/endoplasmic reticular Ca2+ ATPases associated with the KCl-/caffeine- and carbachol-/IP3-sensitive intracellular Ca2+ stores differ from each other, either in types or in their post-translational modification. Such difference might play important role in the regulation of neuronal Ca2+ homeostasis.
Assuntos
Cálcio/metabolismo , Tapsigargina/farmacologia , Atropina/farmacologia , Carbacol/farmacologia , Linhagem Celular Tumoral , Agonistas Colinérgicos/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Fura-2/metabolismo , Humanos , Concentração Inibidora 50 , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Antagonistas Muscarínicos/farmacologia , Neuroblastoma , Potássio/farmacologia , Fatores de TempoRESUMO
We investigated the local anesthetic action on ionic membrane conductance (membrane conductance) and selectivity in membranes formed with neuronal phospholipids in the absence and presence of cholesterol. In membranes without cholesterol, 1 mM bupivacaine and ropivacaine increased the membrane conductance approximately 4.5-fold; and 5 mM lidocaine, ropivacaine and bupivacaine increased the membrane conductance by 2.7-, 2.8- and 22.2-fold, respectively. In the presence of cholesterol, 5 mM ropivacaine had no effect, lidocaine decreased the membrane conductance by 2-fold, and bupivacaine increased the membrane conductance by 17-fold. Local anesthetics did not affect the ion selectivity in membranes without cholesterol, but they all decreased the Na(+) selectivity in membranes with cholesterol. Cholesterol reduced the lidocaine- and ropivacaine-induced membrane conductance increase by eliminating or reversing the Na(+) conductance increase and by lowering the Cl(-) conductance increase. In the absence of cholesterol, 5 mM bupivacaine increased both Na(+) conductance (38-fold) and Cl(-) conductance (19-fold), while in the presence of cholesterol it only increased Cl(-) conductance (26-fold). Of the local anesthetics studied, ropivacaine was the least membrane toxic while bupivacaine was the most toxic.