Combination of heterologous fibrin sealant and bioengineered human embryonic stem cells to improve regeneration following autogenous sciatic nerve grafting repair

Background Peripheral nerve injury is a worldwide clinical problem, and the preferred surgical method for treating it is the end-to-end neurorrhaphy. When it is not possible due to a large nerve gap, autologous nerve grafting is used. However, these surgical techniques result in nerve regeneration at highly variable degrees. It is thus very important to seek complementary techniques to improve motor and sensory recovery. One promising approach could be cell therapy. Transplantation therapy with human embryonic stem cells (hESCs) is appealing because these cells are pluripotent and can differentiate into specialized cell types and have self-renewal ability. Therefore, the main objective of this study was to find conditions under which functional recovery is improved after sciatic nerve neurorrhaphy. We assumed that hESC, either alone or in combination with heterologous fibrin sealant scaffold, could be used to support regeneration in a mouse model of sciatic nerve injury and repair via autografting with end-to-end neurorrhaphy. Methods Five millimeters of the sciatic nerve of C57BL/6 J mice were transected off and rotated 180 degrees to simulate an injury, and then stumps were sutured. Next, we applied heterologous fibrin sealant and/or human embryonic stem cells genetically altered to overexpress fibroblast growth factor 2 (FGF2) at the site of the injury. The study was designed to include six experimental groups comprising neurorrhaphy (N), neurorrhaphy + heterologous fibrin sealant (N + F), neurorrhaphy + heterologous fibrin sealant + doxycycline (N + F + D), neurorrhaphy + heterologous fibrin sealant + wild-type hESC (N + F + W), neurorrhaphy + heterologous fibrin sealant + hESC off (N + F +T), and neurorrhaphy + heterologous fibrin sealant + hESC on via doxycycline (N + F + D + T). We evaluated the recovery rate using Catwalk and von Frey functional recovery tests, as well as immunohistochemistry analysis. Results The experiments indicated...(AU)

Pravastatin induces cell cycle arrest and decreased production of VEGF and bFGF in multiple myeloma cell line

Abstract Multiple myeloma (MM) is a B cell bone marrow neoplasia characterized by inflammation with an intense secretion of growth factors that promote tumor growth, cell survival, migration and invasion. The aim of this study was to evaluate the effects of pravastatin, a drug used to reduce cholesterol, in a MM cell line.Cell cycle and viability were determinate by Trypan Blue and Propidium Iodide. IL6, VEGF, bFGF and TGF were quantified by ELISA and qRT-PCR including here de HMG CoA reductase. It was observed reduction of cell viability, increase of cells in G0/G1 phase of the cell cycle and reducing the factors VEGF and bFGF without influence on 3-Methyl-Glutaryl Coenzyme A reductase expression.The results demonstrated that pravastatin induces cell cycle arrest in G0/G1 and decreased production of growth factors in Multiple Myeloma cell line.

Resumo O Mieloma Múltiplo é uma neoplasia de linfócitos B da medula óssea, caracterizada por inflamação com uma intensa secreção de fatores de crescimento que promovem o aumento do volume do tumor, sobrevivência celular, migração e invasão. O objetivo deste estudo foi avaliar os efeitos da pravastatina, uma droga usada para reduzir o colesterol, em um linhagem de MM. O ciclo celular e viabilidade foram determinadas por Trypan Blue e iodeto de propídio. IL6, VEGF, bFGF e TGF foram quantificadas por ELISA e qRT-PCR, incluindo aqui de HMG CoA redutase. Observou-se a redução da viabilidade das células, aumento de células na fase G0/G1 do ciclo celular e redução no VEGF e bFGF, sem influência na expressão da enzima 3-Metil-Glutaril Coenzima A redutase. Os resultados demonstraram que a pravastatina induz parada no ciclo celular em G0/G1 e diminuição da produção de fatores de crescimento em várias linhas de células de Mieloma.

Pravastatin induces cell cycle arrest and decreased production of VEGF and bFGF in multiple myeloma cell line / Pravastatina induz parada no ciclo celular e diminuição na produção de VEGF e bFGF em linhagem de Mieloma Multiplo

Abstract Multiple myeloma (MM) is a B cell bone marrow neoplasia characterized by inflammation with an intense secretion of growth factors that promote tumor growth, cell survival, migration and invasion. The aim of this study was to evaluate the effects of pravastatin, a drug used to reduce cholesterol, in a MM cell line.Cell cycle and viability were determinate by Trypan Blue and Propidium Iodide. IL6, VEGF, bFGF and TGF were quantified by ELISA and qRT-PCR including here de HMG CoA reductase. It was observed reduction of cell viability, increase of cells in G0/G1 phase of the cell cycle and reducing the factors VEGF and bFGF without influence on 3-Methyl-Glutaryl Coenzyme A reductase expression.The results demonstrated that pravastatin induces cell cycle arrest in G0/G1 and decreased production of growth factors in Multiple Myeloma cell line.(AU)

Resumo O Mieloma Múltiplo é uma neoplasia de linfócitos B da medula óssea, caracterizada por inflamação com uma intensa secreção de fatores de crescimento que promovem o aumento do volume do tumor, sobrevivência celular, migração e invasão. O objetivo deste estudo foi avaliar os efeitos da pravastatina, uma droga usada para reduzir o colesterol, em um linhagem de MM. O ciclo celular e viabilidade foram determinadas por Trypan Blue e iodeto de propídio. IL6, VEGF, bFGF e TGF foram quantificadas por ELISA e qRT-PCR, incluindo aqui de HMG CoA redutase. Observou-se a redução da viabilidade das células, aumento de células na fase G0/G1 do ciclo celular e redução no VEGF e bFGF, sem influência na expressão da enzima 3-Metil-Glutaril Coenzima A redutase. Os resultados demonstraram que a pravastatina induz parada no ciclo celular em G0/G1 e diminuição da produção de fatores de crescimento em várias linhas de células de Mieloma.(AU)

Fator de crescimento fibroblástico 18 (FGF18) modula a expressão de CTGF em CCOs e embriões bovinos produzidos in vitro

