Os autores agradecem a Sra. Risa Otsuka de assistência técnica e experiências de ajuda. Esta pesquisa foi parcialmente financiada pelo Ministério da Educação, Ciência, Esportes e Cultura, Grant-in-Aid para Jovens Cientistas (B), 2010, 22700476, e Memorial Foundation Suzuken 2010 para KT E esse estudo foi suportado por Pesquisa e Desenvolvimento de Simulação da Próxima Geração Integrada da matéria viva, uma parte do Desenvolvimento e Uso do Projeto de próxima geração de supercomputadores do MEXT, e em parte por JST ERATO Projeto Biologia Gás Suematsu.

1. Marcando eritrócitos com isotiocianato de fluoresceína Isómero I (FITC) Todos os protocolos experimentais nós utilizados foram aprovados pelo comitê de ética da Keio University School of Medicine. <ol><li> Anestesiar uma ratinhos dadores, e depois de fazer uma incisão celiotomia retirar sangue total a partir da veia cava inferior com uma seringa de 1 ml contendo pequena quantidade de heparina. O rato foi imediatamente sacrificados por injecção de pentobarbital sódico (100 mg / kg ip). </li><li> Lava-se a sangue total duas vezes com PBS (pH 7,4) por centrifugação durante 5 min a 400 g num rotor de balanço-balde. </li><li> Dissolve-se 10 mg de FITC em 5 ml de 100 mM de Na 2 HPO 4 e filtrá-lo com uma membrana com 0,22-iM poros. </li><li> Num tubo de ensaio misturar 1 ml da solução de FITC com 0,15 ml de 3 mM de glucose, 0,25 ml de 180 mM NaH 2 ml PO 4, e 1,5 de 100 mM de Na 2 HPO 4. Adicionar 0,2 ml de RBC pellet, e toque o tubo para misturar. </li><li> Manter o tubo durante 2 horas a 4 ° C e, em seguida lavar os RBCs duas vezes em PBS coradas. </li><li> Coloque uma pequena quantidade da suspensão de RBC em uma grama corrediça e ver se as RBCs aparência saudável (isto é, que a sua forma e tamanho são normal) e que a intensidade de fluorescência é suficiente. </li><li> Suspender o 0,1 ml de glóbulos vermelhos em 0,9 ml de PBS a pH 7,4, e manter a suspensão a 4 ° C até injecção. </li></ol> 2. Preparação de oxigênio-sensível Dye <ol><li> Dissolve-se 500 mg de BSA em 10 ml de PBS a pH 7,4. </li><li> Adicionar 30 mg de Pd (II)-meso-tetra (4-carboxifenil) porphine (Pd-TCPP) à solução de BSA e agitou-se durante a noite. </li><li> (Opcional) Extrair BSA-bound Pd-TCPP usando cromatografia de fluxo por gravidade ou uma coluna de spin para o separar livre Pd-TCPP. </li><li> Centrifugar a solução para remover não dissolvido Pd-TCPP, e filtrar o sobrenadante com um filtro de membrana com 0,22-iM poros. &lt;/ Li&gt; <li> Loja de 1 ml em tubos de alíquotas a -20 ° C. Evite a repetição de ciclos de congelamento e descongelamento. </li></ol> 3. Preparação dos animais <ol><li> Para a observação microscópica da microcirculação preparar uma placa de plástico com um furo 20 milímetros de diâmetro, e uma fita de 30 mm de quadrado de vidro de cobertura sobre o furo quadrado. </li><li> Anestesiar um mouse, retire a pele e preparar a pele. Coloque um cateter na cauda vão para injecção de drogas usando uma agulha 30 G ligada a um polietileno de 10-cm (PE 10) do cateter preenchido com heparinizado PBS. </li><li> Após a incisão mediana, estender um lóbulo principal do fígado na chapa de plástico, e coloque o mouse em decúbito esternal. </li><li> Preparar pequenas tiras com o envoltório de cozinha (3 mm x 8 mm) e azulejo-los em torno da borda do lobo hepático para inibir a mover-se do fígado com a respiração e mantendo-o a partir de secagem. </li><li> Observe a microcirculação hepática sob um microscópio (com luz transmitida), e confirmar que o bfluxo Lood não tem nenhuma estase no campo de visão, pelo menos, 15 min. </li><li> Lentamente injetar 0,2 ml de hemácias fluorescente etiquetado para a observação do fluxo de sangue ou 0,2 ml de Pd-TCPP solução para medição de pO 2. Esta quantidade de glóbulos vermelhos marcados com FITC serão responsáveis ​​por 1/50 de todos os RBCs em circulação na região visualizada. </li></ol> 4. Visualização do Fluxo de Sangue <ol><li> Excitar o FITC por irradiação com luz da lâmpada de mercúrio que passou através de um filtro passa-banda (450-490 nm) 1. </li><li> Gravar a imagem fluorescente com uma câmara CCD. </li></ol> 5. pO Medição 2 Pd-TCPP fosforescência é relativamente fraca e deve ser detectado com um detector de alta sensibilidade. Todas as experiências precisam de ser realizado numa sala escura. <ol><li> Os picos de absorção de Pd-TCPP são a 410 nm e 532 nm, de modo que o segundo-harmónica comprimento de onda de 532 nm é recomendado para a excitação. Este comprimento de onda pode ser