Nathan R. Tykocki

VasoTracker 2 is a collection of open‐source tools for blood vessel diameter measurement and vascular physiology research, featuring automated diameter tracking software and low‐cost myography hardware components. This blood vessel analysis platform addresses the need for accessible alternatives to expensive commercial vascular imaging systems by providing comprehensive tools for studying vessel reactivity and endothelial function. The software enables multipoint diameter tracking in branched vessels using advanced edge detection algorithms, supporting brightfield microscopy, fluorescence imaging and ultrasound recordings. Automated pressure protocols enable standardized myogenic tone experiments, and both real‐time and offline analysis of pre‐recorded data are supported. The platform's versatility allows researchers to study vascular dynamics across diverse experimental conditions, from isolated vessel preparations to in vivo imaging applications. For ex vivo applications, VasoTracker 2 includes modular, low‐cost open‐source pressure myograph hardware: a confocal‐compatible vessel chamber and a programmable pressure controller, VasoMoto. These components enable construction of a complete pressure myograph system at a significantly reduced cost compared to commercial alternatives. The hardware integrates seamlessly with the diameter measurement software.VasoTracker 2 provides free software and low‐cost hardware for vessel diameter analysis, broadening access to advanced vascular research tools. The platform's open‐source nature allows researchers to modify and extend the system for specific applications. This flexibility, combined with significant cost savings, benefits vascular researchers worldwide. image Key points: VasoTracker 2 is an open‐source platform that combines versatile vessel diameter tracking software with modular low‐cost hardware components for vascular physiology research. The software enables multipoint diameter analysis across brightfield, fluorescence and ultrasound imaging modalities. The hardware includes a confocal‐compatible vessel chamber and programmable pressure controller (VasoMoto) that can be assembled into a complete pressure myograph system. The open‐source approach provides researchers with accessible tools for advanced vascular physiology experiments.

The urinary bladder must remain distensible enough to expand as urine is produced yet strong enough to expel urine without rupture. Disruption in the arrangement of the bladder wall extracellular matrix (ECM) can lead to alterations in mechanical compliance, disrupting normal function and resulting in bladder dysfunction. Previously, we discovered that the mast cell activator compound 48/80 increased detrusor excitability and bladder wall mechanical compliance, resulting in increased urination frequency in mice in a mast cell-independent manner. While the effects on detrusor excitability depended on urothelial prostaglandin production, the mechanisms by which compound 48/80 altered mechanical compliance remained unclear. This study tests the hypothesis that compound 48/80 increases compliance through activation of matrix metalloproteases (MMPs) in the bladder wall. Using the Pentaplanar Reflected Image Macroscopy (PRIM) System and multiphoton microscopy, we measured ex vivo intravesical pressure, mechanical compliance, wall thickness, and permeability in bladders from C57Bl/6 mice before and after exposure to compound 48/80. Intravesical instillation of compound 48/80 increased mechanical compliance and reduced wall thickness without affecting urothelial permeability. This change in compliance was inhibited by the nonspecific MMP inhibitor doxycycline and the MMP-2 inhibitor ARP100. Bladder MMP-2 activity increased, and TIMP-2 activity decreased, after incubation with compound 48/80 as measured by zymography. Together, these findings suggest that compound 48/80 changes bladder mechanical compliance through ECM breakdown caused by rapid activation of MMP-2 and inhibition of TIMP-2. Moreover, these effects occur without altering urothelial permeability, suggesting a novel receptor-mediated mechanism and not a direct effect of compound 48/80 on MMP-2 or TIMP-2.

VasoTracker 2 is an open-source platform for studying blood vessel dynamics, featuring versatile diameter-tracking software and complementary low-cost hardware components. This system surpasses existing tools through accessible, high-resolution analysis across multiple imaging modalities, enabling comprehensive assessment of vascular dynamics in both real-time and pre-recorded experiments. Advanced algorithms enable multi-point diameter tracking in branched vessels, automated pressure-response protocols, and reliable edge detection. The software can assess vessels imaged by brightfield microscopy, fluorescence imaging, and in ultrasound recordings, supporting diverse applications from isolated vessel studies to in vivo assessment. For ex vivo applications, VasoTracker 2 includes modular open-source hardware components that can be used to create a low-cost pressure myograph system: a confocal-compatible vessel chamber and a programmable pressure controller, VasoMoto. By combining powerful analytical capabilities with an open-access approach, VasoTracker 2 provides free software and low-cost hardware alternatives to commercial systems, democratizing access to advanced vascular research tools for scientists worldwide.

