RESUMEN

The in vivo three-dimensional genomic architecture of adult mature neurons at homeostasis and after medically relevant perturbations such as axonal injury remains elusive. Here, we address this knowledge gap by mapping the three-dimensional chromatin architecture and gene expression program at homeostasis and after sciatic nerve injury in wild-type and cohesin-deficient mouse sensory dorsal root ganglia neurons via combinatorial Hi-C, promoter-capture Hi-C, CUT&Tag for H3K27ac and RNA-seq. We find that genes involved in axonal regeneration form long-range, complex chromatin loops, and that cohesin is required for the full induction of the regenerative transcriptional program. Importantly, loss of cohesin results in disruption of chromatin architecture and severely impaired nerve regeneration. Complex enhancer-promoter loops are also enriched in the human fetal cortical plate, where the axonal growth potential is highest, and are lost in mature adult neurons. Together, these data provide an original three-dimensional chromatin map of adult sensory neurons in vivo and demonstrate a role for cohesin-dependent long-range promoter interactions in nerve regeneration.

Asunto(s)

Axones , Cromatina , Cohesinas , Regeneración Nerviosa , Regiones Promotoras Genéticas , Células Receptoras Sensoriales , Animales , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/fisiología , Ratones , Regiones Promotoras Genéticas/genética , Cromatina/metabolismo , Regeneración Nerviosa/genética , Regeneración Nerviosa/fisiología , Axones/metabolismo , Axones/fisiología , Humanos , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Elementos de Facilitación Genéticos/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Nervio Ciático/metabolismo

RESUMEN

BACKGROUND: Peripheral neuropathy (PN) constitutes a dose-limiting side effect of oxaliplatin chemotherapy that often compromises the efficacy of antineoplastic treatments. Sensory neurons damage in dorsal root ganglia (DRG) are the cellular substrate of PN complex molecular origin. Dehydropeptidase-1 (DPEP1) inhibitors have shown to avoid platin-induced nephrotoxicity without compromising its anticancer efficiency. The objective of this study was to describe DPEP1 expression in rat DRG in health and in early stages of oxaliplatin toxicity. To this end, we produced and characterized anti-DPEP1 polyclonal antibodies and used them to define the expression, and cellular and subcellular localization of DPEP1 by immunohistochemical confocal microscopy studies in healthy controls and short term (six days) oxaliplatin treated rats. RESULTS: DPEP1 is expressed mostly in neurons and in glia, and to a lesser extent in endothelial cells. Rats undergoing oxaliplatin treatment developed allodynia. TNF-𝛼 expression in DRG revealed a pattern of focal and at different intensity levels of neural cell inflammatory damage, accompanied by slight variations in DPEP1 expression in endothelial cells and in nuclei of neurons. CONCLUSIONS: DPEP1 is expressed in neurons, glia and endothelial cells of DRG. Oxaliplatin caused allodynia in rats and increased TNF-α expression in DRG neurons. The expression of DPEP1 in neurons and other cells of DRG suggest this protein as a novel strategic molecular target in the prevention of oxaliplatin-induced acute neurotoxicity.

Asunto(s)

Antineoplásicos , Ganglios Espinales , Oxaliplatino , Enfermedades del Sistema Nervioso Periférico , Animales , Oxaliplatino/toxicidad , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/prevención & control , Enfermedades del Sistema Nervioso Periférico/patología , Masculino , Antineoplásicos/toxicidad , Ratas , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Hiperalgesia/prevención & control , Factor de Necrosis Tumoral alfa/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Ratas Sprague-Dawley , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Inflamación/metabolismo , Inflamación/inducido químicamente

RESUMEN

Background: IL-1ß plays a critical role in the pathophysiology of neuroinflammation. The presence of cleaved IL-1ß (cIL-1ß) in the neurons of the dorsal root ganglion (DRG) implicates its function in biological signaling arising from the sensory neuron. This study was conducted to analyze the role of IL-1ß in nociceptive transduction after tissue injury. Methods: A plantar incision was made in C57BL/6 mice, following which immunohistochemistry and RNA scope in situ hybridization were performed at various time points to analyze cIL-1ß, caspase-1, and IL-1 receptor 1 (IL-1R1) expression in the DRG. The effect of intrathecal administration of a caspase-1 inhibitor or regional anesthesia using local anesthetics on cIL-1ß expression and pain hypersensitivity was analyzed by immunohistochemistry and behavioral analysis. ERK phosphorylation was also analyzed to investigate the effect of IL-1ß on the activity of spinal dorsal horn neurons. Results: cIL-1ß expression was significantly increased in caspase-1-positive DRG neurons 5 min after the plantar incision. Intrathecal caspase-1 inhibitor treatment inhibited IL-1ß cleavage and pain hypersensitivity after the plantar incision. IL-1R1 was also detected in the DRG neurons, although the majority of IL-1R1-expressing neurons lacked cIL-1ß expression. Regional anesthesia using local anesthetics prevented cIL-1ß processing. Plantar incision-induced phosphorylation of ERK was inhibited by the caspase-1 inhibitor. Conclusion: IL-1ß in the DRG neuron undergoes rapid cleavage in response to tissue injury in an activity-dependent manner. Cleaved IL-1ß causes injury-induced functional activation of sensory neurons and pain hypersensitivity. IL-1ß in the primary afferent neurons is involved in physiological nociceptive signal transduction.

