RESUMO
PURPOSE: Chronic prostatitis/chronic pelvic pain syndrome causes symptoms that include the frequent and urgent need to urinate, pain or burning during urination and pain radiating to the back, abdomen and/or colorectum. These bladder symptoms suggest that chronic prostatitis/chronic pelvic pain syndrome is associated with sensitization of adjacent organs, termed cross-organ sensitization. The objective of this study was to determine the extent of 1) changes in immunomodulatory mediators in the prostate and bladder after inflammation of the prostate and 2) bladder function and bladder afferent sensitization. MATERIALS AND METHODS: Prostate and bladder histology, immunohistochemistry and expression of immunomodulatory targets were examined weekly after zymosan or vehicle was injected in the dorsal lobe of the mouse prostate. Cystometry, bladder and bladder afferent sensitivity were also assessed weekly. RESULTS: Prostate inflammation induced significant up-regulation in proinflammatory and anti-inflammatory cytokines TNF-α (tumor necrosis factor-α) and IL-10 (interleukin-10), growth factor NGF (nerve growth factor), and T-lymphocyte markers FoxP3, CD4 and CD8 in the prostate and the bladder. Notably, prostatitis significantly increased urinary voiding frequency, induced hypersensitivity to bladder distension and sensitized bladder afferents. We also examined sensory (afferent) co-innervation by injecting retrograde tracers DiI and Fast Blue in the bladder wall and the prostate, respectively. This showed that a significant proportion (approximately 17%) of dorsal root ganglion afferent somata contained tracers from the bladder and the prostate. CONCLUSIONS: These observations support an afferent contribution to chronic prostatitis/chronic pelvic pain syndrome and cross-organ sensitization from prostate to bladder.
Assuntos
Gânglios Espinais/metabolismo , Prostatite/complicações , Doenças da Bexiga Urinária/etiologia , Bexiga Urinária/inervação , Animais , Western Blotting , Doença Crônica , Citocinas/biossíntese , Citocinas/genética , Modelos Animais de Doenças , Gânglios Espinais/patologia , Regulação da Expressão Gênica , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Prostatite/diagnóstico , Prostatite/genética , RNA/genética , Bexiga Urinária/diagnóstico por imagem , Doenças da Bexiga Urinária/diagnóstico , Doenças da Bexiga Urinária/genéticaRESUMO
Isolectin B4-binding (IB4+) dorsal root ganglion (DRG) neurons are distinct from peptidergic DRG neurons in their terminal location in the spinal cord and respective contributions to various classes and modalities of nociception. In DRG neurons innervating the mouse colon (c-DRG neurons), the reported proportion of IB4+ population is inconsistent across studies, and little is known regarding their role in colorectal mechanonociception. To address these issues, in C57BL/6J mice, we quantified IB4+ binding after labeling c-DRG neurons with Fast Blue and examined functional consequences of ablating these neurons by IB4-conjugated saporin. Sixty-one percent of Fast Blue-labeled neurons in the L6 DRG were IB4+, and 95% of these IB4+ c-DRG neurons were peptidergic. Intrathecal administration of IB4-conjugated saporin reduced the proportion of IB4+ c-DRG neurons to 37%, which was due to the loss of c-DRG neurons showing strong to medium IB4+ intensity; c-DRG neurons with weak IB4+ intensity were spared. However, this loss altered neither nociceptive behaviors to colorectal distension nor the relative proportions of stretch-sensitive colorectal afferent classes characterized by single-fiber recordings. These findings demonstrate that more than 1 half of viscerosensory L6 c-DRG neurons in C57BL/6J mouse are IB4+ and suggest, in contrast to the reported roles of IB4+/nonpeptidergic neurons in cutaneous mechanonociception, c-DRG neurons with strong-to-medium IB4+ intensity do not play a significant role in colorectal mechanonociception.
Assuntos
Colo/inervação , Gânglios Espinais/patologia , Lectinas/metabolismo , Neurônios/metabolismo , Dor Visceral/patologia , Vias Aferentes/lesões , Vias Aferentes/fisiologia , Amidinas/metabolismo , Análise de Variância , Animais , Fenômenos Biofísicos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Colo/fisiologia , Técnicas In Vitro , Lectinas/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Fosfopiruvato Hidratase/metabolismo , Estimulação Física , Proteínas Inativadoras de Ribossomos Tipo 1/toxicidade , Saporinas , Canais de Cátion TRPV/metabolismoRESUMO
Chronic nonbacterial prostatitis, characterized by genitourinary pain in the pelvic region in the absence of an identifiable cause, is common in adult males. Surprisingly, the sensory innervation of the prostate and mediators that sensitize its innervation have received little attention. We thus characterized a mouse model of chronic prostatitis, focusing on the prostate innervation and how organ inflammation affects gene expression of putative nociceptive markers in prostate afferent somata in dorsal root ganglia (DRG) and mediators in the prostate. Retrograde tracing (fast blue) from the prostate revealed that thoracolumbar and lumbosacral DRG are the principal sources of somata of prostate afferents. Nociceptive markers (eg, transient receptor potential, TREK, and P2X channels) were upregulated in fast blue-labeled thoracolumbar and lumbosacral somata for up to four weeks after inflaming the prostate (intraprostate injection of zymosan). Prostatic inflammation was evident histologically, by monocyte infiltration and a significant increase in mast cell tryptase activity 14, 21, and 28 days after zymosan injection. Interleukin 10 and NGF were also significantly upregulated in the prostate throughout the 4 weeks of inflammation. Open-field pain-related behaviors (eg, rearing) were unchanged in prostate-inflamed mice, suggesting the absence of ongoing nociception, but withdrawal thresholds to lower abdominal pressure were significantly reduced. The increases in IL-10, mast cell tryptase, and NGF in the inflamed prostate were cotemporaneous with reduced thresholds to probing of the abdomen and upregulation of nociceptive markers in DRG somata innervating the prostate. The results provide insight and direction for the study of mechanisms underlying pain in chronic prostatitis.
