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.

Effects of soy isoflavones extract on the lipid profile and uterus in ovariectomized rats.

AIM: Although soy isoflavones (ISO) have been shown to relief postmenopausal symptoms, it remains inconclusive whether ISO can improve lipid-profile without uterotrophic effects under estrogen-deficiency. Thus, we investigated the effects of ISO on lipid-profile and uterus of ovariectomized (Ovx) rats. MATERIALS AND METHODS: Twenty-five adult rats were Ovx or Sham-operated (Sham) and assigned into five groups: Sham and Ovx groups, administered with vehicle solutions; Ovx-E, treated with 10 µg/kg of 17ß-Estradiol; Ovx-ISO, treated with 200 mg/kg of ISO; Ovx-E + ISO, treated with estradiol + ISO combined. After fifty days of treatments, rats were euthanized and uterine horns were processed for histomorphometry or to collagen fibers and glycosaminoglycans evaluations. Blood samples were collected to evaluate levels of triglycerides, total cholesterol (TC) and its fractions (HDL/VLDL). Data were subjected to statistical analysis (p < .05). RESULTS: Uterus weight was lower in Ovx group than the Sham and Ovx-E groups, whereas it was similar between Ovx and Ovx-ISO groups. Histomorphometry showed atrophic uterus in Ovx and Ovx-ISO groups, whereas uterotrophic effects were noticed in Ovx-E and Ovx-E + ISO groups. Collagen fibers-birefringence was higher in Sham, Ovx, and Ovx-ISO groups than in Ovx-E and Ovx-E + ISO groups. Sulfated glycosaminoglycans content was similar among Sham, Ovx, and Ovx-ISO groups, while it was higher in estrogen-treated groups; total glycosaminoglycans content was similar among groups. TC and HDL was higher in Ovx-ISO group, whereas VLDL and triglycerides levels was higher in Ovx-E + ISO group and similar among other groups. CONCLUSION: Soy isoflavones at 200 mg/kg have slight beneficial effects on lipid-profile without uterotrophic effects in Ovx rats.

Histomorphometric and immunohistochemical changes in interstitial cells and ovarian follicles of rats with polycystic ovaries treated with clomiphene citrate.

OBJECTIVE: To evaluate the histomorphometric and immunohistochemical changes in interstitial cells and ovarian follicles of rats treated with clomiphene citrate during and after induction of permanent estrus. METHODS: Twenty four adult-female rats with regular estrous cycle were equally divided into three groups: (1) GCtrl-at estrous phase. (2) GPCOS-at permanent-estrous phase. (3) GCC-PCOS rats, which remained exposed to 60 days of continuous illumination and treated with Clomiphene Citrate. After that, the animals were euthanized, and the ovaries were removed and processed for paraffin embedding. Sections were stained with H.E. for histomorphometry or subjected to immunohistochemistry for Ki-67 and cleaved caspase-3 detections. RESULTS: The GPCOS showed lack of corpus luteum and several ovarian cysts, as well as interstitial-like cells. The presence of corpus luteum and a significant increase in primary and antral follicles were observed in GCC, which also showed a decrease in the number of ovarian cysts and in the area occupied by interstitial-like cells, as well as a decrease in nuclear volume of interstitial cells. The percentage of cell proliferation was significantly higher in granulosa cells of the GCC. On the other hand, the percentage of apoptosis was significantly higher in the granulosa cells of GPCOS than the GCC. CONCLUSION: The ovaries of rats treated with clomiphene citrate showed a decrease in the number of cysts, an increase in the number of ovarian follicles, the presence of corpus luteum along with a decrease in the nuclear volume in the area occupied by interstitial cells.

Effects of early and late treatments of low-intensity, high-frequency mechanical vibration on bone parameters in rats.

