Ce travail a été soutenu par une subvention de la National Institutes of Health, R01CA142928.

1. Injection intraveineuse de bleu d&#39;Evans dans la veine de la queue de la souris latéral <ol><li> Préparer une solution à 0,5% stérile de bleu d&#39;Evans dans du PBS. Si nécessaire, stériliser par filtration la solution pour éliminer toute particule qui n&#39;a pas dissous. </li><li> Aspirer 200 pi Bleu Evans solution dans une seringue. Évitez toutes les bulles d&#39;air qui auraient échappé dans la seringue. </li><li> Placer les souris qui sont de 8-12 semaines vieux dans un dispositif de retenue pour que l&#39;animal n&#39;est pas librement mobile, mais sa queue peut être manipulé. </li><li> Placez le dispositif de retenue de la souris sur le côté afin de la veine latérale de la queue est facilement visible et sont tournées vers le haut. </li><li> Tenez la queue avec la main non dominante entre le pouce et l&#39;index. </li><li> Insérer l&#39;aiguille (petit calibre, 27-30) à une avance 10-15 degrés d&#39;angle, biseau vers le haut, dans la veine latérale de la queue dans la direction de la tête. Gardez l&#39;aiguille et la seringue parallèlement à la queue. </li><li> Do ne s&#39;applique pas contre-pression pour confirmer le placement approprié car cela pourrait réduire la veine. </li><li> Injecter lentement 200 pi Bleu Evans solution dans la veine de la queue de la souris. </li><li> Observez la facilité avec laquelle les progrès piston, car c&#39;est la preuve de la mise en place correcte de l&#39;aiguille dans la veine. </li><li> Placez la souris de nouveau dans sa cage et l&#39;observer pendant 30 min. </li></ol> 2. Collection d&#39;organes et d&#39;extraction de Bleu d&#39;Evans des organes <ol><li> Sacrifier les souris par dislocation cervicale. Pour Miles fins de dosage dislocation cervicale est recommandée car elle limite l&#39;interférence significative avec la perméabilité vasculaire. Sacrifier toutes les souris en même temps, aussi vite que possible. Travailler avec les cohortes de 6 souris ou moins, car peu de temps après la mort vaisseaux sanguins deviennent plus perméables. </li><li> Placez la souris sur le dos et la broche leurs pieds sur un tableau blanc. </li><li> Ouvrir la cavité abdominale et thoracique pour exposer thorACIC et organes abdominaux. </li><li> Prenez des photos représentatives de montrer les différences dans l&#39;extravasation du bleu d&#39;Evans. Inclure toutes les souris dans le même domaine afin d&#39;avoir des conditions d&#39;éclairage identiques pour toutes les souris. </li><li> Recueillir des organes d&#39;intérêts et de les mettre dans des tubes de 1,5 ml </li><li> Peser un tube vide et apporter la valeur d&#39;équilibre à zéro. </li><li> Transférer l&#39;échantillon de tissu et il pondérer. Répétez l&#39;opération pour tous les échantillons de tissus. Les tissus peuvent être séchés à l&#39;air pour éliminer la variabilité de la teneur en eau entre les différents organes. </li><li> . Ajouter 500 ul de formamide dans chaque tube échantillon de tissu. </li><li> Transférez tous les tubes à un bain d&#39;eau à 55 ° C ou du bloc chauffant. Incuber pendant 24-48 heures pour extraire Evans Blue à partir de tissu. </li></ol> 3. Quantification de Evans Blue extravasé dans le tissu interstitiel <ol><li> Centrifuger le formamide / Bleu Evans mélange de granulés tous les fragments de tissus restants. </li><li> Mesurer l&#39;absorbance à 610nm. Utiliser 500 ul formamide comme vide. </li><li> Calculer ng extravasation du bleu Evans par mg de tissu. </li><li> Tracer toutes les données dans un graphique. </li><li> Effectuer une analyse statistique pour déterminer les différences significatives. </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>

Les auteurs n&#39;ont aucun conflit ou en concurrence intérêts financiers.

La perméabilité vasculaire est un marqueur essentiel pour l&#39;état des vaisseaux sanguins. Augmentation de la perméabilité vasculaire a été montré pour être présent dans plusieurs maladies systémiques telles que le diabète, l&#39;hypertension et les maladies auto-immunes 6,7,8. Augmentation de la perméabilité vasculaire a été montré pour être médiée par la contrainte de cisaillement, des facteurs de croissance tels que le facteur de croissance endothélial vasculaire et le facteur de cro...

