Role of pulmonary blood flow in postpneumonectomy lung growth JOHN
RUSS,
Departments
University
T. MCBRIDE, KEVIN K. KIRCHNER, GERALD of Pediatrics, Nuclear Medicine, and Toxicology, and Derztistry, Rochester, New York 14642
MCBRIDE, JOHN T., KEVIN K. KIRCHNER, GERALD Russ, AND JACOB FINKELSTEIN. Role of pulmonary blood floul in postpneumonectomy lung growth. J. Appl. Physiol. 73(6): 24482451, 1992.-To study the influence of blood flow on postpneumonectomy lung growth, we banded the left caudal lobe pulmonary artery of eight ferrets in such a way that blood flow to the caudal lobe did not increasewhen the right lung was excised 1 wk later. The fraction of the cardiac output received by the right lung before pneumonectomywastherefore directed entirely to the left cranial lobe. Three weeks after pneumonectomy the weight, volume, and protein and DNA contents of the two lobesof the left lung were measuredand comparedwith those of five unoperated animals and eight animals after right pneumonectomyalone. Although its perfusion did not increase after pneumonectomy, the left caudal lobe of banded animals participated in compensatory growth, increasing in weight and protein and DNA contents. Although the cranial lobe of banded animalsreceived 25%more of the cardiac output than the same lobe in pneumonectomized animals, cranial lobe volume and protein and DNA contents in the two groupswere similar. Cauda1lobeswere smaller in banded than in simple pneumonectomized animals and tended to contain lessprotein, whereas the cranial lobes tended to be heavier. We conclude that increasedpulmonary perfusion is not necessaryfor compensatory lung growth in adult ferrets, but it may modify this response. compensatory lung growth; pneumonectomy; pulmonary artery band
UNILATERAL PNEUMONECTOMY triggers a burst of growth in the remaining lung that compensates to some
extent for the tissue and function that have been lost. Similar phenomena follow unilateral nephrectomy and partial hepatectomy (5, 11). Genes and gene products involved in these phenomena have been characterized (1, 4, 11-13, 15), but, surprisingly, the primary stimuli for compensatory growth in any of these organs are not known. Two anatomic constraints unique to the lung have been thought to influence postpneumonectomy lung growth. First, to function efficiently, the lung must fit within the thorax. There is convincing evidence that the stretch to which the remaining lung is subjected plays a role in compensatory lung growth (3, 10, 16). Second, because the entire cardiac output necessarily flows through the lung, the perfusion of the remaining lung instantly increases with partial lung resection. Increased perfusion could influence compensatory lung growth by delivering a greater quantity of nutrients or growth2448
0161-7567/92
$2.00 Copyright
AND
JACOB
of Rochester
FINKELSTEIN School
of Medicine
stimulating factors to the remaining lung, by “washing out” one or more growth-suppressing factors, by increasing the metabolic activity of lung cells, or by physically distending pulmonary vessels. Although a role for increased pulmonary blood flow in compensatory lung growth has been proposed for many years, this hypothesis has never been definitively tested (3, 15). To investigate the role of pulmonary blood flow in compensatory lung growth, we banded the left caudal lobe pulmonary artery of ferrets in such a way that the increased pulmonary blood flow to the left lung after right pneumonectomy was directed entirely to the cranial lobe. Therefore, left caudal lobe perfusion remained at baseline during the period of compensatory lung growth, whereas cranial lobe perfusion increased by a factor of three. METHODS
Young full-grown male ferrets weighing 1,200-1,500 g were anesthetized for surgical procedures with xylazine and ketamine (25 and 2.5 mg/kg ip) and inhaled isoflurane. Five animals served as unoperated controls. In eight animals (pneumonectomy) the right thorax was opened between the fifth and sixth ribs, the four lobes of the right lung were ligated at the hilum and excised, and the incision was closed. In nine animals (band/pneumonectomy), the left caudal lobe pulmonary artery was exposed through an incision in the sixth intercostal space by elevating the lower margin of the cranial lobe (Fig. 1). A 2.3-mm diameter circular band of polyethylene tubing (PE-20) threaded through with a silk suture was passed around the artery, the suture was tied snugly, and the thorax was closed. One week later, a right pneumonectomy was performed. Three weeks after pneumonectomy, animals in all groups were anesthetized with pentobarbital sodium (100 mg/kg ip), and 0.2 ml of a solution of “Tc-labeled macroaggregated albumin was infused into a sublingual vein. The lung was then exposed through a midsternotomy, perfused with buffered saline via the pulmonary artery, and excised. The protocol was approved by the University Committee on Animal Resources. The excised lobes were separated and cannulated, and the volume of each at 25 cmH,O transpulmonary pressure was measured. The airways were trimmed flush with the pleura, and the lobes were blotted dry, weighed, and homogenized in EDTA buffer. One aliquot of each was
