Bisacurone gel ameliorated burn wounds in experimental rats via its anti-inflammatory, antioxidant, and angiogenic properties

Purpose: To investigate putative mechanism of wound healing for chitosan-based bisacurone gel against secondary burn wounds in rats. Methods: A second-degree burn wound with an open flame using mixed fuel (2 mL, 20 seconds) was induced in Sprague Dawley rats (male, 180-220 g, n = 15, each) followed by topical treatments with either vehicle control (white petroleum gel, 1%), silver sulfadiazine (1%) or bisacurone gel (2.5, 5, or 10%) for 20 days. Wound contraction rate and paw withdrawal threshold were monitored on various days. Oxidative stress (superoxide dismutase, glutathione, malondialdehyde, and nitric oxide), pro-inflammatory cytokines (tumour necrosis factor-alpha, interleukins by enzyme-linked immunosorbent assay), growth factors (transforming growth factor-ß, vascular endothelial growth factor C using real time polymerase chain reaction and Western blot assay) levels, and histology of wound skin were assessed at the end. Results: Bisacurone gel showed 98.72% drug release with a 420.90­442.70 cps viscosity. Bisacurone gel (5 and 10%) significantly (p < 0.05) improved wound contraction rate and paw withdrawal threshold. Bisacurone gel attenuated oxidative stress, pro-inflammatory cytokines, and water content. It also enhanced angiogenesis (hydroxyproline and growth factor) and granulation in wound tissue than vehicle control. Conclusions: These findings suggested that bisacurone gel can be a potential candidate to treat burn wounds via its anti-inflammatory, antioxidant, and angiogenic properties.

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.

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.

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

Cordycepin prevents the esophageal stricture formation in the alkali-burn rat model by exerting anti-inflammatory and antifibrotic effects

Purpose To investigate the efficacy of cordycepin, an adenosine analogue, on prevention of esophageal damage and stricture formation due to esophageal caustic burns in rat model comparing with prednisolone. Methods Caustic esophageal burn was introduced by 37.5% of NaOH to distal esophagus. Thirty-two Wistar albino rats were divided in four groups: sham rats undergone laparotomy, treated with 0.9% NaCl; control rats injured with NaOH without cordycepin treatment; cordycepin group injured with NaOH, treated with 20 mg/kg cordycepin; prednisolone group injured with NaOH, treated with 1 mg/kg prednisolone for 28 days. Efficacy was assessed by histopathological and immunohistochemical analysis of esophageal tissues. Results Cordycepin treatment significantly decreased inflammation, granulation tissue and fibrous tissue formation and prevented formation of esophageal strictures shown by histopathological damage score and stenosis indexes compared to control group (p 0.01). These effects are relatively more substantial than prednisolone, probably based on attenuation of elevation of proinflammatory cytokines hypoxia-inducible factor 1-alpha (HIF-1?), tumor necrosis factor alpha (TNF-?), proliferative and fibrotic factor fibroblast growth factor 2 (FGF2) and angiogenic factor vascular endothelial growth factor A (VEGFA) (p 0.05). Conclusions The findings suggest that cordycepin has a complex multifactorial healing process in alkali-burned tissue, more successful than prednisolone in preventing the formation of esophageal strictures and may be used as a therapeutic agent in the acute phase of esophageal alkali-burn.(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)

A importância da família dos fatores de crescimento endotelial vascular na reprodução

O ciclo estral da cadela difere de fêmeas de outras espécies e tem como protagonista principal o corpo lúteo, glândula endócrina transitória que secreta diversos fatores relacionados à sua manutenção e regressão. Um dos fatores chaves que regula a angiogênese é o VEGFA, fator de crescimento endotelial pertencente a uma ampla família. O objetivo do trabalho foi revisar a importância do VEGFA, de suas isoformas, seus receptores e dos fatores de crescimento na angiogênese, na reprodução e no corpo lúteo canino. Sobre as isoformas angiogênicas do VEGFA e sua estruturação em exons e introns, nada se achou na espécie canina. Sugerimos a estrutura das isoformas caninas homologas às do ser humano para posterior validação. Adicionalmente, identificamos a expressão de fatores angiogênicos durante o diestro não gestacional na espécie canina, através da técnica de RNA-seq. Para tal, foram utilizados corpos lúteos provenientes de 18 cadelas que passaram por ovariosalpingohisterectomia (OSH) nos dias 10, 20, 30, 40, 50 e 60 após a ovulação (p.o). Os corpos lúteos coletados foram utilizados para extração de RNA total a partir do protocolo de TRIZOL e posterior análise por RNA-seq, seguindo o protocolo TruSeq RNA Sample Preparation Guide, descrito por Illumina. Identificamos a expressão diferencial do VEGFA, VEGFC, VEGFD, VEGFR1, VEGFR2 e VEGFR3 nas comparações entre o período luteotrófico e regressão luteínica. Os dados demonstram maior expressão de fatores angiogênicos no dia 20 p.o, período relacionados à proliferação celular e manutenção da função do corpo lúteo canino.

