Experimental Studies on The Role of Nitric Oxide (NO) in High Altitude Stress Induced Physiological and Behavioral Changes in Rats

The present study evaluated the effects of acute high altitude stress induced physiological and neurobehavioral changes and the possible involvement of nitric oxide (NO) in the regulation of such effects in rats. High altitude was simulated in a high altitude/hypoxia chamber and the effects of such stress and its modulation by NO-ergic agents were assessed on plasma corticosterone, anxiety and oxidative stress markers in rats. Acute high altitude stress induced reductions in open arm entries and open arm time in the elevated plus maze (EPM) test-suggestive of anxiety like behavior and was associated with increased levels of plasma corticosterone. Assay of brain homogenates showed lowered levels of NO metabolites (NOx) and GSH but elevated MDA. Pretreatment with the NO-mimetic, L-arginine (500 mg/kg) attenuated, whereas, the NOS inhibitor, L-NAME (50 mg/kg) aggravated both behavioral and biochemical changes induced by such high altitude exposure. Further, when combined with restraint stress (RS), these neurobehavioral and biochemical changes were further accentuated when compared to high altitude per se. Similarly, pretreatment with the L-Arginine neutralized the effects of high altitude + RS, whereas, L-NAME potentiated the behavioral and biochemical effects of the combined stressors. These results suggest that acute high altitude stress induced HPA axis activation, anxiogenesis and oxidative stress which were under the regulatory influence of NO. J Physiol Pharmacol 2016; 60(4) : 371–379 *Corresponding author : Prof. Arunabha Ray, Department of Pharmacology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi – 110 007, India, Tel . : +91-11-27662155; Fax: +91-11-27667420, E-mail address: [email protected] (Received on April 12, 2015) Introduction The lowest portion of the atmosphere, troposphere envelopes the earth’s entire surface. As altitude increases baromet r ic p ressure fa l l s w i th in troposphere. With decrease in barometric pressure, partial pressure of oxygen decreases proportionately resulting in a condition called as hypobaric hypoxia (1). Environmental stressors like ascent to high altitude (and the resultant hypoxia) can disrupt the physiological homeostasis of the biological system and lead to a variety of physiological and behavioral responses as well as pathophysiological states. For example, such hypobaric hypoxia is known to disrupt the hemodynamic balance of the cardiorespiratory system and precipitate pulmonary edema (2). The CNS, in particular, is highly vulnerable to such hypoxic insu l ts and could resul t in complex 372 Sharma, Gulati, Ravi and Ray Indian J Physiol Pharmacol 2016; 60(4) (1–2%) of “electron leakage” through the electron transport chain with subsequent direct reduction of molecular oxygen to the superoxide anion (11). These ROS show a high chemical reactivity and the brain, in particular, is highly susceptible to such oxidative damage, due to its high polyunsaturated fatty acid (PUFA) content and a weak anti-oxidant defense system. The balance between pro-oxidant/antioxidant is thought to be crucial in processes like cell death, motor neuron disease, axonal injury and neurodegeneration. The anti-oxidants are also known to directly or indirectly protect the cells from the adverse effects of drugs, xenobiotics, carcinogens as well as toxic radical reactions (12). Earlier studies from our laboratory have shown that NO may act as an important regulatory molecule during emotional stressors like restraint stress, and its interactions with reactive oxygen species have been suggested dur ing such s t ress induced anxiogenesis (13,14,15). In the present study, the possible role of NO was invest igated on h igh a l t i tude st ress induced physiological and neurobehavioral changes in rats. The influence of such stress of high altitude alone and in combination with emotional stressors like restraint stress was evaluated on plasma corticosterone and anxiety like behavior. Further, alteration of oxidative stress markers in the brain during such hypoxic insults were also assessed. The effects of NO modulators on both behavioral and biochemical parameters during such environmental (simulated high altitude) and emotional (restraint) stress were also evaluated for studying possible interactions of NO and oxidative stress. Materials and methods Experimental animals Male Wistar rats (150-200g) were used for the study. The animals (n=6 per group) were maintained under standard laboratory conditions (12 h light/dark cycle at 22±2°C) and had free access to food and water throughout the experiments. Animal care was as per Indian National Science Academy (INSA) Guidelines for Care and Use of Animals in Scientific Research. neurobehaviora l a l terat ions. Neurobehaviora l dysfunctions and high-altitude sickness that include phenomenon like negative mood states and inefficient cogn i t i ve per fo rmance are we l l documented subsequent to high al t i tude exposure (3, 4). Individuals known to spend long-term periods at a high altitude as well as the local residents at high altitude could also develop chronic mountain sickness accompanied with severe symptomatic polycythemia and hypoxemia (5). Acute Mountain Sickness and impaired neurobehavioral functions caused by high altitude exposure consequently are known to affect med ica l ass is tance and mi l i ta ry suppor t in emergency conditions. Emotional and environmental stressors reportedly influence brain function and is known to be a key factor in the genesis of neuropsychiatric disorders. Further, emotional stressors could also compound the effects of environmental stress of high altitude stress on the organism (6). Nitric oxide (NO) is a unique bioregulator molecule with complex physiological effects and having pathophysiological significance. Initially discovered as a vasodilator entity in the cardiovascular system, its role as a neuromodulator has now been widely documented. The changes in the central and peripheral nervous systems consequent on exposure to high altitude simulation may be mediated by the endogenous generat ion of n i t r ic oxide (NO), which is an intercellular messenger and potent vasodilator. NO is synthesized from L-arginine in the mammalian brain as well as in invertebrate neural structures by the enzyme nitric oxide synthase (NOS) (7-9). Stressful conditions also generate reactive oxygen species (ROS). Reactive oxygen species are known to be generated continually as by-products during aerobic metabolism. Some of the various factors involved are exposure to UV light, hypoxia, pollution and many other stressors (10). Strenuous physical activity is also known to increase oxidative stress that might be because of 10 to 15 fold increased oxygen util ization in order to meet the energy demands. This is also coupled with small amount Indian J Physiol Pharmacol 2016; 60(4) Experimental Studies on The Role of Nitric Oxide (NO) 373 The study protocol was approved by the Institutional Animal Ethical Committee (IAEC) of the VPCI.