Dysfunctional Bladder Morphology and Functional Impairments Are Identified in the Alzheimer's Disease APPNL-G-F/NL-G-F Murine Model.

BACKGROUND: While symptoms related to lower urinary tract dysfunction (LUTD) are common in individuals with Alzheimer's disease (AD), pathophysiological links between AD and LUTD remain unclear. OBJECTIVE: This study aimed to investigate whether AD neuropathology would cause autonomic dysfunction along the spinal cord-bladder axis, which could result in alterations in bladder muscle kinetics. METHODS: We utilized APPNL-G-F/NL-G-F knock-in (APP KI) and APPwt/wt (wild-type) mice at two different ages, 4- and 10-month-old, to investigate how AD impacts bladder tissue function by immunohistochemistry, western blotting, and pharmacomyography. RESULTS: We showed that the mucosal layer partially separated from the detrusor in 10-month-old APP KI mouse bladders. Although there was no detectable amyloid deposition in the APP KI bladder, we found amyloid plaques in APP KI lumbar spinal cord. Further immunoblot analysis revealed that tyrosine hydroxylase protein levels were significantly reduced in both 4- and 10-month-old bladder tissues, suggesting reduction of norepinephrine synthesis in APP KI mouse bladders. In contrast, the level of ß2 adrenergic receptor was increased in 4-month-old but not 10-month-old APP KI bladders. In bladder strips, the adrenergic agonist isoproterenol induced increased relaxation in 4- but not 10-month-old APP KI bladders. With 10 Hz electrical field stimulation, 10-month-old APP KI bladder strips were more responsive than wild-type controls, with no differences observed in 4-month-old APP KI bladders. CONCLUSIONS: APP KI mice exhibit LUTD, which is likely arising from amyloid pathology in the spinal cord, and results in maturational declines in presynaptic activity combined with compensatory postsynaptic upregulation.

Loss of resilience contributes to detrusor underactivity in advanced age.

Volume hyposensitivity resulting from impaired sympathetic detrusor relaxation during bladder filling contributes to detrusor underactivity (DU) associated with aging. Detrusor tension regulation provides an adaptive sensory input of bladder volume to the brainstem and is challenged by physiological stressors superimposed upon biological aging. We recently showed that HCN channels have a stabilizing role in detrusor sympathetic relaxation. While mature mice maintain homeostasis in the face of stressors, old mice are not always capable. In old mice, there is a dichotomous phenotype, in which resilient mice adapt and maintain homeostasis, while non-resilient mice fail to maintain physiologic homeostasis. In this DU model, we used cystometry as a stressor to categorize mice as old-responders (old-R, develop a filling/voiding cycle) or old-non-responders (old-NR, fail to develop a filling/voiding cycle; fluctuating high pressures and continuous leaking), while also assessing functional and molecular differences. Lamotrigine (HCN activator)-induced bladder relaxation is diminished in old-NR mice following HCN-blockade. Relaxation responses to NS 1619 were reduced in old-NR mice, with the effect lost following HCN-blockade. However, RNA-sequencing revealed no differences in HCN gene expression and electrophysiology studies showed similar percentage of detrusor myocytes expressing HCN (Ih) current between old-R and old-NR mice. Our murine model of DU further defines a role for HCN, with failure of adaptive recalibration of HCN participation and intensity of HCN-mediated stabilization, while genomic studies show upregulated myofibroblast and fibrosis pathways and downregulated neurotransmitter-degradation pathways in old-NR mice. Thus, the DU phenotype is multifactorial and represents the accumulation of age-associated loss in homeostatic mechanisms.

Cuprizone-mediated demyelination reversibly degrades voiding behavior in mice while sparing brainstem reflex.

Multiple sclerosis (MS) is a chronic, progressively debilitating demyelinating disease of the central nervous system (CNS). Nearly 80% of MS patients experience lower urinary tract dysfunction early in their diagnosis. This significantly affects the quality of life, and in latter stages of disease is a leading cause of hospitalization. Previously, animal models have shown that inflammatory demyelination in the CNS causes profound bladder dysfunction, but the confounding influence of systemic inflammation limits the potential interpretation of the contribution of CNS demyelination to bladder dysfunction. Since the micturition circuit has myelinated neuronal connections in the cortex, brainstem, and spinal cord, we examined alterations in bladder function in the cuprizone model characterized by demyelinating lesions in the cortex and corpus callosum that are independent of T-cell-mediated autoimmunity. Herein, we report that a 4-week dietary cuprizone treatment in C57Bl/6J mice induced alterations in voiding behavior with increased micturition frequency and reduced volume voided, similar to human MS bladder dysfunction. Subsequently, recovery from cuprizone treatment restored normal bladder function. Demyelination and remyelination were confirmed by Luxol Fast Blue staining of the corpus callosum. Additionally, we also determined that an 8-week cuprizone treatment, resulting in chronic demyelination lacking spontaneous remyelination potential, is associated with an exacerbated voiding phenotype. Interestingly, while cuprizone-induced CNS demyelination severely affected conscious (cortical) urinary behavior, the brainstem and spinal cord reflex remained unchanged, as confirmed by urethane-anesthetized cystometry. This is the first study to show that cortical demyelination independent of inflammation can negatively impact urinary function.

