Angiogenesis in the Ischemic Rat Lung

Summary

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.

Abstract

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.

Introduction

Systemic angiogenesis in the lung is well-recognized. In disease states such as asthma ², interstitial pulmonary fibrosis ³, cancer ⁴, and chronic pulmonary thromboembolism ⁵, the systemic vasculature in and surrounding the lung proliferates and invades the pulmonary parenchyma. However, animal models to study this differential activation of the systemic rather than the pulmonary circulation are few. Perhaps the most reproducible model of systemic neovascularization in the lung of the adult mammal is that which occurs after inducing chronic pulmonary artery ischemia. The response to left pulmonary artery obstruction in humans ^5-7, dogs ⁸, pigs ⁹, sheep ¹⁰, guinea pigs ¹¹, rats ^{1, 12, 13}, and mice ¹⁴ is the rapid proliferation of the bronchial artery as well as intercostal arteries. The mechanisms responsible for systemic neovascularization of the lung after pulmonary ischemia are largely unknown and have not been widely studied. The time course of bronchial angiogenesis in the rat after left pulmonary artery obstruction has been carefully described in the histologic work of Weibel ¹. Extending this work in the rat, our laboratory has focused on both the growth factors important in this process as well as the physiologic outcome of this neovasculature in the lung. Results demonstrate the CXC chemokine CINC-3 is elevated early after ischemia and treating rats with a neutralizing antibody to CXCR2, the receptor for CINC-3, attenuates angiogenesis ¹³. The newly established bronchial vasculature 14 days after the onset of pulmonary ischemia was shown to be abnormal with significantly increased protein permeability ¹⁵. Left lung function was not normal showing decreased diffusing capacity and a decrease in lung volume ¹⁵. Although the neovasculature may have contributed to the preservation of lung tissue during chronic pulmonary ischemia, it appears not to be normal and may contribute to a sustained decrease in pulmonary function.

Perhaps one of the most curious aspects of this model relates to the spatial distribution of proliferating blood vessels. Despite the release of growth factors within the pulmonary parenchyma due to ischemia, the neovasculature originates in relatively large upstream bronchial arteries. The normal bronchial artery arises as a small branch from the aorta and invades the airway tree at the carina. Thus the mechanism by which growth factors induce the initial phase of arteriogenesis is not clear. We suggest that the rat, with a vascular anatomy similar to humans, provides a unique opportunity to study the mechanisms responsible for systemic angiogenesis during pulmonary ischemia. Although complete obstruction of the left pulmonary artery is a rare occurrence in human subjects, increased bronchial vascularity appears to be similarly induced in patients whatever the site and size of pulmonary artery obstruction ¹⁶. Thus, we provide a detailed description of the surgical approach to ligate the left pulmonary artery in rats and a means to quantify the magnitude of angiogenesis.

Protocol

All protocols performed on rats have been approved by the Johns Hopkins University Animal Care and Use Committee and in accordance with NIH guidelines. Whenever possible, the animal should be surgically prepped in an area separate from the surgical area to minimize contamination of the surgical site.

1. Anesthesia/analgesia

1. Place rat (Sprague Dawley male rats, 125-150 g; Harlan, Indianapolis, IN) in an induction chamber infused with 3% isoflurane.
2. Place anesthetized rat on surgery board attached to nose cone and ventilator with 3% isoflurane anesthetic. Use sterile technique for all procedures. Drape rat to ensure sterile operating field.

2. Intubation

1. Immobilize appendages in supine position using surgical tape.
2. Remove rat from nose cone, extend tongue with padded forceps.
3. Use 14 gauge intracath with blunted metal stilet as guide. Slide behind tongue, into trachea.
4. Remove metal stilet leaving white plastic intracath in trachea. Ensure the rat is breathing and air is flowing through the tube.
5. Replace nose cone with direct adapter to catheter, connect rat to ventilator (90 breaths/min; 8 ml/kg tidal volume; Rodent Ventilator Model 683, Harvard Apparatus, Holliston, Massachusetts).
6. Apply Puralube (Butler Schein, Dublin, OH) veterinary ointment on eyes.