gerado por um Nd: YAG pulso de laser 2. </li><li> Alimentar o feixe do laser Nd: YAG na porta apropriada no microscópio invertido, e ajustar o ponto de feixe para uma posição central no plano focal. A resolução espacial depende do tamanho do ponto de laser, que é alterado pela passagem do feixe através de um furo de pino. </li><li> Para detectar a fosforescência, anexar um filtro de longo-pass (&gt; 620 nm) e um tubo fotomultiplicador (PMT) para a outra porta do microscópio. O PMT deve ser sensível aos comprimentos de onda vermelhos, especialmente em torno de 700 nm. </li><li> Amostrar o sinal de fosforescência (500 pontos amostrados a 200 kHz) e calcular a constante de tempo de decaimento, encaixando uma função exponencial para a decomposição. </li><li> Para a calibração preparar dois conjuntos de amostras de Pd-TCPP solução com pO 2 150 mmHg e 0 mmHg. Adicionar ditionito de sódio 1% em volume de amostra para produzir ausência de oxigénio. Manter o pH das amostras a 7,4 ea temperaturatura a 37 ° C durante a calibragem. </li><li> Fixar o Stern-Volmer tempo de decaimento constante k q e livre de oxigénio luminescência τ 0 na equação (1) 3. No nosso caso, com Pd-TCPP soluções a 37 ° C e um pH de 7,4, k q é 374 mmHg -1 s -1 e 0 é τ 0,74 ms. Estes parâmetros de medição dependem do equipamento (isto é, a laser, detector, outros dispositivos ópticos) e do método de processamento de sinal, de modo a calibração é necessário em cada sistema de medição 4. τ 0 / τ = ​​1 + k q • τ 0 • pO 2 (1) </li><li> Definir o rato sobre a placa sobre a platina do microscópio, observar a microcirculação hepática, e ajustar a posição do alvo de microvasos até ao ponto de o ponto de laser. </li></ol> 6. Modelo de Hepatite <ol><li> Camundongos rápidos com livre acesso à água durante a noite. </li><li> Prepara-se uma20 mg por ml de solução de acetaminofeno (APAP) em DMSO. </li><li> Injectá-la por via intraperitoneal em 1 ml/100 g de peso corporal (ie, 200 mg / kg de peso corporal). Após a injecção, o rato pode ser dado livre acesso a comida e água 5. </li><li> 24 horas após a injecção, anestesiar o rato e expor o fígado através de uma incisão mediana. Se a hepatite é conseguido, necrose na região pericentral será facilmente visível a olho nu. </li></ol> 7. Os resultados representativos Exemplo, imagens de microcirculação hepática são mostrados na Figura 1, onde (a) é uma imagem de luz de transmissão e (b) é uma imagem de fluorescência. Individuais eritrócitos marcado com FITC são observados no filme de vídeo, e vénulas de portal, sinusóides, e vénulas centrais são reconhecidos por direcções de fluxo de sangue. Como mostrado na Figura 2, o ponto de laser irradiação de um vénula central, através de uma lente objectiva x100 foi aproximdiatamente 10 um de diâmetro. Intravascular pO 2 na microcirculação hepática foi medida em ratinhos C57BL macho / 6 ratinhos, e Figura 3 (a) mostra as relações entre pO 2 e diâmetro do vaso em vénulas de portal e central. A pO 2 gradiente de portal para vênulas centrais indica que liberam oxigênio hemácias de forma eficiente ao passar sinusóides. Como mostrado na Figura 3 (b), a média pO 2 foi de 59,8 mmHg em vasos de portal, 48,2 mmHg em sinusóides, e 38,9 mmHg em vénulas centrais. Quando produzido hepatite aguda, injetando APAP via intraperitoneal, intravascular pO 2 foi significativamente elevado em cada parte da microcirculação hepática: p &lt;0,05 na veia porta, e p &lt;0,01 em sinusóides e veias centrais (Figura 3 (c)). <img src="/files/ftp_upload/3996/3996fig1.jpg" alt="A Figura 1" /> Figura 1. Baixa ampliação-images mostrando microcirculação hepática com (a) a luz transmitida e (b) de fluorescência. <img src="/files/ftp_upload/3996/3996fig2.jpg" alt="A Figura 2" /> Figura 2. Imagem do ponto de laser irradiando uma vênula alvo central para medição pO 2. <img src="/files/ftp_upload/3996/3996fig3.jpg" alt="A Figura 3" /> Figura 3. PO 2 gradiente na microcirculação hepática de camundongos sadios e de camundongos com acetaminofeno-induzida lesão hepática. (A) mostra pO 2 versus diâmetro do vaso para o portal vénulas (PV) e vénulas central (CV), e (b) mostra o gradiente de oxigénio a partir de PV para CV através sinusóides (S). A pO 2 diferença entre PV e CV reflete o consumo de oxigênio de hepatócitos ou outras células do parênquima. (C) mostra que, em condições de hepatite induzida por APAP administração, pO 2 foi significativamente elevados em PV, S, e CV e pO 2gradiente diminuiu, indicando que o consumo de oxigênio hepático foi comprometida.