Asunto(s)

Ganglios Espinales , Interleucina-1beta , Ratones Endogámicos C57BL , Animales , Interleucina-1beta/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Masculino , Caspasa 1/metabolismo , Hiperalgesia/metabolismo , Neuronas/metabolismo , Neuronas/patología , Neuronas/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos , Receptores Tipo I de Interleucina-1/metabolismo , Células del Asta Posterior/metabolismo , Células del Asta Posterior/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo

RESUMEN

Micro and nanoscale patterning of surface features and biochemical cues have emerged as tools to precisely direct neurite growth into close proximity with next generation neural prosthesis electrodes. Biophysical cues can exert greater influence on neurite pathfinding compared to the more well studied biochemical cues; yet the signaling events underlying the ability of growth cones to respond to these microfeatures remain obscure. Intracellular Ca2+ signaling plays a critical role in how a growth cone senses and grows in response to various cues (biophysical features, repulsive peptides, chemo-attractive gradients). Here, we investigate the role of inositol triphosphate (IP3) and ryanodine-sensitive receptor (RyR) signaling as sensory neurons (spiral ganglion neurons, SGNs, and dorsal root ganglion neurons, DRGNs) pathfind in response to micropatterned substrates of varied geometries. We find that IP3 and RyR signaling act in the growth cone as they navigate biophysical cues and enable proper guidance to biophysical, chemo-permissive, and chemo-repulsive micropatterns. In response to complex micropatterned geometries, RyR signaling appears to halt growth in response to both topographical features and chemo-repulsive cues. IP3 signaling appears to play a more complex role, as growth cones appear to sense the microfeatures in the presence of xestospongin C but are unable to coordinate turning in response to them. Overall, key Ca2+ signaling elements, IP3 and RyR, are found to be essential for SGNs to pathfind in response to engineered biophysical and biochemical cues. These findings inform efforts to precisely guide neurite regeneration for improved neural prosthesis function, including cochlear implants.

Asunto(s)

Neuritas , Canal Liberador de Calcio Receptor de Rianodina , Transducción de Señal , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Neuritas/metabolismo , Animales , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Conos de Crecimiento/metabolismo , Conos de Crecimiento/efectos de los fármacos , Señalización del Calcio , Ratas , Propiedades de Superficie , Células Cultivadas , Oxazoles , Compuestos Macrocíclicos

RESUMEN

Maladaptive changes of metabolic patterns in the lumbar dorsal root ganglion (DRG) are critical for nociceptive hypersensitivity genesis. The accumulation of branched-chain amino acids (BCAAs) in DRG has been implicated in mechanical allodynia and thermal hyperalgesia, but the exact mechanism is not fully understood. This study aimed to explore how BCAA catabolism in DRG modulates pain sensitization. Wildtype male mice were fed a high-fat diet (HFD) for 8 weeks. Adult PP2Cmfl/fl mice of both sexes were intrathecally injected with pAAV9-hSyn-Cre to delete the mitochondrial targeted 2 C-type serine/threonine protein phosphatase (PP2Cm) in DRG neurons. Here, we reported that BCAA catabolism was impaired in the lumbar 4-5 (L4-L5) DRGs of mice fed a high-fat diet (HFD). Conditional deletion of PP2Cm in DRG neurons led to mechanical allodynia, heat and cold hyperalgesia. Mechanistically, the genetic knockout of PP2Cm resulted in the upregulation of C-C chemokine ligand 5/C-C chemokine receptor 5 (CCL5/CCR5) axis and an increase in transient receptor potential ankyrin 1 (TRPA1) expression. Blocking the CCL5/CCR5 signaling or TRPA1 alleviated pain behaviors induced by PP2Cm deletion. Thus, targeting BCAA catabolism in DRG neurons may be a potential management strategy for pain sensitization.

Asunto(s)

Aminoácidos de Cadena Ramificada , Dieta Alta en Grasa , Ganglios Espinales , Hiperalgesia , Animales , Ganglios Espinales/metabolismo , Masculino , Hiperalgesia/metabolismo , Aminoácidos de Cadena Ramificada/metabolismo , Ratones , Dieta Alta en Grasa/efectos adversos , Femenino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Canal Catiónico TRPA1/metabolismo , Canal Catiónico TRPA1/genética , Ratones Noqueados , Receptores CCR5/metabolismo , Receptores CCR5/genética

