Early maturation of corpus luteum in rabbits - effect of sildenafil citrate on luteolytic capacity in the early luteal period

Background: Prostaglandin F2 alpha (PGF2 α) binds to the specific receptor (PTGFR) on the corpus luteum (CL) in mammals, inducing regression of the CL structure (luteolysis) and initiating a new cycle. While PGF2 α is effective only on mature CL, the immature CL structure (early luteal phase) resists PGF2 α. In this study, sildenafil citrate, which is used to increase blood flow in the genital organs for treating specific pregnancy issues in women, was administered during the early luteal phase in a rabbit model to test the hypothesis of enhancing blood flow to the CL, thereby promoting earlier maturation and enabling a response to PGF2 α. Materials, Methods & Results: The study was conducted in 2 sub-studies: clinical and molecular. A large number of rabbits were initially included in the sub-studies to ensure a sufficient number of pseudo-pregnant rabbits. Ovulation in rabbits was induced with buserelin acetate and was considered as day 0 of the study. The sub-studies were continued with rabbits whose pseudo-pregnancies were confirmed according to progesterone (P4 ) results. As a result, the studies were continued with a total of 41 pseudo-pregnant New Zealand female rabbits, 21 of which were included in the clinical sub-study and 20 in the molecular sub-study. In both sub-studies, on day 3 of the luteal period, rabbits in the treatment group received 5 mg/kg sildenafil citrate and all rabbits received a single dose of exogenous PGF2 α on day 4 to induce luteolysis. In the clinical sub-study, echotexture and intraovarian blood flow changes in the ovaries were determined by ultrasonography (USG) examination. In the molecular sub-study, the expression changes of Hypoxia Inducible Factor 1 Alpha (HIF1A) and Vascular Endothelial Growth Factor (VEGF) related to angiogenesis, Steroidogenic Acute Regulatory Protein (StAR) related to P4 metabolism, Prostaglandin-Endoperoxide Synthase 2 (PTGS2) related to prostaglandin (PG) mechanism and 15-Hydroxyprostaglandin Dehydrogenase (HPGD) genes at mRNA level were determined using Real Time Polymerase Chain Reaction (RT-PCR) in CL tissues obtained with ovariohysterectomy (OVH) at 1 and 12 h after PGF2 α injection. In addition, blood samples were collected for determine P4 levels from all rabbits. In the clinical sub-study; there was no difference between the groups in mean gray values (MGV), whereas there was a significant decrease in both pulsatile index (PI) and resistance index (RI) values at 40 min after PGF2 α injection (P < 0.05). In the molecular sub-study, it was determined that sildenafil citrate had no significant effect (P > 0.05) on the expression levels 1 and 12 h after PGF2 α injection. According to the results of the molecular sub-study, no significant effect of sildenafil citrate on the mRNA expression levels in the investigated genes was detected (P > 0.05). However, within each group, differences were found according to OVH time after PGF2 α injection. It was observed that PTGS2 and HPGD mRNA expressions decreased at the 12th h compared to the 1st h, while HIF1A expression increased (P < 0.05). Discussion: According to the results obtained from clinical and molecular sub-studies, it was determined that a single dose of sildenafil citrate (5 mg/kg) applied on the 3rd day of the luteal period did not contribute to the maturation process of the CL, did not increase blood flow, and was insufficient to break the resistance of the CL against PGF2 α applied on the 4th day of the luteal period. However, a significant decrease in the PI value at the 40th min after PGF2 α injection suggests that sildenafil citrate has a supportive effect, and that this decrease is also seen in the RI value, suggesting that its effect is insufficient against the vasoconstrictive effect of PGF2 α.

Mesenchymal stem cells have ameliorative effect on the colitis model via Nrf2/HO-1 pathway

Purpose: To evaluate the ameliorative effect of mesenchymal stem cells (MSCs) on acetic acid colitis model via Nrf2/HO-1 pathway in rats. Methods: In this study, 30 rats were divided into three groups. Acute colitis was induced by rectal administration of 4% solution of acetic acid. MSCs were injected intraperitoneally in the treatment group. Results: Increased levels of tumor necrosis factor-α (TNF-α), pentraxin-3, and malondialdehyde (MDA) in colitis group were revealed biochemically. Increased level of TNF-α and decreased levels of Nrf2 and interleukin-10 (IL-10) were observed in rectum tissues. Increased fibrous tissue proliferation, vascularization and inflammatory cell infiltration were described in the colitis group. Significant improvement was observed in MSCs treated group histopathologically. Increased immunopositivity of TNF-α, vascular endothelial growth factor (VEGF) and CD68 markers was observed in the colitis group cells, and decreased level of this positivity was observed in MSCs treated group. Conclusions: Biochemical, histopathological and immunohistochemical results strongly support the ameliorative effect of MSCs against acetic induced colitis model via Nrf2/HO-1 pathway in rats.