Assuntos
Inflamação/imunologia , Nociceptores/metabolismo , Dor/metabolismo , Próstata/imunologia , Próstata/inervação , Prostatite/imunologia , Animais , Comportamento Animal , Doença Crônica , Modelos Animais de Doenças , Gânglios Espinais/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/complicações , Mediadores da Inflamação/metabolismo , Interleucina-10/metabolismo , Masculino , Camundongos , Fator de Crescimento Neural/metabolismo , Dor/etiologia , Dor/psicologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Próstata/patologia , Prostatite/induzido quimicamente , Prostatite/complicações , Prostatite/fisiopatologia , Receptores Purinérgicos P2X/metabolismo , Triptases/metabolismo , Regulação para Cima , Zimosan/toxicidadeRESUMO
Measuring the excitability of individual axons is complicated by the prohibitive difficulty in obtaining intracellular recordings. Here, we present an innovative methodology that enables local excitability to be measured anywhere in a channelrhodopsin (ChR2)-expressing neuron. The approach hinges on activating ChR2 in a spatially and temporally precise manner while recording the resulting spike train from a remote site. We validated this approach in primary afferent neurons (PANs). Initial encoding of somatosensory stimuli relies on transduction of the physical stimulus into a receptor potential and transformation of the receptor potential into a spike train; the transformation process depends on the excitability of the most distal PAN endings but, as explained above, is extraordinarily difficult to study in situ using traditional methods. Using ChR2-based photoactivation, we show 1) that excitability differs between the distal endings and more proximal portions of PAN axons, 2) that the transformation process differs between PANs, and 3) that the transformation process is directly affected by inflammation. Beyond presenting an innovative method by which to study axonal excitability, this study has validated its utility in helping to decipher the earliest stages of somatosensory encoding.
Assuntos
Potenciais de Ação/fisiologia , Axônios/fisiologia , Condução Nervosa/fisiologia , Optogenética/métodos , Estimulação Luminosa/métodos , Animais , Células Cultivadas , Channelrhodopsins , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos EndogâmicosRESUMO
BACKGROUND: Artemin (Artn), a member of the glial cell line-derived growth factor (GDNF) family, supports the development and function of a subpopulation of peptidergic, TRPV1-positive sensory neurons. Artn (enovin, neublastin) is elevated in inflamed tissue and its injection in skin causes transient thermal hyperalgesia. A genome wide expression analysis of trigeminal ganglia of mice that overexpress Artn in the skin (ART-OE mice) showed elevation in nicotinic acetylcholine receptor (nAChR) subunits, suggesting these ion channels contribute to Artn-induced sensitivity. Here we have used gene expression, immunolabeling, patch clamp electrophysiology and behavioral testing assays to investigate the link between Artn, nicotinic subunit expression and thermal hypersensitivity. RESULTS: Reverse transcriptase-PCR validation showed increased levels of mRNAs encoding the nAChR subunits α3 (13.3-fold), ß3 (4-fold) and ß4 (7.7-fold) in trigeminal ganglia and α3 (4-fold) and ß4 (2.8-fold) in dorsal root ganglia (DRG) of ART-OE mice. Sensory ganglia of ART-OE mice had increased immunoreactivity for nAChRα3 and exhibited increased overlap in labeling with GFRα3-positive neurons. Patch clamp analysis of back-labeled cutaneous afferents showed that while the majority of nicotine-evoked currents in DRG neurons had biophysical and pharmacological properties of α7-subunit containing nAChRs, the Artn-induced increase in α3 and ß4 subunits resulted in functional channels. Behavioral analysis of ART-OE and wildtype mice showed that Artn-induced thermal hyperalgesia can be blocked by mecamylamine or hexamethonium. Complete Freund's adjuvant (CFA) inflammation of paw skin, which causes an increase in Artn in the skin, also increased the level of nAChR mRNAs in DRG. Finally, the increase in nAChRs transcription was not dependent on the Artn-induced increase in TRPV1 or TRPA1 in ART-OE mice since nAChRs were elevated in ganglia of TRPV1/TRPA1 double knockout mice. CONCLUSIONS: These findings suggest that Artn regulates the expression and composition of nAChRs in GFRα3 nociceptors and that these changes contribute to the thermal hypersensitivity that develops in response to Artn injection and perhaps to inflammation.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas do Tecido Nervoso/farmacologia , Nociceptores/fisiologia , Receptores Nicotínicos/metabolismo , Gânglio Trigeminal/patologia , Animais , Feminino , Gânglios Espinais/citologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Hexametônio/uso terapêutico , Hiperalgesia/tratamento farmacológico , Hiperalgesia/fisiopatologia , Masculino , Mecamilamina/uso terapêutico , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/toxicidade , Antagonistas Nicotínicos/uso terapêutico , Nociceptores/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/genética , Pele/inervação , Pele/patologiaRESUMO
Modeling visceral pain requires an appreciation of the underlying neurobiology of visceral sensation, including characteristics of visceral pain that distinguish it from pain arising from other tissues, the unique sensory innervation of visceral organs, the functional basis of visceral pain, and the concept of viscero-somatic and viscero-visceral convergence. Further, stimuli that are noxious when applied to the viscera are different than stimuli noxious to skin, muscle, and joints, thus informing model development and assessment. Visceral pain remains an important and understudied area of pain research and basic science knowledge and mechanisms acquired using animal models can translate into approaches that can be applied to the study and development of future therapeutics.
