Department for Clinical Endocrinology and Diseases of Metabolism (Professor A. Querido, M. D.), Academisch Ziekenhuis,

Transcription

1 Department for Clinical Endocrinology and Diseases of Metabolism (Professor A. Querido, M. D.), Academisch Ziekenhuis, Leiden, Holland EFFECTS OF HYPOTHALAMIC LESIONS AND OF COLD ON THYROID ACTIVITY IN THE RAT By L. van Beugen and J. J. van der Werff ten Bosch ABSTRACT The effect was studied of an electrolytic basal midline lesion in the anterior part of the hypothalamus on thyroid activity of rats exposed to environmental temperatures of 24\s=deg\C and 4\s=deg\C. The index of thyroid function used was the biological halflife of thyroidal 131I calculated from the amount of radioactivity released by the gland in seven days. In one experiment, thyroid function was studied at 24\s=deg\C one month after the placement of a lesion and at 4\s=deg\C a month later. In another experiment hypothalamic lesions were made in the middle of a release study, during which some of the animals were kept at 24\s=deg\C and others at 4\s=deg\C. The two experiments yielded comparable results; the biological halflife values were approximately 7\m=1/2\days at 24\s=deg\C and 4 days at 4\s=deg\C in intact or blankoperated rats, and approximately 13 days at 24\s=deg\C and 7 days at 4\s=deg\C in rats with a hypothalamic lesion. Thyroid 127I contents of various groups were identical. It appears that the biological halflife was reduced to approximately half the normal value by exposure to cold, that a lesion caused doubling of the halflife and that summation of the two effects was produced, when a lesioned rat was exposed to cold or when a cold\x=req\ exposed animal was lesioned. The basal midline region of the hypothalamus destroyed by the lesions, whilst appearing to be essential for the maintenance of [00BB]spontaneous[00AB]thyroid activity, does not seem to be essential for the thyroid response to cold. Observations on socalled goitre\x=req\ block lesions are discussed in the light of these findings. There is no doubt that a low environmental temperature may cause modifica tions in the activity of the thyroid gland. It has been shown that the effect of a cold environment on the thyroid gland is mediated by the hypothalamus and the adenohypophysis (von Euler Sc Holmgren 1956; Knigge Sc Bierman 1958). Although it is clear that intact connections between the median eminence

2 of the hypothalamus and the adenohypophysis are essential for thyroid response to cold, little is known about the localisation of hypothalamic structures re quired for the transmission of the cold stimulus. The present work is concerned with the effect of circumscribed lesions in the anterior part of the hypothalamus on the rate of thyroidal 131I release of rats exposed to environmental temperatures of 24 C and 4 C. Parts of this study have been published elsewhere (van Beugen 1960; van Beugen Sc van der Werff ten Bosch 1960). The experiments are reported in two separate parts. A»chronic«experiment deals with rats operated one month before thyroid function studies were carried out. An»acute«experiment is concerned with rats which were operated after thyroid function studies had been started. MATERIALS AND GENERAL METHODS The rats used were adult females of an inbred Great Wistar strain; they were fed a standard commercial diet and water ad libitum. Unless stated otherwise the rats were housed at room temperature, i. e. 24 C (range 2227 C). The daily hours of light were constant throughout Operations were carried out under pentobarbitone sodium anaesthesia in the»chronic«experiment and under ether anaesthesia in the»acute«experiment. Lesions were placed in the anterior part of the hypothalamus with the aid of a stereotaxic machine and a platinumwire glassinsulated electrode with a 1 mm bare tip. Anodal lesions were made by passing 2 ma D. C. for 1 minute, using an indifferent electrode inserted into the rectum. Blankoperated rats differed from the lesioned animals only in that no current was passed when the electrodes were in position. The thyroid I31I counting apparatus which included a scintillation crystal was identical to that previously described (van Beugen Se van der Werff ten Bosch 1961). Biological halflives of thyroidal 131I were calculated from the numbers of counts obtained at an interval of seven days, after correction for background and physical decay, thyroidal 131I falling exponentially over the period studied. Total 127I content of thyroid glands was determined by the method of Barker (1948). After in situ fixation in 10% formalin, the brains were embedded in low viscosity nitrocellulose, serially sectioned and stained with Luxol Fast Blue and cresyl violet (Klüver Se Barrera 1953). Numerical data are presented as means + standard error of the means, to which the Student's test was applied for the calculation of statistical significance. i. Chronic experiment Methods Four weeks after lesions had been made in a group of rats, intact control rats were selected from the same stock in such a way that the two groups of rats were of approximately similar body weight. Both lesioned and control rats then received 5 //c carrierfree 181I diluted in 0.5 ml distilled water intraperitoneally before being given their ration of food. Biological halflife of thyroidal 131I was calculated from the numbers of counts obtained over the thyroid gland 24 hours and 8 days after the

