On the effect of peripheral immune stimulation on depressive-like behaviour and central inflammatory gene expression in rat

Entzündungsreaktionen werden in der Pathophysiologie von Depressionen eine immer größere Rolle zugeschrieben, doch trotz der zunehmenden Forschung in diesem Gebiet sind die zugrunde liegenden Mechanismen noch nicht geklärt. In Tierversuchen wurde gezeigt, dass eine periphere Entzündung nicht nur die zentrale Expression von pro-inflammatorischen Mediatoren erhöht, sondern auch zu Verhalten führt, dass dem depressiver Menschen ähnelt. In dieser Arbeit wurde der Effekt einer peripheren Injektion des Exotoxins Lipopolysaccharid (LPS), das auf der äußeren Membran gramnegativer Bakterien zu finden ist, auf die Genexpression in den Gehirnarealen Amygdala, Hippocampus, Hypothalamus, Prefrontaler Cortex und Striatum in Ratten untersucht. Außerdem wurden die Proteinlevels gewisser Cytokine im Serum der Ratten gemessen. Verhaltensänderungen nach der LPS Injektion wurde in dem erzwungenen Schwimmtest (engl. “forced swim test”, FST) erfasst. Dieser Test misst depressiv-ähnliches Verhalten (engl. “depressive-like behaviour”) in Tieren. LPS wurde den Tieren intraperitoneal injiziert und 6 Stunden später wurde ihr Verhalten im FST gemessen. Eine Stunde danach wurden die Ratten getötet und ihre Gehirne seziert. Um festzustellen, ob der FST an sich die Genexpression im Gehirn verändert, wurden Kontrolltiere, die nicht dem Schwimmtest ausgesetzt wurden, in die Studie inkludiert. mRNA wurde aus den verschiedenen Hirnarealen extrahiert und Veränderungen in der Genexpression durch quantitative Echt-Zeit Polymerasenkettenreaktion (qRT PCR) gemessen. Die periphere Injektion von LPS löste eine erhöhte Genexpression der Cytokine IL-1β und TNFα und der Gewebsprotease MMP-9 und deren Inhibitor TIMP-1 aus. Diese Ergebnisse stehen in signifikanter Korrelation mit dem Anstieg an immobilem Verhalten während des FST (p=0.001 im Hypothalamus für IL-1β and TNFα). Es wurde kein nennenswerter Effekt des erzwungenen Schwimmtests auf die zentrale Genexpression festgestellt, was darauf schließen lässt, dass dieser Test geeignet ist, um die zugrunde liegenden Mechanismen und die entscheidenden Mediatoren im Zusammenspiel von Entzündungsreaktionen und Depressionen zu untersuchen und vielleicht sogar neue therapeutische Ziele in der Behandlung von affektiven Störungen zu identifizieren.Accumulating evidence suggests a role of inflammation in the pathophysiology of depression. However, despite the extensive research in this field, little is known about the underlying mechanisms. Animal experiments prove to be of great help, showing that peripheral inflammation induces central up-regulation of pro-inflammatory mediators and, at the same time, depressive-like behaviour. In this study, the effect of peripheral administration of the exotoxin lipopolysaccharide (LPS) on gene expression in the brain areas amygdala, hippocampus, hypothalamus, prefrontal cortex and striatum, as well as protein levels in the serum of Sprague Dawley rats, was investigated. Furthermore, changes in behaviour were measured in the forced swim test (FST), an animal model to test for depressive-like behaviour. The animals were injected intraperitoneal (i.p.) with LPS and 6 hours later tested in the FST. One hour later, the animals were sacrificed and their brains removed and dissected. To evaluate whether the FST itself changes gene expression in the brain, control animals that were not exposed to the FST were included in the study. mRNA was extracted from the different brain areas and differences in expression levels assessed using quantitative real-time polymerase chain reaction (qRT-PCR). LPS treatment was followed by an up-regulation in gene expression of the cytokines IL-1β and TNFα, and the tissue remodelers MMP-9 and its inhibitor, TIMP-1, which correlated significantly (p=0.001 in hypothalamus for both IL-1β and TNFα) with the observed significant increase in depressive-like behaviour. Exposure to the FST itself didn´t seem to have noteworthy influences on gene-expression, making this test a valuable method to investigate inflammation-related players in depression and maybe identify new targets for the treatment of mood disorders