RESUMO
Urinary bladder pain is a primary symptom associated with interstitial cystitis/painful bladder syndrome. We used systemic injections of cyclophosphamide (CYP), an alkylating antineoplastic agent, to induce cystitis and examine the roles of 2 channels previously demonstrated to be required for inflammatory visceral hyperalgesia: transient receptor potential vanilloid-1 (TRPV1) and ankyrin-1 (TRPA1). Injection of CYP (100 mg/kg, i.p.) every other day for 5 days was accompanied by bladder edema and urothelial ulceration, but without significant plasma extravasation or infiltration of neutrophils. Toluidine blue staining showed a significant increase in the number of degranulated bladder mast cells after CYP treatment. Despite this mild pathology, CYP-treated mice exhibited bladder hyperalgesia 1 day after the final injection that persisted 7 days later. Although many previous studies of visceral hyperalgesia have reported changes in dorsal root ganglion neuron TRPV1 expression and/or function, we found no change in bladder afferent TRPV1 expression or sensitivity on the basis of the percentage of bladder afferents responsive to capsaicin, including at submaximal concentrations. In contrast, the percentage of bladder afferents expressing functional TRPA1 protein (i.e., those responsive to mustard oil) increased â¼2.5-fold 1 day after CYP treatment, and remained significantly elevated 7 days later. Moreover, bladder hyperalgesia was reversed by acute treatment with the TRPA1 antagonist HC-030031 (300 mg/kg, i.p.). Our results indicate that CYP-induced bladder hyperalgesia can be induced without robust inflammation or changes in primary afferent TRPV1. However, significant changes were observed in TRPA1 expression, and blockade of TRPA1 alleviated CYP-induced bladder hyperalgesia.
Assuntos
Cistite Intersticial/metabolismo , Hiperalgesia/metabolismo , Canais de Cátion TRPV/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Bexiga Urinária/metabolismo , Animais , Ciclofosfamida/intoxicação , Cistite Intersticial/induzido quimicamente , Modelos Animais de Doenças , Camundongos , Canal de Cátion TRPA1RESUMO
Menthol and other counterstimuli relieve itch, resulting in an antipruritic state that persists for minutes to hours. However, the neural basis for this effect is unclear, and the underlying neuromodulatory mechanisms are unknown. Previous studies revealed that Bhlhb5(-/-) mice, which lack a specific population of spinal inhibitory interneurons (B5-I neurons), develop pathological itch. Here we characterize B5-I neurons and show that they belong to a neurochemically distinct subset. We provide cause-and-effect evidence that B5-I neurons inhibit itch and show that dynorphin, which is released from B5-I neurons, is a key neuromodulator of pruritus. Finally, we show that B5-I neurons are innervated by menthol-, capsaicin-, and mustard oil-responsive sensory neurons and are required for the inhibition of itch by menthol. These findings provide a cellular basis for the inhibition of itch by chemical counterstimuli and suggest that kappa opioids may be a broadly effective therapy for pathological itch.
