**7. Materials and methods for studying the protective role of exercise against deleterious effects of oxidative stress**

#### **7.1. Animal models**

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) were exposed to chronic combined social isolation and treadmill running. CSITR was achieved by exposing the individually housed rats to the daily treadmill running during 12 weeks [42]. Chronically stressed animals were exposed to treadmill training ran on the treadmill 5 days a week for 12 weeks [22]. During that period, the exercise time was increased from 10 min to 20 min/day and treadmill speed gradually increased from 10 to 20 m/min by the end of the second week, without incline [22]. The animals ran according to the protocol for 10 additional weeks (20 min/day at a speed of 20 m/min, 5 days a week) [22]. The treadmill training protocol used in our studies involved a gradual increase in running intensity and is commonly used in the similar studies [29, 30]. Animals were exposed to a low-intensity treadmill training [31], which is in accordance with the protocol of Erdem et al. [30] who suggested that low exercise intensity is a key factor in the effect of submaximal endurance training on adrenomedullary catecholamine biosynthesis. During the exercise, we monitored the animals continuously. In **IMM group** (n = 10), the animals were exposed to acute stress immobilization, for a period of 2 hours [42]. Immobilization stress was elicited as described by Kvetnansky and Mikulaj [44]. In **CSITR+IMM group** (n = 10), the animals were exposed to CSITR during 12 weeks, and after CSITR, these animals were exposed to additional acute IMM stress for 2 hours [42]. The animals were sacrificed 3 hours after the acute immobilization. Data from literature show that 3 hours after the acute immobilization, changes in gene expression of catecholamine biosynthetic enzymes in the peripheral tissues are expected [43, 61]. To confirm the presence of oxidative stress in chronically stressed animals, we have introduced a **CSI** group. **CSI group** (n = 10) consisted of animals exposed to treatment of chronic social isolation for a period of 12 weeks. The rats were individually housed. The visual and olfactory communication among the isolated rats was reduced to the minimal level. In this group we measured the concentration malondialdehyde (MDA) in the spleen. Measurement of MDA is widely used as an indicator of lipid peroxidation. Increased levels of lipid peroxidation products have been associated with a variety of chronic diseases. The spleens were rapidly dissected and frozen. To avoid potentially confounding acute effects of exercise, animals were sacrificed 48 hours after the last training session, which is in accordance with protocol of Gavrilović et al. [31].

SOD2 (Rn00690587\_g1), CAT (Rn00560930\_m1), and GPx (Rn00577994\_g1). The reference gene (endogenous control) was included in each analysis to correct the differences in the inter-assay amplification efficiency, and all transcripts were normalized to cyclophilin A (Rn00690933\_m1) expression [31]. The results are reported as a fold change relative to the calibrator and normal-

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

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

291

The spleens were homogenized in 0.05 M sodium phosphate buffer (pH 6.65). Subsequently, the protein concentration was determined using bicinchoninic acid (BCA) method (Thermo

The TH, DBH, and PNMT proteins were assayed by Western blot analysis as described previously by Gavrilović et al. [42]. Antibodies used for quantification of proteins were for TH the monoclonal primary antibody against mouse TH (monoclonal antibody against TH from mouse-mouse hybrid cells, clone 2/40/15, dilution 1:5000, Chemicon International, USA); for DBH the anti-dopamine-ß hydroxylase (N-terminal) antibody, sheep (dilution 1:5000, Sigma, USA); for PNMT the polyclonal ant-PNMT primary antibody, rabbit (dilutation 1:1000, Protos Biotech Corporation, USA); and for β-actin the rabbit polyclonal anti-β-actin (ab8227, dilutation 1:5000, Abcam, USA) [31]. After that, the membranes were incubated in the secondary antimouse, anti-rabbit (dilution 1:5000, Amersham ECL™ Western Blotting Analysis System, UK) and anti-sheep (dilution 1:5000, Calbiochem, Germany) antibodies conjugated to horseradish peroxidase [31]. A secondary antibody was then visualized by the Western blotting enhanced chemiluminescent detection system (ECL, Amersham Biosciences, UK) [31]. The result was expressed in arbitrary units normalized in relation to β actin, which is in accor-

Spleen tissues were homogenized in 0.01 N HCl in the presence of EDTA and sodium metabisulfite. Catecholamine concentration in spleen fractions was determined using 3-CAT Research ELISA kits (Labor Diagnostica Nord, Nordhorn, Germany) according to the manufacturer's protocol. Absorbance was determined at 450 nm using a microplate reader (Stat Fax 2100). Concentrations were normalized to 1 g of tissues in homogenate. Values were expressed as ng

Determination of MAO A and MAO B activity was performed using the Amplex Red Monoamine Oxidase Assay (A12214, Molecular Probes, USA), described by Zhou and

oxidase-coupled reaction using N-acetyl-3, 7-dihydroxyphenoxazine (Amplex Red), a highly

O2

. Fluorescence was measured with a fluorometer using

in a horseradish per-

Panchuk-Voloshina [63]. This assay is based on the detection of H2

O2

**7.4. Spleen tissue homogenization and measurement of protein concentration**

ized to cyclophilin A as previously described [31].

