INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

MAJOR LUNG RESECTION, i.e., pneumonectomy, is a simple model that mimics the loss of alveoli in lung disease; we have utilized this model in animals to examine the sources and extent of pulmonary adaptation. The amount of functioning lung units removed is definable and reproducible. Because the remaining lung is normal, adaptive response can be readily measured. Our previous studies show that, in adult dogs after pneumonectomy, adaptation for gas-exchange is achieved mainly through greater utilization of existing physiological reserves in the remaining lung and remodeling of the remaining alveolar septa (8, 22). Compensatory alveolar tissue growth does not occur after resection of the smaller left lung (45% of total) but does occur after resection of the larger right lung (55%), suggesting a threshold of resection above which the remaining lung can no longer compensate effectively through physiological means, and the growth of new alveolar tissue is stimulated (22, 23). Regardless of whether the mechanism of response is physiological reserves or new lung growth, the ultimate structural and functional compensation in adult dogs is limited, reaching no more than 70-80% of the original functional capacity for gas-exchange.

Immature animals generally possess a greater growth potential than adult animals. In immature dogs after left pneumonectomy, Thurlbeck et al. (43) found that the remaining lung undergoes an early accelerated rate of alveolar growth, resulting in an increased alveolar number and volume. However, that study was terminated before full maturity was reached. The only previous long-term study in immature dogs, by Davies et al. (11), questioned whether the acceleration of alveolar growth early after pneumonectomy in the study of Thurlbeck et al. was sustained until maturation; Davies et al. found no clear morphometric evidence of alveolar growth in mature dogs 5 yr after they underwent left pneumonectomy as puppies. No physiological data have been obtained during exercise in immature animals after major lung resection; hence, the extent and functional significance of postpneumonectomy compensation remain undefined. The present study reports the first correlation of long-term functional consequences and structural response in dogs after resection of 55% of lung tissue by right pneumonectomy (R-Pnx) as puppies. Our results indicate that extensive lung resection in immature dogs stimulates a vigorous compensatory growth of alveolar septal tissue that exceeds normal maturational lung growth by a factor of >2, resulting in complete normalization of aerobic capacity and gas-exchange function at maturity.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Experimental groups. The protocol was approved by the Institutional Review Board for Animal Research. Twelve litter-matched pure-bred male foxhounds underwent R-Pnx (n = 6) or right thoracotomy without pneumonectomy (Sham, n = 6) at 2 mo of age. Under isoflurane anesthesia a lateral thoracotomy was performed through the right fifth intercostal space. The right main pulmonary artery and veins were doubly ligated with silk sutures. The right main bronchus was then divided and closed with titanium staples. The thorax was closed in layers after we confirmed hemostasis and checked for air leakage by immersion of the bronchial stump under saline. Residual air in the pleural space was evacuated with a catheter connected to an underwater seal. One animal in the R-Pnx group was lost in the immediate postoperative period because of postpneumonectomy pulmonary edema, leaving five animals in the R-Pnx group. All other dogs were raised to maturity. Physiological and radiological studies were performed at rest under anesthesia during maturation and were previously published (39, 40). One year after surgery, dogs were trained to run freely on a motorized treadmill while wearing a customized leak-free respiratory mask (1) and attachments necessary for ventilatory measurements. Bilateral subcutaneous carotid artery loops were constructed to allow repeated catheterization. Aerobic capacity, ventilation, gas-exchange, and mechanical and hemodynamic function were measured during heavy exercise.

Exercise training. The treadmill speed was kept constant at 6 or 8 miles/h, depending on the preference of the dog. After a warm-up period at 6 miles/h and 0% grade, the treadmill grade was raised by 5% every 3 min up to 60% or 80% of the previously achieved maximal workload. Exercise was sustained for a total of 30 min/day 5 days/wk. Maximal workload and the corresponding O₂ uptake, CO₂ output, heart rate, and respiratory rate were measured at intervals, and the training workloads were adjusted accordingly. Exercise intensity was varied from day to day during training to prevent dogs from anticipating the upcoming workload.

