**Animal Models for Chronic Stress-Induced Oxidative Stress in the Spleen: The Role of Exercise and Catecholaminergic System Stress in the Spleen: The Role of Exercise and Catecholaminergic System**

**Animal Models for Chronic Stress-Induced Oxidative** 

DOI: 10.5772/intechopen.70008

Ljubica Gavrilović, Vesna Stojiljković, Nataša Popović, Snežana Pejić, Ana Todorović, Ivan Pavlović and Snežana B. Pajović Nataša Popović, Snežana Pejić, Ana Todorović, Ivan Pavlović and Snežana B. Pajović

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70008

Ljubica Gavrilović, Vesna Stojiljković,

#### **Abstract**

We examined the effects of daily exercise on the gene expression of catecholamine biosynthetic enzymes (tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH), and phenyl ethanolamine N-methyltransferase (PNMT)), vesicular monoamine transporter 2 (VMAT 2), antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)), concentrations of catecholamines (noradrenaline (NA) and adrenaline (A)) and malondialdehyde (MDA), activities of monoamine oxidase (MAO), and antioxidant enzymes in the spleen of chronically psychosocially stressed rats. Exposure of chronically stressed rats to exercise increased the levels of PNMT protein by 19%, VMAT 2 mRNA by 100%, NA by 160%, and A by 140%; decreased/unchanged MAO enzyme activity; returned concentrations of MDA to control level; and increased CAT and GPx mRNA levels (50% and 150%, respectively). Exercise induced the accumulation of the catecholamines and a decrease of stress-induced oxidative stress in the spleen, which may significantly affect the immune-neuroendocrine interactions in stress conditions. Also, exercise induced the catecholaminergic system and antioxidant defense to become more ready to a novel stressor, which indicates that exercise may induce potentially positive physiological adaptations. Our combined model of chronic social isolation and long-term daily treadmill running in rats may be a good animal model in the research of therapeutic role of exercise in human disease caused by chronic stress.

**Keywords:** treadmill running, chronic social isolation, catecholamine, antioxidant enzymes, spleen, rats

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

#### **1. Introduction**

Many studies have shown that stress disturbs homeostasis, which induces various disorders. A number of diseases and pathological conditions are related to the long-term adaptive response to stress, particularly under conditions of chronic stress when allostasis can shift from a healthy toward a pathological state [1]. Chronic stress induces behavioral, endocrine, and immune changes in animals [2, 3]. It is known that stress affects a rapid rise of plasma and tissue catecholamines, including the spleen [4]. Data from literature indicate that the sympathetic nervous system (SNS) is one of the major pathways involved in immune-neuroendocrine interactions. The regulation of immunity by sympathetic noradrenergic nerve fibers in the lymphoid organs has been demonstrated by the distribution of tyrosine hydroxylase (TH) nerve fibers, by the presence of adrenoreceptors on the immune system cells, and by immunomodulatory role of noradrenaline (NA) [5]. For example, adrenaline (A) and NA produced by sympathetic nerves may modulate cellular function by acting on β-2 adrenergic receptors of B and Th1 cells [6]. In addition, catecholamine biosynthetic enzymes are expressed in the lymphoid organs [7], as well as in neutrophils and macrophages [8]. It is known that normal catecholaminergic turnover results from balance among synthesis, release, and reuptake of catecholamines. Because of the significant role of catecholamines in neuroendocrine-immune network in stress response, detection of regulatory mechanism for catecholamine synthesis, degradation, release, and reuptake in the spleen in conditions provoked by chronic stress is exceptionally relevant in stress biology, due to its potentially negative impact on immune functions and health. Effective management of stress depends on the ability to identify and quantify the effects of various stressors and determine if individual or combined stressors have distinct biological effects [9]. Animal models have contributed considerably to the current understanding of mechanisms underlying the role of stress in health and disease [10]. It is known that animal model of chronic stress isolation (CSI) produces increased concentrations of catecholamines in the plasma and decreased gene expression of catecholamine biosynthetic enzymes in the spleen, which can modulate the immune function [11]. However, very little is known about the impact of long-term exercise on the catecholaminergic turnover and the antioxidant defense system in the spleen of chronically psychosocially stressed rats. Because of the potential therapeutic role of physical exercise, we investigated whether a combined animal model of chronic isolation and treadmill running in rats (CSITR) may be a good animal model for chronic stress research as well as the benefits of exercise on neuroendocrine and immune functions in stress conditions. Our CSITR animal model was achieved by exposing the individually housed rats to the daily treadmill running for a 12-week period. We opted for long-term daily treadmill running because the short intensive physical activity may induce oxidative stress, while it is not the case with sport-specific activity of longer duration [12]. In addition, we exposed the experimental animals to additional acute immobilization stress, because we wanted to examine whether daily treadmill running induced potentially positive adaptations of the splenic catecholaminergic turnover and antioxidant protection in stress conditions.

