We acknowledge the work of Dr. Adlah Sukkar, M.D. in assisting with casting of the lung. This work has been funded by NHLBI, HL088005.

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
<ol>
	<li>Place rat (Sprague Dawley male rats, 125-150 g; Harlan, Indianapolis, IN) in an induction chamber infused with 3% isoflurane.</li>
	<li>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.</li>
</ol>
2. Intubation
<ol>
	<li>Immobilize appendages in supine position using surgical tape.</li>
	<li>Remove rat from nose cone, extend tongue with padded forceps.</li>
	<li>Use 14 gauge intracath with blunted metal stilet as guide. Slide behind tongue, into trachea.</li>
	<li>Remove metal stilet leaving white plastic intracath in trachea. Ensure the rat is breathing and air is flowing through the tube.</li>
	<li>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).</li>
	<li>Apply Puralube (Butler Schein, Dublin, OH) veterinary ointment on eyes.</li>
</ol>
3. Thoracotomy
<ol>
	<li>Place rat right side down, immobilize appendages using surgical tape.</li>
	<li>Shave left side ribcage area.</li>
	<li>Remove excess fur with small vacuum.</li>
	<li>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).</li>
	<li>Make transverse incision with sterile dissecting scissors or sterile scalpel in center of field.</li>
	<li>Blunt dissect through layers of tissue and fat down to ribs (last layer is thin membrane covering ribs).</li>
	<li>Count ribs to determine 3rd intercostal space.</li>
	<li>Using sterile 45 &deg; Graefe forceps, make blunt incision between 3rd and 4th rib.</li>
	<li>Insert rib separators, pull gently creating open void with full visualization of lung, place tape on sutures to keep open.</li>
</ol>
4. Left Pulmonary Artery Ligation
<ol>
	<li>With 90 &deg; Graefe forceps, move left lung back with right hand.</li>
	<li>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.</li>
	<li>Use Dumont pattern #5 45 &deg; 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.</li>
	<li>Separate directly under artery without going through the vessel; smoothly slide the forceps tips held together along the separation.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Tie down occluding the left pulmonary with a square knot, carefully snip remainder of suture.</li>
	<li>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).</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Cut midline along neck, blunt dissect to reveal trachea and left carotid artery (microsphere injection site).</li>
	<li>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.</li>
	<li>Place bottle of microspheres (15 &mu;m crimson polystyrene fluorescent microspheres, 1 x 106 spheres/ml; Invitrogen, Eugene, OR) in water sonicator for 30 sec.</li>
	<li>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.</li>
	<li>Attach Hamilton syringe to 4-way stopcock and infuse microspheres with syringe pump (rate: 500 &mu;l/min; Genie Plus, Kent Scientific, Torrington, CT).</li>
	<li>Remove Hamilton syringe and flush apparatus with 1 ml of heparized saline at 500 &mu;l/min.</li>
	<li>Perform full chest thoracotomy and exsanguinate the rat by severing the inferior vena cava.</li>
	<li>Remove the left lung and other tissues of interest.</li>
	<li>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 &deg;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 &deg;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 &deg;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).</li>
</ol>