Em 1995 a via Hippo foi inicialmente descoberta na Drosofila melanogaster, e desde então diversos pesquisadores vêm tentando entender melhor essa via. A via Hippo está relacionada a processos de proliferação celular, tanto em células foliculares ovarianas como no desenvolvimento embrionário. Os efetores dessa via, YAP e TAZ atuam como coativadores de transcrição de genes de proliferação e sobrevivência celular como CTGF e CYR61. Porém o fator de crescimento de fibroblasto também induz a expressão de CTGF. E segundo estudos recentes foi descoberto que o fator de crescimento fibroblastico 18 (FGF18) está presente na tuba uterina durante o desenvolvimento embrionário inicial. Isso nos levou a pensar que o FGF18 teria alguma função durante o desenvolvimento embrionário. Portanto, o objetivo deste estudo foi determinar se o FGF18 modula a via Hippo através da expressão de genes alvos de proliferação celular (CTGF e CYR61) durante a maturação oocitária e desenvolvimento embrionário inicial. Para isso três experimentos foram realizados. No primeiro experimento complexos cumulus oocito (CCOs) de bovinos foram maturados in vitro durante 6, 12 e 24 horas na presença de concentrações graduais de FGF18(0, 10 e 100ng/mL). No segundo experimento os CCOs foram maturados 3, 6 e 9 horas na presença de 0 ou 100ng/mL de FGF18. No experimento três CCOs foram colocados para maturação e foram divididos em três grupos. Um grupo considerado grupo controle no qual CCOs foram maturados, realizada a fertilização in vitro (FIV) e depois cultivados in vitro (CIV) sem a adição de FGF18. Um grpo no qual 100nf/mL de FGF18 foram adicionados durante as 24 horas da maturação in vitro e epós foi realizado a fertilização e o cultivo em meios sem adição de FGF18. E um terceiro grupos os CCOs foram cultivados (CIV) por sete dias na presença de 100ng/mL de FGF18. Neste grupo a MIV e FIV foram realizadas na ausência de FGF18. Os genes avaliados nos três experimentos foram os genes de proliferação celular, CTGF e CYR61. O experimento um que teve o objetivo de avaliar a adição de concentrações graduais durante a maturação houve um aumento (p<0,05) da expressão gênica para CTGF no grupo tratado com 100ng/mL as 12horas. No experimento dois em que o objetivo era avaliar a concentração de 100ng/mL houve uma diminuição (p<0,05) do grupo tratado em relação ao grupo controle as três horas. E no experimento três em que o objetivo era avaliar a adição de FGF18 durante o desenvolvimento embrionário, houve diferença estatística (p<0,05) do grupo tratado com FGF18 durante a CIV. Desde forma concluímos que FG18 modula a expressão de CTGF em período críticos da maturação nuclear do oócito, da expansão do cumulus e do desenvolvimento embrionário inicial

In 1995, the Hippo pathway was initially discovered in Drosophila melanogaster, and since then several researchers have been trying to better understand this pathway. The Hippo pathway is involved in cell proliferation processes, both in ovarian follicular cells in the early embryonic development. The effectors of this pathway YAP e TAZ act as transcription coactivators of cell proliferation and survival genes such as CTGF and CYR61. However, the fibroblast growth factor also induces CTGF expression. However, recent studies by the group found that fibroblast growth factor 18 (FGF18) is present in the fallopian tube during early embryonic development. This led us to think that FGF18 would have some role during embryonic development. Therefore, the aim of the following study was to determine whether FGF18 modulates the Hippo pathway through the expression of target genes for cell proliferation (CTGF and CYR61) during oocyte maturation and early embryonic development. To acquire the results of the objective, three experiments were carried out, with in vitro maturation (IVM) of cumulus oocyte complexes (COCs) in the first and second experiments, with the addition of FGF18 during IVM, and the structures were collected at different times. In the first experiment, gradual concentrations of FGF18 (0, 10 and 100ng / mL) were added and there was a control group without the addition of FGF18, and 6, 12 and 24 hours of maturation were collected. In the second experiment, a concentration of 100ng / mL of FGF18 was added during IVM, there was a control group and the structures were collected at 0, 3, 6 and 9 hours of maturation. In the experiment, three COCs were put to maturation and were divided into three groups. A group considered a control group in which COCs were matured, with in vitro fertilization (IVF) and then in vitro culture (IVC) without the addition of FGF18. A group in which, during maturation, FGF18 was added only during IVM and afterwards fertilization and culture were carried out in media without the addition of FGF18. In a third group, COCs were put to mature, fertilized and, when put to culture, FGF18 was added in the medium. The genes evaluated in the three experiments were the cell proliferation genes, CTGF and CYR61. In experiment one, which aimed to evaluate the addition of gradual concentrations during maturation, there was an increase (p <0.05) in CTGF gene expression in the group treated with 100ng / mL at 12 hours. In experiment two, in which the objective was to assess the concentration of 100ng / mL, there was a decrease (p <0.05) in the treated group compared to the control group at three hours. And in experiment three in which the objective was to evaluate the addition of FGF18 during embryonic development, there was a statistical difference (p <0.05) in the group treated with FGF18 during IVC. Therefore, we conclude that FGF18 modulates CTGF expression in critical periods of oocyte nuclear maturation, cumulus expansion and early embryonic development in cattle.

Caracterização estrutural do fator de crescimento de fibroblasto-10 (FGF-10) e seu papel na fisiologia folicular ovariana / Structural characterization of the fibroblast growth factor-10 (FGF-10) and its role in ovarian follicular physiology