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BLOOD
FLOW AND POSTPNEUMONECTOMY
Caudal
2449
LUNG GROWTH
tomy. When animals in both groups were considered together, the weight, full inflation volume, and total protein and DNA contents of the left lungs (summed values for the caudal and cranial lobes) increased by 56,50,87, and 49%, respectively, over control values (P < 0.002 for each comparison). Differences in these parameters of left lung growth between pneumonectomized and band/ pneumonectomized animals were not statistically significant. We were able to assess the role of pulmonary blood flow in compensatory lung growth by comparing the cauHeart da1 lobe in pneumonectomized animals (which received FIG. 1. Schematic of left thorax in ferret. Pulmonary artery to left -50% of the cardiac output, a 2-fold increase over baselung leaves heart cranially and gives off several branches to cranial Lobe line) with the same lobe in banded/pneumonectomized before turning caudally to supply caudal lobe. Artery to left caudal lobe is visualized through intercostal incision by elevating lower margin of animals (which experienced no increase in blood flow cranial lobe. Preformed circular length of polyethylene tubing threaded after right pneumonectomy). In banded animals caudal through with a suture is passed around artery and, when the suture is lobe weight and protein and DNA contents were each tied, forms a band with 2.3-mm ID. Band has little effect on baseline greater by 244% compared with controls (Table 1, Fig. 2) perfusion but prevents increase in left caudal lobe perfusion after right despite the fact that the caudal lobe in these animals pneumonectomy. received no greater perfusion than the same lobe in unoperated animals. Caudal lobe volume was 18% greater in used to measure radioactivity for calculation of relative banded animals than in unoperated controls, but this lobar perfusion, and rest was used for triplicate determidifference did not reach statistical significance (P = nations of total protein by the Lowry technique and DNA 0.09). The caudal lobes in banded animals were 17% by a dye-binding assay (9). animals (P = The significance of differences between groups was smaller than those of pneumonectomized 0.05) and tended to have a lower protein content tested with analysis of variance and two-tailed Student’s (P = 0.07). t test. We were able to assess the role of augmented pulmonary blood flow in compensatory lung growth by comparRESULTS ing the cranial lobes of pneumonectomized animals In both unoperated controls and simple right pneu(which received 40% of the cardiac output after surmonectomized animals, blood flow to the left lung was gery) with the cranial lobes of banded animals (which equally divided between the cranial and caudal lobes. In received 77% of the cardiac output, a 3-fold increase over unoperated controls, the left caudal lobes received, on baseline). Compensatory growth of the cranial lobes was average, 28 t 5 (SD)% of the cardiac output, which represimilar in these two groups of animals (Fig. 3). The 37% sented 49.6 t 3.7% of the blood flow to the left lung (Taincrease over unoperated controls in cranial lobe DNA in ble 1). In simple pneumonectomized animals the caudal the pneumonectomized group did not reach staCstica1 lobe received 48.5 + 2.2% of the cardiac output. After significance (P = 0.07) unless the outlying value for one pneumonectomy in banded animals, the caudal lobe re- pneumonectomized animal was excluded (P = 0.02). Alceived 23.3 t 4.8% of the cardiac output, a value that was though there were no statistically significant differences not significantly different from that in controls. The cra- in cranial lobe measurements between the two operated nial lobe in banded animals’therefore received 76.7% of groups, the cranial lobes of banded animals tended to be the cardiac output. In one banded animal, the caudal lobe heavier (P = 0.07). received a negligible fraction (0.4%) of the cardiac output. Although the data for this animal were similar to DISCUSSION those for the other banded animals, they are excluded from subsequent analyses. The possibility that pulmonary blood flow might influCompensatory growth of the left lung occurred in both ence postpneumonectomy lung growth has been recogbanded and unbanded animals by 3 wk postpneumonecnized since the earliest studies of this phenomenon. This TABLE
lobe
1. Mean lobar weights, volumes, perfusions, and protein and DNA contents Left caudal lobe Wt, g
Control PNX P vs. control PABIPNX P vs. control P vs. PNX
. 1,95+0.25 3.07%46
Volume,
ml
32.3tl.9 45.Ot5.0
0.001
0.002
2.8lkO.23
38.3t6.2
0.001 0.2
0.09 0.05
Q, %
Left cranial lobe Protein,
mg
49.6t3.7 48.5k2.2
215H5 454t89
23.3t4.8
366+71
0.001 0.004 0.07
DNA, mg
2ut2 3lk6 0.007 32+9 0.04 0.88
Wt, g
Volume,
ml
2.12t0.37 3.39t0.55 0.001 4.00t0.62
29.5rtz.u 52.Ok6.0 50.9k5.1
0.001 0.07
0.001 0.7
&, %
Protein, mg
DNA,
mg
50.4t3.7 51.5k2.2
28lt2 493t64
76.6k4.8
539t98
2423 33t9 0.07 36t6
0.001 0.3
0.007 0.45
0.001
0.001
Values are means +: SD. &, lobar perfusion as percentage of left lung perfusion; PNX, simple right pneumonectomy; lobe pulmonary artery band/right pneumonectomy.
