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Laboratoire de Chirurgie Experimentale, Paris South University, and Hpital Marie Lannelongue, Le Plessis-Robinson
Laboratoire d'Anesthesie, Paris South University, Le Kremlin-Bicêtre, France
Department of Pathology, McGill University, Montreal, Quebec, Canada
ABSTRACT
Rationale: Pulmonary artery occlusion stimulates angiogenesis in the systemic circulation of the ipsilateral lung and increases systemic- to-pulmonary blood flow. Whether this systemic neovascularization decreases after lung revascularization is unknown.
Objectives: To assess the influence of lung revascularization on anatomy and flow of bronchial vessels supplying a chronically ischemic lung in piglets.
Methods: Piglets were studied before (control) and 5 wk after left pulmonary artery ligation and 5 wk after left pulmonary artery reimplantation into the pulmonary artery trunk. The systemic blood flow to the right and left lungs was measured using colored microspheres, and the bronchial vasculature was assessed using light-microscopic morphometry. Renal and total blood flow, systemic blood pressure, and pulmonary blood pressure were measured in each experimental condition.
Measurements and Main Results: Systemic blood flow to the left lung increased from 0.4 ± 0.1 to 11.5 ± 3.8 ml/min/g (p < 0.05) after left pulmonary artery ligation and returned toward the control value (1.2 ± 0.6 ml/min/g) after revascularization, whereas it remained unchanged in the right lung. The number of bronchial vessels increased twofold in the ligated lung (p = 0.01), and did not decrease after reperfusion; however, vessel diameters decreased markedly. Renal and total blood flows, as well as mean pulmonary and systemic arterial pressures, were similar in the three experimental conditions.
Conclusion: Revascularization after a period of left pulmonary artery occlusion normalizes the systemic blood flow to the left lung and induces partial loss of collateral vessels.
Key Words: bronchial arteries pig pulmonary artery pulmonary thromboembolism
The two vascular beds of the lung exhibit strikingly different responses to pulmonary artery occlusion: the pulmonary vasculature is unable to create collateral vessels bypassing the occlusion, whereas the systemic vasculature generates bronchial- to-pulmonary artery anastomoses that supply the ischemic lung regions. Systemic blood flow to the lung reaches up to 30% of the total cardiac output in patients with chronic thromboembolic obstruction of the pulmonary arteries (1) or in animals with unilateral pulmonary artery ligation (2), as compared with less than 3% in normal subjects. The systemic blood flow increase is ascribable to low downstream pulmonary pressures at sites of bronchopulmonary anastomoses (3) and to remodeling and proliferation of the systemic arterial network (4).
In patients with chronic pulmonary thromboembolic disease, hemoptysis is a common and life-threatening complication related to the development of numerous systemic collaterals. However, hemoptysis has not been reported after pulmonary thromboendarterectomy to achieve reperfusion of previously occluded pulmonary arteries, suggesting that systemic-to-pulmonary vascularization may diminish once circulation is restored in the pulmonary bed. Possible mechanisms may include a decrease in the driving pressure for systemic-to-pulmonary blood flow and/or involution of newly formed vessels.
To test the hypothesis that systemic-to-pulmonary vascularization may diminish once circulation is restored in the pulmonary bed, we investigated anatomic and flow changes in the systemic vessels supplying the left lungs of piglets 5 wk after left pulmonary artery ligation and 5 wk after left lung pulmonary artery reperfusion.
METHODS
Twenty-four male piglets (20.2 ± 3.5 kg) were studied before (control) and 5 wk after left pulmonary artery ligation and 5 wk after left pulmonary artery reperfusion. All procedures were approved by our institutional animal care committee.
Surgical Procedures
The piglets were anesthetized and ventilated at a tidal volume of 15 ml/kg, a respiratory rate of 18 cycles/min, and an FIO2 of 0.5. The intrapericardial left pulmonary artery was ligated through a sternotomy. Five weeks later, a left posterolateral thoracotomy was performed and the left pulmonary artery was reimplanted end-to-side into the pulmonary arterial trunk. Patency of the anastomosis was assessed by visual inspection and pulmonary angiography 5 wk after reperfusion.
Light Microscopy and Morphometry
We killed eight piglets 5 wk after pulmonary artery ligation and eight piglets 5 wk after left pulmonary artery revascularization. The right (control) and left lower lobes of the lungs were fixed. Random sections were taken from the midsagittal slice and examined without knowledge of the experimental group. The mean number of bronchial arteries associated with bronchioles greater than or equal to 220-μm diameter was counted. The degree of luminal occlusion in the bronchial vessels was assessed as the percentage of medial thickness to external radius and categorized as less than 25%, 26 to 50%, 51 to 75%, or more than 75%.
