By Louis Gross
Summary: Historically significant British study from 1924 on the pathological lesions appearing in the nervous system and digestive tract of rats fed vitamin deficient diets. This article demonstrates the seriousness and excellence of early vitamin research. From The Journal of Pathology and Bacteriology, 1924. Lee Foundation for Nutritional Research reprint 24.
[ The following is a transcription of the original Archives document. To view or download the original document, click here.]
The Effects of Vitamin Deficient Diet on Rats, with Special Reference to the Motor Functions of the Intestinal Tract In Vivo and In Vitro
In 1914 Keith1 described the macroscopic and microscopic lesions that he found in a number of large intestines removed at operation for conditions of advanced colonic stasis. In 1921 I had the privilege of examining this material and undertook the experimental study that arose out of the anatomical findings. The salient features of these lesions were as follows:
- Accumulation of large cells with indistinct nuclei and numerous brown granules in their cytoplasm in the stroma under the epithelium and above the muscularis mucosae. They are sharply localized to that area, often bathed in lakes of lymph and strictly limited to the colon.
- Distinct pathological lesions often found in Auerbach’s [myenteric] plexus, ranging from inflammatory cell infiltration and necrosis to extreme fibrosis.
- The epithelium is often flattened toward the lumen and sometimes so laden with brown pigment that the nucleus is seen with difficulty. The latter is often vesicular and appears unstained. Here and there the goblet cells are seen to form bladders and occasionally appear to burst into the reticularis—evidently because a tough film that covers their surface prevents their proper discharge into the lumen.
- Occasional fibrosis of the muscular coats and thickening of the submucous connective tissue.
The accumulation of the pigment-bearing cells in the stroma and the pathological condition of Auerbach’s tissue appeared to me very suggestive. Without going into a lengthy discussion on the neurogenic and myogenic theories of intestinal activity, the fact that this is still an unsettled point makes the discovery of the nervous lesion of particular interest. Clark and I2 have recently furnished additional evidence that Auerbach’s plexus is of prime importance in maintaining the tone of the intestine and in initiating and carrying out peristalsis, which is really a complex wave of altered intestinal tone.
In 1919 McCarrison3 published a report on the intestinal lesions of animals fed on certain vitamin-deficient and otherwise unbalanced diets. Among the most marked changes, which included congestive, necrotic, and inflammatory changes in the mucous membrane and atrophy of the myenteron, he found also degeneration in Auerbach’s plexus. He drew attention to these findings and suggested that vitamin-deficient and ill-balanced diets that can produce such lesions may be a possible source of intestinal stasis in the human.
If degenerative changes of Auerbach’s plexus could be produced in such a way, it seemed to me that here was a method of investigation that should yield interesting results both on the physiology of the intestinal activity as well as on the pathology of constipation, for whether or not the pathological conditions in the colons from cases of obstinate constipation that Keith had described resulted from vitamin deficiencies, the occurrence in both of a characteristic and unusual lesion seemed well worth following up.
My experiments were designed to determine whether pure vitamin deficiencies produce an alteration in intestinal activity in rats and also to determine the general anatomical changes resulting from these deficiencies. The latter investigation was necessary because the descriptions of the pathological changes resulting from vitamin deficiencies that appear in the literature are based for the most part on tissues from dead or dying animals and represent extreme conditions often complicated by inanition. Furthermore, the diets largely used have been grossly unbalanced, so that the results in any case cannot be held to be those following pure vitamin deficiencies. My material is from animals that were not moribund and that were fed on vitamin deficient diets [that were] otherwise well balanced.
Before proceeding to the discussion of my experimental results, it will be essential to give a detailed description of my observations on so-called “normal” control animals. It seems to me that sufficient emphasis has not been laid on this point. In most of the literature dealing with functional and particularly anatomical investigations on vitamin deficiencies, it is more or less assumed that normal control animals are in the greater proportion of cases normal, and little attention is paid to the wide individual variations found, especially upon microscopic examination. As will be seen, however, in the description that follows, laboratory animals—particularly albino rats—show extraordinarily high individual variations and morbidity. It is only after this is fully studied and analyzed in a sufficiently large number of cases that the results following experimental diets assume their true significance, and this is usually strikingly different from the conclusions that would be drawn without such a preliminary study on controls.
B. Technique Employed in These Studies
In all I used 217 rats. The general plan was to divide the rats into groups of six to forty-eight, placing three [each] in a cage. The sexes were divided, and an approximately equal number of bucks and does was used in each experiment. At various intervals the rats were killed for isolated intestine tracings, histological material, and adrenaline estimations. Because hyperadrenalinemia was strongly suggested as responsible for many of the signs and symptoms of vitamin B deficiency,4 some of the groups received daily intraperitoneal injections of adrenaline in an attempt to reproduce the lesions said to be characteristic of this deficiency. The dose injected was 1/10 cc of 1/2000 solution of adrenaline chloride (Parke Davis) per 100 g of rat. Other animals were unilaterally adrenalectomized. It was suggested that if any diminution in intestinal activity were found, this might be accounted for on a basis of general muscular atrophy involving the intestinal musculature. Several groups of rats were accordingly given daily intraperitoneal injections of pituitary extract in the hope that this would tend to cause hypertrophy of the bowel. The dose injected was 1/10 cc Parke Davis pituitrin per 100 g of rat.
During the course of the feeding, notes were taken on the general behavior of the animals; the temperatures and weights were determined twice a week under as similar conditions as possible; and the weight of food intake and character of feces were noted.
The diets I employed were well balanced and were made up according to the formulae shown in Table I. Hereafter in this paper, diet deficient in vitamin A will be referred to as A diet, that deficient in vitamin B as B diet, and in vitamin C as C diet. “P diet” contained, in adequate quantity, all the known vitamins.
Table I. Table of the Diets Used, Showing the Proportions of the Ingredients in Parts by Weight.
|A||heated||hardened fat||Marmite||lemon juice|
*All animals received 50 parts starch, 5 parts salt mixture, and 30 parts water.