The use of occlusive dressings: influence on excisional wound healing in animal model

Purpose: To analyze the influence of occlusive dressing on the healing of excisional skin wounds in mice. Methods: Pre-clinical, comparative, and translational study. Mice were divided into three experimental groups: wounds occluded with hydrocolloid (HD) dressings, transparent polyurethane film (TF) dressings, and without occlusion (WO), monitored at three, six and 14 days, with eight animals each. Closure rate, infiltration of neutrophils and macrophages, measurement of tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) and, histologically, angiogenesis were evaluated. Results: Wound closure was accelerated in the occlusive groups. There was a decrease in TNF-α levels in the HD group when compared to the WO and TF groups. Neutrophils accumulation decreased in the HD group. Increased dosages of macrophages were evidenced in the HD group, compared to the WO and TF groups. Levels of VEGF were increased in the TF and HD groups. Conclusions: It is suggested that the occlusion of wounds modulates the inflammatory response.

Inflammation reduction potential of nanostructured lipid carriers encapsulated with rat's bone marrow cells' lysate / Potencial de redução da inflamação de carreadores lipídicos nanoestruturados encapsulados com lisado de células de medula óssea de ratos

Bone marrow-derived mesenchymal stromal cells (BMSCs) have been used for treating inflammatory disorders. Due to the large size of BMSCs compared to nanoparticles, BMSCs cannot be loaded into the nanoparticles. It is hypothesized that BMSCs lysate loading into the nanocarriers will effectively deliver cellular contents and regulatory elements of BMSCs at the injury site. This study aimed to investigate nanostructured lipid carriers (NLC) loading with BMSCs lysate through basic characterization and morphological analysis. Moreover, this study was mainly designed to investigate the role of NLC loaded BMSCs lysate in reducing inflammation via in-vitro and in-vivoassays. The in-vitro study involves cell viability assays, p53, annexin V and VEGF expression through ELISA and immunocytochemistry, real-time BAX, caspase-3, IL-6, IL-8, TOP2A, PCNA, and Ki-67 gene expression analysis. Additionally, to evaluate in-vivo anti-inflammatory activity, the carrageenan-induced rat paw oedema model was used. In-vitro results showed that NLC loaded BMSCs lysate increased cell viability, decreased apoptosis and pro-inflammatory genes expression and up-regulated angiogenesis and proliferation in H2O2 pre-stimulated cells. Findings of the in-vivo assay also indicated a reduction in rat's paw oedema volume in NLC-loaded BMSCs lysate, and downregulation of BAX, Caspase-3, IL-6, and IL-8 was observed. Enhanced expressions of TOP2A, PCNA, and Ki-67 were obtained. Concluding the results of this study, NLC-loaded BMSCs lysate could reduce inflammation and possibly regenerate damaged tissue mainly via increasing cell viability, angiogenesis and proliferation, and reducing apoptosis and pro-inflammatory cytokines.

Células estromais mesenquimais derivadas da medula óssea (BMSCs) têm sido utilizadas para o tratamento de distúrbios inflamatórios. Devido ao grande tamanho das BMSCs em comparação com as nanopartículas, as BMSCs não podem ser carregadas nas nanopartículas. Supõe-se que o carregamento de lisado de BMSCs no nanocarriers será eficaz na entrega de conteúdos celulares e elementos reguladores de BMSCs no local da lesão. Este estudo teve como objetivo investigar a carga de carreador lipídico nanoestruturado (NLC) com lisado de BMSCs através de caracterização básica e análise morfológica. Além disso, este trabalho foi projetado, principalmente, para investigar o papel do lisado de BMSCs carregado com NLC na redução da inflamação por meio de ensaios anti-inflamatórios in vitro e in vivo. O estudo in vitro envolve ensaios de viabilidade celular, expressão de p53, anexina V e VEGF por ELISA e imunocitoquímica e expressão gênica em tempo real de BAX, caspase-3, IL-6, IL-8, TOP2A, PCNA e Ki-67 . Além disso, para avaliar a atividade anti-inflamatória in vivo,o modelo de edema de pata de rato induzido por carragenina foi utilizado. Os resultados in vitro mostraram que o lisado de BMSCs carregadas com NLC aumentou a viabilidade celular, diminuiu a apoptose e a expressão de genes pró-inflamatórios e aumentou a angiogênese e proliferação em células pré-estimuladas com H2O2. Os achados do ensaio in vivo também indicaram uma redução no volume do edema da pata de rato no lisado de BMSCs carregado com NLC, entretando, foi observada a regulação negativa de BAX, Caspase-3, IL-6 e IL-8. Expressões aumentadas de TOP2A, PCNA e Ki-67 foram obtidas. Assim, concluindo os resultados do estudo, é possível afirmar que o lisado de BMSCs carregado com NLC pode reduzir a inflamação e possivelmente regenerar o tecido danificado principalmente por meio do aumento da viabilidade celular, angiogênese e proliferação e redução da apoptose e citocinas pró-inflamatórias.

