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

**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

284 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

catecholaminergic turnover and antioxidant protection in stress conditions.

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

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 level.

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 risk of cardiovascular diseases [21, 22]. In addition, the gene expression of splenic catecholamine biosynthetic enzymes is decreased after CSI. This might reduce catecholamine synthesis in the spleen and deplete the immunocompetent tissues of catecholamines which cause an impairment of immune response [11]. Key question in adaptive response to stress is how the addition stressor can elicit a variant or altered response depending on prior experience with the current or different stressor. A potentiation of the sympathoadrenal system activity in socially isolated rats upon exposure to novel acute stressors has been reported [23]. The additional acute immobilization does not affect the gene expression of catecholamine biosynthetic enzymes in both auricles of long-term socially isolated rats. This suggests that the response to stress depends on prior experience with stressors [24]. Data from literature indicate a possible adaptation of catecholamine-synthesizing system at the level of gene expression in the heart auricles of chronically socially isolated rats exposed to acute immobilization stress [24]. However, protein levels of catecholamine biosynthetic enzymes in both ventricles of socially isolated rats increased after additional acute stress [25]. With regard to the role of cardiac catecholamines in physiological and pathophysiological processes, it could be hypothesized that increased catecholamine synthesis in the ventricles after acute stress indicates sensitivity of the heart to subsequent stress [25].

It could be concluded that TR shows adaptations that are indicative of chronic stress and that this animal model in rats is good for the study of neuroendocrine and immune functions in

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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**4. Combined animal model of chronic social isolation and long-term** 

Data from literature confirm that exercise has been widely used in the last years with therapeutic and preventive purposes in a series of pathophysiological conditions. Exercise training reduces the risk of developing diseases related to chronic stress. For example, a physically active lifestyle is associated with decreased risks of coronary heart disease and high blood pressure [35]. In addition, in humans, regular exercise has a beneficial impact on depression [36]. It is known that the theory of "cross stressor adaptation hypothesis" suggests that exercise training, as a stressor on the body, may alter responsiveness to other types of stressors [37]. Mueller [38] suggests that exercise training appears to reduce sympathoexcitation to a variety of centrally mediated sympathoexcitatory stimuli. Reduction in sympathoexcitation may contribute, in part, to the reduced incidence of cardiovascular disease in physically active individuals [38]. In addition, physical activity prevents splenic NA depletion, or spillover, typically observed in sedentary rats following periods of intense sympathetic drive [39]. Also, physical activity may prevent stress-induced suppression of splenic immunity by reducing

Treatment of chronic social isolation and long-term daily treadmill running (CSITR) consists of exposing the individually housed Wistar male rats to the daily treadmill running during

Understanding the mechanisms by which CSITR training alters control of the SNS in health and disease could be important for developing new strategies in the prevention and treatment of cardiovascular diseases. Treadmill exercise leads to a decreased gene transcription of catecholamine biosynthetic enzymes in stellate ganglia in stressful conditions. This may suggest the beneficial effects of treadmill exercise on cardiovascular system in stressed

Immobilization is a standardized procedure frequently used as an additional acute stressor and is considered as one of the most intensive stressors that significantly changes gene expression [43]. It is known that immobilization results in well-characterized catecholamine responses [44]. In this model, animals were restrained in a prone position on a board for periods of 120 min [45]. The head was restricted from movement by a metal loop over the nose,

**daily treadmill running in rats and "cross stressor adaptation** 

sympathetic drive to the spleen during stress [40, 41].

**5. Animal model of acute immobilization stress in rats**

and the feet were taped to raise supports with bandage tape [45].

stress conditions.

**hypothesis"**

12 weeks [42].

animals [22].

It could be concluded that animal model of chronic social isolation (CSI) in rats is a good animal model in the research of neuroendocrine and immune functions in stress conditions. Also, the described results indicate the potential application of CSI animal models in understanding of stress in humans.
