Morphogenetic and Proliferative Changes in the Regenerating Lung of the R a t JEAN M. FISHER AND JOHN D. SIMNETT Department of Pathology, University of Newcastle upon Tyne, Englmtd ABSTRACT Unilateral extirpation of the lung in rats is followed by increased mitotic activity in alveolar cells of the contralateral lung, reaching a maximum six to seven days after operation. The response is delayed if the cavity created by the operation is packed with plastic sponge. Unilateral collapse of the lung without removal of tissue also leads to a contralateral mitotic response. Changes in the rate of cell proliferation evidently are not directly dependent on changes in tissue mass and it is suggested that compensatory growth in the lung may be controlled by chemical factors whose local concentration depends on variations in the rate of blood flow. Other changes which follow partial extirpation, observed particularly in the residual tissue of resected lungs, include high rates of proliferation in pleural cells, sub-pleural tissue and bronchial epithelium. It appears that in the regenerating lung new tissue may be formed partly by the proliferation of cells in the main mass of residual tissue and partly by more localized changes in specific tissues. Partial extirpation of organs such as lowing unilateral resection of the lung, the liver (Bucher, '63) and kidney (Goss, compensatory increase in mass of the re'65a) initiates a compensatory response in maining lung did not occur. The response the residual homologous tissues resulting was evidently prevented even though the in near recovery of the original organ mass. lost tissue was not restored, which suggests Observations on the lung show that this that changes in tissue mass may not be organ likewise may undergo some degree the direct stimulus to new growth. It may of compensatory growth (Addis, '28) due, be safely assumed that the amount of at least in part, to an increased rate of cell damaged tissue was not reduced by improliferation (Romanova et al., '67), al- plantation of inert material, in which case though in older animals simple distension the wound hormone hypothesis also beof the alveoli may become increasingly im- comes untenable. In view of its considerportant (Longacre and Johansmann, '40). able theoretical importance we have thereSeveral general hypotheses have been fore sought to confirm Cohn's work using proposed to account for compensatory cell division rates rather than changes in growth. Many such hypotheses assume that the loss of tissue mass is a direct causative tissue mass, as the chief criterion. factor, bringing about changes in organMATERIALS AND METHODS specific growth regulatory factors (BulThe present series of experiments inlough, '65; Burch and Burwell, '65) or changes in the physiological activity of the volved a total of 88 female albino rats remaining tissue (Goss, '65b). Other hypo- aged 84-105 days (mean 115 2 26), theses assume tissue damage to be the within which range the results were unprincipal factor, possibly mediated by affected by age. the release of specific macromolecules Operative procedure for unilateral ("wound hormones") which stimulate cell lobectomy division (Abercrombie, '57; Teir et al., Under ether anaesthesia, the hair of the '67). left thoracic region was shaved, and the Cohn ('39) found that if the thoracic Received Sept. 20, '72. Accepted Mar. 19, '73. cavity was packed with inert material, folANAT. REC., 176: 389396. 389 390 JEAN M. FISHER AND JOHN D. SIMNET area swabbed with antiseptic. A skin incision approximately 3 cm long was made on the left side of the thorax and the pectoral muscles were then deflected and a left intercostal incision made through which the edge of the left lung could be grasped with forceps. The lobe was pulled through the incision, a ligature was made towards the hilar region, and the lobe resected. The mediastinum was allowed to return, and the pleural cavity, pectoral muscles and skin quickly sutured. Recovery from anaesthesia began within five minutes of the end of the operative procedure. tion of cells entering mitosis during the 4 h period of Colcernid arrest. The mean and standard error of the mean were derived for each experimental group. To avoid possible variations in mitosis at different sites within the lung, analyses were made from the distal part of each lobe, avoiding pleural and subpleural regions which were treated separately. OBSERVATIONS Anatomical considerations Removal of the left lobe of the lung lead to a marked mediastinal shift, where the whole heart-lung complex was displaced Restriction of the pleural cavity towards the left pleural cavity (see also with sponge implant Cohn, '39). Pneumonectomy leads to hyA piece of plastic sponge, trimmed to perinflation and a decrease in physiological the size of the left lobe, was boiled in dis- dead space in the contrdateral lung tilled water containing sulphamezathine. (Comroe et al., '62) and the probable On cooling and blotting it was inserted into result of our operation was to provide the the pleural cavity following resection of residual tissue with more space and hence the left lobe, and all wounds closed as a greater potential for expansion and air intake. before. In our experiments insertion of the Sham-operated and control animals sponge implant following lobectomy preIn sham-operated animals bhe left pleu- vented the mediastinal shift, at least for ral cavity was opened, which led to col- several days, and consequently the remainlapse of the lung, whereas the control ani- ing lung was unable to increase its capamals received only a skin incision. In city. Post mortem examination showed, neither case was there removal of tissue. however, that the implant gradually became compressed and displaced to the left, Experimental procedure often with the mediastinum adhering to it. Four hours prior to sacrifice each ani- By 12 days the implant had become infilmal was injected intraperitoneally with trated by