RESUMEN
Target-specific genome editing, using engineered nucleases zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is considered a promising approach to correct disease-causing mutations in various human diseases. In particular, hemophilia A can be considered an ideal target for gene modification via engineered nucleases because it is a monogenic disease caused by a mutation in coagulation factor VIII (FVIII), and a mild restoration of FVIII levels in plasma can prevent disease symptoms in patients with severe hemophilia A. In this study, we describe a universal genome correction strategy to restore FVIII expression in induced pluripotent stem cells (iPSCs) derived from a patient with hemophilia A by the human elongation factor 1 alpha (EF1α)-mediated normal FVIII gene expression in the FVIII locus of the patient. We used the CRISPR/Cas9-mediated homology-directed repair (HDR) system to insert the B-domain deleted from the FVIII gene with the human EF1α promoter. After gene targeting, the FVIII gene was correctly inserted into iPSC lines at a high frequency (81.81%), and these cell lines retained pluripotency after knock-in and neomycin resistance cassette removal. More importantly, we confirmed that endothelial cells from the gene-corrected iPSCs could generate functionally active FVIII protein from the inserted FVIII gene. This is the first demonstration that the FVIII locus is a suitable site for integration of the normal FVIII gene and can restore FVIII expression by the EF1α promoter in endothelial cells differentiated from the hemophilia A patient-derived gene-corrected iPSCs.
Asunto(s)
Factor VIII/metabolismo , Hemofilia A/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Cultivadas , Exones/genética , Factor VIII/genética , Edición Génica , Ingeniería Genética/métodos , Células HEK293 , Hemofilia A/genética , Humanos , Regiones Promotoras Genéticas/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismoRESUMEN
The anterior cingulate cortex (ACC) is a well-known brain area that is associated with pain perception. Previous studies reported that the ACC has a specific role in the emotional processing of pain. Chronic pain is characterized by long-term potentiation that is induced in pain pathways and contributes to hyperalgesia caused by peripheral nerve injury. The mammalian target of rapamycin (mTOR) signaling, which is involved in synaptic protein synthesis, could be a key factor controlling long-term potentiation in neuropathic pain conditions. Until now, there have been no reports that studied the role of mTOR signaling in the ACC involved in neuropathic pain. Therefore, this study was conducted to determine the relationship of mTOR signaling in the ACC and neuropathic pain. Male Sprague-Dawley rats were subjected to cannula implantation and nerve injury under pentobarbital anesthesia. Microinjection with rapamycin into the ACC was conducted under isoflurane anesthesia on postoperative day (POD) 7. A behavioral test was performed to evaluate mechanical allodynia, and optical imaging was conducted to observe the neuronal responses of the ACC to peripheral stimulation. Inhibition of mTOR by rapamycin reduced mechanical allodynia, down-regulated mTOR signaling in the ACC, and diminished the expressions of synaptic proteins which are involved in excitatory signaling, thereby reducing neuropathic pain-induced synaptic plasticity. These results suggest that inhibiting mTOR activity by rapamycin in the ACC could serve as a new strategy for treating or managing neuropathic pain before it develops into chronic pain.
Asunto(s)
Analgésicos/uso terapéutico , Giro del Cíngulo/patología , Neuralgia/tratamiento farmacológico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Analgésicos/farmacología , Animales , Estimulación Eléctrica , Giro del Cíngulo/efectos de los fármacos , Hiperalgesia/complicaciones , Hiperalgesia/patología , Masculino , Microinyecciones , Tejido Nervioso/lesiones , Tejido Nervioso/patología , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Sirolimus/uso terapéutico , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The hyperactive state of sensory neurons in the spinal cord enhances pain transmission. Spinal glial cells have also been implicated in enhanced excitability of spinal dorsal horn neurons, resulting in pain amplification and distortions. Traumatic injuries of the neural system such as spinal cord injury (SCI) induce neuronal hyperactivity and glial activation, causing maladaptive synaptic plasticity in the spinal cord. Recent studies demonstrate that SCI causes persistent glial activation with concomitant neuronal hyperactivity, thus providing the substrate for central neuropathic pain. Hyperactive sensory neurons and activated glial cells increase intracellular and extracellular glutamate, neuropeptides, adenosine triphosphates, proinflammatory cytokines, and reactive oxygen species concentrations, all of which enhance pain transmission. In addition, hyperactive sensory neurons and glial cells overexpress receptors and ion channels that maintain this enhanced pain transmission. Therefore, post-SCI neuronal-glial interactions create maladaptive synaptic circuits and activate intracellular signaling events that permanently contribute to enhanced neuropathic pain. In this review, we describe how hyperactivity of sensory neurons contributes to the maintenance of chronic neuropathic pain via neuronal-glial interactions following SCI.