3 During administration of 131I. Three weeks after the first radioactive dose had been given both groups of rats were removed to a»cold«room with an average temperature of + 4 C (range 25 C). A week later the animals received a second dose of 131I and counts were again obtained after 24 hours and 8 days, the rats remaining in the cold days. for a total of 15 Five months after the lesions had been placed all the animals were killed with ether. Results Thyroid activity at room temperature. exposure to room tempera ture ( + 24 C) intact rats had an average 131I uptake in the thyroid region of 52% of the dose, whilst lesioned rats averaged only 27.9% (Table 1). The biological halflife of 131I was increased in lesioned rats, with an average of 12.7 days as compared with 7.7 days for intact control animals (Table 2). Both parameters used, uptake and release rate, show a markedly decreased activity of the thyroid gland in animals with hypothalamic lesions. Table 1. Thyroid uptake of 131I in 24 h in the chronic experiment. The coldexposed spent 7 days in the cold before administration of the tracer dose. rats had Group No. of rats 24 C % dose No. of rats 4 C % dose mtact lesion ± 5.84* 27.9 ± ± ±2.13 * mean ± SEM. Thyroid activity during cold exposure. After having been in a cold environ ment ( + 4 C) for a week, the 24 hour uptake of 131I averaged 30.5% in lesioned and 43.5 % in intact control rats (Table 1). During the second week of cold exposure the biological halflife averaged 6.6 days in lesioned rats whilst in control animals this figure was 3.7 days (Table 2). Again both para Table 2. Biological halflife of thyroidal 131I in the chronic experiment. The coldexposed rats had spent 7 days in the cold before administration of the tracer dose. Group No. of rats 24 C days 4 C No. of rats days intact lesion ± ± ± ± 0.62

4 Average meters show a marked depression in thyroid activity of lesioned rats as com pared with control animals. It is clear, however, that the biological halflife of thyroid 131I was roughly halved during cold exposure, both in intact and in lesioned rats. Body weight. initial body weight ( ± S. E.) of rats which survived the total experimental period was 143 (± 4.8) g for 10 intact rats and 145 (± 1.3) g for 16 lesioned rats. Final body weights for these two groups of rats averaged 177 (± 6.3) g and 164 (± 6.5) g. Histology of the brain. The brain lesions which were found at histological examination were quite small. Since lesions had been placed five months prior to death it may be assumed that the original lesions had greatly shrunk. Lesions were situated in midline, immediately behind the optic chiasma. Fig. 1 shows a diagram of the lesion site. Two types of lesions could be distinguished. In 14 instances the lesion lay basally (Fig. 2); in 9 of these cases the posterior border of the optic chiasma had been encroached upon whilst the lesion did not involve the optic chiasma in 5 animals. In 7 cases the lesions did not reach down to the floor of the third ventricle, but were situated somewhat higher, ventral to the paraventricular nuclei (Fig. 3). There were no differences in biological halflife between the two groups of rats that could be distin guished on the basis of lesion localisation. In all but two cases the lesion lay well in front of the pituitary stalk. In one exceptional rat biological halflives at 24 C and at 4 C were 6.7 and 4.3 days; the other animal had a halflife of 20.6 days at 24 C and died before the beginning of the cold experiment. Fig. 1. Sagittal diagram through rat brain. The black area indicates approximate localisation and anteroposterior extent of lesions in the»chronic«experiment. CA anterior commissure, CC = = corpus callosum, CM mammillary body, = CO optic chiasma, Fx fornix, HYP hypophysis, PV paraventricular = = = = nucleus.

