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Pelvic floor muscle stretch injury during pregnancy and birth is associated with the incidence of stress urinary incontinence (SUI), a condition that affects 30-60% of the female population and is characterized by involuntary urine leakage during physical activity, further exacerbated by aging. Aging and multiparous rabbits suffer pelvic nerve and muscle damage, resulting in alterations in pelvic floor muscular contraction and low urethral pressure, resembling SUI. However, the extent of nerve injury is not fully understood. Here, we used electron microscopy analysis of pelvic and perineal nerves in multiparous rabbits to describe the extent of stretch nerve injury based on axon count, axon size, myelin-to-axon ratio, and elliptical ratio. Compared to young nulliparous controls, mid-age multiparous animals showed an increase in the density of unmyelinated axons and in myelin thickness in both nerves, albeit more significant in the bulbospongiosus nerve. This revealed a partial but sustained damage to these nerves, and the presence of some regenerated axons. Additionally, we tested whether electrical stimulation of the bulbospongiosus nerve would induce muscle contraction and urethral closure. Using a miniature wireless stimulator implanted on this perineal nerve in young nulliparous and middle age multiparous female rabbits, we confirmed that these partially damaged nerves can be acutely depolarized, either at low (2-5 Hz) or medium (10-20 Hz) frequencies, to induce a proportional increase in urethral pressure. Evaluation of micturition volume in the mid-age multiparous animals after perineal nerve stimulation, effectively reversed a baseline deficit, increasing it 2-fold (p = 0.02). These results support the notion that selective neuromodulation of pelvic floor muscles might serve as a potential treatment for SUI.

Introduction: Women often develop pelvic floor dysfunction due to damage to the pelvic musculature during childbirth; however, the effect on pelvic floor nerves function is less understood. This study used adult rabbits to evaluate the electrophysiological and histological characteristics of the bulbospongiosus (Bsn) and pubococcygeus nerves (Pcn) in multiparity., Methods: Compound nerve action potentials (CNAP) were compared between age-matched nulliparous and multiparous animals and associated to the histological characteristics of myelinated axons from the Bsn and Pcn nerves. The extensor digitorum longus nerve (EDLn) was used as negative control. Data were analyzed with unpaired two-tailed Student's t test or Mann-Whitney U test to determine significant differences between groups., Results: The onset and peak latencies, duration, and conduction velocity of the motor fibers in these pelvic nerves were not significantly different between nulliparous and multiparous animals. However, the peak-to-peak amplitude and area of the CNAP in both Bsn and Pcn were reduced in multiparous rabbits. Histology showed a higher percentage of axons with myelin disorganization caused by multiparity in these pelvic nerves. Together, the data indicate a reduction in the number of functional pelvic axons due to multiparity. As expected, no effect of parity was observed in the EDLn controls., Conclusions: Present findings demonstrated that multiparity affects myelination and consequently conduction properties in the small pelvic floor nerves., (© 2018 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.)

Aims: To characterize the contractile properties of the bulbospongiosus (Bsm), isquiocavernosus (Ism), and pubococcygeus muscles (Pcm), and their involvement in the genesis of vaginal pressure in nulliparous and multiparous rabbits., Methods: Age-matched nulliparous and multiparous rabbits were used to record the isometric contractile responses of each muscle as well as the intravaginal pressure evoked by single square electrical pulses and stimulation trains of ascending frequency. To establish significant differences between groups, two-tail unpaired Student t tests were carried out. The linear correlation between intravaginal pressure and muscle contractile force was analyzed with Pearson correlation tests. For all cases, a P ≤ 0.05 was set as statistically significant., Results: Multiparity decreased the contractile force of Bsm and Ism generated by high-frequency stimulation trains. The normalized force of the Pcm increased when evoked at 1, 4, and 10 Hz while this decreased at higher frequencies (20, 50, and 100 Hz). The contraction of both Bsm and Ism raised particularly the pressure on the perineal vagina while that of the Pcm increased the pressure in the pelvic vagina. Such a functional segregation is still present in multiparous rabbits albeit it was modified., Conclusions: Multiparity induces changes in the contractile responses of Bsm, Ism, and Pcm, which alterates the vaginal pressure., (© 2017 Wiley Periodicals, Inc.)

