Is Atrophic Nonunion a Misnomer - A Hospital-based Prospective Cross-Sectional Study / Pseudoartrose atrófica é um termo incorreto? - Um estudo hospitalar transversal prospectivo

Abstract Objective The present study was conducted to estimate histologically the proportion of avascularity of fracture ends in case of nonunion of long bones. Methods A total of 15 cases of established quiescent nonunion were operated according to the standard protocol and the fracture ends were evaluated histologically. The biopsied tissue was briefly fixed with formalin, embedded with paraffin (FFPE), and 5-micron sections were stained with hematoxylin and eosin according to standard protocols. Immunohistochemistry with anti-CD31 antibody (JC70A clone, DBS) was performed manually using standard protocols. Results All cases of quiescent nonunion were included; radiologically, 2 cases were oligotrophic, and 13 cases were of atrophic nonunion. A total of 20% of the patients were females, 40% were in the age group between 31and 40 years old, and, radiologically, all cases were of atrophic nonunion. All cases showed positivity for CD-31 on immunohistochemistry. The blood vessel density was category I in 13.33% of the cases and category II in 86.67% of the cases. Four cases presented with mild inflammation and two presented with moderate inflammation. The average vessel count was 10 per high power field in the age groups between 20 and 30, 31 and 40, and 41and 50 years old. The age group between 61 and 70 years old showed an average vessel count of 4 per high power field. The difference in the vessel counts of oligotrophic and atrophic nonunion was not significant. No correlation was observed in the density of vessel count and duration of nonunion Conclusion The nomenclature for the classification of nonunion into atrophic, oligotrophic, and hypertrophic needs revision. Our findings do not support that atrophic and oligotrophic nonunion are histologically different.

Resumo Objetivo O presente estudo estimou a proporção de avascularidade histológica das extremidades das fraturas em caso de pseudoartrose de ossos longos. Métodos No total, 15 casos de pseudoartrose quiescente estabelecida foram operados de acordo com o protocolo padrão e as extremidades da fratura foram avaliadas histologicamente. Em resumo, o tecido biopsiado foi fixado em formalina e embebido em parafina (FFPE); secções de 5 mícrons foram coradas com hematoxilina e eosina de acordo com os protocolos padrões. A imunohistoquímica com anticorpo anti-CD31 (clone JC70A, DBS) foi realizada manualmente segundo protocolos padrões. Resultados Todos os casos de pseudoartrose quiescente foram incluídos; 2 eram de pseudoartrose oligotrófica e 13 eram de pseudoartrose atrófica à radiologia. Destes, 20% eram de pacientes do sexo feminino, 40% de indivíduos entre 31 e 40 anos de idade e todos os casos eram de pseudoartrose atrófica à radiologia. Todos os casos eram positivos para CD-31 à imunohistoquímica. A densidade dos vasos sanguíneos era de categoria I em 13,33% dos casos e de categoria II em 86,67%. Quatro casos apresentavam inflamação branda e dois apresentavam inflamação moderada. O número médio de vasos era de 10 por campo de alta potência na faixa etária de 20 a 30, de 31 a 40 e de 41 a 50 anos. A faixa etária de 61 a 70 anos apresentava, em média, 4 vasos por campo de alta potência. A diferença nos números de vasos em pseudoarthroses oligotróficas e atróficas não foi significativa. Não houve correlação entre a densidade de vasos e a duração da pseudoartrose. Conclusão A nomenclatura de classificação da pseudoartrose em atrófica, oligotrófica e hipertrófica precisa ser revista. Nossos achados não indicam que a pseudoartrose atrófica e oligotrófica sejam histologicamente diferentes.

Participação dos neutrófilos durante a implantação embrionária no desenvolvimento da placenta em camundongos / Participation of neutrophils during embryonic implantation in the placental development in mice

A implantação do embrião na parede uterina é um processo complexo que consiste na interação do blastocisto com as células epiteliais do útero, e depende de diferentes tipos celulares do microambiente uterino. Embora a literatura mostre a participação de neutrófilos neste processo, os dados ainda são incipientes para proposição da função exata destas células nos períodos iniciais da gestação. Dados do nosso grupo de pesquisa mostraram que neutrófilos pró-angiogênicos induzem a tolerância gestacional, e que a depleção de neutrófilos durante as fases iniciais da gestação prejudica a implantação do blastocisto e a progressão da gestação. Com base nestes resultados, o presente estudo visou investigar se a depleção de neutrófilos na fase pré-receptiva da janela de implantação do blastocisto altera a morfologia placentária. Para tanto, foi utilizado o modelo de gestação alogênica, onde camundongos fêmeas C57BL/6, após cruzamento com machos Balb/C foram tratadas com anticorpo anti-Ly6G ou isotipo no dia 1,5 da gestação (24 horas após a detecção do plug vaginal) em dose suficiente para manter a depleção de neutrófilos circulantes por 48 horas (200µg/ 500µL; i.p). No final da gestação (dia 18,5), o sangue periférico foi coletado e, em seguida, os animais foram submetidos a laparotomia para retirada da placenta, a qual foi submetida à análise histológica. As análises dos leucócitos circulantes evidenciaram a efetividade do tratamento para depleção de neutrófilos periféricos. A análise histológica mostrou alterações significativas na morfologia da placenta nos animais tratados com anti-Ly6G. Foram detectadas a redução da zona juncional, de células trofoblásticas e de fatores angiogênicos, como fator de crescimento do endotélio vascular (VEGF), e das moléculas de adesão intracelular-1 (ICAM-1) e de plaqueta e endotélio (PECAM-1). Esses dados evidenciam a importância dos neutrófilos nos primeiros dias de gestação para o desenvolvimento da placenta