Assuntos
Dinorfinas/metabolismo , Interneurônios/metabolismo , Inibição Neural/fisiologia , Células do Corno Posterior/metabolismo , Prurido/metabolismo , Prurido/prevenção & controle , Analgésicos Opioides/farmacologia , Analgésicos Opioides/uso terapêutico , Animais , Capsaicina/farmacologia , Dinorfinas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Octreotida/farmacologia , Técnicas de Cultura de Órgãos , Receptores Opioides kappa/agonistas , Medula Espinal/metabolismoRESUMO
The ligand-gated channels transient receptor potential vanilloid 1 (TRPV1) and P2X3 have been reported to facilitate colorectal afferent neuron sensitization, thus contributing to organ hypersensitivity and pain. In the present study, we hypothesized that TRPV1 and P2X3 cooperate to modulate colorectal nociception and afferent sensitivity. To test this hypothesis, we employed TRPV1-P2X3 double knockout (TPDKO) mice and channel-selective pharmacological antagonists and evaluated combined channel contributions to behavioral responses to colorectal distension (CRD) and afferent fiber responses to colorectal stretch. Baseline responses to CRD were unexpectedly greater in TPDKO compared with control mice, but zymosan-produced CRD hypersensitivity was absent in TPDKO mice. Relative to control mice, proportions of mechanosensitive and -insensitive pelvic nerve afferent classes were not different in TPDKO mice. Responses of mucosal and serosal class afferents to mechanical probing were unaffected, whereas responses of muscular (but not muscular/mucosal) afferents to stretch were significantly attenuated in TPDKO mice; sensitization of both muscular and muscular/mucosal afferents by inflammatory soup was also significantly attenuated. In pharmacological studies, the TRPV1 antagonist A889425 and P2X3 antagonist TNP-ATP, alone and in combination, applied onto stretch-sensitive afferent endings attenuated responses to stretch; combined antagonism produced greater attenuation. In the aggregate, these observations suggest that 1) genetic manipulation of TRPV1 and P2X3 leads to reduction in colorectal mechanosensation peripherally and compensatory changes and/or disinhibition of other channels centrally, 2) combined pharmacological antagonism produces more robust attenuation of mechanosensation peripherally than does antagonism of either channel alone, and 3) the relative importance of these channels appears to be enhanced in colorectal hypersensitivity.
Assuntos
Colo/metabolismo , Hipersensibilidade/genética , Dor/genética , Receptores Purinérgicos P2X3/metabolismo , Reto/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Comportamento Animal , Colo/efeitos dos fármacos , Colo/inervação , Hipersensibilidade/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dor/fisiopatologia , Receptores Purinérgicos P2X3/genética , Reto/efeitos dos fármacos , Reto/inervação , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genéticaRESUMO
Visceral afferents expressing transient receptor potential (TRP) channels TRPV1 and TRPA1 are thought to be required for neurogenic inflammation and development of inflammatory hyperalgesia. Using a mouse model of chronic pancreatitis (CP) produced by repeated episodes (twice weekly) of caerulein-induced AP (AP), we studied the involvement of these TRP channels in pancreatic inflammation and pain-related behaviors. Antagonists of the two TRP channels were administered at different times to block the neurogenic component of AP. Six bouts of AP (over 3 wks) increased pancreatic inflammation and pain-related behaviors, produced fibrosis and sprouting of pancreatic nerve fibers, and increased TRPV1 and TRPA1 gene transcripts and a nociceptive marker, pERK, in pancreas afferent somata. Treatment with TRP antagonists, when initiated before week 3, decreased pancreatic inflammation and pain-related behaviors and also blocked the development of histopathological changes in the pancreas and upregulation of TRPV1, TRPA1, and pERK in pancreatic afferents. Continued treatment with TRP antagonists blocked the development of CP and pain behaviors even when mice were challenged with seven more weeks of twice weekly caerulein. When started after week 3, however, treatment with TRP antagonists was ineffective in blocking the transition from AP to CP and the emergence of pain behaviors. These results suggest: (1) an important role for neurogenic inflammation in pancreatitis and pain-related behaviors, (2) that there is a transition from AP to CP, after which TRP channel antagonism is ineffective, and thus (3) that early intervention with TRP channel antagonists may attenuate the transition to and development of CP effectively.
Assuntos
Oximas/uso terapêutico , Dor/prevenção & controle , Pancreatite Crônica/tratamento farmacológico , Piridinas/uso terapêutico , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Amidinas/metabolismo , Analgésicos Opioides/uso terapêutico , Análise de Variância , Animais , Antígenos de Diferenciação/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cálcio/metabolismo , Ceruletídeo/toxicidade , Modelos Animais de Doenças , Progressão da Doença , Comportamento Exploratório/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Monócitos/patologia , Morfina/uso terapêutico , Infiltração de Neutrófilos/efeitos dos fármacos , Gânglio Nodoso/metabolismo , Gânglio Nodoso/patologia , Dor/etiologia , Dor/patologia , Medição da Dor/efeitos dos fármacos , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/complicações , Pancreatite Crônica/patologia , Peroxidase/metabolismo , RNA Mensageiro/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Canal de Cátion TRPA1 , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Fatores de Tempo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Neurturin (NRTN) is a member of the glial cell line-derived neurotrophic factor family of ligands that exerts its actions via Ret tyrosine kinase and GFRα2. Expression of the Ret-GFRα2 coreceptor complex is primarily restricted to the peripheral nervous system and is selectively expressed by sensory neurons that bind the isolectin B(4) (IB(4)). To determine how target-derived NRTN affects sensory neuron properties, transgenic mice that overexpress NRTN in keratinocytes (NRTN-OE mice) were analyzed. Overexpression of NRTN increased the density of PGP9.5-positive, but not calcitonin gene-related peptide-positive, free nerve endings in footpad epidermis. GFRα2-immunopositive somata were hypertrophied in NRTN-OE mice. Electron microscopic analysis further revealed hypertrophy of unmyelinated sensory axons and a subset of myelinated axons. Overexpression of NRTN increased the relative level of mRNAs encoding GFRα2 and Ret, the ATP receptor P2X(3) (found in IB(4)-positive, GFRα2-expressing sensory neurons), the acid-sensing ion channel 2a, and transient receptor potential cation channel subfamily member M8 (TRPM8) in sensory ganglia. Behavioral testing of NRTN-OE mice revealed an increased sensitivity to mechanical stimuli in glabrous skin of the hindpaw. NRTN-OE mice also displayed increased behavioral sensitivity to cool temperature (17°C-20°C) and oral sensitivity to menthol. The increase in cool and menthol sensitivity correlated with a significant increase in TRPM8 expression and the percentage of menthol-responsive cutaneous sensory neurons. These data indicate that the expression level of NRTN in the skin modulates gene expression in cutaneous sensory afferents and behavioral sensitivity to thermal, chemical, and mechanical stimuli.
