RESUMO
AIMS: Previous studies have shown that the activin-binding protein follistatin reduces inflammation in several mouse models of colitis. To determine whether follistatin also has a beneficial effect following bladder inflammation, we induced cystitis in mice using cyclophosphamide (CYP) and examined the relationship between bladder hypersensitivity and bladder follistatin expression. METHODS: Adult female C57BL/6 mice were treated with CYP (100 mg/kg) or vehicle (saline) three times over 5 days. Bladder hypersensitivity was assessed by recording the visceromotor response (VMR) to urinary bladder distension and in vitro single-fiber bladder afferent recording. Follistatin gene expression was measured using qRT-PCR. Immunohistochemistry was employed for further characterization. RESULTS: Bladder hypersensitivity was established by day 6 and persisted to day 14 in CYP-treated mice. On day 14, hypersensitivity was accompanied by increases in follistatin gene expression in the bladder. Follistatin-like immunoreactivity colocalized with laminin, and the percentage of structures in the lamina propria that were follistatin-positive was increased in CYP-treated mice. Exogenous follistatin increased VMR and afferent responses to bladder distension in CYP- but not vehicle-treated mice. CONCLUSIONS: Chronic bladder pain following CYP treatment is associated with increased follistatin expression in the bladder. These results suggest a novel, pro-nociceptive role for follistatin in cystitis, in contrast with its proposed therapeutic role in colitis. This protein has exciting potential as a biomarker and therapeutic target for bladder hypersensitivity. Neurourol. Urodynam. 36:286-292, 2017. © 2015 Wiley Periodicals, Inc.
Assuntos
Cistite/genética , Folistatina/genética , Bexiga Urinária/metabolismo , Animais , Biomarcadores/metabolismo , Ciclofosfamida , Cistite/induzido quimicamente , Cistite/metabolismo , Feminino , Folistatina/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Pain involving thoracic, abdominal, or pelvic organs is a common cause for physician consultations, including one-third of chronic pain patients who report that visceral organs contribute to their suffering. Chronic visceral pain conditions are typically difficult to manage effectively, largely because visceral sensory mechanisms and factors that contribute to the pathogenesis of visceral pain are poorly understood. Mechanistic understanding is particularly problematic in "functional" visceral diseases where there is no apparent pathology and pain typically is the principal complaint. We review here the anatomical organization of the visceral sensory innervation that distinguishes the viscera from innervation of all other tissues in the body. The viscera are innervated by two nerves that share overlapping functions, but also possess notably distinct functions. Additionally, the visceral innervation is sparse relative to the sensory innervation of other tissues. Accordingly, visceral sensations tend to be diffuse in character, are typically referred to nonvisceral somatic structures and thus are difficult to localize. Early arguments about whether the viscera were innervated ("sensate") and later, whether innervated by nociceptors, were resolved by advances reviewed here in the anatomical and functional attributes of receptive endings in viscera that contribute to visceral pain (i.e., visceral nociceptors). Importantly, the contribution of plasticity (i.e., sensitization) of peripheral and central visceral nociceptive mechanisms is considered in the context of persistent, chronic visceral pain conditions. The review concludes with an overview of the functional anatomy of visceral pain processing. © 2016 American Physiological Society. Compr Physiol 6:1609-1633, 2016.
