This work was supported by funds from American Heart Association (A.M.), U.S. Department of Defense (BC074986), and Boston Children&#8217;s Hospital Faculty Career Development Fellowship (T.M., A.M.). The authors thank Amanda Jiang and Elisabeth Jiang for technical assistance.

NOTE: The in vivo animal study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was reviewed and approved by the Animal Care and Use Committee of Boston Children&#8217;s Hospital (Protocol Numbers: 13-10-2526R,14-02-2568R).&#160;All drugs used in this protocol are pharmaceutical grade and these drugs are prepared under sterile conditions.&#160;
1. Fibrin Gel Preparation
<ol>
	<li>Prepare fibrin gel that contains VEGF and bFGF.
	<ol>
		<li>Thaw stock solutions of fibrinogen and thrombin that are stored at -80 &#176;C to room temperature (25 &#176;C).</li>
		<li>Add thrombin (final concentration: 2.5 U/ml), CaCl2 (final concentration: 45 mM), VEGF (final concentration: 0-100 ng/ml) and bFGF (final concentration: 0-100 ng/ml) to the fibrinogen solution (final concentration: 12.5 mg/ml in 0.9% sodium chloride solution 43-45) in a 1.5 ml tube.</li>
		<li>Mix gently by pipetting.</li>
		<li>Gently pipette 200 &#181;l of the mixture onto a sterile plastic dish in a drop-wise fashion using p200 pipette tip.</li>
		<li>Incubate the drops at 37 &#176;C for 30-60 min until they solidify. 
		NOTE: The solidified gel can be kept in the sealed plastic dish at room temperature (25 &#176;C) for several hours before implantation (Figure 1a).</li>
	</ol>
	</li>
	<li>Trim the fibrin gel into approximately 3 x 3 x 3 mm cubes using small surgical scissors before implantation.</li>
</ol>
2. Mouse Preparation
<ol>
	<li>Anesthetize adult mouse (8-12 weeks) by intraperitoneal (IP) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and confirm that the mouse is adequately anesthetized by pinching toe of the mouse.
	<ol>
		<li>Use vet ointment on eyes of the mouse to prevent dryness during the experiment.</li>
	</ol>
	</li>
	<li>Shave fur over left side of the rib cage of the mouse.</li>
	<li>Perform endotracheal intubation of the mouse.</li>
	<li>Place the mouse on the intubation stand angled at 70&#176; and hold mouse in place by hooking its upper incisors over a small rubber band located at the top of the stand.</li>
	<li>Gently retract the tongue to one side using blunt forceps.</li>
	<li>Visualize the larynx with the aid of a fiber-optic gooseneck microscope illuminator.</li>
	<li>Insert endotracheal elastic catheter (21 G) into the trachea.</li>
	<li>Confirm that the mouse is spontaneously breathing in a smooth way (regular 100-150 breaths/min, no paradoxical or shallow respiration).</li>
	<li>Place the mouse in prone position under the dissection microscope.</li>
	<li>Mechanically ventilate the mouse using a rodent ventilator (150 breaths/min and 7 ml/kg tidal volume).</li>
	<li>Count ribs to locate intercostal space between 4th and 5th rib.</li>
	<li>Create a sterile field over the area by thoroughly wiping down with alcohol and Povidone-Iodine. Cover the surgical field adequately with a sterile surgical drape.</li>
</ol>
3. Mouse Surgery
<ol>
	<li>After local injection of 0.25% bupivacaine (200 &#956;l) in the skin, make a transverse skin incision (approximately 1 cm length) over the intercostal space using dissecting scissors.</li>
