Este trabalho foi financiado por uma doação do Instituto Nacional de Saúde, R01CA142928.

1. Injecção intravenosa de azul de Evans na veia da cauda do rato Lateral <ol><li> Prepara-se uma solução de 0,5% de azul de Evans estéril em PBS. Se necessário, esterilizar por filtração da solução para remover qualquer matéria particulada que não se dissolveu. </li><li> Aspirar 200 ul solução azul de Evans para uma seringa. Evitar as bolhas de ar que possam ter escapado para a seringa. </li><li> Coloque ratos que são de 8-12 semanas de idade para um dispositivo de retenção de modo a que o animal não seja livremente móvel, mas a cauda podem ser manipulados. </li><li> Colocar o dispositivo de retenção do rato sobre o seu lado, de modo a veia lateral da cauda é facilmente visível e está virada para cima. </li><li> Segure a cauda com a mão não-dominante entre o polegar eo dedo indicador. </li><li> Inserir a agulha (calibre pequeno, 27-30) a uma antecedência 10-15 graus de ângulo, bisel para cima, para dentro da veia lateral da cauda na direcção da cabeça. Manter o paralelo agulha e seringa para a cauda. </li><li> Do não aplicar pressão para trás para confirmar a colocação correta, pois isso poderia entrar em colapso da veia. </li><li> Lentamente injetar 200 ul Evans solução azul na veia da cauda do rato. </li><li> Observar a facilidade com que os avanços de êmbolo, tal como esta é a prova da correcta colocação da agulha na veia. </li><li> Coloque o mouse em sua gaiola e observá-lo durante 30 minutos. </li></ol> 2. Recolha de órgãos e Extração de azul de Evans dos Órgãos <ol><li> Sacrificar os animais por deslocamento cervical. Para fins de ensaio Miles deslocamento cervical é recomendada, uma vez que limita a interferência significativa com a permeabilidade vascular. Sacrificar todos os ratinhos, ao mesmo tempo, tão rápido quanto possível. Trabalhar com grupos de 6 ratinhos ou menos, porque logo após os vasos sanguíneos se tornam mais permeáveis ​​morte. </li><li> Coloque os ratos em suas costas e fixar os pés em uma placa branca. </li><li> Abra a cavidade abdominal e torácica para expor thorórgãos Acic e abdominal. </li><li> Tire fotos representativas para mostrar diferenças em Evans extravasamento Azul. Incluir todos os ratos no mesmo campo, a fim de ter condições de iluminação idênticas para todos os ratos. </li><li> Coletar órgãos de interesse e colocá-los em tubos de 1,5 ml </li><li> Pesar um tubo vazio e levar o valor do equilíbrio de zero. </li><li> Transfira a amostra de tecido e peso,. Repetir para todas as amostras de tecido. Os tecidos podem ser secos ao ar para eliminar a variabilidade teor de água entre os diferentes órgãos. </li><li> . Adicionar 500 ul de formamida a cada tubo de amostra de tecido. </li><li> Transferir todos os tubos para um banho de água 55 ° C ou bloco de calor. Incubar durante 24-48 h, para extrair Azul de Evans do tecido. </li></ol> 3. Quantificação de azul de Evans extravasado no tecido intersticial <ol><li> Centrifugar a formamida / azul de Evans mistura a pelota quaisquer fragmentos de tecido remanescentes. </li><li> Medir a absorvância a 610nm. Use 500 Formamida ul como em branco. </li><li> Calcular ng Azul de Evans extravasado por mg de tecido. </li><li> Traçar todos os dados em um gráfico. </li><li> Realizar a análise estatística para determinar diferenças significativas. </li></ol>

