The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality.
In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a “second hit” of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.
Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by an increase in pulmonary blood flow, increased vascular resistance, inflammation, and remodeling of small pulmonary blood vessels1. This remodeling usually results in vascular lesions that obstruct and obliterate small pulmonary arteries, causing vasoconstriction and increasing right ventricle afterload2. Few successful pharmacological treatments of PAH exist; as a consequence, PAH-related mortality remains high. Recently, the focus of research on the pathobiology of pulmonary hypertension has moved towards a mechanism of angio-proliferation in which the role of increased pulmonary blood flow is considered as an important trigger in the development of pulmonary vascular remodeling3,4.
Animal models of pulmonary hypertension have provided critical insights that help to explain the pathophysiology of the disease and have served as a platform for drug, cell, gene, and protein delivery. Traditionally, the chronic hypoxia-induced pulmonary hypertension model and the MCT lung injury model have been the main models used to study PAH pathophysiology5. However, they are not sufficient to produce increased pulmonary blood flow and neointimal pattern of remodeling compared to alterations described in human patients. The chronic-hypoxia model in rodents results in thickening of vessel walls with hypoxic vasoconstriction without angio-obliteration of small pulmonary vessels6. Additionally, the hypoxia condition is reversible. Thus, the hypoxia model is also not sufficient to produce severe PAH. The MCT lung injury model does elicit some endothelial dysfunction but the complex vascular obliterative lesions found in humans with severe primary PAH do not develop in the rats2. Additionally, MCT-treated rats tend to die from MCT-induced lung toxicity, veno-occlusive liver disease and myocarditis rather than from PAH2. Finally, the pneumonectomy alone is not sufficient to produce neointimal lesions in the small pulmonary vessels in a short period of time. After the pneumonectomy, there is minimal elevation in pulmonary arterial pressure7. In humans, the pneumonectomy is well tolerated when the contralateral lung is healthy7.
However, the left pneumonectomy procedure combined with MCT or Sugen is advantageous since it mimics increased pulmonary blood flow and results in pulmonary vascular remodeling comparable to severe clinical PAH. The pneumonectomy is performed on the left lung, which has only 1 lobe, rather than on the right, which has four lobes. If the right lung was removed, the animal would be unable to compensate for the respiratory insufficiency. In the pneumonectomy-MCT model, neointimal pattern of remodeling develops in over 90% of operated-animals treated7. Similarly, the combination of Sugen and pneumonectomy results in severe PAH, characterized by angio-obliterative vascular lesions, proliferation, apoptosis, and RV dysfunction8. The left pneumonectomy procedure is also advantageous compared to other surgical procedures to induce PAH. Previously described models in rats to increase pulmonary blood flow to the lungs include the aorto-caval shunt or subclavian-pulmonary artery anastomosis. These models are extremely complicated7,9,10,11. To perform an aorto-caval shunt, the animal's abdomen has to be opened. The shunt is placed in the abdominal aorta, which increases blood flow to all abdominal organs instead of just the lungs, thus, PAH takes much longer to develop. Additionally, it is difficult to determine the blood flow through the shunt, whereas with the pneumonectomy the blood flow to the remaining lung doubles. The subclavian-pulmonary artery anastomosis also has many complications. The flow of arterial blood into the vein can lead to thrombosis of anastomosis and bleeding. Like the aorto-caval shunt, it is difficult to determine the blood flow through the anastomosis. Furthermore, it is an expensive and difficult technique that requires vascular surgical skills. The unilateral left pneumonectomy doubles blood flow and shear stress in the contralateral lung and, in combination with MCT or Sugen, causes the typical hemodynamic and histopathological findings of PAH which is endothelial cell damage8,12.
The novelty of this manuscript is presented in the very detailed and comprehensive surgical protocol of the left pneumonectomy in rats and in the discussion of the technical and physiological challenges of these models. Because this protocol is not currently available, many investigators believe the model is too difficult use. Investigators who have performed the left pneumonectomy have faced high mortality and morbidity rates associated with the unnecessary loss of animals, compromising scientific assessment. Instead, many will use classical models such as MCT injection, chronic-hypoxia, or just the pneumonectomy to create PAH. However, these models are much less effective than the combination of MCT or Sugen with the left pneumonectomy. The primary purpose of this article is to provide the first detailed and reproducible surgical protocol for the left unilateral pneumonectomy in rats and provide the best surgical model of PAH. Combining this protocol for left unilateral pneumonectomy with MCT or SU5416 will allow investigators to create a far more effective and clinically relevant model of severe PAH to study the pathogenesis of this fatal disease.