	<li>After injection of 0.25% bupivacaine (200 &#956;l) into the intercostal muscle, make a muscle incision between the 4th and 5th rib using fine small scissors.</li>
	<li>Insert a dissecting retractor between the ribs to fully visualize the left lung.
	<ol>
		<li>Scrape a small area (1 x 1 mm square) of visceral pleura of the center of left lung using fine forceps.</li>
		<li>Apply gentle pressure on the area using a sterile cotton swab until bleeding and air leaks are completely controlled.</li>
		<li>Put small amount of fresh mixture of fibrinogen/thrombin (fibrin glue) (step 1.1.2. 20 &#181;l) over the area using p200 pipette tip.</li>
	</ol>
	</li>
	<li>Gently place one fibrin gel (step 1.2) using small forceps over the area (Figure 1b).
	<ol>
		<li>Confirm that the gel is well fixed on the area during respiratory movements of the lung.</li>
	</ol>
	</li>
	<li>Make sure that there is neither massive air leaking nor bleeding from the lung.</li>
	<li>Close incisions (muscle and skin layers) with absorbable suture, which does not have to be removed.</li>
	<li>Aspirate the thoracic cavity using 27 G needle and 1 ml syringe to prevent pneumothorax.</li>
	<li>Terminate mechanical ventilation.</li>
</ol>
4. Mouse Recovery
<ol>
	<li>Make sure the mouse is spontaneously breathing in a smooth way (regular 100-150 breaths/min, no paradoxical or shallow respiration).</li>
	<li>IP inject 1 ml of pre-warmed 0.9% NaCl to prevent dehydration.</li>
	<li>Allow mouse to recover on the circulating warm water pad.</li>
	<li>Remove the endotracheal tube after confirming that the mouse has stable breathing.</li>
	<li>Inject Meloxicam (5 mg/kg, subcutaneous injection (SC), for 3 days&#160;as postoperative analgesic.</li>
	<li>Monitor the movements of the mouse carefully at a minimum of 15 min intervals until it is sternal (able to roll onto its stomach and remain upright) and conscious.</li>
	<li>After recovery, return the mouse to a new cage isolated from mice without surgery.</li>
	<li>Monitor the surgical site for signs of infection (redness, swelling, discharge), animal&#8217;s basic biologic functions (food and water intake, urination, defecation, body weight gain) as well as clinical signs of distress (piloerection, reduced locomotion) daily following the surgical procedure.</li>
</ol>
5. Harvesting the Lung
<ol>
	<li>7 to 30 days after implantation, euthanize the mouse using CO2 via source of compressed gas.</li>
	<li>Make an incision between the tip of xyphoid process and the sternal notch (median sternotomy) and harvest whole lung with the implanted gel for histological and biochemical analysis by cutting the trachea and dissecting all connections to the heart, lungs, and trachea.</li>
	<li>Fix implanted gel with lung with 4% paraformaldehyde solution overnight at 4 &#186;C, embed in OCT compound, and take serial step sections of 30 &#181;m thickness.</li>
	<li>Perform histological (hematoxylin and eosin staining) and immunohistochemical analyses (endothelial marker: CD31, epithelial marker: aquaporin (AQP)5 and surfactant protein (SP)-B) using confocal microscope 22,37,40.</li>
	<li>Compile stacks of optical sections (30 &#181;m thick) to form three-dimensional images of lung endothelial and epithelial cells using 3D image analysis software 37.</li>
	<li>Quantify projected areas of newly formed blood vessels using image analysis software 46.</li>
</ol>

