David Marine

Exophthalmos in Thyroidectomized Guinea Pigs by Thyrotropic Substance of Anterior Pituitary, and the Mechanism Involved

We pointed out 1 that chronic, progressive, bilateral exophthalmos, produced in immature (2-3 months old) rabbits, maintained on a diet of alfalfa hay and oats, by the daily intramuscular injection of 0.05-0.1 cc. methyl cyanide, occurs in association with thyroid insufficiency (that is, (a) marked hyperplasia of the thyroid or (b) thyroidectomy). It is obvious, therefore, that exophthalmos is not dependent upon either a normal or abnormal thyroid secretion. Neither do the medulla and cortex of the suprarenal gland have any specific or initial relation to exophthalmos. To obtain further information on the nature of exophthalmos we have produced it in young normal and thyroidectomized guinea pigs by using acetic acid extracts of anterior pituitary powder (Armour) that Spaul, 2 Uhlenhuth and Schwartzbach, 3 and Loeb and Bassett 4 have shown to contain the thyrotropic hormone and with which Loeb and Bassett have produced exophthalmos in guinea pigs. The data are given in the following table. It is clear that acetic acid extracts of ox anterior pituitary powder produce exophthalmos as well in thyroidectomized guinea pigs as in those with intact thyroids. Also, as in the case of methyl cyanide, the exophthalmos does not appear in guinea pigs with intact thyroids until they have developed thyroid hyperplasia (thyroid insufficiency), and to both cyanide and anterior pituitary extract some animals are resistant.

Farther studies on etiology of goitre with particular reference to the action of cyanides.

The immediate cause of thyroid hyperplasia in all probability is a relative or an absolute deficiency of iodine. 1 The fundamental or essential cause of goiter is unknown, but the search for the essential cause, as we have often suggested, appears to resolve itself into determining the cause or causes of the iodine deficiency. As most iodine deficiencies are relative rather than absolute, the search further limits itself largely to determining the factors which create the increased needs of the organism for the iodine containing hormone. The simplest way of increasing the need of the thyroid for iodine would be by depressing the utilization of oxygen in the tissues, and the discovery by Chesney and Webster 2 that the prolonged feeding of cabbage caused thyroid hyperplasia in rabbits appeared to offer a practical means of testing this hypothesis. It has been shown that there are great seasonal and climatic variations in the goitrogenic activity of cabbage, 3 that drying in a current of air or in vacuo causes a loss of the goitrogenic agent, 4 that prolonged steaming does not impair and under certain conditions may increase its goitrogenic power, 5 that boiling for 30 minutes at pH 3.0 (HC1) does not injure it, 6 that the goitrogenic substance may be extracted from cabbage with ether and other ethereal solvents 7 and that this substance is but slightly extracted by prolonged aqueous leaching. Since all the Brassicae so far tested may produce goiter and since mustard oils (isothiocyanates) are the most characteristic constituents of these plants it was thought that their goitrogenic activity might be connected in some way with these substances or with their cyanide precursors. Several of the mustard oils (allyl, ethyl, phenyl) have been fed to rabbits with negative results.

Glycosuria. in rabbits following injections of saline extract of anterior pituitary.

A relation of the anterior pituitary to carbohydrate metabolism has often been suspected on the basis of the frequent occurrence of hyperglycemia and glycosuria in acromegaly, and of anatomical changes in the anterior pituitary in diabetes mellitus, but such a connection was not indicated experimentally until Houssay and Biasotti 1 showed that pancreatic diabetes could be prevented in toads by removal of the anterior pituitary. Subcutaneous implantation of anterior pituitary in such depancreatized toads re-established the diabetes. They were unable to show a similar relation in mammals either by gland implantation or by injections of anterior pituitary extracts, although depancreatized and hypophysectomized dogs survived longer and showed milder diabetes than did depancreatized controls 2 . Evans, Meyer, Simpson, and Reichert 3 reported observations in which they produced glycosuria and polyuria in 2 of 4 dogs by daily injections of the growth hormone fraction of anterior pituitary extracts for 8 or 9 months. We have made somewhat similar observations in rabbits. Hyperglycemia, polyuria, glycosuria, and lipemia developed after injecting a saline extract of ox pituitary daily for 6 or more days, in some instances of surprisingly great intensity. It occurred in each of the 4 rabbits used, though varying in degree. The extract was prepared twice weekly from fresh chilled glands, according to the method of Schockaert 4 except for the measures used in sterilization. It produced typical reactions among which were rapid hypertrophy of the mammary glands with lactation, luteinization of the ovaries, hypertrophy of the thyroid and suprarenal cortex. The rabbits were kept on our stock diet of alfalfa hay and oats. Two were injected with 2 cc. and 2 with 4 cc. daily, equivalent to one-half and one anterior pituitary respectively.

Studies on Simple Goiter Produced by Cabbage and Other Vegetables.

