thyroid activity to changes in the environmental temperature are in all

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1 Z, 1 J. Physiol. (I954) I26, I-28 THE MEASUREMENT AND EXPERIMENTAL MODIFICATION OF THYROID ACTIVITY IN THE RABBIT BY K. BROWN-GRANT, C. VON EULER, G. W. HARRIS AND S. REICHLIN From the Physiological Laboratory, University of Cambridge, and the Department of Neuroendocrinology, Institute of Psychiatry, Maudsley Hospital, London (Received 28 January 1954) There is much evidence indicating that the activity of the thyroid gland is influenced by the central nervous system. The well-known adjustments of thyroid activity to changes in the environmental temperature are in all probability mediated through the nervous system. The final purpose of the present experiments was to investigate the functional relationship between the central nervous system, anterior pituitary gland and thyroid gland. Since several of the techniques required (remote control stimulation, placement of hypothalamic lesions and operative techniques) had already been developed for rabbits, this species was chosen for study. Further, since it was believed that the thyroid gland responds slowly to such stimuli as environmental changes, a method was desired whereby measurements of thyroid activity could be extended for prolonged periods in the conscious animal. In a previous study, Green & Harris (unpublished) had failed to find an increased oxygen consumption in rabbits following prolonged electrical stimulation of the hypothalamus. However, measurements of the gaseous metabolism in the rabbit may not give an accurate index of thyroid activity. Recent developments in the use of radioactive iodine afford a more direct measure of thyroid function, but most of the methods described in the literature are not directly applicable to the conscious rabbit in which repeated measurements are desired. This paper deals with methods of studying thyroid activity, and with the changes in such activity induced by a cold environment, injection of thyrotrophin or thyroxine, and by hypophysectomy. A preliminary report of these results has been made (Brown-Grant, von Euler, Harris & Reichlin, 1953). 1 PHYSIO. CXXVI

2 2 K. BROWN-GRANT AND OTHERS METHODS Female rabbits of mixed stock, weighing kg, were used. Animals were fed ad lib. on a pellet diet (diet no. 18 devised by the Medical Research Council, and made up by the Associated London Flour Millers Ltd.) and tap water throughout the course of the experiments, except for a short period after hypophysectomy when the diet was supplemented with green food. The animals were kept in a temperature-controlled room at approximately 290 C, except for short periods when counts were being made (as described below). Carrier free 131I was obtained from the Atomic Energy Research Establishment, Harwell. The radioactive iodine was made up as a stock solution containing 10 or 20,uc/ml., and in the earlier experiments potassium iodide, 1 /ig/ml., was added as carrier. The dose given at any one time ranged from 0 5 to 12 0 fc, usually 2-0 or 400,c contained in ml. of stock solution, according to the physical decay. Thus the greatest amount of carrier 1271 given at an injection was 10 jg but this was usually ug. Conventional scaling units and preamplifiers were used for counting. All figures quoted in this paper have been corrected for background and isotope decay, but correction for the resolving time of the apparatus was unnecessary at the counting rates obtained. Neck and thigh counts in vivo In order to obtain counts of the radioactivity of the thyroid region, the fur of the rabbit's neck was clipped short with scissors. The neck was not shaved since it was found that shaving produced skin oedema after a few hours. The unanaesthetized animals were placed in hammocks with restraining clamps similar to those described by Harris (1948). The neck stock of the clamp wvas replaced by a padded lead stock which incorporated the Geiger-Muller tube (Fig. 1). To obtain constant positions of individual animals in the stock, and therefore in relation to the Geiger- Muller tube, a scale was mounted on the side of the rack frame and the position of the movable neck stock for each animal was noted. For counting the y-radiation from the neck a G 10 Pb Geiger-Muller tube (20th Century Electronics Ltd.) was used with a window in the lead shield of approximately 40 x 25 mm. The tube was carefully light shielded and showed a background count of about 50/min. The reliability of this method for obtaining such counts was tested in the following manner. A rabbit with neck counts of approximately 800 counts/min was placed in position in the clamp and the time for 400 counts measured with a stopwatch. This was repeated 10 times. The animal was then removed from the clamp, replaced and a further series of ten counts made. This was repeated until ten series of ten counts had been obtained. Analysis of these figures showed that there was no significant difference between the means of ten counts made with the rabbit in the same position and the means of ten counts made with the rabbit in ten changed positions. The procedure finally adopted for obtaining a 'neck count' was as follows. Three or four counts of 2 min duration, removing and replacing the animal between each, were averaged and expressed as counts/min. Analysis of a sample of neck counts taken at random over the range counts/min from thirty-three different rabbits showed the standard error of an individual neck count to be +2-2%, and the standard deviation of the standard error of the neck counts in this group was ±1-13 %. The variation of counting rate due to differences in the positioning of an animal were in fact of the same order as the statistical error expected at these counting rates. The fact that the result of each experiment depends on a curve plotted from many neck counts, and the fact that these points fit a smooth curve with relatively little scatter give further confidence in the method. Observations on any animal were stopped before the counting rate fell to less than 3 times the background, the usual counting range being 5-80 times background. In many early experiments made during the period immediately following the injection of radio-iodine, each count of the neck was followed by a count of the thigh. After counting the neck the rabbit was removed from the hammock, the bladder emptied by manual expression and the animal replaced in the hammock, being held so that the extensor muscle mass of the thigh was in apposition with the window of the Geiger-Muller tube. The counting procedure was as for the neck.

3 THYROID ACTIVITY IN THE RABBIT 3 (a), 2cm (b) Fig. 1. (a) side view and (b) view from in front, of the castle constructed of lead, 0 5 in. thick. A removable neck stock and adjustable nose clamp permit the animal to be placed so that the region of the thyroid gland is in apposition with the upper surface of the Geiger-Mfiller tube. Sponge rubber pads (not illustrated) of suitable size are used under the stock to minimize movement and prevent undue pressure or friction on the upper surface of the animal's neck. This castle replaces the neck stock of the rabbit clamp previously described (see text). 1-2