The authors declare that they have no competing financial interests.

This article introduces a new method to implant biomaterials on the lung surface of living adult mouse. With this system, host lung-derived angiogenesis is successfully recapitulated inside the material. This system allows researchers to explore crosstalk between endothelial cells, other cells (e.g., epithelial cells, mesenchymal cells, immune cells) and various ECM components that are required for local angiogenesis 50-53 and alveolar regeneration 24,54. Although conventional in vivo subcutaneous hydrogel implantation has been used extensively for angiogenesis research 37-39, those methods do not recapitulate organ-specific angiogenesis. This system, in which hydrogel is implanted directly on the lung surface, will enable researchers to explore the roles of the lung-specific microenvironment in angiogenesis and alveolar regeneration in adult mouse lung. These gels can be fabricated from various ECM-rich biomaterials (e.g., collagens, fibrins) that can be supplemented with various chemical factors (e.g., angiogenic factors, growth factors) 55,56, progenitor cells and/or iPS cells. In addition to chemical factors, mechanical forces also control angiogenesis 23,37. The stiffness of fibrin gel changes in a fibrinogen concentration-dependent manner 57 and manipulating the fibrinogen concentration may affect angiogenesis not only through chemical signals but also through physical cues 58,59. Therefore, physicochemical properties of the fibrin gels may need to be optimized carefully to recapitulate physiological organ-specific angiogenesis in the future. Wound healing after scraping the visceral pleura also produces an endogenous fibrin clot, which includes various types of host cells and promotes the healing process and tissue regeneration. This natural clot may interact with the exogenously implanted fibrin gel, and hence control angiogenesis in the implanted gel. Fluorescently labeled fibrinogen may enable researchers to distinguish between natural fibrin clot and implanted fibrin gel and explore these mechanisms. Although this is a powerful method to characterize angiogenesis in adult mouse lungs, application to the study of lung development and diseases in neonatal mice would likely present technical challenges.
The ultimate goal of this study is to recruit functional blood vessels into fibrin gels implanted on diseased lungs and to use the matrix as a medical device to restore functional lung structures. Possible communications between host cells and the vascular and alveolar structures inside the gels as well as the functionality of these structures should be explored in future experiments. Since VEGF levels in the lungs are decreased in patients with BPD 60 and emphysema 61, adding VEGF to the matrix may improve recruitment of blood vessels into the matrix implanted on these diseased lungs. Mechanical properties also differ between healthy and diseased lungs 23,62. For example, expression of matrix metalloproteinases and lysyl oxidase, which control degradation and crosslinking of collagens, respectively, are altered in various lung diseases including COPD and pulmonary fibrosis 63-67. In diseased lungs, certain lineages of progenitors for lung endothelial and epithelial cells are depleted 68. Thus, manipulating these factors (angiogenic factors, ECMs, ECM stiffness) or implanting fibrin gels supplemented with progenitor cells 69 will likely lead to the formation of functional blood vessels inside the matrix and recovery of lung function in various pathological conditions. Since chemical factors can be supplemented inside the fibrin gels to modulate local angiogenesis, this system can also be utilized explore specific environmental cues that may normalize diseased lungs in chronic lung diseases.
In summary, this article introduces a method to implant fibrin hydrogel on the lung surface of living mouse, which enables researchers to characterize lung-specific angiogenesis in vivo. Modification of various factors (e.g., time course, concentrations and combinations of angiogenic factors, various kinds of hydrogels, physicochemical properties of hydrogels) in this system, will unveil the mechanisms of angiogenesis and regeneration in the lung. Thus, this system will significantly advance scientific knowledge of basic vascular biology, tissue engineering, as well as pulmonary medicine.

