Angiogenesis in the Ischemic Rat Lung
February 8th, 2013
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 overall goal of this procedure is to ligate the left pulmonary artery. This is accomplished by first intubating and ventilating an anesthetized rat. Next, a thoracotomy is performed and the left pulmonary artery is ligated.
After the ligation, the chest is closed and the rat is allowed to recover. Ultimately, the extent of neovascularization in the left lung can be assessed by measuring the level of fluorescence in the tissue or by other histological methods. The main advantage of this technique is that it provides an opportunity to study the in vivo development and neovascularization of the bronchial circulation in the lung.
This provides a a, an opportunity to study the complex 3D structure of this new vasculature in a ventilating lung. Now, this is very much different from what is currently done by most investigators who form who, who perform in vitro studies studying endothelial cell proliferation and chemotaxis or tube formation. Generally, individuals new to this procedure will struggle with trying to do the dissection between the left pulmonary artery and the left bronchus.
The only advice I can offer is to practice, to be patient and to view this video frequently to see if there are minor adjustments in the surgical approach. Demonstrating this procedure is John Jenkins, a technician from my laboratory. To begin place an anesthetized rat on a surgery board and attach a nose cone for the continuation of anesthesia, intubate the animal with a plastic catheter and connect the rat to a ventilator.
After applying ointment to the eyes, place the rat on its right side and tape down the appendages. Remove the hair over the surgical area and prepare the skin with repeated scrubs of Betadine and alcohol. When ready, make a transverse incision through the skin.
Next blunt, dissect through the layers of fat and tissue to reveal the rib cage. Once exposed, identify the third intercostal space and use forceps to make a blunt incision between the third and fourth rib. Insert rib separators into the opening and pull the ribs open gently until the lung is visible.
Secure the opening. First, move the left lung back with the right hand. Then with straight forceps, grab the left pulmonary artery and airway with the left.
The left pulmonary artery should lie on top of the airway and the forceps should be perpendicular to the table. Once aligned, properly use carved forceps to separate the artery from the left main stem bronchus at their natural borders. This separation line appears thin and white between the two individual structures.
Taking care not to go through the vessel. Slide the tips of the forceps along the separation. Continue in this manner until the left pulmonary artery lays on the curve of the forceps.
With the left hand gently release the straight forceps and grab a piece of precut suture. Open the curved forceps, cradling the artery enough to grab the suture. Gently pull the suture through in an upward motion relative to the curve of the forceps.
Once through, tighten the suture to occlude the left pulmonary artery and snip away any remaining suture while holding them together with forceps. Suture the ribs. Taking care not to suture the skin loosely.
Tie a square knot that will later close the ribs. Hyperinflate the lung by briefly occluding the ventilator expiratory line, and then tighten the suture. Secure the suture by making another full knot before snipping away the ends.
Remove the end expirator pressure and watch for 30 seconds to ensure that the lungs do not collapse. Lastly, apply five drops. Bupivacaine, close the skin with tissue glue.
Turn off the isof fluorine gas, but continue to ventilate the rat for one to two minutes on room air until spontaneous respiration returns to a steady state. Next, disconnect the tracheal tube from the ventilator and ensure that the rat is breathing spontaneously before removing it. Administer a local analgesic periodically over the next 24 hours.
Lastly, wipe the ointment from the eyes and monitor the animal until it is recovered at desired time. Points after ischemia labeled microspheres can be injected. To assess the magnitude of bronchial perfusion, a small volume infusion pump is used to deliver labeled microspheres into the left carotid artery at a constant rate.
Connect the catheter to a blunt 25 gauge needle and a four-way stopcock. To begin follow the surgical preparations demonstrated earlier. When ready, make an incision over the midline along the neck blunt.
Dissect the tissue to reveal the trachea and left carotid artery. Once isolated past two sutures under the carotid artery, tie the proximal suture firmly. Next, make a small cut in the artery and insert a saline filled catheter.
Secure this in place with the upstream suture. This vascular cast provides a remarkable visualization of bronchial angiogenesis in the lung. 28 days after left pary artery ligation here, an airway section from the left lung three days after ation is shown.
Note the two prominent bronchial vessels filled with red blood cells located within the airway wall. The inset is stained to show proliferating endothelial cells of the bronchial artery. This example shows a left lung section taken 14 days after ligation.
Note the increased size of the bronchial vessels at this time point and the withered pulmonary artery fluorescence used to assess the magnitude of systemic perfusion was virtually undetected in the left lung of naive rats. However, there was a substantial, significant and consistent level measured in the left lung from rats. Studied 14 days after ligation Once mastered.
