Introdução: Diversos estudos sugerem que o estresse nas fases iniciais de desenvolvimento pode induzir alterações persistentes na capacidade do eixo Hipotálamo-Pituitária-Adrenal (HPA) em responder ao estresse na vida adulta. O desequilíbrio do cortisol tem sido identificado como um correlato biológico dos transtornos depressivos. Essas anormalidades parecem estar relacionadas às mudanças na capacidade dos glicocorticóides circulantes em exercer seu feedback negativo na secreção dos hormônios do eixo HPA por meio da ligação aos receptores mineralocorticóides (RM) e glicocorticóides (RG) nos tecidos do eixo HPA. Devido à grande variedade de estressores, assim como os diferentes subtipos de depressão, os achados dos estudos atuais têm sido inconsistentes. Dessa forma, necessitando de mais estudos para que se possa elucidar os mecanismos envolvidos na associação entre o Estresse Precoce (EP) e o desenvolvimento de quadros depressivos. Objetivo: O objetivo deste estudo é avaliar a correlação entre Estresse Precoce e alterações no eixo Hipotálamo-Pituitária-Adrenal e na função dos receptores glicocorticóides e mineralocorticóides em pacientes depressivos. Metodologia: Foram recrutados inicialmente 30 sujeitos divididos em dois grupos: grupo de pacientes com diagnóstico de episódio depressivo atual (n=20) e grupo de controles (n=10). Posteriormente os pacientes foram divididos em outros dois grupos de acordo com o EP, compondo a amostra final por três grupos: grupo de pacientes depressivos com presença de EP (n=13), grupo de pacientes depressivos com ausência de EP (n=7) e grupo de controles (n=10). Os pacientes foram avaliados por meio de Entrevista Clínica de acordo com os critérios diagnósticos do DSM-IV, para a confirmação do diagnóstico. Para avaliação da gravidade dos sintomas depressivos foi aplicada a Escala de Depressão de Hamilton (HAM-D21), sendo incluídos apenas pacientes com HAM-D21 17. A presença de EP foi confirmada através da aplicação do Questionário Sobre Traumas na Infância (QUESI). Foram utilizados também a Escala de Avaliação de Depressão de Montgomery-Asberg (MADRS), o Inventário de Depressão de Beck (BDI), o Inventário de Ansiedade de Beck (BAI), a Escala de Ideação Suicida de Beck (BSI), a Escala de Desesperança de Beck (BHS), a Escala Hospitalar de Ansiedade e Depressão (HAD) e a Escala de Impulsividade de Barratt (BIS-11) para a avaliação de sintomas psiquiátricos. A avaliação endócrina foi controlada por placebo, cego por parte dos controles e pacientes, não randomizado, com desenho de medidas repetidas, onde os efeitos da Fludrocortisona (0.5 mg) e da Dexametasona (0.5 mg) foram avaliados através do cortisol salivar e plasmático. A secreção de cortisol plasmático e salivar foi avaliada nos sujeitos, após a administração de uma cápsula de Placebo, Fludrocortisona e Dexametasona às 22hs do dia anterior. O cortisol salivar foi coletado às 22h, ao acordar, 30 e 60 minutos após acordar e antes da coleta plasmática, nos dias seguintes após os desafios. Resultados: Na amostra de pacientes depressivos e controles, encontramos níveis significativamente menores de cortisol salivar ao acordar após a administração de Placebo nos pacientes depressivos comparados aos controles. Encontramos também uma tendência dos pacientes apresentarem níveis maiores de cortisol salivar ao acordar do que os controles após a administração de Dexametasona. Quando avaliado o cortisol após a administração de Fludrocortisona, os pacientes apresentaram níveis significativamente menores de cortisol salivar 30 minutos após acordar e na Área Sob a Curva (AUC) do que os controles. Além disso, encontramos também uma tendência dos pacientes depressivos apresentarem níveis menores de cortisol salivar 60 minutos após acordar do que os controles. Quando comparados entre pacientes depressivos com presença e