Urothelial cells are first responders to noxious contents of the urine. Our study demonstrates that the urothelium is not only a barrier but also a modulator of urinary bladder smooth muscle phasic activity and contractility independent of immune cell recruitment in response to an inflammatory insult.

Optimal bladder compliance is essential to urinary bladder storage and voiding functions. Calculated as the change in filling volume per change in pressure, bladder compliance is used clinically to characterize changes in bladder wall biomechanical properties that associate with lower urinary tract dysfunction. But because this method calculates compliance without regard to wall structure or wall volume, it gives little insight into the mechanical properties of the bladder wall during filling. Thus, we developed Pentaplanar Reflected Image Macroscopy (PRIM): a novel ex vivo imaging method to accurately calculate bladder wall stress and stretch in real time during bladder filling. The PRIM system simultaneously records intravesical pressure, infused volume, and an image of the bladder in five distinct visual planes. Wall thickness and volume were then measured and used to calculate stress and stretch during filling. As predicted, wall stress was nonlinear; only when intravesical pressure exceeded ~ 15 mmHg did bladder wall stress rapidly increase with respect to stretch. This method of calculating compliance as stress vs stretch also showed that the mechanical properties of the bladder wall remain similar in bladders of varying capacity. This study demonstrates how wall tension, stress and stretch can be measured, quantified, and used to accurately define bladder wall biomechanics in terms of actual material properties and not pressure/volume changes. This method is especially useful for determining how changes in bladder biomechanics are altered in pathologies where profound bladder wall remodeling occurs, such as diabetes and spinal cord injury.

ID 22897 Poster Board 30 Inflammatory hyperalgesia induced by mast cell degranulation releases histamine within the urinary bladder that disrupts the micturition reflex and alters detrusor contractility. Psychological stress also causes lower urinary tract symptoms (frequency, urgency, and hesitancy) in 4-week-old mice with a pathophysiology similar to neurogenic inflammatory hyperalgesia. Most investigations have solely focused on molecular changes to nerve sensitivity instead of direct changes to urinary bladder smooth muscle activity. Recently, our lab found that the urinary bladder smooth muscle of unstressed mice contracted and rapidly desensitized to histamine in an H1 receptor-dependent manner. We hypothesized that social stress increases histamine H1 receptor mRNA expression to prolong histamine-induced urinary bladder smooth contractions.4-week-old C57BL/6 male (N=6) mice were randomly housed with CD1 adult male aggressor mice for 5 minutes, after which both mice were placed in barrier housing for 1 hour. Social stress was repeated with different aggressor mice for 14 days. Afterward, bladders from 6-week-old mice were dissected and cut into strips from non-stressed (N=3) and stressed (N=3) mice to measure isometric contractile force generation and changes in contractility by histamine (200 μM). Individual bladder smooth muscle cells were also dissociated and collected for single-cell qRT-PCR. Counter to our hypothesis, histamine-induced contractions were markedly reduced in stressed as compared to non-stressed mice. Surprisingly, however, single-cell qRT-PCR performed on isolated smooth muscle cells uncovered an upregulation of histamine H3 receptor mRNA in stressed mice that was absent in non-stressed mice. H3 receptors were present and constitutively active in the tissue, as the H3 receptor antagonist ciproxifan (10 μM) recovered the response to histamine in stressed bladders but had no effect in non-stressed bladders. In both conditions histamine-induced contractions rapidly desensitized, implying that H3 receptors do not affect this physiological mechanism. Our data aligns with previous findings that support a role for elevated histamine H3 receptor mRNA in patients with interstitial cystitis. Additionally, our data suggest that the histamine H3 receptor might have a protective role in social stress by preventing histamine-induced changes to smooth muscle contractility in stressed bladders. This research is supported by NIH P20-DK127554 (NRT), NIH R01-DK119615 (NRT & GCM), and NIH R01 Supplement (BMJ).

