Estrogen depletion modulates aortic prothrombotic signaling in normotensive and spontaneously hypertensive female rats.

AIM: In this study, we investigated how platelets and aorta contribute to the creation and maintenance of a prothrombotic state in an experimental model of postmenopausal hypertension in ovariectomized rats. METHODS: Bilateral ovariectomy was performed in both 14-week-old female spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. The animals were kept in phytoestrogen free diet. Vascular parameters, platelet, coagulation and aortic prothrombotic functions and mechanisms were assessed. RESULTS: Exacerbated platelet aggregation was observed in both SHR and WKY animals after ovariectomy. The mechanism was related to aortic COX2 downregulation and reduction in AMP, ADP, and ATP hydrolysis in serum and platelets. A procoagulant potential was observed in plasma from ovariectomized rats and this was confirmed by kallikrein and factor Xa generation in aortic rings. Aortic rings derived from ovariectomized SHR presented a greater thrombin generation capacity compared to equivalent rings from WKY animals. The mechanism involved tissue factor and PAR-1 upregulation as well as an increase in extrinsic coagulation and fibrinolysis markers in aorta and platelets. Aortic smooth muscle cells pre-treated with a plasma pool derived from estrogen-depleted animals developed a procoagulant profile with tissue factor upregulation. This procoagulant profile was dependent on inflammatory signalling, since NFκB inhibition attenuated the procoagulant activity and tissue factor expression. CONCLUSIONS: A prothrombotic phenotype was observed in both WKY and SHR ovariectomized rats being associated with platelet hyperreactivity and tissue factor upregulation in aorta and platelets. The mechanism involves proinflammatory signalling that supports greater thrombin generation in aorta and vascular smooth muscle cells.

Tissue engineering application combining epoxidized natural rubber blend and mesenchymal stem cells in in vivo response.

This study aimed to investigate biocompatibility, integration, and tissue host response of the Poly (Lactic-co-Glycolic acid) (PLGA)/Poly (isoprene) (PI) epoxidized (PLGA/PIepox) innovative scaffold combined with adipose derived mesenchymal stem cells (ADSC). We implanted the scaffold subcutaneously on the back of 18 female rats and monitored them for up to 14 days. When compared to controls, PLGA/PIepox + ADSC demonstrated an earlier vascularization, a tendency of inflammation reduction, an adequate tissue integration, higher cell proliferation, and a tendency of expression of collagen decreasing. However, 14 days post-implantation we found similar levels of CD31, Ki67 and AE1/AE3 in PLGA/PIepox when compared to control groups. The interesting results, lead us to the assumption that PLGA/PIepox is able to provide an effective delivery system for ADSC on tissue host. This animal study assesses PLGA/PIepox + ADSC in in vivo tissue functionality and validation of use, serving as a proof of concept for future clinical translation as it presents an innovative and promising tissue engineering opportunity for the use in tissue reconstruction.

Use of clomiphene citrate protocol for controlled ovarian stimulation impairs endometrial maturity.

INTRODUCTION: Despite recent advances in assisted reproduction techniques and recent knowledge regarding embryo and endometrium quality, implantation and birth rates remain low. The objective of this study was to investigate whether clomiphene citrate alters endometrial maturation in infertile patients. METHODS: In a prospective self-matched cohort study, we assessed the ovulation of women in spontaneous and stimulated cycles (with clomiphene citrate). We determined the ovulation day by ultrasound scanning. In both cycles, we took four blood samples (BS1 - at early proliferative phase, BS2 - at mid proliferative phase, BS3 - after ovulation and BS4 - at mid luteal phase) to determine the serum concentrations of FSH, LH, estradiol and progesterone. We retrieved an endometrial biopsy five days after ovulation, followed by blinded analysis and classification according to Noyes criteria, in both cycles. RESULTS: Twenty-two participants completed the study. There were significant differences in FSH BS3 (p=0.001), in LH BS3 and BS4 (p<0.001 and p=0.049, respectively), in estradiol BS2, BS3 and BS4 (p<0.001, p=0.024 and p<0.001, respectively) and in progesterone BS3 and BS4 (p=0.028 and p<0.001, respectively). Considering Noyes criteria, there was a one-day delay when comparing the stimulated cycle with the spontaneous cycle (p=0.004), and a two-day delay when comparing the stimulated cycle with the biopsy day. CONCLUSION: This study indicates that ovarian stimulation with clomiphene citrate delays the endometrial maturity, and could possibly impair the implantation process due to asynchrony.