4 4 K. BROWN-GRANT AND OTHERS Thyroid content of 131I In order to measure the amount of 131I in the thyroid gland, the animals were killed and a block of tissue containing the thyroid gland and a segment of the trachea and larynx was removed and placed in 2 ml. 2 N-NaOH at 370 C overnight. Precautions were taken against evaporation of the solution during this period, and an aliquot was counted in the manner described below for blood. Blood counts Blood samples (about 1-5 ml.) were collected from the marginal vein ofthe ear into glass-weighing bottles containing a small amount of heparin powder (Roche Products Ltd.) and a small crystal of potassium iodide as carrier. (The radioactivity of the added potassium iodide was negligible.) Duplicate counts were made on each sample using a Perspex cuvette (internal diameter 18 mm) with a base of mica sheet (12.5 u thick), placed immediately above the end window of a,-sensitive Geiger-Muller tube (type EHM 2 S, General Electric Co. Ltd.) within a lead castle. A volume of 0-6 ml. fluid in this type of cuvette was found to act as an infinite thickness sample. Background counts were made for 30 min (approximately 10 counts/min), and usually 1000 counts were made on each blood sample. As a control procedure it was found that a given concentration of l31i in blood or distilled water gave counting rates which were not significantly different. Organicallybound radio-iodine was estimated by precipitating 1 vol. plasma with 5 vol. of 10 % trichloroacetic acid. After centrifuging, the precipitate was washed twice with trichloroacetic acid and dissolved in 2 N-NaOH. The volume of this solution was measured and an aliquot counted. Urine counts After injection of 13'I the animals were placed in metabolism cages and the urine collected in glass flasks. At various intervals the bladder was emptied by manual expression and any urine obtained added to that in the flask. These samples were measured and counted in the same way as blood samples. A given concentration of L31I in urine or distilled water gave counting rates that did not differ significantly. Procedures The anaesthetics used in the following operations were (i) ether (after atropine sulphate 2 mg, s.c.), (ii) ether as a supplement to intravenous chloralose and urethane (2.5 ml./kg body weight of 2 % chloralose and 10 % urethane), (iii) intravenous pentobarbitone sodium (45 mg/kg body weight). Thyroidectomy. The thyroid gland was removed through a lateral skin incision and by retracting but not excising, the infrahyoid muscles. Care was taken to preserve the laryngeal nerves intact. Hypophysectomy. The pituitary gland was removed using the parapharyngeal approach described by Jacobsohn & Westman (1940) modified only by placing the skin incision well laterally to avoid oedema and scarring in the region of the thyroid gland. Following operation the animals were replaced in the constant temperature room (290 C). This high temperature reduced the mortality appreciably as compared with a previous series (Harris, unpublished) kept at ordinary room temperature. Cold exposure. Rabbits were removed from the constant temperature room (290 C) and placed (i) outside the building, protected from wind and rain, but exposed to the prevailing temperature during the winter ( - 2 to 210C) or during the spring (10-25 C), (ii) in colder rooms in the laboratory at 1 or C. The period of cold exposures varied from 23 to 98 hr. Drugs. Thyrotrophic hormone was obtained from Armour and Co., Chicago, U.S.A. (batch numbers PRR3 and R ). This was dissolved in 0 9% sodium chloride solution immediately before use and injected either intravenously, subcutaneously or both. Synthetic L-thyroxine sodium ('Eltroxin', Glaxo Laboratories Ltd.) was injected intraperitoneally in doses of 50 or 100/ig in 0 9 % sodium chloride solution. Potassium iodide (1271) was injected intravenously or subcutaneously dissolved in water or 0-9 % sodium chloride solution. Histology. The thyroid glands were fixed in Susa or 10 % formol, embedded in wax sectioned at 7-10, and stained with haematoxylin and eosin, or haematoxylin and Van Gieson. Hypo-

5 THYROID ACTIVITY IN THE RABBIT 5 physectomized rabbits were perfused with 100 ml. of 1 part of indian ink to 2 parts distilled water through the carotid arteries immediately after death. After fixation in 10% formaldehyde, decalcification (in equal parts 40 % formic acid and 7 % sodium formate solution), and dehydration, a block of tissue containing the hypothalamus and base of skull was embedded in celloidin and serially sectioned at , thickness. The sections were stained with haematoxylin and eosin. RESULTS A. Distribution of 131I in the thyroid gland, blood and urine of rabbits following intravenous injection (a) Thyroid gland The data given in this section regarding accumulation of radioactive iodine by the thyroid gland refer to neck counts made as described above. In most cases observations were begun a few hours after the injection of 131I and continued for a number of days. In these cases the background count derived c., 2 Rabbit 304, EJ= _E 13iL '311 i.v. 12.xii.51 N c ~~ iv l2xii.51 ~~~~Neck -o-o-o- Blood * *. o.] S 1 11Urine E k~0-2 8 z e Time (hr) Fig. 2. The distribution of 131I in the thyroid region, blood and urine of a rabbit following the injection of a small tracer dose of radio-iodine. 3 4c of 131I were injected intravenously at 0 hr. from neck tissues other than the thyroid gland was low, and an approximate correction was made by subtracting the thigh count from the total neck count to obtain what is referred to as the thyroid count. In six experiments the uptake of 1311 by the thyroid was studied in detail during the first 8 hr after injection, as well as over the following days. Since the background count from neck tissues other than the thyroid gland is high relative to the thyroid count during the first hours after injection, a more detailed correction had to be made during this period. Details of this correction factor will be found below. Observations on the concentration of 131I by the thyroid gland have been made on twenty rabbits following intravenous injection of ,uc in thirty-one experiments. Typical results are shown in Fig. 2. It may be seen that the thyroid gland accumulates 1311 rapidly during the first few hours

6 6 K. BROWN-GRANT AND OTHERS o Rabbit 350, 21tc "'I, 2. v.52._ 2C 0 z 150 Time (hr) Fig. 3. Curves showing the radioactivity in the thyroid region of three rabbits following intravenous injection of Note the variability in the time and magnitude of the peak value in the three cases ' z Thyroid gland content of I'll in uc 0-7 Fig. 4. The relationship of thyroid counts/min, as measured with the technique described in the text, to the thyroid content of i3li in uc.

7 THYROID ACTIVITY IN THE RABBIT 7 after injection. This is seen more clearly in Fig. 3. The accumulation of radioactive iodine continues until a peak value is reached between 12 and 48 hr in the majority of cases. From this point onwards the concentration of 131J in the gland falls at a rate which varies in different animals, and under different conditions. With the method used and with the small dose of tracer iodide given, measurable amounts of radioactivity were found in the thyroid gland for periods of 10 days and usually longer. A factor for converting thyroid counts/min into 'tc 131J in the thyroid gland, was found by injecting twelve rabbits with varying doses of 131I. After an interval (48 hr or more), when the contribution to the neck count from nonthyroidal tissue was negligible, neck counts were made, the animal killed immediately and the 131J content of the thyroid gland measured (see Methods). The results so obtained are given in Fig. 4. It was found that thyroid counts/ min, divided by 4300 ( 360, S.D. of 12 observations) gives the thyroid content of 1311 in,tc. 120 E ~80 U V 0 'a V 40 Rabbit 343, 2gc13'l, 13. v Time (hr) Fig. 5. Curves illustrating the fall of 131I in the blood (6) Blood following intravenous injection of radio-iodine. The blood concentration of 131I has been followed in nine rabbits for periods of hr after intravenous injection of radio-iodine. Mixing of 1311 in the blood stream appears to be complete at about 1-1 min when peak values are recorded for radioactivity in venous blood samples. After this time the concentration of radioactive iodine in the blood stream begins to fall rapidly (Fig. 5) as the iodine is passing into the tissue fluids, being excreted by the kidney and taken up by the thyroid gland. About one-quarter of the initial

