Déclaration éthique: Toutes les expériences ont été approuvés par le Comité de protection des animaux et l&#39;utilisation institutionnelle. les soins des animaux était selon les directives institutionnelles. Souris ICR femelle (7 - 8 semaines; 25-30 g) ont été logés dans la climatisation, l&#39;éclairage contrôlé les animaleries de la Faculté de médecine Sackler de l&#39;Université de Tel-Aviv. À terme, les animaux ont été euthanasiés à un surdosage anesthésie. 1. fibré confocale microscopie de fluorescence (FCFM) Calibration <ol><li> Allumer l&#39;appareil. </li><li> Connectez le microsonde (mini0 / 30). </li><li> Calibrer l&#39;appareil selon les instructions du fabricant. </li></ol> 2. Préparation souris for Imaging <ol><li> Anesthetize par une injection sous-cutanée de deux Ketaset (100 mg / kg) et XYL-M2 (6 mg / kg). Confirmez anesthésie appropriée par l&#39;absence de réponse aux pieds-pincement. </li><li> Inciser la peau en dessous de l&#39;aine pour<html> révéler les vaisseaux artériels fémoraux. Gardez le site d&#39;incision humide avec une solution saline suivante incision. </li><li> Chauffer la queue en utilisant un sac (ou un gant) rempli d&#39;eau tiède (pas trop chaude au toucher) pendant environ 30 secondes. Préparer une administration intraveineuse (IV) de dérivation pour l&#39;administration de FITC dextran (agent de contraste) et de sérum physiologique ou un agent chimiothérapeutique, en insérant une aiguille (30 G, 1/2 de pouce) dans la veine caudale et la fixation d&#39;une seringue de 1 ml de ce . Vérifiez la veine est ouverte en injectant une solution saline. REMARQUE: Une administration intraveineuse de FITC dextran (poids moléculaire élevé; 100 ul; 10 mg / ml; 2,000 kDa) facilite la visualisation de la microvascularisation fémoral par FCFM. La doxorubicine (100 ul; 8 mg / kg, l&#39;adriamycine) ou une solution saline sont également plus tard administrés IV dans la veine caudale préchauffé. </li><li> Placez la souris passivement sur une scène de polystyrène. Fixez la souris pour le patin et maintenir la position à l&#39;aide de ruban adhésif chirurgical. </li></ol>title &quot;&gt; 3. Imagerie des fémorales vaisseaux sanguins en FCFM Pendant et après l&#39;administration de la doxorubicine ou Saline NOTE: Le microscope confocale fibrée utilisée dans cette étude se compose de deux unités: (1) la microsonde (mini0 / 30). (2) unité de balayage laser (LSU-488; longueur d&#39;onde 488 nm). <ol><li> Effectuer tous les fuseaux-tours analyses utilisant la longueur d&#39;onde laser nm 488 LSU. NOTE: Le détecteur de l&#39;unité principale détecte la filtré (500-650 nm) fluorescence émise. Les images acquises sont ensuite reconstruits et affichées à un rythme de 12 images / sec. </li><li> Débranchez soigneusement la seringue de l&#39;aiguille et de joindre une nouvelle seringue contenant FITC dextran. Administrer (IV) 100 pi de FITC dextran. </li><li> Mettre la microsonde (mini0 / 30) à un champ de vue approprié et il fixer, après l&#39;ajustement de l&#39;axe z, afin d&#39;obtenir l&#39;image correspondante. Attendez que le signal initial à se estomper jusqu&#39;à ce qu&#39;un signal clair et ciblé est visible. </li><li> Enregistrer un flux sanguin de référence pour une période de stabilisation court (~ 30 sec). Puis connecter à l&#39;aiguille une autre seringue, contenant soit doxorubicine ou une solution saline. IV administrer 100 pi doxorubicine ou une solution saline. </li><li> Surveiller le flux injecté de FITC-dextrane en continu pendant 20 min. Sur le logiciel de FCFM-associé, utilisez le bouton de diamètre sur la règle supérieure afin de mesurer les vaisseaux sanguins et les classer comme faible (&lt;15 um) ou grande (&gt; 15 um). </li><li> Euthanasier l&#39;animal à un surdosage d&#39;anesthésie. </li></ol>

