**10. Conclusions**

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.

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

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

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 exercise in human diseases.