This technique can be done in less than an hour if it is performed properly. After watching this video, you should have a good understanding of how to intubate the rat, perform the thoracotomy, and successfully ligate the pulmonary artery. Depending on your outcome variable, you should be able to cannulate the left carotid artery and infuse labeled microspheres for perfusion analysis.
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Overview
This study outlines a surgical procedure to ligate the left pulmonary artery in rats, promoting bronchial neovascularization. The method allows for the assessment of neovascularization in the left lung through fluorescence measurement and histological analysis.
Key Study Components
Area of Science
- Neuroscience
- Vascular Biology
- Experimental Surgery
Background
- The lung receives blood from both systemic and pulmonary sources.
- Smaller systemic vasculature often shows neovascularization in lung pathologies.
- Cessation of pulmonary blood flow can lead to increased bronchial angiogenesis.
- Understanding these mechanisms is crucial for developing therapeutic strategies.
Purpose of Study
- To investigate the effects of left pulmonary artery ligation on bronchial neovascularization.
- To provide a detailed surgical protocol for inducing ischemia.
- To assess neovascularization through fluorescence and histological methods.
Methods Used
- Intubation and ventilation of anesthetized rats.
- Thoracotomy to access the left pulmonary artery.
- Ligation of the left pulmonary artery.
- Recovery of the rat post-surgery for assessment.
Main Results
- Successful ligation of the left pulmonary artery was achieved.
- Increased neovascularization was observed in the left lung.
- Fluorescence measurements indicated significant changes post-ligation.
- Histological analysis confirmed the development of bronchial circulation.
Conclusions
- The ligation technique effectively induces bronchial neovascularization.
- This model can be used to study lung vascular responses to ischemia.
- Findings may contribute to understanding lung pathologies and therapies.
Frequently Asked Questions
Executive Industry Relevance
Quantitative assessment of angiogenesis in the ischemic rat lung provides a robust in vivo model for evaluating vascular remodeling and neovascularization under pathophysiological conditions. This approach enables mechanistic de-risking of therapeutic hypotheses targeting lung perfusion and supports predictive confidence in early-stage vascular biology programs. The model's translational relevance lies in its ability to mimic systemic arterial responses observed in human pulmonary ischemia.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of angiogenic pathways and cellular mechanisms in a physiologically relevant lung environment.
- Supports functional target validation by quantifying neovascularization following controlled ischemic insult.
- Facilitates mechanistic de-risking for candidate molecules modulating vascular growth or repair.
Screening & Assay Development
- Provides a validated in vivo system for assessing angiogenic response to experimental interventions.
- Enables reproducible quantification of perfusion using labeled microsphere injection and fluorescence readouts.
- Supports assay standardization for comparative evaluation of vascular-modulating compounds.
Translational & Preclinical Research
- Aligns with disease-relevant models of pulmonary ischemia and vascular remodeling.
- Enables continuity from discovery through preclinical validation of angiogenesis-modulating strategies.
- Supports risk-adjusted advancement decisions based on quantitative perfusion and histological endpoints.
Pipeline & Workflow Integration
This model integrates into the discovery-to-preclinical continuum for vascular-targeted therapies, bridging early mechanistic studies and translational research in pulmonary disease.
- Discovery Biology: Supports hypothesis testing on angiogenic mechanisms and systemic vascular adaptation.
- Screening: Delivers reproducible, quantitative perfusion measurements for compound evaluation.
- Analytics: Provides fluorescence and histological outputs for robust statistical comparison of angiogenic responses.
- Translational Research: Models human-relevant vascular remodeling in response to ischemia.
- Enterprise Reuse: Offers a reusable in vivo platform for diverse vascular biology and pulmonary research programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in vascular target validation and mechanistic studies.
- Operational Value: Standardizes in vivo angiogenesis assessment with reproducible protocols and quantitative outputs.
- Strategic Value: Informs go/no-go decisions for vascular-modulating candidates and reduces late-stage biological risk.
- Portfolio Impact: Enables risk-adjusted prioritization of programs targeting pulmonary vascular remodeling.
Implementation Considerations
- Requires expertise in small animal surgery and vascular dissection.
- Demands access to fluorescence imaging and histological analysis infrastructure.
- Necessitates cross-team standardization of surgical and analytical protocols.
- Adaptation may be needed for different animal models or vascular beds.
- Quantification complexity arises from dual vascular supply in the lung.
