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Radioautographic studies of regeneration in the common newt III. Regeneration and repair of the intestine

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Radioautographic Studies of Regeneration in the
Common Newt
Department of Anatomy, The University of Texas Medical Branch,
Galveston, Texas
The intestine of larval and adult frogs
(OSteen, '59; Goodchild, '56) and of the
adult urodele (OSteen, '58) will reform
completely to a normal histological and
functional state following its complete
transection. During the early stages of regeneration a mass of undifferentiated cells,
which has been referred to as a blastema,
accumulates around the transected ends of
the intestine. Associated with this accumulation of cells, many other organs and tissues, such as the liver, fat body, gonads,
and body wall adhere to the areas of injury.
Each tissue which is in contact with the
injured area of the intestine undergoes
marked morphological changes, and frequently, cells from these tissues appear to
separate from the main mass and pass into
the blastema. The blastema also receives
cellular contributions from the fibrous connective tissue of the intestine, the smooth
muscle and the serosa (Goodchild, '56;
O'Steen, '58, '59).
There have been some questions as to
the role of the mucosal epithelium in the
formation of the blastema. Goodchild ('56)
stated that during early stages of regeneration in frogs, mucosal epithelium underwent marked dedifferentiation, and in later
stages in several instances, "new mucosa
appeared to arise de novo from cords of
small cells." The mucosal epithelium of
other adult frogs appeared to reform from
migrations of cells from existing epithelium. In the regeneration of the intestine
of the adult newt, OSteen ('58) reported
dediff erentiation of the pseudostratified
columnar epithelium to a cuboidal and
spindle-shaped character, with some of the
cells apparently separating from the tissue
and passing into the blastema. Clusters of
epithelial cells were observed in the blas-
tema of several intestines. Regeneration of
the mucosa appeared to be by migration of
cells from the adjacent epithelium and
possibly from de novo formation from the
blastema. However, this did not appear to
occur during the regeneration of the intestine of larval anurans (O'Steen, '59) or
in the healing of wounds in stomachs of
larval urodeles (Chiakulas and Lefkowitz,
'60). In these larval forms the mucosal
epithelium was repaired largely, if not entirely, by the migration of epithelial cells
across the wound area. The cells were
morphologically modified during this migration, but there were no indications of
them passing into the blastema.
These reports indicated that other studies
should be made on the cellular behavior in
this tissue and that it would be advantageous if the cells could be selectively
marked so that their proliferation and migration could be followed. Previous studies
(O'Steen and Walker, '60; Patten, '60) demonstrated that intraperitoneal injections
of tritiated thymidine would label the cells
of the mucosa, but would not label to any
extent other tissues of the intestine. Thus,
a series of adult newts were given injections of the isotope, the intestines were
transected, and radioautographs were prepared from a series of regenerating tissues.
This is a report of the results of this investigation.
Intraperitoneal injections of tritiated
thymidine were given to a total of 74 adult
common newts, TTituTus viridescens. The
1 This investigation was supported by
grants from the National Institutes of Health, United
States Public Health Service (RG 7062) and from
Houston Endowment, Inc., and National Muscular
Dystrophy Research Foundation.
animals were divided into two groups, each
animal of Group A receiving 15 uc and of
Group B receiving 2 uc of the isotope.
Ten days after the injection of the isotope, the animals were anesthetized in
MS-222 ( 1: 1000 dilution), an incision was
made in the lateral body wall, and a loop of
the intestine was exposed. A single, complete transection was made through the
intestine and its underlying mesentery at
an area approximately midway between
the pyloric and rectal sphincters. The cut
ends of the intestine were randomly replaced into the coelom, and the incision
was closed with interrupted black silk sutures (size 5-0). Operations were performed in standard Holtfreter’s solution
containing chloramphenicol (30 pg/ml).
Operated animals recovered for a period
of three days in this solution, after which
time they were returned to aged tap water.
