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) in appropriate culture medium (e.g. Dulbecco&#8217;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum and 1% penicillin/streptavidin) at 37 &#176;C until approximately 90% confluent.</li>
	<li>Trypsinize cells, wash them 2 times with PBS, count, and then inject them into the tail veins of isoflurane-anesthetized 10 weeks old female nude rats at 5 million cells per animal, using a syringe with a 27 G needle. Surgical level anesthesia is verified by the lack of reaction to toe pinch.</li>
</ol>
2. Monitoring of Metastases Using MicroCT
<ol>
	<li>Examine rats once a week using a micro-CT/micro-Irradiator, to detect the presence of metastases above approximately 2 mm in diameter in diameter. The micro-CT is commissioned as previously described12.
	<ol>
		<li>Subject rats to 3% isoflurane anesthesia prior to imaging. Confirm deep anesthesia by toe pinch.</li>
		<li>After onset of anesthesia, quickly transfer rats to the imaging cradle in the imaging chamber and connect via a nose-cone to an isoflurane-air mixture at 2.5-3%. Adjust the position of the rat in the cradle in a way that its thorax is positioned in the photon beam of the MicroCT scanner, using the external position controls and laser delimiter on the imaging cradle. Ensure the door to the imaging chamber is closed, to shield the investigator from the gamma rays.</li>
		<li>Control the position of the animal again using the color video camera. Perform a low-resolution CT imaging test run, and use the resulting image to adjust the field of view to the xyz dimensions of the thoracic cavity.</li>
		<li>Image the rat thorax using a 2 mm Al filter at 40 kVp, 2.5 mA, and 0.008 voxel at 7 FPS and return the animal to its cage. Imaging of one animal should take not more than 15 or 20 min. Do not return an animal that has undergone surgery to the company of other animals until fully recovered. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
		<li>Confirm metastases by the appearance of relatively radio-opaque objects that cannot be explained by intrathoracic blood vessels (Figure 1A)</li>
	</ol>
	</li>
</ol>
3. Window Chamber Surgery
<ol>
	<li>Anesthesia, vital signs and tail vein catheterization
	<ol>
		<li>Select animals with presence of metastatic disease. Inject animal with an intraperitoneal dose of 50 mg/kg pentobarbital. Confirm surgical-level anesthesia by toe pinch before proceeding. 
		 Note: Anesthesia protocols should be matched to the respective experimental setup. Pentobarbital was chosen here as a long-acting anesthetic, in order to induce deep anesthesia for lengthy procedures, while offering the option of facile re-dosing. However, loss of animals to overdosing is a common problem with pentobarbital anesthesia. Another option that preserves autonomic reflexes to a larger degree than pentobarbital is ketamine in combination with sedatives such as xylazine or medetomidine, which however permits only for a single re-dosing cycle.</li>
		<li>Shave animals on the side of the body that has the metastatic disease, and in the neck area, using a clipper. Wipe off all remaining loose hair from the skin. After loose hair is removed, apply veterinary ointment to the eyes, to prevent them from drying out. 
		NOTE: Athymic nude rats may have residual hair that requires removal before proceeding with surgical procedures. It is very important to remove all hair thoroughly, as it may interfere with surgical procedures and imaging.</li>
		<li>Fix the animal in a supine position on a metal plate that is placed on a 37 &#176;C water circulated heating pad. The front and hind extremities are fixed on the plate with tape. 
		NOTE: It is useful to control and record vital signs, such as heart rates and arterial blood oxygenation by using a pulse oximeter, throughout surgical and experimental procedures.</li>
	</ol>
	</li>
	<li>Tracheal intubation
	<ol>
		<li>In order to place a catheter for ventilation of the animal, first make a transversal cervical skin incision, followed by the median separation of the longitudinal musculature ventral to the trachea.</li>
		<li>Use repeated opening-to-closing action with a sharp forceps to create a passage for the suture through the dorsal side of the trachea.</li>
		<li>Make a small incision into the trachea on the ventral side, not more than semi-circular, approximately between the second and third tracheal ring. Leave a sufficiently long portion of the trachea exposed on the dorsal surface, to enable fixation of the tracheal catheter.</li>
		<li>Insert a 2.5 to 3.0 mm &#8220;Y&#8221; tracheal cannula into the trachea, and tighten with a 4-0 monofilament suture. Ensure the cannula is connected to a pressure cycled ventilator with a bottle connected to the expiration duct that is filled with 6 cm of water, to maintain positive pulmonary pressure. Inflowing gas should be 100% oxygen, unless experimentally desired otherwise.</li>
		<li>Insert a catheter with a 25-27 G needle, and filled with heparinized saline into one of the tail veins of the rat, and fix in place with tape. 