Respiratory apparatus. The dog breathed through a large two-way respiratory valve (model 2700, Hans Rudolph, Kansas City, MO) connected to a two-way inflatable balloon valve (model 8230, Hans Rudolph) and a 3-liter anesthetic rebreathing bag. The inspiratory port was connected to a screen pneumotachometer (model 3813, Hans Rudolph) and opened to room air or a large meteorologic balloon containing 100% O₂. The expiratory port led to a mixing chamber and a heated screen pneumotachometer. Expired gas concentrations were sampled continuously from the distal end of the mixing chamber by a mass spectrometer (model MGA 1100, Perkin-Elmer). The pneumotachometer-computer system was calibrated by the method of Yeh et al. (51). Rectal temperature, gas concentrations, and electrocardiogram were continuously monitored during exercise. All signals were digitized by computer at 50 Hz. Ventilation, O₂ uptake, CO₂ production, respiratory rate, tidal volume, and heart rate were calculated from mixed expired gas and averaged over a predetermined number of breaths. Expired flow was integrated to obtain volume, which was expressed in BTPS conditions.

Catheterization and hemodynamic measurements during exercise. Percutaneous catheterization of the carotid artery was performed in the awake dog under local anesthesia on the day of study. The catheter was connected to a fluid-filled transducer and a carrier amplifier, and the signals were digitized by computer. Maximal O₂ uptake was measured as defined by Seeherman et al. (37), i.e., the point where O₂ uptake no longer increased with increasing workload and was associated with a continuously rising lactate concentration. Hb concentration was measured spectrophotometrically (Beckman Instruments, Fullerton, CA). Hematocrit was determined with a microcapillary centrifuge. Conventional blood gases were measured (ABL3, Radiometer, Copenhagen, Denmark) and O₂ saturation at body temperature was calculated by using O₂ half-saturation pressures of Hb measured for dog blood.

Rebreathing measurements during exercise. These methods have been reported previously in detail (7, 8). An anesthetic bag was filled with one of two rebreathing gas mixtures containing 9% helium, 0.6% acetylene (C₂H₂), 0.3% C¹⁸O, and 30% O₂-balance N₂ or 90% O₂. The bag volume was selected from the dog's average tidal volume at a given workload plus 200 ml ATPD to prevent collapse of the bag during rebreathing. Exercise consisted of a 5-min warm-up period at 6 miles/h and 0% grade. Then the workload was increased to a preselected level and sustained for ~4 min. At the end of the 3rd min, the balloon valve was automatically switched at a selected end expiration to allow the dog to rebreathe from the anesthetic bag for 5-10 s, depending on the workload. Rebreathing measurements were repeated at each workload with use of each of the two different rebreathing mixtures. Gas concentrations at the mouth were monitored continuously. Diffusing capacity of the lung (DL_CO) and cardiac output were estimated from the exponential rates of disappearance of end-tidal C¹⁸O and C₂H₂, respectively, with respect to helium. Lung volume was estimated from helium dilution. All results were corrected for mixing efficiency by using a method adapted from Hook and Meyer (18). In all dogs, 90% mixing was achieved within four breaths. The Bunsen solubility coefficient for C₂H₂ in blood and tissue was corrected to the measured body temperature and hematocrit, as described by Jibelian et al. (29).

Postmortem studies. On completion of physiological measurements (~18 mo after surgery), the dog was deeply anesthetized with pentobarbital sodium (25 mg/kg iv) and intubated via a tracheostomy. The lungs were collapsed through bilateral intercostal incisions. Simultaneously, an overdose of pentobarbital sodium (100 mg/kg iv) was given, and the lungs were immediately reinflated within the thorax by intratracheal instillation of 2.5% glutaraldehyde buffered in 0.03 M potassium phosphate (pH 7.40, 350 mosM) at a constant hydrostatic pressure of 25 cmH₂O above the highest point of the sternum in the supine position. The lungs and heart were then removed en bloc and immersed in 2.5% glutaraldehyde. Major respiratory muscles were dissected completely, trimmed of extraneous tissue, weighed, and processed for separate analysis.

Diffusing capacity of the lung for O₂ and CO estimated by morphometry. Lung diffusing capacity was calculated for O₂ (DL_O2) and for CO (DL_CO) by a modified version (47) of the previously established morphometric model of Weibel (45). The model describes the gas diffusion path from alveolar air to the binding sites on Hb as two serially linked conductance steps: 1) through the tissue and plasma barrier (Db_O2 or Db_CO) and 2) in the erythrocyte (De_O2 or De_CO)

(1)

(2)

where

(3)

where S_A and S_c are the measured total alveolar and capillary surface area and V_c is the measured total capillary blood volume. K_b is the Krogh diffusion coefficient for O₂ or CO, respectively, in tissue and plasma taken from the literature (49). A set of systematic linear test lines was laid over each electron micrograph. The length of all intercepts of the test line with the barrier, from the epithelial surface to the nearest red cell membrane, was measured with a logarithmic ruler. Intercepts that did not cross the epithelial and the red cell surfaces were not measured. The _hb in a direction perpendicular to the epithelial surface is given by the mean of all reciprocal intercept lengths

(4)

where n is the number of linear intercepts of length l. The factor , derived from stereological principles, was introduced to correct for the mean projection angle (15, 48). The estimate of _hb has been shown to be normally distributed (47).

