INTRODUCTION:

Stress refers to physical, mental or emotional factor which produce deleterious effects on brain. Stress is the major factor that alters the neuronal properties of brain leading to disturbance in the cognitive processes like learning and memory.¹ The highly susceptible areas influenced by the stress and stress hormones are the cognitive areas of the brain which are mainly involved in memory, anxiety and decision making.² Anterior cingulate cortex is one such area which has been consistently involved with depression among adolescents³.

Substantia nigra neurons are also highly sensitive to stress induced neuronal changes.⁴ Restraint or immobilization and electric foot shock are the models of physical stress used in animal experiments .⁵Duration of stress also plays an important role in causing the neuronal damage similar to the nature of stress . Although both acute and chronic stress results in severe neuroendocrine damages, chronic stress is known to hamper memory and reduce neurogenesis.⁶ There are few studies which shows that acute stress enhances the cellular growth by stimulating the growth factor which suggests that stress could enhance neurogenesis.⁷ BALB/C mice are more stress sensitive and have been proposed to be a better model of pathological anxiety. ⁸ As cingulate gyrus and substantia nigra is involved in motivational and additive behaviors,⁹ observation of the effect of stress on these regions will help in understanding the neurobiology of psychiatric disorders. Limbic brain circuitry will still be developing in early adolescent stage, hence any changewhich affects the maturation of the brain during these periods will alter the emotion and behavior. ¹⁰ Maladaptive behaviors in response to threatening stimuli in youth are due to altered early developmental mechanisms, fronto-limbic function and HPA-axis reactivity due to stress.¹¹ Mental distress observed in university students showed that the living condition has strong association with stress. Less social support during this problem facing period, students will end up in mental illness. ¹² Support from family and friends will help to overcome the stress. ¹³Changes in cognition developed due to Chronic stress leads to reduction in weight gain, raised corticosterone level, behavioral problems and development of psychological disorders.¹⁴ This is the main reason why today chronic stress-induced anxiety disorders are becoming the major health concerns. ¹⁵ In the adult’s job stress in work place may affect their health. ¹⁶ Due to work pressure and heavy responsibilities in their work settings they may find difficulty in balancing both work and life. ¹⁷

MATERIALS AND METHODS:

The present experiments were conducted on Albino mice of BALB/C strain. Approval was taken by the institutional animal ethical clearance committee(No: IAEC/KMC/78/2013). Animals were maintained in institutional animal house. Adult age (271 days old) mice of both the sexes were used. These animals were randomly divided into three groups as control (C), restraint stress (RS) and foot shock stress (FSS).

Each group consisted of two sub groups according to duration of stress:

Short duration stress (5 days) and Long duration stress (21 days)

Control group of mice remained in their home cage, whereas the experimental mice of restraint stress group were stressed 6 hrs/ day in a wire mesh restrainer and mice of foot shock stress group were given intermittent electric foot shock using foot shock apparatus for three hours per day for the duration of 5 and 21 days. At the end of stress exposure period behavioral tests were conducted. Animals were sacrificed, brain, suprarenal gland and stomach were processed further for qualitative analysis.

Behavioral Tests:

After the stress exposure period, behavioral tests were conducted in both control and experimental animals. T –Maze test to evaluate spatial learning was conducted as defined by Dunnett et al., 1982. ¹⁸ Passive avoidance tests to assess the memory, as described by the Bures et al., 1983 were followed. ¹⁹

Qualitative Analysis:

Mice were anesthetized a day after the last day of behavioural test, Brain was collected and processed for cresyl violet staining as defined by the Shetty and Turner, 2000. ²⁰ From well stained sections of cingulate gyrus and substantia nigra regions, viable neuronal cell density observed. Suprarenal glands were processed with Haematoxylin and Eosin staining for cortical atrophy analysis.Stomach was dissected to observe the presence of ulcers if any.

Statistical analysis:

The test results were analyzed with using Graph Pad Prism, version 5 (Graph Pad Prism Software inc., USA). One way ANOVA followed by Bonferroni’s test was done and the results were expressed in Mean ± SD. The p value < 0.05 was considered statistically significant.

