RESUMO
This study reports that targeting intrinsically disordered regions of the voltage-gated sodium channel 1.7 (NaV1.7) protein facilitates discovery of sodium channel inhibitory peptide aptamers (NaViPA) for adeno-associated virus-mediated (AAV-mediated), sensory neuron-specific analgesia. A multipronged inhibition of INa1.7, INa1.6, INa1.3, and INa1.1 - but not INa1.5 and INa1.8 - was found for a prototype and named NaViPA1, which was derived from the NaV1.7 intracellular loop 1, and is conserved among the TTXs NaV subtypes. NaViPA1 expression in primary sensory neurons (PSNs) of dorsal root ganglia (DRG) produced significant inhibition of TTXs INa but not TTXr INa. DRG injection of AAV6-encoded NaViPA1 significantly attenuated evoked and spontaneous pain behaviors in both male and female rats with neuropathic pain induced by tibial nerve injury (TNI). Whole-cell current clamp of the PSNs showed that NaViPA1 expression normalized PSN excitability in TNI rats, suggesting that NaViPA1 attenuated pain by reversal of injury-induced neuronal hypersensitivity. IHC revealed efficient NaViPA1 expression restricted in PSNs and their central and peripheral terminals, indicating PSN-restricted AAV biodistribution. Inhibition of sodium channels by NaViPA1 was replicated in the human iPSC-derived sensory neurons. These results summate that NaViPA1 is a promising analgesic lead that, combined with AAV-mediated PSN-specific block of multiple TTXs NaVs, has potential as a peripheral nerve-restricted analgesic therapeutic.
Assuntos
Dependovirus , Canal de Sódio Disparado por Voltagem NAV1.7 , Células Receptoras Sensoriais , Animais , Ratos , Dependovirus/genética , Células Receptoras Sensoriais/metabolismo , Masculino , Humanos , Feminino , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Gânglios Espinais/metabolismo , Ratos Sprague-Dawley , Neuralgia/metabolismo , Neuralgia/genética , Neuralgia/tratamento farmacológico , AnalgesiaRESUMO
Patients with Fabry disease suffer from chronic debilitating pain and peripheral sensory neuropathy with minimal treatment options, but the cellular drivers of this pain are unknown. Here, we propose a mechanism we believe to be novel in which altered signaling between Schwann cells and sensory neurons underlies the peripheral sensory nerve dysfunction we observed in a genetic rat model of Fabry disease. Using in vivo and in vitro electrophysiological recordings, we demonstrated that Fabry rat sensory neurons exhibited pronounced hyperexcitability. Schwann cells probably contributed to this finding because application of mediators released from cultured Fabry Schwann cells induced spontaneous activity and hyperexcitability in naive sensory neurons. We examined putative algogenic mediators using proteomic analysis and found that Fabry Schwann cells released elevated levels of the protein p11 (S100A10), which induced sensory neuron hyperexcitability. Removal of p11 from Fabry Schwann cell media caused hyperpolarization of neuronal resting membrane potentials, indicating that p11 may contribute to the excessive neuronal excitability caused by Fabry Schwann cells. These findings demonstrate that sensory neurons from rats with Fabry disease exhibit hyperactivity caused in part by Schwann cell release of the protein p11.
