RESUMEN
Here, we present evidence showing Piezo1 protein expression in the primary sensory neurons (PSNs) and non-neuronal cells of rat peripheral nervous system. Using a knockdown/knockout validated antibody, we detected Piezo1 immunoreactivity (IR) in â¼60% of PSNs of rat dorsal root ganglia (DRG) with higher IR density in the small- and medium-sized neurons. Piezo1-IR was clearly identified in DRG perineuronal glia, including satellite glial cells (SGCs) and Schwann cells; in sciatic nerve Schwann cells surrounding the axons and cutaneous afferent endings; and in skin epidermal Merkel cells and melanocytes. Neuronal and non-neuronal Piezo1 channels were functional since various cells (dissociated PSNs and SGCs from DRGs, isolated Schwann cells, and primary human melanocytes) exhibited a robust response to Piezo1 agonist Yoda1 by an increase of intracellular Ca2+ concentration ([Ca2+]i). These responses were abolished by non-specific Piezo1 antagonist GsMTx4. Immunoblots showed elevated Piezo1 protein in DRG proximal to peripheral nerve injury-induced painful neuropathy, while PSNs and SGCs from rats with neuropathic pain showed greater Yoda1-evoked elevation of [Ca2+]i and an increased frequency of cells responding to Yoda1, compared to controls. Sciatic nerve application of GsMTx4 alleviated mechanical hypersensitivity induced by Yoda1. Overall, our data show that Piezo1 is widely expressed by the neuronal and non-neuronal cells in the peripheral sensory pathways and that painful nerve injury appeared associated with activation of Piezo1 in PSNs and peripheral glial cells.
Asunto(s)
Neuralgia , Neuroglía , Animales , Humanos , Ratas , Ganglios Espinales/metabolismo , Canales Iónicos/metabolismo , Neuralgia/metabolismo , Neuroglía/metabolismo , Células de Schwann/metabolismo , Nervio Ciático/metabolismo , Células Receptoras Sensoriales/metabolismoRESUMEN
Many patients with chronic pain conditions suffer from depression. The mechanisms underlying pain-induced depression are still unclear. There are critical links of medial prefrontal cortex (mPFC) synaptic function to depression, with signaling through the endocannabinoid (eCB) system as an important contributor. We hypothesized that afferent noxious inputs after injury compromise activity-dependent eCB signaling in the mPFC, resulting in depression. Depression-like behaviors were tested in male and female rats with traumatic neuropathy [spared nerve injury (SNI)], and neuronal activity in the mPFC was monitored using the immediate early gene c-fos and in vivo electrophysiological recordings. mPFC eCB Concentrations were determined using mass spectrometry, and behavioral and electrophysiological experiments were used to evaluate the role of alterations in eCB signaling in depression after pain. SNI-induced pain induced the development of depression phenotypes in both male and female rats. Pyramidal neurons in mPFC showed increased excitability followed by reduced excitability in the onset and prolonged phases of pain, respectively. Concentrations of the eCBs, 2-arachidonoylglycerol (2-AG) in the mPFC, were elevated initially after SNI, and our results indicate that this resulted in a loss of CB1R function on GABAergic interneurons in the mPFC. These data suggest that excessive release of 2-AG as a result of noxious stimuli triggers use-dependent loss of function of eCB signaling leading to excessive GABA release in the mPFC, with the final result being behavioral depression.SIGNIFICANCE STATEMENT Pain has both somatosensory and affective components, so the complexity of mechanisms underlying chronic pain is best represented by a biopsychosocial model that includes widespread CNS dysfunction. Many patients with chronic pain conditions develop depression. The mechanism by which pain causes depression is unclear. Although manipulation of the eCB signaling system as an avenue for providing analgesia per se has not shown much promise in previous studies. An important limitation of past research has been inadequate consideration of the dynamic nature of the connection between pain and depression as they develop. Here, we show that activity-dependent synthesis of eCBs during the initial onset of persistent pain is the critical link leading to depression when pain is persistent.
Asunto(s)
Dolor Crónico/fisiopatología , Depresión/etiología , Endocannabinoides/fisiología , Neuralgia/fisiopatología , Corteza Prefrontal/fisiopatología , Animales , Mapeo Encefálico , Dolor Crónico/complicaciones , Dolor Crónico/tratamiento farmacológico , Dolor Crónico/psicología , Depresión/fisiopatología , Conducta Alimentaria , Femenino , Neuronas GABAérgicas/química , Gabapentina/uso terapéutico , Genes fos , Hiperalgesia/fisiopatología , Hiperalgesia/psicología , Interneuronas/química , Imagen por Resonancia Magnética , Masculino , Neuralgia/complicaciones , Neuralgia/tratamiento farmacológico , Neuralgia/psicología , Nocicepción/fisiología , Prueba de Campo Abierto , Corteza Prefrontal/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/análisis , Neuropatía Ciática/fisiopatología , Neuropatía Ciática/psicología , Organismos Libres de Patógenos Específicos , NataciónRESUMEN
The Sigma-1 receptor (σ1R) is highly expressed in the primary sensory neurons (PSNs) that are the critical site of initiation and maintenance of pain following peripheral nerve injury. By immunoblot and immunohistochemistry, we observed increased expression of both σ1R and σ1R-binding immunoglobulin protein (BiP) in the lumbar (L) dorsal root ganglia (DRG) ipsilateral to painful neuropathy induced by spared nerve injury (SNI). To evaluate the therapeutic potential of PSN-targeted σ1R inhibition at a selected segmental level, we designed a recombinant adeno-associated viral (AAV) vector expressing a small hairpin RNA (shRNA) against rat σ1R. Injection of this vector into the L4/L5 DRGs induced downregulation of σ1R in DRG neurons of all size groups, while expression of BiP was not affected. This was accompanied by attenuation of SNI-induced cutaneous mechanical and thermal hypersensitivity. Whole-cell current-clamp recordings of dissociated neurons showed that knockdown of σ1R suppressed neuronal excitability, suggesting that σ1R silencing attenuates pain by reversal of injury-induced neuronal hyperexcitability. These findings support a critical role of σ1R in modulating PSN nociceptive functions, and that the nerve injury-induced elevated σ1R activity in the PSNs can be a significant driver of neuropathic pain. Further understanding the role of PSN-σ1R in pain pathology may open routes to exploit this system for DRG-targeted pain therapy.
