The Number of Villi in Rat's Jejunum and Ileum: Effect of Normal Growth, Partial Enterectomy, and Tube Feeding

J. Anat. (1972). 111, 2, pp. 283-291 283 With 6 figures Printed in Great Britain

The number of villi in rat's jejunum and ileum: effect of normal growth, partial enterectomy, and tube feeding

J. M. FORRESTER Department of Physiology, Edinburgh University (Accepted 8 January 1972)

INTRODUCTION The villi of the rat small intestine are covered by enterocytes which have a life-span, from their time of origin in the crypts until shedding at the villus tip, of only about one and a half days (Leblond & Stevens, 1948; Bertalanffy, 1960). Their shape varies from one part of the small intestine to another, and even adjacent villi may differ strikingly. In view of these features suggesting a rapidly changing scene, this paper describes a procedure for enumerating the villi in rat jejunum and ileum, and ex- amines the stability of the total number during normal growth, after partial enterectomy, and after tube feeding.

METHODS Locally bred Wistar male rats were used. They were fed on a standard pelleted rat food manufactured in Edinburgh. They always had tap water ad libitum. Enumeration procedure. Rats were killed by inhalation of chloroform in the morn- ing. The position of the suspensory ligament was marked on the small intestine where a band of connective tissue is attached to the intestine at the duodenojejunal junction. Then the small intestine was removed from pylorus to ileocolic valve by gentle traction, and washed through with cold saline (NaCl 0-9 %, w/v). It was weighed, and after removal of the duodenum, was laid in a trough and perfused with Bouin's solution at an outlet pressure of 20 cm of solution for at least 20 minutes (Hromadkova & Skala, 1969). Perfusion rate was of the order of 5 ml/minute. The addition of a stain such as Brilliant Scarlet Red to the solution makes the villi easier to see, but is not essential. The partly fixed intestine was then suspended vertically. Its own weight stretched it less than before fixation; control experiments on portions of the upper part indicated that the length while suspended did not differ from the length prepared horizontally on glass by more than 2 %, and this difference was not considered further. The preparation thus suspended vertically was opened by a single longitudinal cut, and short lengths about 1 cm long were cut at evenly spaced intervals. They were washed gently to remove debris and spread on coverslips with the villous side down; to assist adhesion, silicone fluid MS 200/60,000 cs (Hopkin and Williams, Chadwell Heath, Essex) was used as a mountant. On this the pieces took up their fixed length. They were liable to dry and required prompt examination. Since the density of villi is not uniform around the original circumference of the 284 J. M. FORRESTER gut, a 0-2 cm band was counted right across these opened preparations. Enumera- tion is easy at the lower end of the small intestine, but towards the upper end the pattern is more difficult to discriminate into single units; villi may be wide and sinuous and may overlap each other. The duodenum was excluded for this reason. Dis- crimination was best achieved by using binocular vision through a dissecting microscope, and transmitted light. Counting was done by placing a graticule as close to the villous surface as possible. A surface-ruled graticule (Graticules Ltd., London) lay on the top of the coverslip, ruled surface down, separated from the villi below the coverslip only by the thickness of the slip itself. The view thus obtained of the villi through graticule and coverslip is shown in Fig. 1 (inset). Parallax. Since the villi and graticule were not precisely in the same plane, the area counted differed from that represented by the graticule. The difference was assessed photographically. With the dissecting microscope at its usual working distance and magnification, photographs were made through it of the graticule in two positions one higher than the other by the thickness of a coverslip. The negative images were measured under a travelling microscope and could not be distinguished; it was concluded that the parallax error was less than 1 % linear, or, since area was in question, 2 % in terms of area; it was not further considered. Computation of the totalnumber. From the 14 counts made in each small intestine, the total number was calculated in the way shown in Fig. 1. In this figure the vertical axis represents the number of villi actually counted in each 0-2 cm length of one intestine; each number multiplied by 5 thus gives the number per cm length. The horizontal axis represents the length along the intestine in cm. The total count can be calculated as the area under the plotted line connecting the counts, multiplied by 5 since the count is not over a full cm length but only over a 0-2 cm length. The area is summed as a set of trapezia, which is conveniently done by digital computer. At the upper and lower ends the count was made not right at the end but usually 2 cm away from it, to avoid cannulation damage, so the plotted line is extended horizontally to the end. This procedure introduces a small exaggeration of the total count, because at both ends the number of villi per length is usually falling. Reliability of computation of total number. Six intestines were counted in duplicate, using two sets of separate but closely adjoining pieces. The totals showed a mean difference of *11 % (range 0-02-2 1%). Computation ofarea. An estimate of the area of the serosa is obtainable by measur- ing the width of the fixed preparations at the 14 sites of counting, and then calculat- ing the total area by a procedure analogous to that for total number of villi. Enterectomy. Removal of part of the jejunum was carried out in five rats under pentobarbital anaesthesia, by methods similar to those described by Lambert (1965). The rats were killed between 18 and 30 days later. Tube feeding. Rats were tube fed as described by Farris & Griffith (1962) twice a day, with an interval of at least 6 hours between feeds. The feed consisted of olive oil 4 ml (3 5 ml on the first day) plus 4 ml (3 5 ml on the first day) of a mixture of Com- plan (Glaxo Ltd., Greenford, Middlesex) and glucose. This mixture comprised Com- plan 600 g, glucose 250 g and water 1000 ml, which gave a volume of 1580 ml. The rats still had access to their normal pelleted food, and indeed consumed 50-60 % of their normal intake of this during the first 24 hours of tube feeding, and thereafter Rat intestinal villi 285