<ol>
	<li>Beck, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+NO+synthase:+role+in+cellular+signaling.">Inducible NO synthase: role in cellular signaling.</a> J. Exp. Biol. 202, 645-653 (1999).</li><li>Bertglia, S., Giusti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+nitric+oxide+in+capillary+perfusion+and+oxygen+delivery+regulation+during+systemic+hypoxia.">Role of nitric oxide in capillary perfusion and oxygen delivery regulation during systemic hypoxia.</a> Am. J. Physiol. Heart Circ. Physiol. 288, H525-H531 (2005).</li><li>Miles, A. A., Miles, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+reactions+to+histamine,+histamine-liberator+and+leutaxine+in+the+skin+of+guinea+pigs.">Vascular reactions to histamine, histamine-liberator and leutaxine in the skin of guinea pigs.</a> J. Physiol. (London). 118, 228-257 (1952).</li><li>Weis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability+in+cardiovascular+disease+and+cancer.">Vascular permeability in cardiovascular disease and cancer.</a> Curr. Opin. Hematol. 15, 243-249 (2008).</li><li>Kumar, P., Shen, Q., Pivetii, C. D., Lee, E. S., We, M. H., Yuan, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+endothelial+hyperpermeability:+implications+in+inflammation.">Molecular mechanisms of endothelial hyperpermeability: implications in inflammation.</a> Expert Rev. Mol. Med. 30, 11-19 (2009).</li><li>Viazzi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability,+blood+pressure,+and+organ+damage+in+primary+hypertension.">Vascular permeability, blood pressure, and organ damage in primary hypertension.</a> Hypertens. Res. 31, 873-879 (2008).</li><li>Scheppke, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+vascular+permeability+suppression+by+topical+application+of+a+novel+VEGFR2/Src+kinase+inhibitor+in+mice+and+rabbits.">Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits.</a> J. Clin. Invest. 118, 2337-2346 (2008).</li><li>Blanchet, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+CD34+Leads+To+Exacerbated+Autoimmune+Arthritis+through+Increased+Vascular+Permeability.">Loss of CD34 Leads To Exacerbated Autoimmune Arthritis through Increased Vascular Permeability.</a> J. Immunol. 184, 1292-1299 (2010).</li><li>Dvorak, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell-derived+mediators+of+enhanced+microvascular+permeability,+vascular+permeability+factor/vascular+endothelial+growth+factor,+histamine,+and+serotonin,+cause+leakage+of+macromolecules+through+a+new+endothelial+cell+permeability+organelle,+the+vesiculo-vacuolar+organelle.">Mast cell-derived mediators of enhanced microvascular permeability, vascular permeability factor/vascular endothelial growth factor, histamine, and serotonin, cause leakage of macromolecules through a new endothelial cell permeability organelle, the vesiculo-vacuolar organelle.</a> Chem. Immunol. Allergy. 85, 185-204 (2005).</li><li>Le Guelte, A., Gavard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelial+cell-cell+junctions+in+endothelial+permeability.">Role of endothelial cell-cell junctions in endothelial permeability.</a> Methods Mol. Biol. 763, 265-279 (2011).</li><li>Martins-Green, M., Petreaca, M., Yao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+system+for+in+vitro+detection+of+permeability+in+human+"endothelium".">An assay system for in vitro detection of permeability in human "endothelium".</a> Methods Enzymol. 443, 137-153 (2008).</li></ol>

Os autores não têm nenhum conflito ou concorrentes interesses financeiros.

Permeabilidade vascular é um marcador para o estado fundamental do vaso sanguíneo. Aumento da permeabilidade vascular tem mostrado estar presente em várias doenças sistémicas, incluindo a diabetes, a hipertensão, e doenças auto-imunes 6,7,8. Aumento da permeabilidade vascular tem sido mostrado para ser mediada por tensão de corte, os factores de crescimento tais como o factor de crescimento endotelial vascular e factor de crescimento de fibroblastos, os mediadores inflamatórios tais como histamina, s...