Blastocyst implantation is a complex process, consisting of the interaction between blastocyst and uterine epithelial cells. Also, it is well known that the implantation site resembles an inflammatory response, with a profusion of recruited immune cells into the endometrial stroma and lumen from the blood. The role of macrophages, natural killers, and dendritic cells have been extensively studied, however, the participation of neutrophils in this process remains unclear. Data from our research group showed that pro-angiogenic neutrophils induced gestation tolerance, also peripheral neutrophils depletion at the time of active placental development led to smaller embryo sizes and abnormal placentation in mice. In this context, the present study aimed to investigate whether pharmacological depletion of neutrophils in mice in the blastocyst implantation phase alters placental morphology. Therefore, C7/BL/6 female mice, after mating with Balb/C males, were treated with an anti-Ly6G antibody or isotype on day 1 of gestation (after detection of the vaginal plug) at a dose sufficient to maintain the depletion of circulating neutrophils for 48 hours (200 µg/500µL; i.p). At the end of the gestational day (day 18), peripheral blood was collected, and then the animals were submitted to laparotomy for the placenta removal and subsequent histological analysis. The analysis of circulating leukocytes from neutrophils depleted mice showed a reduction of peripheral neutrophils up to 48 hours after antibody injection. The histological analysis showed significant alterations in the placenta morphology of the animals treated with anti-Ly6G. The morphometric analyses showed a reduction in the size of neutrophils depleted placenta due to diminished junctional zone and reduction of trophoblast cells. Also, it was observed a reduction of vascular endothelial growth factors (VEGF), reduction of adhesion molecules intracell-1 (ICAM-1), and platelets and endothelium (PECAM-1) positive cells in the junctional zone. In conclusion, these data show the importance of neutrophils on the first days of pregnancy for the development of the placenta

Docosahexaenoic fatty acid nanoencapsulated with Anti-PECAM-1 as strategy to increase atherosclerotic plaque stability / Ácido graxo docosahexaenoico nanoencapsulado com anti-PECAM-1 como uma estratégia para aumentar a estabilidade de placas ateroscleróticas

Cardiovascular diseases (CVDs) are the main cause of mortality worldwide, being the ischemic heart disease responsible for 85% of deaths. Atherosclerosis is a chronic inflammation of the arteries that underlies ischemic forms of CVD and involves the innate and adaptive immune systems, from initial fatty streak formation to atherosclerotic plaque ruptures, which defines the beginning and end stages of disease, respectively. Recent research on the reduction of systemic inflammation in order to treat CVD is controversial, since results show that this reduced inflammation can also increase patient susceptibility to general infection. Therefore, new tissue-targeting strategies are necessary. Docosahexaenoic fatty acid (DHA) is a natural bioactive precursor of pro-resolving oxylipins that can reduce inflammation. Based on these factors, the objective of this study was to develop a nanocapsule containing algae oil as a DHA source and apply anti-PECAM-1 on its surface to drive it to the inflamed endothelium. Initially, a surface-functionalized metal-complex multi-wall nanocapsule containing algae oil in its nucleus (MLNC-DHA-a1) was developed. This nanocapsules presented a mean diameter of 163 ± 5 nm, was spherical in shape, showed 94.80% conjugation efficiency using 200 µg/mL of anti-PECAM-1 on the surface, and did not show significant toxicity toward HUVECs at concentrations from 0.14 to 2.90x1011 nanocapsules/mL. The nanocapsules were also stable for 2 h, sufficient time to allow for clinical applications. In cell viability assays, concentrations of 0.14 to 1.40x1011 nanocapsules/mL did not significantly affect the viability of immortalized murine macrophages (RAW 264.7) and U-937 cells after 24, 48, and 72 h of treatment. Finally, macrophages were incubated with 0.75x1011 MLNC-DHA-a1 nanocapsules/mL for 4 h and showed a significant uptake, observed using dark-field hyperspectral microscopy (CytoViva®). Once inside murine macrophages (RAW 264.7), MLNC-DHA-a1 nanocapsules promoted a strong increase in M2 phenotype polarization compared to non-treated control cells. Our results suggest that DHA-enriched algae oil, as part of a lipid core nanocapsules, does not reduce cell viability and improves macrophage phenotype, making it a promising potential therapy for controlling chronic inflammation and healing or stabilizing atherosclerotic plaques

As doenças cardiovasculares (DCVs) são a principal causa de mortalidade no mundo, sendo os eventos isquêmicos responsáveis por 85% das mortes. A aterosclerose é uma inflamação crônica das artérias associada aos eventos isquêmicos das DCVs, na qual o sistema imunológico inato e adaptativo estão envolvidos desde a formação inicial das estrias gordurosas até a ruptura das placas ateroscleróticas. Pesquisas recentes direcionadas à redução da inflamação sistêmica têm mostrado resultados controversos, pois essa abordagem pode aumentar a susceptibilidade do paciente a infecções. Nesse sentido, novas estratégias direcionadas ao tecido lesionado são necessárias. No que se refere a medicamentos anti-inflamatórios ou suplementos alimentares, o ácido docosaexaenóico (DHA) tem sido relatado como um precursor natural de oxilipinas pró- resolutivas. Baseado nesse contexto, o objetivo deste estudo foi desenvolver nanocápsulas contendo óleo de alga como fonte de DHA e vetorizar essas nanopartículas com o anticorpo antiPECAM-1 em sua superfície, visando direcioná-las ao endotélio inflamado. Inicialmente, a nanocápsula multiparede metal-complexa funcionalizada contendo óleo de alga em seu núcleo (MLNC-DHA-a1) foi desenvolvida, apresentando um diâmetro médio de 163 ± 5 nm, formato esférico, onde a eficiência de conjugação do anti-PECAM-1 (200 µg/mL) foi de 94,80% sem toxicidade significativa em HUVECs nas concentrações de 1.14 a 2.9 x 1011 nanocápsulas/mL. As nanocápsulas apresentaram uma estabilidade de 2h, o que representa tempo suficiente para a sua aplicação clínica. A seguir, ensaios de viabilidade celular foram realizados em outras linhagens de células para avaliar a toxicidade das nanocápsulas. As concentrações de 0.14 a 1.40 x 1011 de nanocápsulas/mL não afetaram significativamente a viabilidade celular de macrófagos murinos imortalizados (RAW 264.7) e U-937 após 24, 48 e 72 h de tratamento. Por fim, os macrófagos (RAW 264.7) foram incubados com 0.75 x 1011 MLNC-DHA-a1/mL durante 4 h e apresentam uma captação significativa das nanocápsulas, observada por microscopia hiperespectral de campo escuro (CytoViva®). Uma vez captadas pelos macrófagos murinos imortalizados (RAW 264.7), as nanoformulações MLNC-DHA-a1 promoveram um forte aumento da polarização do fenótipo M2 em comparação com as células controle não tratadas. Nossos resultados sugerem que o óleo de alga rico em DHA presente no núcleo lipídico das nanocápsulas, não reduziu a viabilidade celular e estimulou uma maior polarização de macrófagos para o tipo M2, sendo assim uma terapia potencial para controlar a inflamação crônica e cicatrizar ou estabilizar placas ateroscleróticas