The overall goal of this protocol is to introduce a method to implant fibrin gel on the lung surface of adult mouse, which allows researchers to characterize the molecular mechanisms of lung vascular and alveolar development, and to leverage this knowledge in order to develop biomimetic materials capable of recapitulating physiological lung vascular and alveolar formation to treat various lung diseases.
More than 35 million Americans suffer from chronic lung diseases including chronic obstructive pulmonary disease and pulmonary fibrosis. These patients have long-lasting chronic respiratory symptoms such as shortness of breath, chest tightness, nagging cough, and tiredness, which significantly impair their daily life 1-3. Despite a great amount of effort to develop effective therapies for these lung diseases, currently there is no cure; therefore, quality of life for these patients is poor and economic and human costs are high 4-7. Currently, lung transplantation is the only way to save patients with end-stage chronic lung diseases. However, because of the shortage of transplant donors, high cost, serious complications, and low survival rate 8-11, transplantation is not an optimal approach. Recent rapid progress in tissue engineering techniques has enabled researchers to bioengineer implantable lung by repopulating decellularized whole lung with various types of progenitor cells or induced pluripotent stem (iPS) cells 12,13. However, these bioengineered lungs are functional in host animals only for several hours after implantation 12,14,15. Utilizing biomaterials to regenerate the complex structures and functions of lungs has also been fairly unsuccessful. This may be because key biological processes that govern adult lung regeneration have not been well explored. In the lung, formation of the vascular system is one of the earliest and most important events during development and regeneration 16-21. Newly formed vasculatures in the lung not only deliver oxygen, nutrients and various cell components required for organ formation, but also provide instructive regulatory signals to surrounding cells 22-25. Thus, angiogenesis plays key roles in regenerative alveolarization in adult lungs 24,26,27. In addition, deregulated angiogenesis contributes to chronic lung diseases such as chronic obstructive pulmonary disease (COPD) 28, bronchopulmonary dysplasia (BPD) 21-23, and pulmonary fibrosis 29. Thus, to develop more efficient strategies for engineering lungs or treating chronic lung diseases, it is necessary to understand the fundamental mechanisms of lung-specific angiogenesis.
Each organ displays unique mechanical and chemical properties, which may differ between physiological and pathological conditions 30-33. These organ-specific microenvironments regulate endothelial cell behaviors and orchestrate vascular network formation in an organ-specific manner 24,34-36. Thus, to develop more efficient strategies for lung regeneration, the mechanism underlying lung-specific angiogenesis needs to be understood. While conventional in vivo angiogenesis assays such as subcutaneous hydrogel implantation have been used extensively for angiogenesis research 37-39, those methods do not recapitulate organ-specific angiogenesis. Recently, a novel method to implant Matrigel in an elastic mold on the mouse lung has been developed and shown to successfully recruit blood vessels and lung epithelial cells into the gels 22. This unique approach will allow researchers to explore the mechanism of lung-specific angiogenesis as well as interactions between blood vessels and non-vascular lung cells in physiological and pathological conditions. Since 1) Matrigel is not suitable for clinical application; 2) the elastic mold used to cast the gel may affect interactions between hydrogels and host lung tissue and 3) the elastic mold on the lung potentially causes impairment of lung function and pain during respiration, as a more clinically relevant approach, a 3D fibrin matrix containing angiogenic factors (vascular endothelial growth factor (VEGF)/ basic fibroblast growth factor (bFGF)) has been implanted on the mouse lung without casting in the elastic mold, and has successfully recapitulated host lung-derived angiogenesis. Fibrin gel, polymer fibrils generated from thrombin-cleaved fibrinogen, is known to trap a variety of angiogenic factors such as bFGF and VEGF to accelerate angiogenesis in vivo 40,41. Because of its regenerative ability and biodegradable nature 42, fibrin gel is widely used in the field of tissue engineering.
This article introduces a novel and unique approach to implant fibrin gel on the lung surface of living adult mouse and demonstrates that host lung-derived angiogenesis is recapitulated inside the gels in vivo. This method, which enables researchers to study lung-specific angiogenesis, will likely lead to the development of new therapeutic approaches for various types of lung diseases and significantly advance efforts to successfully regenerate adult lung.

<table><tbody><tr><td>Fibrinogen from human placenta</td><td>Sigma</td><td>F4883</td><td>For fabrication of fibrin gel</td></tr><tr><td>Thrombin from bovine plasma</td><td>Sigma</td><td>T9549</td><td>For fabrication of fibrin gel</td></tr><tr><td>Recombinant mouse VEGF 164</td><td>R&amp;amp;D</td><td>493-MV</td><td>For supplementation to fibrin gel</td></tr><tr><td>Recombinant mouse bFGF</td><td>R&amp;amp;D</td><td>3139-FB</td><td>For supplementation to fibrin gel</td></tr><tr><td>Rodent Intubation Stand</td><td>Braintree Scientific INC</td><td>RIS 100</td><td>For intubation</td></tr><tr><td>Fiber-Optic Light Source</td><td>Fisher Scientific</td><td>12-565-35</td><td>For intubation</td></tr><tr><td>21 G Elastic catheter</td><td>B.Braun</td><td>4251652-02</td><td>For intubation</td></tr><tr><td>MiniVent Ventilator</td><td>Harvard Apparatus</td><td>CGS-8009</td><td>For ventilation</td></tr><tr><td>Stemi DV4 Steromicroscope</td><td>Fisher Scientific</td><td>12-070-515</td><td>For surgey</td></tr><tr><td>Absobable suture</td><td>Ethicon</td><td>PDP304</td><td>Surgical suture</td></tr><tr><td>Antibody against CD31</td><td>BD Biosciences</td><td>553370</td><td>Immunohistochemistry</td></tr><tr><td>Antibody against AQP5</td><td>Abcam</td><td>AB78486</td><td>Immunohistochemistry</td></tr><tr><td>Antibody against SP-B</td><td>Abcam</td><td>AB40876</td><td>Immunohistochemistry</td></tr></tbody></table>

implantation of fibrin gel on mouse lung to study lung-specific angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