Assuntos
Comportamento Animal/fisiologia , Neurturina/metabolismo , Células Receptoras Sensoriais/metabolismo , Pele/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Temperatura Baixa , Masculino , Mentol/farmacologia , Camundongos , Camundongos Transgênicos , Neurturina/genética , Estimulação Física , Pele/inervação , Canais de Cátion TRPM/genéticaRESUMO
Carbohydrate malabsorption such as in lactose intolerance or enteric infection causes symptoms that include abdominal pain. Because this digestive disorder increases intracolonic osmolarity and acidity by accumulation of undigested carbohydrates and fermented products, we tested whether these two factors (hypertonicity and acidity) would modulate colorectal afferents in association with colorectal nociception and hypersensitivity. In mouse colorectum-pelvic nerve preparations in vitro, afferent activities were monitored after application of acidic hypertonic saline (AHS; pH 6.0, 800 mosM). In other experiments, AHS was instilled intracolonically to mice and behavioral responses to colorectal distension (CRD) measured. Application of AHS in vitro excited 80% of serosal and 42% of mechanically-insensitive colorectal afferents (MIAs), sensitizing a proportion of MIAs to become mechanically sensitive and reversibly inhibiting stretch-sensitive afferents. Acute intracolonic AHS significantly increased expression of the neuronal activation marker pERK in colon sensory neurons and augmented noxious CRD-induced behavioral responses. After three consecutive daily intracolonic AHS treatments, mice were hypersensitive to CRD 4-15 days after the first treatment. In complementary single fiber recordings in vitro, the proportion of serosal class afferents increased at day 4; the proportion of MIAs decreased, and muscular class stretch-sensitive afferents were sensitized at days 11-15 in mice receiving AHS. These results indicate that luminal hypertonicity and acidity, two outcomes of carbohydrate malabsorption, can induce colorectal hypersensitivity to distension by altering the excitability and relative proportions of colorectal afferents, suggesting the potential involvement of these factors in the development of abdominal pain.
Assuntos
Colo , Hipersensibilidade , Intolerância à Lactose/fisiopatologia , Mecanotransdução Celular/fisiologia , Reto , Fibras Aferentes Viscerais/fisiologia , Administração Retal , Animais , Comportamento Animal/fisiologia , Colo/inervação , Colo/fisiopatologia , Dilatação/psicologia , Hipersensibilidade/etiologia , Hipersensibilidade/fisiopatologia , Mecanorreceptores/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Estimulação Física/métodos , Reto/inervação , Reto/fisiopatologia , Solução Salina Hipertônica/administração & dosagemRESUMO
Inflammation of the distal bowel is often associated with abdominal pain and hypersensitivity, but whether and which colorectal afferents contribute to the hypersensitivity is unknown. Using a mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, we investigated colorectal hypersensitivity following intracolonic TNBS and associated changes in colorectum and afferent functions. C57BL/6 mice were treated intracolonically with TNBS or saline. Visceromotor responses to colorectal distension (15-60 mmHg) were recorded over 8 wk in TNBS- and saline-treated (control) mice. In other mice treated with TNBS or saline, colorectal inflammation was assessed by myeloperoxidase assay and immunohistological staining. In vitro single-fiber recordings were conducted on both TNBS and saline-treated mice to assess colorectal afferent function. Mice exhibited significant colorectal hypersensitivity through day 14 after TNBS treatment that resolved by day 28 with no resensitization through day 56. TNBS induced a neutrophil- and macrophage-based colorectal inflammation as well as loss of nerve fibers, all of which resolved by days 14-28. Single-fiber recordings revealed a net increase in afferent drive from stretch-sensitive colorectal afferents at day 14 post-TNBS and reduced proportions of mechanically insensitive afferents (MIAs) at days 14-28. Intracolonic TNBS-induced colorectal inflammation was associated with the development and recovery of hypersensitivity in mice, which correlated with a transient increase and recovery of sensitization of stretch-sensitive colorectal afferents and MIAs. These results indicate that the development and maintenance of colorectal hypersensitivity following inflammation are mediated by peripheral drive from stretch-sensitive colorectal afferents and a potential contribution from MIAs.