Hemoperitoneum in a cow with lumbar muscle hemangiosarcoma

Background: Hemangiosarcoma is a malignant neoplasm of endothelial cells with an infiltrative growth pattern. Hemangiosarcomas are frequently reported in canines and rare in felines, sheep, goats, swine, horses and cattle. Few cases of hemangiosarcoma were reported in cattle. In the present report, we describe the clinicopathological findings of a bovine muscle hemangiosarcoma. Case: A 6-year-old, Girolando cow from the Dairy Cattle Sector of the Federal Rural University of Rio de Janeiro (UFRRJ), Seropédica, presented sternal decubitus. Clinical signs were markedly pale mucous membranes, moderate dehydration, respiratory distress, and increased heart rate. The hematological examination revealed intense regenerative anemia. Due to the worsening of the clinical condition, the cow was submitted to euthanasia. The necropsy and collection of various fragments of organs were performed, which were sent to the "Setor de Anatomia Patológica" (SAP-UFRRJ). Tissues were fixed in 10 % buffered formalin, routinely processed for histology and stained with Hematoxylin and Eosin (HE). The external mucous membranes were markedly pale. Multifocal areas of 1.5 x 1.0 cm, irregular and dark red were observed dissecting the quadratus lumborum muscle (hemangiosarcoma) fibers. These neoplasms were associated with an extensive cruoric clot adhered to the muscle fibers. The extensive, red, friable mass measured approximately 76 x 55 x 20 cm on the serous surfaces of the organs of the peritoneal cavity (hemoperitoneum). The spleen was moderately reduced. The bone marrow was markedly pale. Histologically, it was observed that there was an extensive proliferation of endothelial cells in the quadratus lumbar muscle mass dissecting the epimysium and perimysium. Endothelial cells had moderate pleomorphism, organized in vascular channels and forming multifocally solid areas with a significant amount of eosinophilic fibrillar material (fibrin). Sections of muscle neoplasm were subjected to immunohistochemistry with anti-von Willebrand factor primary antibody, which showed a multifocal moderate cytoplasmic immunolabeling of neoplastic endothelial cells. Discussion: There are few reports of striated muscle hemangiosarcoma in cattle. Muscular hemangiosarcomas were reported in a 4-month-old calf in the left cervical trapezius muscle and a 6-year-old Holstein cow with left pelvic limb mass lateral and distal to the knee. Some reports presented hemangiosarcoma in the iliopsoas muscle, left cervical trapezius muscle, pelvic limb muscles and right cervical muscle of the bovine. In the presented report, hemoperitoneum occurred as a result of hemorrhages from muscle hemangiosarcoma. Other studies have demonstrated cavity hemorrhages in joint, pelvic, pleural and cranial cavities associated with hemangiosarcoma. Hemangiosarcoma with regenerative anemia must be distinguished from other diseases that cause anemia. The main differential diagnoses of bovine with anemia are vena cava syndrome, coumarin derivatives poisoning, acute poisoning by Pteridium spp., tick fever, anaplasmosis, babesiosis and trypanosomiasis. Hemangiosarcoma should be differentiated from other lesions as hemangioma, vascular tumor of lymphatic endothelium and perivascular wall tumors. Cases with poorly differentiated morphology should be submitted for immunohistochemistry. In the present hemangiosarcoma case, we have used the von Willebrand factor for immunohistochemistry diagnosis. Expression of angiogenic growth factors such as CD31, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and angiopoietin-1 (Ang-1) have also been used in the diagnosis of vascular proliferation lesions. Hemangiosarcoma in cattle should be included mainly in the differential diagnosis of diseases that cause acute anemia in cattle.

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.

In-vitro production and pre-validation of lyophilized canine platelet-rich plasma for therapeutic use

ABSTRACT: Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF- (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.