Aims: We analyzed the effect of multiparity on the fiber type composition of two skeletal muscles involved in the maintenance of the micturition process, the pelvic pubococcygeus (Pc) and perineal bulbospongiosus (Bs) muscles in nulliparous and multiparous rabbits (Oryctolagus cuniculus)., Methods: We used the basic ATPase and NADH-TR techniques to identify and characterize slow, intermediate, and fast fiber types and glycolitic and oxidative fibers in muscles, respectively., Results: Pc muscles of multiparous rabbits present relatively high percentages of slow and intermediate fibers but a low percentage of fast fibers (P < 0.05) as compared to Pc muscles from nulliparous rabbits, while percentages of glycolytic and oxidative fibers were similar (P > 0.05). Bs muscles of multiparous rabbits had a higher proportion of intermediate and glycolytic fibers (P < 0.05) than muscles of nulliparous. Both, Pc and Bs muscles of nulliparous and multiparous rabbits contain slow fibers with similar large cross sectional area, but fast fibers in multiparous muscles showed small cross sectional area than in nulliparous., Conclusions: Multiparity modified the fiber type composition of Pc and Bs muscles in female rabbits. We propose that the contractile force and the physiological role of both muscles during micturition are affected because of the observed changes in the relative composition of muscle fiber types., (© 2016 Wiley Periodicals, Inc.)

Aim: To determine the extent of damage and regeneration associated with multiparity on the pubococcygeus and bulbospongiosus muscles., Methods: Age-matched virgin nulliparous and multiparous rabbits that were killed at days 3 and 20 after the fourth delivery were used to harvest pubococcygeus and bulbospongiosus muscles. The activity of the lysosomal enzyme β-glucuronidase was used as a muscle damage indicator. The number of immunoreactive myofiber-associated nuclei anti-Pax7, -MyoD, and -myogenin, as well as the anti-desmin immunoreactive area were measured in muscle sections to estimate some regenerative stages. Significant differences were considered at a P ≤ 0.05., Results: The β-glucuronidase activity was increased at postpartum day 20 in the pubococcygeus muscle. This variable was unaltered in the bulbospongiosus muscles of multiparas regardless of the postpartum day on which this was measured. The number of immunoreactive nuclei anti-Pax7 in the pubococcygeus muscle was similar between nulliparas and multiparas, whilst those of anti-MyoD and anti-myogenin were increased at postpartum days 3 and 20. The same was true for these latter three markers evaluated in the bulbospongiosus muscles, supporting an ongoing regeneration. The desmin-positive percentage of muscle area per field was increased at postpartum day 20 in the pubococcygeus muscle, whilst such an increment was seen at postpartum days 3 and 20 in the bulbospongiosus muscles., Conclusions: Damage and regeneration of the pubococcygeus and bulbospongiosus muscles are differently influenced by multiparity in rabbits. This could rely on the anatomical location, metabolism, myofiber composition, and muscle exertion during pregnancy and/or the delivery of each muscle., (© 2014 Wiley Periodicals, Inc.)

In rodents, vaginal distention after delivery or experimental manipulation affects innervations as well as the amount of striated/smooth musculature or collagen in both the urethra and vagina. These changes are associated with modifications in excretory and reproductive processes. Although successive and consecutive vaginal deliveries (multiparity) affect the contractile and functional properties of the female lower urogenital tract (LUT), its impact on LUT morphometry, including persistency, has been barely studied. The caudal urethra (CU) and cranial (V1) and caudal (V2) pelvic vaginal regions were excised from young and adult nulliparous (YN and AN, respectively) and multiparous (YM and AM, respectively) rabbits. Tissues were histologically processed and stained with Masson's trichrome. The thickness of the tissue layers and areas covered by tissue components were measured and compared using two-way ANOVA followed by Student-Newmann-Keuls tests to determine statistical differences (P ≤ 0.05). Compared to YN, YM, and AN tissues showed a reduction in the thickness of the epithelium, as well as in areas covered by striated musculature, collagen, and blood vessels of the LUT. In comparison with YM, only some morphometric changes were recovered in the AM group. Our study shows that multiparity and age can be associated with epithelial and muscular atrophy of urethral and vaginal walls. The morphometry of the LUT between young and adult female rabbits varies with multiparity. These findings may help to better understand the effects of multiparity on young and adult females and its correlation with the development of pelvic dysfunctions., (© 2014 Wiley Periodicals, Inc.)