Assuntos
Colite , Colo , Hipersensibilidade , Reto , Ácido Trinitrobenzenossulfônico , Fibras Aferentes Viscerais/fisiologia , Administração Retal , Animais , Colite/etiologia , Colite/metabolismo , Colite/fisiopatologia , Colo/inervação , Colo/fisiopatologia , Modelos Animais de Doenças , Hipersensibilidade/etiologia , Hipersensibilidade/fisiopatologia , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Inflamação/induzido quimicamente , Mecanorreceptores/fisiologia , Mecanotransdução Celular , Camundongos , Camundongos Endogâmicos C57BL , Estimulação Física/métodos , Reto/inervação , Reto/fisiologia , Reto/fisiopatologia , Solução Salina Hipertônica/administração & dosagem , Fatores de Tempo , Ácido Trinitrobenzenossulfônico/administração & dosagem , Ácido Trinitrobenzenossulfônico/metabolismoRESUMO
Irritable bowel syndrome (IBS) is characterized as functional because a pathobiological cause is not readily apparent. Considerable evidence, however, documents that sensitizing proinflammatory and lipotoxic lipids, mast cells and their products, tryptases, enteroendocrine cells, and mononuclear phagocytes and their receptors are increased in tissues of IBS patients with colorectal hypersensitivity. It is also clear from recordings in animals of the colorectal afferent innervation that afferents exhibit long-term changes in models of persistent colorectal hypersensitivity. Such changes in afferent excitability and responses to mechanical stimuli are consistent with relief of discomfort and pain in IBS patients, including relief of referred abdominal hypersensitivity, upon intra-rectal instillation of local anesthetic. In the aggregate, these experimental outcomes establish the importance of afferent drive in IBS, consistent with a larger literature with respect to other chronic conditions in which pain is a principal complaint (e.g., neuropathic pain, painful bladder syndrome, fibromyalgia). Accordingly, colorectal afferents and the environment in which these receptive endings reside constitute the focus of this review. That environment includes understudied and incompletely understood contributions from immune-competent cells resident in and recruited into the colorectum. We close this review by highlighting deficiencies in existing knowledge and identifying several areas for further investigation, resolution of which we anticipate would significantly advance our understanding of neural and neuro-immune contributions to IBS pain and hypersensitivity.
Assuntos
Síndrome do Intestino Irritável/fisiopatologia , Animais , Doença Crônica , Colo/imunologia , Colo/inervação , Colo/fisiopatologia , Citocinas/imunologia , Feminino , Humanos , Síndrome do Intestino Irritável/complicações , Síndrome do Intestino Irritável/imunologia , Linfócitos/imunologia , Macrófagos/imunologia , Masculino , Mastócitos/imunologia , Camundongos , Inflamação Neurogênica/imunologia , Inflamação Neurogênica/fisiopatologia , Dor/etiologia , Ratos , Reto/imunologia , Reto/inervação , Reto/fisiopatologiaRESUMO
Afferent input contributes significantly to the pain and colorectal hypersensitivity that characterize irritable bowel syndrome. In the present study, we investigated the contributions of mechanically sensitive and mechanically insensitive afferents (MIAs; or silent afferents) to colorectal hypersensitivity. The visceromotor response to colorectal distension (CRD; 15-60 mmHg) was recorded in mice before and for weeks after intracolonic treatment with zymosan or saline. After CRD tests, the distal colorectum with the pelvic nerve attached was removed for single-fiber electrophysiological recordings. Colorectal afferent endings were located by electrical stimulation and characterized as mechanosensitive or not by blunt probing, mucosal stroking, and circumferential stretch. Intracolonic zymosan produced persistent colorectal hypersensitivity (>24 days) associated with brief colorectal inflammation. Pelvic nerve muscular-mucosal but not muscular mechanosensitive afferents recorded from mice with colorectal hypersensitivity exhibited persistent sensitization. In addition, the proportion of MIAs (relative to control) was significantly reduced from 27% to 13%, whereas the proportion of serosal afferents was significantly increased from 34% to 53%, suggesting that MIAs acquired mechanosensitivity. PGP9.5 immunostaining revealed no significant loss of colorectal nerve fiber density, suggesting that the reduction in MIAs is not due to peripheral fiber loss after intracolonic zymosan. These results indicate that colorectal MIAs and sensitized muscular-mucosal afferents that respond to stretch contribute significantly to the afferent input that sustains hypersensitivity to CRD, suggesting that targeted management of colorectal afferent input could significantly reduce patients' complaints of pain and hypersensitivity.