Asunto(s)
Neuralgia , Receptores sigma , Animales , Ganglios Espinales/metabolismo , Neuralgia/genética , Neuralgia/terapia , Ratas , Ratas Sprague-Dawley , Receptores sigma/genética , Receptores sigma/metabolismo , Células Receptoras Sensoriales/metabolismo , Receptor Sigma-1RESUMEN
The complexity of neuropathic pain and its associated comorbidities, including dysautonomia, make it difficult to treat. Overlap of anatomical regions and pharmacology of sympathosensory systems in the central nervous system (CNS) provide targets for novel treatment strategies. The dorsal periaqueductal gray (dPAG) is an integral component of both the descending pain modulation system and the acute stress response and is critically involved in both analgesia and the regulation of sympathetic activity. Local manipulation of the endocannabinoid signaling system holds great promise to provide analgesia without excessive adverse effects and also influence autonomic output. Inhibition of fatty acid amide hydrolase (FAAH) increases brain concentrations of the endocannabinoid N-arachidonoylethanolamine (AEA) and reduces pain-related behaviors in neuropathic pain models. Neuropathic hyperalgesia and reduced sympathetic tone are associated with increased FAAH activity in the dPAG, which suggests the hypothesis that inhibition of FAAH in the dPAG will normalize pain sensation and autonomic function in neuropathic pain. To test this hypothesis, the effects of systemic or intra-dPAG FAAH inhibition on hyperalgesia and dysautonomia developed after spared nerve injury (SNI) were assessed in male and female rats. Administration of the FAAH inhibitor PF-3845 into the dPAG reduces hyperalgesia behavior and the decrease in sympathetic tone induced by SNI. Prior administration of the CB1 receptor antagonist AM281, attenuated the antihyperalgesic and sympathetic effects of FAAH inhibition. No sex differences were identified. These data support an integrative role for AEA/CB1 receptor signaling in the dPAG contributing to the regulation of both hyperalgesia behavior and altered sympathetic tone in neuropathic pain.
Asunto(s)
Neuralgia , Disautonomías Primarias , Femenino , Masculino , Animales , Ratas , Endocannabinoides/farmacología , Hiperalgesia/tratamiento farmacológico , Sustancia Gris Periacueductal/metabolismo , Receptor Cannabinoide CB1 , Amidohidrolasas/metabolismo , Neuralgia/tratamiento farmacológico , Alcamidas Poliinsaturadas/uso terapéuticoRESUMEN
OBJECTIVES: Dorsal root ganglion (DRG) stimulation is effective in treating chronic pain. While burst stimulation has been proven to enhance the therapeutic efficacy in spinal cord stimulation, currently only a tonic stimulation waveform is clinically used in DRG stimulation. We hypothesized that burst DRG stimulation might also produce analgesic effect in a preclinical neuropathic pain model. We evaluated both the therapeutic effects of burst DRG stimulation and the possible effects of DRG stimulation upon inflammation within the DRG in a preclinical neuropathic pain model. MATERIALS AND METHODS: Rats received either a painful tibial nerve injury or sham surgery. Analgesic effects of DRG stimulation were evaluated by testing a battery of evoked pain-related behaviors as well as measuring the positive affective state associated with relief of spontaneous pain using conditioned place preference. Histological evidence for neuronal trauma or neuroinflammation was evaluated. RESULTS: All of the waveforms tested (20 Hz-tonic, 20 Hz-burst, and 40 Hz-burst) have similar analgesic effects in sensory tests and conditioned place preference. Long-term DRG stimulation for two weeks does not change DRG expression of markers for nerve injury and neuroinflammation. CONCLUSIONS: DRG stimulation using burst waveform might be also suitable for treating neuropathic pain.