Assuntos
Dor Visceral/fisiopatologia , Animais , Humanos , Neurônios Aferentes/fisiologia , Vísceras/inervaçãoRESUMO
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
The sensory innervation of the distal colorectum includes mechanically insensitive afferents (MIAs; â¼25%), which acquire mechanosensitivity in persistent visceral hypersensitivity and thus generate de novo input to the central nervous system. We utilized an optogenetic approach to bypass the process of transduction (generator potential) and focus on transformation (spike initiation) at colorectal MIA sensory terminals, which is otherwise not possible in typical functional studies. From channelrhodopsin2-expressing mice (driven by Advillin-Cre), the distal colorectum with attached pelvic nerve was harvested for ex vivo single-fiber recordings. Afferent receptive fields (RFs) were identified by electrical stimulation and tested for response to mechanical stimuli (probing, stroking, and stretch), and afferents were classified as either MIAs or mechanosensitive afferents (MSAs). All MIA and MSA RFs were subsequently stimulated optically and MIAs were also tested for activation/sensitization with inflammatory soup (IS), acidic hypertonic solution (AHS), and/or bile salts (BS). Responses to pulsed optical stimuli (1-10 Hz) were comparable between MSAs and MIAs whereas 43% of MIAs compared with 86% of MSAs responded tonically to stepped optical stimuli. Tonic-spiking MIAs responded preferentially to AHS (an osmotic stimulus) whereas non-tonic-spiking MIAs responded to IS (an inflammatory stimulus). A significant proportion of MIAs were also sensitized by BS. These results reveal transformation as a critical factor underlying the differences between MIAs (osmosensors vs. inflammatory sensors), revealing a previously unappreciated heterogeneity of MIA endings. The current study draws attention to the sensory encoding of MIA nerve endings that likely contribute to afferent sensitization and thus have important roles in visceral pain.
Assuntos
Colo/inervação , Mecanotransdução Celular , Neurônios Aferentes/fisiologia , Reto/inervação , Animais , Ácidos e Sais Biliares/farmacologia , Colo/citologia , Feminino , Transdução de Sinal Luminoso , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/metabolismo , Optogenética , Pressão Osmótica , Reto/citologia , Rodopsina/genética , Rodopsina/metabolismoRESUMO
Using immunohistochemical techniques, we characterized changes in the expression of several neurochemical markers in lumbar 4-sacral 2 (L4-S2) dorsal root ganglion (DRG) neuron profiles (NPs) and the spinal cord of BALB/c mice after axotomy of the L6 and S1 spinal nerves, major tributaries of the pelvic (targeting pelvic visceral organs) and pudendal (targeting perineum and genitalia) nerves. Sham animals were included. Expression of cyclic AMP-dependent transcription factor 3 (ATF3), calcitonin gene-related peptide (CGRP), transient receptor potential cation channel subfamily V, member 1 (TRPV1), tyrosine hydroxylase (TH) and vesicular glutamate transporters (VGLUT) types 1 and -2 was analysed seven days after injury. L6-S1 axotomy induced dramatic de novo expression of ATF3 in many L6-S1 DRG NPs, and parallel significant downregulations in the percentage of CGRP-, TRPV1-, TH- and VGLUT2-immunoreactive (IR) DRG NPs, as compared to their expression in uninjured DRGs (contralateral L6-S1-AXO; sham mice); VGLUT1 expression remained unaltered. Sham L6-S1 DRGs only showed a small ipsilateral increase in ATF3-IR NPs (other markers were unchanged). L6-S1-AXO induced de novo expression of ATF3 in several lumbosacral spinal cord motoneurons and parasympathetic preganglionic neurons; in sham mice the effect was limited to a few motoneurons. Finally, a moderate decrease in CGRP- and TRPV1-like-immunoreactivities was observed in the ipsilateral superficial dorsal horn neuropil. In conclusion, injury of a mixed visceral/non-visceral nerve leads to considerable neurochemical alterations in DRGs matched, to some extent, in the spinal cord. Changes in these and potentially other nociception-related molecules could contribute to pain due to injury of nerves in the abdominopelvic cavity.
Assuntos
Gânglios Espinais/metabolismo , Neurônios/metabolismo , Nervo Pudendo/metabolismo , Medula Espinal/metabolismo , Fator 3 Ativador da Transcrição/metabolismo , Animais , Axotomia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Regulação para Baixo , Genitália/inervação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neurônios Motores/metabolismo , Pelve/inervação , Períneo/inervação , Canais de Cátion TRPV/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Regulação para Cima , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
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
This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum-nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.
