Effect of nonsteroidal anti-inflammatory drugs on pelvic floor muscle regeneration in a preclinical birth injury rat model.

BACKGROUND: Pelvic floor muscle injury is a common consequence of vaginal childbirth. Nonsteroidal anti-inflammatory drugs are widely used postpartum analgesics. Multiple studies have reported negative effects of these drugs on limb muscle regeneration, but their impact on pelvic floor muscle recovery following birth injury has not been explored. OBJECTIVE: Using a validated rat model, we assessed the effects of nonsteroidal anti-inflammatory drug on acute and longer-term pelvic floor muscle recovery following simulated birth injury. STUDY DESIGN: Three-month old Sprague Dawley rats were randomly assigned to the following groups: (1) controls, (2) simulated birth injury, (3) simulated birth injury+nonsteroidal anti-inflammatory drug, or (4) nonsteroidal anti-inflammatory drug. Simulated birth injury was induced using a well-established vaginal balloon distension protocol. Ibuprofen was administered in drinking water (0.2 mg/mL), which was consumed by the animals ad libitum. Animals were euthanized at 1, 3, 5, 7, 10, and 28 days after birth injury/ibuprofen administration. The pubocaudalis portion of the rat levator ani, which, like the human pubococcygeus, undergoes greater parturition-associated strains, was harvested (N=3-9/time point/group). The cross-sectional areas of regenerating (embryonic myosin heavy chain+) and mature myofibers were assessed at the acute and 28-day time points, respectively. The intramuscular collagen content was assessed at the 28-day time point. Myogenesis was evaluated using anti-Pax7 and anti-myogenin antibodies to identify activated and differentiated muscle stem cells, respectively. The overall immune infiltrate was assessed using anti-CD45 antibody. Expression of genes coding for pro- and anti-inflammatory cytokines was assessed by quantitative reverse transcriptase polymerase chain reaction at 3, 5, and 10 days after injury. RESULTS: The pubocaudalis fiber size was significantly smaller in the simulated birth injury+nonsteroidal anti-inflammatory drug compared with the simulated birth injury group at 28 days after injury (P<.0001). The median size of embryonic myosin heavy chain+ fibers was also significantly reduced, with the fiber area distribution enriched with smaller fibers in the simulated birth injury+nonsteroidal anti-inflammatory drug group relative to the simulated birth injury group at 3 days after injury (P<.0001), suggesting a delay in the onset of regeneration in the presence of nonsteroidal anti-inflammatory drugs. By 10 days after injury, the median embryonic myosin heavy chain+ fiber size in the simulated birth injury group decreased from 7 days after injury (P<.0001) with a tight cross-sectional area distribution, indicating nearing completion of this state of regeneration. However, in the simulated birth injury+nonsteroidal anti-inflammatory drug group, the size of embryonic myosin heavy chain+ fibers continued to increase (P<.0001) with expansion of the cross-sectional area distribution, signifying a delay in regeneration in these animals. Nonsteroidal anti-inflammatory drugs decreased the muscle stem cell pool at 7 days after injury (P<.0001) and delayed muscle stem cell differentiation, as indicated by persistently elevated number of myogenin+ cells 7 days after injury (P<.05). In contrast, a proportion of myogenin+ cells returned to baseline by 5 days after injury in the simulated birth injury group. The analysis of expression of genes coding for pro- and anti-inflammatory cytokines demonstrated only transient elevation of Tgfb1 in the simulated birth injury+nonsteroidal anti-inflammatory drug group at 5 but not at 10 days after injury. Consistently with previous studies, nonsteroidal anti-inflammatory drug administration following simulated birth injury resulted in increased deposition of intramuscular collagen relative to uninjured animals. There were no significant differences in any outcomes of interest between the nonsteroidal anti-inflammatory drug group and the unperturbed controls. CONCLUSION: Nonsteroidal anti-inflammatory drugs negatively impacted pelvic floor muscle regeneration in a preclinical simulated birth injury model. This appears to be driven by the negative impact of these drugs on pelvic muscle stem cell function, resulting in delayed temporal progression of pelvic floor muscle regeneration following birth injury. These findings provide impetus to investigate the impact of postpartum nonsteroidal anti-inflammatory drug administration on muscle regeneration in women at high risk for pelvic floor muscle injury.

Mechanisms governing protective pregnancy-induced adaptations of the pelvic floor muscles in the rat preclinical model.