1

1

(5)

1

1

1

1

1

(6)

Statistical analysis. Values are means ± SE. Physiological data were analyzed with respect to O₂ uptake or cardiac output, and the slopes and intercepts were compared between groups by ANOVA. Postmortem data from the remaining lung of R-Pnx animals were compared separately from those in the left lung and both lungs of Sham animals by ANOVA using STATVIEW (version 4.5, Abacus Concepts, Berkeley, CA). Differences among groups were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Exercise measurements of gas-exchange. Table 1 shows the physiological data during maximal exercise. There was no difference in body weight between groups. Maximal O₂ uptake was similar between groups. End-expiratory and end-inspiratory lung volumes were lower in R-Pnx dogs, but only the reduction in end-expiratory lung volume reached statistical significance. There were no significant differences in hematocrit, mean PA_O2, and arterial blood gases between groups. There were also no significant differences in minute ventilation, cardiac output, stroke volume, and DL_CO between groups. Blood lactate concentration increased similarly with increasing O₂ uptake in both groups (Fig. 1). The relationships of arterial O₂ saturation and alveolar-arterial PO₂ difference to O₂ uptake during exercise are similar in both experimental groups (Fig. 2). One Sham animal developed significant declines in arterial O₂ saturation during moderate exercise due to hypoventilation; the alveolar-arterial PO₂ difference of this animal at a given O₂ uptake was in keeping with that of other Sham animals. The relationships of DL_CO to pulmonary blood flow, estimated by the rebreathing technique, were also similar between groups (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Relationships of arterial lactate concentration to O2 uptake during exercise. There was no significant difference between groups. R-Pnx, right pneumonectomy.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Top: relationship of arterial O2 saturation to O2 uptake during exercise. There was no significant difference between groups. Bottom: relationship of alveolar-arterial PO2 gradient to O2 uptake during exercise. There was no significant difference between groups.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Left: relationship between lung diffusing capacity for CO (DLCO) and pulmonary blood flow measured by a rebreathing technique at rest and during exercise. There was no significant difference between groups. Right: DLCO estimated by a morphometric model in left lung of animals after R-Pnx compared with that in left lung as well as both lungs of control (Sham) animals. Symbols represent individual animals within each group; bars represent group means. * Significantly different from Sham left (P < 0.0001).

Postmortem measurements. Morphometric data were available from three animals in the R-Pnx group and six animals in the Sham group. Two animals in the R-Pnx group died before the terminal experiment, and their lungs could not be adequately fixed. Figure 4 shows the similar light-microscopic appearance of the gas-exchange region in the two experimental groups. Tables 2 and 3 show the morphometric measurements of the remaining left lung in animals after R-Pnx compared with those in the left lung and both lungs of control animals. In both groups, volume of the intact lung measured by immersion displacement was significantly larger than volume of the sectioned lung measured by the Cavalieri principle (Table 3), confirming that alveolar septa were fixed under tension, but the two measurements varied in parallel between the groups. Volume of the left lung after R-Pnx increased more than twofold to equal that of two lungs in control animals (Table 3). Except for a higher type I epithelial volume after R-Pnx, volume densities of septal structures and surface densities of alveoli and capillaries were not different between groups (Table 2). Absolute volume of septal structures and surface areas of alveoli and capillaries after R-Pnx were also not different from those in both lungs of control animals (Table 3). Thus alveolar septal tissue volume of the left lung after R-Pnx exceeded that of the control left lung by a factor of 2.69; this compensatory tissue proliferation involved all tissue components of the septum: volumes of epithelium, interstitium, and endothelium increased 2.6-, 2.76-, and 2.64-fold, respectively. Capillary blood volume was correspondingly higher by 2.7-fold after R-Pnx. The _hb was similar between groups (Table 2). As a result of the increased surface areas and capillary blood volume, DL_O2 and DL_CO of the remaining lung after R-Pnx estimated by morphometry increased 2.62-fold to a level similar to that in both lungs of control animals (Table 3).