3. Thoracotomy

1. Place rat right side down, immobilize appendages using surgical tape.
2. Shave left side ribcage area.
3. Remove excess fur with small vacuum.
4. Make sterile field by wiping down with alcohol followed by Povidone-Iodine swabstick (Dynarex Corporation, Orangebur, NY). Repeat this process two more times (for a total of three scrubs).
5. Make transverse incision with sterile dissecting scissors or sterile scalpel in center of field.
6. Blunt dissect through layers of tissue and fat down to ribs (last layer is thin membrane covering ribs).
7. Count ribs to determine 3^rd intercostal space.
8. Using sterile 45 ° Graefe forceps, make blunt incision between 3^rd and 4^th rib.
9. Insert rib separators, pull gently creating open void with full visualization of lung, place tape on sutures to keep open.

4. Left Pulmonary Artery Ligation

1. With 90 ° Graefe forceps, move left lung back with right hand.
2. Using Dumont pattern #5 straight forceps, grab left pulmonary artery and airway with left hand. Left pulmonary artery will lay on top of airway. Ensure that forceps are directly perpendicular to the table. Additionally, it is most effective to pick up the left pulmonary artery/ left mainstem bronchus at the most distal position (closest to parenchyma). Push the ventilating left lung aside with this left hand maneuver.
3. Use Dumont pattern #5 45 ° curved forceps to separate the left pulmonary artery from the left mainstem bronchus at their natural borders. This separation line appears thin and white between the two individual structures.
4. Separate directly under artery without going through the vessel; smoothly slide the forceps tips held together along the separation.
5. Continue until tips of forceps have visibly separated the left pulmonary artery and the left mainstem bronchus. Only a small point of tip needs to be fully through. If blood can be visualized on the tip of the forceps, then it is not fully through and ligation should not be attempted. Once through the space, the left pulmonary artery will lay on the curve of the forceps. Hold in this position.
6. Gently release straight blunt forceps grip (left hand) and grab piece of a pre-cut suture (~2-3 inches; polypropylene suture size 6-0; Myco Medical, Cary, NC).
7. Open curved forceps cradling left pulmonary artery and grab suture. Gently pull suture through space between left pulmonary artery and left mainstem bronchus in an upward motion relative to the curve of the forceps.
8. Tie down occluding the left pulmonary with a square knot, carefully snip remainder of suture.
9. Close ribs using blunt forceps to hold rib and suture twice with polypropylene (blue monofilament) size 4-0 attached to a 19 mm, 3/8 circle reverse cutting needle (Myco Medical, Cary, NC ) in a hemostat, being careful not to suture skin (only ribs).
10. Complete a loose square knot, inflate the lung, place on positive end-expiratory pressure (PEEP; 2-5 cmH₂O), hyperinflate lung then secure knot tightly, and make another full knot before snipping remainder of suture. Remove from PEEP and visualize for 30 sec to ensure that the lung does not collapse. Apply 5 drops Bupivicaine (APP Pharmaceuticals, Schaumbur, IL).
11. Close skin by placing tissue glue on wound and push skin together using back end of cotton tip applicator. Give Bupivicaine (2.0 mg/kg subcutaneous) at the site of incision subsequently every 8 hr for 24 hr or until the animal resumes normal activity. As the subcutaneous layer in this area is very thin, it is not easy to suture on its own. When the tissue glue is applied and the skin is pushed together it also closes the subcutaneous layer.
12. Turn off isoflurane gas but continue to ventilate the rat for 1-2 min on room air until voluntary movement returns. Disconnect the tracheal tube from the ventilator and ensure that the rat is breathing spontaneously before removing it.
13. Wipe off Puralube from eyes with cotton swab and monitor movement and recovery. Inject buprenorphine hydrochloride (0.05 mg/kg intraperitoneal, Butler Schein, Dublin OH). Continue delivery of analgesic every 12 hr for 48 hr after surgery.

