mice, rats and rabbits produces a temporary fall in the absolute number of

Transcription

1 328 J. Physiol. (I950) III, I2.492:6I2.II2 PITUITARY GLAND AND BLOOD LYMPHOCYTES BY H. F. COLFER, J. DE GROOT AND G. W. HARRIS From the Physiological Laboratory, University of Cambridge (Received 6 December 1949) It has been shown that injection of adrenocorticotrophic hormone (A.C.T.H.) in mice, rats and rabbits produces a temporary fall in the absolute number of blood lymphocytes (Dougherty & White, 1943, 1944). This response would appear to be mediated by the adrenal cortex, for injection of extracts of the adrenal cortex produce a similar lymphopenia, whereas injection of A.C.T.H. into adrenalectomized animals has no effect (Dougherty & White, 1944). These results have been confirmed by other workers. For details and discussion see recent reviews by Valentine, Craddock & Lawrence (1948), and White (1949). It thus seemed possible that the production of a temporary lymphopenia might be used as an indicator of the secretion of A.C.T.H. by the anterior pituitary gland. In preliminary experiments electrical stimulation of various regions of the hypothalamus and pituitary gland was performed by the remote control method in unanaesthetized rabbits to see whether a lymphopenia might be so produced. For this method of stimulation (Harris, 1948 a) it was necessary to immobilize the rabbits in clamps for periods up to 2 hr. These experiments were stopped, however, when it was found that the emotional stress involved by immobilizing the animals in clamps (without electrical stimulation) was sufficient to produce the lymphopenic response. This paper deals with the part played by the pituitary gland in this reaction to emotional stress. The effects of electrical stimulation of the hypothalamus and hypophysis by a technique that does not require immobilization of the animal form a further report. METHODS Blood count8. Adult rabbits of both sexes, body weight 2-3 kg., were used. The total white cell count was made using the Thoma form of haemocytometer, diluting the blood ten times with 1% acetic acid coloured with gentian violet, and counting the number of cells in 0-02 cu.mm. of diluent. The differential count was made by counting a total of at least 200 cells (stained with Leishman's stain) under a * in. oil immersion objective. It was found convenient to use an electric counting device, foot operated, in recording the number of cells. The figures obtained in this way from counts on forty-five normal rabbits are: total white blood corpuscles per cu.mm. 10, (s.e. of mean of sixty-three observations) (Dougherty & White, ,053 ± 1730/cu.mm.), and absolute lymphocytes per cu.mm _ 161 (63) (Dougherty & White, /cu.mm.).

2 PITUITARY AND LYMPHOCYTES 329 Emotional stre8s. In the preliminary experiments the animals were placed in restraining clamps, as described by Harris (1948a), for periods of 1 or 2 hr. In all later experiments the emotional stress stimulus was administered by passing a faradic current, from an inductorium, through two needle electrodes inserted subcutaneously over the lumbar vertebrae (O'Connor & Verney, 1942). In most experiments this stimulus was administered twice, for periods of 5 min. (1 min. on, 1 min. off), with half an hour interval. The strength of the stimulus used was that which just produced definite signs of fright on the part of the animal, such as crying, struggling, tachyeardia, and polypnoea. The full procedure was as follows. The rabbit, which had been left undisturbed overnight, was removed from its cage and the initial blood sample taken from the marginal vein of the ear. Two needle electrodes were then inserted under the skin and the faradic stimulus administered. Further blood samples were taken hourly for 3 hr. and usually again at the sixth hour, the animal being replaced in its cage in the intervals. Operative procedures. Hypophysectomy was performed by the parapharyngeal method of Jacobsohn & Westman (1940), which was found to be very satisfactory. The first few days after operation the animals were often disinclined to take food, and during this period many died from hypoglycaemia on the second or third day, in spite of glucose administration. The mortality of the animals that survived the first few post-operative days, and started eating again, was very low. Denervation of the adrenal glands was performed through a peritoneal approach and involved removal of the splanchnic nerves and upper part of the lumbar sympathetic chain on both sides. Histology. The routine procedure consisted of killing the animal with chloroform, injecting the carotid arteries with 150 c.c. of a one in three dilution of indian ink, fixing the head in formalin, decalcifying in Bensley's solution, and embedding a block of tissue containing the base of the skull, sella turcica and hypothalamus in celloidin. Serial sections, u. thick, were cut and stained with haemalum and eosin. RESULTS Emotional stress stimulus in normal rabbits In seven preliminary experiments on five rabbits the animals were placed in immobilizing clamps for periods of 2 hr. It was found that this immobilization produced a lymphopenia in all cases. In order to standardize the emotional stimulus the method of O'Connor & Verney (1942) was adopted in most of the later experiments. It was found that the maximum drop in blood lymphocytes occurred at about the third hour after the emotional stress stimulus. In these and the later experiments the results have been expressed by giving the number of lymphocytes per cu.mm. at the third hour as a percentage of the initial count. Thus a result of 100% indicates no change in the lymphocyte count at the third hour after the emotional stress stimulus, whilst a result of 50% indicates a drop in blood lymphocytes to one-half the initial number. It has been assumed that in this way figures are obtained which are comparable between animals showing a difference in their initial (resting) lymphocyte counts. In all of forty-four experiments on forty-two normal rabbits a temporary lymphopenia was observed to follow emotional stress. The lymphocyte counts at the third hour were 69-2% ± 1-8 (44) of the initial counts. After a further 3 hr. the lymphocyte count was returning, or had returned, to the initial level. Of these forty-two normal rabbits, male. thirty-one were female and eleven were No sex difference in the lymphopenic response to emotional stress was 22-2