Formação e manutenção de barreiras permeáveis ​​selectivos são essenciais para o desenvolvimento do órgão apropriado e 1,2 desempenho. Linha de células endoteliais do lúmen do vaso sanguíneo e formar uma barreira semi-permeável, que é essencial para o transporte selectivo entre o sangue e o espaço intersticial de todos os órgãos. Uma barreira de permeabilidade adequada é mantida através apertados célula-a-célula junções que são estritamente controladas por factores de crescimento, citoquinas e moléculas de stress outros produtos 3. O rompimento da barreira de células endoteliais pode resultar em aumento da permeabilidade vascular e de fuga. Estes efeitos são observados em vários estados de doença e a compreensão da sinalização sublinhado molecular requer métodos multidisciplinares 4,5. Neste artigo, descreve um método in vivo para medir a permeabilidade vascular utilizando um modelo de rato. O ensaio que se descreve, também conhecido como ensaio de Miles, é uma bem establtada método para testar a permeabilidade vascular in vivo. O ensaio baseia-se no facto de que, sob condições fisiológicas basais, a albumina não atravessa a barreira endotelial. Azul de Evans, um corante azóico com elevada afinidade para a albumina, é injectado na corrente sanguínea de um animal experimental e, sob condições fisiológicas, é esperado para ser restringida dentro dos vasos sanguíneos. Quando um estímulo permeabilidade vascular é adicionado, quer por aplicação tópica ou sistemicamente, os vasos sanguíneos começar a vazar proteína e, assim, também o azul de Evans, que se liga à albumina. Isto resulta numa coloração rápida azulada dos tecidos que têm vasos permeáveis. Injecção com êxito do corante na veia da cauda do rato lateral é crítica para o bom resultado do experimento. Técnica de injecção da veia da cauda requer prática extensiva e deverá ser dominadas antes de se iniciar a experiência. Permeabilidade vascular é altamente dependente da idade e do peso do umImal, então quando comparando diferentes linhagens de camundongos, é imperativo que os ratos ou outros assuntos de teste têm perto de datas de nascimento e peso idênticos. Outros factores que influenciam a permeabilidade da barreira endotelial são condições ambientais, tais como temperatura, humidade, e muito importante, o stress manuseamento do rato. Devido ao grande número de fatores que podem influenciar o resultado do experimento, é sempre aconselhável que o experimento é repetido pelo menos três vezes e análise estatística realizada. Este ensaio pode ser utilizado para comparar a permeabilidade ou vaso de ratos que foram geneticamente modificadas, bem como os ratos com diferentes antecedentes genéticos. A permeabilidade pode ser determinada na presença ou na ausência de um estímulo, dependendo da função do gene, que é modulada. Este ensaio também pode ser usado ou para testar o efeito de diferentes compostos sobre a permeabilidade do vaso.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> REAGENTES </td></tr><tr><td> Azul de Evans </td><td> SIGMA </td><td> E2129 </td><td></td></tr><tr><td> FORMAMIDA </td><td> INVITROGEN </td><td> 15515-026 </td><td></td></tr><tr><td> PBS </td><td></td><td></td><td> Fosfato 0,2 M 1,5 M NaCl pH 7,4 </td></tr><tr><td></td><td></td><td></td><td> EQUIPAMENTOS </td></tr><tr><td> ESPECTROFOTÔMETRO </td><td> EPPENDORF </td><td> 952000006 </td><td></td></tr><tr><td> Dispositivo de retenção MOUSE </td><td> Harvard Apparatus </td><td> 340012 </td><td></td></tr><tr><td> SERINGA </td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> AGULHAS </td><td> BD </td><td> 305106 </td><td> O calibre da agulha depende do tamanho do animal. </td></tr><tr><td> BALANÇO </td><td> Denver Instrument </td><td> TP-64 </td><td></td></tr></tbody></table>

