Shift from Pro- to Anti-Inflammatory Phase in Pelvic Floor Muscles at Postpartum Matches Histological Signs of Regeneration in Multiparous Rabbits.

Background and Objectives: Pelvic floor muscles (PFM) play a core role in defecation and micturition. Weakening of PFM underlies urogynecological disorders such as pelvic organ prolapse and stress urinary incontinence. Vaginal delivery damages PFM. Muscle trauma implies an inflammatory response mediated by myeloid cells, essential for subsequent recovery. Molecular signaling characterizing the pro-inflammatory phase shifts M1 macrophages to M2 macrophages, which modulate muscle repair. The present study aimed to evaluate histological characteristics and the presence of M1 and M2 macrophages in bulbospongiosus (Bsm) and pubococcygeus muscles (Pcm). Materials and Methods: Muscles from young nulliparous (N) and multiparous rabbits on postpartum days three (M3) and twenty (M20) were excised and histologically processed to measure the myofiber cross-sectional area (CSA) and count the centralized myonuclei in hematoxylin-eosinstained sections. Using immunohistochemistry, M1 and M2 macrophages were estimated in muscle sections. Kruskal-Wallis or one-way ANOVA testing, followed by post hoc tests, were conducted to identify significant differences (p < 0.05). Results: The myofiber CSA of both the Bsm and Pcm of the M3 group were more extensive than those of the N and M20 groups. Centralized myonuclei estimated in sections from both muscles of M20 rabbits were higher than those of N rabbits. Such histological outcomes matched significant increases in HLA-DR immunostaining in M3 rabbits with the CD206 immunostaining in muscle sections from M20 rabbits. Conclusions: A shift from the pro- to anti-inflammatory phase in the bulbospongiosus and pubococcygeus muscles of multiparous rabbits matches with centralized myonuclei, suggesting the ongoing regeneration of muscles.

Effects of Ferulic Acid Supplementation on Growth Performance, Carcass Traits and Histochemical Characteristics of Muscle Fibers in Finishing Pigs.

FA dietary supplementation on the growth performance, carcass traits and histochemical characteristics of the Longissimus thoracis muscle from finishing pigs was investigated. Four hundred and twenty pigs were used in this study, and 105 animals (with five replicate pens and 21 pigs per pen) were assigned to one of four treatments: basal diet (BD) without additives (C-); BD + 10 ppm ractopamine hydrochloride + 0.97% lysine (C+); BD + 25 ppm of FA (FA); and BD + 25 ppm of FA + 0.97% lysine (FA-Lys). Dietary supplementation with FA or ractopamine increased both the average daily gain (14%) and loin muscle area (19%), while fat deposition decreased by 53%, in comparison with C- (p < 0.05). The growth performance of pigs treated with FA was similar to those of ractopamine (p > 0.05). The histochemical analysis showed that FA and C+ treatments induced a shift in muscle fiber types: from fast fibers to intermediate (alkaline ATPase) and from oxidative to glycolytic fibers. Muscle tissues from animals treated with FA or ractopamine had a lower cross-sectional area and a greater number of muscle fibers per area (p < 0.05). Findings regarding growth performance and carcass traits indicate that FA supplementation at 25 ppm without extra-lysine can replace the use of ractopamine as a growth promoter in finishing pigs.

Targeted neuromodulation of pelvic floor nerves in aging and multiparous rabbits improves continence.

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.

Chronic Undernutrition Differentially Changes Muscle Fiber Types Organization and Distribution in the EDL Muscle Fascicles.

