EVALUATION OF MILD STRESS INDUCED DEPRESSION IN RATS

International Standard Serial Number (ISSN): 2319-8141 1|P ag e International Journal of Universal Pharmacy and Bio Sciences 5(2): March-April 2016 INTERNATIONAL JOURNAL OF UNIVERSAL PHARMACY AND BIO SCIENCES IMPACT FACTOR 2.093*** ICV 5.13*** RESEARCH ARTICLE …………!!! Pharmaceutical Sciences EVALUATION OF MILD STRESS INDUCED DEPRESSION IN RATS Ms. Shruti B. Patil* and Mr. Rahul S. Nikam*, Ms. Vaibhavi N. Garge, Dr.Vilasrao J. Kadam Department of Pharmacology and Toxicology, Bharati Vidyapeeth’s College of Pharmacy, C. B. D. Belapur, Navi Mumbai-400614, Maharashtra, India. KEYWORDS: Mild stress, anhedonia, locomotor activity. ABSTRACT Depression is a common disorder observed in plenty of the population. Chronic mild stress is a well-established model, whereas acute mild stress For Correspondence: Ms. Vaibhavi N. Garge * Address: Associate Professor of has never been tried. The present study investigates behavioural, physiological and biochemical parameters of depression in rats as an aspect of mild stress induced depression. Sucrose preference test, adrenal Pharmacology, Bharati Vidyapeeth’s College of gland weight, body weight, locomotor activity and brain serotonin levels Pharmacy, C. B. D. were evaluated on the stress induced rats. Findings from the study indicate Belapur, Navi Mumbai- that sucrose consumption as a result of anhedonia and locomotor activity 400614, Maharashtra, India. of stressed rats decreased, brain serotonin levels were elevated and there was no significance difference in body weight of stressed and normal control rats. Full Text Available On www.ijupbs.com 2|P ag e International Standard Serial Number (ISSN): 2319-8141 1. INTRODUCTION: One of the most frequently occurring mental health problems incorporate depression. It accounts for approximately to 1 in 5 primary care consultations. At any given time, 10-30% of women and 7-15% of men are affected by depression, indicating that it affects women twice as frequently as men. The reason for gender variation being effect of specific sex hormones, anatomical differences of brain, dysregulation of hypothalamic-pituitary-adrenocortical (HPA) axis and fluctuating neurotrophins expression levels in brain resulting from stress leading to development of depression. The latest national psychiatric survey shows that approximately 17% of population has depression. [1,2] 1.1. Role of stressors in depression[1]: Psychosocial stressors are strategic depression induction parameters which can be used in rats to reciprocate the depression seen in humans. The social stressors such as physical exhaustion, excessive workload, sudden lifestyle changes, and lack of sleep present the stress response seen in humans which arise from stressful events in everyday life. The depression induced in rats can be extrapolated in that induced in humans. 1.2. Role of stress in depression[2,3]: Psychosocial stressors mentioned above play vital role in onset of depression. These stressors have direct effect on cognitive functions and neurotransmitters. The stressors act on humans with already elicited depression 2.5 times more likely than the control ones [1].The prominent association between stressors and depression has been confirmed by genetic studies[9]. 1.3. Depression neurobiology[1,3,4]: Major areas affected by depression are prefrontal and cingulated cortex, hippocampus, striatum, amygdala and thalamus as per the studies conducted. Neurotransmitters mainly involved in depression are Serotonin, Dopamine and Norepinephrine. Neocortex and hippocampus control measures of depression such as hopelessness, worthlessness, suicidality, while striatum and amygdala control memory and hence as a result mediate anhedonia (decreased reward pleasure activity), anxiety. Neurovegetative symptoms of depression are hypo or hypersomnia, loss or gain of appetite, loss of interest in pleasurable activities. 1.4. Brief introduction of the article: The present article focuses on induction of mild stress in rats. The stress procedure is followed for 14 days, followed by evaluation of the same. The evaluation parameters were behavioural, physiological and biochemical. The behavioural parameters include open field test, sucrose preference test, physiological tests include measurement of adrenal gland weight and body weight, while biochemical parameter include measurement of brain serotonin Full Text Available On www.ijupbs.com 3|P ag e International Standard Serial Number (ISSN): 2319-8141 level.[4] The biochemical parameter was evaluated to correlate the findings of behavioural and physiological evaluations. It is worth mentioning that one of the depression evaluation parameters, i.e., sucrose preference test doesn’t give consistent results. Not all the experimentations on depression have found reduction in consumption of sucrose solution, the result varies with laboratories, experiments and animal strains. 