<ol>
	<li>Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+stages+in+the+development+of+collateral+circulation+to+the+lung+in+the+rat.">Early stages in the development of collateral circulation to the lung in the rat.</a> Circulation Research. 8, 353-376 (1960).</li><li>Li, X., Wilson, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+vascularity+of+the+bronchial+mucosa+in+mild+asthma.">Increased vascularity of the bronchial mucosa in mild asthma.</a> Am. J. Respir. Crit. Care Med. 156, 229-233 (1997).</li><li>Turner-Warwick, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precapillary+systemic-pulmonary+anastomoses.">Precapillary systemic-pulmonary anastomoses.</a> Thorax. 18, 225-237 (1963).</li><li>Muller, K. M., Meyer-Schwickerath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchial+arteries+in+various+stages+of+bronchogenic+carcinoma.">Bronchial arteries in various stages of bronchogenic carcinoma.</a> Pathol. Res. Pract. 163, 34-46 (1978).</li><li>Remy-Jardin, M., Duhamel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+Collateral+Supply+in+Patients+with+Chronic+Thromboembolic+and+Primary+Pulmonary+Hypertension:+Assessment+with+Multi-Detector+Row+Helical+CT+Angiography.">Systemic Collateral Supply in Patients with Chronic Thromboembolic and Primary Pulmonary Hypertension: Assessment with Multi-Detector Row Helical CT Angiography.</a> Radiology. , 274-281 (2005).</li><li>Karsner, H., Ghoreyeb, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+in+infarction:+The+circulation+in+experimental+pulmonary+embolism.">Studies in infarction: The circulation in experimental pulmonary embolism.</a> J. Exp. Med. 18, 507-522 (1913).</li><li>Endrys, J., Hayat, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+bronchopulmonary+collaterals+and+collateral+blood+flow+in+patients+with+chronic+thromboembolic+and+primary+pulmonary+hypertension.">Comparison of bronchopulmonary collaterals and collateral blood flow in patients with chronic thromboembolic and primary pulmonary hypertension.</a> Heart. 78, 171-176 (1997).</li><li>Virchow, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uber+die+Standpunkte+in+den+Wissenschaftlichen+Medizin.">Uber die Standpunkte in den Wissenschaftlichen Medizin.</a> Virchow Archiv. 1, 1-19 .</li><li>Fadel, E., Mazmanian, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+reperfusion+injury+after+chronic+or+acute+unilateral+pulmonary+artery+occlusion.">Lung reperfusion injury after chronic or acute unilateral pulmonary artery occlusion.</a> Am. J. Respir. Crit. Care Med. 157, 1294-1230 (1998).</li><li>Charan, N. B., Carvalho, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+bronchial+circulatory+system+after+unilateral+pulmonary+artery+obstruction.">Angiogenesis in bronchial circulatory system after unilateral pulmonary artery obstruction.</a> J. Appl. Physiol. 82, 284-291 (1997).</li><li>Shi, W., Hu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+reactivity+of+pulmonary+vessels+in+postobstructive+pulmonary+vasculopathy.">Altered reactivity of pulmonary vessels in postobstructive pulmonary vasculopathy.</a> J. Appl. Physiol. 88, 17-25 (2000).</li><li>Shi, W., Giaid, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+reactivity+to+endothelin+of+pulmonary+arteries+in+long-term+post-obstructive+pulmonary+vasculopathy+in+rats.">Increased reactivity to endothelin of pulmonary arteries in long-term post-obstructive pulmonary vasculopathy in rats.</a> Pulm. Pharmacol. Ther. 11, 189-196 (1998).</li><li>Sukkar, A., Jenkins, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+CXCR2+Attenuates+Bronchial+Angiogenesis+in+the+Ischemic+Rat+Lung.">Inhibition of CXCR2 Attenuates Bronchial Angiogenesis in the Ischemic Rat Lung.</a> J. Appl. Physiol. 104, 1470-1475 (2008).</li><li>Mitzner, W., Lee, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+the+mouse+lung.">Angiogenesis in the mouse lung.</a> Am. J. Pathol. 157, 93-101 (2000).</li><li>Wagner, E. M., Jenkins, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+and+vascular+function+during+chronic+severe+pulmonary+ischemia.">Lung and vascular function during chronic severe pulmonary ischemia.</a> J. Appl. Physiol. 110, 538-544 (2011).</li><li>Remy-Jardin, M., Bouaziz, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchial+and+nonbronchial+systemic+arteries+at+multi-detector+row+CT+angiography:+comparison+with+conventional+angiography.">Bronchial and nonbronchial systemic arteries at multi-detector row CT angiography: comparison with conventional angiography.</a> Radiology. 233, 741-749 (2004).</li><li>Baluk, P., Tammela, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+persistent+lymphatic+vessel+hyperplasia+in+chronic+airway+inflammation.">Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation.</a> J. Clin. Invest. 115, 247-257 (2005).</li><li>Bailey, S. R., Boustany, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+vascular+reactivity+and+vascularisation+in+human+chronic+airway+disease.">Airway vascular reactivity and vascularisation in human chronic airway disease.</a> Pulm. Pharmacol. Ther. 22, 417-425 (2009).</li></ol>

No conflicts of interest declared.

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. ...

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.

<table border="1">
	<tbody>
		<tr>
			<td>Reagents:</td>
			<td>Company</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>buprenorphine hydrochloride, Puralube</td>
			<td>Butler Schein</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>bupivicaine</td>
			<td>APP Pharmaceuticals</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Povidone-Iodine swabstick</td>
			<td>Dynarex Corporation</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>polypropylene suture size 6-0, 3/8 circle reverse cutting needle</td>
			<td>Myco Medical</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PE20 tubing</td>
			<td>Becton Dickinson</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 &mu;m crimson polystyrene fluorospheres</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml Hamilton glass syringe</td>
			<td>Hamilton Company</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Equipment:</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Genie Plus syringe pump</td>
			<td>Kent Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence Spectrophotometer</td>
			<td>Digilab</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rodent Ventilator Model 683</td>
			<td>Harvard Apparatus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 1. Table of specific reagents and equipment.</td>
		</tr>
	</tbody>
</table>

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.

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...

Watch this Scientific Journal Video about Angiogenesis in the Ischemic Rat Lung at JoVE.com

Angiogenesis in the Ischemic Rat Lung

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 ...

Research

JoVE Journal

Medicine

15.5K Views.  Johns Hopkins University. 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.

Video: Angiogenesis in the Ischemic Rat Lung

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

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 ...

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.

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 ...

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

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

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

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

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

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart.
There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipient&#8217;s infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft.
A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.

We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

Real-Time Monitoring of Cellular Dynamics in Mouse Lung Transplantation

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...