		NOTE: Ensure patency of the tail vein catheter throughout the procedure by repeatedly injecting a small amount of heparinized saline into the tail vein. Also, oro-tracheal intubation, i.e. the guidance of a tracheal tube via the mouth of the anesthetized animal and past the larynx into the trachea, is a possible alternative to the tracheotomy procedure that is described here. However,this method requires special training and experience, to avoid damage to the trachea, and also to preclude the accidental cannulation of the esophagus. </li>
	</ol>
	</li>
	<li>Application of pulmonary window
	<ol>
		<li>Remove the skin from the side of the chest where the metastatic disease is located, by creating an incision, and then detaching the skin using blunt dissection. 
		 Note: The skin can be excised and removed subsequent to detachment</li>
		<li>Proceed by dissecting the two layers of overlaying musculature (pectoralis, serratus, and latissimus dorsi), but leaving the intercostal muscles intact. Create a perforation in the chest cavity of approximately 1.5 cm in diameter, by removing portions of typically two adjacent ribs. Ideally, locate the perforation in of the region of the sixth and seventh ribs.</li>
		<li>Osteotomy:
		<ol>
			<li>To minimize bleeding and damage to the lung surface, tightly hold the rib to be cut with a toothed surgical forceps during cutting. Using surgical scissors, cut the medial side of the rib first, at an angle of approximately 45&#176;, leaving the pointed side of the remaining rib bone pointing outside.</li>
			<li>Subsequently, cut the lateral side of the rib bone in a similar manner, again leaving the pointed side of the rib bone pointing outwards, to prevent damage to the lung surface.</li>
			<li>Repeat the procedure for the adjacent rib, then cut the intercostal muscles and remove the excised piece. During this procedure, maintain lung pressure in a way that mechanical interaction between the lung surface and the rib cage is minimized. Do this by appropriately regulating inspiration pressure on the ventilator.</li>
		</ol>
		</li>
		<li>Insertion of the window:
		<ol>
			<li>Insert a custom made lung window, consisting of a coverglass that is attached to a Plexiglas socket (Figure 1B). Attach the window to the socket by gluing, or by applying a very small amount of vacuum grease. Insert the window in a way that surface metastases are located close to the center of the window. If necessary, adjust the inserted hole to bring the micrometastasis to the center of the window by enlarging the hole slightly to the respective side. 
			NOTE: Metastatic disease on the pleural surface can be identified as clearly recognizable white dots or areas within the otherwise pink-to-salmon colored healthy parts of the lung surface that appear predominantly along the fissures. While micrometastases can occur on other areas of the pleural exterior, the investigated cell lines almost always display surfacing micrometastases in the perforated area, once metastatic disease can be radiologically detected.</li>
			<li>After inserting the socket into the perforation, and creating direct contact with the visceral pleura of the lung, suture the edges of the window frame to the surrounding intercostal muscle, using 4.0 monofilament suture (Figure 1C). Use a slight increase of inspiration pressure on the ventilator to help residual air to escape and to create a seal. 
			 Note: The respiration rate of rats can vary greatly, depending on the status of anesthesia, status of excitement or anxiety, oxygen concentration of inspired air (FiO2), etc. It is recommendable to adjust the respiration rate between 70 and 90 bpm. The inspiration pressure should be adjusted with caution and not be set to more than approx. 8 cm H2O (0.6 mmHg), to avoid damage to the lung surface.</li>
		</ol>
		</li>
		<li>Position the animal in a custom designed restrainer that is designed to eliminate Z-directional movement (Figure 1D) on a steel plate that is positioned on a thermostatic (electrical) heating blanket, under a fluorescence microscope. Control the animal&#8217;s body temperature using a rectal thermistor. Adjust the screws of the animal restrainer, and the inspiration pressure on the ventilator to achieve optimal control of lateral movement. 
		 Note: Natural breathing in mammals involves all three directions of lung movement and chest extension: bilateral, dorso-ventral, and cranio-caudal. In order to preserve the natural breathing movement as much as possible, it is important to minimize Z-directional compression to the necessary extent. Because the Z-dimensional restraint has the potential to introduce artifacts that may affect blood flow and other parameters, it is advisable to keep conditions constant during series of repeated measurements in the same animal.</li>
	</ol>
	</li>
</ol>
4. Imaging and Measurement of Microcirculatory Blood Flow
<ol>
	<li>Collect red blood cells via cardiac puncture and label them with DiI (1,1=-dioctadecyl-3,3,3=,3=-tetramethyl-indocarbocyanine perchlorate), as described previously 10.</li>
