Rachelle L. Prantil

Uropathic Observations in Mice Expressing a Constitutively Active Point Mutation in the 5-HT3A Receptor Subunit

Mutant mice with a hypersensitive serotonin (5-HT)3A receptor were generated through targeted exon replacement. A valine to serine mutation (V13′S) in the channel-lining M2 domain of the 5-HT3A receptor subunit rendered the 5-HT3 receptor ∼70-fold more sensitive to serotonin and produced constitutive activity when combined with the 5-HT3B subunit. Mice homozygous for the mutant allele (5-HT3Avs/vs) had decreased levels of 5-HT3A mRNA. Measurements on sympathetic ganglion cells in these mice showed that whole-cell serotonin responses were reduced, and that the remaining 5-HT3 receptors were hypersensitive. Male 5-HT3Avs/vs mice died at 2-3 months of age, and heterozygous (5-HT3Avs/+) males and homozygous mutant females died at 4-6 months of age from an obstructive uropathy. Both male and female 5-HT3A mutant mice had urinary bladder mucosal and smooth muscle hyperplasia and hypertrophy, whereas male mutant mice had additional prostatic smooth muscle and urethral hyperplasia. 5-HT3A mutant mice had marked voiding dysfunction characterized by a loss of micturition contractions with overflow incontinence. Detrusor strips from 5-HT3Avs/vs mice failed to contract to neurogenic stimulation, despite overall normal responses to a cholinergic agonist, suggestive of altered neuronal signaling in mutant mouse bladders. Consistent with this hypothesis, decreased nerve fiber immunoreactivity was observed in the urinary bladders of 5-HT3Avs/vs compared with 5-HT3A wild-type (5-HT3A+/+) mice. These data suggest that persistent activation of the hypersensitive and constitutively active 5-HT3A receptor in vivo may lead to excitotoxic neuronal cell death and functional changes in the urinary bladder, resulting in bladder hyperdistension, urinary retention, and overflow incontinence.

Experimental system for ex vivo measurement of murine aortic stiffness

While vascular stiffness is universally studied using pulse wave velocity, this method overestimates the stiffness of small calibre blood vessels. We have developed and rigorously validated an ex vivo system for measuring stiffness of the mouse aorta. The system consists of a temperature-controlled tissue bath, a pressurization loop and a helium-neon laser micrometer. We harvested thoracic aortas from 8 (n = 56), 11 (n = 6) and 14 (n = 6) week male C57BL/6J mice, mounted them within a tissue chamber and applied an intraluminal pressure waveform while measuring mid vessel outer diameter. Vessel stiffness (E(p), mmHg) was calculated from the pressure-diameter response. Vessels were then stained for endothelial cells, smooth muscle cells, elastin fibres and collagen. The data indicate highly reproducible stiffness measurements in 8 week mice (E(p) = 602.4 +/- 160.2; p = 0.934), age-related stiffening between 11 and 14 week mice (11 week E(p) = 646.9 +/- 62.4, 14 week E(p) = 795.4 +/- 87.5, p = 0.008), and a morphologically intact vessel wall. These results represent the first ex vivo measurements of murine aortic stiffness and illustrate that our methods are feasible and reliable. Since we demonstrate that the system is sensitive to age-related stiffening and does not damage the vessel, this approach is useful for investigating the pathophysiology of vascular disease from biomechanical and histological perspectives.

Effects of Diabetes Mellitus on the Biomechanical Properties of the Female Rat Urethra in the Passive State

Patients with diabetes mellitus (DM) suffer impaired lower urinary tract dysfunctions. The purpose of the current study was to evaluate the effects of DM on the passive biomechanical properties of the female rat urethra. DM was induced by injection of streptozotocin. Urethras were excised and mounted in an ex-vivo testing system. EDTA was added to the bath to inactivate smooth muscle. Continuous outer diameter measurements were made at proximal, middle, and distal portions of the urethra with a laser micrometer during stepwise increases of static, intraurethral pressure (0 to 20 mmHg). Compliance and beta stiffness were calculated from measured data. Healthy urethras served as controls. Statistical comparisons were made via ANOVA. The control tissue was most compliant proximally and decreased significantly along the length. This compliance gradient vanished with DM. A significant decrease in compliance and increase in beta stiffness was noted for 10 wk DM compared to controls. These findings suggest that DM has a large effect on the biomechanical properties of the urethra.Copyright

Effects of diabetes mellitus on the biomechanical properties of the female rat urethra

The effects of diabetes mellitus (DM) on bladder function are well described, but little is known of effects on the outlet function of the urethra. In the present study, biomechanical properties of urethras from DM and healthy female rats were compared. At 3, 5, and 10 weeks following streptozotocin induction of DM, urethras were excised from anesthetized rats maintaining in vivo length, mounted onto tees at in vivo length in an established ex vivo vascular testing system, and maintained in an oxygenated circulating bath of physiological media at 37/spl deg/C. Stepwise intraurethral pressure increments from 0-20 mmHg were applied and diameters were simultaneously recorded at proximal, mid, and distal positions. Urethras from age-matched rats served as controls. There was a proximal-distal decreasing gradient in compliance in normal urethras, and a progressive increase in beta stiffness from 3-10 weeks following DM in the mid and proximal regions to values indistinguishable from the distal. Increasing urethral stiffness in DM results in a collapse of the compliance gradient leading to increased outlet resistance for a bladder in which contractility is already compromised. Thus, DM effects on the urethra interact with those on the bladder to further compromise lower urinary tract function.

Establishing a system for the study of urethral biomechanical function

By applying experimental concepts and principles originally established in the study of blood vessel biomechanics, we are exploring various aspects of lower urinary tract function, dysfunction, and treatment effects on tissue properties and performance. We demonstrate here the feasibility of using a modified ex vivo vascular perfusion apparatus for urethral functional studies. Through administration of various pharmacologic agents, and against applied luminal pressures, we demonstrate control over urethral smooth and striated muscle contractile elements. The ability to measure such physiologic responses provides the basis for performing novel functional studies pertaining to the role of each component in overall urethral function and biomechanics. Therefore, the use of this system to evaluate ex vivo physiology and biomechanical properties will advance the understanding of underlying principles of both normal and abnormal urethral function.