RESUMO: O plasma rico em plaquetas (PRP) é uma alternativa terapêutica promissora, pois as plaquetas são ricas em fatores de crescimento com ação na regeneração de tecidos. No entanto, o PRP fresco não pode ser armazenado por longos períodos. Esse trabalho teve como objetivo desenvolver um protocolo de obtenção de PRP liofilizado canino capaz de manter a viabilidade pós reconstituição. Portanto, foram testados diversos protocolos de extração e liofilização. Para validação do PRP canino liofilizado foi analisada a concentração dos fatores de crescimento VEGF e TGF- antes e após o processo de liofilização, a estrutura tridimensional do PRP liofilizado reconstituído em forma de gel por microscopia eletrônica e seu efeito in vitro na proliferação de células-tronco mesenquimais. Os resultados demonstraram que o PRP liofilizado apresentou concentrações terapêuticas adequadas dos fatores de crescimento VEGF e TGF- (9,1pg/ml e 6161,6pg/ml, respectivamente). O gel de PRP reconstituído após liofilização apresentou uma durabilidade in vitro de 10 dias, sua estrutura tridimensional mostrou-se semelhante ao PRP fresco e proporcionou intensa proliferação de células-tronco mesenquimais durante o cultivo. O teste de imunogenicidade não demonstrou evidências de reação imune. Os achados foram promissores, sugerindo a possibilidade de uso de PRP canino liofilizado para o mercado. Novos estudos in vivo e in vitro deverão ser conduzidos para comprovação terapêutica.

Effect of low-level laser therapy on the membrane induced by the Masquelet technique at an orthotopic site in rabbits

ABSTRACT Purpose: To evaluate the low-level laser therapy (LLLT) on the membrane induced by the Masquelet technique in rabbits. Methods: Twelve Norfolk rabbits at approximately 3 months of age were used. A 1-cm segmental defect was induced in both radii, which were filled with polymethylmethacrylate cylinder. LLLT was used postoperatively in the bone defect of one of the forelimbs every 48 hours for 15 days. Six rabbits were euthanatized on third and sixth postoperative weeks. Results: In both forelimbs, radiographs showed new bone growth from radius cut ends on the third postoperative week and more advanced stage on the sixth postoperative week. Ultrasound showed induced membrane one week after the surgery. Histologically, there were no significant differences in the semi-quantitative score of inflammation intensity, total number of blood vessels, bone metaplasia, and collagen. The average thicknesses were 2,050.17 and 1,451.96 μm for control membranes and 2,724.26 and 2,081.03 μm for irradiated membranes, respectively, on third and sixth postoperative weeks. Vascular endothelial growth factor A (VEGF-A) and platelet derived growth factor (PDGF) expression were present in the induced membranes of control and irradiated forelimbs, but there was no significant difference. Conclusions: Based on assessment methods, it was not possible to demonstrate the effect of LLLT on the induced membrane.

Effects of ergosteroside combined risedronate on fracture healing and BMP-2, BMP-7 and VEGF expression in rats

Purpose To evaluate the effect of ergosterol combined with risedronate on fracture healing. Methods Sixty male Sprague Dawley fracture model rats were assigned into group A (n=20), group B (n=20), and group C (n=20) at random. All rats were fed by gavage until their sacrifice as it follows: group A with ergosteroside and risedronate, group B with risedronate, and group C with saline solution. At weeks 2 and 4, 10 rats of each group were sacrificed. Healing effect and bone tissue changes in the fractures site were assessed by using hematoxylin and eosin stain histology. Enzyme-linked immunosorbent assay was used to detect the expression of serum bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-7 (BMP-7), and vascular endothelial growth factor (VEGF). Reverse transcriptase polymerase chain reaction was applied to detect the expression of osteoprotegerin (OPG) mRNA, osteocalcin (OCN) mRNA and core-binding factor subunit-?1 (CBF-?1) mRNA. Results In terms of serum BMP-2, BMP-7, and VEGF expression at weeks 2 and 4 after gavage, group A < group B < group C (P<0.05). At week 4 after gavage, serum VEGF expression in the three groups harbored positive relationship with serum BMP-2 and BMP-7 expression (P<0.05). Regarding serum OPG, OCN and CBF-?1 mRNA expression at weeks 2 and 4 after gavage, group A

Altrenogest during early pregnancy modulates uterine glandular epithelium and endometrial growth factor expression at the time implantation in pigs