Background: Interest in folliculogenesis has grown extensively in recent years. Nevertheless, several aspects of follicular activity are still poorly understood. Thus, in vitro culture of ovarian follicles using new substances has been established as a very viable model, enhancing the prospects for a better understanding of follicular activity. Among the family members of the fibroblast growth factor (FGFs), FGF-10 has received recent attention for its ability to regulate the development of ovarian follicles and oocyte maturation. Given the relevance of FGF-10 in the folliculogenesis process, this review aimed to describe the structural features, expression and the main biological effects of FGF-10 on the development of ovarian follicles in mammals. Review: Along this work, it was shown aspects related to structural characterization of FGF-10 and its receptors, as well as FGF-10 expression in different cell types, emphasizing its importance to follicular development. FGF-10 is a paracrine member of the family of FGFs, and is characterized by promoting biological responses via cell surface receptors (FGFRs) of tyrosine kinase-type. Of these receptors, FGFR-1, FGFR-2 and FGFR-3 may undergo alternative transcriptional arrangements, enabling the formation of two isoforms (b and c) that have varying degrees of affinity for the various FGFs. Thus, seven FGFR proteins (FGFRs 1b, 1c, 2b, 2c, 3b, 3c and 4) with different binding specificities are generated from the four FGFR genes. The FGFRs transmit intracellular signals after binding with the ligand through the phosphorylation of tyrosine, which activates various transduction patterns in the cytoplasm. The signal transduction of FGF-10 may occur through three main pathways: protein of rat sarcoma (Ras)/MAPK, PLCg/Ca2+ and phosphatidylinositol-3 kinase (PI3K)/ protein kinase B (Akt), which are involved in the transmission of biological signals, leading to cellular proliferation and differentiation. FGF-10 mRNA expression was detected in the ovarian stroma, oocyte and theca cells of preantral and antral follicles. On the other hand, the expression of mRNA for FGF-10 receptors was found in, granulosa cells, theca cells, cumulus cells and oocytes. Although FGFs are widely distributed in different tissues and cell types, the importance and function of FGFs in the ovary are still poorly documented. FGF-10 has been shown to be an important mediator of mesenchymal and epithelial cell interactions during follicle development, promoting follicular survival, activation and growth. Besides the action on folliculogenesis, FGF-10 was recently identified as a growth factor able to improve oocyte competence. However, in antral follicles, the presence of FGF-10 is associated with increased follicular atresia, which matches its anti-estrogenic action. Discussion: From this review, we can conclude that FGF-10 is an important regulator of female reproduction. This growth factor acts in follicle survival, oocyte maturation, expansion of cumulus cells and proliferation of granulosa/theca cells through direct and/or indirect actions in the control of folliculogenesis. Furthermore, FGF-10 seemed to have different effects throughout the follicular development. However, it is necessary to perform additional studies that may provide a better understanding about the importance of FGF-10 during folicullogenesis.(AU)

Caracterização estrutural do fator de crescimento de fibroblasto-10 (FGF-10) e seu papel na fisiologia folicular ovariana / Structural characterization of the fibroblast growth factor-10 (FGF-10) and its role in ovarian follicular physiology

Background: Interest in folliculogenesis has grown extensively in recent years. Nevertheless, several aspects of follicular activity are still poorly understood. Thus, in vitro culture of ovarian follicles using new substances has been established as a very viable model, enhancing the prospects for a better understanding of follicular activity. Among the family members of the fibroblast growth factor (FGFs), FGF-10 has received recent attention for its ability to regulate the development of ovarian follicles and oocyte maturation. Given the relevance of FGF-10 in the folliculogenesis process, this review aimed to describe the structural features, expression and the main biological effects of FGF-10 on the development of ovarian follicles in mammals. Review: Along this work, it was shown aspects related to structural characterization of FGF-10 and its receptors, as well as FGF-10 expression in different cell types, emphasizing its importance to follicular development. FGF-10 is a paracrine member of the family of FGFs, and is characterized by promoting biological responses via cell surface receptors (FGFRs) of tyrosine kinase-type. Of these receptors, FGFR-1, FGFR-2 and FGFR-3 may undergo alternative transcriptional arrangements, enabling the formation of two isoforms (b and c) that have varying degrees of affinity for the various FGFs. Thus, seven FGFR proteins (FGFRs 1b, 1c, 2b, 2c, 3b, 3c and 4) with different binding specificities are generated from the four FGFR genes. The FGFRs transmit intracellular signals after binding with the ligand through the phosphorylation of tyrosine, which activates various transduction patterns in the cytoplasm. The signal transduction of FGF-10 may occur through three main pathways: protein of rat sarcoma (Ras)/MAPK, PLCg/Ca2+ and phosphatidylinositol-3 kinase (PI3K)/ protein kinase B (Akt), which are involved in the transmission of biological signals, leading to cellular proliferation and differentiation. FGF-10 mRNA expression was detected in the ovarian stroma, oocyte and theca cells of preantral and antral follicles. On the other hand, the expression of mRNA for FGF-10 receptors was found in, granulosa cells, theca cells, cumulus cells and oocytes. Although FGFs are widely distributed in different tissues and cell types, the importance and function of FGFs in the ovary are still poorly documented. FGF-10 has been shown to be an important mediator of mesenchymal and epithelial cell interactions during follicle development, promoting follicular survival, activation and growth. Besides the action on folliculogenesis, FGF-10 was recently identified as a growth factor able to improve oocyte competence. However, in antral follicles, the presence of FGF-10 is associated with increased follicular atresia, which matches its anti-estrogenic action. Discussion: From this review, we can conclude that FGF-10 is an important regulator of female reproduction. This growth factor acts in follicle survival, oocyte maturation, expansion of cumulus cells and proliferation of granulosa/theca cells through direct and/or indirect actions in the control of folliculogenesis. Furthermore, FGF-10 seemed to have different effects throughout the follicular development. However, it is necessary to perform additional studies that may provide a better understanding about the importance of FGF-10 during folicullogenesis.

Efeito do fator de crescimento de fibroblasto-10 na produção in vitro de embriões bovinos

O presente estudo visou avaliar o efeito do FGF10 durante a pré-maturação e/ou maturação in vitro (MIV) de ovócitos na produção in vitro de embriões bovinos. No primeiro experimento, os ovócitos foram maturados na presença ou não de FGF10. No segundo experimento, foi avaliado o efeito da suplementação de FGF10 durante a pré-maturação e/ou maturação, utilizando cinco grupos: T1: ovócitos maturados por 22h (controle); T2: ovócitos pré-maturados por 22h e maturados por 22h; T3: ovócitos pré-maturados por 22h e maturados por 22h com FGF10; T4: ovócitos pré-maturados por 22h com FGF10 e maturados por 22h; T5: pré-maturados por 22h com FGF10 e maturados por 22h com FGF10. Em ambos os experimentos foram avaliadas a maturação nuclear, a expansão das células do cumulus, a produção embrionária e a expressão dos genes: KRT8, PLAC8, CD9, PAG2, HSPB1 e MSH6 em embriões no D7 (experimento 1) ou D8 (experimento 2). Os dados de maturação nuclear e desenvolvimento embrionário foram analisados pelo teste do Qui-quadrado, e os da expansão das células do cumulus e expressão gênica por análise de variância, sendo P<0,05 considerado significativo. No primeiro experimento, a suplementação com FGF10 durante a MIV não afetou (P>0,05) nenhum dos parâmetros avaliados. No segundo experimento, após a maturação, a expansão em todos os tratamentos submetidos à pré-maturação, independente da presença de FGF10 (P<0,05), foi menor do que no grupo controle. A presença de FGF10 na pré-maturação/maturação não aumentou a quantidade de embriões sendo a taxa de blastocisto em D7 similar (P>0,05) entre T2 (43,7%), T3 (38,7%), T4 (39,6%) e T5 (47,3%). Entretanto, nos grupos T2 e T5 a taxa de blastocisto no D7 foi similar ao controle (53,4%). Todos os grupos apresentaram taxas de embriões eclodidos do D8 semelhantes. O nível de transcritos para o PLAC8 foi superior no grupo T4 quando comparado ao T1, os demais grupos foram iguais entre si. Já para o gene MSH6, somente foi detecta diferença no nível de transcritos entre os grupos T5 e T1, sendo inferior no T5. Conclui-se que a presença de FGF10 durante a MIV e/ou pré-MIV não tem efeito benéfico na produção e a qualidade dos embriões. Da mesma forma, a pré-maturação, independente da presença do FGF10, afeta a expansão das célullas do cumulus mas não tem efeito na produção embrionária.

