Preclinical experimental stress studies: Protocols, assessment and comparison

Behavioural pharmacology

Preclinical experimental stress studies: Protocols, assessmentand comparison

Anjana Bali, Amteshwar Singh Jaggi nQ1

Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala 147002, India

a r t i c l e i n f o

Article history:Received 18 July 2014Received in revised form8 October 2014Accepted 9 October 2014

KeywordsQ10 :StressCorticosteroneImmobilizationRestrainElectric foot shockACTHSleep

a b s t r a c t

Stress is a state of threatened homeostasis during which a variety of adaptive processes are activated toproduce physiological and behavioral changes. Preclinical models are pivotal for understanding thesephysiological or pathophysiological changes in the body in response to stress. Furthermore, these modelsare also important for the development of novel pharmacological agents for stress management. Thewell described preclinical stress models include immobilization, restraint, electric foot shock and socialisolation stress. Stress assessment in animals is done at the behavioral level using open field, socialinteraction, hole board test; at the biochemical level by measuring plasma corticosterone and ACTH; atthe physiological level by measuring food intake, body weight, adrenal gland weight and gastriculceration. Furthermore the comparison between different stressors including electric foot shock,immobilization and cold stressor is described in terms of intensity, hormonal release, protein changesin brain, adaptation and sleep pattern. This present review describes these preclinical stress protocols,and stress assessment at different levels.

& 2014 Published by Elsevier B.V.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Stress protocols employed in preclinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.1. Immobilization stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.1.1. Acute immobilization stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.1.2. Chronic immobilization stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.2. Restrain stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.2.1. Acute restrain stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.2.2. Chronic restrain stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.3. Electric foot shock-induced stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.3.1. Electric foot shock as physical stressor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.3.2. Electric foot shock as psychological stressor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.4. Social isolation stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53. Stress assessment in preclinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.1. Behavioral tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.1.1. Open field exploration test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.1.2. Hole board test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.1.3. Social interaction test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.2. Biochemical parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.2.1. Plasma corticosterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.2.2. Adrenocorticotrophin hormone (ACTH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.2.3. Dissociation of ACTH and glucocorticoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3.3. Physiological parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

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http://dx.doi.org/10.1016/j.ejphar.2014.10.0170014-2999/& 2014 Published by Elsevier B.V.

n Corresponding author. Mobile: þ91 9501016036.E-mail addresses: [email protected] (A. Bali),

[email protected] (A.S. Jaggi).

Please cite this article as: Bali, A., Jaggi, A.S., Preclinical experimental stress studies: Protocols, assessment and comparison. Eur JPharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.10.017i

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3.3.1. Food intake and body weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.3.2. Adrenal weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.3.3. Gastric ulceration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4. Comparison between electric foot shock, immobilization and cold stressor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74.1. Intensity, hormonal release and protein changes in brain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74.2. Adaptation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74.3. Sleep pattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Future prospective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Uncited references . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1. Introduction

Hans Selye, Father of stress, described stress as a non-specificphenomenon representing the intersection of symptoms producedby a wide variety of noxious agents. Selye employed variousconditions, including fasting, extreme cold, and others as stressorsto produce the representative stress response, and defined that thedeterminants of the stress response are non-specific (Selye, 1950,1998). However, John Wayne Mason modified the original conceptof Selye by incorporating the importance of psychosocial stressorsand the emotional aspects of stress. He defined stress response asa specific hormonal, behavioral and physiological response ratherthan the non-specific response as advocated by Selye (Mason,1968, 1970). The stress response is an orchestrated process whichinvolves various mechanisms for physiological and metabolicadjustments in the body to cope with the demands of a homeo-static challenge (Bali and Jaggi, 2013). These changes occur at thepsychological (emotional and cognitive), behavioral (fight andflight), and biological level (altered autonomic and neuro-endocrine function). According to Richard Lazarus, an appraisalof an event is essential for a psychosocial situation to be stressful,and argued that cognitive processes of appraisal are central indetermining whether a situation is potentially threatening or not.In other words, stress response develops when the demands onthe organism perceived by the individual exceed its ability to cope(Lazarus, 1966).

Acute stress triggers cascade of biological events mainly due toactivation of two major pathways i.e. hypothalamic–pituitary–adrenal (HPA) axis and sympathetic adreno-medullary system.However, repeated exposure of a same stressor is generallyassociated with general adaptation syndrome (a phase of resis-tance to the homologous stressful condition). However, duringpersistent exposure of stress, the initial “adaptive response” maychange to “maladaptation” in which biological and behavioralresponses are counter-productive to the organism. Furthermore,repeated exposure of same stressor (chronic stress) sensitizes theHPA axis and exposure to novel, unpredictable stressor may causelong lasting dysregulation of the HPA axis (Gray et al., 2013;Herman, 2013). Accordingly, over-activation of these systemsduring persistent stress tends to produce the deleterious effectsin the body. In fact, the lack of turning off the physiologicalresponse to a stressor (when it is not required) produces allostaticload/overload leading to the development of pathophysiologicalchanges (Karatsoreos and McEwen, 2011; McEwen, 2007). Stresshas been postulated to be involved in the pathophysiology of avariety of diseasesQ11 including anxiety, depression, dementia andother diseases (Gareri et al., 2000; Rasheed et al., 2010; Yadin andThomas, 1993).

Animal models are pivotal for understanding the pathophysio-logical changes occurring in the disease and for the development ofnovel pharmacological agents for stress management. Accordingly,

diverse animal models, acute as well as chronic, have been createdto simulate the stress condition in animals akin to humans. Theseinclude immobilization (Bhatia et al., 2011; Rabasa et al., 2011),restraint Q12(Jackson and Moghaddam, 2006), electric foot shock (Baliet al., 2013; Rabasa et al., 2011) and social isolation Q13stress (Serraet al., 2008) and have been used more frequently by differentscientists to evaluate the anti-stress activity of pharmacologicalinterventions. The assessment of stress in animals as a part ofresearch activity is also of paramount importance and is done atvarious levels including the behavioral, biochemical and physiolo-gical levels. Stress-induced animal behavioral assessment is usuallydone using several well-established behavioral paradigms, includingopen field test, social interaction test and hole board test. Quanti-tative estimation of plasma corticosterone and ACTH levels at thebiochemical level; while determination of body weight, adrenalgland weight, and gastric ulceration at the physiological level is alsocarried by different scientists as indices Q3of stress (Bhatia et al., 2011;Kumar et al., 2010; Manchanda et al., 2011). The present reviewdiscusses the different experimental stress protocols in animals andstress assessment at biochemical, behavioral and physiologicallevels.

2. Stress protocols employed in preclinical studies

There have been a number of preclinical models for inducing stressin animals (Jaggi et al., 2011). However, more commonly employedmodels in preclinical studies include immobilization, restraint, electricfoot shock, social isolation stress and are described below.

2.1. Immobilization stress

Kvetnansky and coworkers developed a gold standard protocolfor inducing stress in laboratory animals by immobilizing rats/mice for a stipulated period of time. Thereafter, it has become oneof the most frequently employed stress models for rodents. Atypical immobilization procedure involves fixing the four limbs ofmouse/rat in the prone position on the plain board with anadhesive tape. The head is also fixed with a metal loop over theneck area to limit the head motion. This model has been widelyused to induce both acute as well as chronic stress by varying theduration of immobilization stress. Immobilization is a complexstressor and has both physical as well as psychological dimensions.The struggling and muscular exertion during immobilization is anintense form of physical exercise. During the first exposure toimmobilization stress, rats or mice struggle vigorously to freethemselves for 5–10 min. After that time, they stop struggling andremain motionless. However, on repeated immobilization expo-sures for many days, the animals tend to develop signs ofhabituation to stressor and stop struggling much earlier. Thestruggling and muscular exertion in the process to free itself

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comprises the physical components of stress. On the other hand,limited movement during immobilized position and exposure inan open area comprises the psychological stress (Kvetnansky andMikulai, 1970).

Immobilization is well tolerated by laboratory rats and mice, and isused in chronic stress studies extending over weeks or even months.This stressor is sufficiently intense to activate the stress-responsivesystem in the body, including HPA axis and the sympathetic nervoussystem. This type of physical and psychological stress is particularlyuseful for studying stress-induced neurodegeneration, post-traumaticdisorders and deregulation of the immune system (Southwick et al.,1994). Immobilization may also be combined with other stressors,such as cold temperatures or even placing the immobilization board inshallow water such that the animal is partially submerged. However,the limitation of the model is that in this model the intensity of thestressor cannot be altered, as with other stresses like foot-shock stressor cold stress.

2.1.1. Acute immobilization stressA single episode of immobilizationQ4 typically lasts for 120–

150 min for acute stress induction (Bhatia et al., 2011; Kumaret al., 2011; Kvetnansky and Mikulai, 1970). Scientists have variedthe immobilization time periods for specific investigation. Garcıa

and coworkers subjected the rats to immobilization stress for20 min, 1 h and 2 h to study the effects of stressor duration andintensity on post-stress recovery (García et al., 2000). Othersscientists have employed stressor of 2 h duration in mice (Bhatiaet al., 2011), 3 h duration in rats (Takayama et al., 1986); 6 h in Q14mice (Goyal and Kumar, 2007) to investigate the anti-stress effectsof different pharmacological agents including components of plantorigin. Other research studies involving variable time of immobi-lization stress include employment of 1 h stress in mice to studythe adaptive response of HPA axis (Rabasa et al., 2011), 2.5 h stressto study the cognition enhancing actions of acute stress in mice(Das et al., 2000), 3 h stress to study the effect of stress ontesticular steroidogenesis in adult rats (Orr et al., 1994), 4 h stressto evaluate the significance of pain sensitivity for the resistance toimmobilization stress in rats (Zarubina and Shabanov, 2012) and4 h stress to determine the effects of odorants on serum levels ofadrenocorticotropic hormone (ACTH) and corticosterone in rats(Komori et al., 2003) (Table 1).

2.1.2. Chronic immobilization stressThere have been many studies on rats and mice to examine the

effects of chronic immobilization stress on various aspects of stressresponse including disease development pattern. Different

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Table 1Different acute and chronic immobilization/restraint stress protocols in preclinical studies to study different aspects of stress.

S.no.

Duration Species Objective of study References

Immobilization stress

1. Acuteimmobilization

(a) 20 min, 1 h and2 h

Rat To study the effects of stressor duration and intensity on post-stress recovery García et al. (2000)

(b) 2 h, 3.5 h, 6 h Mice To investigate the antistress effects of different pharmacological agents including componentsof plant origin

Bhatia et al. (2011), Ha Q9et al. (2014),Goyal and Kumar (2007)

(c) 1 h Mice To study the HPA axis response during stress Rabasa et al. (2011)(d) 2.5 h Mice To study the cognition enhancing actions of acute stress Das et al. (2000)(e) 3 h Rat To study the effect of stress on testicular steroidogenesis Orr et al. (1994)(f) 4 h Rat To evaluate the significance of pain sensitivity for the resistance to immobilization stress Zarubina and Shabanov (2012)(g) 4 h Rat To determine the effects of odorants on serum levels of adrenocorticotropic hormone (ACTH)

and corticosteroneKomori et al. (2003)

2. Chronicimmobilization

(a) 1 h for 5–14 days Mice To study the adaptive response of HPA axis response in chronic stress model Rabasa et al. (2011), Haleem et al.(2013)

(b) 2.5 h for 5 days Mice To evaluate the cognitive dysfunction of chronic stress Das et al. (2000)(c) 2 h for 10 days Rat To determine the association of stress recovery with the changes in the mesolimbic brain

regionsLucas et al. (2011)

(d) 1 h for 14 days Mice To study the development of anxiety and depression in mice Xing et al. (2013)(e) 20 min for 21

daysRat To study effects of stress on serum ghrelin levels Elbassuoni (2013)

(f) 1 h for 22 days Rat To evaluate the effects of stress and estradiol on pain tolerance Cruthirds et al. (2011)(h) 1 h for 30 days Rat To evaluate the cardiovascular risk of stress exposure Baptista et al. (2014)

Restraint stress3. Acute restraint

stress(a) 15 min and 2 h Mice To investigate the changes in stress responsivity with time of restraint Zimprich et al. (2014)(b) 15 min Rat To investigate the effects of mild calorie restriction on anxiety and HPA axis responses to stress Kenny et al. (2014)(c) 60 min Rat To evaluate the activity of the HPA axis in adolescent rat offspring Xu et al. (2014)(d) 3 h Rat To evaluate the effects of physical stress on serum cortisol level Jameel et al. (2014)(e) 3.5 h Rat To study the beneficial effects of sodium cromoglycate and diethyldithiocarbamic acid in acute

stress-induced behavioral alterations in ratsManchanda et al. (2011)

4. Chronicrestraint stress

(a) 60 min/d for7 days

Mice to evaluate the stress adaptive process Miyagawa et al. (2014)

(b) 1 h/d for 21 days Rat to investigate the effect of stress on spatial learning and memory Abidin et al. (2004)(c) 60 min stress for

20 daysRat to evaluate the nociceptive response HeidariOranjaghi et al. (2012)

(d) 6 h/d for 21 days Rat to study the effects of stress on glial activity in the rostral ventromedial medulla Imbe et al. (2004)

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scientists have employed variable time periods ranging from 5 to22 days to induce chronic stress of varying degree in rats and mice(Bhatia et al., 2011; Larco et al., 2012). The different research-basedstudies employing immobilization stressor for chronic stressinclude immobilization for 1 h daily for 5–14 days in mice tostudy the adaptive response of HPA axis in chronic stress model(Haleem et al., 2013; Rabasa et al., 2011), 2.5 h daily for 5 con-secutive days to evaluate the cognitive dysfunction of chronicstress in mice (Das et al., 2000), 2 h daily for 10 days to determinethe association of stress recovery with the changes in the meso-limbic brain regions (Lucas et al., 2011), 1 h daily for 14 days tostudy the development of anxiety and depression in mice (Xinget al., 2013), 20 min daily for 21 days to study effects of stress onserum ghrelin levels inQ15 rats (Elbassuoni, 2013), 1 h daily for 22days to evaluate the effects of stress and estradiol on paintolerance (Cruthirds et al., 2011), 1 h for 30 days to evaluate thecardiovascular risk of stress exposure (Baptista et al., 2014)(Table 1).

2.2. Restrain stress

Restrain stress is a modified form of immobilization stress inwhich animals are not allowed to move for a specified period oftime. The neural and endocrine responses have described thatrestrain stress is less intense stressor than immobilization. Never-theless, restrain stress is one of more commonly employed modelfor the induction of acute as well as chronic stress in rats (Jaggiet al., 2011; Kumari et al., 2007, Manchanda et al., 2011). Restraintinvolves placing the test animal in a well-ventilated plastic tube ora wire-mesh container. Although the animal's range of movementis severely limited, the limbs are not secured and the animalremains within an enclosed area (Southwick et al., 1994). Restrainstress is generally induced by placing the rats individually in the5.5-cm-diameter and 18-cm-long semi-cylindrical, acrylic restrai-ner with air holes for variable period including 2–6 h (Kaur et al.,2010; Manchanda et al., 2011; Uramoto et al., 1990). However,there have been variations among different models of this cate-gory with respect to difference in the size of restrainers with use ofother restrainers of size 25�9�7 cm3 with a 1.5-cm-diameterhole at one far end for breathing in mice (Das et al., 2000).