The Effect of Garlic and Voluntary Exercise on Cardiac Angiogenesis in Diabetes: The Role of MiR-126 and MiR-210 / O Efeito do Alho e de Exercícios Físicos Voluntários na Angiogênese Cardíaca no Diabetes: O Papel do miRNA-126 e do miRNA-210

Abstract Background: Diabetes mellitus (DM) is one of the major risk factors for cardiovascular disease, leading to endothelial dysfunction and angiogenesis impairment . MiR-126 and miR-210 support angiogenic response in endothelial cells. Objective: The present study sought to explore the effect of garlic and voluntary exercise, alone or together, on miR-126 and miR-210 expressions and cardiac angiogenesis in rats with type 1 diabetes. Methods: Male Wistar rats were divided into five groups (n = 7): Control, Diabetes, Diabetes+Garlic, Diabetes+Exercise, and Diabetes+Garlic+Exercise. Diabetes was induced in the animals by streptozotocin (ip, 50 mg/kg). The rats were then fed raw fresh garlic homogenate (250 mg/kg) or were subjected to voluntary exercise, or to combined garlic and voluntary exercise for 6 weeks. MiR-126 and miR-210 expressions in the myocardium were determined by real time PCR, and the serum lipid profile was measured by enzymatic kits. Angiogenesis was evaluated by immunostaining for PECAM-1/ CD31 in the myocardium. Results: Diabetes reduced both cardiac miR-126 expression and angiogenesis (p < 0.05). On the other hand, there was a miR-210 expression increase in the myocardium of diabetic animals (p < 0.001). However, those effects reversed either with garlic or voluntary exercise (p < 0.01). Moreover, treating diabetic rats with garlic and voluntary exercise combined had an additional effect on the expressions of miR-126 and miR-210 (p < 0.001). Furthermore, both voluntary exercise and garlic significantly improved serum lipid profiles (p < 0.001). Conclusion: The induction of diabetes decreased angiogenesis in the myocardium, whereas our treatment using long-term voluntary exercise and garlic improved myocardial angiogenesis. These changes were possibly owing to the enhancement of myocardial miR-126 and miR-210 expressions.

Resumo Fundamento: O diabetes mellitus (DM) é um dos principais fatores de risco para doenças cardiovasculares, levando à disfunção endotelial e inibição da angiogênese. O miRNA-126 e o miRNA-210 promovem a resposta angiogênica em células endoteliais. Objetivo: O presente estudo buscou explorar o efeito do alho e de exercícios físicos voluntários, isoladamente ou em conjunto, nas expressões do miRNA-126 e do miR-210 e na angiogênese cardíaca em ratos com diabetes tipo 1. Métodos: Ratos Wistar machos foram divididos em cinco grupos (n = 7): Controle, Diabetes, Diabetes+Alho, Diabetes+Exercícios e Diabetes+Alho+Exercícios. Introduziu-se diabetes nos animais por estreptozotocina (ip, 50 mg/kg). Os ratos foram então alimentados com homogenato de alho fresco cru (250 mg/kg), ou foram submetidos a exercícios voluntários, ou a uma combinação de alho e exercícios voluntários, durante 6 semanas. As expressões do miRNA-126 e do miRNA-210 no miocárdio foram determinadas por PCR em tempo real, e o perfil lipídico sérico foi medido por kits enzimáticos. A angiogênese foi avaliada por imunocoloração por PECAM-1/CD31 no miocárdio Resultados: O diabetes reduziu a expressão do miRNA-126 cardíaco e da angiogênese (p < 0,05). Por outro lado, houve um aumento da expressão do miRNA-210 no miocárdio dos animais diabéticos (p < 0,001). No entanto, tais efeitos foram revertidos com alho ou exercícios voluntários (p < 0,01). Além disso, o tratamento de ratos diabéticos conjuntamente com alho e exercícios voluntários teve um efeito adicional sobre as expressões do miRNA-126 e do miRNA-210 (p < 0,001). Além disso, tanto os exercícios voluntários quanto o alho melhoraram significativamente os perfis lipídicos séricos (p < 0,001). Conclusões: A indução de diabetes diminuiu a angiogênese no miocárdio, enquanto nosso tratamento com exercícios voluntários de longa duração e alho melhorou a angiogênese miocárdica. Estas alterações devem-se, possivelmente, ao aumento das expressões do miRNA-126 e do miRNA no miocárdio.

Garlicin Post-Conditioning Suppresses Adhesion Molecules in a Porcine Model of Myocardial Ischemia-Reperfusion Injury / 中国结合医学杂志

OBJECTIVES@#To evaluate whether garlicin post-conditioning can attenuate myocardial ischemiareperfusion injury in a catheter-based porcine model of acute myocardial infarction (AMI) by affecting adhesion molecules integrin β1/CD29 and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31).@*METHODS@#Twenty-two swine were devided into 3 groups: 6 in a sham-operation group, and 8 each in the model and garlicin groups. AMI porcine model was established in the model and garlicin groups. The distal parts of the left anterior descending coronary artery in the animals of the model and garlicin groups were occluded by dilated balloon for 2 h, followed by reperfusion for 3 h. Garlicin (1.88 mg/kg) was injected over a period of 1 h, beginning just before reperfusion, in the garlicin group. Real-time polymerase chain reaction, immunohistochemistry and Western blot were carried out to detect mRNA and protein expressions of CD29 and CD31 3 h after reperfusion.@*RESULTS@#Hematoxylin-eosin staining showed a better myocardial structure in the garlicin group after reperfusion. Compared to the model group, garlicin inhibited both the mRNA and protein expression of CD29 and CD31 in reperfusion area and no-reflflow area (P<0.05 respectively).@*CONCLUSIONS@#Garlicin post-conditioning induced cardio-protection against myocardial ischemia-reperfusion injury in this catheter-based porcine model of AMI. The cardio-protective effect of garlicin is possibly owing to suppression of production of CD29 and CD31, by inhibition of the mRNA expression of CD29 and CD31.