Chesney, Webster and Clawson 1 , 2 , 3 have shown that when fresh cabbage (Brassica oleracea) is fed to rabbits as the principal food, clinically detectable thyroid hyperplasia occurs in 2 or 3 months, and with continued cabbage feeding very large goiters (up to 45 gm.) have been produced. They further showed that the development of goiter was associated with a lowering of the metabolism and that the administration of small amounts of iodine readily raises the metabolic rate in goiterous rabbits and prevents thyroid hyperplasia. In these respects, as well as histologically, the thyroid hyperplasia produced by feeding cabbage is identical with that of simple or endemic goiter. We have confirmed these findings. Boiling or steaming cabbage for 30 minutes increases its capacity to produce thyroid hyperplasia. Steaming for 15 minutes is less effective and steaming for 60 minutes renders it neither more nor less potent than for 30 minutes. Steamed cabbage from which 60% of the weight is removed as press juice is practically as effective as whole cabbage when fed in calori-equivalent amounts. Hashed fresh cabbage has very little capacity to produce thyroid hyperplasia, while hashed steamed cabbage fully retains this quality. In our hands the press juice of 500 gm. of steamed cabbage (300 cc.) when fed daily to rabbits whether whole or concentrated in vacuo was only slightly effective. Rabbits previously iodized and fed with fresh cabbage up to 75 calories per kg. per day (3 gm. of cabbage are equivalent to approximately 1 calorie) developed palpable thyroids in about 30 days, while with the same amount of steamed cabbage only 10 to 15 days were necessary. The lowering of the metabolic rate to the thyroidectomy level (from about 2.4 to 1.75 calories per kg. per hour) would also occur in about half the time required if fresh cabbage were used.

Parathyroid hypertrophy and hyperplasia in fowls

Physiological overgrowth of the parathyroid glands in mammals has been very rarely observed. Erdheim, 1 Bauer 2 and Strada 3 have recently described its occurrence in man in association with some cases of osteomalacia. Three instances of undoubted general parathyroid enlargement in bitches in association with lactation have come under my observation. In the reports of partial removal and of transplantation of mammalian parathyroids, particularly in dogs, one of the most characteristic features has been the absence of any noteworthy compensatory enlargement of the remaining portion within the time limits in which other tissues, like the thyroid, heart muscle, kidney, etc., react to artificially induced insufficiencies. In the course of some experiments with the thyroid gland in fowls in 1910, I observed several instances of marked enlargement of the parathyroids independent of the changes occurring in the thyroid glands. These parathyroid changes were found in fowls which had been fed with maize and wheat for periods of 2 to 6 months. The observations were repeated in 1911, 1912, and 1913, with similar results. Since calcium temporarily relieves the symptoms of parathyroid tetany in mammals, and since maize and wheat contain very little calcium, it was thought possibly the parathyroid overgrowth might be a result of a calcium deficiency, and if this was so, calcium might exert some protective actionagainst parathyroid overgrowth. Calcium hydroxide, calcium lactate, calcium carbonate (as chalk and crushed oyster shells), magnesium carbonate, strontium carbonate, sodium citrate, sulphuric acid, neutral sulphur and sodium hydroxide have been given in the diet of maize and wheat for periods of one, two and three months. One hundred and ten fowls have been used. No detectable inhibition of the parathyroid overgrowth could be detected in the fowls given magnesium carbonate, strontium carbonate, sulphuric acid, neutral sulphur, sodium citrate or sodium hydroxide,-the growth being as marked as in the controls.

Production of Goiter and Exophthalmos in Rabbits by Administration of Cyanide

We have shown that several of the organic cyanides 1 when injected subcutaneously into rabbits daily for 3 or more weeks produce thyroid hyperplasia. Methyl cyanide produces the greatest thyroid reactions, and young rabbits (3 to 4 months) are more reactive than adults to the same doses per kg. Chronic bilateral exophthalmos has occurred in a significant number of prepubertal rabbits after methyl cyanide has been given for varying lengths of time. Exophthalmos has developed as early as the 20th day after beginning daily injections of 0.1 cc. of methyl cyanide in 3 months old rabbits. Of the 2 breeds used (Dutch and Belgian) we have observed it most frequently in young Dutch rabbits. It has not been detected in adults (6 months and over) in either strain. Representative instances are given in Table I. So far we have observed only moderate degrees of exophthalmos. It occurs earliest in those rabbits which develop thyroid hyperplasia the quickest and appears to be proportional to the degree of thyroid hyperplasia. Rabbits that have failed to develop thyroid hyperplasia, or rabbits in which the hyperplasia has been slight have not developed exophthalmos. When exophthalmos is developing the rabbits become nervous and fidgety. This is frequently associated with soft feces and an increase in the volume of urine. No attempt has been made to determine what rôle the hormones of the posterior pituitary and chromaffin tissue may play in the production and maintenance of the exophthalmos. There is, however, some hypertrophy and great hyperemia of the medulla of the suprarenals in these animals.

Effect of ascorbic acid on thyroid and suprarenals of guinea pigs.