8 8 K. BROWN-GRANT AND OTHERS blood radio-iodine is still present 10 hr after injection and about one-twelfth after 48 hr. Thereafter the rate of loss from the blood is much slower. The proportion of radioactive iodine in organic combination in the blood (P.B.I.) has been investigated at varying times after injection of 1311 in four rabbits. 5-10% of the total radioactivity may be present in this fraction as early as 5 hr after injection, and 80% or more after 70 hr. (c) Urine The percentage of the injected dose of 131I excreted in the urine in the first 24 hr after injection has been determined in eighteen experiments in fourteen rabbits. An average of 55-3 (+ 15-7S.D.) % of the dose administered was excreted in this time. In four rabbits the concentration of 1311 in the urine has been studied in more detail for periods of hr, and the curve representing the excretion found to be roughly parallel to that of the blood concentration (see Fig. 2). (d) Application of the above studies to the measurement of thyroid activity The aim of the present investigation was to obtain an accurate method of measuring changes in thyroid activity. A method was required that would permit repeated observations on any one animal over a period of several weeks. The danger of radiation damage, however, limits the amount of radio-iodine that can be safely injected each time. Many methods are in clinical use for measuring thyroid activity (see Myant, 1952). In considering the application of these methods to the rabbit, it was felt that those based on the rate of disappearance of 1311 from the blood, or excretion of 131J in the urine, involved too many variables apart from thyroid activity and were indeed found to be too insensitive to reveal minor changes in thyroid function. It was in fact observed in experiments on thyroidectomized rabbits (see below) that the rate of disappearance of 131J from the blood did not alter markedly after thyroidectomy. The rate of appearance of organically bound 131I in the blood would appear to offer a more direct and sensitive measure, but a few observations showed that the dose of 131I necessary to produce satisfactory levels of P.B. 131I in the blood was too large to enable repeated observations to be made on the same rabbit. The determination of the radio-iodine content of the thyroid gland at various time intervals after injection of 1311 appears to be the most direct and convenient method of measuring thyroid activity in the rabbit. However, a study of the general shape of the curve representing the 131I content of the gland following radio-iodine administration gave little support to this view, in that the position and magnitude of the peak value was variable in different animals and in the same animal from time to time. Also a few experiments on animals that had been exposed to a cold environment, or injected with

9 THYROID ACTIVITY IN THE RABBIT 9 thyrotrophic hormone previous to injection of 131I, gave no consistent change in the position or magnitude of the peak value of the thyroid content of 131I. It is clear that the general pattern of the thyroid curve is due to the combined effects of two processes: first, the accumulation of 131I from the blood, and secondly the secretion of radioactive thyroid hormone back into the blood. It seemed reasonable then to attempt to measure these two factors as independently as possible by studying the rate of uptake of 131I by the thyroid during the first few hours after injection, and by studying the rate of decline of the 1311 content of the thyroid at a time when the uptake of injected 131I from the blood had fallen to a negligible value. These two methods, and the results obtained by their use, are described in the next two sections (B and C) of this paper. B. Measurement of thyroid activity by 131I uptake In order to measure the rate of uptake of 131I by the thyroid gland, observations were made of the neck count during the first 5 hr after intravenous injection of radio-iodine. Intravenous injection of 131I into a rabbit already in position over the Geiger-Muller tube showed that the neck count rose immediately. It was clear that at this time the thyroid could have accumulated only negligible amounts of 131J, and that this high count was nearly all due to the radio-iodine in the blood of the great vessels of the neck. This high initial figure is probably due to the veins of the neck being distended by the horizontal position of the animal and some slight pressure of the restraining neck clamp. It thus became necessary to see if an accurate correction factor could be obtained which would allow extraction of the thyroid count from the total neck count under these circumstances. For this purpose a study was made of a series of thyroidectomized rabbits. (a) Thyroidectomized rabbits Ten experiments were performed on five thyroidectomized rabbits days after thyroidectomy, when the neck wound had healed and oedema of the neck tissues had subsided. Each animal was injected intravenously with 2)uc 131I and the neck counts, blood counts and thigh counts followed for periods up to 6 hr. The first neck count was started 1 min after giving the injection and immediately after this was completed a blood sample was taken and a count made of the thigh. This procedure was repeated approximately 30, 60, 90, 150, 210, 270 and 360 min after giving These observations were made on two separate occasions on each animal. Curves of these results were plotted in the following manner. The ratio between the first neck count and the first blood count was determined, and all blood counts multiplied by this neck/blood factor. Similarly, the ratio between

10 10 K. BROWN-GRANT AND OTHERS the first neck count and the first thigh count was determined and all thigh counts multiplied by this neck/thigh factor. These values, together with those obtained for the neck counts, were then plotted against time as the abscissa (Fig. 6). From the method of plotting, the three curves start from the same initial level. The blood curve then falls in the typical manner so that by the end of 6 hr only between one-third and one-half of the initial blood 131J is still present. The neck count of these thyroidectomized rabbits falls in a similar manner, in each case the neck curve being parallel with, and slightly higher than, the blood curve. The thigh curve, on the other hand, often shows a rise in counts for the first hour followed by a fall, so that after the second hour this curve is also parallel with the blood and neck curves but at a considerably higher level. These results were uniform in the ten experiments. 800 Rabbit 306, thyroidectomized,, 600 2}.Lc 1311 i.v _ Time (min) Fig. 6. Change in blood, neck and thigh radioactivity following intravenous injection of 131I in the thyroidectomized rabbit. These curves have been plotted so that they start from the same initial level. Note the parallelism in the loss of radio-iodine from the blood and neck tissues. Since the neck curves of these animals and the blood curves showed good correlation in each case, it was thought reasonable to use the blood count at any time multiplied by the neck/blood factor estimated as soon as possible after the injection of 131J, as a suitable correction for obtaining the thyroid count from the total neck count in the normal rabbit. Thus Tt = N1-B (Bi N where T, = thyroid count at time t, N = neck count at time t, B1= blood count at time t, No= neck count a few min after injection, Bo= blood count a few min after injection. The time at which the initial neck count was made and the initial blood sample taken varied slightly in different experiments. The neck count was started 1 min after completion of the injection of radio-iodine and

11 THYROID ACTIVITY IN THE RABBIT 1 1 took about 6- min to perform (three 2 min counts). The mid-point of the middle count was taken arbitrarily as the time of the neck count. As soon as possible after the neck count the blood sample of 1-5 ml. was collected. It was found that plotting the blood curve and extrapolating to the time of the initial neck count gave a figure for the neck/blood ratio that was slightly smaller than the figure found if the obtained values were used. However, the difference was within the experimental error, and since the tendency was to increase the difference between the neck curve and the calculated blood curve in the thyroidectomized rabbit, the extrapolated figure was not used. A possible source of error in obtaining the initial neck count lies in the uptake of 131I by the thyroid during the first 7' min required to make the count. This error would be increased under conditions of increased thyroid activity and would result in an underestimate of the thyroid uptake of 131J. The neck and thigh counts of the thyroidectomized rabbit are due to radioiodine in the blood and in the tissue fluids. Since the neck count is slightly, and the thigh count considerably, higher than the corresponding 'corrected' blood count, it seems likely that the proportion of counts derived from 131I in the tissue fluids of the neck forms a relatively small fraction, whereas it is appreciably greater in the thigh. The passage of radio-iodine from the blood vessels into the tissue fluids of the thigh may be readily observed during the first hour after injection of 131J when the total thigh counts may increase even though the 131I in the blood is falling rapidly. The above method of obtaining the extrathyroidal background of the neck from the blood count has been used for calculating thyroid counts from the total neck counts in the experiments described in the next section. (b) 131J uptake by the thyroid gland Twenty-seven experiments were performed on sixteen rabbits in which the rate of uptake of 131I by the thyroid gland was determined. Five rabbits were used more than once. The general procedure was as follows: the rabbits received an intravenous injection of 2 uc 131I and neck counts and blood samples were taken during the next 5 hr, at approximately 4, 16, 36, 64, 100, 144, 196 and 256 min after injection. In a few cases observations were extended up to 24 hr or more. For each experiment curves were plotted showing total neck counts, blood counts multiplied by initial neck/blood ratio, and thyroid counts estimated as described above (Fig. 7). In repeated experiments on the same rabbit, at intervals of 3 days to several weeks, residual radioactivity was often found to be present in the thyroid gland at the beginning of the subsequent experiment. In these cases a neck count was made before the second experiment was begun, and the appropriate correction applied to the counts obtained thereafter.