Comparison of Improving Effects for Diabetic Erectile Dysfunction according to the Anti-Glycemic Agents: Phlorizin and Insulin

PURPOSE: To compare the improving effects of diabetic erectile dysfunction with two anti-glycemic agents; phlorizin and insulin. MATERIALS AND METHODS: Sixty Sprague-Dawley rats were divided into four groups (n=15 in each group): normal control (C), untreated diabetic rats (D), and diabetic rats treated by phlorizin (P) or insulin (I). Ten weeks after the diabetic induction using an injection of streptozotocin (55 mg/kg), four weeks of diabetic control was conducted. Erectile response, Western blot, and immunohistochemistry were assessed. RESULTS: During the experiment, the C-group showed continuous weight gain, while the other groups suffered from weight loss. After start of diabetic control, the body weight of I-group was increased; whereas, there was no meaningful change in the P-group. Meanwhile, comparable blood glucose levels were achieved in the P- and I-groups. The erectile response was markedly decreased in the D-group, whereas the P- and I-groups were similar as good as the C-group. In addition, D-group showed the significant decrease in the cavernosal smooth muscle content and increased apoptosis. Platelet endothelial cell adhesion molecule-1 protein expression, phosphorylation of endothelial nitric oxide synthase and myosin phosphatase target subunit 1 were significantly distorted in the D-group, while the P- and I-groups were comparable with the C-group. CONCLUSIONS: Phlorizin treatment resulted in the improvement of erectile function as same as insulin despite the lack of anabolic weight gains. These results suggest that control of blood glucose level rather than a type of anti-glycemic agents is more important for the prevention and treatment of diabetic erectile dysfunction

Utilidad de tinción de tricrómico de Masson en la cuantificación de densidad media vascular en mucosa oral normal, displasia epitelial y carcinoma oral de células escamosas / Utility of Masson's trichrome stain in the quantification of mean vascular density in normal oral mucosa, epithelial dysplasia and oral squamous cell carcinoma

RESUMEN: El objetivo de este estudio fue valuar la utilidad del uso de la tinción de Tricrómico de Masson (TM) en la cuantificación de la densidad media vascular (DMV) en Mucosa Oral Normal (MON), Displasia Epitelial Oral (DEO) y Carcinoma Oral de Células Escamosas (COCE). Estudio descriptivo de serie de casos. Se analizaron 17 muestras de MON, 15 muestras de DEO y 16 de COCE, teñidas con TM. Para determinar su utilidad, se compararon con las mismas muestras analizadas con técnica de inmunohistoquímica contra CD31. La cuantificación de la DMV se realizó en las 3 áreas de mayor vascularización de cada muestra. Se determinó la DMV según diagnóstico mediante la tinción TM e inmunohistoquímica contra CD31, y se calculó la correlación entre ambos. La DMV cuantificada con TM y contra CD31 difiere según el diagnóstico, observándose un aumento de la DMV al malignizarse el diagnóstico. No se encontraron diferencias al comparar la DMV cuantificada con TM y contra CD31. La correlación de la DMV analizado por TM y contra CD31 es significativa y moderada. La cuantificación de vasos sanguíneos es posible mediante la tinción de TM en muestras de MON, DEO y COCE, con una correlación moderada con la inmunohistoquímica contra CD31.

SUMMARY. The objective of this study was to evaluate the utility of Masson's Trichrome (TM) staining in the quantification of the mean vascular density (DMV) in samples of normal oral mucosa (MON), oral epithelial dysplasia (ODE) and oral squamous cell carcinoma (COCE). The design - a descriptive study of case series. We analyzed 17 samples of MON, 15 samples of DEO and 16 samples of COCE, stained with TM. To determine usefulness, we compared and analyzed the same samples, either stained with TM or with immunohistochemical technique against CD31. Quantification of the DMV was performed in the 3 areas of greatest vascularization in each sample. DMV was determined according to diagnosis by TM staining and immunohistochemistry against CD31, and the correlation between the two was then calculated. DMV quantified with TM and against CD31 differs according to the diagnosis, with an increase in DMV upon malignant diagnosis. No differences were found when comparing DMV quantified with TM and against CD31. The correlation of the DMV analyzed by TM and against CD31 is significant and moderate. Quantification of blood vessels is possible by TM staining in samples of MON, DEO and COCE. TM staining is moderately correlated with immunohistochemistry against CD31.

Histochemical and immunohistochemical evaluation of angiogenesis in rabbits (Oryctolagus cuniculus) submitted to skin grafts associated with platelet-rich plasma / Histoquímica e imunohistoquíca na avaliação da angiogênese em coelhos submetidos ao emprego de enxertos cutâneos associado com plasma rico em plaquetas