5 Fig. 2. Transverse section through rat hypothalamus, showing basal lesion in the midline. The paraventricular nuclei appear at the dorsal end of the lesion; the optic tracts may be seen laterally. Transverse section through Fig. 3. hypothalamus, showing midline lesion above the chiasma. The base of the brain is intact. rat optic

6 2. Acute experiment Methods Twentyfive rats housed at room temperature received 10 //c carrierfree 131I diluted in 0.5 ml distilled water intraperitoneally. Twentyfour hours and 8 days later counts of thyroid 131I were obtained. On the eighth day after 131I administration each animal was assigned at random to one of three groups. Six rats were only anaesthetised with ether, in 11 rats a lesion was placed in the hypothalamus whilst 8 animals underwent a blankoperation. These procedures were carried out during the afternoon, counts of the rats having been taken in the morning. Again seven days later the counting procedure was repeated, so that a second biological halflife could be calculated for each individual rat. A group of 19 other rats went through a similar procedure. These animals had. however, been placed in the»cold«room one week before the administration of 131I, and remained there for a total of 22 days, except for the time required for counting and operations. All rats were killed with ether immediately after the last counts had been obtained. The adrenal glands were removed and weighed fresh. The thyroid glands were removed for the determination of total 127I content. Results Thyroid activity at room temperature. Table 3 shows values for average biological halflives of the three groups of rats. The values for the control period do not differ significantly, as might be expected since the groups did not then differ from the experimental point of view. The biological halflife was some what increased by simple anaesthesia = (P ) and by a blankoperation = (P ). The lesions, however, caused a very marked change, i. e. doubling of the biological halflife. Table 3. Biological halflife of thyroidal 131I at 24 C in the acute experiment. Group No. of rats pre»operative«days post»operative anaesthesia blankoperation lesion 6 8 II 6.6 ± ± ± ± ± ± 1.10 Thyroid activity during cold exposure (Table 4). The control figures for biological halflife during cold exposure were significantly smaller than those obtained in rats kept at room temperature (compare Tables 4 and 3). The differences between the»preoperative«values of the three groups that were subsequently formed were not statistically significant. It can be seen that simple anaesthesia and a blankoperation caused no significant change in the

7 Thyroid Table 4. Biological halflife of thyroidal 131I at 4 C in the acute experiment. The rats had spent 7 days in the cold before administration of the tracer dose. Group No. of rats pre»operative«days post»operative«anaesthesia blankoperation 4.2 ± ± ± ± ± ± 0.59 biological halflife of thyroid 131I. A lesion in the hypothalamus depressed thyroid activity so that the postoperative biological halflife was double that obtained before the operation. In the cold, therefore, as at room temperature, hypothalamic lesions caused a marked slowing of thyroidal 131I release. In all groups, however, including the lesioned rats, the»postoperative«halflife value obtained during cold exposure was approximately 60 % of the»post operative«figure obtained at room temperature. Thyroid mi (Tables 5 and 6). content of 127I at the time of death, i. e. at the end of the second halflife period, was found to average 7.5 micrograms for the combined groups of anaesthetised and blankoperated animals, both for animals kept at room temperature and for those exposed to +4 C for the preceding three weeks. The values for lesioned animals showed no significant differences from these control figures at either temperature. Body weight and adrenal weights. Initial body weights, obtained one week before 131I was administered, and the body weights at the time of death, 22 days later, are recorded in Tables 5 and 6. There were no statistically signi ficant differences between groups of animals exposed to identical environ mental temperature. The data on adrenal weight, expressed in mg per 100 g body weight, show that animals exposed to cold had larger adrenal glands than those kept at room temperature. The hypothalamic lesions do not seem to have affected the weight of the adrenal glands. Table 5. Data on rats kept at room temperature (acute experiment). Group No. of rats thyroid I27I (micrograms) body weight (g) initial final adrenals mg/100 g body weight anaesthesia blankoperation lesion 8 1 I 8.5 ± ± ± ± ± ± ± ± ± ± ± ± 1.2