ausência de EP e controles, encontramos uma tendência dos pacientes depressivos com ausência de EP apresentarem níveis menores de cortisol salivar ao acordar após Placebo do que os controles. As médias dos níveis de cortisol salivar ao acordar não diferiram entre os pacientes com presença de EP e os controles e entre os pacientes do grupo presença e ausência de EP. Com relação aos níveis de cortisol salivar após a administração de Dexametasona entre pacientes depressivos com presença e ausência de EP e controles, os pacientes depressivos com ausência de EP apresentaram níveis significativamente maiores de cortisol salivar ao acordar do que os controles. Encontramos também uma tendência dos pacientes com ausência de EP apresentarem níveis maiores de cortisol salivar ao acordar do que os pacientes com presença de EP, porém não foram encontradas diferenças significativas entre os pacientes com presença de EP e os controles. Conclusão: Nossos dados demonstram uma hipoatividade do eixo HPA nos pacientes depressivos. Além disso, estes achados sugerem que esta desregulação do eixo HPA se deva em parte a uma diminuição da sensibilidade dos RG e uma hiperativação dos RM nos pacientes depressivos. No entanto, quando comparados pacientes depressivos com presença e ausência de Estresse Precoce, os desafios com agonistas seletivos como a Dexametasona (agonista RG) e a Fludrocortisona (agonista RM) não foram capazes de detectar esta diferença fisiopatológica e distinguir entre os diferentes tipos de psicopatologia. Dessa forma, estes resultados sugerem que estudos com um agonista misto (RG/RM) como a Prednisolona teriam potencial para distinguir os pacientes depressivos com presença de Estresse Precoce. / Introduction: Several studies suggest that stress in early stages of development can induce persistent changes in the ability of the Hypothalamic-Pituitary-Adrenal (HPA) axis to respond to stress in adulthood. The imbalance of cortisol has been identified as a biological correlate of depressive disorders. These abnormalities seem to be related to changes in the ability of circulating glucocorticoids to practice their negative feedback on the secretion of HPA axis hormones through connecting to the mineralocorticoid receptor (MR) and glucocorticoid (GR) in the tissues of HPA axis. Due to the wide variety of stressors, as well as the different subtypes of depression, the findings of current studies have been inconsistent. Thus, more studies need to be able to elucidate the mechanisms involved in the association between Early Life Stress (ELS) and the development of depression. Objective: The objective this study is to evaluate the correlation between of Early Life Stress and changes in Hypothalamic-Pituitary-Adrenal axis and at receptors function glucocorticoid and mineralocorticoid in depressive patients. Methodology: We recruited 30 subjects initially divided into two groups: patients with current depressive episode (n =20) and control group (n = 10) Subsequently, patients were divided into two groups according to the ELS, making the final sample of three groups: depressive patients with ELS (n =13) group of depressive patients without ELS (n=7) and control group (n=10). Patients were evaluated by clinical interview according to the diagnostic criteria of DSM-IV to confirm the diagnosis. To evaluate the severity of depressive symptoms was applied to the Hamilton Depression Scale (HAM-D21), and included only patients with HAM-D21 17. The presence of ELS was confirmed by the Childhood Trauma Questionnaire (CTQ). We also used the Depression Rating Scale Montgomery-Asberg (MADRS), the Beck Depression Inventory (BDI), the Beck Anxiety Inventory (BAI), the Scale for Suicide Ideation Beck (BSI), the Scale Beck Hopelessness (BHS), the Hospital Anxiety and Depression Scale (HADS) and the Barratt Impulsiveness