On Neuroimmunology and Brain Function: Experimental and Clinical Studies

The immune system has been implicated in the mechanisms underlying many psychiatric disorders. Immune mediators are expressed in the central nervous system (CNS) not only in response to harmful stimuli, but also in a constitutive manner, and serve as important plasticity factors during development. There is complex bidirectional communication between the immune system and the CNS throughout life which is based on interactions between neurotransmitters, neuroendocrine hormones, cytokines, and their respective receptors. Exploring the interplay between brain, behaviour and immunity is central to our understanding of the pathology of psychiatric morbidity. The aim of this thesis is to investigate the role of some aspects of the immune system in several psychiatric conditions, both in experimental and clinical contexts. We used the Flinders sensitive line (FSL), a genetic animal model of depression, to study central gene expression of markers related to immune response and neurotransmission following immune stimulation and antidepressant treatment. Several genes were found to be expressed differently in rats displaying depressive-like behaviour compared to their controls (Paper I), a finding that we replicated in Paper II. Additionally, we showed that antidepressant treatment with escitalopram altered expression of several genes, notably the astrocyte-derived protein S100B, and the serotonin receptor 5-HT2A, in the amygdala and hypothalamus (Paper II), two brain regions that have been shown to be of relevance for the effect of antidepressant treatment. Our results support the use of the FSL model for studying the role of these immune-related markers in depression and antidepressant treatment. In the clinical studies included in this thesis, we found that genetic variants in immune-related genes were associated with neuropsychiatric traits and the volume of certain brain regions. The gene encoding the NF-kB inhibitor-like protein 1 (NFKBIL1) was found to be associated with autistic-like traits, as well as with language impairment in a cohort from the general population (Paper III). We further investigated the effect of genetic variation in the gene coding for interleukin-1beta (IL1B) on the volume of several brain regions in a case-control population of patients diagnosed with bipolar disorder (Paper IV). Genotype distribution did not differ between patients and controls, suggesting that variants in IL1B may not be associated with bipolar disorder. However, we found associations between IL1B polymorphisms and the volume of the putamen in the left hemisphere in patients and controls, suggesting that genetic variation in IL1B may influence neurodevelopment. In conclusion, this thesis demonstrates associations between immune mediators and mental functions, as well as altered brain development in humans. Also, insight is gained into the use of the FSL animal model for investigating the impact of the immune system for depression. Taken together, our findings confirm the importance of the immune system for the development of psychiatric disorders

Expression of inflammatory markers in a genetic rodent model of depression

The complex bidirectional communication between the central nervous system and the peripheral immune system is of possible relevance for both normal brain functions and the development of psychiatric disorders. The aim of this investigation was to study central expression of inflammatory markers in a genetic rat model of depression (the Flinders sensitive line (FSL) and its control, the Flinders resistant line (FRL)). A peripheral immune activation was induced by lipopolysaccharide (LPS) in order to investigate possible differences in immune reactions between the two rat lines. To confirm behavioural differences between the rat lines the forced swim test was performed, a test to assess depressive-like behaviour. Expression of candidate inflammatory genes was measured in amygdala, hippocampus, hypothalamus, prefrontal cortex and striatum using quantitative real time PCR. Our results show, for the first time, significantly lower central expression of the glial-specific protein S100B and complement factor C3 in several brain regions of the FSL rats compared to controls, both at baseline and after peripheral immune stimulation. No significant differences in immune responses to LPS were observed between the rats lines. Both S100B and C3 have been suggested to be of relevance for brain development and plasticity as well as brain disorders. These proteins may be of importance for the behavioural differences between the FSL and FRL rats, and this model may be useful in studies exploring the influence of the immune system on brain function

Expression of inflammatory markers in a genetic rodent model of depression

The complex bidirectional communication between the central nervous system and the peripheral immune system is of possible relevance for both normal brain functions and the development of psychiatric disorders. The aim of this investigation was to study central expression of inflammatory markers in a genetic rat model of depression (the Flinders Sensitive line (FSL) and its control, the Flinders Resistant line (FRL)). A peripheral immune activation was induced by lipopolysaccharide (LPS) in order to investigate possible differences in immune reactions between the two rat lines. To confirm behavioural differences between the rat lines the forced swim test was performed, a test to assess depressive-like behaviour. Expression of candidate inflammatory genes was measured in amygdala, hippocampus, hypothalamus, prefrontal cortex and striatum using quantitative real time PCR. Our results show, for the first time, significantly lower central expression of the glial-specific protein S100B and complement factor C3 in several brain regions of the FSL rats compared to controls, both at baseline and after peripheral immune stimulation. No significant differences in immune responses to LPS were observed between the rats lines. Both S100B and C3 have been suggested to be of relevance for brain development and plasticity as well as brain disorders. These proteins may be of importance for the behavioural differences between the FSL and FRL rats, and this model may be useful in studies exploring the influence of the immune system on brain functions