an in vivo assay to test blood vessel permeability

Este método baseia-se na injecção intravenosa de azul de Evans nos ratinhos como modelo animal de teste. Azul de Evans é um corante que se liga a albumina. Sob condições fisiológicas o endotélio é impermeável à albumina, de modo do azul de Evans encadernados restos de albumina restrito dentro dos vasos sanguíneos. Em condições patológicas que promovem o aumento da permeabilidade vascular células endoteliais parcialmente perder seus contatos próximos e com o endotélio torna-se permeável a pequenas proteínas, como a albumina. Esta condição permite o extravasamento de azul de Evans dos tecidos. Um endotélio saudável impede o extravasamento do corante nos tecidos vizinhos vascularizados. Órgãos com aumento da permeabilidade irá mostrar um aumento significativo em comparação com coloração azul órgãos com endotélio intacto. O nível de permeabilidade vascular pode ser avaliado por simples visualização ou por medição quantitativa do corante incorporado por miligrama de tecido de controlo versus animal experimental / tecido. Dois powerful aspectos deste ensaio são a simplicidade e características quantitativas. O Evans Blue, podem ser extraídos a partir de tecidos por incubação de uma quantidade específica de tecido em formamida. Evans Blue é a absorvância máxima a 620 nm e a absorvência é mínima a 740 nm. Utilizando uma curva padrão para o azul de Evans, medições da densidade óptica pode ser convertida em miligrama de corante capturado por miligrama de tecido. A análise estatística deve ser usado para avaliar diferenças significativas na permeabilidade vascular.

Foi utilizado um ensaio in vivo para testar a permeabilidade vazamento navio em ratinhos de 8-12 semanas de idade. Este teste é útil para comparar a permeabilidade vascular relativa entre animais de fundo genético diferente ou em uma única estirpe de ratos submetidos a tratamentos que afectam a vasculatura. Os nossos resultados mostram que a estirpe de murganhos geneticamente modificados, criados no nosso laboratório tem um endotélio mais permeável em comparação com ratinhos de tipo selvagem. ...

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An in vivo Assay to Test Blood Vessel Permeability

Estamos apresentando um<em&gt; In vivo</em&gt; Ensaio para testar a permeabilidade dos vasos sanguíneos. Este ensaio baseia-se na injecção intravenosa de uma visualização do corante e subsequente da sua difusão para os espaços intersticiais.

Um<em&gt; In vivo</em&gt; Ensaio para ensaio de permeabilidade dos vasos sangüíneos

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

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An in vivo Assay to Test Blood Vessel Permeability

63.9K visualizações.  Fox Chase Cancer Center. Estamos apresentando um In vivo Ensaio para testar a permeabilidade dos vasos sanguíneos. Este ensaio baseia-se na injecção intravenosa de uma visualização do corante e subsequente da sua difusão para os espaços intersticiais.

Vídeo: An in vivo Assay to Test Blood Vessel Permeability

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Induzida pela quimioterapia Vascular Toxicidade - em tempo real<em&gt; In vivo</em&gt; Imagem de Impairment Vessel

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Medicina

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

Ex Vivo Intestinal Sacs para Avaliar mucosa Permeabilidade de modelos de doença gastrointestinal

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Ensaios In Vitro para Avaliar Formação e Perturbação de Embarque de Barreira de Sangue-Cérebro

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

A abordagem de amostragem de 4 embarcações para estudos integrativos de fisiologia placentária humana<em&gt; In Vivo</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Re-Arterialized transplante de fígado parcial de rato com na vivo orientado a embarcação 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

Um método In Vivo para avaliar a barreira de sangue do intestino e metabolismo hepático da Microbiota produtos

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Using an Automated Hirschberg Test App to Evaluate Ocular Alignment

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing the difference in corneal reflection generated by the phone camera flash relative to the iris center based on high resolution images, the app can measure misalignment with a much higher precision than the naked eye performing the Hirschberg test. It has been validated in a previous clinical evaluation study by comparing to the clinical gold standard-prism and alternate cover test. The goal of this article is to describe the testing techniques regarding how to use the app to measure ocular alignment for different fixation distances, without or with cover to break fusion, as well as angle kappa, so that users can use the app to perform equivalent tests typically done in clinic using prisms.

We present a protocol of using a smartphone app to perform Hirschberg test for measuring manifest and intermittent ocular misalignment (strabismus) under near and far fixation conditions.

Usando um aplicativo de teste hirschberg automatizado para avaliar o alinhamento ocular

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

Medição do fluxo sanguíneo strial em Cochlea de rato utilizando uma janela de vaso aberto e microscopia de fluorescência intravital

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

Corte de precisão Fatias pulmonares como uma ferramenta eficiente para ex vivo estrutura de vasos pulmonares e estudos de contratibilidade

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...