The butter was mixed with cod liver oil amounting to 2 percent of its bulk. The heated casein was prepared by heating shallow layers of casein in a dry oven for 24 hours at a temperature of 110 centigrade. The extracted casein was prepared by extracting casein with boiling, 95-percent alcohol for 4 hours. Marmite is a commercial preparation of autolyzed yeast. The hardened fat used was hydrogenated cottonseed oil. The salt mixture was as follows, in parts by weight:
|[Mineral] Salt||Amount [part by weight]|
|Sodium acid phosphate||10.41|
Except where the brain was wanted for examination, the rat was killed by a blow on the head with a metal rod. A forelimb was immediately amputated as close to the trunk as possible, and the animal was bled into a clean glass receptacle. The blood could thus be used for various tests.
As general fixative for tissues, I used a 10-percent solution of neutralized formalin in physiological saline. This fixative has many shortcomings, but in view of the large amount of varied material it was necessary to handle, it was chosen for its expediency. For special purposes osmic vapor was used, according to the method of Cramer.5
C. Observations on Forty-Eight “Normal” Control Rats
The rats were purchased from dealers and weighed 125 g or over [each]. It would have been much more advisable to have bred my own rats, but unfortunately neither time nor facility made this possible. A small number of rats used for special experiments, as described in section E, were bred in the laboratory. The animals were under preliminary observation for at least one week, usually three weeks, and if there was no obvious evidence of disease, they were then put under the experimental conditions.
The rats were active, displayed considerable interest in life, especially during the feeding hour, and ate with avidity. Their fur was glossy; the animals felt sleek and supple to the touch, and their dispositions were on the whole good. The average time under observation was eight weeks. Weights and temperatures were taken once a week unless for some special purpose it was deemed advisable to make the readings more frequent. The observations were made as far as possible under the same conditions.
During the course of this work, two rats developed “head-trouble,” which also occurred in several rats on other diets, due to acute suppurative mastoiditis, sometimes bilateral. Occasionally, this went on to meningitis and cerebellar abscess. The symptoms always commenced with the rat keeping its head on one side. In the most advanced condition, the rat kept rolling over and over and making desperate attempts to clutch the cage wall so as to keep itself in one position.
In Figure 1 is seen the temperature chart, made from the compiled averages of twenty unselected rats (ten bucks and ten does) on P diet. [Temperatures for rats on A diet, B diet, and C diet are also shown.] The upper dotted line represents the upper extremes in temperature, and the lower dotted line the lower extremes. There is a great deal of individual variation, some of which no doubt is due to pathological lesions.
Figure 1. [Figure showing average temperatures of rats on each of the experimental diets. See original for data.]
In Figure 2 is seen the weight curve, made from the compiled averages of twenty unselected male rats on P diet. [Weight curve for rats on A diet, B diet, and C diet also shown.] Note that there is a steady increase in weight for the P-diet animals.
Figure 2. [Figure showing growth curves of rats on each of the experimental diets. See original for data.]
With the exception of the two cases of mastoiditis referred to, not one of the forty-eight “normal” rats autopsied showed clinical evidence of morbidity. After the animals had been under observation for the desired length of time, they were killed in the manner already described, and the tissues were generally in the fixative within fifteen minutes after death.
In Table II are shown the gross autopsy findings on the contents of the alimentary tract of these forty-eight rats. I have deliberately gone into some detail in this table in order to demonstrate the great individual variations and also to serve as a baseline for observations that are to follow. It will be seen that the gross anatomical findings in this respect do not differ very widely between the averages of forty-eight unselected cases and the average of sixteen normal rats that showed at autopsy no gross evidence of abnormality in the organs. Two rats showed a thickened cecum, to one of which there was an adhesion of the meso-testis of the same side.
Table II. Gross Autopsy Findings in the Alimentary Tract with Reference to Its Contents, Expressed as Percent of Incidence of Arbitrary Values. [See original for data.]
Thirty-one percent of lungs showed evidence of gross inflammation. The thyroid showed a moderate variation in size but otherwise no gross lesion. The retro-ileocecal glands were markedly enlarged in 7 percent of cases and moderately enlarged in 30 percent. For the rest several livers showed an occasional, small, yellowish nodule, which turned out upon microscopic examination to be a localized granuloma resembling a gumma. An occasional cyst of Taenia crassicollis was also seen. The livers and spleens varied somewhat in size but were otherwise apparently normal. The kidney and heart showed no gross lesions. The adrenals are considered in detail in a separate publication.21 The thymus showed some variation in shape but was on the whole of good size. The intestine of 9 percent of these rats contained the flatworm Hymenolepis murina.
Microscopically, the following organs were examined: thyroid, parathyroid, thymus, heart, lungs, liver, spleen, kidney, adrenal, testis, vesiculae seminales, ovary, retro-ileocecal glands, mesenteric glands, striated muscle, first part of duodenum, mid-jejunum, terminal ileum, ileocecal junction, first part of ascending colon, and descending colon, just above the brim of the pelvis. In all some 2600 slides were examined. Tables III to VIII summarize the results in contrast with the experimental tissues. It is evident that there is hardly a lesion produced in these experimental animals that is not found in some of the normals.
The great variability of the thyroid has been pointed out by many investigators, but it is to be noted that 4 percent of my “normal” rats showed acute inflammatory lesions, and 12 percent showed distinct evidence of some fibrosis (Table III).
Table III. Microscopic Findings in the Thyroid, Expressed as Percent of Incidence. [See original for data.]
In the gross anatomical inspection, 31 percent of rats showed some lung lesion. The microscope, however, revealed that 65 percent of my control rats showed some evidence of inflammation (Table IV). In 39 percent the lesions were evidently healed. The remaining 26 percent showed conditions that ranged from bronchiectatic abscesses to cheesy granulomata. In some the lesions were small and localized, in others lobular, and in several lobar. It is to be noticed that without the microscopic examination, 69 percent of my rats would have been passed as normal insofar as the lungs were concerned.