Fiber type composition, organization, and distribution are key elements in muscle functioning. These properties can be modified by intrinsic and/or extrinsic factors, such as undernutrition and injuries. Currently, there is no methodology to quantitatively analyze such modifications. On one hand, we propose a fractal approach to determine fiber type organization, using the fractal correlation method in software Fractalyse. On the other hand, we applied the kernel methodology from machine learning to build radial-basis functions for the spatial distribution of fibers (distribution functions), by dividing into square cells a two-dimensional binary image for the spatial distribution of fibers from a muscle fascicle and mounting on each cell a radial-basis function in such a way that the sum of all cell functions creates a smooth version of the fiber histogram on the cell grid. The distribution functions thus created belong in a reproducing kernel Hilbert space which permits us to regard them as vectors and measure distances and angles between them. In the present study, we analyze fiber type organization and distribution in fascicles (F2, F3, F4, and F5) of the extensor digitorum longus muscle (EDLm) from control and undernourished male rats. Fibers were classified according to the ATPase activity in slow, intermediate, and fast. Then, (x, y) coordinates of fibers were used to build binary images and distribution functions for each fiber type and both conditions. The fractal organization analysis showed that fast and intermediate fibers, from both groups, had a fractal organization within the four fascicles, i.e., the fiber assembly is distributed in clusters. We also show that chronic undernutrition altered the organization of fast fibers in the F3, although it still is considered a fractal organization. Distribution function analysis showed that each fiber type (slow, intermediate, and fast) has a unique distribution within the fascicles, in both conditions. However, chronic undernutrition modified the intra-fascicular fiber type distributions, except in the F2. Altogether, these results showed that the methodology herein proposed allows for analyzing fiber type organization and distribution modifications. On the other side, we show that chronic undernutrition alters not only the fiber type composition but also the organization and distribution, which could affect the muscle functioning, and ultimately, its behavior (e.g., locomotion).

Signs of damage in pelvic floor muscles at the end of pregnancy in rabbits.

INTRODUCTION AND HYPOTHESIS: Temporary effects to pelvic floor muscles are linked to impairments in micturition, particularly stress urinary incontinence (SUI), during pregnancy. We hypothesize that bulbospongiosus (Bsm) and pubococcygeus (Pcm) are differently damaged in primigravid and primiparous rabbits. METHODS: Twenty-four rabbits allocated evenly (n = 6) into nulliparous, pregnant, and primiparous groups on postpartum days 3 (P3) and 20 (P20) were used to evaluate the myofiber cross-sectional area (CSA), ß-glucuronidase activity, and anti-3-nitrotyrosine (anti-3-NTyr) immunoreactivity in Bsm and Pcm muscles. Appropriate statistical tests were done to determine significant differences among groups (P ≤ 0.05). RESULTS: The average CSA of Bsm was not significantly different, albeit a high percentage of myofibers was enlarged in late-pregnant and primiparous rabbits on P3; ß-glucuronidase activity and indirect parameter of muscle damage was also higher. These variables did not change in the Pcm muscle during the different reproductive stages. In contrast, the 3-NTyr immunoreactivity, an indicator of oxidative damage, was increased on P3 for Pcm myofibers and P20 for myofibers of both muscles. CONCLUSIONS: Our findings demonstrate reliable signs of damage to Bsm and Pcm muscles in young female rabbits passing different reproductive stages. Damage to the Bsm muscles as detected at the end of pregnancy persisted after delivery. This was not the case for Pcm muscles, in which damage seems to appear after delivery.

Multiparity affects conduction properties of pelvic floor nerves in rabbits.

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.

Differential effect of chronic undernutrition on the fiber type composition of fascicles in the extensor digitorum longus muscles of the rat.