Low-intensity, high-frequency mechanical vibration (LHMV) has shown to increase bone formation. However, studies comparing the effectiveness of early- and late-treatments of LHMV to counteract bone loss have not been documented. This study was designed to compare the effects of early- and late-treatments of LHMV (at 30 Hz/0.6 g, 20 min per day/five days per week, for 12 weeks) on bone parameters in ovariectomized (Ovx) rats. Thirty days after ovariectomy, 40 adult rats were randomly divided into four groups: GI (early control group); GII treated with LHMV 3 weeks after Ovx (early treatment); GIII (late control group) and GIV treated with LHMV twelve weeks after Ovx (late treatment). Bone mineral density (BMD) was analyzed before Ovx and after treatments. Then, animals were killed, and the femurs were collected and their length and diaphysis diameter were measured; the distal femurs were taken and processed for histomorphometry and polarized light microscopy for collagen fibers analysis or subjected to immunohistochemistry of cleaved caspase-3 in osteocytes. Statistical analysis was done by ANOVA followed by the Bonferroni post hoc test (p < 0.05). BMD was similar among the groups before Ovx, but after treatments, it was significantly higher in GII and GIV compared with their control groups (p < 0.05). Femur length and cortical bone thickness were similar among the groups, but the diaphysis diameter of GII was higher compared with GI. Trabecular bone area was higher in the vibrated groups, but it was greater in GII (p < 0.05). Also, the vibrated groups showed the higher content collagen fibers and lower presence apoptotic osteocytes (positive caspase-3 immunoreactivity) when compared with the other groups (p < 0.05). These results suggest that both early- and late-treatments with LHMV counteract bone loss, being the early treatment more effective than the late treatment.

Effects of melatonin and metformin on the ovaries of rats with polycystic ovary syndrome.

OBJECTIVE: To study the combined and isolated effects of melatonin and metformin in the ovarian tissue of rats with PCOS. DESIGN: Experimental study using a rat model of PCOS induced by continuous light exposure. INTERVENTION(S): Forty adult female rats were divided into 5 groups: physiological estrus phase (Sham); permanente estrus with PCOS induced by continuous lighting exposure for 60 consecutive days (control); with PCOS treated with melatonin; with PCOS treated with metformin; with PCOS treated with melatonin + metformin. After 60 days of treatments, all rats were killed, and ovaries were collected and processed for paraffin-embedding. Formalin-fixed paraffin-embedded sections were stained with hematoxylin and eosin or subjected to immunohistochemistry for proliferation (Ki-67) and apoptosis (cleaved caspase 3) detection markers. SETTING: Federal University of São Paulo, Brazil. ANIMALS: Forty adult female Wistar rats (Rattus norvegicus albinus). MAIN OUTCOME MEASURE(S): Number of corpus luteum and ovarian cysts, number of ovarian follicles (primary and antral follicles), number of interstitial cells, percentage of ovarian follicles (primary and antral follicles), and of interstitial cells immunostained to cleaved caspase-3 and Ki-67. RESULTS: Absence of corpus luteum, a higher number of cysts, and increased nuclear volume and area of interstitial cells, along with a decrease in primary and antral follicle numbers, were noticed in the control group compared with the Sham group. Melatonin and metformin treatments attenuated these effects, although the combined treatment did not mitigate the increased number of cysts and ovaries induced by PCOS. An increase in theca interna cell apoptosis was observed in the control group, whereas melatonina and metformin treatments reduced it significantly. A higher percentage of caspase-3-immunostained granulosa cells was noted in the Sham and all treated groups compared with the control group; no aditive effects on ovarian cell apoptosis were observed in the combined treatment. The percentage of Ki-67- immunostained granulosa cells was significantly higher in the control group compared with the Sham group. However, the combined treatment, not melatonin and metformin alone, mitigated this effect. A higher percentage of Ki-67-immunostained interstitial cells was observed in all treated groups compared with the Sham and control groups, whereas no additive effects in that immunoreactivity were observed in the combined treatment. CONCLUSIONS: Melatonin and metformin may improve ovarian function in rats with PCOS. The combined melatonin and metformin treatment is more effective in attenuating excessive granulosa cell proliferation, but it is not more effective in improving ovarian function than these drugs applied alone in rats with PCOS.