Measurement of Systemic-to-Pulmonary Blood Flow
Systemic blood flow to the lung was measured in eight piglets using 15-μm colored microspheres (Bioseb Chaville, Chaville, France) in the control condition (yellow), 5 wk after left pulmonary artery ligation (white), and 5 wk after revascularization of the left lung (violet). The piglets were anesthetized and ventilated. After thoracotomy, cardiac output was measured using a flow probe and systemic and pulmonary artery pressures using direct puncture of the aorta and main pulmonary artery, respectively. A bolus of 3 · 106 microspheres in 2 ml of saline was injected into the left atrium over 15 s. Blood was drawn from the aorta using an aspirating pump (5 ml in 80 s) beginning 10 s before the injection. The number of microspheres in aortic blood was taken as the reference. After the animals were killed, three biopsies per lobe, each weighing 300 to 500 mg, were collected. High-performance liquid chromatography was used for dye measurement and subsequent microsphere number calculation (5) without knowledge of the experimental conditions. The systemic blood flow to each lung per gram of tissue (s) was calculated as follows: s = MSt · Qref/Mref/Wt, where MSt was the number of microspheres in the tissue sample; Qref, the reference flow rate determined by the calibrated aspirating pump; Mref, the reference number of microspheres; and Wt, the weight of the tissue sample. The blood flow values of the tissue samples were averaged for each animal in each condition. A similar procedure was used to measure renal blood flow. Systemic blood flow supplying the lung was also assessed using nonselective thoracic aortography.
Statistical Analysis
All results are expressed as means ± SEM. One-way analysis of variance followed by the Fisher test for multiple comparisons was done using Statview II (Abacus Concept, Berkeley, CA). Probability values less than 0.05 were considered significant.
RESULTS
After left lung revascularization, all animals survived, with normal patency of the pulmonary artery anastomosis by visual inspection and pulmonary angiography (Figure 1). Total and renal blood flow values, mean pulmonary and systemic arterial pressures, and pulmonary vascular resistances were similar in the three experimental conditions (Table 1). Although the differences were not statistically different, the animals tended to have higher pulmonary arterial pressures after pulmonary artery ligation, as well as after revascularization. This was likely caused by the redistribution of the pulmonary blood flow in the right lung after ligation and the increase in pulmonary vascular resistance of the left lung after revascularization due to the development of postobstructive vasculopathy (2).
Light Microscopy and Morphometry
Qualitatively, the vessels, airways, and parenchyma of the right lobes in both the ligated and reperfused groups were unremarkable except for a few foci of chronic inflammation. The bronchial vessels were small and normally distributed around the airways (Figure 2). In contrast, in the lungs with pulmonary arterial ligation, the bronchial vessels were increased in number and size. These vessels were thin walled; their distribution was irregular; and they encircled the pulmonary arteries, veins, and bronchi. In the revascularized lungs, the bronchial vessels had far smaller diameters and thicker walls than in the ligated lungs (Figure 2); partial or nearly total occlusion of the lumen was seen.
The morphometric data corroborated the qualitative descriptive findings. As shown in Figure 3, the number of bronchial vessels per 100 μm of subepithelial basement membrane was increased twofold 5 wk after ligation (5.9 ± 2.7 vs. 2.8 ± 1.9, p = 0.01), and this increase was still present 5 wk after reperfusion. In contrast, as shown in Figure 4, there was a substantial decrease in vessel size leading to an increase in the calculated percentage of medial thickness in the revascularized group as compared with the controls and ligated group.
Systemic Blood Flow to the Lung
Ligation of the left pulmonary artery or revascularization of the left lung had no effect on renal blood flow or on systemic blood flow to the right lung (Table 1 and Figure 5). However, although the differences were not statistically different the animals tended to have higher systemic blood flow to the right lung after pulmonary artery ligation as well as after revascularization as compared with control condition. Systemic blood flow to the left lung was increased about 30-fold 5 wk after left pulmonary artery ligation but decreased after left lung pulmonary artery reperfusion, dropping near the control value within 5 wk (Table 1 and Figure 5). Left lung systemic blood flows in the control condition and after left lung pulmonary artery reperfusion were not significantly different from right lung systemic blood flow. All animals studied 5 wk after left pulmonary ligation exhibited back-bleeding distal to the site of pulmonary artery ligation, indicating retrograde pulmonary blood flow through bronchopulmonary anastomoses. A fine meshwork of vessels distal to the pulmonary occlusion and dilated bronchial artery was consistently visible on aortic arteriograms of the left lung after ligation but not after left lung revascularization. In addition to bronchial arteries, intercostal arteries contributed to the development of systemic collaterals.