Asunto(s)
Neuralgia/fisiopatología , Neuroglía/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Neuralgia/etiología , Traumatismos de la Médula Espinal/complicaciones , Sinapsis/fisiologíaRESUMEN
Spinal synaptic plasticity is believed to drive central sensitization that underlies the persistent nature of neuropathic pain. Our recent data showed that synaptic plasticity in the dorsal horn is cell type specific: intense afferent stimulation produced long-term potentiation (LTP) in excitatory spinothalamic tract neurons (STTn), whereas it produced long-term depression (LTD) in inhibitory GABAergic interneurons (GABAn). In addition, reactive oxygen species (ROS) were shown to be involved in LTP in STTn (STTn-LTP) and in LTD in GABAn (GABAn-LTD). This study examined the roles of 2 biologically important ROS--superoxide [·O2] and hydroxyl radicals [·OH]--in neuropathic mechanical hyperalgesia and cell type-specific spinal synaptic plasticity. The [·O2] donor induced stronger mechanical hyperalgesia than the [·OH] donor in naive mice. The [·O2] scavenger showed greater antihyperalgesic effect than [·OH] scavengers in the spinal nerve ligation (SNL) mouse model of neuropathic pain. In addition, the [·O2] donor induced both STTn-LTP and GABAn-LTD, but the [·OH] donor induced only GABAn-LTD. On the other hand, the [·O2] scavenger inhibited STTn-LTP and GABAn-LTD induction in naive mice and alleviated SNL-induced potentiation in STTn and depression in GABAn. The [·OH] scavenger, however, inhibited depression in GABAn but did not interfere with potentiation in STTn. These results indicate that mechanical hyperalgesia in SNL mice is the result of the combination of STTn-LTP and GABAn-LTD. Behavioral outcomes compliment electrophysiological results which suggest that [·O2] mediates both STTn-LTP and GABAn-LTD, whereas [·OH] is involved primarily in GABAn-LTD.
Asunto(s)
Neuronas GABAérgicas/fisiología , Radical Hidroxilo/metabolismo , Neuralgia/patología , Plasticidad Neuronal/fisiología , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Vías Aferentes/fisiopatología , Animales , Óxidos N-Cíclicos/farmacología , Modelos Animales de Enfermedad , Depuradores de Radicales Libres/uso terapéutico , GABAérgicos/farmacología , Neuronas GABAérgicas/efectos de los fármacos , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Hiperalgesia/patología , Hiperalgesia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuralgia/tratamiento farmacológico , Plasticidad Neuronal/efectos de los fármacos , Marcadores de Spin , Nervios Espinales/lesiones , Nervios Espinales/patología , Potenciales Sinápticos/efectos de los fármacos , Potenciales Sinápticos/fisiología , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
Remyelination via the transplantation of oligodendrocyte precursor cells (OPCs) has been considered as a strategy to improve the locomotor deficits caused by traumatic spinal cord injury (SCI). To date, enormous efforts have been made to derive OPCs from human pluripotent stem cells (hPSCs), and significant progress in the transplantation of such cells in SCI animal models has been reported. The current methods generally require a long period of time (>2 months) to obtain transplantable OPCs, which hampers their clinical utility for patients with SCI. Here we demonstrate a rapid and efficient method to differentiate hPSCs into neural progenitors that retain the features of OPCs (referred to as OPC-like cells). We used cell sorting to select A2B5-positive cells from hPSC-derived neural rosettes and cultured the selected cells in the presence of signaling cues, including sonic hedgehog, PDGF and insulin-like growth factor-1. This method robustly generated neural cells positive for platelet-derived growth factor receptor-α (PDGFRα) and NG2 (~90%) after 4 weeks of differentiation. Behavioral tests revealed that the transplantation of the OPC-like cells into the spinal cords of rats with contusive SCI at the thoracic level significantly improved hindlimb locomotor function. Electrophysiological assessment revealed enhanced neural conduction through the injury site. Histological examination showed increased numbers of axon with myelination at the injury site and graft-derived myelin formation with no evidence of tumor formation. Our method provides a cell source from hPSCs that has the potential to recover motor function following SCI.