Table IV. Microscopic Findings in Lungs, Expressed as Percent of Incidence. [See original for data.]
Twenty-three percent of the livers show inflammatory foci (Table V). These varied from slight, focal polymorphonuclear accumulations to degenerative necrotic foci to distinct granulomata. The lesions in the liver gave me the impression of being metastatic embolic infections of the same nature as those that involved the lungs. Whether these were due to retrograde emboli from the lungs or to clumps of bacteria that had passed the systemic circulation and been caught in the liver or, finally, to independent foci from an intestinal lesion, I cannot say. The latter source, however, was suggested very strongly in a number of cases where the liver lesions seemed to bear a relation to the extent of the intestinal granulomatous ulcers that, as will be seen in Table VIII, were found in the ileocecal region of 19 percent of control rats.
Table V. Microscopic Findings in the Liver, Expressed as Percent of Incidence. [See original for data.]
The fixative that I used is particularly bad for the kidney, so it was difficult to define artifact [here] from genuine lesion. Nevertheless, 6 percent of rats showed cloudy swelling, and in 21 percent casts were seen in the collecting tubules; these were usually hyaline (Table VI).
Table VI. Microscopic Findings in the Kidney, Expressed as Percent of Incidence. [See original for data.]
The spleen (Table VII) is a notoriously variable organ, and I have shown elsewhere6 that it is unwise to draw conclusions from its histology unless similar age periods are being compared. Since my rats were more or less of the same age, there [is] some significance in the changes found.
Table VII. Microscopic Findings in the Spleen, Expressed as Percent of Incidence. [See original for data.]
Eighteen percent of the control rats showed a moderate vacuolation of the cells of the pancreas. Five percent of cases showed acute inflammatory infiltrations, and 3 percent sclerosis of the pancreas. In no case was a lesion of the islets of Langerhans found.
The testes of 17 percent of the rats presented here and there tubules that showed faulty spermatogenesis and irregular arrangement of the germinal cell layers. The bulk of the tissue, however, appeared quite normal. In 100 percent of the vesiculae seminales, the lining epithelium was cylindrical. The vesicles were well filled with secretion. In 24 percent of the ovaries, occasional ova showed degenerative changes; the majority of the Graafian follicles, however, were normal.
By far the most marked intercurrent intestinal lesion was seen on the cecal aspect of the ileocecal region (Table VIII). Here, in 61 percent of the cases, collections of inflammatory cells were found in varying grades of severity. In 30 percent this was mild and consisted of localized accumulations of polymorphonuclear leucocytes and plasma cells lying on the muscularis mucosae and immediately beneath it. In 12 percent of cases, this lesion was more severe and extensive, forming in fact a well-marked and quite evident infiltration. From this all grades of extension and severity led, up to conditions of marked ulceration. The base of these ulcers consisted of markedly fibrosed granulomatous tissue covered with a layer of necrotic debris and thickly infiltrated with cells of acute inflammation. This lesion frequently extended through all the coats of the intestine locally. Ulceration occurred in 19 percent of control animals. It is perhaps no coincidence that 61 percent of control animals showed some form of localized cecal inflammation and that 65 percent of the same animals showed some evidence of a lung lesion. The two conditions were usually found in the same rat.
Table VIII. Percent Incidence of Inflammatory Lesions in the Cecal Aspect of the Ileocecal Region. [See original for data.]
Moreover, after some experience the cecal inflammation could be anticipated by inspection of the retro-ileocecal glands. These glands were found enlarged under 75 percent of the ulcers. Besides these ulcers 25 percent of the other inflammatory foci showed enlarged glands. In only 6 percent of cases that showed enlarged retro-ileocecal glands did I fail to find a microscopic inflammatory focus in the cecum. The main cause of the enlargement of the gland is lymph stasis. Although over 20 percent of the glands showed some evidence of inflammation, this was in no way commensurate with the extent of the intestinal lesion.
The remainder of the intestine showed few changes. These are best considered when dealing with the results of vitamin deficiencies.
It may be argued that my control animals are so grossly abnormal that they can hardly serve as a baseline, but, as I have stated earlier, though my rats were by no means of especially selected stock, they represent the sort of animals used in most experiments of the kind now being carried out on the effects of vitamin deficiencies on adult animals. Moreover, I took reasonable precautions to determine clinically whether the rats were abnormal, and it was only at autopsy and upon careful microscopic examination that the lesions described were found.
It is my intention to make a similar statistical examination of adult rats bred under my observation. I have no doubt that the incidence of morbidity will be lower, perhaps considerably lower, but the point that I wish to make is that unless gross and histological examinations are carried out in detail and on a large number of control animals, the experimental results lose much of their validity. Perhaps this point seems to be so obvious as to require no emphasis, but I have found on the whole that the control animals used in a considerable number of experiments quoted in the literature are either of an astonishingly healthy stock or else they have not had as searching an examination as my results indicate to be necessary.
We are now in a position to examine the lesions produced by feeding vitamin deficient diets to rats.
D. Observations on Rats Fed on Vitamin Deficient Diets
Studies on the pathology of vitamin deficiencies have largely been in two specialized directions, namely, rickets and beriberi. As neither of these questions concerns us here, I shall make no attempt to review the enormous literature. Accounts of the pathology based on only a small number of animals are unfortunately of little value, and I must leave out of account those researches on the effect of diets unbalanced in respects other than vitamin content as well as those whose microscopic results were obtained from tissues removed from animals that were brought to the point of death on the experimental diet. This leaves the present field of investigation practically untouched. I shall, however, mention several isolated papers of special interest in relation to the topics under discussion.
The nineteen rats fed on A diet behaved on the whole like normal rats. It was only in the latter weeks of the experiment that they were noticeably affected with “colds” in a number of cases. As a general rule the food intake was fair compared with the normal [rats]. Their average weights (Figure 2) showed a flattening of the growth curve. After the fourth week, there was a distinct drop. The temperature chart (Figure 1) shows no significant difference from the normal. The average length of time during which the rats were on this diet was six weeks.
The thirty-four rats on B diet showed the most marked clinical manifestations of vitamin deficiency. They were rarely kept on this diet longer than six weeks, for it was my object to avoid pronounced disturbances that cause a general metabolic upset with secondary phenomena. The average time on this diet was five weeks. The first thing noticed was the refusal to eat food, which may occur immediately and certainly within a few days. Almost as striking is the loss in weight that commenced soon after the inception of the experiment (Figure 2). This is, however, not directly due to the lowered food intake, for even if the food consumption is increased, as by addition of flavoring agents to the diet, no growth results in the absence of vitamin B (Drummond7).
The temperature suffers a gradual decline and in about three to four weeks is well below the normal (Figure 1). By this time the rat has usually become so thin that its ribs are easily felt. The fur becomes shaggy, the hair standing on end. The rat assumes a rather characteristic pose, sitting with its back hunched up, and the slightest unusual sound causes it to start or jump. If the diet is kept on, the rat develops great weakness of the hind limbs, extreme emaciation, and incoordination. If the balance of the rat is disturbed, as by swinging, and it is then placed on the floor, it rolls over and over helplessly for a time. The clinical picture of vitamin B deficiency in adult rats is thus totally different from that produced by deficiency of vitamin A and, as will be seen, of vitamin C. One of the vitamin B deficient rats developed acute suppurative mastoiditis.
The twenty rats on C diet exhibited clinically no characteristic difference from the normal. Their temperatures were on the whole somewhat higher than normal (Figure 1). The weight curve follows very closely the normal for the first few weeks on the diet; later it flattens out and drops (Figure 2). The average length of time during which the rats were on this diet was seven weeks. Because rats do not display the florid picture of scurvy that is so characteristically shown by guinea pigs on vitamin C deficient diet, it has been held by some observers that rats require no vitamin C in their diet. Harden and Zilva8 as well as Drummond9 are, however, of the opinion that rats that are deprived of vitamin C do not thrive. This I can confirm for adult rats.
The thyroid, heart, kidney, and striated muscle showed more or less the same variability in the three vitamin deficiencies as in the normal. The livers of vitamin B deficient rats decreased in weight, the decrease being fairly proportional to the length of time on the diet. The incidence of small spleens was considerably greater than normal in the three vitamin deficiencies, being greatest in vitamin B deficiency, next in C, and last in A deficiency. The latter diet showed the greatest incidence of large spleens, probably on account of the greater incidence of infection. The thymus showed the most striking change, presenting invariably a marked atrophy in vitamin B deficiency that has already been pointed out by numerous observers.
The lungs showed gross lesions in 63 percent of vitamin A deficient rats, 23 percent of B deficient [rats], and 42 percent of C deficient animals—compared with 31 percent in the controls. As opposed to 9 percent incidence of flatworms (usually Hymenolepis murina) in normal rats, 22 percent of vitamin A deficient rats, 18 percent of B deficient rats, and 30 percent of C deficient rats harbored these parasites in their intestines. Enlarged retro-ileocecal glands were found in 37 percent of control rats, 23 percent of vitamin A deficient rats, 38 percent of B deficient rats, and 20 percent of C deficient rats.
Table II brings out some points in the gross content of the intestinal tract that are of interest in connection with some in vivo experiments on motor function, which will be described below. It is seen that the overdistended stomach so often attributed to vitamin B deficiency is in fact found more frequently in normal rats, as is to be expected from their higher food intake.
Peyer’s patches were less prominent in vitamin B deficient rats; otherwise the small intestine showed on the whole little difference from the normal immediately after death, the possible exception being the somewhat higher incidence of gas in the intestine of vitamin A deficient rats. It is necessary to emphasize the words “immediately after death” because, even in half an hour, fermentation set up in the intestinal contents produces often in vitamin A [deficient rats]—and probably also in B deficient animals—more ballooning than is seen in normal rats. This is probably due to differences in bacterial flora or in thickness of intestinal musculature or in both. As will be shown later, evidence is adduced that suggests a thinning of intestinal muscles.
Two rats on vitamin C deficiency presented thickened ceca. This was also found in one vitamin A deficient rat, where it was the seat of an adhesion to the meso-testis of the same side. Otherwise, with the exception that the cecum of vitamin B deficient rats was never found empty or small, this viscus [sic] showed on the whole little gross difference from the normal. The ascending colon showed a considerable increase in incidence of distension in vitamin B deficient rats and to a lesser extent in vitamin A deficient rats. The descending colon showed in both vitamin A and B deficiency a considerably greater incidence of distension with feces. Fifty percent of control rats and 65 percent of C deficient rats showed a completely empty descending colon. This was empty in only 22 percent of vitamin A deficient rats and in 20 percent of B deficient rats (see page 43 below).
Gross observations on the alimentary tract show, therefore, no striking differences between normal and vitamin deficient rats. When, however, the motor functions are studied, a marked difference is seen.
Microscopic examination showed some increased incidence of cells lying between the alveoli of the thyroid gland in vitamin B deficiency (Table III). The cells are probably thyroid epithelial cells that are not arranged in glandular form. Vitamin B deficiency is also associated more frequently than normal with somewhat larger and more distended alveoli. Otherwise the variations in microscopic appearance of the thyroid gland from rats on the various vitamin deficiencies differ in no essentials from the controls. This is also true for the parathyroids.
Table IV brings out the striking fact that vitamin A deficiency presented in every case some form of lung lesion. Of these 74 percent were active acute conditions. This is in sharp contrast to the other vitamin deficiencies. The microscopic appearances of these lesions were on the whole similar to those already described for the control rats.
The interesting points to be noticed in the liver (Table V) are the great congestion found in vitamin A deficiency and the hyaline appearance of the cells frequently found in vitamin B deficiency. It is possible that the latter appearance is due to the low store of glycogen that P. Eggleton and I have found associated with this condition. There is a slightly higher incidence of inflammatory foci in the livers of vitamin A deficient rats.
The kidneys (Table VI) showed a considerable increased incidence of vacuolation of the convoluted tubules nearest the pyramids and also in the more marked congestion in vitamin A deficiency. Cloudy swelling was most frequently seen in the kidneys of vitamin B deficient rats.
Bloody, atrophic spleens (Table VII) were encountered most frequently in vitamin B deficient rats. The atrophy involves chiefly the pulp cells and not, as has been stated, the lymphoid tissue. The spleens of vitamin A deficient rats showed a huge increase of thickened hyaline blood vessels and of pigment. The findings in the spleen must, however, be taken with great caution on account of the great changes that the constituents of the spleen undergo with age (Gross6).
The pancreas showed a greatly increased incidence of vacuolation in vitamin B deficiency (63 percent as opposed to 18 percent in the control rats). The vacuoles were usually much more prominent as well. Forty-three percent of the vitamin C deficient rats and 21 percent of the vitamin A deficient rats also showed vacuolation. No lesions were found in the islets of Langerhans for any of the vitamin deficiencies. Some observations on the carbohydrate metabolism can be found elsewhere.21
All three vitamin deficiencies resulted in an increased incidence of atrophy in the testes (60 percent in vitamin A deficiency, 61 percent in B deficiency, and 50 percent in C deficiency—compared with 17 percent in controls). This atrophy varied in degree, from a marked shriveling up of the seminiferous tubules, with almost complete disappearance of cellular contents, to disorganization of the cell layers and cessation of spermatogenesis, as first described by Drummond.7 A curious finding, most frequently seen in vitamin B deficiency, is the presence in the tubules of large, spherical giant cells with many small, clear, bladder-like nuclei. The vesiculae seminales showed in all three deficiencies a flatter epithelium. In vitamin B deficiency and vitamin C deficiency the epithelium frequently showed pyknotic nuclei surrounded by a clear zone.
The ovary showed in all three deficiencies a considerably higher incidence of Graafian follicles undergoing complete degeneration (80 percent in vitamin A deficiency and 75 percent in both B and C deficiencies—as opposed to 24 percent in controls). Frequently, all that remained of the follicle was a narrow layer of the stratum granulosum, the cavity being filled with necrotic debris.
The findings in the intestine were on the whole rather disappointing. A moderate atrophy of the reticular cells of the mucosa was best seen in the descending colon of vitamin B deficient rats (43 percent, as opposed to 6 percent in controls). In the duodenum of 2 percent of control rats, a more or less granular exudate (or transudate) containing occasional lymphocytes was seen lying immediately under the epithelium of the tips of the villi. This condition was seen in 16 percent of vitamin B deficient rats, 13 percent of A deficient rats, and 5 percent of C deficient rats.
It is interesting to note that a few pigment cells resembling those found in the human colon are seen in the ascending colon of the rat. These lie, however, under the muscularis mucosae and occur only in 2 percent of control rats, 6 percent of vitamin [A] deficient rats, 3 percent of B deficient rats, and none of the C deficient rats examined microscopically.
Ulceration was found in the ascending colon of 6 percent of control rats and 3 percent of vitamin B deficient rats. The ileocecal region showed lesions similar to those already described in the control rats and in approximately the same proportion of incidence. The only interesting point noticed in the microscopic sections of the retro-ileoccecal glands was the increased incidence of pigment-bearing cells in the sinuses of the glands from vitamin B deficient rats. It is to be remembered that cells somewhat similar in appearance are found in the colon from cases of stasis in the human. These vitamin B deficient rats suffer, as will be seen later, from considerable stasis.
In 16 percent of vitamin B deficient rats, the Peyer’s patches were seen to undergo regressive changes. These consisted in elongation and clumping of the lymphocytes, which stained very deeply with hematoxylin. Hyaline connective tissue strands were seen between these clumps. Lesser grades of this lesion were encountered in 2 percent of control rats. With regard to Auerbach’s plexus, I must confess that I have not been able to confirm McCarrison’s observations. With the fixative and stains that I employed, it was impossible to determine how much of the disorganization found was due to artifact and how much was actually a pathological lesion.
The lesions specific to vitamin deficiencies that I found in the adult rats are thus seen to be extremely few. Atrophy of the thymus in the case of vitamin B is the only special lesion. The other changes, such as atrophy of the spleen pulp, slight atrophy of subepithelial intestinal reticularis, etc., are found in a certain proportion of control animals and show an increased incidence in the various vitamin deficiencies. The rather marked vacuolation so frequently seen in the pancreas of the vitamin B deficient rat is worthy of note and undoubtedly has some bearing on the function of the organ under these conditions.
Cramer10 has recently called attention to the extreme atrophy that occurs in the intestine of vitamin A deficient rats. I can only assume that the discrepancy in our results is due either to the fact that the rats that he employed were young and growing, whereas mine were adult, or to a difference in the length of time on the diet.
E. Investigations on the Effects of Vitamin Deficient Diets on the Motor Functions of the Intestinal Tract In Vivo and In Vitro
The in vivo studies on the effect of vitamin deficiencies were carried out on animals that I had bred in this laboratory. In every case the control experiment was carried out on the same animal, and at the end of the experiment each rat was killed and autopsied in order to rule out intercurrent lesions.
The method that I finally adopted was to use rats weighing approximately 100 g. These were given a small ball of diet weighing 1 g, with which 5 mg of finely powdered animal charcoal were thoroughly mixed. The animal was then placed on a specially designed metabolism apparatus (Gross and Connell11) that automatically timed and separated the excreta. With this apparatus it was possible to estimate within an hour the time taken for the first appearance and for the disappearance of the charcoal in the feces.
It is essential to use a weighed amount of charcoal, for Connell and I have found that the time taken for the charcoal to disappear apparently varies—in the same animal—directly with the amount given. The first appearance, however, is independent of this factor. Another point to be noticed is that the length of time it takes for the last trace of charcoal to disappear from the feces varies inversely, more or less, with the age, or rather size, of the animal. That is to say, for the same amount of charcoal, a smaller animal takes longer to free its intestine from the last traces than does a larger one. For this reason it is important to sandwich the experiment in between two control experiments.
The first appearance of charcoal is usually sharp and made out with very little difficulty. The endpoint is somewhat more difficult to determine. If there is any doubt as to whether the color of the scybalum in question is due to bile or to charcoal, it is a good plan to receive the feces into vessels containing a small quantity of water. This keeps them moist and soft until it is desired to examine them. If the scybalum is smeared over a piece of rough, white paper, the black mark left by charcoal is easily distinguishable from the bile stain. If there is further doubt, the pellet may be mushed up in a test tube containing several cubic centimeters of 25-percent sulfuric acid. This digests and floats to the surface the main constituents of the feces without charring them and allows the charcoal to settle as a distinct, black deposit.
The observations recorded here are based on the results of investigations on eighteen rats that were found normal at autopsy. The length of time necessary for the first appearance and disappearance of charcoal while the animal was on P diet was found for each rat; this was then determined for the experimental diet and, finally, for P diet again. The vitamin deficient diets tested were A and B. C diet was not tried since time did not permit.
The average time taken for the charcoal to make its first appearance was enormously increased in the case of vitamin B deficiency. It was considerably shortened in vitamin A deficiency. Two typical examples will illustrate this point.
Rat 390 on P diet took 9 hours to show the first trace of charcoal; in 65 hours this had entirely disappeared from the feces. The same rat on B diet showed the first appearance of charcoal in 12 hours; after 16 days the feces still showed a considerable amount of charcoal, and it was only after administering Marmite for 8 days that the last traces of charcoal disappeared. The rat was then fed on P diet for a day and tested. The charcoal first appeared in 10 hours and disappeared in 5 days. Figure 3 shows this result diagrammatically. It is noteworthy that this rat was by no means ill when it was first given B diet.
Figure 3. Rate of Passage of Food Through the Alimentary Tract. [Figure showing bowel transit times for rat 390 on experimental and control diets. See original for data.]
Immediately after the first timing experiments on P diet were over, two days more on P diet were allowed to elapse after the last appearance of the charcoal in order to make certain that the last traces had been emptied. The rat was then placed on B diet for one day, after which the second timing observations were made. The food intake of the rat was considerably lowered, especially towards the middle of the experiment. A change too was noted in the feces, both in quality and quantity. They became large and puffy and somewhat harder; at the same time, there was a considerable diminution in their number, so that whereas this rat passed on an average thirteen to fourteen scybala a day while on normal diet, during the second week on B deficiency [diet], it passed on an average two a day. Occasionally, 24 hours or more elapsed without the passage of feces, a very unusual occurrence for a rat.
Figure 4. Effect of Vitamin B Deficiency on the Number of Scybala Passed Daily and the Result of Administering Marmite. [Figure showing daily fecal output of rat 390 on vitamin B deficient diet. See original for data.]
Figure 4 shows the effect on this rat of adding 0.2 g Marmite daily after the sixteenth day on B deficiency diet. B diet was continued, and the amount given was deliberately kept the same as what the rat had been taking daily during the B deficiency. Nevertheless, as is seen, after a lag of three days the number of scybala passed daily rapidly increased, until the former normal level was reached. To determine whether the Marmite had a local stimulating effect on the bowel—a fact that did not seem probable a priori since its effect became manifest only after three days—I tested the effect of isotonic and hypertonic solutions of this substance on the isolated rat’s intestine. They produced an inhibition and fall in tone even in as low a concentration as 1 in 150,000.
It might be argued that the stasis produced by vitamin B deficiency was more apparent than real and that the delay in disappearance of the charcoal was due to diminished intake, with consequent diminished output. While this may explain the smaller number of scybala passed, it does not account for the delay in expulsion of the intestinal contents such as they are. Moreover, as I have shown, the simultaneous administration of Marmite to the same rat on the same diet, kept to the same quantity as the intake while on B deficiency, counteracted the delay in the expulsion of the intestinal contents, as evidenced by the charcoal. Finally, the action of the Marmite is certainly not a local one, for, apart from its inhibitory effect on the isolated intestine and the comparatively long time (for a laxative) before its effects were produced, it caused no diarrhea in rats fed on either normal [P] diet or B diet.
The salt content of the 10-percent solution that I actually administered by mouth was under 3 percent. It seems to me, therefore, that these experiments prove that vitamin B is essential for the maintenance of the normal motor functioning of the intestine. I have no doubt that in the normal economy of the bowel, other factors—such as roughage—also play an important part, for in some timing experiments in which I studied the effects of powdered agar, I found that this indigestible material does stimulate the activity of the bowel. Furthermore, in studying the effects of foods such as carrots, I found that the speed with which this food passed through the alimentary tract suggested that stimulating substances other than roughage and vitamins may also serve as exogenous regulators of intestinal activity.
Autopsies performed on rats killed during stasis showed the charcoal accumulated in the cecum, which, along with the colon (section D above), is probably the first to suffer from the result of vitamin B deprivation. The most remarkable point is that the intestine seems to feel the need of vitamin B almost immediately, thus bearing out the conclusions of previous observers as to the inability of higher organisms to store this vitamin in appreciable quantity. In this connection it is interesting to note that brewer’s yeast is used as a laxative by the farmers in the north of France (Herbomez12).
Reeves,13 working on six cases of constipation in human subjects, was able to cure four and relieved the remaining two by the administration of yeast. Smith14 found that when human subjects are kept on a diet of bread, milk, and cheese, they have a tendency to become constipated. Adding yeast to the diet, however, relieves the constipation. Recently, Murlin and Mattill9 have reviewed the literature on the subject and performed a number of experiments on the laxative properties of yeast using human subjects and dogs for the purpose. Their criteria of constipation were increased water content, bulk, and nitrogen content of the stools. They found that boiled yeast had a weaker laxative action [than uncooked yeast].