connective tissue, blood cells and Colcemid (0.1 mg/ 10 g body weight). Sub- macrophages. sequently the intact right lungs and the Changes in the residual tissue of left lung stumps were dissected out, large resected lungs lobes being cut into two or three pieces, Following the resection of' lung lobes and placed in 0.45% sodium chloride for 15 minutes to swell the mitotic figures some viable tissue always remained. (Simnett and Heppleston, '66) after which Fig. 1 The bronchial epithelium of an unop the material was fixed in Worcester's fluid, erated control animal. The proliferative rate is sectioned at 5 and stained in Weigert's very low so that mitotic figures are rarely seen. x 500. haematoxylin and eosin. Fig. 2 Bronchial epithelium within the trauTwo sample areas for each animal were matized zone of a partially amputated left lung scanned at magnification X 1000 (oil im- 3 days after operation. The epithelial cells have mersion) and the numbers of arrested enlarged and become very basophilic, and mitotic activity has increased considerably (arrows inmetaphases occurring in alveolar wall pop- dicate the mitotic figures). x 500: ulations were recorded together with the Fig. 3 The periphery of the lung in a control average number of nuclei for each sample animal. The pleural epithelium is composed of calculated from random nuclear counts. squamous cells, and alveolar spaces extend to the periphery of the lung. x 150. The mitotic incidence (MI) was expressed extreme Fig. 4 The thickened pleural surface of a as the number of arrested metaphases per partially amputated lung four days after opera10' nuclei, which represented the propor- tion. x 150. REGENERATION 3N RAT LUNG 391 392 JEAN M. FISHER A N D JOHN D. SIMNET Within 24 hours an acute irklammatory response developed in the area adjacent to the cut surface. Initially there was an accumulation of granulocytes, lymphocytes and macrophages followed by the appearance of multinucleated cells and a build-up of fibroblast-like cells, By seven days such inflammatory areas were largely transformed into scar tissue. There was a notable increase in mitotic activity of bronchial epithelium adjacent to the cut surface which was first observed 24 hours after the operation and was maintained for several days (figs. 1, 2). The following estimates for mitotic incidence in bronchial cells were both based on ten sample counts each containing lo3 cells: MI in control bronchial epithelium, 290/ lo5; MI in bronchial epithelium two days after lobectomy, 5900/105. Structural changes occurred at the surface of the resected lobe and since they were also observed in intact lung lobes (vide infra) the possibility that they were due to the migration of cells over the cut surface can therefore be excluded. These changes appear to comprise two distinct processes: a transformation of the pleura from a single layer of squamous cells to a rapidly dividing population of cubical cells comprising several layers and simultaneously the proliferation and thickening of the alveolar tissue immediately under the pleural basement membrane (figs. 3, 4). The boundary between the two layers became indistinct and the following values for mitotic activity in the surface region (see figs. 5, 6 ) are therefore composite figures which include pleural and alveolar cells: MI/105 at three days, 771 2 120; at six days, 1557 3- 523 and at 12 days 1086 2 493. Pleural thickening and mitotic activity did Fig. 5 The pleural region of a partially amputated lung on the 4th day followinq uartial lobectomy. The pleura, containing many proliferating cells (arrows) forms a layer several cells deep. x 500. Fig. 6 The pleural region of the contralateral lung seven days after lobectomy and sponge implantation. The subpleural alveolar tissue forms a thickened layer of cells many of which are in mitosis (arrows). X 500. REGENERATION 393 IN RAT LUNG not affect the entire intact surface and no regular pattern in its localization was discernible. Localized pleural changes were also observed in the resected lung from animals with sponge implants. sponge implants. Mitotic counts as high as 3980/105 were recorded in this thickened area. Partial lobectomy produced a marked increase in overall alveolar mitotic activity while sponge implantation delayed the reChanges in the contralateral lung sponse (fig. 7). For the purpose of statisfollowing lobectomy tical analysis the data were expressed as a No inflammatory response was observed, series of regression lines covering the penor was there any increase in bronchial riod of 0-6 days and from six days until the mitosis. Localized thickening of the pleura end of the experiment, each line calculated and peripheral alveolar tissue, as de- assuming linear regression. Significance scribed above, occurred in the intact lung of differences between the slopes of inlobes both in simple lobectomy experi- dividual lines was calculated by comparments and in lobectomized animals with ing the regression coefficient using Student’s t test. There was no change observed in lobectomized animals until two days after operation following which the MI increased to a maxium of 961 -c- 59/10’ or 160% of the unoperated control value (600 -C 49/105) (p < 0.001). Following this there was a decrease in MI, the significance of which ( p < 0.001) was indicated by a change in slope of the regression line. In contrast the alveolar cell MI in animals with sponge implants gradually decreased up to six days (p < 0.001) by comparison with simple lobectomy after which there was a rapid increase in MI (for the change of slope in the regression line, p < 0.01) which by 12 days was 177% of the unoperated control (p < 0.005). u 4j L CONTROLS at 2 DAYS:GOOt: 49 SHAM-OPERATEDat 2 D A Y 3 9 5 6 2 95 01 0 I I 4 8 DAYS AFTER LOBECTOMY 11 % Fig. 7 Changes in mitotic activity in the contralateral lung following different experimental 0 -the mitotic incidence in the treatments. undamaged contralateral lung of partially lobectomized