Assuntos
Colo/inervação , Doenças Funcionais do Colo/induzido quimicamente , Mecanotransdução Celular/fisiologia , Neurônios Aferentes/fisiologia , Doenças Retais/induzido quimicamente , Reto/inervação , Animais , Colo/efeitos dos fármacos , Mecanorreceptores/fisiologia , Camundongos , Estimulação Física , Reto/efeitos dos fármacos , Zimosan/toxicidadeRESUMO
The nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) families of growth factors regulate the sensitivity of sensory neurons. The ion channels transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential channel, subfamily A, member 1 (TRPA1), are necessary for development of inflammatory hypersensitivity and are functionally potentiated by growth factors. We have shown previously that inflamed skin exhibits rapid increases in artemin mRNA with slower, smaller increases in NGF mRNA. Here, using mice, we show that, in inflamed colon, mRNA for both growth factors increased with a pattern distinct from that seen in skin. Differences were also seen in the pattern of TRPV1 and TRPA1 mRNA expression in DRG innervating inflamed skin and colon. Growth factors potentiated capsaicin (a specific TRPV1 agonist) and mustard oil (a specific TRPA1 agonist) behavioral responses in vivo, raising the question as to how these growth factors affect individual afferents. Because individual tissues are innervated by afferents with unique properties, we investigated modulation of TRPV1 and TRPA1 in identified afferents projecting to muscle, skin, and colon. Muscle and colon afferents are twice as likely as skin afferents to express functional TRPV1 and TRPA1. TRPV1 and TRPA1 responses were potentiated by growth factors in all afferent types, but compared with skin afferents, muscle afferents were twice as likely to exhibit NGF-induced potentiation and one-half as likely to exhibit artemin-induced potentiation of TRPV1. Furthermore, skin afferents showed no GDNF-induced potentiation of TRPA1, but 43% of muscle and 38% of colon afferents exhibited GDNF-induced potentiation. These results show that interpretation of afferent homeostatic mechanisms must incorporate properties that are specific to the target tissue.
Assuntos
Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Canais de Cátion TRPV/fisiologia , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Cálcio/metabolismo , Toxina da Cólera/metabolismo , Colite/induzido quimicamente , Colite/metabolismo , Colo/metabolismo , Citocinas/genética , Citocinas/metabolismo , Dermatite/etiologia , Dermatite/metabolismo , Modelos Animais de Doenças , Corantes Fluorescentes/metabolismo , Adjuvante de Freund/efeitos adversos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculos/metabolismo , Vias Neurais/fisiologia , Peroxidase/metabolismo , RNA Mensageiro/metabolismo , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Pele/metabolismo , Canal de Cátion TRPA1 , Fatores de Tempo , Aglutininas do Germe de Trigo/metabolismoRESUMO
In adult mammals, the phenotype of half of all pain-sensing (nociceptive) sensory neurons is tonically modulated by growth factors in the glial cell line-derived neurotrophic factor (GDNF) family that includes GDNF, artemin (ARTN) and neurturin (NRTN). Each family member binds a distinct GFRα family co-receptor, such that GDNF, NRTN and ARTN bind GFRα1, -α2, and -α3, respectively. Previous studies revealed transcriptional regulation of all three receptors in following axotomy, possibly in response to changes in growth factor availability. Here, we examined changes in the expression of GFRα1-3 in response to injury in vivo and in vitro. We found that after dissociation of adult sensory ganglia, up to 27% of neurons die within 4 days (d) in culture and this can be prevented by nerve growth factor (NGF), GDNF and ARTN, but not NRTN. Moreover, up-regulation of ATF3 (a marker of neuronal injury) in vitro could be prevented by NGF and ARTN, but not by GDNF or NRTN. The lack of NRTN efficacy was correlated with rapid and near-complete loss of GFRα2 immunoreactivity. By retrogradely-labeling cutaneous afferents in vivo prior to nerve cut, we demonstrated that GFRα2-positive neurons switch phenotype following injury and begin to express GFRα3 as well as the capsaicin receptor, transient receptor potential vanilloid 1(TRPV1), an important transducer of noxious stimuli. This switch was correlated with down-regulation of Runt-related transcription factor 1 (Runx1), a transcription factor that controls expression of GFRα2 and TRPV1 during development. These studies show that NRTN-responsive neurons are unique with respect to their plasticity and response to injury, and suggest that Runx1 plays an ongoing modulatory role in the adult.
Assuntos
Sistema Nervoso Periférico/lesões , Células Receptoras Sensoriais/fisiologia , Pele/inervação , Animais , Sequência de Bases , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Primers do DNA , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Hibridização In Situ , Camundongos , Sistema Nervoso Periférico/fisiopatologia , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Pele/fisiopatologia , Canais de Cátion TRPV/metabolismo , Regulação para CimaRESUMO
BACKGROUND & AIMS: The transient receptor potential (TRP) channels TRPV1 and TRPA1 have each been associated with regulation of efferent properties of primary afferent neurons that initiate neurogenic inflammation and are required for the development of inflammatory hyperalgesia. To evaluate the role of these channels in producing pain during pancreatic inflammation, we studied pancreatic nodose ganglion (NG) and dorsal root ganglion (DRG) sensory neurons (identified by content of retrograde tracer) and behavioral outcomes in a mouse model of acute pancreatitis. METHODS: Pancreatic inflammation was induced by 8 hourly injections of cerulein (50 µg/kg). The extent of inflammation, pancreatic neuron TRP channel expression and function and excitability, and pain-related behaviors were evaluated over the course of the following week. RESULTS: Histology and myeloperoxidase activity confirmed pancreatic inflammation that was associated with increased excitability and messenger RNA expression of the TRP channels in NG and DRG pancreatic neurons. Calcium imaging of pancreatic NG and DRG neurons from mice given cerulein revealed increased responses to TRP agonists. TRPV1 and TRPA1 antagonists attenuated cerulein-induced pain behaviors and pancreatic inflammation; they had a synergistic effect. CONCLUSIONS: Pancreatic inflammation significantly increased the expression and functional properties of TRPV1 and TRPA1, as well as the excitability of pancreatic sensory neurons in vagal and spinal pathways. TRP channel antagonists acted synergistically to reverse pancreatic inflammation and associated pain behaviors; reagents that target interactions between these channels might be developed to reduce pain in patients with acute pancreatitis.