<ol>
	<li>Chow, A. Y., 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=Anthracyclines+cause+endothelial+injury+in+pediatric+cancer+patients:+a+pilot+study.">Anthracyclines cause endothelial injury in pediatric cancer patients: a pilot study.</a> J Clin Oncol. 24 (6), 925-928 (2006).</li><li>Nuver, J., 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=Acute+chemotherapy-induced+cardiovascular+changes+in+patients+with+testicular+cancer.">Acute chemotherapy-induced cardiovascular changes in patients with testicular cancer.</a> J Clin Oncol. 23 (36), 9130-9137 (2005).</li><li>Vos, F. Y., 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=Endothelial+cell+effects+of+cytotoxics:+balance+between+desired+and+unwanted+effects.">Endothelial cell effects of cytotoxics: balance between desired and unwanted effects.</a> Cancer Treat Rev. 30 (6), 495-513 (2004).</li><li>Kerbel, R. S., 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='Accidental'+anti-angiogenic+drugs.+anti-oncogene+directed+signal+transduction+inhibitors+and+conventional+chemotherapeutic+agents+as+examples.Eur.">'Accidental' anti-angiogenic drugs. anti-oncogene directed signal transduction inhibitors and conventional chemotherapeutic agents as examples.Eur.</a> J Cancer. 36 (10), 1248-1257 (2000).</li><li>Soultati, A., 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=Endothelial+vascular+toxicity+from+chemotherapeutic+agents:+preclinical+evidence+and+clinical+implications.">Endothelial vascular toxicity from chemotherapeutic agents: preclinical evidence and clinical implications.</a> Cancer Treat Rev. 38 (5), 473-483 (2012).</li><li>Tempelhoff, G. 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=Blood+coagulation+during+adjuvant+epirubicin/cyclophosphamide+chemotherapy+in+patients+with+primary+operable+breast+cancer.">Blood coagulation during adjuvant epirubicin/cyclophosphamide chemotherapy in patients with primary operable breast cancer.</a> J Clin Oncol. 14 (9), 2560-2568 (1996).</li><li>Ben Aharon, I., 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=Doxorubicin-induced+vascular+toxicity--targeting+potential+pathways+may+reduce+procoagulant+activity.">Doxorubicin-induced vascular toxicity--targeting potential pathways may reduce procoagulant activity.</a> PLoS One. 8 (9), e7515 (2013).</li><li>Laemmel, E., 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=Fibered+confocal+fluorescence+microscopy+(Cell-viZio)+facilitates+extended+imaging+in+the+field+of+microcirculation.+A+comparison+with+intravital+microscopy.">Fibered confocal fluorescence microscopy (Cell-viZio) facilitates extended imaging in the field of microcirculation. A comparison with intravital microscopy.</a> J Vasc Res. 41 (5), 400-411 (2004).</li><li>Al-Gubory, K. H., Houdebine, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+green+fluorescent+protein-expressing+cells+in+transgenic+animals+using+fibred+confocal+fluorescence+microscopy.">In vivo imaging of green fluorescent protein-expressing cells in transgenic animals using fibred confocal fluorescence microscopy.</a> Eur J Cell Biol. 85 (8), 837-845 (2006).</li><li>Bar-Joseph, H., 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=In+vivo+bioimaging+as+a+novel+strategy+to+detect+doxorubicin-induced+damage+to+gonadal+blood+vessels.">In vivo bioimaging as a novel strategy to detect doxorubicin-induced damage to gonadal blood vessels.</a> PLoS One. 6 (9), e23492 (2011).</li><li>Kaushal, V., Kaushal, G. P., Mehta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+toxicity+of+anthracyclines+on+cultured+endothelial+cells.">Differential toxicity of anthracyclines on cultured endothelial cells.</a> Endothelium. 11 (5-6), 253-258 (2004).</li><li>Kim, E. J., 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=Doxorubicin-induced+platelet+cytotoxicity:+a+new+contributory+factor+for+doxorubicin-mediated+thrombocytopenia.">Doxorubicin-induced platelet cytotoxicity: a new contributory factor for doxorubicin-mediated thrombocytopenia.</a> J Thromb Haemost. 7 (7), 1172-1183 (2009).</li><li>Walsh, J., Wheeler, H. R., Geczy, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+tissue+factor+on+human+monocytes+by+cisplatin+and+adriamycin.">Modulation of tissue factor on human monocytes by cisplatin and adriamycin.</a> Br J Haematol. 81 (4), 480-488 (1992).</li><li>Kotamraju, S., 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=Doxorubicin-induced+apoptosis+in+endothelial+cells+and+cardiomyocytes+is+ameliorated+by+nitrone+spin+traps+and+ebselen.+Role+of+reactive+oxygen+and+nitrogen+species.">Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species.</a> J Biol Chem. 275 (43), 33585-33592 (2000).</li><li>Vasquez-Vivar, J., 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=Endothelial+nitric+oxide+synthase-dependent+superoxide+generation+from+adriamycin.">Endothelial nitric oxide synthase-dependent superoxide generation from adriamycin.</a> Biochemistry. 36 (38), 11293-11297 (1997).</li></ol>