Possible role of annexin A1/FPR2 pathway in COX2/NLRP3 inflammasome regulation in alveolar bone cells of estrogen-deficient female rats with diabetes mellitus.

BACKGROUND: Annexin A1 (ANXA1) and the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome play important roles in bone remodeling. However, expression profiles of these factors in bone cells under diabetes mellitus (DM) and estrogen-deficient conditions are poorly understood. This study investigated the immunoexpression of ANXA1 and its formyl peptide receptor 2 (FPR2), as well as NLRP3 inflammasome mediators, during remodeling of the alveolar process in diabetic and estrogen-deficient rats. METHODS: Twenty adult female Wistar rats were divided into four groups (n = 5): Sham-operated (SHAM) and ovariectomized (OVX) rats received a vehicle solution, and SHAM and OVX rats were intraperitoneally administered 60 mg/kg/body weight (BW) of streptozotocin (STZ) to induce DM (SHAM-Di and OVX-Di groups). After 7 weeks, the rats were euthanized and their maxillae were fixed in phosphate-buffered 4% formaldehyde and embedded in paraffin. Sections were stained with hematoxylin/eosin (H&E) and picrosirius red or subjected to immunohistochemical detection of ANXA1, FPR2, NLRP3, interleukin-1ß (IL-1ß), and cyclooxygenase-2 (COX2). RESULTS: Estrogen deficiency and DM were associated with deleterious effects in bone tissue, as evidenced by a lower number of osteocytes and higher number of empty lacunae in the SHAM-Di and OVX-Di groups compared to the nondiabetic groups. Both diabetic groups showed a smaller vascular area and weaker collagen fiber birefringence intensity in alveolar bone tissue. A significantly higher number of ANXA1/FPR2-positive osteoblasts, osteocytes, and osteoclasts was accompanied by a significantly higher number of these cells immunolabeled for COX2, NLRP3, and IL-1ß in the diabetic and OVX groups, especially in both estrogen-deficient and diabetic rats. CONCLUSION: These results indicate a possible role for the ANXA1/FPR2 pathway as a fine-tuning/anti-inflammatory regulator to counterbalance exacerbated COX2/NLRP3/IL-1ß activation in bone cells during bone remodeling under estrogen deficiency and DM.

Additional Insights Into the Role of Osteocalcin in Osteoblast Differentiation and in the Early Steps of Developing Alveolar Process of Rat Molars.

This study investigated whether osteocalcin (OCN) is present in osteoblast precursors and its relationship with initial phases of alveolar process formation. Samples of maxillae of 16-, 18-, and 20-day-old rat embryos (E16, E18, and E20, respectively), and 05-, 10-, and 15-day-old postnatal rats (P05, P10, and P15, respectively) were fixed and embedded in paraffin or araldite. Immunohistochemistry for osterix (Osx), alkaline phosphatase (ALP), and OCN detection was performed and the number of immunolabelled cells was computed. Non-decalcified sections were subjected to the von Kossa method combined with immunohistochemistry for Osx or OCN detection. For OCN immunolocalization, samples were fixed in 0.5% glutaraldehyde/2% formaldehyde and embedded in LR White resin. The highest number of ALP- and OCN-immunolabelled cells was observed in dental follicle of E16 specimens, mainly in basal portions of dental alveolus. In corresponding regions, osteoblasts in differentiation adjacent to von Kossa-positive bone matrix exhibited Osx and OCN immunoreactivity. Ultrastructural analysis revealed OCN immunoreactive particles inside osteoblast in differentiation, and in bone matrix associated with collagen fibrils and within matrix vesicles, at early stages of alveolar process formation. Our results indicate that OCN plays a role in osteoblast differentiation and may regulate calcium/phosphate precipitation during early mineralization of the alveolar process.

Effects of the healing activity of rosemary-of-Chapada (Lippia gracilis Schauer) on cutaneous lesions in rats.