This study aimed to evaluate the effect of FGF10 during pre-maturation and in vitro maturation (IVM) of bovine oocytes on in vitro embryo production (IVP). In the first experiment oocytes were matured in the presence or not of FGF10. In the second experiment, the effect of FGF10 during prematuration and/or maturation was evaluated using 5 experimental groups: T1: oocytes matured for 22h (control); T2: oocytes prematured for 22h and matured for 22h; T3: oocytes prematured for 22h and matured for 22h with FGF10; T4: oocytes prematured with FGF10 for 22h and matured for 22h; T5: oocytes prematured for 22h with FGF10 and matured for matured 22h with FGF10. In both experiments, nuclear maturation, cumulus cells expansion, embryo production and the relative expression of genes: KRT8, PLAC8, CD9, PAGE2, HSPB1 and MSH6 were evaluated. Data of nuclear maturation and embryo development were analyzed by chi-square test and those of cumulus cells expansion and gene expression by analysis of variance. P <0.05 was considered statistically significant. There was no effect of FGF10 supplementation during IVM on any of the parameters evaluated in the first experiment. On the second experiment after maturation a lower cumulus expansion was observed in all treatments submitted to prematuration, irrespective of FGF10 presence, than the control. The supplementation of prematuration or maturation medium with FGF10 did not cause an increase in embryo production on D7, being the blastocyst rate similar among T2 (43.7%), T3 (38.7%), T4 (39.6%) e T5 (47.3%). However, blastocyst rate was similar between T2, T5 and control (53, 4%). All groups had similar hatching rates on D8. Transcripts level for PLAC8 was higher on T4 compared to T1, and similar for the other groups. For MSH6 gene, differences in transcripts level were only observed between T5 and T1. It can be concluded that presence of FGF10 during IVM and/or prematuration have not affected embryo production or quality. Prematuration, regardless presence of FGF10, affected cumulus expansion but not embryo production.

S100ß and fibroblast growth factor-2 are present in cultured Schwann cells and may exert paracrine actions on the peripheral nerve injury / S100ß e fator de crescimento de fibroblasto-2 estão presentes nas células de Schwann cultivadas e exercem ações parácrinas na lesão do nervo

PURPOSE: The neurotrophic factor fibroblast growth factor-2 (FGF-2, bFGF) and Ca++ binding protein S100ß are expressed by the Schwann cells of the peripheral nerves and by the satellite cells of the dorsal root ganglia (DRG). Recent studies have pointed out the importance of the molecules in the paracrine mechanisms related to neuronal maintenance and plasticity of lesioned motor and sensory peripheral neurons. Moreover, cultured Schwann cells have been employed experimentally in the treatment of central nervous system lesions, in special the spinal cord injury, a procedure that triggers an enhanced sensorymotor function. Those cells have been proposed to repair long gap nerve injury. METHODS: Here we used double labeling immunohistochemistry and Western blot to better characterize in vitro and in vivo the presence of the proteins in the Schwann cells and in the satellite cells of the DRG as well as their regulation in those cells after a crush of the rat sciatic nerve. RESULTS: FGF-2 and S100ß are present in the Schwann cells of the sciatic nerve and in the satellite cells of the DRG. S100ß positive satellite cells showed increased size of the axotomized DRG and possessed elevated amount of FGF-2 immunoreactivity. Reactive satellite cells with increased FGF-2 labeling formed a ring-like structure surrounding DRG neuronal cell bodies.Reactive S100ß positive Schwann cells of proximal stump of axotomized sciatic nerve also expressed higher amounts of FGF-2. CONCLUSION: Reactive peripheral glial cells synthesizing FGF-2 and S100ß may be important in wound repair and restorative events in the lesioned peripheral nerves.(AU)

OBJETIVO: O fator neurotrófico fator de crescimento de fibroblastos-2 (FGF-2, bFGF) e a proteína ligante de Ca++ S100ß são expressos pelas células de Schwann dos nervos e por células satélites do gânglio da raiz dorsal (GRD). Estudos recentes indicam a importância das moléculas nos mecanismos parácrinos relacionados à manutenção neuronal e à plasticidade de neurônios periféricos motores e sensoriais. Além disso, células de Schwann cultivadas têm sido empregadas experimentalmente no tratamento de lesões no sistema nervo central, especialmente na lesão da medula espinal, a qual mostrou uma melhora da função sensoriomotora. Estas células são ainda propostas no reparo do nervo lesado com perda de tecido. MÉTODOS: Usamos a dupla marcação imunohistoquímica e o Western blot para caracterizar melhor in vitro e in vivo a presença das proteínas nas células de Schwann e nas células satélites do GRD assim como sua regulação nessas células após a compressão do nervo ciático de ratos. RESULTADOS: FGF-2 e S100ß estão presentes nas células de Schwann do nervo ciático e nas células satélites do GRD. Células satélites do GRD axotomizado positivas para S100ß possuíam quantidade aumentada de imurreatividade da FGF-2. Células satélites reativas apresentando maior quantidade de FGF-2 formaram um anel ao redor dos corpos neuronais do GRD. Células de Schwann do coto proximal à axotomia do nervo ciático e positivas para S100ß também expressaram quantidades aumentadas de FGF-2. CONCLUSÃO: As células gliais periféricas ao sintetizar FGF-2 e S100ß podem ser importantes no reparo de cicatrização e em eventos restaurativos nas lesões do nervo.(AU)

