The authors would like to thank Drs. Yuan Hao and Kaifeng Liu for their technical support. This work was supported by an NIH 1R01 HL150106-01A1, the Parker B. Francis Fellowship, and the Pulmonary Hypertension Association Aldrighetti Research Award to Dr. Ke Yuan.

All animal care was in accordance with the guidelines of Boston Children&#39;s Hospital and the Institutional Animal Care and Use Committee approved protocols. The mice used in this study are wild type C57/B6 mice and Cdh5-CreERT2 x Ai14 tdTomato crossed mice.
1. Preparation of solutions
<ol>
	<li>Prepare phosphate buffer solution (1x PBS) and 2% agarose solution required during the experiment in advance.
	<ol>
		<li>Mix 2 g agarose powder into 100 mL of autoclaved water. Heat it in the microwave a few seconds at a time until the solution is clear.</li>
		<li>Place the solution in a water bath at 42 &#176;C until use. 
		&#8203;NOTE: Both PBS and 2% agarose can be stored at room temperature for weeks.</li>
	</ol>
	</li>
</ol>
2. Extraction of the mouse lung
<ol>
	<li>Euthanize the mouse by isoflurane overdose. Achieve isoflurane overdose by placing a small amount of isoflurane on tissue paper in the lower level and placing the mouse in the upper level in a desiccator. The mouse remains in the desiccator until unconscious.</li>
	<li>Ensure death of the mouse by applying inferior pressure on the neck and pulling caudal on the tail. Bring the mouse to the dissecting surface.</li>
	<li>Place the mouse in the supine position and secure it into place taping paws and nose to table with adhesive tape.</li>
	<li>Spray the ventral surface of the mouse with 70% ethanol and wipe off the excess ethanol.</li>
	<li>Lift the abdominal skin of the mouse with forceps at the location of the bladder and cut at the midline with surgical scissors, moving superiorly to the cervical region, exposing the trachea.</li>
	<li>Lift the underlying fascial layer with tweezers and cut with surgical scissors to expose visceral organs and mediastinal compartment, again cutting inferiorly to superior.</li>
	<li>Cut the sternum at the midline to expose the heart and the lung.</li>
	<li>Using blunt dissection, detach the diaphragm from the liver and abdominal compartment.</li>
	<li>Locate the inferior vena cava (IVC) under the intestines and cut the IVC.</li>
	<li>Locate the right ventricle. 
	NOTE: The right ventricle will have a lighter appearance compared to the left ventricle and the intraventricular septum should be visualized, separating the right ventricle from the left ventricle.</li>
	<li>Inject 0.5 mL of 1% fractionated heparin to the right ventricle with a 25-30 G butterfly needle and then flush slowly but steadily with 10-15 mL of 1x PBS (Figure 1A).
	<ol>
		<li>Use caution not to insert the needle through the heart and inject heparin into the mediastinal cavity. 
		NOTE: The injection of 1x PBS turns the lung tissue white. The fluid extravasating from the abdominal aorta should be clear and colorless and the liver may become white in appearance after successful flushing.</li>
	</ol>
	</li>
	<li>Locate the larynx and dissect the surrounding fascia and tissue using blunt tip tweezers.</li>
	<li>Place the surgical suture under the trachea and loosely tie a surgical knot.</li>
	<li>Place a small hole in the trachea superior to string and canulate with 20 G blunt-ended needle.</li>
	<li>Tighten the suture around the needle and trachea using a surgical knot.</li>
	<li>Inject 2.5-4 mL of agarose into the trachea and monitor for inflation of the lung.</li>
	<li>After agarose is instilled, pour cold, chilled 1x PBS over the lung to solidify agarose.</li>
	<li>Cut the trachea superior to surgical suture and dissect the lung and heart from the mediastinum by removing any adhesions and fascia.</li>
	<li>After solidification, place in 1x DMEM (enough to submerge tissue) in a Petri dish to keep on ice. Immediately slice one lobe using a vibratome machine (Figure 1B).</li>
</ol>
3. Precision cut lung slices
<ol>
	<li>Attach the sample to the platform with superglue, keeping the medial side of the sample facing up.</li>
	<li>Fill the sample container with 1x PBS, ensuring that the sample is completely submerged.</li>
	<li>Fill the container surrounding the sample container with ice to keep surrounding PBS and sample cold.</li>
