Effects of Food Ingestion on the Cell Proliferation Kinetics in the Canine Fundic Mucosa

Vol. 61, No.3 Print ed in U.S.A .

GASTROENTEROLOGY

Copyright© 1971 by The Williams & Wilkins Co.

EFFECTS OF FOOD INGESTION ON THE CELL PROLIFERATION KINETICS IN THE CANINE FUNDIC MUCOSA G. WILLEMS, M.D. , Y. VANSTEENKISTE, AND PH. SMETS, M.D. Laboratory of Experimental Surgery, Saint-Pierre Hospital, and Department of Scientific Computation, Medica( School, University of Brussels, Brussels, Belgium

Biopsies of the canine fundic mucosa were performed after 36 hr of fasting and at various intervals after food ingestion. In vitro incubation of the mucosal specimens with tritiated thymidine and autoradiography was utilized to observed deoxyribonucleic acid-synthesizing cells. The mitotic activity and the height of the foveolae were estimated simultaneously in the same specimens. The deoxyribonucleic acid synthetic index was considerably increased between the 12th and the 20th hr after food ingestion, whereas a higher mitotic index was observed only between the 20th and the 24th hr. The foveolar height, abruptly reduced after the meal, began to increase again after the 20th hr, almost attaining preprandial levels at the 24th hr as a consequence of this wave of proliferative activity. allow adequate study of dynamic processes and the use of radioactive deoxyribonucieic acid (DNA) precursors is proving increasingly valuable for following these processes at a cellular level. 8 In this work, we investigated the effect of food ingestion on both DNA synthetic index and mitotic index in the canine fundic mucosa. In vitro autoradiography with tritiated thymidine was utilized, its uptake being indicative of DNA-synthetizing cells even in incubated mucosal specimens. 9 Previously, it had been demonstrated that in vitro labeling with this precursor affords a valuable means to estimate proliferative parameters in the fundic mucosa. 10

Autoradiographic analysis following administration of tritiated thymidine has provided information regarding the cell renewal in the gastric mucosa of man, 1• 2 dog, 3 and various small rodents. •. 5 There exist, however, only a few details concerning the physiological variations of the cell kinetics in the stomach. 6 In the rat gastric mucosa, higher mitotic indexes were observed after feeding. 7 This might arise either from a lengthening of the time that progenitor cells spend into mitosis or, on the contrary, from a shortening of the generative cycle and, hence, from a stimulation of the cell proliferation. Actually, classical histological techniques alone do not

Materials and Methods

Received January 4, 1970. Accepted March 31, 1971. Address requests for reprints to: Dr. G. Willems, Department of Surgery, H6pital Saint-Pierre, Rue Haute, 322, 1000- Brussels, Belgium This work was supported in part by the Fonds de Ia Recherche Scientifique Medicale. Dr. G. Willems and Dr. Ph. Smets are "Charge de Recherches" of the Fonds National de Ia Recherche Scientifique. The authors express their thanks to Dr. J. De Graef and to Dr. P. Galand of the University of Brussels for their generous and adequate advice.

Four adult mongrel dogs weighing from 10 to 12 kg were used. Gastric nylon cannulae were placed surgically, permitting repeated biopsies of the gastric mucosa. 11 A 6-week period of recovery was allowed after surgery. Biopsy specimens, ranging from 8 to 12 mm in length were taken through the cannula from the anterior or posterior wall of the corpus fundic mucosa. Taking specimens from the mucosal areas in contact with the cannula or in proximity of previous biopsies was carefully avoided by using a human rectoscope to control the biopsy 323

324

WILLEMS ET AL.