The estrous cycle of bitches differs from that of other species and its main protagonist is the corpus luteum, a transient endocrine gland that secretes several factors related to its maintenance and regression. One of the main factors that regulate angiogenesis is VEGFA, an endothelial growth factor belonging to a large family. The aim of the study was to shed light on the importance of VEGFA, its isoforms and receptors in angiogenesis, reproduction and canine corpus luteum. The angiogenic isoforms of this growth factor have been investigated for its structure in exons and introns, and nothing has been found about this conformation in dogs. Here we suggest the structure of canine isoforms homologous to that of humans for further validation and we seek to identify the expression of angiogenic factors during non-gestational diestrus in the canine species, using the RNA-seq technique. For this purpose, corpus luteum of 18 bitches submitted to ovariosalpingohisterectomy (OSH) were used on days 10, 20, 30, 40, 50 and 60 after ovulation (p.o). The collected corpus luteum were used for extraction of total RNA using the TRIZOL protocol and subsequent analysis by RNA-seq, following the protocol TruSeq RNA Sample Preparation Guide, described by Illumina. We identified the differential expression of VEGFA, VEGFC, VEGFD, VEGFR1, VEGFR2 and VEGFR3 when comparing the luteotrophic and regression periods. The data demonstrate the active participation of angiogenic factors on day 20 post ovulation, a period related to cell proliferation and maintenance of canine corpus luteum function.

IDENTIFICATION AND FUNCTIONAL ANNOTATION OF GENES RELATED TO HORSES PERFORMANCE: FROM GWAS TO POST-GWAS

O desempenho atlético de cavalos é uma característica influenciada por um grande número de genes. Porém, essa característica tem sido pouco estudada no âmbito da genética molecular, havendo poucos genes candidatos identificados para o desempenho atlético em cavalos. Objetivou-se com este estudo: 1 / identificar genes candidatos associados a adaptações fisiológicas no condicionamento físico de cavalos através de uma revisão sistemática e 2 / construir redes de genes a partir dos genes candidatos identificados, com o objetivo de destacar os genes mais candidatos ao desempenho de cavalos. Uma revisão sistemática realizada por dois avaliadores independentes buscou artigos revisados por pares usando 20 combinações de palavras-chave. A partir disso, nove artigos foram selecionados e definidos como grupos para análise funcional. Um total de 669 genes candidatos foram identificados e usados para análises adicionais. Redes de gene-processos biológicos de cada grupo de genes foram construídas destacando processos associados ao desempenho de cavalos(por exemplo, regulação da pressão arterial sistêmica por vasopressina; regulação da polimerização e despolimerização de actina; secreção de glicocorticoides). Além disso, fatores de transcrição associados a genes candidatos foram identificados. Com base nos processos biológicos(por exemplo, regulação do fator de crescimento endotelial vascular e desenvolvimento do sistema esquelético) e evidências da revisão da literatura, conseguimos identificar os principais FTs relacionados ao desempenho de cavalos em cada grupo, o que nos permitiu construir uma rede gene-FT destacando o FTe a maioria dos genes candidatos ao desempenho de cavalos. A partir da rede gene-FT, pudemos observar quatro FTs principais (TFAP2A, ARNT, EGR1eSP1) e53 genes candidatos (por exemplo, PPP4R2, PDLRN3, IFNAR1e LOC100071438) relacionados ao desempenho em equinos.

The athletic performance of horses is a trait influenced by a large number of genes. However, this trait has been little studied in the field of molecular genetics, with few candidate genes identified for athletic performance in horses. The objective of this study was to: 1/identify candidate genes associated with physiological adaptations in the physical conditioning of horses via systematic review, and 2/build gene networks from the identified candidate genes aiming to highlight the most candidate genes for horse performance. A systematic review performed by two independent judges search for peer-reviewed articles using 20 combinations of keywords. From that, nine articles were selected and defined as a group for functional analysis. A total of 669candidate genes identified and used for further analyses. Gene networks of biological processes from each group set of genes were constructed highlighting processes associated with horse performance (e.g. regulation of systemic arterial blood pressure by vasopressin; regulation of actin polymerization and depolymerization; and glucocorticoid secretion). Besides, transcription factors associated with candidate genes were identified. Based on biological process (e.g. Regulation of vascular endothelial growth factor and Skeletal system development) and evidences from literature review, we were able to identify the main TFs related to horse performance in each group, which allowed us to construct a gene-TF network highlighting the TF and most candidate genes for horse performance. From the gene-TF network we were able to observe four main TFs (TFAP2A, ARNT, EGR1, and SP1) and 53most candidate genes (e.g. PPP4R2, PDLRN3, IFNAR1, and LOC100071438) related to horse performance.

