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Abstract Vincristine (VCR) is one of the most widely used chemotherapy agents in treating pediatric cancer. Nonetheless, it is known to cause dose-dependent neurotoxicity which can impact virtually every organ system. Despite its widespread use, the precise impact of VCR on the lower urinary tract (LUT) remains inadequately elucidated. Our initial clinical and translational investigations suggest a sex-specific influence of childhood VCR exposure on LUT function. Thus, the current study aimed to investigate the late effects of systemic VCR exposure on LUT physiology and the underlying mechanisms, focusing on dosage and male-sex, employing juvenile CD-1 mice as a model. Male mice subjected to VCR exhibited augmented functional bladder capacity accompanied by frequent non-void contractions during awake cystometry, alongside mast cell accumulation within the bladder, compared to the saline-treated control group. Noteworthy functional changes were observed in bladder strips from the VCR group, including decreased nerve-mediated contraction, heightened contractile responses to cholinergic and purinergic agonists, enhanced responsiveness to histamine—primarily via histamine receptor 1 (Hrh1)—and an augmented relaxation effect with compound 48/80 (a mast cell degranulator), relative to the control group. Significant changes in gene expression levels associated with neuroinflammation and nociception were observed in both the bladder and lumbosacral dorsal root ganglia (Ls-DRG) of the VCR group. These findings suggest that VCR exposure during childhood, particularly in males, triggers neuroimmune responses in the bladder and Ls-DRG, amplifying responsiveness to neurotransmitters in the bladder, thereby contributing to LUT dysfunction characterized by a mixed bladder phenotype as a late effect during survivorship.

Purpose: To investigate potential beneficial effects of tocotrienols which have been suggested to inhibit hypoxia-inducible factor (HIF) pathway, on partial bladder outlet obstruction (PBOO)-induced bladder pathology. Materials and Methods: PBOO was surgically created in juvenile male mice. Sham-operated mice were used as controls. Animals received daily oral administration of either tocotrienols (T3) or soybean oil (SBO, vehicle) from day 0 to 13 post-surgery. Bladder function was examined in vivo by void spot assay. At 2 weeks post-surgery, the bladders were subjected to physiological evaluation of detrusor contractility in vitro using bladder strips, histology by H&E staining and collagen imaging, and gene expression analyses by quantitative PCR. Results: A significant increase in the number of small voids was observed after 1 week of PBOO compared to the control groups. At 2 weeks post-surgery, PBOO+SBO mice showed a further increase in the number of small voids, which was not observed in PBOO+T3 group. PBOO-induced decrease in detrusor contractility was similar between two treatments. PBOO induced bladder hypertrophy to the same degree in both SBO and T3 treatment groups, however, fibrosis in the bladder was significantly less prominent in the T3 group than the SBO group following PBOO (1.8- vs. 3.0-fold increase in collagen content compared to the control). Enhanced levels of HIF target genes in the bladders were observed in PBOO+SBO group, but not in PBOO+T3 group compared to the control. Conclusions: Oral tocotrienol treatment reduced the progression of urinary frequency and bladder fibrosis by suppressing HIF pathways triggered by PBOO.

Abstract Vincristine (VCR) is one of the most common chemotherapy agents used in pediatric oncology. Despite the well-known VCR-induced peripheral neuropathy, potential impacts of VCR on lower urinary tract (LUT) function remain poorly defined. We investigated the effects of systemic VCR exposure in childhood on LUT function by using juvenile mice treated with VCR (4 mg/kg) or saline and evaluated at 5 weeks later. VCR induced a decreased urinary frequency with increased functional bladder capacity and non-void contractions. There were no changes in detrusor contractility between the groups. VCR exposure caused sexual dimorphic changes; in females, increased intravesical pressure at micturition and downregulations of a major player in bladder afferent firing, Htr3b, in the bladders, and Cav1.2 in the lumbosacral dorsal root ganglia (Ls-DRG), while male mice displayed increases in bladder compliance and detrusor activity, upregulations of IL-2, Trpa1 and Itga1 in the bladders and neuroinflammation-related genes, P2×4, P2×7, IL-2 and CD68 in the Ls-DRG. These results suggest that that systemic VCR exposure caused sensory neuropathy via sex-dimorphic mechanisms, leading to altered LUT function. These changes might clinically present as gender-specific signs or symptoms of LUT dysfunction, and follow-up urological assessment may be of benefit for pediatric cancer patients treated with VCR.

Abstract Bladder and bowel dysfunction (BBD) is a common yet underdiagnosed paediatric entity that describes lower urinary tract symptoms (LUTS) accompanied by abnormal bowel patterns manifested as constipation and/or encopresis. LUTS usually manifest as urgency, urinary frequency, incontinence, and urinary tract infections (UTI). Despite increasing recognition of BBD as a risk factor for long-term urinary tract problems including recurrent UTI, vesicoureteral reflux, and renal scarring, the mechanisms underlying BBD have been unclear, and treatment remains empirical. We investigated how constipation affects the lower urinary tract function using a juvenile murine model of functional constipation. Following four days of functional constipation, animals developed LUTS including urinary frequency and detrusor overactivity evaluated by awake cystometry. Physiological examination of detrusor function in vitro using isolated bladder strips, demonstrated a significant increase in spontaneous contractions without affecting contractile force in response to electrical field stimulation, carbachol, and KCl. A significant upregulation of serotonin receptors, Htr2a and Htr2c, was observed in the bladders from mice with constipation, paralleled with augmented spontaneous contractions after pre-incubation of the bladder strips with 0.5 µM of serotonin. These results suggest that constipation induced detrusor overactivity and increased excitatory serotonin receptor activation in the urinary bladder, which contributes to the development of BBD.

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.

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.

Abstract Background Previously published results from our laboratory identified a mechano-gated two-pore domain potassium channel, TREK-1, as a main mechanosensor in the smooth muscle of the human urinary bladder. One of the limitations of in vitro experiments on isolated human detrusor included inability to evaluate in vivo effects of TREK-1 on voiding function, as the channel is also expressed in the nervous system, and may modulate micturition via neural pathways. Therefore, in the present study, we aimed to assess the role of TREK-1 channel in bladder function and voiding patterns in vivo by using TREK-1 knockout (KO) mice. Methods Adult C57BL/6 J wild-type (WT, N = 32) and TREK-1 KO (N = 33) mice were used in this study. The overall phenotype and bladder function were evaluated by gene and protein expression of TREK-1 channel, in vitro contractile experiments using detrusor strips in response to stretch and pharmacological stimuli, and cystometry in unanesthetized animals. Results TREK-1 KO animals had an elevated basal muscle tone and enhanced spontaneous activity in the detrusor without detectable changes in bladder morphology/histology. Stretch applied to isolated detrusor strips increased the amplitude of spontaneous contractions by 109% in the TREK-1 KO group in contrast to a 61% increase in WT mice (p ≤ 0.05 to respective baseline for each group). The detrusor strips from TREK-1 KO mice also generated more contractile force in response to electric field stimulation and high potassium concentration in comparison to WT group (p ≤ 0.05 for both tests). However, cystometric recordings from TREK-1 KO mice revealed a significant increase in the duration of the intermicturition interval, enhanced bladder capacity and increased number of non-voiding contractions in comparison to WT mice. Conclusions Our results provide evidence that global down-regulation of TREK-1 channels has dual effects on detrusor contractility and micturition patterns in vivo. The observed differences are likely due to expression of TREK-1 channel not only in detrusor myocytes but also in afferent and efferent neural pathways involved in regulation of micturition which may underly the “mixed” voiding phenotype in TREK-1 KO mice.