STUDIES OF SMALL BOWEL ADAPTATION AFTER INTESTINAL RESECTION IN THE RAT

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1 GASTROENTEROLOGY 1971 by The Williams & Wilkins Co. Vol. 60, No.1 Printed in U. S. A. STUDIES OF SMALL BOWEL ADAPTATION AFTER INTESTINAL RESECTION IN THE RAT ELLIOT WESER, M.D., AND MARY H. HERNANDEZ, B.A. Department of Physiology and Medicine, Section of Gastroenterology, University of Texas Medical School at San Antonio, San Antonio, Texas Fasting rats were killed 5 to 6 weeks after a 50-cm resection of either proximal or distal small bowel or sham operation. Remaining intestinal segments appeared increased in both thickness and diameter. The mean villous height was greater in proximal and distal bowel remnants of resected animals than in comparable segments of sham animals of the same age and weight. The number of epithelial cells per!j. of villous surface increased in proximal and distal villi. Lactase, sucrase, and maltase activities were significantly lower in homogenates of both whole mucosa and isolated villous epithelial cells collected from proximal remnants. With the exception of sucrase, disaccharidase activity was also significantly lower in whole mucosa and isolated epithelial cells collected from distal remnants. Isolated epithelial cells collected from proximal and distal remnants were incubated with C 14 -labeled glucose and C 14 -labeled leucine. The ratios of glucose or leucine counts per min per ml of cell water to counts per min per ml of incubation medium indicated active transport and were equal to ratios obtained from cells collected from control segments. However, when uptake of glucose and leucine was determined as a function of the number of cells, the net transport of both substrates was reduced in cells collected from the distal remnant. Glucose transport in everted gut sacs prepared from proximal remnants was significantly less than control segments. In the early steady state after small bowel resection, the remaining segment shows hyperplasia with an increase in the number of epithelial cells lining the villous surface. These presumably less mature and smaller cells have diminished disaccharidase and transport activity. In earlier studies we have seen that morphological and functional adaptation occur in the remaining intestine after extensive small bowel resection in the rat. 1-6 Some authors have found this response only true in the small bowel segment dis- Received March 16, Accepted August 5, This work was presented in part at the southern section meeting of the American Federation for Clinical Research, February 1, 1969, New Orleans, Louisiana. Address requests for reprints to: Dr. Elliot Weser, University of Texas Medical School at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas tal to the site of anastomosis. 7 The intestinal mucosal mass increases with heightening of the villi and circumferential enlargement of the small bowel. 2, 3. 5, 6 Whether previously reported functional changes, such as an increase in glucose transport, 6 are related to a greater number of epithelial cells lining the villi (hyper- This investigation was supported in part by Research Grant AM l from the National Institute of Arthritis and Metabolic Diseases and from an award by the Morrison Trust of San Antonio. The authors wish to express their appreciation to Miss Victoria Po for her technical assistance.

2 70 WESER AND HERNANDEZ Vol. 60, No. 1 plasia) or to a greater transport capacity of individual epithelial cells is not clear. In this paper, the effects of villous hyperplasia on epithelial cell disaccharidase activity and glucose and leucine transport were studied in rats following intestinal resection. It is suggested than an increase in the number of epithelial cells accounts for any increase in functional activity. Materials and Methods Operative technique. Under ether anesthesia, young male Sprague-Dawley rats, weighing around 150 g, underwent resection of 50 em of either proximal or distal small intestine. Proximally, the duodenum was identified and, beginning with a point 5 cm from the ligament of Treitz, measurements were made with a centimeter ruler without stretching the bowel. Similarly, a point 5 cm proximal to the ileocecal value was used for measurement. The remaining bowel was joined in an end-to-end anastamosis ensuring patency of the lumen. Following surgery, the rats were fed a standard rat chow diet ad lib (Ralston Purina