Multiparity and Aging Impact Chondrogenic and Osteogenic Potential at Symphyseal Enthesis: New Insights into Interpubic Joint Remodeling.

Pregnancy and childbirth cause adaptations to the birth canal to allow for delivery and fast recovery. To accommodate delivery through the birth canal, the pubic symphysis undergoes changes that lead to the interpubic ligament (IpL) and enthesis formation in primiparous mice. However, successive deliveries influence joint recovery. We aimed to understand tissue morphology and chondrogenic and osteogenic potential at symphyseal enthesis during pregnancy and postpartum in primiparous and multiparous senescent female mice. Morphological and molecular differences were found at the symphyseal enthesis among the study groups. Despite the apparent incapacity to restore cartilage in multiparous senescent animals, the symphyseal enthesis cells are active. However, these cells have reduced expression of chondrogenic and osteogenic markers and are immersed in densely packed collagen fibers contiguous to the persistent IpL. These findings may indicate alterations of key molecules in the progenitor cell population maintenance of the chondrocytic and osteogenic lineages at the symphyseal enthesis in multiparous senescent animals, possibly compromising the mouse joint histoarchitecture recovery. This sheds light on the distention of the birth canal and the pelvic floor that may play a role in pubic symphysis diastasis (PSD) and pelvic organ prolapse (POP), both in orthopedic and urogynecological practice in women.

HSP70 of Leishmania amazonensis alters resistance to different stresses and mitochondrial bioenergetics.

The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.

Dact gene expression profiles suggest a role for this gene family in integrating Wnt and TGF-ß signaling pathways during chicken limb development.

BACKGROUND: Dact gene family encodes multifunctional proteins that are important modulators of Wnt and TGF-ß signaling pathways. Given that these pathways coordinate multiple steps of limb development, we investigated the expression pattern of the two chicken Dact genes (Dact1 and Dact2) from early limb bud up to stages when several tissues are differentiating. RESULTS: During early limb development (HH24-HH30) Dact1 and Dact2 were mainly expressed in the cartilaginous rudiments of the appendicular skeleton and perichondrium, presenting expression profiles related, but distinct. At later stages of development (HH31-HH35), the main sites of Dact1 and Dact2 expression were the developing synovial joints. In this context, Dact1 expression was shown to co-localize with regions enriched in the nuclear ß-catenin protein, such as developing joint capsule and interzone. In contrast, Dact2 expression was restricted to the interzone surrounding the domains of bmpR-1b expression, a TGF-ß receptor with crucial roles during digit morphogenesis. Additional sites of Dact expression were the developing tendons and digit blastemas. CONCLUSIONS: Our data indicate that Dact genes are good candidates to modulate and, possibly, integrate Wnt and TGF-ß signaling during limb development, bringing new and interesting perspectives about the roles of Dact molecules in limb birth defects and human diseases.

Avian extraintestinal Escherichia coli exhibits enterotoxigenic-like activity in the in vivo rabbit ligated ileal loop assay.

Avian pathogenic Escherichia coli (APEC) strains harbor a number of virulence genes and cause extraintestinal diseases, such as septicemia, swollen-head syndrome, salpingitis, and omphalitis in poultry. APEC strains are not known to cause intestinal diseases. Herein, for the first time, it is reported that APEC strains were able to induce an enterotoxigenic-like effect in rabbit ligated ileal loops. Strain SEPT362 caused cell detachment of the intestinal villi, which also showed a flattened and wilted appearance, but the integrity of the tight junctions was maintained. Additionally, this strain did not adhere to enterocytes in vivo, although adhesin encoding genes ( fimH, csgA, lpfA2-3, and ECP) were present while other lpfA types, sfa, afa, papC, and ral genes were not. This enterotoxigenic-like activity was conserved after thermal treatment of the supernatant at 65°C but not at 100°C. Moreover, experiments based on filtering with different molecular weight cut-off (MWCO) pore sizes demonstrated that the component associated with the observed biological effect has a molecular weight >100 kDa. Blast search and polymerase chain reaction assays for known E. coli virulence factors showed that strain SEPT362 harbors the gene encoding for the toxin EAST-1 and the serine protease autotransporter (SPATE) Tsh, but is negative for genes encoding for the toxins LT-I, STh, STp, Stx1, Stx2, CNF-1, CNF-2, CDT and the SPATEs Sat, Pic, Vat, SigA, SepA, EatA, EspP, or EspC. A cloned copy of the tsh gene in E. coli K-12 was also tested and was shown to have an enterotoxic effect. These results suggest that APEC might induce fluid accumulation in the rabbit gut. The Tsh autotransporter seems to be one of the factors associated with this phenotype.

