Corticotropin-releasing hormone and pituitary-adrenocortical responses in chronically stressed rats

Brain corticotropin-releasing hormone (CRH) concentration and pituitary adreno-cortical responses were examined in chronically stressed rats: body restraint stress (6 h/day) for 4 or 5 weeks. Stressed rats showed a reduction in weight gain. CRH concentration in the median eminence and the rest of the hypothalamus were not different between control and chronically immobilized rats. The anterior pituitary adenocorticotropic hormone (ACTH) concentration was elevated in chronically stressed rats, whereas plasma ACTH and corticosterone levels did not differ from the control values. The median eminence CRH concentration was reduced to the same extent at 5 min after onset of ether exposure (1 min) in chronically immobilized rats and controls. However, plasma ACTH and corticosterone showed greater responses to ether stress in chronically immobilized rats than in control rats. Plasma ACTH and corticosterone responses to exogenous CRH were not different between control and chronically immobilized rats, while the response to arginine vasopressin (AVP) was significantly greater in chronically immobilized rats. These results suggest that chronic stress caused an increase in the ACTH-secreting mechanism and that pituitary hypersensitivity to vasopressin might at least be partly responsible for this.     

Nitric oxide mediates the interleukin-1beta- and nicotine-induced hypothalamic-pituitary-adrenocortical response during social stress.

We investigated the role of nitric oxide (NO) in the interleukin 1beta (IL-1beta) and nicotine induced hypothalamic-pituitary-adrenal axis (HPA) responses, and a possible significance of CRH and vasopressin in these responses under basal and social stress conditions. Male Wistar rats were crowded in cages for 7 days prior to treatment. All compounds were injected i.p., nitric oxide synthase (NOS) inhibitors, alpha-helical CRH antagonist and vasopressin receptor antagonist 15 min before IL-1beta or nicotine. Identical treatment received control non-stressed rats. Plasma ACTH and serum corticosterone levels were measured 1 h after IL-1beta or nicotine injection. L-NAME (2 mg/kg), a general nitric oxide synthase (NOS) inhibitor, considerably reduced the ACTH and corticosterone response to IL-1beta (0.5 microg/rat) the same extent in control and crowded rats. CRH antagonist almost abolished the nicotine-induced hormone responses and vasopressin antagonist reduced ACTH secretion. Constitutive endothelial eNOS and neuronal nNOS inhibitors substantially enhanced the nicotine-elicited ACTH and corticosterone response and inducible iNOS inhibitor, aminoguanidine, did not affect these responses in non-stressed rats. Social stress significantly attenuated the nicotine-induced ACTH and corticosterone response. In crowded rats L-NAME significantly deepened the stress-induced decrease in the nicotine-evoked ACTH and corticosterone response. In stressed rats neuronal NOS antagonist did not alter the nicotine-evoked hormone responses and inducible NOS inhibitor partly reversed the stress-induced decrease in ACTH response to nicotine. These results indicate that NO plays crucial role in the IL-1beta-induced HPA axis stimulation under basal and social stress conditions. CRH and vasopressin of the hypothalamic paraventricular nucleus may be involved in the nicotine induced alterations of HPA axis activity. NO generated by eNOS, but not nNOS, is involved in the stress-induced alterations of HPA axis activity by nicotine.     

Prenatal exposure to dexamethasone alters hippocampal drive on hypothalamic-pituitary-adrenal axis activity in adult male rats