The testis of the mice C57/BL6 offspring in adulthood have alterations due to maternal caffeine consumption

To investigate the effects of the maternal caffeine consumption during pregnancy to adult male testis mice offspring. METHODS: Twenty pregnant mice were divided into control group (c) and caffeine group (cf). dams received daily saline or 20 mg/kg of caffeine subcutaneously. Male offspring were monitored daily until 13th week. The testis were used to evaluate both the proliferation (pcna) and apoptosis (bax); leptin receptor (ob-r); aromatase; follicle stimulating hormone (fshr), luteinizing hormone (lhr) and androgen receptors (ar); steroidogenic acute regulatory protein (star); vascular endothelial growth factor (vegf) and estrogen receptors (erα and erβ) by western blotting. Serum concentrations of testosterone, estradiol and leptin were measured. RESULTS: There was a significant reduction in food intake and the body mass gain (p<0.05) in the cf ; pcna (p=0.01), fshr (p=0.02), star (p=0.0007), vegf (p=0.009), ar (p=0.03) in the cf. while an increase were note in bax (p=0.01), ob-r (p=0.02), lhr (p=0.04) and in the aromatase (p=0.03) in the cf. only erα and erβ were not changed by maternal caffeine. The serum testosterone levels in the cf offspring were 90% lower than in the c offspring (p=0.04). CONCLUSION: Maternal caffeine consumption has a role and alters the testis of the offspring in adulthood.(AU)

Survival, growth, and hormone production in vitro from preantral follicles of crossbred female goats (Saanen × Anglo-Nubian)

The present study aimed to evaluate the survival, growth, antrum formation, oocyte extrusion, and hormone production during in vitro culture of caprine preantral follicles isolated from pure-breed (Saanen) or crossbreed (F1 generation: ½ Saanen + ½ Anglo-Nubian) goats. Secondary follicles (diameter: 150-250 µm) from Saanen or crossbreed (Saanen × Anglo-Nubian) goats were isolated from the ovarian cortex by microdissection and cultured in vitro for 18 days in α-modified minimum essential medium (α-MEM+) supplemented with vascular endothelial growth factor and increasing concentrations of follicle-stimulating hormone. Every six days follicular morphology, growth, antrum formation, and follicular extrusion were evaluated. In addition, on days 2, 6, 12, and 18 of culture the medium samples were collected and stored at -20°C for further measurement of estradiol and progesterone. The follicular survival, antrum formation, and oocyte extrusion were analyzed by the Chi square test. Follicular diameter and hormone assays were compared using the Kruskal-Wallis test. Survival rates, growth, antral follicle formation, and oocyte extrusion of preantral follicles cultured in vitro were similar between the different genetic groups. The production of estradiol and progesterone indicated the maintenance of cell viability throughout the culture. In conclusion, preantral follicles from pure-breed or crossbreed goats can be used with the same efficiency for in vitro culture of isolated caprine preantral follicles.

Survival, growth, and hormone production in vitro from preantral follicles of crossbred female goats (Saanen × Anglo-Nubian)

The present study aimed to evaluate the survival, growth, antrum formation, oocyte extrusion, and hormone production during in vitro culture of caprine preantral follicles isolated from pure-breed (Saanen) or crossbreed (F1 generation: ½ Saanen + ½ Anglo-Nubian) goats. Secondary follicles (diameter: 150-250 µm) from Saanen or crossbreed (Saanen × Anglo-Nubian) goats were isolated from the ovarian cortex by microdissection and cultured in vitro for 18 days in α-modified minimum essential medium (α-MEM+) supplemented with vascular endothelial growth factor and increasing concentrations of follicle-stimulating hormone. Every six days follicular morphology, growth, antrum formation, and follicular extrusion were evaluated. In addition, on days 2, 6, 12, and 18 of culture the medium samples were collected and stored at -20°C for further measurement of estradiol and progesterone. The follicular survival, antrum formation, and oocyte extrusion were analyzed by the Chi square test. Follicular diameter and hormone assays were compared using the Kruskal-Wallis test. Survival rates, growth, antral follicle formation, and oocyte extrusion of preantral follicles cultured in vitro were similar between the different genetic groups. The production of estradiol and progesterone indicated the maintenance of cell viability throughout the culture. In conclusion, preantral follicles from pure-breed or crossbreed goats can be used with the same efficiency for in vitro culture of isolated caprine preantral follicles.(AU)

AVALIAÇÃO COMPARATIVA DA EFICÁCIA NA OBTENÇÃO DO PLASMA RICO EM PLAQUETAS (PRP) PRODUZIDO A PARTIR DE SANGUE DE MUAR (Equus caballus X Equus asininus) E EQUINOS (Equus caballus) SADIOS.