Effects of microcurrent stimulation on hyaline cartilage repair in immature male rats (Rattus norvegicus).

BACKGROUND: In this study, we investigate the effects of microcurrent stimulation on the repair process of xiphoid cartilage in 45-days-old rats. METHODS: Twenty male rats were divided into a control group and a treated group. A 3-mm defect was then created with a punch in anesthetized animals. In the treated group, animals were submitted to daily applications of a biphasic square pulse microgalvanic continuous electrical current during 5 min. In each application, it was used a frequency of 0.3 Hz and intensity of 20 µA. The animals were sacrificed at 7, 21 and 35 days after injury for structural analysis. RESULTS: Basophilia increased gradually in control animals during the experimental period. In treated animals, newly formed cartilage was observed on days 21 and 35. No statistically significant differences in birefringent collagen fibers were seen between groups at any of the time points. Treated animals presented a statistically larger number of chondroblasts. Calcification points were observed in treated animals on day 35. Ultrastructural analysis revealed differences in cell and matrix characteristics between the two groups. Chondrocyte-like cells were seen in control animals only after 35 days, whereas they were present in treated animals as early as by day 21. The number of cuprolinic blue-stained proteoglycans was statistically higher in treated animals on days 21 and 35. CONCLUSION: We conclude that microcurrent stimulation accelerates the cartilage repair in non-articular site from prepuberal animals.

Obesity increases blood-brain barrier permeability and aggravates the mouse model of multiple sclerosis.

Obesity-induced insulin resistance (OIR) has been associated with an increased prevalence of neurodegenerative disorders such as multiple sclerosis. Obesity results in increased blood-brain barrier (BBB) permeability, specifically in the hypothalamic regions associated with the control of caloric intake. In obesity, the chronic state of low-grade inflammation has been implicated in several chronic autoimmune inflammatory disorders. However, the mechanisms that connect the inflammatory profile of obesity with the severity of experimental autoimmune encephalomyelitis (EAE) are poorly defined. In this study, we show that obese mice are more susceptible to EAE, presenting a worse clinical score with more severe pathological changes in the spinal cord when compared with control mice. Analysis of immune infiltrates at the peak of the disease shows that high-fat diet (HFD)- and control (chow)-fed groups do not present any difference in innate or adaptive immune cell compartments, indicating the increased severity occurs prior to disease onset. In the setting of worsening EAE in HFD-fed mice, we observed spinal cord lesions in myelinated regions and (blood brain barrier) BBB disruption. We also found higher levels of pro-inflammatory monocytes, macrophages, and IFN-γ+CD4+ T cells in the HFD-fed group compared to chow-fed animals. Altogether, our results indicate that OIR promotes BBB disruption, allowing the infiltration of monocytes/macrophages and activation of resident microglia, ultimately promoting CNS inflammation and exacerbation of EAE.

Changes of large molecular weight hyaluronan and versican in the mouse pubic symphysis through pregnancy.