ID 26334 Poster Board 32 Background: Alteration in wall stiffness and bladder compliance can disrupt normal bladder function and lead to lower urinary tract symptoms (LUTS). Urinary bladder fibrosis due to inflammation is implicated in many clinical cases of LUTS. Mast cell activation represents a key target to understand the fibrotic inflammatory pathways within the bladder wall. Our lab has previously shown that mast cell activation using Compound 48/80 increases mechanical compliance and detrusor contractility, and prostaglandins and matrix metalloproteases (MMPs) released from mast cells can directly alter the mechanical properties of the bladder wall. Blocking the release of prostaglandins blocks the increase in both mechanical compliance and detrusor contractility; however, the specific MMPs responsible for driving changes in compliance are unknown. Thus, in this study we pharmacologically investigated the role of specific MMPs in driving the increase in compliance upon mast cell activation. We hypothesized that MMP-2 is present in the urinary bladder wall and is responsible for the increase in mechanical compliance caused by mast cell degranulation. Methods: Whole mouse bladders from C57Bl/6 (wild-type) mice were dissected and mounted in the custom-designed pentaplanar reflected image macroscopy (PRIM) system for simultaneous measurement of transient pressure events, intravesical pressure, bladder volume, wall stress, and wall stretch during ex vivo bladder filling. Wall compliance was derived as a measure of the stress versus stretch relationship using the pressure-volume curves and associated wall geometry gathered from imaging data. Bladders were exposed to Compound 48/80 (10 μg/ml) in the presence of vehicle, the broad-spectrum MMP inhibitor marimastat (10 μM), or the MMP2 inhibitor ARP-100 (10μM) during ex vivo filling. MMP-2 protein expression in whole urinary bladder protein isolates was also determined using western blot analysis. Results: Western blot analysis showed that MMP-2 is present in the bladder wall in both detrusor and urothelium layers. Both marimastat and ARP-100 completely abolished the increase in compliance caused by Compound 48/80. However, neither altered the increase in amplitude of transient pressure events. Conclusions: These findings suggest that the effects of mast cell activation are dual in nature: mast cell activation causes a release of prostaglandins that directly alter detrusor contractility and also increase wall compliance by activating MMP-2. Increases in excitability and profound bladder wall remodeling also occur in minutes after degranulation, suggesting these mechanisms are initiated rapidly after inflammatory insult. Support/Funding Information: R01DK119615 (NRT) and P20DK127554 (SR and NRT).

A balance between stiffness and compliance is essential to normal bladder function, and changes in the mechanical properties of the bladder wall occur in many bladder pathologies. These changes are often associated with the release of basic secretagogues that in turn drive the release of inflammatory mediators from mast cells. Mast cell degranulation by basic secretagogues is thought to occur by activating an orphan receptor, Mas-related G protein-coupled receptor B2 (Mrgprb2). We explored the effects of the putative mast cell degranulator and Mrgprb2 agonist Compound 48/80 on urinary bladder wall mechanical compliance, smooth muscle contractility, and urodynamics, and if these effects were mast cell dependent. In wild-type mice, Mrgprb2 receptor mRNA was expressed in both the urothelium and smooth muscle layers. Intravesical instillation of Compound 48/80 decreased intermicturition interval and void volume, indicative of bladder overactivity. Compound 48/80 also increased bladder compliance while simultaneously increasing the amplitude and leading slope of transient pressure events during ex vivo filling and these effects were inhibited by the Mrgprb2 antagonist QWF. Surprisingly, all effects of Compound 48/80 persisted in mast cell-deficient mice, suggesting these effects were independent of mast cells. These findings suggest that Compound 48/80 degrades extracellular matrix and increases urinary bladder smooth muscle excitability through activation of Mrgprb2 receptors located outside of mast cells. Thus, the pharmacology and physiology of Mrgprb2 in the urinary bladder is of potential interest and importance in terms of treating lower urinary tract dysfunction.

Optimal bladder compliance is essential to urinary bladder storage and voiding functions. Calculated as the change in filling volume per change in pressure, bladder compliance is used clinically to characterize changes in bladder wall biomechanical properties that associate with lower urinary tract dysfunction. But because this method calculates compliance without regard to wall structure or wall volume, it gives little insight into the mechanical properties of the bladder wall during filling. Thus, we developed Pentaplanar Reflected Image Macroscopy (PRIM): a novel ex vivo imaging method to accurately calculate bladder wall stress and stretch in real time during bladder filling. The PRIM system simultaneously records intravesical pressure, infused volume, and an image of the bladder in 5 distinct visual planes. Wall thickness and volume were then measured and used to calculate stress and stretch during filling. As predicted, wall stress was nonlinear; only when intravesical pressure exceeded ~ 15 mmHg did bladder wall stress rapidly increase with respect to stretch. This method of calculating compliance as stress vs stretch also showed that the mechanical properties of the bladder wall remain similar in bladders of varying capacity. This study demonstrates how wall tension, stress and stretch can be measured, quantified, and used to accurately define bladder wall biomechanics in terms of actual material properties and not pressure/volume changes. This method is especially useful for determining how changes in bladder biomechanics are altered in pathologies where profound bladder wall remodeling occurs, such as diabetes and spinal cord injury.