PURPOSE: To evaluate the effects of rosemary leaf essential oil-based ointments on the healing of rat skin lesions. METHODS: Sixty adult male rats, with dorsal excisional skin wounds made surgically under anesthesia, were divided into three groups (n = 20): Sham group (untreated wounds); control group (CG, wounds treated with vehicle); and essential oil (EO) treated group (wounds treated with essential oil-based ointments), administered topically once daily. Skin wounds were evaluated at 4, 7, 14, and 21 days after EO or vehicle treatments. Lesions were analyzed macroscopically for the contraction degree. Formalin-fixed paraffin-embedded sections of skin wounds were used for histopathological evaluation. RESULTS: Macroscopic evaluation showed wounds edges with thin crust without firmness and yellowish color, along with an improvement in wound contraction in EO group when compared to the other groups. A reduced inflammatory reaction, along with newly formed small diameter capillaries and more organized and elongated collagen fibers, were more frequently observed in EO group than in the other groups. Moreover, blood vessel number and collagen fibers density were significantly higher in EO group. CONCLUSIONS: Skin lesion treatment with rosemary leaf essential oil-based ointments accelerates the initial stages of healing, reduces inflammation, and increases angiogenesis, collagen fibers density, and wound contraction in rats.

Effect of hyperbaric oxygenation on random rat skin flaps vascularization.

PURPOSE: To evaluate the effect of hyperbaric oxygenation (HBO) on angiogenesis in random rat skin flaps, by immunoexpression of vascular endothelial growth factor A (VEGF-A). METHODS: Forty adult rats were divided into four groups: GE) epilated; GE/HBO) epilated subjected to HBO; GER) epilated submitted to dorsal skin flap; GER/HBO) epilated subjected to dorsal skin flap + HBO. HBO was performed with rats inside a chamber under atmosphere close to 100% oxygen and pressure of 2.4 absolute atmospheres, 2h per day during seven consecutive days. GE and GER groups were placed in the hyperbaric chamber without HBO. Then, under anesthesia, skin flaps were removed and separated into three portions relative to pedicle fixation. The samples were fixed in formalin and processed for paraffin embedding. Histological sections were submitted to immunohistochemistry for VEGF-A detection. The number of immunostained-blood vessels were counted under light microscopy. RESULTS: GE and GE/HBO groups showed normal and similar skin morphology in the three flap portions. A fibrin-leukocyte crust, along with denatured collagen and intense leukocyte infiltrate, was mainly observed in the dermis of the medial and distal flap portions of GER group. Meanwhile, the GER/HBO group presented more regions with intact collagen and small areas of leukocyte infiltrate in the three flap regions. VEGF-A-immunostained blood vessels were largely seen in all regions of GE and GE/HBO groups, whereas no significant differences were found between these groups. A decrease in vascularization was noticed in GER and GER/HBO groups, which was more evident in the most distal portion of the flaps. However, the number of VEGF-A-immunostained blood vessels in GER/HBO group was significantly higher when compared to GER group. CONCLUSIONS: Hyperbaric oxygenation was associated with increased angiogenesis and improved viability of rat skin flaps.

Immunoexpression pattern of autophagy mediators in alveolar bone osteoclasts following estrogen withdrawal in female rats.

It is known that estrogen deficiency increases osteoclast formation and activity. Autophagy, a cell survival pathway, has been shown to be crucial for osteoclast function. However, little is known about the effects of estrogen depletion on osteoclast autophagy. Here, we evaluated the effects of estrogen deficiency in the immunoexpression of autophagy mediators in alveolar bone osteoclasts of ovariectomized rats. Twelve adult female rats were ovariectomized (OVX-group) or SHAM-operated (SHAM-group). After three weeks, the rats were euthanized and maxillary fragments containing alveolar bone of the first molars were processed for light microscopy or transmission electron microscopy (TEM). Paraffin-sections were subjected to the TRAP method (osteoclast marker) or to the immunohistochemical detections of beclin-1, LC3α, and p62 (autophagy mediators); araldite-sections were processed for TEM. The number of TRAP-positive osteoclasts and the number of immunolabeled-multinucleated cells (MNCs) along the alveolar bone surface of the first molar were computed. The number of TRAP-positive osteoclasts and the number of beclin-1-, LC3α- and p62-immunolabelled osteoclasts were significantly higher in OVX-group than the SHAM-group. MNCs were frequently located juxtaposed to Howship lacunae along the alveolar bone surface, indicating that these cells are osteoclasts. TEM revealed osteoclasts exhibiting autophagosomes. Our data indicate that autophagy plays an important role during estrogen deficiency-induced osteoclastogenesis. Thus, our results contribute to a better understanding on the role of autophagy on osteoclasts under estrogenic deficiency, and reinforce the idea that modulation of autophagy may be a useful tool to inhibit excessive oral bone resorption in post-menopausal women.