During pregnancy, the mouse pubic symphysis undergoes expansion and remodeling resulting in formation of a flexible and elastic interpubic ligament allowing passage of a term fetus. In the current study, we sought to identify and characterize components of the extracellular matrix that likely play an important role in elongation and flexibility of the interpubic ligament during parturition. Mouse pubic symphyses and interpubic ligaments collected at time points during pregnancy and postpartum were utilized to evaluate collagen type, collagen content, processing and solubility, matricellular protein, and proteoglycan expression and quantitative assessment of all glycosaminoglycans. These studies revealed increased gene expression for hyaluronan synthase 1, hyaluronan synthase 2, and versican on Gestation Day 18 as well as a decline in protein expression for the versican-degrading protease a disintegrin-like and metalloprotease with thrombospondin type 1 (ADAMTS1) motif. These findings suggest that the primary mediators of increased elongation and flexibility of the interpubic ligament at term result from increased synthesis and reduced metabolism of viscoelasticity-promoting molecules such as high molecular weight hyaluronan and versican.

Elastic fiber assembly in the adult mouse pubic symphysis during pregnancy and postpartum.

Impairment of pelvic organ support has been described in mice with genetic modifications of the proteins involved in elastogenesis, such as lysyl oxidase-like 1 (LOXL1) and fibulin 5. During pregnancy, elastic fiber-enriched pelvic tissues are modified to allow safe delivery. In addition, the mouse pubic symphysis is remodeled in a hormone-controlled process that entails the modification of the fibrocartilage into an interpubic ligament (IpL) and the relaxation of this ligament. After first parturition, recovery occurs to ensure pelvic tissue homeostasis. Because ligaments are the main supports of the pelvic organs, this study aimed to evaluate elastogenesis in the IpL during mouse pregnancy and postpartum. Accordingly, virgin, pregnant, and postpartum C57BL/6 mice were studied using light, confocal, and transmission electron microscopy as well as Western blots and real-time PCR. Female mice exhibited the separation of the pubic bones and the formation, relaxation, and postpartum recovery of the IpL. By the time the IpL was formed, the elastic fibers had increased in profile length and diameter, and they consisted of small conglomerates of amorphous material distributed among the bundles of microfibrils. Our analyses also indicated that elastin/tropoelastin, fibrillin 1, LOXL1/Loxl1, and fibulin 5 were spatially and temporally regulated, suggesting that these molecules may contribute to the synthesis of new elastic fibers during IpL development. Overall, this work revealed that adult elastogenesis may be important to assure the elasticity of the pelvic girdle during preparation for parturition and postpartum recovery. This finding may contribute to our understanding of pathological processes involving elastogenesis in the reproductive tract.

Oral tolerance and OVA-induced tolerogenic dendritic cells reduce the severity of collagen/ovalbumin-induced arthritis in mice.

Dietary proteins play an important role in the regulation of systemic immune response, in a phenomenon known as oral tolerance (OT). To evaluate the effects of OT on a murine model of type II collagen (CII) plus ovalbumin (OVA)-induced arthritis (CIA), mice were fed with OVA either before or after CIA induction. OT significantly reduced the paw edema and synovial inflammation, as well as serum levels of anti-CII, the ex vivo proliferation and inflammatory cytokine production by spleen cells from CIA mice. The frequencies of Foxp3(+) and IL-10(+) cells were higher, whereas IFNγ(+) cells and IL-17(+) cells were lower, among gated CD4(+) spleen T cells from tolerized CIA mice than in those from non-tolerized CIA mice. Adoptive transfer of tolerogenic dendritic cells (DCs) before CIA induction mimics the effects observed in the OT. We demonstrate here that bystander suppression induced by OT can modify the course of CIA and tolerogenic DCs play a role this phenomenon.

High iNOS mRNA and protein localization during late pregnancy suggest a role for nitric oxide in mouse pubic symphysis relaxation.

