Histomorphometric analysis of the endometrium in an ectopic model of endometriosis in mice.

Objective: Evaluate histomorphometry of ectopic and eutopic endometrial tissues in receptor mice. Method: Eighteen female Balb/c were divided into 3 groups, 6 animals each: GI Control, no procedure; GII - Sham, animals that had the same procedures as GIII without receiving the ectopic endometrial implant. Instead, they received saline solution; GIII - endometriosis model, animals had surgical intervention with an ectopic endometrial implant. GI and GIII mice were treated with 17ß-estradiol, 100 µg/kg each. All animals were euthanized to collect uterine horns, which were fixed in 4% paraformaldehyde, embedded in paraffin, stained with Hematoxilin and Eosin and submitted to histomorphometric analyzes. Data underwent one-way ANOVA followed by Tukey's test. Results: Local tissue growth, showing important lesions and adhesions, as well as dark cysts were noticed. In GIII group, there was an increase in number of blood vessels and glands (GIII ≥ GI and GIII p > .001). Thickening of the GIII endometrial epithelial was also evident (GIII ≥ GI and GIII. p > .001). We also noticed an increase in the number of eosinophils (GIII (GIII ≥ GI and GIII. p > .001). Conclusion: Easy to perform model, capable of reproducing morphological endometriosis characteristics. From our findings, there was an increase of endometrial thickness as well as an increase in the eosinophils population.

Isoflavone Protects the Renal Tissue of Diabetic Ovariectomized Rats via PPARγ.

Diabetes associated with post-menopause is related to a worse condition of kidney disease. Taking into consideration that this disorder may be regulated by estrogenic mediators, we evaluated the renal protective effect of isoflavone. We investigated the role of the PPARγ in the pathogenesis of the disease. For this study, we used diabetic female rats in a postmenopausal model through ovariectomy. The animals were treated with isoflavone or 17ß-estradiol. A dosage was administered to bring on blood glycemia, and through immunohistochemistry, we evaluated the immunoreactivity of PPARγ in the endometrium and renal tissue. We analyzed the immunoreactivity of renal injury molecule KIM-1 and the collagen and glycogen densities in the kidney. Through bioinformatics analysis, we observed PPARγ and COL1A1 gene expression under the influence of different glucose doses. In particular, we observed that isoflavone and 17ß-estradiol regulate blood glycemia. Renal injury was inhibited by isoflavone, observed by a reduction in KIM-1, along with glycogen accumulation. These benefits of isoflavone may be associated with PPARγ overexpression in the kidneys and endometrium of diabetic ovariectomized rats.

Spatio-temporal immunolocalization of VEGF-A, Runx2, and osterix during the early steps of intramembranous ossification of the alveolar process in rat embryos.

Vascular endothelial growth factor A (VEGF-A) is expressed by several cell types and is a crucial factor for angiogenic-osteogenic coupling. However, the immunolocalization of VEGF-A during the early stages of the alveolar process formation remains underexplored. Thus, we analyzed the spatio-temporal immunolocalization of VEGF-A and its relationship with Runt-related transcription factor 2 (Runx2) and osterix (Osx) during the early steps of intramembranous ossification of the alveolar process in rat embryos. Embryo heads (E) of 16, 18 and 20-day-old rats were processed for paraffin embedding. Histomorphometry and immunohistochemistry to detect VEGF-A, Runx2, and Osx (osteoblast differentiation markers) were performed. The volume density of bone tissue including bone cells and blood vessels increased significantly in E18 and E20. Cells showing high VEGF-A immunoreactivity were initially observed within a perivascular niche in the ectomesenchyme; afterwards, these cells were diffusely located near bone formation sites. Runx2-and Osx-immunopositive cells were observed in corresponded regions of cells showing strong VEGF-A immunoreactivity. Although these immunostained cells were observed in all specimens, this immunolocalization pattern was more evident in E16 specimens and gradually decreased in E18 and E20 specimens. Double immunofluorescence labelling showed intracellular co-localization of Osx and VEGF-A in cells surrounding the developing alveolar process, indicating a crucial role of VEGF-A in osteoblast differentiation. Our results showed VEGF-A immunoexpression in osteoblasts and its precursors during the maxillary alveolar process formation of rat embryos. Moreover, the VEGF-A-positive cells located within a perivascular niche at the early stages of the alveolar process development suggest a crosstalk between endothelium and ectomesenchymal cells, reinforcing the angiogenic-osteogenic coupling in this process.