12 12 K. BROWN-GRANT AND OTHERS Two methods were used to give an index of thyroid activity from the data of each experiment. First, the technique of Stanley & Astwood (1947) was used, whereby the uptake of radioactive iodine by the thyroid was plotted against ltime. The results on the rabbit largely confirm the findings of Stanley & Astwood on man in that the points so plotted roughly fit a straight line (Fig. 7). It was then possible to take the slope of this line, or 'accumulation Rabbit 343, / 2j2c 131 i.v. 15.iii.52 / 8 i52 A I /II If Time (min). Fig. 7. Two experiments on the same rabbit showing the constancy of the 'accumulation gradient '. The curves show the rise in radioactivity in the neck and the fall in radioactivity in the blood following injection of 2 luc 1311 at 0 hr. The ' accumulation gradient ' of the thyroid is obtained by subtracting the correction factor (obtained from the blood counts, see text) from the total neck counts and plotting against (V\T). gradient', as an index of thyroid activity. The second method was to calculate the clearance values of 1311 from the blood by the thyroid, by calculating the thyroid increment of 131I per unit concentration in the blood for each 10 min interval of the experiment. It was noted that the clearance values so obtained were approximately constant during the first 40 min after 131I injection, but then gradually decreased. It seems likely that this apparent fall in clearance values may be due to the secretion of radioactive 1311 in organic combination into the blood.

13 THYROID ACTIVITY IN THE RABBIT 13 The results obtained in different animals under similar experimental conditions varied widely. However, the results obtained in each of the five rabbits tested more than once, remained constant over periods of several weeks (Fig. 7). C. Measurement of thyroid activity by 131I output Measurement of thyroid activity by a study of the rate of release of 131I into the blood (as part of the thyroid hormone) has been performed by a similar method to that used for the rat by Wolff (1951), Albert (1951) and Perry (1951) Rabbit 343, release curve, t\ ~~~~~~~~~~~~2ikc I'll, 21. iv. 52 E 1000 X 0 z Soo~~~~~o 500_ O Time (hr) Fig. 8. Changes in thyroid radioactivity starting 48 hr after injection of L311 plotted semi-logarithmically. Rabbits were injected intravenously or subcutaneously with 2 or 4,uc 131I. After a period of 48 hr the thyroid content of 131I had usually passed peak value and the blood content of 131I had fallen to insignificant levels. At this time the background due to 1311 in the general neck tissues and other tissues of the body was found to be negligible and the neck count therefore gives a measure of the radioactive iodine content of the thyroid gland. Neck counts were then followed on these animals, observations being made twice daily for periods up to 22 days. It was found that the 1311 content of the thyroid gland decreased exponentially with time. This has been observed in more than 250 experiments on over

14 14 K. BROWN-GRANT AND OTHERS eighty rabbits and is related to the fact that 1311-labelled hormone is being continuously replaced by non-labelled hormone. A typical result is illustrated in Fig. 8. Plotted semi-logarithmically the points fall on a straight line the slope of which represents the ratio of the amount of hormone secreted per unit time to the total amount in the gland. Provided therefore that the amount of hormone in the gland remains constant, changes in the slope will be related to changes in thyroid activity. TABLE 1. The distribution of the rates of loss of thyroidal Data obtained from 100 release curves in 30 normal rabbits. (Average 17-6 %/day). % loss/day No. of curves co~ (9)) x2 co I ( 2 ( 00 ", Time in weeks after arrival in the laboratory Fig. 9. Histogram to show the decrease in the rate of loss of radio-iodine from the thyroid that occurs in the first 12 weeks after arrival of the animals in the laboratory. This figure summarizes sixty-two experiments in a group of twelve rabbits. The number of animals studied at each time interval is given in brackets. The rate of loss at each period is compared with the rate at 2-4 weeks at which time an observation was made on every rabbit. The slope of the release curves observed in different animals varied widely. Expressed as percentage loss of thyroidal radio-iodine per day the variation observed in 100 experiments on thirty normal rabbits was 4-40 %/day (Table 1). The slope of the curve also varied in the same animal at different times after arrival in the laboratory. Repeated experiments on a group of twelve rabbits showed that the percentage loss of radio-iodine per day, in successive experiments, decreased over a 2- to 3-month period after arrival (Fig. 9). Radiation damage can be excluded as a cause for this phenomenon, since rabbits maintained in the laboratory several weeks before experiment showed a similar slow rate of 1311 release as though subjected to repeated

15 THYROID ACTIVITY IN THE RABBIT 15 experiments over this period. It seems likely that the decreasing values observed are related in part to an increase in the hormone content of the thyroid gland, due to an increase in dietary iodine intake. Evidence for this view is derived from the results observed following administration of potassium iodide. (1) Two rabbits given potassium iodide (10 mg s.c.) on the day of arrival showed a constant loss of radio-iodine of 3-3 and 4-5 %/day over the following 2 weeks. Control rabbits, not injected with potassium iodide, showed a loss of 25-1 %/day (± 73 S.D. of nine observations) over the same period after arrival. (2) Six rabbits were given potassium iodide in varying doses ( mg s.c. or i.v.) during release curves. Two rabbits with high rates of 1311 release from the thyroid (probably indicating iodine-poor glands) showed marked slowing in the rate of 131I output following potassium iodide administration (1.4 and 12-5 mg s.c.). One rabbit, also with a high rate of release, showed no effect following potassium iodide (0-125 mg s.c.). Three rabbits with low rates of release from the thyroid (probably indicating iodine-rich glands) showed no effect following injection of potassium iodide (0.45 and 0 50 mg s.c. and 5.0 mg i.v.). The above observations are also of interest in regard to the possibility that the slope of the release curve is significantly influenced by a reaccumulation of 131I derived from the break-down of radioactive hormone. If such were the case an apparent increased rate of release of 131J might be expected to follow administration of potassium iodide (Perry, 1951). This has not been observed. The part played by reaccumulation of 131J in determining the slope of the release curve Radioactive hormone secreted by the thyroid is partly excreted in the faeces and partly broken down in the tissues with liberation of inorganic 131I. This latter is partly excreted in the urine and partly reaccumulated by the thyroid gland. Therefore the measured loss of thyroidal radioactivity per day will represent the difference between the loss of 131I as radioactive hormone and the uptake of 1311, derived from degraded hormone. For the purpose of the present study it is important to know the extent of this reaccumulation. (a) Data derived from a study of thyroid: renal sharing of 131I, and the excretion of 1311 by the kidney during a release curve Thyroid: renal sharing. Five normal rabbits were injected intravenously with 7-7 c 131I. One hour later the animals were killed and the amount of 1311 in the urine and thyroid measured. (The urine excreted in the one hour period was collected, added to that present in the bladder after killing, and to washings of the urinary tract.) Expressing the 1311 in the thyroid as a per-

