Cellular and extracellular vaginal changes following murine ovarian removal.

Loss of estrogen as a result of aging, pelvic cancer therapy, genetics, or eating disorders affects numerous body systems including the reproductive tract. Specifically, a chronic hypoestrogenic state fosters debilitating vaginal symptoms like atrophy, dryness, and dyspareunia. Current treatment options, including vaginal estrogen and hyaluronan (HA), anecdotally improve symptoms, but rectifying mechanisms are largely understudied. In order to study the hypoestrogenic vaginal environment, in particular the extracellular matrix (ECM), as well as understand the mechanisms behind current treatments and develop new therapies, we characterized a reliable and reproducible animal model. Bilateral ovariectomies (OVX) were performed on 9-week-old CD1 mice. After 1 month of estrogen loss due to ovarian removal, a phenotype that is similar to human vaginal tissue in an estrogen reduced state was noted in mice compared to sham-operated controls. The uterine to body weight ratio decreased by 80% and vaginal epithelium was significantly thinner in OVX compared to sham mice. Estrogen signaling was altered in OVX, but submucosal ERα localization did not reach statistical differences. HA localization in the submucosal area was altered and CD44 expression decreased in OVX mice. Collagen turn-over was altered following OVX. The inflammation profile was also disrupted, and submucosal vaginal CD45+ and F4/80+ cell populations were significantly reduced in the OVX mice. These results show altered cellular and molecular changes due to reduced estrogen levels. Developing new treatments for hypoestrogenic vaginal symptoms rely on better understanding of not only the cellular changes, but also the altered vaginal ECM environment. Further studies using this mouse model has the potential to advance women's vaginal health treatments and aid in understanding the interplay between organ systems in both healthy, aged, and diseased states.

Unveiling Vaginal Fibrosis: A Novel Murine Model Using Bleomycin and Epithelial Disruption.

Objective: Develop, validate, and characterize a fibrotic murine vaginal wound healing model using bleomycin instillations and epithelial disruption. Approach: We tested the effect of repeated bleomycin instillations with mucosal layer disruption on induction of vaginal fibrosis. Tissue samples collected at various time points were analyzed for fibrosis-related gene expression changes and collagen content. Results: Low (1.5U/kg) and high-dose (2.5U/kg) bleomycin instillations alone did not induce fibrosis, but when high-dose bleomycin was combined with epithelial disruption, increased pro-fibrotic gene expression and trichrome staining were observed. To evaluate spatial and temporal changes in the ECM structure and gene expression, tissue samples were collected at 1 day, 3 weeks, and 6 weeks after bleomycin and epithelial disruption. Data analyses revealed a significant decrease in matrix metabolizing genes and an increase in pro-fibrotic genes and inhibitors of matrix metabolizing genes in the bleomycin plus epithelial disruption group at 3 weeks. Elevated levels of the profibrotic genes Acta2 , Col1a1 , and Col3a were exclusively detected in this group at 3 weeks, and trichrome staining confirmed increased collagen content after 3 weeks. Hydroxyproline levels showed a tendency towards elevation at 3 weeks (p=0.12) and 6 weeks (p=0.14), indicating fibrosis manifestation at 3 weeks and resolution by 6 weeks post-instillation and epithelial disruption. Innovation: We combined bleomycin instillations with epithelial disruption to induce fibrosis and understand the mechanisms of the vaginal repair process. Conclusions: Epithelial disruption combined with bleomycin induces murine vaginal fibrosis within three weeks, characterized by increased collagen synthesis. Remarkably, the vaginal tissue fully recovers within six weeks, elucidating the regenerative capacity of the vagina.

Liver-specific deletion of mechanistic target of rapamycin does not protect against acetaminophen-induced liver injury in mice.

