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1.
Am J Physiol Renal Physiol ; 326(6): F957-F970, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38601986

RESUMO

Stretch-activated two-pore domain K+ (K2P) channels play important roles in many visceral organs, including the urinary bladder. The TWIK-related K+ channel TREK-1 is the predominantly expressed K2P channel in the urinary bladder of humans and rodents. Downregulation of TREK-1 channels was observed in the urinary bladder of patients with detrusor overactivity, suggesting their involvement in the pathogenesis of voiding dysfunction. This study aimed to characterize the long-term effects of TREK-1 on bladder function with global and smooth muscle-specific TREK-1 knockout (KO) mice. Bladder morphology, bladder smooth muscle (BSM) contractility, and voiding patterns were evaluated up to 12 mo of age. Both sexes were included in this study to probe the potential sex differences. Smooth muscle-specific TREK-1 KO mice were used to distinguish the effects of TREK-1 downregulation in BSM from the neural pathways involved in the control of bladder contraction and relaxation. TREK-1 KO mice developed enlarged urinary bladders (by 60.0% for males and by 45.1% for females at 6 mo; P < 0.001 compared with the age-matched control group) and had a significantly increased bladder capacity (by 137.7% at 12 mo; P < 0.0001) and compliance (by 73.4% at 12 mo; P < 0.0001). Bladder strips isolated from TREK-1 KO mice exhibited decreased contractility (peak force after KCl at 6 mo was 1.6 ± 0.7 N/g compared with 3.4 ± 2.0 N/g in the control group; P = 0.0005). The lack of TREK-1 channels exclusively in BSM did not replicate the bladder phenotype observed in TREK-1 KO mice, suggesting a strong neurogenic origin of TREK-1-related bladder dysfunction.NEW & NOTEWORTHY This study compared voiding function and bladder phenotypes in global and smooth muscle-specific TREK-1 KO mice. We found significant age-related changes in bladder contractility, suggesting that the lack of TREK-1 channel activity might contribute to age-related changes in bladder smooth muscle physiology.


Assuntos
Hipertrofia , Camundongos Knockout , Contração Muscular , Músculo Liso , Canais de Potássio de Domínios Poros em Tandem , Bexiga Urinária , Animais , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Domínios Poros em Tandem/deficiência , Bexiga Urinária/fisiopatologia , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Músculo Liso/metabolismo , Músculo Liso/fisiopatologia , Músculo Liso/patologia , Masculino , Feminino , Envelhecimento/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fatores Etários , Micção
2.
PLoS One ; 17(12): e0278918, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36490282

RESUMO

Multiple sclerosis (MS) often leads to the development of neurogenic lower urinary tract symptoms (LUTS). We previously characterized neurogenic bladder dysfunction in a mouse model of MS induced by a coronavirus, mouse hepatitis virus (MHV). The aim of the study was to identify genes and pathways linking neuroinflammation in the central nervous system with urinary bladder (UB) dysfunction to enhance our understanding of the mechanisms underlying LUTS in demyelinating diseases. Adult C57BL/6 male mice (N = 12) received either an intracranial injection of MHV (coronavirus-induced encephalomyelitis, CIE group), or sterile saline (control group). Spinal cord (SC) and urinary bladders (UB) were collected from CIE mice at 1 wk and 4 wks, followed by RNA isolation and NanoString nCounter Neuroinflammation assay. Transcriptome analysis of SC identified a significantly changed expression of >150 genes in CIE mice known to regulate astrocyte, microglia and oligodendrocyte functions, neuroinflammation and immune responses. Two genes were significantly upregulated (Ttr and Ms4a4a), and two were downregulated (Asb2 and Myct1) only in the UB of CIE mice. Siglec1 and Zbp1 were the only genes significantly upregulated in both tissues, suggesting a common transcriptomic link between neuroinflammation in the CNS and neurogenic changes in the UB of CIE mice.


