**8. Results**

#### **8.1. Changes of the TH, DBH, PNMT, CREB, and VMAT 2 mRNA levels and TH, DBH, and PNMT protein levels in the spleen**

The animals exposed to CSITR showed a decreased level of TH mRNA by 22% (p < 0.05, Tukey test, **Figure 1a**), DBH mRNA by 11% (p < 0.05, Tukey test, **Figure 1b**), PNMT mRNA by 29% (p < 0.05, Tukey test, **Figure 1c**), CREB mRNA by 69% (p < 0.01, Tukey test, **Figure 1d**), and increased levels of VMAT 2 mRNA by 100% (p < 0.01, Tukey test, **Figure 1e**) and PNMT protein by 19% (p < 0.05, Tukey test, **Figure 2c**), whereas levels of TH and DBH protein (**Figure 2a** and **b**) were unchanged compared with the controls.

IMM stress does not change significantly gene expression of catecholamine biosynthetic enzymes (**Figures 1a**–**c** and **2a**–**c**) and levels of VMAT 2 mRNA (**Figure 1e**) 3 hours after immobilization. However, the additional exposure of CSITR animals to acute immobilization stress led to increased levels of PNMT protein by 33% (p < 0.05, Tukey test **Figure 2c**) and VMAT 2 mRNA by 100% (p < 0.01, Tukey test, **Figure 1e**) 3 hours after immobilization.

#### **8.2. Changes of the NA and A concentrations in the spleen**

excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm. Monoamine oxidase activ-

Malondialdehyde concentration in the spleen fractions was determined using Spectrophotometric Assay for Malondialdehyde BIOXYTECH® MDA-586 (OXIS Health Products, Inc., USA) according to the manufacturer's protocol. The MDA-586 method is based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole, with MDA at 45°C. Malondialdehyde

SOD, CAT, GPx, and GR activities were determined using methods previously described by Stojiljković et al. [64]. Determination of total SOD activity was performed using Oxis Bioxytech SOD-525 Assay (Oxis International, Inc., Portland, OR, USA). CAT activity was determined by the method of Beutler [59], and GPx activity was assessed using the Oxis Bioxytech GPx-340 Assay (Oxis International, Inc., Portland, OR, USA). The final result for enzyme activity was

The data are presented as means ± S.E.M. Differences of gene expression (mRNA and protein levels) of catecholamine biosynthetic enzymes (TH, DBH, and PNMT); levels of CREB, VMAT 2, SOD 1, SOD 2, CAT, and GPx mRNA; concentration of NA, A, and MDA; as well as enzyme activities (MAO A, MAO B, total SOD, CAT, and GPx) in the spleen were analyzed by one-way ANOVA. The effects of CSITR and IMM compared to control animals, as well as the effects of CSITR+IMM compared to CSITR, were tested by Tukey post-hoc test. Statistical

Correlations of mRNA levels, protein levels, hormone levels, and enzyme activity were ana-

**8.1. Changes of the TH, DBH, PNMT, CREB, and VMAT 2 mRNA levels and TH, DBH,** 

The animals exposed to CSITR showed a decreased level of TH mRNA by 22% (p < 0.05, Tukey test, **Figure 1a**), DBH mRNA by 11% (p < 0.05, Tukey test, **Figure 1b**), PNMT mRNA by 29% (p < 0.05, Tukey test, **Figure 1c**), CREB mRNA by 69% (p < 0.01, Tukey test, **Figure 1d**), and increased levels of VMAT 2 mRNA by 100% (p < 0.01, Tukey test, **Figure 1e**) and PNMT protein by 19% (p < 0.05, Tukey test, **Figure 2c**), whereas levels of TH and DBH protein (**Figure 2a**

lyzed by the Pearson test, using the Sigma Plot v10.0 (with SigmaStat integration).

ity was expressed as U/mg of protein.

**7.8. Malondialdehyde measurement**

**7.9. Antioxidant enzyme activities**

significance was accepted at p < 0.05.

**and PNMT protein levels in the spleen**

and **b**) were unchanged compared with the controls.

**7.10. Data analysis**

**8. Results**

concentration was expressed as μM/mg of protein.

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

expressed as units per milligram of protein (U/mg).

CSITR significantly increased the spleen concentrations of NA by 160% (p < 0.01, Tukey test, **Figure 3a**) and A by 140% (p < 0.01, Tukey test, **Figure 3b**), compared with control animals. The significant positive correlation was found between the levels of PNMT protein and A concentration in the spleen of animals exposed to CSITR (Pearson R = 0.631, p < 0.05, **Figure 4a**).

The exposure of the control animals to acute immobilization stress significantly increased NA concentration by 250% (p < 0.01, Tukey test, **Figure 3a**) and A concentration by 240%

**Figure 1.** Effects of CSITR and CSITR+IMM models on tyrosine hydroxylase (TH) [a], dopamine-ß-hydroxylase (DBH) [b], phenylethanolamine N-methyltransferase (PNMT) [c], cAMP response element binding (CREB) [d], and vesicular monoamine transporter 2 (VMAT2) [e] mRNA levels in the spleen. Data are shown as mean ± SEM of 10 rats. Symbols: +p < 0.05, ++p < 0.01 CSITR animals compared to control animals (Tukey test) and ##p < 0.01 CSITR+IMM animals compared to CSITR animals (Tukey test).

**Figure 2.** Effects of CSITR and CSITR+IMM models on tyrosine hydroxylase (TH) [a], dopamine-ß-hydroxylase (DBH) [b], and phenylethanolamine N-methyltransferase (PNMT) [c] protein levels in the spleen. Data are shown as mean ± SEM of 10 rats. Symbols: +p < 0.05 CSITR animals compared to control animals (Tukey test) and #p < 0.05 CSITR+IMM animals compared to CSITR animals (Tukey test).

(p < 0.01, Tukey test, **Figure 3b**), whereas the additional acute immobilization of CSITR animals decreased NA concentration by 17% (p < 0.05, Tukey test, **Figure 3a**) and increased A concentration by 15% (p < 0.05, Tukey test, **Figure 3b**) 3 hours after immobilization. The significant positive correlation was found between the levels of PNMT protein and A concentration in the spleen of animals exposed to CSITR+IMM (Pearson R = 0.721, p < 0.05, **Figure 4b**). However, the significant negative correlation was found between the levels of NA concentration and A concentration in the spleen of animals exposed to CSITR+IMM (Pearson R = −0.661, p < 0.05, **Figure 4c**).

of immobilization. The additional acute immobilization of CSITR animals increased enzyme activities of MAO A by 116% (p < 0.01, Tukey test, **Figure 5a**) and MAO B by 107% (p < 0.01,

**Figure 3.** Effects of CSITR and CSITR+IMM models on the concentration of noradrenaline (NA) [a] and adrenaline (A) [b] in the spleen. Data are shown as mean ± SEM of 10 rats. Symbols: ++p < 0.01 CSITR animals compared to control animals (Tukey test); **\*\***p < 0.01 IMM animals compared to control animals (Tukey test); and #p < 0.05 CSITR+IMM animals

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Chronic social isolation (CSI) significantly increased concentrations of MDA by 21% (p < 0.05, Tukey test, **Figure 6**) compared with control animals. The animals exposed to CSITR showed

The exposure of the control animals to acute immobilization stress significantly increased MDA concentration by 26% (p < 0.05, Tukey test, **Figure 6**), whereas the additional acute immobilization of CSI animals increased MDA concentration by 50% (p < 0.01, Tukey test,

Tukey test, **Figure 5b**) 3 hours after the cessation of immobilization.

unchanged levels of MDA compared with control animals (**Figure 6**).

**8.4. Changes of the MDA concentrations in the spleen**

compared to CSITR animals (Tukey test).

#### **8.3. Changes of the MAO A and MAO B activity in the spleen**

The animals exposed to CSITR showed a decreased enzyme activity of MAO B by 34% (p < 0.05, Tukey test, **Figure 5b**), whereas enzyme activity of MAO A (**Figure 5a**) was unchanged, compared with control animals.

IMM stress significantly increased the enzyme activities of MAO A by 1000% (p < 0.001, Tukey test, **Figure 5a**) and MAO B by 376% (p < 0.001, Tukey test, **Figure 5b**) 3 hours after the cessation

**Figure 3.** Effects of CSITR and CSITR+IMM models on the concentration of noradrenaline (NA) [a] and adrenaline (A) [b] in the spleen. Data are shown as mean ± SEM of 10 rats. Symbols: ++p < 0.01 CSITR animals compared to control animals (Tukey test); **\*\***p < 0.01 IMM animals compared to control animals (Tukey test); and #p < 0.05 CSITR+IMM animals compared to CSITR animals (Tukey test).

of immobilization. The additional acute immobilization of CSITR animals increased enzyme activities of MAO A by 116% (p < 0.01, Tukey test, **Figure 5a**) and MAO B by 107% (p < 0.01, Tukey test, **Figure 5b**) 3 hours after the cessation of immobilization.

#### **8.4. Changes of the MDA concentrations in the spleen**

(p < 0.01, Tukey test, **Figure 3b**), whereas the additional acute immobilization of CSITR animals decreased NA concentration by 17% (p < 0.05, Tukey test, **Figure 3a**) and increased A concentration by 15% (p < 0.05, Tukey test, **Figure 3b**) 3 hours after immobilization. The significant positive correlation was found between the levels of PNMT protein and A concentration in the spleen of animals exposed to CSITR+IMM (Pearson R = 0.721, p < 0.05, **Figure 4b**). However, the significant negative correlation was found between the levels of NA concentration and A concentration in the spleen of animals exposed to CSITR+IMM

**Figure 2.** Effects of CSITR and CSITR+IMM models on tyrosine hydroxylase (TH) [a], dopamine-ß-hydroxylase (DBH) [b], and phenylethanolamine N-methyltransferase (PNMT) [c] protein levels in the spleen. Data are shown as mean ± SEM of 10 rats. Symbols: +p < 0.05 CSITR animals compared to control animals (Tukey test) and #p < 0.05 CSITR+IMM

The animals exposed to CSITR showed a decreased enzyme activity of MAO B by 34% (p < 0.05, Tukey test, **Figure 5b**), whereas enzyme activity of MAO A (**Figure 5a**) was unchanged, com-

IMM stress significantly increased the enzyme activities of MAO A by 1000% (p < 0.001, Tukey test, **Figure 5a**) and MAO B by 376% (p < 0.001, Tukey test, **Figure 5b**) 3 hours after the cessation

(Pearson R = −0.661, p < 0.05, **Figure 4c**).

animals compared to CSITR animals (Tukey test).

pared with control animals.

**8.3. Changes of the MAO A and MAO B activity in the spleen**

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Chronic social isolation (CSI) significantly increased concentrations of MDA by 21% (p < 0.05, Tukey test, **Figure 6**) compared with control animals. The animals exposed to CSITR showed unchanged levels of MDA compared with control animals (**Figure 6**).

The exposure of the control animals to acute immobilization stress significantly increased MDA concentration by 26% (p < 0.05, Tukey test, **Figure 6**), whereas the additional acute immobilization of CSI animals increased MDA concentration by 50% (p < 0.01, Tukey test,

**Figure 4.** The correlation between PNMT protein level and concentrations of A and NA in the spleen of animals exposed to chronic social isolation and daily treadmill running, as well as of animals exposed to additional acute 2h immobilization stress after chronic social isolation and daily treadmill running (Pearson). **(**a) The correlation in the levels of PNMT protein and A concentrations in the spleen of animals exposed to CSITR (Pearson). (b) The correlation in the levels of PNMT protein and A concentrations in the spleen of animals exposed to CSITR+IMM (Pearson). (c) The correlation between NA and A concentrations in the spleen of animals exposed to CSITR+IMM (Pearson).

**Figure 6**) 3 hours after the cessation of immobilization. Also, the additional acute immobilization of CSITR animals increased MDA concentration by 16% (p < 0.05, Tukey test, **Figure 6**) 3 hours after the cessation of immobilization.

> IMM stress does not change mRNA levels of SOD 1, SOD 2, and CAT (**Figure 7a**–**c**) as well as enzyme activity of total SOD and CAT (**Figure 8a** and **b**) 3 hours after the cessation of immobilization. However, IMM treatment significantly increased mRNA levels of GPx by 20% (p < 0.05, Tukey test, **Figure 7d**) as well as enzyme activity of GPx by 135% (p < 0.01, Tukey test, **Figure 8c**) 3 hours after the cessation of immobilization. The additional acute immobilization of CSITR animals increased mRNA levels of SOD 1 by 37% (p < 0.05, Tukey test, **Figure 7a**), SOD 2 by 115% (p < 0.01, Tukey test, **Figure 7b**), CAT by 57% (p < 0.05, Tukey test, **Figure 7c**), and GPx by 18% (p < 0.05, Tukey test, **Figure 7d**) as well as enzyme activities of total SOD by 68% (p < 0.05, Tukey test, **Figure 8a**), CAT by 13% (p < 0.05, Tukey test, **Figure 8b**), and GPx by

> **Figure 5.** Effects of CSITR and CSITR+IMM models on the enzyme activity of the monoamine oxidase A (MAO A) [a] and monoamine oxidase B (MAO B) [b] in the spleen. Data are shown as mean ± SEM of 10 rats. Symbols: +p < 0.05 CSITR animals compared to control animals (Tukey test), **\*\*\***p < 0.001 IMM animals compared to control animals (Tukey test),

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and ##p < 0.01 CSITR+IMM animals compared to CSITR animals (Tukey test).

576% (p < 0.01, Tukey test, **Figure 8c**) 3 hours after the cessation of immobilization.

#### **8.5. Changes of the SOD 1, SOD 2, CAT, and GPx mRNA levels as well as total SOD, CAT, and GPx activity in the spleen**

The animals exposed to CSITR showed unchanged levels of SOD 1 and SOD 2 mRNA (**Figure 7a** and **b**), as well as significantly increased levels of CAT mRNA by 50% (p < 0.05, Tukey test, **Figure 7c**) and GPx mRNA by 150% (p < 0.01, Tukey test, **Figure 7d**) compared with control animals. However, CSITR treatment significantly decreased the enzyme activities of total SOD by 36% (p < 0.05, Tukey test, **Figure 8a**) and GPx by 30% (p < 0.05, Tukey test, **Figure 8c**) compared with control animals, whereas CAT activity remained unchanged (**Figure 8b**).

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**Figure 5.** Effects of CSITR and CSITR+IMM models on the enzyme activity of the monoamine oxidase A (MAO A) [a] and monoamine oxidase B (MAO B) [b] in the spleen. Data are shown as mean ± SEM of 10 rats. Symbols: +p < 0.05 CSITR animals compared to control animals (Tukey test), **\*\*\***p < 0.001 IMM animals compared to control animals (Tukey test), and ##p < 0.01 CSITR+IMM animals compared to CSITR animals (Tukey test).

**Figure 6**) 3 hours after the cessation of immobilization. Also, the additional acute immobilization of CSITR animals increased MDA concentration by 16% (p < 0.05, Tukey test, **Figure 6**) 3

**Figure 4.** The correlation between PNMT protein level and concentrations of A and NA in the spleen of animals exposed to chronic social isolation and daily treadmill running, as well as of animals exposed to additional acute 2h immobilization stress after chronic social isolation and daily treadmill running (Pearson). **(**a) The correlation in the levels of PNMT protein and A concentrations in the spleen of animals exposed to CSITR (Pearson). (b) The correlation in the levels of PNMT protein and A concentrations in the spleen of animals exposed to CSITR+IMM (Pearson). (c) The

The animals exposed to CSITR showed unchanged levels of SOD 1 and SOD 2 mRNA (**Figure 7a** and **b**), as well as significantly increased levels of CAT mRNA by 50% (p < 0.05, Tukey test, **Figure 7c**) and GPx mRNA by 150% (p < 0.01, Tukey test, **Figure 7d**) compared with control animals. However, CSITR treatment significantly decreased the enzyme activities of total SOD by 36% (p < 0.05, Tukey test, **Figure 8a**) and GPx by 30% (p < 0.05, Tukey test, **Figure 8c**) compared with control animals, whereas CAT activity remained unchanged

**8.5. Changes of the SOD 1, SOD 2, CAT, and GPx mRNA levels as well as total SOD,** 

correlation between NA and A concentrations in the spleen of animals exposed to CSITR+IMM (Pearson).

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hours after the cessation of immobilization.

**CAT, and GPx activity in the spleen**

(**Figure 8b**).

IMM stress does not change mRNA levels of SOD 1, SOD 2, and CAT (**Figure 7a**–**c**) as well as enzyme activity of total SOD and CAT (**Figure 8a** and **b**) 3 hours after the cessation of immobilization. However, IMM treatment significantly increased mRNA levels of GPx by 20% (p < 0.05, Tukey test, **Figure 7d**) as well as enzyme activity of GPx by 135% (p < 0.01, Tukey test, **Figure 8c**) 3 hours after the cessation of immobilization. The additional acute immobilization of CSITR animals increased mRNA levels of SOD 1 by 37% (p < 0.05, Tukey test, **Figure 7a**), SOD 2 by 115% (p < 0.01, Tukey test, **Figure 7b**), CAT by 57% (p < 0.05, Tukey test, **Figure 7c**), and GPx by 18% (p < 0.05, Tukey test, **Figure 7d**) as well as enzyme activities of total SOD by 68% (p < 0.05, Tukey test, **Figure 8a**), CAT by 13% (p < 0.05, Tukey test, **Figure 8b**), and GPx by 576% (p < 0.01, Tukey test, **Figure 8c**) 3 hours after the cessation of immobilization.

**Figure 6.** Effects of CSITR and CSITR+IMM models on the concentration of malondialdehyde (MDA) in the spleen. Data are shown as mean ± SEM of 10 rats. Symbols: +p < 0.05 CSI animals compared to control animals (Tukey test), **\***p < 0.05 IMM animals compared to control animals (Tukey test), §§p < 0.01 CSI+IMM animals compared to CSI animals, and #p < 0.05 CSITR+IMM animals compared to CSITR animals (Tukey test).

**9. Discussion**

It is known that chronic social isolation induces a reduction of gene expression of noradrenaline biosynthetic enzymes in the spleen [11]. Since the data from literature confirm that the treadmill running stimulates concomitantly peripheral catecholamine secretion and central noradrenergic activity, i.e., NA turnover and release [65], it was tentative to expect that treadmill running would change the splenic catecholamine synthesis of chronically psychosocially stressed rats. However, the results presented in this chapter show that the treadmill running does not lead to further modulation of gene expression of splenic noradrenaline biosynthetic enzymes (TH and DBH) and that reduced level of CREB mRNA coincides with the reduced TH and DBH mRNA levels of chronically psychosocially stressed rats. Also, the treadmill running does not change levels of splenic TH and DBH protein of chronically stressed rats. This finding indicates the decrease of de novo synthesis of NA in the spleen and that the CREB plays a major role in regulating the expression of TH and DBH genes during treadmill running, which is in accordance with the reports of Erdös et al. [13]. Therefore, the treadmill exercise does not affect the synthesis of splenic NA biosynthetic enzymes of chronically stressed rats. Although levels of splenic noradrenaline biosynthetic enzymes are unchanged,

**Figure 8.** Effects of CSITR and CSITR+IMM models on total superoxide dismutase (SOD) [a], catalase (CAT) [b], and glutathione peroxidase GPx [c] enzyme activity in the spleen. Data are shown as mean ± SEM of 10 rats. Symbols: +p < 0.05 CSITR animals compared to control animals (Tukey test), **\*\***p < 0.01 IMM animals compared to control animals (Tukey

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test), and #p < 0.05, ##p < 0.01 CSITR+IMM animals compared to CSITR animals (Tukey test).

**Figure 7.** Effects of CSITR and CSITR+IMM models on CuZn superoxide dismutase (SOD1) [a], Mn superoxide dismutase (SOD2) [b], catalase (CAT) [c], and glutathione peroxidase GPx [d] mRNA levels in the spleen. Data are shown as mean ± S.E.M. of 10 rats. Symbols: +p < 0.05, ++p < 0.01 CSITR animals compared to control animals (Tukey test), **\***p < 0.05 IMM animals compared to control animals (Tukey test), and #p < 0.05, ##p < 0.01 CSITR+IMM animals compared to CSITR animals (Tukey test).

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**Figure 8.** Effects of CSITR and CSITR+IMM models on total superoxide dismutase (SOD) [a], catalase (CAT) [b], and glutathione peroxidase GPx [c] enzyme activity in the spleen. Data are shown as mean ± SEM of 10 rats. Symbols: +p < 0.05 CSITR animals compared to control animals (Tukey test), **\*\***p < 0.01 IMM animals compared to control animals (Tukey test), and #p < 0.05, ##p < 0.01 CSITR+IMM animals compared to CSITR animals (Tukey test).

#### **9. Discussion**

**Figure 7.** Effects of CSITR and CSITR+IMM models on CuZn superoxide dismutase (SOD1) [a], Mn superoxide dismutase (SOD2) [b], catalase (CAT) [c], and glutathione peroxidase GPx [d] mRNA levels in the spleen. Data are shown as mean ± S.E.M. of 10 rats. Symbols: +p < 0.05, ++p < 0.01 CSITR animals compared to control animals (Tukey test), **\***p < 0.05 IMM animals compared to control animals (Tukey test), and #p < 0.05, ##p < 0.01 CSITR+IMM animals compared to CSITR

**Figure 6.** Effects of CSITR and CSITR+IMM models on the concentration of malondialdehyde (MDA) in the spleen. Data are shown as mean ± SEM of 10 rats. Symbols: +p < 0.05 CSI animals compared to control animals (Tukey test), **\***p < 0.05 IMM animals compared to control animals (Tukey test), §§p < 0.01 CSI+IMM animals compared to CSI animals, and #p

< 0.05 CSITR+IMM animals compared to CSITR animals (Tukey test).

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animals (Tukey test).

It is known that chronic social isolation induces a reduction of gene expression of noradrenaline biosynthetic enzymes in the spleen [11]. Since the data from literature confirm that the treadmill running stimulates concomitantly peripheral catecholamine secretion and central noradrenergic activity, i.e., NA turnover and release [65], it was tentative to expect that treadmill running would change the splenic catecholamine synthesis of chronically psychosocially stressed rats. However, the results presented in this chapter show that the treadmill running does not lead to further modulation of gene expression of splenic noradrenaline biosynthetic enzymes (TH and DBH) and that reduced level of CREB mRNA coincides with the reduced TH and DBH mRNA levels of chronically psychosocially stressed rats. Also, the treadmill running does not change levels of splenic TH and DBH protein of chronically stressed rats. This finding indicates the decrease of de novo synthesis of NA in the spleen and that the CREB plays a major role in regulating the expression of TH and DBH genes during treadmill running, which is in accordance with the reports of Erdös et al. [13]. Therefore, the treadmill exercise does not affect the synthesis of splenic NA biosynthetic enzymes of chronically stressed rats. Although levels of splenic noradrenaline biosynthetic enzymes are unchanged, concentration of NA in the spleen of chronically stressed animals exposed to daily exercise is increased. This finding indicates exogenous source of NA in the spleen of chronically stressed rats exposed to daily exercise. These findings strengthen the idea that the sympathetic nervous system (SNS) participates in the NA response to CSITR, which is in accordance with results of Blandino et al. [66], who have confirmed that the noradrenergic system plays an integral role in modulations of splenic IL-1 beta response to stress. In addition, exposure of chronically stressed rats to daily treadmill running reduces PNMT mRNA level. However, CSITR treatment leads to continuous accumulation of PNMT protein catalyzing the conversion of NA to A, suggesting the possibility of the conversion of sympathetic neurotransmitter NA to A in the spleen (**Figure 4a**). This is indicated by significant positive correlation between the levels of PNMT protein and A in the spleen. It is known that catecholamine via adrenergic receptors induces modulation of many immune functions like splenic cytokine production [4]. Moreover, catecholamines might be stored into vesicles by VMAT or degraded by MAO and catechol-O-methyltransferase (COMT) [67]. Expression of VMAT, which plays an important role in the transport of newly synthesized catecholamines into vesicles, positively correlated with norepinephrine levels in both T and B cells which might suggest increased capacity for intracellular catecholamine production [68]. Endogenous catecholamines can modulate function of lymphocytes themselves by a paracrine and autocrine pathway [69]. O'Donnell et al. [70] found that increase of catecholamine levels coincided with reduction of splenic B and NK cells and a concomitant increase in T cells. As reported in this chapter, the treadmill running increases splenic VMAT 2 gene expression, and that increased level of VMAT 2 mRNA coincides with the increased splenic NA and A levels of chronically psychosocially stressed adult rats. A high splenic VMAT 2 transcript level suggests increased capacity of the splenic catecholamines. Therefore, exercise induces accumulation of catecholamines in the spleen of chronically stressed rats, indicating higher readiness of catecholaminergic system to a novel stressor (**Figure 1e**). Brown et al. [71] found that endogenous catecholamines might further initiate intracellular oxidation and apoptosis. However, daily treadmill running does not change enzyme activity of MAO A and decreases enzyme activity of MAO B in the spleen of chronically stressed rats (**Figure 5**). Decreased or unchanged enzyme activities of MAOs indicate that daily treadmill running decreases catecholamine degradation of chronically stressed rats. Therefore, these results indicate that the treadmill running induces accumulation of the splenic catecholamines and that the SNS probably plays a major role in accumulation of the splenic catecholamines in chronically stressed rats.

The decreased oxidative stress resulting from chronic training may originate from the elevated antioxidant system [75]. Powers et al. [76] observed that different combinations of intensity (low, moderate, and high) and duration (30, 60, and 90 min/day) produced different effects on the regulation of the antioxidant enzymes SOD, CAT, and GPx in the left ventricle. Exposure of chronically stressed rats to daily treadmill running induces an increase in CAT and GPx mRNA levels, while SOD1 and SOD2 mRNA levels remain unchanged (**Figure 7**). It is known that the adaptive response of the antioxidant system is specific to either the type of tissue or the different antioxidant systems involved [77, 78]. Ordonez et al. [79] found that a 12-week exercise significantly increased erythrocyte glutathione peroxidase activity which resulted in reduced oxidative damage. Sprint training caused an increase in the cardiac activity of glutathione redox cycle-related enzymes (GPx and GR) without inducing any changes in glutathione S-transferases (GST) and SOD activities or glutathione (GSH) levels in the myocardium [80]. It is important to notice that in CSITR the level of CAT activity remains unchanged, whereas total SOD and GPx activities are decreased (**Figure 8**). After 12 weeks of training process, changes in mRNA levels of antioxidant enzymes are not consistent with the changes in enzyme activities in the spleen of chronically stressed rats. Discrepancies between mRNA levels and activities may be related to differences in mRNA stability or translational efficiency [81]. García-López et al. [82] suspect that it is possible that the expressions of antioxidant enzymes mRNA were initially upregulated and then downregulated. In addition, regulation of expression might act on individual mRNAs to block their translation and thereby lead to their degradation [82]. Therefore, message degradation may be the primary target of regulation of expression [82]. Discrepancies between mRNA levels and activities of MnSOD may be in a kinase/phosphatase signal transduction pathway that may exert a fine control over posttranscriptional regulation of MnSOD expression [83]. In addition, CAT may be inactivated by its substrate, hydrogen peroxide, due to formation of complex II or complex III of CAT at high peroxide concentrations [84]. Nilakantan et al. [85] found that NO or NO-derived products inhibit both CAT and GPx enzyme activities. The results presented in this chapter confirm that daily treadmill running induces high splenic antioxidant enzyme transcript levels probably for immediate translation whenever necessary in chronically stressed rats, which is in accordance with the results of García-López et al. [82]. A high splenic CAT and GPx transcript levels suggest that exercise could induce the antioxidant

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defense system to become more ready to a novel stressor.

To confirm whether exercise is optimal stimulus to regulate expression levels of splenic catecholamines and antioxidant enzymes and whether the exposure of chronically stressed rats to daily treadmill running induces potentially positive adaptations of the splenic catecholamines and antioxidant protection, this chapter discusses the effects of additional acute immobilization stress. Detection of regulatory mechanism for catecholamine metabolism and antioxidant protection in the spleen in conditions provoked by the additional acute immobilization of chronically stressed animals exposed to daily exercise is exceptionally relevant in stress biology, because of the significant role of catecholamines and oxidative stress in modulation of immune function. The acute immobilization (IMM) and additional acute immobilization (CSITR+IMM) do not affect the synthesis of splenic noradrenaline biosynthetic enzymes (TH and DBH) 3 hours after a termination of immobilization stimulus (**Figure 2**). Also, acute

Chronic social isolation significantly increases concentrations of MDA in the spleen (**Figure 6**). The literature data confirm that exercise training has beneficial effects on oxidative stress and antioxidant defense systems in multiple organs [72]. Meguid et al. [73] showed a significant decrease in serum level of malondialdehyde (MDA) in Down syndrome individuals after treadmill exercise for 3 months. Exposure of chronically stressed rats to daily treadmill running induces return of MDA concentration in the spleen to basal level (**Figure 6**). This confirms that the chronic exercise training induces adaptations that decrease stress-induced oxidative stress. It is in line with the reports of Belviranli et al. [74], who observed that chronic exercise has protective role because the decreased oxidative damage is associated with improved aerobic metabolism induced by physical training.

The decreased oxidative stress resulting from chronic training may originate from the elevated antioxidant system [75]. Powers et al. [76] observed that different combinations of intensity (low, moderate, and high) and duration (30, 60, and 90 min/day) produced different effects on the regulation of the antioxidant enzymes SOD, CAT, and GPx in the left ventricle. Exposure of chronically stressed rats to daily treadmill running induces an increase in CAT and GPx mRNA levels, while SOD1 and SOD2 mRNA levels remain unchanged (**Figure 7**). It is known that the adaptive response of the antioxidant system is specific to either the type of tissue or the different antioxidant systems involved [77, 78]. Ordonez et al. [79] found that a 12-week exercise significantly increased erythrocyte glutathione peroxidase activity which resulted in reduced oxidative damage. Sprint training caused an increase in the cardiac activity of glutathione redox cycle-related enzymes (GPx and GR) without inducing any changes in glutathione S-transferases (GST) and SOD activities or glutathione (GSH) levels in the myocardium [80]. It is important to notice that in CSITR the level of CAT activity remains unchanged, whereas total SOD and GPx activities are decreased (**Figure 8**). After 12 weeks of training process, changes in mRNA levels of antioxidant enzymes are not consistent with the changes in enzyme activities in the spleen of chronically stressed rats. Discrepancies between mRNA levels and activities may be related to differences in mRNA stability or translational efficiency [81]. García-López et al. [82] suspect that it is possible that the expressions of antioxidant enzymes mRNA were initially upregulated and then downregulated. In addition, regulation of expression might act on individual mRNAs to block their translation and thereby lead to their degradation [82]. Therefore, message degradation may be the primary target of regulation of expression [82]. Discrepancies between mRNA levels and activities of MnSOD may be in a kinase/phosphatase signal transduction pathway that may exert a fine control over posttranscriptional regulation of MnSOD expression [83]. In addition, CAT may be inactivated by its substrate, hydrogen peroxide, due to formation of complex II or complex III of CAT at high peroxide concentrations [84]. Nilakantan et al. [85] found that NO or NO-derived products inhibit both CAT and GPx enzyme activities. The results presented in this chapter confirm that daily treadmill running induces high splenic antioxidant enzyme transcript levels probably for immediate translation whenever necessary in chronically stressed rats, which is in accordance with the results of García-López et al. [82]. A high splenic CAT and GPx transcript levels suggest that exercise could induce the antioxidant defense system to become more ready to a novel stressor.

concentration of NA in the spleen of chronically stressed animals exposed to daily exercise is increased. This finding indicates exogenous source of NA in the spleen of chronically stressed rats exposed to daily exercise. These findings strengthen the idea that the sympathetic nervous system (SNS) participates in the NA response to CSITR, which is in accordance with results of Blandino et al. [66], who have confirmed that the noradrenergic system plays an integral role in modulations of splenic IL-1 beta response to stress. In addition, exposure of chronically stressed rats to daily treadmill running reduces PNMT mRNA level. However, CSITR treatment leads to continuous accumulation of PNMT protein catalyzing the conversion of NA to A, suggesting the possibility of the conversion of sympathetic neurotransmitter NA to A in the spleen (**Figure 4a**). This is indicated by significant positive correlation between the levels of PNMT protein and A in the spleen. It is known that catecholamine via adrenergic receptors induces modulation of many immune functions like splenic cytokine production [4]. Moreover, catecholamines might be stored into vesicles by VMAT or degraded by MAO and catechol-O-methyltransferase (COMT) [67]. Expression of VMAT, which plays an important role in the transport of newly synthesized catecholamines into vesicles, positively correlated with norepinephrine levels in both T and B cells which might suggest increased capacity for intracellular catecholamine production [68]. Endogenous catecholamines can modulate function of lymphocytes themselves by a paracrine and autocrine pathway [69]. O'Donnell et al. [70] found that increase of catecholamine levels coincided with reduction of splenic B and NK cells and a concomitant increase in T cells. As reported in this chapter, the treadmill running increases splenic VMAT 2 gene expression, and that increased level of VMAT 2 mRNA coincides with the increased splenic NA and A levels of chronically psychosocially stressed adult rats. A high splenic VMAT 2 transcript level suggests increased capacity of the splenic catecholamines. Therefore, exercise induces accumulation of catecholamines in the spleen of chronically stressed rats, indicating higher readiness of catecholaminergic system to a novel stressor (**Figure 1e**). Brown et al. [71] found that endogenous catecholamines might further initiate intracellular oxidation and apoptosis. However, daily treadmill running does not change enzyme activity of MAO A and decreases enzyme activity of MAO B in the spleen of chronically stressed rats (**Figure 5**). Decreased or unchanged enzyme activities of MAOs indicate that daily treadmill running decreases catecholamine degradation of chronically stressed rats. Therefore, these results indicate that the treadmill running induces accumulation of the splenic catecholamines and that the SNS probably plays a major role in accumulation of the

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

Chronic social isolation significantly increases concentrations of MDA in the spleen (**Figure 6**). The literature data confirm that exercise training has beneficial effects on oxidative stress and antioxidant defense systems in multiple organs [72]. Meguid et al. [73] showed a significant decrease in serum level of malondialdehyde (MDA) in Down syndrome individuals after treadmill exercise for 3 months. Exposure of chronically stressed rats to daily treadmill running induces return of MDA concentration in the spleen to basal level (**Figure 6**). This confirms that the chronic exercise training induces adaptations that decrease stress-induced oxidative stress. It is in line with the reports of Belviranli et al. [74], who observed that chronic exercise has protective role because the decreased oxidative damage is associated with improved aero-

splenic catecholamines in chronically stressed rats.

bic metabolism induced by physical training.

To confirm whether exercise is optimal stimulus to regulate expression levels of splenic catecholamines and antioxidant enzymes and whether the exposure of chronically stressed rats to daily treadmill running induces potentially positive adaptations of the splenic catecholamines and antioxidant protection, this chapter discusses the effects of additional acute immobilization stress. Detection of regulatory mechanism for catecholamine metabolism and antioxidant protection in the spleen in conditions provoked by the additional acute immobilization of chronically stressed animals exposed to daily exercise is exceptionally relevant in stress biology, because of the significant role of catecholamines and oxidative stress in modulation of immune function. The acute immobilization (IMM) and additional acute immobilization (CSITR+IMM) do not affect the synthesis of splenic noradrenaline biosynthetic enzymes (TH and DBH) 3 hours after a termination of immobilization stimulus (**Figure 2**). Also, acute immobilization (IMM) does not change the level of PNMT mRNA and PNMT protein 3 hours after the termination of immobilization stimulus (**Figure 2**). Wong et al. [86] reported that PNMT protein and enzyme activity changes require additional time of approximately 18–20 hours to reach maximum stimulated levels. Three hours after the additional acute immobilization (CSITR+IMM), the increased synthesis of splenic PNMT protein (**Figure 2**) affects the increase of A (**Figure 3**) in the spleen of chronically stressed animals exposed to daily exercise. These data raise the possibility that 3 hours after additional acute stress, the spleen only converts sympathetic neurotransmitter NA to A of chronically stressed animals exposed to daily exercise. This is confirmed by the significant positive correlation between the levels of PNMT protein and A (**Figure 4b**), as well as negative correlation between the levels of NA and A in the spleen (**Figure 4c**). The acute immobilization (IMM) triggers an exaggerated elevation of splenic catecholamines, while the additional acute immobilization (CSITR+IMM) elevates only splenic A in chronically stressed animals exposed to daily exercise. This data confirm that the chronically stressed animals exposed to daily exercise show high readiness to convert sympathetic neurotransmitter NA to A. In addition, significantly elevated levels of VMAT 2 mRNA 3 hours after additional acute immobilization in chronically stressed animals exposed to daily exercise were found (**Figure 1**). Chronically stressed animals exposed to daily exercises have statistically less significant activation of MAO enzymes after additional acute immobilization compared with the animals exposed only to acute immobilization stress (**Figure 5**). These results confirm that the additional acute immobilization (CSITR+IMM) reveals high readiness of chronically stressed animals exposed to daily exercise for the accumulation of splenic A.

to a novel additional acute stressor by increased antioxidant protection. The readiness of the chronically stressed organism exposed to exercise to respond to a heterotypic stressor by an exaggerated expression of splenic antioxidant enzymes is an important adaptive phenomenon of the antioxidant defense system. Therefore, these results confirm that exercise have an

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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The exposure of chronically stressed rats to daily exercise induces the increase in the synthesis of splenic PNMT protein catalyzing the conversion of sympathetic neurotransmitter NA to A. In addition, the increased levels of splenic VMAT 2 mRNA and decreased/unchanged MAO enzyme activity suggest that daily exercise leads to accumulation of splenic catecholamines in chronically stressed rats. The accumulation of the splenic catecholamines provoked by exercises may have an important impact on the immune-neuroendocrine interactions in stress conditions. The return of the splenic MDA concentrations to basal levels confirms that exercise may decrease stress-induced oxidative stress, while the increased splenic antioxidant enzyme (CAT and GPx) transcript levels suggest that exercise could induce the antioxidant defense system to become more ready to a novel stressor, which indicates that exercises may repair oxidative damage in chronically stressed rats. Moreover, it can be concluded that exposure of chronically stressed rats to daily exercise causes high splenic antioxidant enzyme transcript levels and catecholamine levels and that the exercise can be beneficial, inducing an

The remarkable anatomical and physiological similarities between humans and animals, particularly mammals, have prompted researchers to investigate a large range of mechanisms and assess novel therapies in animal models before applying their discoveries to humans [88]. Our combined model of chronic social isolation and long-term daily treadmill running may be a good animal model in the research of the preventive role of exercise on neuroendocrine and immune functions in stress conditions, suggesting the potential application of CSITR animal model in understanding of human stress, as well as the potential therapeutic role of

This work was supported by the Ministry of Education and Science of the Republic of Serbia,

The authors report no conflict of interest. The authors alone are responsible for the content

important protective role in the splenic antioxidant defense system.

adaptive response to possibly other stressors that may be encountered later.

**10. Conclusions**

exercise in human diseases.

**Acknowledgements**

**Conflict of interest**

and writing of the paper.

Contract No.III 41027 and No.III 41022.

Additionally, it was proven that 3 hours after the acute immobilization, concentration of splenic MDA increased, which is in accordance with the reports of Belviranli et al. [74], who showed that the acute stress triggers oxidative stress. The acute immobilization (IMM) does not change either the levels of SOD 1, SOD 2, and CAT mRNA (**Figure 7**) or the total SOD and CAT enzyme activity (**Figure 8**) in the spleen 3 hours after a termination of immobilization stimulus. This finding is in line with the reports of Pajović et al. [16], who confirm that the acute immobilization does not change the levels of SOD enzyme activity. The increased oxidative stress produces inhibitory effects on CAT and SOD activity, which is evident from decreased enzyme activity of CAT and SOD (**Figure 8**) and increased concentration of MDA (**Figure 6**). These results are in accordance with the reports of Haider et al. [87] who showed that increased oxidative stress produced inhibitory effects on CAT activity. However, the acute immobilization (IMM) increases only the levels of mRNA and enzyme activity of GPx (**Figures 7** and **8**), but that increase was not sufficient to reduce oxidative stress. These results, together with the above mentioned data, confirm that acute immobilization induces oxidative stress. In addition, elevated levels of MDA 3 hours after the cessation of immobilization in chronically stressed animals exposed to daily exercise are observed (**Figure 6**). However, additional acute immobilization (CSITR+IMM) induces an increase of SOD 1, SOD 2, CAT, and GPx mRNA (**Figure 7**), as well as total SOD, CAT, and GPx enzyme activity (**Figure 8**) 3 hours after the cessation of immobilization in chronically stressed animals exposed to daily exercise. These data suggest high readiness of splenic antioxidant enzymes to repair or prevent damage by reactive oxygen species in chronically stressed animals exposed to daily exercise after additional acute immobilization stress. This could mean that exercise may condition physiological systems to "expect" a problem and, therefore, be more ready to respond to a novel additional acute stressor by increased antioxidant protection. The readiness of the chronically stressed organism exposed to exercise to respond to a heterotypic stressor by an exaggerated expression of splenic antioxidant enzymes is an important adaptive phenomenon of the antioxidant defense system. Therefore, these results confirm that exercise have an important protective role in the splenic antioxidant defense system.