transporter (VMAT) 2) and degradation (monoamine oxidase (MAO)), as well as the concentrations of catecholamines (NA and A) in the spleen of chronically psychosocially stressed adult rats. Transcription factor cAMP response element-binding protein (CREB) plays a major role in regulation of TH and DBH gene expression during exercise [13]. This chapter discusses the effect of physical exercise on the level of CREB mRNA in the spleen of chronically stressed rats. As the rise in catecholamine catabolism results in increased reactive oxygen species (ROS) production, we measured the concentration of malondialdehyde (MDA), as well as gene expression and activity of the antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)). Also, we examined how the additional acute immobilization stress changed the mentioned parameters. In the study, we presumed that physical exercise in chronically stressed rats may induce the potentially positive physiological adaptations of the splenic catecholaminergic turnover, as well as antioxidant protection and

Animal Models for Chronic Stress-Induced Oxidative Stress in the Spleen: The Role of Exercise...

http://dx.doi.org/10.5772/intechopen.70008

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Detecting regulatory physiological mechanism by which physical activity changes catecholaminergic turnover and antioxidant defense system in the spleen in conditions provoked by chronic stress is important in the prevention of immune diseases caused by chronic stress. Also, these results may confirm whether CSITR could be a good animal model in the search for beneficial impact of exercise on neuroendocrine and immune functions in stress conditions.

Many authors have confirmed that animal models are essential to biological research. Chronic individual housing of rats, frequently termed "isolation stress," represents a very strong psychosocial stress [3, 14], which can induce neuroendocrine changes [15] and increased activity of the antioxidant defense system [16, 17] in animals. Also, isolation stress affects different behavioral processes in animals. For example, social isolation led to a reduced duration of grooming and a prolonged latency period to the onset of grooming [18]. In addition, social interactions are an important source of human stress. Social isolation has deleterious effects on health and therefore is regarded as one of the most relevant causes of diseases in mamma-

Animal models of chronic social isolation (CSI) consisted of 11-week-old Wistar male rats that were subjected to social isolation, with a single animal per cage for 12 or 3 weeks [11, 15]. The visual and olfactory communication among the isolated rats was reduced to the minimal

It is known that exposure of an organism to a social isolation leads to the engagement of several hormonal and neurotransmitter systems in the stress response. Chronic social isolation of adult rat males produces a depletion of brain catecholamine stores but no changes in heart auricles and adrenal glands [15]. In addition, CSI of adult rat males decreases the gene expression of catecholamine biosynthetic enzymes in the adrenal medulla [20] and increases concentrations of catecholamines in the plasma [11]. Also, CSI induces an increase in the gene expression of noradrenaline biosynthetic enzymes in stellate ganglia, which may be connected to the increased

**2. Animal model of chronic social isolation in rats**

lian species [14]. For example, it is a risk factor for human depression [19].

oxidative damage repair.

level.

We investigated how long-term daily 20 min treadmill running affected the gene expression of key enzymes involved in catecholamine biosynthesis (TH, dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT)), storage (vesicular monoamine transporter (VMAT) 2) and degradation (monoamine oxidase (MAO)), as well as the concentrations of catecholamines (NA and A) in the spleen of chronically psychosocially stressed adult rats. Transcription factor cAMP response element-binding protein (CREB) plays a major role in regulation of TH and DBH gene expression during exercise [13]. This chapter discusses the effect of physical exercise on the level of CREB mRNA in the spleen of chronically stressed rats. As the rise in catecholamine catabolism results in increased reactive oxygen species (ROS) production, we measured the concentration of malondialdehyde (MDA), as well as gene expression and activity of the antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)). Also, we examined how the additional acute immobilization stress changed the mentioned parameters. In the study, we presumed that physical exercise in chronically stressed rats may induce the potentially positive physiological adaptations of the splenic catecholaminergic turnover, as well as antioxidant protection and oxidative damage repair.

Detecting regulatory physiological mechanism by which physical activity changes catecholaminergic turnover and antioxidant defense system in the spleen in conditions provoked by chronic stress is important in the prevention of immune diseases caused by chronic stress. Also, these results may confirm whether CSITR could be a good animal model in the search for beneficial impact of exercise on neuroendocrine and immune functions in stress conditions.