Scientific Pierce, USA), described by Stich [62].

dance with protocol of Gavrilović et al. [31].

**7.6. Concentrations of catecholamines**

of catecholamine per g of tissues.

sensitive and stable probe for H2

**7.7. Monoamine oxidase enzyme activities**

**7.5. Western blot analysis**

#### **7.2. Spleen tissue homogenization, RNA isolation, and cDNA synthesis**

Total RNAs were isolated from 0.08 g spleen tissues by using TRIZOL reagent (Invitrogen, USA) as described previously by Gavrilović et al. [31]. Reverse transcription was performed using Ready-To-Go You-Prime First-Strand Bead (Amersham Biosciences, UK) and pd (N)6 Random Hexamer (Amersham Biosciences, UK) primer according to the manufacturer's protocol, which is in accordance with protocol of Gavrilović et al. [31].

#### **7.3. Quantitative real-time PCR**

TH, DBH, PNMT, CREB, VMAT2, CuZn SOD (SOD1), Mn SOD (SOD2), CAT, and GPx mRNA levels were quantified by quantitative real-time RT-PCR as described previously by Gavrilović et al. [42]. TaqMan PCR assays were carried out using Assay-on-Demand Gene Expression Products (Applied Biosystems, USA) for TH (Rn00562500\_m1), DBH (Rn00565819\_m1), PNMT (Rn01495589\_g1), CREB (Rn01441386\_g1), VMAT2 (Rn00564688\_m1), SOD1 (Rn00566938\_m1), SOD2 (Rn00690587\_g1), CAT (Rn00560930\_m1), and GPx (Rn00577994\_g1). The reference gene (endogenous control) was included in each analysis to correct the differences in the inter-assay amplification efficiency, and all transcripts were normalized to cyclophilin A (Rn00690933\_m1) expression [31]. The results are reported as a fold change relative to the calibrator and normalized to cyclophilin A as previously described [31].

#### **7.4. Spleen tissue homogenization and measurement of protein concentration**

The spleens were homogenized in 0.05 M sodium phosphate buffer (pH 6.65). Subsequently, the protein concentration was determined using bicinchoninic acid (BCA) method (Thermo Scientific Pierce, USA), described by Stich [62].

#### **7.5. Western blot analysis**

were exposed to chronic combined social isolation and treadmill running. CSITR was achieved by exposing the individually housed rats to the daily treadmill running during 12 weeks [42]. Chronically stressed animals were exposed to treadmill training ran on the treadmill 5 days a week for 12 weeks [22]. During that period, the exercise time was increased from 10 min to 20 min/day and treadmill speed gradually increased from 10 to 20 m/min by the end of the second week, without incline [22]. The animals ran according to the protocol for 10 additional weeks (20 min/day at a speed of 20 m/min, 5 days a week) [22]. The treadmill training protocol used in our studies involved a gradual increase in running intensity and is commonly used in the similar studies [29, 30]. Animals were exposed to a low-intensity treadmill training [31], which is in accordance with the protocol of Erdem et al. [30] who suggested that low exercise intensity is a key factor in the effect of submaximal endurance training on adrenomedullary catecholamine biosynthesis. During the exercise, we monitored the animals continuously. In **IMM group** (n = 10), the animals were exposed to acute stress immobilization, for a period of 2 hours [42]. Immobilization stress was elicited as described by Kvetnansky and Mikulaj [44]. In **CSITR+IMM group** (n = 10), the animals were exposed to CSITR during 12 weeks, and after CSITR, these animals were exposed to additional acute IMM stress for 2 hours [42]. The animals were sacrificed 3 hours after the acute immobilization. Data from literature show that 3 hours after the acute immobilization, changes in gene expression of catecholamine biosynthetic enzymes in the peripheral tissues are expected [43, 61]. To confirm the presence of oxidative stress in chronically stressed animals, we have introduced a **CSI** group. **CSI group** (n = 10) consisted of animals exposed to treatment of chronic social isolation for a period of 12 weeks. The rats were individually housed. The visual and olfactory communication among the isolated rats was reduced to the minimal level. In this group we measured the concentration malondialdehyde (MDA) in the spleen. Measurement of MDA is widely used as an indicator of lipid peroxidation. Increased levels of lipid peroxidation products have been associated with a variety of chronic diseases. The spleens were rapidly dissected and frozen. To avoid potentially confounding acute effects of exercise, animals were sacrificed 48 hours after

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

the last training session, which is in accordance with protocol of Gavrilović et al. [31].