RESUMEN

Traumatic spinal cord injury (tSCI) has complex pathophysiological events that begin after the initial trauma. One such event is fibroglial scar formation by fibroblasts and reactive astrocytes. A strong inhibition of axonal growth is caused by the activated astroglial cells as a component of fibroglial scarring through the production of inhibitory molecules, such as chondroitin sulfate proteoglycans or myelin-associated proteins. Here, we used neural precursor cells (aldynoglia) as promoters of axonal growth and a fibrin hydrogel gelled under alkaline conditions to support and guide neuronal cell growth, respectively. We added Tol-51 sulfoglycolipid as a synthetic inhibitor of astrocyte and microglia in order to test its effect on the axonal growth-promoting function of aldynoglia precursor cells. We obtained an increase in GFAP expression corresponding to the expected glial phenotype for aldynoglia cells cultured in alkaline fibrin. In co-cultures of dorsal root ganglia (DRG) and aldynoglia, the axonal growth promotion of DRG neurons by aldynoglia was not affected. We observed that the neural precursor cells first clustered together and then formed niches from which aldynoglia cells grew and connected to groups of adjacent cells. We conclude that the combination of alkaline fibrin with synthetic sulfoglycolipid Tol-51 increased cell adhesion, cell migration, fasciculation, and axonal growth capacity, promoted by aldynoglia cells. There was no negative effect on the behavior of aldynoglia cells after the addition of sulfoglycolipid Tol-51, suggesting that a combination of aldynoglia plus alkaline fibrin and Tol-51 compound could be useful as a therapeutic strategy for tSCI repair.

Asunto(s)

Axones , Fibrina , Ganglios Espinales , Animales , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Axones/metabolismo , Axones/efectos de los fármacos , Fibrina/metabolismo , Hidrogeles/química , Hidrogeles/farmacología , Ratas , Glucolípidos/farmacología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/citología , Movimiento Celular/efectos de los fármacos

RESUMEN

AIMS: Poly (ADP-ribose) polymerase (PARP) has been extensively investigated in human cancers. Recent studies verified that current available PARP inhibitors (Olaparib or Veliparib) provided clinical palliation of clinical patients suffering from paclitaxel-induced neuropathic pain (PINP). However, the underlying mechanism of PARP overactivation in the development of PINP remains to be investigated. METHODS AND RESULTS: We reported induction of DNA oxidative damage, PARP-1 overactivation, and subsequent nicotinamide adenine dinucleotide (NAD+) depletion as crucial events in the pathogenesis of PINP. Therefore, we developed an Olaparib PROTAC to achieve the efficient degradation of PARP. Continuous intrathecal injection of Olaparib PROTAC protected against PINP by inhibiting the activity of PARP-1 in rats. PARP-1, but not PARP-2, was shown to be a crucial enzyme in the development of PINP. Specific inhibition of PARP-1 enhanced mitochondrial redox metabolism partly by upregulating the expression and deacetylase activity of sirtuin-3 (SIRT3) in the dorsal root ganglions and spinal cord in the PINP rats. Moreover, an increase in the NAD+ level was found to be a crucial mechanism by which PARP-1 inhibition enhanced SIRT3 activity. CONCLUSION: The findings provide a novel insight into the mechanism of DNA oxidative damage in the development of PINP and implicate PARP-1 as a possible therapeutic target for clinical PINP treatment.

Asunto(s)

Daño del ADN , Mitocondrias , Neuralgia , Paclitaxel , Poli(ADP-Ribosa) Polimerasa-1 , Animales , Masculino , Ratas , Modelos Animales de Enfermedad , Daño del ADN/efectos de los fármacos , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , NAD/metabolismo , Neuralgia/inducido químicamente , Neuralgia/metabolismo , Neuralgia/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Paclitaxel/toxicidad , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo

RESUMEN

Individuals suffering from diabetic polyneuropathy (DPN) experience debilitating symptoms such as pain, paranesthesia, and sensory disturbances, prompting a quest for effective treatments. Dipeptidyl-peptidase (DPP)-4 inhibitors, recognized for their potential in ameliorating DPN, have sparked interest, yet the precise mechanism underlying their neurotrophic impact on the peripheral nerve system (PNS) remains elusive. Our study delves into the neurotrophic effects of DPP-4 inhibitors, including Diprotin A, linagliptin, and sitagliptin, alongside pituitary adenylate cyclase-activating polypeptide (PACAP), Neuropeptide Y (NPY), and Stromal cell-derived factor (SDF)-1a-known DPP-4 substrates with neurotrophic properties. Utilizing primary culture dorsal root ganglia (DRG) neurons, we meticulously evaluated neurite outgrowth in response to these agents. Remarkably, all DPP-4 inhibitors and PACAP demonstrated a significant elongation of neurite length in DRG neurons (PACAP 0.1 µM: 2221 ± 466 µm, control: 1379 ± 420, p < 0.0001), underscoring their potential in nerve regeneration. Conversely, NPY and SDF-1a failed to induce neurite elongation, accentuating the unique neurotrophic properties of DPP-4 inhibition and PACAP. Our findings suggest that the upregulation of PACAP, facilitated by DPP-4 inhibition, plays a pivotal role in promoting neurite elongation within the PNS, presenting a promising avenue for the development of novel DPN therapies with enhanced neurodegenerative capabilities.

