ACTH-treated Mice Research Articles

Search for factors contributing to resistance to the electroconvulsive seizure treatment model using adrenocorticotrophic hormone-treated mice

Approximately one third of patients with depression remain treatment resistant with existing antidepressants, suggesting that the currently-available antidepressants cannot induce appropriate responses in the brains of all patients. Long-term exposure to adrenocorticotrophic hormone (ACTH) has been proposed as a model that mimics at least some aspects of clinical treatment-resistant depression in rodents. The purpose of this study was to explore potential causes of antidepressant treatment resistance using the chronic ACTH-treated mouse model. We subjected ACTH-treated mice to a rodent model of electroconvulsive therapy, i.e., electroconvulsive seizure (ECS), which induces various molecular and cellular changes, including in gene expression and adult neurogenesis in the hippocampus. First, behavioral effect of repeated ECS in the forced swim test (FST) was examined. In our experimental setting, ACTH-treated mice showed resistance to the antidepressant-like effect of ECS in the FST. We then examined which cellular and molecular changes induced by ECS were attenuated by ACTH administration. Chronic ACTH treatment suppressed the increase of gene expression such as of Bdnf, Npy, and Drd1 induced by ECS in the hippocampus. In contrast, there was no difference in ECS-induced promotion of the early neurogenetic process in the hippocampus between ACTH-treated and control mice. Our results suggest the possibility that impaired neuromodulation and monoamine signaling in the hippocampus are among the factors contributing to antidepressant treatment resistance.

Improving Effects of Peptides on Brain Malfunction and Intranasal Delivery of Those Derivatives to the Brain

This review focuses on the anti-dementia and antidepressant-like effects of peptides including glucagon-like peptide (GLP)-1, GLP-2, neuromedin U (NmU), and oxytocin, and the intranasal delivery of these peptides to the brain. Intracerebroventricularly administered GLP-1, NmU, and oxytocin improved impairment of learning and memory in mice treated with lipopolysaccharide or β-amyloid protein. GLP-1 also improved impairment of learning and memory in juvenile diabetes model rats. On the other hand, GLP-2 exhibited antidepressant-like effects in mice during the forced-swim test, which were associated with 5-HT1A, α2, β1, and D2 receptors. GLP-2 also exerted antidepressant-like effects in adrenocorticotropic hormone (ACTH)-treated mice through restoration of the hypothalamic-pituitary-adrenal-axis and neurogenesis in the subgranular zone of the dentate gyrus. Because intracerebroventricular administration is invasive and the peptides are unable to penetrate the blood-brain barrier, we introduced our new method of intranasal administration to deliver the peptides to the brain. We prepared a GLP-2 derivative containing cell-penetrating peptides (CPPs) and a penetration accelerating sequence (PAS). Intranasally administered PAS-CPPs-GLP-2 was distributed throughout the brain, and exhibited antidepressant-like effects in both naive and ACTH-treated mice. The derivatives of GLP-1, NmU, and oxytocin with the PAS and CPPs were also distributed throughout the brain after intranasal administration, and improved impairment of learning and memory. We confirmed that our peptide derivatives were effectively delivered into the brain by intranasal administration. As such, these derivatives may be useful for the clinical treatment of psychiatric and neurological diseases.

Mechanisms of Salt‐Sensitive Hypertension in a Mouse Model of ACTH‐Dependent Cushing Syndrome

