Effect of regularity of exposure to chronic immobilization stress on the circadian pattern of pituitary adrenal hormones, growth hormone, and thyroid stimulating hormone in the adult male rat

Psychoneuroendocrinology, Vol.

18, No. I, pp. 67-77, 1993

0306-4530/93 $6.00 + .00 © 1993 Pergamon Press Ltd.

Printed in the U.S.A.

EFFECT OF REGULARITY OF EXPOSURE TO CHRONIC IMMOBILIZATION STRESS ON THE CIRCADIAN PATTERN OF PITUITARY A D R E N A L HORMONES, GROWTH HORMONE, A N D THYROID STIMULATING HORMONE IN THE A D U L T MALE RAT OCTAVI MARTf, 1 AMADEU GAVALD,~, 1 TRINIDAD JOLfN, 2 and ANTONIO ARMARIO I IDepartament de Biologia Cel.lular i de Fisiologia, Unitat de Fisiologia Animal, Facultat de Ci~ncies, Universitat Aut6noma de Barcelona, Bellaterra, Barcelona, Spain; and 2Instituto de Investigaciones Biom6dicas, CSIC, Madrid, Spain

(Receioed 3 April 1992; in final form 13 July 1992)

SUMMARY Circadian variation of serum levels of adrenocorticotropin hormone (ACTH), corticosterone, growth hormone (GH), and thyroid-stimulating hormone (TSH) were studied in three groups of adult male rats exposed to chronic intermittent immobilization stress (IMO) for 2 hr daily under different schedules. IMO resulted in reduced food intake, body weight loss, and increased adrenal weight. ACTH levels were not affected but corticosterone levels were increased in all IMO rats as compared to control ones during the diurnal phase of the circadian cycle. IMO decreased serum GH and TSH levels but the circadian pattern of secretion was influenced in a complex way depending on the specific pattern of daily exposure to IMO. Differences observed between the IMO groups were not caused by differences in food intake because its circadian rhythm was very similar in all IMO groups. These results suggest that regularity of exposure to immobilization alters in a complex fashion circadian GH and TSH rhythms.

INTRODUCTION WHEREAS THE BIOLOGICAL e f f e c t s o f a c u t e s t r e s s h a v e b e e n e x t e n s i v e l y s t u d i e d a n d a r e n o w q u i t e well c h a r a c t e r i z e d , t h e effects o f c h r o n i c s t r e s s h a v e r e c e i v e d less a t t e n t i o n until r e c e n t l y , d e s p i t e t h e f a c t t h a t c h r o n i c s t r e s s is m o r e f r e q u e n t t h a n a c u t e s t r e s s . N o n e t h e l e s s , it has b e e n s h o w n t h a t c h r o n i c s t r e s s i n d u c e s i m p o r t a n t b e h a v i o r a l , n e u r o chemical, and endocrine disturbances different from those caused by acute stress (Adell et al., 1988; A n i s m a n et al., 1981; A r m a r i o et al., 1990; D i l s a v e r & A l e s s i , 1987; G a r c f a M f i r q u e z & A r m a r i o , 1987; N u k i n a et al., 1987; O r r et al., 1990; R i e g l e , 1973; R o s e l l i n i Address correspondence and reprint requests to: Octavi Marti, Departament de Biologia Cel.lular i de Fisiologia, Unitat de Fisiologia Animal, Facultat de Ci~ncies, Universitat Autbnoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. 67