Effects of estrogen deficiency followed by streptozotocin-induced diabetes on periodontal tissues of female rats.

Although both estrogen deficiency and diabetes contribute to periodontal tissue deterioration, the combined effects of these conditions on periodontium is unknown. Thus, we analyzed the combined effects of ovariectomy followed by streptozotocin (STZ)-induced diabetes on periodontal tissues of rats. Twenty adult rats were ovariectomized (OVX) or SHAM-operated (SHAM). After 3 weeks, the rats received an intraperitoneal injection of STZ (60 mg/kg/body weight) to induce diabetes or vehicle (blank) solution. The groups were assigned as follows (n = 5): SHAM-vehicle (SHAM), OVX-vehicle (OVX), SHAM + STZ (SHAM-Di), and OVX + STZ (OVX-Di). Seven weeks post-diabetes induction, the rats were euthanized. Blood samples were collected for glucose measurements and maxillae were processed for paraffin embedding. Sections stained with hematoxylin/eosin, Masson's trichrome, and picrosirius-red were used for alveolar bone loss and collagen fiber analysis in the lamina propria. Immunohistochemistry was performed for runt-related transcription factor 2 (Runx2), matrix metalloproteinase 9 (MMP-9), and tryptase detection. Alveolar bone loss and fewer collagen fibers were observed in the OVX-Di group, collagen fibers with irregular organization, and MMP-9 immunoreactivity were more evident in diabetic groups, and MMP-9-positive osteoclasts on alveolar bone surface were noticed in all groups. The OVX-Di group showed lower Runx2 immunoreactivity (osteoblast formation marker), and more tryptase-positive cells (mast cell marker) in the alveolar bone marrow. Our results indicate that estrogen depletion, followed by STZ-induced diabetes, promotes periodontal tissue deterioration that is more evident than both interventions applied alone. Furthermore, our results points to a possible participation of bone-derived mast cells in this process.

The influence of phytoestrogens or estrogens on the proliferation of the rat endocervical mucosa.

OBJECTIVE: Although estrogen therapy is widely used against post-menopausal symptoms, it can present adverse effects, including endometrial cancer. Soy isoflavones are considered a possible alternative to estrogen therapy. However, there are still concerns whether isoflavones exert trophic effects on the uterine cervix. To evaluate the histomorphometric and immunohistochemical alterations in the uterine cervix of ovariectomized rats treated with soy isoflavones (Iso). METHODS: Fifteen adult Wistar rats were ovariectomized (Ovx) and divided into three groups: Group I (Ovx), administered with vehicle solution; Group II (OVX-Iso), administered with concentrated extract of Iso (150 mg/kg) by gavage; and Group III (OVX-E2), treated with 17ß-estradiol (10 µg/kg), subcutaneously. After 30 days of treatments, the uterine cervix was fixed in 10% formaldehyde and processed for paraffin-embedding. Sections were stained with Hematoxylin and eosin for morphological and morphometric studies or subjected to immunohistochemistry for detections of Ki-67 and vascular endothelial growth factor-A (Vegf-A). The data obtained were subjected to statistical analysis (p ≤ 0.05). RESULTS: We noted an atrophic uterine cervix in GI, whereas it was more voluminous in GII and even more voluminous in GIII. The thickness of the cervical mucosa was significantly higher in GIII, as compared to GI and GII. The cell proliferation (Ki-67) was significantly elevated in the estradiol and isoflavones treated groups, whereas Vegf-A immunoexpression was significantly higher in GIII, as compared to groups GII and GI. CONCLUSIONS: Soy isoflavones cause less trophic and proliferative effects in the uterine cervix of rats as compared to estrogen.