Several studies have shown that chronic low food consumption alters the composition and metabolism of the extensor digitorum longus muscle (EDLm) fiber types. EDLm is constituted by four independent fascicles (F2-F5) of different sizes; their constitution and metabolism, however, as well as how chronic undernourishment affects these is virtually unknown. Thus, the aim of this study is to evaluate the relative fiber type composition and metabolism of each independent fascicle in the EDLm, using control and chronically undernourished young male rats by using the alkaline ATPase and NADH-TR histochemical techniques. Our results indicate that all control fascicles showed a higher percentage of intermediate fibers (P<0.001), except for F5, which had a higher percentage of fast fibers (P<0.001). After chronic undernutrition, the proportion of intermediate fibers decreased in F4 (P<0.05) and increased in F5 (P<0.001), whereas fast fibers decreased in F3 (P<0.05) and F5 (P<0.001). When we investigated fiber metabolism we found that F3 and F4 had a similar composition (mainly glycolytic), whereas F2 and F5 predominantly contained oxidative fibers. All fascicles of chronic undernourished rats showed a general decrease in oxidative fibers (P>0.05), except for F3, in which oxidative fibers increased (P<0.05). After determining the possible predominant metabolism expressed in intermediate fibers, we propose that chronic undernutrition induces the transformation of fast-glycolytic to intermediate-oxidative/glycolytic fibers, mainly in F3 and F5. Our observations confirm that chronic undernourishment differentially affects the fiber types of each fascicle in the EDLm, which could alter their individual physiological contractile properties.

Fiber type composition of pubococcygeus and bulbospongiosus striated muscles is modified by multiparity in the rabbit.

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.

Differential damage and repair responses of pubococcygeus and bulbospongiosus muscles in multiparous rabbits.

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 ß-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 ß-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.

Aromatase expression is linked to estrogenic sensitivity of periurethral muscles in female rabbits.

Beyond its role in the conversion of androgens to estrogens, the expression of aromatase could influence on the estrogenic signalling in targeted tissues. Considering the well-defined biochemical and physiological differences between the pubococcygeus (Pcm) and bulbospongiosus (Bsm) muscles in female rabbits, it is presently hypothesized that the aromatase expression is differentially linked to the estrogen sensitivity of each muscle. To this end, serum estradiol levels and the aromatase expression, presence of ERα and ERß and morphometry were evaluated in the Pcm and Bsm of female rabbits allocated in control, ovariectomized (OVX) and OVX treated with estradiol benzoate (OVX + EB) groups. Aromatase expression was high in the Pcm. Independently to serum estradiol, ovariectomy increased aromatase expression in the Pcm while decreased it in the Bsm. The EB treatment avoided the effect of ovariectomy only in the Pcm. The number of immunoreactive nuclei anti-ERα and anti-ERß was high in the Pcm of OVX and OVX + EB rabbits, while those in the Bsm remained unchanged. The number of peripheral nuclei per fibre and the cross-sectional area-to-myonucleus ratio were modified only in the Pcm. Our findings support aromatase expression in the Pcm, and Bsm of rabbits is differentially linked to estrogenic sensitivity of each muscle.

Fiber type characterization of striated muscles related to micturition in female rabbits.

Pelvic and perineal striated muscles are relevant for reproduction and micturition in female mammals. Damage to these muscles is associated with pelvic organ prolapse and stress urinary incontinence. The fiber type composition of skeletal muscle influences the susceptibility for damage and/or regeneration. The aim of the present study was to determine the fiber type composition of a perineal muscle, the bulbospongiosus, and a pelvic muscle, the pubococcygeus. Both muscles were harvested from adult female rabbits (8-10 months old). NADH-TR (nicotinamide adenine dinucleotide tetrazolium reductase) histochemistry was undertaken to identify oxidative and glycolytic muscle fibers. Alkaline (pH 9.4) ATP-ase (actomyosin adenosine triphosphatase) histochemistry was used to classify type I, type IIb or type IIa/IId muscle fibers. Results showed that the content of glycolytic fibers in the bulbospongiosus muscle was higher than that of oxidative fibers. Meanwhile, the opposite was true for the pubococcygeus. In the bulbospongiosus muscle, the content of type IIb muscle fibers was higher than that of type I, but was similar to that of type IIa/IId. In contrast, the content of each fiber type was similar in the pubococcygeus muscle. The relative proportion of fibers in bulbospongiosus and pubococcygeus muscles is consistent with their function during voiding and storage phases of micturition.