Evidence that Melatonin Increases Inhibin Beta-A and Follistatin Gene Expression in Ovaries of Pinealectomized Rats.

Melatonin plays an important role in the regulation of ovarian function including oocyte maturation in different mammalian species. Many studies indicate that melatonin has an impact on the ovarian function of a variety of ovarian cells. However, the information on the exact mechanism and involved hormones is low. To evaluate inhibin beta-A (INHBA) and follistatin (FST) expression in the ovaries of pinealectomized rats treated with melatonin, thirty adult female Wistar rats were randomized into three groups of ten animals each: group 1 (GSh), sham-operated controls receiving vehicle; group 2 (GPx), pinealectomized animals receiving vehicle; and group 3 (GPxMe), pinealectomized animals receiving replacement melatonin (1.0 mg/kg body weight. It was assumed that each animal drank 6.5 ± 1.2 ml per night and weighs approximately 300 g.) for 60 consecutive days. The ovaries were collected for mRNA abundance and protein of INHBA and FST by qRT-PCR and immunohistochemical analyses, respectively. Treatment with melatonin resulted in the upregulation of INHBA and FST genes in the ovarian tissue of the melatonin-treated animals (GPxMe), when compared with GPx. These findings were then confirmed by analyzing the expression of protein by immunohistochemical analyses, which revealed higher immunoreactivity of INHBA and FST in GPxMe animals in the follicular cells compared with GSh and GPx rats. Melatonin increases the expression of INHBA and FST in the ovaries of pinealectomized female rats.

Metformin improves ovarian follicle dynamics by reducing theca cell proliferation and CYP-17 expression in an androgenized rat model.

BACKGROUND: Metformin influences insulin receptor signaling, which might interfere with the proliferation of ovarian follicular structures and steroidogenesis. We hypothesize that reductions in glucose and insulin levels might interfere with CYP-17 expression and histomorphological changes in an androgenized rat model. The aim of this study was to analyze the effect of metformin on CYP-17 expression, follicular dynamics, and proliferative parameters in neonatally androgenized female rats. METHODS: Thirty-six newborn rats were randomly allocated to the following three groups on the third day of life: control (CG, n = 12), androgenized (GA, n = 12), and androgenized + metformin (GAmet, n = 12). The GA and GAmet animals were administered 0.1 mL of testosterone propionate (1.25 mg/animal) diluted in castor oil (vehicle) in a single dose; the CG rats received a subcutaneous injection of the vehicle in the dorsum. After 90 days, gavage treatment was initiated, distilled water was administered to the CG and GA rats, and metformin (150 mg/kg) was administered to the GAmet animals. The treatment was administered daily for six weeks. Following anesthesia, blood was drawn for biochemical measurements, and the ovaries were removed for histological and immunohistochemical analyses of Ki67, VEGFA and CYP17 expression. The glucose and insulin levels were also measured. RESULTS: The comparison of the GA and GAmet animals revealed that metformin decreased the weight as well as the glucose and insulin levels, slowed the proliferation of the theca interna and interstitial cells, as evidenced by Ki-67 and VEGF-A expression, and diminished CYP17 expression in the analyzed ovarian structures. In addition, metformin reduced the number of degenerating follicles and interstitial cells and improved angiogenesis. CONCLUSION: Metformin improves the carbohydrate metabolism, reduces proliferation, and decreases CYP-17 expression in the follicular structures of androgenized rats.

IGF-1R and Leptin Expression Profile and the Effects of Metformin Treatment on Metabolic and Endocrine Parameters in PCOS Mice.