View larger version (110K): [in this window] [in a new window]   Fig. 4.   Light-microscopic appearance of lung from 1 Sham animal (A) and 1 animal after R-Pnx (B). Scale bar, 100 µm. Magnification ×180.

Correlation of DL_CO estimated by physiological and morphometric methods. The mean DL_CO estimated by morphometry in each group agreed well with the highest DL_CO obtained by rebreathing during exercise (Fig. 3, right). In the present study, individual estimates of DL_CO by the two methods differed by 5-20%; the mean ratio of DL_CO measured by rebreathing to DL_CO measured by morphometry for all immature dogs (n = 9) is 1.05 ± 0.05 (SE). In Fig. 5 the correlation between the two methods is shown for all animals we have studied to date, including dogs after left pneumonectomy as adults, after R-Pnx as adults, and adult Sham dogs, in addition to the immature dogs reported here. There is a highly significant correlation (r = 0.803). The mean ratio of DL_CO measured by rebreathing to DL_CO measured by morphometry for all dogs (n = 21) is 1.10 ± 0.04 (SE).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Correlation between DLCO measured by rebreathing method at peak exercise and by morphometry postmortem in animals subjected to R-Pnx as puppies (Sham, ; R-Pnx, ) and as adults (Sham, ; R-Pnx, ; left pneumonectomy, *). Solid line, regression line through all data points. DLCO(morphometry) = 0.90 DLCO(rebreathing) + 1.62, r = 0.803. Dashed line, identity. Data from adult dogs are from Hsia et al. (22, 23).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Summary of results. Immature dogs responded vigorously to major lung resection. One year after removal of 55% of lung by R-Pnx, aerobic capacity and pulmonary gas-exchange function measured up to maximal exercise were completely normal. Normalization of gas-exchange function resulted from a remarkable compensatory lung growth in excess of normal developmental lung growth that involved all cellular compartments of the alveolar septa and returned alveolar tissue volumes and surface area completely to normal. In both groups, estimates of lung diffusing capacity by a morphometric method postmortem agreed well with that by a physiological rebreathing method at peak exercise, supporting the belief that structural changes in the remaining lung mediate the increased capacity for alveolar-capillary gas-exchange. Even though morphometric data were obtained from only three animals after pneumonectomy, the magnitude of change was large and consistent (>2-fold in most measurements) as well as statistically significant.

Compensatory response to pneumonectomy in adult animals. There are three potential mechanisms for augmenting diffusing capacity after pneumonectomy: 1) recruitment of incompletely used pulmonary capillaries, 2) remodeling of existing alveolar-capillary membrane to enhance gas diffusion, and 3) growth of new alveolar tissue and capillaries. After pneumonectomy the entire cardiac output is directed through one lung; hence, effective pulmonary blood flow per unit of lung at any workload is doubled compared with control animals. The greater effective blood flow can open previously collapsed capillaries or distend open capillaries and can potentially redistribute red cell traffic in a more homogeneous fashion. After pneumonectomy the effective ventilation per unit of lung at any given workload is similarly increased. A higher effective ventilation can cause greater stretching or unfolding of the alveolar membrane and thus augment gas-exchange surface. These functional changes lead to an increase in DL_CO of the remaining lung in the absence of any intrinsic structural alteration (8). We previously reported that, in adult dogs after left pneumonectomy (45% lung resection), adaptation for gas-exchange is achieved primarily through these physiological mechanisms that recruit existing reserves of diffusing capacity in the remaining lung (8). Remodeling of the remaining alveolar structure also occurred after left pneumonectomy, i.e., enlargement of the alveolar air spaces and thinning of the alveolar tissue barrier, leading to a lower resistance to gas diffusion and further increasing DL_CO (22). Alveolar tissue volume of the remaining lung did not increase after left pneumonectomy; i.e., we have found no evidence of compensatory alveolar growth. Alveolar tissue growth did occur, however, in adult dogs after resection of the larger right lung (55% of total) (23). Because there is no intrinsic difference between the two lungs, these data suggest a threshold of resection (~50%) below which the remaining lung structure can adapt adequately without new tissue growth to maintain an acceptable level of function; above this threshold the remaining structure can no longer compensate effectively, and regenerative growth of new alveolar units is stimulated (23). Regardless of the mechanism of response (physiological recruitment, structural remodeling, or growth), the ultimate functional compensation in adult dogs is limited; i.e., at 1 yr after left pneumonectomy or R-Pnx, arterial O₂ saturation, diffusing capacity, pulmonary hemodynamics, cardiac output, and pulmonary mechanics at maximal exercise remain abnormal; compensation reached no more than 70-80% of the normal values (19-21, 24-27).