	<li>Turn on the vibratome and adjust to the desired settings below.
	<ol>
		<li>Set the thickness to 300um.</li>
		<li>Set the frequency to 100 Hz.</li>
		<li>Set the amplitude to 0.6 mm.</li>
		<li>Set the speed to 5 &#181;m/s.</li>
	</ol>
	</li>
	<li>Using an Allen wrench, turn the blade holder into the safe position and open the jaws to insert a blade.</li>
	<li>Tighten the jaws with an Allen wrench and turn the blade holder into the appropriate position for slicing.</li>
	<li>Place the sample and the platform onto the box and slide in front of the blade.</li>
	<li>Fill the box around the sample platform with 1x PBS until the sample is submerged.</li>
	<li>Bring the vibratome blade up to the edge of the block and manually lower the blade until it is even with the top of the sample.</li>
	<li>Confirm the appropriate settings and run the vibratome (Figure 2).</li>
	<li>As fresh slices are cut, remove the samples from the PBS and place them into a sterile Petri dish with 10 mL of 1x DMEM containing 1x antibiotic-antimycotic.</li>
	<li>When finished, retract the blade entirely and then use the Allen wrench to raise the blade into a safe position.</li>
	<li>Store the samples in 1x DMEM in an incubator at 37 &#176;C with viability preserved for up to 10 days (see viability experiment below).</li>
</ol>
4. Example vasoconstrictor experiment
<ol>
	<li>Place a vibratome slice on a microscope slide.</li>
	<li>Before placing it on the slide, use a cleaning wipe to get rid of the excess medium (PBS).</li>
	<li>Place the sample under phase-contrast microscopy and use 10-20x magnification to identify a vessel.</li>
	<li>Put 500 &#181;L of vasoconstrictors, such as 60 mM KCl or 1 &#181;M Endothelin-1 to completely submerge the slide.</li>
	<li>Observe and capture the video by recording for 30 s-60 s (Video 1).</li>
</ol>
5. Preparing the tissue for RNA or protein lysis on PCLS
<ol>
	<li>Before beginning the experiment, fill a small polystyrene foam box with 1 L of liquid nitrogen.</li>
	<li>Place two small microcentrifuge tubes (1.5 mL) in the container with liquid nitrogen before starting. 
	NOTE: Liquid nitrogen should cool outside of the tubes, however, not be inside. Be sure to handle liquid nitrogen with care and do not expose the skin to liquid nitrogen. Use forceps and gloves to place and remove tubes from the box.</li>
	<li>Place 4-5 fresh vibratome slices in a mortar bowl.</li>
	<li>Pour 5-10 mL of liquid N2 on top of the slices in the mortar bowl.</li>
	<li>Quickly use the pestle to grind the flash freeze slices into powder before liquid nitrogen evaporates. 
	NOTE: Tissue should condense into a fine powder. If not, add additional liquid nitrogen until achieved.</li>
	<li>Using a steel laboratory scooper (prechilled with liquid nitrogen), scoop the powder into the two chilled microcentrifuge tubes.</li>
	<li>Lyse the powder by adding 500 &#181;L of RNA extraction reagent to proceed with RNA isolation or 500 &#181;L of RIPA buffer (contains 1x protease inhibitors) to proceed with protein lysis.</li>
	<li>Store the samples at -80 &#176;C until additional RNA isolation, BCA measurement, and western blot.</li>
</ol>
6. Determining viability
<ol>
	<li>After vibratome preparation, place two lung slices into a 24-well culture plate with 450 &#181;L of DMEM media and 50 &#181;L of cell viability reagent (ensure that the tissue is submerged entirely).</li>
	<li>Place the samples back into 5% CO2 at 37 &#176;C incubator overnight with minimal exposure to light.</li>
	<li>In the morning, observe the solution for color change. The blue solution turns pink when the tissue is viable.</li>
</ol>
7. Preservation of cell labeling
<ol>
	<li>Treat a transgenic Cdh5-CreERT2 crossed with Ai14 tdTomato mouse with an IP injection of Tamoxifen (2 mg/day) for a total of 5 days (total dose 10 mg Tamoxifen) to induce tdTomato label Cre positive cells.</li>
	<li>One week after injection, prepare the lungs using the above steps 1 to 3.</li>
	<li>After preparation of the precision-cut lung slice, place the tissue onto a microscope slide and place a coverslip on top of the tissue.</li>
	<li>Use a confocal microscope to detect tdTomato staining (using excitation wavelength 555 nm).</li>
</ol>