Alzheimer's disease amyloidogenesis is linked to altered lower urinary tract physiology.

AIMS: While most Alzheimer's disease (AD) research emphasizes cognitive and behavioral abnormalities, lower urinary tract symptoms (LUTS) are observed in a third of AD patients, contributing to morbidity, poor quality of life, and need for institutionalization. Alzheimer's disease-associated urinary dysfunction (ADUD) has been assumed to be due to cognitive decline alone. While mouse studies have suggested that bladder innervation and voiding behavior may be altered in AD models, technical challenges precluded voiding reflex assessments. This study seeks to establish a mouse model of ADUD, and it seeks to characterize the noncognitive sequelae involved in AD-pathology associated alterations in the voiding reflex. METHODS: Having developed techniques permitting the assessment of bladder volume, pressure, and flow in mice, we now provide evidence of alterations in involuntary bladder control and increased response heterogeneity in a transgenic amyloidosis mouse model of AD using cystometry and tissue pharmacomyography. Tg-APP/PS1DE9 (PA) mice and their wild-type (WT) littermates (n = 6-8 per group) were used before plaque onset in the PA mice (4-6 months) and after plaque accumulation in the PA mice (8-10 months) in comparison to their WT control littermates. RESULTS: Novel findings include data suggestive of sphincteric discoordination, with pharmacological evidence of altered adrenergic mechanisms. CONCLUSIONS: Together, these data highlight the importance of addressing noncognitive sequelae of AD and offer novel translational insights into the debilitating impact of AD on LUTS and incontinence.

The hyperpolarization-activated, cyclic nucleotide-gated channel resides on myocytes in mouse bladders and contributes to adrenergic-induced detrusor relaxation.

Control of urinary continence is predicated on sensory signaling about bladder volume. Bladder sensory nerve activity is dependent on tension, implicating autonomic control over detrusor myocyte activity during bladder filling. Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are known contributors to bladder control, but their mechanism of action is not well understood. The lack of a definitive identification of cell type(s) expressing HCN in the bladder presents a significant knowledge gap. We recently reported a complete transcriptomic atlas of the C57BL/6 mouse bladder showing the dominant HCN paralog in mouse bladder, Hcn1, is limited to a subpopulation of detrusor smooth myocytes (DSMs). Here, we report details of these findings, along with results of patch-clamp experiments, immunohistochemistry, and functional myobath/tension experiments in bladder strips. With the use of a transgenic mouse expressing fluorescence-tagged α-smooth muscle actin, our data confirmed location and function of DSM HCN channels. Despite previous associations of HCN with postulated bladder interstitial cells, neither evidence of specific interstitial cell types nor an association of nonmyocytes with HCN was discovered. We confirm that HCN activation participates in reducing sustained (tonic) detrusor tension via cAMP, with no effect on intermittent (phasic) detrusor activity. In contrast, blockade of HCN increases phasic activity induced by a protein kinase A (PKA) blocker or a large-conductance Ca2+-activated K+ (BK) channel opener. Our findings, therefore, suggest a central role for detrusor myocyte HCN in regulating and constraining detrusor myocyte activity during bladder filling.

Distinct profiles of cellular senescence-associated gene expression in the aged, diseased or injured central nervous system.

The molecular process of cellular senescence, which is known to contribute to aging, has been implicated in several diseases of the central nervous system (CNS). The purpose of this study was to generate an unbiased survey of cellular senescence gene expression with whole brain tissues using a standardized, curated set of 88 genes associated with cellular senescence. We performed a comparative analysis of aged brains with two CNS disease models; the 5xFAD mouse model of Alzheimer's disease, and cuprizone-induced CNS demyelination. Each experimental group could be distinguished from the others by expression of unique subsets of cellular senescence genes, with minimal overlap between each group. Gene ontology analyses identified unique processes within cellular senescence among each group. To examine how these changes translate to the human condition, we interrogated gene expression data from publicly available databases of human aging and AD cases which also corroborated our finding that cellular senescence gene expression changes in AD differ significantly from healthy aging, although the changes in human did not always correlate with the murine models. These data provide important insight on the common and unique global changes in expression of cellular senescence genes in the CNS accompanying aging, injury or disease. Future studies may define, using more refined cellular assays, the specific cellular phenotype differences, and how disparate drivers of unique disease pathologies all seemingly culminate in a common activation of cellular senescence.

