Original Papers Digestion 19: 277-283 (1979)
Effect of Chronic Ethanol Consumption on Mucosal Morphology and Mitotic Index in the Rat Small Intestine1 S. Zucoloto and M.A. Rossi Department of Pathology, Laboratory of Experimental Pathology, Medical School of Ribeirao Preto University of S3o Paulo, RibeiriSo Preto, Sao Paulo
Key Words. Ethanol • Intestinal mucosa • Mitosis • Rat Abstract. Rats were maintained 16 weeks on a well-balanced semisynthetic solid diet supple
In an earlier study, Baraona etal. (1), work ing with an experimental model in which mature albino rats were given ethanol added into a totally liquid nutritionally adequate diet for a relatively short period (3 - 4 weeks), found shorter jejunal villi with hypoplasia of absorp tive cells, reduction of jejunal enzyme activities, and increased cellularity and number of mitosis
1 This work is part of the Doctor Thesis of Sergio Zucoloto presented to the Medical School of Ribeirao Preto in June 1977.
in the jejunal and ileal crypts of ethanol-fed rats as compared to pair-fed controls. As the villus changes were less pronounced in the ileum than in the jejunum, they suggested that the intes tinal changes could result from topical effect of ethanol. On the other hand, it is known that luminal contents play an important role in the control of mucosal morphology, epithelial replacement, and function of the intestine, either directly through chemical, physical, and mechanical actions of foodstuffs, or indirectly by way of different mechanisms, such as altered bacterial
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 6:01:21 AM
mented with ethanol which comprised 35% of total calories. A control group was pair-fed the same basic diet with sucrose replacing ethanol isocalorically. Striking changes in mucosal morphology and mitotic index were observed in the jejunum and ileum of ethanol-fed rats in comparison to pair-fed controls. Furthermore, it is significant that these changes were more pronounced in the ileum than in the jejunum. Since ethanol is almost completely absorbed in the stomach and upper intestine, under the conditions of this study, we propose that, apart from a possible topical toxic effect of ethanol, there appear to be other separate possible causes of the extensive small intestinal changes found in ethanol-fed rats. The first is that the changes are due to injurious effects of blood-borne ethanol; secondly, the changes could be a functional adaptation due to altered luminal nutrition as a consequence of the introduction of ethanol in the diet.
flora or altered release of local or systemic hormones (11). In view of these facts, we planned to inves tigate the effect of a prolonged period of etha nol consumption on mucosal morphology and mitotic index in the small intestine of otherwise well-nourished rats fed a nutritious solid diet supplemented with ethanol added to the drink ing fluid, thus mimicking the conditions under which man consumes ethanol. Results of an ultrastructural investigation on this material has been published previously (12).
Materials and Methods Male Wistar albino rats, weighing an average of 52 g, were obtained from the breeding colony at the Medical School of Ribeirao Preto. They were assigned randomly into two groups: group 1 (ethanol diet) 15 rats, and group 2 (pair-fed controls) - 15 rats. The animals were individually housed in wire cages with raised bottoms and fed solid food in stainless steel feeding dishes and liquid in Richter graduated drinking tubes. They were weighed thrice weekly and their dietary consumption recorded daily. All rats were fed on a well-balanced semisynthetic diet, which composi tion in weight was casein 23%, soybean oil 15%, su crose 24%, dextrose 18%, agar 4%, vitamin mixture (Vitamin Diet Fortification Mixture, Nutritional Bio chemicals Corporation, Cleveland, Ohio) 10%, salt mixture (Association of Official and Agricultural Chemists, Washington, D.C.) 4%, choline 1.8%, and cystein 0.2% (1 g of diet provides 4.32 kcal). The rats in group 1 received the basic solid diet and a solution of 32% (v/v) ethanol and 25% (w/v) sucrose in water (1 ml of solution provides 2.81 kcal) ad libitum, ac cording to Porta and Gomez-Dumm (7). The animals in group 2 were pair-fed with those in group 1 and received equal amounts of the basic diet and the same volume of water plus sucrose isocaloric with the etha nol-sucrose solution consumed by the ethanol rats. Sucrose was added to the water in the form of a 25% (w/v) solution (1 ml of solution provides 1 kcal) and also to the solid diet, in order to increase palatability and intake. Caloric percentages were calculated from weight percentages based on standard physiological