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Fig. 1. The villus count at various points along one rat small intestine. Inset: villi as seen for counting. Graticule: 0 5 mm.

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3 0 +t , @aS 2 - .__W _- - - - - ^ 3 >1 2 4 6 8 10 12 0 l l l l l 4 8 12 16 20 24 Feeds Fig. 2. Time course of changes in rat small intestine during tube feeding. Broken line represents control level. Vertical bars represent standard errors (single observations are also shown, at 9 and 12 days). ~~~~~~~Days 286 J. M. FORRESTER much less. The tube feed given did not contain enough of every known nutritional requirement for rats (Warner, 1962) and included about 6-2 times as much fat as the normal daily intake of pelleted food intake by the rats. Preliminary experiments indicated that nearly all (95 Go) of the 7-7 g of fat given daily was being absorbed by the second and subsequent days of tube feeding. No steps were taken to avoid coprophagy (Barnes, Fiala, McGhee & Brown, 1957) and so it is not known whether one or more cycles ofintestinal transit were required for this high rate of fat absorption. Rate ofgrowth of small intestine during the tube feeding. In a preliminary experi- ment, wet weight and dry weight (48 hours at 75 °C) of small intestine were measured in groups of six rats at various stages in tube feeding, in order to estimate the rate of growth of the small intestine. Groups were similar in body weight: means ranged from 260 to 272 g, with standard errors of < 6 g. Control groups of rats ate normal pelleted food, except the night before killing, when they each received one tube feed of 3.5 ml olive oil plus 3 5 ml Complan mixture; on the night before killing, neither the control nor the experimental rats were allowed access to pelleted food. The results appear in Fig. 2 and indicate that after 6 days (i.e. 12 tube feeds) wet weight of small intestine had increased by over 400 and dry weight by nearly 50 %. Serosal area is not known, since dry weight was to be measured and so the intestine could not be fixed. The increase is rapid, comparable to the increase in weight of the remaining kidney after unilateral nephrectomy (Jackson & Levine, 1928; Mason & Ewald, 1965), and can be attributed to the change in time course and rate of food intake (Fatbry, 1969) together with the high fat load. Ten or twelve tube feeds (5-6 days) were used when studying the number of villi.

RESULTS Normal growth. The number of villi in the jejunum and ileum (taken together) of 34 normal rats of various body weights is shown in Fig. 3. The mean number is 127600. There is no evidence of an increase in the number with increasing body weight, and linear regression analysis shows no correlation between the two. It might be supposed that the number shows some tendency to decline at the lowest weights examined. But in these very young rats the villi are still finger-shaped (Verzar & McDougall, 1936) and not of the wider form found in the adult; it looked probable that some overlapping was occurring, obstructing precise counting and causing some shorter villi to be missed. Partial enterectomy. The number of villi in the portion of jejunum examined, and the number found remaining at subsequent killing, are shown in Fig. 4. It is apparent that there is no evidence of any increase in the number of villi. Yet in four of the five rats the remaining part of the small intestine had probably increased markedly in serosal area. From observations on normal rats (Fig. 5) it is possible to determine the normal relation between body weight and serosal area of the small intestine. Both were plotted logarithmically, since this produces a better fit for the regression line (see Huxley, 1932; Gould, 1966). From estimates thus produced of the area of small intestine remaining after partial enterectomy, and from the area measured at death 18-30 days later, the values in Table 1 were derived, and suggest that the remainder changed in serosal area by between minus 6 % and plus 670. Rat intestinal villi 287 a 150000 -.*- ..'--+2 S.D.

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I 0I I 0 I 100 200 300 400 500 600 Body weight (g) Fig. 3. Total number of villi in jejunum plus ileum of 34 normal male rats of various body weights.

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0' 200 250 300 350 Body weight (g) Fig. 4. Number of villi excised (upper segment of each column) and found at death (lower segment) in five rats of the body weights shown. Mean ± 2 S.D. for normal rats is shown, from Fig. 3. Numbers within columns are rat numbers in Table 1.

Tube feeding. The results are shown in Table 2. The tube feeding produced no significant change in the number of villi, although there was a significant (P < 0-05) 15 % increase in serosal area. There was also a highly significant (P < 0001) 13 % increase in the fixed length. This change raises the possibility of deciding whether the increase in length is selective and occurs predominantly in one part of the small intestine rather than in another. But calculation of the percentage of total villi found in each segment failed to show any significant differences, suggesting that the elonga- tion is uniform along the length of the intestine. I9 A NA II I 288 J. M. FORRESTER 250 _ 200 .* / * 14 0

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60 60 1 I 60 1 00 300 500 Body weight (g) Fig. 5. Relation between serosal area of small intestine and body weight in 27 normal male rats. Logarithmic scales. The calculated regression line is shown. Table 1. Changes in serosal area of remainder after partial enterectomy Rat number 1 2 3 4 5 Time between partial enter- 18 23 30 30 30 ectomy and killing (days) Serosal area excised (cm2) 27 36 34 37 35 Calculated remainder (cm2) 110 118 115 129 122 Area at killing (cm2) 167 171 193 122 149 Increase (M) 51 46 67 -6 22

Table 2. Changes after tube feeding (means and standard errors) Controls Tube fed Change (n= 6) (n= 6) (%) P Body weight (g) 264-8 3-3 262-2 + 1-7 NS Fixed length of jejunum+ 108±2-6 123±1-4 +13 < 0001 ileum (cm) Serosal area (cm2) 160±8 3 183±3 3 +15 < 0-05 Number ofvilli 129000±3000 135000±7000 - NS

DISCUSSION The reliability of the measurement procedure indicates that individual deviations from the mean count of 127 600 are largely real and not simply due to errors of measurement. The mean count is comparable with the counts of Clarke (1971), who found 146000 in the adult rat, since he was able to include the duodenum in his enumeration. Clarke gave evidence of a 7 % lower count in rats weighing 20-40 g. The smaller .Rat intestinal villi 289 30

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0 100 200 300 400 Body weight (g) Fig. 6. Food intake of normal male rats. Values derived from means for two litters as they grew. Line drawn by eye. rats here examined (about 60 g body weight) gave counts not significantly different from those of heavier rats. At the other end of life, no evidence appears here of any decline with increasing age, but probably only about half of the natural life-span has been examined. Evidently the full normal complement of villi is laid down in the rat before 60 g body weight is reached, at about 4 weeks of postnatal life. Villi first appear at 20 days gestation (Kameraad, 1942), and are present at birth in the colon as well as in the small intestine, but after the first 4 weeks of life the number in the small intestine hardly changes over the wide span of body weights examined. Body weight increases perhaps tenfold after thefirstmonth, but the work of the intestine does not increase in proportion, since food intake only rises by a factor of about two and a half times (Fig. 6) up to a body weight of over 400 g, and is probably steady above that weight. Each villus is covered by cells originating from (on average) about 15 crypts, and the constant number of villi might depend on a constant number of crypts. But the number of crypts per villus appears to change through life (Clarke, 1971), which makes this hypothesis untenable. Similarly, the constant number might depend on a constant pattern of blood or lymph vessels. If this is so, then it is understandable that the finger-like villi of the young rat are not superseded by new shapes differently distributed, but rather enlarge laterally to become the blade-like or even convoluted villi of the adult. Ferguson, Maxwell & Carr (1969) described a similar change in human babies. But Miller et al. (1970) believe from their findings on the villous vascular pattern that two leaf-like villi can fuse. The present results suggest that such a process is not numerically important during normal growth, unless perhaps villi fuse and new villi are formed in a fashion so well-balanced that the number present at any age remains fairly constant. The situation resembles that in the rat kidney, where the total number of glomeruli reaches a plateau at about 100 days of postnatal life and maintains it to 350 days 19-2 290 J. M. FORRESTER (Arataki, 1926a). It is clear from Arataki's report that the plateau would have been reached earlier if morphologically immature (but identifiable) glomeruli had been included by him. Both Arataki (1926a) and Moore & Hellman (1930) detected a loss of glomeruli in the ageing rat, above 350 days old. But the general growth of their rats had little resemblance to that of rats under modern conditions; it was much slower, and after 350 days of age their animals actually lost a considerable amount of weight, a phenomenon now unusual (McCay, Crowell & Maynard, 1935; Dunn, Murphy & Rockland, 1947). The senile loss of glomeruli they found may in fact have been a consequence of indifferent nutrition rather than of old age. Tube feeding and partial enterectomy do not appear to affect the number of intestinal villi. Flint (1912) came to the same conclusion about partial enterectomy from his observations on dogs. Similarly, Arataki (1926b) and Moore & Hellman (1930) found that unilateral nephrectomy did not provoke new formation of glomeruli in the remaining kidney. But it is probable that in one situation a rat can produce a new villi: when a part of the intestinal mucosa alone has been removed. Such new growth of villi after mucosal damage has been demonstrated in the small intestine of the cat by Florey & Harding (1935) and McMinn & Mitchell (1954). But there is no information on how the numbers of new crypts and villi that re-form in a denuded mucosal area may compare with the normal number in a similar area.