Assuntos
Modelos Animais de Doenças , Doença de Fabry , Células de Schwann , Células Receptoras Sensoriais , Animais , Masculino , Ratos , Células Cultivadas , Doença de Fabry/metabolismo , Doença de Fabry/fisiopatologia , Proteômica , Células de Schwann/metabolismo , Células Receptoras Sensoriais/metabolismo , Feminino , Ratos Sprague-DawleyRESUMO
BACKGROUND: Peripheral and central nociceptive sensitization is a critical pathogenetic component in osteoarthritis (OA) chronic pain. T-type calcium channel 3.2 (CaV3.2) regulates neuronal excitability and plays important roles in pain processing. We previously identified that enhanced T-type/CaV3.2 activity in the primary sensory neurons (PSNs) of dorsal root ganglia (DRG) is associated with neuropathic pain behavior in a rat model of monosodium iodoacetate (MIA)-induced knee OA. PSN-specific T-type/CaV3.2 may therefore represent an important mediator in OA painful neuropathy. Here, we test the hypothesis that the T-type/CaV3.2 channels in PSNs can be rationally targeted for pain relief in MIA-OA. METHODS: MIA model of knee OA was induced in male and female rats by a single injection of 2 mg MIA into intra-knee articular cavity. Two weeks after induction of knee MIA-OA pain, recombinant adeno-associated viruses (AAV)-encoding potent CaV3.2 inhibitory peptide aptamer 2 (CaV3.2iPA2) that have been characterized in our previous study were delivered into the ipsilateral lumbar 4/5 DRG. Effectiveness of DRG-CaV3.2iPA2 treatment on evoked (mechanical and thermal) and spontaneous (conditioned place preference) pain behavior, as well as weight-bearing asymmetry measured by Incapacitance tester, in the arthritic limbs of MIA rats were evaluated. AAV-mediated transgene expression in DRG was determined by immunohistochemistry. RESULTS: AAV-mediated expression of CaV3.2iPA2 selective in the DRG-PSNs produced significant and comparable mitigations of evoked and spontaneous pain behavior, as well as normalization of weight-bearing asymmetry in both male and female MIA-OA rats. Analgesia of DRG-AAV-CaV3.2iPA1, another potent CaV3.2 inhibitory peptide, was also observed. Whole-cell current-clamp recordings showed that AAV-mediated CaV3.2iPA2 expression normalized hyperexcitability of the PSNs dissociated from the DRG of MIA animals, suggesting that CaV3.2iPA2 attenuated pain behavior by reversing MIA-induced neuronal hyperexcitability. CONCLUSIONS: Together, our results add therapeutic support that T-type/CaV3.2 in primary sensory pathways contributes to MIA-OA pain pathogenesis and that CaV3.2iPAs are promising analgesic leads that, combined with AAV-targeted delivery in anatomically segmental sensory ganglia, have the potential for further development as a peripheral selective T-type/CaV3.2-targeting strategy in mitigating chronic MIA-OA pain behavior. Validation of the therapeutic potential of this strategy in other OA models may be valuable in future study.
Assuntos
Neuralgia , Osteoartrite do Joelho , Animais , Modelos Animais de Doenças , Feminino , Gânglios Espinais/metabolismo , Ácido Iodoacético/toxicidade , Masculino , Osteoartrite do Joelho/metabolismo , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismoRESUMO
ABSTRACT: Ample data support a prominent role of peripheral T-type calcium channels 3.2 (Ca V 3.2) in generating pain states. Development of primary sensory neuron-specific inhibitors of Ca V 3.2 channels is an opportunity for achieving effective analgesic therapeutics, but success has been elusive. Small peptides, especially those derived from natural proteins as inhibitory peptide aptamers (iPAs), can produce highly effective and selective blockade of specific nociceptive molecular pathways to reduce pain with minimal off-target effects. In this study, we report the engineering of the potent and selective iPAs of Ca V 3.2 from the intrinsically disordered regions (IDRs) of Ca V 3.2 intracellular segments. Using established prediction algorithms, we localized the IDRs in Ca V 3.2 protein and identified several Ca V 3.2iPA candidates that significantly reduced Ca V 3.2 current in HEK293 cells stably expressing human wide-type Ca V 3.2. Two prototype Ca V 3.2iPAs (iPA1 and iPA2) derived from the IDRs of Ca V 3.2 intracellular loops 2 and 3, respectively, were expressed selectively in the primary sensory neurons of dorsal root ganglia in vivo using recombinant adeno-associated virus (AAV), which produced sustained inhibition of calcium current conducted by Ca V 3.2/T-type channels and significantly attenuated both evoked and spontaneous pain behavior in rats with neuropathic pain after tibial nerve injury. Recordings from dissociated sensory neurons showed that AAV-mediated Ca V 3.2iPA expression suppressed neuronal excitability, suggesting that Ca V 3.2iPA treatment attenuated pain by reversal of injury-induced neuronal hypersensitivity. Collectively, our results indicate that Ca V 3.2iPAs are promising analgesic leads that, combined with AAV-mediated delivery in anatomically targeted sensory ganglia, have the potential to be a selective peripheral Ca V 3.2-targeting strategy for clinical treatment of pain.
Assuntos
Analgesia , Aptâmeros de Peptídeos , Canais de Cálcio Tipo T , Neuralgia , Ratos , Humanos , Animais , Dependovirus , Manejo da Dor , Células HEK293 , Ratos Sprague-Dawley , Gânglios Espinais/metabolismo , Neuralgia/tratamento farmacológico , Células Receptoras Sensoriais/metabolismo , Analgésicos/uso terapêutico , Aptâmeros de Peptídeos/farmacologia , Peptídeos/uso terapêutico , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo T/metabolismoRESUMO
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.
