Effect of acute stress on serum cortisol level in female wistar rats

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1 Original article Effect of acute stress on serum cortisol level in female wistar rats Dr Mohammed Khaleel Jameel *, Dr Anuradha Rajiv Joshi ** *MSc (Medical Physiology) Ex PG Student,** Associate Professor Department of Physiology, Bharati Vidyapeeth Deemed University Medical College, Pune , Maharashtra, India Corresponding author: Dr Anuradha Rajiv Joshi Abstract Aims: Stress can be defined according to Psychoneuroimmunology experts as environmental events (real or perceived) that perturb one s psychological or physiological homeostasis or balance. (1) Acute stress triggers release of several hormones in animals. Severity of discomfort experienced by the animal is directly proportional to an increase in glucocorticoid concentration. (2) The study was planned to estimate serum cortisol levels in Wistar female rats after exposure to acute stress. Methodology: Randomly selected 6 Wistar female rats weighing gm were used for the study. Animals were given stress by forced swim test and restraint test. Serum cortisol level was measured before and after the tests by ELISA. Results: Analysis of the results was done by students paired t test. After forced swim test and after restraint test serum cortisol levels were significantly more in female rats. Also serum cortisol levels were more after restraint test than forced swim test but no statistically significant difference was observed. Conclusions: An increase in serum cortisol level was observed after acute stress. Key words: Stress, Wistar rat, Forced swim test,, Serum Cortisol,Restraint test Introduction animal are obvious. (5) During stress, normal The ability of an organism to respond and adapt to circadian pattern of hypothalamic pituitary adrenal environmental and homeostatic challenges is very (HPA) axis is altered.this has adverse effects on important for its continued survival. The first health. More is the intensity of stress more is the classification of the stress concept was derived from activation of the HPA axis. (6) Even a mild acute Bernard s steady-state of the milieu intérieur. The stressor is capable of increasing glucocorticoid (GC) sympathoadrenal system was identified as an internal levels in rats. (7) Irrespective of gender animal s well regulator. (3) Stress was defined by Selye as the being is affected by stress. (8,9) Serum cortisol nonspecific response of the body to any demand concentrations have been used as an marker of stress made upon it.(4) Stress affects various systems of in many studies. (8,9) Thus this study was planned to body such as immune system, cardiovascular system see effect of acute stress on serum cortisol level in etc. Levels of catecholamines, adrenocorticotrophic female Wistar rats. hormone and glucocorticoids are increased during Materials and methods stress response. (5) Normally adaptation of animal Approval by institutional animal ethical committee occurs by physiological and behavioral modifications was taken before initialion of the project. 6 adult to predictable situations. But when the situation is not female wistar rats were used. predictable, changes in the endocrine status of an 109

2 Animals were exposed to forced swim test and restraint test with a gap of of 15 days. Tests were carried out between 1p.m. to 4 p.m. Forced swim test: It is the tendency of the living being to escape or avoid a noxious stimuli/condition. If the animal is not able to escape the stressful stimuli or it feels threatened, the animal will show stress response. This principle is used for developing forced swimming model for inducing stress in laboratory animals. The glass cylinder was filled with water (25 0 C). Rat was placed in water filled cylinder and allowed to step on moving wheel for survival. Rat was judged to be immobile wherever it remained floating in water in a slightly upright position, making no movements except trying to keep its head above water surface. (8,2) Restraint stress test: Animals were isolated in a restraint transparent tube for 4 hours. (8) Blood was collected in all the animals before and after each test. The samples were taken at fixed timings. 2 ml of blood sample was collected by retro orbital puncture with all aseptic precautions in a plain vacutainer. It was allowed to clot for 30 minutes at room temperature. Serum was separated by centrifugation. Serum samples were stored at C and were subjected for estimation of serum cortisol. (2) The hormone was estimated by automated chemiluminescence immunoassay system Alpha Prime LS, France by using Cortisol ELISA kit (DRG,USA). This immunoassay kit allows for in vitro quantitative determination of endogenic cortisol concentration in serum. Competitive inhibition enzyme immunoassay technique has high sensitivity and excellent specificity for estimation of cortisol levels in rats. Observations and Results: Average weight of the female wistar rats was gm. Table 1: Effect of acute forced swim test on serum cortisol in female Wistar rats Before forced After forced swim Parameter swim test test Serum cortisol (Mean +SD) < 0.05 * P < 0.05* --Statistically significant Table No. 1 shows statistically significant (P< 0.05*) increase in serum cortisol level after forced swim test in female Wistar rats. 110