8 Table 6. Data C The rats had spent 7 days in the cold kept before administration of the tracer dose, i. e. 22 days at time of death. on at 4 rats No. of Group rats (acute experiment). anaesthesia blank 6.7 ± 1.01* operation lesion of four rats body weight (g) thyroid 127I (micrograms) ± 0.93 ± 0.25 adrenals initial final mg/100 g body weight 124 ± ± ± ± ± ± ± ± ± 2.1 only. the brain. The lesions of the different animals were highly uniform in localisation and size (Fig. 4). They were cystic and involved the lumen and the walls of the third ventricle. The lesions were basal in the posterior border of the optic chiasma or immediately behind this structure; Histology of Fig. 4. Transverse sections through a single rat hypothalamus, to show localisation of lesions in the»acute«experiment. A lesion in midline above the optic chiasma, which is behind the chiasma the lesion broadens and involves slightly damaged at this level. the base of the brain; the paraventricular nuclei are divided by the cystic third ventricle. C more posteriorly the lesion no longer involves the base of the brain. D at its posterior end the lesion consists mainly of a widened third ventricle; the insertion of the pituitary stalk appears to be undamaged.

9 posteriorly they extended to just before the pituitary stalk, but in this region the lesions no longer involved the floor of the third ventricle. The dorsal extension of the lesions was limited to the ventral edge of the paraventricular nuclei; in some instances these nuclei were separated by a dilated third ven tricle, whilst in one case the medial portion of one paraventricular nucleus had been destroyed. Laterally the lesions were contained within a boundary passing halfway between the midline and the fornix and mammillothalamic tract. DISCUSSION The present paper is concerned with two aspects of experimental thyroid physiology, i. e. the influence of a lesion in the anterior part of the hypo thalamus and the influence of a cold environment on thyroid activity. It is pertinent, before discussing these aspects, to discuss briefly the parameter of thyroid activity employed in this work. 1. Validity of biological halflife as an index of thyroid function The biological halflife of 131I accumulated by and still present in the thyroid gland 24 h after administration of a tracer dose was used as an index of thyroid gland activity. At room temperature a constant mean value of about 7 days has been obtained in a large number of intact animals discussed in this and in a previous paper (van Beugen Sc van der Werff ten Bosch 1961). In the present work the placement of a lesion in the hypothalamus was followed by a lengthening of the biological halflife to 1214 days, both in a chronic (lesion made long before) and in an acute (lesion made during release study) experiment. Also, in both of these experiments and in the previous work referred to above, exposure of intact rats to an environmental temperature of +4 C caused a shortening of the biological halflife to approximately 4 days. Since the thyroid glands of rats submitted to these various procedures in an acute experiment contained identical amounts of thyroid 127I it may be assumed that differences in biological halflife reflected differences in thyroid hormone secretion rate. In comparison with the data of other workers (e. g. 2 days; D'Angelo 1958) a»normal«biological halflife of 7 days in the rat appears quite long. This difference is, perhaps, attributable to the use made of thiouracil preparations by most of the workers who have studied 131I release in the rat. It is known that thiouracil derivatives raise the rate of 131I release by the thyroid gland (Wolff 1951; Perry 1951). Other investigations which did not involve the use of goitrogenic drugs have yielded biological halflives of 3.3 days (Wolff 1951) and 7.2 days (Florsheim 1958).

10 2. Effect of cold exposure The enhancing influence of a cold environment on thyroid activity has been described by many authors (for references see: van Beugen Sc van der Werff ten Bosch 1961). The present work confirms previous findings of a marked increase in the rate of 131I release by the thyroid gland. BrownGrant (1956 a) showed that in the rat, the increase in the rate of release started within a few hours of the beginning of the period of cold exposure. The experiments described here were so arranged that some adaptation to the cold environment could have taken place before thyroid studies were started. They show that the thyroid response to cold is not a transient shortlived effect. It was of interest to find that all groups of rats in the acute experiment which were exposed to cold showed a significant rise in adrenal weight when compared with their appropriate controls kept at room temperature. This may indicate an adrenal gland response to the low environmental temperature, and since adrenocortical activation would tend to depress thyroid activity (BrownGrant et al. 1954; BrownGrant 1956 è), the rise in thyroid activity noted becomes of even greater significance. 3. Effect of basal lesions in anterior part of hypothalamus It is clear from the data presented that a basal lesion in the anterior part of the hypothalamus causes a depression in the rate of 131I release from the thyroid gland, resulting in a biological halflife of thyroidal 131I approximately twice as long as in intact or blankoperated controls. This effect of a lesion is apparent both in a chronic experiment, in which the lesions were placed a number of weeks prior to thyroid function studies, and in an acute experiment, in which the lesions were made in the middle of a release rate study. In the acute experiment the lesions were fairly large, and since the lesioning para meters were identical in both experiments the small size of the lesions in rats of the chronic experiment may be attributed to shrinkage of the original lesion. The results in terms of»spontaneous«thyroid activity and thyroid response to cold of the two different sets of animals were identical. In the chronic ex periment the lesions had also caused a depression in thyroid 131I uptake. Other workers have reported, in animals with hypothalamic lesions, a re duction in thyroid uptake of 131I in the dog (Ganong et al. 1955) and the ferret (Donovan Sc van der Werff ten Bosch 1959) and in the thyroid uptake and release of 131I in the rat (D'Angelo Sc Traum 1956; Florsheim 1958); there is fair agreement on the localisation of the lesionarea essential for such an effect being between the optic chiasma, the paraventricular nuclei and the infundibulum. Similar lesions have been reported to prevent the goitrogenic effect of propylthiouracil (PTU) in the rat (Greer 1951, 1952, 1955; Bogdanove