BACKGROUND: Acetaminophen (APAP) overdose can cause liver injury and liver failure, which is one of the most common causes of drug-induced liver injury in the United States. Pharmacological activation of autophagy by inhibiting mechanistic target of rapamycin (mTOR) protects against APAP-induced liver injury likely via autophagic removal of APAP-adducts and damaged mitochondria. In the present study, we aimed to investigate the role of genetic ablation of mTOR pathways in mouse liver in APAP-induced liver injury and liver repair/regeneration. METHODS: Albumin-Cre (Alb-Cre) mice, mTORf/f and Raptorf/f mice (C57BL/6J background) were crossbred to produce liver-specific mTOR knockout (L-mTOR KO, Alb Cre+/-, mTORf/f) and liver-specific Raptor KO (L-Raptor, Alb Cre+/-, Raptor f/f) mice. Alb-Cre littermates were used as wild-type (WT) mice. These mice were treated with APAP for various time points for up to 48 h. Liver injury, cell proliferation, autophagy and mTOR activation were determined. RESULTS: We found that genetic deletion of neither Raptor, an important adaptor protein in mTOR complex 1, nor mTOR, in the mouse liver significantly protected against APAP-induced liver injury despite increased hepatic autophagic flux. Genetic deletion of Raptor or mTOR in mouse livers did not affect APAP metabolism and APAP-induced c-Jun N-terminal kinase (JNK) activation, but slightly improved mouse survival likely due to increased hepatocyte proliferation. CONCLUSIONS: Our results indicate that genetic ablation of mTOR in mouse livers does not protect against APAP-induced liver injury but may slightly improve liver regeneration and mouse survival after APAP overdose.

Animal Models and Alternatives in Vaginal Research: a Comparative Review.

While developments in gynecologic health research continue advancing, relatively few groups specifically focus on vaginal tissue research for areas like wound healing, device development, and/or drug toxicity. Currently, there is no standardized animal or tissue model that mimics the full complexity of the human vagina. Certain practical factors such as appropriate size and anatomy, costs, and tissue environment vary across species and moreover fail to emulate all aspects of the human vagina. Thus, investigators are tasked with compromising specific properties of the vaginal environment as it relates to human physiology to suit their particular scientific question. Our review aims to facilitate the appropriate selection of a model aptly addressing a particular study by discussing pertinent vaginal characteristics of conventional animal and tissue models. In this review, we first cover common laboratory animals studied in vaginal research-mouse, rat, rabbit, minipig, and sheep-as well as human, with respect to the estrus cycle and related hormones, basic reproductive anatomy, the composition of vaginal layers, developmental epithelial origin, and microflora. In light of these relevant comparative metrics, we discuss potential selection criteria for choosing an appropriate animal vaginal model. Finally, we allude to the exciting prospects of increasing biomimicry for in vitro applications to provide a framework for investigators to model, interpret, and predict human vaginal health.

An Avant-Garde Model of Injury-Induced Regenerative Vaginal Wound Healing.

Objective: To design and validate a novel murine model of full-thickness (FT) vaginal wound healing that mirrors postinjury tissue repair and underscores the impact of estrogen signaling-driven healing kinetics, inflammation, and neovascularization. Approach: Five-week-old female CD1 mice were subjected to two 1-mm FT wounds. To assess wound healing kinetics, vaginas were harvested at 6, 12, 18, 24, 48, and 72 h and 7 days postinjury. Wounds from all time points were analyzed by hematoxylin and eosin and trichrome to, respectively, assess the rate of wound closure and tissue deposition. Inflammatory leukocyte (CD45), neutrophil (Ly6G), and macrophage (F480 and CD206) infiltration was examined by immunohistochemistry (IHC) and the resulting anti-inflammatory M2 (CD206)/total (F480) macrophage ratio quantified. Neovascularization (CD31) and estrogen receptor-α (ERα) expression levels were similarly determined by IHC. Results: We observed rapid healing with resolution of mucosal integrity by 48 h (p < 0.05), and overall neutrophils and polarized type 2 macrophages (M2) apexed at 12 h and reduced to near control levels by day 7 postinjury. Tissue repair was virtually indistinguishable from the surrounding vagina. CD31+ vessels increased between 12 h and day 7 and ERα trended to decrease at 12 h postinjury and rebound at day 7 to uninjured levels. Innovation: A proof-of-concept murine model to study vaginal wound healing kinetics and postinjury regenerative repair in the vagina was developed and verified. Conclusion: We surmise that murine vaginal mucosal repair is accelerated and potentially regulated by estrogen signaling through the ERα, thus providing a cellular and molecular foundation to understand vaginal healing responses to injury.

