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Regeneration of the lung in the dog
Regeneration of the lung in the dog A. Brugarolas, M.D., and H. Takita, M.D., Buffalo, N. Y.
Itomy t is known that after a pulmonary lobecthe remaining lung lobes expand and fill the entire hemithorax of the same side. To what extent this lung expansion is due to emphysematous dilatation or to formation of new lung tissue is not known, although it has been assumed that compensatory emphysema occurs. The studies of Montgomery! on pulmonary wound healing demonstrated that, a few days after surgical injury, a local regenerative process appears in the edges of the wound. The pleural surface first covers the wound in a few days. Subsequently, budding of the bronchial and vascular structures is observed, and after several weeks the pulmonary architecture is completely restored. This experiment was conceived to investigate whether the lung has the capacity to regenerate parenchyma de novo after excision of a large portion of the pulmonary tissue. It was hypothesized that three conditions must be met to provide stimulus for the regeneration of the lung: (1) a certain degree of hypoxemia and acidosis due to respiratory insufficiency; (2) a stump of pulmonary parenchyma with a section of the bronchus and pulmonary artery and venous circulation; and (3) virtual intraFrom the Department of Surgery and Experimental Surgery Roswell Park Memorial Institute, 666 Elm Street, Buffalo, N. Y. 14203. Supported in part by U. S. Public Health Service Grant No. RR05648-06. Received for publication July 3, 1972.
thoracic space subjected to adequate ventilatory pressures and movement. A model accomplishing these three requirements was developed in the adult dog, and the data are presented. Materials and methods Adult mongrel dogs, weighing from 10 to 15 kilograms were allocated to the following experiments: Experiment 1: Lung regeneration. Three dogs first underwent left subtotal pneumonectomy, which consisted of upper and middle bilobectomy and excision of most of the lower lobe; a small stump of pulmonary tissue, measuring 3 by 3 by 3 em., was left at the hilus. This stump contained bronchus, pulmonary artery, and pulmonary vein to maintain pulmonary circulation and ventilation. When the dogs recovered from this operation, they were taken out of the cages for daily active exercises. Contralateral (right) upper middle lobectomy was performed 2, 3, and 4 months later, respectively. Experiment 2: Control-acute experiment. The surgical procedures of Experiment 1, i.e., left subtotal pneumonectomy and contralateral upper and middle bilobectomy, were performed in one stage on 1 dog. Experiment 3: Control-total left pneumonectomy. Two dogs first underwent a left total pneumonectomy. Postoperatively, they were taken out of the cages for daily
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active exercises as in Experiment 1. After 2 and 4 months, respectively, contralateral bilobectomy was carried out. All dogs were anesthetized with intravenous Nembutal (30 mg. per kilogram), intubated, and ventilated with room air via a Harvard respirator. Thoracotomies were done through an incision in the fifth intercostal space. The pulmonary lobes were removed after suture ligation of the pulmonary artery and vein with 5-0 silk and suture of the bronchial stump with 4-0 silk. The pulmonary stump that remained after most of the lower lobe had been wedged was closed with two continuous layers of 6-0 silk. During all operations the cardiac output (CO) was monitored by an electromagnetic transducer placed in the ascending aorta and connected to a continuous recording tape. Pulmonary arterial pressure (PAP) was monitored during the operations as well. The pulmonary vascular resistance was calculated in units as follows: PVR
.
Unit
PAP = --.
