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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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’s Hospital (Protocol Numbers: 13-10-2526R,14-02-2568R). All drugs used in this protocol are pharmaceutical grade and these drugs are prepared under sterile conditions. 

1. Fibrin Gel Preparation

1. Prepare fibrin gel that contains VEGF and bFGF.
   1. Thaw stock solutions of fibrinogen and thrombin that are stored at -80 °C to room temperature (25 °C).
   2. 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.
   3. Mix gently by pipetting.
   4. Gently pipette 200 µl of the mixture onto a sterile plastic dish in a drop-wise fashion using p200 pipette tip.
   5. Incubate the drops at 37 °C for 30-60 min until they solidify.
      NOTE: The solidified gel can be kept in the sealed plastic dish at room temperature (25 °C) for several hours before implantation (Figure 1a).
2. Trim the fibrin gel into approximately 3 x 3 x 3 mm cubes using small surgical scissors before implantation.

2. Mouse Preparation

1. 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.
   1. Use vet ointment on eyes of the mouse to prevent dryness during the experiment.
2. Shave fur over left side of the rib cage of the mouse.
3. Perform endotracheal intubation of the mouse.
4. Place the mouse on the intubation stand angled at 70° and hold mouse in place by hooking its upper incisors over a small rubber band located at the top of the stand.
5. Gently retract the tongue to one side using blunt forceps.
6. Visualize the larynx with the aid of a fiber-optic gooseneck microscope illuminator.
7. Insert endotracheal elastic catheter (21 G) into the trachea.
8. Confirm that the mouse is spontaneously breathing in a smooth way (regular 100-150 breaths/min, no paradoxical or shallow respiration).
9. Place the mouse in prone position under the dissection microscope.
10. Mechanically ventilate the mouse using a rodent ventilator (150 breaths/min and 7 ml/kg tidal volume).
11. Count ribs to locate intercostal space between 4^th and 5^th rib.
12. 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.

3. Mouse Surgery

1. After local injection of 0.25% bupivacaine (200 μl) in the skin, make a transverse skin incision (approximately 1 cm length) over the intercostal space using dissecting scissors.
2. After injection of 0.25% bupivacaine (200 μl) into the intercostal muscle, make a muscle incision between the 4^th and 5^th rib using fine small scissors.
3. Insert a dissecting retractor between the ribs to fully visualize the left lung.
   1. Scrape a small area (1 x 1 mm square) of visceral pleura of the center of left lung using fine forceps.
   2. Apply gentle pressure on the area using a sterile cotton swab until bleeding and air leaks are completely controlled.
   3. Put small amount of fresh mixture of fibrinogen/thrombin (fibrin glue) (step 1.1.2. 20 µl) over the area using p200 pipette tip.
4. Gently place one fibrin gel (step 1.2) using small forceps over the area (Figure 1b).
   1. Confirm that the gel is well fixed on the area during respiratory movements of the lung.
5. Make sure that there is neither massive air leaking nor bleeding from the lung.
6. Close incisions (muscle and skin layers) with absorbable suture, which does not have to be removed.
7. Aspirate the thoracic cavity using 27 G needle and 1 ml syringe to prevent pneumothorax.
8. Terminate mechanical ventilation.

4. Mouse Recovery

1. Make sure the mouse is spontaneously breathing in a smooth way (regular 100-150 breaths/min, no paradoxical or shallow respiration).
2. IP inject 1 ml of pre-warmed 0.9% NaCl to prevent dehydration.
3. Allow mouse to recover on the circulating warm water pad.
4. Remove the endotracheal tube after confirming that the mouse has stable breathing.
5. Inject Meloxicam (5 mg/kg, subcutaneous injection (SC), for 3 days as postoperative analgesic.
6. 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.
7. After recovery, return the mouse to a new cage isolated from mice without surgery.
8. Monitor the surgical site for signs of infection (redness, swelling, discharge), animal’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.

5. Harvesting the Lung

1. 7 to 30 days after implantation, euthanize the mouse using CO₂ via source of compressed gas.
2. 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.
3. Fix implanted gel with lung with 4% paraformaldehyde solution overnight at 4 ºC, embed in OCT compound, and take serial step sections of 30 µm thickness.
4. 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.
5. Compile stacks of optical sections (30 µm thick) to form three-dimensional images of lung endothelial and epithelial cells using 3D image analysis software ³⁷.
6. Quantify projected areas of newly formed blood vessels using image analysis software ⁴⁶.

Results

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...

Discussion

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 <...

Materials

List of materials used in this article

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