3 330 H. F. COLFER, J. DE GROOT AND G. W. HARRIS found, the average results in the female rabbits being 6840/ (32), and in the male rabbits / (12). Two experiments on adult rabbits that had been gonadectomized when a few weeks old, gave results of 53 % (castrate male) and 64% (spayed female). These figures suggest that the gonads are not directly involved in the reaction. A study of the other blood cells, following an emotional stress stimulus, showed that a granulocytosis occurred in the majority of cases, and that the erythrocyte count remained unaltered. This latter observation may be taken as evidence that the lymphopenia was not due to changes in blood volume. Emotional stress stimulus in hypophysectomized rabbits The operation of hypophysectomy is followed by a fall in the resting level of blood lymphocytes. In nine rabbits the preoperative lymphocyte count per cu.mm. was (18), and on the first post-operative day (9). In those animals which survived, the pre-operative level of lymphocytes was regained within 1-4 weeks. A similar reduction in the blood lymphocytes has been observed to follow exposure of the pituitary gland without removal (in blank operations), and also following operations to denervate the adrenal glands. Therefore this reaction appears associated with trauma rather than a specific loss of the pituitary gland, and could be interpreted as being part of the picture of the alarm reaction described by Selye (1946). Fourteen rabbits were submitted to emotional stress stimuli following the operation for hypophysectomy. During the first two post-operative weeks, twenty-six experiments were performed on twelve of these rabbits, and in no case was a definite lymphopenic response observed. Previous to operation ten of these animals had been tested, and responded normally. After the second post-operative week seventeen experiments were performed on eight rabbits, and it was found that four of these animals showed a continued lack of response, whilst the other four animals showed a gradual return of the response to emotional stress. Microscopic examination of serial sections through the sella turcica revealed the hypophysectomy to have been incomplete in the four cases that showed a return of the response. It was of interest that the pituitary fragments left in situ in these animals still retained, or had reacquired, vascular connexion with the tuber cinereum through the hypophysial portal vessels (P1. 1). At first it seemed possible that the loss of lymphopenic response in the early post-operative days was associated with the low resting level of blood lymphocytes. This is unlikely as (a) injection of anterior pituitary extract or adrenocorticotrophic hormone still elicited the usual lymphopenic response in these animals (see below), and (b) two rabbits in which the pituitary gland had been exposed but not damaged, and which showed a similar fall in the resting lymphocyte level after operation, still responded with the usual acute decrease

4 THE JOURNAL OF PHYSIOLOGY, VOL. 111, Nos. 3 & 4 PLATE 1 Microphotograph of a midline sagittal section through hypothalamus and base of skull (including sella turcica) of an incompletely hypophysectomized rabbit (180). The vascular system of this rabbit was perfused with indian ink immediately after death. Note that the residual fragment of anterior pituitary tissue (P.) is connected to the tuber cinereum (T.C.) of the hypothalamus (H.) by blood vessels. The outline of the sella turcica is indicated by the dotted line. Magnification, x 12. To face p. 330

