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Regeneration of the gastric mucosa in the rat.

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University of Alabama Medical Center,
Birmingham, Alabama
Several investigators (Harvey, '07 ; Ferguson, '28; Grant,
'45; Gunter, '50; and others) have studied regeneration of
the stomach mucosa after injuring it in various ways. I n general they agree on most of the events occurring during the
process of repair, but there are some areas of disagreement
and some statements made in connection with the process may
be questioned. Thus Gunter ('50) states that in regeneration
of the mucosa in the cat mitoses do not occur for several days
or until the defect is completely covered. If this were true, it
would be contrary to general conclusions made in regard to
mitotic activity that the activity is increased when there is a
loss of cells or a depletion of certain cellular products (Hunt,
'51) and that in the stomach increased activity normally occurs within 24 hours (Hunt, '57).
There is also an area of disagreement as to the specificity
of the cell types in the stomach mucosa. According to Stevens
and Leblond ( ' 5 3 ) , the cell types of the stomach are fixed and
foveolar surface cells do not transform to mucous neck cells
or the latter into other types. On the other hand, evidence
has been presented (Hunt, '57 ; and Hunt and Hunt, '57) that
such transformations do occur and that the mucous neck cells
serve largely to replenish short-lived parietal cells. Most of
the past experimental work such as Harvey's ('07) and Ferguson's ('28) also offer strong support for the idea of transformation of one cell type to another.
I n order to reexamine some of these controversial or uncertain points, the regeneration of the gastric mucosa of rats
has been studied after defects were produced. The mitotic
activity during repair has been noted especially, as well as the
sequence of events during the development of new glands.
Furthermore, animals of different ages were used to see if the
regenerative process was slowed as the animals became older.
Regeneration of the mucosa was studied in 26 Long-Evans
rats all but 7 of which were males. Sixteen animals were 81
to 141 days of age (average 117) and 10 were 364 t o 823 days
(average 563). The time allowed for regeneration after making the injury was as follows: 4 hrs. (1r a t ) , 24 hrs. (2 rats),
48 hrs. ( a ) , 72 hrs. (a), 4 days (a), 5 days ( l ) ,7 days (a),
8 days (1), 10 days (1), 11days (1), 14 days (1), 16 days (1),
18 days (1),19 days (1), 21 days (1), 23 days (1), 33 days (1),
34 days (l), 36 days (l),47 days (l), 62 days (1).
To produce the defect in the mucosa, animals were anesthetized with nembutal and, after exteriorizing the stomach,
an incision of about 1em was made in the fore part, which is
lined with stratified squamous epithelium. This opening allowed a portion of the glandular stomach to be everted and an
area approximately 2 X 6 mm was cauterized with a “Hyfrecator” electric dessicator. The work mas done under a
dissecting microscope having an ocular micrometer. Eecause
of contractions the area cauterized sometimes varied 0.2 or
0.3 mm more o r less than intended. I n each animal the same
area parallel t o the juncture line of the forestomach and 1 to
3 mm from it was injured.
Since the presence of food was found to be a factor affecting
mitotic activity (Hunt, ’57), animals were fasted 48 hours
before being sacrificed. After decapitation of the animal the
stomach was removed, slit along the lesser curvature and forestomach, stretched over and pinned to cork and fixed in
Zenker-formol or cold absolute ethanol-form01 (9 : 1) mixture.
Sections (6 p ) were cut at right angles to the long axis of the
wound and stained with the Periodic Acid-Schiff (PAS) procedure of McManus.
First day o f regeneratiom. The injury 24 hours after dessication measured 7 X 1.3 to 2 mm (fig. 2). This was after fixation and partial dehydration, and since the size of the injury
did not differ appreciably from that calculated to have been
made originally, the stretching of the stomach over cork during
fixation maintained the original dimensions. Furthermore,
measurements of the width of the defect in the sections indicate
that appreciable shrinkage from imbedding did not occur.
Figure 2 shows that the margin of the defect is not as sharp
as that seen shortly after the injury was made (fig.1) but
rolls in slightly from the unaffected area to the dark floor
of the defect.