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& Seligman, 1976; Zebrowska-Lupina et al., 1991). Consequences of chronic exposure to stressors are dependent on particular characteristics of the stressors such as intensity and duration. However, other more psychological properties of stressors such as controllability and predictability are important (Abbott et al., 1984). In particular, it has been reported that regularity of exposure to chronic stress is important in determining its influence on physiological variables such as corticosterone, glucose, and free fatty acids (Quirce et al., 1981; Ramade & Bayle, 1984, 1985). Laboratory rats showed a well known circadian rhythmicity in activity, food and water intake and circulating corticosterone, growth hormone (GH), and thyroid-stimulating hormone (TSH) levels (Armario & Jolin, 1986; Kimura & Tsai, 1984; Krieger & Hauser, 1978; Mosier & Jansons, 1987; Ottenweller & Hedge, 1982; Tannenbaum et al., 1976). Since exposure to a severe stressor is usually done during the light phase of the cycle, when animals are resting, it is possible that chronic exposure to stressors and the particular schedule of exposure to them have important effects on circadian patterns. Although few attempts have been made to study this possibility, alterations in the normal rhythm of corticosterone have been described after exposure to electric footshock for 3 days in the ewe (Przekop et al., 1985), or after intense stress caused by hemorrhagic shock and surgical trauma (Levine et al., 1980). The present work was undertaken to study whether regularity of exposure to chronic immobilization stress alters the physiological response to stress and exerts differential effects on the circadian pattern of food intake and pituitary-adrenal (PA) hormones, GH and TSH.

MATERIALS A N D METHODS

Animals and Stress Adult male Sprague-Dawley rats approximately 80 days old at the beginning of the experiments were used. They were kept under standard conditions (lights on from 0730h to 1930h, temperature 22 -+ I°C) for at least 1 week before and throughout the whole experimental period. Rats had free access to tap water and food. The stress procedure has been described elsewhere (Kvetnansky & Mikulaj, 1970). Briefly, rats were immobilized in a wooden board in a prone position by attaching their four limbs to metal mounts with adhesive tape. Head movements were restricted with two metal loops behind the neck of the rat. Experiment 1 The rats were housed three or four per cage and randomly assigned to four experimental groups: 1 = Unstressed rats (controls); 2 = rats immobilized 2 hr per day, beginning at 0830h (IMO 1); 3 = rats immobilized always at 0915h for a variable number of hours per day (between 0 and 5), the average number of hours per day being 2 (IMO 2); and 4 = rats immobilized 2 hr per day, the stress beginning each day at a randomly chosen time between 0900h and 1800h (IMO 3). All rats from a given cage received the same chronic treatment. Chronic stress lasted for 15 days, six sessions per week. Body weight and food intake were measured every 2 to 4 days. In order to avoid the consequences

CHRONIC STRESS AND ANTERIOR PITUITARY HORMONES

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different IMO sessions could have on the variables under study on the day of sacrifice, the IMO session began at 0830h and lasted for 2 hr in all stressed groups the day before sacrifice. On day 16, five to six rats from each experimental group were quickly killed by decapitation without stress at 0830, 1400, 1900, and 2400h. The rats were killed in a separate room within 15 sec after they had been taken from the animal room. At lights off, killing procedure was performed under a dim red light. After decapitation, the adrenal glands were quickly removed, trimmed of fat, and weighed. Trunk blood was collected into plastic tubes on ice-cold baths, and the serum obtained by centrifugation was aliquoted and frozen at -20°C until hormone measurements were performed.

Experiment 2 In an additional experiment, the same experimental protocol as in Experiment 1 was followed, except that the rats were housed one per cage. On the 12th day of chronic treatment, the IMO session began at 0830h and lasted for 2 hr in all stressed groups. On the following day, no stress was applied and the circadian rhythm of food intake was studied by weighing the amount of food put into the feeders and measuring the amount of food remaining in the cages at 0800, 1400, 1900, 2400, and 0800h. Hormone Analysis and Statistics Serum hormone levels were measured by radioimmunoassay (RIA). Analysis of corticosterone was carried out as described elsewhere (Armario & Castellanos, 1984), except that rabbit serum against corticosterone-3-OCMO (BioClin, UK) was used. ACTH was determined by a double antibody RIA, in which we used human ~:SI-ACTH (DuPont NEN Res. Products, USA) as a tracer, ACTHI_24 (Sigma, USA) as the standard, and rabbit antiserum against human ACTH (hACTH22VO2) kindly provided by the NIDDK through the National Hormone and Pituitary Program (University of Maryland, School of Medicine). Aprotinin (Trasylol) was kindly provided by Bayer (Barcelona, Spain). GH and TSH were also determined by double antibody RIAs with the rat GH and rat TSH kits kindly donated by the NIDDK. Values of these two hormones are given in terms of rGHRP-I and r-TSH-RP-2 standards, respectively. All the samples to be compared were processed in the same assay in order to avoid interassay variations. Intra-assay coefficients of variation were less than 8%, except for ACTH (12%). Both two-way and one-way ANOVAs were used for statistical analysis of data from Experiment 1. After one-way ANOVAs, post hoc comparisons of means were carried out with the Student-Newman-Keuls (SNK) test. MANOVA with repeated measures and Student's t-test were used for the analysis of circadian rhythm of food intake in Experiment 2. For statistical analysis of food intake, the cage was considered as the experimental unit. When necessary, data were log-transformed to achieve homogeneity of variances. RESULTS The one-way ANOVAs showed significant effects of chronic stress on final body weight gain (p < .00005), food intake (p < .00005) and both absolute (p < .001) and relative (p < .0001) adrenal weights. Post hoc SNK tests revealed that the three IMO groups showed reduced food intake and body weight, as compared to control rats. All

