Name: angiogenesis in the ischemic rat lung
Uploaded: 2013-02-08T13:00:00
Description: Watch this Scientific Journal Video about Angiogenesis in the Ischemic Rat Lung at JoVE.com

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 o...

<ol>
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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 ...

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