Assuntos
Dor Abdominal , Acetanilidas/farmacologia , Acrilamidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Pancreatite , Purinas/farmacologia , Canais de Cátion TRPV/imunologia , Canais de Potencial de Receptor Transitório/imunologia , Dor Abdominal/tratamento farmacológico , Dor Abdominal/etiologia , Dor Abdominal/imunologia , Doença Aguda , Animais , Comportamento Animal/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Gânglios Espinais/citologia , Gânglios Espinais/imunologia , Gânglios Espinais/metabolismo , Expressão Gênica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gânglio Nodoso/citologia , Gânglio Nodoso/imunologia , Gânglio Nodoso/metabolismo , Pâncreas/imunologia , Pâncreas/inervação , Pancreatite/complicações , Pancreatite/tratamento farmacológico , Pancreatite/imunologia , Técnicas de Patch-Clamp , Canal de Cátion TRPA1 , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Reactive oxygen species (ROS) scavengers have been shown to relieve persistent pain; however, the mechanism is not clearly understood. Superoxide produced from mitochondrial oxidative phosphorylation is considered the major source of ROS in neurons during excitation where mitochondrial superoxide levels are normally controlled by superoxide dismutase (SOD-2). The present study hypothesizes that capsaicin-induced secondary hyperalgesia is a consequence of superoxide build-up in spinal dorsal horn neurons and SOD-2 is a major determinant. To test this hypothesis, the spinal levels of SOD-2 activity, inactivated SOD-2 proteins, and mitochondrial superoxide were measured and correlated to the levels of capsaicin-induced secondary hyperalgesia in mice with and without SOD-2 manipulations. The data suggest that superoxide accumulation is a culprit in the abnormal sensory processing in the spinal cord in capsaicin-induced secondary hyperalgesia. Our studies also support the notion that SOD-2 nitration is a critical mechanism that maintains elevated superoxide levels in the spinal cord after capsaicin treatment. Finally, our findings suggest a therapeutic potential for the manipulation of spinal SOD-2 activity in pain conditions.
Assuntos
Mitocôndrias/metabolismo , Dor/metabolismo , Dor/patologia , Medula Espinal/ultraestrutura , Análise de Variância , Animais , Antioxidantes/metabolismo , Capsaicina/efeitos adversos , Ditiocarb/administração & dosagem , Ditiocarb/análogos & derivados , Relação Dose-Resposta a Droga , Pé/fisiopatologia , Sequestradores de Radicais Livres/administração & dosagem , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Hiperalgesia/prevenção & controle , Masculino , Metaloporfirinas/administração & dosagem , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Dor/induzido quimicamente , Medição da Dor/métodos , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Medula Espinal/patologia , Estilbamidinas , Superóxido Dismutase/deficiência , Superóxido Dismutase/metabolismoRESUMO
Recent studies indicate that reactive oxygen species (ROS) are critically involved in persistent pain primarily through spinal mechanisms, thus suggesting ROS involvement in central sensitization. To investigate ROS involvement in central sensitization, the effects of ROS scavengers and donors on pain behaviors were examined in mice. Capsaicin- induced hyperalgesia was used as a pain model since it has 2 distinctive pain components, primary and secondary hyperalgesia representing peripheral and central sensitization, respectively. Capsaicin (25 microg/5 microl) was injected intradermally into the left hind foot. Foot withdrawal frequencies in response to von Frey filament stimuli were measured and used as an indicator of mechanical hyperalgesia. The production of ROS was examined by using a ROS sensitive dye, MitoSox. Mice developed primary and secondary mechanical hyperalgesia after capsaicin injection. A systemic or intrathecal post-treatment with either phenyl-N-tert-butylnitrone (PBN) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1 oxyl (TEMPOL), ROS scavengers, significantly reduced secondary hyperalgesia, but not primary hyperalgesia, in a dose-dependent manner. Pretreatment with ROS scavengers also significantly reduced the magnitude and duration of capsaicin-induced secondary hyperalgesia. On the other hand, intrathecal injection of tert-butylhydroperoxide (t-BOOH, 5 microl), a ROS donor, produced a transient hyperalgesia in a dose-dependent manner. The number of MitoSox positive dorsal horn neurons was increased significantly after capsaicin treatment. This study suggests that ROS mediates the development and maintenance of capsaicin-induced hyperalgesia in mice, mainly through central sensitization and that the elevation of spinal ROS is most likely due to increased production of mitochondrial superoxides in the dorsal horn neurons.