Histochemical staining consists of a set of specific chemical reactions of structures or tissue-endogenous substances. Immunohistochemistry allows verification of proteins in tissues related to biological and pathological factors. The standardization of methods to assess angiogenesis resulting from formation of new blood vessels in procedures with stimulants is important to facilitate the implementation of research as well as to assist interpretation of data. In rabbits some markers of angiogenesis antibodies in the skin are not standardized because of cross-reactions that may occur because the antibodies are made from such animals.The aim of this study was to analyze the immunohistochemical methods through dyes and immunohistochemical markers angiogenesis in rabbits (Oryctolagus cuniculus) having undergone reconstructive surgery with skin grafts associated with plasma angiogenesis stimulator rich in platelets, in order to evaluate which method would be better to visualize the vessels, as well as to evaluate which antibody would promote better immunostaining, and find the differences between the methods and to standardize the methodology to be applied in experiments using rabbits. Sixteen rabbits were used, split into two groups of eight animals: Gprp (plasma rich in platelets) and Gc (control, saline solution, 9%). The same technique of reconstructive surgery using graft mesh was performed on each rabbit. The groups differed only in the application of platelet-rich plasma before the surgical wound synthesis. Samples for evaluation of angiogenesis were collected 15 days after the surgical procedure. The dyes Hematoxylin & Eosin and Masson's Trichrome were used in the histochemical study to evaluate vascular proliferation. Markers CD31, CD34 and Caveolin-1 was used for the immunohistochemical study. The evaluation between the groups (Gprp and Gc) in regard to the categorical variable (vascular proliferation intensity) used the Kruskal-Wallis test with p values equal to or less than 0.05 being considered significant. The immunohistochemistry was subjected to analysis of variance for a completely randomized design, with two groups and five repetitions (medium) and 5% significance level. Multiple comparison of groups resulted in the Tukey test (p=0.05) used. The amount of vascular proliferation assessed by histochemical method HE and Masson's Trichrome was found to be a significant variable in Gprp when compared with group Gc. When evaluating the methods used, there was no significant difference. There was no difference in the three markers which were used for correlating microvessels; however, there was more intense staining of vessels when Caveolin-1 Antibody was used. This caused intense marking of the capillaries and small vessels, as well as of larger vessels. When using CD31 and CD34, the same was observed, but it was not as intense as with Caveolin-1; though some cases showed sincere and discreet marking. The results of this study demonstrated that the histochemical methods performed are effective for semi-quantitative assessment of angiogenesis. The immunohistochemical comparison of Caveolin-1, CD31, and CD34 as markers of angiogenesis in rabbits showed that both antibodies could immunostain the newly formed vessels; but the Caveolin-1 showed better immunostaining in small and medium-sized vessels, as well as a minor presence in the background. Although not specific markers for angiogenesis, they can be used as immunohistochemical markers of vascular endothelium in rabbits.(AU)

Colorações histoquímicas consistem de um conjunto de reações químicas específicas das estruturas ou substâncias endógenas do tecido. Logo a Imunohistoquímica permite observar proteínas presentes nos tecidos relacionadas com fatores determinantes do comportamento biológico e patológico. A padronização dos métodos que avaliam a angiogênese decorrente de procedimentos que utilizam substâncias estimulantes à formação de novos vasos são importantes, a fim de facilitar a execução das pesquisas, bem como auxiliar na interpretação dos dados, visto que em coelhos alguns anticorpos marcadores de angiogênese na pele ainda não são padronizadas em virtude das reações cruzadas que podem ocorrer devido aos anticorpos serem confeccionados a partir de tais animais. Objetivou-se analisar os métodos histoquímicos por meio das colorações e imunohistoquímicas com marcadores de angiogênese em coelhos (Oryctolagus cuniculus) submetidos ao emprego de enxertos cutâneos associado com estimulador de angiogênese plasma rico em plaquetas, a fim de avaliar qual método seria melhor para visualização dos vasos, bem como avaliar qual anticorpo promoveria melhor imunomarcação, buscando-se assim encontrar a diferenças entre os métodos e padronizar a metodologia a ser aplicada em experimentos que utilizem coelhos. Utilizou-se 16 coelhos, separados em dois grupos com oito animais, compreendendo os grupos Gprp (plasma rico em plaquetas) e Gc (controle, solução fisiológica 0,9%). Em todos os animais foi realizada a mesma técnica de cirurgia reconstrutiva de enxertia do tipo malha, os grupos diferiram apenas a aplicação do plasma rico em plaquetas antes da síntese da ferida cirúrgica. As amostras para avaliação da angiogênese foram coletadas após 15 dias do procedimento cirúrgico. Utilizou-se no estudo histoquímico as colorações Hematoxilina & Eosina e Tricrômico de Masson para avaliação da proliferação vascular, e os anticorpos CD31 e CD34 e Caveolina - 1 para avaliação imunohistoquímica. A comparação entre os grupos (Gprp e Gc) em relação à variável categórica (intensidade de proliferação vascular) foi utilizado o teste de Kruskal-Wallis, com valores de p iguais ou inferiores a 0,05 foram considerados significativos. Os dados imuno-histoquímico foram submetidos à análise de variância para um delineamento inteiramente ao acaso, com 2 grupos e 5 repetições (médias) e nível de significância de 5%. Nas comparações múltiplas das médias dos grupos, utilizou-se o teste de Tukey (α = 0,05). A intensidade de proliferação vascular avaliada pelo método histoquímico HE e Tricômico de Masson encontrou-se que tal variável foi significativa no Gprp, quando comparado com o Gc. Avaliando os métodos utilizados não houve diferença significativa. A contagem microvascular (MVC) realizada com os diferentes marcadores (Caveolina-1, CD31 e CD34) foi significativa no Gprp. Correlacionando a contagem microvascular dos três marcadores utilizados não houve diferença significativa, no entanto observou-se marcação mais intensa dos vasos utilizando o anticorpo Caveolina-1, sendo intensa a marcação dos capilares, vasos de pequeno calibre, bem como em vasos maiores. Nas avaliações de CD31 e CD34 observou que houve imunomarcação dos vasos, porém não foi intensa como a Caveolina-1, alguns casos apresentaram fundo, bem como marcação discreta. Os resultados encontrados neste estudo evidenciaram os métodos histoquímicos são eficazes para avaliação semiquantitativa da angiogênese. A comparação imunohistoquímicas da Caveolina-1, CD31 e CD34 como marcadores de angiogênese em coelhos evidenciaram que ambos os anticorpos são capazes de imunomarcar os vasos neoformados, porém a Caveolina-1 apresentou melhor imunomarcação de vasos de pequeno e médio calibre, bem como menor presença de fundo, embora não seja um marcador específico para angiogênese pode ser utilizada como marcador imunohistoquímico de endotélio vascular em coelhos.(AU)

Qualitative analysis of the viability of autogenous fat grafts grafted in different environments of interstitial pressure. Preliminary results and description of a new experimental model in mini-pigs

Abstract Purpose: To evaluate the feasibility of an experimental model of autologous fat graft (AFG) in different interstitial pressure (IP) environments. Methods: Three mini-pigs(Minipig-BR) with age of 8 months (weight: 25-30 kg) were used. AFG were collected from the bucal fat pad, and grafted in the intramuscular pocket (biceps femoralis muscle). IP model was based on a fusiform ressection followed by primary closure "under tension". A blood pressure catheter located in the intramuscular region connected to a pressure module was applied to quantify IP. Results: The mean operative time was 236 min (210 - 272 min). All the AFG and muscular segments were removed successfully. Average interstitial pressure CP and H were 3 and 10.6 mmHg respectively. The AFG were biopsied for histopathological analysis 30 days after graft. Hematoxylin-eosin staining and immunohistochemical analyzes (TNF-alpha, CD31 and Perilipine with monoclonal antibodies) were employed. Conclusion: The data show that minipigs model could be used as a recipient site for autologous fat graft techniques and allow the development of studies to explore the AFG intake and pathophysiology response.