sites. The biopsies were performed after a 36-hr period of fasting and 4, 8, 12, 16, 20, and 24 hr after ingestion of 250 mg of ground meat by the same 36-hr fasted animals. The experiment was divided into three parts (table 1) to avoid nocturnal influences and the possible effect of wounding the mucosa on the progress of the epithelial cells through the cell cycle phases. Control biopsies were taken at 9 AM, 1 PM, and 5 PM, and at different days without food ingestion in order to investigate potential hourly or daily variations. As in previous experiments, 10 · 12 each mucosal specimen was rapidly divided into 1-mm thick lamellae and then incubated for 15 min in a shaking water bath at 37 C in 25-ml flasks containing 2 ml of Eagle's medium (General Biochemicals, Chagrin Falls, Ohio) , supplemented with 10% calf serum and Ha-thymidine at a concentration of 10 !J.C per ml. The pH was adjusted to 7.2 to 7.4 with a 5% sodium bicarbonate solution and maintained with carbogen bubbling during incubation. After the end of incubation, the specimens were washed in 0.9 % NaCl, fixed in Bouin's solution for 24 hr, embedded in paraffin and cut at 3- to 4-IJ. thickness. Sections were stained with periodic acid-Schiff (PAS) and then covered with nuclear emulsion (llford K 5 in gel form). The autoradiographs were developed after 3-week exposure at 4 C and counterstained with hematoxylin. In each specimen, labeling index, i.e., the percentage of cells in DNA synthesis, and miTABLE

1. Tim e schedule of the experiments

on 4 dogs Experi rnent 11

Hour of

feedin gb

Hour of bi opsy

Time in terval after feedi ng

hr

1

9:05AM

2

9 : 00 PMc

3 4

9 : 00 AMC

5

Fasted controls Fasted controls

{9:00AM 1:00PM 5: 00PM

36 4

{9:00AM 1:00PM 5 : 00PM 9:00AM 9 : 00 AM

12 16 20 24 36

{1:00PM

36

5 :00PM

40

8

a One week elapsed between the experiments. • At the time of food ingestion, all dogs were fasted for 36 hr. c Day before the biopsy.

Vol. 61, No.3

totic index were counted on a total of 20 glands cut along their whole length, the total cell count in each specimen ranging from 1393 to 2087 epithelial cells. For these determinations, analyses were restricted to that portion of the gland where the greatest number of labeled cells is known to occur. As extensively described elsewhere, a. 10 this site of maximal proliferation is defined in each gland by taking as reference position the limit between the foveolar cells and the mucous neck cells. These latter are easily recognizable from the former by the pink, foamy appearance of their intracellular mucus after PAS reaction. 11 • Ia Starting from this limit, the glandular cells are numbered upwards and downwards, the cell located exactly above this point being termed position + 1, the next cell toward the mouth of the crypt position + 2, etc. In the fundic glands of fasted dogs, 96% of the labeled cells are known to occur between position -5 and position +15. a. 10 This was verified in our experiment by counting the average proportion of labeled cells situated between these limits on a total of 1000 labeled cells in the controls (250 in each specimen) as well as in the specimens taken 16 hr after food ingestion. The height of the foveolae was expressed as the number of cell positions between the deepest foveolar cell and the superficial epithelium. The mean foveolar height was recorded in each specimen on a total of 20 glands cut along their whole length. For the statistical analysis, the proportions (labeling and mitotic indexes) were submitted to the arc sine transformation. 14 The data came from a two-way layout with one observation per cell. The analysis of variance, 1 5 weighted for the transformed proportions and unweighted for the foveolar heights, was then performed and the differences either between the average response of the 4 dogs and the average response at each time interval were tested.

Results Both labeling index (fig. 1) and mitotic index increased in the 4 animals after ingestion, but an important lag period elapsed before the response (fig. 2). The mean labeling index tested with the transformed data varied from its initial value at the 12th hr (P < 0.01) whereas the mitotic index rose only at the 20th hr (P < 0.01) when the labeling index was already diminishing. The statistical analysis showed that, in the control experiments without food ingestion, the fluctuations from dog to dog and from

Septemb er 1971

EFFECTS OF FOOD INGESTION ON CELL PROLIFERATION

325

FIG. 1. A, autora diography of fundic mucosa. Control specimen taken in a 36-hr fasted animal and incubated during 15 min with 10 p.c per ml 3 H-thymidine. Note labeled cells in the depth of the foveolae (periodic acidSchiff, hematoxylin, X 400) . B, autoradiography of fundic mucosa. Specimen taken 12 hr after food ingestion and incubated as in A . Note the increased labeling index and the desquamated mucosal surface (periodic acidSchiff, hematoxylin, X 400).

day to day were negligible and that the observed labeling or mitotic indexes were not correlated with the hour of the biopsy. The proportion of labeled cells encountered in the glandular site of maximal proliferation to the whole labeled cell population was similar in the controls (96.5%) and in the specimens taken 16 hr after food ingestion (94.9%). Contrary to fasted controls, labeled cells were occasionally seen in the superficial epithelium of these latter specimens. The foveolar height was abruptly reduced after feeding (P < 0.01), remained then at the same level until the 20th hr and was increasing at the 24th hr (P < 0.01) (fig. 2). Discussion The concomitant observation of labeled nuclei and of mitotic figures in the mucosa permits the identification and quantitation of cells going through the DNA synthetic phase and through the mitotic phase of the

cell cycle, the other cells being either in a premitotic (G:J or in a postmitotic (G 0 or G 1) resting phase. The present analyses of DNA synthetic activity revealed significant increases in proliferative activity between the 12th and the 20th hr after food ingestion, while examination of the mitotic indices provided evidence of significant increase only between the 20th and 24th hr after eating. Since labeling and mitotic indexes increased successively, a slowing down of the cells, when going through one of the cell cycle phases, cannot be involved and actually only stimulated cell turnover can explain the phenomenon. Variations of the kinetic parameters caused by diurnal influences or day to day variations cannot explain our results, since they were not significant in the control experiments without feeding. A direct effect of wounding the mucosa on cell proliferation was improbable with the experimental procedure used. It has been shown that a mitotic response after an experimental de-

WILLEMS ET AL.

326

. ..,

20

c

12

16

20

24h

20

24 h

20

24h

Time intuval

.,,

.lol

/

.:; 08 !: 06

~ ~ ~ t-1--+---+----+-! 0

12

16

Time interval

.

~

u. 10

12

16

Timc interval

FIG. 2. Mean labeling index, mitotic index, and foveolar height in canine fundic mucosa at various intervals after food ingestion. Labeling and mitotic indexes are expressed as a percentage, the vertical bars represent the confidence limit at 95%. The foveolar heights are expressed in numbers of cell positions between the mucous neck and the luminal surface(± standard error) .

feet in the gastric mucosa does occur in an area within 2 to 2.5 mm adjacent to the wound, but such an effect is preceded by a lag period of 20 hr. 16 This lag period exceeds the maximal 8-hr interval which we used between our biopsies. Moreover, mucosal samples were not taken from sites adjacent to previous biopsies. The effects of gastric cannulation were not explored, but sampling the mucosal area in contact with the cannula was always avoided. Although the mitotic index has been shown to fluctuate from one crypt to another, 7 the large size of the mucosal specimens and the high total cell count provided a reliable pattern of the proliferative parameters in the mucosa.

Vol. 61, No.3

Under the effect of food ingestion, a considerable but not uniformly distributed loss of cells from the mucosal surface is known to occur. 17 The observed reduction of the average foveolar height probably reflects such a postprandial shedding. As previously expected by Grant et al., 18 a regenerative process follows this cell loss. Maintenance of the normal mucosal pattern depends on the adequacy of the proliferative response. The increase in gastric mitoses following food ingestion in fasted rats 7 or after feeding spicy pickles to man 19 is in agreement with our results. The remarkable time shift between labeling and mitotic peaks accounts for a particular model of proliferative response: a cohort of resting G 1 or G 0 cells is likely to have abruptly entered the S phase after a lag period of approximately 12 hr. After their DNA synthesis had been completed, their passing through the mitosis phase was reflected by the retarded mitotic peak and finally resulted in an increase of the foveolar cell population and, thus, of the foveolar height. The observation of a 3 H-thymidine uptake by some superficial epithelial cells in the specimens obtained after food ingestion might suggest abnormalities in the spatial distribution of DNA-synthesizing cells, similar to those reported by Winawer and Lipkin 20 in the case of atrophic gastritis in man. However, the number of cell positions between these labeled superficial cells and the mucous neck of the nearest gland never exceeded the normal. Therefore, we assumed that their abnormal localization resulted from a simple shortening of the foveola at the expense of its most superficial, nondividing segment, rather than from an extension of the glandular site of proliferation. Since our experimental animals had to be fasted for a prolonged period (36 hr) prior to feeding, unless previous meals would have interfered, we were unable to analyze the real physiological variations in normal feeding conditions. Our data, however, support the conclusion that refeeding of animals after prolonged fasting is fol-