Asunto(s)
Diferenciación Celular , Células Precursoras de Oligodendrocitos/citología , Células Precursoras de Oligodendrocitos/trasplante , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/trasplante , Traumatismos de la Médula Espinal/cirugía , Animales , Antígenos/metabolismo , Axones/metabolismo , Escala de Evaluación de la Conducta , Línea Celular , Células Cultivadas , Modelos Animales de Enfermedad , Miembro Posterior , Humanos , Masculino , Vaina de Mielina/fisiología , Proteoglicanos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factores de TiempoRESUMEN
Chronic compression of dorsal root ganglion (CCD) results in neuropathic pain. We investigated the role of spinal GABA in CCD-induced pain using rats with unilateral CCD. A stereological analysis revealed that the proportion of GABA-immunoreactive neurons to total neurons at L4/5 laminae I-III on the injured side decreased in the early phase of CCD (post-CCD week 1) and then returned to the sham-control level in the late phase (post-CCD week 18). In the early phase, the rats showed an increase in both mechanical sensitivity of the hind paw and spinal WDR neuronal excitability on the injured side, and such increase was suppressed by spinally applied muscimol (GABA-A agonist, 5 nmol) and baclofen (GABA-B agonist, 25 nmol), indicating the reduced spinal GABAergic inhibition involved. In the late phase, the CCD-induced increase in mechanical sensitivity and neuronal excitability returned to pre-CCD levels, and such recovered responses were enhanced by spinally applied bicuculline (GABA-A antagonist, 15 nmol) and CGP52432 (GABA-B antagonist, 15 nmol), indicating the regained spinal GABAergic inhibition involved. In conclusion, the alteration of spinal GABAergic inhibition following CCD and leading to a gradual reduction over time of CCD-induced mechanical hypersensitivity is most likely due to changes in GABA content in spinal GABA neurons.
Asunto(s)
Antagonistas del GABA/uso terapéutico , Ganglios Espinales/fisiopatología , Hiperalgesia/tratamiento farmacológico , Compresión de la Médula Espinal/tratamiento farmacológico , Médula Espinal/fisiopatología , Animales , Conducta Animal/efectos de los fármacos , Bencilaminas/uso terapéutico , Bicuculina/uso terapéutico , Antagonistas de Receptores de GABA-A/uso terapéutico , Antagonistas de Receptores de GABA-B/uso terapéutico , Miembro Posterior/inervación , Miembro Posterior/patología , Hiperalgesia/etiología , Hiperalgesia/fisiopatología , Masculino , Dimensión del Dolor/efectos de los fármacos , Ácidos Fosfínicos/uso terapéutico , Ratas , Ratas Sprague-Dawley , Compresión de la Médula Espinal/etiología , Compresión de la Médula Espinal/fisiopatología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Cation-specific epithelial receptors on the tongue have been well demonstrated. However, active regions along the nucleus of the solitary tract (NST) for cations Na(+), K(+), NH4(+) are still unclear, even though the best responses of NST neurons to taste stimuli vary depending on the cell. In the present study, the spatial distribution patterns of cation-specific active regions in the NST are investigated. The tongues of urethane-anesthetized Sprague-Dawley rats (n = 25) were stimulated with artificial saliva (control), 0.5 M NaCl, 1.0 M NaCl, 0.5 M KCl, and 0.3 M NH(4) Cl. Then, the three-dimensional positions of c-Fos-like-immunoreactive (cFLI) cells in the NST were generated. The spatial distributions of cFLI cells in the NST were compared among five taste stimulations. cFLI cells were observed throughout the NST, irrespective of the stimulus; however, the intermediate-medial central regions of the NST had higher numbers of cFLI cells than the other regions in all taste stimulations. Analysis of images revealed that the activated regions in the NST differed significantly depending on the cations. The intermediate-dorsal-central region and the caudal-ventral region were activated by a 0.5 M concentration of sodium, the rostral-ventral region and the intermediate-dorsal/ventral region were activated by a 1.0 M concentration of sodium, the intermediate-dorsal/ventral region was activated by potassium ions, and the rostral-ventral region and the intermediate-ventral central region were activated by ammonium ions. These results suggest that the responses of NST cells to cation salt ions are regulated differentially.