PAEYPNX,
left caudal
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2450
BLOOD
FLOW AND POSTPNEUMONEGTOMY
k
0 V PL w &I WEIGHT
VOLUME
PROTEIN
LEFT CAUDAL
DNA
LOBE
FIG. 2. Weight, full inflation volume, and total protein and DNA contents of left caudal lobe of control (open bars), pneumonectomized (hatched bars), and pulmonary artery-banded/pneumonectomized (cross-hatched bars) animals as a percent of mean control values. Error bars indicate 95% confidence intervals. After right pneumonectomy, caudal lobes of pneumonectomized animals received 49% of cardiac output, whereas those of banded/pneumonectomized animals received only 23%. Values for 2 experimental groups were all significantly greater than control values (P < 0.01) except that difference in lobar volume between banded/pneumonectomized and control animals did not reach statistical significance (P = 0.09). Lobar volume was greater in pneumonectomized (increased blood flow) vs. banded/pneumonectomized (baseline blood flow) animals, and protein content tended to be greater.
hypothesis gained support when Simnett et al. (21) reported that an extract of lung contained one or more tissue-specific factors that inhibited proliferation of lung epithelial cells in culture. Such factors, called “chalanes,” had been described previously in other tissues (22). According to this theory, increased perfusion of the remaining lung could wash out endogenously produced, lung-specific chalones, thereby allowing lung cells to proliferate. Cellular proliferation and compensatory growth would then continue until the mass of tissue reached the level at which its production of ohalones again suppressed cellular proliferation. Fisher and Simnett (6) and Simnett (20) subsequently argued that compensatory lung growth in mice after unilateral lung collapse (no lung tissue had been excised) supported this theory, bu t the hypothesis has never been tested directly. Blood flow might also encourage compensatory lung growth by delivering growth-stimulating factors or by increasing the metabolic activity of the cells of the remaining lung. Although gas exchange is a passive process as far as the lung is concerned, the nonrespiratory functions of pulmonary endothelial cells might well be increased after pneumonectomy. Such mechanisms have been postulated to play a role in compensatory renal (5) and hepatic (4) growth, but the data are unclear; in neither of these organs is the relationship between bl .ood flow and compensatory growth straightforward. It is also possible that the “erectile” effect of vascular distension by increased blood flow might promote growth (23). Both clinical and experimental observations suggest that pulmonary blood flow influences postnatal lung growth. In individuals with congenital absence of one pulmonary artery, the affected lung is small and radiographically hyperlucent but does enlarge during childhood and may be remarkably similar in overall structu re and mechanical function to the n.ormally p erfused lung (14) .
LUNG GROWTH
Haworth et al. (8) ligated the right pulmonary artery of newborn piglets and found that the right lung was small at maturity with the expected number of alveoli, whereas the left lung was large with an increased number of alveoli. Rinaldi et al. (17) banded the left pulmonary artery in immature rats and observed no detectable change in lung volumes but did observe an increase in alveolar size in both lungs. Tartter and Goss (22) reported that ligation of the right pulmonary artery in rats was followed by a rapid increase in the wet weight of the left lung, which was 40% of the increase that followed right pneumonectomy. These investigators, however, measured only wet lung weight, and the data did not exclude possible influences of a decrease in right lung volume or an increase in ventilation or stretch of the left lung after right pulmonary artery ligation. There is no information on the mechanisms underlying the effects of blood flow on normal lung growth. Our study is the first in which the influence of pulmonary blood flow is directly assessed during postpneumonectomy lung growth. Our major finding was that increased pulmonary blood flow is not a necessary or critical stimulus for compensatory lung growth: the left caudal lobes of banded animals increased in weight and DNA and protein contents when compared with unoperated control animals despite the fact that lobar pulmonary blood flow remained at normal or even slightly subnormal levels. Although the increase in cau da1 lobe volume over unoperated controls did not reach statistical significance, the consistency of the other observations suggests that compensatory growth did occur. This conclusion is also supported by comparing the caudal and cranial lobes of banded animals. This comparison is interesting not only because the perfusion of the two lobes was so different but also because these two lobes would have been exposed to similar changes in ventilation and mean distending pressure. The fact that the overall response of the two lobes was similar further suggests that blood flow is not a critical stimulus for compensatory lung growth. This observation should be interpreted with caution, however, because patterns of compensatorv growth of various lobes may differ (19).
FIG. 3. Same parameters as in Fig. 2 for cranial lobes of 3 groups of animals. All values for each of operated groups were greater than control values (P < 0.01) except that greater DNA content in pneumonectomized animals vs. controls did not reach statistical significance (see text). Cranial lobe weight tended to be greater in banded/pneumonectomized (75% of cardiac output) vs. pneumonectomized (50% of cardiac output) animals (P = 0.07).