Blocking peripheral drive from colorectal afferents by subkilohertz dorsal root ganglion stimulation.

ABSTRACT: Clinical evidence indicates dorsal root ganglion (DRG) stimulation effectively reduces pain without the need to evoke paresthesia. This paresthesia-free anesthesia by DRG stimulation can be promising to treat pain from the viscera, where paresthesia usually cannot be produced. Here, we explored the mechanisms and parameters for DRG stimulation using an ex vivo preparation with mouse distal colon and rectum (colorectum), pelvic nerve, L6 DRG, and dorsal root in continuity. We conducted single-fiber recordings from split dorsal root filaments and assessed the effect of DRG stimulation on afferent neural transmission. We determined the optimal stimulus pulse width by measuring the chronaxies of DRG stimulation to be below 216 µs, indicating spike initiation likely at attached axons rather than somata. Subkilohertz DRG stimulation significantly attenuates colorectal afferent transmission (10, 50, 100, 500, and 1000 Hz), of which 50 and 100 Hz show superior blocking effects. Synchronized spinal nerve and DRG stimulation reveals a progressive increase in conduction delay by DRG stimulation, suggesting activity-dependent slowing in blocked fibers. Afferents blocked by DRG stimulation show a greater increase in conduction delay than the unblocked counterparts. Midrange frequencies (50-500 Hz) are more efficient at blocking transmission than lower or higher frequencies. In addition, DRG stimulation at 50 and 100 Hz significantly attenuates in vivo visceromotor responses to noxious colorectal balloon distension. This reversible conduction block in C-type and Aδ-type afferents by subkilohertz DRG stimulation likely underlies the paresthesia-free anesthesia by DRG stimulation, thereby offering a promising new approach for managing chronic visceral pain.

Animal modeling of lower urinary tract dysfunction associated with multiple sclerosis: Part I: Justification of the mouse model for MS research.

Lower urinary tract symptoms and dysfunction (LUTS/LUTD) contribute to loss of quality of life, morbidity, and need for medical intervention in most patients with multiple sclerosis (MS). Although MS is an inflammatory neurodegenerative disease, clinical manifestations including continence control disorders have traditionally been attributed to the loss of neural signaling due to neurodegeneration. Clinical approaches to MS-LUTS/LUTD have focused on addressing symptoms in the context of urodynamic dysfunctions as pathophysiologic understandings are incomplete. The mouse model provides a useful research platform for the discovery of more detailed molecular, cellular, and tissue-level knowledge of the disease and its clinical manifestations. The aim of this two-part review is to provide a state-of-the-art update on the use of the mouse model for MS research, with a focus on lower urinary tract symptoms. Part I presents a summary of the current understanding of MS pathophysiology, the impact on lower urinary tract symptoms, and briefly introduces the types of mouse models available to study MS. Part II presents the common animal models that are currently available to study MS, their mechanism, relevance to MS-LUTS/LUTD and their urinary pathophysiology, advantages, and disadvantages.

Aging Changes in Bladder Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels Are Associated With Increasing Heterogeneity of Adrenergic/Mucosal Influence on Detrusor Control in the Mouse.

A geroscience-informed approach to the increasing prevalence of bladder control problems in older adults requires understanding the impact of aging on dynamic mechanisms that ensure resilience in response to stressors challenging asymptomatic voluntary control over urine storage and voiding. Bladder control is predicated on sensory neural information about bladder volume. Modulation of volume-induced bladder wall tensions by autonomic and mucosal factors controls neural sensitivity to bladder volume. We hypothesized that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels integrate these factors and thereby mediate adrenergic detrusor tension control. Furthermore, loss of HCN expression compromises that integration and could result in loss of precision of detrusor control. Using a life-span mouse model, reverse transcription quantitative real-time PCR and pharmacologic studies in pretensioned intact and mucosa-denuded bladder strips were made. The dominant hcn1 expression declines with maturation and aging; however, aging is also associated with increased variance around mean values. In strips from Mature animals, isoproterenol had less effect in denuded muscle strips than in intact strips, and HCN blockade diminished isoproterenol responsiveness. With aging, variances about mean response values significantly increased, paralleling hcn1 expression. Our findings support a role for HCN in providing neuroendocrine/paracrine integration and suggest an association of increased heterogeneity of HCN expression in aging with reductions in response precision to neuroendocrine control. The functional implication is an increased risk of dysfunction of brainstem/bladder regulation of neuronal sensitivity to bladder volume. This supports the clinical model of the aging bladder phenotype as an expression of loss of resilience, and not as emerging bladder pathology with aging.