Melatonin may prevent or reverse polycystic ovary syndrome in rats.

OBJECTIVE: To evaluate the ovarian effects of melatonin (Mel) in a rat model of polycystic-ovary-syndrome (PCOS) before and after permanent estrus induction. METHODS: Thirty-two adult-female rats with regular estrous cycle were equally divided into four groups: 1) GCtrl - at estrous phase. 2) GPCOS - at permanent-estrous phase. 3) GMel1 - treated for 60 days with Mel (0.4 mg/Kg) during permanent estrus induction and 4) GMel2 - rats with PCOS and treated for 60 days with Mel. After that, the animals were euthanized, and the ovaries were removed and processed for paraffin embedding. Sections were stained with H.E. for histomorphometry or subjected to immunohistochemistry for Ki-67 and cleaved caspase-3 (Casp-3) detections. RESULTS: The GPCOS showed lack of corpus luteum and several ovarian cysts, as well as interstitial-like cells. The presence of corpus luteum and a significant increase in primary and antral follicles were observed in Mel-treated groups, which also showed a decrease in the number of ovarian cysts and in the area occupied by interstitial-like cells. These results were more evident in GMel1. The percentage of Ki-67-positive cells was significantly higher in the Mel-treated groups, mainly in the GMel2, as compared to GPCOS. On the other hand, the percentage of Casp-3-positive cells was significantly lower in granulosa cells of GMel1, whereas it was significantly higher in the interstitial-like cells of GMel2, in comparison to GPCOS. CONCLUSION: Melatonin administration prevents the permanent estrus state in the PCOS rat model. This effect is more efficient when melatonin is administered before permanent estrus induction.

The role of ischemic preconditioning in the expression of apoptosis-related genes in a rat model of intestinal ischemia-reperfusion injury.

PURPOSE: To analyze the effects of ischemic preconditioning (IPC) in the expression of apoptosis-related genes in rat small intestine subjected to ischemia and reperfusion. METHODS: Thirty anesthetized rats underwent laparotomy and were drive into five groups: control (CG); ischemia (IG); ischemia and reperfusion (IRG); IPC and ischemia (IG+IPC); IPC and ischemia and reperfusion (I/RG+IPC). Intestinal ischemia was performed by clamping the superior mesenteric artery for 60 minutes, whereas reperfusion lasted for 120 minutes. IPC was carried out by one cycle of 5 minutes of ischemia followed by 10 minutes of reperfusion prior to the prolonged 60-minutes-ischemia and 120-minutes-reperfusion. Thereafter, the rats were euthanized and samples of small intestine were processed for histology and gene expression. RESULTS: Histology of myenteric plexus showed a higher presence of neurons presenting pyknotic nuclei and condensed chromatin in the IG and IRG. IG+IPC and I/RG+IPC groups exhibited neurons with preserved volume and nuclei, along with significant up-regulation of the anti-apoptotic protein Bcl2l1 and down-regulation of pro-apoptotic genes. Moreover, Bax/Bcl2 ratio was lower in the groups subjected to IPC, indicating a protective effect of IPC against apoptosis. CONCLUSION: Ischemic preconditioning protect rat small intestine against ischemia/reperfusion injury, reducing morphologic lesions and apoptosis.

Effects of estrogen status in osteocyte autophagy and its relation to osteocyte viability in alveolar process of ovariectomized rats.