O uso do Plasma Rico em Plaquetas (PRP) autólogo apresenta resultados positivos em diversas enfermidades na medicina equina, tais como tendinites e desmites, no tratamento de feridas, osteoartrite e na cicatrização de fraturas. No entanto, a falta de padronização dos protocolos de preparo do PRP torna difícil a avaliação dos resultados clínicos. O objetivo desse estudo foi avaliar comparativamente a eficácia de obtenção do plasma rico em plaquetas (PRP) utilizando dois protocolos distintos, produzido a partir de sangue de muares (Equus caballus x Equus asininus) e equinos (Equus caballus) sadios. Para este estudo, foram coletados 54mL de sangue total de 19 equídeos sadios, sendo 8 equinos da raça Quarto de Milha, e 11 Muares subdivididos em 12 tubos a vácuo com citrato de sódio a 3,2%. As amostras de cada grupo foram submetidas ao protocolo I com descanso de 2 horas no final do processamento do PRP e o protocolo II com 2 horas de descanso antes do processamento do PRP. A força g e o tempo de centrifugação foram as mesmas utilizados para ambos os protocolos. Após as centrifugações foram aliquotadas 2mL do plasma e congelados para análise do VEGF (Fator de Crescimento Endotelial Vascular) (estágio A e B) e o PRP (estágio C) por meio de ELISA. Verificou-se que a concentração plaquetária no grupo do protocolo II dos equinos foi superior ao grupo do protocolo I (p< 0,01; 95%), enquanto que nos muares, o protocolo I apresentou uma média de plaquetas no PRP de 816,9 x 103/µL ± 177,50 e para o protocolo II uma média foi de 769,1 x 103/µL ± 212,20, não havendo diferenças significativas. Ao mensurar os níveis de VEGF nos diferentes estágios da produção do PRP, os mesmos não apresentaram diferenças significativas. Desta forma concluímos que não houve ativação plaquetária no processamento do PRP. O protocolo II é o mais eficaz na concentração do PRP em equinos e não difere quanto à eficácia para os muares. Ao comparar as espécies, os muares forneceram o PRP mais concentrado que dos equinos.

Autologous platelet rich plasma (PRP) has been used with positive results in equine veterinary medicine to treat conditions such as tendonitis, desmitis, in wound healing, osteoarthritis and fracture healing. However, lack of standardization for PRP preparation protocols make it difficult to evaluate clinical results. This study evaluates platelet yield using two different PRP preparation protocols from blood obtained from healthy horses (Equus caballus) and mules/jacks (Equus caballus x Equus asininus). Fifty four millilitres of whole blood were collected into twelve 3.2% sodium citrate evacuated tubes from 19 animals, eight horses and eleven mules/jacks. The samples were subjected to different procedural protocols (I and II) using the same g force and centrifugation time but with different moments of sample resting. After the centrifugations, 2mL of the plasma were aliquoted and frozen for analysis of VEGF (Vascular Endothelial Growth Factor) (stage A and B) and PRP (stage C) by ELISA. Mean platelet counts in protocol II were significantly higher than protocol I for the horses (p< 0.01; 95%) while there were no significant differences (p = 0.49) between protocols for the mules (protocol I mean PRP platelet count 816.900 x 103.000/µL ± 177.500; protocol II mean PRP platelet count 769.100 x 103.000/µL ± 212.200). When measuring VEGF levels at the different stages of PRP production, there were no significant differences, indicating there was no platelet activation and degranulation during processing. In conclusion, protocol II for PRP preparation produces a higher yield for horses but not for mules. When species were compared, mules offered a higher platelet yield than horses.