Total RNAs were isolated from 0.08 g spleen tissues by using TRIZOL reagent (Invitrogen, USA) as described previously by Gavrilović et al. [31]. Reverse transcription was performed using Ready-To-Go You-Prime First-Strand Bead (Amersham Biosciences, UK) and pd (N)6 Random Hexamer (Amersham Biosciences, UK) primer according to the manufacturer's pro-

TH, DBH, PNMT, CREB, VMAT2, CuZn SOD (SOD1), Mn SOD (SOD2), CAT, and GPx mRNA levels were quantified by quantitative real-time RT-PCR as described previously by Gavrilović et al. [42]. TaqMan PCR assays were carried out using Assay-on-Demand Gene Expression Products (Applied Biosystems, USA) for TH (Rn00562500\_m1), DBH (Rn00565819\_m1), PNMT (Rn01495589\_g1), CREB (Rn01441386\_g1), VMAT2 (Rn00564688\_m1), SOD1 (Rn00566938\_m1),

**7.2. Spleen tissue homogenization, RNA isolation, and cDNA synthesis**

tocol, which is in accordance with protocol of Gavrilović et al. [31].

**7.3. Quantitative real-time PCR**

The TH, DBH, and PNMT proteins were assayed by Western blot analysis as described previously by Gavrilović et al. [42]. Antibodies used for quantification of proteins were for TH the monoclonal primary antibody against mouse TH (monoclonal antibody against TH from mouse-mouse hybrid cells, clone 2/40/15, dilution 1:5000, Chemicon International, USA); for DBH the anti-dopamine-ß hydroxylase (N-terminal) antibody, sheep (dilution 1:5000, Sigma, USA); for PNMT the polyclonal ant-PNMT primary antibody, rabbit (dilutation 1:1000, Protos Biotech Corporation, USA); and for β-actin the rabbit polyclonal anti-β-actin (ab8227, dilutation 1:5000, Abcam, USA) [31]. After that, the membranes were incubated in the secondary antimouse, anti-rabbit (dilution 1:5000, Amersham ECL™ Western Blotting Analysis System, UK) and anti-sheep (dilution 1:5000, Calbiochem, Germany) antibodies conjugated to horseradish peroxidase [31]. A secondary antibody was then visualized by the Western blotting enhanced chemiluminescent detection system (ECL, Amersham Biosciences, UK) [31]. The result was expressed in arbitrary units normalized in relation to β actin, which is in accordance with protocol of Gavrilović et al. [31].

#### **7.6. Concentrations of catecholamines**

Spleen tissues were homogenized in 0.01 N HCl in the presence of EDTA and sodium metabisulfite. Catecholamine concentration in spleen fractions was determined using 3-CAT Research ELISA kits (Labor Diagnostica Nord, Nordhorn, Germany) according to the manufacturer's protocol. Absorbance was determined at 450 nm using a microplate reader (Stat Fax 2100). Concentrations were normalized to 1 g of tissues in homogenate. Values were expressed as ng of catecholamine per g of tissues.

### **7.7. Monoamine oxidase enzyme activities**

Determination of MAO A and MAO B activity was performed using the Amplex Red Monoamine Oxidase Assay (A12214, Molecular Probes, USA), described by Zhou and Panchuk-Voloshina [63]. This assay is based on the detection of H2 O2 in a horseradish peroxidase-coupled reaction using N-acetyl-3, 7-dihydroxyphenoxazine (Amplex Red), a highly sensitive and stable probe for H2 O2 . Fluorescence was measured with a fluorometer using excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm. Monoamine oxidase activity was expressed as U/mg of protein.

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.

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

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

293

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).

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

#### **7.8. Malondialdehyde measurement**

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 concentration was expressed as μM/mg of protein.

#### **7.9. Antioxidant enzyme activities**

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

#### **7.10. Data analysis**

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 significance was accepted at p < 0.05.

Correlations of mRNA levels, protein levels, hormone levels, and enzyme activity were analyzed by the Pearson test, using the Sigma Plot v10.0 (with SigmaStat integration).