PART 2: Mouse models for multiple sclerosis research.

Lower urinary tract symptoms and dysfunction (LUTS/LUTD) contribute to loss of quality of life, morbidity, and need for medical intervention in most patients with multiple sclerosis (MS). Although MS is an inflammatory neurodegenerative disease, clinical manifestations including continence control disorders have traditionally been attributed to the loss of neural signaling due to neurodegeneration. Clinical approaches to MS-LUTS/LUTD have focused on addressing symptoms in the context of urodynamic dysfunctions as pathophysiologic understandings are incomplete. The mouse model provides a useful research platform for discovery of more detailed molecular, cellular, and tissue-level knowledge of the disease and its clinical manifestations. The aim of this two-part review is to provide a state-of-the-art update on the use of the mouse model for MS research, with a focus on lower urinary tract symptoms. Part I presents a summary of current understanding of MS pathophysiology, the impact on lower urinary tract symptoms, and briefly introduces the types of mouse models available to study MS. Part II presents the common animal models that are currently available to study MS, their mechanism, relevance to MS-LUTS/LUTD and their urinary pathophysiology, advantages and disadvantages.

Should we routinely assess psychological morbidities in idiopathic lower urinary tract dysfunction: ICI-RS 2019?

AIMS: Psychological morbidities play a major role in idiopathic lower urinary tract dysfunction (iLUTD). The aim of the Think Tank (TT) was to discuss the relevance of psychological morbidities in idiopathic LUTD over the life span, including overactive bladder (OAB) or dysfunctional voiding (DV) and methods of assessment. METHODS: The paper is based on a selective review of the literature and in-depth discussions, leading to research recommendations regarding the assessment of psychological morbidities in iLUTD on children and adults held during the TT of the International Consultation on Incontinence Research Society in 2019. RESULTS: Psychological comorbidities affect the health behaviors and treatment outcomes in patients with iLUTD. Both clinically relevant comorbid mental disorders, as well as subclinical psychological symptoms have a major impact and negatively influence incontinence treatment. Research is needed to elucidate mechanisms underlying iLUTD and psychological comorbidities. Clinical studies are needed to determine how perception generation and cognition impacts on the relationship of urinary perceptions, symptoms, and objective urodynamic function. Due to high psychological comorbidity rates, screening with validated, generic questionnaires for emotional and behavioral disorders in children with nocturnal enuresis, daytime urinary incontinence, and fecal incontinence is recommended. Brief screening is recommended for all adults with iLUTD, especially with OAB and DV, who are refractory to treatment. CONCLUSIONS: Due to the high rate and relevance in clinical practice, screening for psychological comorbidities is recommended for all age groups. The research recommendations of this TT may be followed to improve the assessment of psychological morbidities in iLUTD.

Best practices for cystometric evaluation of lower urinary tract function in muriform rodents.

AIMS: Rodent cystometry has provided valuable insights into the impact of the disease, injury, and aging on the cellular and molecular pathways, neurologic processes, and biomechanics of lower urinary tract function. The purpose of this white paper is to highlight the benefits and shortcomings of different experimental methods and strategies and to provide guidance on the proper interpretation of results. METHODS: Literature search, selection of articles, and conclusions based on discussions among a panel of workers in the field. RESULTS: A range of cystometric tests and techniques used to explore biological phenomena relevant to the lower urinary tract are described, the advantages and disadvantages of various experimental conditions are discussed, and guidance on the practical aspects of experimental execution and proper interpretation of results are provided. CONCLUSIONS: Cystometric evaluation of rodents comprises an extensive collection of functional tests that can be performed under a variety of experimental conditions. Decisions regarding which approaches to choose should be determined by the specific questions to be addressed and implementation of the test should follow standardized procedures.

Can we improve our diagnosis of impaired detrusor contractility in women? An ICI-RS 2019 proposal.