Asunto(s)

Neuropatías Diabéticas , Inhibidores de la Dipeptidil-Peptidasa IV , Ganglios Espinales , Proyección Neuronal , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Animales , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Proyección Neuronal/efectos de los fármacos , Neuropatías Diabéticas/tratamiento farmacológico , Neuropatías Diabéticas/metabolismo , Ratones , Neuropéptido Y/metabolismo , Neuropéptido Y/farmacología , Quimiocina CXCL12/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Linagliptina/farmacología , Dipeptidil Peptidasa 4/metabolismo , Fosfato de Sitagliptina/farmacología , Células Cultivadas , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Oligopéptidos

RESUMEN

Bone cancer pain (BCP) represents a prevalent symptom among cancer patients with bone metastases, yet its underlying mechanisms remain elusive. This study investigated the transcriptional regulation mechanism of Kv7(KCNQ)/M potassium channels in DRG neurons and its involvement in the development of BCP in rats. We show that HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes, which encode Kv7(KCNQ)/M potassium channels in dorsal root ganglion (DRG), contributes to the sensitization of DRG neurons and the pathogenesis of BCP in rats. Also, HDAC2 requires the formation of a corepressor complex with MeCP2 and Sin3A to execute transcriptional regulation of kcnq2/kcnq3 genes. Moreover, EREG is identified as an upstream signal molecule for HDAC2-mediated kcnq2/kcnq3 genes transcription repression. Activation of EREG/EGFR-ERK-Runx1 signaling, followed by the induction of HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes in DRG neurons, leads to neuronal hyperexcitability and pain hypersensitivity in tumor-bearing rats. Consequently, the activation of EREG/EGFR-ERK-Runx1 signaling, along with the subsequent transcriptional repression of kcnq2/kcnq3 genes by HDAC2 in DRG neurons, underlies the sensitization of DRG neurons and the pathogenesis of BCP in rats. These findings uncover a potentially targetable mechanism contributing to bone metastasis-associated pain in cancer patients.

Asunto(s)

Neoplasias Óseas , Dolor en Cáncer , Receptores ErbB , Ganglios Espinales , Histona Desacetilasa 2 , Canal de Potasio KCNQ2 , Animales , Histona Desacetilasa 2/metabolismo , Histona Desacetilasa 2/genética , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ2/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias Óseas/patología , Ratas , Dolor en Cáncer/genética , Dolor en Cáncer/metabolismo , Dolor en Cáncer/patología , Receptores ErbB/metabolismo , Receptores ErbB/genética , Canal de Potasio KCNQ3/genética , Canal de Potasio KCNQ3/metabolismo , Transcripción Genética , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3/genética , Transducción de Señal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Femenino , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Ratas Sprague-Dawley , Sistema de Señalización de MAP Quinasas/genética

RESUMEN

Endometriosis is a chronic inflammatory disease that causes debilitating pelvic pain in women. Macrophages are considered to be key players in promoting disease progression, as abundant macrophages are present in ectopic lesions and elevated in the peritoneum. In the present study, we examined the role of GATA6+ peritoneal macrophages on endometriosis-associated hyperalgesia using mice with a specific myeloid deficiency of GATA6. Lesion induction induced the disappearance of TIM4hi MHCIIlo residential macrophages and the influx of increased Ly6C+ monocytes and TIM4lo MHCIIhi macrophages. The recruitment of MHCIIhi inflammatory macrophages was extensive in Mac Gata6 KO mice due to the severe disappearance of TIM4hi MHCIIlo residential macrophages. Ki67 expression confirmed GATA6-dependent proliferative ability, showing different proliferative phenotypes of TIM4+ residential macrophages in Gata6f/f and Mac Gata6 KO mice. Peritoneal proinflammatory cytokines were elevated after lesion induction. When cytokine levels were compared between Gata6f/f and Mac Gata6 KO mice, TNFα at day 21 in Gata6f/f mice was higher than in Mac Gata6 KO mice. Lesion induction increased both abdominal and hind paw sensitivities. Gata6f/f mice tended to show higher sensitivity in the abdomen after day 21. Elevated expression of TRPV1 and CGRP was observed in the dorsal root ganglia after ELL induction in Gata6f/f mice until days 21 and 42, respectively. These results support that peritoneal GATA6+ macrophages are involved in the recruitment and reprogramming of monocyte-derived macrophages. The extensive recruitment of monocyte-derived macrophages in Mac Gata6 KO mice might protect against inflammatory stimuli during the resolution phase, whereas GATA6 deficiency did not affect lesion initiation and establishment at the acute phase of inflammation. GATA6+ residential macrophages act to sustain local inflammation in the peritoneum and sensitivities in the neurons, reflecting endometriosis-associated hyperalgesia.