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BLOOD FLOW AND PUSTPNEUMONECTOMY On the other hand, comparison of banded animals to unbanded anim .als after pneumonectomy su ggests that the level of perfusion may modify compensatory growth. Although the only statistically significant difference between the two groups of operated animals was a smaller caudal lobe volume in banded animals, several other comparisons nearly reached statistical significance (greater cranial lobe weight and lower caudal lube protein content in banded animals). The lack of statistically significant differences does not establish that the responses in the two groups were identical, particularly as confidence intervals were wide. The direction of most differences between the banded and simple pneumonectomized animals is compatible with the hypothesis that increased perfusion might augment compensatory growth. We have used young adult animals for these and other studies because of their greater size and because compensatory lung growth is not confounded by ongoing normal growth of the lung and thorax. Although the lung may not grow after pneumonectomy in mature animals of some species (15), our findings demonstrate that postpneumonectomy lung growth does occur in young adult male ferrets. It is possible that the results would differ in immature animals or in other species. Ligation of a pulmonary artery in immature animals is associated with the development of extensive collaterals between the bronchial and pulmonary circulation, which may influence subsequent lung growth (7). Collaterals would presumably have been less likely to develop in these animals in which caudal lobe blood flow remained at near-normal levels. A persistent difference in perfusion between the cranial and caudal lobes was apparent when the thorax of banded animals was opened. Compensatory lung-growth is a complex phenomenon that may involve alterations in lung s&u&&e and function in addition tu changes in mass, size, and cell number. Considerable controversy still surrounds such basic questions as the way in which alveolar geometry is altered by the compensatory response to partial lung resection in animals of different ages. Our measurements of overall lung volume, weight, and biochemistry do not address possible influences of perfusion on these important aspects of compensatory lung growth. This work was supported in part by a Career Investigator Award of the American Lung Association and National Heart, Lung, and Blood Institute Grant I-IL-29842 (to J. T. McBride) and a Parker B. Francis Fellowship (to K. K, Kirchner). Present address of K. K. Kirchner: Pulmonary Div., The Children’s Hospital, 1056 E. 19th Ave., Denver, CO 80218. Address for reprint requests: J. T. McBride, Box 667, Univ. of Rochester Medical Center, Rochester, NY 14642. Received 6 December 1991; accepted in final form 22 June 1992. REFERENCES 1. ALCORN, J. A., J. P. FEITELBERG, AND D. A. BRENNER. Transient induction of C-jun during hepatic regeneration. Hepatology 11: 909-915,199O.
2. BULLOUGH, tive review. 3. CAGLE, P.
LUNG GROWTH
2451
W. S. Mitotic and functional homeostasis:
a speculaRes. 25: 1683-1727, 1983. W. M. THURLBECK. Postpneumonectomy comlung growth. Am. Rev. Respir. Dis. 138: 1314-1326,1988. R. I?., B. L. LILJEQUIST, AND K. F. BARTIZAL. Depressed Cancer T., AND
pensatory 4. CORNELL, liver regeneration after partial hepatectomy of germ-free, athymic and lipopolysaccharide-resistant mice. Hepatology 11: 916-922, 1990.
FINE, L. Biology of renal hypertrophy.
Kidney
Int.
29: 619-634,
1986. FISHER,
J. M., AND J. D. SIMNETT. Morphogenetic and proliferative changes in the regenerating lung of the rat. Anat. Rec. 176: 389-396,1973. HAWORTH,
S. G., M. DE LEVAL, AND F. J. MACARTNEY. How the left lung is perfused after ligating the left pulmonary artery in the pig at birth: clinical implications for the hypoperfused lung. Cardiouasc. Res. 15: 214-226, 1981. 8. HAWORTH, S. G., S. A. MCKENZIE,
AND M. L. FITZPATRICK. Alveolar development after ligation of left pulmonary artery in newborn pig: clinical relevance to unilateral pulmonary artery. Thorax 36:
938-943, 1981. 9. LEBARCA, C., AND
K. PAIGEN. A simple, rapid, and sensitive DNA assay procedure. Anat. Biochem. 102: 344-352, 1980. J. T. Lung volumes after an increase in lung distension 10. MCBRIDE, in pneumonectomized ferrets. J. Appl. Physiol. 67: 1418-1421? 1989. 11. MICHALOPOULOS, G. K. Liver regeneration: nisms of growth control. FASEB J. 4: 176-187, 12. MOHN, K. L., T. M. LAZ, A. E. MELBY, AND R.
molecular
mecha-
1990. J. TAUB.
Immediateearly gene expression differs between regenerating liver, insulinstimulated H-35 cells, and mitogen-stimulated Balb/c 3T3 cells. J.
Biol. Chem. 265: 21914-21921, 1990. 13. NORMAN, J. T., R. E. BOHMAN, G. FISCHMANN, MCDONOUGH, D. SLAMON, AND L. G. FINE.