Kallikrein-kinin system and oxidative stress in cisplatin-induced ovarian toxicity.

Kallikrein-kinin system (KKS) is involved in vascular reactivity and inflammatory response to cytotoxic drugs. Since cisplatin is a widely used chemotherapy and its cytotoxic mechanism can trigger inflammation and oxidative damage, in this work we evaluated the role of KKS in an animal model of cisplatin-induced ovarian toxicity. Biomarkers of ovarian stem cells, activity of KKS, inflammation and oxidative damage were measured in ovarian tissue of C57BL/6 female mice treated with vehicle or cisplatin (2.5 mg/kg). Cisplatin group presented greater number of atretic follicles, and lower numbers of antral and total viable follicles. Ki67, DDX4 and OCT-4 markers were similar between groups. Cisplatin triggered plasma and ovarian tissue kallikrein generation; and increased expression of bradykinin receptors B1 and B2. Neutrophil and macrophage infiltration markers increased. Superoxide anion generation also increased, while reduced glutathione levels decreased. These results suggest that KKS is activated and contributes to ovarian injury during cisplatin treatment.

Ovarian tissue vitrification is more efficient than slow freezing to preserve ovarian stem cells in CF-1 mice.

OBJECTIVE: The aim of this study was to investigate the efficacy of protocols for mice ovary cryopreservation to compare the differences in Mouse Vasa Homologue expression (a germline cell marker) and ovarian viability after vitrification or slow freezing. METHODS: Female CF1 mice aged 40-45 days were randomly divided into three groups: Control, vitrification or slow freezing. Their ovaries were surgically removed, rinsed in saline solution and cryopreserved. For vitrification, we used a commercial protocol and for slow freeze, we used 1.5 M ethylene glycol (EG) as cryoprotectant. After that, the ovaries were processed for histological an immunohistochemical analysis, and counting of primordial, primary, pre-antral and antral follicles. RESULTS: No significant difference was found in the proportion of high-quality primordial, primary and pre-antral follicles after thawing/warming in the slow freezing and vitrification groups. The immunohistochemistry for MVH antibody demonstrated that the slow freeze group had a higher number of unmarked cells (p=0.012), indicating a harmful effect on the MVH expression in the ovarian tissue, where the cell structure is complex. CONCLUSION: Although both protocols indicated similar results in the histological analysis of follicular counts, the vitrification protocol was significantly better to preserve ovarian stem cells, an immature germ cell population. These cells are able to self-renew having regeneration potential, and may be effective for the treatment of ovarian failure and consequently infertility.

Adipose-derived stem cells improve full-thickness skin grafts in a rat model.