Fator de crescimento fibroblástico básico e seus receptores em relação à atividade proliferativa na placenta bubalina em diferentes fases da gestação / Basic fibloblast growth factor and its receptors in relation to proliferative activity in the buffalo placenta during gestation

Estudou-se a distribuição espaço-temporal do fator de crescimento fibroblástico básico (bFGF), do receptor 1 do fator de crescimento fibroblástico (FGFR1) e do receptor 2 do fator de crescimento fibroblástico (FGFR2) na placenta bubalina, correlacionando-a à proliferação celular. Para a detecção do bFGF, FGFR1, FGFR2 e antígeno Ki-67, colheram-se 12 placentas de búfalas nos terços inicial, médio e final da gestação, em abatedouros, e realizaram-se testes de imunoistoquímica. Detectou-se e avaliou-se a expressão do bFGF, do FGFR1, do FGFR2 e do antígeno Ki-67 ao longo da gestação. No compartimento fetal da placenta, observaram-se correlações positivas entre a expressão do bFGF e Ki-67, entre FGFR1 e Ki-67 e entre FGFR2 com Ki-67 (r=0,313, 0,358 e 0,384, respectivamente). No epitélio e estroma maternos observaram-se altas correlações entre FGFR1 e Ki-67 (r=0,739 e r=0,511, respectivamente). Os resultados sugerem envolvimento do bFGF, FGFR1 e FGFR2 na proliferação do trofoblasto enquanto no compartimento materno da placenta bubalina apenas o FGFR1 atuaria como modulador dessa atividade.(AU)

The space-temporal expression of basic fibroblast growth factor (bFGF), fibroblast growth factor receptor 1 (FGFR1) and fibroblast growth factor receptor 2 (FGFR2) in buffalo placenta and correlation to proliferative activity was studied. For the localization of bFGF, FGFR1, FGFR2 and Ki-67, 12 buffalo placentas from initial, middle and final gestational thirds were collected and immunohistochemistry tests were performed. Expression of bFGF and its receptors was detected and analyzed from the initial third until the end of gestation. In the fetal compartment, positive correlations were observed between the expression of bFGF and Ki-67, FGFR1 and Ki-67, besides FGFR2 and Ki-67 (r=0.313, 0.358 and 0.384, respectively). High correlations were found between FGFR1 and Ki-67 in maternal epithelium and stroma (r=0.789 and r=0.511, respectively). The results suggest that bFGF, FGFR1 and FGFR2 may be involved in the modulation of trophoblast proliferation, whereas maternal compartment proliferation in the buffalo placenta would only be modulated by FGFR1.(AU)

Effects of the basic fibroblast growth factor and its anti-factor in the healing and collagen maturation of infected skin wound / Efeitos do fator de crescimento de fibroblastos básico e do seu anti-fator na cicatrização e maturação do colágeno de feridas infectadas de pele

PURPOSE: The infection is one of the main factors that affect the physiological evolution of the surgical wounds. The aim of this work is to evaluate the effects of fibroblast growth factor (FGFâ) and anti-FGFâ in the healing, synthesis and maturation of collagen when topically used on infected skin wounds of rats. METHODS: An experimental study was perfomed in 60 male Wistar rats. All animals were divided in two groups (A and B). Each group was divided in three subgroups A1, B1; A2, B2 and A3, B3. After anesthesia with pentobarbital, two open squared wounds (1cm²), 4cm distant to each other, were done in the dorsal skin of all the rats. In group A (n=30) the wounds were contaminated with multibacterial standard solution, and in group B(n=30) the wounds were maintained sterile. These wounds were named F1 (for inflammation analysis) and F2 (for collagen study). The open wounds of A1 and B1 rats were topically treated with saline solution, A2 and B2 were treated with FGFâ and subgroups A3 and B3 were treated with FGFâ and anti-FGFâ. The rats were observed until complete epitelization of F2 wounds for determination of healing time and the expression of types I and III collagen, using Picro Sirius Red staining. Inflammatory reaction in F1 wounds was studied using hematoxilineosin staining. The three variable was measured by the Image Pro-Plus Média Cybernetics software. The statistical analysis was performed by ANOVA and Tukey test, considering p<0.05 as significant. RESULTS: It was observed that infection retarded significantly (p<0.05) the time of wound scarring and the topical application of FCFb reverted the inhibition of healing caused by bacteria. The inflammatory reaction was greater in the subgroup B2 than in B1 and A3, and the difference was significant (p<0.05). It was observed greater expression of type I collagen in all the subgroups treated with FCFb, when compared with the untreated subgroups. Type III collagen was significantly...(AU)

OBJETIVO: Avaliar os efeitos do fator de crescimento de fibroblastos básico (FCFâ) e do anti-FCFâ na cicatrização e maturação do colágeno em feridas infectadas na pele de ratos. MÉTODOS: Um estudo experimental foi realizado em 60 ratos Wistar, divididos em dois grupos (A e B). Cada grupo foi divididos em 03 subgrupos A1,B1; A2,B2 e A3,B3. Após anestesia com pentobarbital sódico intraperitoneal, foram feitas duas feridas abertas de 1cm² na pele no dorso distando 4cm uma da outra. Essas feridas foram denominadas feridas F1 (para análise inflamatória) e F2 (para estudo do colágeno). No grupo A(n=30), as feridas foram contaminadas com solução multibateriana e no grupo B (n=30) as feridas não foram contaminadas. As feridas receberam tratamento tópico com aplicação única. Nos subgrupos A1 e B1 foram tratadas com solução salina tópica, as dos subgrupos A2 e B2 foram tratadas com o FCFâ e nos subgrupos A3 e B3 foram tratadas com FCFâ e com o anti-FCFâ. Os dados formam analisados pelos testes ANOVA de Tukey, considerando p<0,05 como significante. RESULTADOS: A infecção retardou de modo significante o tempo de cicatrização e a aplicação do FCFâ foi capaz de reverter a inibição da cicatrização provocada pela infecção(p<0.05). A resposta inflamatória foi maior nos grupos tratados com o FCFâ, e a aplicação do anti-FCFâ inibiu a reação inflamatória(p<0.05). Houve aumento significante dos colágenos tipo I e III em todos os subgrupos tratados com FCFâ, comparando com os não tratados, sendo a expressão do tipo I mais intensa do que do tipo III (p<0.05). A aplicação do anti-FCFâ inibiu a expressão das moléculas do colágeno. CONCLUSÕES: O FCFâ foi capaz de acelerar a cicatrização de feridas abertas infectadas e contribui para a maturação do colágeno, ao aumentar a expressão do colágeno tipo I, fenômeno que foi atenuado pela ação do anti-FCFâ.(AU)

