Ex vivo biomechanical, functional, and immunohistochemical alterations of adrenergic responses in the female urethra in a rat model of birth trauma

Abstract

Birth trauma and pelvic injury have been implicated in the etiology of stress urinary incontinence (SUI). This study aimed to assess changes in the biomechanical properties and adrenergic-evoked contractile responses of the rat urethra after simulated birth trauma induced by vaginal distension (VD). Urethras were isolated 4 days after VD and evaluated in our established ex vivo urethral testing system that utilized a laser micrometer to measure the urethral outer diameter at proximal, middle, and distal positions. Segments were precontracted with phenylephrine (PE) and then exposed to intralumenal static pressures ranging from 0 to 20 mmHg to measure urethral compliance. After active assessment, the urethra was rendered passive with EDTA and assessed. Pressure and diameter measurements were recorded via computer. Urethral thickness was measured histologically to calculate circumferential stress-strain response and functional contraction ratio (FCR), a measure of smooth muscle activity. VD proximal urethras exhibited a significantly increased response to PE compared with that in controls. Conversely, proximal VD urethras had significantly decreased circumferential stress and FCR values in the presence of PE, suggesting that VD reduced the ability of the proximal segment to maintain smooth muscle tone at higher pressures and strains. Circumferential stress values for VD middle urethral segments were significantly higher than control values. Histological analyses using antibodies against general (protein gene product 9.5) and sympathetic (tyrosine hydroxylase) nerve markers showed a significant reduction in nerve density in VD proximal and middle urethral segments. These results strongly suggest that VD damages adrenergic nerves and alters adrenergic responses of proximal and middle urethral smooth muscle. Defects in urethral storage mechanisms, involving changes in adrenergic regulation, may contribute to stress urinary incontinence induced by simulated birth trauma.

Fig. 1.

A depiction of the regimen for smooth muscle activation via phenylephrine (PE). Initially, a baseline outer diameter (OD) was achieved at 8 mmHg, and a nitric oxide synthase inhibitor (NOSi) was added to inhibit endogenous release of nitric oxide. PE was added to contract urethral smooth muscle via α adrenergic receptors; representative tracing for control (top) and vaginal distension (VD; bottom) proximal segments is shown. Average %OD response values are summarized in Table 1.

Fig. 2.

Pressure-diameter curves generated in the active state (after precontraction with PE) for the proximal (top, left), middle (top, right), and distal (bottom) segments in control and VD preparations. Error bars represent SE. Proximal control urethras were significantly stiffer in pressures ranging from 14 to 20 mmHg (indicated by *P < 0.05). Middle control urethras were significantly more compliant than VD middle segments from 2 to 12 mmHg (indicated by *P < 0.05).

Fig. 3.

Functional contraction ration (FCR) value for proximal segments for both control and VD urethras. *P < 0.05, significant difference between control and VD for the proximal FCR values at 18 and 20 mmHg. Error bars represent SE.

Fig. 4.

Active circumferential stress-strain response for control (top; n = 7), and VD (bottom; n = 7) urethras. Error bars represent SE.

Fig. 5.

Active proximal and middle circumferential stress values for control and VD groups derived for low circumferential strains in proximal (top) and middle urethral segments. *P < 0.001, significance for comparisons between the control and VD groups at low strains ranging from 0.03 to 0.12 for proximal and the entire low strain range for middle segments. Error bars represent SE.

Fig. 6.

Immunofluorescence staining in control (A and C) and VD (B and D) proximal urethras for PGP 9.5 (A and B) and tyrosine hydroxylase (C and D). Images are partial cross sections of the urethra acquired at ×20 magnification. Positive staining is indicated by white areas in the grayscale images. Quantitative measurements of the staining are provided in Table 2.