CO
Arterial blood gases were determined daily after operation for 10 days and at weekly intervals thereafter. Chest x-ray films were obtained at monthly intervals. Pulmonary angiograms and autopsy pathologic studies supplemented the evaluation of the results. Results Experiment 1: Lung regeneration. The 3 dogs tolerated the left subtotal pneumonectomy without complications. Three weeks later, arterial blood gases were within normal limits, and chest x-ray films demonstrated a small radiolucent shadow at the hilus of the left lung, which indicated that the pulmonary stump was patent. At 2, 3, and 4 months, the dogs underwent right bilobectomies. At the time of operation, the pulmonary arterial pressure increased 25 per cent above the base-line values. The pulmonary vascular resistance was twice the base line at the end of the procedure. Before the thoracotomy was closed, blood
gases were P0 2 60 mm. Hg, Pco, 56 mm. Hg, and pH 7.30. The first day after the operation, P0 2 increased to 80 mm. Hg, Pco, was 48 mm. Hg, and pH was 7.32. Two weeks after the operation, blood gases were within normal limits. One month after the right bilobectomy, the dogs appeared in good condition, tolerating moderate exercise well. The performance of the dogs improved steadily until they had normal exercise tolerance. Autopsies were performed on 2 dogs. One of them died 2 months after right bilobectomy when a right-third lobectomy was attempted. When the right chest was opened, the dog developed cardiac arrest due to malfunction of the ventilator. Resuscitation was given and the heartbeat resumed, although the animal remained decerebrate. Nevertheless, a third lobectomy was performed. At the end of the procedure, the pulmonary arterial pressure increased 50 per cent above base-line values, and the pulmonary vascular resistance was four times the base line. Arterial blood gases were P0 2 54 mm. Hg, Pco, 62 mm. Hg, and pH 7.19. The dog was maintained under assisted respiration and died 48 hours later. Another dog was put to death 3 months after the right bilobectomy. The third dog of this group is alive 7 months after the right bilobectomy. This dog is able to run without distress, and its physical performance is considered to be normal. Pulmonary angiograms of the autopsied dogs demonstrated formation of new vessels when compared with the angiograms obtained after the left subtotal pneumonectomy. The new vessels, which appeared to have regenerated from the stumps of the ligated lobar pulmonary artery and vein (Fig. 1), reached the periphery of the lung. At autopsy, the pulmonary stumps which remained after the left subtotal pneumonectomy measured about four times their previous size. The pulmonary parenchyma was well aerated, and no bullous dilatation was observed. The elasticity of the lung was considered normal. Random sections of the peripheral and
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Regeneration of lung
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February. 1973
Fig. 1. Pulmonary arteriograms of the left pulmonary stump before (A) and after (8) regeneration. The newly formed vessels reach the lung surface.
hilar portions of the lung were studied microscopically. Hematoxylin and eosin stain demonstrated a normal pulmonary configuration and architecture. Control lung consisted of the pulmonary tissue obtained at the time of the first operation and subsequent lobectomies. The alveolar diameters of the regenerated lung were similar to those of the controls. There were no signs of emphysema, broken septae, or increase in the thickness of the alveolar septae. The arteries and veins were not unlike those of the normal lung microscopically. Bronchial buds, with columnar epithelium, muscularis mucosae, and smooth muscle layering were identified. Random counts in the microscopic field in different sections demonstrated a similar number of alveoli in the regenerated and control lung (Fig. 2). Examination of the heart revealed no hypertrophy or infarction. Experiment 2: Control-acute experiment. The dog died on the operating table with assisted respiration within 3 hours of the left subtotal pneumonectomy and right bilobectomy. The pulmonary arterial pres-
sure increased sharply up to 80 per cent above base-line values immediately after the occlusion of the pulmonary artery branches to the right upper and middle lobes. The pulmonary vascular resistance increased to ten times above base-line values. Cardiac output decreased , and peripheral arterial pressure decreased progressively to 50 mm. Hg. Arterial blood gases demonstrated acidosis, hypoxia, and hypercapnia. Terminal values were P0 2 26 mm. Hg, reo, 80 mm. Hg, and pH 6.89. Experiment 3: Control-total left pneumonectomy. Two dogs tolerated the left pneumonectomy without complications . A mild respiratory acidosis followed this operation, but 3 weeks later the blood-gas values were within normal limits. The dogs underwent a right upper and middle bilobectomy 2 and 4 months after the left pneumonectomy, respectively. Both dogs died on the operating table with assisted respiration within 6 hours of the operation. The pulmonary arterial pressure increased 60 per cent above the base line, the pulmonary vascular resistance increased to five times the
The Journol of
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Thoracic and Card iova scular Surgery
:" .