2. MATERIALS AND METHODS: 2.1. Animals: Male (n=36) Albino Wistar rats of the age 45 days were used for this experiment (all rats having same age), acquired from Bombay Veterinary College, Mumbai, Maharashtra. The average weight of rats was 135±5 gm. The rats were kept in well ventilated room, housed in polypropylene cages, maintained at 12-hour light/dark cycle (lights on at 8.00 AM) and relative humidity being between 48-52%. They were well fed with commercial diet (Amrut Laboratories) and purified water ad libitum. The experimental protocol was structured such as to reduce the number of animals and sufferings and was approved by IAEC (Institutional Animal Ethics Committee), the protocol number being IAEC/PR/2014-15/05-06, as per CPCSEA (Committee for the Purpose of Care and Supervision on Experimental Animals). 2.2. Chemicals and Reagents: All the reagents obtained were used for biochemical analysis of depression. The reagents namely HCl, n-Butanol, n-Heptane, Mercaptoethanol, o-Phthalaldehyde and Boric acid were procured from SD Fine-Chem Ltd., Mumbai, all being Analytical Grade reagents. 2.3. Experimental Procedure: Stress procedure was confirmed and designed by literature survey. Rats were divided into group of six, three rats per cage were housed. The groups were named as normal control and disease control, the stress procedure being applied to disease control groups. The normal control ones were undisturbed and received normal food and water supply, while the disease control ones were subjected to the stress regime. The stress procedure was followed for 14 days and was applied at random so as not to make the regime predictable to animals. A simple randomization procedure was applied. The following stressors were used as an induction procedure[14]: (i) Grouped housing of animals (12 hrs): It was carried out by placing 6 animals per cage, measurement of the cages being 36cm×21cm×14cm, for 12 hrs. The principle of this stressor was to induce stress by over-crowding the cages and not allowing the rats to move because of very less space available. Full Text Available On www.ijupbs.com 4|P ag e International Standard Serial Number (ISSN): 2319-8141 (ii) Cage soiling (12 hrs): Cage soiling was performed by pouring 250mL of water into every cage containing 3 animals and left the cages damp for 12 hrs, rational for this stressor being damp bedding. The dampness due to water brings stress to rats. (iii)Cage tilting (24 hrs): Cage tilting was accomplished by tilting the cages at an angle of 45º, held by a support, each cage containing 3 rats. The purpose of this stressor is to make the rats struggle for food and water, at the same time not making them deprived of it. The struggling point is the stressor agent. The schedule of stress procedure is as follows: Days of Induction Stressors Duration Day 1 Grouped housing 12 hrs Day 2 Cage tilting 24 hrs Day 3 Cage soiling 12 hrs Grouped housing 12 hrs Day 4 Cage tilting 24 hrs Day 5 Grouped housing 12 hrs Cage soiling 12 hrs Day 6 Cage tilting 24 hrs Day 7 Cage soiling 12 hrs Grouped housing 12 hrs Day 8 Cage tilting 24 hrs Day 9 Grouped housing 12 hrs Cage soiling 12 hrs Day 10 Cage tilting 24 hrs Day 11 Cage soiling 12 hrs Grouped housing 12 hrs Day 12 Cage tilting 24 hrs Day 13 Grouped housing 12 hrs Cage soiling 12 hrs Cage tilting 24 hrs Day 14 Table 1. Stress induction regime Full Text Available On www.ijupbs.com International Standard Serial Number (ISSN): 2319-8141 5|P ag e 3. PARAMETERS[3]: 3.1. Behavioural parameters: (i) Locomotor activity: Depression is known to cause immobility in rats[7].Actophotometer is the instrument which has photo electric cells at the base of the rectangular arena (40×40×20 cm) which are connected in series with counter. Movement of rat cuts the infra-red beam which records a count on the dial.[8] Before each evaluation rats were subjected to 10 minutes of acclimatisation and in next 10 minutes they are evaluated, which was carried out between 08:00 hours and 11:00 hours on 0th, 7th and 14th day. Locomotor Activity Evaluation 500 Normal control Disease Control Mobility 400 300 200 100 14 ay D ay D D ay 0 7 0 Figure 1. Effect of stress on behavioural parameter, locomotor activity. Bars represent mean±S.E.M. of 6 rats, p<0.05. (ii) Sucrose Preference Test [9]: Rats exposed to variety of mild stressors show reduced preference towards consumption of sucrose solution. 1% Sucrose solution and plain water are presented to rats in the cage for 24 hours to evaluate their preference. To avoid positional preference towards solution bottle, positions of bottles were switched after 12 hours, such that in previous spot where the sucrose solution bottle was placed, now there comes the plain water bottle. No water or food deprivation was applied before the test. The formula for sucrose preference is as follows: % Sucrose Preference= [Total sucrose consumed (g)/ Total fluid consumed (g)] × 100 Full Text Available On www.ijupbs.com International Standard Serial Number (ISSN): 2319-8141 6|P ag e Sucose Preference Test %Sucrose preference 0.8 Normal Control Disease Control 0.6 0.4 0.2 14 ay D D ay 0 0.0 Figure 2.Effect of stress on reward function, sucrose preference test. Bars represent mean±S.E.M. of 6 rats, p<0.05. 