The sections show that the mucosa including its muscularis
was completely destroyed and only necrotic o r charred tissue
remains in the 2 mm gap. There is no apparent damage to the
muscularis externa but the subrnucosa shows vascular changes
of inflammation and numerous blood cells. Since no glandular
remnants occur in the floor of the wound, all regeneration
would have to come from the viable cells at the margin.
At the margin there is no marked change in the appearance
of the foveolae o r glands or the epithelial cells, except for
an almost complete disappearance of mucous neck cells for
about a millimeter, o r slightly more in some places, from the
The mitotic activity, however, in the marginal zone is definitely increased. I n the young (112 day) rat there is an
average of 16.5 mitoses per millimeter of surface in an area
within 2 to 2.5 mm adjacent to the wound. More peripherally
the average is 0.8 per millimeter which is approximately the
same as in a normal series of animals fasted 48 hours (Hunt,
5 7 ) . In the old (738 day) rat an average of 1 2 mitoses per
millimeter occurs near the wound which is probably not significantly less than that of the young rat. The dividing cells
are located 75 to 150 p from the surface in the deep part of
the foveolae or glandular necks. Most of the dividing cells
have a few PAS-positive granules like those of the surface and
foveolar cells but a few have none.
Second day of regeNeratiorz. Two days after dessication
of the mucosa the injured area appears as in figures 3 and 7.
The mucosa has been completely destroyed in an area approximately 1 3/4 x 5 to 6 mm which corresponds closely to
that measured when the injury was made. I n the section
(fig. 7), made in the plane of the arrow of figure 3 a slight
rolling inward of the mucosa along the margin of the wound
is visible. On the left side (arrow, fig. 7) may be seen a thin
layer of epithelial surface cells growing over the denuded
connective tissue f o r a distance of 60 to 70 p. I n other parts
of the wound these tongues of cells have a length of as much
as 280 to 350 p. The cells look superficially like a syncytium,
frequently have inclusions of cellular debris and contain little
o r no PAS-positive premucin granules characteristic of the
surface cells. They have no visible attachment to the underlying connective tissue and will soon begin to invaginate to
form fovcolae as shown in figure 13 or extend deeply towards
the muscularis mucosae along the viable cells a t the margin
of the wound. The unusual invasive character of relatively
undifferentiated stomach cells has been discussed by Grant
and Ivy ( '55).
I n the 113 day rat, foveolae are two or three times as deep
(220 to 260 p) as usual in an area 2 to 2 1/2 mm adjacent to
the wound. Beyond this, foveolae are typical and like those
outside of the regenerating areas of figures 10 and 11 where
they are 80 to 90 p deep. The area of deeper foveolae corresponds to that where more intense mitotic activity occurs
on the first day of regeneration.
In a zone 200 to 300 p wide next to the injured area there is
a marked change in the appearance of many of the cells. The
cytoplasm is more basophilic probably because of increased
amounts of ribonucleic acid, and many cells contain no visible
PAS-positive premucin. These cells are 11/2 to 2 p larger in
diameter and the larger nuclei contain more prominent nucleoli
than usual (cells “ b ” near center of fig. 12). Mucous neck
cells which usually occur in this region of larger cells are
absent. Deeper, where they do not normally occur, they appear
to have replaced zymogenic cells. Harvey (’07) and Ferguson ( ’28) believe that the zymogenic cells differentiate into
mucous neck cells but this is very difficult to prove. Just peripheral to the 200 p marginal zone, mucous neck cells have increased in number just deep to the foveolae (left side, fig. 7).
No mucous neck cells were present in this zone on the first
Mitoses occur frequently in the large basophilic cells of
the marginal zone and in the deep foveolar cells containing
a small amount of surface premucin but none were observed at
this time in the mucous neck cells adjacent to the wound. The
deep foveolar cells shouring mitoses occur in the 113 day rat
for a distance of a millimeter from the wound with an average
activity of 15 mitoses per millimeter. I n the 355 day rat increased mitotic activity occurs in a somewhat wider zone and
the foveolae are not so deep as in the younger animal. This
might be evidence that the younger animals respond more
quickly and intensively to the regenerative stimulus but,
if so, the older ones apparently make up the deficit within a
day or so.
