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 enter- ectomy, 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 indi- cated that the length while suspended did not differ from the length prepared hori- zontally 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 micros- cope, 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 285 400 *9..fifi0iAl bO c cu iuk.. E u 200 F- " C-L ' ^' t ^ ^ 1M..-....I s~~~~~~~~A Jejunum IleumI 0 L 0 41 82 cm Fig. 1. The villus count at various points along one rat small intestine. Inset: villi as seen for counting. Graticule: 0 5 mm. 15 r 0 9*1 bO 4 t I -c lo k .5 3: 5 - - 0 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.