J. W. BOWEN, A. Patterns of mRNA expression during early cell growth differ in kidney epithelial cells destined to undergo compensatory hypertrophy versus regenerative hyperplasia. Proc. Natl. Acad. Sci. USA 85: 6768-6772, 1988. 14. OAKLEY, J. C., G. GLICK, AND R. M. MCCREDIE. Congenital absence of a pulmonary artery. Am. J. Med. 34: 264-271,1963. 15. RANNELS, D. E. Role of physical forces in compensatory growth of the lung. Am. J. Physiol. 257 (Lung CeZZ.Mol. Physiol. 1): L179L189, 1989. 16. RANNELS, D. E., AND S. R. RANNELS. Compensatory growth of the lung following partial pneumonectomy. Exp. Lung Res. 14: 157182,1988. 17. RINALDI, MCGREGOR.
M., A. HISLOP, N. J. ODOM, S. G. HAWORTH, AND C. G. A. Surgical factors affecting growth potential of the immature rat lung. Eur. J. Cardiothorac. Surg. 5: 218-222, 1991. 18. RUSSO, L. A., AND H. E. MORGAN. Control of protein synthesis and ribosome formation in rat heart. Diabetes A4etab. Reu. 5: 31-47, 1989. 19. SEKHON, H. S., C. M SMITH, AND W. M. THURLBECK. Chronic hypoxia and hyperoxia-induced compensatory lung structure alterations following left pneumonectomy (Abstract). Am. Reu. Respir. Dis. 143: A400, 1991. 20. SIMNETT, J. D. Stimulation of cell division following collapse of the lung. Anat. Rec. 180: 681-686, 1974. 21. SIMNETT, J. D., J. M. FISHER, AND A. G. HEPPLESTON.
unilateral
Tissue-specific inhibition of lung alveolar cePfmitosis in organ culture. Nature Land. 223: 944-946,1969. 22. TARTTER, P. I., AND R. J. GOSS. Compensatory pulmonary hypertrophy after incapacitation of one lung in the rat. J. Thorae. Cczrdiovast. Surg. 111: 267-277, 1973. 23. VOGEL, W. M., C. S. APSTEIN, AHN. Acute alterations in left
L. L. BRIGGS, W. H. GUSCH, AND J. ventricular diastolic chamber stiffness. Role of “erectile” effects of coronary arterial pressure and flow in normal and damaged hearts. Circ. Res. 51: 465-478, 1982.
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RUSS,
Departments
University
T. MCBRIDE, KEVIN K. KIRCHNER, GERALD of Pediatrics, Nuclear Medicine, and Toxicology, and Derztistry, Rochester, New York 14642
MCBRIDE, JOHN T., KEVIN K. KIRCHNER, GERALD Russ, AND JACOB FINKELSTEIN. Role of pulmonary blood floul in postpneumonectomy lung growth. J. Appl. Physiol. 73(6): 24482451, 1992.-To study the influence of blood flow on postpneumonectomy lung growth, we banded the left caudal lobe pulmonary artery of eight ferrets in such a way that blood flow to the caudal lobe did not increasewhen the right lung was excised 1 wk later. The fraction of the cardiac output received by the right lung before pneumonectomywastherefore directed entirely to the left cranial lobe. Three weeks after pneumonectomy the weight, volume, and protein and DNA contents of the two lobesof the left lung were measuredand comparedwith those of five unoperated animals and eight animals after right pneumonectomyalone. Although its perfusion did not increase after pneumonectomy, the left caudal lobe of banded animals participated in compensatory growth, increasing in weight and protein and DNA contents. Although the cranial lobe of banded animalsreceived 25%more of the cardiac output than the same lobe in pneumonectomized animals, cranial lobe volume and protein and DNA contents in the two groupswere similar. Cauda1lobeswere smaller in banded than in simple pneumonectomized animals and tended to contain lessprotein, whereas the cranial lobes tended to be heavier. We conclude that increasedpulmonary perfusion is not necessaryfor compensatory lung growth in adult ferrets, but it may modify this response. compensatory lung growth; pneumonectomy; pulmonary artery band
UNILATERAL PNEUMONECTOMY triggers a burst of growth in the remaining lung that compensates to some
extent for the tissue and function that have been lost. Similar phenomena follow unilateral nephrectomy and partial hepatectomy (5, 11). Genes and gene products involved in these phenomena have been characterized (1, 4, 11-13, 15), but, surprisingly, the primary stimuli for compensatory growth in any of these organs are not known. Two anatomic constraints unique to the lung have been thought to influence postpneumonectomy lung growth. First, to function efficiently, the lung must fit within the thorax. There is convincing evidence that the stretch to which the remaining lung is subjected plays a role in compensatory lung growth (3, 10, 16). Second, because the entire cardiac output necessarily flows through the lung, the perfusion of the remaining lung instantly increases with partial lung resection. Increased perfusion could influence compensatory lung growth by delivering a greater quantity of nutrients or growth2448
0161-7567/92
$2.00 Copyright
AND
JACOB
of Rochester
FINKELSTEIN School
of Medicine