Culture of rat corpus cavernosal endothelial cells using modified immunomagnetic beads and cloning / 中华男科学杂志

Objective@#To search for the methods of isolating, purifying and culturing corpus cavernosal endothelial cells (CCECs) from SD rats, observe their growth characteristics, and providing seed cells for the study of erectile dysfunction (ED).@*METHODS@#The corpus cavernosal tissue from the SD rat was digested with 0.1% elastase, followed by purification of CCECs with immunomagnetic beads. After further amplification, monoclonal CCECs were sorted out with the cloning cylinder and their morphological and proliferative characteristics were observed. The von Willebrand factor (VWF) in the CCECs was identified by immunofluorescence staining, the CD31 molecule detected by immumohistochemistry, the purity of the CCECs determined by flow cytometry, and the proliferation of the cells measured with CCK-8 and growth curves.@*RESULTS@#After 7 days of purification and culture, the CCECs were fused into a monolayer under the inverted phase-contrast microscope, arranged like flagstones. The growth curves showed that the CCECs were in latency with a low growth rate at 1－2 days, in the logarithmic growth phase with a rapid rate at 3－4 days, and into the platform phase around the 6th day. VWF was positively expressed in the CCECs with much green fluorescence, and so was CD31 with a large number of brownish particles. The positive rate of the CCECs which were labelled with the VWF purified with magnetic beads combined with cloning cylinders was up to (91.9±3.75)%.@*CONCLUSIONS@#High-purity rat CCECs can be cultured successfully using immunomagnetic beads combined with cloning cylinders, with stable proliferation and passage in the endothelial cell medium.

Effects of Next-Generation Low-Energy Extracorporeal Shockwave Therapy on Erectile Dysfunction in an Animal Model of Diabetes

PURPOSE: Gene therapy, stem cell therapy, and low-energy extracorporeal shockwave therapy (ESWT) have been investigated as treatments for refractory erectile dysfunction (ED), but inconclusive evidence has been obtained. We investigated the effect of a next-generation electromagnetic cylinder ESWT device on an animal model of ED. MATERIALS AND METHODS: Diabetes mellitus (DM)-induced rats were divided into 3 groups: group 1, control; group 2, DM; and group 3, DM+ESWT. Rats were treated with ESWT 3 times a week for 2 weeks. After the treatment course, intracavernous pressure was measured and the corpus cavernosum and cavernous nerve were evaluated. RESULTS: In the DM group, all parameters predicted to be significantly lower in the ED model had statistically significantly decreased (p < 0.01). As a measurement of erectile function, intracavernous pressure was evaluated. The DM+ESWT group exhibited significantly restored erectile function compared to the DM group (p < 0.05). Moreover, ESWT treatment restored smooth muscle content, as assessed by Masson's trichrome staining (p < 0.05). Finally, corporal tissue and the dorsal nerve were evaluated by immunohistochemistry, Western blotting, and ELISA. After ESWT treatment, vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), platelet endothelial cell adhesion molecule-1, cyclic guanosine monophosphate, and neuronal nitric oxide synthase (nNOS) expression levels were restored to levels in the DM group (p < 0.05). CONCLUSIONS: Electromagnetic cylinder ESWT device resulted in increased VEGF, nNOS, and eNOS expression; reduced smooth muscle atrophy; and increased endothelial cell regeneration in a DM-associated ED model. Our data suggest that safe and effective application could be possible in future clinical studies.

Inflammatory response of human dental pulp to at-home and in-office tooth bleaching

ABSTRACT Tooth bleaching is a technique of choice to obtain a harmonious smile, but bleaching agents may damage the dental pulp. Objective: This study evaluated the inflammatory responses of human dental pulp after the use of two bleaching techniques. Material and Methods: Pulp samples were collected from human third molars extracted for orthodontic reasons and divided into three groups: control - no tooth bleaching (CG) (n=7); at-home bleaching with 15% carbamide peroxide (AH) (n = 10), and in-office bleaching with 38% hydrogen peroxide (IO) (n=12). Pulps were removed and stained with hematoxylin-eosin for microscopic analysis of inflammation intensity, collagen degradation, and pulp tissue organization. Immunohistochemistry was used to detect mast cells (tryptase+), blood vessels (CD31+), and macrophages (CD68+). Chi-square, Kruskal-Wallis, and Mann Whitney tests were used for statistical analysis. The level of significance was set at p<.05. Results: The inflammation intensity and the number of macrophages were significantly greater in IO than in AH and CG (p<0.05). The results of CD31+ (blood vessels per mm2) were similar in CG (61.39±20.03), AH (52.29±27.62), and IO (57.43±8.69) groups (p>0.05). No mast cells were found in the pulp samples analyzed. Conclusion: In-office bleaching with 38% hydrogen peroxide resulted in more intense inflammation, higher macrophages migration, and greater pulp damage then at-home bleaching with 15% carbamide peroxide, however, these bleaching techniques did not induce migration of mast cells and increased the number of blood vessels.