Abstract This study evaluated the effects of supplying altrenogest from day 6-12 of pregnancy on the endometrial glandular epithelium, corpora lutea (CL) morphology, and endometrial and CL gene expression. A total of 12 crossbred females (Landrace × Large White) were used. The females were assigned to 4 treatments according to a random design with a 2 × 2 factorial arrangement, with two categories (sow or gilt) and two treatments (non-treated and treated with altrenogest). On day 6 of pregnancy, animals were allocated to one of the following groups: non-treated (NT, n = 6; 3 sows and 3 gilts), and (T, n = 6; 3 sows and 3 gilts) treated daily with 20 mg of altrenogest, from day 6-12 of pregnancy. All animals were euthanized on day 13 of pregnancy. All CLs were individually weighed, and their volume were determined. The endometrial glandular density (GD), mean glandular area (MGA), and vascular density (VD) were determined by histomorphometric and immunohistochemical analyses. Endometrium samples were collected and analyzed by qRT-PCR to evaluate the abundance of transcripts for VEGF and IGF-I. Females in the T group had higher MGA (P < 0.05) compared to the NT group. There was no effect of treatment on GD or VD for both experimental groups. Sows in the T group had augmented expression of IGF-I (P < 0.05). Progestagen had no detrimental effect on CL morphology. In conclusion, altrenogest improves the uterine environment during the peri-implantation period in pigs without compromising corpora lutea development.

The influence of adipocyte-derived stem cells (ASCs) on the ischemic epigastric flap survival in diabetic rats

ABSTRACT Purpose: To assess the effects of adipocyte-derived stem cell (ASC)-injection on the survival of surgical flaps under ischemia in diabetic rats. Methods: Diabetes was induced in 30 male Wistar rats using streptozotocin (55 mg/kg). After eight weeks, epigastric flap (EF) surgery was performed. The animals were divided into control (CG), medium-solution (MG), and ASC groups. The outcomes were: the survival area (SA), the survival/total area rate (S/TR), and expression levels (EL) of genes: C5ar1, Icam1, Nos2, Vegf-a. Results: In the ASC group, compared to CG, we observed improved flap SA (CG-420 mm2 vs. ASC-720 mm2; p=0.003) was observed. The S/TR analysis was larger in the ASC group (78%) than the CG (45%). This study showed an increase in the Vegf-a EL in the ASC group (2.3) vs. CG (0.93, p=0.0008). The Nos2 EL increased four-fold in the ASC group compared to CG, and C5ar1 EL decreased almost two-fold in the ASC group vs. the CG (p=0.02). There was no difference among the groups regarding Icam1 EL. Compared to the MG, the ASC group had a bigger flap SA (720 mm2 vs. 301 mm2, respectively), a bigger S/TR (78% vs. 32%, p=0.06, respectively) and increased EL of Vegf-a (2.3 vs. 1.3, respectively). No difference between ASC-group and MG was seen regarding Nos2 (p=0.08) and C5ar1 (p=0.05). Conclusions: This study suggests that ASCs increase the survival of EF under IR in diabetic rats.

In-vitro production and pre-validation of lyophilized canine platelet-rich plasma for therapeutic use / Produção e validação de plasma rico em plaquetas canino liofilizado para fins de utilização terapêutica

Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-β, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF-β (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.(AU)

O plasma rico em plaquetas (PRP) é uma alternativa terapêutica promissora, pois as plaquetas são ricas em fatores de crescimento com ação na regeneração de tecidos. No entanto, o PRP fresco não pode ser armazenado por longos períodos. Esse trabalho teve como objetivo desenvolver um protocolo de obtenção de PRP liofilizado canino capaz de manter a viabilidade pós reconstituição. Portanto, foram testados diversos protocolos de extração e liofilização. Para validação do PRP canino liofilizado foi analisada a concentração dos fatores de crescimento VEGF e TGF-β antes e após o processo de liofilização, a estrutura tridimensional do PRP liofilizado reconstituído em forma de gel por microscopia eletrônica e seu efeito in vitro na proliferação de células-tronco mesenquimais. Os resultados demonstraram que o PRP liofilizado apresentou concentrações terapêuticas adequadas dos fatores de crescimento VEGF e TGF- β (9,1pg/ml e 6161,6pg/ml, respectivamente). O gel de PRP reconstituído após liofilização apresentou uma durabilidade in vitro de 10 dias, sua estrutura tridimensional mostrou-se semelhante ao PRP fresco e proporcionou intensa proliferação de células-tronco mesenquimais durante o cultivo. O teste de imunogenicidade não demonstrou evidências de reação imune. Os achados foram promissores, sugerindo a possibilidade de uso de PRP canino liofilizado para o mercado. Novos estudos in vivo e in vitro deverão ser conduzidos para comprovação terapêutica.(AU)

In-vitro production and pre-validation of lyophilized canine platelet-rich plasma for therapeutic use / Produção e validação de plasma rico em plaquetas canino liofilizado para fins de utilização terapêutica

Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-β, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF-β (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.(AU)