Company, St. Louis, Mo.). During the week after resection, a slight weight loss was noted; thereafter, the rate of weight gain was approximately equal to shamoperated and normal rats. Morphology. Five to 6 weeks after intestinal resection, 23 rats (14 resected distally and 9 resected proximally) were killed by decapitation. Eleven weight- and age-matched sham animals served as controls. The proximal or distal bowel segments were rapidly removed, rinsed with cold saline, and everted over a glass rod. In resected and control rats, comparable l-cm segments of everted intestine were obtained at 20 cm from the pylorous and 20 cm from the ileocecal junction. After fixing in 10% formalin, perpendicular sections were prepared from the middle of the segments and stained with hematoxylin and eosin. Morphology was examined by light microscopy without knowing the experimental origins of the tissue. The mean height of the three tallest villi was obtained by using an eyepiece micrometer. In addition, the mean number of epithelial cells between the base of the villus (crypt opening) and villus tip was determined by direct count in the same three villi. In other experiments 35-cm segments of small intestine distal to a point 10 cm from the pylorous were removed from 14 rats resected distally (proximal remnant) and everted over a glass rod. Segments of equal length from 11 rats resected proximally were similarly prepared. These segments were measured proximal to a point 5 cm from the ileocecal junction (distal remnant). Comparable proximal and distal control segments were obtained from 17 and 15 control animals respectively. Intestinal epithelial cells were isolated and collected according to the method of Perris.8 The cell pellet was gently suspended in normal saline (1 : 40, v Iv) and cell counts were performed in duplicate in a counting chamber using phase contrast microscopy. Only distinguishable epithelial cells were counted, usually as individual cells but occasionally in rafts of cells. The mean value was expressed as the number of epithelial cells per mg of protein of isolated cells. Protein was determined by the method of Lowry et al. 9 Duplicate counts varied within 12%. The cell counts were related to the amount of protein in the cell pellet to indicate the protein content of individual epithelial cells. Disaccharidase activity. The isolated epithelial cells were homogenized in chilled distilled water and disaccharidase activity was measured according to the method of Dahlqvist. 10 (One unit of disaccharidase activity equals 1 JLmole of disaccharide hydrolyzed per min.) In similar segments of bowel removed as indicated above, the mucosa was scraped off with a steel spatula, homogenized in iced distilled water, and disaccharidase activity determined. Transport studies. The absorptive capacity of resected intestine was compared with that of control intestine using individual epithelial cells and everted gut sacs. Isolated epithelial cells were incubated under oxygen at 37 C in 50 ml of Krebs-Ringer phosphate buffer containing either 28.7 mm glucose and 0.5 JLc of glucose-l-c1 4 and, in some instances, phlorizin, 10-4 M, or 2 mm leucine and 0.5 p,c of leucine-l_c 14. After 5 min, the incubation mixture was rapidly chilled to 3 C and centrifuged at 5000 rpm for 5 min. The cell pellet was homogenized (1: 10) in iced distilled water. Aliquots of 0.5 ml of homogenized cells and 0.5 ml of incubation medium were placed in counting vials containing 15 ml of phosphor and counted in a liquid scintillation spectrometer. The phosphor was a mixture of 0.4% 2,5- diphenyloxazole, 0.005% 1, 4-bis[2-(5ophenyloxazolyl) 1 benzene, and 5% thixotropic gel in toluene. Quenching, which was corrected by the channels ratio method, was fairly constant for all of the samples. The ratio of counts per min per ml of cells to counts per min per ml of

3 Jarwn.ry 1971 SMALL BOWEL ADAPTATION 71 medium was determined according to the method of Eggermont and Loeb.!! Ratios significantly greater than 1 were interpreted as indicating active transport. The radioactivity present in the cells was extracted and subjected to descending paper chromatography using ethanol and water (7: 3, v Iv) as the solvent. In triplicate experiments, a mean of 67% (range, 50 to 90%) was recovered as and 64% (range, 50 to 81%) recovered as leucine. At a distance of 15 to 20 cm from the pylorous, everted gut sacs, 2 cm in length, were removed from distally resected and control rats. The sac was filled with 0.8 ml of Krebs Ringer phosphate buffer containing 11 mm glucose and 8 mllc of glucose-l-c!4. Glucose transport was measured according to the technique of Crane and Wilson.!2 At the conclusion of each experiment the dry weight of the sac was determined. g l ~ c o s e WHOLE MUCOSA >-. ~ PIO.OOI P<O.05 1-" ~ ~ 1-"- ~ 20 " ~ ~ «a u ~ «c 10 -'::> >c ~ ~ I - ~ u"- «" U J 40 ~ If) «G. " a:.. u ~ :::J 'c 20 If)::> ISOLATED CELLS P(005 n.s. Proximal Distal PrOllimel Distal P(O.OOl n.s. P(0.OO5 n.s. Results Morphology. Five to 6 weeks after proximal or distal resection, the remaining intestinal segment appeared increased in both thickness and diameter. These Prollimal Distal Pr... i... Distal T P<O.05 PIO.05 P=005 PI002 TABLE 1. Intestinal adaptation to small bowel resection Proximal control (11) Proximal remnant (14) P.. Distal control (11) Distal remnant (9).. P.... Mean ± SD. Villous height' Surface epithelium ~ cellsl. 323 ± ± ± ± 0.10 < 0.02 < ± ± ± ± 0.D7 < 0.01 <0.02 TABLE 2. Isolation of rat intestinal epithelial cells Proximal control (17) Proximal remnant (14) P.... Distal control (15) Distal remnant (11) P M'ean ± SD. No. of cells' mg - I protein 1520 ± ± 895 NS 1817 ± ± 884 < 0.05 Proximal Distal Proximal Distal FIG. 1. Disaccharidase activity in intestinal mucosa and isolated epithelial cells. Open bars represent control segments; shaded bars represent remnants after intestinal resection. One standard deviation is shown above the bar. n, number of animals. changes were more striking for the distal remnant. As shown in table 1, the villous height in the proximal remnant increased from 343 ± 67 (mean ± standard deviation) to 423 ± 90!l (P < 0.02). In the distal remnant, the villous height was 491 ± 169 Il compared with 265 ± 45!l in controls (P < 0.01). Related to these changes was a significantly greater number of epithelial cells per unit length of villous (table 1). In proximal villi, epithelia:l cells increased from 0.23 ± 0.06 to 0.34 ± 0.10 cell per!l (P < 0.01) and in distal villi from 0.28 ± 0.06 to 0.35 ± 0.07 cell per!l (P < 0.02).

4 72 WESER AND HERNANDEZ Vol. 60, No.1 ISOLATED CELLS '"..J..J UJ U.. ~ 20! '" Z :l P<O.025 P(0.OO5 P(O.OOI n.s. P(O.02 P(O.OOI Pro)(imal Distal LACTASE ACTIVITY SUCRASE ACTIVITY MALTASE ACTIVITY FIG. 2. Disaccharidase activity per 10 epithelial cells. Open bars represent control segmtnts; shaded bar& represent remnants after intestinal resection. One standard deviation is shown above the bar. n, number of animals. TABLE 3. Ratios of disaccharidase activity in rat intestinal epithelial cells Sucrase to Maltase tc Maltase to lactase lactase sucrase Proximal control (21) 3.6 ± 1.9" 13.8 ± ± 1.6 Proximal remnant (13) 3.5 ± ± ± 3.0 Distal control (21) 2.0 ± ± ± 5.8 Distal remnant (14) 2.6 ± ± ± 1.5 p... <0.02 "Mean ± SD. The number of epithelial cells per mg of protein recovered from the proximal remnant was not significantly different from that recovered from proximal control segments (table 2). On the other hand, 2410 ± 844 epithelial cells per mg of protein were recovered from the distal remnant compared with 1790 ± 595 cells per mg of protein from distal control segments (P < 0.05). Disaccharidase activity. Disaccharidase activity for lactase, sucrase, and maltase was significantly reduced in homogenates of whole mucosa and epithelial cells collected from the proximal remnant (fig. 1). Not only was specific disaccharidase activity (units per g of protein) decreased, but also activity per 10 6 epithelial cells (fig. 2). Lactase and maltase, but not sucrase activity, were also decreased in whole mucosa and epithelial cells recovered from the distal remnant (figs. 1 TABLE 4. Uptake of glucose and leucine by rat intestinal epithelial cells Origin of cells Glucose-l-CI 4 Leucine-l-C U cpm/ml cells/cpm/ml medium Proximal control. 1.5 ± 0.1" 2.2 ± 0.4 (13)b (7) Proximal remnant 1.4 ± ± 0.5 (10) (7) p... NS NS Distal control. 1.4 ± ± 0.7 (13) (7) Distal remnant 1.3 ± ± 0.5 (11) (7) P. NS NS a Mean ± SD. b Numbers in parentheses indicate number of animals. TABLE 5. Uptake of glucose and leucine by rat intestinal epithelial cells Origin of cells Proximal control (9) Proximal remnant (8) p Distal control (10). Distal remnant (9) p "Mean ± SD ± ± 4.3 NS ± ± 5.0 <0.001.m/ld' cells Leucineo 6.9 ± ± 2.7 NS 10.0 ± ± 2.2 <0.05 and 2). The ratios of sucrase to lactase, maltase to lactase, and maltase to sucrase were the same for cells collected both from the proximal remnant and the proximal controls (table 3). The same was true

5 January 1971 SMALL BOWEL ADAPTATION 73 TABLE 6. Glucose transport in rat everted gut sac after distal small bowel resection Glucose transport" Dry weight of sac" "m/min/cm Proximal control (11) ± 0.01 Proximal remnant (11) ± P. "m/min/g mg 2.25 ± ± ± ± 13 <0.01 <0.01 a Mean ± SD. for cells from the distal remnant except for the ratio of maltase to sucrase which reflects the reduced maltase activity without a reduction in sucrase activity. Glucose and leucine transport. The ratio of glucose-1-c 4 counts per min per ml of cell water to counts per min per ml of incubation medium was not greater than 1 when phlorizin was present in the medium. In the absence of phlorizin, ratios greater than 1 were obtained in all cell preparations and indicated a net transport of glucose (table 4). Cells collected from the proximal and distal remnants gave ratios of 1.4 ± 0.2 and 1.3 ± 0.1 respectively, and they were not significantly different from control cells. Similarly, no significant differences were found for leucine ratios in cells collected from either proximal or distal remnants (table 4). However, when uptake of glucose and leucine was determined as a function of the number of cells, the net transport of both substrates was reduced in cells collected from the distal remnant (table 5). Glucose transport in gut sacs. Glucose transport by everted gut sacs prepared from proximal remnants was ± J.Illlole per min per cm and was not significantly different from the control value of ± 0.01 JLmole per min per cm (table 6). The dry weight of gut sacs from proximal remnants was twice as much as controls. Therefore, glucose transport was 1.08 ± 0.45 J.Illlole per min per g for sacs from proximal remnants and significantly less than the control value of 2.25 ± 0.75 JLmole per min per g (P < 0.01). Discussion Previous studies in the rat have shown that following small bowel resection the remaining intestine undergoes an increase in mucosal mass with enlargement of intestinal villi. 3, 5,6,13 This compensatory change is more marked in the ileum than in the jejunum and reaches a maximum 4 weeks after resection. 5, 6 Thereafter, there are no further increments in villous height compared with controls. For this reason we chose a period from 5 to 6 weeks after resection to perform our initial studies. Our results are consistent with these previous observations. Not only are the villi taller in the remaining segments of intestine, but there is an increased number of epithelial cells per unit length of villous, suggesting that the increase in villous height results from epithelial cell hyperplasia. Observations in man have suggested a similar mucosal hyperplasia after extensive small bowel resection, although there was no detectable change in villous 14 SIze. Earlier studies in the rat have demonstrated a continuously accelerated epithelial cell turnover with shortened life spans of individual epithelial cells in remaining intestine after resection. 13, 15 This increase in cell renewal is most marked in the ileum and least in the duodenum. These findings have suggested the existence of a humoral factor, "intestinal epithelial growth hormone," which may be released after resection. 15 In the present study, a greater number of epithelial cells per mg of protein was isolated from the distal remnant. This may be explained by a more rapid cell renewal rate resulting in a greater population of younger cells with reduced protein content. We do not know why similar results statistically were not found for cells obtained from proximal remnants. The total number of available epithelial cells is probably more in distal remnants because of the greater increase

6 74 WESEx AND HERNANDEZ Vol. 60, No.1 in villous height and this may influence the recovery of isolated epithelial cells. It has been shown that after resection the intestinal mucosa may acquire characteristics of metabolic immaturity. Whereas normal rats depend on energy supplied by oxidative phosphorylation in order to transport vitamin A, resected animals utilize energy supplied by anaerobic glycolytic phosphorylation.1 6 This alteration in metabolism is a characteristic of fetal tissue and rapidly dividing immature cells. 