Zucoloto/Rossi
fuel values, i.e., 4 kcal/g carbohydrate or protein, 9 kcal/g fat, and 7.1 kcal/g ethanol. After 16 weeks on test the rats were sacrificed in light ether anesthesia by exsanguination from the aor ta, between 3 and 5 p.m. Small segments (1.5-2.5 cm) of proximal jejunum and distal ileum were rapidly removed, 2 -3 cm distal to the ligament of Treitz, and 2- 3 cm proximal to the ileocecal valve, respectively. These segments were placed serosal side down on a piece of cardboard, immersed in Bourn's fixative (75 ml of saturated aqueous picric acid, 25 ml of 40% formaldehyde solution, and 5 ml of glacial acetic acid) for 72 h, and embedded in paraffin. The tissues were then longitudinally sectioned at 4 #rm in the plane perpendicular to the serosa, and stained with PAShematoxylin. The histological material was coded so that examination could be done blindy by two investi gators. For the present purpose, 6 rats from both ethanolfed and control groups were selected at random. The following measurements were made using a micro meter scale (14): (A) length of the apex of the villus to the base of the crypt; (B) depth of the crypt; and (C) height of the enterocytes from the middle onethird of the villus. Measurements A and B were made at 80 X magnification and measurement C was made at 200 X magnification. 25 measurements were per formed for each segment from each rat. Other meth ods for the quantitative examination of the jejunal and ileal mucosae were carried out on medium power light microscopic fields (X 320): (a) a 100-point ocular (Integrating eyepiece II, Zeiss, Oberkochen, FRG) was used to estimate the volume fraction of two mucosal components by counting the fraction of all points lying over epithelium and lamina propria (the muscularis mucosa was disregarded); and (b) a 15-line ocular (Wild Heerbrugg Ltd., Heerbrugg, Switzerland) was employed to determine an index related to the surface-to-volume of the mucosa (s/v) by counting the number of times the lines cut the mucosal surface (s) and the number of end points of the lines lying over the mucosa (v) (4, 18). At least 12 fields of jejunum and ileum from each rat were examined. Counting of epithelial cells nuclei visualized on both sides of villi and crypts were performed in at least 20 properly oriented villi and crypts of jejunum and ileum from each animal. The mitotic index was estimated in the same material counting the mean percent of cells in mitosis (2). Dividing (cells seen in mid- and late prophase, metaphase, anaphase and telophase) and non-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 6:01:21 AM
278
279
Ethanol on Rat Small Intestine
Table I. Average caloric intakes, final body weights, and growth rates of ethanol-fed and pair-fed control rats (mean ± SEM) Group
Rats Caloric intake, kcal/day/rat carbo hydrate
fat
Final body weight g
Growth rate g/day/rat
protein
ethanol
total
22.4 ± 0.57 (34.9)
64.3 ± 1.78 290.3 ± 13.9 1.99 t 0.10
Ethanol
15
26.4 ± 0.79 9.2 ± 0.47 (41.0) (14.3)
6.3 ± 0.32 (9.8)
Pair-fed
15
48.8 ± 1.24 9.2 ± 0.47 (75.9) (14.3)
6.3 ± 0.32 (9.8)
64.3 ± 1.78
280.6 ± 8.23 1.92 ±0.60
dividing cells, to a total of 1,500 cells/segment, were counted differentially. All these measurements were made where crypt-villus units were cut longitudinally. Representative fragments from the median lobe of the liver were frozen and sections cut and stained with Sudan III to demonstrate lipid. Additional fragments were processed for conventional light microscopy. Statistical analysis of the results was made using Student’s paired t test (13). Data are presented as mean ± SEM.
Results
Only 1 animal died in the first week of the experimental period from pneumonia, being im mediately replaced. Rats fed ethanol remained in good health, with no signs of nutritional deficiencies. The average daily consumption of calories as carbohydrate, fat, protein, and ethanol for both groups are given in table 1. The percentage pro portions of dietary caloric components in the final regimen (basic diet plus ethanol-sucrose solution) consumed by rats of the ethanol group during the experimental period were as follows: ethanol 35%, carbohydrate 41%, fat 14%, and protein 10%. They consumed 11.03 g ethanol/kg body weight/day. The composition
of the diet of the pair-fed control group was the same, except that the ethanol-derived calories were replaced by sucrose. The lipid content was relatively low and the protein level probable adequate. The average daily intake of pro tein/100 g of body weight by rats from both groups was 0.53. The protein/kcal ratio (mg of protein/kcal) was 24.5 for both ethanol-fed and pair-fed control group, which is above the mini mum of 22 (17). The evaluated consumption of vitamins, minerals, and lipotropes was more than the recommended daily allowances. The lipotropic value of the final regimens (expressed in mg choline/100 kcal) was 184. Further, dur ing the experimental period all rats grew well, at the same growth (1.92 + 0.06 g/day/rat in the ethanol fed rats and 1.99 ± 0.10 g/day/rat in the pair-fed controls) (table I). They gained weight continuously and no difference was found at any time between weights of rats in group 1 and rats in group 2. With relation on the morphologic studies our results showed that: (l)T h e mean villus length and the mean crypt depth of the jejunum were found to be unaltered by chronic ethanol consumption (479.9 ± 25.2 and 205.5 ± 13.8 jam, respective-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 6:01:21 AM
The percentage proportions of dietary caloric components in each group are given in parentheses.
Zucoloto/Rossi
280
Table II. Effect of chronic ethanol consumption on villus length, crypt depth and epithelial cell count (mean ± SEM) Villus length
Crypt epithelial cell count
depth
Mm
Mm
epithelial cell count
Jejunum Ethanol-fed Pair-fed controls Significance
479.9 ± 25.2 519.1 ± 33.1 NS
134.2 * 6.1 140.6 ± 2.9 NS
205.5 ± 13.8 198.9 ± 7.7 NS
62.2 ± 1.3 63.2 ± 0.8 NS
Ileum Ethanol-fed Pair-fed controls Significance
304.9 ± 16.4 355.0 ± 28.6 p
Effect of Chronic Ethanol Consumption on Mucosal Morphology and Mitotic Index in the Rat Small Intestine1 S. Zucoloto and M.A. Rossi Department of Pathology, Laboratory of Experimental Pathology, Medical School of Ribeirao Preto University of S3o Paulo, RibeiriSo Preto, Sao Paulo
Key Words. Ethanol • Intestinal mucosa • Mitosis • Rat Abstract. Rats were maintained 16 weeks on a well-balanced semisynthetic solid diet supple
In an earlier study, Baraona etal. (1), work ing with an experimental model in which mature albino rats were given ethanol added into a totally liquid nutritionally adequate diet for a relatively short period (3 - 4 weeks), found shorter jejunal villi with hypoplasia of absorp tive cells, reduction of jejunal enzyme activities, and increased cellularity and number of mitosis
1 This work is part of the Doctor Thesis of Sergio Zucoloto presented to the Medical School of Ribeirao Preto in June 1977.
in the jejunal and ileal crypts of ethanol-fed rats as compared to pair-fed controls. As the villus changes were less pronounced in the ileum than in the jejunum, they suggested that the intes tinal changes could result from topical effect of ethanol. On the other hand, it is known that luminal contents play an important role in the control of mucosal morphology, epithelial replacement, and function of the intestine, either directly through chemical, physical, and mechanical actions of foodstuffs, or indirectly by way of different mechanisms, such as altered bacterial
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 6:01:21 AM
mented with ethanol which comprised 35% of total calories. A control group was pair-fed the same basic diet with sucrose replacing ethanol isocalorically. Striking changes in mucosal morphology and mitotic index were observed in the jejunum and ileum of ethanol-fed rats in comparison to pair-fed controls. Furthermore, it is significant that these changes were more pronounced in the ileum than in the jejunum. Since ethanol is almost completely absorbed in the stomach and upper intestine, under the conditions of this study, we propose that, apart from a possible topical toxic effect of ethanol, there appear to be other separate possible causes of the extensive small intestinal changes found in ethanol-fed rats. The first is that the changes are due to injurious effects of blood-borne ethanol; secondly, the changes could be a functional adaptation due to altered luminal nutrition as a consequence of the introduction of ethanol in the diet.
flora or altered release of local or systemic hormones (11). In view of these facts, we planned to inves tigate the effect of a prolonged period of etha nol consumption on mucosal morphology and mitotic index in the small intestine of otherwise well-nourished rats fed a nutritious solid diet supplemented with ethanol added to the drink ing fluid, thus mimicking the conditions under which man consumes ethanol. Results of an ultrastructural investigation on this material has been published previously (12).
