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Morphogenetic and proliferative changes in the regenerating lung of the rat.

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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. S. 1965 Mitotic a d functional
homeostasis: a speculative review. Cancer Res.,
25: 1683-1727.
B u x h , P. R. J., and R. G. Burwell 1965 Self
and not self
a clonal induction approach to
immunology. Q . Rev. Biol., 40: 252-279.
Cameron, R. C. 1967 Inflammation and repair.
In: Pathology. S. L. Robbins, ed. W. B.
Saunders, Philadelphia and London, pp. 31-73.
Cohn, R. 1939 Factors affecting the postnatal
growth of the lung. Anat. Rec., 75: 195-205.
Comroe, J. H.,R. E. Forstcr, A. B. Dubois, W. A.
Briscoe and E. Carlsen 1962 The lung.
Clinical physiology and pulmonary function
tests. Second ed. Year Book Medical Publishers
Inc., Chicago.
Goss, R. J. 1964 Adaptive Growth. Logos Press,
London.
1965a Kinetics of cornpcnsatory growth.
Q. Rev. Biol., 40: 123-146.
1965b The functional demand theory
of growth regulation. In: Regeneration in Animals and Related Problems. V. Kiortsis and
H. A. L. Trampusch, eds. North-Holland Publishing Co., Amstcrdam, pp. 444-451.
Longacre, J. J., and R. Johansmann 1940 An
experimcntal study of the fate of the remaining lung following total pneumonectomy.
J. Thoracic Surg., 10: 131-137.
-
395
Malt, R. A. 1969 Compensatory growth of the
kidney. New England J. Med., 28: 144G1459.
Montgomery, G. L. 1943 Healing of experimental wounds of lung. Brit. J. Surg., 31:
292-299.
Overton, J. 1955 Mitotic responses in a m phibian epidermis to feeding and grafting.
J. EXP. ZOO^., 130: 433-483.
Romanova, L. K., E. M. Leikina and K. K. Antipova 1967 Nucleic acid synthesis and mitotic activity during development of compensatory hypertrophy of the lungs in rats. Bull.
Exp. Biol. Med., 63: 96-100.
Rushmer, R. F. 1965 The artcrial system. In:
Physiology and Biophysics. Arteries and
arterioles. T. C. Ruch and H. D. Patton. eds.
W. B. Saunders, Philadelphia and London, pp.
600-618.
Simnett,-J. D. 1964 Histocompatibility in the
Platanna, Xenopus laevis laevis (Daudin), following nuclear transplantation. Exp. Cell Res.,
33: 232-239.
Simnett, J. D.,J. M. Fisher 2nd A. G. Heppleston
1969 Tissuespecific inhibition of lung alveolar
mitosis in organ culture. Nature, 223: 944-946.
Teir, H., A. Lahtiharju, A. Alho and K-J.Forsell
1967 Autoregulation of growth by tissue
breakdown products. In: Control of cellular
growth in adult organisms. H. Teir and
T. Rytomaa, eds. Academic Press, London and
New York, pp. 67-81.
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