Solutions in which the animals were kept
were changed daily. All animals were kept
in a constant temperature room (22°C) at
all times.
Animals from the two groups were killed
at one, 8, 10, 14, 30, and 45 days after
the operation. After fixation for 24 hours
in Carnoy’s fluid, the operated area of the
intestine was excised and embedded in
paraffin. Longitudinal sections were cut
serially at 7 u. Radioautographs were prepared using the technique of Messier and
Leblond ( ’ 5 7 ) , modified by Walker (’59).
The deletion of the celloidin step, as recently described by Kopriwa et al. (’60),
was made in the later autoradiographs.
Although various exposure periods for
slides coated with emulsion were tested,
the results reported here are based on a
40 day period.
Quantitative data are expressed as the
number of radioactive nuclei in randomly
selected groups of 100 cells counted.
A comparison of the radioautographs
prepared from the intestines of animals of
Group A (15 uc tritiated thymidine) and of
Group B ( 2 pc tritiated thymidine) indicated that the general pattern of labeling
in both groups was the same. Tritiated
thymidine was incorporated into the nuclei
of mucosal epithelial cells and the nuclei
of granular and agranular leucocytes. Only
scattered, infrequent nuclei were labeled in
the serosa and loose connective tissue of
the intestine. Although the pattern of radioactive cell distribution was the same in
both groups, the frequency of labeled cells
was less in Group B than in Group A. In
radioautographs of tissues from the first
day after the operation, the number of
radioactive mucosal epithelial cells averaged 40 per 100 cells in Group A and 24
per 100 cells in Group B. These averages
were based on counts of 600 cells in the
“cell nests” (after Patten and Andrews, ’54)
and in the surface epithelium (figs. 1, 2).
The frequency of labeled cells also varied
from animal to animal within each group.
Some animals had relatively few radioactive mucosal epithelial cells (5-15 cells
per loo), but others had many nuclei distinctly labeled (over 50 per 100). The intensity of labeling of mucosal cells varied
within each group, that is, some cells had
many silver granules over each nucleus,
while others had only a few granules (10
to 20 per nucleus). In general, there were
fewer silver granules superimposed on the
nuclei of cells of Group B, as compared
with Group A.
During the early stages of regeneration
(1 to 14 days after operation), there was
an accumulation of undifferentiated cells
forming the blastema. The mucosal epithelial cells began to morphologically dedifferentiate and migrate at the cut surfaces
of the intestine, as previously described in
detail by O’Steen (’58). The blastema during its early development was composed
of unlabeled cells resembling fibroblasts
and a mixture of labeled and unlabeled
mononuclear and polymorphonuclear leucocytes. The mucosal epithelial cells near
this accumulation of blastema had changed
from a pseudostratified columnar to a simple cuboidal shape and appeared to be migrating out into the blastema (fig. 3). The
distal end of the migrating extension was
composed of a mass of polygonal cells
and was not a simple epithelium (figs. 3 ,
4 ) . Labeled nuclei and mitoses were observed in these mucosal extensions (fig. 4).
Frequently, cells apparently separated from
these extensions and contributed to the
blastema. In some intestines clusters of
labeled cells were seen in the blastema
(figs. 5, 6 ) .
During later stages from the time of
blastema formation until complete regeneration of the intestine, the pattern of Iabeling remained the same as in the earlier
stages. Epithelial extensions of the cephalic and caudal ends of the intestine
continued to elongate until contact was
made and functional regeneration was
complete (O'Steen, ' 5 8 ) . Labeled nuclei
were concentrated in the entire extent of
the mucosal epithelium; in addition, labeled nuclei were scattered throughout the
blastema. By 30 days after transection, all
labeled cells had only a few silver granules
remaining over the nucleus, indicating that
the cells which were heavily labeled during
early regeneration had been dividing,
thereby diluting the nuclear content of incorporated isotope.
Finally, complete histological regeneration returned the intestine to its normal
structural and functional condition (fig.