Materials and Methods Male Wistar albino rats, weighing an average of 52 g, were obtained from the breeding colony at the Medical School of Ribeirao Preto. They were assigned randomly into two groups: group 1 (ethanol diet) 15 rats, and group 2 (pair-fed controls) - 15 rats. The animals were individually housed in wire cages with raised bottoms and fed solid food in stainless steel feeding dishes and liquid in Richter graduated drinking tubes. They were weighed thrice weekly and their dietary consumption recorded daily. All rats were fed on a well-balanced semisynthetic diet, which composi tion in weight was casein 23%, soybean oil 15%, su crose 24%, dextrose 18%, agar 4%, vitamin mixture (Vitamin Diet Fortification Mixture, Nutritional Bio chemicals Corporation, Cleveland, Ohio) 10%, salt mixture (Association of Official and Agricultural Chemists, Washington, D.C.) 4%, choline 1.8%, and cystein 0.2% (1 g of diet provides 4.32 kcal). The rats in group 1 received the basic solid diet and a solution of 32% (v/v) ethanol and 25% (w/v) sucrose in water (1 ml of solution provides 2.81 kcal) ad libitum, ac cording to Porta and Gomez-Dumm (7). The animals in group 2 were pair-fed with those in group 1 and received equal amounts of the basic diet and the same volume of water plus sucrose isocaloric with the etha nol-sucrose solution consumed by the ethanol rats. Sucrose was added to the water in the form of a 25% (w/v) solution (1 ml of solution provides 1 kcal) and also to the solid diet, in order to increase palatability and intake. Caloric percentages were calculated from weight percentages based on standard physiological
Zucoloto/Rossi
fuel values, i.e., 4 kcal/g carbohydrate or protein, 9 kcal/g fat, and 7.1 kcal/g ethanol. After 16 weeks on test the rats were sacrificed in light ether anesthesia by exsanguination from the aor ta, between 3 and 5 p.m. Small segments (1.5-2.5 cm) of proximal jejunum and distal ileum were rapidly removed, 2 -3 cm distal to the ligament of Treitz, and 2- 3 cm proximal to the ileocecal valve, respectively. These segments were placed serosal side down on a piece of cardboard, immersed in Bourn's fixative (75 ml of saturated aqueous picric acid, 25 ml of 40% formaldehyde solution, and 5 ml of glacial acetic acid) for 72 h, and embedded in paraffin. The tissues were then longitudinally sectioned at 4 #rm in the plane perpendicular to the serosa, and stained with PAShematoxylin. The histological material was coded so that examination could be done blindy by two investi gators. For the present purpose, 6 rats from both ethanolfed and control groups were selected at random. The following measurements were made using a micro meter scale (14): (A) length of the apex of the villus to the base of the crypt; (B) depth of the crypt; and (C) height of the enterocytes from the middle onethird of the villus. Measurements A and B were made at 80 X magnification and measurement C was made at 200 X magnification. 25 measurements were per formed for each segment from each rat. Other meth ods for the quantitative examination of the jejunal and ileal mucosae were carried out on medium power light microscopic fields (X 320): (a) a 100-point ocular (Integrating eyepiece II, Zeiss, Oberkochen, FRG) was used to estimate the volume fraction of two mucosal components by counting the fraction of all points lying over epithelium and lamina propria (the muscularis mucosa was disregarded); and (b) a 15-line ocular (Wild Heerbrugg Ltd., Heerbrugg, Switzerland) was employed to determine an index related to the surface-to-volume of the mucosa (s/v) by counting the number of times the lines cut the mucosal surface (s) and the number of end points of the lines lying over the mucosa (v) (4, 18). At least 12 fields of jejunum and ileum from each rat were examined. Counting of epithelial cells nuclei visualized on both sides of villi and crypts were performed in at least 20 properly oriented villi and crypts of jejunum and ileum from each animal. The mitotic index was estimated in the same material counting the mean percent of cells in mitosis (2). Dividing (cells seen in mid- and late prophase, metaphase, anaphase and telophase) and non-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 6:01:21 AM