5 PITUITARY AND LYMPHOCYTES 331 after emotional stress. It is probable that the negative responses of the incompletely hypophysectomized rabbits in the first two post-operative weeks were due to lack of vascular connexions to the retained fragments of the gland. TABLE 1. To show the effect of emotional stress on blood lymphocytes following the operation for hypophysectomy. The figures given express the blood lymphocytes at the third hour following the emotional stimulus as a percentage of the initial count. Note that in the completely hypophysectomized animals no definite lymphopenic response follows emotional stress, and that in some cases even a lymphocytosis occurs Rabbit , castrate male , spayed female st week 107, 102, , 103, , , , , 144 Lymphopenic response Post-operative 2nd week , rd week ~~~I , 115, 125, 112 C , 108 C I, incompletely hypophysectomized; C, completely hypophysectomized th week and later _ Operative result C 81 I 57 I C 87 I - C - ~~C - C 98 C - ~~C To see whether the more peripheral mechanism underlying the lymphopenic response to stress was still capable of reacting after hypophysectomy, the response of operated animals to intravenous injections of anterior pituitary extracts was studied. The results of these experiments are given in Table 2, TABLE 2. Preoperative The lymphopenic response following injection of crude anterior pituitary extract or purified adrenocorticotrophic hormone Lymphocyte count 3 hr. after injection expressed as percentage of initial Rabbit i.v. injection count 180, I 3 mg. crude ant. pit. ext , I 3 mg. crude ant. pit. ext , C 500 pg. A.C.T.H , C 250,Ug. A.C.T.H , C 500 jig. A.C.T.H , C, S 500 pg. A.C.T.H. 58 I, incompletely hypophysectomized; C, completely hypophysectomized; S, spayed. and it may be seen that two incompletely hypophysectomized, and three completely hypophysectomized rabbits gave definite lymphopenic responses

6 332 H. F. COLFER, J. DE GROOT AND G. W. HARRIS after injection of crude anterior pituitary extract and purified A.C.T.H., respectively. These observations were made during the first two post-operative weeks when all animals failed to show a response to the emotional stress stimulus. The granulocytosis, which was seen to accompany the lymphopenia after stress in the majority of normal rabbits, still occurred in the hypophysectomized animals. The relationship of the adrenal medulla Since the adrenal medulla is excited by emotion, and adrenaline is thought to exert a stimulating effect on the anterior pituitary gland and on the adrenal cortex (Vogt, 1944, 1945; Long, 1947), it was decided to investigate the effect of intravenous injection of adrenaline on the blood lymphocytes, and the effect of denervation of the adrenal glands on the lymphopenic response following emotional stress. Injection of adrenaline. Intravenous administration of 1 c.c. of 1/150,000 solution of adrenaline hydrochloride in seven normal rabbits resulted in a definite lymphopenic response in one case (66 %), one doubtful response (87 %) and five negative responses (107, 99, 101, 98, 109%). Control injections of 0-9 % sodium chloride made intravenously into six rabbits failed to produce a lymphopenic response (97, 102, 102, 101, 97, 96%). Emotional stress response after denervation of the adrenal glands. In four rabbits the adrenal glands were denervated. In six experiments on these animals the lymphopenic response (66, 60, 85, 72, 62, 70 %) to the usual emotional stress stimulus did not differ significantly from the response in normal animals. DISCUSSION It is well known that emotional stress produces a lymphopenia in mice and rats (Harlow & Selye, 1937; Elmadjian & Pincus, 1945; Latta & Nelson, 1948) and man (Pincus, 1947; Mikkelsen & Hutchens, 1948). Little work appears to have been done on this reaction in the rabbit. Cheng (1930) and Nice & Katz (1936) report that restraint in the abdomen-up position or a subcutaneous faradic stimulus in the rabbit results in a leucopenia which according to Cheng is a lymphopenia. In the present work it has beeen found that restraint or subcutaneous faradism in rabbits results in a lymphopenia which is generally associated with a granulocytosis. The assumption is usually made that stress lymphopenia is due to activation of the anterior pituitary: adrenal cortex mechanism. No detailed study has been made of the part played by the pituitary gland in this reaction. As a first step in such a study it was thought important to see whether hypophysectomy abolished the response. There can be no doubt that such is the case in the rabbit, and the control experiments in which pituitary extract was injected into hypophysectomized animals show that the peripheral mechanism is still