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Não temos nada a divulgar.

Fornecimento de oxigênio aos tecidos é um papel essencial da microcirculação, e um monte de papéis descrever doenças relacionadas à anatomia microvascular e reologia 6. O fluxo de sangue hepático é uma combinação de sangue arterial e venoso. Cerca de 30% do fluxo sanguíneo para o fígado é bem oxigenado sangue fornecido pela artéria hepática e os outros 70%, fornecido pela veia portal, é o fluxo venoso mal o sangue oxigenado a partir do baço e do tracto gastrointestinal 7. Doenças...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Pd (II) meso-Tetra (4-carboxifenil) porphine </td><td> Frontier Scientific, Inc. </td><td> PdT790 </td></tr><tr><td> Isotiocianato de fluoresceína isómero I </td><td> Sigma-Aldrich Co. </td><td> F7250 </td></tr><tr><td> Acetaminophen </td><td> Sigma-Aldrich Co. </td><td> A7085 </td></tr></tbody></table> <table border="1"><tbody><tr><td> Nome do equipamento </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td></td><td></td><td></td><td> Equipamento </td></tr><tr><td> tubo fotomultiplicador </td><td> Hamamatsu Photonics KK </td><td> H10722-20 </td></tr></tbody></table>

visualization and analysis of blood flow and oxygen consumption in hepatic microcirculation: application to an acute hepatitis model