It seems to me that the rate with which the intestine clears itself of a given amount of inactive pigment is a much more certain measure of the motor activity of the bowel and that the experimental results I have produced can hardly be called by any other name than “constipation.” I am using this term to indicate a symptom that I am quite well aware may have its origin in a variety of causes. The results obtained by the use of yeast, although not so clear-cut as the experiments that I present here and more open to criticism, nevertheless bear out my contention that lack of vitamin B is followed by a stasis in the alimentary tract and a stasis so produced is relieved by the exhibition of vitamin B.
Rat 396 illustrates the effect of deprivation of vitamin A on the motor functions of the intestinal tract. This rat was somewhat younger, hence it took 6 days to empty the bowel of the last trace of charcoal on the P diet. It took 6 hours for the first appearance. Two days after the last appearance of charcoal, A diet was given, and the following day timing observations were commenced. The charcoal appeared in 5 hours and disappeared in 16 hours. Fearing that this was due to some error in technique, the timing was repeated on A diet. This time it took 23 hours for the charcoal to disappear. A subsequent test with P diet in the usual way showed a first appearance of charcoal in 9 hours and a disappearance in 8 days (Figure 3). The feces remained more or less constant in number or, perhaps in several cases, showed a slight increase. This was repeated on a number of rats.
In both vitamin A and vitamin B deficiencies, particularly in the former, the feces were gray and somewhat lacking in bile pigments. This was more noticeable in comparing the dried scybala. That food rich in vitamin B contains substances that stimulate the flow of bile and pancreatic juice was shown by Voegtlin and Myers.16 It is seen, therefore, that vitamin A deficiency, although undoubtedly associated with an alteration in the secretory functions of the alimentary tract, caused no initial stasis but rather a more rapid peristalsis.
The question that now arose was, what structures in the intestinal tract are involved in this stasis? I have shown that histological evidence on this point is very uncertain. I attempted no comparisons of the thickness of the muscle because this varies so enormously according to the state of tonus or stretching of the intestine when fixed. Such comparisons are only possible where differences are very marked. This was not the case in the sections that I examined.
I therefore attempted to determine whether there was any difference between the work done by the isolated intestine from vitamin deficient rats and that from normal rats, estimating the work done in arbitrary dynamic units. I hoped this would give me a clue as to the amount or state of the musculature. The rat to be examined was killed in the manner described, the abdomen was immediately opened, and slips of the first part of the duodenum, terminal ileum, and ascending colon (always taken at the same points) were removed, wrapped in gauze dampened with Ringer solution, and placed on ice. This caused a tonic contraction of the smooth muscle. At fixed intervals thereafter, 4 cm of the different portions of the intestine were removed and placed into a bath containing 250 cc of Tyrode’s solution of tested pH and kept at a constant temperature of 39 degrees C. After an interval of 15 minutes, a tracing was taken of the rhythmic movements of the slip. No oxygen was used.
Several points in connection with this technique deserve mention. First, by placing the intestine on ice, I subject each intestine examined to as nearly as possible the same degree of terms. Measured sections under this condition are therefore fairly comparable, providing that due allowance is made for the differences in thickness due to the differences in size of the animal. Second, by avoiding the use of oxygen, I eliminate the differences that alterations in the supply of oxygen cause (Gross and Clark2). Third, given the same leverage of the writing point and the same weight of the lever, the product of amplitude and frequency is a measure of the work done by the muscle. Assuming that this work varies with the musculature, amongst other things, and assuming that the musculature is a function of the weight of the rat, I attempted to reduce the work done by the various intestines to the same proportion by means of a formula. Needless to say, this method can give no more than an approximation of the differences of the actual work done, and differences to be significant must be fairly large. The formula I used was:
(amplitude x frequency)/[(highest body weight/100)1/3]2
(Note: The internal and external diameters of the gut, it is assumed, vary roughly as the diameters of the rat. Further, the power of the muscle will depend on the volume of muscle employed in the experiments—other things such as stretching, temperature, etc., being equal. This volume will be given by the product of length and cross section. The cross section will be given by the formula π (r12– r22), where d1 and d2 are the internal and external diameters of the gut, respectively. This expression is a linear function of the square of the diameter of the rat. The length of the gut is constant (4 cm), hence the volume of gut employed is proportional to the square of the diameter of the rat; the diameter of the rat, in turn, is proportional to the cube root of the rat weight. Hence the volume of the gut employed is proportional to weight 2/3 [sic]. Each must be reduced to the volume corresponding to that given by a rat of unit weight (100 g). Hence the actual power measured by amplitude x frequency must be divided by [(highest body weight/100)1/3]2. And the relative power is therefore given by (amplitude x frequency)/[(highest body weight/100)1/3]2.)
Table IX shows the factors obtained for the duodenum and ileum under the several experimental diets and conditions. [See original for data.]
Table IX. Units of Work Performed by Unit Portions of Isolated Small Intestine from Rats on the Experimental Diets. [See original for data.]
The following points are brought out:
- The work factor for the duodenum is considerably lower in the vitamin deficiencies.
- Whereas the ileum work factor is lower than that of the duodenum in the control animals, this order is reversed in each of the deficient groups. This may have some bearing on Alvarez’s theory of dynamic gradient.17
- With the exception of the ileum in vitamin A deficient rats, daily intraperitoneal injections of pituitrin caused no obvious difference in the work factor.
- Daily intraperitoneal injections of adrenaline were associated, contrary to what was expected, with an increase in the work factor, which was astonishingly high in vitamin B deficient rats.
The only other observation that might be of interest in this connection is the greater and more rapid response that the duodenum and ileum of a number of vitamin B deficient rats gave to a concentration of 1 in 500,000 pilocarpine nitrate in the bath as compared with that given by other vitamin-deficient and normal rats.
The colon, on account of its irregular and sluggish motility, did not lend itself to a study of this nature.
If these experiments are a true index of the work power of the intestinal muscle, one may be allowed to conclude that vitamin deficiencies on the whole diminish this power in the first part of the small intestine and alter the relations of the dynamic gradient. This must be an index of either atrophy or other qualitative changes in the muscular or neuromuscular apparatus.