Asunto(s)
Vías Aferentes/fisiología , Cationes/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Núcleo Solitario/metabolismo , Gusto/efectos de los fármacos , Compuestos de Amonio , Análisis de Varianza , Animales , Relación Dosis-Respuesta a Droga , Masculino , Neuronas/metabolismo , Potasio , Ratas , Ratas Sprague-Dawley , Sodio , Núcleo Solitario/citología , Núcleo Solitario/efectos de los fármacos , Gusto/fisiologíaRESUMEN
We investigated the role of peripheral NMDA receptors (NMDARs) in antidromic nerve stimulation-induced tactile hypersensitivity outside the skin area innervated by stimulated nerve. Tetanic electrical stimulation (ES) of the decentralized L5 spinal nerve, which induced enlargement of plasma extravasation, resulted in tactile hypersensitivity in the L4 plantar dermatome of the hind-paw. When intraplantar (i.pl.) injection was administered into the L4 dermatome before ES, NMDAR and group-I metabotropic Glu receptor (mGluR) antagonists and group-II mGluR agonist but not AMPA/kainate receptor antagonist prevented ES-induced hypersensitivity. I.pl. injection of PKA or PKC inhibitors also prevented ES-induced hypersensitivity. When the same injections were administered after establishment of ES-induced hypersensitivity, hypersensitivity was partially reduced by NMDAR antagonist only. In naïve animals, i.pl. Glu injection into the L4 dermatome induced tactile hypersensitivity, which was blocked by NMDAR antagonist and PKA and PKC inhibitors. These results suggest that the peripheral release of Glu, induced by antidromic nerve stimulation, leads to the expansion of tactile hypersensitive skin probably via nociceptor sensitization spread due to the diffusion of Glu into the skin near the release site. In addition, intracellular PKA- and PKC-dependent mechanisms mediated mainly by NMDAR activation are involved in Glu-induced nociceptor sensitization and subsequent hypersensitivity.
Asunto(s)
Hiperalgesia/etiología , Receptores de N-Metil-D-Aspartato/fisiología , Nervio Tibial/fisiología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Maleato de Dizocilpina/farmacología , Estimulación Eléctrica , Ácido Glutámico/fisiología , Isoquinolinas/farmacología , Masculino , Proteína Quinasa C/fisiología , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Sulfonamidas/farmacologíaRESUMEN
Administration of cocaine increases locomotor activity by enhancing dopamine transmission. To explore the peripheral mechanisms underlying acupuncture treatment for drug addiction, we developed a novel mechanical acupuncture instrument (MAI) for objective mechanical stimulation. The aim of this study was to evaluate whether acupuncture inhibition of cocaine-induced locomotor activity is mediated through specific peripheral nerves, the afferents from superficial or deep tissues, or specific groups of nerve fibers. Mechanical stimulation of acupuncture point HT7 with MAI suppressed cocaine-induced locomotor activity in a stimulus time-dependent manner, which was blocked by severing the ulnar nerve or by local anesthesia. Suppression of cocaine-induced locomotor activity was elicited after HT7 stimulation at frequencies of either 50 (for Meissner corpuscles) or 200 (for Pacinian corpuscles) Hz and was not affected by block of C/Aδ-fibers in the ulnar nerve with resiniferatoxin, nor generated by direct stimulation of C/Aδ-fiber afferents with capsaicin. These findings suggest that HT7 inhibition of cocaine-induced locomotor activity is mediated by A-fiber activation of ulnar nerve that originates in superficial and deep tissue.