DISCUSSION
Occlusion of a pulmonary artery to one lung stimulates angiogenesis in the systemic circulation of that lung and increases the systemic-to-pulmonary blood flow (4). The present study in piglets with ligation of the left pulmonary artery showed that the systemic blood flow to the left lung returned to normal within 5 wk after pulmonary revascularization and that the newly formed systemic vessels in the same lung underwent partial involution.
As previously shown in other species (2, 4, 6, 7), proliferation of the systemic blood vessels supplying the left lung was found 5 wk after left pulmonary artery ligation by gross examination and thoracic aortography. Although we ligated the pulmonary artery through a sternotomy, thus avoiding the development of pleuropulmonary adhesions, intercostal arteries contributed to the vessel proliferation. All animals had back-bleeding distal to the site of pulmonary artery ligation, indicating retrograde systemic-to-pulmonary blood flow. The morphologic changes in the intrapulmonary bronchial arteries in our piglets resembled those reported in the canine lung (2): the number and diameter of bronchial vessels were increased in the ligated lungs as compared with the contralateral lungs. Systemic blood flow to the lung was measured using the colored microspheres reference sample technique, and our findings were consonant with previous microspheres studies in anesthetized animals (8–10). The 30-fold increase in systemic blood flow to the ligated left lung was within the range of values found in chronic pulmonary artery occlusion (2, 4) and contrasted with the absence of significant changes in right lung, kidney, or total blood flow values. A significant fraction of left ventricle injected microspheres is shunted through peripheral anastomoses and is subsequently trapped in the pulmonary circulation leading to overestimation of bronchial blood flow in the nonoccluded lung (2, 4). This could obviously bias results in a direction opposite to our results and therefore does not affect the interpretation of our finding.
It was unknown until now whether the anatomic and flow changes in the systemic pulmonary vasculature were reversible after pulmonary revascularization. Ex vivo studies (3, 11) indicated that systemic-to-pulmonary flow diminished when pulmonary vascular downstream pressures increased. We therefore hypothesized that systemic-to-pulmonary blood flow would decrease after pulmonary revascularization in piglets with long-standing ligation of the left pulmonary artery. To test this hypothesis, we reimplanted the left pulmonary artery into the pulmonary artery trunk 5 wk after pulmonary artery ligation and then waited 5 additional wk. We found that systemic-to-pulmonary blood flow had decreased toward control values in the revascularized lungs. Consistent with the microsphere measurement results, thoracic aortograms showed loss of systemic-to-pulmonary collateral vessels. The normalization of systemic-to-pulmonary blood flow was associated with subtotal occlusion of numerous bronchial arteries in the revascularized lung consistent with the increase in ratio of medial thickness to external radius (Figure 2 and 4), although the number of bronchial vessels remained increased. In aggregate, these findings indicate complete functional, and partial anatomical, normalization of the systemic vasculature as soon as 5 wk after revascularization. It is reasonable to assume that a greater degree of vessel involution would have been found had we examined animals after longer revascularization periods. Similar vessel involution occurs in various circumstances, including morphogenesis, the postpartum period, and recanalization of occluded coronary arteries (12–14).
The mechanism probably involves a decrease in the driving forces across the systemic-to-pulmonary anastomoses after restoration of antegrade pulmonary blood flow, leading to gradual decreases in systemic-to-pulmonary blood flow, bronchial artery wall shear stress, and bronchial artery lumen diameter. The change in wall shear stress may link the diameter changes in larger systemic vessels to those in distal intrapulmonary bronchial microvessels: if a distal vessel decreases in diameter, the resulting flow reduction causes a decrease in shear stress in proximal vessels, whose diameter therefore diminishes also (15). Nonhemodynamic factors may also contribute to the changes seen after restoration of pulmonary blood flow; they may include metabolic stimuli and the balance between pro- and antiangiogenic factors.
This study has important clinical implications. The development of an extensive systemic-to-pulmonary collateral network puts patients with chronic pulmonary thromboembolic disease at risk for life-threatening hemoptysis (16). An important question is whether this risk persists after reperfusion of the pulmonary arteries. Our study provides the first evidence that systemic-to-pulmonary blood flow diminishes after pulmonary artery revascularization. Thus, our results suggest that pulmonary thromboendarterectomy may prevent hemoptysis in patients with chronic pulmonary thromboembolic disease.
Involution of collateral blood vessels occurs in various vascular beds after recanalization of an occluded artery. Similarly, we found that reestablishment of the pulmonary circulation after a period of pulmonary artery occlusion normalized the systemic collateral blood flow to the reperfused lung and led to partial anatomic involution of the newly formed collateral vessels.
FOOTNOTES
Supported by the Association Chirurgicale pour le Developpement et l’Amelioration des Techniques de Depistage et de Traitement des Maladies Cardio-vasculaires.
Originally Published in Press as DOI: 10.1164/rccm.200506-894OC on October 20, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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