Assuntos
Infecções por Coronavirus , Sintomas do Trato Urinário Inferior , Esclerose Múltipla , Bexiga Urinaria Neurogênica , Animais , Masculino , Camundongos , Sistema Nervoso Central , Coronavirus , Infecções por Coronavirus/complicações , Infecções por Coronavirus/genética , Perfilação da Expressão Gênica , Sintomas do Trato Urinário Inferior/genética , Camundongos Endogâmicos C57BL , Esclerose Múltipla/complicações , Esclerose Múltipla/genética , Esclerose Múltipla/virologia , Vírus da Hepatite Murina/genética , Proteínas de Ligação a RNA , Bexiga Urinária , Bexiga Urinaria Neurogênica/genética
3.
PLoS One ; 17(1): e0262769, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35077502

RESUMO

The study investigated the cellular and molecular mechanisms in the peripheral nervous system (PNS) underlying the symptoms of urologic chronic pelvic pain syndrome (UCPPS) in mice. This work also aimed to test the feasibility of reversing peripheral sensitization in vivo in alleviating UCPPS symptoms. Intravesical instillation of vascular endothelial growth factor A (VEGFA) was used to induce UCPPS-like symptoms in mice. Spontaneous voiding spot assays and manual Von Frey tests were used to evaluate the severity of lower urinary tract symptoms (LUTS) and visceral hypersensitivity in VEGFA-instilled mice. Bladder smooth muscle strip contractility recordings (BSMSC) were used to identify the potential changes in myogenic and neurogenic detrusor muscle contractility at the tissue-level. Quantitative real-time PCR (qPCR) and fluorescent immunohistochemistry were performed to compare the expression levels of VEGF receptors and nociceptors in lumbosacral dorsal root ganglia (DRG) between VEGFA-instilled mice and saline-instilled controls. To manipulate primary afferent activity, Gi-coupled Designer Receptors Exclusively Activated by Designer Drugs (Gi-DREADD) were expressed in lumbosacral DRG neurons of TRPV1-Cre-ZGreen mice via targeted adeno-associated viral vector (AAVs) injections. A small molecule agonist of Gi-DREADD, clozapine-N-oxide (CNO), was injected into the peritoneum (i. p.) in awake animals to silence TRPV1 expressing sensory neurons in vivo during physiological and behavioral recordings of bladder function. Intravesical instillation of VEGFA in the urinary bladders increased visceral mechanical sensitivity and enhanced RTX-sensitive detrusor contractility. Sex differences were identified in the baseline detrusor contractility responses and VEGF-induced visceral hypersensitivity. VEGFA instillations in the urinary bladder led to significant increases in the mRNA and protein expression of transient receptor potential cation channel subfamily A member 1 (TRPA1) in lumbosacral DRG, whereas the expression levels of transient receptor potential cation channel subfamily V member 1 (TRPV1) and VEGF receptors (VEGFR1 and VEGFR2) remained unchanged when compared to saline-instilled animals. Importantly, the VEGFA-induced visceral hypersensitivity was reversed by Gi-DREADD-mediated neuronal silencing in lumbosacral sensory neurons. Activation of bladder VEGF signaling causes sensory neural plasticity and visceral hypersensitivity in mice, confirming its role of an UCPPS biomarker as identified by the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) research studies. Pharmacogenetic inhibition of lumbosacral sensory neurons in vivo completely reversed VEGFA-induced pelvic hypersensitivity in mice, suggesting the strong therapeutic potential for decreasing primary afferent activity in the treatment of pain severity in UCPPS patients.


Assuntos
Analgésicos/uso terapêutico , Dor Crônica/tratamento farmacológico , Região Lombossacral/inervação , Percepção da Dor/efeitos dos fármacos , Dor Pélvica/tratamento farmacológico , Células Receptoras Sensoriais/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Feminino , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Fator A de Crescimento do Endotélio Vascular/farmacologia
4.
Front Cell Neurosci ; 15: 782275, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35095422