Asunto(s)
Terapia por Acupuntura , Vías Aferentes/efectos de los fármacos , Trastornos Relacionados con Cocaína/terapia , Puntos de Acupuntura , Vías Aferentes/fisiopatología , Animales , Conducta Animal/efectos de los fármacos , Capsaicina/farmacología , Trastornos Relacionados con Cocaína/fisiopatología , Diterpenos/farmacología , Estimulación Eléctrica , Locomoción/efectos de los fármacos , Masculino , Fibras Nerviosas Mielínicas/efectos de los fármacos , Fibras Nerviosas Amielínicas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Fármacos del Sistema Sensorial/farmacología , Nervio Cubital/efectos de los fármacosRESUMEN
The spontaneous axon regeneration of damaged neurons is limited after spinal cord injury (SCI). Recently, mesenchymal stem cell (MSC) transplantation was proposed as a potential approach for enhancing nerve regeneration that avoids the ethical issues associated with embryonic stem cell transplantation. As SCI is a complex pathological entity, the treatment of SCI requires a multipronged approach. The purpose of the present study was to investigate the functional recovery and therapeutic potential of human MSCs (hMSCs) and polymer in a spinal cord hemisection injury model. Rats were subjected to hemisection injuries and then divided into three groups. Two groups of rats underwent partial thoracic hemisection injury followed by implantation of either polymer only or polymer with hMSCs. Another hemisection-only group was used as a control. Behavioral, electrophysiological and immunohistochemical studies were performed on all rats. The functional recovery was significantly improved in the polymer with hMSC-transplanted group as compared with control at five weeks after transplantation. The results of electrophysiologic study demonstrated that the latency of somatosensory-evoked potentials (SSEPs) in the polymer with hMSC-transplanted group was significantly shorter than in the hemisection-only control group. In the results of immunohistochemical study, ß-gal-positive cells were observed in the injured and adjacent sites after hMSC transplantation. Surviving hMSCs differentiated into various cell types such as neurons, astrocytes and oligodendrocytes. These data suggest that hMSC transplantation with polymer may play an important role in functional recovery and axonal regeneration after SCI, and may be a potential therapeutic strategy for SCI.
RESUMEN
Homogeneous culture of neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) would provide a powerful tool for biomedical applications. However, previous efforts to expand mechanically dissected neural rosettes for cultivation of NPCs remain concerns regarding non-neural cell contamination. In addition, several attempts to purify NPCs using cell surface markers have not demonstrated the expansion capability of the sorted cells. In the present study, we show that polysialic acid-neural cell adhesion molecule (PSA-NCAM) is detected in neural rosette cells derived from hPSCs, and employ PSA-NCAM as a marker for purifying expandable primitive NPCs from the neural rosettes. PSA-NCAM-positive NPCs (termed hNPC(PSA-NCAM+)) were isolated from the heterogeneous cell population of mechanically harvested neural rosettes using magnetic-based cell sorting. The hNPC(PSA-NCAM+) extensively expressed neural markers such as Sox1, Sox2, Nestin, and Musashi-1 (80â¼98% of the total cells) and were propagated for multiple passages while retaining their primitive characteristics in our culture condition. Interestingly, PSA-NCAM-negative cells largely exhibited characteristics of neural crest cells. The hNPC(PSA-NCAM+) showed multipotency and responsiveness to instructive cues towards region-specific neuronal subtypes in vitro. When transplanted into the rat striatum, hNPC(PSA-NCAM+) differentiated into neurons, astrocytes, and oligodendrocytes without particular signs of tumorigenesis. Furthermore, Ki67-positive proliferating cells and non-neural lineage cells were rarely detected in the grafts of hNPC(PSA-NCAM+) compared to those of neural rosette cells. Our results suggest that PSA-NCAM-mediated cell isolation provides a highly expandable population of pure primitive NPCs from hPSCs that will lend themselves as a promising strategy for drug screening and cell therapy for neurodegenerative disorders.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Células-Madre Neurales/metabolismo , Ácidos Siálicos/metabolismo , Animales , Encéfalo/citología , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Femenino , Humanos , Separación Inmunomagnética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/trasplante , Células-Madre Neurales/citología , Células-Madre Neurales/trasplante , Neuronas/citología , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Trasplante de Células MadreRESUMEN
