Les auteurs remercient Mme Risa Otsuka pour l&#39;assistance technique et les expériences d&#39;aide. Cette recherche a été partiellement soutenu par le Ministère de l&#39;Education, la Science, des Sports et de la Culture, Grant-in-Aid pour jeunes scientifiques (B), 2010, 22700476 et Suzuken Memorial Foundation 2010 pour KT et cette étude a été soutenue par la recherche et le développement de la simulation de la prochaine génération intégrée de la matière vivante, une partie de la mise au point et l&#39;utilisation du projet superordinateur de la prochaine génération du MEXT, et en partie par JST ERATO Suematsu projet Biologie gaz.

1. Étiquetage érythrocytes avec l&#39;isothiocyanate de fluorescéine isomère I (FITC) Tous les protocoles expérimentaux que nous avons utilisées ont été approuvées par le Comité de protection des animaux de Keio University School of Medicine. <ol><li> Anesthésier un souris donneuses, et après avoir fait une incision coeliotomie retirer le sang total de la veine cave inférieure avec une seringue de 1 ml contenant petite quantité d&#39;héparine. La souris a été immédiatement euthanasié par injection de pentobarbital sodique (100 mg / kg ip). </li><li> Laver le sang total deux fois avec du PBS (pH 7,4) par centrifugation pendant 5 min à 400 g dans un rotor à oscillation-seau. </li><li> Dissolvez 10 mg de FITC dans 5 ml de 100 mM de Na 2 HPO 4 et filtrer avec une membrane ayant des pores de 0,22 um. </li><li> Dans un tube à essai mélanger 1 ml de la solution de FITC avec 0,15 ml de 3 mM de glucose, 0,25 ml de 180 mM NaH 2 PO 4 ml, et 1,5 de 100 mM de Na 2 HPO 4. Ajouter 0,2 ml de la RColombie-Britannique à granulés, et appuyez sur le tube pour mélanger. </li><li> Garder le tube pendant 2 heures à 4 ° C, puis laver les hématies colorées dans du PBS deux fois. </li><li> Mettez une petite quantité de la suspension de RBC sur une herbe diapositive et de voir si les globules rouges semblent en bonne santé (à savoir que leur forme et la taille sont normaux) et que l&#39;intensité de fluorescence est suffisante. </li><li> Suspendre l&#39;0,1 ml de globules rouges dans 0,9 ml de PBS à pH 7,4, et de garder la suspension à 4 ° C jusqu&#39;à l&#39;injection. </li></ol> 2. Préparation de l&#39;oxygène colorant sensible <ol><li> Dissoudre 500 mg de BSA dans 10 ml de PBS à pH 7,4. </li><li> Ajouter 30 mg de Pd (II)-méso-tétra (4-carboxyphényl) porphine (Pd-TCPP) à la solution de SAB et agitation pendant une nuit. </li><li> (Facultatif) Extrait BSA lié Pd-TCPP par écoulement par gravité en utilisant la chromatographie ou une colonne de centrifugation pour le séparer de libre-Pd TCPP. </li><li> Centrifuger la solution pour éliminer non dissous Pd-TCPP, et filtrer le surnageant avec un filtre à membrane ayant des pores de 0,22 pm. &lt;/ Li&gt; <li> Magasin de 1 ml aliquotes dans des tubes à -20 ° C. Éviter la répétition des cycles gel-dégel. </li></ol> 3. Préparation des animaux <ol><li> Pour l&#39;observation microscopique de la microcirculation de préparer une plaque en plastique avec un trou de 20 mm de diamètre, et la bande d&#39;un verre de 30 mm carrés couverture carrée sur le trou. </li><li> Anesthésier une souris, retirez la fourrure, et préparer la peau. Placer un cathéter dans la queue vain l&#39;injection de drogues en utilisant une aiguille 30 G connecté à un polyéthylène de 