The first point that deserves emphasis is the fact that, apart from such specific lesions as rickets, beriberi, and scurvy, pure vitamin deficiencies produce in the adult rat remarkably few characteristic lesions. Similar conclusions have been arrived at by Simonnet18 and Lumiere19 on pigeons, and by Karr20 on dogs.
If any conclusions applicable to man are to be drawn from dietary deficiencies in animals, the rat is not a very unsatisfactory animal since its diet is very much of the same nature as that of man and the anatomy of its intestinal tract shows an extraordinary resemblance to that of the primates. The results following a variation in the diet of the rat can therefore be applied to man with more justification than can those produced in birds or even in herbivorous or graminivorous mammals.
I have attempted to show the advisability of performing autopsies on all cases where conclusions are to be drawn from dietary experiments on rats because these animals are very prone to morbid conditions. I have drawn attention to the necessity of performing histological observations on tissues taken from animals immediately after death in order that the results may be considered valid. Finally, I cannot commend too highly the practice of breeding one’s own animals for experimentation.
The main object of this research was to determine whether or not pure vitamin deficiencies can be a source of intestinal stasis. This has proved positive for a deficiency of vitamin B. Although I have been unable to find unmistakable lesions in Auerbach’s plexus produced by any of the vitamin deficiencies, the in vivo experiments showed a marked stasis immediately following the elimination of Marmite from the diet. I have shown that Marmite has no local stimulating effect on the isolated rat’s intestine. Vitamin A deficiency produces an intestinal disturbance that manifests itself in a hurrying through of intestinal contents. Both of these vitamin deficiencies are accompanied by a qualitative difference in the feces. I have not studied the effects of vitamin C deficiency on the motor functions of the intestine of the rat in vivo.
Early in this paper, I drew attention to the subepithelial accumulation of pigment-bearing cells that are characteristically found in great numbers in the colon removed at operation for conditions of advanced stasis. It is interesting to note that the retro-ileocecal glands of vitamin B deficient rats alone showed a definitely increased incidence of pigment-bearing cells. Whether these cells are identical with those found in the human colon I cannot at present say. It is clear that the latter are the result of a chronic, long, drawn-out condition, whereas those that I have found in rats occurred in relatively acute conditions of stasis.
Whether vitamin deficiencies play a role as an etiological factor of some forms of intestinal stasis in everyday life is not a matter that I desire to discuss here. Personally, I am inclined to believe that they do, and sufficient evidence has been produced in this paper to make the consideration of vitamin B deficiency an important factor in the prevention of this condition, especially since more attention is being paid to vitamins A and C as etiological factors of morbid conditions met with in everyday life. If it should turn out that the present so-called vitamin B corresponds to more than one factor, as has recently been suggested by a number of observers, I cannot predict in which factor the “anti-stasis” element will be found.
I wish to acknowledge here my great indebtedness to Sir Arthur Keith, to Professors E.H. Starling and J.C. Drummond, and to Dr. Katherine H. Coward for constant help and advice as well as for the privilege of carrying out this work in their laboratories. The expenses in connection with this research were aided by grants from the Medical Research Council and the British Medical Association, London.
By Louis Gross, Beit Memorial Research Fellow, Laboratories of the Royal College of Surgeons, London, and the Institute of Physiology, University College, London. Reprinted from The Journal of Pathology and Bacteriology, Vol. XXVII, 1924, by the Lee Foundation for Nutritional Research.
1. Keith. A. Brit. Journ. Surg., vol. ii, p. 576, 1914.
2. Gross, L., and Clark. A.J. Journ. Physiol., vol. lvii, p. 457, 1923.
3. McCarrison, R. Brit. Med. Journ., vol. ii. pp. 36–39, 1919; Brit. Med. Journ., vol. i, p. 823, 1920.
4. Gross, L. Proc. Roy. Soc. Med., vol. xv (Surgery section, Proctology subsection), pp. 71–73, 1922.
5. Cramer, W. Journ. Physiol., vol. lii, Proc. xiii, 1918,
6. Gross, L. Journ. Med. Res., vol. xxxix, pp. 311–38, 1919.
7. Drummond, J.C. Biochem. Journ., vol. xii, pp. 25–41, 1918.
8. Harden, A., and Zilva, S.S. Biochem. Journ., vol. xii, p. 408, 1918.
9. Drummond, J.C. Biochem. Journ., vol. xiii, pp. 77–80, 1919.
10. Cramer, W. Lancet, vol. i, pp. 1046–50, 1923.
11. Gross, L., and Connell, S.J.B. Journ. Physiol., vol. lvii., Proc. lx, 1923.
12. Herbomez. Paris Thesis, 1904.
13. Reeves. N.Y. Med. Journ., vol. cxv, p. 637, 1922.
14. Smith. Journ. Lab. Clin. Med., vol. vii, p. 473, 1922.
15. Murlin, J.R., and Mattill, H.A. Amer. Journ. Physiol., vol. lxiv, pp. 75–96, 1923.
16. Voegtlin, C., and Myers, J.C. Journ. Phar. and Exper. Ther., vol. xiii, p. 301, 1919.
17. Alvarez, W.C. The Mechanics of the Digestive Tract. P.B. Hoeber, New York, 1922.
18. Simonnet, H. Compt. Rend. Soc. Biol., vol. lxxxiii. p. 1508, 1920.
19. Lumiere, A. Paris Med., vol. x, p. 474, 1920.
20. Karr, W.G. Journ. Biol. Chem., vol. xliv, pp. 255 and 277, 1920.
21. Gross, L. Biochem. Journ., vol. xvii, p. 569, 1923.
Reprint No. 24
Lee Foundation for Nutritional Research
Milwaukee, Wisconsin 53201
Note: Lee Foundation for Nutritional Research is a nonprofit, public-service institution, chartered to investigate and disseminate nutritional information. The attached publication is not literature or labeling for any product, nor shall it be employed as such by anyone. In accordance with the right of freedom of the press guaranteed to the Foundation by the First Amendment of the U.S. Constitution, the attached publication is issued and distributed for informational purposes.