Assuntos
Colo/inervação , Reto/inervação , Células Receptoras Sensoriais/fisiologia , Potenciais de Ação/fisiologia , Animais , Estimulação Elétrica , Masculino , Camundongos , Junção Neuromuscular/fisiologia , Neurônios Aferentes/fisiologia , Dor/fisiopatologia , Pelve , Nervos Esplâncnicos/fisiologiaRESUMO
Stretch-sensitive afferents comprise â¼33% of the pelvic nerve innervation of mouse colorectum, which are activated by colorectal distension and encode visceral nociception. Stretch-sensitive colorectal afferent endings respond tonically to stepped or ramped colorectal stretch, whereas dissociated colorectal dorsal root ganglion neurons generally fail to spike repetitively upon stepped current stimulation. The present study investigated this difference in the neural encoding characteristics between the soma and afferent ending using pharmacological approaches in an in vitro mouse colon-nerve preparation and complementary computational simulations. Immunohistological staining and Western blots revealed the presence of voltage-gated sodium channel (NaV) 1.6 and NaV1.7 at sensory neuronal endings in mouse colorectal tissue. Responses of stretch-sensitive colorectal afferent endings were significantly reduced by targeting NaV1.6 using selective antagonists (µ-conotoxin GIIIa and µ-conotoxin PIIIa) or tetrodotoxin. In contrast, neither selective NaV1.8 (A803467) nor NaV1.7 (ProTX-II) antagonists attenuated afferent responses to stretch. Computational simulation of a colorectal afferent ending that incorporated independent Markov models for NaV1.6 and NaV1.7, respectively, recapitulated the experimental findings, suggesting a necessary role for NaV1.6 in encoding tonic spiking by stretch-sensitive afferents. In addition, computational simulation of a dorsal root ganglion soma showed that, by adding a NaV1.6 conductance, a single-spiking neuron was converted into a tonic spiking one. These results suggest a mechanism/channel to explain the difference in neural encoding characteristics between afferent somata and sensory endings, likely caused by differential expression of ion channels (e.g., NaV1.6) at different parts of the neuron.
Assuntos
Colo/fisiologia , Gânglios Espinais/fisiologia , Mecanorreceptores/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.6/fisiologia , Reto/fisiologia , Potenciais de Ação , Animais , Colo/inervação , Colo/metabolismo , Gânglios Espinais/metabolismo , Masculino , Mecanorreceptores/metabolismo , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.8/fisiologia , Estimulação Física , Reto/inervação , Reto/metabolismoRESUMO
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: A low-level inflammation has been hypothesized to mediate visceral hypersensitivity in functional bowel disorders that persist after or even in the absence of gut inflammation. We aimed to test the efficacy of a steroidal anti-inflammatory treatment, and identify local inflammatory molecules mediating post- and non-inflammatory colorectal hypersensitivity using two mouse models. METHODS: Visceromotor responses to colorectal distension were quantified as a measure of colorectal sensitivity. On day 1, mice received intracolonic saline (control), trinitrobenzenesulfonic acid (postinflammatory on day 15), or acidified hypertonic saline (non-inflammatory). Colorectal sensitivity before (day 10) and after (day 15) 4-day dexamethasone (Dex) treatment was compared, and colonic gene expression of inflammatory molecules was quantified. KEY RESULTS: Dexamethasone effectively inhibited gene expression of inflammatory molecules such as interleukin (IL)-1ß and mast cell protease-1 in the colon, but did not attenuate colorectal hypersensitivity in either model. Gene expression of inflammatory molecules in the colon did not differ between control and the non-inflammatory model, but the postinflammatory model showed increased IL-10 and tight junction protein 2, and decreased IL-6, transforming growth factor (TGF)-ß, a precursor of ß-endorphin, occludin, and mucin 2. While no common molecule explained colorectal hypersensitivity in these models, hypersensitivity was positively correlated with TGF-ß2 mRNA in control, and with IL-1ß, inhibin ßA, and prostaglandin E2 synthase in the Dex-treated postinflammatory model. In the non-inflammatory model, cyclooxygenase-2 mRNA was negatively correlated with colorectal sensitivity. CONCLUSIONS & INFERENCES: These results suggest that persistent functional colorectal hypersensitivity is mediated by condition-specific mediators whose gene expression in the colon is not inevitably sensitive to steroidal anti-inflammatory treatment.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Colo/metabolismo , Hiperalgesia/metabolismo , Inflamação/metabolismo , Dor Visceral/metabolismo , Animais , Colo/efeitos dos fármacos , Dexametasona/farmacologia , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Masculino , Manometria , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da PolimeraseRESUMO
