Name: automated measurement of microcirculatory blood flow velocity in pulmonary metastases of rats
Uploaded: 2014-11-30T14:00:00
Description: Watch this Scientific Journal Video about Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats at JoVE.com

The scientific advice of Drs. Timothy McMahon and Siqing Shan is appreciated. The presenters thank Drs. David Kirsch and Patricia Steeg for the generous gift of the fluorescently labeled Mouse Sarcoma and metastatic MDAMB-231 cells, respectively. This work was funded in part by the U.S. Defense Advanced Research Projects Agency (DARPA) Prime Award Number N66001-10-C-2134, and in part by the Department of Radiation Oncology, Duke University Medical Center.

NOTE: Toutes les procédures liées aux animaux décrits dans le présent protocole ont été préalablement approuvé par le soin et l&#39;utilisation institutionnelle animaux Comité Duke University (DUIACUC). 1. Culture cellulaire et cancer Injection <ol><li> Cultiver des cellules cancéreuses métastatiques marquées par fluorescence (par exemple des cellules de cancer du sein MDAMB231-GFP humaines, don du Dr. Patricia Steeg, NCI, et les cellules de sarcome de la souris YFP-é...

<ol>
	<li>Billingsley, K. G., 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=Pulmonary+metastases+from+soft+tissue+sarcoma:+analysis+of+patterns+of+diseases+and+postmetastasis+survival.">Pulmonary metastases from soft tissue sarcoma: analysis of patterns of diseases and postmetastasis survival.</a> Ann Surg. 229, 602-610 (1999).</li><li>Rashid, O. M., Takabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+the+role+of+surgery+in+the+management+of+breast+cancer+lung+metastasis.">The evolution of the role of surgery in the management of breast cancer lung metastasis.</a> J Thorac Dis. 4, 420-424 (2012).</li><li>Mayer, A., Vaupel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia,+lactate+accumulation,+and+acidosis:+siblings+or+accomplices+driving+tumor+progression+and+resistance+to+therapy.">Hypoxia, lactate accumulation, and acidosis: siblings or accomplices driving tumor progression and resistance to therapy.</a> Advances in experimental medicine and biology. 789, 203-209 (2013).</li><li>Okunieff, P., O'Dell, W., Zhang, M., Zhang, L., Maguire, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+oxygen+measurements+and+personalized+medicine.">Tumor oxygen measurements and personalized medicine.</a> Advances in experimental medicine and biology. 765, 195-201 (2013).</li><li>Palmer, G. M., 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+optical+molecular+imaging+and+analysis+in+mice+using+dorsal+window+chamber+models+applied+to+hypoxia,+vasculature+and+fluorescent+reporters.">In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters.</a> Nature protocols. 6, 1355-1366 (2011).</li><li>Palmer, G. M., 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=Optical+imaging+of+tumor+hypoxia+dynamics.">Optical imaging of tumor hypoxia dynamics.</a> Journal of biomedical optics. 15, (2010).</li><li>Funakoshi, N., 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=A+new+model+of+lung+metastasis+for+intravital+studies.">A new model of lung metastasis for intravital studies.</a> Microvasc Res. 59, 361-367 (2000).</li><li>Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+assessment+of+pulmonary+vascular+responses.">Real-time imaging assessment of pulmonary vascular responses.