7). By this time the only evidence of the
blastema was seen as scattered cells in the
submucosa of some intestines. Radioactive
cells in the tissues in this area did not vary
in pattern of distribution from those seen
in areas adjacent to the region of the operation (fig. 8). In these regenerated intestines at 30 to 45 days after the transection,
65 to 90% of the mucosal epithelial cells
in the regenerated zone had a light label
as evidenced by few silver granules over
each nucleus. The number of radioactive
epithelial cells decreased to normal in
areas away from this zone. All radioactive
nuclei in the regenerated zone appeared to
be overlaid with a similar number of silver
granules. In adjacent areas of unoperated
intestine some nuclei were overlaid with
many silver granules, while others had
only a few granules. Only rare radioactive
nuclei were observed in the smooth muscle,
submucosal connective tissue, or serosa.
An exception to the above distribution
of labeled cells frequently was seen in the
cells of the serosa. Radioautographs of 4
completely regenerated intestines (30 to
45 days) had labeled serosal cells. However, these were observed only in the regenerated zone approximately two to three
millimeters on either side of the site of
the transection.
The use of a radioactive isotope for the
study of cell dynamics raises the question
related to radiation damage of the tissue
and its subsequent influence on the behavior of the cells. The isotope used in
this study, tritiated thymidine, has received
special consideration concerning radiation
damage since it is incorporated into the
nucleus of premitotic cells during the synthesis of DNA. Damage to spermatogonial
cells has been demonstrated in the mouse
following injections of this isotope (Johnson and Cronkite, '59). In addition, Sauer
and Walker ('61) have described cytological changes characteristic of radiation
damage in chick embryos that have been
injected with tritiated thymidine. However, in the present study, radioautographs
of transected intestines of newts from both
Group A and Group B showed no cytological evidence that radiation damage had
occurred in the tissues, and any influence
of the isotope that might have taken place
was not sufficient to cause an interference
with the normal chronological sequence of
events. Not only did Group A, which received the large dose ( 1 5 uc), show no difference in time of regeneration when compared with Group B ( 2 uc), but there was
also no difference in time period when both
groups were compared with uninjected animals ( O'Steen, '58).
In the present study, it was possible to
selectively label a single tissue component
of the intestine, namely the mucosal epithelium, and thereby to follow the behavior
of cells of this tissue in relation to other
cell types. Injections of tritiated thymidine
were given 10 days prior to the transection
so that cells stimulated to divide by the
injury of transection would not be labeled
by the same dose designed to label the
mucosal epithelium. It had been previously
demonstrated that an injection of a large
dose of the isotope (15 uc) remains in the
blood stream of newts for periods exceeding 5 days and that it can label cells preparing to divide as a result of an injury
made at that time (O'Steen and Walker,
'60, '61; O'Steen, '61).
The mucosal epithelium played a primary role in the initial covering of the
transected ends of the intestine and later
in the completion of regeneration. Labeled
cells were observed in the cell nests and in
the surface epithelium, which migrated out
over and covered the wounded surfaces
(figs. 1, 2). The extensions of mucosal
epithelium (figs. 3, 4 ) resembled those described in the digestive tracts of mammals
(Hunt, '58; Johnson and McMinn, '60) in
their shape and in the presence of mitoses.
However, the primary site of epithelial cell
division was in the cell nests in unoperated
areas adjacent to the transection (Dawson,
'27; Patten and Andrew, '54; OSteen and
Walker, '60). Functional regeneration of
the intestine, that is, the stage at which
food could again pass through the site of
transection, was accomplished primarily by
the extension of the epithelial outgrowths
from the two cut ends of the intestine until
they met and fused. As the outgrowths
migrated through the blastema, single labeled cells and clusters of radioactive cells
were observed in the blastema (figs. 5, 6 ) .