Assuntos
Neuralgia , Receptores sigma , Animais , Gânglios Espinais/metabolismo , Neuralgia/genética , Neuralgia/terapia , Ratos , Ratos Sprague-Dawley , Receptores sigma/genética , Receptores sigma/metabolismo , Células Receptoras Sensoriais/metabolismo , Receptor Sigma-1RESUMO
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.
Assuntos
Neuralgia , Traumatismos dos Nervos Periféricos , Analgésicos , Animais , Gânglios Espinais/fisiologia , Neuralgia/metabolismo , Neuralgia/terapia , Traumatismos dos Nervos Periféricos/metabolismo , Ratos , Ratos Sprague-Dawley , Nervo TibialRESUMO
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.
Assuntos
Dor Crônica/fisiopatologia , Depressão/etiologia , Endocanabinoides/fisiologia , Neuralgia/fisiopatologia , Córtex Pré-Frontal/fisiopatologia , Animais , Mapeamento Encefálico , Dor Crônica/complicações , Dor Crônica/tratamento farmacológico , Dor Crônica/psicologia , Depressão/fisiopatologia , Comportamento Alimentar , Feminino , Neurônios GABAérgicos/química , Gabapentina/uso terapêutico , Genes fos , Hiperalgesia/fisiopatologia , Hiperalgesia/psicologia , Interneurônios/química , Imageamento por Ressonância Magnética , Masculino , Neuralgia/complicações , Neuralgia/tratamento farmacológico , Neuralgia/psicologia , Nociceptividade/fisiologia , Teste de Campo Aberto , Córtex Pré-Frontal/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/análise , Neuropatia Ciática/fisiopatologia , Neuropatia Ciática/psicologia , Organismos Livres de Patógenos Específicos , NataçãoRESUMO
Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER) chaperones implicated in neuropathic pain. Here we examine if the Sig-1R may relate to neuropathic pain at the level of dorsal root ganglia (DRG). We focus on the neuronal excitability of DRG in a "spare nerve injury" (SNI) model of neuropathic pain in rats and find that Sig-1Rs likely contribute to the genesis of DRG neuronal excitability by decreasing the protein level of voltage-gated Cav2.2 as a translational inhibitor of mRNA. Specifically, during SNI, Sig-1Rs translocate from ER to the nuclear envelope via a trafficking protein Sec61ß. At the nucleus, the Sig-1R interacts with cFos and binds to the promoter of 4E-BP1, leading to an upregulation of 4E-BP1 that binds and prevents eIF4E from initiating the mRNA translation for Cav2.2. Interestingly, in Sig-1R knockout HEK cells, Cav2.2 is upregulated. In accordance with those findings, we find that intra-DRG injection of Sig-1R agonist (+)pentazocine increases frequency of action potentials via regulation of voltage-gated Ca2+ channels. Conversely, intra-DRG injection of Sig-1R antagonist BD1047 attenuates neuropathic pain. Hence, we discover that the Sig-1R chaperone causes neuropathic pain indirectly as a translational inhibitor.
Assuntos
Genoma , Neuralgia/genética , Receptores sigma/metabolismo , Animais , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Retículo Endoplasmático/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Tecido Nervoso/lesões , Tecido Nervoso/patologia , Membrana Nuclear/metabolismo , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-fos/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Receptores sigma/agonistas , Receptores sigma/genética , Canais de Translocação SEC/metabolismo , Transcrição Gênica , Receptor Sigma-1RESUMO
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.
Assuntos
Aptâmeros de Peptídeos/genética , Canais de Cálcio Tipo N/metabolismo , Dependovirus/genética , Terapia Genética/métodos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas do Tecido Nervoso/genética , Neuralgia/terapia , Animais , Aptâmeros de Peptídeos/química , Aptâmeros de Peptídeos/metabolismo , Bloqueadores dos Canais de Cálcio/química , Dependovirus/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Domínios Proteicos , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Dependovirus/fisiologia , Proteína Glial Fibrilar Ácida/genética , Neuroglia/metabolismo , Transgenes , Animais , Dependovirus/genética , Gânglios/fisiologia , Gânglios/virologia , Gânglios Espinais/fisiologia , Gânglios Espinais/virologia , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Transdução Genética , TropismoRESUMO
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.