Asunto(s)
Neuralgia , Traumatismos de los Nervios Periféricos , Analgésicos , Animales , Ganglios Espinales/fisiología , Neuralgia/metabolismo , Neuralgia/terapia , Traumatismos de los Nervios Periféricos/metabolismo , Ratas , Ratas Sprague-Dawley , Nervio TibialRESUMEN
OBJECTIVE: The objective of this preclinical study was to examine the responses of the brain to noxious stimulation in the presence and absence of different modes of spinal cord stimulation (SCS) using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD-fMRI). MATERIALS AND METHODS: Sprague-Dawley rats were randomized to groups based on the mode of SCS delivered which included tonic stimulation (n = 27), burst stimulation (n = 30), and burst-cycle stimulation (n = 29). The control (sham) group (n = 28) received no SCS. The SCS electrode was inserted between T10 and T12 spinal levels prior to fMRI session. The experimental protocol for fMRI acquisition consisted of an initial noxious stimulation phase, a treatment phase wherein the SCS was turned on concurrently with noxious stimulation, and a residual effect phase wherein the noxious stimulation alone was turned on. The responses were statistically analyzed through paired t-test and the results were presented as z-scores for the quantitative analysis of the fMRI data. RESULTS: The treatment with different SCS modes attenuated the BOLD brain responses to noxious hindlimb stimulation. The tonic, burst, and burst-cycle SCS treatment attenuated BOLD responses in the caudate putamen (CPu), insula (In), and secondary somatosensory cortex (S2). There was little to no corresponding change in sham control in these three regions. The burst and burst-cycle SCS demonstrated greater attenuation of BOLD signals in CPu, In, and S2 compared to tonic stimulation. CONCLUSION: The high-resolution fMRI study using a rat model demonstrated the potential of different SCS modes to act on several pain-matrix-related regions of the brain in response to noxious stimulation. The burst and burst-cycle SCS exhibited greater brain activity reduction in response to noxious hindlimb stimulation in the caudate putamen, insula, and secondary somatosensory cortex compared to tonic stimulation.
Asunto(s)
Neuralgia , Estimulación de la Médula Espinal , Animales , Encéfalo/diagnóstico por imagen , Imagen por Resonancia Magnética , Ratas , Ratas Sprague-Dawley , Médula Espinal/diagnóstico por imagenRESUMEN
The monosodium iodoacetate knee osteoarthritis model has been widely used for the evaluation of osteoarthritis pain, but the pathogenesis of associated chronic pain is not fully understood. The T-type calcium channel 3.2 (CaV3.2) is abundantly expressed in the primary sensory neurons, in which it regulates neuronal excitability at both the somata and peripheral terminals and facilitates spontaneous neurotransmitter release at the spinal terminals. In this study, we investigated the involvement of primary sensory neuron-CaV3.2 activation in monosodium iodoacetate osteoarthritis pain. Knee joint osteoarthritis pain was induced by intra-articular injection of monosodium iodoacetate (2 mg) in rats, and sensory behavior was evaluated for 35 days. At that time, knee joint structural histology, primary sensory neuron injury, and inflammatory gliosis in lumbar dorsal root ganglia, and spinal dorsal horn were examined. Primary sensory neuron-T-type calcium channel current by patch-clamp recording and CaV3.2 expression by immunohistochemistry and immunoblots were determined. In a subset of animals, pain relief by CaV3.2 inhibition after delivery of CaV3.2 inhibitor TTA-P2 into sciatic nerve was investigated. Knee injection of monosodium iodoacetate resulted in osteoarthritis histopathology, weight-bearing asymmetry, sensory hypersensitivity of the ipsilateral hindpaw, and inflammatory gliosis in the ipsilateral dorsal root ganglia, sciatic nerve, and spinal dorsal horn. Neuronal injury marker ATF-3 was extensively upregulated in primary sensory neurons, suggesting that neuronal damage was beyond merely knee-innervating primary sensory neurons. T-type current in dissociated primary sensory neurons from lumbar dorsal root ganglia of monosodium iodoacetate rats was significantly increased, and CaV3.2 protein levels in the dorsal root ganglia and spinal dorsal horn ipsilateral to monosodium iodoacetate by immunoblots were significantly increased, compared to controls. Perineural application of TTA-P2 into the ipsilateral sciatic nerve alleviated mechanical hypersensitivity and weight-bearing asymmetry in monosodium iodoacetate osteoarthritis rats. Overall, our findings demonstrate an elevated CaV3.2 expression and enhanced function of primary sensory neuron-T channels in the monosodium iodoacetate osteoarthritis pain. Further study is needed to delineate the importance of dysfunctional primary sensory neuron-CaV3.2 in osteoarthritis pain.