Formation et le maintien des barrières perméables sélectives sont essentiels pour le développement des organes appropriée et 1,2 performance. Les cellules endothéliales tapissent la lumière des vaisseaux sanguins et de former une barrière semi-perméable qui est essentiel dans le transport sélectif entre le sang et l&#39;espace interstitiel de tous les organes. Une barrière de perméabilité suffisante est maintenue par serrées cellule à cellule jonctions qui sont strictement contrôlées par des facteurs de croissance, des cytokines et des molécules de stress autres produits 3. Rupture de la barrière de cellules endothéliales peut entraîner une plus grande perméabilité et une fuite vasculaire. Ces effets sont observés dans divers états pathologiques et la compréhension de la signalisation moléculaire souligner nécessite des méthodes pluridisciplinaires 4,5. Dans cet article, nous décrivons une méthode in vivo pour mesurer la perméabilité des vaisseaux en utilisant un modèle de souris. Le test que nous décrivons, également connu sous le nom d&#39;analyse Miles, est un bien établMINE Procédé pour tester la perméabilité vasculaire in vivo. Le test est basé sur le fait que, dans des conditions physiologiques basales, l&#39;albumine ne traverse pas la barrière endothéliale. Bleu d&#39;Evans, un colorant azoïque avec une forte affinité pour l&#39;albumine, est injecté dans le flux sanguin d&#39;un animal expérimental, et dans des conditions physiologiques, il est prévu d&#39;être limité à l&#39;intérieur des vaisseaux sanguins. Lorsqu&#39;un stimulus perméabilité vasculaire est ajouté, par voie topique ou systémique, les vaisseaux sanguins commencent à couler protéine et donc, aussi le bleu Evans qui est lié à l&#39;albumine. Cela se traduit par une coloration bleutée des tissus rapide dont les navires perméables. Injection réussie du colorant dans la veine de la queue de la souris latéral est essentiel pour la bonne réussite de l&#39;expérience. Technique d&#39;injection queue veine exige de la pratique approfondie et doivent être maîtrisées avant de commencer l&#39;expérience. La perméabilité des vaisseaux est fortement dépendante de l&#39;âge et du poids de l&#39;unimal, donc lorsque l&#39;on compare différentes souches de souris, il est impératif que les souris ou les sujets de test ont d&#39;autres proches de la date de naissance et le poids identiques. Parmi les autres facteurs qui influent sur la perméabilité de la barrière endothéliale sont des conditions environnementales comme la température, l&#39;humidité, et c&#39;est très important, le stress de manipulation de la souris. En raison de la multitude de facteurs qui peuvent influencer le résultat de l&#39;expérience, il est toujours conseillé que l&#39;expérience est répétée au moins trois fois et l&#39;analyse statistique effectuée. Ce test peut être utilisé ou pour comparer la perméabilité des vaisseaux de souris qui ont été génétiquement modifiés, ainsi que les souris de différentes origines génétiques. On peut évaluer la perméabilité à la présence ou l&#39;absence d&#39;un stimulus, en fonction de la fonction du gène qui est modulé. Ce test peut également être utilisé ou pour tester l&#39;effet de différents composés sur la perméabilité des vaisseaux.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> REACTIF </td></tr><tr><td> Evans Blue </td><td> SIGMA </td><td> E2129 </td><td></td></tr><tr><td> FORMAMIDE </td><td> INVITROGEN </td><td> 15515-026 </td><td></td></tr><tr><td> PBS </td><td></td><td></td><td> Phosphate 0,2 M 1,5 M de NaCl pH 7,4 </td></tr><tr><td></td><td></td><td></td><td> ÉQUIPEMENT </td></tr><tr><td> SPECTROPHOTOMÈTRE </td><td> EPPENDORF </td><td> 952000006 </td><td></td></tr><tr><td> DISPOSITIF DE RETENUE SOURIS </td><td> HARVARD APPARATUS </td><td> 340012 </td><td></td></tr><tr><td> SERINGUE </td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> AIGUILLES </td><td> BD </td><td> 305106 </td><td> La jauge de l&#39;aiguille dépend de la taille de l&#39;animal. </td></tr><tr><td> SOLDE </td><td> Denver Instrument </td><td> TP-64 </td><td></td></tr></tbody></table>