Estrogen maintains osteocyte viability, whereas its deficiency induces osteocyte apoptosis. As autophagy is important for osteocyte viability, we hypothesized whether the anti-apoptotic effect of estrogen is related to autophagy in osteocytes. Thirty adult female rats were sham-operated (SHAM) or ovariectomized (OVX). After three weeks, twelve rats of SHAM and OVX groups were killed before treatment (basal period), whereas the remaining rats received estrogen (OVXE) or vehicle (OVX) for 45 days. Fragments of maxilla containing alveolar process of the first molars were embedded in paraffin or Araldite. Paraffin-sections were stained with hematoxylin/eosin for histomorphometry, or subjected to the silver impregnation method for morphological analysis of osteocyte cytoplasmic processes. Autophagy was analyzed by immunohistochemical detections of beclin-1, MAP-LC3α and p62, whereas apoptosis was evaluated by immunohistochemical detections of cleaved caspase-3 and BAX, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) method and by ultrastructural analysis. Araldite-semithin sections were subjected to the Sudan-black method for detection of lipids. OVX-basal group showed high frequency of caspase-3-, TUNEL- and p62-positive osteocytes accompanied with low frequency of beclin-1- and MAP-LC3α-positive osteocytes. At 45 days, OVXE group exhibited higher number of osteocytes, higher frequency of beclin-1- and MAP-LC3α-positive osteocytes, and lower frequency of caspase-3, BAX-, TUNEL- and p62-positive osteocytes than OVX group. Significant reduction in bone area was observed in the OVX compared to OVXE and SHAM groups. The highest frequency of Sudan-Black-positive osteocytes and osteocytes with scarce cytoplasmic processes, or showing apoptotic features were mainly observed in OVX groups. Our results indicate that estrogen deficiency decreases autophagy and increases apoptosis, whereas estrogen replacement enhances osteocyte viability by inhibiting apoptosis and maintaining autophagy in alveolar process osteocytes. These results suggest that the anti-apoptotic effect of estrogen may be, at least in part, related to autophagy regulation in osteocytes.

Osteoporosis and autophagy: What is the relationship?

Autophagy is a survival pathway wherein non-functional proteins and organelles are degraded in lysosomes for recycling and energy production. Therefore, autophagy is fundamental for the maintenance of cell viability, acting as a quality control process that prevents the accumulation of unnecessary structures and oxidative stress. Increasing evidence has shown that autophagy dysfunction is related to several pathologies including neurodegenerative diseases and cancer. Moreover, recent studies have shown that autophagy plays an important role for the maintenance of bone homeostasis. For instance, in vitro and animal and human studies indicate that autophagy dysfunction in bone cells is associated with the onset of bone diseases such as osteoporosis. This review had the purpose of discussing the issue to confirm whether a relationship between autophagy dysfunction and osteoporosis exits.

Mast cell concentration and skin wound contraction in rats treated with Ximenia americana L.

PURPOSE: To evaluate wound contraction and the concentration of mast cells in skin wounds treated with wild plum (Ximenia americana) essential oil-based ointment in rats. METHODS: Sixty rats were submitted to two cutaneous wounds in the thoracic region, on the right and left antimeres. Thereon, they were divided into three groups: GX (wounds treated once a day with hydro alcoholic branch extract of Ximenia americana), GP (wounds that received vehicle), and GC (wounds without product application). Wounds were measured immediately after the injury as well as 4, 7, 14 and 21 days post-topical application of the extract. At these days, five rats from each group were euthanatized. Thereafter, samples were fixed in 10% formaldehyde and processed for paraffin embedding. Sections were stained with H.E, Masson's Trichrome and toluidine blue for morphological, morphometrical and histopathological analysis, under light microscopy. The degree of epithelial contraction was measured and mast cell concentrations were also evaluated with an image analyzer (Image Pro-plus®software) . RESULTS: The extract treated group showed lower mast cell concentrations in the 4th day of lesion, as compared to GP (GX GP = GC; p<0.05) . CONCLUSION: Ointment containing 10% X. americana induces a decrease in mast cell concentration, at the beginning of the healing process, and promotes early skin wound contraction in rats.