Remodeling and relaxation of the mouse pubic symphysis (PS) are central events in parturition. The mouse PS remodels in a hormone-controlled process that involves the modification of the fibrocartilage into an interpubic ligament (IpL), followed by its relaxation prior to parturition. It is recognized that nitric oxide synthase (NOS) and consequently nitric oxide (NO) generation play important roles in extracellular matrix modification, and may promote cytoskeleton changes that contribute to the remodeling of connective tissue, which precedes the onset of labor. To our knowledge, no studies thus far have investigated inducible nitric oxide synthase (iNOS) expression, protein localization, and NO generation in the mouse PS during pregnancy. In this work, we used a combination of the immunolocalization of iNOS, its relative mRNA expression, and NO production to examine the possible involvement of iNOS in remodeling and relaxation of the mouse IpL during late pregnancy. The presence of iNOS was observed in chondrocytes and fibroblast-like cells in the interpubic tissues. In addition, iNOS mRNA and NO production were higher during preterm labor on Day 19 of pregnancy (D19) than NO production on D18 or in virgin groups. The significant increase in iNOS mRNA expression and NO generation from the partially relaxed IpL at D18 to the completely relaxed IpL at D19 may indicate that NO plays an important role in late pregnancy during relaxation of the mouse IpL.

Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. METHODS: We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. RESULTS: ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. CONCLUSION: These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.

Relaxation of the mouse pubic symphysis during late pregnancy is not accompanied by the influx of granulocytes.

In some animals, such as mice and guinea pigs, a hormonally controlled mechanism increases the flexibility of the pubic symphysis and enhances the cervical remodeling necessary for safe delivery. Cervical ripening during pregnancy is associated with a paradoxical influx of leukocytes. However, the changes in cell metabolism during relaxation of the mouse pubic symphysis for delivery have not been extensively studied. In this work, we used light microscopy and transmission and scanning electron microcopy, as well as immunohistochemistry and Western blotting for MMP-8, to investigate the involvement of granulocytes or resident stromal cells in the relaxation of the virgin pubic symphysis during late pregnancy (days 18 and 19, before delivery) in vivo and in explanted joints. MMP-8 was studied because this collagenase is a hallmark for cervical ripening associated with the influx of granulocytes during late pregnancy. Extensive dissolution and disorganization of the extracellular matrix was seen around fibroblastic-like cells in late pregnancy. In contrast to the cervix (positive control), morphological and immunohistochemical analyses revealed that there was no characteristic cellular inflammatory response in the interpubic tissue. Staining for MMP-8 was observed in chondroid and fibroblastic-like cells of virgin and relaxed interpubic ligament, respectively. However, no granulocytes were seen during the extensive remodeling of the pubic joint in late pregnancy. These results indicate that constitutive stromal cells may have an important role in tissue relaxation during remodeling of the pubic symphysis in pregnancy.

Time-dependent regulation of morphological changes and cartilage differentiation markers in the mouse pubic symphysis during pregnancy and postpartum recovery.

Animal models commonly serve as a bridge between in vitro experiments and clinical applications; however, few physiological processes in adult animals are sufficient to serve as proof-of-concept models for cartilage regeneration. Intriguingly, some rodents, such as young adult mice, undergo physiological connective tissue modifications to birth canal elements such as the pubic symphysis during pregnancy; therefore, we investigated whether the differential expression of cartilage differentiation markers is associated with cartilaginous tissue morphological modifications during these changes. Our results showed that osteochondral progenitor cells expressing Runx2, Sox9, Col2a1 and Dcx at the non-pregnant pubic symphysis proliferated and differentiated throughout pregnancy, giving rise to a complex osteoligamentous junction that attached the interpubic ligament to the pubic bones until labour occurred. After delivery, the recovery of pubic symphysis cartilaginous tissues was improved by the time-dependent expression of these chondrocytic lineage markers at the osteoligamentous junction. This process potentially recapitulates embryologic chondrocytic differentiation to successfully recover hyaline cartilaginous pads at 10 days postpartum. Therefore, we propose that this physiological phenomenon represents a proof-of-concept model for investigating the mechanisms involved in cartilage restoration in adult animals.

Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts

Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.(AU)

Plesiomonas shigelloides exports a lethal cytotoxic-enterotoxin (LCE) by membrane vesicles.

Plesiomonas shigelloides isolated from water in Brazil was previously described as a hemorrhagic heat-labile cytotoxic-enterotoxin producer. We purified this toxin from culture supernatants using ion metallic affinity chromatography (IMAC) followed by molecular exclusion chromatography. The pure toxin presented molecular mass of 50kDa and isoelectric point (pI) around 6.9 by 2D electrophoresis. When injected intravenously, the purified cytotoxic-enterotoxin induced also severe spasms followed by sudden death of mice. Hence, we entitled it as lethal cytotoxic-enterotoxin (LCE). The presence of membrane vesicles (MVs) on cell surfaces of P. shigelloides was observed by scan electron microscopy (SEM). From these MVs the LCE toxin was extracted and confirmed by biological and serological assays. These data suggest that P. shigelloides also exports this cytotoxic-enterotoxin by membrane vesicles, a different mechanism of delivering extra cellular virulence factors, so far not described in this bacterium.

Effects of hyperbaric oxygen on Pseudomonas aeruginosa susceptibility to imipenem and macrophages.

BACKGROUND: The seriousness to treat burn wounds infected with Pseudomonas aeruginosa led us to examine whether the effect of the carbapenem antibiotic imipenem is enhanced by hyperbaric oxygen (HBO). MATERIALS & METHODS: The effects of HBO (100% O2, 3 ATA, 5 h) in combination with imipenen on bacterial counts of six isolates of P. aeruginosa and bacterial ultrastructure were investigated. Infected macrophages were exposed to HBO (100% O2, 3 ATA, 90 min) and the production of reactive oxygen species monitored. RESULTS: HBO enhanced the effects of imipenen. HBO increased superoxide anion production by macrophages and likely kills bacteria by oxidative mechanisms. CONCLUSION: HBO in combination with imipenem can be used to kill P. aeruginosa in vitro and such treatment may be beneficial for the patients with injuries containing the P. aeruginosa.

Effectiveness of hyperbaric oxygen for experimental treatment of schistosomiasis mansoni using praziquantel-free and encapsulated into liposomes: assay in adult worms and oviposition.

The treatment of schistosomiasis depends on a single drug: praziquantel (PZQ). However, this treatment presents limitations such as low and/or erratic bioavailability that can contribute to cases of tolerance. Improvements to the available drug are urgently needed and studies with a controlled system of drug release, like liposomes, have been gaining prominence. The present study evaluated the activity and synergy between liposomal-praziquantel (lip.PZQ) and hyperbaric oxygen therapy (HBO). Mice received doses of 60 or 100mg/kg PZQ or lip.PZQ, 50 days post-infection, and after the treatment, were exposed to HBO (3 atmosphere absolute - ATA) for 1h. The viability of adult worms and oviposition were analyzed, by necropsy and Kato-Katz examination performed after 15 days of treatment. A concentration of 100mg/kg of lip.PZQ+HBO was more effective (48.0% reduction of worms, 83.3% reduction of eggs/gram of feces) and 100% of the mice had altered of oograms (indicating interruption of oviposition) compared to other treatments and to the Control group (infected and untreated). It is known that PZQ requires participation of the host immune system to complete its antischistosomal activity and that HBO is able to stimulate the immune system. The drug became more available in the body when incorporated into liposomes and, used with HBO, the HBO worked as an adjuvant. This explains the decreases of oviposition and worms recovered form hepatic portal system.

Evaluation of the effects of electrical stimulation on cartilage repair in adult male rats.