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TABLE

O. MARTi et al.

I, EFFECT

OF VARIOUS

Experiment 1

Initial body weight (g) (n = 12-16)

Control IMO 1 IMO 2 IMO 3

396.2 386.6 389.1 390.1

Experiment 2 Control IMO 1 IMO 2 IMO 3

--- 8.0 - 7.3 --- 11.6 ± 8.5

(n = 6) 377.0 369.6 366.8 360.7

- 13.5 --+ 5.3 --+ 8.0 --- 1 0 . 2

IMO

SCHEDULES

Final weight increment (g) (n = 12-16) 32.0 -54.9 -55.0 -42.7

± ± -

2.2 5.1" 4.7* 3.8*

(n = 6) 46.6 -30.4 -46.7 -47.2

± 2.6 - 3.9* --- 2.5",t ± 3.0*,t

ON SOME PHYSIOLOGICAL

VARIABLES

Adrenal weight Food intake (g/rat/day) (n = 6-8) 23.0 14.6 14.0 15.3

± -

0.7 0.7* 0.6* 0.3*

Absolute (rag) (n = 6-7) 57.7 70.6 72.5 70.5

± 3.0 ± 3.5* -+ 3.1" ± 2.6*

Relative (rag/100 g) (n = 6-7) 13.4 20.9 21.7 21.3

--- 0.7 ± 1.0" -+ 1.3" ± 0.6*

n = 6) 25.8 16.5 14.6 15.9

- 0.8 ± 0.4* ± 0.5* --- 0.9*

See Materials and Methods section for details. Mean -+ SEM are represented. The number of animals per group or the number of cages controlled (food intake) are in parentheses. *p < .05 vs. controls, t p < .05 vs. IMO 1 (Student-Newman-Keuls, SNK).

IMO groups also showed adrenal hypertrophy, both in absolute and relative terms. The changes were of the same magnitude, regardless of the treatment (Table I). Figures 1A and 1B show the effect of the different chronic IMO schedules on the PA axis. The t w o - w a y A N O V A revealed significant effects of chronic treatment and time (p < .001 in both cases) on serum corticosterone. The interaction between the two main factors was also significant (p = .001). In contrast, only a significant effect of time (p < .001) on serum A C T H was found. When serum corticosterone levels of the different chronic treatments were compared at each time, the S N K test revealed that serum corticosterone levels were higher in all groups of IMO rats as compared to control rats at 0830h and 1400h, but not at 1900h and 2400h. Figures 2A and 2B show the effect of chronic IMO on the circadian pattern of T S H and GH. The two-way A N O V A showed that both chronic treatment and time significantly modified serum G H and T S H levels (p < .001 in both cases). The interaction chronic treatment by time was also highly significant (p < .001) for both hormones. Post-hoc comparison of individual groups at each time revealed a complex effect of the different IMO schedules on T S H and G H (see Fig. 2). In Experiment 2, one-way A N O V A s revealed significant effect of chronic stress on food intake (p < .0001) and final weight gain (p < .0001). A Student-Newman-Keuls (SNK) test showed that chronic IMO resulted in diminished food intake with no differences between the three groups of IMO. Body weight loss was significant in all IMO groups, but it was slightly greater in IMO 2 and IMO 3 rats than in IMO 1 rats (Table I).