RESULTS:

Behavioral tests:

1. T-maze test:

Restraint and foot shock stressed animals in short duration stress had no significant impairment in spatial learning. While the animals exposed to long duration stress, showed significant impairment as there was more percentage bias because of less number of alternations and also there was less percentage of correct response in comparison with their age matched control.(Fig 1 a,b,c)

Fig.1. a and b: Bar diagram showing number of alternations and percentage bias in spontaneous alternation task performance; Mean ± SD is shown, n = 6 mice in each group. Control (C); Restraint stress (RS); Foot shock stress (FSS). C vs RS* p<0.05; C vs FSS ^$$ p <0.01. (one way ANOVA followed by Bonferroni`s post-test)

Fig. 1. c: Bar diagram showing percentage of correct response in rewarded alternation task performance; Mean ± SD is shown, n = 6 mice in each group. Control (C); Restraint stress (RS); Foot shock stress (FSS). C vs RS* p<0.05; C vs FSS ^$p <0.05. (one way ANOVA followed by Bonferroni`s post-test).

2. Passive avoidance test:

During memory retention test after 24 hours of exploration animals of stressed group showed poor memory retention compared to their age matched control group except restraint stressed animals in short duration stress. Foot shock stressed animals in short duration stress group spent greater time in dark compartment and more number of crossing across the compartment indicating reduced memory retention capacity compared to their age matched controls group. In long duration stressed animals showed poor memory retention in both stressed group (RS and FSS) spending greater time in dark compartment and increased number of crossing compared to their age matched controls group. (Fig.2. a and b)

Fig.2.a and b: Bar diagram showing the total time spent in a small compartment and total number of crossings during passive avoidance retention test. Mean ± SD is shown, n = 6 mice in each group. Control (C); Restraint stress (RS); Foot shock stress (FSS). C vs RS* p<0.05; C vs FSS ^$p <0.05; C vs FSS ^$$ p <0.01. (one way ANOVA followed by Bonferroni`s post-test).

3. Qualitative analysis:

i) Neuronal cell density of viable neurons in cingulate gyrus:

The cresyl violet stained sections from the cingulate gyrus showed significantly less number of viable neurons in 5days stress group compared to their age matched control group (both RS and FSS). (Photo.3a and b)

Fig.3 aandb: Photomicrographs of the Cingulate gyrus region of Adult group. Cc:corpus collosum. Note the decreased number of viable neurons and cell srinkage in restraint and footshock stressed groups compared to their age matched control groups respectively(100x;Cresyl violet stain).

ii) Neuronal cell density of viable neuron in substantia nigra:

Qualitative analysis of neurons from the cresyl violet stained sections of substantia nigra showed significant decrease in number of viable neurons in 5days stressed group compared to their age matched control group (both RS and FSS). (Fig.3c and d)

Fig.3.c and d: Photomicrographs of the Substantia nigra region of Adult group. CC: crus cerebri. Note the decreased number of viable neurons and cell shrinkage in restraint and footshock stressed groups compared to their age matched control groups respectively (100x;Cresyl violet stain).

4. Suprarenal glands:

The Haematoxylin and eosin-stained sections of the suprarenal glands showed significant hypertrophy of the cortex in stressed group compared to their age matched control group. (Fig.4.a andb)

Fig.4.a and b: Photomicrographs of the Suprarenal gland of Adult group.. Note the hypertrophy of suprarenal cortex in restraint and footshock stressed groups compared to their age matched control groups respectively(100x;Haematoxylin and Eosin stain).

5. Gastric ulcers:

In Adult age group of animals the gastric mucosa observed under dissection microscope showed ulcerations in stressed mice. These results indicate and confirm the effect of stress on mice. (Fig.5.a and b)

Fig.5.a and b: Photomicrographs of the gastric mucosa of Adult group. Note the presence of gastric ulcers (epethelial eroding) in restraint and footshock stressed groups compared to their age matched control groups respectively.

DISCUSSION:

Stress is our body's response to physical, emotional, or psychological strain. Neuronal damages due to stress depend upon the duration (acute and chronic) and type of stress (physical and psychological). Early neuronal damage and altered dendritic arborizations of cingulate gyrus and substantia nigra will affects the afferent projections of these neurons which subsequently alters the normal functioning of these brain areas. Impairment in spatial learning and poor memory retention observed in stressed group clearly shows the stress induced damage to the cognitive areas of the brain and its impact on learning and memory.

Restraint stress in rats showed structural alterations, ²¹ significant decrease in the total number of the neurons and glial cells, ²² reduction in the number of surviving neurons of cingulate gyrus. ²³The histopathological changes observed in our study suggesting cell necrosis/apoptosis and significant reduction in viable neuronal cell density reveals the amount of neurodegenerative changes in the stressed group.