Effects of the healing activity of rosemary-of-Chapada (Lippia gracilis Schauer) on cutaneous lesions in rats

Purpose: To evaluate the effects of rosemary leaf essential oil-based ointments on the healing of rat skin lesions. Methods: Sixty adult male rats, with dorsal excisional skin wounds made surgically under anesthesia, were divided into three groups (n = 20): Sham group (untreated wounds); control group (CG, wounds treated with vehicle); and essential oil (EO) treated group (wounds treated with essential oil-based ointments), administered topically once daily. Skin wounds were evaluated at 4, 7, 14, and 21 days after EO or vehicle treatments. Lesions were analyzed macroscopically for the contraction degree. Formalin-fixed paraffin-embedded sections of skin wounds were used for histopathological evaluation. Results: Macroscopic evaluation showed wounds edges with thin crust without firmness and yellowish color, along with an improvement in wound contraction in EO group when compared to the other groups. A reduced inflammatory reaction, along with newly formed small diameter capillaries and more organized and elongated collagen fibers, were more frequently observed in EO group than in the other groups. Moreover, blood vessel number and collagen fibers density were significantly higher in EO group. Conclusions: Skin lesion treatment with rosemary leaf essential oil-based ointments accelerates the initial stages of healing, reduces inflammation, and increases angiogenesis, collagen fibers density, and wound contraction in rats.

Effect of hyperbaric oxygenation on random rat skin flaps vascularization

ABSTRACT Purpose: To evaluate the effect of hyperbaric oxygenation (HBO) on angiogenesis in random rat skin flaps, by immunoexpression of vascular endothelial growth factor A (VEGF-A). Methods: Forty adult rats were divided into four groups: GE) epilated; GE/HBO) epilated subjected to HBO; GER) epilated submitted to dorsal skin flap; GER/HBO) epilated subjected to dorsal skin flap + HBO. HBO was performed with rats inside a chamber under atmosphere close to 100% oxygen and pressure of 2.4 absolute atmospheres, 2h per day during seven consecutive days. GE and GER groups were placed in the hyperbaric chamber without HBO. Then, under anesthesia, skin flaps were removed and separated into three portions relative to pedicle fixation. The samples were fixed in formalin and processed for paraffin embedding. Histological sections were submitted to immunohistochemistry for VEGF-A detection. The number of immunostained-blood vessels were counted under light microscopy. Results: GE and GE/HBO groups showed normal and similar skin morphology in the three flap portions. A fibrin-leukocyte crust, along with denatured collagen and intense leukocyte infiltrate, was mainly observed in the dermis of the medial and distal flap portions of GER group. Meanwhile, the GER/HBO group presented more regions with intact collagen and small areas of leukocyte infiltrate in the three flap regions. VEGF-A-immunostained blood vessels were largely seen in all regions of GE and GE/HBO groups, whereas no significant differences were found between these groups. A decrease in vascularization was noticed in GER and GER/HBO groups, which was more evident in the most distal portion of the flaps. However, the number of VEGF-A-immunostained blood vessels in GER/HBO group was significantly higher when compared to GER group. Conclusions: Hyperbaric oxygenation was associated with increased angiogenesis and improved viability of rat skin flaps.

Biology of Bone Tissue: Structure, Function, and Factors That Influence Bone Cells.

Bone tissue is continuously remodeled through the concerted actions of bone cells, which include bone resorption by osteoclasts and bone formation by osteoblasts, whereas osteocytes act as mechanosensors and orchestrators of the bone remodeling process. This process is under the control of local (e.g., growth factors and cytokines) and systemic (e.g., calcitonin and estrogens) factors that all together contribute for bone homeostasis. An imbalance between bone resorption and formation can result in bone diseases including osteoporosis. Recently, it has been recognized that, during bone remodeling, there are an intricate communication among bone cells. For instance, the coupling from bone resorption to bone formation is achieved by interaction between osteoclasts and osteoblasts. Moreover, osteocytes produce factors that influence osteoblast and osteoclast activities, whereas osteocyte apoptosis is followed by osteoclastic bone resorption. The increasing knowledge about the structure and functions of bone cells contributed to a better understanding of bone biology. It has been suggested that there is a complex communication between bone cells and other organs, indicating the dynamic nature of bone tissue. In this review, we discuss the current data about the structure and functions of bone cells and the factors that influence bone remodeling.