Scale (BIS-11) for the assessment of severity psychiatric symptoms. Endocrine evaluation was placebo-controlled, blinded by the patients and controls, non-randomized design with repeated measures, where the effects of Fludrocortisone (0.5 mg) and dexamethasone (0.5 mg) were assessed using salivary cortisol and plasma. The secretion of plasma cortisol and salivary was evaluated in the subjects, after administration of a capsule of Placebo, Fludrocortisone and Dexamethasone to 22hs the previous day. The salivary cortisol was collected at 22h, on waking, 30 and 60 minutes after waking and before plasma collection in the following days after the challenges. Results: In these sample of depressed patients and controls, we found significantly lower levels of salivary cortisol around waking after administration of Placebo in depressed patients than controls. We also found a trend for patients to have higher levels of salivary cortisol than controls on awakening after administration of Dexamethasone. When measured cortisol after administration of Fludrocortisone, patients showed significantly lower levels of salivary cortisol 30 minutes after waking and the Area Under the Curve (AUC) than controls. In addition, we also found a tendency for depressed patients showed lower levels of salivary cortisol 60 minutes after awakening than controls. When compared between depressed patients with and without ELS and controls, we found a tendency for depressed patients without ELS presented lower levels of salivary cortisol on awakening after Placebo than controls. The mean salivary cortisol levels on waking did not differ between patients with ELS and controls and between patients with and without ELS. The levels of salivary cortisol after Dexamethasone administration between depressed patients with and without ELS and controls, depressed patients without ELS had significantly higher levels of salivary cortisol on awakening than controls. We also found a trend for patients without Early Life Stress have higher levels of salivary cortisol upon waking than patients with Early Life Stress, but there were no significant differences between patients with Early Life Stress and controls. Conclusion: Our data show a hypoactivity of the HPA axis in depressed patients. Moreover, these findings suggest that this dysregulation HPA axis is partly due to a decrease the sensitivity of RG and a hyperactivation of MR in patients depressive. However, when compared depressed patients with and without Early Life Stress, the challenges with selective agonists as the Dexamethasone (agonist GR) and Fludrocortisone (agonist MR) were not able to detect this difference pathophysiological and distinguish between the different types of psychopathology. Thus, these results suggest that studies with a mixed agonist (GR/MR) such as Prednisolone have potential to distinguish of depressive patients with Early Life Stress.

This thesis examines the influence of stress on the activity of hippocampal CA1 area. The main task was to determine whether the stress load affects the changes of the local metabolism of glucocorticoids, and whether the levels of corticosteroid receptors in the CA1 hippocampus are modulated in response to stress. In order to answer these questions, the experiments were carried out using three different rat strains - Fisher, Lewis and Wistar which differ in their activities of hypothalamic-pituitary-adrenal axis. Our results demonstrate that stress has no effect on expression of MR mRNA. Conversely, stress reduces the levels of GR mRNA in CA1 area of the dorsal hippocampus. Moreover, we confirmed that the Lewis and Wistar rats didn't change metabolism of glucocorticoids after stress response. By the Fisher rats increased levels of 11β-HSD1 mRNA expression and therefore increased the metabolism of corticosterone.