Asunto(s)
Benzamidas/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/metabolismo , Neuralgia/metabolismo , Osteoartritis de la Rodilla/metabolismo , Piperidinas/farmacología , Células Receptoras Sensoriales/metabolismo , Factor de Transcripción Activador 3/metabolismo , Animales , Escala de Evaluación de la Conducta , Benzamidas/uso terapéutico , Bloqueadores de los Canales de Calcio/uso terapéutico , Difosfatos/toxicidad , Modelos Animales de Enfermedad , Ganglios Espinales/metabolismo , Imidazoles/toxicidad , Inmunohistoquímica , Inflamación/metabolismo , Masculino , Nociceptores/metabolismo , Osteoartritis de la Rodilla/inducido químicamente , Osteoartritis de la Rodilla/patología , Osteoartritis de la Rodilla/fisiopatología , Piperidinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Nervio Ciático/efectos de los fármacos , Células Receptoras Sensoriales/patología , Regulación hacia ArribaRESUMEN
Transient receptor potential ankyrin 1 (TRPA1) is well documented as an important molecule in pain hypersensitivity following inflammation and nerve injury and in many other cellular biological processes. Here, we show that TRPA1 is expressed not only by sensory neurons of the dorsal root ganglia (DRG) but also in their adjacent satellite glial cells (SGCs), as well as nonmyelinating Schwann cells. TRPA1 immunoreactivity is also detected in various cutaneous structures of sensory neuronal terminals, including small and large caliber cutaneous sensory fibers and endings. The SGC-expressed TRPA1 is functional. Like DRG neurons, dissociated SGCs exhibit a robust response to the TRPA1-selective agonist allyl isothiocyanate (AITC) by an increase of intracellular Ca2+ concentration ([Ca2+]i). These responses are abolished by the TRPA1 antagonist HC030031 and are absent in SGCs and neurons from global TRPA1 null mice. SGCs and neurons harvested from DRG proximal to painful tissue inflammation induced by plantar injection of complete Freund's adjuvant show greater AITC-evoked elevation of [Ca2+]i and slower recovery compared to sham controls. Similar TRPA1 sensitization occurs in both SGCs and neurons during neuropathic pain induced by spared nerve injury. Together, these results show that functional TRPA1 is expressed by sensory ganglia SGCs, and TRPA1 function in SGCs is enhanced after both peripheral inflammation and nerve injury, and suggest that TRPA1 in SGCs may contribute to inflammatory and neuropathic pain.
Asunto(s)
Inflamación/patología , Neuralgia/metabolismo , Neuralgia/patología , Neuroglía/patología , Células Receptoras Sensoriales/patología , Canal Catiónico TRPA1/metabolismo , Animales , Tamaño de la Célula , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Isotiocianatos , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Ratas Sprague-Dawley , Células de Schwann/metabolismo , Células Receptoras Sensoriales/metabolismoRESUMEN
BACKGROUND: Dorsal root ganglion field stimulation is an analgesic neuromodulation approach in use clinically, but its mechanism is unknown as there is no validated animal model for this purpose. The authors hypothesized that ganglion stimulation is effective in reducing pain-like behaviors in preclinical chronic pain models. METHODS: The authors provided ganglion stimulation or spinal cord stimulation to rats with traumatic neuropathy (tibial nerve injury), or osteoarthritis induced by intraarticular knee monosodium iodoacetate, or without injury (naïve). Analgesia was evaluated by testing a battery of pain-related reflexive, functional, and affective behaviors. RESULTS: In rats with nerve injury, multilevel L4 and L5 ganglion stimulation decreased hypersensitivity to noxious mechanical stimulation more (area under curve, -1,447 ± 423 min × % response; n = 12) than single level ganglion stimulation at L4 ([-960 ± 251 min × % response; n = 8; P = 0.012] vs. L4 and L5), and L5 ([-676 ± 295 min × % response; n = 8; P < 0.0001] vs. L4 and L5). Spontaneous pain-like behavior, evaluated by conditioned place preference, responded to single L4 (Pretest [-93 ± 65 s] vs. Test [87 ± 82 s]; P = 0.002; n = 9), L5 (Pretest [-57 ± 36 s] vs. Test [137 ± 73 s]; P = 0.001; n = 8), and multilevel L4 and L5 (Pretest: -81 ± 68 s vs. Test: 90 ± 76 s; P = 0.003; n = 8) ganglion stimulation. In rats with osteoarthritis, multilevel L3 and L4 ganglion stimulation reduced sensitivity to knee motion more (-156 ± 28 min × points; n = 8) than L3 ([-94 ± 19 min × points in knee bend test; n = 7; P = 0.002] vs. L3 and L4) or L4 ([-71 ± 22 min × points; n = 7; P < 0.0001] vs. L3 and L4). Conditioned place preference during osteoarthritis revealed analgesic effectiveness for ganglion stimulation when delivered at L3 (Pretest [-78 ± 77 s] vs. Test [68 ± 136 s]; P = 0.048; n = 9), L4 (Pretest [-96 ± 51 s] vs. Test [73 ± 111 s]; P = 0.004; n = 9), and L3 and L4 (Pretest [-69 ± 52 s; n = 7] vs. Test [55 ± 140 s]; P = 0.022; n = 7). CONCLUSIONS: Dorsal root ganglion stimulation is effective in neuropathic and osteoarthritic preclinical rat pain models with peripheral pathologic origins, demonstrating effectiveness of ganglion stimulation in a placebo-free setting and justifying this model as a suitable platform for mechanistic studies.