Salt‐sensitive blood pressure (BP) reflects underlying renal and vascular (endothelial) dysfunction. People with abnormal glucocorticoid homeostasis, such as in Cushing syndrome and obesity, are often salt‐sensitive but the underlying mechanisms are not clearly defined1,2. Here, we used a mouse model to examine the effect of ACTH excess on blood pressure and salt‐sensitivity. To test this, radio telemetry was used to track BP longitudinally and measure the effect of high salt diet before and after chronic ACTH treatment in C57BL/6J mice (n=6). Data are mean ± SD. After 7 days of baseline measurements, mean BP was 105.8 ± 6.5mmHg before being placed on a high salt diet (3 % Na+ by weight) for a further 7 days. BP increased ~12 mmHg over the first 3 days before declining to 113.1 ± 7.2mmHg. BP returned to 107.7 ± 10.4mmHg, similar to baseline, after a 5‐day washout period. Mice were then treated with ACTH (2.5μg/day). ACTH increased mean BP to 115.5 ± 5.0mmHg with a flattening of the diurnal rhythm following 13 days infusion. When placing these mice on a high salt diet, mean BP increased and stabilised to 136.1 ± 13.4mmHg after 14 days. These data show that chronic ACTH excess contributes to salt‐sensitivity. To investigate potential mechanisms of salt‐sensitivity, the acute pressure natriuresis (PN) response of renal artery function were examined in separate cohorts of mice treated with either vehicle or ACTH (2.5μg/day; n=6 per group) for 14 days. PN response was induced in anaesthesised mice (120mg/kg Inactin) by sequential arterial ligation. BP increased ~40mmHg following ligations in both vehicle and ACTH groups. In vehicle‐treated mice, fractional sodium excretion increased from 0.2 ± 0.2 to 5.0 ± 4.2μmol/min. The natriuretic response was significantly blunted in ACTH‐treated mice, reaching 2.1 ± 1.1μmol/min at its peak. We next investigated vascular function in ACTH‐excess ex vivo by wire myography in isolated mesenteric and renal arteries. Vascular reactivity to phenylephrine (PHE), acetylcholine (ACh) and sodium nitroprusside (SNP) was assessed. ACTH‐excess caused vascular dysfunction in the renal artery but not in the mesenteric artery. ACTH‐excess resulted in a reduction in sensitivity to vasoconstrictor PHE (p=0.0005) and endothelium dependent (ACh) and endothelium independent (SNP) vasodilators (p=0.0128 and p=0.0002, respectively). These data show that chronic glucocorticoid excess contributes to salt‐sensitivity with potential mechanisms behind this being impairment of the acute natriuretic and diuretic response to increased BP and renal vascular dysfunction.Support or Funding InformationSupported by British Heart Foundation PhD studentships FS/16/54/32730 & FS/15/63/32033.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Immunohistochemical determination of the site of antidepressant-like effects of glucagon-like peptide-2 in ACTH-treated mice

The intracerebroventicular administration (i.c.v.) of glucagon-like peptide-2 (GLP-2) had antidepressant-like effects on saline-treated mice in the forced-swim test. The GLP-2 treatment (3μg, i.c.v.) for 6days, but not that of imipramine had antidepressant-like effects on adrenocorticotropic hormone (ACTH)-treated mice. The immunohistochemical detection of the c-fos protein (Fos) revealed that the administration of GLP-2 induced Fos-immunoreactivity (Fos-IR) in the dorsomedial hypothalamic nucleus in saline-treated and ACTH-treated mice, and also in the hippocampal dentate gyrus in ACTH-treated mice, but not in saline-treated mice. In contrast, Fos-IR in the paraventricular nucleus of the hypothalamus decreased after the administration of GLP-2 to ACTH-treated mice. In ACTH-treated mice, the chronic administration of GLP-2 affected hippocampal neurogenesis, in addition to Fos-IR in hypothalamic GABAergic neurons and corticotrophin-releasing factor-containing neurons. These results suggest that GLP-2 acts on specific brain regions to regulate stress conditions, and induces antidepressant-like effects under imipramine-resistant conditions, which may be associated with the modulation of the hypothalamic–pituitary–adrenal-axis.

Glucagon-like peptide-2-induced memory improvement and anxiolytic effects in mice

We investigated the effectiveness of glucagon-like peptide-2 (GLP-2) on memory impairment in lipopolysaccharide (LPS)-treated mice, and anxiety-like behavior in adrenocorticotropic hormone (ACTH)-treated mice. In the Y-maze test, LPS (10 µg/mouse, i.c.v.) significantly decreased spontaneous alternation, which was prevented by pretreatment with GLP-2 (0.01–0.3 µg/mouse, i.c.v.). The GLP-2 treatment just before the Y-maze test also improved LPS-induced memory impairment. Continuous treatment with GLP-2 (3 µg/mouse, i.c.v.) had no effect on the open-field test in saline-treated or ACTH-treated mice. Chronic ACTH treatment did not cause anxiogenic effects in the elevated plus-maze test. GLP-2 showed weak anxiolytic-like effects in the elevated plus-maze test in ACTH-treated, but not saline-treated mice. Moreover, GLP-2 increased 5-HT, but not 5-HIAA and tryptophan hydroxylase 2 levels in the amygdala of ACTH-treated mice. Pharmacological depletion of 5-HT prevented the anxiolytic effects of GLP-2. These results suggest that GLP-2 protected and improved memory function in LPS-treated mice, and also had anxiolytic effects due to changes in the 5-HT system.