Our analyses of three human induced pluripotent stem cell (hiPSC) and six human embryonic stem cell (hESC) lines showed marked variability in differentiation potential into specific lineages, which often hampers their differentiation into specific cell types or cell lineages of interest. Simultaneous inhibition of both Activin/Nodal and BMP pathways with small molecules, SB431542 and dorsomorphin (DM), respectively, promoted significant neural differentiation from all human pluripotent stem cell (hPSC) lines tested, regardless of their differentiation propensity. On the contrary, differentiation into other cell lineages and the number of undifferentiated cells were significantly reduced after differentiation by the dual inhibition. These results demonstrate that innate differentiation propensity of hPSCs could be overcome, at least in part, by modulation of intracellular signaling pathways, resulting in efficient generation of desirable cell types, such as neural cells.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , Células Madre Pluripotentes Inducidas/citología , Neuronas/citología , Western Blotting , Línea Celular , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiologíaRESUMEN
In this study, we examined whether topical treatment of glutamate receptor antagonists attenuate hyperexcitability of lumbar spinal dorsal horn neurons following low thoracic hemisection spinal cord injury in rats. Four weeks after spinal hemisection, neuronal activity in response to mechanical stimuli applied on the peripheral receptive field was significantly increased in three different phenotypes of lumbar spinal dorsal horn neurons: wide dynamic range (WDR), low threshold (LT) and high threshold (HT). Topical application of MK-801 (NMDA receptor antagonist, 50 microg) significantly attenuated the activity of WDR, but not LT and HT neurons; whereas, NBQX (AMPA receptor antagonist, 0.5 and 1 microg) significantly attenuated neuronal activity in all three phenotypes of neurons (*p<0.05). However, MCPG (group I/II metabotropic glutamate receptor antagonist, 100 microg) had no effect. The present study, in the context of previous work, suggests that ionotropic glutamate receptor activation play critical roles in the maintenance of neuronal hyperexcitability and neuropathic "below-level" pain behavior following spinal hemisection injury.
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Células del Asta Posterior/fisiología , Receptores AMPA/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Masculino , Estimulación Física , Células del Asta Posterior/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Vértebras TorácicasRESUMEN
In the present study, we have examined whether spinal hemisection injury induces changes in the electrophysiological properties of thalamic ventral posteriorlateral (VPL) neurons in rats. Male Sprague-Dawley rats were subjected to unilateral spinal cord injury by transverse hemisection at the T13 spinal segment. Four weeks after the T13 spinal hemisection, the injured rats displayed robust allodynic behaviors on both sides of hindpaws compared to sham controls (P < 0.05). Extracellular recordings taken 4 weeks after the hemisection revealed that wide dynamic range (WDR) neurons had significantly increased spontaneous and brush-, pressure-, and pinch-evoked activities, respectively, on both sides of the thalamic VPL regions (P < 0.05). In contrast, low threshold (LT) neurons showed only an increase in the brush-evoked activity compared to sham controls (P < 0.05). However, afterdischarge activity in both types of neurons showed no changes. In addition, both sides of the thalamic VPL regions showed higher incidences of WDR neurons. In conclusion, our data demonstrate that spinal unilateral injury induces bilaterally increased evoked activity in thalamic VPL neurons.