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The authors have no conflicts of interest to disclose.

In this manuscript, an enhanced method to produce high-resolution images of murine lung tissue that preserves the vascular structure and optimizes experimental flexibility is described, specifically using the application of PCLS to obtain microslices of lung tissue that can be viewed in three dimensions with preserved contractility of the vasculature. Using the viability reagent, the protocol demonstrates that carefully prepared and preserved slices can retain viability for more than a week. Preserved viability of the mi...

Advances in the preparation and imaging of lung tissue that preserves cellular components without sacrificing anatomical structure provide a detailed understanding of pulmonary diseases. The ability to identify proteins, RNA, and other biological compounds while maintaining physiological structure offers vital information on the spatial arrangement of cells that can broaden the understanding of the pathophysiology in numerous pulmonary diseases. These detailed images can lead to a better understanding of pulmonary vascular diseases, such as pulmonary artery hypertension, when applied to animal models, potentially leading to improved therapeutic strategies.
Despite advances in technology, obtaining high-quality images of murine lung tissue remains a challenge. The respiratory cycle is driven by a negative intrathoracic pressure generated during inhalation1. When traditionally obtaining biopsies and preparing lung samples for imaging, the negative pressure gradient is lost resulting in the collapse of the airway and vasculature, which no longer represents itself in its present state. To achieve realistic images reflective of current conditions, the pulmonary airways must be reinflated, and the vasculature perfused, changing the dynamic lung into a static fixture. The application of these distinct techniques allows preservation of structural integrity, pulmonary vasculature, and cellular components, including immune cells such as macrophages, allowing lung tissue to be viewed as close to its physiological state as possible.
Precision cut lung slicing (PCLS) is an ideal tool for studying the anatomy and physiology of pulmonary vasculature2. PCLS provides detailed imaging of the lung tissue in three dimensions while preserving structural and cellular components. PCLS has been used in animal and human models to allow for live, high-resolution images of cellular functions in three dimensions, making it an ideal tool to study potential therapeutic targets, measure small airway contraction and study the pathophysiology of chronic lung diseases such as COPD, ILD, and lung cancer3. Using similar techniques, the exposure of PCLS samples to vasoconstrictors can preserve lung structure and vessel contractility, replicating in vitro conditions. Along with preserving contractility, prepared samples can undergo additional analysis such as RNA sequencing, Western blot, and flow cytometry when prepared correctly. Finally, reporter color labeled cells marked with tdTomato fluorescence after lung harvest can preserve labeling after preparing microslices, making it ideal for cell tracking studies. The integration of these techniques provides a sophisticated model preserving the spatial arrangement of cells and vessel contractility that can lead to a more detailed understanding of the signaling cascades and potential therapeutic options in pulmonary vasculature disease.
In this manuscript, PCLS murine lung tissue is exposed to vasoconstrictors, demonstrating preserved structural integrity and vessel contractility. The study demonstrates that the tissue prepared and handled appropriately can remain viable for 10 days. The study also demonstrates the preservation of cells with endogenous fluorescence (tdTomato), allowing samples to provide high-resolution images of the pulmonary vasculature and architecture. Finally, ways to handle and prepare tissue slices for RNA measurement and Western blot to investigate underlying mechanisms have been described.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5cc of fractionated heparin in syringe</td>
			<td>BD</td>
			<td>100 USP units per mL</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>Corning</td>
			<td>&#160;21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>20 1/2 inch gauge blunt end needle for trachea cannulation</td>
			<td>Cml Supply</td>
			<td>90120050D</td>
			<td></td>
		</tr>
		<tr>
			<td>30cc syringe</td>
			<td>BD</td>
			<td>309650</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Anti solution</td>
			<td>Gibco</td>
			<td>15240096</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated vibrating blade microtome</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Viability Reagent (alamarBlue)</td>
			<td>Thermofisher</td>
			<td>DAL1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal</td>
			<td>Zeiss</td>
			<td>880</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium and GLutaMAX, supplemented with 10% FBS, 1% Pen/Strep</td>
			<td>Gibco</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelin-1</td>
			<td>Sigma</td>
			<td>E7764</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar and Pestle</td>
			<td>Amazon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA lysis and extraction buffer</td>
			<td>Thermoscientific</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical suture 6/0</td>
			<td>FST</td>
			<td>18020-60</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen, Thermofisher</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Low Melting Point Agarose</td>
			<td>Invitrogen</td>
			<td>16520050</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica Biosystems</td>
			<td>VT1200 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Winged blood collection set (Butterfly needle) 25-30G</td>
			<td>BD</td>
			<td>25-30G</td>
			<td></td>
		</tr>
	</tbody>
</table>

precision cut lung slices as an efficient tool for ex vivo pulmonary vessel structure and contractility studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

When added to cells or tissue, the viability reagent is modified by the reducing environment of viable tissue and turns pink/red, becoming highly fluorescent. The representative color changes detected from day 0-1 and day 9-10 are demonstrated in Figure 3. As noted, the solution started blue and turned pink overnight, demonstrating viability. Color change typically occurs within 1-4 h; however, a longer time may be necessary. To assay for viability, a plate reader was used to determine the a...

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Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

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

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5.6K Views.  Boston Children's Hospital. Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

Video: Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Combining Volumetric Capnography And Barometric Plethysmography To Measure The Lung Structure-function Relationship

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the lung. Barometric plethysmography is a classic technique to evaluate the lung volume with a long history of clinical use. Volumetric capnography utilizes the profile of exhaled carbon dioxide to determine the volume of the conducting airways, or dead space, and provides an index of airways homogeneity. These techniques may be used independently, or in combination to evaluate the dependence of airways volume and homogeneity on lung volume. This paper provides detailed technical instructions to replicate these techniques and our representative data demonstrates that the airways volume and homogeneity are highly correlated to lung volume. We also provide a macro for the analysis of capnographic data, which can be modified or adapted to fit different experimental designs. The advantage of these measures is that their advantages and limitations are supported by decades of experimental data, and they can be made repeatedly in the same subject without expensive imaging equipment or technically advanced analysis algorithms. These methods may be particularly useful for investigators interested in perturbations that change both the functional residual capacity of the lung and airways volume.

Here, we describe two measures of pulmonary function &#8211; barometric plethysmography, which allows the measurement of lung volume, and volumetric capnography, a tool to measure the anatomic dead space and airways uniformity. These techniques may be used independently or combined to assess airways function at different lung volumes.

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

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Chapters in this video

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Combining Volumetric Capnography And Barometric Plethysmography To Measure The Lung Structure-function Relationship

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases