published by Hektoen and SchuIhof (1), and since the appearance

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1 ON THE INNERVATION AND SECRETORY PATH OF THE THYROID GLAND. BY C. S. HICKS (Beit Memorial Fellow). (From the Biochemistry Laboratories, Cambridge, and Physiology Department, University of Adelaide.) IN an attempt to develop a precipitin test for the presence of thyroid secretion in the blood of "hyperthyroid" patients, it was found that thyreoglobulin prepared by Oswald's method, acted as a powerful antigen when injected into the blood of the rabbit. The present work had already passed its preliminary stages in conjunction with chemical work in the same field (2) prior to knowledge of the first paper on thyreoglobulin published by Hektoen and SchuIhof (1), and since the appearance of an interim report(3) has been further extended. The present paper includes all work up to the present, and serves both as an extension and a corroboration of the work of Hektoen and Schulhof who, however, worked with markedly goitrous animals (2). On the assumption that thyroxin would doubtless be secreted into the blood or lymph, combined with the globulin originally shown by Oswald to contain the total activity of the gland, it was proposed to apply the precipitin test for thyreoglobulin to the examination of blood and lymph from the thyroid in the intact animal. Most of the work on the innervation and control of secretory activity of the thyroid gland has given rise to indirect, or contradictory results capable of interpretation according to prejudice. The anatomy of the nerve supply is clear-namely, that sympathetic nerves to both vessels and gland tissue travel by the superior laryngeal nerve to be distributed along the superior thyroid arteries(4), but removal of the nerve supply seems to leave the gland function unimpaired. Using tests for iodine in the thyroid venous blood van Dyke(5) obtained negative results. Cannon(6) showed that electrical changes in the gland could be produced by stimulation of the thyroid nerves, but his work on the anastomosis of phrenic and sympathetic producing changes in gland structure, has not been corroborated (7). His further work on stimulation of the

2 SECRETION OF THYREOGLOBULIN. 199 nerve supply to the gland and its effect on the denervated heart(8) raises the difficulty of explaining the observed rapid increase in cardiac rate, all the evidence so far available pointing to delayed action on the part of thyroid secretory products. The possible route of the secretion has been decided only on histological and cytological grounds (9). Preliminary work on the lymph drainage of the thyroid gland in the dog was begun under the guidance of Dr F. C. Mann at the Mayo Institute of Experimental Medicine, Rochester, Minn., in the summer of Thyroids from recently killed animals were rapidly washed with water (while being torn into small pieces) until almost colourless. The fragments, free from all fat and connective tissue, were then ground lightly with incinerated beach sand in normal saline containing thymol dissolved in toluol. The mixture was allowed to stand at O0 C. for three days until the thyreoglobulin had dissolved in the saline, and was then thoroughly filtered (see Hektoen and Schulhof). The filtrate was then treated with an equal volume of saturated ammonium sulphate and the precipitate well washed with 50 p.c. ammonium sulphate in normal saline, and re-precipitated, the operation being twice repeated. The precipitate was then dialysed against distilled water in a collodion sac until free from ammonium salts as determined by nesslerising the dialysate. The solution was then evaporated rapidly in vacuo to dryness and solutions of thyreoglobulin made up by dissolving the dried globulin in normal saline to 1 p.c. concentration. 10 c.c. injections of this solution were made into the ear vein of the rabbit on four successive days, repeating the operation after an interval of ten days. During the week following the last injection the titre of the serum reached its maximum of 1: 80,000. Serum precipitins, arising from serum contamination in the antigen, were removed by adding to the antiserum equal parts of dog serum in saline, standing for four hours and centrifuging off the precipitate so formed. Such antiserum gave no precipitate when treated further with dog serum. The tests were made by the layer method, the antiserum being added below the surface of the solution to be tested, the results being read by suitable light after one hour at room temperature. Progressively diluted antigen was used in order to avoid failure of reaction due to high concentration. Collection of Lymph. Warmed ether was carefully administered by means of a tracheal cannula and the main lymphatic vessels of the neck were displayed by blunt dissection and ligated as low in the neck as possible. The lymph from the gland drains by several chanels to a