Alterations in the profile of blood neutrophil membrane receptors caused by in vivo adrenocorticotrophic hormone actions.

Elevated levels of adrenocorticotrophic hormone (ACTH) mobilize granulocytes from bone marrow into the blood, although these neutrophils are refractory to a full migratory response into inflamed tissues. Here, we investigated the dependence of glucocorticoid receptor activation and glucocorticoid-regulated protein annexin A1 (ANXA1) on ACTH-induced neutrophilia and the phenotype of blood neutrophil after ACTH injection, focusing on adhesion molecule expressions and locomotion properties. ACTH injection (5 μg ip, 4 h) induced neutrophilia in wild-type (WT) mice and did not alter the elevated numbers of neutrophils in RU-38486 (RU)-pretreated or ANXA1(-/-) mice injected with ACTH. Neutrophils from WT ACTH-treated mice presented higher expression of Ly6G⁺ANXA1(high), CD18(high), CD62L(high), CD49(high), CXCR4(high), and formyl-peptide receptor 1 (FPR1(low)) than those observed in RU-pretreated or ANXA1(-/-) mice. The membrane phenotype of neutrophils collected from WT ACTH-treated mice was paralleled by elevated fractions of rolling and adherent leukocytes to the cremaster postcapillary venules together with impaired neutrophil migration into inflamed air pouches in vivo and in vitro reduced formyl-methionyl-leucyl-phenylalanine (fMLP) or stromal-derived factor-1 (SDF-1α)-induced chemotaxis. In an 18-h senescence protocol, neutrophils from WT ACTH-treated mice had a higher proportion of ANXAV(low)/CXCR4(low), and they were less phagocytosed by peritoneal macrophages. We conclude that alterations on HPA axis affect the pattern of membrane receptors in circulating neutrophils, which may lead to different neutrophil phenotypes in the blood. Moreover, ACTH actions render circulating neutrophils to a phenotype with early reactivity, such as in vivo leukocyte-endothelial interactions, but with impaired locomotion and clearance.

Glucagon-like peptide-2 but not imipramine exhibits antidepressant-like effects in ACTH-treated mice

We investigated the effectiveness of glucagon-like peptide-2 (GLP-2) against refractory depression in adrenocorticotropic hormone (ACTH)-treated mice as a model of tricyclic antidepressant (TCA)-resistant depression. Chronic ACTH treatment (0.45mg/kg, s.c., 14 days) weakened the antidepressant-like effects of imipramine (20mg/kg, i.p., 6 days) in the forced-swim test (FST). Conversely, GLP-2 (3μg/mice, i.c.v., 6 days) induced antidepressant-like effects in the ACTH-treated mice in the FST. ACTH-treatment increased basal serum corticosterone levels, with an additional increase induced by the FST. Imipramine or GLP-2 had no effect on the basal corticosterone level, but GLP-2 attenuated the additional increase caused by the FST. Moreover, GLP-2 increased 5-HT levels, but not 5-HIAA. These results suggest that GLP-2 induced antidepressant-like effects under imipramine-resistant conditions through increase in 5-HT levels.

Regulation of renal organic anion transporter expression and function in ACTH‐treated mice

Our previous renal microarray studies indicated differential expression of genes encoding organic anion transporters in a mouse model of ACTH‐dependent Cushing syndrome (Physiological Genomics 40: 158, 2010). In the current study, we used quantitative PCR to show significantly (P<0.01) reduced expression of genes encoding OATP‐1 (slco1a1), OAT2 (slc22a7) and OAT3 (slc22a8) in the kidneys of ACTH‐treated mice (n=6/6). In separate groups of mice, a 14‐day treatment with ACTH (n=5) caused hypertension, polyuria and a four‐fold increase in uric acid excretion, compared to controls (n=6). This is consistent with down‐regulation of OAT3, which contributes to the reabsorption of uric acid. ACTH‐treated mice also had a significantly reduced renal clearance of p‐aminohippurate (cPAH), a prototypical substrate of the OAT system. Since cPAH was not affected by co‐administration of probenecid, it is likely that organic anions normally secreted via this pathway are largely excreted by filtration during chronic glucocorticoid excess. These data indicate that organic anion transport in the kidney is physiologically regulated by corticosteroids. Since several classes of drugs are excreted via this pathway, our data have therapeutic implications in conditions of steroid excess.