16 16 K. BROWN-GRANT AND OTHERS centage of that excreted in the urine gives figures of 18-7, 19-0, 14*1, 21*2 and 18-1 % (average 18-2 %) for the five animals, i.e. a thyroid: kidney sharing of approximately 1: renal excretion during release curve. Six normal rabbits were injected with 10-30,uc 131I, and 48 hr later a release curve started. During the 3rd-5th days of the curve, the urine was collected for 24 or 48 hr periods and the 131I in the urine measured. The animals were then killed, the 131I content of the thyroid measured and the value obtained compared with the neck count of the individual animal. In this way a precise factor was obtained allowing conversion of 'neck counts/min' into 'thyroid content of 131I in jic', for each animal. The data obtained, and the calculation of the reaccumulation of 1311 as a percentage of that secreted as hormonal l31j per day, are given in Table 2. It may be seen that an average of 102 % of the amount of 131I secreted as hormone is taken up again by the thyroid. Reaccumulation of l3si by the thyroid 1311 in,uc Measured reaccumulated Total loss loss of by thyroid of thyroid TABLE 2. % thyroid 131I in plc during 24 hr 131I in,uc loss/day 131I in pc in urine period(s) during 24 hr % reaccuof thyroid during 24 hr during 24 hr _ 18-2 period(s) mulation 131I period(s) period(s) 100 =b +d =d/e x 100 Rabbit (a) (b) (c) (d) (e) (f) * * *09 0* * * *4 Av. 102 (b) Data derived from a study using radioactive thyroxine The experiments reported here were performed in collaboration with Dr J. G. Gibson and will be reported in extenso elsewhere (Brown-Grant & Gibson, unpublished). Four normal rabbits were injected with a tracer (2 uc) dose ofradio-thyroxine (1 jug). The peak values of 131I in the thyroid (as measured with a scintillation counter, in vivo) were found to occur about 30 hr after injection and represented 5-2, 6-4, 9*7 and 10-2 (average 7.9) % of the administered radioactivity. After 7 days an average of 46-5% of the administered radioactivity had been excreted in the urine, and 52-5 % in the faeces. Assuming a 1:5 ratio for thyroid: renal sharing of iodide this would indicate that J x 46*5 = 9-3 % of the administered radioactivity was accumulated by the thyroid. This figure is

17 THYROID ACTIVITY IN THE RABBIT17 probably too high, but it is in good agreement with the average figure representing the uptake as measured at 30 hr. (c) Data derivedfrom the studies of thyroid clearance During the period hr after injection of 131I into a rabbit (a period equivalent to the first 24 hr of a release curve) the inorganic l3lj concentration in the blood has been found to be 0-67 x 10-5 uc/ml. blood for each 1,c of 1311 injected (unpublished data, Brown-Grant). For the purpose of the present discussion this figure probably errs on the high side since it is likely that some of this inorganic 1311 is residual from the initial injection as well as being derived from the breakdown of radioactive hormone. However, since the loss of thyroidal 131I from the rabbit's thyroid, as measured with the present technique, is exponential after the 48th hr from injection in the majority of animals, it would seem reasonable to use this figure. Clearance values of blood 131I by the rabbit thyroid have been obtained (Brown-Grant & Gibson, unpublished data) during the period 5-65 min after injection of radioactive iodine. Over this period the release of radioactive hormone is probably negligible and the clearance values obtained have been found constant in any one animal. An average figure for the rabbit gland is 0-48 ml. blood cleared of 131I/min. From the data that the inorganic 1311 concentration of the blood hr after injection of 2,uc 131I is 1-34 x J0-5,uc/ml. blood, and that the thyroid is clearing 0-48 ml. blood/min it may be calculated that the reaccumulation of radioactivity by the thyroid during this period is ,uc/ day. An average figure for the measured loss of radioactivity over this same period is 0 08 jlc. In the presence of ,uc reaccumulated the true loss would be zc. It may be calculated that reaccumulation occurs of 10-4 % of the 1311 released from the thyroid as labelled hormone. The data derived from the three different types of experiments, described in (a), (b) and (c) above, indicate that only about 10% of the 131J that leaves the thyroid as radioactive hormone eventually recirculates to the gland. Experimental modification of thyroid activity The duration of a single release curve is about 2 weeks, which is sufficient to allow periods of control observations before and after any experimental procedure. Changes in slope during any one experiment are immediately related to changes in the rate of release of thyroid hormone, and thus afford a direct measure of thyroid activity. This technique has been used to study the effects of injection of thyrotrophic hormone (TSH) or thyroxine, of exposure to cold and of hypophysectomy on thyroid activity. (a) Thyrotrophic hormone Seven normal and ten hypophysectomized rabbits have received doses of *0 mg of TSH (in terms of mg Armour preparation PRR 3, used 2 PHYSIO. CXXVI

18 18 K. BROWN-GRANT AND OTHERS in the earlier experiment; in terms of mg U.S.P. equivalent in the later experiments in which preparation R was used). Daily injections were made subcutaneously, intravenously or both, for 1 or 2 days, during the course of forty-two experiments. Three of the hypophysectomized animals died following intravenous injection of TSH (8 mg U.S.P. equivalent). In each of the other animals a marked acceleration of the rate of release of 131J from the thyroid was observed (Fig. 10) Rabbit 344, TSH [2zc 1311, 23. iv mg s.c. -TF C 0 E 0 " 2000N zn N 0 N 1000~~~~~~~~ Time (hr) Fig. 10. The effect of TSH on the release of 1311 from the thyroid gland of the rabbit. A marked acceleration in the output of 1311 is observed within a few hours of a single injection of TSH. This latent period has been investigated in detail by making neck counts at intervals of j--1 hr, for 6-8 hr, after subcutaneous injection of a single dose of TSH. In two normal and three hypophysectomized rabbits a clear deviation from the expected line was observed within 1 hr in three cases, in 1-2 hr in one case, and in 2-3 hr in the other case (Fig. 11). The magnitude of the response is roughly proportional to the dose of TSH over the range '0 mg (U.S.P. equivalent). For example, a dose of 35 mg of TSH resulted in an 89% fall of the thyroid content of 131I and a dose of 0*5 mg (U.S.P. equivalent) in a fall of 8-7 %. In the hypophysectomized animal the duration of action of a single subcutaneous injection of TSH (0* mg U.S.P. equivalent) is hr (Fig. 11). In the normal animal this period is followed by one in which the output of thyroidal 1311 is completely inhibited. This inhibition appears to be as complete as that following injection of thyroxine (see below) and usually of 2-3 days' duration. It is