INTRODUCTION: Widely accepted consensus terminology and calculations of detrusor contractility in females do not exist but may be useful. We report the output of a proposal session at the International Consultation on Incontinence Research Society meeting 2019, addressing the title topic. METHODS: Three formal presentations and a lively discussion addressed several questions including: which is the optimal cutoff value of female bladder voiding efficiency during uroflow to suspect obstruction or detrusor underactivity? Is there a definition of pure underactive and pure obstructed voiding in females? Is there a place to distinguish those relatively obstructed from those relatively underactive females especially in those cases of equivocal obstruction? Current measures of contractility were reviewed for their usefulness in women. RESULTS: No recommendation for a specific index or calculation can be made based on current knowledge. "Contractility" may be context-dependent regarding clinical care, clinical prognostication, and physiologic research. CONCLUSIONS: This group proposes that context-sensitive definitions of "Contractility" deserve attention by international leadership. Cooperative clinical and physiologic expertise will be needed to achieve this goal. Following initial recommendations based on expert opinion, the development of final definitions and measures of contractility should be iterative, based upon validation studies to be considered as part of the definitional process.

The aging bladder phenotype is not the direct consequence of bladder aging.

AIMS: The prevalence of urinary dysfunction increases with age, yet therapies are often suboptimal. Incomplete understanding of the linkages between system, organ, and tissue domains across lifespan remains a knowledge gap. If tissue-level changes drive the aging bladder phenotype, parallel changes should be observed across these domains. In contrast, a lack of inter-domain correlation across age groups would support the hypothesis that urinary performance is a measure of the physiologic reserve, dependent on centrally-mediated adaptive mechanisms in the aging system. METHODS: Male and female mice across four age groups underwent sequential voiding spot assays, pressure/flow cystometry, bladder strip tension studies, histology, and quantitative PCR analyses. The primary objective of this study was to test the impact of age on the cortical, autonomic, tissue functional and structural, and molecular domains, and identify inter-domain correlations among variables showing significant changes with age within these domains. RESULTS: Behavior revealed diminished peripheral voiding and spot size in aged females. Cystometry demonstrated increased postvoid residual and loss of volume sensitivity, but the preservation of voiding contraction power, with almost half of oldest-old mice failing under cystometric stress. Strip studies revealed no significant differences in adrenergic, cholinergic, or EFS sensitivity. Histology showed increased detrusor and lamina propria thickness, without a change in collagen/muscle ratio. Adrb2 gene expression decreased with age. No consistent inter-domain correlations were found across age groups. CONCLUSIONS: Our findings are consistent with a model in which centrally-mediated adaptive failures to aging stressors are more influential over the aging bladder phenotype than local tissue changes.

HCN as a Mediator of Urinary Homeostasis: Age-Associated Changes in Expression and Function in Adrenergic Detrusor Relaxation.

The Hyperpolarization activated, cyclic nucleotide gated (HCN) channel is a candidate mediator of neuroendocrine influence over detrusor tonus during filling. In other tissues, HCN loss with aging is linked to declines in rhythmicity and function. We hypothesized that HCN has an age-sensitive expression profile and functional role in adrenergic bladder relaxation. HCN was examined in bladders from young (2-6 months) and old (18-24 months) C57BL/6 female mice, using qRT-PCR, RNAScope, and Western blots. Isometric tension studies were conducted using bladder strips from young wild-type (YWT), old wild-type (OWT), and young HCN1 knock-out (YKO) female mice to test the role HCN in effects of ß-adrenergic stimulation. Hcn1 is the dominant HCN isoform RNA in the mouse bladder wall, and is diminished with age. Location of Hcn RNA within the mouse bladder wall is isoform-specific, with HCN1 limited to the detrusor layer. Passively-tensioned YWT bladder strips are relaxed by isoproterenol in the presence of HCN function, where OWT strips are relaxed only in the presence of HCN blockade. HCN has an age-specific expression and function in adrenergic detrusor relaxation in mouse bladder strips.

Report and Research Agenda of the American Geriatrics Society and National Institute on Aging Bedside-to-Bench Conference on Urinary Incontinence in Older Adults: A Translational Research Agenda for a Complex Geriatric Syndrome.

The American Geriatrics Society, with support from the National Institute on Aging and other funders, held its ninth Bedside-to-Bench research conference, entitled "Urinary Incontinence in the Older Adult: A Translational Research Agenda for a Complex Geriatric Syndrome," October 16 to 18, 2016, in Bethesda, Maryland. As part of a conference series addressing three common geriatric syndromes-delirium, sleep and circadian rhythm disturbance, and urinary incontinence-the series highlighted relationships and pertinent clinical and pathophysiological commonalities between these conditions. The conference provided a forum for discussing current epidemiology, basic science, and clinical and translational research on urinary incontinence in older adults; for identifying gaps in knowledge; and for developing a research agenda to inform future investigative efforts. The conference also promoted networking involving emerging researchers and thought leaders in the field of incontinence, aging, and other fields of research, as well as National Institutes of Health program personnel.