The effects of short term stress on the central and peripheral nitrergic system

Abstract

The mammalian stress response is generated by several integrated neural systems that allow rapid adaptation to homeostatic disturbances. This is principally mediated through the hypothalamic-pituitary-adrenal axis which mobilises energy reserves by adrenal glucocorticoids. This in turn accelerates cellular metabolism which consequently increases free radical formation through the mitochondrial electron transport chain. Excess generation of free radicals such as reactive oxygen and nitrogen species (RNS) may potentially cause damage to fatty acids, proteins, and DNA by oxidative and nitrosative stress. Nitric oxide is a ubiquitous signalling molecule belonging to the family of RNS and is endogenously synthesised from ʟ-arginine by nitric oxide synthase (NOS). Three distinct subtypes of NOS have been identified, with the neuronal and endothelial isoforms being constitutively expressed enzymes responsible for synaptic signalling and smooth muscle relaxation. The inducible isoform is primarily present in cells of the inflammatory immune system where its activity is transcriptionally induced by cytokines and other inflammatory agents and thus, is thought to play an important role in immunomodulation. Recently, a number of studies have demonstrated that the nitrergic system have significant implications in neuropsychiatric disorders including anxiety and depression. Specifically, chronic stress-induced hippocampal neuronal NOS upregulation is responsible for glucocorticoid receptor downregulation, a factor linked to the development of depressive disorders. Although stress and nitrergic signalling are intrinsically linked in pathological states, little is known about the acute physiology between these two systems. Therefore, the primary aim of this thesis was to establish the profile of nitrosative changes in peripheral blood and brain regions known to be impacted by stress following an acute psychological challenge. The observed changes were subsequently investigated by employing a pharmacological intervention using a novel stress-alleviating and anti-inflammatory endocannabinoid enhancer. To achieve the aims for this thesis, Male Wistar rats aged 5-7 weeks postnatal were maintained under control conditions or subjected to acute stress. Blood samples were collected at time-points immediately before, during, and following treatment in order to estimate the levels of stress hormones and redox parameters including oxidative/nitrosative status, glutathione and glutathione disulphide ratio, and lipid peroxidation. Post mortem neural tissue was collected and specific brain regions were isolated for the determination of nitrosative status, NOS enzymatic activity, and relative gene expression of stress and nitrergic-related genes. Following the establishment of these measurements, a separate group of animals was used to examine the effects of endocannabinoid modulation on the nitrergic and inflammatory-related indicators following acute stress. We found that exposure to a single episode of psychological stress causes early and marked changes to both oxidative and nitrosative status sufficient to induce oxidative damage in peripheral blood. Within the hippocampus, there was a rapid increase in levels of nitric oxide due to an initial and transient increase in the activity of calcium-dependent constitutive NOS following acute stress. Transcriptional activation of inducible NOS following stress resulted in sustained elevations in nitric oxide within the hippocampus. In comparison, there was a delayed stress-induced increase in striatal nitrosative status which was mainly driven by the inducible isoform. Furthermore, preventing endocannabinoid hydrolytic degradation with a fatty acid amide hydrolase inhibitor, PF-3845, successfully suppressed hippocampal inducible NOS upregulation and the associated neuroinflammation following acute stress exposure. In conclusion, this thesis has established an acute profile of stress-induced central and peripheral nitrergic activation. By demonstrating changes in the oxidative and nitrosative systems, and linking these to indices of cellular oxidative damage, it has confirmed the involvement of the nitrergic system in the rapid redox response to stress. Short term restraint stress also rapidly increases hippocampal nitrergic activity involving both the constitutive and inducible isoforms of NOS while striatal nitrosative status following stress is predominately inflammatory-mediated. The amelioration of these stress-induced modifications in the hippocampus via central augmentation of endogenous cannabinoid signalling could serve as an alternative treatment for stress-related disorders.