Third, f o u r t h and fifth days of regeneration. No striking
changes in the regenerative sequence occur during the third,
fourth and fifth days but some characteristics are noted which
are significantly different from those of the second day.
Grossly the injured area appears only about haIf as wide
as it was earlier (fig. 4). This is due in part to an overgrowth
of the mucosa, especially its more superficial region which
bulges over the denuded connective tissue (fig. 8). Consequently the gap in the muscularis mucosae is 50 to 100%
greater than the gap between the edges of the mucosa. Glands
a t the margin of the wound slant sharply and lie almost
By the third day there is a great increase in the mucous
neck cells and they form a band of cells 140 to 1701.1deep
and 1.4mm wide just peripheral to the area of injury (fig.
8, “m”).
Close to the wound there is usually a flattened tongue of
cells continuous with the surface mucous cells (arrow, fig. 8).
These “tongue” cells not only spread over the denuded surface but appear to invaginate, so that along the margin of
the wound, in many cases, are gland-like formations with
very flat cells having little or no presecretion. These glands
frequently have expanded lumina as seen to the left in figures
8 and 13. Near this area is a narrow zone 70 to 100 I.I wide
having large basophilic cells. Deep t o this zone and appearing under low power to be continuous with the band of
mucous neck cells is an area where the glands are made up
entirely of mucous neck cells. These glands extend all the way
to the muscularis mucosae and are the most recently regenerated ones. Peripheral to these, on the fifth day at least, there
are glands having parietal cells mixed with mucous neck
cells. The mucous neck cells are not normally present in
such a position.
Mitotic activity is notably different in these stages compared to the second day stage of regeneration. From the
third day on, unusual mitotic activity is confined mostly to a
narrow 1 0 0 zone
close to the wound. Most mitoses in this
zone occur in the somewhat larger basophilic cells o r deep
foveolar cells but now they also occur in the mucous neck
cells. I n this region several dividing mucous neck cells may
be seen in a single high power field. I n the zone 1 to 2mm
beyond the marginal one the dividing mucous neck cells
have also increased. I n the 5 animals (three to 5 days after
injury) the average number of mitoses per millimeter is 0.7
compared with a control number of 0.1 per millimeter taken
from a series reported previously (Hunt, ’57). Since mitoses
were not observed in the mucous neck cells of this region
prior to the third day, this amounts t o a considerably increased activity.
Sevem t o 14 d a y s regeneration. During this period the
wound becomes completely covered with regenerated epithelium. I n figure 9, showing the area of regeneration on
the tenth day, it may be seen that a thin layer of cells has
bridged the gap, the slight defect being an artifact of preparation. The final time of covering varies somewhat probably due largely to the slight differences in the original defect.
I n all cases, however, the defects are closed by the 14th day
of regeneration. No evidence of a significant difference because of age is found.
I n figure 9 the defect in the muscularis mucosae is about
1 (between arrows) and most of the epithelial formations above this area are believed to have regenerated. The
darkly stained glands at the margin of the thin layer of
epithelium are composed entirely of mucous neck cells. The
larger basophilic cells present at earlier stages of regeneration are no longer present and instead, in the deep part of
the foveolae, are cells with a small amount of surface type
premucin. Deep to these cells are ones appearing to be
transitional from surface to mucous neck cells.
The mucous neck, transitional and deep foveolar cells all
divide but divisions of mucous neck cells are much more
numerous than usual (fig. 15). Consequently, many buds
form at the base and sides of the newly regenerated glands
composed of mucous neck cells, which explains the appearance
of branched glands seen a t these and later stages (fig. 14).
To the periphery of the area of purely mucous neck cell
glands are glands with a few parietal cells mixed with mucous
neck cells. These appear about where the mucosa reaches a
normal thickness and it is difficult t o decide at this stage
whether such glands are regenerated or undestroyed ones.
S i x t e e n t o 6 2 days of regeneration. After the defect
has been completely covered, the subsequent sequence of
events of regeneration is much the same as occurred at
the margin of the defect previously, except that earlier
steps progressively disappear from the picture as the period of regeneration is extended. Thus at 18 days the layer
of flat epithelium has disappeared and all the surface cells
have an accumulation of mucus. There are shallow foveolae
from which arise glands composed entirely of mucous neck
cells (fig. 10). These are within a narrow zone three-fourths
of a millimeter wide. More peripherally in a zone that has
evidently regenerated, since it overlies the defect in the
muscularis mucosae, the glands are a mixture of mucous
neck and parietal cells. At 36 days (fig. 11) the mucosa is
fairly well established, being centrally about 280 p in thickness, of which 7 0 p represents the depth of the glands. The
glands here are no longer purely mucous neck cells but a
mixture of mucous neck and parietal cells (fig. 17). To the
sides but under the sloping portion of the regenerated area,
the mucosa is quite normal with glands having zymogenic
cells as well as the other types.
The reappearance of zymogenic cells is seemingly the last
step in regeneration and occurs by further differentiation of
the mucous neck cells when they attain a certain depth in the
mucosa. The elongated, longitudinally cut gland in figure 16,
“g” contains cells near the foveola which are mucous neck
cells in character. Proceeding towards the right the PASstaining material in the cytoplasm of the glandular cells
gradually diminishes and the cells assume the characteristics
of zymogenic cells with basal ergastoplasm and an apical
presecretion zone.
Further regeneration was followed in two animals to the
47th and 62nd day. By the 62nd day the mucosa at the site
of injury has still not attained the full thickness but otherwise it looks essentially normal. The degree of regeneration
in the animal killed the 47th day is not much different than
that of the animal killed on the 36th day.
The mitotic activity a t the regeneration site in these more
advanced stages of regeneration does not seem to be appreciably greater than elsewhere in the stomach except for
the greater activity in the mucous neck cells, where almost
twice as many mitoses as usual occur.
I n general the process of regeneration of the stomach
mucosa of the rat does not differ from that described for
the dog (Ferguson, ’28) o r the cat (Gunter, ’50). I n all
three animals there is a formation of a thin migrating layer
of epithelial cells and subsequently from it foveolae and
glands differentiate. I n Gunter ’s description of the regenrrative process, however, he failed to see any mitotic activity
until after the gap in the mucosa had been closed, an observation which would lead to the conclusion that mitosis had
little t o do with the early phase of regeneration. This idea
is perpetuated in Greep’s “Histology” ( ’54) which states
that “mitoses occur only after complete covering of the
denuded area has been accomplished. ”
Contrary to that statement, the present experiments show
that by far the greatest mitotic activity occurs the first day
after injury and before there is any migration of cells. Within
a distance of 2 or 3 m m from the wound there are at that
time 10 to 20 times as many mitoses as elsewhere in the
stomach. On subsequent days before complete covering of
the defect, mitotic activity remains high in a narrow zone
next to the wound. After covering, mitoses are not appreciably greater than usual except in the mucous neck cells. The
discrepancy between our results and those of Gunter, as far
as mitotic activity is concerned, may be readily explained
by his lack of early stages and perhaps his unfamiliarity
with normal mitotic activity in the stomach. Gunter’s earliest
stage was 48 hours after making the defect.
The increased mitotic activity within a 2mm area around
the defect correlates well with the normal activity occurring
in the stomach mucosa following ingestion of food (Hunt, ’57).
I n those experiments it was found that 20 to 24 hours after
refeeding, following a 48 hour fast, there were concentrations
of mitoses with an average distance apart of 4mm. Thus
the mitotic stimulus appears to be in localized areas and
spreads out from a center for about 2mm o r about the same
distance as occurs from the margin of a defect. From those
observations it was concluded that the stimulus for mitosis
is the depletion of cellular products, which when sufficiently
concentrated act to inhibit mitotic activity. The present experiments support that conclusion.
The rate of regeneration is not significantly different in
the young compared to the old animals. Previously it was
found (Hunt, '57) that the mitotic activity in the stomach
after refeeding did not vary with the age of the animal, so
that unless some pathological condition exists it would be
expected that the regenerative rate a t different ages would
remain constant.