RESUMO

Chronic neuropathic pain leads to long-term changes in the sensitivity of both peripheral and central nociceptive neurons. Glial fibrillary acidic protein (GFAP)-positive glial cells are closely associated with the nociceptive neurons including astrocytes in the central nervous system (CNS), satellite glial cells (SGCs) in the sensory ganglia, and non-myelinating Schwann cells (NMSCs) in the peripheral nerves. Central and peripheral GFAP-positive cells are involved in the maintenance of chronic pain through a host of inflammatory cytokines, many of which are under control of the transcription factor nuclear factor κB (NFκB) and the enzyme cyclooxygenase 2 (COX2). To test the hypothesis that inhibiting GFAP-positive glial signaling alleviates chronic pain, we used (1) a conditional knockout (cKO) mouse expressing Cre recombinase under the hGFAP promoter and a floxed COX2 gene to inactivate the COX2 gene specifically in GFAP-positive cells; and (2) a tet-Off tetracycline transactivator system to suppress NFκB activation in GFAP-positive cells. We found that neuropathic pain behavior following spared nerve injury (SNI) significantly decreased in COX2 cKO mice as well as in mice with decreased glial NFκB signaling. Additionally, experiments were performed to determine whether central or peripheral glial NFκB signaling contributes to the maintenance of chronic pain behavior following nerve injury. Oxytetracycline (Oxy), a blood-brain barrier impermeable analog of doxycycline was employed to restrict transgene expression to CNS glia only, leaving peripheral glial signaling intact. Signaling inactivation in central GFAP-positive glia alone failed to exhibit the same analgesic effects as previously observed in animals with both central and peripheral glial signaling inhibition. These data suggest that the NFκB-COX2 signaling pathway in NMSCs is necessary for the maintenance of neuropathic pain in vivo.

5.
Sci Rep ; 10(1): 14181, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843670

RESUMO

Glial fibrillary acidic protein expressing (GFAP+) glia modulate nociceptive neuronal activity in both the peripheral nervous system (PNS) and the central nervous system (CNS). Resident GFAP+ glia in dorsal root ganglia (DRG) known as satellite glial cells (SGCs) potentiate neuronal activity by releasing pro-inflammatory cytokines and neuroactive compounds. In this study, we tested the hypothesis that SGC Gq-coupled receptor (Gq-GPCR) signaling modulates pain sensitivity in vivo using Gfap-hM3Dq mice. Complete Freund's adjuvant (CFA) was used to induce inflammatory pain, and mechanical sensitivity and thermal sensitivity were used to assess the neuromodulatory effect of glial Gq-GPCR activation in awake mice. Pharmacogenetic activation of Gq-GPCR signaling in sensory SGCs decreased heat-induced nociceptive responses and reversed inflammation-induced mechanical allodynia via peripheral adenosine A1 receptor activation. These data reveal a previously unexplored role of sensory SGCs in decreasing afferent excitability. The identified molecular mechanism underlying the analgesic role of SGCs offers new approaches for reversing peripheral nociceptive sensitization.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Hiperalgesia/prevenção & controle , Inflamação/fisiopatologia , Neuroglia/enzimologia , Nociceptividade/fisiologia , Receptor A1 de Adenosina/fisiologia , Receptor Muscarínico M3/fisiologia , Animais , Benzilatos/farmacologia , Clozapina/análogos & derivados , Clozapina/farmacologia , Adjuvante de Freund/toxicidade , Genes Sintéticos , Temperatura Alta , Hiperalgesia/fisiopatologia , Inflamação/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Agonistas Muscarínicos/farmacologia , Neuroglia/fisiologia , Nortropanos/farmacologia , Regiões Promotoras Genéticas , Agonistas do Receptor Purinérgico P1/farmacologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , Receptor A1 de Adenosina/efeitos dos fármacos , Receptor Muscarínico M3/efeitos dos fármacos , Receptor Muscarínico M3/genética , Receptores Acoplados a Proteínas G , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Teofilina/análogos & derivados , Teofilina/farmacologia , Tato , Xantinas/farmacologia
6.
Front Syst Neurosci ; 12: 69, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30687029