We aim to assess the effects of metformin treatment on metabolic and endocrine parameters and genes expression related to the insulin-responsive pathway in polycystic ovary syndrome (PCOS). This study comprises twenty-eight obese mice divided into three metformin-treated groups for seven and twenty days and eight nonobese and nontreated ones. We found a significant decrease in glycemia after metformin treatment at days seven and twenty. However, we did not observe differences in body weight measurement. Histologically, after twenty days we observed follicular development with regression of androgenic effects. Levels of IGF-1R protein expression were low after twenty days of treatment, but LEP proteins showed an overexpression in the ovarian stroma. We assessed the IGF-1R and LEP mRNAs levels; data showed a significant overexpression of LEP after seven days of treatment, while the IGF-1R was downregulated. Metformin therapy seems to exert a beneficial effect on histological and anovulatory features, reducing follicular number and pyknosis formation, possibly involved in the reversion of androgenic stimulus. Expression of IGF-1 and LEPR indicates a relevant role in androgenic features reversion present in PCOS, hormonal equilibrium, body weight regulation, and glucose metabolism, therefore, under phenotype obesity and infertility regulation in this model.

Adipose tissue-derived stem cell therapy in rat cryopreserved ovarian grafts.

The preliminary results of ovarian transplantation in clinical practice are encouraging. However, the follicular depletion caused by ischemic injury is a main concern and is directly related to short-term graft survival. Cell therapy with adipose tissue-derived stem cells (ASCs) could be an alternative to induce early angiogenesis in the graft. This study aimed to evaluate ASCs therapy in rat cryopreserved ovarian grafts. A single dose of rat ASC (rASCs) or vehicle was injected into the bilateral cryopreserved ovaries of twelve adult female rats immediately after an autologous transplant. Daily vaginal smears were performed for estrous cycle evaluation until euthanasia on postoperative day 30. Follicle viability, graft morphology and apoptosis were assessed. No differences were found with respect to estrous cycle resumption and follicle viability (P>0.05). However, compared with the vehicle-treated grafts, the morphology of the ASCs-treated grafts was impaired, with diffuse atrophy and increased apoptosis (P<0.05). ASCs direct injected in the stroma of rat cryopreserved ovarian grafts impaired its morphology although may not interfere with the functional resumption on short-term. Further investigations are necessary to evaluated whether it could compromise their viability in the long-term.

Effects of melatonin on ovarian follicles.

OBJECTIVE: To evaluate the histomorphometry and expression of Ki-67 and c-kit in ovarian follicles of pinealectomized or melatonin-treated pinealectomized rats. STUDY DESIGN: Forty adult rats were randomly divided into four groups of 10 animals: Group I - control; Group II - sham-pinealectomized; Group III - pinealectomized (Px), and Group IV - Px treated with melatonin (10µg/night, per animal). After two months' treatment, on the night of proestrous, the animals were placed in metabolic cages for night urine collection and subsequent measurement of 6-sulfatoxymelatonin (6-SMT). The rats were anesthetized, blood samples were taken for estrogen and progesterone determinations, and they were then euthanized. The ovaries were dissected out for further histological and immunohistochemical analyses. Data were first submitted to analysis of variance (ANOVA) complemented with the Tukey-Kramer test for multiple comparisons (P<0.05). RESULTS: The urinary levels of 6-SMT and serum progesterone were lower in the Px group (GIII). Exogenous melatonin treatment restored both blood melatonin and 6-SMT urinary levels. The histomorphometric data in Group III revealed a significant increase of degenerating antral and non-antral follicles with regard to the other groups. In addition no corpora lutea were observed in this group. No significant differences were noticed regarding the number of corpora lutea among the other groups (I, II and IV), but the number of cells and the thickness of the theca interna of Px animals (Group III) were higher than in the other groups. Conversely, the density of progesterone receptors (fmol/g) in the ovaries of Group III was significantly lower than in the other groups. CONCLUSION: Our data indicate that melatonin exerts a role on the maintenance of a proper follicular function, and is thus important for ovulation and progesterone production.

Effects of metoclopramide-induced hyperprolactinemia on the prolactin receptor of murine endometrium.