The procedures described below have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Icahn School of Medicine at Mount Sinai. All rats received humane care in compliance with the Mount Sinai “Guide for the Care and Use of Laboratory Animals”.
1. Preparation for Surgery
2. Preparation and Intubation of Rats
3. Preparation of Sterile Environment
4. Left Pneumonectomy Surgical Procedure
5. Post-operative Recovery
6. Administration of “Second Hit” MCT or Sugen
7. Terminal Harvest
According to the accepted classification system, pulmonary hypertension is characterized by a mean pulmonary artery pressure (mPAP) exceeding the upper limits of normal pulmonary artery pressure (i.e., 25 mm Hg). In the pneumonectomy + MCT group, severe PAH developed by day 21 with an increased mPAP (Figure 1). The mPAP is calculated by the formula:
Systolic and diastolic RV and PA pressures and were measured with the pressure catheter in the main pulmonary artery connected to the ADVantage PV system. The mean pulmonary arterial pressures (mPAP) was calculated using the formula above. In the control group (n=20), the mean PA pressure was 18.6±1.76 mm Hg (Figure 1). In the pneumonectomy + MCT group (n=30), the mPAP increased 2.25 times more compared to the control group (41.9±2.89 mm Hg) (Figure 1). In the pneumonectomy + Sugen group (n=30), the mPAP was three times higher than in the control group (53±6.60 mm Hg). In both the MCT and Sugen groups, the RVSP was much higher compared to the control group (Figure 1).
Histopathology of rat lung tissue was performed using hematoxylin and eosin staining followed by imaging with an optical light microscope. In the normal lung, there is space between the alveoli and the alveolar structures are apparent. The vessels are clear and of normal thickness (Figure 2A). In the PAH lung, there is evidence of remodeling, thickening of vessel walls, severe constriction of vessels, inflammation and focal pulmonary arteritis (Figure 2B, C).
Figure 1: Severe pulmonary arterial hypertension in the left pneumonectomy combined with MCT model. Significant differences in mPAP and RVSP between the control group and pneumonectomy + MCT group and the control group and pneumonectomy + Sugen group. Data are presented as mean SEM. The p-values were calculated using a one-way ANOVA and Tukey post hoc test. Please click here to view a larger version of this figure.
Figure 2: Representative photomicrographs of H&E staining of lung tissue. (A) Normal lung tissue. In the normal rat lung, the arteries (red arrow) are open and the vessel walls are normal sized. (B) Pathological vascular remodeling in pulmonary arterial hypertension rats treated with pneumonectomy and MCT. The lung shows focal pulmonary arteritis and inflammation, concentric medial thickening of the vessel walls (white arrow), and concentric intimal thickening of vessel walls (black arrow), resulting in severely constricted vessels. (C) Pathological vascular remodeling in PAH rats treated with pneumonectomy and Sugen. These lungs also show focal pulmonary arteritis and inflammation, concentric medial thickening of vessel walls (white arrow), and concentric intimal thickening of vessel walls (black arrow). The lumens of these vessels are severely constricted and/or entirely closed. Please click here to view a larger version of this figure.
In PAH-affected lungs, vascular proliferation with neointimal formation and obliteration of the pulmonary arteries result in severe hemodynamic changes, right ventricular failure and early mortality7,8. The changes to the vessel walls increase resistance to blood flow, increasing arterial and right ventricular pressure. In the early stages of PAH, usually 3 weeks after administering MCT or Sugen, rats developed nonspecific histological changes like medial hypertrophy, adventitial thickening, and muscularization of the small arteries and arterioles. These changes are potentially reversible. In the later stages, about 6–8 weeks after administering MCT or Sugen, the rat's lungs have neointimal and plexiform vasculopathy that obstruct and obliterate medium and small pulmonary arteries and arterioles, cellular intimal proliferation, and concentric intimal fibrosis. Plexiform lesions usually consist of a plexus of channels lined by endothelial cells and myofibroblasts. In many cases, these changes are associated with interstitial edema and fibrosis, thromboembolic obstruction of distal small pulmonary arteries, partial recanalization of a thrombosed muscular vessels, and fibrinoid necrosis12.