RESUMO

Early activation of transcription factors is one of the epigenetic mechanisms contributing to the induction and maintenance of chronic pain states. Previous studies identified the changes in a number of nociception-related genes, such as calcitonin gene-related peptide (CGRP), substance P (SP), and brain-derived neurotropic factor (BDNF) in the pelvic organs after transient colonic inflammation. The gene and protein expression of these neuropeptides could be modulated by transcription factors Methyl-CpG-binding protein 2 (Mecp2) and cAMP response element-binding protein (CREB). In this study, we aimed to evaluate time-dependent changes in the expression levels of Mecp2 and CREB in the lumbosacral (LS) spinal cord and sensory ganglia after inflammation-induced pelvic pain in rat. Adult Sprague-Dawley rats were treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce transient colonic inflammation. LS (L6-S2) spinal cord segments and respective dorsal root ganglias (DRGs) were isolated from control and experimental animals at 1, 2, 6, 24 h and 3 days post-TNBS treatment. Immunohistochemical (IHC) labeling and Western blotting experiments were performed to assess the expression of Mecp2, CREB and their phosphorylated forms. Total Mecp2 expression, but not phosphorylated p-Mecp2 (pS421Mecp2) expression was detected in the cells of the spinal dorsal horn under control conditions. Colonic inflammation triggered a significant decrease in the number of Mecp2-expressing neurons in parallel with elevated numbers of pS421Mecp2-expressing cells at 2 h and 6 h post-TNBS. The majority of Mecp2-positive cells (80 ± 6%) co-expressed CREB. TNBS treatment caused a transient up-regulation of CREB-expressing cells at 1 h post-TNBS only. The number of cells expressing phosphorylated CREB (pS133CREB) did not change at 1 h and 2 h post-TNBS, but was down-regulated by three folds at 6 h post-TNBS. Analysis of DRG sections revealed that the number of Mecp2-positive neurons was up-regulated by TNBS treatment, reaching three-fold increase at 2 h post-TNBS, and eight-fold increase at 6 h post-TNBS (p ≤ 0.05 to control). These data showed early changes in Mecp2 and CREB expression in the dorsal horn of the spinal cord and sensory ganglia after colonic inflammation, suggesting a possible contribution Mecp2 and CREB signaling in the development of visceral hyperalgesia and pelvic pain following peripheral inflammation.

7.
JCI Insight ; 2(2): e90565, 2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-28138563

RESUMO

The sympathetic nervous system (SNS) accelerates heart rate, increases cardiac contractility, and constricts resistance vessels. The activity of SNS efferent nerves is generated by a complex neural network containing neurons and glia. Gq G protein-coupled receptor (Gq-GPCR) signaling in glial fibrillary acidic protein-expressing (GFAP+) glia in the central nervous system supports neuronal function and regulates neuronal activity. It is unclear how Gq-GPCR signaling in GFAP+ glia affects the activity of sympathetic neurons or contributes to SNS-regulated cardiovascular functions. In this study, we investigated whether Gq-GPCR activation in GFAP+ glia modulates the regulatory effect of the SNS on the heart; transgenic mice expressing Gq-coupled DREADD (designer receptors exclusively activated by designer drugs) (hM3Dq) selectively in GFAP+ glia were used to address this question in vivo. We found that acute Gq-GPCR activation in peripheral GFAP+ glia significantly accelerated heart rate and increased left ventricle contraction. Pharmacological experiments suggest that the glial-induced cardiac changes were due to Gq-GPCR activation in satellite glial cells within the sympathetic ganglion; this activation led to increased norepinephrine (NE) release and beta-1 adrenergic receptor activation within the heart. Chronic glial Gq-GPCR activation led to hypotension in female Gfap-hM3Dq mice. This study provides direct evidence that Gq-GPCR activation in peripheral GFAP+ glia regulates cardiovascular functions in vivo.