Compensatory response to pneumonectomy in immature animals. Although the physiological reserves of diffusing capacity must also have been recruited in immature dogs after pneumonectomy, their postpneumonectomy adaptive response is primarily characterized by active alveolar proliferation. This compensatory proliferative tissue response has been well documented in rats and rabbits (3-6, 33-36, 38, 42, 44), but the rodent data could not be directly extrapolated to large animals because of the continuous growth pattern of the rodent; i.e., its epiphyses never close (12). A few studies have examined large immature animals (11, 30, 43), but none has previously addressed the long-term postpneumonectomy functional outcome at maturity by exercise studies. Short-term physiological studies by Ford et al. (13), Arnup et al. (2), and Thurlbeck et al. (43) in immature dogs 11-15 wk after left pneumonectomy showed normalization of lung volumes (2, 13) and a marked compensatory increase in alveolar number (43). The only long-term study was a series by Wilcox et al. (50), Pimmel et al. (32), and Davies et al. (11) in immature beagles after left pneumonectomy. At 1 yr after pneumonectomy, lung volume and DL_CO measured at rest were normal (50). However, structural studies of the remaining lung performed in these same animals 5 yr after left pneumonectomy demonstrated no increase in the number of alveoli compared with the same lung of control animals (11). Hence, the physiological and structural data from this series of animals are at variance and also differ from the short-term data of Thurlbeck et al. (43). Davies et al. (11) suggested that the acceleration of lung growth early after pneumonectomy may be transient and is not sustained up to maturity. Later studies by Johnson et al. (30) in beagles, 7-9 mo after left pneumonectomy as puppies, reported significant increases in resting lung diffusing capacity, alveolar septal tissue volume, and alveolar surface area of the remaining lung consistent with compensatory lung growth; however, the nature of the increase in septal tissue volume and the extent of functional compensation at exercise were not studied.

Comparison of response in immature and adult dogs. We demonstrate here that postpneumonectomy compensation in gas-exchange is indeed sustained in immature dogs, eventually yielding a functional capacity of the remaining lung more than twice that of the control left lung at maturity; alveolar-capillary gas-exchange is restored completely to normal (Fig. 2). Not only is gas-exchange normal at rest, but the pattern of recruitment of pulmonary capillaries with increasing blood flow during exercise is also normal (Fig. 3). The result is a normal slope and intercept of the relationship between DL_CO and pulmonary blood flow (_c) during exercise, i.e., a normal ratio of DL_CO to _c at any exercise load. This ratio is an important parameter that determines the adequacy of end-capillary O₂ saturation according to the Bohr integral (31). In contrast, postpneumonectomy adaptive mechanisms restore only ~50% of the functional deficit in DL_CO at a given workload in adult dogs after R-Pnx (23); these animals show persistent reductions in DL_CO and DL_CO/_c at peak exercise, causing arterial O₂ saturation to fall prematurely as exercise load increases (26).

Correlation of DL_CO measured by physiological and morphometric methods. Diffusing capacity estimated from structural parameters by a morphometric model (45, 47) is thought to represent the maximum possible diffusing capacity on the basis of the assumptions that 1) the alveolar-capillary anatomy determines the upper limit of gas-exchange and 2) the distribution of capillary red blood cells found postmortem is similar to the in vivo distribution during exercise. Because the alveolar-capillary bed is not fully recruited at rest, DL_CO estimated by the morphometric model is consistently higher than that measured by physiological methods at rest (9, 10). We showed previously in adult dogs that when morphometric DL_CO is compared with physiological DL_CO measured at peak exercise in the same animals, the agreement is much closer (22, 28). Combining all mature and immature dogs studied by these two techniques (Fig. 5), we found that physiological estimates are on average 10% higher than morphometric estimates. This is a remarkable agreement, considering the disparate approaches and assumptions involved in the two techniques.

Conclusions. We conclude that the loss of >50% of lung tissue as a result of surgical pneumonectomy in immature animals leads to strong compensatory growth of the remaining alveolar tissue that exceeds normal maturational growth and fully reconstitutes the size of the pulmonary alveolar-capillary network. On reaching somatic maturity, alveolar gas-exchange capacity during exercise is fully maintained.