'.
\
Fig. 2. Photomicrographs (orig. mag. x200) of the periphery of the left lung . A, Normal left lung at the time of left subtotal pneumonectomy. B, Regenerated left lung at the end of the experiment. Note the similarity of the bronchial branches, small vessels, capillaries, and alveolar dimensions.
original values, and blood gases demonstrated a progressive deterioration toward respiratory acidosis. Peripheral arterial pressure gradually decreased. The term inal arterial blood gases were Po" 36 mm. Hg, Pco, 69 mm. Hg, and pH 7.01. Discussion After Gibbon's- report, it was known that the occlusion of more than 60 per cent of the pulmonary artery is fatal. It is apparent that the dogs in both control groups died from cardiorespiratory failure due to a 75 per cent reduction in the lung volume and pulmonary vascular bed. The survival of the dogs in the other group (lung regeneration) suggests that the pulmonary vascular bed increased to the minimum level that will be tolerated. The subsequent improvement of the arterial blood gases confirms this hypothesis, indicating that the major part of the regeneration probably takes place during the early postoperative period. However, the gradual
improvement of performance of the dogs indicates that the regeneration is a continuing process which may occur for a longer period of time if active exercise triggers the level of adaptation. Our experiment demonstrated that the lung is capable of regeneration with anatomic and functional integrity in the dog. Further studies in the subhuman primate are currently being made to obtain pertinent data with respect to the maximal regenerative capacity prior to clinical application of this finding. The authors wish to express their gratitude to Dr . Edith Sproul for her continued interest and for her expert advice in the review of the pathologic specimens . REFERENCES Montgomery, G. L.: Healing of Experimental Wounds of Lung , Br. J. Surg. 31: 292, 1943. 2 Gibbon, J. H " Hopkinson, M., and Churchill, E. D.: Ch anges in the Circulation Produced by Gradual Occlusion of the Pulmonary Artery, J. Clin. Invest. 11: 543, 1932.
Itomy t is known that after a pulmonary lobecthe remaining lung lobes expand and fill the entire hemithorax of the same side. To what extent this lung expansion is due to emphysematous dilatation or to formation of new lung tissue is not known, although it has been assumed that compensatory emphysema occurs. The studies of Montgomery! on pulmonary wound healing demonstrated that, a few days after surgical injury, a local regenerative process appears in the edges of the wound. The pleural surface first covers the wound in a few days. Subsequently, budding of the bronchial and vascular structures is observed, and after several weeks the pulmonary architecture is completely restored. This experiment was conceived to investigate whether the lung has the capacity to regenerate parenchyma de novo after excision of a large portion of the pulmonary tissue. It was hypothesized that three conditions must be met to provide stimulus for the regeneration of the lung: (1) a certain degree of hypoxemia and acidosis due to respiratory insufficiency; (2) a stump of pulmonary parenchyma with a section of the bronchus and pulmonary artery and venous circulation; and (3) virtual intraFrom the Department of Surgery and Experimental Surgery Roswell Park Memorial Institute, 666 Elm Street, Buffalo, N. Y. 14203. Supported in part by U. S. Public Health Service Grant No. RR05648-06. Received for publication July 3, 1972.