stimulating factors to the remaining lung, by “washing out” one or more growth-suppressing factors, by increasing the metabolic activity of lung cells, or by physically distending pulmonary vessels. Although a role for increased pulmonary blood flow in compensatory lung growth has been proposed for many years, this hypothesis has never been definitively tested (3, 15). To investigate the role of pulmonary blood flow in compensatory lung growth, we banded the left caudal lobe pulmonary artery of ferrets in such a way that the increased pulmonary blood flow to the left lung after right pneumonectomy was directed entirely to the cranial lobe. Therefore, left caudal lobe perfusion remained at baseline during the period of compensatory lung growth, whereas cranial lobe perfusion increased by a factor of three. METHODS
Young full-grown male ferrets weighing 1,200-1,500 g were anesthetized for surgical procedures with xylazine and ketamine (25 and 2.5 mg/kg ip) and inhaled isoflurane. Five animals served as unoperated controls. In eight animals (pneumonectomy) the right thorax was opened between the fifth and sixth ribs, the four lobes of the right lung were ligated at the hilum and excised, and the incision was closed. In nine animals (band/pneumonectomy), the left caudal lobe pulmonary artery was exposed through an incision in the sixth intercostal space by elevating the lower margin of the cranial lobe (Fig. 1). A 2.3-mm diameter circular band of polyethylene tubing (PE-20) threaded through with a silk suture was passed around the artery, the suture was tied snugly, and the thorax was closed. One week later, a right pneumonectomy was performed. Three weeks after pneumonectomy, animals in all groups were anesthetized with pentobarbital sodium (100 mg/kg ip), and 0.2 ml of a solution of “Tc-labeled macroaggregated albumin was infused into a sublingual vein. The lung was then exposed through a midsternotomy, perfused with buffered saline via the pulmonary artery, and excised. The protocol was approved by the University Committee on Animal Resources. The excised lobes were separated and cannulated, and the volume of each at 25 cmH,O transpulmonary pressure was measured. The airways were trimmed flush with the pleura, and the lobes were blotted dry, weighed, and homogenized in EDTA buffer. One aliquot of each was
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Physiological
Society
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BLOOD
FLOW AND POSTPNEUMONECTOMY
Caudal
2449
LUNG GROWTH
tomy. When animals in both groups were considered together, the weight, full inflation volume, and total protein and DNA contents of the left lungs (summed values for the caudal and cranial lobes) increased by 56,50,87, and 49%, respectively, over control values (P < 0.002 for each comparison). Differences in these parameters of left lung growth between pneumonectomized and band/ pneumonectomized animals were not statistically significant. We were able to assess the role of pulmonary blood flow in compensatory lung growth by comparing the cauHeart da1 lobe in pneumonectomized animals (which received FIG. 1. Schematic of left thorax in ferret. Pulmonary artery to left -50% of the cardiac output, a 2-fold increase over baselung leaves heart cranially and gives off several branches to cranial Lobe line) with the same lobe in banded/pneumonectomized before turning caudally to supply caudal lobe. Artery to left caudal lobe is visualized through intercostal incision by elevating lower margin of animals (which experienced no increase in blood flow cranial lobe. Preformed circular length of polyethylene tubing threaded after right pneumonectomy). In banded animals caudal through with a suture is passed around artery and, when the suture is lobe weight and protein and DNA contents were each tied, forms a band with 2.3-mm ID. Band has little effect on baseline greater by 244% compared with controls (Table 1, Fig. 2) perfusion but prevents increase in left caudal lobe perfusion after right despite the fact that the caudal lobe in these animals pneumonectomy. received no greater perfusion than the same lobe in unoperated animals. Caudal lobe volume was 18% greater in used to measure radioactivity for calculation of relative banded animals than in unoperated controls, but this lobar perfusion, and rest was used for triplicate determidifference did not reach statistical significance (P = nations of total protein by the Lowry technique and DNA 0.09). The caudal lobes in banded animals were 17% by a dye-binding assay (9). animals (P = The significance of differences between groups was smaller than those of pneumonectomized 0.05) and tended to have a lower protein content tested with analysis of variance and two-tailed Student’s (P = 0.07). t test. We were able to assess the role of augmented pulmonary blood flow in compensatory lung growth by comparRESULTS ing the cranial lobes of pneumonectomized animals In both unoperated controls and simple right pneu(which received 40% of the cardiac output after surmonectomized animals, blood flow to the left lung was gery) with the cranial lobes of banded animals (which equally divided between the cranial and caudal lobes. In received 77% of the cardiac output, a 3-fold increase over unoperated controls, the left caudal lobes received, on baseline). Compensatory growth of the cranial lobes was average, 28 t 5 (SD)% of the cardiac output, which represimilar in these two groups of animals (Fig. 3). The 37% sented 