BACKGROUND: The intrinsic properties of pelvic soft tissues in women who do and do not sustain birth injuries are likely divergent. However, little is known about this. Rat pelvic floor muscles undergo protective pregnancy-induced structural adaptations-sarcomerogenesis and increase in intramuscular collagen content-that protect against birth injury. OBJECTIVE: We aimed to test the following hypotheses: (1) the increased mechanical load of a gravid uterus drives antepartum adaptations; (2) load-induced changes are sufficient to protect pelvic muscles from birth injury. STUDY DESIGN: The independent effects of load uncoupled from the hormonal milieu of pregnancy were tested in 3- to 4-month-old Sprague-Dawley rats randomly divided into the following 4 groups, with N of 5 to 14 per group: (1) load-/pregnancy hormones- (controls), (2) load+/pregnancy hormones-, (3) reduced load/pregnancy hormones+, and (4) load+/pregnancy hormones+. Mechanical load of a gravid uterus was simulated by weighing uterine horns with beads similar to fetal rat size and weight. A reduced load was achieved by unilateral pregnancy after unilateral uterine horn ligation. To assess the acute and chronic phases required for sarcomerogenesis, the rats were sacrificed at 4 hours or 21 days after bead loading. The coccygeus, iliocaudalis, pubocaudalis, and nonpelvic tibialis anterior musles were harvested for myofiber and sarcomere length measurements. The intramuscular collagen content was assessed using a hydroxyproline assay. An additional 20 load+/pregnancy hormones- rats underwent vaginal distention to determine whether the load-induced changes are sufficient to protect from mechanical muscle injury in response to parturition-associated strains of various magnitude. The data, compared using 2-way repeated measures analysis of variance followed by pairwise comparisons, are presented as mean±standard error of mean. RESULTS: An acute increase in load resulted in significant pelvic floor muscle stretch, accompanied by an acute increase in sarcomere length compared with nonloaded control muscles (coccygeus: 2.69±0.03 vs 2.30±0.06 µm, respectively, P<.001; pubocaudalis: 2.71±0.04 vs 2.25±0.03 µm, respectively, P<.0001; and iliocaudalis: 2.80±0.06 vs 2.35±0.04 µm, respectively, P<.0001). After 21 days of sustained load, the sarcomeres returned to operational length in all pelvic muscles (P>.05). However, the myofibers remained significantly longer in the load+/pregnancy hormones- than the load-/pregnancy hormones- in coccygeus (13.33±0.94 vs 9.97±0.26 mm, respectively, P<.0001) and pubocaudalis (21.20±0.52 vs 19.52±0.34 mm, respectively, P<.04) and not different from load+/pregnancy hormones+ (12.82±0.30 and 22.53±0.32 mm, respectively, P>.1), indicating that sustained load-induced sarcomerogenesis in these muscles. The intramuscular collagen content in the load+/pregnancy hormones- group was significantly greater relative to the controls in coccygeus (6.55±0.85 vs 3.11±0.47 µg/mg, respectively, P<.001) and pubocaudalis (5.93±0.79 vs 3.46±0.52 µg/mg, respectively, P<.05) and not different from load+/pregnancy hormones+ (7.45±0.65 and 6.05±0.62 µg/mg, respectively, P>.5). The iliocaudalis required both mechanical and endocrine cues for sarcomerogenesis. The tibialis anterior was not affected by mechanical or endocrine alterations. Despite an equivalent extent of adaptations, load-induced changes were only partially protective against sarcomere hyperelongation. CONCLUSION: Load induces plasticity of the intrinsic pelvic floor muscle components, which renders protection against mechanical birth injury. The protective effect, which varies between the individual muscles and strain magnitudes, is further augmented by the presence of pregnancy hormones. Maximizing the impact of mechanical load on the pelvic floor muscles during pregnancy, such as with specialized pelvic floor muscle stretching regimens, is a potentially actionable target for augmenting pregnancy-induced adaptations to decrease birth injury in women who may otherwise have incomplete antepartum muscle adaptations.

Muscle stem cells and fibro-adipogenic progenitors in female pelvic floor muscle regeneration following birth injury.

Pelvic floor muscle (PFM) injury during childbirth is a key risk factor for pelvic floor disorders that affect millions of women worldwide. Muscle stem cells (MuSCs), supported by the fibro-adipogenic progenitors (FAPs) and immune cells, are indispensable for the regeneration of injured appendicular skeletal muscles. However, almost nothing is known about their role in PFM regeneration following birth injury. To elucidate the role of MuSCs, FAPs, and immune infiltrate in this context, we used radiation to perturb cell function and followed PFM recovery in a validated simulated birth injury (SBI) rat model. Non-irradiated and irradiated rats were euthanized at 3,7,10, and 28 days post-SBI (dpi). Twenty-eight dpi, PFM fiber cross-sectional area (CSA) was significantly lower and the extracellular space occupied by immune infiltrate was larger in irradiated relative to nonirradiated injured animals. Following SBI in non-irradiated animals, MuSCs and FAPs expanded significantly at 7 and 3 dpi, respectively; this expansion did not occur in irradiated animals at the same time points. At 7 and 10 dpi, we observed persistent immune response in PFMs subjected to irradiation compared to non-irradiated injured PFMs. CSA of newly regenerated fibers was also significantly smaller following SBI in irradiated compared to non-irradiated injured PFMs. Our results demonstrate that the loss of function and decreased expansion of MuSCs and FAPs after birth injury lead to impaired PFM recovery. These findings form the basis for further studies focused on the identification of novel therapeutic targets to counteract postpartum PFM dysfunction and the associated pelvic floor disorders.

Autonomous Extracellular Matrix Remodeling Controls a Progressive Adaptation in Muscle Stem Cell Regenerative Capacity during Development.

Muscle stem cells (MuSCs) exhibit distinct behavior during successive phases of developmental myogenesis. However, how their transition to adulthood is regulated is poorly understood. Here, we show that fetal MuSCs resist progenitor specification and exhibit altered division dynamics, intrinsic features that are progressively lost postnatally. After transplantation, fetal MuSCs expand more efficiently and contribute to muscle repair. Conversely, niche colonization efficiency increases in adulthood, indicating a balance between muscle growth and stem cell pool repopulation. Gene expression profiling identified several extracellular matrix (ECM) molecules preferentially expressed in fetal MuSCs, including tenascin-C, fibronectin, and collagen VI. Loss-of-function experiments confirmed their essential and stage-specific role in regulating MuSC function. Finally, fetal-derived paracrine factors were able to enhance adult MuSC regenerative potential. Together, these findings demonstrate that MuSCs change the way in which they remodel their microenvironment to direct stem cell behavior and support the unique demands of muscle development or repair.