Objectives: (1) To describe the morphology of paravaginal ganglia and the neurochemical pattern of their neurons in virgin rabbits. (2) To analyze the effects of multiparity, primiparity and late pregnancy on morphometry of ganglia and their neurons., Study Design: The morphology and neurochemical pattern of paravaginal ganglia were described in virgin nulliparous Chinchilla breed rabbits. Acetylcholinesterase histochemistry, Masson's trichrome, and immunohistochemistry for cholinergic and adrenergic neurons, and estrogen receptors (ERs) were undertaken. The area covered by ganglia (ganglionic area), the number of neurons, and the neuron soma area were measured in multiparas (4 consecutive and successive deliveries) and age-matched nulliparas. The same variables were measured in primiparous, late-pregnant, and age-matched nulliparous rabbits., Results: Paravaginal ganglia were adjoined to the dorsolateral walls of the pelvic vagina. Their neurons were mostly cholinergic and expressed ERα and ERβ. Multiparity increased the ganglionic area and reduced the number of ganglionic neurons. Late pregnancy transiently reduced the neuron soma area, which was coincident with a low expression of ERs., Conclusion: Multiparity but not primiparity affected the morphometry of ganglia. The hormonal state present in late pregnancy alters the neuron soma area and ER expression. Our findings support the notion that reproduction influences the morphometry of the pelvic plexus., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Objective: To determine changes in morphometry and expression of oestrogen receptors (OR) in the pubococcygeus and bulbospongiosus muscles, and the concentration of serum oestradiol associated with multiparity., Study Design: Twelve chinchilla-breed female rabbits were divided into multiparas who had undergone four consecutive deliveries and age-matched virgin nulliparas. Pubococcygeus and bulbospongiosus muscles were surgically removed from each rabbit and processed histologically. Fibre cross-sectional area, number of peripheral nuclei, and expression of ORα and ORβ were measured for each muscle. Serum samples were obtained and the concentration of serum oestradiol was quantified., Results: Multiparity increased (p ≤ 0.05) fibre cross-sectional area and the number of peripheral nuclei per fibre in pubococcygeus muscle, but not in bulbospongiosus muscle. Expression of both ORα and ORβ was high (p ≤ 0.05) in both muscles from multiparous rabbits. A rise in serum oestradiol was measured at the end of the second pregnancy, which was absent (p ≤ 0.05) at the end of the fourth pregnancy. The concentration of serum oestradiol was similar (p > 0.05) in nulliparous and multiparous rabbits., Conclusions: Multiparity caused morphometric changes in pubococcygeus muscle but not in bulbospongiosus muscle. As OR expression was high for both muscles, some properties related to fibre composition or muscle physiology could be affected. The finding that serum oestradiol was not elevated at the end of the fourth pregnancy could be related to changes in pelvic and perineal muscles associated with multiparity., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Temporal and coordinated activation of pelvic- (pubococcygeous) and perineal- (bulbospongiosus and ischiocavernosus) striated muscles occurs during micturition in female rabbits. We have hypothesized that the coordinated activation of pelvic and perineal muscles is modified during the micturition of young multiparous rabbits. Young virgin and multiparous female chinchilla rabbits were used to simultaneously record cystometrograms and electromyograms of the pubococcygeous, ischocavernosus, and bulbospongiosus muscles. Bladder function was assessed using standard urodynamic variables. The temporal coordination of pelvic- and perineal-striated muscle activity was changed in multiparous rabbits. The cystometrogram recordings were different than those obtained from virgin rabbits, as seen in alterations of the threshold volume, the residual volume, the voiding duration, and the maximum pressure. In rabbits, we find that multiparity causes uncoordinated activity of pubococcygeous, ischiocavernosus, and bulbospongiosus muscles and modifies the urodynamics.