O fator de crescimento de fibroblasto básico melhora a cicatrização de anastomoses duodenais em ratos

OBJECTIVE: Analysis of histopatological alterations, and pressure resistence in duodenal anastomosis treated with basic fibroblast growth factor (FGF-b). METHODS: Twenty Wistar rats were submited to a transversal duodenal section and subsequent anastomosis. They were randomly separated into four groups of five rats each: A1 and A2 (experimentals), in which FCFb was applied over the anastomosis; in B1 and B2 (controls), saline was use at the anastomosis site. The rodents were killed with an anesthetics overdosis, according to the following protocol: A1, B1 on 5th postoperative day and A2, B2 on the 7th one. A pressure resistence test of the anastomosis was done. The digitalized system Image ProPlus was used in order to analyse the mean density of the histopatological elements of healing duodenal tissues. RESULTS: All the rats survived without complications. In the group A1 the intraluminal pressure was 52± 14,4 mmHg and in group A2 it was 140± 34,8 mmHg. In the group B1 the pressure reached 33,6± 15,2 mmHg and in B2 it reached 105± 30,3. In group A1 the mean density of histopatological elements was 93± 9,3 and in A2 it was 181,8± 27,6. In the control groups B1 and B2 the mean densitys were 67,6± 16,7 and 101± 12,9 respectively. The statistical analysis detected a significant difference between the data of the experimental and control groups (p 0,05). CONCLUSION: the topical use of FGFb was able to enhance the resistence of duodenal anastomosis in rats observed and evaluated after five and seven postoperative days. The FCFb enhanced the neovascularization and the mean density of collagen, fibroblasts and inflammatory cells in the healing tissues.

OBJETIVOS: Avaliar as alterações histológicas, e o ganho de resistência em anastomoses duodenais tratadas com fator de crescimento de fibroblasto básico (FGFb). MÉTODOS: Vinte ratos da raça Wistar foram submetidos a secção transversal do duodeno, seguida de anastomose. Os animais foram divididos em 4 grupos de 5 animais cada: A1 e A2 (experimentais), nos quais foi aplicado FCFb sobre a anastomose logo após seu término; e B1 e B2 (controles), nos quais foi administrada solução salina sobre a zona de anastomose. Os roedores foram mortos com superdose de anestésico, sendo A1, B1 no 5º dia e A2, B2 no 7º dia de pós-operatório. Foi feita avaliação quanto à resistência das anastomoses à pressão e análise da densidade média dos achados histopatológicos com auxílio do sistema digitalizado Image proPlus. RESULTADOS: No grupo A1 a pressão suportada pelas anastomoses foi de 52± 14,4 mmHg e no grupo A2 140± 34,8 mmHg. Em B1 a pressão atingiu 33,6± 15,2 mmHg e as anastomoses do grupo B2 suportaram pressão 105± 30,3. No grupo A1 a densidade média dos elementos histopatológicos foi de 93± 9,3 e A2 atingiu 181,8± 27,6. Nos grupos de controle B1 e B2 as densidades médias foram 67,6± 16,7 e 101± 12,9 respectivamente. A análise estatística revelou diferença significante entre nos dados dos grupos experimentais e controles (p 0,05). CONCLUSÃO: a aplicação tópica do FCFb foi capaz de aumentar a resistência das feridas do duodeno suturadas e observadas após 5 e 7 dias de evolução. Estimulou a neovascularização, a formação de fibroblastos e de fibras colágenas, melhorando os escores histológicos em relação ao controle.

O fator de crescimento de fibroblasto básico melhora a cicatrização de anastomoses duodenais em ratos

OBJECTIVE: Analysis of histopatological alterations, and pressure resistence in duodenal anastomosis treated with basic fibroblast growth factor (FGF-b). METHODS: Twenty Wistar rats were submited to a transversal duodenal section and subsequent anastomosis. They were randomly separated into four groups of five rats each: A1 and A2 (experimentals), in which FCFb was applied over the anastomosis; in B1 and B2 (controls), saline was use at the anastomosis site. The rodents were killed with an anesthetics overdosis, according to the following protocol: A1, B1 on 5th postoperative day and A2, B2 on the 7th one. A pressure resistence test of the anastomosis was done. The digitalized system Image ProPlus was used in order to analyse the mean density of the histopatological elements of healing duodenal tissues. RESULTS: All the rats survived without complications. In the group A1 the intraluminal pressure was 52± 14,4 mmHg and in group A2 it was 140± 34,8 mmHg. In the group B1 the pressure reached 33,6± 15,2 mmHg and in B2 it reached 105± 30,3. In group A1 the mean density of histopatological elements was 93± 9,3 and in A2 it was 181,8± 27,6. In the control groups B1 and B2 the mean densitys were 67,6± 16,7 and 101± 12,9 respectively. The statistical analysis detected a significant difference between the data of the experimental and control groups (p 0,05). CONCLUSION: the topical use of FGFb was able to enhance the resistence of duodenal anastomosis in rats observed and evaluated after five and seven postoperative days. The FCFb enhanced the neovascularization and the mean density of collagen, fibroblasts and inflammatory cells in the healing tissues.