Assuntos
Gânglios Simpáticos/metabolismo , Frequência Cardíaca , Coração/inervação , Contração Miocárdica , Neuroglia/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adrenalectomia , Animais , Feminino , Gânglios Simpáticos/citologia , Proteína Glial Fibrilar Ácida/metabolismo , Hipotensão/metabolismo , Camundongos , Camundongos Transgênicos , Norepinefrina/metabolismo , Farmacogenética , Receptores Adrenérgicos beta 1/metabolismo , Simpatectomia Química , Sistema Nervoso Simpático/metabolismo
8.
Cell Calcium ; 55(1): 1-16, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24262208

RESUMO

Astrocyte Gq GPCR and IP3 receptor-dependent Ca(2+) elevations occur spontaneously in situ and in vivo. These events vary considerably in size, often remaining confined to small territories of astrocyte processes called "microdomains" and sometimes propagating over longer distances that can include the soma. It has remained unclear whether these events are driven by constitutive (basal) GPCR signaling activity, neuronal action potential-dependent or quantal vesicular release, or some combination of these mechanisms. Here, we applied manipulations to increase or inhibit neuronal vesicular neurotransmitter release together with low-level stimulation of Schaffer collaterals in acute mouse hippocampal slices in an effort to determine the mechanisms underlying spontaneous astrocyte Ca(2+) events. We found no significant change in spontaneous microdomain astrocyte Ca(2+) elevations when neuronal action potentials were significantly enhanced or blocked. The astrocyte Ca(2+) activity was also not affected by inhibitors of group I mGluRs. However, blockade of miniature neurotransmitter release using Bafilomycin A1 significantly reduced the frequency of microdomain astrocyte Ca(2+) elevations. We then tested whether astrocyte Ca(2+) microdomains can be evoked by low intensity SC stimulation. Importantly, microdomains could not be reproduced even using single, low intensity pulses to the SCs at a minimum distance from the astrocyte. Evoked astrocyte Ca(2+) responses most often included the cell soma, were reduced by group I mGluR antagonists, and were larger in size compared to spontaneous Ca(2+) microdomains. Overall, our findings suggest that spontaneous microdomain astrocyte Ca(2+) elevations are not driven by neuronal action potentials but require quantal release of neurotransmitter which cannot be replicated by stimulation of Schaffer collaterals.


Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cálcio/metabolismo , Macrolídeos/farmacologia , Microdomínios da Membrana/metabolismo , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Potenciais de Ação/fisiologia , Animais , Astrócitos/citologia , Sinalização do Cálcio/fisiologia , Inibidores Enzimáticos/farmacologia , Ácido Glutâmico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Neurotransmissores/metabolismo , Técnicas de Patch-Clamp , Células Piramidais/citologia
9.
J Physiol ; 591(22): 5599-609, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24042499

RESUMO

Glial fibrillary acidic protein (GFAP)-expressing cells (GFAP(+) glial cells) are the predominant cell type in the central and peripheral nervous systems. Our understanding of the role of GFAP(+) glial cells and their signalling systems in vivo is limited due to our inability to manipulate these cells and their receptors in a cell type-specific and non-invasive manner. To circumvent this limitation, we developed a transgenic mouse line (GFAP-hM3Dq mice) that expresses an engineered Gq protein-coupled receptor (Gq-GPCR) known as hM3Dq DREADD (designer receptor exclusively activated by designer drug) selectively in GFAP(+) glial cells. The hM3Dq receptor is activated solely by a pharmacologically inert, but bioavailable, ligand (clozapine-N-oxide; CNO), while being non-responsive to endogenous GPCR ligands. In GFAP-hM3Dq mice, CNO administration increased heart rate, blood pressure and saliva formation, as well as decreased body temperature, parameters that are controlled by the autonomic nervous system (ANS). Additionally, changes in activity-related behaviour and motor coordination were observed following CNO administration. Genetically blocking inositol 1,4,5-trisphosphate (IP3)-dependent Ca(2+) increases in astrocytes failed to interfere with CNO-mediated changes in ANS function, locomotor activity or motor coordination. Our findings reveal an unexpectedly broad role of GFAP(+) glial cells in modulating complex physiology and behaviour in vivo and suggest that these effects are not dependent on IP3-dependent increases in astrocytic Ca(2+).


Assuntos
Sistema Nervoso Autônomo/metabolismo , Sistema Nervoso Autônomo/fisiologia , Neuroglia/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Cálcio/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Ligantes , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Atividade Motora/fisiologia , Neuroglia/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Transdução de Sinais/fisiologia
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