5. Left Carotid Artery Cannulation

To assess the magnitude of bronchial perfusion of the ischemic left lung at desired time points after left pulmonary artery ligation, inject labeled microspheres through the left carotid artery into the aortic arch. Prepare rats as above 1-2.

1. Cut midline along neck, blunt dissect to reveal trachea and left carotid artery (microsphere injection site).
2. Insert catheter filled with heparinized saline, blunt tip of PE20 tubing (Becton Dickinson, Sparks MD) into vessel connected to 25 g needle, 4-way stopcock, 1 ml syringe.
3. Place bottle of microspheres (15 μm crimson polystyrene fluorescent microspheres, 1 x 10⁶ spheres/ml; Invitrogen, Eugene, OR) in water sonicator for 30 sec.
4. Remove bottle, vortex and draw up 0.5 ml (500,000 microspheres) into a 1 ml Hamilton glass syringe (Hamilton Company, Reno, NV) through a 20 g needle.
5. Attach Hamilton syringe to 4-way stopcock and infuse microspheres with syringe pump (rate: 500 μl/min; Genie Plus, Kent Scientific, Torrington, CT).
6. Remove Hamilton syringe and flush apparatus with 1 ml of heparized saline at 500 μl/min.
7. Perform full chest thoracotomy and exsanguinate the rat by severing the inferior vena cava.
8. Remove the left lung and other tissues of interest.
9. To extract microspheres from tissue, take the entire left lung of the rat after exsanguination and place it in 2M KOH (4-6 ml). Place in a 55 °C water bath and leave overnight for tissue digestion. Add Tween 80 (0.25%) to wash the beads, vortex (10 sec) and centrifuge (2,000 rpm; 20 °C for 10 min). Remove the supernatant, add 2-ethoxyethyl acetate (1 ml), vortex and let stand for 1 hr. Vortex the suspension and centrifuge (2,000 rpm; (20 °C for 10 min). Remove the 2-ethoxyethyl acetate aqueous layer containing the fluorescence, place in a cuvette and measure using a Hitachi F-2500 fluorescence spectrophotometer (excitation 612/emission 618; Digilab, Holliston, MA).

Results

Vascular cast: Results of the effects of left pulmonary artery ischemia in the rat are depicted in Figure 1. Shown is a methacrylate cast of the bronchial vasculature and the extensive vascularity of the left airway tree 28 days after LPAL. To obtain this cast, the systemic vasculature was injected with a methacrylate mixture (red), retrograde into the descending aorta and the trachea was cannulated and injected with a white silicon based material. This vascular cast provides a remarkable visuali...

Discussion

Left pulmonary artery ligation in all species tested leads to robust systemic neovascularization of the ischemic lung. We have presented the details of the surgical approach in a rat model. Our results produced by vascular casting, histopathology, and in vivo labeling demonstrate that bronchial arteries proliferate and perfuse the pulmonary parenchyma. Thus, the mechanisms of bronchial angiogenesis can be studied in an animal model that parallels the human condition of chronic pulmonary thromboembolism. ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
buprenorphine hydrochloride, Puralube	Butler Schein
bupivicaine	APP Pharmaceuticals
Povidone-Iodine swabstick	Dynarex Corporation
polypropylene suture size 6-0, 3/8 circle reverse cutting needle	Myco Medical
PE20 tubing	Becton Dickinson
15 μm crimson polystyrene fluorospheres	Invitrogen
1 ml Hamilton glass syringe	Hamilton Company
Equipment:
Genie Plus syringe pump	Kent Scientific
Fluorescence Spectrophotometer	Digilab
Rodent Ventilator Model 683	Harvard Apparatus
Table 1. Table of specific reagents and equipment.