Asunto(s)
Ganglios Espinales , Osteoartritis , Animales , Hiperalgesia , Dolor , Ratas , Ratas Sprague-DawleyRESUMEN
Transmission of pain signals from primary sensory neurons to secondary neurons of the central nervous system is critically dependent on presynaptic voltage-gated calcium channels. Calcium channel-binding domain 3 (CBD3), derived from the collapsin response mediator protein 2 (CRMP2), is a peptide aptamer that is effective in blocking N-type voltage-gated calcium channel (CaV2.2) activity. We previously reported that recombinant adeno-associated virus (AAV)-mediated restricted expression of CBD3 affixed to enhanced green fluorescent protein (EGFP) in primary sensory neurons prevents the development of cutaneous mechanical hypersensitivity in a rat neuropathic pain model. In this study, we tested whether this strategy is effective in treating established pain. We constructed AAV6-EGFP-CBD3A6K (AAV6-CBD3A6K) expressing a fluorescent CBD3A6K (replacing A to K at position 6 of CBD3 peptide), which is an optimized variant of the parental CBD3 peptide that is a more potent blocker of CaV2.2. Delivery of AAV6-CBD3A6K into lumbar (L) 4 and 5 dorsal root ganglia (DRG) of rats 2 weeks following tibial nerve injury (TNI) induced transgene expression in neurons of these DRG and their axonal projections, accompanied by attenuation of pain behavior. We additionally observed that the increased CaV2.2α1b immunoreactivity in the ipsilateral spinal cord dorsal horn and DRG following TNI was significantly normalized by AAV6-CBD3A6K treatment. Finally, the increased neuronal activity in the ipsilateral dorsal horn that developed after TNI was reduced by AAV6-CBD3A6K treatment. Collectively, these results indicate that DRG-restricted AAV6 delivery of CBD3A6K is an effective analgesic molecular strategy for the treatment of established neuropathic pain.
Asunto(s)
Aptámeros de Péptidos/genética , Canales de Calcio Tipo N/metabolismo , Dependovirus/genética , Terapia Genética/métodos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas del Tejido Nervioso/genética , Neuralgia/terapia , Animales , Aptámeros de Péptidos/química , Aptámeros de Péptidos/metabolismo , Bloqueadores de los Canales de Calcio/química , Dependovirus/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/fisiopatología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Dominios Proteicos , Ratas , Ratas Sprague-DawleyRESUMEN
Recombinant adeno-associated viral (AAV)-mediated therapeutic gene transfer to dorsal root ganglia (DRG) is an effective and safe tool for treating chronic pain. However, AAV with various constitutively active promoters leads to transgene expression predominantly to neurons, while glial cells are refractory to AAV transduction in the peripheral nervous system. The present study evaluated whether in vivo satellite glial cell (SGC) transduction in the DRG can be enhanced by the SGC-specific GFAP promoter and by using shH10 and shH19, which are engineered capsid variants with Müller glia-prone transduction. Titer-matched AAV6 (as control), AAVshH10, and AAVshH19, all encoding the EGFP driven by the constitutively active CMV promoter, as well as AAV6-EGFP and AAVshH10-EGFP driven by a GFAP promoter (AAV6-GFAP-EGFP and AAVshH10-GFAP-EGFP), were injected into DRG of adult male rats. Neurotropism of gene expression was determined and compared by immunohistochemistry. Results showed that injection of AAV6- and AAVshH10-GFAP-EGFP induces robust EGFP expression selectively in SGCs, whereas injection of either AAVshH10-CMV-EGFP or AAVshH19-CMV-EGFP into DRG resulted in a similar in vivo transduction profile to AAV6-CMV-EGFP, all showing efficient transduction of sensory neurons without significant transduction of glial cell populations. Coinjection of AAV6-CMV-mCherry and AAV6-GFAP-EGFP induces transgene expression in neurons and SGCs separately. This report, together with our prior studies, demonstrates that the GFAP promoter rather than capsid tropism determines selective gene expression in SGCs following intraganglionic AAV delivery in adult rats. A dual AAV system, one with GFAP promoter and the other with CMV promoter, can efficiently express transgenes selectively in neurons versus SGCs.
Asunto(s)
Dependovirus/fisiología , Proteína Ácida Fibrilar de la Glía/genética , Neuroglía/metabolismo , Transgenes , Animales , Dependovirus/genética , Ganglios/fisiología , Ganglios/virología , Ganglios Espinales/fisiología , Ganglios Espinales/virología , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Transducción Genética , TropismoRESUMEN
OBJECTIVES: Electrical stimulation of the dorsal root ganglion (DRG), referred to here as ganglionic field stimulation (GFS), is effective in reducing clinical pain, probably by interrupting transmission of afferent impulse trains on sensory neurons as they pass through the DRG. We therefore tested whether efferent impulse trains conveyed by sensory neurons, which contribute to neurogenic inflammation, may also be interrupted by GFS. MATERIALS AND METHODS: Collagen-induced arthritis, a model of clinical rheumatoid arthritis, was initiated in rats concurrently with the insertion of an electrode for GFS at the fourth lumbar DRG. Continuous GFS (20 Hz pulse rate, current at 80% of the motor threshold) was initiated 6 days later and continued for 14 days. Plantar pain sensitivity, ankle arthritis score, and dimensions of the foot and ankle were determined one hour after termination of GFS. RESULTS: The foot/ankle contralateral to GFS developed hypersensitivity to threshold and noxious mechanical stimulation, swelling, and high arthritis score, all of which were normalized in the foot/ankle ipsilateral with GFS. Histology showed GFS limited joint destruction. Electrophysiological recording showed GFS can block efferent impulse trains. CONCLUSIONS: Our findings show that GFS can reduce neurogenic inflammation and the resulting joint damage in a model of rheumatoid arthritis, probably by blocking the transit of impulse trains through the DRG. GFS may have clinical utility in limiting joint destruction in inflammatory arthritis such as rheumatoid arthritis.