Há uma grande discrepância entre a oferta ea demanda de oxigênio no fígado, porque o consumo de oxigênio hepático é relativamente elevada, mas cerca de 70% do suprimento sanguíneo hepático é pouco sangue oxigenado da veia porta derivado do trato gastrointestinal e do baço. O oxigénio é entregue a hepatócitos de sangue que flui a partir de um ramo terminal da veia porta a uma vênula central através sinusóides, e isto faz um gradiente de oxigénio em lóbulos hepáticos. O gradiente de oxigénio é um parâmetro importante física que envolve a expressão de enzimas a montante ea jusante, na microcirculação hepática, mas a falta de técnicas de medição de consumo de oxigénio na microcirculação hepática atrasou a elucidação dos mecanismos relacionados com o metabolismo de oxigénio no fígado. Por conseguinte, utilizado marcado com FITC eritrócitos para visualizar a microcirculação hepática e utilizado laser assistida phosphorimetry para medir a pressão parcial de oxigénio no microvasos aí. Noncontact e medição óptico contínuo pode quantificar velocidades de fluxo de sangue, diâmetros dos vasos e gradientes de oxigênio relacionadas com o consumo de oxigênio no fígado. Em um modelo de hepatite aguda que fizemos através da administração de paracetamol para ratos, observamos pressão de oxigênio aumentou em ambos os vênulas portal e central, mas um gradiente de diminuição de oxigênio nos sinusóides, indicando que a necrose dos hepatócitos na zona pericentral poderia mudar a pressão de oxigênio e afetar a expressão da enzima na zona periportal. Em conclusão, os nossos métodos ópticos para hemodinâmica hepáticas de medição e consumo de oxigênio pode revelar mecanismos relacionados à doença hepática.

Watch this Scientific Journal Video about Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model at JoVE.com

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

Um sistema óptico foi desenvolvido para visualizar a microcirculação hepática com eritrócitos marcados com FITC e para medir a pressão parcial de oxigénio no microvasos com laser assistida phosphorimetry. Este método pode ser usado para investigar os mecanismos fisiológicos e patológicos, analisando a estrutura microvascular, o diâmetro, a velocidade de fluxo de sangue, e tensão de oxigénio.

Visualização e análise do fluxo sangüíneo e consumo de oxigênio na microcirculação hepática: aplicação para um modelo Hepatite Aguda

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

visualization-analysis-blood-flow-oxygen-consumption-hepatic

Research

JoVE Journal

Medicine

12.5K visualizações.  Keio University. Um sistema óptico foi desenvolvido para visualizar a microcirculação hepática com eritrócitos marcados com FITC e para medir a pressão parcial de oxigénio no microvasos com laser assistida phosphorimetry. Este método pode ser usado para investigar os mecanismos fisiológicos e patológicos, analisando a estrutura microvascular, o diâmetro, a velocidade de fluxo de sangue, e tensão de oxigénio.

Vídeo: Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

Modelo de tumor hepático murino Bioluminescent

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

Medicina

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

Heterotópico Rat auxiliar Liver Transplantation com fluxo regulado Portal Vein Arterialização na Insuficiência Hepática Aguda

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

O coelho Modelo Blood-shunt para o Estudo da aguda e tardia Sequelas de hemorragia subaracnóidea: Aspectos Técnicos

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

Contraste melhorado Ecografia de Avaliação da Medula Espinhal fluxo de sangue em Experimental lesão medular

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Oxigênio-Glucose Privação e reoxigenação como um<em&gt; In Vitro</em&gt; Isquemia e reperfusão Lesão modelo para estudar Disfunção Barreira Hematoencefálica

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

Um Modelo aguda da retina para a Avaliação sanguíneos da retina barreira Descumprimento e potenciais fármacos para o tratamento

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Análise otimizada de<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; A esteatose hepática

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Um modelo de isquemia cardíaca crônica em suínos, usando um Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Analisando a taxa de consumo de oxigênio em Mouse culto principal Cardiomyocytes Neonatal utilizando um analisador de fluxo extracelular

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.