It is well known fact that conjunctivitis and xserophthalmia are the most noticeable examples of change in the epithelial surfaces of the body brough about by lack of vitamin A. Wolbach and Howe in 1925 found that the specific tissue change due to deprivation of fat soluble vitamin A is replacement of various
epithelia by stratified squamous keratinizing epithelium. Lesions of the stomach and intestine in association with vitamin A deficiency are described by Fridericaia, Pappenheimer and Jensen. Other workers state that a number of nutritional factors are concerned with the maintencance of an intact gastric mucosa
in experimental animals.
From various sources information has been accumulated with demonstrates that ulceration of the gastric mucosa may occur in deficiencies of protein, vitamin A, cystine, choline, riboflavin, pyridoxine, pantothenic acid, tocopherols, calcium, or simply or calories.
Recent reports indicate that gastric ulcers are not the result of these deficiencies but occur evenwhen the essential nutrient is supplied. Knowledge about peptic ulceration resulting from nutritional deficiency is in a confused state. Brunschwig and Rasmussea observed hemorrhagic erosions and ulcerations in the rumen and mucosa of the glandular stomach of rats fed various experimental diets. They felt that the lesions which developed should be regarded as "starvation reaction" and were not due to inadequate quantities of vitamins. It is well known that certain situations are capable of inducing gastrointestestinal ulcers in both man and experimental animals. Stress situations have been reported to produce definite physical changes in animals.
Selye reports that one of the consistent signs of acute stress is gastrointestinal erosions. He found that several factors were involved in the production of the signs of stress. Age: older rats reacted more readily to alarm stimulus than younger rats. Presence of the adrenals and hypophysis: either adrenalectomy or hypophysectomy reduced the resistance of the rat to the alarmstimulus. Starvation: fasting was found to be a particularly effective means of increasing the effect of any stress agent. Ershoff reported that under the stress of prolonged exposure cold, rats on a purified diet deficient in vitamin A were depleted of their liver vitamin A stores more rapidly than rats kept at room temperature.
Clark and Colburn report that the total vitamin A content of the liver of rats treated with cortisone decreases steadily throughout the course of treatment. However, there has not been any report made on the influence of stress situation to the gastric mucosa of vitamin A deficient animal. This experimental study is made
(1) to observe the influence of ACTH or of "shaking stress" on the gastric mucosa of vitamin A deficient rats.
(2) To observe the relation of vitamin A deficient diet to growth, urinary 17-ketosteroid, uropepsin and a correlation between the excretion of 17-ketosteroid, uropepsin and the gastric mucosal change under the influence of stress.
Six experiments were performed.
Experiment Ⅰ: To study the effect of ACTH on the gastric mucosa of adult vitamin A deficient rats.
Experiment Ⅱ: to study the effect of vitamin A free diet upon the gastric mucosa of adult rats, without the administration of ACTH.
Experiment Ⅲ: To study the effect of "shaking stress"* on the gastric mucosa of adult vitamin A deficient rats.
Experiment Ⅳ: To study the effect of "shaking stress" on the gastric mucosa of young vitamin A deficient rats.
Experiment Ⅴ: To observe the change of gastric mucosa immediately after the application of "shaking stress" on young vitamin A deficient rats.
Experiment Ⅵ: To study the relationship between the frequency and duration of application of "shaking stress" and the extent of gastric mucosal change of adult vitamin A defcient rats.
The male albino rats weighing 30gms to 150gms were used. These were placed in individual metabolic cages. The basal diet consisted of low protein and low fat: containg 9% protein, 3 per cent fat, 70 per cent carbohydrate. Vitamin A free diet in the first three experiments was made by removing the butter and cod liver oil from the basal diet replacing them with the equal amount of olive oil. The control groups in Exp. Ⅰ and Ⅱ. were fed on the basal diet. In Exp. Ⅲ, the control group was fed on the vitamin A free diet plus 400 units of vitamin A per 100 gm of diet. In the Experiment Ⅳ, Ⅴ, Ⅵ, the study groups were fed on vitamin A free and fat free diet which meant that butter and cod liver oil were removed from the basal diet without replacing with olive oil. Control groups in these experiments were given the afore mentioned diet plus 400 units of vitamin A per 100 gm of diet.
The twenty-four hour urinary 17-ketosteroid and uropepsin excretion were determined before and after the administration of ACTH or of "shaking stress".
The body weight of rats were recorded weekly. At the end of the experiments, the rats were sacrificed, and the gastric mucosa was examined. The blood samples were drawn at the time of sacrifice(decapitation), the liver was removed and vitamin A and carotene level in the blood and liver were determined.
1. The Growth
The rats in vitamin A free diet showed slow growth and some of them did not grow after one month of feeding while the control group continued to grow on the basal diet. This was more significant among the younger rats born of mother rats fed on vitamin A free diet.
During the administration of ACTH or application of "shaking stress" the growth rate was inhibited temporarily.
2. Vitamin A and carotene level in the plasma and liver.
In Experiment Ⅱ, the average vitamin A content of the plasma was 16.6-19.0 mcg./dl. in the adult rats fed on vitamin A free diet whereas the control group had 30.0-30.6 mcg./dl. indicating approximately a 50 per cent decrease of vitamin A content in the plasma of study group. Administration of ACTH in both groups did not influence the vitamin A content in the plasma.
In Experiment V, young rats born of mother rats fed on vitamin A free diet were used. The vitamin A content of the plasma in the study group was 7.2 mcg./dl. indicating a marked decrease, approximately 80 per cent of vitamin A level in the study group.
Carotene level in the plasma was determined in Experiment Ⅱ and Ⅴ, but it was negligible. The vitamin A and carotene content in the liver was measured in Experiment Ⅱ. The average vitamin A content in the liver was 1,500-2,055 mcg. per cent in the study goup and 11,857-14,605 mcg. per cent in the control group. The carotene level in the liver was also negligible as in the plasma.
3. Twenty-four hour urinary 17-ketosteroid excretion.
The mean 17-ketosteroid excretion prior to the administration of ACTH or application of "shaking stress" was 0.09mgm/24 hr/rat in the study group and 0.08 mgm/24 hr/rat in the control group indicating that there was practically no difference between the two groups. However, following the administration of rapid acting ACTH, the urinary 17-ketosteroid excretion the study group increased to 0.30mgm/24 hr/rat showing three times more than the level before the administration of ACTH.
The control group had 0.18 mgm/24 hr/rat following the ACTH administration showing approximately two times of the level before the administration of ACTH. The long acting ACTH did not influence the urinary 17-retosteroid excretion.
Following the appilcation of single "shaking stress" with frequency of 300 per minute for three hours, the urinary 17-ketosteroid excretion was 0.36 mgm/24 hr/rat in the study group and 0.38 mgm/24 hr/rat in the control group. This means the urinary 17-ketosteroid excretion increased approximately four times of the level before the administration of "shaking stress." The slow shaking, 150 per minute for three hours daily for four days did not influence the urinary 17-ketosteroid excretion.
4. Uropepsin Output.
The mean uropepsin output prior to the administration of ACTH or application of "shaking stress" in the study group was 16 units/24 hr/rat and 14 units/24 hr/rat in the control group indicating no remarkable difference in the both group.
The excretion of uropepsin increased markedly in the study group following the administration of ACTH. When the single dose of 3 units of ACTH was given, the uropepsin excretion was average 75 units/24 hr/rat and when 6.0 units of ACTH was given daily for four days, the average daily excretion of uropepsin was 65 units/24hr/rat. However, in the control group, there was no appreciable change of uropepsin excretion with the same amount of ACTH administration.
Following the application of "shaking stress" the mean uropepsin excretion in the study group was 33 units/24 hr/rat which is approximately two times of the level before the application of "shaking stress." In the control group, there was no appreciable increase of uropepsin excretion.
5. Change of Gastric Mucosa.
A. In experiment Ⅰ and Ⅱ, the gastric mucosal change was not demonstrable in the study group fed on vitamin A free diet for one month to three months. However, when ACTH was administered in these group, 4 of 8 rats developed gastric lesions consists of small brownish superficial ulcers of the glandular stomach. In the control group these changes weeere very minimal and observed in only one of eight rats following the administration of ACTH.
B. In experiment Ⅲ, adult rats weighing about 180 gm were subjected to mechanical shaking with frequency of 300 per minute for three hours followed by 150-200 per minute shaking three hours daily for 15 days. The frequency of shaking had to be reduced because the rats could not tolerate 300 per minute oscillation. Two of eight rats on vitamin A deficient diet revealed mucosal congestion and bleeding, one had gastric ulcer with congestion and bleeding, one rat developed gastric ulcer, However, the control group did not demonstrate any remarkablemuscosal change of stomach.
C. In experiment Ⅳ, young rats weighing between 88-120 gm were subjected to mechanical shaking with frequency of 150 per minute for three hours daily for four days followed by 240 per minute shaking for three hours daily for another four days. The animals were sacrificed ten days after the last mechanical shaking, the gastric mucosa was studied. Gastric ulcer was demonstrated in the glandular stomach in both study and control groups, one each. However, gastric bleeding and erosion were observed in five of eight study group and two of eight control group.
D. In experiment Ⅴ, the young rats born of vitamin A deficient mother rats were subjected to single mechanjcal shaking of per minute for five hours. These were sacrificed 12 hours after the completion of mechanical shaking. The average body weight of study group at the time of this study was 72 gm and the control group was 153 gm. The gastric ulcer was not demonstrated in the study or control group.
However, severe diffuse gastric congestion and bleeding were demonstrated in the mucosa of the glandular stomach of all rats of the study group where as the control group showed very mild congestion of the mucosa in one instance, and mild bleeding in another rat.
E. In experiment Ⅵ, the relationship was observed between the frequency and duration of mechanical shaking and the incidence and severity of the gastric mucosal change.
(ⅰ) when the mechanical shaking of 180 per minute three hours daily for 11 days was applied, the study group showed a mild mucosal hemorrhage and erosion in the glandular portion, but in the control group, the gastric mucosal change was not found.
(ⅱ) The mechanical shaking of 200 per minute, five hours in the first day, 220 per minute shaking six hours on the second day, 240 per minute seven hours in the third day and 280 per minute for 6 hours in the fourth day were given, and the animals were sacrificed within 24 hours after the termination of the shaking for the study of their gastric mucosa.
Bleeding and erosion where observed in both study and control groups to a much more sever degree than the group where milder shaking was applied.
Sever gastric ulcer was demonstrated in the fore-stomach of one rat this study group whereas severe degree than the group where milder shaking was applied.
Sever gastric ulcer was demonstrated in the fore-stomach of onne rat this study group whereas no gastric ulcer was noted in the control group.
(ⅲ) When the mechanical shaking of 280/min/5hrs int he first day, 280/min/7hrs daily for five days was applied, gastric ulcer developed in the fore-stomach of both study and control group, two of three animals in the both groups. However, the severity of gastric lesion was more marked among the study group.
The ulcers induced by mechanical shaking was larger than that produce by ACTH administration and was mainly in the forestomach. Bleeding and erosion were observed in the glandular part of stomach. The ulcers induced by the administration of ACTH was mainly in the glandular stomach and these ulcers were smaller and more superficial than that of ulcers produced by mechanical shaking.
1. The rats fed on vitamin A free diet for one month to three months did not produce gastric lesions. However, when the ACTH was administered, small superficial ulcers were demonstrated in the mucosa of the glandular stomach in the both group. The extent and incidence of gastric lesions were more pronounced in the study group.
2. Application of mechanical shaking stress induced gastric ulcers in the glandular portion of stomach and forestomach and it was more pronounced in the study group. The severity and incidence of gastric lesion was parallel to the frequency and duration of the application of shaking stress. The response to the "shaking stress" was more pronounced among the young rats.
3. The urinary 17-ketosteroid excretion was increased when rapid acting ACTH or "stronger shaking" stress were applied in both study and control groups. However, the long acting ACTH gel or milder "shaking stress" did not produce any remarkable change of urinary 17-ketosteroid excretion.
4. Twenty-four hour uropepsin output increased four to five times among the study group after administration of rapid acting ACTH and two to three times after the application of mechanical shaking.
5. When ACTH was administered to the study group, twenty-four hour uropepsin output increased four to fivetimes of that before the administration of ACTH. The application of "shaking stress." The control group did not show any significant increase in uropepsin excretion with the administration of ACTH or application of "shaking stress."
6. The growth rate of study group began to decline about one month after the initiation of vitamin A free diet feeding while the control group continued to grow. This was more marked among the young rats. The young rats born of mother rat fed on vitamin A free diet showed remarkable decline of growth.