Z38 - Second Harmonic Generation Imaging of Pelvic Floor Muscles in an Animal Model of Vaginal Birth Injury

Introduction: : The pelvic floor muscles (PFMs) are skeletal muscles that mechanically support the pelvic and abdominal organs and aid in the maintenance of pelvic floor function. During vaginal birth, PFMs must accommodate and facilitate passage of the fetus through the birth canal, requiring them to contract and stretch to a large degree. PFM injury resulting from vaginal birth is common, particularly for the first vaginal birth, and is associated with pelvic floor dysfunction later in life, often after the onset of menopause. PFM injury can come in the form of a defect in the belly of the muscle or avulsion (which can be full or partial, uni- or bilaterial) from the pubic rami. Avulsions affect the PFM region referred to as enthesis, as there is not a true tendon between PFMs and pubic rami. To better understand the mechanisms behind PFM defects and avulsions resulting from vaginal delivery, we aimed to evaluate rat PFM structure across various anatomical regions in a vaginal birth injury model. We compared collagen architecture visualized via second harmonic generation (SHG) imaging in enthesial and muscle belly regions and between simulated birth injury (SBI) and control (no SBI) conditions, hypothesizing that SBI would be associated with more aligned collagen fibers in both regions. This study evaluated the rat pubocaudalis (PCa) specifically, which is most comparable to pubovisceralis, the component of the human PFM complex that experiences the largest parturition-associated strains).

Materials and

Methods: : Three-month old female Sprague-Dawley rats were randomly assigned to the control (unperturbed) or SBI group. SBIs were conducted using a well-established vaginal distention protocol, in which a catheter balloon is inserted into the vagina, inflated to a volume of 5mL, and secured in place with a weight attached for 2 hours to simulate the circumferential and downwards distention associated with vaginal delivery. The animals were euthanized; the catheter balloons remained inflated and in place in the SBI group throughout the fixation process to ascertain tissue-level changes occurring when the strain is applied to PFMs. Pelvises were harvested en bloc with the muscles attached to the pelvic bones; fixed in 10% formalin for eight days; and halved. Each hemipelvis was separated from the spine and all soft tissues other than PFMs were carefully removed, leaving PFMs intact and exposed such that the enthesial and muscle belly regions could be imaged. With the medial side facing upward, hemipelvises were individually secured to 65mm culture dishes with gridded bottoms, which ensured consistent sample placement and orientation. SHG microscopy was used to visualize and quantify collagen architecture, specifically fiber alignment. The alignment measurement ranges from 0 to 1, where smaller values indicate less aligned and larger values indicate more aligned fibers. CT-Fire determined fiber angles while a custom Python script calculated alignment. Multiple images were captured in the enthesial and muscle belly regions. Data were compared between experimental groups using a one-tailed Student t-test.

Results, Conclusions, and Discussions:: SHG images obtained from 3 control and 4 animals who underwent SBI were quantified. Results are given as the combination of data from left and right PFMs. Images of the left PFMs were mirrored (flipped about the vertical axis) before fiber orientations and alignment were calculated, making the left and right PFM data comparable. Our analysis shows that collagen fiber alignment was greater in the SBI group compared to the controls in both the entheses and the muscle belly regions (Figure 1). The difference in the fiber alignment was significantly different in the muscle belly region (p=0.027), while approaching statistical significance for the enthesis (p=0.066). These findings indicate that parturition-related strains imposed on the PFMs increase collagen fiber alignment, supporting our hypothesis regarding how the birth injury model would impact collagen architecture. As a transversely isotropic tissue composed partially of collagen (e.g. tendons and skeletal muscle) is loaded uniaxially, collagen fibers will uncrimp and be pulled to more closely align with the axis of loading. This process is referred to as collagen recruitment, and contributes to the nonlinear stress-strain response of such biological tissues. The 5mL volume was clearly enough to induce collagen recruitment, though acute injury was not observed in either the enthesis or muscle belly regions of any sample. Further investigation into how collagen fibers are engaged and then stretched in the vaginal balloon distention birth injury model and what degree of collagen fiber alignment, recruitment, and stretch signifies tissue injury in each region will lead to a better understanding of the mechanisms of maternal birth injuries. Additionally, this approach allows us to study both the PFM entheses and muscle belly regions, which will be needed to elucidate mechanisms of muscle defects and avulsions specifically. Future directions of this work include increasing the sample size, quantifying the impact of SBI on the muscle fibers in the muscle belly region, and utilizing these methods in a pregnant animal model and to evaluate tissue structure post-partum.

Acknowledgements and/or References (Optional)::