September 1971

EFFECTS OF FOOD INGESTION ON CELL PROLIFERA T!ON

lowed by a delayed but considerable stimulation of the cell turnover in the fundic mucosa. REFERENCES 1. McDonald WC, Trier JS, Everett NB: Cell proliferation and migration in the stomach, duodenum, and rectum of man: radioautographic studies. Gastroenterology 46:405-412, 1964 2. Lipkin M: Cell proliferation in the gastrointestinal tract of man. Fed Proc 24:10-15, 1965 3. Willems G, Vansteenkiste Y, Verbeustel S: Autoradiographic study of cell renewal in the fundic mucosa of fasting dogs. Acta Anat (in press) , 1971 4. Messier B: Radioautographic evidence for the renewal of the mucous cells in the gastric mucosa of the rat. Anat Rec 136:242-257, 1959 5. Creamer B, Shorter RG, Bamforth J: The turnover and shedding of epithelial cells. Part 1: the turnover in the gastrointestinal tract. Gut 2:110118, 1961 6. Gerard A: Regeneration cellulaire dans Ia muqueuse gastrique du chien sous l'effet de divers stimulants et de diverses drogues ulcerigenes. Bioi Gastroent 1:5-14, 1969 7. Hunt TE: Mitotic activity in the gastric mucosa of the rat after fasting and refeeding. Anat Rec 127: 539-550, 1957 8. Hughes WL, Bond VP, Brecher G, eta!: Cellular proliferation in the mouse as revealed by autoradiography with tritiated thymidine. Proc Nat Acad Sci USA 44:476-483, 1958 9. Deschner D, Lewis CM, Lipkin M: In vitro study of human rectal epithelial cells. J Clin Invest 42: 1922-1928, 1963 10. Willems G, Galand P, Chretien J : Autoradio-

11.

12.

13.

14.

15. 16. 17.

18.

19.

20.

327

graphic studies on cell population kinetics in dog gastric and rectal mucosa. A comparison between in vitro and in vivo methods. Lab Invest 23:635639, 1970 Gerard A: Etudes histochimiques et histophysiologiques des glycoproteines de Ia muqueuse gastrique chez le chien. Arch Bioi (Liege) 79 :1-104, 1968 Galand P , Mainguet P , Arguello M, et al: In vitro autoradiographic studies of cell proliferation in the gastrointestinal tract of man. J Nucl Med 9: 37-39, 1968 Stevens CE, Leblond CP: Renewal of the mucous cells in the gastric mucosa of the rat. Anat Rec 115:213-246, 1953 Freeman MF, Tukey JW: Transformations related to the angular and the square root. Ann Math Statist 21:607- 611, 1959 Scheffe H: The Analysis of Variance, New York, Wiley and Sons Inc, 1959 Hunt TE: Regeneration of the gastric mucosa in the rat. Anat Rec 131:193-211, 1959 Grant R: Conditions under which the epithelial cells of the gastric mucosa are shed and disintegrate. Canad Med Ass J 51:577- 579, 1944 Grant R, Grossman MF, Ivy AC: Histological changes in the gastric mucosa during digestion and their relationship to mucosal growth. Gastroenterology 25:218-231, 1953 McDonald WC, Anderson FH, Hashimoto S: Histological effects of certain pickles on the human gastric mucosa. Canad Med Ass J 96:1521-1525, 1967 Winawer SJ, Lipkin M: Cell proliferation kinetics in the gastrointestinal tract of man. IV: Cell renewal in the intestinalized gastric mucosa. J Nat Cancer lnst 42 :9-17, 1969