These results correspond with those previously reported in studies on the regeneration in the intestine of common newts
(OSteen, '58), but they do not agree with
observations on wound healing and regeneration in larval amphibians. O'Steen
('59) did not observe any epithelial cells
passing into the blastema of regenerating
intestines of larval anurans ( R a m clamitans). Chiakulas and Lefkowitz ('60) concluded from their studies on wound healing of the stomach in Amblystoma larvae
that the mucosal cells did not dedifferentiate and did not contribute cells to the
blastema. However, they did indicate that
mucosal epithelial cells changed from a
columnar to a cuboidal shape during their
migration in the wound area. In both the
larval anuran (O'Steen, '59) and larval
urodele (Chiakulas and Lefkowitz, '60) the
mucosa was repaired as the result of epithelial migrations. Some basic differences
between the adult and larval intestinal regeneration were obvious and might account
for variations in epithelial cell behavior.
Wound healing following longitudinal incisions in the stomach wall cannot be completely correlated with intestinal regeneration following transection, as was stated
by Chiakulas and Lefkowitz ('60). They
observed a rapid mucosal healing before
the blastema actually formed. Another
basic difference was that intestines of lar-
val anurans were greatly modified by being
tightly coiled into a limited space in the
coelomic cavity. Loops of this coil retained
their alignment even after transection of
several loops, and this alignment and the
close apposition of the cut surfaces possibly led to a more rapid regeneration of
the mucosal epithelium in these animals
(OSteen, '59), as compared with adults.
Mucosal regeneration sometimes preceded
blastema formation in the anuran larvae
as in the u r d e l e larvae. In some adult intestines, large masses of blastema accumulated prior to mucosal regeneration. Any
tissue coming in contact with this blastema
underwent to varying degrees a dissociation of its cellular components. This dissociation of tissues as a result of contact
with the blastema was observed in the
liver, fat body, gonads, and body wall in
earlier studies (O'Steen, '58, '59; Chiakulas
and Lefkowitz, '60). It is possible that
the blastema might have effected dissociation in the mucosal epithelium associated
with it, and these cells then would pass
into the blastema.
In completely regenerated intestines, almost every cell in the newly formed mucosal epithelium was lightly labeled, and
the cells of the submucosa and smooth
muscle were only infrequently radioactive
(fig. 8). Thus, the newly differentiated
tissues had essentially the same relative
number of radioactive and non-radioactive
cells as corresponding tissues in uninjured
areas and in unoperated intestines. Apparently, new mucosal epithelium was derived from pre-existing epithelium, whereas new muscle and connective tissue were
derived from cells of pre-existing unlabeled
tissues. Therefore, the cytoplasmic dedifferentiation that occurred at the site of
injury did not result in a return of these
cells to totipotency with subsequent random distribution of them among newly
differentiating tissues. Instead, there appeared to be a non-random assortment of
redifferentiating cells according to tissue
of origin, at least in the case of epithelium versus connective tissue and smooth
The incorporation of tritiated thymidine
into the nuclei of serosal cells represented
an exception to the normal pattern of labeling in the intestine. In the normal, unop-
erated intestine and in uninjured areas of
operated intestines, only scattered, infrequent radioactive cells were observed in
the serosa. However, in the zone of regeneration, all serosal cells were labeled in
several animals. This was seen only during later stages of renegeration (30 to 45
days after operation). The results did not
indicate a definite explanation for this distribution of radioactive cells, but other investigators have suggested possibilities.
McMinn ('60) and Johnson and McMinn
( ' 6 0 ) , in the reviews on wound healing,
stated that wounds in serous membranes
were probably repaired by metaplasia of
underlying connective tissue cells and from
cells that desquamated from adjacent surfaces. Brunschwig and Robbins ('54), after
experimental observations on the regeneration of peritoneum in mammals, concluded
that peritoneum was replaced simultaneously over the entire serosal wound and
that migration of cells from adjacent intact
mesothelium did not contribute to the healing processes. The latter investigators suggested that any cell of mesoblastic origin
might form peritoneum, mentioning specifically macrophages of the wound exudate and cells in the peritoneal cavity.