Ecografia modo-b e Doppler na avaliação das artérias epigástricas superficiais no neoplasma mamário canino

A hemodinâmica do fluxo sanguíneos dos neoplasmas mamários caninos (NMC) podem sofrer interferências de acordo com suas características biomecânicas. Para verificar o impacto do NMC no fluxo dos vasos que suprem o tecido doente (n=43), cadelas adultas foram submetidas ao exame ecográfico modo-B e Doppler dos NMC, artérias epigástricas superficiais cranial (AESCr) e caudal (AESCa) e linfonodos axilar e inguinal. As fêmeas caninas estudadas foram avaliadas a partir de exame clínico, laboratorial e de imagem e foram incluídas no estudo apenas os estádios oncológicos I, II e III. Os animais controle (n=20) foram submetidos as mesmas avaliações. Para o estudo inicial foram formados dois grupos de NMC (benigno, n=36 e maligno, n=100). Os neoplasmas foram analisados quanto ao tamanho, ecotextura, vascularização, velocidade de fluxo (Vfl) e índice de resistividade (IR) e foi detectado que a ecotextura pode influenciar diretamente a dinâmica vascular. Foram também mensurados (neoplasma maligno, n=37 e cadelas hígidas, n=13) o fator de crescimento endotelial vascular (VEGF), interleucina-8 (Il-8) e estradiol (E2), sendo observada uma correlação positiva entre a concentração sérica do VEGF e a hemodinâmica dos vasos neoplásicos e o incremento nos níveis séricos da Il-8 no neoplasma maligno heterogenio. Não foi observado nenhuma influência do E2. E por fim, verificou-se a interferência (neoplasma maligno, n=43) do número de neoplasmas por cadeia mamária, do estádio oncológico, da ecotextura, da vascularização do neoplasma e do grau histológico, nas Vfl e IR das AESCr e AESCa. Também, foi observado maior impacto na AESCa devido ao maior número de neoplasmas abdominais e inguinais, bem como fatores biomecânicos do neoplasma, a partir da imagem ecográfica, modificando as Vfl e IR das principais artérias que suprem o grupo de mamas frequentemente acometida. Embora existisse diferença entre a hemodinâmica das AESCr e AESCa através da análise de imagem, não foi permitido uma conclusão sobre a significância dessa diferença, o que impulsiona outros trabalhos que possam verificar a importância clínica desse achado. A ecografia modo-B e Doppler permitiu estudar o impacto do neoplasma maligno sobre as AESCr e AESCa.

The objectives of this study were to evaluate the hemodynamics of cranial and caudal superficial epigastric arteries on Doppler ultrasound, identify the ultrasound characteristics of the vessels and of the tumor texture, measure the levels of vascular endothelial growth factor (VEGF) and interleukin-8 of canine patients with mammary carcinomas, and predict the biological behavior of mammary carcinoma based on the hemodynamic variations of superficial epigastric arteries in early cancer stages. For this, the flow velocity and resistive index of the cranial and caudal superficial epigastric arteries of 63 female canines were evaluated by Doppler ultrasound. The mammary tumor group comprised 43 canines with the histopathological diagnosis of mammary carcinoma, whereas the control group comprised 20 healthy canine. The vascular dynamics was significantly different between the two groups, and the number of tumor lesions affected the vessel flow rate. In addition, the echotexture and presentation and distribution of the vessels affected the resistive index in the mammary carcinoma group. Fifty other female canines were divided into two groups (13 healthy canines and 37 canines with mammary carcinoma) to evaluate the clinical usefulness of the serum measurement of VEGF, interleukin-8, and estradiol, combined with ultrasound examination of malignant tumors. The results demonstrated the strong correlation of echotexture with interleukin-8 levels and the positive correlation between tumor vessel flow rate and VEGF levels. These findings indicate a relationship between the tumor environment and expression of interleukin-8 as well as the effect of flow dynamics on VEGF levels. Finally, 142 mammary nodules or masses were evaluated by ultrasound to assess tumor flow dynamics and differentiate between benign and malignant mammary tumors. The biodynamics of internal vessels was significantly different between benign and malignant tumors, with systolic and diastolic velocities varying according to tumor size and echotexture. Therefore, the joint evaluation of tumor size and texture and of the flow velocity of internal tumor vessels may be useful for differentiating between benign and malignant mammary tumors. The present study presents an important application of ultrasound imaging in veterinary oncology and demonstrates the effect of hemodynamics on cranial and caudal superficial epigastric arteries, evidenced by Doppler ultrasound, in canine mammary carcinoma, particularly when associated with the analysis of tumor size and texture, and of the presentation and distribution of tumor vessels. These findings confirm that mechanical factors of tumors interfere in the hemodynamics of superficial epigastric arteries. These results indicate the importance of evaluating the arteries that irrigate the affected mammary chain together with other factors predictive of tumor malignancy, including tumor size, heterogeneous echotexture of the tumor stroma, heterogeneous presentation and complex distribution of tumor vascularization, and serum levels of VEGF and interleukin-8.