Assuntos
Citocinas/líquido cefalorraquidiano , Gânglios Espinais , Hiperalgesia/líquido cefalorraquidiano , Hiperalgesia/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Neuralgia/líquido cefalorraquidiano , Neuralgia/terapia , Medula Espinal , Animais , Modelos Animais de Doenças , Gânglios Espinais/citologia , Masculino , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologiaRESUMO
Painful nerve injury disrupts Ca2+ signaling in primary sensory neurons by elevating plasma membrane Ca2+-ATPase (PMCA) function and depressing sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) function, which decreases endoplasmic reticulum (ER) Ca2+ stores and stimulates store-operated Ca2+ entry (SOCE). The extracellular matrix glycoprotein thrombospondin-4 (TSP4), which is increased after painful nerve injury, decreases Ca2+ current (ICa) through high-voltage-activated Ca2+ channels and increases ICa through low-voltage-activated Ca2+ channels in dorsal root ganglion neurons, which are events similar to the effect of nerve injury. We therefore examined whether TSP4 plays a critical role in injury-induced disruption of intracellular Ca2+ signaling. We found that TSP4 increases PMCA activity, inhibits SERCA, depletes ER Ca2+ stores, and enhances store-operated Ca2+ influx. Injury-induced changes of SERCA and PMCA function are attenuated in TSP4 knock-out mice. Effects of TSP4 on intracellular Ca2+ signaling are attenuated in voltage-gated Ca2+ channel α2δ1 subunit (Cavα2δ1) conditional knock-out mice and are also Protein Kinase C (PKC) signaling dependent. These findings suggest that TSP4 elevation may contribute to the pathogenesis of chronic pain following nerve injury by disrupting intracellular Ca2+ signaling via interacting with the Cavα2δ1 and the subsequent PKC signaling pathway. Controlling TSP4 mediated intracellular Ca2+ signaling in peripheral sensory neurons may be a target for analgesic drug development for neuropathic pain.
Assuntos
Sinalização do Cálcio/fisiologia , Células Receptoras Sensoriais/metabolismo , Nervos Espinhais/lesões , Trombospondinas/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Citoplasma/metabolismo , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Feminino , Gânglios Espinais/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Camundongos da Linhagem 129 , Camundongos Knockout , Neuralgia/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Proteína Quinase C/metabolismo , Ratos Sprague-Dawley , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trombospondinas/genéticaRESUMO
Transferring genetic molecules into the peripheral sensory nervous system to manipulate nociceptive pathophysiology is a powerful approach for experimental modulation of sensory signaling and potentially for translation into therapy for chronic pain. This can be efficiently achieved by the use of recombinant adeno-associated virus (rAAV) in conjunction with nociceptor-specific regulatory transgene cassettes. Among different routes of delivery, direct injection into the dorsal root ganglia (DRGs) offers the most efficient AAV-mediated gene transfer selectively into the peripheral sensory nervous system. Here, we briefly discuss the advantages and applications of intraganglionic microinjection, and then provide a detailed approach for DRG injection, including a list of the necessary materials and description of a method for performing DRG microinjection experiments. We also discuss our experience with several adeno-associated virus (AAV) options for in vivo transgene expression in DRG neurons.
Assuntos
Dependovirus/genética , Gânglios Espinais/metabolismo , Vetores Genéticos/administração & dosagem , Animais , Dor Crônica/terapia , Dependovirus/metabolismo , Técnicas de Transferência de Genes , Terapia Genética , Masculino , Microinjeções , Ratos , Ratos Sprague-Dawley , Transdução GenéticaRESUMO
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.
Assuntos
Analgesia , Gânglios Espinais/transplante , Células-Tronco Mesenquimais/citologia , Neuralgia/terapia , Neurônios Aferentes/citologia , Analgesia/métodos , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Gânglios Espinais/metabolismo , Masculino , Transplante de Células-Tronco Mesenquimais , Neuralgia/genética , Neuralgia/metabolismo , Manejo da Dor/métodos , Ratos Sprague-Dawley , Nervos Espinhais/metabolismoRESUMO
Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca(2+) channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca(2+) currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0µM) reduced depolarization-induced ICa by 16-30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by the efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total ICa. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent.
