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: All animal related procedures described in this protocol have been previously approved by the Duke University Institutional Animal Care and Use Committee (DUIACUC).
1. Cancer Cell Culture and Injection
<ol>
	<li>Cultivate fluorescently labeled metastatic cancer cells (e.g. human MDAMB231-GFP breast cancer cells, gift from Dr. Patricia Steeg, NCI, and YFP-labeled mouse sarcoma cells, gift from Dr. David Kirsch, Duke University Medical Center, Department of Radiation Oncology) i...

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A model is presented that is feasible to image changes in microcirculatory blood flow and other dynamic processes in pulmonary metastases of rats, using intravital microscopy and computational blood flow analysis. While other methods exist to perform microscopy on exposed lungs in open ribcages of rodents, this model is also the first to image pulmonary metastases through a chest wall perforation in a closed-chest setting. Using this method, the feasibility is shown to measure pharmacologically induced changes in microci...

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.

The vasculature in solid tumors is known to differ significantly from normal blood supply, showing greater degrees of tortuosity, and higher intervascular distances13. Accordingly, blood flow tracks in experimental pulmonary breast cancer and sarcoma metastases have irregular shapes and large intervascular gaps (Figure 2A, lower two panels) when compared to the normal pulmonary microcirculation (Figure 2A, upper panel). In a previous study, the ability of the pulmonary window ...

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.

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

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8.4K visualizações.  Duke University Medical Center. 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.