Asunto(s)
Neuronas/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Núcleos Talámicos Ventrales/fisiología , Animales , Estimulación Eléctrica , Potenciales Evocados Somatosensoriales , Miembro Posterior/fisiología , Masculino , Dolor/fisiopatología , Estimulación Física , Ratas , Ratas Sprague-DawleyRESUMEN
We developed a method for the efficient generation of functional dopaminergic (DA) neurons from human embryonic stem cells (hESCs) on a large scale. The most unique feature of this method is the generation of homogeneous spherical neural masses (SNMs) from the hESC-derived neural precursors. These SNMs provide several advantages: (i) they can be passaged for a long time without losing their differentiation capability into DA neurons; (ii) they can be coaxed into DA neurons at much higher efficiency than that from previous reports (86% tyrosine hydroxylase-positive neurons/total neurons); (iii) the induction of DA neurons from SNMs only takes 14 days; and (iv) no feeder cells are required during differentiation. These advantages allowed us to obtain a large number of DA neurons within a short time period and minimized potential contamination of unwanted cells or pathogens coming from the feeder layer. The highly efficient differentiation may not only enhance the efficacy of the cell therapy but also reduce the potential tumor formation from the undifferentiated residual hESCs. In line with this effect, we have never observed any tumor formation from the transplanted animals used in our study. When grafted into a parkinsonian rat model, the hESC-derived DA neurons elicited clear behavioral recovery in three behavioral tests. In summary, our study paves the way for the large-scale generation of purer and functional DA neurons for future clinical applications.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Dopamina , Células Madre Embrionarias/citología , Neuronas/citología , Neuronas/trasplante , Animales , Trasplante de Células , Modelos Animales de Enfermedad , Humanos , Métodos , Enfermedad de Parkinson/terapia , RatasRESUMEN
In this study, we examined whether a competitive AMPA receptor antagonist, NBQX, attenuates mechanical allodynia and hyperexcitability of spinal neurons in remote, caudal regions in persistent central neuropathic pain following spinal cord injury in rats. Spinal cord injury was produced by unilateral T13 transverse spinal hemisection, from dorsal to ventral, in male Sprague Dawley rats (200-250 g). Mechanical thresholds were measured behaviorally, and the excitability of wide-dynamic-range (WDR) dorsal horn neurons in the lumbar cord (L4-L5) was measured to assess central neuropathicpain. On postoperation day (POD) 28 after spinalhemisection, mechanical thresholds were significantly decreased in both injured (ipsilateral) and noninjured (contralateral) hindpaws compared with preinjury and sham control, respectively (P < 0.05). Intrathecal administration of NBQX (0.25, 0.5, 1 mM) significantly reversed the decreased mechanical thresholds in both hindpaws, dose dependently (P < 0.05). The excitability of WDR neurons was significantly enhanced on both sides of the lumbar dorsal horn 28 days following spinal hemisection (P < 0.05). The hyperexcitability of WDR neurons was attenuated by topical administration of NBQX (0.125, 0.25, 0.5, 1 mM), dose dependently (P < 0.05). Regression analysis indicated that at least three molecules of NBQX bond per receptor complex, and are needed to achieve inhibition of WDR hyperexcitability. In conclusion, our study demonstrates that the AMPA receptor plays an important role in behaviors related to the maintenance of central neuropathic pain below the level of spinal cord injury.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/administración & dosificación , Neuralgia/tratamiento farmacológico , Células del Asta Posterior/metabolismo , Quinoxalinas/administración & dosificación , Receptores AMPA/antagonistas & inhibidores , Traumatismos de la Médula Espinal/fisiopatología , Animales , Axotomía , Miembro Posterior/inervación , Inyecciones Espinales , Masculino , Neuralgia/etiología , Neuralgia/fisiopatología , Estimulación Física , Células del Asta Posterior/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores AMPA/efectos de los fármacos , Recuperación de la Función/efectos de los fármacos , Traumatismos de la Médula Espinal/complicacionesRESUMEN
This study was performed to determine which of uninjured lumbar 4 (L4) C-afferents and injured L5 afferents was important for the generation of mechanical hypersensitivity following L5 spinal nerve ligation-and-cut (SNLC, modified spinal nerve ligation) in the rat. The mechanical hypersensitivity established following L5 SNLC was completely abolished 6 weeks after local capsaicin treatment of the sciatic nerve or L4 spinal nerve. At this stage, a substantial number of capsaicin-sensitive C-afferents were eliminated without any loss of A-afferents in the L4 spinal segment, suggesting that the capsaicin-sensitive L4 C-afferents are a major contributor to L5 SNLC-produced mechanical hypersensitivity. The peripheral terminals of L4 C-afferents are active in maintaining mechanical hypersensitivity, even long after L5 SNLC. When capsaicin-sensitive L4 C-afferents were previously eliminated, the induction of L5 SNLC-produced hypersensitivity was partly prevented. Thus, capsaicin-sensitive L4 C-afferents are crucial for the induction and maintenance of mechanical hypersensitivity in the L5 SNLC model. Also, when capsaicin-sensitive L4 C-afferents were previously eliminated, L5 SNLC still produced a partial mechanical hypersensitivity for a 1- to 2-week maintenance period with a several-day delay. This mild hypersensitivity was prevented by the previous L5 dorsal rhizotomy, implying an involvement of inputs from injured L5 afferents in the maintenance of hypersensitivity at the earlier stage. The results suggest that uninjured C-afferents, most likely C-polymodal nociceptors, are necessary for the induction and maintenance of neuropathic pain, and that afferent inputs, presumably from injured Abeta-fibers, also contribute to the maintenance at an earlier stage.
