Mechanical impact of parturition-related strains on rat pelvic striated sphincters

Abstract

Aims: To define the operational resting sarcomere length (L_s ) of the female rat external urethral sphincter (EUS) and external anal sphincter (EAS) and to determine the mechanism of parturition-related injury of EUS and EAS using a simulated birth injury (SBI) vaginal distention model.

Methods: EUS and EAS of 3-month-old Sprague-Dawley control and injured rats were fixed in situ, harvested, and microdissected for L_s measurements and assessment of ultrastructure. EUS and EAS function was determined at baseline, and immediately and 4 weeks after SBI, using leak point pressure (LPP) and anorectal manometry (ARM), respectively. Operational L _s was compared to species-specific optimal L _s using one sample Student's t test. Data (mean ± SD) were compared between groups and time points using repeated measures one-way analysis of variance, followed by Tukey's post hoc pairwise comparisons, with significance set to 0.05.

Results: The operational resting L_s of both sphincters (EUS: 2.09 ± 0.07 µm, EAS: 2.02 ± 0.03 µm) was significantly shorter than optimal rat L_s of 2.4 µm. Strains imposed on EUS and EAS during SBI resulted in significant sarcomere elongation and disruption, compared with the controls (EUS: 3.09 ± 0.11 µm, EAS: 3.37 ± 0.09 µm). Paralleling structural changes, LPP and ARM measures were significantly lower immediately (LPP: 21.5 ± 1.0 cmH₂ O, ARM: 5.1 ± 2.31 cmH₂ O) and 4 weeks (LPP: 27.7 ± 1.3cmH₂ O, ARM: 2.5 ± 1.0 cmH₂ O) after SBI relative to the baseline (LPP: 43.4 ± 8.5 cmH₂ O, ARM: 8.2 ± 2.0 cmH₂ O); P < 0.05.

Conclusions: Analogous to humans, the short resting L_s of rat EUS and EAS favors their sphincteric function. The insult experienced by these muscles during parturition leads to sarcomere hyperelongation, myofibrillar disruption, and dysfunction of the sphincters long-term.

Keywords: birth injury; external anal sphincter; external urethral sphincter; fecal incontinence; rat; stress urinary incontinence.

Figure 1.. Length-tension curve of the rat skeletal muscle.

A sarcomere is represented schematically by the thick (myosin) and thin (actin) horizontal lines, and vertical lines (Z-disks). The maximum active force is produced at the optimal L_s (2.4 μm in the rat), marked by asterisk. For both external urethral (white rectangle) and anal (black circle) sphincters, the operational resting L_s is shorter than the optimal L_s, placing them on the ascending limb of the length-tension curve. Such muscle design allows a higher active and passive force production in response to small strains.

Figure 2.. Mechanical response of the intrinsic structural components of external urethral (EUS) and external anal (EAS) sphincters to parturition-related strains.

Simulated birth injury using vaginal distention with 3 and 5 mL volumes resulted in substantial sarcomere hyperelongation of both sphincters, compared to sarcomere length (L_s) in uninjured controls (A,B). Intrapartum (IP) L_s was analogous to intact pubic symphysis, validating our experimental approach (C,D). Simulated birth injury performed in rats with disarticulated pubis symphysis (SP), resulted in progressive EUS sarcomere hyperelongation with rising distention volume due to strains imposed along the entire length of EUS (C). Conversely, EAS L_s decreased when pubis symphysis was disarticulated (SP) due to a more rostral position of the distention balloon (D). Results are presented as mean ± standard deviation. N=4/group. *Significantly different P values derived from one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc testing with significance level set to 5%.

Figure 3.. Transmission electron microscopy images of external urethral (EUS) and external anal (EAS) sphincters.

Normal striation pattern with aligned sarcomeres is observed in the control muscles (A,C). Disruption of normal muscle microstructure is observed in response to simulated birth injury, as evident from distortion of Z-lines and misalignment of adjacent sarcomeres outlined by black ovals (B,D). Scale bar is 5 μm.

Figure 4.. In vivo measurement of external urethral sphincter (EUS) function.

Leak point pressures (LPP), determined longitudinally, demonstrate substantial decrease caused by simulated birth injury, compared to uninjured values. LPPs were obtained at baseline (A,B); acutely following simulated birth injury (A,C); and after a 4-week recovery period (A,D). Individual animals are represented by differently shaped and pattern symbols (N=5). The acute and 4-week time points are significantly different compared to baseline (P<0.05). P-values derived from repeated measures one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc testing with significance level set to 5%.

Figure 5.. In vivo measurement of external anal sphincter (EAS) function.

Anorectal manometry, determined longitudinally, demonstrates substantial decrease in pressures generated by EAS contraction, following simulated birth injury relative to uninjured values. Anorectal manometry was performed at baseline (A,B); acutely following simulated birth injury (A,C); and after a 4-week recovery period (A,D). The other functional EAS parameters measured by anorectal manometry did not change (E-G). Individual animals are represented by differently shaped and pattern symbols (N=5). The acute and 4-week time points are significantly different compared to baseline and each other (P<0.05). P-values derived from repeated measures one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc testing with significance level set to 5%.