19 THYROID ACTIVITY IN THE RABBIT 19 probably related to suppression of the animal's own TSH secretion as a result of the raised concentration of circulating thyroid hormone due to the exogenous TSH. In one experiment a second dose of TSH was given during the period of inhibition following the initial dose, and produced a typical acceleration of 131I release. It appears unlikely therefore that the period of inhibition is due to inability of the thyroid gland to respond to TSH. Rabbit 542, 0 5 mg TSH s.c. A, 600 v jc 1311, 24. vi. 53 O 4? 6000 hypophysectomized, r tzt_ 5o V, n 0~ ~ ~ ~ ~ ~ Time (hr) Fig. 11. The effect of TSH on the release of 13SI from the thyroid gland of the hypophysectomized rabbit. (b) Thyroxine Nineteen rabbits have received doses of 50 or 100,ug L-thyroxine per day for one or more days, during the course of twenty-two experiments. In each case a prompt and marked inhibition of the release of 131I from the thyroid was observed (Fig. 12). Following a single dose of thyroxine complete inhibition of thyroidal 1311 release was observed in twenty out of twenty-two experiments for periods of hr. In the other two experiments the release was not completely inhibited but was markedly reduced from 10-8 and 23-8 %/day to 1-7 and 1-9 %/day respectively. The latent period of the inhibitory response to a dose of thyroxine may be measured by extrapolating the line through the points obtained during the preliminary control period and the line through the points during the period of inhibition. The interval between the administration of thyroxine and the point of intersection of these two lines is taken to represent the latent period of the inhibitory action of thyroxine. The latent period determined in this way is probably too high but was found to be less than 2 hr in twelve cases, and less than 4 hr in a further six cases, and 5 and 8 hr in the other two cases. 2-2

20 20 K. BROWN-GRANT AND OTHERS The duration of the inhibitory effect of thyroxine varied with the dose. For example, in one rabbit 50 and 100 l g thyroxine were given on two separate occasions during a single experiment. The duration of the inhibition produced by 50,tg was 22 hr and by 100,ug 54 hr. By repeated daily injections of 100l,g the inhibition was prolonged for 10 days, and in no case was there any sign of 'escape' from the inhibitory action. To see whether the inhibitory effect of thyroxine on thyroid release of 131J was due to an effect on the thyroid gland, three rabbits were given TSH whilst their output was inhibited by a previous dose of thyroxine. In each case a typical response to the TSH was observed t2 ~~~~~~~~~~~~~Rabbit 367. release curve, L-Thyroxine 4tic I'll, 3. xi \ 100gg i.p. 0 0 U u z Fig. 12. soo Time (hr) The effect of thyroxine on the release of 1311 from the thyroid gland of the rabbit. In five experiments on five rabbits the duration of the inhibition following a single dose of loo1g thyroxine was accurately determined and an average figure of 77 hr obtained (52, 54, 60, 100 and 110 hr). Assuming that the administered thyroxine effectively replaces the animal's own secretion for the period of inhibition, an approximate figure of 35,ug L-thyroxine was obtained as equivalent to the daily hormonal output. (c) Cold Twenty-six rabbits have been taken from the temperature-controlled room (29 C) and exposed to a colder environment during the course of thirty-five experiments. In seventeen experiments a marked increase in the rate of output of 1311 from the thyroid gland was observed (Fig. 13), the average rate of output being approximately 24 times that of the preceding control period. There were

21 THYROID ACTIVITY IN THE RABBIT 21 eight experiments in which no change in rate of output occurred, seven experiments in which the responses were of doubtful significance and three experiments in which a definite decrease in the rate of 131I release from the thyroid was observed. The latent period of the thyroid response to the cold stimulus was short. In nine experiments in which a sufficient number of points on the curve were available to allow extrapolation, the average latent period was estimated at 4 hr. In all seventeen cases, however, the response was apparent by 24 hr. The increased thyroid activity was usually maintained Cold _ \ ~~~~~~Rabbit 367, l a2,c '3'1,18.xi z 200 I I I I I Time (hr) Fig. 13. The effect of cold on the release of 131I from the thyroid gland of the rabbit. at a constant level throughout the period of cold exposure, but in some cases tended to fall after an initial increase though not to the level seen in the control periods. Most animals which responded to cold by an increased 131I output showed a clear inhibition on being returned to the warm room. It was of interest that exposure to moderate degrees of cold appeared to be a greater stimulus to thyroid activity than exposure to more intense cold. Ten of the twelve rabbits exposed to a temperature of C showed an increased thyroidal 131I output as compared with two out of seven rabbits exposed to a temperature of 1 C. The only three rabbits in the series to show inhibition of thyroid activity following cold were those placed in the cold room at 1 C and subjected to a continuous draught of air at this temperature.

22 22 K. BROWN-GRANT AND OTHERS (d) Hypophysectomy Fourteen hypophysectomized rabbits have been studied. Final histological examination showed that ten rabbits were completely, and four incompletely, hypophysectomized. The results from the latter four have been discarded in the following account. Five animals were hypophysectomized during the course of a release curve. In all cases a prompt and permanent decrease of the rate of release of thyroidal 131J was observed (Fig. 14) _ AX Rabbit 542, release curve, Hypophysectomy 2,Lc 131, 14. v. 53 Z 0 Fig l 40 l Time (hr) The effect of hypophysectomy on the release of 1311 from the thyroid gland of the rabbit. In order to obtain a sufficiently high thyroid content of 131J it was necessary to administer doses 4-7 times as great as those given to normal animals. It was found that the percentage of the injected dose present in the thyroid gland (calculated from the neck counts and the conversion factor) hr after injection was markedly lower (8-45 / S.D. of twenty observations) than that observed in normal animals (22X25% S.D. of seventy-nine observations). The data regarding the rate of release of 131I before and at varying times after hypophysectomy is summarized in Table 3. It is clear that in the hypophysectomized animals a reduced rate of release of thyroid 131J was maintained until the animals were killed. The average percentage loss per day of thyroidal radio-iodine in these rabbits was found to be 3.4% (compared with a pre-operative figure of 26-8%). In 100 observations on thirty other normal rabbits no case was observed of a percentage loss per day of less than 4 %, and in only eight cases a loss of less than 8% (Table 1).