OBJECTIVE: To evaluate the effects of metoclopramide-induced hyperprolactinemia on the prolactin receptor of murine endometrium. DESIGN: Experimental study using the RNA extraction to detect tissue prolactin receptor isoforms by reverse-transcriptase polymerase chain reaction (RT-PCR). SETTING: University-based laboratory. ANIMAL(S): Seventy-two female swiss albino mice (Mus musculus), approximately 100 days old, were divided into six 12-animal groups: (GI) nonoophorectomized mice given vehicle; (GII) nonoophorectomized mice treated with metoclopramide; (GIII) oophorectomized mice treated with metoclopramide; (GIV) oophorectomized mice treated with metoclopramide and 17beta-estradiol; (GV) oophorectomized mice treated with metoclopramide and micronized progesterone; (GVI) oophorectomized mice treated with metoclopramide and a solution of 17beta-estradiol and micronized progesterone. INTERVENTION(S): Drugs were administered for 50 days. Following euthanasia, the middle portions of the uterine horns were removed, sectioned, and immediately frozen for RT-PCR procedures. Blood was collected for the dosage of prolactin and serum estrogen and progesterone using radioimmune assay. MAIN OUTCOME MEASURE(S): Identification of uterine prolactin receptor isoforms. RESULT(S): The PRL receptor and its isoform L were identified only in GI (control group) and GII (metoclopramide), the two groups with nonoophorectomized animals. The amount of PRL receptor mRNA and that of its isoform L from GII were the largest. No other isoforms of the prolactin receptor were identified in any of the groups. CONCLUSION(S): Our results suggest that replacement of estrogen and progestin may not increase the mRNA of endometrial PRL receptor in metoclopromide-induced hyperprolactinemia in rats after castration.

Zafirlukast pocket delivery impairs the capsule healing around textured implants in rats.

BACKGROUND: The aim of this study was to investigate the effects of zafirlukast on capsule thickness, collagen fiber density, and myofibroblast cell count of the healing tissue around silicone textured implants in rats. METHODS: Thirty-six male Wistar rats were divided (n = 18) into two groups. In one group, two parallel incisions (1.5 cm long) were made into the right and left sides of the spine. Two pockets were then created in which shell-shaped textured implants were inserted. The left-side pocket was injected with 0.2 ml of saline solution (SSG) and the right-side pocket with a dose of 1.25 mg/kg of zafirlukast (ZLG). The other 18 rats (sham, SG) had only one pocket created, followed by the placement of an implant and injection of 0.2 ml of saline solution. The rats were euthanized on the 7th, 35th, or 90th days followed by careful dissection of the implant. The capsules and peri-implant tissues were prepared for histologic analysis. An ANOVA test and Tukey test were applied (p < 0.05). RESULTS: ZL was effective in impairing the capsule thickness on the 35th and 90th days compared to the other two groups (sham and saline). Not only was it effective in impairing the collagen density on the 35th and 90th days, but it also showed the same effect in the SSG (systemic); fewer myofibroblasts were counted on the 90th day in the ZLG compared to the SG group; the number of myofibroblasts was significantly lower in the ZLG than in the SSG. CONCLUSIONS: Pocket delivery of one dose of Zafirlukast was effective in impairing capsule formation around the textured implant.

Does gestrinone antagonize the effects of estrogen on endometrial implants upon the peritoneum of rats?

OBJECTIVE: To evaluate the effects of estrogen treatment in combination with gestrinone on an experimental rat model of endometriosis. METHODS: Uterine transplants were attached to the peritoneum of female Wistar rats via a surgical autotransplantation technique. The implanted area was measured during the proestrus phase and after hormonal treatment. We performed morphometric analysis and examined the macroscopic and morphometric alterations of endometrial implants after hormonal treatment in ovariectomized rats. RESULTS: The high dose of estrogen caused macroscopic increases in the endometrial implant group compared with other groups, which were similar to increases in the proestrus phase. The low dose showed morphometric development of implants, such as an increase in number of endometrial glands, leukocyte infiltration and mitosis. Gestrinone antagonized both doses of estrogen. CONCLUSION: Our findings suggest that gestrinone antagonizes estrogen's effects on rat peritoneal endometrial implants.

Ultrastructural evaluation of gingival glycosaminoglycans in ovariectomized rats: effects of steroid hormone therapy.