an in vivo assay to test blood vessel permeability

Cette méthode est basée sur l&#39;injection intraveineuse de bleu d&#39;Evans dans la souris comme modèle animal d&#39;essai. Bleu Evans est un colorant qui se lie albumine. Dans les conditions physiologiques de l&#39;endothélium est imperméable à l&#39;albumine, de sorte Evans bleus assortis de restes d&#39;albumine limitée dans les vaisseaux sanguins. Dans des conditions pathologiques qui favorisent l&#39;augmentation des cellules endothéliales vasculaires perméabilité partiellement perdent leurs contacts étroits et l&#39;endothélium devient perméable à de petites protéines comme l&#39;albumine. Cette condition permet d&#39;extravasation de bleu d&#39;Evans dans les tissus. Un endothélium sain empêche extravasation du colorant dans les tissus vascularisés voisins. Organes ayant une perméabilité accrue montrera nettement augmenté par rapport à coloration bleue des organes avec endothélium intact. Le niveau de perméabilité vasculaire peut être évaluée par simple visualisation ou par mesure quantitative du colorant constituée par milligramme de tissu de contrôle par rapport à des animaux d&#39;expérimentation / tissu. Deux PoweRFUL aspects de ce test sont sa simplicité et caractéristiques quantitatives. Colorant bleu Evans peut être extrait à partir de tissus en incubant une quantité spécifique d&#39;un tissu en le formamide. Maximale d&#39;absorbance Evans Blue est à 620 nm et un minimum d&#39;absorption est à 740 nm. À l&#39;aide d&#39;une courbe d&#39;étalonnage pour le bleu Evans, mesures de densité optique peut être transformé en colorant milligramme capturé par milligramme de tissu. L&#39;analyse statistique doit être utilisée pour évaluer des différences significatives dans la perméabilité vasculaire.

Nous avons utilisé un test de perméabilité in vivo pour tester les fuites navire dans des souris âgées de 8-12 semaines. Ce test est utile pour comparer la perméabilité relative vasculaire entre les animaux de fond génétique différent ou dans une seule souche de souris soumises à des traitements qui affectent le système vasculaire. Nos résultats montrent que la souche de souris génétiquement modifiées, nous avons créé dans notre laboratoire dispose d&#39;un endothélium plus perméabl...

Watch this Scientific Journal Video about An in vivo Assay to Test Blood Vessel Permeability at JoVE.com

An in vivo Assay to Test Blood Vessel Permeability

Nous présentons une<em&gt; In vivo</em&gt; Test afin de tester la perméabilité des vaisseaux sanguins. Ce test est basé sur l&#39;injection intraveineuse d&#39;un colorant et visualisation ultérieure de sa diffusion dans les espaces interstitiels.

Un<em&gt; In vivo</emTest&gt; Tester la perméabilité des vaisseaux sanguins

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 vues.  Fox Chase Cancer Center. Nous présentons une In vivo Test afin de tester la perméabilité des vaisseaux sanguins. Ce test est basé sur l'injection intraveineuse d'un colorant et visualisation ultérieure de sa diffusion dans les espaces interstitiels.

Vidéo: 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.

Induits par la chimiothérapie vasculaire Toxicité - en temps réel<em&gt; In vivo</em&gt; Imagerie de déficience navire

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

Recherche

Médecine

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 intestinale Sacs pour évaluer la perméabilité de la muqueuse dans les modèles de maladies gastro-intestinales

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.

Appauvrissement des cellules de souris de xénogreffes de tumeurs humaines améliore de manière significative l&#39;analyse en aval des cellules cibles

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.

Essais in vitro pour évaluer la formation et la perturbation du vaisseau en forme de barrière de sang-cerveau

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.

L&#39;approche d&#39;échantillonnage à 4 navires pour les études intégratives de la physiologie du plaidoyer humain<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-artérialisé Transplantation de foie partielle de Rat in vivo axée sur le navire 70 % hépatectomie

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.

Une méthode In Vivo pour l’évaluation de la barrière intestinale-sang et le métabolisme hépatique du microbiote produits

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.

Utilisation d’une application de test Hirschberg automatisée pour évaluer l’alignement oculaire

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.

Mesure du débit sanguin strial dans la cochlée de souris à l’aide d’une fenêtre vasculaire ouverte et d’une microscopie à fluorescence intravitale

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.

Les tranches pulmonaires coupées avec précision comme outil efficace pour les études ex vivo sur la structure et la contractilité des vaisseaux pulmonaires

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