17, 18 Our findings of a reduction of disaccharidase activity in whole mucosa and isolated cells from resected animals (except for sucrase in distal remnants) are consistent with a younger cell population. The lack of change for sucrase is at present unexplained. In general, the reduction of disaccharidase activity was the same for all of the enzymes measured with the above-noted exception. The ratio of sucrase to lactase, maltase to lactase, and maltase to sucrase did not change after resection. Despite the general decrease in disaccharidase activity, no changes have been found in the ultrastructure of the epithelial cells and, specifically, the microvilli. 19 Histochemical studies of several other enzymes in the absorptive epithelium after intestinal resection have not shown any changes. 6 However, an increase in staining reactions for nonspecific esterase in the capillaries and macrophages of the lamina propria was found. It has also been reported that intestinal cholinesterase activity is diminished after intestinal resection, and this may play a role in reducing permeability of epithelial cells. I Perfusion studies in rat and man have indicated that the absorption of glucose per unit length of intestinal lumen is increased after small bowel resection. 6, 20 This increase is proportional to the height of the villi. Using everted gut sacs, we found that, when glucose transport was measured as a function of weight of the segment, there was a reduction of transport in segments from distally resected animals. Although the muscular coat of the bowel may thicken slightly in the jejunal segment after distal resection, the epithelial changes are more striking and account for most of the increase in weight. 7 It is possible, therefore, that the reported increase in absorption of glucose after small bowel resection reflects an increase in the number of absorbing epithelial cells which, individually, may have a reduced glucose transport capacity. REFERENCES 1. Loran MR, Althausen TL: Hypertrophy and changes in the cholinesterase activities of the intestine, erythrocytes, and plasma after 'partial' resection of the small intestine of the rat. Arner J Physiol 193: , Bochkov ND: Morphological changes in the jejunum and ileum of rats after wide resection of the small intestine. Bull Exp Bioi Med 47: , Booth CC, Evans KT, Menzies T, et al: Intestinal hypertrophy following partial resection of the small bowel in the rat. Brit J Surg 46: , Nygaard K: Resection of the small intestine in rats. I. Nutritional status and adaptation of fat and protein absorption. Acta Chir Scand 132: , Nygaard K: Resection of the small intestine in rats. III. Morphological changes in the intestinal tract. Acta Chir Scand 133: , Dowling RH, Booth CC: Structural and functional changes following small intestinal resection in the rat. Clin Sci 32: , Nylander G, Olerud S: Intestinal adaptation following extensive resection in the rat. Acta Chir Scand 123:51-56, Perris AD: Isolation of the epithelial cells of the rat small intestine. Canad J Biochem 44: , Lowry OH, Rosebrough NJ, Farr AL, et al.: Protein measurement with the Folin phenol reagent. J Bioi Chern 193: , Dahlqvist A: Method for assay of intestinal disaccharidases. Anal Biochem 7:18-25, Eggermont G, Loeb H: Glucose-galactose intolerance. Lancet 2: , Crane RK, Wilson TH: In vitro method for the study of the rate of intestinal absorption of sugars. J Appl PhysioI12: , Loran MR, Althausen TL: Cellular proliferation of intestinal epithelia in the rat two months after partial resection of the ileum. J Biophys Biochem Cytol 7: , 1960

7 January 1971 SMALL BOWEL ADAPTATION Porus RL: Epithelial hyperplasia following massive small bowel resection in man. Gastroenterology 48: , Loran MR, Crocker TT: Population dynamics of intestinal epithelia in the rat two months after partial resection of the ileum. J Cell Bioi 19: , Loran MR, Althausen TL: Transport of vitamin A in vitro across normal isolated rat intestine and intestine subject to 'partial' resection. Arner J Physiol 197: , Blecher M, White AJ: Effects of various steroids and metabolic inhibitors on the incorporation of glycine-2-c 14 into total proteins and nucleic acids of normal and malignant lymphocytes in vitro. J Bioi Chern 233: , Ville CA, Hagerman DO: Effect of oxygen deprivation on the metabolism of fetal and adult tissues. Arner J Physiol 194: , Skala MD, Konradova V: Hypertrophy of the small intestine after its partial resection in the rat: ultrastructure of the intestinal epithelium. Arner J Dig Dis 14: , Dowling RH, Booth CC: Functional compensation after small bowel resection in man. Lancet 2: , 1966