OBJETIVOS: Avaliar as alterações histológicas, e o ganho de resistência em anastomoses duodenais tratadas com fator de crescimento de fibroblasto básico (FGFb). MÉTODOS: Vinte ratos da raça Wistar foram submetidos a secção transversal do duodeno, seguida de anastomose. Os animais foram divididos em 4 grupos de 5 animais cada: A1 e A2 (experimentais), nos quais foi aplicado FCFb sobre a anastomose logo após seu término; e B1 e B2 (controles), nos quais foi administrada solução salina sobre a zona de anastomose. Os roedores foram mortos com superdose de anestésico, sendo A1, B1 no 5º dia e A2, B2 no 7º dia de pós-operatório. Foi feita avaliação quanto à resistência das anastomoses à pressão e análise da densidade média dos achados histopatológicos com auxílio do sistema digitalizado Image proPlus. RESULTADOS: No grupo A1 a pressão suportada pelas anastomoses foi de 52± 14,4 mmHg e no grupo A2 140± 34,8 mmHg. Em B1 a pressão atingiu 33,6± 15,2 mmHg e as anastomoses do grupo B2 suportaram pressão 105± 30,3. No grupo A1 a densidade média dos elementos histopatológicos foi de 93± 9,3 e A2 atingiu 181,8± 27,6. Nos grupos de controle B1 e B2 as densidades médias foram 67,6± 16,7 e 101± 12,9 respectivamente. A análise estatística revelou diferença significante entre nos dados dos grupos experimentais e controles (p 0,05). CONCLUSÃO: a aplicação tópica do FCFb foi capaz de aumentar a resistência das feridas do duodeno suturadas e observadas após 5 e 7 dias de evolução. Estimulou a neovascularização, a formação de fibroblastos e de fibras colágenas, melhorando os escores histológicos em relação ao controle.

Ação do fator de crescimento de fibroblasto básico na cicatrização da aponeurose abdominal de ratos

OBJECTIVE: As little is known about the use of basic Fibroblast Growth Factor (FCFb) in the healing of abdominal aponeurosis, an experimental work was done to study it. METHODS: Twenty Wistar rats randomly separated in two groups were used. They were anesthetised with pentobarbital 20 mg/Kg intraperitonealy, submited to a 4cm median laparotomy and the aponeurosis was sutured with nylon 5-0. In group I 5mg of FCFb was applied over the sutured aponeurosis end the group II received saline 0,9% over it. After seven days the rats were killed by an overdose of anesthetic. A transversal 1,5 cm wideness sample of aponeurosis was submited to tensil strenght test, using the assay device EMIC MF500. Biopsies from the sutured tissue were processed and colored by HE and Masson trichromic. The histopatologic data were quantitated and transformed into mean density by the digital equipment Image Pro-plus. The Student t test was used and the differences were significant when p 0,05. RESULTS: In group I the the mean tensil strength was 1.103± 103,39gf and the mean density of histopatologic data was 226± 29,32. In group II (control) the tensil strenght was 791,1± 92, 77gf and the histolopatogical density was 114,1± 17,01. The differences were statisticaly significant (p 0,01). CONCLUSION: In conclusion, our data show that FCFb induces an increase of tensil strenght of sutured aponeurosis of rats and turns better the histopatological parameters of wound healing.

OBJETIVO: Trabalho realizado em ratos com o objetivo de estudar o efeito do Fator de Crescimento de Fibroblastos básico (FCFb) na cicatrização da aponeurose abdominal. MÉTODOS: Foram usados 20 ratos Wistar separados aleatoriamente em 2 grupos iguais. Os animais foram anestesiados com pentobarbital sódico na dose de 20 mg/Kg por via intraperitoneal e submetidos a laparotomia mediana de 4 cm, cuja camada aponeurótica foi suturada com mononylon 5-0. No grupo I foi aplicada a dose de 5mg de FCFb sobre a sutura da aponeurose. No grupo II (controle) foi aplicada solução salina 0,9% sobre a linha se sutura. Após observação por 7 dias os animais foram mortos com superdose de anestésico. A camada aponeurótica com 1,5 cm de largura foi submetida a teste de resistência à tensão empregando a Máquina de Ensaios EMIC MF500. Biópsias das zonas de sutura foram processadas e coradas com HE e o tricômico de Masson. Os achados histopatológicos foram quantificados através de sistema digital (Image pro-plus) de captura e processamento de imagens. Os dados obtidos foram analisados pelo teste T com significância 0,05. RESULTADOS: Nos animais do grupo I (experimental) a zona de sutura da camada aponeurótica suportou a carga de 1.103± 103,39gf. A quantificação dos dados histopatológicos desse grupo atingiu a densidade média 226± 29,32. No grupo II (controle) a carga suportada pela zona de sutura foi de 791,1± 92,77 gf. Quando foram comparadas as médias das resistências à tensão dos dois grupos, observou-se uma diferença significante (p 0,01). O exame histopatológico das lâminas desse grupo relevou densidade média 114,1± 17,01, correspondendo a uma diferença significante quando comparadas as médias dos dois grupos (p 0,01). CONCLUSÃO: Os dados permitem concluir que o FCFb contribuiu para aumentar a resistência da aponeurose suturada e para melhorar os parâmetros histopatológicos da cicatrização.

Ação do fator de crescimento de fibroblasto básico na cicatrização da aponeurose abdominal de ratos

OBJECTIVE: As little is known about the use of basic Fibroblast Growth Factor (FCFb) in the healing of abdominal aponeurosis, an experimental work was done to study it. METHODS: Twenty Wistar rats randomly separated in two groups were used. They were anesthetised with pentobarbital 20 mg/Kg intraperitonealy, submited to a 4cm median laparotomy and the aponeurosis was sutured with nylon 5-0. In group I 5mg of FCFb was applied over the sutured aponeurosis end the group II received saline 0,9% over it. After seven days the rats were killed by an overdose of anesthetic. A transversal 1,5 cm wideness sample of aponeurosis was submited to tensil strenght test, using the assay device EMIC MF500. Biopsies from the sutured tissue were processed and colored by HE and Masson trichromic. The histopatologic data were quantitated and transformed into mean density by the digital equipment Image Pro-plus. The Student t test was used and the differences were significant when p 0,05. RESULTS: In group I the the mean tensil strength was 1.103± 103,39gf and the mean density of histopatologic data was 226± 29,32. In group II (control) the tensil strenght was 791,1± 92, 77gf and the histolopatogical density was 114,1± 17,01. The differences were statisticaly significant (p 0,01). CONCLUSION: In conclusion, our data show that FCFb induces an increase of tensil strenght of sutured aponeurosis of rats and turns better the histopatological parameters of wound healing.