Asunto(s)
Hiperalgesia/etiología , Hiperalgesia/fisiopatología , Fibras Nerviosas Amielínicas , Neuronas Aferentes , Nervios Espinales/lesiones , Analgésicos no Narcóticos/farmacología , Animales , Capsaicina/farmacología , Ganglios Espinales/patología , Ligadura , Región Lumbosacra , Masculino , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/fisiopatología , Conducción Nerviosa , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/patología , Nociceptores/fisiopatología , Ratas , Ratas Sprague-Dawley , Rizotomía , Nervio Ciático/efectos de los fármacos , Nervios Espinales/efectos de los fármacos , Factores de TiempoRESUMEN
In this study, we investigated the role of the spinal GABAergic system in central neuropathic painlike outcomes following spinal cord injury (SCI) produced by a spinal hemitransection at T13 of the rat. After SCI, mechanical allodynia develops bilaterally in both hind paws of the rat, lasting longer than 40 days, as evidenced by an increase in paw withdrawal frequency in response to a weak von Frey filament. In naive rats, intrathecal (i.t.) administration in the lumbar spinal cord of GABAA and GABAB receptor antagonists, bicuculline (1-5 microg) and phaclofen (0.1-5 microg), respectively, causes a dose-dependent increase in the magnitude of mechanical allodynia. The SCI-induced mechanical allodynia in both hind-paws is attenuated by i.t. administration in the lumbar spinal cord of GABAA or GABAB receptor agonists, muscimol (1 microg) or baclofen (0.5 microg), respectively. In electrophysiological experiments, rats with SCI show a bilateral increase in hyperexcitability in response to natural stimuli in wide dynamic range (WDR) neurons in the lumbar spinal dorsal horn. The topical application of muscimol (1 microg) or baclofen (0.5 microg) onto the lumbar cord surface reduce the SCIinduced increased responsiveness of WDR neurons. Inhibitory effects of muscimol and baclofen on both the behavioral mechanical allodynia and the hyperexcitability in WDR neuron with SCI compared to controls, were antagonized by pre-treatment of bicuculline (10 microg) and phaclofen (5 microg), respectively. This study provides behavioral and electrophysiological evidence for the important role of the loss of spinal inhibitory tone, mediated by activation of both GABAA and GABAB receptors, in the development of central neuropathic pain following SCI.
Asunto(s)
Dolor/metabolismo , Receptores de GABA/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Médula Espinal/metabolismo , Animales , Antagonistas del GABA/farmacología , Masculino , Dolor/etiología , Dolor/prevención & control , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Ratas , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Traumatismos de la Médula Espinal/complicacionesRESUMEN
We hypothesized that neuropeptides released from the peripheral terminals of primary afferents play an important role in mechanical hyperalgesia after peripheral nerve injury. Nerve injury was performed on rats with lumbar 5 spinal nerve lesion (L5 SNL), which was preceded by L5 dorsal rhizotomy (L5 DR) to avoid the potential central effects induced by L5 SNL through the L5 dorsal root. L5 DR produced a short-lasting (<6 days) decrease in paw withdrawal threshold (PWT) while the following L5 SNL produced a persistent (>42 days) PWT decrease. When intraplantar injection to the affected hind paw was given immediately before L5 SNL, antagonists for both neurokinin 1 (NK1) and calcitonin gene-related peptide 1 (CGRP1) receptors delayed the onset of the PWT decrease for 2-4 days. However, when the same injection was given after L5 SNL, CGRP1, but not NK1, receptor antagonist reversed the decreased PWT for 105 min. It is suggested that peripherally released neuropeptides contribute to the generation of neuropathic pain, with substance P and CGRP contributing to its induction phase, but only CGRP to its maintenance phase.