10 cm (PE 10) cathéter rempli de PBS hépariné. </li><li> Après incision médiane, d&#39;étendre un lobule principale du foie sur la plaque en plastique et placez la souris en décubitus sternal. </li><li> Établir des feuillets d&#39;une pellicule de petites cuisine (3 mm x 8 mm) et la tuile entre eux autour du bord du lobe hépatique d&#39;inhiber le déplacement du foie avec la respiration et l&#39;empêcher de séchage. </li><li> Observez la microcirculation hépatique sous un microscope (à lumière transmise), et de confirmer que le bdébit lood n&#39;a pas la stase dans le champ de vision pendant au moins 15 min. </li><li> Injecter lentement 0,2 ml de globules rouges marqués par fluorescence pour l&#39;observation du flux sanguin ou 0,2 ml de Pd-TCPP solution pour pO 2 de mesure. Cette quantité de globules rouges marqués au FITC comptera pour 1/50 de tous les globules rouges en circulation dans la région visualisée. </li></ol> 4. Visualisation de l&#39;écoulement du sang <ol><li> Exciter l&#39;FITC en l&#39;irradiant avec de la lumière lampe à mercure qui a traversé un filtre passe-bande (450-490 nm) 1. </li><li> Enregistrez l&#39;image fluorescente avec une caméra CCD. </li></ol> 5. pO 2 Mesure Pd-TCPP phosphorescence est relativement faible et devrait être détectés avec un détecteur à haute sensibilité. Toutes les expériences doivent être effectuées dans une pièce sombre. <ol><li> Les pics d&#39;absorption de Pd-TCPP sont à 410 nm et 532 nm, de sorte que la longueur d&#39;onde de seconde harmonique à 532 nm est recommandé pour l&#39;excitation. Cette longueur d&#39;onde peut être générée par un laser Nd: YAG impulsion laser 2. </li><li> Faites passer le faisceau de laser Nd: YAG dans le port approprié sur le microscope inversé, et d&#39;ajuster le point de faisceau à une position centrale dans le plan focal. La résolution spatiale dépend de la taille du spot laser, ce qui est changé en passant le faisceau à travers un trou d&#39;épingle. </li><li> À détecter la phosphorescence, attacher un filtre passe-long (&gt; 620 nm) et un tube photomultiplicateur (PMT) à l&#39;autre port du microscope. Le PMT devrait être sensible aux longueurs d&#39;onde rouges, en particulier autour de 700 nm. </li><li> Échantillonner le signal de phosphorescence (500 points échantillonnés à 200 kHz) et calculer la constante de temps de la décroissance en ajustant une fonction exponentielle de la désintégration. </li><li> Pour l&#39;étalonnage préparer deux ensemble d&#39;échantillons de Pd-TCPP solution avec pO 2 150 mmHg et 0 mmHg. Ajouter dithionite de sodium 1% en volume à utiliser pour produire l&#39;absence d&#39;oxygène. Maintenir le pH des échantillons à 7,4 et la températureture à 37 ° C pendant l&#39;étalonnage. </li><li> Fixer le Stern-Volmer constante de temps k q et de l&#39;oxygène sans décroissance de la luminescence τ 0 dans l&#39;équation (1) 3. Dans notre cas, avec Pd-TCPP solutions à 37 ° C et un pH de 7,4, k q est de 374 mmHg -1 s -1 et τ 0 est de 0,74 ms. Ces paramètres de mesure dépendent de l&#39;équipement (c.