The sequence of events during regeneration provides evidence that one cell type changes to another. On the first
and second days most divisions occur in surface type cells
deep in the foveolae near the wound. During this time no
increase in mitotic activity of the mucous neck cells occurs
and close to the wound they decrease in number for the first
24 hours. By the second day, however, and before they show
increased mitotic activity they become more numerous. By
the third day they are greatly increased in number and only
then do they have more mitoses than usual. There is scarcely
any alternative to the conclusion that they have come mainly
from the previously dividing, deep foveolar cells, most of
which show a small amount of surface type premucin. Furthermore, there is a gradation in appearance from deep
foveolar to mucous neck cells, and in many instances it is
impossible to decide whether the cell should be classed as
a deep foveolar o r a mucous neck cell.
Subsequent to the tenth day, the full sequence of regeneration may be followed from the wound outward and it is
then possible to see evidence of a further transformation of
cell types. At 10 and 18 days (figs. 9 and 10) new foveolae
and glands develop from relatively undifferentiated surface
cells like those in figure 13. J u s t peripheral to this zone
all of the cells in the glands are of the mucous neck cell
type as in figure 15. Beyond this, but in an area which
previously consisted entirely of mucous neck cells, are glands
with both mucous neck and parietal cells (fig. 14). I n the
oldest of the regenerated glands are zymogenic cells in
addition to the others. Thus the same conclusion is reached
as that of Ferguson (’28) and Gunter (’50) that the surface
type cell may become a mucous neck cell and it in turn may
become a parietal or zymogenic cell by further differentiation. Such a conclusion is contrary to that of Stevens and
Leblond ( ’ 5 3 ) who believe there is a specificity of cell types.
Whether there is a dedifferentiation of parietal or zymogenic cells to mucous neck cells is more difficult to decide.
Harvey (’07) in studying the histological changes after gastroenterostomies in dogs was convinced that the zymogenic
cells transformed to mucous neck cells for a distance of
7mm from the cut. I n the rat the only evidence that such
a transformation takes place is the appearance of mucous
neck cells in the fundic portion of glands in a region close
to, but not necessarily adjoining, the site of injury. These
occur in a region where mucous neck cells do not normally
occur and they may be transformed from the zymogenic
cells. There could have been, however, an unusual proliferation of foveolar and mucous neck cells at this site which
might have provided the mucous neck cells for these glands.
With such a possibility the decision of whether there is a
dediff erentiation of zymogenic cells remains uncertain.
1. Defects in the stomach mucosa were produced by cauterization with a “Hyfrecator” electric dessicator and regeneration was subsequently observed from the first to the sixtysecond day.
2. Regeneration occurs by migration of nndamaged cells
over the defect and by ingrowth along the wound margin.
3. Within a 2mm zone adjacent to the wound mitotic
activity on the first day of regeneration is much greater
than elsewhere.
4. After the second day markedly increased mitotic activity is confined to a narrow 100 p wide zone adjacent to the
5. Division of cells occurs primarily in deep foveolar cells
with a small amount of premucin or in enlarged and seemingly
undifferentiated basophilic cells. After the third day, when
mucous neck cells are more numerous near the wound, mitoses
increase in them.
6. After an initial migration of cells to form a flat tongue
of epithelium there are invaginations to form foveolae and
7. It is concluded that surface cells transform into mucous
neck cells and the latter to parietal cells. Later in the regenerative process mucous neck cells also become zymogenic cells.
8. It is also concluded that the rate of regeneration is
not significantly different in young and older animals.
A. N. 1928 A cytological study of the regeneration of gastric
glands following t h e experimental removal of large areas of mucosa.
Am. J. Anat., 48: 403-441.
GRANT, R. 1945 Rate of replacement of the surface epithelial cells of the
gastric mucosa. Anat. Rec., 91: 175-185.
GRANT,R., AND A. C. IVY 1955 The abnormal repair and invasive growths
following the implantation of hydrocarbon carcinogens i n the gastric
submucosa of the rat. Gastroent., 2 9 : 199-218.
GREEP, R. 0. 1954 Histology. Blakiston Co., New York.
GUNTER,G. S. 1950 A histological investigation of the healing of acute gastric
ulceration i n the cat. Gastroent., 15: 708-717.