49.6 t 3.7% of the blood flow to the left lung (Taincrease over unoperated controls in cranial lobe DNA in ble 1). In simple pneumonectomized animals the caudal the pneumonectomized group did not reach staCstica1 lobe received 48.5 + 2.2% of the cardiac output. After significance (P = 0.07) unless the outlying value for one pneumonectomy in banded animals, the caudal lobe re- pneumonectomized animal was excluded (P = 0.02). Alceived 23.3 t 4.8% of the cardiac output, a value that was though there were no statistically significant differences not significantly different from that in controls. The cra- in cranial lobe measurements between the two operated nial lobe in banded animals’therefore received 76.7% of groups, the cranial lobes of banded animals tended to be the cardiac output. In one banded animal, the caudal lobe heavier (P = 0.07). received a negligible fraction (0.4%) of the cardiac output. Although the data for this animal were similar to DISCUSSION those for the other banded animals, they are excluded from subsequent analyses. The possibility that pulmonary blood flow might influCompensatory growth of the left lung occurred in both ence postpneumonectomy lung growth has been recogbanded and unbanded animals by 3 wk postpneumonecnized since the earliest studies of this phenomenon. This TABLE
lobe
1. Mean lobar weights, volumes, perfusions, and protein and DNA contents Left caudal lobe Wt, g
Control PNX P vs. control PABIPNX P vs. control P vs. PNX
. 1,95+0.25 3.07%46
Volume,
ml
32.3tl.9 45.Ot5.0
0.001
0.002
2.8lkO.23
38.3t6.2
0.001 0.2
0.09 0.05
Q, %
Left cranial lobe Protein,
mg
49.6t3.7 48.5k2.2
215H5 454t89
23.3t4.8
366+71
0.001 0.004 0.07
DNA, mg
2ut2 3lk6 0.007 32+9 0.04 0.88
Wt, g
Volume,
ml
2.12t0.37 3.39t0.55 0.001 4.00t0.62
29.5rtz.u 52.Ok6.0 50.9k5.1
0.001 0.07
0.001 0.7
&, %
Protein, mg
DNA,
mg
50.4t3.7 51.5k2.2
28lt2 493t64
76.6k4.8
539t98
2423 33t9 0.07 36t6
0.001 0.3
0.007 0.45
0.001
0.001
Values are means +: SD. &, lobar perfusion as percentage of left lung perfusion; PNX, simple right pneumonectomy; lobe pulmonary artery band/right pneumonectomy.
PAEYPNX,
left caudal
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2450
BLOOD
FLOW AND POSTPNEUMONEGTOMY
k
0 V PL w &I WEIGHT
VOLUME
PROTEIN
LEFT CAUDAL
DNA
LOBE
FIG. 2. Weight, full inflation volume, and total protein and DNA contents of left caudal lobe of control (open bars), pneumonectomized (hatched bars), and pulmonary artery-banded/pneumonectomized (cross-hatched bars) animals as a percent of mean control values. Error bars indicate 95% confidence intervals. After right pneumonectomy, caudal lobes of pneumonectomized animals received 49% of cardiac output, whereas those of banded/pneumonectomized animals received only 23%. Values for 2 experimental groups were all significantly greater than control values (P < 0.01) except that difference in lobar volume between banded/pneumonectomized and control animals did not reach statistical significance (P = 0.09). Lobar volume was greater in pneumonectomized (increased blood flow) vs. banded/pneumonectomized (baseline blood flow) animals, and protein content tended to be greater.
hypothesis gained support when Simnett et al. (21) reported that an extract of lung contained one or more tissue-specific factors that inhibited proliferation of lung epithelial cells in culture. Such factors, called “chalanes,” had been described previously in other tissues (22). According to this theory, increased perfusion of the remaining lung could wash out endogenously produced, lung-specific chalones, thereby allowing lung cells to proliferate. Cellular proliferation and compensatory growth would then continue until the mass of tissue reached the level at which its production of ohalones again suppressed cellular proliferation. Fisher and Simnett (6) and Simnett (20) subsequently argued that compensatory lung growth in mice after unilateral lung collapse (no lung tissue had been excised) supported this theory, bu t the hypothesis has never been tested directly. Blood flow might also encourage compensatory lung growth by delivering growth-stimulating factors or by increasing the metabolic activity of the cells of the remaining lung. Although gas exchange is a passive process as far as the lung is concerned, the nonrespiratory functions of pulmonary endothelial cells might well be increased after pneumonectomy. Such mechanisms have been postulated to play a role in compensatory renal (5) and hepatic (4) growth, but the data are unclear; in neither of these organs is the relationship between bl .ood flow and compensatory growth straightforward. It is also possible that the “erectile” effect of vascular distension by increased blood flow might promote growth (23). Both clinical and experimental observations suggest that pulmonary blood flow influences postnatal lung growth. In individuals with congenital absence of one pulmonary artery, the affected lung is small and radiographically hyperlucent but does enlarge during childhood and may be remarkably similar in overall structu re and mechanical function to the n.ormally p erfused lung (14) .