Glucocorticoids are essential for normal hypothalamic-pituitary-adrenal (HPA) axis activity; however, recent studies warn that exposure to excess endogenous or synthetic glucocorticoid during a specific period of prenatal development adversely affects HPA axis stability. We administered dexamethasone (DEX) to pregnant rats during the last week of gestation and investigated subsequent HPA axis regulation in adult male offspring in unrestrained and restraint-stressed conditions. With the use of real-time PCR and RIA, we examined the expression of regulatory genes in the hippocampus, hypothalamus, and pituitary, including corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), glucocorticoid receptors (GR), mineralcorticoid receptors (MR), and 11-beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1), as well as the main HPA axis hormones, adrenal corticotropic hormone (ACTH) and corticosterone (CORT). Our results demonstrate that the DEX-exposed group exhibited an overall change in the pattern of gene expression and hormone levels in the unrestrained animals. These changes included an upregulation of CRH in the hypothalamus, a downregulation of MR with a concomitant upregulation of 11beta-HSD-1 in the hippocampus, and an increase in circulating levels of both ACTH and CORT relative to unrestrained control animals. Interestingly, both DEX-exposed and control rats exhibited an increase in pituitary GR mRNA levels following a 1-h recovery from restraint stress; however, the increased expression in DEX-exposed rats was significantly less and was associated with a slower return to baseline CORT compared with controls. In addition, circulating levels of ACTH and CORT as well as hypothalamic CRH and hippocampal 11beta-HSD-1 expression levels were significantly higher in the DEX-exposed group compared with controls following restraint stress. Taken together, these data demonstrate that late-gestation DEX exposure in rats is associated with persistent changes in both the modulation of HPA axis activity and the HPA axis-mediated response to stress.     

睡眠中间断低氧对大鼠下丘脑-垂体-肾上腺轴和生长激素的影响

目的:探讨睡眠中间断低氧对大鼠下丘脑-垂体-肾上腺轴和生长激素水平的影响.方法:大鼠分别给予吸入空气,持续低氧和间断低氧气体,在1 d,3 d,7 d和30 d后测定下丘脑促肾上腺皮质激素释放激素(CRH)和生长激素释放激素(GHRH)mRNA水平,并测定30d后血浆CRH,GHRH,促肾上腺皮质激素(ACTH)和皮质酮水平,分析其间的变化关系.结果:与对照组比较,在低氧后1 d,3 d,7 d后大鼠下丘脑CRH mRNA升高,GHRH mRNA降低,在30 d后,间断低氧组下丘脑CRH mRNA升高,GHRH mRNA降低,而持续低氧组则接近正常.间断低氧30 d后,血浆CRH、ACTH,皮质酮均升高,GHRH降低,而生长激素没有明显变化.结论:大鼠睡眠中慢性间断低氧可以引起下丘脑-垂体-肾上腺轴激素水平升高,反馈调节紊乱,可引起GHRH分泌抑制.     

维吾尔药神香草调节哮喘大鼠下丘脑-垂体-肾上腺轴功能紊乱的实验研究

目的：观察维吾尔药（维药）神香草对哮喘大鼠模型神经内分泌免疫网络若干组分的影响。方法：取雄性健康Wistar大鼠50只,随机分为正常对照组,哮喘模型组,神香草低、中、高剂量治疗组。采用致敏和雾化的方法制备哮喘模型。采用酶联免疫吸附试验（ELISA）方法检测血清中白介素-4（IL^4）、白介素-6（IL-6）、干扰素-γ（IFN-γ）、皮质酮（CORT）水平;放免法检测血浆中促肾上腺皮质激素（ACTH）的含量;采用实时定量-聚合酶链反应（RT—PCR）法测定下丘脑促肾上腺皮质激素释放激素（CRH）mRNA表达水平。结果：哮喘反复发作时,大鼠下丘脑-垂体-肾上腺（HPA）轴紊乱,哮喘大鼠下丘脑CRHmRNA表达和血浆ACTH无明显变化,实验各组血清CORT水平升高（P〈0．05）,神香草高剂量组血清CORT含量高于低剂量组（P〈0．05）。细胞因子IFN-γ无明显变化,IL-6、IL-4有下降趋势（P〈0．05）。结论：哮喘反复发作的大鼠存在NEI网络的紊乱;神香草可以增强下丘脑．垂体．肾上腺皮质（HPA）轴的功能,改善细胞因子的平衡。这些可能是其治疗哮喘的机制之一。     

Illuminating the interrelated immune and endocrine adaptations after multiple exposures to short immobilization stress by in vivo blocking of IL-6

