RESUMO
Lower urinary tract or voiding disorders are prevalent across all ages and affect >40% of adults over 40 years old, leading to decreased quality of life and high health care costs. The pontine micturition center (PMC; i.e., Barrington's nucleus) contains a large population of neurons that localize the stress-related neuropeptide, corticotropin-releasing hormone (CRH) and project to neurons in the spinal cord to regulate micturition. How the PMC and CRH-expressing neurons in the PMC control volitional micturition is of critical importance for human voiding disorders. To investigate the specific role of CRH in the PMC, neurons in the PMC-expressing CRH were optogenetically activated during in vivo cystometry in unanesthetized mice of either sex. Optogenetic activation of CRH-PMC neurons led to increased intermicturition interval and voided volume, similar to the altered voiding phenotype produced by social stress. Female mice showed a significantly more pronounced phenotype change compared with male mice. These effects were eliminated by CRH-receptor 1 antagonist pretreatment. Optogenetic inhibition of CRH-PMC neurons led to an altered voiding phenotype characterized by more frequent voids and smaller voided volumes. Last, in a cyclophosphamide cystitis model of bladder overactivity, optogenetic activation of CRH-PMC neurons returned the voiding pattern to normal. Collectively, our findings demonstrate that CRH from PMC spinal-projecting neurons has an inhibitory function on micturition and is a potential therapeutic target for human disease states, such as voiding postponement, urinary retention, and underactive or overactive bladder.SIGNIFICANCE STATEMENT The pontine micturition center (PMC), which is a major regulator of volitional micturition, is neurochemically heterogeneous, and excitatory neurotransmission derived from PMC neurons is thought to mediate the micturition reflex. In the present study, using optogenetic manipulation of CRH-containing neurons in double-transgenic mice, we demonstrate that CRH, which is prominent in PMC-spinal projections, has an inhibitory function on volitional micturition. Moreover, engaging this inhibitory function of CRH can ameliorate bladder hyperexcitability induced by cyclophosphamide in a model of cystitis. The data underscore CRH as a novel target for the treatment of voiding dysfunctions, which are highly prevalent disease processes in children and adults.
Assuntos
Núcleo de Barrington/fisiologia , Hormônio Liberador da Corticotropina/metabolismo , Micção/fisiologia , Vias Aferentes/fisiologia , Animais , Proteínas Arqueais/genética , Núcleo de Barrington/citologia , Channelrhodopsins/genética , Hormônio Liberador da Corticotropina/genética , Ciclofosfamida/toxicidade , Cistite/induzido quimicamente , Cistite/tratamento farmacológico , Cistite/fisiopatologia , Feminino , Genes Reporter/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/fisiologia , Optogenética , Fotoquímica , Proteínas Recombinantes/genética , Medula Espinal/fisiologia , Urodinâmica , VoliçãoRESUMO
Protein kinase C (PKC) and large conductance Ca(2+)-activated potassium channels (BK) are downregulated in the detrusor smooth muscle (DSM) in partial bladder outlet obstruction (PBOO). DSM from these bladders display increased spontaneous activity. This study examines the involvement of PKC in the regulation of spontaneous and evoked DSM contractions and whether pharmacologic inhibition of PKC in normal DSM contributes to increased detrusor excitability. Results indicate the PKC inhibitor bisindolylmaleimide 1 (Bim-1) prevented a decline in the amplitude of spontaneous DSM contractions over time in vitro, and these contractions persist in the presence of tetrodotoxin. Bim-1 also reduced the basal DSM tone, and the ability to maintain force in response to electrical field stimulation, but did not affect maximum contraction. The PKC activator phorbol-12,13-dibutyrate (PDBu) significantly reduced the amplitude and increased the frequency of spontaneous contractions at low concentrations (10 nM), while causing an increase in force at higher concentrations (1 µM). Preincubation of DSM strips with iberiotoxin prevented the inhibition of spontaneous contractions by PDBu. The BK channel openers isopimaric acid and NS1619 reduced the Bim-1-induced enhancement of spontaneous contractions in DSM strips. Our data suggest that PKC has a biphasic activation profile in the DSM and that it may play an important role in maintaining the quiescent state of the normal bladder during storage through the effects on BK channel, while helping to maintain force required for bladder emptying. The data also suggest that PKC dysfunction, as seen in PBOO, contributes to detrusor overactivity.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Proteína Quinase C/metabolismo , Bexiga Urinária/fisiologia , Animais , Benzimidazóis/farmacologia , Ácidos Carboxílicos/farmacologia , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Fenantrenos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Coelhos , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismoRESUMO
AIMS: We set out to characterize the voiding phenotypes of male mice to a water avoidance stress (WAS) protocol and compare the molecular changes with those induced by surgically induced partial bladder outlet obstruction (pBOO). METHODS: Six-week-old male Swiss Webster mice housed with sibling littermates were individually placed on a platform centered in the middle of a water filled basin for 1 hr daily for 4 weeks. A non stressed cohort of sibling littermates served as controls. Measured end points included voiding frequency, voided volume, bladder mass, and in vivo cystometry. Molecular end points included myosin heavy chain (MHC) isoform distribution by PCR, and nuclear translocation of hypoxia inducible factor (HIF1α) and the nuclear factor of activated T-cells (NFAT) by gel shift assay. These molecular endpoints were compared with samples from male mice undergoing anatomic pBOO. RESULTS: WAS resulted in increased average voided volumes and bladder mass, and a decrease in voiding frequency (P < 0.05). The slower MHC A isoform was only expressed in the pBOO group that developed severe hypertrophy. Gel shift assays revealed substantial increases in HIF1-α nuclear translocation in the group subjected to pBOO that developed severe hypertrophy but minimal changes in the pBOO group that developed minimal hypertrophy and the swim stress groups. CONCLUSIONS: The WAS model induces moderate bladder wall hypertrophy in the absence of any surgical manipulation.
Assuntos
Comportamento Animal , Estresse Psicológico/complicações , Obstrução do Colo da Bexiga Urinária/complicações , Bexiga Urinária/fisiopatologia , Transtornos Urinários/etiologia , Micção , Urodinâmica , Água , Transporte Ativo do Núcleo Celular , Animais , Modelos Animais de Doenças , Ensaio de Desvio de Mobilidade Eletroforética , Hipertrofia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fatores de Transcrição NFATC/metabolismo , Fenótipo , Reação em Cadeia da Polimerase , Estresse Psicológico/genética , Estresse Psicológico/metabolismo , Estresse Psicológico/fisiopatologia , Estresse Psicológico/psicologia , Fatores de Tempo , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Obstrução do Colo da Bexiga Urinária/genética , Obstrução do Colo da Bexiga Urinária/metabolismo , Obstrução do Colo da Bexiga Urinária/fisiopatologia , Transtornos Urinários/genética , Transtornos Urinários/metabolismo , Transtornos Urinários/fisiopatologia , Transtornos Urinários/psicologiaRESUMO
We hypothesized that the calcineurin-nuclear factor of activated T-cells (NFAT) pathway is activated following partial bladder outlet obstruction (pBOO), which would allow for pharmacologic treatment to prevent the ensuing bladder wall hypertrophy. Using a model of pBOO in male mice, we were able to demonstrate increased nuclear importation of the transcription factors NFAT and myocyte enhanching factor 2 both of which are under control of calcineurin in both the whole bladder wall as well as the urothelium. We further confirmed that this pathway was activated using transgenic mice containing an NFAT-luciferase reporter construct. Mice were randomized following pBOO to treatment with or without cyclosporine A (CsA), a known inhibitor of calcineurin. The bladder-to-body mass ratio (mg bladder wt/g body wt) of 0.95 ± 0.03 in shams increased to 3.1 ± 0.35 following pBOO, and it dropped back to 1.7 ± 0.22 in the CsA+ group (P < 0.001). Luciferase values (RLU) of 1,130 ± 133 in shams increased to 2,010 ± 474 following pBOO and were suppressed to 562 ± 177 in the CsA+ group (P < 0.05). The myosin heavy chain mRNA (A/B) isoform ratio of 0.07 ± 0.03 in shams increased to 1.04 ± 0.19 following pBOO but it diminished to 0.24 ± 0.1 in the CsA+ group (P < 0.001). In vitro whole organ physiology studies demonstrated improved responses in those bladders from mice treated with CsA. The mRNAs for all four known calcineurin-responsive NFAT isoforms are expressed in the bladder wall, although NFATc(3) and NFATc(4) predominate. Both NFATc3 and NFATc4 are expressed in urothelial as well as smooth muscle cells. We conclude that pBOO activates the calcineurin-NFAT pathway and that CsA treatment decreased bladder hypertrophy, shifted the pattern of myosin isoform mRNA expression back toward that seen in normal controls, and resulted in improved in vitro whole organ performance.