71

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Concerning the circadian rhythm of food intake, we first compared the three IMO groups in order to explore possible differences between these groups. Since no significant effect of the various IMO schedules was found, the three groups were pooled and compared with control rats. In this case, the MANOVA showed significant effect of chronic IMO (p < .001) and time (p < .001). The interaction was also highly significant (p = .001). When control and IMO rats were compared at each time using the Student's t-tests, statistical difference was reached only in the period comprised between 1900h and 2400h (p = .001) (Fig. 3). DISCUSSION In accordance with previous results (Armario et al., 1988; Daniels-Severs et al., 1973; Stone & Platt, 1982), chronic stress reduced food intake and body weight gain and caused adrenal hypertrophy. The effect was the same in all IMO groups. Although body weight loss of IMO 1 rats in Experiment 2 was lower than that of IMO 2 and IMO 3 rats, we were unable to observe differences in the body weight loss using the same IMO schedule in similar experimental designs either in Experiment 1 or in other four studies (unpublished data). This suggests that these gross physiological variables are not substantially affected by the particular IMO schedule. With regard to the PA axis, the most interesting finding was the fact that all IMO groups showed higher diurnal levels of corticosterone than control rats, with no changes in ACTH levels. The dissociation between ACTH and corticosterone could be due to the

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high sensitivity of the adrenal to ACTH at low levels of this hormone, so that minor changes in ACTH could have not been detected by RIA. However, this possibility seems unlikely to us because we have never found increased ACTH levels in chronic IMO rats in several previous experiments run in our laboratories. Alternatively, this might be due, at least in part, to the increased adrenocortical responsiveness to ACTH observed in chronically stressed rats, as reported by us and others (Armario et al., 1985, 1988; Riegle, 1973). The reason for this enhanced adrenal response to ACTH is not well understood, but there are several possibilities. First, although our results could be explained by a decrease in plasmatic clearance rate of corticosterone, this seems unlikely, as N6meth et al. (N6meth & Vigas, 1973; N6meth et al., 1975) observed no change in the rate of disappearance of corticosterone from plasma in rats chronically subjected to trauma. In addition, we have found that serum levels of corticosterone 2 hr after exogenous corticosterone administration did not differ between control and chronic IMO rats (unpublished results). Second, certain factors present in plasma, like 7-MSH, are reported to be potential enhancers of the effects of ACTH on the adrenal gland (Reader et al., 1982). Third, some authors have suggested a direct role for corticotropin releasing factor (CRF) in the control of the adrenocortical activity, by a mechanism independent of pituitary ACTH release. Thus, Van Oers and Tilders (1991) showed that intravenous (IV) administration of a highly specific monoclonal antibody to rat CRF reduced corticosterone levels in the morning, without modifying ACTH levels. Similarly, Andreis et al. (1991) found that intraperitoneal (IP) administration of CRF increased blood levels of corticosterone in hypophysectomized rats, with no rise in circulating levels of ACTH. Nevertheless, the relevance of these factors to explain the changes we observed in adrenocortical secretion after chronic stress remains unknown. In chronically stressed pigeons, Ramade and Bayle (1984, 1985) have described an anticipatory peak of corticosterone before acute exposure to stress. This phenomenon does not appear to be relevant to explain our results, because the chronic IMO-induced increase in corticosterone levels was similar in all IMO groups irrespective of the regularity of exposure to the stressor. In addition, Ramade and Bayle (1984) reported that the anticipatory peak observed in pigeons was drastically dependent on a strictly regular daily exposure to the stressor. Regardless of the mechanisms responsible for the dissociation between ACTH and corticosterone in chronic IMO rats, the finding that increased serum corticosterone was observed at lights on but not at lights off in the three chronic IMO groups is noteworthy. We have not found any evidence for a shift in the circadian corticosterone pattern after chronic IMO, and therefore other alternative explanations are needed. Since in normal rats the adrenal responsiveness to ACTH changes throughout the day, it appears possible that enhanced adrenocortical responsiveness to ACTH might be restricted, through unknown mechanisms, to those hours during which the animals are usually subjected to stress. Since the rat is a nocturnal species, it would be useful to compare the effect of chronic IMO in rats stressed at lights on and in rats stressed at lights off. With regard to GH and TSH levels, the low values found in stressed animals could have been due, at least in part, to the reduced food intake caused by IMO as both