Emerging technologies in pediatric gynecology: new paradigms in women's health care.

PURPOSE OF REVIEW: The current review highlights the complexity of the pediatric and adolescent gynecology subspecialty as well as the recent and exciting opportunities for innovation within the field. RECENT FINDINGS: The opportunities for concept, treatment, instrument, and knowledge-transfer innovation to better serve the specific needs of pediatric gynecology patients include novel approaches to neovagina creation using magnets, improving postoperative vaginal wound healing through newly designed and degradable vaginal stents, and complex Mullerian reconstructive surgical planning using virtual reality immersive experiential training. SUMMARY: There is a significant window of opportunity to address the needs of pediatric, adolescent and adult gynecological patients with new innovative concepts and tools.

C57BL/6 Substrains Exhibit Different Responses to Acute Carbon Tetrachloride Exposure: Implications for Work Involving Transgenic Mice.

Biological differences exist between strains of laboratory mice, and it is becoming increasingly evident that there are differences between substrains. In the C57BL/6 mouse, the primary substrains are called 6J and 6N. Previous studies have demonstrated that 6J and 6N mice differ in response to many experimental models of human disease. The aim of our study was to determine if differences exist between 6J and 6N mice in terms of their response to acute carbon tetrachloride (CCl4) exposure. Mice were given CCl4 once and were euthanized 12 to 96 h later. Relative to 6J mice, we found that 6N mice had increased liver injury but more rapid repair. This was because of the increased speed with which necrotic hepatocytes were removed in 6N mice and was directly related to increased recruitment of macrophages to the liver. In parallel, enhanced liver regeneration was observed in 6N relative to 6J mice. Hepatic stellate cell activation occurred earlier in 6N mice, but there was no difference in matrix metabolism between substrains. Taken together, these data demonstrate specific and significant differences in how the C57BL/6 substrains respond to acute CCl4, which has important implications for all mouse studies utilizing this model.

Reproductive age-associated fibrosis in the stroma of the mammalian ovary.

Under normal physiological conditions, tissue remodeling in response to injury leads to tissue regeneration without permanent damage. However, if homeostasis between synthesis and degradation of extracellular matrix (ECM) components is altered, fibrosis - or the excess accumulation of ECM - can disrupt tissue architecture and function. Several organs, including the heart, lung and kidney, exhibit age-associated fibrosis. Here we investigated whether fibrosis underlies aging in the ovary - an organ that ages chronologically before other organs. We used Picrosirius Red (PSR), a connective tissue stain specific for collagen I and III fibers, to evaluate ovarian fibrosis. Using bright-field, epifluorescence, confocal and polarized light microscopy, we validated the specific staining of highly ordered PSR-stained fibers in the ovary. We next examined ovarian PSR staining in two mouse strains (CD1 and CB6F1) across an aging continuum and found that PSR staining was minimal in ovaries from reproductively young adult animals, increased in distinct foci in animals of mid-to-advanced reproductive age, and was prominent throughout the stroma of the oldest animals. Consistent with fibrosis, there was a reproductive age-associated increase in ovarian hydroxyproline content. We also observed a unique population of multinucleated macrophage giant cells, which are associated with chronic inflammation, within the ovarian stroma exclusively in reproductively old mice. In fact, several genes central to inflammation had significantly higher levels of expression in ovaries from reproductively old mice relative to young mice. These results establish fibrosis as an early hallmark of the aging ovarian stroma, and this altered microenvironment may contribute to the age-associated decline in gamete quality.

Differential effects of hyaluronan synthase 3 deficiency after acute vs chronic liver injury in mice.