HARVEY,B. C. H. 1907 A study of the structure of the gastric glands of
the dog and of the changes which they undergo after gastroenterostomy
and occlusion of the pylorus. Am. J. Anat., G: 207-243.
HUNT,T. E. 1951 The effect of hypophyseal extract on mitotic activity of
the rat hypophysis. Anat. Rec., 111: 713-726.
1957 Mitotic activity in the gastric mucosa of t h e rat after
fasting and refeeding. Anat. Rec., 187: 539-550.
HUNT,T. E., AND E . A. HUNT 1957 The relation of mast cells and adrenal
glands t o t h e development of gastric ulcers. Anat. Bee., 127: 311-312.
C. E., AND C. P. LEBLOND 1953 Renewal of the mucous cells i n the
gastric mucosa of the rat. Anat. Rec., 115: 231-245.
1 Wound 4 liourv after cautcrization. b’orestouiacli t.o left. X 4.2.
2 Oiic? day after cauterizittioii. Milliirieter vetile to right. X 4.2.
3 Two days after cauterization. Edgcn~sliglitly rolled towarcls floor o f c1cfc.c.t.
Arrow indicate8 plane of Hcwtioii ti8 secw in figure 5. X 4.9.
R f t h day of regeiierritioa. Gap ill 1iiurOv:i re(1iiccd to :ibout 1 mm. Arrow
indicates plane of section, figure 8. X 4.2.
Truth clay of repieration. Floor o f drfrct. c*orcred aa iu section, figtire 9.
Thirty-sixth day of regeiicrntion. Arrow iiidicrtea plnitc of seetioil, figure 30.
Rcvtioii showi1ig defect iii itiucos:i two il:r
To11gue of cells at arro\v. x 23.
Mucsos:~ 011 fifth d:iy of rcgcwcr;ition.
ifter injury. N o t r deep foveohc.
b’oveo1:ie deep atid niucous neck cc~lls
numerous. Toiipic~of cells :it :irrow. X 29.
Mticosa o n tenth day of ~cgeiieration. Foveo1:ie not so deep and niucous
neck cells riot so iiunieroiis as in figure 8. Arrows indieate a p p r o s i m a t r
extent of original tlcfcct. X 29.
10 11lncos:1 oii eighteciitli t1:i.v of ~ c g e n c r ; ~ t i o nMost
recently rcgenerntetl gl:rntls
coniposed of niucoiis i i c ~ kcc119. X 29.
11 Mucos:~ o n thirt
tli t h y of rcgciirxratioit. Ceiitr:il regeiier:Ltioii zone about
oiie third normal thicknes.; wit11 deep foveolae arid glaiids of niucous neck
ant1 parietal cells. x 23.
T l l ( J 3 l \ h E. H L S T
1'LATb; 3
12 Hiicas:t :it iiirirgiii of ilefwt on xwoiid d t y of rcgeiiertitioii. Eiikirgcd
Lasopliilic cdla (1)) nrid iiunicroua mitotic figures. x 871.
13 Eiglitli d:ty of regeiicrutioii. (irowth of toiigiie of rpitlieliuui o w r d(wii1etl
coiiiiectire tiaxue :itid its I)egiuiiiiig invagination (nrroivj. Soti. grtiilnril
diii,ii~utiono f iiiucia iii surface cells goiiig froiii the left to tlic free iiitirgiii
o f tongue*.
14 MU(WH:I,sixteciitli day of rc+ymwitioii. S o t c brniiclial gltuids coiiiposrtl
entirely of aiucous iicck cells (tirrow). x il.
18 Glaiicls coiiiposccl of iiiii~ouaiieek ccllx. 1)ivicliiig c d l x (arrowsj.
x 493.
16 Miicosti, tliirty-fourtli tltiy of regeneration. S o t e longitucliiinlly cut gl:liitl
(gj with iiiucous iiwk cc*llx at left und trtiiisitiou to zyiiiogeiiic cells to riglit
of guide liac. x 71.
17 Mucosel glniids, thirtpaixtli doy of regemration, xliowiiig iiiiieous iicrk tiiitl
parietal ccllv but uo zyiiiugciiic eella; fovcolar cells ribovc uail right. x 393.
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