O plasma rico em plaquetas (PRP) é uma alternativa terapêutica promissora, pois as plaquetas são ricas em fatores de crescimento com ação na regeneração de tecidos. No entanto, o PRP fresco não pode ser armazenado por longos períodos. Esse trabalho teve como objetivo desenvolver um protocolo de obtenção de PRP liofilizado canino capaz de manter a viabilidade pós reconstituição. Portanto, foram testados diversos protocolos de extração e liofilização. Para validação do PRP canino liofilizado foi analisada a concentração dos fatores de crescimento VEGF e TGF-β antes e após o processo de liofilização, a estrutura tridimensional do PRP liofilizado reconstituído em forma de gel por microscopia eletrônica e seu efeito in vitro na proliferação de células-tronco mesenquimais. Os resultados demonstraram que o PRP liofilizado apresentou concentrações terapêuticas adequadas dos fatores de crescimento VEGF e TGF- β (9,1pg/ml e 6161,6pg/ml, respectivamente). O gel de PRP reconstituído após liofilização apresentou uma durabilidade in vitro de 10 dias, sua estrutura tridimensional mostrou-se semelhante ao PRP fresco e proporcionou intensa proliferação de células-tronco mesenquimais durante o cultivo. O teste de imunogenicidade não demonstrou evidências de reação imune. Os achados foram promissores, sugerindo a possibilidade de uso de PRP canino liofilizado para o mercado. Novos estudos in vivo e in vitro deverão ser conduzidos para comprovação terapêutica.(AU)

Valuation of angiogenesis in bovine xenografts implanted in intracorporal sites of rabbits as models of in-vivo bioreactors / Avaliação da angiogênese em xenoenxertos bovinos implantados em sítios intracorpóreos de coelhos como modelos de biorreatores in vivo

The aim of this study was to evaluate neovascularization of bovine xenografts implanted in intracorporeal sites of rabbits (bioreactors). 30 rabbits were used, divided into 6 groups, according to the evaluation time (7, 15, 30, 45, and 60 days); each animal received xenogenic implants in 3 different intracorporeal sites (A1 - omentum bag; A2 - intermuscular space of quadriceps femoris; A3 - subperiosteal of ilium bone). Histological assessments graded the presence of angiogenesis, the number of inflammatory cells, newly formed bone tissue, and the presence of giant cells. Histological analyses showed intense angiogenesis in all implanted xenografts. Presence of inflammatory infiltrate and giant cells at the A1 implant site and presence of bone neoformation at the A3 implant site were noted. Degeneration of implants and formation of a fibrous capsule were noted. When comparing the interaction of the site with the days of evaluation, statistical analysis showed a significant difference (p≤0.05) in any time of neovascularization analysis. The vascular endothelial growth factor (VEGF) and inflammatory cells of the omentum in its structure, may have contributed to the greater presence of neovessels and inflammatory cells, a fact that may indicate functionality as a possible bone substitute.(AU)

O objetivo deste estudo foi avaliar a neovascularização de xenoenxertos bovinos implantados em sítios intracorpóreos de coelhos (biorreatores). Foram utilizados 30 coelhos, os quais foram divididos em seis grupos, de acordo com o tempo de avaliação (sete, 15, 30, 45 e 60 dias); cada animal recebeu implantes xenogênicos em três diferentes sítios intracorpóreos (A1 - bolsa de omento; A2 - espaço intermuscular do quadríceps femoral; A3 - subperiosteal do osso ílio). Avaliações histológicas classificaram a presença de angiogênese, o número de células inflamatórias, de tecido ósseo neoformado e a presença de células gigantes. As análises histológicas mostraram intensa angiogênese em todos os xenoenxertos implantados. Observou-se presença de infiltrado inflamatório e células gigantes no local do implante A1 e presença de neoformação óssea no local do implante A3. Ao mesmo tempo, a degeneração dos implantes e a formação de uma cápsula fibrosa foram observadas. Ao comparar a interação do local com os dias de avaliação, a análise estatística mostrou diferença significativa (P≤0,05) em qualquer momento da análise de neovascularização. O fator de crescimento endotelial vascular (VEGF) e as células inflamatórias do omento em sua estrutura podem ter contribuído para a maior presença de neovasos e células inflamatórias, fato que pode indicar funcionalidade como possível substituto ósseo.(AU)

Altrenogest during early pregnancy modulates uterine glandular epithelium and endometrial growth factor expression at the time implantation in pigs