thoracic space subjected to adequate ventilatory pressures and movement. A model accomplishing these three requirements was developed in the adult dog, and the data are presented. Materials and methods Adult mongrel dogs, weighing from 10 to 15 kilograms were allocated to the following experiments: Experiment 1: Lung regeneration. Three dogs first underwent left subtotal pneumonectomy, which consisted of upper and middle bilobectomy and excision of most of the lower lobe; a small stump of pulmonary tissue, measuring 3 by 3 by 3 em., was left at the hilus. This stump contained bronchus, pulmonary artery, and pulmonary vein to maintain pulmonary circulation and ventilation. When the dogs recovered from this operation, they were taken out of the cages for daily active exercises. Contralateral (right) upper middle lobectomy was performed 2, 3, and 4 months later, respectively. Experiment 2: Control-acute experiment. The surgical procedures of Experiment 1, i.e., left subtotal pneumonectomy and contralateral upper and middle bilobectomy, were performed in one stage on 1 dog. Experiment 3: Control-total left pneumonectomy. Two dogs first underwent a left total pneumonectomy. Postoperatively, they were taken out of the cages for daily
187
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Brugarolas and Takita
The Jeurnol of Thorocic ond Cordiovosculor Surgery
active exercises as in Experiment 1. After 2 and 4 months, respectively, contralateral bilobectomy was carried out. All dogs were anesthetized with intravenous Nembutal (30 mg. per kilogram), intubated, and ventilated with room air via a Harvard respirator. Thoracotomies were done through an incision in the fifth intercostal space. The pulmonary lobes were removed after suture ligation of the pulmonary artery and vein with 5-0 silk and suture of the bronchial stump with 4-0 silk. The pulmonary stump that remained after most of the lower lobe had been wedged was closed with two continuous layers of 6-0 silk. During all operations the cardiac output (CO) was monitored by an electromagnetic transducer placed in the ascending aorta and connected to a continuous recording tape. Pulmonary arterial pressure (PAP) was monitored during the operations as well. The pulmonary vascular resistance was calculated in units as follows: PVR
.
Unit
PAP = --.
CO
Arterial blood gases were determined daily after operation for 10 days and at weekly intervals thereafter. Chest x-ray films were obtained at monthly intervals. Pulmonary angiograms and autopsy pathologic studies supplemented the evaluation of the results. Results Experiment 1: Lung regeneration. The 3 dogs tolerated the left subtotal pneumonectomy without complications. Three weeks later, arterial blood gases were within normal limits, and chest x-ray films demonstrated a small radiolucent shadow at the hilus of the left lung, which indicated that the pulmonary stump was patent. At 2, 3, and 4 months, the dogs underwent right bilobectomies. At the time of operation, the pulmonary arterial pressure increased 25 per cent above the base-line values. The pulmonary vascular resistance was twice the base line at the end of the procedure. Before the thoracotomy was closed, blood
gases were P0 2 60 mm. Hg, Pco, 56 mm. Hg, and pH 7.30. The first day after the operation, P0 2 increased to 80 mm. Hg, Pco, was 48 mm. Hg, and pH was 7.32. Two weeks after the operation, blood gases were within normal limits. One month after the right bilobectomy, the dogs appeared in good condition, tolerating moderate exercise well. The performance of the dogs improved steadily until they had normal exercise tolerance. Autopsies were performed on 2 dogs. One of them died 2 months after right bilobectomy when a right-third lobectomy was attempted. When the right chest was opened, the dog developed cardiac arrest due to malfunction of the ventilator. Resuscitation was given and the heartbeat resumed, although the animal remained decerebrate. Nevertheless, a third lobectomy was performed. At the end of the procedure, the pulmonary arterial pressure increased 50 per cent above base-line values, and the pulmonary vascular resistance was four times the base line. Arterial blood gases were P0 2 54 mm. Hg, Pco, 62 mm. Hg, and pH 7.19. The dog was maintained under assisted respiration and died 48 hours later. Another dog was put to death 3 months after the right bilobectomy. The third dog of this group is alive 7 months after the right bilobectomy. This dog is able to run without distress, and its physical performance is considered to be normal. Pulmonary angiograms of the autopsied dogs demonstrated formation of new vessels when compared with the angiograms obtained after the left subtotal pneumonectomy. The new vessels, which appeared to have regenerated from the stumps of the ligated lobar pulmonary artery and vein (Fig. 1), reached the periphery of the lung. At autopsy, the pulmonary stumps which remained after the left subtotal pneumonectomy measured about four times their previous size. The pulmonary parenchyma was well aerated, and no bullous dilatation was observed. The elasticity of the lung was considered normal. Random sections of the peripheral and
Volume 65 Number 2
Regeneration of lung
189
February. 1973
Fig. 1. Pulmonary arteriograms of the left pulmonary stump before (A) and after (8) regeneration. The newly formed vessels reach the lung surface.