11 thyroid & Halmi 1953; Greer Sc Erwin 1954; D'Angelo Sc Traum 1956; Florsheim 1958) and guinea pig (Bogdanove Se D'Angelo 1959). In many, though not all, rats such lesions were found to prevent compensatory hypertrophy of the thyroid gland after removal of three quarters of this structure, although such lesions did not prevent the increased rate of 13II release by the remaining portion of thyroid tissue (Reichlin 1957, 1960). On the basis of these experiments it has been assumed that the anterior part of the hypothalamus may be sensitive to lowered levels of circulating thyroid hormone, and that destruction of this area may prevent the response of the pituitary axis to the low levels of circulating thyroid hormone resulting from the administration of PTU or from the removal of a large portion of the thyroid gland. Table 7. Mean thyroid weights (mg/100 g body weight) in rats with or without a hypothalamic lesion and/or after propylthiouraciladministration. weeks between operation and PTU days of PTU intact No PTU PTU No PTU PTU 34** over 4* Calculated from data of Greer (1955). Data of D'Angelo Se Traum (1956). Calculated from data of Florsheim (1958). PTUtreated rats from his Table 2, untreated from his Table 3. Lesioned animals included are those noted to be»severely affected«or to possess a»goiter block«. However, the data presented on the socalled goitreblock lesions do not seem to warrant the conclusion that the lesions had produced a goitreblock. Table 7 summarizes some of the salient figures from the literature. These particular data were selected because the papers from which they were taken are the only ones which give thyroid weights for the four groups of animals: intact without and with PTU and lesioned without and with PTU. It is clear that PTU caused marked difference thyroid gland enlargement in intact animals, and that a occurred between intact rats and lesioned rats which had been treated with PTU. An important point, which seems to have been overlooked in these studies, is the effect of a lesion itself on thyroid weight. It may be seen from the table that in all instances the hypothalamic lesion had caused a fall in thyroid weight. It may also be noted that in all instances PTUadministration