23 TABLE 3. THYROID ACTIVITY IN THE RABBIT Rate of release of thyroidal 131I (as % loss/day) in rabbits before and after hypophysectomy Percentage loss thyroidal 131I/day Weeks after operation Rabbit, no. Preoperative < , , 300, 30* , 28-5, 27-5, < * < , 33*0 1-6 < <1.0 1*5 - < <1.0 1* < *5 < DISCUSSION A convenient and accurate method of measuring variations in thyroid activity is one employing 131I, although the danger of radiation damage to the thyroid has to be considered. It has been reported (Feller, Chaikoff, Taurog & Jones, 1949) that thyroid function in the rat was not disturbed up to 10 days after the injection of 24 or 30tc of 131J, and no evidence of damage to the thyroid gland or other tissues of the rat was observed for periods up to 8 months after a single injection of 18 uc of 131I (Goldberg, Chaikoff, Lindsay & Feller, 1950; Goldberg & Chaikoff, 1950). Further, the calculations of Myant (1953) suggest that the thyroid of the rabbit does not suffer radiation damage following four injections of 12 ltc 131I each. It therefore seemed reasonable to use doses of 2 or 4/1c of 1311 for repeated experiments in the normal rabbit. A microscopic study of the thyroid glands of our animals has revealed no sign of radiation damage. Since it has been established that emotional stress affects thyroid activity in the rabbit (Brown-Grant, Harris & Reichlin, 1954), the question arises as to whether the routine confinement of conscious rabbits in the hammocks for the purpose of making neck counts influences the rate of release of 131I by the gland. Any such influence is unlikely for the following reasons. (1) The curves representing the output of 131I by the thyroid gland were smooth and there was no correlation between the slope of the curve over any period of time and the frequency or grouping of the neck counts during the same period. (2) As far as can be judged by their behaviour, rabbits quickly became accustomed to the procedures involved in making neck counts. (3) Emotional stress stimuli (such as immobilization) must be continued for at least several hours before a detectable change in the rate of 131I output occurs (Brown-Grant et al. 1954).

24 24 K. BROWN-GRANT AND OTHERS The use of measurements of thyroid uptake of 131I as a valid index of thyroid activity requires the assumption that the radio-iodine taken up by the gland is not passed back into the blood as part of the thyroid hormone during the period of measurement. Calculation of clearance values, that is thyroid increment per unit blood concentration, shows that these values begin to decrease after min. The most likely explanation is that at this time 131I is being returned from the thyroid to the blood stream as part of the thyroid hormone (Myant, Pochin & Goldie, 1949). A similar explanation probably holds true for the flattening of the 'accumulation gradient' as reported by Stanley & Astwood (1947) for man, and as seen in the present work on the rabbit. The measurement of the release of 131I from the thyroid gland affords a better index of thyroid activity than measurement of 131J uptake for several reasons. The uptake of radio-iodine is only indirectly related to the rate of hormone secretion, whereas the release of 131I is directly related to hormone secretion in that the loss of 131I from the thyroid gland is due to loss of labelled thyroid hormone. Uptake methods are in general affected by transient variations in iodide content of the blood, and by changes in renal function and the iodide space of the body. These factors do not influence the rate of release of 1311 to any appreciable extent. Uptake methods based on the thyroid clearance of 1311 are not influenced by changes in extra-thyroidal iodine metabolism, but repeated control experiments are necessary. Measurement of the rate of release of 131J is a simpler technique, and provides a continuous measure of thyroid activity, thus allowing control and experimental periods to be incorporated in a single experiment. It would seem clear therefore that measurement of the output of 131I from the thyroid gland affords a more convenient means of studying changes in thyroid activity than measurement of 131J uptake. The slope of the curve representing the rate of 131J output from the thyroid varies considerably in different animals, and in any one animal tends to decrease in the weeks following arrival in the laboratory. It is most likely that this is related to differences in the thyroid gland content of In the iodine-poor thyroid the proportion of the total hormone content lost per day and the percentage loss of 131J per day are high, and vice versa in the iodinerich gland (Stanbury, Brownell, Riggs, Perinetti, Del Castillo & Itoiz, 1952; Fish, Carlin & Hickey, 1952; Riggs, 1952). The above interpretation agrees well with the results observed after administration of potassium iodide. A further factor determining the slope of 131I release curves might be the reaccumulation of 131I from degraded thyroid hormone. However, the results obtained from three different types of experiment indicate that only about 10 % of the 131J released from degraded thyroid hormone is reaccumulated by the thyroid. The average rate of loss of thyroid radioactivity, as measured, is

25 THYROID ACTIVITY IN THE RABBIT % per day. Assuming a 10% reaccumulation of radioactivity, the true figure representing the loss would be about 19% per day, a difference which is insignificant compared with the changes observed to follow the various experimental procedures studied in the present work. Also, although reaccumulation of 1311 may occur to a slight degree, the changes in slope of the release curves could not be explained by variation in reaccumulation rate since this factor would act in a direction opposite to those observed. For example the decreased release of 1311 seen after administration of thyroxine could not be explained by a diminished rate of reaccumulation of radio-iodine. Administration of thyrotrophic hormone, or thyroxine, cold exposure and hypophysectomy had the expected effects on the rate of release of 1311 from the thyroid gland. A striking feature of the effects produced by thyrotrophic hormone, thyroxine and cold was the rapidity of action. The latent period for the action of thyrotrophic hormone, injected subcutaneously, was found to be 1-2 hr which is in agreement with the figures of 2-3 hr obtained by Wolff (1951) using a similar method in the rat, and may be compared with the figure of 8 hr given by Stanley & Astwood (1947) using an uptake method in man. The latent period for the action of thyroxine, injected intraperitoneally, was found to be about 2 hr. Wolff (1951) noted that thyroxine inhibited the release of 1311 within 24 hr in the rat and gave data suggestive of a much shorter latent period. Stanley (1948) found a latent period for the action of thyroxine of more than 10 hr and less than 24 hr in man, as judged by 1311 uptake. The latent period observed in the rabbit includes the time taken for absorption of thyroxine from the peritonea] cavity, and for the TSH concentration in the blood to fall. Thus the time taken for a raised blood thyroxine concentration to inhibit pituitary secretion is less than 2 hr. The latent period for thyroid activation by cold exposure was less than 4 hr. These short time intervals found to precede changes in pituitary thyrotrophic secretion in response to thyroxine or cold are comparable with the time relationships found for the reflex release of adrenocorticotrophin (Colfer, de Groot & Harris, 1950) and gonadotrophin (Fee & Parkes, 1929) in the rabbit. It is also of interest that changes in pituitary activity may be reflected in the thyroid within 1 hr whereas changes in thyroid activity take very much longer to produce changes in the metabolic rate. Thyrotrophic hormone administration produces a marked increase in thyroid activity, which is followed in the normal rabbit by a period of inhibition. This inhibition is probably due to suppression of endogenous TSH secretion by the excess circulating thyroid hormone, and is similar to that described in man by Perlmutter, Weisenfeld, Slater & Wallace (1952). The finding that individual rabbits may become refractory to the effect of TSH following repeated injections is in agreement with many previous observations using other criteria of thyroid function (Thompson, 1941).