As stated previously, rats in other PAH models do not develop vascular obliteration of the small vessels, neointimal and plexiform lesions, and high PA and RV pressures. Treatment with the chronic-hypoxia PAH model was found to produce some thickening of vessels but the vessels remain open and there are no vascular obliterative lesions and little inflammation5. RVSP and mPAP values are slightly elevated compared to control5. Similarly, treatment with either MCT or pneumonectomy resulted in pulmonary arterial pressures that were not significantly higher than control rats and slightly remodeled vessels7,9.
Conversely, we found that the left pneumonectomy combined with MCT or Sugen is an effective model to create severe PAH. Compared to the control group, the mean pulmonary arterial pressure (mPAP) and the right ventricular systolic pressure (RVSP) in rats with severe PAH nearly doubled (Figure 1). Additionally, these rats developed plexiform lesions, concentric medial and intimal thickening, thromboembolic obstruction of distal small pulmonary arteries, inflammation, and very elevated pulmonary artery and right ventricular pressures (Figure 2). Compared to the chronic-hypoxia, MCT injection, and pneumonectomy only models of PAH performed in other studies, the pneumonectomy combined with MCT or Sugen model creates a clinically relevant condition. Furthermore, this model, if performed correctly, has a nearly 0% intra-operative mortality rate and only a 10% mortality rate post-recovery from failure of the right lung to compensate respiratory insufficiency. Higher rates of either intra-operative or post-recovery mortality is usually indicative that human or equipment error has taken place.
In order to successfully perform the left pneumonectomy and create PAH, there are several critical steps in this protocol that must be completed. First, it is very important to monitor the animal's oxygen saturation, capnography, and counting heart rate throughout the entire procedure to ensure the animal is breathing and his heart is beating. Monitoring oxygen saturation further confirms proper endotracheal intubation. Intubating the animal is absolutely necessary for this procedure. When the animal's chest cavity is opened, the negative pressure that normally exists in the thoracic cavity is shifted to atmospheric positive pressure. Thus, rats need to be provided with positive pressure via artificial ventilation. Without proper intubation, the animal's lungs will collapse from the positive atmospheric pressure.
The surgical incision has to be made laterally or posterolaterally and in the third intercostal space. Depending on the rat's anatomy, the surgeon may have to open a different intercostal space in order to access the lung and visualize the vessels. Using other approaches will make it very difficult to reach the left pulmonary artery. When opening the chest cavity, it is very important to use an electrocautery pen to stop bleeding from the skin, muscles, and surrounding vessels. If the investigator skips this step, the animal will lose blood and less oxygen will circulate. The incision must be a minimum of 2 cm long so that there is enough space to take out the entire lung when ligating. Otherwise, the investigator will tear the tissue when trying to remove the lung from a small opening. It is also essential to use atraumatic forceps when moving the lung in order to avoid rupturing the tissue and hemorrhaging. The pulmonary artery is closed first to prevent bleeding from manipulation of the lungs. The investigator should be very careful when ligating the vessels to avoid closing the left azygos vein or tearing the vessels otherwise, the animal will die. Additionally, it is much easier for the surgeon to use titanium hemoclips rather than sutures to ligate the left pulmonary artery, left main bronchus, and left pulmonary veins with a suture. Because the investigator has to make a relatively long incision in the intercostal space, it is necessary to close the intercostal muscles and ribs with sutures. Once the chest is closed, air has to be evacuated from the chest to restore negative pressure and it is important to prevent contralateral lung and heart distortion with a closed pneumothorax.
Finally, recovery is one of the most critical steps after the rat pneumonectomy. A thoracotomy is considered a very painful surgery and analgesics are essential to promote adequate ventilation, improve lung excursions in the post-operative period, and reduce pain. Rats should be recovered in sternal position to maximize lung inflation. After 10 min, the investigator can decrease the ventilation rate to help stimulate the animal to breathe on its own and wake up. If the animal becomes cyanotic and oxygen saturation levels decrease, it is necessary to increase the oxygen, ventilation rate, and increase tidal volume to 5 mL or more depending on the capacity of the ventilator. Extubation of the rats should be done as late as possible, once the animal is fully awake. The investigator may put the extubated rat into a chamber with just flowing oxygen to further aid recovery.