3 Table 2: Effect of acute restraint stress on serum cortisol in female Wistar rats Before restraint After restraint stress Parameter stress Serum cortisol < ** (Mean +SD) P < 0.001**--Highly significant Table No.2 shows statistically highly significant (P< 0.001**) increase in serum cortisol level after restraint stress in female Wister rats. Table 3: Comparison of serum cortisol levels after forced swim test and after Restraint stress in female rats Parameter Serum cortisol (Mean +SD) After forced swim test After restraint test > 0.05 P > Not significant Table No.3 shows comparison of serum cortisol levels after forced swim test and after restraint stress in female rats. No statistically significant difference is observed. Discussion: exhaustion. (11).Comparable results were also This study showed effect of acute stress on serum observed in male rats.(2) cortisol level in female Wistar rats. Table 2 shows significant increase in serum cortisol As shown in Table1 serum cortisol levels were levels after acute restraint stress in female rats. Renée significantly more after forced swim test in female Grimée et al. (12) observed fast and transient rats. Contarteze RVL et al (10) observed that HPA increase in plasma catecholamines and a slow but axis stimulation is directly proportional to duration of more sustained increase in plasma corticosterone the activity, during acute forced swim test. In another (CORT) levels, in rats after immobilization for 5, 15 study glucocorticoid levels were increased when rats and 30 min. Rats immobilized for 15 min were exposed to forced swim test till complete 1110

4 demonstrated tremendous increase in plasma ACTH and plasma CORT levels. Similar findings have been observed by Young Ju Cho et al. (13) after exposing the rats for acute restraint stress for one and half hours. In another study 1 h immobilization stress (IS) was imposed to rats at the beginning of the dark period, i.e., when the animals started to be active. Serum concentration of corticosterone was also increased by 56% at the end of the IS and was close to baseline level 4 h later. (14) Table 3 shows comparison of increased serum cortisol levels amongst two tests. Serum cortisol levels are not increased significantly after comparison amongst forced swim test and restraint test in female rats. These observations have indicated that both the tests exhibited comparable amount of stress in female rats. It has been also observed that animals exposed to any one type of stress also showed high levels of CRF. (15) Statistically significant increase in levels of serum cortisol after acute stress suggests that hypothalamicpituitary-adrenocortical (HPA) axis is activated. This feedback mechanism is extremely important for servival with stress.(16). Many studies showed that any situation which is new or unpredictable most likely activates the HPA axis. (16,17,18) Conclusion: Our study showed effect of acute stress on serum cortisol level. Stress due to exhaustive physical activity or without any physical activity affects release of cortisol. Acknowledgements: The authors are thankful to Dr J S Dawane, Department of Pharmacology and Dr M K Padwal, Department of Biochemistry for their help. References: 1. Kopin IJ. Definitions of stress and sympathetic neuronal responses. Annals of The New York Academy of Sciences. 1995;771: Mohammed Khaleel Jameel, Anuradha Rajiv Joshi, Jayashree Dawane, Meghana Padwal, ARJoshi, VAPandit et al Effect of Various Physical Stress Models on Serum Cortisol Level in Wistar Rats.Journal of Clinical and Diagnostic Research Mar, Vol-8(3): Philip E Cryer. Physiology and pathophysiology of the human sympathoadrenal neuroendocrine system. New England Journal of Medicine. 1980;303(8): Selye H. Forty years of stress research: principal remaining problems and misconceptions. Canadian Medical Association Journal. 1976; 115: Matteri RL, Carroll JA, Dyer CJ. Neuroendocrine responses to stress. In: Moberg GP, Mench JA, editors. The biology of animal stress. CABI Publishing, p E. Möst, R. Palme Hormones as indicators of stress Domestic Animal Endocrinology 2002;23: Gambarana C. Experimental protocols for the study of stress in animals and humans. Nutrients, stress and medical disorders. (Eds) Yehuda S and Mostofsky D Human press Inc Totowa. 2005; Elizabeth A. Younga,b, James Abelsona, Stafford L. Lightman. Cortisol pulsatility and its role in stress regulation and health. Frontiers in Neuroendocrinology 2004;25 :

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