SUMMARY A procedure for enumerating the number of villi in the male rat's small intestine (excluding duodenum) is described. The mean number of 127 600 appears to remain unchanged as body weight rises from 60 g to over 600 g. After partial enterectomy, the remaining villi do not become more numerous. Tube feeding causes rapid growth of the small intestine, but no increase in the number of villi. Professor W. E. Watson gave constant encouragement; while on leave, Dr J. Hadjiminas, Reader in Physiology at Athens University gave help which included performing the partial enterectomies; and Professor Alan Muir provided helpful criticism. REFERENCES ARATAKI, M. (1926a). On the postnatal growth of the kidney, with special reference to the number and size of the glomeruli (albino rat). American Journal ofAnatomy 36, 399-436. ARATAKI, M. (1926b). Experimental researches on the compensatory enlargement of the surviving kidney after unilateral nephrectomy. American Journal of Anatomy 36, 437-450. BARNES, R. H., FIALA, G., McGHEE, B. & BROWN, A. (1957). Prevention of coprophagy in the rat. Journal ofNutrition 63, 489-498. BERTALANFFY, F. D. (1960). Mitotic rates and renewal times of the digestive tract epithelia in the rat. Acta anatomica 40, 130-148. CLARKE, R. M. (1971). Does the number of intestinal villi grow with the rat? Journal ofAnatomy 109, 352. DUNN, M. S., MURPHY, E. A. & ROCKLAND, L. B. (1947). Optimal growth of the rat. Physiological Reviews 27, 72-94. FABRY, P. (1969). Feeding Pattern and Nutritional Adaptations. London: Butterworth. FARRIS, E. J. & GRIFFITH, J. Q. (Eds.) (1962). The Rat in Laboratory Investigation, 2nd edn. New York: Hafner. FERGUSON, A., MAXWELL, J. D. & CARR, K. (1969). Progressive changes in the small intestinal villus pattern with increasing length of gestation. Journal ofPathology 99, 87-91. Rat intestinal villi 291 FLINT, J. M. (1912). The effect of extensive resections of the small intestine. Bulletin of Johns Hopkins Hospital 23, 127-143. FLOREY, H. W. & HARDING, H. E. (1935). Healing of artificial defects of the duodenal mucosa. Journal of Pathology and Bacteriology 40, 211-218. GOULD, S. J. (1966). Allometry and size in ontogeny and phylogeny. Biological Reviews 41, 587-640. HROMA'DKOVAk, V. & SKALA, 1. (1969). Factors influencing the assessment of size of the mucosal surface and length of the small intestine in rats. Digestion 1, 149-158. HUXLEY, J. S. (1932). Problems of Relative Growth. New York: Dial Press. JACKSON, C. M. & LEVINE, N. M. (1928). Rate and character of compensatory renal hypertrophy after unilateral nephrectomy in young albino rats. Anatomical Record 41, 323-333. KAMERAAD, A. (1942). Development of the G.I. tract of the rat. I. Histogenesis of the epithelium of the stomach, small intestine and pancreas. Journal ofMorphology 70, 323-351. LAMBERT, R. (1965). Surgery of the Digestive System in the Rat (transl. B. Julien). Springfield: Thomas. LEBLOND, C. P. & STEVENS, C. E. (1948). The constant renewal of the intestinal epithelium in the albino rat. Anatomical Record 100, 357-371. MCCAY, C. M., CROMWELL, M. F. & MAYNARD, L. A. (1935). The effect of retarded growth upon the length of life span and upon the ultimate body size. Journal ofNutrition 10, 63-78. MCMINN, R. M. H. & MITCHELL, J. E. (1954). The formation of villi following artificial lesions of the mucosa in the small intestine of the cat. Journal ofAnatomy 88, 99-107. MASON, R. C. & EWALD, B. H. (1965). Studies on compensatory renal hypertrophy. I. Effect of unilateral ureteral ligation and transection. Proceedings of the Society for Experimental Biology and Medicine of New York 120, 210-214. MILLER, D. S., RAHMAN, M. A., TANNER, R., MATHAN, V. I. & BAKER, S. J. (1970). The vascular archi- tecture of the different forms of small intestinal villi in the rat. Scandinavian Journal ofGastroenterology 4, 477-482. MOORE, R. A. & HELLMAN, L. S. (1940). The effect of unilateral nephrectomy on the senile atrophy of the kidney in the white rat. Journal ofExperimental Medicine 51, 51-57. VERZAR, F. & MCDOUGALL, E. J. (1936). Absorption from the Intestine. London: Longman. WARNER, R. G. (1962). In Nutrient Requirements ofLaboratory Animals. National Academy of Sciences, New York: National Research Council publication 990.