This study evaluated the effects of supplying altrenogest from day 6-12 of pregnancy on the endometrial glandular epithelium, corpora lutea (CL) morphology, and endometrial and CL gene expression. A total of 12 crossbred females (Landrace × Large White) were used. The females were assigned to 4 treatments according to a random design with a 2 × 2 factorial arrangement, with two categories (sow or gilt) and two treatments (non-treated and treated with altrenogest). On day 6 of pregnancy, animals were allocated to one of the following groups: non-treated (NT, n = 6; 3 sows and 3 gilts), and (T, n = 6; 3 sows and 3 gilts) treated daily with 20 mg of altrenogest, from day 6-12 of pregnancy. All animals were euthanized on day 13 of pregnancy. All CLs were individually weighed, and their volume were determined. The endometrial glandular density (GD), mean glandular area (MGA), and vascular density (VD) were determined by histomorphometric and immunohistochemical analyses. Endometrium samples were collected and analyzed by qRT-PCR to evaluate the abundance of transcripts for VEGF and IGF-I. Females in the T group had higher MGA (P < 0.05) compared to the NT group. There was no effect of treatment on GD or VD for both experimental groups. Sows in the T group had augmented expression of IGF-I (P < 0.05). Progestagen had no detrimental effect on CL morphology. In conclusion, altrenogest improves the uterine environment during the peri-implantation period in pigs without compromising corpora lutea development.(AU)

The protective effect of Ganoderma lucidum on testicular torsion/detorsion-induced ischemia-reperfusion (I/R) injury

Purpose To investigate the protective effect of Ganoderma lucidum on testicular torsion/detorsion (T/D)-induced ischemia-reperfusion (I/R) injury. Methods Thirty male Wistar albino rats were randomly categorized into 3 groups: Group 1: sham, Group 2 ( T/D): 2,5 hours of ischemia and 7 days of reperfusion, Group 3 (T/D+ G. lucidum ): 2,5 hours of ischemia and 7 days of reperfusion and 7 days of 20 mg/kg via gastric gavage G. lucidum polysaccharides per day. Biochemical assays of Malondialdehyde (MDA), superoxide dismutase (SOD), Catalase (CAT), Glutathione (GSH) levels , histopathology and expression levels of VEGF and Bcl-2 with immunohistochemical methods were examined in testicular tissue. Results G. lucidum treatment was found to have prevented the T/D-induced I/R injury by decreasing MDA levels of the testis. SOD, CAT and GSH activities were decreased in group 2, while they were increased in group 3 (p<0.001) and significant improvement in the tube diameter was observed in group 3. Bcl-2-positive germinal cells were lowered in group 3 compared to the group 2. VEGF expression showed an increase in group 2, whereas it decreased in group 3. Conclusion The antioxidant G. lucidum is thought to induce angiogenesis by reducing the apoptotic effect in testicular torsion-detorsion.(AU)

Changrui enema inhibits inflammation-induced angiogenesis in acute radiation proctitis by regulating NF-kB and VEGF

Purpose Changrui enema, a traditional Chinese medicine prescription, is used as a supplementary treatment for acute radiation proctitis (ARP). Herein we explored the inhibition effects of Changrui enema on NF-kB and VEGF in ARP mice. Methods A total of 120 C57BL/6 mice were divided randomly into normal mice group, ARP mice group, western medicine enema group (dexamethasone combined with gentamicin), and Changrui enema group. ARP mice were established by pelvic local irradiation. The expression of IL-1beta, NF-kB, VEGF, AQP1, AQP3, p-ERK1/2 and p-JNK was determined by immunohistochemistry or western blot. Results The study firstly found that Changrui enema alleviated ARP mice. The expression of IL-1beta, NF-kB, VEGF, AQP1 and p-ERK1/2 was increased in ARP mice, and was reserved by Changrui enema. However, the expression of AQP3 and p-JNK was decreased in ARP mice, and was up-regulated by Changrui enema. Conclusions Changrui enema is an effective treatment with fewer side effects for ARP. The mechanism of Changrui enema may be related to the inhibition of inflammation-induced angiogenesis. Changrui enema inhibits IL-1beta and NF-kB expression as well as VEGF expression. Interestingly, AQP1 promotes angiogenesis, while AQP3 inhibits inflammation. Changrui enema probably inhibits AQP1 expression by down-regulating p-ERK1/2, and improves AQP3 expression by up-regulating p-JNK.(AU)