Asunto(s)

Endometriosis , Factor de Transcripción GATA6 , Macrófagos Peritoneales , Animales , Femenino , Ratones , Citocinas/metabolismo , Modelos Animales de Enfermedad , Endometriosis/inmunología , Endometriosis/patología , Endometriosis/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/inmunología , Factor de Transcripción GATA6/metabolismo , Factor de Transcripción GATA6/genética , Hiperalgesia/etiología , Hiperalgesia/metabolismo , Hiperalgesia/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Peritoneo/patología , Peritoneo/inmunología , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética

RESUMEN

The majority of somatosensory DRG neurons express GABAA receptors (GABAAR) and depolarise in response to its activation based on the high intracellular chloride concentration maintained by the Na-K-Cl cotransporter type 1 (NKCC1). The translation of this response to peripheral nerve terminals in people is so far unclear. We show here that GABA (EC50 = 16.67µM) acting via GABAAR produces an influx of extracellular calcium in approximately 20% (336/1720) of isolated mouse DRG neurons. In contrast, upon injection into forearm skin of healthy volunteers GABA (1mM, 100µl) did not induce any overt sensations nor a specific flare response and did not sensitize C-nociceptors to slow depolarizing electrical sinusoidal stimuli. Block of the inward chloride transporter NKCC1 by furosemide (1mg/100µl) did not reduce electrically evoked pain ratings nor did repetitive GABA stimulation in combination with an inhibited NKCC1 driven chloride replenishment by furosemide. Finally, we generated a sustained period of C-fiber firing by iontophoretically delivering codeine or histamine to induce tonic itch. Neither the intensity nor the duration of histamine or codeine itch was affected by prior injection of furosemide. We conclude that although GABA can evoke calcium transients in a proportion of isolated mouse DRG neurons, it does not induce or modify pain or itch ratings in healthy human skin even when chloride gradients are altered by inhibition of the sodium coupled NKCC1 transporter.

Asunto(s)

Furosemida , Ganglios Espinales , Voluntarios Sanos , Hiperalgesia , Miembro 2 de la Familia de Transportadores de Soluto 12 , Ácido gamma-Aminobutírico , Humanos , Animales , Ratones , Ácido gamma-Aminobutírico/metabolismo , Masculino , Adulto , Furosemida/farmacología , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Hiperalgesia/metabolismo , Hiperalgesia/inducido químicamente , Hiperalgesia/fisiopatología , Femenino , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Dolor Agudo/metabolismo , Dolor Agudo/fisiopatología , Calcio/metabolismo , Receptores de GABA-A/metabolismo , Prurito/inducido químicamente , Prurito/metabolismo , Prurito/fisiopatología , Adulto Joven

RESUMEN

The relationship between transcription and protein expression is complex. We identified polysome-associated RNA transcripts in the somata and central terminals of mouse sensory neurons in control, painful (plus nerve growth factor), and pain-free conditions (Nav1.7-null mice). The majority (98%) of translated transcripts are shared between male and female mice in both the somata and terminals. Some transcripts are highly enriched in the somata or terminals. Changes in the translatome in painful and pain-free conditions include novel and known regulators of pain pathways. Antisense knockdown of selected somatic and terminal polysome-associated transcripts that correlate with pain states diminished pain behavior. Terminal-enriched transcripts included those encoding synaptic proteins (e.g., synaptotagmin), non-coding RNAs, transcription factors (e.g., Znf431), proteins associated with transsynaptic trafficking (HoxC9), GABA-generating enzymes (Gad1 and Gad2), and neuropeptides (Penk). Thus, central terminal translation may well be a significant regulatory locus for peripheral input from sensory neurons.

Asunto(s)

Dolor , Células Receptoras Sensoriales , Animales , Células Receptoras Sensoriales/metabolismo , Ratones , Masculino , Femenino , Dolor/metabolismo , Biosíntesis de Proteínas , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/genética , Glutamato Descarboxilasa/metabolismo , Glutamato Descarboxilasa/genética , Polirribosomas/metabolismo , Ratones Endogámicos C57BL , Ganglios Espinales/metabolismo

RESUMEN

Inflammatory arthritis leads to peripheral nerve sensitization, but the therapeutic effect is often unsatisfactory. Our preliminary studies have found that in mice with inflammatory arthritis, the use of ionotropic glutamate receptor antagonists can produce a good analgesic effect without altering foot swelling, suggesting that pain relief may be related to the improvement of neuropathic pain. However, the underlying mechanisms remain unclear. To further investigate the effects of neuropathic pain medications on inflammatory arthritis and the impact of the ionotropic glutamate receptor NR2B subunit (NR2B) on inflammatory arthritis, this study employed gabapentin (GBP) treatment on the inflammatory arthritis mouse model (the adjuvant induced arthritis, AIA), and we found a significant reduction in pain. Further studies revealed that in AIA, the expression levels of NR2B, TRPV1, pain-related molecules (substance P, PGE2), inflammatory cytokines (IL-1, IL-6, TNF-α, and GM-CSF) and Ca2+ were elevated in the foot and dorsal root ganglia (DRG). GBP treatment was able to influence the downregulation of the expression levels of NR2B, TRPV1, pain-related molecules, inflammatory cytokines and Ca2+. Mechanistic studies have shown that GBP treatment affects the downregulation of NR2B, and the downregulation of NR2B expression leads to the downregulation of TRPV1, pain-related molecules and inflammatory cytokines, thereby alleviating pain. These results suggest that in peripheral sensitization caused by AIA, GBP can play a role in improving pain, and NR2B may be a key target of peripheral nerve sensitization induced by inflammatory arthritis. GBP provides a theoretical basis for the clinical treatment of inflammatory arthritis.

