Neuroendocrinology 29: 90-99 (1979)

Effects of Chronic Immobilization Stress on Pituitary Hormone Secretion, on Hypothalamic Factor Levels, and on Pituitary Responsiveness to LHRH and TRH in Female Rats P. Du Ruisseau, Y. Taché, P. Brazeau1 and R. Collu Centre de Recherche Pédiatrique, Hôpital Sainte-Justine and Research Centre, Montreal General Hospital, Montreal, P.Q.

Key Words. Chronic stress • AP hormones • LHRH • TRH • SRIF

1 Scholar of the Medical Research Council of Can­ ada.

Received: June 29, 1978 Accepted: January 10, 1979

It has recently been published that chronic intermittent stress in female [9, 25] and in male rats [23] elicits an inverse relationship be­ tween the secretion of ACTH and those of GH, LH, PRL, and to a lesser degree, of FSH.

Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 7/11/2018 1:47:15 PM

Abstract. The effects of 8 h of daily immobilization applied for 10 consecutive days on pituitary and plasma levels of anterior pituitary (AP) hormones as well as on TRH, LHRH and somatostatin (SRIF) content in the hypothalamus, amygdala and forebrain were studied in adult female rats. The in vivo pituitary responsiveness to intraventricular injections of LHRH combined with TRH was also tested in groups of rats submitted to the same stress procedure. Long-term intermittent immobilization stress markedly reduced GH, LH, PRL and TSH plasma levell, while FSH titers were not modified. Parallel to these variations, FSH and LH pituitary levels increased, while those of GH, PRL and TSH remained unchanged. No modifi­ cation of hypothalamic or amygdala TRH, LHRH or SRIF content have been observed, whereas forebrain TRH levels decreased significantly. In nonstressed animals, LHRH + TRH significantly increased LH and TSH plasma levels and decreased those of PRL, FSH and GH. In immobilized rats, the hypothalamic factors further stimulated the secretion of LH and TSH, released FSH and PRL, and had a greater inhibiting effect on plasma GH levels. These results indicate that chronic stress-induced decreases in plasma AP hormone levels are not associated with a diminished pituitary responsiveness to releasing factors, nor with modification of hypothalamic factor content. Furthermore, the enhanced liberation of LH,and the release of FSH after LHRH + TRH administration during stress could be secondary to the increased pituitary content of these hormones.

pituitary Hormones and Hypothalamic Factors during Chronic Stress

Materials and Methods Animals Adult female Charles River CD® rats (Canadian Breeding Farms and Laboratories Ltd., St. Constant, Que.), weighing 200 g, were maintained ad libitum on Purina laboratory chow (Mondou Inc. Montreal, Que.) and tap water. They were housed 5/cage under condi­ tions of controlled lighting (from 06:00 to 18:00 h) and temperature (2 4 ± 2 “C). The estrous cycle was monitored by daily vaginal smears, and in all experi­ ments control animals were used at diestrus, since chronically stressed rats arc in a state of persistent diestrus.

In vivo Pituitary Responsiveness to Releasing Factors A chronic intraventricular (IVT) cannula was im­ planted in 8 groups of 10 rats under Nembutal anes­ thesia (Abbott, 50 mg/kg IP) according to the method of Hayden et al. [13] 48 h prior to the experiment. Four groups were submitted to immobilization stress 8 h daily for 10 consecutive days as previously de­ scribed [25] and the remaining four groups were left undisturbed. 20 min before the end of the last im­ mobilization period one stressed and one nonstressed group were injected in the right ventricle of the brain with saline (10 ,ul) while the remaining 6 groups re­ ceived LHRH (Ayerst, 250, 500 or 750 ng) and TRH (Abbott, 1,2.5 or 5 //g) diluted in saline. The animals were killed by decapitation 20 min after the injection, around 17:00 h. Trunk blood was collected in heparin­ ized tubes, centrifuged immediately, and the plasma stored at -20 °C for subsequent analysis. Individual plasma samples were assayed in duplicate for GH, PRL, LH, FSH and TSH levels. The IVT route of administration of releasing hor­ mones was chosen not because it is more effective than the IV, but rather because it can be used without additional stress for the animals such as a SC or an IV injection in the tail vein would have caused. Levels o f A P Hormones and Hypothalamic Factors One group of 12 rats was immobilized 8 h/day for 10 consecutive days while a control group was left undisturbed. At the end of the last immobilization period, the animals of both groups were decapitated. Trunk blood was collected in heparinized tubes, cen­ trifuged and the plasma stored at -20°C for subsequent analysis. The pituitaries were weighed after removal of the posterior lobe and homogenized in 5 ml of 1% BSA-phospho-saline buffer. The brains were rapidly removed, cleaned of blood with cold saline and im­ mediately frozen on dry ice for subsequent determina­ tion of LHRH, TRH and SRIF levels in the hypo­ thalamus, amygdala and forebrain. Hypothalamic and forebrain sections were obtained by the technique of Glowinsky and Iversen [12]. The forebrain section con­ tained both frontal and some temporal cortex, as well as some anterior diencephalic tissue. Amygdaloid nu­ clei were included in a region 3 mm deep between the sulcus rhinalis of the temporal lobe and the optic tracts. The rostral border of the amygdala was limited by a cut in the temporal lobe at the level of the optic

Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 7/11/2018 1:47:15 PM

With regard to the pattern of endocrine response to repeated stress, it appears also that the magnitude and duration of the mor­ phologic, and to some extent of the hormonal changes, are related to the severity of the stressors [23], The ability of the pituitary to simultaneously release increased amounts of several trophic hormones after combined hemiextirpation of several glands [9] and the unchanged hormonal responses to repeated immobilization in dexamethasone-treated rats [24] rule out the possibility that a causal re­ lationship exists between enhanced ACTH secretion and the suppression of other pitu­ itary hormones. In order to evaluate whether the major hormonal effects of long-term stress are ex­ erted at the pituitary and/or the hypothalamic level, hypophyseal and plasma concentrations of anterior pituitary (AP) hormones and the hypothalamic, amygdala and forebrain con­ tent of TRH, LHRH and SRIF were deter­ mined in chronically immobilized female rats. The AP response to intraventricular injections of LHRH combined with TRH was also tested in chronically stressed rats. A preliminary re­ port of this work has already been published [8, 21].

91

Du Ruisseau/Tache/Brazeau/Collu

92

chiasm and the caudal border by a cut just anterior to the mammillary bodies. The hypothalamus and fore­ brain were further cut into 2 equal halves and the right and left amygdala were handled separately. The individual parts were weighed and homogenized with either 10 vol of methanol (for TRH extraction) or with 1 ml of 2 N acetic acid (for LHRH and SR1F extrac­ tion). After centrifugation for 10 min at 1,500 g of the methanol extract, the supernatants were dried over­ night under an air stream, and then were resuspended in buffer solution (0.01 M phosphate, 0.15 M NaCl, pH 7.5) containing 1% egg white for hypothalamic factor assay. The 2 N acetic acid extracts were cen­ trifuged as described above and the supernatants were assayed. Pituitary Hormone and Hypothalamic Factor Determinations The plasma and pituitary concentrations of GH, PRL, FSH and TSI-I were measured with radioim­ munoassay kits supplied by NIAMDD, and plasma LH levels were determined according to the method of Niswender et at. [17], The values of these hormones are expressed in terms of their respective NIAMDD-RatRP-1 standards. TRH, LHRH and SRIF were mea­ sured with highly specific double antibody radioim­ munoassays using ( l25I) iodoTRH, LHRH and SRIF. Anti-TRH [5] and anti-LHRH [10] were produced in New Zealand white rabbits immunized with TRH and

LHRH coupled to bovine serum albumin (BSA). AntiSRIF was produced in sheep immunized with SRIF coupled to methylated BSA [11]. In each experiment, the hormonal assays were performed in single batches to avoid interassay variability. Statistical Probabilities Statistical probabilities were calculated using one­ way analysis of variance or Student’s t test

Results Effects o f Chronic Stress on Pituitary Hormone and Hypothalamic Factor Levels

Daily immobilization stress (8 h/day) for 10 days, significantly decreased TSH (-36%), PRL (-58%), LH (-78%) and GH (-91%) plasma levels but did not modify those of FSH. In chronically restrained rats a marked increase in pituitary levels of FSH (207%) and LH (198%) was observed, while those of TSH, PRL and GH were not modified by the stress regimen (table I).

TSH Pituitary levels, /

Effects of chronic immobilization stress on pituitary hormone secretion, on hypothalamic factor levels, and on pituitary responsiveness to LHRH and TRH in female rats.

Neuroendocrinology 29: 90-99 (1979) Effects of Chronic Immobilization Stress on Pituitary Hormone Secretion, on Hypothalamic Factor Levels, and on Pi...
1MB Sizes 0 Downloads 0 Views