The question of morphological versus
physiological dedifferentiation of cells during the regeneration of amphibian limbs
has recently been investigated using radioautography (Riddiford, '60; Hay and Fischman, '61; and OSteen and Walker, '61),
with results which indicated that an epithelium, specifically the epidermis, does
not contribute cells to the limb blastema,
but does act by migrating and covering the
newly regenerated appendage. The results
of the present study provide further evidence that an epithelial cell of an adult
amphibian does not become totipotent during the formation of an undifferentiated
mass of cells, namely the blastema, from
an aggregation of tissues.
1. The regeneration of the intestine of
the adult newt was investigated using the
technique of radioautography following the
intraperitoneal injection of tritiated thymidine. Cell renewal systems in the newt
were thereby labeled. In the intestine this
involved the mucosal epithelium and leucocytes.
2. During the early stages of regeneration, the mucosal epithelium migrated over
the cut ends of the intestine. Mitoses and
labeled cells preparing to divide were seen
in these extensions of epithelium.
3 . Some radioactive mucosal cells, either
singly or in clusters, were observed in the
4. After the intestines had completely
regenerated, the distribution of radioactive
cells in the newly formed zone was the
same as in uninjured areas or in unoperated intestines, at least, in the case of
mucosal epithelium, smooth muscle, and
connective tissue. This indicated a nonrandom assortment of cells during regeneration of these tissues.
5. Radioautographs of several regenerated intestines had radioactive cells in
the newly formed serosa. A proposal is
suggested to explain this observation.
6. The results provide further evidence
that cells from adult tissues, particularly
epithelia, do not become totipotent, but
instead are only morphologically dedifferentiated during blastema formation.
Brunschwig, A., and C. F. Robbins 1954 Regeneration of peritoneum : experimental observations and clinical experience in radical
resections of intra-abdominal cancer. XVe
Congr. SOC.internat. Chir., Bruxelles, 756-765.
Chiakulas, J. J., and L. Lefkowitz 1960 The regeneration of smooth muscle during healing of
urodele gut wounds. Dev. Biol., 2: 446-460.
Dawson, A. B. 1927 On the role of the so-called
intestinal glands of Necturus with a note on
mucin formation. Trans. Amer. Micro. SOC.,
46: 1-14.
Goodchild, C. G. 1956 Reconstitution of the
digestive tract in the adult leopard frog, Runa
pipiens Schreber. J. Exp. Zool., 131: 301-327.
Hay, E. D., and D. A. Fischman 1961 Origin
of the blastema in regenerating limbs of the
newt, TrituTuS viridescens. An autoradiographic
study using tritiated thymidine to follow cell
proliferation and migration. Dev. Biol., 3: 2659.
Hunt, T. E. 1958 Regeneration of the gastric
mucosa in the rat. Anat. Rec., 131: 193-212.
Johnson, F. R., and R. M. H. McMinn 1960
The cytology of wound healing of body surfaces in mammals. Biol. Rev., 35: 364-412.
Johnson, H. A., and E. P. Cronkite 1959 The
effect of tritiated thymidine on mouse spermatogonia. Radiat. Res., 1 1 : 825-831.
mon newt. I. Phvsioloeical
Kopriwa, B., B. Messier and C . P. Leblond 1960
- reeeneration.
137: 501-509.
Technical progress in the coating technique
1961 Radioautographic studies of refor radioautography. Anat. Rec., 136: 340-341.
generation in the common newt. 11. RegeneraMcMinn, R. M. H. 1960 The cellular anatomy
tion of the forelimb. Ibid., 139: 547-555.
of experimental wound healing. Ann. Roy. Coll.