CHRONIC STRESS AND ANTERIOR PITUITARY HORMONES

75

hormones are very sensitive to reduced food intake (Armario & Jolfn, 1986; Moberg et al., 1975). H o w e v e r , the three IMO groups showed similar total amount of food ingested and circadian rhythm of food intake. At present, no other factor than light appears to synchronize circadian T S H rhythmicity (Armario & Jolin, 1986; Ottenweiler & Hedge, 1982). However, we have found no circadian T S H rhythmicity in IMO 3 rats (one-way Kruskall-Wallis A N O V A , p -- .4926). This suggests that the specific pattern of exposure to chronic IMO might have a role in the control of circadian T S H rhythms, or that frequent changes in the time the animals are exposed to stress might be, in some respects, more noxious than regular exposure, resulting in a higher degree of T S H inhibition. Although chronic IMO diminishes GH, the circadian rhythm of serum G H in all IMO rats, as measured at the indicated time clocks, was roughly parallel to that of controls rats. This finding suggests that the zeitgeber of G H secretion, which is commonly associated to REM sleep in the sleep-wakefulness cycle rather than to food intake (Kimura & Tsai, 1984; Martino et al., 1985; Mitsugi & Kimura, 1985; Moore-Ede, 1986; Mosier & Jansons, 1987; Tannenbaum et al., 1976) is not substantially affected by chronic regular and irregular immobilization stress. Nevertheless, chronic IMO could have induced changes in the ultradian G H secretion pattern. This possibility cannot be discarded, due to the low frequency of blood sampling used in the present experiment. In summary, the present results demonstrate that the effects of chronic stress on resting hormone levels depend on the time point at which the animals are killed. Intermittent chronic exposure to a severe stressor such as IMO, regardless of the regularity of exposure, causes an increase in serum corticosterone levels in the diurnal phase of the day only. This increase was not associated to enhanced A C T H release, so that other mechanisms should be involved. In addition, chronic stress provokes a decrease in plasma levels of G H and T S H probably due, at least in part, to stress-induced anorexia. H o w e v e r , chronic IMO rats present alterations in the T S H and G H circadian rhythms apparently linked, in a complex way, to the particular IMO schedule.

Acknowledgments: Octavi Marti is a fellow of the Formacio d'Investigadors programme of the Universitat Aut6noma de Barcelona. This work was partially supported by grants from DG1CYT86/0030, FISss 87/1232, FISss 88/1016, and C1RIT-AR/88. REFERENCES Abbott BB, Schoen LS, Badia P (1984) Predictable and unpredictable shock: Behavioral measures of aversion and physiological measures of stress. Psychol Bull 96:45-71. Adell A, Garcfa-Mfirquez C, Armario A, Gelpf E (1988) Chronic stress increases serotonin and noradrenaline in rat brain and sensitizes their responses to a further acute stress. J Neurochem 50:1678-1681. Andreis PG, Neri G, Nussdorfer GG ( 199 I) Corticotropin-releasing hormone (CRH) directly stimulates corticosterone secretion by the rat adrenal gland. Endocrinology 128:1198-1200. Anisman H, Kokkinidis L, Sklar LS (1981) Contribution of neurochemical change to stress-induced behavioral deficits. In: Cooper SJ (Ed) Theory in Psyc'hopharmacology, Vo! l. Academic Press, London, pp 65-102. Armario A, Campmany L, L6pez-Calder6n A, Joli'n T (1990) Chronic stress reduced GH response to the serotonin agonist 5-methoxy N,N-dimethyltriptamine but did not alter pituitary-adrenal, prolactin, or TSH responses in the rat. Stress Med 6:133-139.

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