**3. Animal model of long-term treadmill running in rats**

Physical exercise produces modulation of neuroendocrine and immune functions [26] and increases the activity of the antioxidant defense system [27]. Long-term treadmill running in rats is forced exercise which has the propensity to induce both psychological and physical stress [28].

Long-term treadmill running animal model (TR) consists of 11-week-old Wistar male rats that are exposed to long-term treadmill running. Long-term treadmill running is achieved by the rats' daily running on the treadmill for a period of 12 weeks. The treadmill running intensity is gradually increased from week to week and from the initial 10 min—10m/min up to 20 min—20m/min at 00 incline [22, 29, 30]. Animals are being exposed to treadmill training 5 days a week for 12 weeks [22].

Treadmill running may induce physiological adaptations, which can be reflected in increased plasma catecholamine concentration, as well as in the change of the synthesis of catecholamine biosynthetic enzymes in rats [31]. It is a very strong stressor, which activates the sympathoadrenomedullary system and increases the synthesis of splenic PNMT protein catalyzing the conversion of NA to A, which both can modulate the immune functions [31]. It is known that cardiovascular diseases, such as hypertension and heart failure, are often associated with sympathetic nervous system overreactivity [32, 33]. The increase of the noradrenaline biosynthetic enzyme expression in stellate ganglia, which causes the increase of plasma NA levels, due to chronic forced running, may play a role in the growing risk for cardiovascular diseases [22, 34].

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 stress conditions.