Asunto(s)

Analgésicos , Gabapentina , Receptores de N-Metil-D-Aspartato , Animales , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Gabapentina/farmacología , Masculino , Ratones , Analgésicos/farmacología , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Canales Catiónicos TRPV/metabolismo , Citocinas/metabolismo , Artritis/tratamiento farmacológico , Artritis/metabolismo , Artritis/inducido químicamente

RESUMEN

Sexual dimorphism among mammals includes variations in the pain threshold. These differences are influenced by hormonal fluctuations in females during the estrous and menstrual cycles of rodents and humans, respectively. These physiological conditions display various phases, including proestrus and diestrus in rodents and follicular and luteal phases in humans, distinctly characterized by varying estrogen levels. In this study, we evaluated the capsaicin responses in male and female mice at different estrous cycle phases, using two murine acute pain models. Our findings indicate that the capsaicin-induced pain threshold was lower in the proestrus phase than in the other three phases in both pain assays. We also found that male mice exhibited a higher pain threshold than females in the proestrus phase, although it was similar to females in the other cycle phases. We also assessed the mRNA and protein levels of TRPV1 in the dorsal root and trigeminal ganglia of mice. Our results showed higher TRPV1 protein levels during proestrus compared to diestrus and male mice. Unexpectedly, we observed that the diestrus phase was associated with higher TRPV1 mRNA levels than those in both proestrus and male mice. These results underscore the hormonal influence on TRPV1 expression regulation and highlight the role of sex steroids in capsaicin-induced pain.

Asunto(s)

Capsaicina , Dolor , Canales Catiónicos TRPV , Animales , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética , Capsaicina/farmacología , Masculino , Femenino , Ratones , Dolor/metabolismo , Dolor/genética , Hormonas Esteroides Gonadales/metabolismo , Ciclo Estral/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglio del Trigémino/metabolismo , Ganglio del Trigémino/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Caracteres Sexuales , ARN Mensajero/metabolismo , ARN Mensajero/genética

RESUMEN

Lysophosphatidic acids (LPAs) evoke nociception and itch in mice and humans. In this study, we assessed the signaling paths. Hydroxychloroquine was injected intradermally to evoke itch in mice, which evoked an increase of LPAs in the skin and in the thalamus, suggesting that peripheral and central LPA receptors (LPARs) were involved in HCQ-evoked pruriception. To unravel the signaling paths, we assessed the localization of candidate genes and itching behavior in knockout models addressing LPAR5, LPAR2, autotaxin/ENPP2 and the lysophospholipid phosphatases, as well as the plasticity-related genes Prg1/LPPR4 and Prg2/LPPR3. LacZ reporter studies and RNAscope revealed LPAR5 in neurons of the dorsal root ganglia (DRGs) and in skin keratinocytes, LPAR2 in cortical and thalamic neurons, and Prg1 in neuronal structures of the dorsal horn, thalamus and SSC. HCQ-evoked scratching behavior was reduced in sensory neuron-specific Advillin-LPAR5-/- mice (peripheral) but increased in LPAR2-/- and Prg1-/- mice (central), and it was not affected by deficiency of glial autotaxin (GFAP-ENPP2-/-) or Prg2 (PRG2-/-). Heat and mechanical nociception were not affected by any of the genotypes. The behavior suggested that HCQ-mediated itch involves the activation of peripheral LPAR5, which was supported by reduced itch upon treatment with an LPAR5 antagonist and autotaxin inhibitor. Further, HCQ-evoked calcium fluxes were reduced in primary sensory neurons of Advillin-LPAR5-/- mice. The results suggest that LPA-mediated itch is primarily mediated via peripheral LPAR5, suggesting that a topical LPAR5 blocker might suppress "non-histaminergic" itch.

Asunto(s)

Hidroxicloroquina , Ratones Noqueados , Prurito , Receptores del Ácido Lisofosfatídico , Animales , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Prurito/inducido químicamente , Prurito/metabolismo , Prurito/genética , Prurito/tratamiento farmacológico , Ratones , Hidroxicloroquina/farmacología , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Masculino , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Lisofosfolípidos/metabolismo , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos

RESUMEN

BACKGROUND: Peripheral nerve injury is a challenging orthopedic issue in clinical management that often leads to limb dysfunction or even disability in severe cases. A thorough exploration of the repair process of peripheral nerve injury and the underlying mechanism contributes to formulate more effective therapeutic strategies. METHODS: In the present study, we established a sciatic nerve transection injury model in Sprague-Dawley (SD) rats. A 12-week compensatory repair of sciatic nerve transection injury using a chitin cannula for small gap anastomosis was then performed via sleeve jointing the proximal common peroneal nerve to the distal tibial nerve and common peroneal nerve, with a 2 mm interval. Compensatory repair via small gap amplification was observed via gross observation of nerve specimen, osmic acid staining, and electrophysiological stimulation of sciatic nerve branches of the tibial and common peroneal nerve. Rat limbs were observed, and the functional recovery of effector muscles of the gastrocnemius and tibialis anterior muscles was assessed through weighing the muscle wet weight, Hematoxylin and Eosin (H&E) staining, and muscle strength detection. H&E staining, Masson staining, and toluidine blue staining were performed to observe the morphological changes of the dorsal root ganglion. Positive expressions of key proteins involved in the Phosphatase and tensin homologue deleted on chromosome ten (PTEN)-protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, including PTEN, AKT, mTOR, Toll-like receptor 4 (TLR4), and Caspase9 in the dorsal root ganglion during compensatory repair of sciatic nerve after injury via small gap amplification, were detected by immunohistochemical staining. RESULTS: It is found that the compensatory repair of sciatic nerve transection injury using a chitin cannula for small gap anastomosis via sleeve jointing effectively restored the continuity, number of myelinated nerve fibers, and nerve conduction velocity. It promoted toe abduction recovery, improved muscle fiber morphology and increased the wet weight and muscle strength of the gastrocnemius muscle and tibialis anterior muscle. Moreover, it increased the number of neurons and nerve fibers, and improved their morphology. Downregulated PTEN, TLR4, and Caspase9 in the dorsal root ganglia and upregulated AKT and mTOR were observed after small gap amplification than those of the transection injury group, which were closer to those of the control group. CONCLUSIONS: Compensatory repair of sciatic nerve transection injury using a chitin cannula for small gap anastomosis via sleeve jointing can restore the morphology and function of the sciatic nerve, effector muscles, and corresponding dorsal root ganglia by activating the PTEN-AKT/mTOR signaling pathway in the dorsal root ganglia. Our findings provide novel therapeutic targets for peripheral nerve injuries.

Asunto(s)

Ganglios Espinales , Regeneración Nerviosa , Transducción de Señal , Animales , Masculino , Ratas , Modelos Animales de Enfermedad , Ganglios Espinales/metabolismo , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfohidrolasa PTEN/metabolismo , Ratas Sprague-Dawley , Nervio Ciático/lesiones , Neuropatía Ciática/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

Endometriosis is an estrogen-dependent inflammatory gynecological disease whose pathogenesis is unclear. C-C motif chemokine ligand 18 (CCL18), a chemokine, is involved in several inflammatory diseases. In this study, we aimed to investigate the role of CCL18 in endometriosis and its underlying mechanisms. Human endometrium and peritoneal fluid were obtained from women with and without endometriosis for molecular studies. The expression level of CCL18 in each tissue sample was examined by RNA sequencing analysis, quantitative PCR analysis and immunohistochemistry staining. The effects of CCL18 on cell migration, tube formation and neurite growth were investigated in vitro using primary endometrial cells, human umbilical vein endothelial cells (HUVECs) and dorsal root ganglion (DRG) neurons, respectively. Moreover, the development of endometriosis in mice was studied in vivo by blocking CCL18. CCL18 was shown to be overexpressed in endometrial foci and peritoneal fluid in women with endometriosis and was positively correlated with endometriosis pain. In vitro, CCL18 promoted the migration of ectopic endometrial cells, tube formation of HUVECs, and nerve outgrowth of DRG neurons. More importantly, inhibition of CCL18 significantly suppressed lesion development, angiogenesis, and nerve infiltration in a mouse model of endometriosis. In conclusion, CCL18 may play a role in the progression of endometriosis by increasing endometrial cell migration and promoting neuroangiogenesis.

Asunto(s)

Movimiento Celular , Quimiocinas CC , Endometriosis , Endometrio , Células Endoteliales de la Vena Umbilical Humana , Neovascularización Patológica , Endometriosis/metabolismo , Endometriosis/patología , Femenino , Humanos , Animales , Endometrio/metabolismo , Endometrio/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Ratones , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Quimiocinas CC/metabolismo , Adulto , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Líquido Ascítico/metabolismo , Líquido Ascítico/patología , Ratones Endogámicos C57BL

RESUMEN

Peripheral nerve injury (PNI) often leads to retrograde cell death in the spinal cord and dorsal root ganglia (DRG), hindering nerve regeneration and functional recovery. Repetitive magnetic stimulation (rMS) promotes nerve regeneration following PNI. Therefore, this study aimed to investigate the effects of rMS on post-injury neuronal death and nerve regeneration. Seventy-two rats underwent autologous sciatic nerve grafting and were divided into two groups: the rMS group, which received rMS and the control (CON) group, which received no treatment. Motor neuron, DRG neuron, and caspase-3 positive DRG neuron counts, as well as DRG mRNA expression analyses, were conducted at 1-, 4-, and 8-weeks post-injury. Functional and axon regeneration analyses were performed at 8-weeks post-injury. The CON group demonstrated a decreased DRG neuron count starting from 1 week post-injury, whereas the rMS group exhibited significantly higher DRG neuron counts at 1- and 4-weeks post-injury. At 8-weeks post-injury, the rMS group demonstrated a significantly greater myelinated nerve fiber density in autografted nerves. Furthermore, functional analysis showed significant improvements in latency and toe angle in the rMS group. Overall, these results suggest that rMS can prevent DRG neuron death and enhance nerve regeneration and motor function recovery after PNI.