Alterações morfológicas e vasculares do corpo lúteo durante a gestação inicial de conceptos bovinos clonados por SCNT com diferentes fenótipos gestacionais

A clonagem bovina por transferência nuclear de células somáticas ainda apresenta problemas ligados às elevadas perdas gestacionais. Porém, estas gestações têm sido bons modelos para estudos de fisiopatologia gestacional. Estudos prévios mostraram a ocorrência de três fenótipos após a transferência de embriões (TE) clonados por, sendo eles: (1) Gestações normais (CNG) com presença do embrião (E), vesícula embrionária (VE) e corpo lúteo (CL), (2) Gestações anembrionadas (CAG) com presença da VE e CL, e ausência do E, e (3) Receptoras com CL persistente (CPCL). O objetivo então, foi avaliar as alterações morfovasculares do CL ao longo 35 dias após a ovulação nesses três fenótipos comparando-os com gestações de embriões produzidos in vivo (grupo controle; GC). A hipótese a ser testada foi que a morfofuncionalidade do CL é dependente do grau de desenvolvimento do concepto. Vacas Bos indicus foram utilizadas como receptoras de embriões no dia 7. Exames de ultrassonografia (US) nos modos B e Doppler colorido foram realizados nas receptoras a cada dois dias no primeiro mês de gestação para análise das características morfovasculares do CL. No dia 35, as receptoras foram abatidas para coleta de amostras de tecido luteínico destinados à morfometria e Western-Blot (WB) para quantificação do fator de crescimento endotelial vascular (VEGF) e seu receptor 2 (VEGF-R2). Foram realizados 14 ciclos de transferência de embrião em tempo fixo (TETF) onde 120 receptoras receberam 79 embriões clonados e 41 embriões produzidos in vivo. Foram obtidas doze gestações no grupo CNG (15,19%), uma no CAG (1,27%), quatro no CPCL (5,06%) e nove no GC (21,95%). Os dados ultrassonográficos não diferindo entre os grupos (p>0,05), porém mostraram uma diminuição do diâmetro e da área do CL ( 2mm e 0,5 cm² respectivamente; p<0,05) entre os dias 10 e 20 e a vascularização do CL aumentou desde o dia 6 até o dia 24 (30%; p<0,05). Um fato relevante foi a diminuição (0,5 cm²; p<0,05) da área de vascularização luteínica do grupo CNG quando comparado ao GC nos dias 32 e 34. In situ os dados de CL mostraram volume (cm³) e diâmetro (mm) (p=0,007) maiores em GC e menores em CNG. O 11 comprimento do embrião CNG teve médias em torno de 2 mm maiores que em GC (p=0,0131) de 26 a 34 dias de gestação. Pela morfometria, os grupos não diferiram entre si (p>0,05) quanto as médias das proporções de células luteínicas pequenas e grandes. Pelo WB, a quantidade relativa de VEGF no CNG obteve os maiores resultados quando comparada (p=0,025) aos outros grupos e de VEGF-R2 mostrou maiores em CPCL (p=0,006). Apesar CAG e CPCL foram semelhantes entre si, o grupo CNG mostra grandes diferenças em relação a GC quanto às características luteínicas. Em suma, este estudo fornece as bases para novas investigações sobre os aspectos que regem a interação entre o desenvolvimento do concepto e a morfofuncionalidade do CL durante a gestação inicial em bovinos.