LUNG GROWTH
Haworth et al. (8) ligated the right pulmonary artery of newborn piglets and found that the right lung was small at maturity with the expected number of alveoli, whereas the left lung was large with an increased number of alveoli. Rinaldi et al. (17) banded the left pulmonary artery in immature rats and observed no detectable change in lung volumes but did observe an increase in alveolar size in both lungs. Tartter and Goss (22) reported that ligation of the right pulmonary artery in rats was followed by a rapid increase in the wet weight of the left lung, which was 40% of the increase that followed right pneumonectomy. These investigators, however, measured only wet lung weight, and the data did not exclude possible influences of a decrease in right lung volume or an increase in ventilation or stretch of the left lung after right pulmonary artery ligation. There is no information on the mechanisms underlying the effects of blood flow on normal lung growth. Our study is the first in which the influence of pulmonary blood flow is directly assessed during postpneumonectomy lung growth. Our major finding was that increased pulmonary blood flow is not a necessary or critical stimulus for compensatory lung growth: the left caudal lobes of banded animals increased in weight and DNA and protein contents when compared with unoperated control animals despite the fact that lobar pulmonary blood flow remained at normal or even slightly subnormal levels. Although the increase in cau da1 lobe volume over unoperated controls did not reach statistical significance, the consistency of the other observations suggests that compensatory growth did occur. This conclusion is also supported by comparing the caudal and cranial lobes of banded animals. This comparison is interesting not only because the perfusion of the two lobes was so different but also because these two lobes would have been exposed to similar changes in ventilation and mean distending pressure. The fact that the overall response of the two lobes was similar further suggests that blood flow is not a critical stimulus for compensatory lung growth. This observation should be interpreted with caution, however, because patterns of compensatorv growth of various lobes may differ (19).
FIG. 3. Same parameters as in Fig. 2 for cranial lobes of 3 groups of animals. All values for each of operated groups were greater than control values (P < 0.01) except that greater DNA content in pneumonectomized animals vs. controls did not reach statistical significance (see text). Cranial lobe weight tended to be greater in banded/pneumonectomized (75% of cardiac output) vs. pneumonectomized (50% of cardiac output) animals (P = 0.07).
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BLOOD FLOW AND PUSTPNEUMONECTOMY On the other hand, comparison of banded animals to unbanded anim .als after pneumonectomy su ggests that the level of perfusion may modify compensatory growth. Although the only statistically significant difference between the two groups of operated animals was a smaller caudal lobe volume in banded animals, several other comparisons nearly reached statistical significance (greater cranial lobe weight and lower caudal lube protein content in banded animals). The lack of statistically significant differences does not establish that the responses in the two groups were identical, particularly as confidence intervals were wide. The direction of most differences between the banded and simple pneumonectomized animals is compatible with the hypothesis that increased perfusion might augment compensatory growth. We have used young adult animals for these and other studies because of their greater size and because compensatory lung growth is not confounded by ongoing normal growth of the lung and thorax. Although the lung may not grow after pneumonectomy in mature animals of some species (15), our findings demonstrate that postpneumonectomy lung growth does occur in young adult male ferrets. It is possible that the results would differ in immature animals or in other species. Ligation of a pulmonary artery in immature animals is associated with the development of extensive collaterals between the bronchial and pulmonary circulation, which may influence subsequent lung growth (7). Collaterals would presumably have been less likely to develop in these animals in which caudal lobe blood flow remained at near-normal levels. A persistent difference in perfusion between the cranial and caudal lobes was apparent when the thorax of banded animals was opened. Compensatory lung-growth is a complex phenomenon that may involve alterations in lung s&u&&e and function in addition tu changes in mass, size, and cell number. Considerable controversy still surrounds such basic questions as the way in which alveolar geometry is altered by the compensatory response to partial lung resection in animals of different ages. Our measurements of overall lung volume, weight, and biochemistry do not address possible influences of perfusion on these important aspects of compensatory lung growth. This work was supported in part by a Career Investigator Award of the American Lung Association and National Heart, Lung, and Blood Institute Grant I-IL-29842 (to J. T. McBride) and a Parker B. Francis Fellowship (to K. K, Kirchner). Present address of K. K. Kirchner: Pulmonary Div., The Children’s Hospital, 1056 E. 19th Ave., Denver, CO 80218. Address for reprint requests: J. T. McBride, Box 667, Univ. of Rochester Medical Center, Rochester, NY 14642. Received 6 December 1991; accepted in final form 22 June 1992. REFERENCES 1. ALCORN, J. A., J. P. FEITELBERG, AND D. A. BRENNER. Transient induction of C-jun during hepatic regeneration. Hepatology 11: 909-915,199O.
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