26 26 K. BROWN-GRANT AND OTHERS Thyroxine administration was followed by a prompt and complete inhibition of thyroid activity in twenty out of twenty-two experiments. The duration of this inhibition varied with the total dose of thyroxine and was followed by a return to the previous rate of 131I release. That this effect is not due to the iodine content of thyroxine is shown by the fact that iodine as potassium iodide, in doses equivalent to the iodine content of the thyroxine used, either had no effect on the release curve or resulted in a slight but permanent slowing. From the duration of the inhibition following a single dose of thyroxine it is possible to obtain a rough estimate (35 jug/day) of the absolute rate of secretion of the thyroid, in terms of L-thyroxine. It is probable that the figure so obtained is too high, since no account was taken of the relatively rapid excretion of excess thyroxine (see Gross & Pitt-Rivers, 1952). It is likely that repeated small doses of thyroxine would give a lower figure and afford a simple rapid method of estimating thyroid secretion rate in the rabbit, as described by Perry (1951) for the rat. Exposure to moderate degrees of cold appeared to be more effective in stimulating thyroid activity in the rabbit than exposure to more intense cold. This finding is difficult to explain, but if confirmed by later work the possibility should be considered that intense cold acts both as a stress stimulus, which in general inhibits thyroid activity (Brown-Grant et al. 1954), as well as a specific stimulus for the thyroid. That severe cold may inhibit thyroid activity has been shown previously by Williams, Jaffe & Kemp (1949). Hypophysectomy promptly and permanently slows the release of 131I from the rabbit's thyroid, as in the rat (Wolff, 1951; Randall, Lorenz & Albert, 1951). It was of interest that the uptake of 131I by the thyroid of hypophysectomized rabbits (as measured by the content of hr after injection) appeared to be less reduced by the operation than the rate of 13jI output. This finding may indicate that the uptake of iodine is less immediately dependent on pituitary function than is the output of thyroid hormone, but the possibilities must also be considered that the observation reflects only a decreased rate of iodine turnover in the thyroid, or a decreased rate of iodine excretion in the urine, following hypophysectomy. In conclusion it may be said that the method of plotting 'release curves' in the rabbit forms a simple and reliable technique for the study of thyroid activity and offers many advantages over those involving measurement of 131I uptake. SUMMARY 1. The general distribution of 131I, after intravenous injection in the rabbit, has been studied. 2. Methods for measuring thyroid activity in the conscious rabbit are described. A method based on the release of 131J from the thyroid gland was found to have many advantages over that involving measurement of 131J uptake.

27 THYROID ACTIVITY IN THE RABBIT Using the release method, the effects of administration of thyrotrophic hormone and thyroxine, cold exposure and hypophysectomy on thyroid activity in the rabbit have been studied. It gives us great pleasure to acknowledge the many kindnesses of Dr (then Prof.) E. D. Adrian and Prof. Sir Bryan Matthews during the first part of this work. Our gratitude is due to Mr R. R. W. Dye, Mr G. J. Christofinis, Mr W. Piper and Mr D. Canwell for valuable technical assistance, and for the preparation of histological material. Thyrotrophic hormone was obtained through the kindness of Armour and Co., Chicago. The expenses of this research were in part defrayed by grants to one of us (G. W. H.) from the Medical Research Council and from the Foulerton Gift Fund of the Royal Society. We should also like to acknowledge the grant of a Research Studentship from the Medical Research Council (to K. B.-G.), a personal grant from the British Council (to C. v. E.) and a Fellowship from the Commonwealth Fund (to S. R.). REFERENCES ALBERT, A. (1951). The in vivo determination of the biological decay of thyroidal radio-iodine. Endocrinology, 48, BROWN-GRANT, K., VON EULER, C., HARRIS, G. W. & REICHLIN, S. (1953). Thyroid activity in the rabbit. In Thyroid Gland, Mem. Soc. Endocr. 1, BROWN-GRANT, K., HARRIS, G. W. & REICHLrN, S. (1954). The effect of emotional and physical stress on thyroid activity in the rabbit. J. Physiol. 126, COLFER, H. F., DE GROOT, J. & HARRIS, G. W. (1950). Pituitary gland and blood lymphocytes. J. Physiol. 111, FEE, A. R. & PARKES, A. S. (1929). Studies on ovulation. 1. The relation of the anterior pituitary body to ovulation in the rabbit. J. Physiol. 67, FELLER, D. D., CHAIROFF, I. L., TAUROG, A. & JONES, H. B. (1949). The changes induced in iodine metabolism of the rat by internal radiation of its thyroid bv 131J. Endocrinology, 45, FISH, W. A., CARLIN, H. & HIcREY, F. C. (1952). The thyroidal uptake, retention and iodine pool in rats under controlled iodide diet. Endocrinology, 51, GOLDBERG, R. C. & CHAIKOFF, I. L. (1950). The cytological changes that occur in the anterior pituitary glands of rats injected with various doses of 131I and their significance in the estimation of thyroid function. Endocrinology, 46, GOLDBERG, R. C., CHAIKOFF, I. L., LINDSAY, S. & FELLER, D. D. (1950). Histopathological changes induced in the normal thyroid and other tissues of the rat by internal radiation with various doses of radioactive iodine. Endocrinology, 46, GROSS, J. & PITT-RIVERS, R. (1952). Experimental study of thyroid metabolism with radioactive iodine. Brit. med. Bull. 8, HARRIS, G. W. (1948). Electrical stimulation of the hypothalamus and the mechanism of neural control of the adenohypophysis. J. Physiol. 107, JACOBSORN, D. & WESTMAN, A. (1940). A parapharyngeal method of hypophysectomy in rabbits. Acta physiol. 8cand. 1, MYANT, N. B. (1952). Radio-iodine tests of thyroid function in man. Brit. med. Bull. 8, MYANT, N. B. (1953). The uptake of radio-iodine by the rabbit's thyroid measured in vivo. J. Physiol. 120, MYANT, N. B., POCHN, E. E. & GOLDIE, E. A. C. (1949). The plasma iodide clearance rate of the human thyroid. Clin. Sci. 8, PERLMUTTER, M., WEISENFELD, S., SLATER, S. & WALLACE, E. Z. (1952). Study of mechanism of inhibition of thyroid gland induced by ingestion of thyroid substance. J. clin. Endocr. 12, PERRY, W. F. (1951). A method for measuring thyroid hormone secretion in the rat with its application to the bioassay of thyroid extracts. Endocrinology, 48, RANDALL, R. V., LORENZ, N. & ALBERT, A. (1951). The effect of hypophysectomy on the biological decay of thyroidal radioiodine. Endocrinology, 48, RIGGs, D. S. (1952). Quantitative aspects of iodine metabolism in man. Pharmacol. Rev. 4,

28 28 K. BROWN-GRANT AND OTHERS STANBBURY, J. B., BROWNELL, G. L., RIGGS, D. S., PERINETTI, M., DEL CASTILLO, E. & ITOIZ, J. (1952). The iodine deficient human thyroid gland. J. clin. Endocr. 12, STANLEY, M. M. (1948). Cited by RABEN, M. S. & ASTWOOD, E. B. (1949) in: The use of radioiodine in physiological and clinical studies on the thyroid gland. J. clin. Invest. 28, STANLEY, M. M. & AsTwoOD, E. B. (1947). Determination of relative activities of antithyroid compounds in man by means of radioactive iodine. Endocrinology, 41, THOMPSON, K. W. (1941). Antihormones. Physiol. Rev. 21, WILLIAMS, R. H., JAFFE, H. & KEMP, C. (1949). Effect of severe stress upon thyroid function. Amer. J. Physiol. 159, WOLFF, J. (1951). Some factors that influence the release of iodine from the thyroid gland. Endocrinology, 48,