To examine whether host lung-derived vascular formation is recapitulated inside the biomaterials implanted on the lung, fibrin gels supplemented with major angiogenic factors VEGF and bFGF (0, 10 and 100 ng/ml each) were implanted on the surface of living mouse lungs as reported using Matrigel 22. Fibrin gels 47 that contain these angiogenic growth factors were fabricated as shown in Figure 1a. After thoracotomy, a small area of the left lung surface was scraped using forceps and the fabricated fibrin gel was implanted on the lung of the adult mouse using a small amount of fibrin glue, which is FDA approved and widely used as an effective sealant to stop air leaks and reduce bleeding in lung surgery 48,49 (Figure 1b). Most mice recovered without severe respiratory symptoms (e.g., pneumothorax, respiratory distress). Seven days after implantation, mice were euthanized and lungs were harvested. Implanted fibrin gel was incorporated into the host lung 7 days after implantation (Figure 1c). 3D reconstruction of confocal fluorescence images has shown that host-derived CD31-positive endothelial cells formed vascular networks inside the gels 7 days after implantation in a VEGF/bFGF dose-dependent way (Figure 2a, c). Type I (AQP5 positive) and type II (SP-B positive) lung epithelial cells were also recruited along newly formed blood vessels inside the gels that were supplemented with higher concentrations of VEGF and bFGF (each 100 ng/ml) (Figure 2a). H&#38;E staining of histological sections revealed that other types of host cells also migrated into the gel 7 days after implantation (Figure 2b). These findings suggest that host lung-derived regenerative vascular networks are successfully constructed inside the fibrin gels that are supplemented with angiogenic factors and implanted on the surface of adult mouse lung.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/52012/52012fig1highres.jpg" width="600" /> 
Figure 1: (a) Fibrin gel prepared before implantation. (b) Fibrin gel implanted over the scraped visceral pleura of the left lung (arrowheads). (c) Implanted fibrin gel (arrowheads) incorporated into the host lung 7 days after implantation. Scale bars 1 mm.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/52012/52012fig2highres.jpg" width="600" /> 
Figure 2: (a) Fluorescence micrographs showing formation of vascular networks (CD31 positive; green) and recruited type I (AQP5-positive; magenta) or type II (SP-B positive; magenta) lung epithelial cells inside the fibrin gel supplemented with various concentrations of VEGF and bFGF (0, 10 and 100 ng/ml each) 7 days after implantation. Dashed lines indicate the interface between implanted fibrin gel and host lung. Scale bar: 20 &#181;m. (b) Light micrograph of H&#38;E staining showing infiltration of host cells into the fibrin gel 7 days after implantation. Arrows indicate the interface between the gel and host lung. Scale bar: 20 &#181;m. (c) Graph showing projected areas of newly formed blood vessels in the fibrin gels that are supplemented with various concentrations of VEGF and bFGF (0, 10 and 100 ng/ml each) 7 days after implantation.

Watch this Scientific Journal Video about Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis at JoVE.com

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

implantation-fibrin-gel-on-mouse-lung-to-study-lung-specific

Nanyang Technological University

Research

JoVE Journal

Medicine

10.0K Views.  Boston Children's Hospital and Harvard Medical School. Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse. 

Video: Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Developmental Biology

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

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 ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

Cancer Research

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Immunology and Infection

Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor shortages and posttransplant complications persist. Bioengineered lungs, integrating patient-specific cells into decellularized animal scaffolds, present a promising alternative. Despite progress in using bioengineered lungs in animal models, functionality and structure remain immature. This protocol addresses a critical barrier in organ bioengineering: the need for a cost-effective experimental platform. By using mouse models instead of larger animals like rats or swine, researchers can significantly reduce the resources required for each experiment, accelerating research progress. 
The protocol outlines a detailed procedure for lung bioengineering using mouse heart-lung blocks and human primary cells, focusing on isolation strategy for the mouse heart-lung block, decellularization, bioreactor setup, perfusion-based organ culture, and orthotopic transplantation of bioengineered lungs. This mouse-scale platform not only reduces experimental costs but also provides a viable framework for optimizing cell types and numbers for recellularization, testing different cell types using histological and molecular methods, and ensuring blood flow post-transplantation. The method holds potential for broad applications, including studying cell interactions in three-dimensional culture conditions, cell-matrix interactions, and ex vivo cancer modeling, thereby advancing the field of organ bioengineering.

This paper describes how to create bioengineered mouse lungs using decellularization and recellularization methods. It also details subsequent orthotopic lung transplantation.

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor ...

Bioengineering

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

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