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 <...

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 border="0" cellpadding="0" cellspacing="0" width="956">
	<tbody>
		<tr height="48">
			<td height="48" style="height:48px;width:343px;">Name of Material/ Equipment</td>
			<td style="width:177px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:289px;">Comments/Description</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fibrinogen from human placenta</td>
			<td>Sigma</td>
			<td>F4883</td>
			<td>For fabrication of fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Thrombin from bovine plasma</td>
			<td>Sigma</td>
			<td>T9549</td>
			<td>For fabrication of fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Recombinant mouse VEGF 164</td>
			<td>R&#38;D</td>
			<td>493-MV</td>
			<td>For supplementation to fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Recombinant mouse bFGF</td>
			<td>R&#38;D</td>
			<td>3139-FB</td>
			<td>For supplementation to fibrin gel</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:343px;">Rodent Intubation Stand</td>
			<td style="width:177px;">Braintree Scientific INC</td>
			<td style="width:147px;">RIS 100</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fiber-Optic Light Source</td>
			<td style="width:177px;">Fisher Scientific</td>
			<td>12-565-35</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">20G Elastic catheter</td>
			<td>B.Braun</td>
			<td>4251652-02</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:343px;">MiniVent Ventilator</td>
			<td style="width:177px;">Harvard Apparatus</td>
			<td style="width:147px;">CGS-8009</td>
			<td>For ventilation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Stemi DV4 Steromicroscope</td>
			<td style="width:177px;">Fisher Scientific</td>
			<td>12-070-515</td>
			<td>For surgey</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Absobable suture</td>
			<td>Ethicon</td>
			<td>PDP304</td>
			<td>Surgical suture</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against CD31</td>
			<td>BD Biosciences</td>
			<td align="right">553370</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against AQP5</td>
			<td style="width:177px;">Abcam</td>
			<td>AB78486</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against SP-B</td>
			<td style="width:177px;">Millipore</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 th...

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

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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 ...

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

Treatment of Osteochondral Defects in the Rabbit&#039;s Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

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 ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Methods Article

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