3.2. Physiological parameters: (i) Body weight: Body weight could be a parameter to evaluate depression. [1]Rats' body weight was evaluated on 0t and 14th day. Body Weight Evaluation Body weight (gram) 300 Normal control Disease Control 200 100 14 ay D D ay 0 0 Figure 3. Effect of stress on body weight. Bars represent mean±S.E.M. of 6 rats, p<0.05. Full Text Available On www.ijupbs.com International Standard Serial Number (ISSN): 2319-8141 7|P ag e (ii) Weight of adrenal gland [1,2]: Stress is known to cause Cortical (Adrenal Cortex) hypertrophy mediated through HPA axis. Weight of adrenal glands of rats in each group were measured post euthanasia. 40 Normal Control Disease Control 30 20 10 0 on tr ol C e is ea s D N or m al C on tr ol Adrenal gland weight(mg) Adrenal Gland Weight Evaluation Figure 4.Effect of stress on adrenal glands (leads to hypertrophy of adrenal glands). Bars represent mean±S.E.M. of 6 rats, p<0.05. 3.3. Biochemical parameters: (i) Brain 5-HT Levels[12,13]: One of the theories of depression is 5-HT theory, according to which in the state of depression, 5-HT levels in the neuronal synapse are decreased. [10] One of the areas of brain affected in depression is prefrontal cortex.[1,3,4] After rats were sacrificed, slices of prefrontal cortex were prepared and 10 mg of tissue was weighed and brain 5-HT levels were determined using fluorimetry assay.[11 Full Text Available On www.ijupbs.com International Standard Serial Number (ISSN): 2319-8141 8|P ag e B r a in S e r o t o n in L e v e ls E v a lu a t io n C o n c e n t r a t io n (n g /1 0 m g ) 4 N o rm a l C o n tro l D is e a s e C o n tro l 3 2 1 1 0 Figure 5. Effect of stress on brain serotonin levels. Bars represent mean±S.E.M. of 6 rats, p<0.05. 3.3. Statistical Analysis: For statistical analysis, GraphPad Prism 5 software was used. Values of mean, standard error of mean (S.E.M.) were obtained from n number of animals. Data was represented as mean±S.E.M. of n number of animals, p value less than 0.05 was considered as significant. 4. RESULT AND DISCUSSION: As seen in the graph of figure 1 the mobility of the rats was evaluated as a parameter of behavioural despair. The mobility was checked for normal control as well as disease control rats and compared against 0th day, 7th day and 14th day. The activity decreased as the mild stress induction proceeded is what could be predicted from the graph (p<0.05). The sucrose preference test is an evaluation of rats as a reward function. As depicted in the graph, sucrose preference was measured between normal control and disease control rats. The formula applied to the values obtained gave the percent sucrose consumed, which graphically gave an interpretation of the result. As seen in figure 2 the sucrose preference decreased as the stress induction proceeded (p<0.05). Due to depression the body weight may increase or decrease as a result of modification in body homeostasis. The data presented in figure 3 implies that the body weight decreased as a result of stress (p<0.05). Full Text Available On www.ijupbs.com 9|P ag e International Standard Serial Number (ISSN): 2319-8141 The mild stress causes hypertrophy of the adrenal cortex which is depicted in the graph of figure 4. The adrenal gland weight was elevated in case of disease control rats when compared to normal control rats (p<0.05). The biochemical parameter evaluated was brain serotonin levels. Due to stress there was decrease in serotonin levels majorly in the prefrontal cortex. The portion was removed and evaluated for its serotonin levels and the disease control rats were found to have lesser amount of serotonin, than the normal control ones, in brain. ACKNOWLEDGEMENT: This project wouldn’t be complete without the support of and facilities provided by our college, BharatiVidyapeeth’s College of Pharmacy, Navi Mumbai. REFERENCES: 1. Giancarlo Lucca et al., Chronic Mild Stress Paradigm Reduces Sweet Food Intake in Rats without Affecting Brain Derived Neurotrophic Factor Protein Levels, Current Neurovascular Research, 2008, 5(4):208,209. 2. Ritabrata Banerjee et al., Effect of Chronic stress and Antidepressant Treatment on Behavioural, Physiological and Neurochemical Aspects in Male and female Rats, Al Ameen Journal of Medical Science, 2012, 5(2):172. 3. Professor Hardeland et al., Behavioral and Pharmacological Validation of Chronic Social Stress as a Model of Depressive-like symptoms in Rats, 2006:3-5. 4. Nina Dedic et al., Mouse Model of Depression, Psychiatric Disorders- Trends and Developments, 2012:186,188. 5. 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