Évaluation de la toxicité vasculaire induite par la chimiothérapie est difficile en raison de la difficulté à visualiser la dynamique de la vascularisation en réponse à un stimuli en temps réel. De nombreuses études cliniques ont mis en cause que plusieurs chimiothérapies causent lésion vasculaire directe, mais le mécanisme et les caractéristiques de cette toxicité reste à élucider. Nous avons établi un temps réel, in vivo plate-forme d&#39;imagerie moléculaire pour évaluer la toxicité pote...

Les données cliniques indiquent que plusieurs classes de chimiothérapies provoquent une variété de pathologies vasculaires qui se manifestent par un phénomène de Raynaud, l&#39;hypertension, myocardialinfarction, attaque vasculaire cérébrale et hépatique veino-occlusivedisease 1,2. «Médicaments anti-angiogéniques &#39;accidentelle&#39;&quot; est un terme relativement nouveau, qui décrit des agents chimiothérapeutiques classiques qui agissent comme inhibiteurs de l&#39;angiogenèse possibles, même si elles pas à l&#39;origine développé à cet effet 3-5 mais conçus pour éliminer les cellules tumorales en imposant aussi peu &quot;garantie dommages &quot;pour les cellules normales que possible trois. Plusieurs chimiothérapies ont été implicite que vasculo-toxiques comme observé dans les études cliniques utilisant des biomarqueurs sériques. Parmi ceux-ci sont des agents (tels que le cyclophosphamide), les composés du platine tels que le cisplatine () et anthracyclines 1,2,5-7 alkylation. Complications cardiovasculaires aiguës peuvent se produire en result de la toxicité vasculaire induite par la chimiothérapie. Ils peuvent évoluer vers des affections chroniques comme l&#39;athérosclérose et considération pour le risque accru de morbidité vasculaire tard. Pourtant, en dépit des preuves cliniques de montage, il ya un manque d&#39;études désignés soulignant le mécanisme de toxicité vasculaire et par conséquent, de plus amples éclaircissements de la pathogénie exacte qu&#39;ils infligent est justifiée. Un défi majeur dans révélant le mécanisme de toxicité vasculaire induite par la chimiothérapie découle de la complexité de l&#39;enquête la fonction vasculaire in vivo. Nous décrivons ici une plate-forme de haute résolution en imagerie moléculaire in vivo chez la souris qui permet de capturer le flux sanguin et des caractéristiques de navires. Cette plate-forme facilite la détection des effets directs induits par le traitement vasculaires: en temps réel, ainsi que de les suivre sur une période de temps dans les mêmes individus.