Assuntos
Calcineurina/farmacologia , Obstrução do Colo da Bexiga Urinária/tratamento farmacológico , Bexiga Urinária/efeitos dos fármacos , Animais , Calcineurina/uso terapêutico , Inibidores de Calcineurina , Ciclosporina/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Cadeias Pesadas de Miosina/metabolismo , Fatores de Transcrição NFATC/biossíntese , Tamanho do Órgão/efeitos dos fármacos , Bexiga Urinária/anatomia & histologiaRESUMO
Prolonged residence of mice in spaceflight is a scientifically robust and ethically ratified model of muscle atrophy caused by continued unloading. Under the Rodent Research Program of the National Aeronautics and Space Administration (NASA), we assayed the large-scale mRNA and metabolomic perturbations in the quadriceps of C57BL/6j male mice that lived in spaceflight (FLT) or on the ground (control or CTR) for approximately 4 weeks. The wet weights of the quadriceps were significantly reduced in FLT mice. Next-generation sequencing and untargeted mass spectroscopic assays interrogated the gene-metabolite landscape of the quadriceps. A majority of top-ranked differentially suppressed genes in FLT encoded proteins from the myosin or troponin families, suggesting sarcomere alterations in space. Significantly enriched gene-metabolite networks were found linked to sarcomeric integrity, immune fitness, and oxidative stress response; all inhibited in space as per in silico prediction. A significant loss of mitochondrial DNA copy numbers in FLT mice underlined the energy deprivation associated with spaceflight-induced stress. This hypothesis was reinforced by the transcriptomic sequencing-metabolomics integrative analysis that showed inhibited networks related to protein, lipid, and carbohydrate metabolism, and adenosine triphosphate (ATP) synthesis and hydrolysis. Finally, we discovered important upstream regulators, which could be targeted for next-generation therapeutic intervention for chronic disuse of the musculoskeletal system. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.
Assuntos
Atrofia Muscular , Músculo Quadríceps/patologia , Voo Espacial , Ausência de Peso , Animais , Masculino , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro , Ausência de Peso/efeitos adversosRESUMO
Several studies have anecdotally reported the occurrence of altered urinary voiding patterns in rodents exposed to social stress. A recent study characterized the urodynamic and central changes in a rat model of social defeat. Here, we describe a similar voiding phenotype induced in mice by social stress and in addition we describe potential molecular mechanisms underlying the resulting bladder wall remodeling. The mechanism leading to the altered voiding habits and underlying bladder phenotype may be relevant to the human syndrome of dysfunctional voiding which is thought to have a psychological component. To better characterize and investigate social stress-induced bladder wall hypertrophy, FVB mice (6 wk old) were randomized to either social stress or control manipulation. The stress involved repeated cycles of a 1-h direct exposure to a larger aggressive C57Bl6 breeder mouse followed by a 23-h period of barrier separation over 4 wk. Social stress resulted in altered urinary voiding patterns suggestive of urinary retention and increased bladder mass. In vivo cystometry revealed an increased volume at micturition with no change in the voiding pressure. Examination of these bladders revealed increased nuclear expression of the transcription factors MEF-2 and NFAT, as well as increased expression of the myosin heavy chain B isoform mRNA. BrdU uptake was increased within the urothelium and lamina propria layers in the social stress group. We conclude that social stress induces urinary retention that ultimately leads to shifts in transcription factors, alterations in myosin heavy chain isoform expression, and increases in DNA synthesis that mediate bladder wall remodeling. Social stress-induced bladder dysfunction in rodents may provide insight into the underlying mechanisms and potential treatment of dysfunctional voiding in humans.