Bovine cloning by somatic cell nuclear transfer still presents obstacles related to high pregnancy loss. However, these pregnancies have been good models for the study of gestational pathophysiological. Previous studies have shown the occurrence of three pregnancy phenotypes after cloned embryo transfer (ET), which are: (1) Normal pregnancies (CNG) with the presence of the embryo (E), embryonic vesicle (EV) and corpus luteum (CL), (2) Anembryonic gestation (CAG) in the presence of EV and CL and absence of E, and (3) Embryo recipient with persistent CL (CPCL). The aim of this study was to evaluate the morphovascular changes of CL over the first 35 days of gestation after TE in these three phenotypes, comparing them with pregnancies obtained with in vivo produced embryos (control group; GC). The hypothesis to be tested was that CLs morphofunctionality is dependent on the degree of fetus development. Bos indicus cows were used as embryos recipient on day 7. Ultrasound (US) in the B and Doppler modes were performed every two days in the first month of gestation to examine CL morphovascular characteristics. On day 35, recipients were slaughtered and luteal tissue samples collected for morphometry and Western-Blot (WB) for quantification of vascular endothelial growth factor (VEGF) and its receptor 2 (VEGF-R2). Fourteen Fixed-time embryo transfer (FTET) cycles were performed in witch 120 recipients received 79 cloned embryos and 41 embryos produced in vivo. This study obtained twelve pregnancies in the CNG group (15.19%), one in the CAG (1.27%), four in CPCL (5.06%) and nine in the control group (21.95%). Ultrasonographic data showed no difference between groups, but a slight decrease of diameter and CL area (p<0.05) between days 10 and 20 and luteal vascularization increased in all groups studied from day 6 to day 24 (p <0,05). A relevant fact was the decrease of the luteal vascularization area of CNG group from day 28 compared to the control group on days 32 and 34. In situ CL data showed volume (cm³) and diameter (mm) (p=0.007) higher in CG than in CNG. The embryo had greater average length (p=0.0131) in CNG group than in GC from day 26 to day 34 of gestation. By morphometry analysis, no significant differences were found (p>0.05) between the proportions of cell types among groups. At WB, the relative amount of VEGF CNG obtained the highest values and VEGF-R2 shown higher amounts in CPCL group (p=0.006). Although CAG and CPCL were similar to each other, the CNG group showed large differences from GC within the luteal characteristics. In summary, this study 13 provides the basis for further research on the aspects that govern the interaction between the development of the conceptus and CL morphofunctionality during early pregnancy in cattle

AVALIAÇÃO DO FATOR DE CRESCIMENTO ENDOTELIAL VASCULAR (VEGF) E MACRÓFAGOS ASSOCIADOS AO TUMOR (TAM) EM CARCINOMAS MAMÁRIOS DE CADELAS

No microambiente tumoral, os macrófagos podem estimular a proliferação das células malignas, mediante produção de fatores de crescimento que favorecem a angiogênese. Embora a relação entre angiogênese e macrófagos tenha sido reconhecida em diversas pesquisas envolvendo tumores mamários de mulheres, esta relação na espécie canina permanece sob investigação. O objetivo deste estudo foi avaliar a marcação de macrófagos associados ao tumor (TAM) e do fator de crescimento endotelial vascular (VEGF) em carcinomas mamários de cadelas mediante técnica de imuno-histoquímica. Foram selecionados do setor de patologia do Hospital Veterinário da Universidade de Franca/SP (UNIFRAN), 99 blocos de parafina de carcinomas mamários de cadelas. As amostras foram submetidas a reações de imuno-histoquímica com os anticorpos anti-VEGF (VG-1, Abcam) e anti-macrófago (MAC 387, DBS). Para a determinação do VEGF, foi realizada a soma dos escores dos percentuais de células tumorais coradas cujo percentual de marcação variou de um a quatro, sendo 1: até 25% das células positivas, 2: 25 a 50% das células positivas, 3: 50 a 75% das células positivas e 4: mais de 75% das células positivas. Para a quantificação de macrófagos, avaliou-se o número absoluto de células positivas que se apresentaram entremeadas ao tumor. Os resultados foram avaliados,por análise de variância seguida pelo teste de Tukey, Kruskal-Wallis, seguido pelo teste de Dunns e teste t de Student, considerando-se nível de significância de 5%. Comparando-se os escores de marcação do VEGF, observou-se que carcinomas tubulares, papilares e carcinomas em tumor misto apresentaram médias de escores superiores ao carcinoma sólido (p<0,001). Avaliando-se as contagens de macrófagos entre os tipos histológicos, os carcinomas tubulares apresentaram média (13,52±16,38) superior aos carcinomas sólidos (2,77±3,59) (p<0,01). Contudo, não houve diferença na contagem de VEGF e de macrófagos entre os graus histológicos I, II e III (p>0,05). Quando categorizados em dois grupos de arcação de VEGF (50% e >50%), a média de TAM foi maior nos animais com marcação acima de 50% de VEGF (p=0,002). Conclui-se que, apesar do VEGF e de macrófagos não terem se apresentado aumentados nos tumores considerados agressivos, ambos demonstraram estar relacionados, uma vez que marcações de VEGF acima de 50% revelaram maiores contagens de macrófagos nas neoplasias mamárias caninas.