BACKGROUND/AIMS: To evaluate the behavior of glycosaminoglycans (GAGs) in rat gingiva and the effects of lack of sexual steroids and the hormonal therapy with estrogen and dexamethasone (DEX). METHODS: 40 female rats were divided into four groups: GI: animals in permanent estrus; GII: ovariectomized (OVX) animals + vehicle; GIII: OVX animals treated with 17beta-estradiol benzoate (10 microg/kg), and GIV: OVX animals treated with 17beta-estradiol benzoate (10 microg/kg) + DEX (3 mg/kg). After treatment, the gingiva was removed and its GAGs content was evaluated by electronic microscopy after stained by cuprolinic blue technique. RESULTS: The electron-microscopic data showed that low values of chondroitin sulfate were found in castrated animals (35.05 +/- 3.58%) compared to other groups (GI: 41.17 +/- 1.13; GIII: 48.04 +/- 2.60; GIV: 49.09 +/- 2.68%). In contrast, the amount of dermatan sulfate in GII (57.70 +/- 2.50%) was higher than in the other groups (GI: 46.12 +/- 1.30; GIII: 42.65 +/- 2.98; GIV: 42.68 +/- 5.43%). CONCLUSIONS: GAGs may be influenced by estradiol, and DEX did not seem to antagonize the role of estradiol in the GAGs of gingiva. The histotypical structure of gingiva is related to the amount of chondroitin sulfate. Consequently, the estrogen therapy may be important for gingival health.

Morphological and morphometric analysis of the adrenal cortex of androgenized female rats.

AIM: We evaluated the thickness of the adrenal cortex zones of female rats androgenized to mimic polycystic ovary syndrome. METHODS: Forty-four female virgin Wistar-Hannover rats were divided into two groups: controls (n = 17) and animals which received testosterone propionate on the 2nd day of life (n = 27). At 90 days of life, after confirmation of persistent estrus, the animals were sacrificed, and the adrenal cortex zones were evaluated. Student's t test and Levene's test were used in the statistical analysis (p < 0.05 considered significant). RESULTS: The adrenal glands of the androgenized rats were more voluminous and had a more intensely vascularized zona reticularis than the control animals. The mean thicknesses of zona glomerulosa and zona reticularis in the androgenized rats were 58.4 and 730.7 mum, respectively, significantly thicker than the values in the control group (45.0 and 328.3 mum, respectively). CONCLUSION: Zona reticularis and zona glomerulosa of the androgenized female rats were significantly thicker than those of the control animals.

The effects of soy extract on the uterus of castrated adult rats.

OBJECTIVE: The aim of this study was to evaluate the effects of different doses of a standardized soy extract on the uterus of castrated rats. METHODS: Fifty-six adult castrated female Wistar rats were randomly divided into seven groups (eight animals in each) that received: GI--drug vehicle (propylene glycol); GII--soy extract 10mg/kg per day; GIII--soy extract 50mg/kg per day; GIV--soy extract 100mg/kg per day; GV--soy extract 300mg/kg per day; GVI--soy extract 600mg/kg per day; GVII-conjugated equine estrogens (CEE) 200microg/kg per day. After 21 days of treatment, all animals were sacrificed and fragments of the uterine horns were immediately removed, fixed in 10% formaldehyde and submitted to routine histological techniques for morphometric study. The endometrial cell proliferation index was determined with the PCNA antibody PC-10 and expressed as the percentuals of the PCNA-positive nuclei relative to the total countings. Other fragments were immediately frozen in liquid nitrogen for RNA extraction and VEGF analysis using RT-PCR technique. RESULTS: The minimal dose of soy extract that produced a significant increase of the morphometric parameters was 100mg/kg (GIV). The maximum effects on endometrial and myometrial morphometry were detected in the groups treated with 300 and 600mg/kg of soy extract (groups V and VI) and CEE (GVII). The expression of PCNA in the endometrial epithelium and stroma was increased by treatment with 100-600mg/kg per day of soy extract (groups IV-VI) or with CCE (group VII). Doses equal to or higher than 50mg/kg of soy extract (groups III-VI) and CEE stimulated the expression of VEGF. CONCLUSION: The treatment of adult castrated rats during 21 days with doses of 100mg/kg per day or higher of soy extract may determine significant proliferation in the endometrium and myometrium.

Metoclopramide-induced hyperprolactinaemia caused marked decline in pinopodes and pregnancy rates in mice.