OBJETIVO: Trabalho realizado em ratos com o objetivo de estudar o efeito do Fator de Crescimento de Fibroblastos básico (FCFb) na cicatrização da aponeurose abdominal. MÉTODOS: Foram usados 20 ratos Wistar separados aleatoriamente em 2 grupos iguais. Os animais foram anestesiados com pentobarbital sódico na dose de 20 mg/Kg por via intraperitoneal e submetidos a laparotomia mediana de 4 cm, cuja camada aponeurótica foi suturada com mononylon 5-0. No grupo I foi aplicada a dose de 5mg de FCFb sobre a sutura da aponeurose. No grupo II (controle) foi aplicada solução salina 0,9% sobre a linha se sutura. Após observação por 7 dias os animais foram mortos com superdose de anestésico. A camada aponeurótica com 1,5 cm de largura foi submetida a teste de resistência à tensão empregando a Máquina de Ensaios EMIC MF500. Biópsias das zonas de sutura foram processadas e coradas com HE e o tricômico de Masson. Os achados histopatológicos foram quantificados através de sistema digital (Image pro-plus) de captura e processamento de imagens. Os dados obtidos foram analisados pelo teste T com significância 0,05. RESULTADOS: Nos animais do grupo I (experimental) a zona de sutura da camada aponeurótica suportou a carga de 1.103± 103,39gf. A quantificação dos dados histopatológicos desse grupo atingiu a densidade média 226± 29,32. No grupo II (controle) a carga suportada pela zona de sutura foi de 791,1± 92,77 gf. Quando foram comparadas as médias das resistências à tensão dos dois grupos, observou-se uma diferença significante (p 0,01). O exame histopatológico das lâminas desse grupo relevou densidade média 114,1± 17,01, correspondendo a uma diferença significante quando comparadas as médias dos dois grupos (p 0,01). CONCLUSÃO: Os dados permitem concluir que o FCFb contribuiu para aumentar a resistência da aponeurose suturada e para melhorar os parâmetros histopatológicos da cicatrização.

Membrana amniótica preservada em glicerina no reparo de feridas cutâneas de membros locomotores de eqüinos

In the present experimental study, the use of the equine amniotic membrane preserved in glvcerol 98%, at room temperature, in wounds with secound intention healing of equine limbs were evaluated. Surgical wounds of the size of 9.6cm² were made on the fetiock joint, medial aspect of the proximal third of the metacarpus and lateral aspect of the medium third of the metatarsus ofboth limbs offive adult horses, making a total of thirty wounds. Two experimental groups were made with fifteen wounds in each group, where one of these groups was treated with amniotic membrane, and the other, the control group, treated only with a damp gauze changed every 48 hours. In all the wounds the procedure was to evaluate the measurement of the área, degree of secretion, development of exuberant granulation tissue and the complete time of epitheliazation. Bacteriologic and histopatologic exams were made in a sample of the treated and contrai groups. The biochemical determination of proteins and the identification of fibroblastic growth factor were realized in the amnionic membrane. In all the treated wounds were observed shorter healing time, less formation of exuberant granulation tissue, smaller incidence of pathogenic bacterias and a smaller increase of the wound area.

Neste estudo experimental, avaliou-se o uso de membrana amniótica eqüina preservada em glicerina 98%, à temperatura ambiente, em feridas com cura por segunda intenção, nos membros locomotores de eqüinos. Foram provocadas cirurgicamente feridas de 9,6cm² na face lateral da articulação metacarpo-falangeana, face medial do terço proximal do metacarpo e face lateral do terço médio do metatarso de ambos os membros locomotores de cinco eqüinos adultos, perfazendo total de trinta feridas. Foram constituídos dois grupos experimentais com quinze feridas cada, sendo um destes grupos tratado com membrana amniótica, e o outro, grupo controle, tendo recebido apenas gaze umidecida em solução fisiológica. Os curativos foram trocados a cada 48horas, tendo sido a membrana amniótica e a gaze umidecida substituídas. Em todas as feridas foram mensurados a área, o grau de secreção, a formação de tecido de granulação exuberante e o tempo para a epitelização se completar. Realizou-se também exame bacteriológico e histopatológico em um grupo de feridas, tratadas e controle. Na membrana amniótica, fez-se determinação bioquímica de proteínas e identificação de fator de crescimento de fibroblasto. Em todas as feridas tratadas, observou-se: menor tempo para cicatrização, menor formação de tecido de granulação exuberante, menor incidência de bactérias patológicas e menor aumento na área total.

Membrana amniótica preservada em glicerina no reparo de feridas cutâneas de membros locomotores de eqüinos

In the present experimental study, the use of the equine amniotic membrane preserved in glvcerol 98%, at room temperature, in wounds with secound intention healing of equine limbs were evaluated. Surgical wounds of the size of 9.6cm² were made on the fetiock joint, medial aspect of the proximal third of the metacarpus and lateral aspect of the medium third of the metatarsus ofboth limbs offive adult horses, making a total of thirty wounds. Two experimental groups were made with fifteen wounds in each group, where one of these groups was treated with amniotic membrane, and the other, the control group, treated only with a damp gauze changed every 48 hours. In all the wounds the procedure was to evaluate the measurement of the área, degree of secretion, development of exuberant granulation tissue and the complete time of epitheliazation. Bacteriologic and histopatologic exams were made in a sample of the treated and contrai groups. The biochemical determination of proteins and the identification of fibroblastic growth factor were realized in the amnionic membrane. In all the treated wounds were observed shorter healing time, less formation of exuberant granulation tissue, smaller incidence of pathogenic bacterias and a smaller increase of the wound area.

Neste estudo experimental, avaliou-se o uso de membrana amniótica eqüina preservada em glicerina 98%, à temperatura ambiente, em feridas com cura por segunda intenção, nos membros locomotores de eqüinos. Foram provocadas cirurgicamente feridas de 9,6cm² na face lateral da articulação metacarpo-falangeana, face medial do terço proximal do metacarpo e face lateral do terço médio do metatarso de ambos os membros locomotores de cinco eqüinos adultos, perfazendo total de trinta feridas. Foram constituídos dois grupos experimentais com quinze feridas cada, sendo um destes grupos tratado com membrana amniótica, e o outro, grupo controle, tendo recebido apenas gaze umidecida em solução fisiológica. Os curativos foram trocados a cada 48horas, tendo sido a membrana amniótica e a gaze umidecida substituídas. Em todas as feridas foram mensurados a área, o grau de secreção, a formação de tecido de granulação exuberante e o tempo para a epitelização se completar. Realizou-se também exame bacteriológico e histopatológico em um grupo de feridas, tratadas e controle. Na membrana amniótica, fez-se determinação bioquímica de proteínas e identificação de fator de crescimento de fibroblasto. Em todas as feridas tratadas, observou-se: menor tempo para cicatrização, menor formação de tecido de granulação exuberante, menor incidência de bactérias patológicas e menor aumento na área total.