Le stress en début de vie (ELS) est associé à une susceptibilité accrue au développement de troubles liés au stress, tels que le trouble dépressif majeur (TDM). L'amygdale latérale (AL), une région du cerveau importante pour la régulation des comportements émotionnels et cognitifs, est vulnérable aux effets du ELS. Cependant, les mécanismes par lesquels l'ELS altère le comportement ne sont pas très bien définis. Auparavant, de nombreuses études se sont concentrées sur les mécanismes neuronaux qui sous-tendent les troubles comportementaux induits par le stress, mais le rôle des cellules gliales dans ce circuit reste indéterminé. Pourtant, les astrocytes, un type de cellule gliale, sont des déterminants clés du comportement. Nous avons donc cherché à identifier le rôle des astrocytes dans les effets de l'ELS sur le comportement dépendant de l'AL. Pour ce faire, nous avons utilisé un modèle de rongeur avec séparation maternelle, limitation de la litière et de la nidification pour reproduire les effets de l'ELS sur le cerveau en développement afin d’évaluer ses effets à long terme sur les astrocytes et le comportement dépendant de l'amygdale latérale. Bien que l'ELS n'ait pas eu d'influence sur le comportement anxieux des souris, ce dernier a altéré de manière significative la détection des menaces, un processus cognitif qui implique la capacité de distinguer avec précision un son menaçant précédemment appris (le stimulus conditionné) d'un son non menaçant dans un contexte nouveau. De plus, la diminution de la sensibilité au stress des astrocytes par la suppression des récepteurs glucocorticoïdes astrocytaires a amélioré de manière significative la fonction cognitive chez les souris ELS et naïves. Globalement, nos résultats suggèrent que les astrocytes jouent un rôle central dans la régulation des effets de l'ELS sur les troubles cognitifs. Ces données soulignent l'importance des astrocytes comme cibles thérapeutiques potentielles pour atténuer le dysfonctionnement cognitif, un symptôme omniprésent de la psychopathologie. / Early Life Stress (ELS) is associated with an enhanced susceptibility to the development of stress-related disorders, such as major depressive disorder (MDD). The lateral amygdala (LA), a brain region important for the regulation of emotive and cognitive behaviours is vulnerable to the effects of ELS. However, the mechanisms by which ELS impairs behaviour are poorly defined. Previously, research has focused on the neuronal mechanisms underlying stress-induced behavioural impairments, however the role of glial cells in this circuitry remains undetermined. Astrocytes, a type of glial cell, are key determinants of behaviour. Hence, we aimed to identify the role of astrocytes in the effects of ELS on LA-dependent behaviour. To accomplish this, we used a rodent model of maternal separation and limited bedding and nesting to replicate the effects of ELS on the developing brain by assessing its long-term effects on astrocytes and lateral-amygdala dependent behaviour. Although ELS did not influence anxiety-like behaviour in mice, ELS significantly impaired threat-detection, a cognitive process involving the ability to accurately distinguish between a previously learned threatening tone (the conditioned stimulus) and a non-threatening tone in a novel context. Additionally, decreasing astrocyte stress sensitivity by deleting astrocyte glucocorticoid receptors significantly enhanced cognitive function in both ELS and naïve mice. Overall, our results suggest that astrocytes are pivotal in the regulation of the effects of ELS on cognitive impairment. This data highlights the importance of astrocytes as potential therapeutic targets for mitigating cognitive dysfunction, a pervasive symptom of psychopathology.