Assuntos
Canais de Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Gânglios Espinais/citologia , Células Receptoras Sensoriais/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Masculino , Potenciais da Membrana/fisiologia , Neurônios Aferentes/metabolismo , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacosRESUMO
We previously demonstrated safe and reliable gene transfer to the dorsal root ganglion (DRG) using a direct microinjection procedure to deliver recombinant adeno-associated virus (AAV) vector. In this study, we proceed to compare the in vivo transduction patterns of self-complementary (sc) AAV6 and AAV8 in the peripheral sensory pathway. A single, direct microinjection of either AAV6 or AAV8 expressing EGFP, at the adjusted titer of 2×10(9) viral particle per DRG, into the lumbar (L) 4 and L5 DRGs of adult rats resulted in efficient EGFP expression (48±20% for AAV6 and 25±4% for AAV8, mean ± SD) selectively in sensory neurons and their axonal projections 3 weeks after injection, which remained stable for up to 3 months. AAV6 efficiently transfers EGFP to all neuronal size groups without differential neurotropism, while AAV8 predominantly targets large-sized neurons. Neurons transduced with AAV6 penetrate into the spinal dorsal horn (DH) and terminate predominantly in superficial DH laminae, as well as in the dorsal columns and deeper laminae III-V. Only few AAV8-transduced afferents were evident in the superficial laminae, and spinal EGFP was mostly present in the deeper dorsal horn (lamina III-V) and dorsal columns, with substantial projections to the ventral horn. AAV6-mediated EGFP-positive nerve fibers were widely observed in the medial plantar skin of ipsilateral hindpaws. No apparent inflammation, tissue damage, or major pain behaviors were observed for either AAV serotype. Taken together, both AAV6 and AAV8 are efficient and safe vectors for transgene delivery to primary sensory neurons, but they exhibit distinct functional features. Intraganglionic delivery of AAV6 is more uniform and efficient compared to AAV8 in gene transfer to peripheral sensory neurons and their axonal processes.
Assuntos
Dependovirus/genética , Gânglios Espinais/metabolismo , Vetores Genéticos/genética , Animais , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Masculino , Ratos , Células Receptoras Sensoriais/metabolismoRESUMO
The T-junction of sensory neurons in the dorsal root ganglion (DRG) is a potential impediment to action potential (AP) propagation towards the CNS. Using intracellular recordings from rat DRG neuronal somata during stimulation of the dorsal root, we determined that the maximal rate at which all of 20 APs in a train could successfully transit the T-junction (following frequency) was lowest in C-type units, followed by A-type units with inflected descending limbs of the AP, and highest in A-type units without inflections. In C-type units, following frequency was slower than the rate at which AP trains could be produced in either dorsal root axonal segments or in the soma alone, indicating that the T-junction is a site that acts as a low-pass filter for AP propagation. Following frequency was slower for a train of 20 APs than for two, indicating that a cumulative process leads to propagation failure. Propagation failure was accompanied by diminished somatic membrane input resistance, and was enhanced when Ca(2+)-sensitive K(+) currents were augmented or when Ca(2+)-sensitive Cl(-) currents were blocked. After peripheral nerve injury, following frequencies were increased in axotomized C-type neurons and decreased in axotomized non-inflected A-type neurons. These findings reveal that the T-junction in sensory neurons is a regulator of afferent impulse traffic. Diminished filtering of AP trains at the T-junction of C-type neurons with axotomized peripheral processes could enhance the transmission of activity that is ectopically triggered in a neuroma or the neuronal soma, possibly contributing to pain generation.
Assuntos
Potenciais de Ação/fisiologia , Células Receptoras Sensoriais/fisiologia , Nervos Espinhais/lesões , Nervos Espinhais/fisiopatologia , Animais , Comportamento Animal , Gânglios Espinais/fisiologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The plasma membrane Ca2+-ATPase (PMCA) is the principal means by which sensory neurons expel Ca2+ and thereby regulate the concentration of cytoplasmic Ca2+ and the processes controlled by this critical second messenger. We have previously found that painful nerve injury decreases resting cytoplasmic Ca2+ levels and activity-induced cytoplasmic Ca2+ accumulation in axotomized sensory neurons. Here we examine the contribution of PMCA after nerve injury in a rat model of neuropathic pain. RESULTS: PMCA function was isolated in dissociated sensory neurons by blocking intracellular Ca2+ sequestration with thapsigargin, and cytoplasmic Ca2+ concentration was recorded with Fura-2 fluorometry. Compared to control neurons, the rate at which depolarization-induced Ca2+ transients resolved was increased in axotomized neurons after spinal nerve ligation, indicating accelerated PMCA function. Electrophysiological recordings showed that blockade of PMCA by vanadate prolonged the action potential afterhyperpolarization, and also decreased the rate at which neurons could fire repetitively. CONCLUSION: We found that PMCA function is elevated in axotomized sensory neurons, which contributes to neuronal hyperexcitability. Accelerated PMCA function in the primary sensory neuron may contribute to the generation of neuropathic pain, and thus its modulation could provide a new pathway for peripheral treatment of post-traumatic neuropathic pain.
