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

Abstract

BackgroundPelvic floor muscle (PFM) injury is a common consequence of vaginal childbirth. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used postpartum analgesics. Multiple studies report negative effects of these drugs on limb muscle regeneration. The impact of NSAIDs on PFM recovery following birth injury has not been explored. ObjectiveUsing the validated rat model, we assessed the effects of NSAIDs on acute and longer-term PFM recovery following simulated birth injury (SBI). Study DesignThree-month old Sprague-Dawley rats were randomly assigned to (1) controls; (2) SBI; (3) SBI + NSAID; or (4) NSAID group. SBI was induced using a well-established vaginal balloon distention protocol. Ibuprofen was administered in drinking water (0.2 mg/ml) ad lib. Animals were sacrificed at 1,3,5,7,10,28 days post birth injury/ibuprofen administration. The pubocaudalis portion of the rat levator ani, which, like the human pubococcygeus, experiences larger parturition-associated strains, was harvested (N=3-9/timepoint/group). The cross-sectional areas of regenerating (embryonic myosin heavy chain (eMyHC)+) and mature myofibers were assessed at the acute and 28-day timepoints, respectively. The intramuscular collagen content was assessed at the 28-day timepoint. 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 qRT-PCR at 3,5,10 days post-injury. ResultsThe pubocaudalis fiber size was significantly smaller in the SBI+NSAID compared to SBI group at 28 days post-injury, P<0.0001. The median size of eMyHC+ fibers was also significantly reduced, with fiber area distribution enriched with smaller fibers in SBI+NSAID compared to SBI group at 3 days post-injury (P<0.0001), suggesting a delay in the onset of regeneration in the presence of NSAIDs. By 10 days post-injury, the median eMyHC+ fiber size in the SBI group decreased from 7 days post-injury (P<0.0001) with a tight cross-sectional area distribution, indicating nearing completion of this state of regeneration. However, in the SBI+NSAID group, the size of eMyHC+ fibers continued to increase (P<0.0001) with expansion of the cross-sectional area distribution, signifying a delay in regeneration in these animals. NSAID decreased the muscle stem cell pool at 7 days post-injury (P<0.0001) and delayed muscle stem cell differentiation, as indicated by persistently elevated number of myogenin+ cells 7 days post-injury (P<0.05). In contrast, the proportion of myogenin+ cells returned to baseline by 5 days post-injury in the SBI group. The analysis of expression of genes coding for pro- and anti-inflammatory cytokines demonstrated only transient elevation of Tgfb1 in SBI+NSAID group at 5 but not at 10 days post-injury. Consistent with previous studies, SBI+NSAID resulted in increased deposition of intramuscular collagen compared to uninjured animals. There were no significant differences in any outcomes of interest between the NSAID group and the unperturbed controls. ConclusionsNSAIDs negatively impact PFM regeneration in the preclinical SBI model. The above appears to be driven by the negative impact of NSAIDs on pelvic muscle stem cell function, resultant in delayed temporal progression of PFM regeneration following birth injury. These novel findings provide impetus to investigate the impact of postpartum NSAID administration on muscle regeneration in women at high risk for PFM injury.