Present study showed that both long and short duration stress restraint and footshock type resulted in decreased number of neurons may be due to stress induced reduction of neurogenesis,²⁴ by impairing cell proliferation and increasing cell apoptosisor may be due altered number and maturation of young neurons.

Death of nigral dopaminergic neurons in mice which underwent chronic stress ²⁵ evident from the stained sections of substantia nigra pars compacta region showing numerous degenerated neurons, which were darkly stained, shrunken, and with fragmented nuclei. ²⁶ Similarly results of our study also shows the significant decline in viable neuronal cell density observed in the cresyl violet stained sections of substantia nigra in stress.

Animals exposed to long duration stress spontaneous alteration test showed less number of alterations in choosing the arms of T Maze and in rewarded alteration test animals showed less percentage of correct response to find the reward. These animals have not learnt from the previous exploration trial about alternation of reward in each trial. However, these deficits were not recorded in short duration stressed mice. In passive avoidance apparatus, during retention test, long duration stressed mice showed severe memory impairment as they spent more time in small compartment and more number of crossing compared to their age matched control. Severe neurodegeneration of cingulate gyrus neurons may be the one of reasons for this memory impairment as these animals are entering and spending time in small compartment though they have received footshock in the smaller compartment at the end of exploration. In short duration stress group only in foot shock stressed mice slight memory impairment was seen while restraint stressed mice of short duration stress group spent lesser time in the small compartment suggesting memory retention.

Studies have reported cingulate gyrus as an essential portion of the limbic system, which is concerned with emotion, learning, and memory. ²⁷ Learning deficit is observed in repeated identical movements in lesions of pars compacta. It also plays significant role in spatial recognition otherwise known as ‘spatial learning’. Because of its connections to dorsal striatum, it is involved in spatial or episodic-like memory functions. ²⁸ Neurons of the midbrain are the main source of dopamine in the brain and are thought to be involved in reward-seeking behavior.²⁹ Stress induced long-lasting changes in the behavior of an organism results in structural and functional impairment of brain circuitry. Within individual synapses, neurons, and neuronal circuits, stores information’s and maintains to guide the behavior of an organism. This mechanism is called synaptic plasticity which allows the organism to adapt to its constantly evolving environment,.These mechanisms become deregulated under long bouts of physical or psychological stress where the connectivity between the regions of the brain becomes unstable, resulting in altered behaviors.³⁰

In the current study we have observed the cortical hypertrophy and also gastric ulceration in mice exposed to both restraint and foot-shock stress when compared to their age matched control groups.These results indicate and confirm the effect of stress on mice. Hypertrophy of cells of the zona fasciculata (ZF) and zona reticularis results from stress induced increased ACTH. ³² Modifications in mechanisms maintaining homeostasis in the adrenal gland is found (decrease in cellular proliferation) on exposure to chronic immobilization stress .³³Suprarenal cortical hypertrophy seen in stained sections on exposure to stress found in our study is comparable to similar reports.

Loss of epithelial lining and areas of erosion observed glandular part of the stomach. ³⁴ Significant increase in ulcer index seen in stress induced rats ³⁵ gastric ulceration observed in present study are the indicator of stress effect on stomach.

Sever neurodegenerative changes, learning and memory deficit, suprarenal hypertrophy and gastric ulcerations on prolonged exposure to stress observed in our study proves that stress can lead to adverse effect on prolonged acquaintance. Appropriate measures should be taken at initial stages to reduce the stress in young generations like students and young employees to cope up with their stress by conducting various extracurricular activities.³⁶ Corporate sectors should maintain healthy status of working area,³⁷ as good health among employees will result in higher productivity ³⁸ Practice of Yoga therapy and meditation techniques are one of the best upcoming stress reduction methodologies which will help people to overcome the stress. ³⁹

CONCLUSION:

Cognitive areas of the brain are prone to stress as there was lesser number of viable neurons in both cingulate gyrus and substantia nigra of stress exposed animals in comparison to their age matched control animals. Severe neurodegenerative changes were observed on prolonged exposure to stress which proves that stress, whether it is physical or psychological can lead to adverse effect on prolonged acquaintance.