Assuntos
Fatores de Regulação Miogênica/metabolismo , Fatores de Transcrição NFATC/metabolismo , Estresse Psicológico/fisiopatologia , Bexiga Urinária/fisiopatologia , Retenção Urinária/psicologia , Animais , Sinalização do Cálcio , Fatores de Transcrição MEF2 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Retenção Urinária/fisiopatologia , MicçãoRESUMO
Repeated exposure to social stress shifts the voiding phenotype in male mice leading to bladder wall remodeling and is associated with increased expression of the stress neuropeptide, corticotropin-releasing factor (CRF) in Barrington's nucleus neurons. In these studies, we set out to determine if the voiding phenotype could recover upon removal from the stressor. Male mice were exposed for 1h daily to an aggressor and the voiding phenotype was assessed at one month followed by randomization to three groups. One group underwent immediate sacrifice. Two groups were allowed a one month recovery from the social stress exposure with or without the addition of fluoxetine (1.2mg/ml) in their drinking water and repeat voiding patterns were measured prior to sacrifice. Social stress significantly increased bladder mass, bladder mass corrected for body weight, voided volumes, and decreased urinary frequency. The abnormal voiding phenotype persisted after a 1month recovery with no effect from the addition of fluoxetine. CRF mRNA in Barrington's nucleus was increased by social stress and remained elevated following recovery with no effect from the addition of fluoxetine. The mRNA and protein expression for the alpha 1 chains of type 1 and type III collagen was unchanged across all groups suggesting that changes in the extracellular matrix of the bladder are not responsible for the voiding phenotype. This persisting voiding dysfunction correlates with the persistent elevation of CRF mRNA expression in Barrington's nucleus.
Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Comportamento Social , Estresse Psicológico/fisiopatologia , Bexiga Urinária/fisiopatologia , Micção/fisiologia , Animais , Antidepressivos de Segunda Geração/farmacologia , Núcleo de Barrington/efeitos dos fármacos , Núcleo de Barrington/metabolismo , Núcleo de Barrington/patologia , Colágeno/metabolismo , Fluoxetina/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Proteoma , RNA Mensageiro/metabolismo , Distribuição Aleatória , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/patologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/patologia , Micção/efeitos dos fármacosRESUMO
BACKGROUND: Dexamethasone (Dex) limits and all-trans-retinoic acid (RA) promotes alveolarization. While structural changes resulting from such hormonal exposures are known, their functional consequences are unclear. METHODS: Neonatal rats were treated with Dex and/or RA during the first two weeks of life or were given RA after previous exposure to Dex. Morphology was assessed by light microscopy and radial alveolar counts. Function was evaluated by plethysmography at d13, pressure volume curves at d30, and exercise swim testing and arterial blood gases at both d15 and d30. RESULTS: Dex-treated animals had simplified lung architecture without secondary septation. Animals given RA alone had smaller, more numerous alveoli. Concomitant treatment with Dex + RA prevented the Dex-induced changes in septation. While the results of exposure to Dex + RA were sustained, the effects of RA alone were reversed two weeks after treatment was stopped. At d13, Dex-treated animals had increased lung volume, respiratory rate, tidal volume, and minute ventilation. On d15, both RA- and Dex-treated animals had hypercarbia and low arterial pH. By d30, the RA-treated animals resolved this respiratory acidosis, but Dex-treated animals continued to demonstrate blood gas and lung volume abnormalities. Concomitant RA treatment improved respiratory acidosis, but failed to normalize Dex-induced changes in pulmonary function and lung volumes. No differences in exercise tolerance were noted at either d15 or d30. RA treatment after the period of alveolarization also corrected the effects of earlier Dex exposure, but the structural changes due to RA alone were again lost two weeks after treatment. CONCLUSION: We conclude that both RA- and corticosteroid-treatments are associated with respiratory acidosis at d15. While RA alone-induced changes in structure andrespiratory function are reversed, Dex-treated animals continue to demonstrate increased respiratory rate, minute ventilation, tidal and total lung volumes at d30. Concomitant treatment with Dex + RA prevents decreased septation induced by Dex alone and results in correction of hypercarbia. However, these animals continue to have abnormal pulmonary function and lung volumes. Increased septation as a result of RA treatment alone is reversed upon discontinuation of treatment. These data suggest that Dex + RA treatment results in improved gas exchange likely secondary to normalized septation.