Asunto(s)

Muerte Celular , Modelos Animales de Enfermedad , Ganglios Espinales , Regeneración Nerviosa , Traumatismos de los Nervios Periféricos , Nervio Ciático , Animales , Ganglios Espinales/metabolismo , Ratas , Nervio Ciático/lesiones , Traumatismos de los Nervios Periféricos/terapia , Masculino , Ratas Sprague-Dawley , Neuronas/metabolismo , Magnetoterapia/métodos , Recuperación de la Función , Neuronas Motoras/metabolismo , Neuronas Motoras/fisiología

RESUMEN

Neuroimmune cross-talk participates in intestinal tissue homeostasis and host defense. However, the matrix of interactions between arrays of molecularly defined neuron subsets and of immunocyte lineages remains unclear. We used a chemogenetic approach to activate eight distinct neuronal subsets, assessing effects by deep immunophenotyping, microbiome profiling, and immunocyte transcriptomics in intestinal organs. Distinct immune perturbations followed neuronal activation: Nitrergic neurons regulated T helper 17 (TH17)-like cells, and cholinergic neurons regulated neutrophils. Nociceptor neurons, expressing Trpv1, elicited the broadest immunomodulation, inducing changes in innate lymphocytes, macrophages, and RORγ+ regulatory T (Treg) cells. Neuroanatomical, genetic, and pharmacological follow-up showed that Trpv1+ neurons in dorsal root ganglia decreased Treg cell numbers via the neuropeptide calcitonin gene-related peptide (CGRP). Given the role of these neurons in nociception, these data potentially link pain signaling with gut Treg cell function.

Asunto(s)

Péptido Relacionado con Gen de Calcitonina , Ganglios Espinales , Neuroinmunomodulación , Nociceptores , Linfocitos T Reguladores , Canales Catiónicos TRPV , Células Th17 , Animales , Ratones , Péptido Relacionado con Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/genética , Neuronas Colinérgicas/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Microbioma Gastrointestinal , Intestinos/inmunología , Intestinos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Nocicepción , Nociceptores/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Células Th17/inmunología , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética

RESUMEN

A large portion of neuropathic pain suffering patients may also concurrently experience neuropathic itch, with a negative impact on the quality of life. The limited understanding of neuropathic itch and the low efficacy of current anti-itch therapies dictate the urgent need of a better comprehension of molecular mechanisms involved and development of relevant animal models. This study was aimed to characterize the itching phenotype in a model of trauma-induced peripheral neuropathy, the spared nerve injury (SNI), and the molecular events underlying the overlap with the nociceptive behavior. SNI mice developed hyperknesis and spontaneous itch 7-14 days after surgery that was prevented by gabapentin treatment. Itch was associated with pain hypersensitivity, loss of intraepidermal nerve fiber (IENF) density and increased epidermal thickness. In coincidence with the peak of scratching behavior, SNI mice showed a spinal overexpression of IBA1 and GFAP, microglia and astrocyte markers respectively. An increase of the itch neuropeptide B-type natriuretic peptide (BNP) in NeuN+ cells, of its downstream effector interleukin 17 (IL17) along with increased pERK1/2 levels occurred in the spinal cord dorsal horn and DRG. A raise in BNP and IL17 was also detected at skin level. Stimulation of HaCat cells with conditioned medium from BV2-stimulated SH-SY5Y cells produced a dramatic reduction of HaCat cell viability. This study showed that SNI mice might represent a model for neuropathic itch and pain. Collectively, our finding suggest that neuropathic itch might initiate at spinal level, then affecting skin epidermis events, through a glia-mediated neuroinflammation-evoked BNP/IL17 mechanism.

Asunto(s)

Modelos Animales de Enfermedad , Neuralgia , Enfermedades Neuroinflamatorias , Prurito , Animales , Prurito/metabolismo , Prurito/patología , Neuralgia/metabolismo , Neuralgia/etiología , Ratones , Masculino , Enfermedades Neuroinflamatorias/metabolismo , Humanos , Gabapentina/farmacología , Interleucina-17/metabolismo , Ratones Endogámicos C57BL , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/patología , Células HaCaT , Microglía/metabolismo , Microglía/efectos de los fármacos , Hiperalgesia/metabolismo , Proteínas de Microfilamentos/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Asta Dorsal de la Médula Espinal/efectos de los fármacos , Proteínas de Unión al Calcio