Intermittent psychological stress was induced in adult rats by 2 h/day of immobilization stress for 4 days, with or without blocking the function of IL-6 by using an anti-IL-6 antibody. Basal concentrations of serum corticosterone, IL-1beta, IL-6, and TNF-alpha were assessed 24 h after the last intervention, as were levels of glucocorticoid receptors (GR) and activities of glucocorticoid-inducible enzymes (tyrosine aminotransferase and glutamine synthetase) in muscle and liver. Whole blood cultures were used to assess both spontaneous and LPS-induced reactivity of peripheral blood mononuclear cells. Stress increased corticosterone concentration in a manner partially modulated by IL-6. Serum IL-1beta concentration was downregulated during stress when IL-6 was blocked (P < 0.01). LPS-induced IL-6 secretion by peripheral blood mononuclear cells in vitro correlated positively with serum IL-1beta concentration in antibody-treated groups, independently of stress (R = 0.70 in nonstressed and R = 0.78 in stressed rats; both P < 0.05), whereas serum corticosterone concentration correlated positively with LPS-induced secretion of IL-6 only in control rats (R = 0.66; P < 0.05). Reductions in liver GR levels indicated independent effects of stress (34.5%) and anti-IL-6 antibody (16.7%) and additive effects for both (62.5%). Similar results are reported for vastus muscle. Conversely, stress increased tyrosine aminotransferase and glutamine synthetase activities in muscle and liver with a significant (P < 0.05) effect of anti-IL-6 antibody only seen in stressed livers. In conclusion, IL-6 plays a role in maintaining circulating IL-1beta concentration after multiple exposures to stress, thus promoting a continued elevation of corticosterone release; in peripheral tissues, IL-6 antagonizes the effects of glucocorticoids, especially at the level of GR concentration.     

Central injection of morphine stimulates plasma corticosterone and interleukin (IL)-6 and IL-6 R mRNAs in the pituitary and adrenals in adjuvant-induced arthritis.

Adjuvant-induced arthritis (AA) in the rat is a T-cell mediated, chronic inflammatory stress in which circulating interleukin (IL)-6 levels are elevated. In addition, there are profound neuroendocrine changes associated with the development of hind-paw inflammation which have major implications for the ability of the rat to respond the to stress. Central injection of morphine is also able to increase circulating IL-6 concentration in control animals. In the present study we have determined the effects of a single injection of morphine into the lateral ventricle of control and AA animals on plasma corticosterone levels, on changes in plasma corticosterone and on IL-6 and IL-6 receptor mRNAs in the pituitary and adrenal gland. IL-6 and IL-6 receptor mRNAs were increased in the anterior pituitary of AA rats given moprhine compared with saline-treated AA rats. In the adrenal cortex, IL-6 mRNA was unaltered and IL-6 receptor mRNA was significantly decreased under these same conditions. AA rats were unable to mount corticosterone response to acute stress but were able to respond to acute stimulation with e.g. LPS. In the present study we found a sustained increase in plasma corticosterone in control animals which was still significantly elevated 2 hours following morphine injection, with a further significant increase in AA rats. These data suggest that alternative systems distinct from those activated in response to acute stress are activated by morphine in the AA animals. The similarity with the sustained increase in corticosterone following LPS injection suggest that either similar pathways are involved, or that central opiates may be involved in mediating HPA axis response to stress.     

Hyperglycemia does not increase basal hypothalamo-pituitary-adrenal activity in diabetes but it does impair the HPA response to insulin-induced hypoglycemia