To investigate the effects of heterologous adipose-derived stem cells (ADSCs) on autologous full-thickness skin grafts, we designed a first-intention healing model using Wistar rats. We harvested and sutured two full-thickness skin grafts in the dorsal recipient beds of 15 rats, randomized into three groups. In the treatment group, 1 × 106 ADSCs resuspended in saline solution (200 µL) were administered subcutaneously to the skin graft. The control group received only saline solution subcutaneously, whereas the negative control group did not receive any treatment. Compressive dressings were maintained until postoperative day 5. The grafts were assessed by two observers, who checked for the presence of epidermolysis on day 14. Planimetry showed the relative areas of normal skin, redness, ulceration, and contraction. Graft samples were obtained on day 14 and stained with hematoxylin and eosin and Masson's trichrome. Epidermal analysis evaluated thickening, keratosis, acanthosis, hydropic degeneration, and inflammatory infiltrate. Dermal evaluation investigated the absence of hair follicles, granulation tissue formation, presence of inflammatory infiltrate, and collagen deposition. Immunohistochemistry was performed for dermal anti-VEGF and epidermal anti-Ki-67 staining. The ADSC group presented better macroscopic aspects, lower incidence of epidermolysis, and less loss of hair follicles. In addition, the ADSC group presented the lowest frequency of histopathological changes in the dermis and epidermis, as well as the largest subcutaneous and granulation tissue VEGF averages and the weakest Ki-67 staining of the epidermal basal layer. Subcutaneous administration of ADSCs may improve the integration of skin grafts, reducing the deleterious effects of ischemia and reperfusion injury.

Cell therapy for chemically induced ovarian failure in mice.

Cell therapy has been linked to an unexplained return of ovarian function and fertility in some cancer survivors. Studies modeling this in mice have shown that cells transplantation generates donor-derived oocytes in chemotherapy-treated recipients. This study was conducted to further clarify the impact of cell transplantation from different sources on female reproductive function after chemotherapy using a preclinical mouse model. Methods. Female mice were administered 7.5 mg/kg cisplatin followed by cell transplantation (one week later) using GFP+ female cell donors. For cell tracking, adipose derived stem cell GFP+ (ADSC), female germline stem cell GFP+/MVH+ (FGSC), or ovary cell suspension GFP+ mice were transplanted into cisplatin-treated wild-type recipients. After 7 or 14 days animals were killed and histological analysis, IHQ for GFP cells, and ELISA for estradiol were performed. Results. Histological examinations showed that ADSC, ovary cell suspension, and FGSC transplant increase the number of follicles with apparent normal structure in the cells recipient group euthanized on day 7. Cell tracking showed GFP+ samples 7 days after transplant. Conclusion. These data suggest that intraovarian injection of ADSCs and FGSC into mice with chemotherapy-induced ovarian failure diminished the damage caused by cisplatin.

Development of different degrees of elastase-induced emphysema in mice: a randomized controlled experimental study

Introduction: Mouse models of emphysema are important tools for testing different therapeutic strategies. The aim of this study was to develop a mouse model of emphysema induced by different doses of elastase in order to produce different degrees of severity. Methods: Thirty female mice (C57BL/6) were used in this study. Different doses of porcine pancreatic elastase were administered intratracheally once a week for four weeks, as follows: 0.1 U (n=8), 0.15 U (n=7), and 0.2 U (n=7). Control mice (n=8) received 50 microL of sterile saline solution intratracheally. Lung mechanics were analyzed by plethysmography. Mean linear intercept and volume fraction occupied by collagen and elastic fibers were determined. Results: An increase in lung resistance was observed with 0.2 U of elastase [median (P-25-P75): 2.02 (1.67; 2.34) cmH2O.s/mL], as well as a decrease in tidal volume and minute ventilation. Peak expiratory flow increased significantly in the groups treated with 0.15 U and 0.2 U of elastase. Mean linear intercept was higher with 0.15 U and 0.2 U of elastase, with destruction of alveolar walls [median (P-25-P75): 30.31 (26.65-43.13) microm and 49.49 (31.67-57.71) microm respectively]. The volume fraction occupied by collagen and elastic fibers was lower in the group receiving 0.2 U of elastase. Conclusion: Four intratracheal instillations of 0.2 U of elastase once a week induced changes in lung function and histology, producing an experimental model of severe pulmonary emphysema, whereas 0.15 U resulted in only histological changes.