Transcriptional and physiological responses to chronic ACTH treatment by the mouse kidney

We investigated the effects on urinary steroid and electrolyte excretion and renal gene expression of chronic infusions of ACTH in the mouse. ACTH caused a sustained increase in corticosteroid excretion; aldosterone excretion was only transiently elevated. There was an increase in the excretion of deoxycorticosterone, a weak mineralocorticoid, to levels of physiological significance. Nevertheless, we observed neither antinatriuresis nor kaliuresis in ACTH-treated mice, and plasma renin activity was not suppressed. We identified no changes in expression of mineralocorticoid target genes. Water turnover was increased in chronic ACTH-treated mice, as were hematocrit and hypertonicity: volume contraction is consistent with high levels of glucocorticoid. ACTH-treated mice exhibited other signs of glucocorticoid excess, such as enhanced weight gain and involution of the thymus. We identified novel ACTH-induced changes in 1) genes involved in vitamin D (Cyp27b1, Cyp24a1, Gc) and calcium (Sgk, Calb1, Trpv5) metabolism associated with calciuria and phosphaturia; 2) genes that would be predicted to desensitize the kidney to glucocorticoid action (Nr3c1, Hsd11b1, Fkbp5); and 3) genes encoding transporters of enzyme systems associated with xenobiotic metabolism and oxidative stress. Although there is evidence that ACTH-induced hypertension is a function of physiological cross talk between glucocorticoids and mineralocorticoids, the present study suggests that the major changes in electrolyte and fluid homeostasis and renal function are attributable to glucocorticoids. The calcium and organic anion metabolism pathways that are affected by ACTH may explain some of the known adverse effects associated with glucocorticoid excess.

Blood pressure and vascular reactivity in ACTH‐treated mice with ouabain‐resistant α2 Na‐K‐ATPase

We have shown that alterations in the ouabain-binding characteristics of the α1 and α2 subunits of the Na-K-ATPase (NKA) can influence cardiovascular function in vivo. Notably, we have reported that ACTH-induced hypertension is prevented in mice expressing a mutant, ouabain-resistant α2NKA isoform. To explore this ACTH-resistance, we evaluated regional vascular resistance in ouabain-resistant α2NKA (α2R) and ouabain-sensitive α2NKA mice (wild type: WT). Mice were treated for 5–6 days with ACTH, or sham treated, then prepared for blood pressure and flow measurements. In untreated mice, there were no differences in blood pressure, renal and mesenteric blood flow or vascular resistance under baseline conditions, and sensitivity to phenylephrine (PE) was comparable between the two groups. In ACTH-treated mice, resting blood pressure was lower in α2R than in WT, confirming that hypertension is prevented in these mutants. Following ACTH-treatment, renal and mesenteric blood flow decreased significantly in α2R, but not in WT mice. Calculations of vascular resistance confirmed that resting vascular resistance was paradoxically elevated in the hypertension-resistant α2R mice. PE-responsiveness was also enhanced in the ACTH-treated α2R mice compared to untreated, whereas PE-responsiveness was not altered by ACTH in WT. These data suggest that the anti-hypertensive phenotype of ouabain-resistant α2NKA mice occurs in spite of increased reactivity in these vascular beds.

Deleterious effects of adrenocorticotrophic hormone administration during late pregnancy upon offspring somatic, neurological, and sexual development in mice.