Host inflammatory response to polypropylene implants: insights from a quantitative immunohistochemical and birefringence analysis in a rat subcutaneous model

ABSTRACT Objectives To describe acute and sub acute aspects of histological and immunohistochemical response to PP implant in a rat subcutaneous model based on objective methods. Materials and Methods Thirty rats had a PP mesh subcutaneously implanted and the same dissection on the other side of abdomen but without mesh (sham). The animals were euthanized after 4 and 30 days. Six slides were prepared using the tissue removed: one stained with hematoxylin-eosin (inflammation assessment); one unstained (birefringence evaluation) and four slides for immunohistochemical processing: IL-1 and TNF-α (pro-inflammatory cytokines), MMP-2 (collagen metabolism) and CD-31 (angiogenesis). The area of inflammation, the birefringence index, the area of immunoreactivity and the number of vessels were objectively measured. Results A larger area of inflammatory reaction was observed in PP compared to sham on the 4th and on the 30th day (p=0.0002). After 4 days, PP presented higher TNF (p=0.0001) immunoreactivity than sham and no differences were observed in MMP-2 (p=0.06) and IL-1 (p=0.08). After 30 days, a reduction of IL-1 (p=0.010) and TNF (p=0.016) for PP and of IL-1 (p=0.010) for sham were observed. Moreover, area of MMP-2 immunoreactivity decreased over time for PP group (p=0.018). Birefringence index and vessel counting showed no differences between PP and sham (p=0.27 and p=0.58, respectively). Conclusions The implantation of monofilament and macroporous polypropylene in the subcutaneous of rats resulted in increased inflammatory activity and higher TNF production in the early post implant phase. After 30 days, PP has similar cytokines immunoreactivity, vessel density and extracellular matrix organization.

Extracellular signal-regulated kinase signaling pathway regulates the endothelial differentiation of periodontal ligament stem cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of extracellular signal-regulated kinase (ERK) signaling pathway on the endothelial differentiation of periodontal ligament stem cells (PDLSC).</p><p><b>METHODS</b>Human PDLSC was cultured in the medium with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) to induce endothelial differentiation. Endothelial inducing cells was incubated with U0126, a specific p-ERK1/2 inhibitor. PDLSC from one person were randomly divided into four groups: control group, endothelial induced group, endothelial induced+DMSO group and endothelial induced+U0126 group. The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0, 1, 3, 6 and 12 hours during endonthelial induction. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) after a 7-day induction. The proportion of CD31(+) to VE-cadherin(+) cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction. The measurement data were statistically analyzed.</p><p><b>RESULTS</b>Phosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively, during the endothelial induction (P<0.01). The mRNA expressions of CD31 and VEGF in induced+U0126 group were decreased to 0.09±0.18 and 0.49±0.17, which were both significantly different with those in induced group (P<0.05). The proportion of CD31(+) to VE-cadherin(+) cells of induced+U0126 group were decreased to 5.22±0.85 and 3.56±0.87, which were both significantly different with those in induced group (P<0.05). In Matrigel assay, the branching points, tube number and tube length were decreased to 7.0±2.7, 33.5±6.4, and (15 951.0±758.1) pixels, which were all significantly different with those in induced group (P<0.05).</p><p><b>CONCLUSIONS</b>The endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSC.</p>

Neamine inhibits growth of pancreatic cancer cells in vitro and in vivo / 华中科技大学学报（医学）（英德文版）

Neamine, a non-toxic derivative of neomycin, has recently been shown to have antitumor activities in various types of cancers. However, its effect on pancreatic cancer is still unknown. The study aimed to investigate its antitumor activity on pancreatic cancer and the underlying mechanisms. MTT assay was used to observe the effect of neamine on angiogenin (ANG)-induced AsPC-1 cell proliferation. Tissue microassay and immunofluorescence staining were used to detect the expression of ANG and its nuclear translocation, respectively. Tumor xenografts were established by subcutaneous inoculation of AsPC-1 pancreatic cancer cells into the right flanks of nude mice, and neamine was injected subcutaneously. Immunohistochemistry was done to observe the expression of ANG, CD31 and Ki-67 in tumor xenografts. It was found that neamine blocked the nuclear translocation of ANG effectively and inhibited ANG-induced AsPC-1 cell proliferation in a dose-dependent manner. Neamine had anti-tumor effects on AsPC-1 xenograft models. Consistently, neamine reduced the expression levels of ANG, Ki-67 and CD31 in tumor xenografts. It was concluded that neamine may be a promising agent for treatment of pancreatic cancer.

Expression of PECAM-1 and E-Cadherin in the Umblical Cords of Gestational Diabetic Mothers / Expresión de PECAM-1 y E-cadherina en los Cordones Umbilicales de Madres con Diabetes Gestacional

The purpose of this study is to examine the changes in the umbilical cord in women diagnosed with gestational diabetes mellitus In this study, as a control group human placental tissues from normotensive pregnancies was collected from diabetic women at 28­35 weeks of gestation. Gestational diabetes (n= 20) and normal umbilical cord (n= 20) for a total of 40 units were received.GDM groups compared to the control group was significantly higher values was detected (p<0.01). In GDM group, light microscopy showed erosion of the endothelium and complete rupture of theumbilicalvessels resulting in extravasation of blood within Wharton's jelly. it was observed that the cytoplasmic fragments and cell infiltration of the spill to the subepithelial layer of apoptotic cell PECAM-1 positive reaction showed. E-Cadherin in endothelial side surface of diabetes group showed weak expression in the nucleus and showed positive reaction in smooth muscle.

El objetivo fue examinar los cambios que presenta el cordón umbilical de mujeres con diagnóstico de diabetes mellitus gestacional (DMG). Se incluyeron en el grupo control muestras de tejidos placentarios humanos de embarazos normotensos y de mujeres diabéticas de entre 28­35 semanas de gestación. Las muestras se divieron en cordones umbilicales con cambios de DMG (n= 20) y cordones umbilicales normales (n= 20), constituyendo un total de 40 muestras. El grupo de DMG, en comparación con el grupo control, presentó valores significativamente más elevados (p<0,01). En el grupo de DMG, la microscopía óptica demostró la erosión del endotelio y la ruptura completa de los vasos umbilicales, resultando en la extravasación de sangre dentro de la gelatina . Se observaron fragmentos citoplasmáticos e infiltración celular de la capa subepitelial de células apoptóticas mostró una reacción positiva a PECAM-1. En el grupo de DMG, la E-cadherina de la superficie lateral endotelial mostró una expresión débil en el núcleo y una reacción positiva en el músculo liso.