	<li>Inject 300 &#181;l of labeled red blood cells into the tail vein of the rat before the window chamber surgery is done, to avoid first pass adhesion effects to the glass window. Eliminate any air bubbles in the syringe or catheter, as introduction of air into the vein will inhibit any further injections.</li>
	<li>Image blood flow with a microscope CCD camera at -40 &#176;C chip cooling temperature, and approximately 100X overall resolution (i.e. with a 10X microscope objective, and 10X pre-camera ocular). Use standard Rhodamine/ TRITC filter sets (excitation 450-490 nm, emission &#62;515 nm). Record the actual frame rate and pixel resolution of the resulting image sequences. Record at least 200 (ideally around 300) images per stack, to ensure successful analysis of flow velocity.</li>
	<li>Replenish fluid loss in the animal by injecting approximately 1 ml of saline i.p. every hr. 
	NOTE: Experimental settings that involve an intervention, e.g. a drug that modifies the blood flow velocity in a pulmonary cancer metastasis, requires repeated measurements of blood flow velocity in pulmonary metastatic cancer. For these extended experiments, it is important to replenish the animal with sufficient fluid.</li>
	<li>Euthanize the animals by infusion of 3 ml of 3N KCl into the tail vein</li>
	<li>Evaluate the image stacks using a published, publically available Matlab-based computer algorithm that will create flow velocity grayscale and color encoded maps for all blood flow traces 10,11. Subsequently evaluate the resulting grayscale images by using commercial or publicly available image analysis software, such as Image J, after thresholding off values that indicate no movement of blood cells, i.e. no active vasculature.</li>
</ol>

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The authors have nothing to disclose.

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. ...

automated-measurement-microcirculatory-blood-flow-velocity-pulmonary

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.4K Views.  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.

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

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

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

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

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

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 contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

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

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

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

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

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

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of endothelial function can be achieved by employing the flow-mediated dilation technique, typically performed in the brachial or superficial femoral artery. Briefly, a blood pressure cuff placed distal to an ultrasound probe is inflated to a suprasystolic pressure, which results in limb ischemia. After 5 min of occlusion the cuff is deflated, resulting in reactive hyperemia and increases in wall shear rate that signal vasodilatory molecules to be released from the endothelium eliciting vasodilation. Despite the thousands of studies performing flow-mediated dilation in humans, surprisingly, no studies have performed this technique non-invasively in living rats. Considering the recent shift in focus to translational research, the establishment of guidelines for non-invasive measurement of flow-mediated dilation in rats and other rodents would be extremely valuable. In the following article, a protocol is presented for the non-invasive measurement of flow-mediated dilation in brachial and superficial femoral arteries of rats, as those sites are most commonly measured in humans.

Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats.

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of ...

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

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

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

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...

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

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a "second hit" of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.

The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality.

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

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

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

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

Summary

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Chapters in this video

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

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

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

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

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

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

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats