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

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

It is known that the acute immobilization stressor (IMM) triggers an exaggerated elevation of the plasma catecholamines [42]. One of the key questions in adaptive response to stress is how the additional acute stressor can provoke a variant or altered response depending on prior experience with the current or a different stressor [43]. Additional acute immobilization increases plasma catecholamines in animals previously exposed to chronic social isolation (CSI+IMM) [46] and in animals previously exposed to long-term treadmill running (TR+IMM) [34]. This could mean that prior experience may condition physiological systems to "expect" a problem and, therefore, be more ready to respond to a novel additional acute stressor [43]. In addition, immobilization stress more significantly elevates TH and DBH protein levels in the stellate ganglia in rats previously exposed to long-term treadmill running (TR+IMM). Continuous accumulation of their proteins is an adaptation on applied stress regime [34]. Also, chronically stressed rats exposed to novel stressors exhibit exaggerated responses in gene expression of PNMT enzyme catalyzing conversion of NA to A [34]. The increased release of A from stellate ganglia during the additional acute immobilization in rats previously exposed to long-term treadmill running may be caused by the increased synthesis of PNMT. Increased levels of PNMT enzyme in stellate ganglia may have pathophysiological impact, especially on the cardiovascular system, since A is a powerful β2 adrenergic receptor agonist [34]. Also, the heterotypic novel additional acute immobilization stressor elevates the plasma catecholamines but not excessively in the animals previously exposed to CSITR [42]. This finding might be explained by the quality and especially by the intensity of the stressor used. The novel stressors elicit exaggerated responses in prestressed animals, when the novel stressor is of equal or greater intensity or duration and/or it is repeated [43]. Animals exposed to CSITR are already prepared to manage the new situation evoked by a novel stressor, and the exaggerated response is not necessary [42]. Animals exposed to CSITR treatment have statistically more significant expression of TH, DBH, and PNMT genes in the adrenal medulla after additional acute immobilization stress compared with the animals exposed to acute immobilization stress [42]. The increased catecholamine synthesis in the adrenal medulla of chronically stressed animals after additional acute stress is an important adaptive phenomenon of the sympathoadrenomedullary system in rats [43].

activation, and negative feedback, which in turn promote the proinflammatory cytokines [47]. The increased proinflammatory cytokines ultimately cause inflammation, which may induce various diseases [47]. For example, the proinflammatory cytokines alter the metabolic processes of neurotransmitters [48], whose secretion suppression and the reuptake block activity

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

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

289

During oxidative stress high concentrations of ROS modify nucleoside triphosphates which are incorporated into the DNA during DNA synthesis and may give rise to mutations. Mutations in the genes of regulatory enzymes, transporters, and receptors of the neurotransmitters in the central nervous system (CNS) have been associated with aggression [49].

Mutation in human MAO A gene is associated with impulsive aggression in male humans [50], juvenile delinquency [51], impulsivity [52], and female panic and depressive disorders [53, 54]. In addition, mutations in the TH and DA receptor 4 genes influence impulsivity [55– 57], and polymorphism in the glutamate transporter (VGLUT) gene is significantly associated with increased "aggression to strangers" [58]. Polymorphism in the tryptophan hydroxylase (Tph2) gene as a causal factor in 5-hydroxytryptamine (5-HT) deficiency is associated with

Genetically modified mouse and rat models are used in the research of human diseases. It is known that the low activity of MAO A enzyme consequently increases catecholamine levels [49]. Reduced levels of the MAO A enzyme, as well as increased NA levels, were observed in aggressive men [49]. MAO A knockout mice showed increased aggression in adulthood [60]

**7. Materials and methods for studying the protective role of exercise** 

Eleven-week-old Wistar male rats were maintained under standard laboratory conditions with water and food ad libitum and kept three to four per cage [25]. The care was taken to minimize the pain and discomfort of the animals according to the recommendations of the Ethical Committee of the Vinča Institute of Nuclear Sciences [25], Belgrade, Serbia, which follows the guidelines of the registered "Serbian Society for the Use of Animals in Research and Education." Animals were divided into four groups in accordance with our previous protocol [42]. The **control group** (n = 10) was not exposed to stress. The animals in **CSITR group** (n = 10)

and for this reason were used in the research of behavioral disorders.

**against deleterious effects of oxidative stress**

play a role in the pathogenesis of depression [47].

**6.2. Genetically modified models**

*6.2.1. Target genes*

depression [59].

**7.1. Animal models**

*6.2.2. Transgenic mouse/rat models*

#### **6. Other animal models**

#### **6.1. Chemical manipulation**

Data from literature indicate that chronic stress produces the activation of SNS and hypothalamus-pituitary-adrenal (HPA) axis. In our previous study, we found that exposure of rats to daily treadmill running increased plasma concentrations of NA, A, and ACTH and decreased CORT concentration [34]. It is known that stress hormones via adrenergic and glucocorticoid receptors of immune cells inhibit secretion of the proinflammatory cytokines, such as interleukin 1 beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and interferon-y (INF-γ), while promoting the secretion of the anti-inflammatory cytokines, such as interleukin-4 (IL-4), interleukin-10 (IL-10), and interleukin-13 (IL-13) [47]. Lasting stress exposure induces HPA "fatigue," glucocorticoid resistance, nuclear factor kappa B (NF-Kb) activation, and negative feedback, which in turn promote the proinflammatory cytokines [47]. The increased proinflammatory cytokines ultimately cause inflammation, which may induce various diseases [47]. For example, the proinflammatory cytokines alter the metabolic processes of neurotransmitters [48], whose secretion suppression and the reuptake block activity play a role in the pathogenesis of depression [47].

#### **6.2. Genetically modified models**

During oxidative stress high concentrations of ROS modify nucleoside triphosphates which are incorporated into the DNA during DNA synthesis and may give rise to mutations. Mutations in the genes of regulatory enzymes, transporters, and receptors of the neurotransmitters in the central nervous system (CNS) have been associated with aggression [49].

#### *6.2.1. Target genes*

It is known that the acute immobilization stressor (IMM) triggers an exaggerated elevation of the plasma catecholamines [42]. One of the key questions in adaptive response to stress is how the additional acute stressor can provoke a variant or altered response depending on prior experience with the current or a different stressor [43]. Additional acute immobilization increases plasma catecholamines in animals previously exposed to chronic social isolation (CSI+IMM) [46] and in animals previously exposed to long-term treadmill running (TR+IMM) [34]. This could mean that prior experience may condition physiological systems to "expect" a problem and, therefore, be more ready to respond to a novel additional acute stressor [43]. In addition, immobilization stress more significantly elevates TH and DBH protein levels in the stellate ganglia in rats previously exposed to long-term treadmill running (TR+IMM). Continuous accumulation of their proteins is an adaptation on applied stress regime [34]. Also, chronically stressed rats exposed to novel stressors exhibit exaggerated responses in gene expression of PNMT enzyme catalyzing conversion of NA to A [34]. The increased release of A from stellate ganglia during the additional acute immobilization in rats previously exposed to long-term treadmill running may be caused by the increased synthesis of PNMT. Increased levels of PNMT enzyme in stellate ganglia may have pathophysiological impact, especially on the cardiovascular system, since A is a powerful β2 adrenergic receptor agonist [34]. Also, the heterotypic novel additional acute immobilization stressor elevates the plasma catecholamines but not excessively in the animals previously exposed to CSITR [42]. This finding might be explained by the quality and especially by the intensity of the stressor used. The novel stressors elicit exaggerated responses in prestressed animals, when the novel stressor is of equal or greater intensity or duration and/or it is repeated [43]. Animals exposed to CSITR are already prepared to manage the new situation evoked by a novel stressor, and the exaggerated response is not necessary [42]. Animals exposed to CSITR treatment have statistically more significant expression of TH, DBH, and PNMT genes in the adrenal medulla after additional acute immobilization stress compared with the animals exposed to acute immobilization stress [42]. The increased catecholamine synthesis in the adrenal medulla of chronically stressed animals after additional acute stress is an important adaptive phenom-

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

enon of the sympathoadrenomedullary system in rats [43].

Data from literature indicate that chronic stress produces the activation of SNS and hypothalamus-pituitary-adrenal (HPA) axis. In our previous study, we found that exposure of rats to daily treadmill running increased plasma concentrations of NA, A, and ACTH and decreased CORT concentration [34]. It is known that stress hormones via adrenergic and glucocorticoid receptors of immune cells inhibit secretion of the proinflammatory cytokines, such as interleukin 1 beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and interferon-y (INF-γ), while promoting the secretion of the anti-inflammatory cytokines, such as interleukin-4 (IL-4), interleukin-10 (IL-10), and interleukin-13 (IL-13) [47]. Lasting stress exposure induces HPA "fatigue," glucocorticoid resistance, nuclear factor kappa B (NF-Kb)

**6. Other animal models**

**6.1. Chemical manipulation**

Mutation in human MAO A gene is associated with impulsive aggression in male humans [50], juvenile delinquency [51], impulsivity [52], and female panic and depressive disorders [53, 54]. In addition, mutations in the TH and DA receptor 4 genes influence impulsivity [55– 57], and polymorphism in the glutamate transporter (VGLUT) gene is significantly associated with increased "aggression to strangers" [58]. Polymorphism in the tryptophan hydroxylase (Tph2) gene as a causal factor in 5-hydroxytryptamine (5-HT) deficiency is associated with depression [59].

#### *6.2.2. Transgenic mouse/rat models*

Genetically modified mouse and rat models are used in the research of human diseases. It is known that the low activity of MAO A enzyme consequently increases catecholamine levels [49]. Reduced levels of the MAO A enzyme, as well as increased NA levels, were observed in aggressive men [49]. MAO A knockout mice showed increased aggression in adulthood [60] and for this reason were used in the research of behavioral disorders.