Behavioral tests also reveals that the severity of memory and learning deficit increased with the duration of stress than type of stress.Severity of memory impairment was more on prolonged exposure to stress, which in turn proves that different durations of stress influence on the adult endocrinal and behavioral reactivity to stress. Cognitive deficit in adulthood has an impact on throughout the lifespan and on prolonged exposure results in stress related anxiety, depression and other psychiatric disorders. Study upon stress is very much needed to provide a stress-free environment to our younger generation to build a healthy society.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

REFERENCES:

1. Jeansok, Kim and Diamond M, The stressed hippocampus, synaptic plasticity and lost memories. Nature reviews Neuroscience. 2002; 3: 453-462 https://doi.org/10.1038/nrn849

2. Mcewen BS. Physiology and Neurobiology of Stress and Adaptation: Central Role of the Brain. Physiol Rev. 2007; 87: 873-904. https://doi.org/10.1152/physrev.00041.2006

3. Carrie, Mastena, et al. Subgenual anterior cingulate responses to peer rejection: A marker of adolescents’ risk for depression. Dev psychopathol. 2011; 23(1): 283-92. https://doi.org/10.1017/S0954579410000799

4. Everds NE, et al. Interpreting Stress Responses during Routine Toxicity Studies. Toxicologic Pathology. 2012; 1-55: 1533-1601. https://doi.org/10.1177/0192623312466452

5. Babu PB et al. Longitudinal study on the effect of restraint stress during preweaning period on postnatal growth of body and constituent parts in albino mice. IJPMBS. 2012; 1(2): 2278 – 5221.

6. Imperato A, et al, Puglisi-Allegra S et.al. Repeated stressful experiences differently affect limbic dopamine release during and following stress. Brain Res.1992; 17: 194–199. https://doi.org/10.1016/0006-8993(92)90274-D

7. Kirby ED, et al. Acute stress enhances adult rat hippocampal neurogenesis and activation of new-born neurons via secreted astrocytic FGF2. eLife. 2013; 2: e00362 https://doi.org/10.7554/eLife.00362

8. Mahony CM, et al. Restraint stress induced brain activation patterns in two strains of mice differing in their anxiety behaviour. EPub. 2010 1; 213(2): 148-54 https://doi.org/10.1016/j.bbr.2010.04.038

9. Deroche V, et al. Individual differences in cocaine self-administration: dose-response and ratio response study. Soc Neurosci Abstr. 1995; 21:767-769.

10. Moriah ET, et al. Stress-induced activation of the HPA axis predicts connectivity between subgenual cingulate and salience network during stress in adolescents. Journal of Child Psychology and Psychiatry. 2011; 52(10): 1026-1034. https://doi.org/10.1111/j.1469-7610.2011.02422.x

11. Choi HS, et al. Anxiolytic effects of Herbal Ethanol Extract from Gynostemma pentaphyllumin Mice after Exposure to Chronic Stress. Molecules. 2013; 18(4): 4342-4356. https://doi.org/10.3390/molecules18044342

12. Sailaja Busi, Zewdie Oltaye, Ezez Aklile, Tesfaw Ayenew, Berihum Yimam, Kefyalew Wubie, Addis Kindie, Sinit Tesfaye. Prevalence and determinants of Mental distress among University students, College of Medicine and Health sciences in Hawassa University, Ethiopia Asian J. Nur. Edu. And Reserch. 2016;6(3):356-360 http://dx.doi.org/10.5958/2349-2996.2016.00067.7

13. Amol Bhagwat Kanade, Shubhangi Sarwan, Pradnya Said, Suvarna Kadam, Gokul Dhakne, Pramod Gore. A study to asses the academic stress and coping strategies used among the undergraduate nursing students from selected colleges of Pune city. Asian J Nursing education and research 2021;11(2):183-188 DOI: 10.5958/2349-2996.2021.00045.8

14. Brown SM, et al. Mild, Short-term Stress Alters Dendritic Morphology in Rat Medial Prefrontal Cortex. Cerebral Cortex. 2005; 15:1714—1722. https://doi.org/10.1093/cercor/bhi048

15. Cryan JF, et al. Differential behavioral effects of the antidepressants reboxetine, fluoxetine, and moclobemide in a modified forced swim test following chronic treatment. Psychopharmacology (Berl). 2005; 182(3): 335–344. https://doi.org/10.1007/s00213-005-0093-5

16. V.Bidnur, R D Patil. A study on perceived over qualification, Job satisfaction and job stress of garge scale and medium scale executive with reference to Sangli, Kolhapur and Satara M.I.D.C area. Asian J. management 2(3): July-sept 2011 page 104-107.