In previous publications from this laboratory1-5 it has been shown that fresh plant and fruit juices contain an antigoitrous agent when tested on immature rabbits maintained on a goitrogenic diet of alfalfa hay and oats. (The goitrogenic effect of this diet is greatly enhanced by cyanide.) It was shown that this antigoitrous effect was in general inversely proportional to the iodine reducing capacity and that treating the fresh juice with copper salts destroyed or lessened this antigoitrous effect. Our data further indicated that the antigoitrous agent was probably identical with ascorbic acid but because of the lack of crystalline ascorbic acid on the one hand and the presence of iodine (also an antigoitrous agent), on the other, in all these extracts we were unable to prove it. About 2 years ago one of us 6 simplified Szent-Györgyis and Waugh and Kings method of extracting ascorbic acid and by utilizing iris leaves, which are extremely rich in ascorbic acid, has prepared sufficient quantities of this hormone in crystalline form for these experiments, which were carried out as follows: Young guinea pigs weighing between 150 and 250 gm. were placed on a diet of alfalfa hay and rolled oats. (This diet produces scurvy in from 15 to 20 days. Pigs maintained on this diet when injected with the thyrotropic factor develop more marked exophthalmos and thyroid hyperplasia and more quickly than when given greens daily in addition.) They were injected with 1/2 to 2 cc. of ox anterior pituitary extract prepared after Loeb and Bassetts method and given 25 to 100 mg. of crystalline ascorbic acid by mouth daily. The pigs were sacrificed at various intervals and the principal data of 22 are given in Table I.

Further Studies on the Exophthalmos in Rabbits Produced by Methyl Cyanide.

Chronic, progressive, bilateral exophthalmos has been produced in more than 150 prepubertal rabbits maintained on a diet of alfalfa hay and oats by the daily intramuscular injection of 0.05-0.1 cc. of methyl cyanide. 1 , 2 Feeding fresh vegetables markedly inhibits its development. Exophthalmos may appear as early as the 14th day and as late as the 60th day after beginning the cyanide injections. Males are much more susceptible and some breeds (Dutch) are more susceptible than others (Belgian). The degree of exophthalmos obtained has been highly variable in rabbits of the same age, sex and breed, but it is always proportional to the degree of thyroid hyperplasia (goiter) present. We believe it is established that the initial cause of the exophthalmos is dependent primarily upon an increased tonicity and spastic contraction of the smooth muscles of the orbit and eyelids which are brought about by stimulation through the autonomic nervous system. 1. Dividing the cervical sympathetic trunk in 3 rabbits below the superior cervical ganglion definitely diminished the exophthalmos on the operated side when the animals were quiet, and particularly reduced the spasm of the lower eyelid. If the animal was disturbed, however, the exophthalmos became as marked as on the intact side. 2. Removal of the superior cervical ganglion permanently abolished the exophthalmos on that side in 6 animals. 3. Curetting the medulla of both suprarenals was without effect on existing exophthalmos in 3 animals, nor did it hasten or delay the onset of exophthalmos. Curetting the right suprarenal medulla and removing the left suprarenal gland in 18 rabbits was also without definite effect. 4. Thyroidectomy in 13 prepubertal rabbits hastened the onset of exophthalmos and increased it when performed after exophthalmos had developed.

Effect of Ergotamine Tartrate on the Heat Production of Normal and Thyroidectomized Rabbits.

There are available in atropine and pilocarpine a good paralyzing and a good stimulating drug for the parasympathetic nervous system. For the sympathetic nervous system we have in adrenalin a powerful stimulant, but as yet no proven drug which paralyzes the sympathetic or neutralizes the action of adrenalin. Recently through the work of Stoll 1 a very promising drug, ergotamine (ergotoxin of Dale 2 ), has become available in sufficient amounts and purity to work with. This drug has been shown to have many activities opposite to those of adrenalin. For example, it lowers blood sugar, 3 body temperature and heat production, especially in case of exophthalmic goiter. The only observations on the effect of ergotamine on heat production in animals which we have been able to find are those of Bouckaert, 4 who noted a rapid fall in heat production following the injection of ½ mg. of ergotamine tartrate in a thyroidized dog from +26.4 to −19 per cent. This same observer was unable to obtain a reduction in a normal dog. It seemed so unlikely that a drug which lowers metabolism in thyroidized animals should not also lower metabolism of normal animals that we were led to repeat this experiment, using rabbits and doses of 1/8, 1/4 and 1/2 mg. of ergotamine tartrate∗ given subcutaneously. In the control rabbits 1 cc. of 0.9 salt solution was injected subcutaneously. The results are given in the following table : Ergotarnine tartrate in the doses used causes a striking fall in heat production in normal rabbits without the occurrence of detectable untoward effects. The metabolism lowering effect of ergotamine tartrate was evident within an hour after its injection (the exact time of onset was not determined as our shortest observation period was 1 hour) and may last more than 8 hours.

Influence of Gonads on Exophthalmos in Rabbits

Summary Male rabbits develop exophthalmos more frequently than females. This difference is independent of the thyroid gland. Exophthalmos develops most frequently in rabbits about the age of puberty (4–5 months). Gonadectomy greatly reduces the incidence even in thyroidectomized rabbits. Oestrone (menformon, theelin), pituitrin-S and adrenalin in the dosages and method of administration used neither produce, nor modify existing, exophthalmos.