This study describes the organization of mature hyaline xiphoid cartilage during repair in animals submitted to electrical current stimulation. Twenty male Wistar rats, 90 days old, were divided into a control group (CG) and a treated group (TG). A cylindrical full-thickness cartilage defects were created with a 3-mm punch in anesthetized animals. After 24h, TG received daily applications of a continuous electrical current (1Hz/20µA) for 5min. The animals were sacrificed after 7, 21 and 35 days for structural analysis. In CG, the repair tissue presented fibrous characteristics, with fibroblastic cells being infiltrated and permeated by blood vessels. Basophilic foci of cartilage tissue were observed on day 35. In TG, the repair tissue also presented fibrous characteristics, but a larger number of thick collagen fibers were seen on day 21. A large number of cartilaginous nests were observed on day 35. Cell numbers were significantly higher in TG. Calcification points were detected in TG on day 35. There was no difference in elastic fibers between groups. Ultrastructural analysis revealed the presence of chondrocyte-like cells in CG at all time points, but only on days 21 and 35 in TG. The amount of cuprolinic blue-stained proteoglycans was higher in TG on day 35. Microcurrent stimulation accelerates the repair process in non-articular hyaline cartilage.

In vivo and in vitro Leishmania amazonensis infection induces autophagy in macrophages.

Autophagy is the primary mechanism of degradation of cellular proteins and at least two functions can be attributed to this biological phenomenon: increased nutrient supply via recycling of the products of autophagy under nutrient starvation; and antimicrobial response involved in the innate immune system. Many microorganisms induce host cell autophagy and it has been proposed as a pathway by which parasites compete with the host cell for limited resources. In this report we provide evidence that the intracellular parasite Leishmania amazonensis induces autophagy in macrophages. Using western blotting, the LC3II protein, a marker of autophagosomes, was detected in cell cultures with a high infection index. Macrophages infected with L. amazonensis were examined by transmission electronic microscopy, which revealed enlarged myelin-like structures typical late autophagosome and autolysosome. Other evidence indicating autophagy was Lysotracker red dye uptake by the macrophages. Autophagy also occurs in the leishmaniasis skin lesions of BALB/c mice, detected by immunohistochemistry with anti-LC3II antibody. In this study, autophagy inhibitor 3-methyladenine (3MA) reduced the infection index, while autophagy inductors, such as rapamycin or starvation, did not alter the infection index in cultivated macrophages, suggesting that one aspect of the role of autophagy could be the provision of nutritive support to the parasite.

Recovery of the pubic symphysis on primiparous young and multiparous senescent mice at postpartum.

It has been observed that parturition has a significant effect on female skeletal architecture and that age alters musculoskeletal tissues and their functions. We therefore hypothesized that multiparity affects the recovery of the pubic symphysis in senescent mice at postpartum and the morphology of the interpubic tissues. The pubic symphysis of primiparous young, virgin senescent (VS) and multiparous senescent (MS) Swiss mice was examined by light microscopy, transmission electron microscopy, morphometric analysis and immunohistochemistry. The mouse pubic symphysis was remodeled during the first pregnancy: the cellular phenotype and morphology changed to ensure a structurally safe birth canal, followed by recovery of the interpubic articulation after birth. The morphology of the pubic symphysis in the VS group was maintained in a state similar to that observed in virgin young mice. In contrast, MS mice exhibited an interpubic ligament characterized by extended fibrocyte-like cells, an opened interpubic articulation gap, compacted and thin collagen fibrils and scarce galectin-3-positive cells. Thus, we found that the cellular and extracellular characteristics of the pubic symphysis were altered by multiparity in senescent mice. These particular tissue characteristics of the MS group might be associated with an impaired recovery process at postpartum. Thus, a better understanding of the alterations that occur in the birth canal, including the pubic symphysis, due to multiparity in reproductively aged mice may contribute to our comprehension of the biological mechanisms that modify the skeleton and pelvic ligaments and even play a role in the murine model of pelvic organ prolapse.