<table border="0" cellpadding="0" cellspacing="0" width="1395">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:579px;">Name of Material/ Equipment</td>
			<td style="width:423px;">Company</td>
			<td style="width:213px;">Catalog Number</td>
			<td style="width:183px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">general anesthesia (100mg/kg ketaset and 6mg/kg XYL-M2).&#160;</td>
			<td>Fort Dodge Animal Health, IA, USA and Biove Laboratories, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">a depilatory cream (Veet)</td>
			<td>&#160;ReckittBenckiser, Bristol, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A 30-gauge, 1/2-inch needle attached to 1 ml syringe&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC dextran at a volume of 100 &#181;l (10 mg/ml; FD2000S, MW 2000000 Dalton)&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxorubicin (8 mg/kg, Adriamycin)</td>
			<td>Teva, Israel</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paclitaxel (1.2 mg/kg, Medexel)&#160;</td>
			<td>Taro, Israel</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

chemotherapy-induced vascular toxicity - real-time in vivo imaging of vessel impairment

Certaines classes de chimiothérapies peuvent exercer changements vasculaires aiguës qui peuvent évoluer dans des conditions à long terme qui peuvent prédisposer le patient à un risque accru de morbidité vasculaire. Pourtant, bien que les preuves cliniques de montage, il ya un manque d&#39;études claires de toxicité vasculaire et donc l&#39;étiologie d&#39;un groupe hétérogène de vasculaires / troubles cardiovasculaires reste à élucider. En outre, le mécanisme qui peut expliquer la toxicité vasculaire peut différer complètement les principes de la cardiotoxicité induite par la chimiothérapie, qui est liée à une lésion directe des myocytes. Nous avons établi un temps réel, in vivo plate-forme d&#39;imagerie moléculaire pour évaluer la toxicité vasculaire aigu potentiel des thérapies anti-cancéreuses. Nous avons mis en place une plate-forme d&#39;in vivo, l&#39;imagerie moléculaire à haute résolution chez la souris, adapté pour visualiser la vascularisation au sein des organes confinés et blo de référenceod navires dans les mêmes individus, alors que chaque individu servent de son propre contrôle. les parois des vaisseaux sanguins ont été altérées après l&#39;administration de la doxorubicine, représentant un mécanisme unique de toxicité vasculaire qui peut être le cas au début de blessures des organes cibles. Ici, la méthode de microscopie confocale à fluorescence (FCFM) imagerie basée fibrée est décrite, qui fournit un mode innovant de comprendre les phénomènes physiologiques aux niveaux cellulaire et sub-cellulaire dans les matières animales.

En imagerie continue vivo au temps réel Appareil de formation d&#39;image utilisée ici est une haute définition, microscope confocal fibrée, équipé d&#39;une sonde qui permet la visualisation de la vascularisation et de sa réponse à divers stimuli comme la chimiothérapie. Cette méthode est peu invasive depuis bien qu&#39;il puisse faciliter l&#39;imagerie des vaisseaux ou organes profonds, il nécessite une petite incision de la sonde. Les faisceaux de sonde sont con...

Watch this Scientific Journal Video about Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment at JoVE.com

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 ...

chemotherapy-induced-vascular-toxicity-real-time-vivo-imaging-vessel

Research

JoVE Journal

Medicine

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

7.3K vues.  Tel Aviv University. 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.

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

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Multispectrale en temps réel d&#39;imagerie par fluorescence pour la détection peropératoire du ganglion sentinelle en oncologie gynécologique

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Recherche

Médecine

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Evaluation de la fixation des nanoparticules dans les tumeurs en temps réel par imagerie intravitale

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

En temps réel d&#39;imagerie numérique de leucocytes-endothélial interaction dans l&#39;ischémie-reperfusion (IRI) du muscle Cremaster Rat

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

An in vivo Assay to Test Blood Vessel Permeability

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 physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

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 ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Transplantation dans la chambre antérieure de l&#39;oeil pour longitudinale, non-invasive<em&gt; In vivo</em&gt; Imagerie avec une seule cellule de résolution en temps réel

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

En temps réel d&#39;imagerie à rayons X du poumon Volumes fluide dans néonatale Souris Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rose Bengale Photothrombosis par imagerie optique confocal<em&gt; In Vivo</em&gt;: Un modèle de l&#39;unité des maladies du navire

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Time-lapse Extended Imagerie intravitale du temps réel multicellulaire Dynamics dans le microenvironnement de la tumeur

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

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 ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Analyse en temps réel la pression-volume de l’infarctus aigu du myocarde chez la souris

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...