Asunto(s)
Artritis Reumatoide/patología , Artritis Reumatoide/fisiopatología , Estimulación Eléctrica , Ganglios Espinales/fisiología , Animales , Artritis Experimental/patología , Artritis Experimental/fisiopatología , Modelos Animales de Enfermedad , Inflamación/fisiopatología , Masculino , Ratas , Ratas WistarRESUMEN
OBJECTIVE: The mechanisms of dorsal root ganglion (DRG) stimulation for chronic pain remain unclear. The objective of this work was to explore the neurophysiological effects of DRG stimulation using computational modeling. METHODS: Electrical fields produced during DRG stimulation were calculated with finite element models, and were coupled to a validated biophysical model of a C-type primary sensory neuron. Intrinsic neuronal activity was introduced as a 4 Hz afferent signal or somatic ectopic firing. The transmembrane potential was measured along the neuron to determine the effect of stimulation on intrinsic activity across stimulation parameters, cell location/orientation, and membrane properties. RESULTS: The model was validated by showing close correspondence in action potential (AP) characteristics and firing patterns when compared to experimental measurements. Subsequently, the model output demonstrated that T-junction filtering was amplified with DRG stimulation, thereby blocking afferent signaling, with cathodic stimulation at amplitudes of 2.8-5.5 × stimulation threshold and frequencies above 2 Hz. This amplified filtering was dependent on the presence of calcium and calcium-dependent small-conductance potassium channels, which produced a hyperpolarization offset in the soma, stem, and T-junction with repeated somatic APs during stimulation. Additionally, DRG stimulation suppressed somatic ectopic activity by hyperpolarizing the soma with cathodic or anodic stimulation at amplitudes of 3-11 × threshold and frequencies above 2 Hz. These effects were dependent on the stem axon being relatively close to and oriented toward a stimulating contact. CONCLUSIONS: These results align with the working hypotheses on the mechanisms of DRG stimulation, and indicate the importance of stimulation amplitude, polarity, and cell location/orientation on neuronal responses.
Asunto(s)
Simulación por Computador , Terapia por Estimulación Eléctrica , Ganglios Espinales/fisiología , Neuralgia/fisiopatología , Neuronas/fisiología , Animales , Análisis de Elementos Finitos , HumanosRESUMEN
Targeted dorsal root ganglion (DRG) electrical stimulation (i.e. ganglionic field stimulation - GFS) is an emerging therapeutic approach to alleviate chronic pain. Here we describe blood oxygen-level dependent (BOLD) functional magnetic resonance imaging (fMRI) responses to noxious hind-limb stimulation in a rat model that replicates clinical GFS using an electrode implanted adjacent to the DRG. Acute noxious sensory stimulation in the absence of GFS caused robust BOLD fMRI response in brain regions previously associated with sensory and pain-related response, such as primary/secondary somatosensory cortex, retrosplenial granular cortex, thalamus, caudate putamen, nucleus accumbens, globus pallidus, and amygdala. These regions differentially demonstrated either positive or negative correlation to the acute noxious stimulation paradigm, in agreement with previous rat fMRI studies. Therapeutic-level GFS significantly attenuated the global BOLD response to noxious stimulation in these regions. This BOLD signal attenuation persisted for 20minutes after the GFS was discontinued. Control experiments in sham-operated animals showed that the attenuation was not due to the effect of repetitive noxious stimulation. Additional control experiments also revealed minimal BOLD fMRI response to GFS at therapeutic intensity when presented in a standard block-design paradigm. High intensity GFS produced a BOLD signal map similar to acute noxious stimulation when presented in a block-design. These findings are the first to identify the specific brain region responses to neuromodulation at the DRG level and suggest possible mechanisms for GFS-induced treatment of chronic pain.
Asunto(s)
Analgesia , Terapia por Estimulación Eléctrica/métodos , Ganglios Espinales/fisiología , Oxígeno/sangre , Dolor/fisiopatología , Animales , Electrodos Implantados , Pie , Miembro Posterior , Imagen por Resonancia Magnética , Masculino , Manejo del Dolor , Ratas , Ratas Sprague-Dawley , Estimulación de la Médula EspinalRESUMEN
Nerve damage can induce a heightened pain response to noxious stimulation, which is termed hyperalgesia. Pain itself acts as a stressor, initiating autonomic and sensory effects through the dorsal periaqueductal gray (dPAG) to induce both sympathoexcitation and analgesia, which prior studies have shown to be affected by endocannabinoid signaling. The present study addressed the hypothesis that neuropathic pain disrupts autonomic and analgesic regulation by endocannabinoid signaling in the dPAG. Endocannabinoid contents, transcript levels of endocannabinoid signaling components, and catabolic enzyme activity were analyzed in the dPAG of rats at 21 days after painful nerve injury. The responses to two nerve injury models were similar, with two-thirds of animals developing hyperalgesia that was maintained throughout the postinjury period, whereas no sustained change in sensory function was observed in the remaining rats. Anandamide content was lower in the dPAG of rats that developed sustained hyperalgesia, and activity of the catabolic enzyme fatty acid amide hydrolase (FAAH) was higher. Intensity of hyperalgesia was correlated to transcript levels of FAAH and negatively correlated to heart rate and sympathovagal balance. These data suggest that maladaptive endocannabinoid signaling in the dPAG after nerve injury could contribute to chronic neuropathic pain and associated autonomic dysregulation. This study demonstrates that reduced anandamide content and upregulation of FAAH in the dPAG are associated with hyperalgesia and reduced heart rate sustained weeks after nerve injury. These data provide support for the evaluation of FAAH inhibitors for the treatment of chronic neuropathic pain.