-à-laser, détecteur, d&#39;autres dispositifs optiques) et la méthode de traitement du signal, afin d&#39;étalonnage est nécessaire dans chaque système de mesure 4. τ 0 / τ = ​​1 + k q • τ 0 • pO 2 (1) </li><li> Régler la souris sur la plaque sur la platine du microscope, observer la microcirculation hépatique, et d&#39;ajuster la position de la cible des microvaisseaux au point de la tache laser. </li></ol> 6. Modèle de l&#39;hépatite <ol><li> Souris rapide avec un accès gratuit à l&#39;eau durant la nuit. </li><li> Préparer un20 mg par ml de solution d&#39;acétaminophène (APAP) dans le DMSO. </li><li> Injecter par voie intrapéritonéale à elle 1 ml/100 g de poids corporel (soit 200 mg / kg de poids corporel). Après l&#39;injection, la souris peut être donné libre accès à la nourriture et l&#39;eau 5. </li><li> 24 heures après l&#39;injection, anesthésier la souris et d&#39;exposer le foie en faisant une incision médiane. Si l&#39;hépatite est atteint, la nécrose dans la région péricentral sera facilement visible à l&#39;œil nu. </li></ol> 7. Les résultats représentatifs Des exemples d&#39;images de la microcirculation hépatique sont représentés à la figure 1, où (a) est une image de lumière transmise et (b) est une image de fluorescence. Individuels érythrocytes marqués au FITC sont observées dans le film vidéo, et des veinules de portail, sinusoïdes, et des veinules centrales sont reconnus par les directions d&#39;écoulement de sang. Comme le montre la figure 2, le spot laser irradiant une veinule centrale à travers une lentille de l&#39;objectif x100 était Approximimmédiatement 10 microns de diamètre. Intravasculaire pO 2 dans la microcirculation hépatique a été mesuré dans C57BL mâle / 6 souris, et la figure 3 (a) montre les relations entre pO 2 et le diamètre des vaisseaux dans les veinules portales et centrale. Le gradient de pO 2 à partir du portail aux veinules centrales indique que les globules rouges libèrent de l&#39;oxygène de manière efficace tout en passant par des sinusoïdes. Comme le montre la figure 3 (b), la moyenne était de 59,8 pO 2 mmHg dans les vaisseaux, 48,2 mmHg portail de sinusoïdes, et de 38,9 mmHg dans les veinules centrales. Lorsque nous avons produit une hépatite aiguë par injection intrapéritonéale APAP, intravasculaire pO 2 a été significativement plus élevées dans chaque partie de la microcirculation hépatique: p &lt;0,05 dans les veines portes et p &lt;0,01 dans les sinusoïdes et les veines centrales (figure 3 (c)). <img src="/files/ftp_upload/3996/3996fig1.jpg" alt="Figure 1" /> Figure 1. Faible grossissement images montrant la microcirculation hépatique, avec (a) la lumière transmise et (b) de fluorescence. <img src="/files/ftp_upload/3996/3996fig2.jpg" alt="Figure 2" /> Figure 2. Image de spot laser irradiant une veinule ciblée central pour pO 2 de mesure. <img src="/files/ftp_upload/3996/3996fig3.jpg" alt="Figure 3" /> Figure 3. PO 2 gradient dans la microcirculation hépatique des souris témoins sains et des souris atteintes d&#39;une lésion hépatique induite par l&#39;acétaminophène. (A) montre pO 2 par rapport diamètre du vaisseau pour le portail veinules (PV) et le centre de veinules (CV), et (b) montre le gradient d&#39;oxygène à partir de PV pour CV par sinusoïdes (s). La pO 2 différence entre PV et CV reflète la consommation d&#39;oxygène des hépatocytes ou d&#39;autres cellules du parenchyme. (C) montre que dans des conditions hépatite induite par l&#39;administration APAP, pO 2 a été significativement plus élevée dans les PV, S, et le CV et la pO 2gradient a diminué, ce qui indique que la consommation d&#39;oxygène hépatique a été compromise.