</a> Proc Am Thorac Soc. 8, 458-465 (2011).</li><li>Tabuchi, 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=Precapillary+oxygenation+contributes+relevantly+to+gas+exchange+in+the+intact+lung.">Precapillary oxygenation contributes relevantly to gas exchange in the intact lung.</a> Am J Respir Crit Care Med. 188, 474-481 (2013).</li><li>Hanna, G., 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=Automated+measurement+of+blood+flow+velocity+and+direction+and+hemoglobin+oxygen+saturation+in+the+rat+lung+using+intravital+microscopy.+American+journal+of+physiology.">Automated measurement of blood flow velocity and direction and hemoglobin oxygen saturation in the rat lung using intravital microscopy. American journal of physiology.</a> Lung cellular and molecular physiology. 304, 86-91 (2013).</li><li>Fontanella, A. N., 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=Quantitative+mapping+of+hemodynamics+in+the+lung,+brain,+and+dorsal+window+chamber-grown+tumors+using+a+novel,+automated+algorithm.">Quantitative mapping of hemodynamics in the lung, brain, and dorsal window chamber-grown tumors using a novel, automated algorithm.</a> Microcirculation. , (2013).</li><li>Newton, 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=Commissioning+a+small-field+biological+irradiator+using+point,+2D,+and+3D+dosimetry+techniques.">Commissioning a small-field biological irradiator using point, 2D, and 3D dosimetry techniques.</a> Medical physics. 38, 6754-6762 (2011).</li><li>Vaupel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironmental+physiology+and+its+implications+for+radiation+oncology.">Tumor microenvironmental physiology and its implications for radiation oncology.</a> Seminars in radiation oncology. 14, 198-206 (2004).</li><li>Tabuchi, A., Mertens, M., Kuppe, H., Pries, A. R., Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+murine+pulmonary+microcirculation.">Intravital microscopy of the murine pulmonary microcirculation.</a> J Appl Physiol. 104, 338-346 (2008).</li><li>Fingar, V. H., Taber, S. W., Wieman, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+for+the+study+of+pulmonary+microcirculation:+determination+of+pulmonary+edema+in+rats.">A new model for the study of pulmonary microcirculation: determination of pulmonary edema in rats.</a> J Surg Res. 57, 385-393 (1994).</li><li>Manzoor, A. A., Schroeder, T., Dewhirst, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-stop-shop+tumor+imaging:+buy+hypoxia,+get+lactate+free.">One-stop-shop tumor imaging: buy hypoxia, get lactate free.</a> The Journal of clinical investigation. 118, 1616-1619 (2008).</li><li>Evans, S. M., 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=Imaging+and+analytical+methods+as+applied+to+the+evaluation+of+vasculature+and+hypoxia+in+human+brain+tumors.">Imaging and analytical methods as applied to the evaluation of vasculature and hypoxia in human brain tumors.</a> Radiation research. 170, 677-690 (2008).</li><li>Radiloff, D. 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=The+combination+of+theophylline+and+endothelin+receptor+antagonism+improves+exercise+performance+of+rats+under+simulated+high+altitude.">The combination of theophylline and endothelin receptor antagonism improves exercise performance of rats under simulated high altitude.</a> Journal of applied physiology. 113, 1243-1252 (2012).</li></ol>