Proximal type of epithelioid sarcoma: a rare aggressive tumor presenting simultaneously in spine and pelvis

A 40-year-old female presented with bilateral lower limb weakness with bladder and bowel incontinence. MRI study revealed a destructive lesion involving the D7 vertebral body and a large tumor in the gluteal muscles invading the right iliac blade. A histological examination demonstrated a tumor comprising of rounded to ovoid pleomorphic epithelioid cells with marked cytological atypia. Tumor cells expressed CD 34, vimentin and focally pancytokeratin but were negative for CD31, EMA, SMA, WT1 and LCA. A D6-7 laminectomy with posterior decompression was done. Postoperatively, external beam radiotherapy was given. However, the patient deteriorated rapidly with no neurological improvement. Epitheiliod sarcomas and their recently described proximal variant, by virtue of being an exceedingly unusual tumor are often misdiagnosed or diagnosed late beyond the stage of salvage. This report highlights the histopathology and that need to be analyzed to correctly diagnose this entity

The protein expression of heme oxygenase-1 and platelet endothelial cell adhesion molecules-1 in human coronary artery endothelial cell induced by zinc oxide nanoparticle / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the protein expression of heme oxygenase-1 (HO-1) and platelet endothelial cell adhesion molecules-1 (PECAM-1) in human coronary artery endothelial cells induced with Zinc Oxide Nanoparticle (ZnO-NPs).</p><p><b>METHODS</b>MTT assay was used to determine the cell viability of ZnO-NPs. Levels of HO-1 and PECAM-1 protein in culture supernatants were measured using ELISA after human coronary artery endothelial cells were treated with different concentrations (0, 10, 20, 40µg/ml) of ZnO-NPs for 24 h.</p><p><b>RESULTS</b>The cell viability of human coronary artery endothelial cells in each group was 89.76%, 83.61%, 63.10%, 53.20%, 48.11%, 42.35%, 38.06%, 25.44% respectively when treated with different concentrations of ZnO-NPs (12.5, 25, 50, 70, 80, 90, 100, 200µg/ml). Protein levels of HO-1 (ng/L) in each group were 0.041±0.011, 0.512±0.076, 0.906±0.059, 1.062±0.089 respectively after the stimulation of different concentrations of ZnO-NPs (0, 10, 20, 40µg/ml). Comparisons in each group were statistically significant (P < 0.05). Protein levels of PECAM-1 (µg/L) in each group were 7.966 ± 0.046, 7.993 ± 0.036, 8.629 ± 0.052, 8.811 ± 0.039 respectively after the stimulation of different concentrations of ZnO-NPs (0, 10, 20, 40 µg/ml). Compared with the control group, protein levels of PECAM-1 increased (P < 0.05) when the concentration of ZnO-NPs was 20µg/ml or 40 µg/ml.</p><p><b>CONCLUSION</b>ZnO-NPs stimulation could inhibit the viability of human coronary artery endothelial cells and upregulate the protein expression of HO-1 and PECAM-1.</p>

Characterization of a novel mouse model with genetic deletion of CD177

Neutrophils play an essential role in the innate immune response to infection. Neutrophils migrate from the vasculature into the tissue in response to infection. Recently, a neutrophil cell surface receptor, CD177, was shown to help mediate neutrophil migration across the endothelium through interactions with PECAM1. We examined a publicly available gene array dataset of CD177 expression from human neutrophils following pulmonary endotoxin instillation. Among all 22,214 genes examined, CD177 mRNA was the most upregulated following endotoxin exposure. The high level of CD177 expression is also maintained in airspace neutrophils, suggesting a potential involvement of CD177 in neutrophil infiltration under infectious diseases. To determine the role of CD177 in neutrophils in vivo, we constructed a CD177-genetic knockout mouse model. The mice with homozygous deletion of CD177 have no discernible phenotype and no significant change in immune cells, other than decreased neutrophil counts in peripheral blood. We examined the role of CD177 in neutrophil accumulation using a skin infection model with Staphylococcus aureus. CD177 deletion reduced neutrophil counts in inflammatory skin caused by S. aureus. Mechanistically we found that CD177 deletion in mouse neutrophils has no significant impact in CXCL1/KC- or fMLP-induced migration, but led to significant cell death. Herein we established a novel genetic mouse model to study the role of CD177 and found that CD177 plays an important role in neutrophils.

Fuzheng Huayu recipe promotes the fenestration of capillarization in liver sinusoidal endothelial cells / 中南大学学报(医学版)

OBJECTIVE@#To explore the effect of Fuzheng Huayu (FZHY) recipe on the fenestration of capillarization in liver sinusoidal endothelial cells (LSECs).
@*METHODS@#Ten Sprague Dawley (SD) rats were fed with 0.46 g/kg FZHY powder by intragastric administration. Two hours later, a second gavage were given to the rats. The serum from rat heart at 1 hour after second gavage was collected (FZHY group, n=10). Another ten SD rats was administrated with distilled water through the same process and served as the control (control group, n=10). The serum from both groups were separately diluted with Dulbecco minimum essential medium (DMEM) for 10% and served as the culture medium for LSECs. At the different conditions, the vWF and CD31 expressions were detected by immunocytochemistry and Western blot, while the changes of LSECs fenestrae structure were observed under scanning electron microscopy.
@*RESULTS@#1) Immunocytochemistry and Western blot showed that the vWF and CD31 protein levels were lower in LSECs in the FZHY group than those in the control group. The gray levels of vWF and CD31 protein were 0.548±0.020 and 0.262±0.010 in the FZHY group, and 0.845±0.090 and 0.383±0.010 in the control group respectively, with statistical significant difference (t=5.18, 9.61, both P<0.05). 2) The results from scanning electron microscopy showed that the fenestration of LSECs was closed and almost lost in the control group, but many fenestra appeared in the LSECs in the FZHY group.
@*CONCLUSION@#FZHY recipe can suppress the expression of vWF and CD31, increase the fenestrae on the LSECs surface and reverse the capillarization of LSECs.