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Nous n&#39;avons rien à communiquer.

Fournir de l&#39;oxygène vers les tissus est un rôle essentiel de la microcirculation, et beaucoup de documents décrivent les maladies liées à l&#39;anatomie et la rhéologie des microvaisseaux 6. Le flux sanguin du foie est une combinaison de sang artériel et veineux. Environ 30% du flux sanguin vers le foie est bien oxygéné du sang fourni par l&#39;artère hépatique et l&#39;autre 70%, fourni par la veine porte, est la sortie du sang mal oxygéné veineux de la rate et du tractus gastro 7.</su...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Pd (II) méso-tétra (4-carboxyphényl) porphine </td><td> Frontier Scientific, Inc </td><td> PdT790 </td></tr><tr><td> Isothiocyanate de fluorescéine isomère I </td><td> Sigma-Aldrich Co. </td><td> F7250 </td></tr><tr><td> L&#39;acétaminophène </td><td> Sigma-Aldrich Co. </td><td> A7085 </td></tr></tbody></table> <table border="1"><tbody><tr><td> Nom de l&#39;équipement </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td></td><td></td><td></td><td> Équipement </td></tr><tr><td> Tube photomultiplicateur </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

Il ya un écart considérable entre l&#39;offre et la demande en oxygène dans le foie, car la consommation d&#39;oxygène hépatique est relativement élevé, mais environ 70% de l&#39;approvisionnement sanguin hépatique est mal oxygéné sang de la veine portail dérivé du tractus gastro-intestinal et la rate. L&#39;oxygène est livrée à des hépatocytes par le sang s&#39;écoulant d&#39;une branche terminale de la veine porte à une veinule centrale via sinusoïdes, ce qui rend un gradient d&#39;oxygène en lobules hépatiques. Le gradient d&#39;oxygène est un paramètre important physique qui implique l&#39;expression d&#39;enzymes en amont et en aval de la microcirculation hépatique, mais le manque de techniques pour mesurer la consommation d&#39;oxygène dans la microcirculation hépatique a retardé l&#39;élucidation des mécanismes relatifs à métabolisme de l&#39;oxygène dans le foie. On a donc utilisé marqué FITC érythrocytes de visualiser la microcirculation hépatique et utilisé assistée par laser Phosphorimétrie pour mesurer la pression partielle d&#39;oxygène dans le microvaisseaux là. Noncontact et continue de mesure optique permet de quantifier les vitesses d&#39;écoulement de sang, les diamètres des navires, et les gradients d&#39;oxygène liés à la consommation d&#39;oxygène dans le foie. Dans un modèle de l&#39;hépatite aiguë nous avons fait en administrant de l&#39;acétaminophène pour les souris que nous avons observées pression d&#39;oxygène a augmenté dans les deux veinules portales et central, mais un gradient d&#39;oxygène a diminué dans les sinusoïdes, indiquant que la nécrose des hépatocytes dans la zone péricentral pourrait modifier la pression d&#39;oxygène et affecter l&#39;expression de l&#39;enzyme dans la zone périportale. En conclusion, nos méthodes optiques pour l&#39;hémodynamique hépatique de mesure et de la consommation d&#39;oxygène peut révéler des mécanismes liés à la maladie hépatique.

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

Un système optique est développé pour visualiser la microcirculation hépatique avec érythrocytes FITC marqués et pour mesurer la pression partielle d&#39;oxygène dans le microvaisseaux avec assistée par laser Phosphorimétrie. Cette méthode peut être utilisée pour étudier les mécanismes physiologiques et pathologiques en analysant la structure microvasculaire, le diamètre, la vitesse d&#39;écoulement du sang, et la tension d&#39;oxygène.

Visualisation et analyse du flux sanguin et la consommation d&#39;oxygène dans la microcirculation hépatique: Application à un modèle de l&#39;hépatite aiguë

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 vues.  Keio University. Un système optique est développé pour visualiser la microcirculation hépatique avec érythrocytes FITC marqués et pour mesurer la pression partielle d'oxygène dans le microvaisseaux avec assistée par laser Phosphorimétrie. Cette méthode peut être utilisée pour étudier les mécanismes physiologiques et pathologiques en analysant la structure microvasculaire, le diamètre, la vitesse d'écoulement du sang, et la tension d'oxygène.

Vidéo: 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.

Modèle murin Bioluminescent tumeurs hépatiques

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

Recherche

Médecine

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.

Hétérotopique auxiliaire Rat Liver Transplantation avec un débit régulé veine porte artérialisation dans l&#39;insuffisance hépatique aiguë

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.

Le lapin de sang-shunt modèle pour l&#39;étude de aiguë et les séquelles tardives de hémorragie méningée: Aspects techniques

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 échographie amélioré pour l&#39;évaluation de la moelle épinière flux sanguin dans la partie expérimentale Traumatisme médullaire

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.

Oxygène-glucose privation et réoxygénation comme un<em&gt; In Vitro</em&gt; Ischémie-reperfusion modèle pour étudier la dysfonction barrière hémato-encéphalique

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.

Un modèle aigu rétinienne pour l&#39;évaluation sanguin rétinien barrière Breach et potentiels médicaments pour le traitement

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.

Analyse optimisée des<em&gt; In Vivo</em&gt; et<em&gt; In vitro</em&gt; Stéatose hépatique

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.

Un modèle d’ischémie cardiaque chronique chez les porcs à l’aide d’un Constrictor Améroïde

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.

Analyse des taux de consommation d’oxygène dans les Cardiomyocytes néonatals cultivés primaires de souris à l’aide d’un analyseur de Flux extracellulaire

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.