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
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
Using specific riboprobes, we characterized the expression of vesicular glutamate transporter (VGLUT)1-VGLUT3 transcripts in lumbar 4-5 (L4-5) dorsal root ganglions (DRGs) and the thoracolumbar to lumbosacral spinal cord in male BALB/c mice after a 1- or 3-day hindpaw inflammation, or a 7-day sciatic nerve axotomy. Sham animals were also included. In sham and contralateral L4-5 DRGs of injured mice, VGLUT1-, VGLUT2- and VGLUT3 mRNAs were expressed in â¼45%, â¼69% or â¼17% of neuron profiles (NPs), respectively. VGLUT1 was expressed in large and medium-sized NPs, VGLUT2 in NPs of all sizes, and VGLUT3 in small and medium-sized NPs. In the spinal cord, VGLUT1 was restricted to a number of NPs at thoracolumbar and lumbar segments, in what appears to be the dorsal nucleus of Clarke, and in mid laminae III-IV. In contrast, VGLUT2 was present in numerous NPs at all analyzed spinal segments, except the lateral aspects of the ventral horns, especially at the lumbar enlargement, where it was virtually absent. VGLUT3 was detected in a discrete number of NPs in laminae III-IV of the dorsal horn. Axotomy resulted in a moderate decrease in the number of DRG NPs expressing VGLUT3, whereas VGLUT1 and VGLUT2 were unaffected. Likewise, the percentage of NPs expressing VGLUT transcripts remained unaltered after hindpaw inflammation, both in DRGs and the spinal cord. Altogether, these results confirm previous descriptions on VGLUTs expression in adult mice DRGs, with the exception of VGLUT1, whose protein expression was detected in a lower percentage of mouse DRG NPs. A detailed account on the location of neurons expressing VGLUTs transcripts in the adult mouse spinal cord is also presented. Finally, the lack of change in the number of neurons expressing VGLUT1 and VGLUT2 transcripts after axotomy, as compared to data on protein expression, suggests translational rather than transcriptional regulation of VGLUTs after injury.
Assuntos
Gânglios Espinais/metabolismo , Neurônios/metabolismo , Medula Espinal/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/análise , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Axotomia , Membro Posterior , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo , Nervo Isquiático/lesões , Proteína Vesicular 1 de Transporte de Glutamato/análise , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/análise , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/análiseRESUMO
Using patch-clamp techniques, we studied the plasticity of acid-sensing ion channels (ASIC) and transient receptor potential V1 (TRPV1) channel function in dorsal root ganglia (DRG) neurons retrogradely labeled from the bladder. Saline (control) or cyclophosphamide (CYP) was given intraperitoneally on days 1, 3, and 5. On day 6, lumbosacral (LS, L6-S2) or thoracolumbar (TL, T13-L2) DRG were removed and dissociated. Bladders and bladder DRG neurons from CYP-treated rats showed signs of inflammation (greater myeloperoxidase activity; lower intramuscular wall pH) and increased size (whole cell capacitance), respectively, compared with controls. Most bladder neurons (>90%) responded to protons and capsaicin. Protons produced multiphasic currents with distinct kinetics, whereas capsaicin always triggered a sustained response. The TRPV1 receptor antagonist A-425619 abolished capsaicin-triggered currents and raised the threshold of heat-activated currents. Prolonged exposure to an acidic environment (pH range: 7.2 to 6.6) inhibited proton-evoked currents, potentiated the capsaicin-evoked current, and reduced the threshold of heat-activated currents in LS and TL bladder neurons. CYP treatment reduced density but not kinetics of all current components triggered by pH 5. In contrast, CYP-treatment was associated with an increased current density in response to capsaicin in LS and TL bladder neurons. Correspondingly, heat triggered current at a significantly lower temperature in bladder neurons from CYP-treated rats compared with controls. These results reveal that cystitis differentially affects TRPV1- and ASIC-mediated currents in both bladder sensory pathways. Acidification of the bladder wall during inflammation may contribute to changes in nociceptive transmission mediated through the TRPV1 receptor, suggesting a role for TRPV1 in hypersensitivity associated with cystitis.