Ziel: Gewinnung neuer Erkenntnisse auf dem Gebiet der Glucocorticoidforschung. Die Arbeit gliedert sich in zwei Teile: 1. Klinische Studie zu Wirkungen und Nebenwirkungen einer niedrig bis mittelhoch dosierten Methylprednisolon(MP)-Therapie bei Patienten mit entzündlich-rheumatischen Erkrankungen. 2. Durchflusszytometrische Untersuchungen mit humanen PBMC mit dem Ziel, membranständige Glucocorticoidrezeptoren (mGCR) nachzuweisen. Methodik: 1. In einer klinischen Studie wurden zwei Patientengruppen mit jeweils 20 Patienten miteinander verglichen. Alle Patienten hatten entzündlich-rheumatische Erkrankungen und bekamen eine MP-Therapie über mindestens ein Jahr. Die Dosierungen in der ersten Gruppen entsprachen einer low-dose GC-Therapie, die Patienten in der zweiten Gruppe bekamen eine medium-dose GC-Therapie. Erwünschte, unerwünschte Wirkungen sowie die Lebensqualität der Patienten wurden erhoben. 2. Humane PBMC wurden durchflusszytometrisch untersucht. Es wurden konventionelle Färbemethoden sowie eine hoch-sensitive Liposomenfärbung zur Detektion spezifischer membranständiger Antigene angewandt. Ergebnisse: 1. In den meisten Fällen waren die relativ niedrigen Dosierungen von MP geeignet, die Krankheitsaktivität der entzündlich-rheumatischen Erkrankung wirksam zu kontrollieren. Einzelne Exazerbationen waren allerdings zu verzeichnen. Bei den meisten unerwünschten Wirkungen zeigten sich keine Unterschiede zwischen den Dosisgruppen. Osteoporosetypische Rückenschmerzen traten signifikant höher in der oberen Dosisgruppe auf (p=0,04), bei dem erhöhten Augeninnendruck zeigte sich eine Tendenz (p=0,1). Häufige Nebenwirkungen auch bei niedrigen Dosierungen waren: Unterblutungen der Haut und Pergamenthaut (76,2 % bzw. 73,8 % aller Patienten) bzw. eine Cushing-Symptomatik (61,9 % aller Patienten). 2. Mit der Liposomen-Färbetechnik ließen sich erstmals mGCR auf humanen PBMC systematisch nachweisen. Bis zu 5 % der B-Lymphozyten und bis zu 7 % der Monozyten exprimierten mGCR bei Gesunden. Stimulationen des Immunsystems durch Impfungen oder eine aktive rheumatoide Arthritis führten zu einer deutlichen Erhöhung des Anteils mGCR-positiver Monozyten auf über 20 %. Schlussfolgerungen: 1. Niedrig bis mittelhoch dosierte Therapien mit MP können effektiv die Aktivität von entzündlich-rheumatischen Erkrankungen kontrollieren. Die unerwünschten Effekte sind vermutlich dosisabhängig, für die meisten ist jedoch nicht relevant, ob mit einer low-dose oder einer medium-dose Therapie behandelt wird. 2. mGCR werden auf humanen PBMC unter physiologischen Bedingungen exprimiert. Unter bestimmten immunologischen Bedingungen werden sie hochreguliert. Herkunft und Funktion der Rezeptoren müssen noch genauer geklärt werden. / Purpose: Gaining new knowledge in glucocorticoid research. The dissertation consists of two parts: 1. Clinical study on effects and side-effects of a low-dose / medium-dose therapy with methylprednisolone (MP) in patients with inflammatory rheumatic diseases. 2. Flowcytometric investigation of human PBMC in order to detect membrane-bound glucocorticoid-receptors (mGCR). Methods: 1. In a clinical study two groups of patients - 20 patients each - were compared. All patients had inflammatory rheumatic diseases and recieved MP-therapy for at least one year. The first group recieved a low-dose GC-therapy, the second group a medium-dose GC-therapy. 2. Human PBMC were examined. We used conventional and high-sensitive liposome staining technique for the detection of specific membrane-bound antigens. Results: 1. In most cases rather low dosages of MP were able to control the disease activitiy of inflammatory rheumatic diseaeses. However, we observed disease exacerbation in some cases. Most side-effects showed the same characteristics in both groups. There was a significant higher appearance of typical osteoporotic back pain in the higher dosage group (p=0,04) and a tendency to higher intraophtalmic pressure in this group (p=0,1). Common side effects with even low dosages were: skin hematoma and thin skin (76,2 % and 73,8 % respective) and a Cushing-Syndrome (61,9 % of all patients). 2. With the liposome staining technique we showed for the first time systematically mGCR on human PBMC. Up to 5 % of B-lymphocytes and 7 % of monocytes presented mGCR in healthy blood donors. Stimulation of the immunological system by vaccination or in case of an active rheumatoid arthritis led to a marked increase of mGCR-positive monocytes to more than 20 %. Conclusions: 1. Low-dose and medium-dose methylprednisolone therapy can effectivly control the activity of inflammatory rheumatic diseases. The side effects are probably dose-dependent. However, for most side effects it doesn''t matter if patients are treated with a low-dose or a medium-dose therapy. 2. mGCR are expressed on human PBMC under physiological conditions and are up-regulated under certain immunological conditions. The function of these receptors has to be examined more profoundly.