Patten, S. F., Jr. 1960 Renewal of the intestine
Surg. Eng., 26: 245-260.
epithelium of the urodele. Exp. Cell Res., 20:
Messier, B., and C. P. Leblond 1957 Prepara638-641.
tion of coated radioautographs by dipping secPatten, S. F., and W. Andrew 1954 Replacetions i n fluid emulsion. Proc. SOC.Exp. Biol.,
ment and differentiation of the intestinal epi96: 7-10.
thelium in the urodele. Anat. Rec., 118: 338.
O’Steen, W. K. 1958 Regeneration of the in- Riddiford, L. M. 1960 Autoradiographic studies
testine in adult urodeles. J. Morph., 103: 435of tritiated thymidine infused into the blastema
of the early regenerate in the adult newt, Tri.1959 Regeneration and repair of the
turus. J. Exp. Zool., 144: 25-32.
intestine in Rana clamitans larvae. J . E X ~ . Sauer, M. E., and B. E. Walker 1961 Radiation
Zool., 141: 449476.
injury resulting from nuclear labeling with
1961 Non-random distribution of radiotritiated thymidine i n the chick embryo. Radiat.
active cells during regeneration of the adult
Res., 14: 633-642.
newt intestines. Anat. Rec., 139: 261.
Walker, B. E. 1959 Radioautographic observaOSteen, W. K., and B. E. Walker 1960 Radiotions on regeneration of transitional epithelium.
autographic studies of regeneration i n the comTex. Rep. Biol. Med., 17: 375-384.
All figures are photomicrographs of radioautographs of longitudinal sections of regenerating intestines from animals of Group A, which received 15 pc of tritiated thymidine i n a
single intraperitoneal dose. Exposure time was 40 days. All sections were stained with
Harris’ hematoxylin and eosin.
1 General view of intestine adjacent to the region of regeneration, showing the typical
pattern of labeling. Radioactive cells are only in the mucosal epithelium. Many nuclei
show heavy labels (black nuclei), while others that are lightly labeled cannot be distinguished a t this magnification. Lumen of the intestine (L). Ten days after oFeration.
x 100.
Higher magnification of the same section as i n figure 1. This shows that some surface
epithelial cells ( E ) are heavily labeled (many silver granules over the nucleus), while
others in the surface epithelium and in the cell nests ( C N ) are lightly labeled. x 600.
3 A prolongation of mucosal epithelium can be seen extending out into the area of the
early blastema ( B ) . The extension, which is surrounded by fibrocytes and blood elements, originated from the cephalic cut end of the intestine which is out of the field
to the right. Heavily labeled cells can be seen in the mucosal epithelium (arrows). Ten
days after operation. x 100.
Higher magnification of the mucosal epithelial extension seen in figure 3. Radioactive
cells are seen among unlabeled cells in the epithelium. One group of these cells appeared
to be separating from the tip of the extension and passing into the blastema (arrow).
Also note the structural modification in the columnar epithelium and the presence
of polymorphonuclear leucocytes i n the area. x 400.
W. Keith OSteen and Bruce E. Walker
A cluster of labeled cells (arrow) in the blastema of a regenerating
intestine. Radioactivity cannot be distinguished at this magnification
(refer to fig. 6 for higher magnification). The lumen ( L ) of the operated cephalic end of the intestine can be seen a t the lower part of
the figure. Twenty days after operation. X 100.
Higher magnification of the cluster of radioactive cells indicated in
figure 5. Twenty days after operation. X 600.
General view of a histologically complete, regenerated intestine. The
region where the intestine was originally transected is indicated by
the arrows. All tissue components of the intestine have reformed at
this site. The luinina of the cephalic ( A ) and caudal ( B ) ends of
the intestine are filled with black India ink, which was injected to give
a n indication of the degree of regeneration. No blastema was observed
a t this time. Thirty days after operation. x 100.
A section through the area of regeneration i n a completely regenerated intestine. Radioactive nuclei are concentrated in the mucosal
epithelium. Thirty days after operation. x 470.
W. Keith O’Steen and Bruce E. Walker
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intestinal, common, radioautography, newt, repair, regenerative, studies, iii
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