Assuntos
Canais Iônicos Sensíveis a Ácido/fisiologia , Cistite/fisiopatologia , Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPV/fisiologia , Bexiga Urinária/inervação , Animais , Ciclofosfamida , Cistite/induzido quimicamente , Masculino , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPV/efeitos dos fármacosRESUMO
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
PURPOSE: VGLUTs, which are essential for loading glutamate into synaptic vesicles, are present in various neuronal systems. However, to our knowledge the expression of VGLUTs in neurons innervating the bladder has not yet been analyzed. We studied VGLUT1, VGLUT2 and VGLUT3 in mouse bladder neurons. MATERIALS AND METHODS: We analyzed the expression of VGLUT1, VGLUT2 and calcitonin gene-related peptide by immunohistochemistry in the retrograde labeled primary afferent and autonomic neurons of BALB/c mice after injecting fast blue in the bladder wall. To study VGLUT3 we traced the bladder of transgenic mice, in which VGLUT3 is identified by enhanced green fluorescent protein detection. RESULTS: Most bladder dorsal root ganglion neurons expressed VGLUT2. A smaller percentage of neurons also expressed VGLUT1 or VGLUT3. Co-expression with calcitonin gene-related peptide was only observed for VGLUT2. Occasional VGLUT2 immunoreactive neurons were seen in the major pelvic ganglia. Abundant VGLUT2 immunoreactive nerves were detected in the bladder dome and trigone, and the urethra. VGLUT1 immunoreactive nerves were discretely present. CONCLUSIONS: We present what are to our knowledge novel data on VGLUT expression in sensory and autonomic neurons innervating the mouse bladder. The frequent association of VGLUT2 and calcitonin gene-related peptide in sensory neurons suggests interactions between glutamatergic and peptidergic neurotransmissions, potentially influencing commonly perceived sensations in the bladder, such as discomfort and pain.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Gânglios Espinais/metabolismo , Bexiga Urinária/inervação , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Animais , Sistema Nervoso Autônomo/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/genética , Imunofluorescência , Regulação da Expressão Gênica , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Neurônios Aferentes/metabolismo , Neurônios Aferentes/fisiologia , Sensibilidade e Especificidade , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Bexiga Urinária/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/genéticaRESUMO
Previous studies in rat and mouse documented that a subpopulation of dorsal root ganglion (DRG) neurons innervating non-visceral tissues express tyrosine hydroxylase (TH). Here we studied whether or not mouse DRG neurons retrogradely traced with Fast Blue (FB) from colorectum or urinary bladder also express immunohistochemically detectable TH. The lumbar sympathetic chain (LSC) and major pelvic ganglion (MPG) were included in the analysis. Previously characterized antibodies against TH, norepinephrine transporter type 1 (NET-1) and calcitonin gene-related peptide (CGRP) were used. On average, â¼14% of colorectal and â¼17% of urinary bladder DRG neurons expressed TH and spanned virtually all neuronal sizes, although more often in the medium-sized to small ranges. Also, they were more abundant in lumbosacral than thoracolumbar DRGs, and often coexpressed CGRP. We also detected several TH-immunoreactive (IR) colorectal and urinary bladder neurons in the LSC and the MPG, more frequently in the former. No NET-1-IR neurons were detected in DRGs, whereas the majority of FB-labeled, TH-IR neurons in the LSC and MPG coexpressed this marker (as did most other TH-IR neurons not labeled from the target organs). TH-IR nerve fibers were detected in all layers of the colorectum and the urinary bladder, with some also reaching the basal mucosal cells. Most TH-IR fibers in these organs lacked CGRP. Taken together, we show: (1) that a previously undescribed population of colorectal and urinary bladder DRG neurons expresses TH, often CGRP but not NET-1, suggesting the absence of a noradrenergic phenotype; and (2) that TH-IR axons/terminals in the colon or urinary bladder, naturally expected to derive from autonomic sources, could also originate from sensory neurons.
Assuntos
Colo/inervação , Gânglios Espinais/citologia , Neurônios/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismo , Bexiga Urinária/inervação , Amidinas/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neurônios Aferentes/fisiologia , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Pelve/inervaçãoRESUMO
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.