7 PITUITARY AND LYMPHOCYTES 333 capable of being stimulated, and producing a lymphopenia, for at least 10 days after hypophysectomy. These results are in general agreement with those of Sayers & Sayers (1948), who studied the relationship of stress to the content of sudanophilic substance and ascorbic acid in the adrenal cortex of the rat. It seems likely that emotional stress excites the adenohypophysis through the mediation of the hypophysial portal vessels (Harris, 1948b) and it is significant, in this respect, that the incompletely hypophysectomized rabbits were found to possess vascular connexions between the tuber cinereum and retained pituitary tissue (Pl. 1). The possibility that emotional stress stimulates the anterior pituitary gland via the splanchnic nerves, adrenal medulla and adrenaline secretion, according to the general hypothesis of Long (1947), is thought to be unlikely since a normal stress lymphopenia occurred after adrenal denervation and since intravenous injection of adrenaline did not usually elicit a lymphopenia. The lymphopenic response is the quickest indicator of possible anterior pituitary stimulation available at present. For experimental work this reaction has the obvious advantages of (i) short time relations, (ii) easy and quick measurement, (iii) absence of anaesthesia and operative interference, and (iv) easy repetition of experiment in the same animal. Care must be taken in using lymphopenia as an indicator of pituitary activity that the animal suffers no incidental emotional stress throughout the experiment and has been quiet and at rest for the previous 12 hr. Provided that precautions are observed it is thought that the lymphopenic response will be of great value in elucidating the factors governing anterior pituitary secretion. SUMMARY 1. Emotional stress (restraint or subcutaneous faradic shocks) in normal rabbits resulted in a marked absolute lymphopenia which was generally accompanied by an absolute granulocytosis. The lymphopenia was maximum at about the third hour after the stimulus. 2. Emotional stress in hypophysectomized rabbits failed to produce any lymphopenia, though the granulocytosis was not abolished. Intravenous injection of pituitary extract still evoked a lymphopenia after hypophysectomy. 3. Denervation of the adrenal glands did not alter the lymphopenic response to stress. Intravenous injection of adrenaline did not produce a lymphopenia in the majority of animals. It is concluded that the adrenal medulla plays little part in the reaction. 4. The lymphopenic response is the quickest indicator of anterior pituitary activity known at present. We should like to thank Dr J. D. Fisher of Armour and Company, Chicago, for making available to us a supply of purified adrenocorticotrophic hormone. We are also grateful to Mr R. R. W. Dye for his valuable technical assistance.

8 334 H. F. COLFER, J. DE GROOT AND G. W. HARRIS REFERENCES Cheng, S. C. (1930). Amer. J. Hyg. 11, 449. Dougherty, T. F. & White, A. (1943). Science, 98, 367. Dougherty, T. F. & White, A. (1944). Endocrinology, 35, 1. Elmadjian, F. & Pincus, G. (1945). Endocrinology, 37, 47. Harlow, C. M. & Selye, H. (1937). Proc. Soc. exp. Biol., N.Y., 38, 141. Harris, G. W. (1948a). J. Physiol. 107, 418. Harris, G. W. (1948b). Physiol. Rev. 28, 139. Jacobsohn, D. & Westman, A. (1940). Acta Physiol. Scand. 1, 71. Latta, J. S. & Nelson, W. W. (1948). Amer. J. Anat. 82, 321. Long, C. N. H. (1947). Fed. Proc. 6, 461. Mikkelsen, W. P. & Hutchens, T. T. (1948). Endocrinology, 42, 394. Nice, L. B. & Katz, H. L. (1936). Amer. J. Phy8iol. 117, 571. O'Connor, W. J. & Verney, E. B. (1942). Quart. J. exp. Phy8iol. 31, 393. Pincus, G. (1947). Rec. Progr. Horm. Be8. 1, 123. Sayers, G. & Sayers, M. A. (1948). Rec. Progr. Horm. Res. 2, 81. Selye, H. (1946). J. din. Endocrinol. 6, 117. Valentine, W. M., Craddock, C. G. & Lawrence, J. S. (1948). Blood, 3, 729. Vogt, M. (1944). J. Physiol. 103, 317. Vogt, M. (1945). J. Physiol. 104, 60. White, A. (1949). Ann. Rev. Phy8iol. 11, 355.