Benefits of pentoxifylline for skin flap tissue repair in rats

Purpose To assess the action of pentoxifylline, administered by subcutaneous route, on skin flap tissue repair in rats, and to verify the histological aspects and biomarkers. Methods Thirty-two male Wistar rats were divided into four groups: control (CT) and treated with pentoxifylline (P1, P3 and P5). Modified McFarlane technique flap was used. Ten days later, the animals were euthanized and the areas of viable and necrotic tissue were evaluated. Hematoxylin/eosin staining was used to assess the morphometric characteristics of the number of vessels and epithelial thickness. Picrosirius red was used to assess collagen density. VEGF and TGF-beta1 levels on the skin flap and serum of the animals were measured by the ELISA method. Results The macroscopic evaluation of the skin flap dimensions showed reduced necrotic tissue in the pentoxifylline (p < 0.05) treated groups. There was an increase in angiogenesis and reepithelization, demonstrated by analyses with an increased number of vessels (p 0.05), VEGF and epithelial thickness. Fibrogenic effect showed decreased collagen density and TGF-1 in the skin flap and serum. Conclusion The benefits of pentoxifylline administered by subcutaneous route, at dose 100 mg/kg, which was effective to improve the survival of skin flap by acting on tissue repair components, stimulating angiogenesis and reepithelization, in addition to reducing fibrogenesis.(AU)

Paeonia lactiflora improves ovarian function and oocyte quality in aged female mice

Although ovarian aging is a key cause of decreased ovarian function and oocyte quality, it remains a problem in infertility treatment. Therefore, this study is aimed to investigate whether Paeonia lactiflora (PL), a herb improves ovarian function and oocyte quality using aged female mice. C57BL/6 female mice aged 8 months were treated orally every day with PL of 26.5 mg/kg (n=7) and 53 mg/kg (n=7) of body weight for 4 weeks using an oral zoned needle. The control group (n=7) was treated with normal saline. Ovaries and serum were collected for the H&E stain and the evaluation of reactive oxygen species (ROS) levels, respectively. In the second experiment, female mice were orally administered with PL (26.5 mg/kg: n=12, 53 mg/kg: n=12, control: n=12) and then superovulated with PMSG and hCG, and mated with male mice. Zygotes were retrieved and cultured for 4 days. Ovaries were provided for examination of expressions of genes associated with angiogenesis (VEGF and visfatin), anti-aging (Sirt1 and Sirt2), and follicular development (c-Kit, BMP-15, and GDF-9). PL significantly increased numbers of surviving follicles (primordial, primary, secondary, and antral), numbers of zygotes retrieved, embryo development rate, and ovarian expression of VEGF, visfatin, c-Kit, BMP-15, and GDF-9 at both doses. However, ovarian expression of Sirt1 and Sirt2 was increased at 53.0 mg/kg of PL. ROS levels were not affected by PL. These results suggest that PL may possess beneficial effects regarding ovarian function and oocyte quality, possibly by activation of ovarian angiogenesis and follicular development.

Ozonized solutions favor the repair of experimentally induced skin wounds in rats / Soluções ozonizadas favorecem o reparo de feridas cutâneas experimentalmente induzidas em ratos

This study aimed to evaluate and compare the effects of ozonized solutions on tissue wound repair in rats. Treatments consisted of ozonized water (GA), 0.9% sodium chloride (GCL), ozonized oil (GO), and 0.2% allantoin cream (GAL). The morphometric evaluation showed that wounds of the GA group presented a higher degree of retraction (p<0.05) at three and eight days of treatment (37.96 and 84.81%, respectively). Picrosirius red staining showed that groups GA and GO presented higher deposition (p<0.05) of type I collagen at 15 and 22 days of treatment, respectively. The neovascularization was higher in wounds of group GO on days 3, 8, and 15 (p<0.05), with higher VEGF immunostaining. (p<0.05). Thus, ozonized water enhances wound retraction and assists in the maturation and remodeling phase, while ozonized oil promotes higher neovascularization during tissue repair and higher deposition of type I collagen from the third week of treatment.(AU)

O objetivo deste estudo foi avaliar e comparar os efeitos de soluções ozonizadas sobre o reparo tecidual de feridas em ratos. Foram realizados os tratamentos: água ozonizada (GA), cloreto de sódio 0,9% (GCL), óleo ozonizado (GO) e creme de alantoína 0,2% (GAL). À avaliação morfométrica, as feridas do grupo GA apresentaram maior grau de retração (p<0,05) aos três e oito dias de tratamento (37,96% e 84,81%, respectivamente). A coloração de picrosirius red mostrou que os grupos GA e GO apresentaram maior deposição (p<0,05) de colágeno do tipo I aos 15 e aos 22 dias de tratamento, respectivamente. Já a variável neovascularização foi maior (p<0,05) nas feridas do grupo GO nos dias três, oito e 15, o que fora ratificado à imunoistoquímica, com maior imunomarcação de VEGF nas feridas do grupo GO (p<0,05). Conclui-se que a água ozonizada potencializa a retração da ferida e auxilia na fase de maturação e remodelamento, enquanto o óleo ozonizado promove maior neovascularização durante o reparo tecidual e maior deposição de colágeno do tipo I a partir da terceira semana de tratamento.(AU)