17. Uma M.H Is work life balance important.Asian J. Management 2016:7(2): 153-155 http://dx.doi.org/10.5958/2321-5763.2016.00023.8

18. Dunnet SB, et al. Septal transplants restore maze learning in rats with fornix fimbria lesions. Brain research.1982; 251(2): 335-348. https://doi.org/10.1016/0006-8993(82)90751-X

19. Bures J,et al. Tecniques for study of brain and behaviour NY: Elsevier Science Publishers BV, Inc, Amsterdam/New York. 1983.

20. Shetty AK and Turner DA. Fetal hippocampal grafts containing CA3 cells restore host hippocampal glutamate decarboxylase-positive interneuron numbers in a rat model of temporal lobe epilepsy. J Neurosci. 2000; 20: 8788–8801. https://doi.org/10.1523/JNEUROSCI.20-23-08788.2000

21. Zhu MY, et al. Exogenous agmatine has neuroprotective effect against restraint-induced structural changes in the rat brain. Eur J Neurosci. 2009; 27(6): 1320-1332. https://doi.org/10.1111/j.1460-9568.2008.06104.x

22. Noorafshan A, et al. Curcumin and sertraline prevent the reduction of the number of neurons and glial cells and the volume of rats’ medial prefrontal cortex induced by stress. Acta Neurobiol Exp (Wars). 2014; 74(1): 44-53.

23. Rahman A, et al. Disruption of the Blood–Brain Barrier and Neuronal Cell Death in Cingulate Cortex, Dentate Gyrus, Thalamus, and Hypothalamus in a Rat Model of Gulf-War Syndrome. Neurobiology of Disease. 2002; 10: 306–326. https://doi.org/10.1006/nbdi.2002.0524

24. Kim J and Diamond M. The stressed hippocampus, synaptic plasticity and lost memories. Nature reviews neuroscience. 2002; 3: 453-462 https://doi.org/10.1038/nrn849

25. Pablos RMD,et al. Chronic stress, enhances microglia activation and exacerbates death of nigral dopaminergicneurons under conditions of inflammation. Journal of Neuroinflammation. 2014; 11-34. https://doi.org/10.1186/1742-2094-11-34

26. Babu PB, et al. "Longitudinal Study on the Effect of Restraint Stress during Preweaning Period on Postnatal Growth of Body and Constituent Parts in Albino Mice," International Journal of Pharma Medicine and Biological Sciences. 2012; 1(2): 80-90.

27. Hadland KA and Rushworth MF. The effect of cingulate lesions on social behaviour and emotion. Neuropsychologia. 2013; 41(8): 919-931. https://doi.org/10.1016/S0028-3932(02)00325-1

28. Claudio DA, et al. "Evidence for the substantia nigra pars compacta as an essential component of a memory system independent of the hippocampal memory system". Neurobiology of learning and memory. 2003; 79 (3): 236–42. https://doi.org/10.1016/S1074-7427(03)00008-X

29. Nauta, Haring J, et al. "Efferent projections of the sub-thalamic nucleus: An auto-radiographic study in monkey and cat". The Journal of Comparative Neurology. 1978; 180 (1): 1–16. https://doi.org/10.1002/cne.901800102

30. Carrión AO and Pŏppel E. Dopamine, learning, and reward-seeking behaviour. Acta NeurobiolExp (Wars). 2007; 67(4): 481-8.

31. Prakash N and Wurst W. Development of dopaminergic neurons in the mammalian brain. Cell. Mol. Life Sci. 2006; 63: 187-206. https://doi.org/10.1007/s00018-005-5387-6

32. Iversen SD and Iversen LL. Dopamine: 50 years in perspective. Trends Neurosci. 2007; 30: 188-193. https://doi.org/10.1016/j.tins.2007.03.002

33. Elizalde N, et al. Long lasting behavioural effects and recognition memory deficit induced by chronic mild stress in mice; effect of anti-depressant treatment. Psychopharmacology. 2008; 199(1): 1-14. https://doi.org/10.1007/s00213-007-1035-1

34. Ryan L, et al. Enriched environment prevents chronic stress induced spatial learning and memory deficits. Behav Brain.2008; 187(1):41-47. https://doi.org/10.1016/j.bbr.2007.08.025