BACKGROUND: The impact of hyperprolactinaemia on endometrial function, along with embryo implantation, has been the subject of discussion. This article examines whether experimental hyperprolactinaemia can affect mouse ovarian function, endometrial pinopodes and embryo implantation. METHODS: For pinopode analysis, 60 female mice were randomly divided into two groups: control (vehicle) and experimental [metoclopramide (MCP) 200 microg per day]. Injections were given subcutaneously for 50 days, and then, normally cycling females were housed with male mice for copulation during proestrus. The animals were killed on the fifth day following coitus when the antimesometrium portions of the uterine horns were removed for endometrial analysis. Blood was collected for prolactin (PRL) determination. In the second experiment, 60 female mice were used to evaluate the ovarian function by measuring estrogen and progesterone levels and counting luteal bodies and oocytes in the oviduct and uterus during estrus. RESULTS: The highest pregnancy rates and the largest population of pinopodes were both found in the vehicle group (P<0.01). Estrogen and progesterone levels in MCP-treated mice were lower than those in control mice (P<0.05). Also, the number of implantations was significantly lower in the MCP-treated group compared with the vehicle group after embryo transfer (P<0.001). CONCLUSION: PRL seems to have suppressive effects on ovarian function and the number of pinopodes; conceivably, hyperprolactinaemia has a negative effect on mouse embryo implantation.

Experimental model of double wounds on the rats back , in order to study the skin cicatrization process on rat treated with cellulose coat

To the present study were used males rats (Rattus Norvegicus, var. Albinus, Rodentia Mammalia) EPM-1 Wistar, which came from Biotério Central da UNIFESP-EPM. Each animal was submitted to two wound of circular shape, made with a "punch", with superficial cuts of 2 cm diameter; located on medium line of dorsal region. In the present study, one wound was re-covered with the cellulose coat and the other serves as a control. This experimental form, makes it possible to us realize different studies such as macroscopic aspects of the wounds as well morphometric and morphological parameters analysis.

Foram utilizados para o presente estudo, ratos machos (Rattus Norvegicus, var. Albinus, Rodentia Mammalia), da linhagem EPM-1 Wistar, oriundos do Biotério Central da UNIFESP-EPM.Cada animal foi submetido a duas lesões de formato circular, realizadas através de um "punch", com superfície de corte de dois centímetros de diâmetro; localizadas na linha média da região dorsal. No presente estudo uma das lesões foi recoberta com a película de celulose e a outra servindo de controle. Esta forma de modelo experimental, nos possibilita realizarmos estudos quanto ao aspecto macroscópico das lesões (contratura e fechamento das feridas), bem como a análise de parâmetros morfométricos e morfológicos, dentre outros.

Efeito do diclofenaco de sódio na cicatrização da parede abdominal de ratos

The aim of the experiment was to do a valuation of the effect of the sodium diclofenac on the collagenous sinthesis in abdominal wall healing. It was used 40 male Wistar rats to do longitudinal laparotomies with a standardized technique, divided into 2 groups: One without the drug (control group) and another group wich were administrated sodium diclofenac (3mg/kg) every day for 4 days. In the 7th and 14th postoperative day, 10 animals of each group were submitted to euthanasia and the front abdominal wall involving the scar was removed to be prepared to hystological analysis. The segments were prepared with Hematoxilyn-Eosin and Picrosirius Red F3BA, in order to do either a general view of healing process or a quantitative valuation of collagenous. The resultant data were submitted to statistical analysis. It was conclued that the abdominal wall scar in rats treated with sodium diclofenac had less collagenous fibres in the 7th and 14th postoperative days than rats from the control group.

O objetivo deste trabalho foi avaliar o efeito do diclofenaco de sódio na cicatrização da parede abdominal de ratos. Foram utilizados 40 (quarenta) ratos machos Wistar, submetidos a laparotomia com técnica padronizada, distribuidos em dois grupos, um grupo controle e outro onde se administrou o diclofenaco de sódio - 3 mg/kg/dia, via intramuscular durante 4 (quatro) dias consecutivos. Ao sétimo e ao décimo quarto dias de pós operatório, respectivamente, fora realizada a eutanásia, retirando-se a camada músculo-facial abdominal envolvendo a cicatriz operatória para a realização do estudo histológico. Os segmentos foram corados por Hematoxilina-Eosina e Picrosirius Red F3BA, sendo feita observação qualitativa do processo cicatricial e quantitativa do colágeno. Os resultados encontrados foram analisados estatisticamente. Concluiu-se que a cicatriz da parede abdominal de rato tratado com o diclofenaco de sódio apresenta menor quantidade de fibras colágenas no 7 FONT FACE="Symbol">° /font> e 14 FONT FACE="Symbol">° /font> dias de pós-operatório, quando comparado a animal do grupo controle.