BACKGROUND: Hyaluronan (HA) is a ubiquitous extracellular matrix (ECM) glycosaminoglycan synthesized by three different enzymes, hyaluronan synthase (HAS)1, 2, and 3. HA synthesis mediated by HAS3 promotes inflammation and is pathogenic in animal models of human lung and intestinal disease. Liver fibrosis is a common endpoint to chronic liver injury and inflammation for which there is no cure. Although plasma HA is a commonly used biomarker for liver disease, if and how HA contributes to disease pathogenesis remains unclear. Here, we tested the hypothesis that HA synthesized by HAS3 enhances inflammation and fibrosis. To test this hypothesis, we exposed wild-type or Has3-/- mice to carbon tetrachloride (CCl4) once (acute) or ten (chronic) times. RESULTS: HAS3-deficient mice exhibited increased hepatic injury and inflammatory chemokine production 48 h after acute CCl4; this was associated with a threefold reduction in plasma HA levels and alterations in the proportions of specific molecular weight HA polymer pools. Hepatic accumulation of fibrosis-associated transcripts was also greater in livers from HAS3-deficient mice compared to controls after acute CCl4 exposure. Surprisingly, fibrosis was not different between genotypes. Hepatic matrix metalloproteinase (MMP)13 mRNA and MMP13 activity was greater in livers from Has3-null mice after chronic CCl4; this was prevented by a MMP13-specific inhibitor. Collectively, these data suggest that Has3, or more likely HA produced by HAS3, limits hepatic inflammation after acute injury and attenuates MMP13-mediated matrix metabolism after chronic injury. CONCLUSIONS: These data suggest that HA should be investigated further as a novel therapeutic target for acute and chronic liver disease.

Moderate (2%, v/v) Ethanol Feeding Alters Hepatic Wound Healing after Acute Carbon Tetrachloride Exposure in Mice.

Wound healing consists of three overlapping phases: inflammation, proliferation, and matrix synthesis and remodeling. Prolonged alcohol abuse can cause liver fibrosis due to deregulated matrix remodeling. Previous studies demonstrated that moderate ethanol feeding enhances liver fibrogenic markers and frank fibrosis independent of differences in CCl4-induced liver injury. Our objective was to determine whether or not other phases of the hepatic wound healing response were affected by moderate ethanol after CCl4 exposure. Mice were fed moderate ethanol (2% v/v) for two days and then were exposed to CCl4 and euthanized 24-96 h later. Liver injury was not different between pair- and ethanol-fed mice; however, removal of necrotic tissue was delayed after CCl4-induced liver injury in ethanol-fed mice. Inflammation, measured by TNFα mRNA and protein and hepatic Ly6c transcript accumulation, was reduced and associated with enhanced hepatocyte apoptosis after ethanol feeding. Hepatocytes entered the cell cycle equivalently in pair- and ethanol-fed mice after CCl4 exposure, but hepatocyte proliferation was prolonged in livers from ethanol-fed mice. CCl4-induced hepatic stellate cell activation was increased and matrix remodeling was prolonged in ethanol-fed mice compared to controls. Taken together, moderate ethanol affected each phase of the wound healing response to CCl4. These data highlight previously unknown effects of moderate ethanol exposure on hepatic wound healing after acute hepatotoxicant exposure.

Identifying Novel Targets for Treatment of Liver Fibrosis: What Can We Learn from Injured Tissues which Heal Without a Scar?

The liver is unique in that it is able to regenerate. This regeneration occurs without formation of a scar in the case of non-iterative hepatic injury. However, when the liver is exposed to chronic liver injury, the purely regenerative process fails and excessive extracellular matrix proteins are deposited in place of normal liver parenchyma. While much has been discovered in the past three decades, insights into fibrotic mechanisms have not yet lead to effective therapies; liver transplant remains the only cure for advanced liver disease. In an effort to broaden the collection of possible therapeutic targets, this review will compare and contrast the liver wound healing response to that found in two types of wound healing: scarless wound healing of fetal skin and oral mucosa and scar-forming wound healing found in adult skin. This review will examine wound healing in the liver and the skin in relation to the role of humoral and cellular factors, as well as the extracellular matrix, in this process. While several therapeutic targets are similar between fibrotic liver and adult skin wound healing, others are unique and represent novel areas for hepatic anti-fibrotic research. In particular, investigations into the role of hyaluronan in liver fibrosis and fibrosis resolution are warranted.