Gênero e manifestação de stress em hipertensos / Gender and the manifestation of stess in patients hypertensive

Embora estudos sustentem maior vulnerabilidade ao stress na população feminina, há escassez de dados sobre a população de hipertensos. Realizou-se um estudo transversal para verificar a diferença na prevalência de sintomas de stress entre os gêneros, por meio da aplicação do Inventário de Sintomas de Stress de Lipp e de um questionário de dados sociodemográficos. Os resultados não mostram diferença estatisticamente significativa concernente à presença de sintomas de stress entre as amostras masculina (n=33) e feminina (n=70). Contudo, mais homens apresentaram escores na fase de resistência (60,6 por cento), enquanto a fase de quase-exaustão preponderou entre as mulheres: 41,4 por cento versus 15,2 por cento dos homens. Considerando sintomas físicos e psicológicos no geral, independente de predominância de fase, as mulheres relataram ter significativamente mais sintomas psicológicos de stress nos escores referentes às fases de resistência, quase-exaustão e à fase de exaustão (p<0,05). Esses achados podem auxiliar no planejamento de abordagens de controle de stress.

Even though studies support the idea of greater vulnerability to stress in the female population, there is a dearth of studies related to hypertensive patients. A cross-sectional study was conducted with the aim of verifying the difference in the prevalence of stress symptoms in a male and female sample of hypertensive patients, using Lipp's Adult Stress Symptoms Inventory and a socio-demographic questionnaire. There was no statistically significant difference in respect of the presence of stress symptoms between males (n=33) and females (n=70). However, men obtained higher scores in the resistance phase (60.6 percent), while women obtained higher scores in the near-exhaustion phase (41.4 percent versus 15.2 percent of men). Considering overall physical and psychological symptoms, irrespective of the predominant phase, women reported significantly higher psychological stress symptoms than men in terms of the scores in the resistance and near-exhaustion phases, as well as the scores in the exhaustion phase (p<0.05). These findings may help in the planning of stress-management strategies.

Gênero e manifestação de stress em hipertensos / Gender and the manifestation of stess in patients hypertensive

Embora estudos sustentem maior vulnerabilidade ao stress na população feminina, há escassez de dados sobre a população de hipertensos. Realizou-se um estudo transversal para verificar a diferença na prevalência de sintomas de stress entre os gêneros, por meio da aplicação do Inventário de Sintomas de Stress de Lipp e de um questionário de dados sociodemográficos. Os resultados não mostram diferença estatisticamente significativa concernente à presença de sintomas de stress entre as amostras masculina (n=33) e feminina (n=70). Contudo, mais homens apresentaram escores na fase de resistência (60,6 por cento), enquanto a fase de quase-exaustão preponderou entre as mulheres: 41,4 por cento versus 15,2 por cento dos homens. Considerando sintomas físicos e psicológicos no geral, independente de predominância de fase, as mulheres relataram ter significativamente mais sintomas psicológicos de stress nos escores referentes às fases de resistência, quase-exaustão e à fase de exaustão (p<0,05). Esses achados podem auxiliar no planejamento de abordagens de controle de stress.(AU)

Even though studies support the idea of greater vulnerability to stress in the female population, there is a dearth of studies related to hypertensive patients. A cross-sectional study was conducted with the aim of verifying the difference in the prevalence of stress symptoms in a male and female sample of hypertensive patients, using Lipp's Adult Stress Symptoms Inventory and a socio-demographic questionnaire. There was no statistically significant difference in respect of the presence of stress symptoms between males (n=33) and females (n=70). However, men obtained higher scores in the resistance phase (60.6 percent), while women obtained higher scores in the near-exhaustion phase (41.4 percent versus 15.2 percent of men). Considering overall physical and psychological symptoms, irrespective of the predominant phase, women reported significantly higher psychological stress symptoms than men in terms of the scores in the resistance and near-exhaustion phases, as well as the scores in the exhaustion phase (p<0.05). These findings may help in the planning of stress-management strategies.(AU)