Recently, we established that hypothalamo-pituitary-adrenal (HPA) and counterregulatory responses to insulin-induced hypoglycemia were impaired in uncontrolled streptozotocin (STZ)-diabetic (65 mg/kg) rats and insulin treatment restored most of these responses. In the current study, we used phloridzin to determine whether the restoration of blood glucose alone was sufficient to normalize HPA function in diabetes. Normal, diabetic, insulin-treated, and phloridzin-treated diabetic rats were either killed after 8 days or subjected to a hypoglycemic (40 mg/dl) glucose clamp. Basal: Elevated basal ACTH and corticosterone in STZ rats were normalized with insulin but not phloridzin. Increases in hypothalamic corticotrophin-releasing hormone (CRH) and inhibitory hippocampal mineralocorticoid receptor (MR) mRNA with STZ diabetes were not restored with either insulin or phloridzin treatments. Hypoglycemia: In response to hypoglycemia, rises in plasma ACTH and corticosterone were significantly lower in diabetic rats compared with controls. Insulin and phloridzin restored both ACTH and corticosterone responses in diabetic animals. Hypothalamic CRH mRNA and pituitary pro-opiomelanocortin mRNA expression increased following 2 h of hypoglycemia in normal, insulin-treated, and phloridzin-treated diabetic rats but not in untreated diabetic rats. Arginine vasopressin mRNA was unaltered by hypoglycemia in all groups. Interestingly, hypoglycemia decreased hippocampal MR mRNA in control, insulin-, and phloridzin-treated diabetic rats but not uncontrolled diabetic rats, whereas glucocorticoid receptor mRNA was not altered by hypoglycemia. In conclusion, despite elevated basal HPA activity, HPA responses to hypoglycemia were markedly reduced in uncontrolled diabetes. We speculate that defects in the CRH response may be related to a defective MR response. It is intriguing that phloridzin did not restore basal HPA activity but it restored the HPA response to hypoglycemia, suggesting that defects in basal HPA function in diabetes are due to insulin deficiency, but impaired responsiveness to hypoglycemia appears to stem from chronic hyperglycemia.     

Electrical stimulation of afferent vagus nerve induces IL-1beta expression in the brain and activates HPA axis

Possible roles of the afferent vagus nerve in regulation of interleukin (IL)-1beta expression in the brain and hypothalamic-pituitary-adrenal (HPA) axis were examined in anesthetized rats. Levels of IL-1beta mRNA and protein in the brain were measured by comparative RT-PCR and ELISA. Direct electrical stimulation of the central end of the vagus nerve was performed continuously for 2 h. The afferent stimulation of the vagus nerve induced increases in the expression of mRNA and protein levels of IL-1beta in the hypothalamus and the hippocampus. Furthermore, expression of corticotropin-releasing factor mRNA was increased in the hypothalamus 2 h after vagal stimulation. Plasma levels of ACTH and corticosterone were also increased by this stimulation. The present results indicate that activation of the afferent vagus nerves itself can induce production of IL-1beta in the brain and activate the HPA axis. Therefore, the afferent vagus nerve may play an important role in transmitting peripheral signals to the brain in the infection and inflammation.     

Dehydration-induced drinking decreases Fos expression in hypothalamic paraventricular neurons expressing vasopressin but not corticotropin-releasing hormone

Water-restricted (WR) rats exhibit a rapid suppression of plasma corticosterone following drinking. The present study monitored Fos-like immunoreactivity (Fos) to assess the effect of WR-induced drinking on the activity of vasopressin (VP)-positive magnocellular and parvocellular neurons and corticotropin-releasing hormone (CRH)-positive parvocellular neurons in the paraventricular nucleus of the hypothalamus. Adult male rats received water for 30 min (WR) in the post meridiem (PM) each day for 6 days and were killed without receiving water or at 1 h after receiving water for 15 min. In WR rats, Fos increased in VP magnocellular and parvocellular neurons but not CRH neurons. After drinking, Fos was reduced in VP magnocellular and parvocellular neurons but did not change in CRH neurons. To assess the severity of osmotic stress, rats were sampled throughout the final day of WR. Plasma osmolality, hematocrit and plasma VP were increased throughout the day before PM rehydration, and plasma ACTH and corticosterone were elevated at 1230 and 1430, respectively, showing that WR activates hypothalamic-pituitary-adrenal activity during the early PM before the time of rehydration. To determine the effects of WR-induced drinking on CRH neurons activated by acute stress, WR rats underwent restraint. Restraint increased plasma ACTH and corticosterone and Fos in CRH neurons; although rehydration reduced plasma ACTH and Fos expression in VP neurons, Fos in CRH neurons was not affected. These results suggest that inhibition of VP magnocellular and parvocellular neurons, but not CRH parvocellular neurons, contributes to the suppression of corticosterone after WR-induced drinking.     