If the technique is executed correctly and the aforementioned considerations are addressed, performing a left unilateral pneumonectomy combined with MCT or Sugen creates a reliable model of severe PAH than MCT alone, hypoxia or other methods. When the pneumonectomy is performed correctly, the animal survives, the procedure has a short duration (15–30 min), and the investigator does not need specific vascular surgical skills. Furthermore, the investigator is able to successfully create PAH. The limitations of this method are that a thoracotomy is an invasive procedure, endotracheal intubation is required, and the investigator does need some general surgical skills. The neointimal pulmonary vascular occlusive lesions and the pronounced increases in pulmonary artery pressure similar to human pulmonary hypertension patients are evident in rats after the combination of pneumonectomy with MCT or SU5416 injection7,8,9. The present model is a reliable method to study the role of pulmonary overflow in the contralateral lung and flow-induced pulmonary hypertension.
This protocol may be useful for studying other diseases. It is possible to use this model to study the compensatory growth of pulmonary tissue in the contralateral lung. This technique can also be used to study and develop treatments for right ventricular failure (RVF). Severe right ventricle hypertrophy develops in animals that develop neointimal lesions from increased arterial pressure7. RVF causes death in about 70% of patients with PAH13. Increasing pulmonary blood flow can be useful for studying and developing treatments for patients who suffer from congenital cardiac diseases as well.
The authors have nothing to disclose.
This manuscript was supported by NIH grant 7R01 HL083078-10 grants from the American Heart Association AHA-17SDG33370112 and from the National Institutes of Health NIH K01 HL135474 to Y.S. and from the National Institutes of Health R01 HL133554 to L.H.
Surgical Blade | Bard-Parker | 371215 | Incision |
Forane (Isoflurane, USP) | Baxter | NDC 10019-360-40 | anesthesia |
BD Angiocath 16 G | BD | 381157 | intubation tube, chest tube |
BD 1 mL Insulin Syringe | BD | 329652 | administer buprinex post-operatively |
Biogel Surgeons Surgical Gloves | Biogel | 30460-01 | sterile surgical gloves |
Wahl BravMini+ Trimmer | Braintree Scientific | CLP-41590 P | shave surgical site |
SU5416 | Cayman Chemical | 13342 | Sugen |
Fiber Optic Illuminator | Cole-Parmer | EW-41723-02 | light for intubation |
Surgipro II 4-0 Suture | Covidien | VP831X | Closing intercostal muscles |
Polysorb 5-0 Suture | Covidien | GL-885 | Closing skin |
Medium Slide Top Induction Chamber | DRE Veterinary | 12570 | oxygen & isoflurane delivery |
DRE Compact 150 Rodent Anesthesia Machine | DRE Veterinary | 373 | oxygen & isoflurane delivery |
Small Vessel Cauterizer Kit | Fine Science Tools | 18000-00 | cauterizer to minimize bleeding |
VentElite Small Animal Ventilator | Harvard Apparatus | 55-7040 | ventilator |
MouseSTAT Jr | Kent Scientific | MSTAT-JR | pulse oximeter & heart rate monitor |
Mouse Paw Pulse Oximeter Sensor | Kent Scientific | SPO2-MSE | pulse oximeter & heart rate paw sensor |
PhysioSuite RightTemp | Kent Scientific | PS-02 | temperature pad |
PVP Prep Solution | Medline | MDS093944 | Cleaning surgical site |
Poly-lined Drape | Medline | NON21002Z | cover animal |
3 mL syringe | Medline | SYR103010 | administer fluids post-operatively |
Microsurgical Kits, Integra | Miltex | 95042-540 | surgical tools: plain wire speculum, double-ended probe, McPherson-Vannas Iris scissors straight, straight iris scissors |
Hemostatic forceps – Micro-Jacobson-Mosquito | Miltex | 17-2602 | mosquito |
Buprenorphrine HCl 0.3 mg/mL | Par Pharmaceutical | NDC 42023-179-01 | Pain relief |
Cooley-Mayo curved scissors | Pilling | 352090 | Large scissors |
Gerald Tissue forceps | Pilling | 351900 | forceps |
Wangesnsteen Tissue Forceps | Pilling | 342929 | atraumatic forceps |
Pilling Thin Vascular Needle Holder | Pilling | 354962DG | needle holder |
Crotaline | Sigma-Aldrich | C2401-1G | MCT |
Surflash 20 G IV Catheter | Terumo | SR*FF2051 | For pressure reading during organ harvest |
ADVantage PV System with 1.2 Fr Catheter | Transonic Inc | ADV500 | Record pulmonary artery and right ventricle pressure |
Medium Hemoclip | Weck | 523700 | ligate vessels |
Open Ligating Clip Applicator; Medium, curved | Weck Horizon | 237081 | hemoclip applicator |
Surgical Microscope | Zeiss OPMI MD | 1808 | magnification |