Asunto(s)
Amidohidrolasas/metabolismo , Endocannabinoides/metabolismo , Frecuencia Cardíaca , Neuralgia/fisiopatología , Sustancia Gris Periacueductal/enzimología , Animales , Regulación Enzimológica de la Expresión Génica , Masculino , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
Injury-induced neuropathic pain remains a serious clinical problem. Recent studies indicate that bone marrow stromal cells (BMSCs) effectively attenuate chronic neuropathic pain in animal models. Here, we examined the therapeutic effect of intrathecal administration of BMSCs isolated from young (1-month-old) rats on pain hypersensitivity induced by tibial nerve injury. Cerebrospinal fluid (CSF) was collected and analyzed to examine the effect of BMSC administration on the expression of 67 soluble factors in CSF. A sustained remission in injury-induced mechanical hyperalgesia was observed in BMSC-treated rats but not in control animals. Engrafted BMSCs were observed in spinal cords and dorsal root ganglia at 5 weeks after cell injection. Injury significantly decreased the levels of six soluble factors in CSF: intercellular adhesion molecule 1 (ICAM-1), interleukin-1ß (IL-1ß), IL-10, hepatocyte growth factor (HGF), Nope protein, and neurogenic locus notch homolog protein 1 (Notch-1). Intrathecal BMSCs significantly attenuated the injury-induced reduction of ICAM-1, IL-1ß, HGF, IL-10, and Nope. This study adds to evidence supporting the use of intrathecal BMSCs in pain control and shows that this effect is accompanied by the reversal of injury-induced reduction of multiple CSF soluble factors. Our findings suggest that these soluble factors may be potential targets for treating chronic pain.
Asunto(s)
Citocinas/líquido cefalorraquídeo , Ganglios Espinales , Hiperalgesia/líquido cefalorraquídeo , Hiperalgesia/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Neuralgia/líquido cefalorraquídeo , Neuralgia/terapia , Médula Espinal , Animales , Modelos Animales de Enfermedad , Ganglios Espinales/citología , Masculino , Ratas , Ratas Sprague-Dawley , Médula Espinal/citologíaRESUMEN
BACKGROUND: Gαi-interacting protein (GINIP) is expressed specifically in dorsal root ganglion (DRG) neurons and functions in modulation of peripheral gamma-aminobutyric acid B receptor (GBR). Genetic deletion of GINIP leads to impaired responsiveness to GBR agonist-mediated analgesia in rodent. It is, however, not defined whether nerve injury changes GINIP expression. RESULTS: Immunolabeling with validated antibody revealed GINIP expression in ~40% of total lumbar DRG neurons in normal adult rats. GINIP immunoreactivity was detected in ~80% of IB4-positive (nonpeptidergic) and ~30% of CGRP-positive (peptidergic) neurons. GINIP immunoreactivity in the spinal cord dorsal horn was colabeled with IB4 and partially with CGRP. In addition, GINIP was expressed in DRG neurons immunopositive for GBR1, GBR2, Gαi(s), and Gαo and was also extensively colabeled with multiple nociceptive neuronal markers, including Trpv1, NaV1.7, CaV2.2α1b, CaV3.2α1b, TrkA, and Trek2. Peripheral nerve injury by L5 spinal nerve ligation significantly decreased the proportion of GINIP immunoreactivity-positive neurons from 40 ± 8.4% to 0.8 ± 0.1% (p < 0.01, mean ± SD, four weeks after spinal nerve ligation) and the total GINIP protein to 1.3% ± 0.04% of its basal level (p < 0.01, n = 6 animals in each group, two weeks after spinal nerve ligation) in the ipsilateral L5 DRGs. CONCLUSION: Our results show that GINIP is predominantly expressed by small nonpeptidergic nociceptive neurons and that nerve injury triggers loss of GINIP expression. Signal transduction roles of GINIP may be diverse as it colabeled with various subgroups of nociceptive neurons. Future studies may investigate details of the signaling mechanism engaged by GINIP, as well as the pathophysiological significance of lost expression of GINIP in neuropathic pain.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuralgia/metabolismo , Neuralgia/patología , Neuronas/metabolismo , Neuronas/patología , Nociceptores/metabolismo , Traumatismos de los Nervios Periféricos/patología , Animales , Axotomía , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Masculino , Traumatismos de los Nervios Periféricos/metabolismo , Terminales Presinápticos/metabolismo , Transporte de Proteínas , Ratas Sprague-DawleyRESUMEN
The present study was undertaken to examine whether variations in endocannabinoid signaling in the dorsal periaqueductal gray (dPAG) are associated with baseline autonomic nerve activity, heart rate, and blood pressure. Blood pressure was recorded telemetrically in rats, and heart rate and power spectral analysis of heart rate variability were determined. Natural variations from animal to animal provided a range of baseline values for analysis. Transcript levels of endocannabinoid signaling components in the dPAG were analyzed, and endocannabinoid content and catabolic enzyme activity were measured. Higher baseline heart rate was associated with increased anandamide content and with decreased activity of the anandamide-hydrolyzing enzyme, fatty acid amide hydrolase (FAAH), and it was negatively correlated with transcript levels of both FAAH and monoacylglycerol lipase (MAGL), a catabolic enzyme for 2-arachidonoylglycerol (2-AG). Autonomic tone and heart rate, but not blood pressure, were correlated to levels of FAAH mRNA. In accordance with these data, exogenous anandamide in the dPAG of anesthetized rats increased heart rate. These data indicate that in the dPAG, anandamide, a FAAH-regulated lipid, contributes to regulation of baseline heart rate through influences on autonomic outflow.