278
279
Ethanol on Rat Small Intestine
Table I. Average caloric intakes, final body weights, and growth rates of ethanol-fed and pair-fed control rats (mean ± SEM) Group
Rats Caloric intake, kcal/day/rat carbo hydrate
fat
Final body weight g
Growth rate g/day/rat
protein
ethanol
total
22.4 ± 0.57 (34.9)
64.3 ± 1.78 290.3 ± 13.9 1.99 t 0.10
Ethanol
15
26.4 ± 0.79 9.2 ± 0.47 (41.0) (14.3)
6.3 ± 0.32 (9.8)
Pair-fed
15
48.8 ± 1.24 9.2 ± 0.47 (75.9) (14.3)
6.3 ± 0.32 (9.8)
64.3 ± 1.78
280.6 ± 8.23 1.92 ±0.60
dividing cells, to a total of 1,500 cells/segment, were counted differentially. All these measurements were made where crypt-villus units were cut longitudinally. Representative fragments from the median lobe of the liver were frozen and sections cut and stained with Sudan III to demonstrate lipid. Additional fragments were processed for conventional light microscopy. Statistical analysis of the results was made using Student’s paired t test (13). Data are presented as mean ± SEM.
Results
Only 1 animal died in the first week of the experimental period from pneumonia, being im mediately replaced. Rats fed ethanol remained in good health, with no signs of nutritional deficiencies. The average daily consumption of calories as carbohydrate, fat, protein, and ethanol for both groups are given in table 1. The percentage pro portions of dietary caloric components in the final regimen (basic diet plus ethanol-sucrose solution) consumed by rats of the ethanol group during the experimental period were as follows: ethanol 35%, carbohydrate 41%, fat 14%, and protein 10%. They consumed 11.03 g ethanol/kg body weight/day. The composition
of the diet of the pair-fed control group was the same, except that the ethanol-derived calories were replaced by sucrose. The lipid content was relatively low and the protein level probable adequate. The average daily intake of pro tein/100 g of body weight by rats from both groups was 0.53. The protein/kcal ratio (mg of protein/kcal) was 24.5 for both ethanol-fed and pair-fed control group, which is above the mini mum of 22 (17). The evaluated consumption of vitamins, minerals, and lipotropes was more than the recommended daily allowances. The lipotropic value of the final regimens (expressed in mg choline/100 kcal) was 184. Further, dur ing the experimental period all rats grew well, at the same growth (1.92 + 0.06 g/day/rat in the ethanol fed rats and 1.99 ± 0.10 g/day/rat in the pair-fed controls) (table I). They gained weight continuously and no difference was found at any time between weights of rats in group 1 and rats in group 2. With relation on the morphologic studies our results showed that: (l)T h e mean villus length and the mean crypt depth of the jejunum were found to be unaltered by chronic ethanol consumption (479.9 ± 25.2 and 205.5 ± 13.8 jam, respective-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 6:01:21 AM
The percentage proportions of dietary caloric components in each group are given in parentheses.
Zucoloto/Rossi
280
Table II. Effect of chronic ethanol consumption on villus length, crypt depth and epithelial cell count (mean ± SEM) Villus length
Crypt epithelial cell count
depth
Mm
Mm
epithelial cell count
Jejunum Ethanol-fed Pair-fed controls Significance
479.9 ± 25.2 519.1 ± 33.1 NS
134.2 * 6.1 140.6 ± 2.9 NS
205.5 ± 13.8 198.9 ± 7.7 NS
62.2 ± 1.3 63.2 ± 0.8 NS
Ileum Ethanol-fed Pair-fed controls Significance
304.9 ± 16.4 355.0 ± 28.6 p