hilar portions of the lung were studied microscopically. Hematoxylin and eosin stain demonstrated a normal pulmonary configuration and architecture. Control lung consisted of the pulmonary tissue obtained at the time of the first operation and subsequent lobectomies. The alveolar diameters of the regenerated lung were similar to those of the controls. There were no signs of emphysema, broken septae, or increase in the thickness of the alveolar septae. The arteries and veins were not unlike those of the normal lung microscopically. Bronchial buds, with columnar epithelium, muscularis mucosae, and smooth muscle layering were identified. Random counts in the microscopic field in different sections demonstrated a similar number of alveoli in the regenerated and control lung (Fig. 2). Examination of the heart revealed no hypertrophy or infarction. Experiment 2: Control-acute experiment. The dog died on the operating table with assisted respiration within 3 hours of the left subtotal pneumonectomy and right bilobectomy. The pulmonary arterial pres-
sure increased sharply up to 80 per cent above base-line values immediately after the occlusion of the pulmonary artery branches to the right upper and middle lobes. The pulmonary vascular resistance increased to ten times above base-line values. Cardiac output decreased , and peripheral arterial pressure decreased progressively to 50 mm. Hg. Arterial blood gases demonstrated acidosis, hypoxia, and hypercapnia. Terminal values were P0 2 26 mm. Hg, reo, 80 mm. Hg, and pH 6.89. Experiment 3: Control-total left pneumonectomy. Two dogs tolerated the left pneumonectomy without complications . A mild respiratory acidosis followed this operation, but 3 weeks later the blood-gas values were within normal limits. The dogs underwent a right upper and middle bilobectomy 2 and 4 months after the left pneumonectomy, respectively. Both dogs died on the operating table with assisted respiration within 6 hours of the operation. The pulmonary arterial pressure increased 60 per cent above the base line, the pulmonary vascular resistance increased to five times the
The Journol of
1 90
Brugarolas and Takita
Thoracic and Card iova scular Surgery
:" .
'.
\
Fig. 2. Photomicrographs (orig. mag. x200) of the periphery of the left lung . A, Normal left lung at the time of left subtotal pneumonectomy. B, Regenerated left lung at the end of the experiment. Note the similarity of the bronchial branches, small vessels, capillaries, and alveolar dimensions.
original values, and blood gases demonstrated a progressive deterioration toward respiratory acidosis. Peripheral arterial pressure gradually decreased. The term inal arterial blood gases were Po" 36 mm. Hg, Pco, 69 mm. Hg, and pH 7.01. Discussion After Gibbon's- report, it was known that the occlusion of more than 60 per cent of the pulmonary artery is fatal. It is apparent that the dogs in both control groups died from cardiorespiratory failure due to a 75 per cent reduction in the lung volume and pulmonary vascular bed. The survival of the dogs in the other group (lung regeneration) suggests that the pulmonary vascular bed increased to the minimum level that will be tolerated. The subsequent improvement of the arterial blood gases confirms this hypothesis, indicating that the major part of the regeneration probably takes place during the early postoperative period. However, the gradual
improvement of performance of the dogs indicates that the regeneration is a continuing process which may occur for a longer period of time if active exercise triggers the level of adaptation. Our experiment demonstrated that the lung is capable of regeneration with anatomic and functional integrity in the dog. Further studies in the subhuman primate are currently being made to obtain pertinent data with respect to the maximal regenerative capacity prior to clinical application of this finding. The authors wish to express their gratitude to Dr . Edith Sproul for her continued interest and for her expert advice in the review of the pathologic specimens . REFERENCES Montgomery, G. L.: Healing of Experimental Wounds of Lung , Br. J. Surg. 31: 292, 1943. 2 Gibbon, J. H " Hopkinson, M., and Churchill, E. D.: Ch anges in the Circulation Produced by Gradual Occlusion of the Pulmonary Artery, J. Clin. Invest. 11: 543, 1932.