Elevated corticosterone and inhibition of ACTH responses to CRH and ether in the neonatal rat: effect of hypoxia from birth

Hypoxia is a common cause of neonatal morbidity and mortality. We have previously demonstrated a dramatic ACTH-independent activation of adrenal steroidogenesis in hypoxic neonatal rats, leading to increases in circulating corticosterone levels. The purpose of the present study was to determine if this ACTH-independent increase in corticosterone inhibits the ACTH response to acute stimuli. Neonatal rats were exposed to normoxia (control) or hypoxia from birth to 5 or 7 days of age. At the end of the exposure, plasma ACTH and corticosterone were measured before and after either ether vapors were administered for 3 min or CRH (10 microg/kg) was given intraperitoneally. Thyroid function, pituitary pro-opiomelanocortin (POMC) mRNA and ACTH content, and hypothalamic corticotropin-releasing hormone (CRH), neuropeptide Y (NPY), and AVP mRNA were also assessed. Hypoxia led to a significant increase in corticosterone without a large increase in ACTH, confirming previous studies. The ACTH responses to ether or CRH administration were almost completely inhibited in hypoxic pups. Hypoxia did not affect the established regulators of the neonatal hypothalamic-pituitary-adrenal axis, including pituitary POMC or ACTH content, hypothalamic CRH, NPY, or AVP mRNA (parvo- or magnocellular), or thyroid function. We conclude that hypoxia from birth to 5 or 7 days of age leads to an attenuated ACTH response to acute stimuli, most likely due to glucocorticoid negative feedback. The neural and biochemical mechanism of this effect has yet to be elucidated.     

Role of hypothalamic inputs in maintaining pituitary-adrenal responsiveness in repeated restraint

The role of hypothalamic structures in the regulation of chronic stress responses was studied by lesioning the mediobasal hypothalamus or the paraventricular nucleus of hypothalamus (PVH). Rats were acutely (60 min) and/or repeatedly (for 7 days) restrained. In controls, a single restraint elevated the plasma adrenocorticotropin (ACTH), corticosterone, and prolactin levels. Repeated restraint produced all signs of chronic stress, including decreased body and thymus weights, increased adrenal weight, basal corticosterone levels, and proopiomelanocortin (POMC) mRNA expression in the anterior pituitary. Some adaptation to repeated restraint of the ACTH response, but not of other hormonal responses, was seen. Lesioning of the mediobasal hypothalamus abolished the hormonal response and POMC mRNA activation to acute and/or repeated restraint, suggesting that the hypothalamo-pituitary-adrenal axis activation during repeated restraint is centrally driven. PVH lesion inhibited the ACTH and corticosterone rise to the first restraint by approximately 50%. In repeatedly restrained rats with PVH lesion, the ACTH response to the last restraint was reduced almost to basal control levels, and the elevation of POMC mRNA level was prevented. PVH seems to be important for the repeated restraint-induced ACTH and POMC mRNA stimulation, but it appears to partially mediate other restraint-induced hormonal changes.     

Immunoneutralization of corticotropin-releasing hormone prevents the diurnal surge of ACTH

To determine whether CRH is required for the evening rise in plasma ACTH, rats were injected at 0800 hr with CRH antiserum (anti-CRH) or normal rabbit serum (NRS). Blood samples were taken through venous catheters at 0800 hr before treatment and at 1300, 1700, and 2100 hr. Plasma was assayed for immunoreactive ACTH and corticosterone. There was no significant difference in pretreatment values between the two groups. Immunoneutralization of CRH abolished the rise in plasma ACTH seen at 1700 hr in the NRS group but had little effect on earlier levels. The diurnal elevation in plasma corticosterone continued after anti-CRH treatment, but peak levels occurred earlier. Plasma ACTH and corticosterone were significantly correlated at the time of the diurnal surge, but not at 0800 hr or 1300 hr in the NRS controls or at any time point in the anti-CRH group. These results suggest that CRH is required for the diurnal surge of plasma ACTH. They also confirm previous observations by others that the adrenal cortex does not require active CRH or a diurnal surge of ACTH in order to exhibit a significant diurnal increase in secretion of corticosterone, and that factors other than CRH may be relatively more active than CRH in regulation of ACTH secretion during the time of circadian inactivity.