35. Bali A, et al. Investigations into mild electric foot shock stress-induced cognitive enhancement: possible role of angiotensin neuropeptides. Journal of the Renin-Angiotensin- Aldosterone System. 2013; 14(3): 197–203. https://doi.org/10.1177%2F1470320312456579

36. Christoffel DJ, et al. Structural and synaptic plasticity in stress-related disorders. Rev Neurosci. 2011; 22(5): 535–549. https://doi.org/10.1515/RNS.2011.044

37. Everds NE, et al. Interpreting Stress Responses during Routine Toxicity Studies. Toxicologic Pathology. 2012; 1-55: 1533-1601.

38. Everds NE, et al. Interpreting stress responses during routine toxicity studies: a review of the biology, impact, and assessment. Toxic Pathol. 2013; 41(4): 560-614. https://doi.org/10.1177%2F0192623312466452

39. Romanini MC,et al. Chronic Stress Effects on Adrenal Cortex Cellular Proliferation in Pregnant Rats. Int. J. Morphol. 2011; 29(4): 1148-1157. http://dx.doi.org/10.4067/S0717-95022011000400013

40. Pellegrini A, et al. Stress-induced morphohistochemical and functional changes in rat adrenal cortex, testis and major salivary glands. Histo Chem J. 1998; 30(10): 695-701. https://doi.org/10.1023/A:1003493921112

41. Nachankar RS, et al. Prevention of Cold Stress Induced Adrenal Hypertrophy by Spirulina platensis. Acta Horticulturae. 2005; 6: 680. http://dx.doi.org/10.17660/ActaHortic.2005.680.14

42. Kulkarni MP and Juvekar AR. Attenuation of Acute and Chronic Restraint Stress-induced Perturbations in Experimental Animals by Nelumbo nucifera Gaertn. Indian J Pharm Sci. 2008; 70(3): 327–332. https://dx.doi.org/10.4103%2F0250-474X.42982

43. Dhanraj T.S,, Murugaiah K, Sarmila . Antiulcer activity of Mukia maderasapatana on Srtess induced in rats. Asian J.Res Sci. 2(2): April-june 2012: page 52-54

44. Stepanichev M,et al Rodent Models of Depression: Neurotrophic and Neuroinflammatory Biomarkers. Bio Med Research International. 2014; 1-20. https://doi.org/10.1155/2014/932757

45. Bagchi D, et al. Stress, diet, alcohol-induced oxidative gastrointestinal mucosal injury in rats and protection by bismuth subsalicylate. J.Appl.toxicol. 1998; 18(1): 3-13. https://doi.org/10.1002/(SICI)1099-1263(199801/02)18:1%3C3::AID-JAT461%3E3.0.CO;2-K

46. Lipika Mondal. A co-relational study on job related stress,stress reaction and coping strtergies adopted by nursing personenel providing care to PLHIVs at selected Medical college and Hospital. Kolkatta. Asian J, Nursing Education and Reserch.2020: 10(2): 227-234. http://dx.doi.org/10.5958/2349-2996.2020.00049.X

47. Sanket V. Bhingardive, T. Sivabalan. Job stress and coping strategies adopted by employees working at private university, Asian J.Nur.edu. and Research.2017 :7(3); 341-344. http://dx.doi.org/10.5958/2349-2996.2017.00068.4

48. P Radha. Stress Management. Asian J. Management 2(4): Oct-Dec,2011 page 202-203.

49. Bhavya Jose (Shimly T. Thomas), Sr Sajeena . Effectiveness of yoga therapy on stress management and concentration among students of selected schools in kerala. Asian J. Nur. Edu.andReserch.2017:7(3); 299-304. http://dx.doi.org/10.5958/2349-2996.2017.00062.3

50. Asmitha vala, Vijaya Nancy Rani,Jeenath Justin Doss. K. A study to asses the Effectiveness of Meditation Tecniques on stress Reduction among college students Harivadana College of Rajkot. Int. J. Adv. Nur. Management. 2017 :5(1);73-76. http://dx.doi.org/10.5958/2454-2652.2017.00016.6

Received on 29.05.2021 Modified on 27.10.2021

Research J. Pharm. and Tech 2022; 15(11):5260-5266.

DOI: 10.52711/0974-360X.2022.00886