3 200 C. S. HICKS. node near the angle of the jaw, and above this the trunks draining the head were ligated, the filling of the lymphatics after the lower ligature increasing the visibility of the otherwise extremely delicate vessels. It is clear that the ligated vessels will contain thyroid lymph plus some neck lymph, and a cannula inserted in this vessel supplied the material to be tested. See Carlson and Woelfel for details of technique(lo). Lymph was collected for a period of three hours, during which time the anssthesis was uniform and lightly maintained. From the two glands a total amount of 0*4 c.c. of lymph was obtained in the above period, representing an average flow of 1*6 c.c. per gland per 24 hours. Test. The lymph was mixed with an equal volume of 1: 250 dog serum in 0'9 p.c. sodium chloride solution and after standing for four hours at room temperature, the precipitate was removed by centrifuging at 3000 r.p.m. The dilutions of lymph in saline were arranged in small sedimentation tubes, and to 0*2 c.c. in each tube was added carefully at the bottom of the tube 0-2 c.c. antiserum. After standing for an hour at the room temperature these tubes were examined for precipitate at the plane of contact of the solution. Table I shows the result obtained with animal A compared with pure antigen solution. TABLE I. Thyreoglobulin Thyroid Blood solution lymph serum Anti-dog thyreoglobulin serum alone 1: 80,000 1: 260 1: 120 Same after adding equal vol. 1: 250 dog serum and centrifuging 1: 60,000 1: 20 No action TABLE II. Thyroid Thyroid Arterial Dog lymph blood blood A 1:20 1:15 No action B 1:12 1:15 C 1:18 1:18 D 1:24 1:20 E 1:30 1:18 NOTE. Serum precipitins removed before testing. It is clear that thyroid lymph and venous blood contains a substance which reacts with an antiserum against dog thyreoglobulin, while this substance is absent from arterial blood, or too dilute to react. In the case of animals D and E 60 minims of the B.P. tincture of iodine in capsules had been administered before the operation. This was done to further test the accuracy of an observation originally made by Mann, and further confirmed by Mann and Hicks during the preliminary work to this study, that iodine administration caused an increase in lymph

4 SECRETION OF THYREOGLOBULIN. 201 flow from the thyroid in the dog. In the present case the same observation was made-the lymphatics stood out more plainly after ligaturing, and felt tense, and the flow was more plentiful than in the other cases, being 0*5 and 0*6 c.c. per gland respectively. It will be noted that there is an apparent slight increase in the strength of the reaction with antiserum, but the extent of the experiment warrants no further conclusion. Further work is necessary to establish this observation as being constant, and is in progress at present. Stimulation of the cervical sympathetic(11) was carried out and the lymph from the stimulated side compared with that from the other side in animals B, C, D and E. No change in titre could be established either between the lymph of the same side before and after stimulation, or between the stimulated and non-stimulated sides. It would appear from these results that thyreoglobulin finds its way more by the blood stream than by the lymphatics, the rate of flow in the latter being very slow compared with that of the blood in the veins, and the concentration being of the same order in both. No conclusion can be drawn as to the effect of possible secretory fibres in the sympathetic nerve supply to the thyroid, although it might be urged that the effect might be slower than the nature of the experiment could detect. These results confirm those of Carlson, Hektoen and Schulhof (12). SUMMARY AND CONCLUSIONS. 1. Secretion of thyreoglobulin into blood and lymph has been studied in dogs with normal thyroids. 2. Thyreoglobulin as an index of thyroid secretion passes from the thyroid gland to both lymphatics and veins, chiefly by the latter route. 3. Electrical stimulation of the sympathetic nerve supply to the thyroid of the dog produces no change in thyroid secretion rate as measured by the output of thyreoglobulin determined by the quantitative precipitin reaction of the latter. 4. Administration of iodine appears to increase the flow of lymph from the apical lymphatics of the thyroid gland as well as to increase the content of thyreoglobulin therein. This latter is a conclusion based on experience with four animals and is tentative pending further work on the subject. My thanks are due to Dr F. C. Mann of the Mayo Foundation, Rochester, Minn., and also to Dr G. Scott Williamson of the Dunn Laboratory, St Bartholomew's Hospital, who secured for me the dog

5 S. HICKS. thyroids used in the preparation of the thyreoglobulin for these experiments. The work has been carried out during the tenure of a Beit Memorial Fellowship for Medical Research, and was done under the auspices of the Medical Research Council. REFERENCES. 1. Hektoen and Schulhof. Journ. Amer. Med. Assoc. 80. p Hicks. Journ. Chem. Soc p and Ibid Hicks. Dissertation, Cambridge Anderson. Amer. Journ. Anat. 13. p van Dyke. Amer. Journ. Physiol. 56. p Cannon and Cassell. Ibid. 41. p Cannon. Ibid. 41. p Cannon and Smith. Ibid. 60. p Bensley. Amer. Joum. Anat. 19. p Carlson and Woelfel. Amer. Journ. Physiol. 26. p Ascher and Flack. Centralblatt f. Physiol Carlson, Hektoen and Schulhof. Amer. Journ. Physiol. 71. p