On présente un modèle qui est possible à l&#39;évolution de l&#39;image dans le débit sanguin microcirculatoire et d&#39;autres processus dynamiques dans des métastases pulmonaires de rat, en utilisant la microscopie intravitale et l&#39;analyse de la circulation sanguine calcul. Alors que d&#39;autres méthodes existent pour effectuer la microscopie sur les poumons exposés en cages thoraciques ouvertes de rongeurs, ce modèle est également le premier à l&#39;image des métastases pulmonaires par une perforatio...

The lung is one of the most important target organs of metastatic disease, and because this condition is difficult to treat successfully with chemo- and radiation therapy, a cure is still rare1,2. Specific pathophysiological and microcirculatory features of solid primary and metastatic tumors, such as microregional hypoxia, diffusion limitation and inefficient tumor vasculature, greatly contribute to their resistance to anticancer treatment3,4. Due to the microscopic scale and dynamic nature of parameters such as microvascular blood flow, intravital microscopy of the tumor in the living animal has become a very important research tool in the field5. While intravital microscopy models have been applied to tumors in different organ sites, including the metastatic lung within an open rib cage, no protocol has been developed yet for the research of pulmonary metastases in a physiologically preserving, closed-chest environment6,7. Such an endeavor is particularly hampered by the necessity to surgically access the rib cage without affecting the overall function of the lung7-9. Recently, a method was introduced to image pulmonary microcirculatory blood flow in a close-chest setting in live rats, using fluorescence intravital microscopy10. This protocol enables the systematic quantification of blood flow velocity from injected, fluorescently labeled red blood cells, using computerized analysis, while keeping the animal physiologically stable and preserving the integrity of the lung11. In this present study, it is shown how this technology can be modified to image and quantify microcirculatory blood flow in tail vein-inoculated pulmonary metastases on the pleural surface in the immunocompromised rat. This model is also the first one to study metastatic lung tumors in a closed-chest intravital microscopy setting.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Athymic nude rats</td>
			<td style="width:71px;">Charles River</td>
			<td style="width:119px;">Strain code 316</td>
			<td style="width:167px;">Female 10 week-old athymic nude rats</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micro-CT/micro-Irradiator&#160;</td>
			<td style="width:71px;">Precision X-ray Inc.</td>
			<td style="width:119px;">Xrad 225Cx</td>
			<td>Use MicroCT to detect metastases</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DiI (1,1=-dioctadecyl-3,3,3=,3=-tetramethyl-indocarbocyanine perchlorate)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">468495-100MG</td>
			<td>Mix 100 ul packed red blood cells with 100 ul of 0.5 mg/ml DiI in 200 proof ethanol, 2 ml of 5% dextrose solution in water, and fill up to a 10-ml final volume with saline</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Rodent ventilator</td>
			<td style="width:71px;">Kent Scientific</td>
			<td style="width:119px;">TOPO Small Animal Ventilator</td>
			<td>Device is important to maintain positive lung pressure after application of pneumothorax</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axioskop fluorescence microscope upright</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Microscope for intravital imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Andor CCD camera</td>
			<td style="width:71px;">Andor</td>
			<td style="width:119px;">iXonEM 885</td>
			<td>CCD camera for live imaging of blood flow</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pulse oximeter</td>
			<td style="width:71px;">StarrLife</td>
			<td style="width:119px;">MouseOx</td>
			<td>Pulse oximeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescence microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Fluorescence microscope</td>
		</tr>
	</tbody>
</table>

automated measurement of microcirculatory blood flow velocity in pulmonary metastases of rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

La vascularisation dans les tumeurs solides est connu pour différer considérablement de l&#39;approvisionnement en sang normale, montrant de plus grands degrés de tortuosité et les distances intervasculaires plus élevés 13. En conséquence, les pistes de la circulation sanguine dans le cancer du sein et de métastases pulmonaires sarcome expérimentales ont des formes irrégulières et de grandes lacunes intervasculaires (figure 2A, deux panneaux inférieurs) par rapport à la microcirc...

Watch this Scientific Journal Video about Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats at JoVE.com

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Mesure automatisée du flux sanguin Microcirculatory vitesse dans métastases pulmonaire des rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

Research

JoVE Journal

Medicine

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

La mesure in vivo de la surface de la couche de souris pulmonaire endothéliale

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Recherche

Médecine

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Mesures microscopie intravitale Vidéo de la rétine débit sanguin chez les souris

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

Mesure automatisée de l&#39;emphysème pulmonaire et petites remodelage des voies aériennes dans la cigarette de fumée souris exposées

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

L&#39;augmentation de l&#39;artère pulmonaire pulsatile débit améliore l&#39;hypertension pulmonaire hypoxique chez les porcelets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Transcutanée Microcirculatory Imagerie en nouveau-nés prématurés

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Un modèle de Cardiac Remodeling Grâce à constriction de l&#39;aorte abdominale chez les rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats

La pneumonectomie gauche combiné avec Monocrotaline ou Sugen comme un modèle d’Hypertension pulmonaire chez le rat

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

Enregistrement simultané de l’électrocardiographie et envahissantes sang pression mesure chez les Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Évaluation de la réserve de flux coronaire après la réperfusion d'ischémie myocardique chez les rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...