Asunto(s)
Sistema Nervioso Autónomo/fisiología , Presión Sanguínea/fisiología , Endocannabinoides/metabolismo , Retroalimentación Fisiológica/fisiología , Frecuencia Cardíaca/fisiología , Sustancia Gris Periacueductal/metabolismo , Animales , Masculino , Ratas , Ratas Sprague-Dawley , Descanso/fisiologíaRESUMEN
BACKGROUND: Cell-based therapy may hold promise for treatment of chronic pain. Mesenchymal stem cells (MSCs) are readily available and robust, and their secretion of therapeutic peptides can be enhanced by genetically engineering. We explored the analgesic potential of transplanting bone marrow-derived MSCs that have been transduced with lentivectors. To optimize efficacy and safety, primary sensory neurons were targeted by MSC injection into the dorsal root ganglia (DRGs). RESULTS: MSCs were transduced using lentivectors to express enhanced green fluorescent protein (EGFP) or to co-express the analgesic peptide glial cell line-derived neurotrophic factor (GDNF) and EGFP by a viral 2A bicistronic transgene cassette. Engineered MSCs were injected into the 4(th) lumbar (L4) and L5 DRGs of adult allogeneic rats to evaluate survival in the DRGs. MSCs were detected by immunofluorescence staining up to 2-3 weeks after injection, distributed in the extracellular matrix space without disrupting satellite glial cell apposition to sensory neurons, suggesting well-tolerated integration of engrafted MSCs into DRG tissue. To examine their potential for inhibiting development of neuropathic pain, MSCs were injected into the L4 and L5 DRGs ipsilateral to a spinal nerve ligation injury. Animals injected with GDNF-engineered MSCs showed moderate but significant reduction in mechanical allodynia and hyperalgesia compared to controls implanted with MSCs expressing EGFP alone. We also observed diminished long-term survival of allografted MSCs at 3 weeks, and the development of a highly-proliferating population of MSCs in 12% of DRGs after transplantation. CONCLUSIONS: These data indicate that genetically modified MSCs secreting analgesic peptides could potentially be developed as a novel DRG-targeted cell therapy for treating neuropathic pain. However, further work is needed to address the challenges of MSC survival and excess proliferation, possibly with trials of autologous MSCs, evaluation of clonally selected populations of MSCs, and investigation of regulation of MSC proliferation.
Asunto(s)
Analgesia , Ganglios Espinales/trasplante , Células Madre Mesenquimatosas/citología , Neuralgia/terapia , Neuronas Aferentes/citología , Analgesia/métodos , Animales , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Ganglios Espinales/metabolismo , Masculino , Trasplante de Células Madre Mesenquimatosas , Neuralgia/genética , Neuralgia/metabolismo , Manejo del Dolor/métodos , Ratas Sprague-Dawley , Nervios Espinales/metabolismoRESUMEN
Thrombospondin-4 (TSP4) belongs to a family of large, oligomeric extracellular matrix glycoproteins that mediate interactions between cells and interactions of cells with underlying matrix components. Recent evidence shows that TSP4 might contribute to the generation of neuropathic pain. However, there has been no systematic examination of TSP4 expression in the dorsal root ganglia (DRG) after injury. This study, therefore, investigates whether TSP4 protein level is changed in DRG after injury following spinal nerve ligation (SNL) and spared nerve injury in rats by performing Western blotting, immunohistochemistry, and immunocytochemistry. After nerve ligation, TSP4 protein level is upregulated in the axotomized somata of the fifth lumbar (L5) DRG. There is substantial additional TSP4 in the nonneuronal compartment of the L5 DRG that does not costain for markers of satellite glia, microglia, or Schwann cells and appears to be in the interstitial space. Evidence of intracellular overexpression of TSP4 persists in neurons dissociated from the L5 DRG after SNL. These findings indicate that, following peripheral nerve injury, TSP4 protein expression is elevated in the cytoplasm of axotomized sensory neurons and in the surrounding interstitial space.