Assuntos
Axotomia , Membrana Celular/enzimologia , Neuralgia/enzimologia , Neuralgia/patologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Células Receptoras Sensoriais/enzimologia , Nervos Espinhais/patologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuralgia/fisiopatologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/patologia , Trocador de Sódio e Cálcio/metabolismo , Nervos Espinhais/efeitos dos fármacos , Nervos Espinhais/enzimologia , Nervos Espinhais/fisiopatologia , Tapsigargina/farmacologiaRESUMO
BACKGROUND: Lentivector-mediated gene delivery into the dorsal root ganglion (DRG) is a promising method for exploring pain pathophysiology and for genetic treatment of chronic neuropathic pain. In this study, a series of modified lentivector particles with different cellular promoters, envelope glycoproteins, and viral accessory proteins were generated to evaluate the requirements for efficient transduction into neuronal cells in vitro and adult rat DRG in vivo. RESULTS: In vitro, lentivectors expressing enhanced green fluorescent protein (EGFP) under control of the human elongation factor 1α (EF1α) promoter and pseudotyped with the conventional vesicular stomatitis virus G protein (VSV-G) envelope exhibited the best performance in the transfer of EGFP into an immortalized DRG sensory neuron cell line at low multiplicities of infection (MOIs), and into primary cultured DRG neurons at higher MOIs. In vivo, injection of either first or second-generation EF1α-EGFP lentivectors directly into adult rat DRGs led to transduction rates of 19 ± 9% and 20 ± 8% EGFP-positive DRG neurons, respectively, detected at 4 weeks post injection. Transduced cells included a full range of neuronal phenotypes, including myelinated neurons as well as both non-peptidergic and peptidergic nociceptive unmyelinated neurons. CONCLUSION: VSV-G pseudotyped lentivectors containing the human elongation factor 1α (EF1α)-EGFP expression cassette demonstrated relatively efficient transduction to sensory neurons following direct injection into the DRG. These results clearly show the potential of lentivectors as a viable system for delivering target genes into DRGs to explore basic mechanisms of neuropathic pain, with the potential for future clinical use in treating chronic pain.
Assuntos
Envelhecimento/metabolismo , Gânglios Espinais/metabolismo , Técnicas de Transferência de Genes , Lentivirus/genética , Animais , Comportamento Animal , Linhagem Celular Transformada , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Injeções , Glicoproteínas de Membrana/metabolismo , Músculos/metabolismo , Dor/metabolismo , Dor/patologia , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Regiões Promotoras Genéticas/genética , Vírus da Raiva/metabolismo , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismo , Transdução Genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Nociception modulates heart rate (HR) and mean arterial pressure (MAP), suggesting their use of HR and MAP as indicators of pain in animals. We explored this with telemetric recording in unrestrained control and neuropathic (spinal nerve ligation) rats. Plantar stimulation was performed emulating techniques commonly used to measure pain, specifically brush stroke, von Frey fiber application, noxious pin stimulation, acetone for cooling, and radiant heating, while recording MAP, HR, and specific evoked somatomotor behaviors (none; simple withdrawal; or sustained lifting, shaking, and grooming representing hyperalgesia). Pin produced elevations in both HR and MAP, and greater responses accompanied hyperalgesia behavior compared to simple withdrawal. Von Frey stimulation depressed MAP, and increased HR only when stimulation produced hyperalgesia behavior, suggesting that minimal nociception occurs without this behavior. Brush increased MAP even when no movement was evoked. Cold elevated both HR and MAP whether or not there was withdrawal, but MAP increased more when withdrawal was triggered. Heating, consistently depressed HR and MAP, independent of behavior. Other than a greater HR response to pin in animals made hyperalgesic by injury, cardiovascular events evoked by stimulation did not differ between control and neuropathic animals. We conclude that (a) thermoregulation rather than pain may dominate responses to heat and cooling stimuli; (b) brush and cooling stimuli may be perceived and produce cardiovascular activation without nocifensive withdrawal; (c) sensations that produce hyperalgesia behavior are accompanied by greater cardiovascular activation than those producing simple withdrawal; and (d) von Frey stimulation lacks cardiovascular evidence of nociception except when hyperalgesia behavior is evoked.