animals. A is the linear regression line from zero to six days, and B the linear regression line from six days to the end of the experiment. -0the mitotic incidence in the undamaged contralateral lung of partially lobectomized and sponge-implanted animals; C is the linear regression line from zero to six days, and D is the linear regression line from six days to the end of the experiment. Related pairs of regression lines have been joined by a curve, and their theoretical points of intersection indicated by dotted lines. Controls received a simple skin incision. Sham-operated animals received a thoracic incision causing ipsilateral callapse of the lung. - Changes in the lungs of sham-operated animals There was an increase in the number of granulocytes and lymphocytes in the collapsed lung while thickening at the pleural surface, such as described above, was also observed. Two days after operation the MI in collapsed alveolar tissue was 772 & 201/105 though this was not shown to be significantly different (p = 0.1-0.05) from untreated control tissue (600 -C 49/10’). The non-collapsed functional lobes were of normal histological appearance but two days after contralateral collapse the alveolar tissue showed an increased MI (956 2 95/105) which was significantly higher ( p < 0.005) than in untreated controls (600 & 49/105). DISCUSSION Alveolar cell mitotic activity increased 394 JEAN M. FISHER AND JOHN D. SIMNET following partial extirpation of the lung but as reported by Romanova et al. ('67) the response was slower than in the liver (Bucher, '63) or kidney (Goss, '65a) where the peak MI usually occurs between one and three days after operation. The mitotic response in alveolar tissue was considerably delayed by packing the thoracic cavity with inert material, which substantiates Cohn's ('39) report that restoration of tissue mass could thus be prevented. Compensatory growth can evidently be suppressed even after a considerable reduction of tissue mass while conversely the mitotic response can be initiated even where the tissue mass remains unaltered, as in the case of the contralateral lobes following partial collapse of the lung. It follows that compensation cannot be controlled by factors whose production is directly dependent on tissue mass. Wound hormones may likewise be excluded as major controlling factors since the response was delayed even when damaged tissue was present, or was initiated in the absence of damaged tissue. While the above conclusions do not necessarily invalidate existing hypotheses for the mechanism of growth control they do suggest that there are additional unidentified factors which are of paramount importance. One such factor may be the rate of blood flow. In a variety of tissues inflammation, which is associated with dilatation of blood vessels, is accompanied by increased mitotic activity (Cameron, '67), this phenomenon being apparent in grafts of skin (Overton, '55; Simnett, '64) and of embryonic tissues (Chopra and Simnett, '70). Following partial extirpation of the liver (Benacerraf et al., '57) and kidney (Malt, '69) the rate of blood flow in the remaining tissue increases, while in the lung blood may be diverted to fully-functional areas (Rushmer, '65) in which, as shown by the contralateral lung following partial collapse, there is an associated increase in cell division rate. The degree of inflation plays a major part in regulating the pulmonary circulation (Rushmer, '65) which may explain why the sponge implants, which prevent distension of the contralateral lung, delay the mitotic response. There is evidently a close association between changes in vascularization and changes in mitotic activity, but unless a causal relationship can be established any further development of the hypothesis remains speculative. However, it is unlikely that the increased blood supply could stimulate cell division by the direct provision of extra nutrients or oxygen, since there is no evidence that the rate of cell proliferation is normally limited by metabolic requirements. A more likely hypothesis is that increased vascularization may cause a more rapid clearance of locally produced mitotic control factors which have been shown to exist in a number of organs including the lung (Simnett, Fisher and Heppleston, '69). Very high rates of mitosis were observed in bronchial epithelium adjacent to wounds. It is probable that this provides for the regeneration of new bronchial tissue, but a second function may be to form a population of cells which eventually differentiate into new alveolar tissue, as has been described in local wounds in the lungs of cats (Montgomery, '43). The response in alveolar cell proliferation following ablation or collapse, though highIy significant, amounted to less than a two fold increase over the control values which is much less than in many other compensating organs, such as the liver where increases of up to 200 fold have been described (Goss, '64). Very high rates of cell division were, however, observed in the thickened sub-pleural layer of intact lungs and we therefore suggest that compensatory growth in this organ has two contributory elements: the proliferation of cells in the main mass of the tissue and a much more dramatic but localized response in the outer layers which results in a population of new cells which may eventually differentiate into extra functional tissue. LITERATURE C1E D Abercrombie, M. 1957 Localized formation of new tissue in an adult mammal. Symp. SOC. Exp. Biol., 11: 235-254. Addis, T. 1928 Compensatory hypertrophy of the lung after unilateral pneumonectomy. J. Exp. Med., 47: 51-56. Benacerraf, B., D.Bilbey, G. Biozzi, B. N. Hdpern and C. Stiffel 1957 The measurement of liver blood flow in partially hepatectomized animals. J. Physiol., 136: 287-293. REGENERATION IN RAT LUNG Bucher, N. L. R. 1963 Regeneration of mammalian liver. Int. Rev. Cytol., 15: 245400. Bullough, W. 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