The influence of administration of adrenocorticotrophic hormone (ACTH) during days 12-17 of pregnancy upon somatic, neurological, and neuromuscular development of offspring in mice was studied. The effects upon the onset of puberty in female offspring was also examined. Litters from mice given the higher of two doses of ACTH (1 IU/day or 8 IU/day) showed lower body weights at birth and weaning than controls. This treatment also increased pre- and postnatal mortality rates, although not significantly. Litters from mice treated with either dose of ACTH showed retarded development of the forelimb and hindlimb grasp reflexes, the body righting reflex, the auditory startle response, and eye opening. Although ear opening was delayed in litters from ACTH-treated mice, results did not achieve statistical significance. Study of female offspring housed in small groups revealed that one indicator of puberty, vaginal opening, was delayed in female offspring of ACTH-treated mice. Experiments were conducted to identify factors mediating this syndrome: ACTH did not depress maternal food intake or alter the length of pregnancy, therefore fetal undernutrition or premature birth can be excluded as mediating factors. All litters were fostered to untreated mice to control for postnatal factors influencing development. As ACTH cannot cross the placenta, the syndrome is likely to result from in utero exposure to abnormally high concentrations of glucocorticosteroids of maternal origin. It is concluded that such alterations to the fetal environment can exert a deleterious influence upon somatic, neurological, and sexual development, and that hormones of the maternal pituitary-adrenocortical axis may naturally act to regulate general development of the fetus.

An ACTH-induced renal glomerular lesion in the mouse: immunofluorescence microscopy.

Mice were injected with adrenocorticotropic hormone (ACTH) and the glomerular lesion that was induced was studied by light and immunofluorescence microscopy. By light microscopy, kidneys from ACTH-treated mice showed typical ACTH-induced glomerular lesions. Immunofluorescence of kidneys from ACTH-treated mice revealed intense staining for IgG and IgM in the extraglomerular mesangium (EGM), in Bowman's space, and in the ascending thick limb of Henle near the macula densa. Staining for immunoglobulins was unchanged after treatment with acid buffer. Immunoreactivity for complement (C3) was confined largely to the EGM and Bowman's space. Staining for albumin was almost exclusively in Bowman's space or the peripheral glomerular tuft in discrete aggregates. The above patterns of IgG, IgM, C3 and albumin were seen in control mice, although much less frequently. The results show that in mice, treatment with ACTH results in the increased accumulation of plasma proteins in the juxtaglomerular apparatus (JGA). This effect may reflect a role for the JGA in the normal clearing of plasma proteins from the glomerulus and/or directly from the blood.

Effects of Adrenocorticotropin on Pregnancy and Prolactin in Mice*

The effect of chronic ACTH administration on pregnancy and serum PRL levels was studied in 40 adrenalectomized and 71 intact house mice. Three periods of gestation were examined: days 1–8 (series I), day 8 to parturition (series II), and day 1 to parturition (series III). Day 1 of gestation was determined by the presence of a vaginal plug. Mice received daily sc injections of 4 U of ACTH or the gelatin vehicle between 0900–1100 h. Bilateral adrenalectomy was performed 7 days before mating, and adrenalectomized mice were maintained on 50 μg cortisone acetate/day. In series I, ACTH treatment reduced maternal body weight, uterine weight, and nidation sites in intact (P < 0.01) and adrenalectomized mice (P < 0.05) compared to their respective controls. In series II, ACTH treatment reduced maternal body weight, uterine weight, litter size and pup size in intact mice (P < 0.01). No significant differences were observed among adrenalectomized ACTH-treated mice and adrenalectomized controls in this series. In ser...

Opiate antagonist counteracts reproductive inhibition by porcine acth extract

The effects of naloxone, an opiate receptor antagonist, on the maturation of female mice chronically pretreated with ACTH from weaning, were examined. Naloxone treatment, begun at 40 days of age, blocked ACTH-induced delay of puberty. Naloxone also reversed decreases in weights of the reproductive organs produced by ACTH. Naloxone treatment also resulted in estrous cycling and increased the number of ACTH-treated mice with ovarian corpora lutea. These changes are reflected in increased LH levels in ACTH-naloxone treated vs. ACTH-saline treated animals. Naloxone alone may increase reproductive function. Effects of ACTH on spleen and thymus weights, presumably via the adrenals, were partially reversed by naloxone. ACTH may have a different mechanism in adrenal function as compared to reproductive function.

Decreased aggressive behavior in the offspring of ACTH-treated mice

ACTH administration to pregnant mice, at both 1- and 8-IU dosages, altered the subsequent aggressive behavior of the offspring. The proportion, intensity, and duration of fighting in males were decreased in comparison to those in controls. A second experiment found that the female offspring of ACTH-treated pregnant animals also were deficient in the aggressive behavior initiated by exposure to testosterone propionate in adulthood. The possible long-term effects of the ACTH treatment on androgen responsiveness and brain chemistry are discussed.