Mammary gland tumors are the most common malignancies in dogs. In the tumor 8 microenvironment, macrophages can stimulate the proliferation of malignant cells through production of growth factors that promote angiogenesis. Although the 10 relationship between angiogenesis and macrophages has been recognized in several studies involving mammary tumors in women, this relationship remains under 12 investigation in dogs. The objective of this study was to evaluate the immunostaining of 13 tumor-associated macrophages (TAM) and vascular endothelial growth factor (VEGF) in 14 mammary carcinomas of female dogs by means of immunohistochemistry technique. Ninety-nine 99 paraffin blocks of mammary carcinomas of female dogs were selected 16 from Veterinary Hospital of the University of Franca / SP (UNIFRAN). The samples under went immunohistochemical reactions with antibodies anti-macrophage (MAC 387, 18 DBS) and anti-VEGF (VG-1, Abcam). macrophages quantification was evaluated by the absolute number of positive cells around the tumor. VEGF analysis was performed by 20 percentage scores as follow: 1: up to 25% of positive cells, 2: 25 to 50% of positive cells 21 3: 50-75% of positive cells and 4: more than 75% of the positive cells. Analysis of 22 variance plus Tukey's test or Kruskal-Wallis plus Dunn's test was performed considering 23 5% significance level. Macrophages counts between histologic types showed higher 24 mean of tubular carcinomas (13,52 ± 16,38) that the solid carcinomas (2,77 ± 3,59) 25 (p<0,01). When comparing VEGF scores, it was observed that tubular, papillary and 26 mixed tumor carcinomas showed higher mean scorethat the solid carcinoma (p<0,001). 27 However, there was no difference to macrophages count or VEGF scores means 28 between tumor grades I, II and III (p> 0,05). When classified into two groups VEGF 29 labeling (50% and> 50%), the TAM mean was higher in animals with VEGF labelling 30 above 50% (p = 0,002). In conclusion, although VEGF and macrophages were not 31 increased in aggressive tumors, both markers could be related, since VEGF labeling over 50% showed higher macrophages density in canine mammary tumors.

Effect of thalidomide on the healing of colonic anastomosis, in rats / Efeito da talidomida na cicatrização de anastomoses colônicas em ratos

PURPOSE: Thalidomide, because of its anti-inflammatory properties, as re-emerged as an option for the treatment of Crohn's disease refractory to standard therapy. We studied the effect of thalidomide on the healing of colonic anastomosis. METHODS: Sixty male rats (Rattus norvegicus), were divided into 3 groups of 20 animals each, respectively receiving 0.5 or 1.0 mg/kg thalidomide by the oral route for 7 days, or saline solution (control). All animals were submitted to continuous end-to-end anastomosis with 6-0 Prolene sutures. After sacrifice the anastomoses were analyzed macroscopically and submitted to determination of hydroxyproline, to histology and to immunohistochemistry for metalloproteinase 1, metalloproteinase 1 inhibitor and vascular endothelial growth factor (VEGF). RESULTS: Statistical analysis of the data showed no significant difference in macroscopic aspect or hydroxyproline determination (p= 0.5403). In the immunohistochemical analysis, the following p values were obtained: p = 0.5817 for VEGF, p = 0.1854 for metalloproteinase 1, and p = 0.0023 for metalloproteinase 1 inhibitor, with this last value being considered statistically significant. CONCLUSION: We conclude that thalidomide influenced collagen maturation. There was a stronger action of metalloproteinases, possibly indicating a negative tendency for the healing process.(AU)

OBJETIVO: Sabe-se que agentes farmacológicos podem influenciar no processo de cicatrização. A talidomida, devido às suas propriedades antiinflamatórias, tem ressurgido como uma opção no tratamento da doença de Crõhn refratária à terapêutica convencional. Neste trabalho, estudamos o efeito da talidomida na cicatrização de anastomoses colônicas no rato. MÉTODOS: Foram utilizados 60 animais Rattus norvegius, com peso médio de 300g. Organizou-se 3 grupos de 20 animais, sendo um grupo controle (AC), um grupo (BD), com administração de talidomida 0,5 mg/kg por 7 dias e um grupo (AD) com administração de talidomida 1,0 mg/kg por 7 dias. Foi realizada anastomose término-terminal contínua de prolene 6-0. O sacrifício foi no 7º. dia pós operatório e as anastomoses foram analisadas quanto a aspecto macroscópico, dosagem de hidroxiprolina, histologia em hematoxilina-eosina e imuno-histoquímica para metaloproteinase 1, inibidor de metaloproteinase 1 e VEGF. RESULTADOS: Não houve diferença estatisticamente significante para a observação macroscópica e para dosagem de hidroxiprolina (p=0,5403). Na análise imunohistoquímica, para VEGF houve p=0,5817, para metaloproteinase 1, p=0,1854 e para inibidor de metaloproteinase, p=0,0023, considerado estatisticamente significante. CONCLUSÃO: Concluímos que a talidomida influenciou na maturação do colágeno. Notou-se maior ação das metaloproteinases, que pode significar uma tendência negativa para o processo cicatricial.(AU)