Assuntos
Dexametasona/farmacologia , Alvéolos Pulmonares/crescimento & desenvolvimento , Alvéolos Pulmonares/fisiopatologia , Tretinoína/farmacologia , Acidose Respiratória/induzido quimicamente , Acidose Respiratória/patologia , Animais , Animais Recém-Nascidos , Dexametasona/efeitos adversos , Feminino , Pulmão/efeitos dos fármacos , Pulmão/crescimento & desenvolvimento , Pulmão/fisiopatologia , Masculino , Gravidez , Alvéolos Pulmonares/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Testes de Função Respiratória , Tretinoína/efeitos adversosRESUMO
INTRODUCTION: To study the pathophysiology of dysfunctional voiding, we have previously developed a model of stress-induced voiding dysfunction. We have shown that cyclosporine A (CsA), an inhibitor of the Ca(2+)-calmodulin complex, can prevent social stress-induced urinary retention. However, treatment with cyclosporine has not had an effect on the increase in the stress peptide corticotrophin-releasing factor (CRF) in Barrington's nucleus, which is involved in the micturition pathway. OBJECTIVE: We now investigate whether cyclosporine administered after stress can reverse the abnormal voiding phenotype, and whether it has effects on the bladder wall itself, or on the stress response within Barrington's nucleus. MATERIALS AND METHODS: Six-week old Swiss-Webster mice were exposed to aggressor males for 1 h a day, followed by 23 h of barrier separation. In a long-term trial, 1 month of stress was followed by single-cage housing for 6 months. In a separate CsA reversal trial, mice either received CsA in drinking water or had plain drinking water during 1 month of single-cage housing during recovery. Bladder contractile function was examined on a Guth myograph. Nuclear translocation of myocyte enhancing factor (MEF)-2 and NFAT (nuclear factor of activated T cells) in the bladder was assessed using electrophoretic mobility shift assays (EMSAs). The expression of CRF was determined in Barrington's nucleus using in situ hybridization. RESULTS: Voiding dysfunction persisted for up to 6 months after stress exposure while mice recovered in single-cage housing. In the CsA reversal trial, voiding patterns improved when they received CsA in water during single-cage housing following stress, whereas those that underwent single-cage housing alone had persistent abnormal voiding (Fig. A). There was no difference between CRF levels in Barrington's nucleus between reversal groups (p = 0.42) (Fig. B), possibly indicating a direct effect on the bladder rather than a persistent stress effect. There were no differences in the contractility of bladder wall muscle. CsA decreased the nuclear translocation of MEF-2 and NFAT induced by stress (Fig. C,D). CONCLUSION: CsA reverses stress-induced urinary retention, but does not change the stress-induced CRF increase in Barrington's nucleus. Furthermore, bladder smooth muscle contractility is unchanged by CsA; however, there are changes in the levels of the downstream transcription factors MEF-2 and NFAT. We suspect that additional CsA responsive neural changes play a pivotal role in the abnormal voiding phenotype following social stress.