12 thyroid has been followed by an increase in the weight of thyroid glands of lesioned rats. The data of D'Angelo Sc Traum show that the thyroid weight had in creased to approximately 400 % after PTU in both intact and lesioned animals. The data of Florsheim indicate a doubling of thyroid weight after PTU in both groups. The data of Greer show that the thyroid weight increase was con siderably larger in intact than in lesioned animals. It is possible that the dif ference between the data of Greer and the other two sets of data is attributable of lesions and the onset to: a. the shorter period of time between the placement of PTUadministration, and: b. the shorter period of PTUadministration in Greer s experiments. With the passage of time, the depressant effect of a hypo thalamic lesion on the weight of the thyroid gland may become more marked, as the data in the table seem to suggest. The effect of PTU on thyroid weight may become more marked as the duration of PTUadministration increases; Purves (1960) has noted that the goitrogenic effect of PTU does not begin to occur until the thyroid follicles have lost most of their colloid. This discussion of the goitreblock experiments is relevant here in view of a discussion on the influence of a hypothalamic lesion on the thyroid response to cold. The figures in Tables 2, 3 and 4 clearly show that roughly speaking the biological halflife of thyroidal 1S1I is doubled by a lesion and halved by cold exposure, whilst the combined procedures of lesioning and cold exposure result in a summation of the two effects. These findings were obtained in both the chronic experiment, in which the effect of cold was superimposed upon the effect of a lesion, and in the acute experiment, in which the reverse sequence prevailed. It would thus appear that in this investigation the two variables studied, a lesion in the hypothalamus and exposure to cold, were effective independently. The data in Table 7 are suggestive of a similar indépendance of the effect of a lesion and the effect of PTU. This corollary is, perhaps, not perfect because the parameter employed to study the effect of PTU, change in weight of the thyroid gland, is subject to specific conditions such as the time required to obtain thyroid gland atrophy (after a lesion) or hypertrophy (after PTU). In summary, then, lesions located basally in the anterior part of the hypo thalamus cause a reduction both in thyroid 131I release rate and in thyroid weight. Such lesions do not appear to inhibit the percentual change in the biological halflife of thyroidal 18,I which normally follows upon exposure to a cold environment, whereas the data on PTUinduced goitre seem to indicate a similar normal percentual change in thyroid weight whether a lesion is present or not. It is concluded from these findings and arguments that the anterior basal part of the hypothalamus destroyed in these experiments is essential for the maintenance of normal thyroid gland activity. Nevertheless this part of the hypothalamus does not seem essential for the response of the pituitary axis to a low environmental temperature or to PTU.

13 ACKNOWLEDGEMENTS The authors are indebted to Prof. A. Querido for his advice and encouragement, and to Miss M. Duisterhof, Miss F. Dantuma, Miss J. S. Cijsouw, Mr. W. Zuidervaart and Mr. A. Huisman for their technical assistance. The expenses of this research were in part defrayed by grants from the National Health Organisation T. N. 0., The Hague. REFERENCES Barker S. B.: J. biol. Chem. 173 (1948) 715. van Beugen L.: Studies concerning the central nervous control of thyroid activity, M. D. Thesis, Leiden (1960). van Beugen L. Sc van der Werff ten Bosch J. J.: First International Congress of Endo crinology, Advance abstracts of short communications. Acta endocr. (Kbh.) Suppl. 51 (1960) 95. van Beugen L. Se van der Werff ten Bosch J. J.: Acta endocr. (Kbh.) 37 (1961) 470. Bogdanove E. M. Se D'Angelo S..: Endocrinology 64 (1959) 53. Bogdanove E. M. Sc Halmi N. S.: Endocrinology 53 (1953) 274. BrownGrant K.: J. Physiol. 131 (1956 ) 52. BrownGrant K.: J. Physiol. 131 (1956 b) 58. BrownGrant., Harris G. W. Se Reichlin S.: J. Physiol. 126 (1954) 41. D'Angelo S..: J. Endocr. 17 (1958) 286. D'Angelo S. A. Se Traum R. E.: Endocrinology 59 (1956) 593. Donovan B. T. Se van der Werff ten Bosch J. J.: J. Physiol. 147 (1959) 93. von Euler C. Se Holmgren B.: J. Physiol. 131 (1956) 137. Florsheim W. H.: Endocrinology t52 (1958) 783. Ganong W. F., Frederickson D. S. Se Hume D. M.: Endocrinology 57 (1955) 355. Greer M..: Proc. Soc. exp. Biol. (N. Y.) 77 (1951) 603. Greer M..: J. clin. Endocr. 12 (1952) Greer M..: Endocrinology 57 (1955) 755. Greer M. A. Se Erwin H. L: J. clin. Invest. 33 (1954) 938. Klüver H. Se Barrera E.: J. Neuropath, exp. Neurol. 12 (1953) 400. Knigge K. M. Se Bierman S. M.: Amer. J. Physiol. 192 (1958) 625. Perry W. F.: Endocrinology 48 (1951) 643. Purves H. D.: First International Congress of Endocrinology, Advance abstracts of symposiums lectures. Acta endocr. (Kbh.) Suppl. 50 (1960) 21. Reichlin S.: Endocrinology 60 (1957) 567. Reichlin S.: Endocrinology 66 (I960) 327. Wolff J.: Endocrinology 48 (1951) 284. Received on May 6th, 1961.