2.2.1. Acute restrain stressResearchers have employed variable acute stress protocols to

investigate different aspects of stress and these include 15 min and2 h restrain stress to investigate the changes in stress responsivitywith time of restraint (development of hypersensitivity in 15 minrestrain stress and hyposensitivity in 2 h restrain stress) (Zimprichet al., 2014); 15 min stress to investigate the effects of mild calorierestriction on anxiety and HPA axis responses to stress in the malerat (Kenny et al., 2014); 60 min of prenatal stress to evaluate theactivity of the HPA axis in adolescent rat (Xu et al., 2014); 3 h stressto evaluate the effects of physical stress on serum cortisol level inrat (Jameel et al., 2014); 3.5 h stress to study the beneficial effectsof sodium cromoglycate and diethyldithiocarbamic acid in acutestress-induced behavioral alterations in rats (Manchanda et al.,2011) (Table 1).

2.2.2. Chronic restrain stressChronic stress sessions usually last for 7–30 days with different

stress protocols including 60 min/day for 7 days to study the stressadaptive process in mice (Miyagawa et al., 2014); 1 h/day for 21 daysto investigate the effect of stress on spatial learning and memory inrats (Abidin et al., 2004); 60 min stress for 20 days to evaluate thenociceptive response in rats (HeidariOranjaghi et al., 2012); 6 h/dayfor 21 days to study the effects of stress on glial activity in the rostral

ventromedial medulla in rat (Imbe et al., 2004). Restraint has alsobeen employed in the development of an animal model of long-termweightlessness. This model involves continuous restriction of themovement of laboratory rats for as long as 60 days to simulate thephysiological changes occurring during long-term space flight. How-ever, the restraint apparatus is modified such that the animal can gainaccess to food and water. This model is similar to studies withhumans in which long-term bed rest has been used as a model ofweightlessness associated with space flight (Sulzman, 1996) (Table 1).

2.3. Electric foot shock-induced stress

Electric foot shock stressor includes both physical as well asemotional components and it is used as direct (physical stress) andindirect stressor (psychological stress). It has been mainly usedwith varying degree to produce mild as well severe stress of bothacute and chronic in nature. Electric foot shock paradigm mainlycomprises acute or chronic exposures of foot shocks with variableintensity and of different duration on electrified grid floor in anelectric foot shock apparatus.

2.3.1. Electric foot shock as physical stressorA direct application of electric foot shock to animals produces

physical stress to induce behavioral and other changes in the bodyorgans. The major advantage of this stressor over other commonlyused immobilization and restrain stress is that its intensity,duration and frequency may be varied to induce stress of variabledegree. Accordingly, different scientists have varied the stressintensity, duration and frequency to study different aspects ofstress.

2.3.1.1. Electric foot shock-induced acute stress. Four electric shocksof 0.8 mA intensity and 2 s duration delivered at inter-stimulusinterval of 1 min have been shown to significantly triggeranhedonia-like behavior to produce depression in rats (Enkelset al., 2010). The exposure to acute electric foot shocks with anintensity of 3 mA, duration of 200 ms and a frequency of 1/s over a5-min period have been shown to significantly increase theplasma corticosterone levels in rats (Retana-Márquez et al.,2003). Rabasa et al. (2011) employed electric foot shocks of0.5 mA intensity for moderate stress or 1.5 mA intensity forsevere shock for 1 h with one shock every 60 s in a regularschedule to investigate the stress adaptive response of HPA axis.120 electric foot shocks each of 0.15 mA intensity and 5 s durationover a period of 1-h session (every 30 s) was employed toinvestigate the effects of stress in altering the humoral andcellular immune function (Brevet et al., 2010). Six foot shocks of1 mA intensity of 750 ms duration within a period of 2 min havebeen employed to study transcriptional changes in brain (Santhaet al., 2013) (Table 2).

2.3.1.2. Electric foot shock induced chronic stress. A stress protocolcomprising delivery of 60 unpredictable shocks of 0.8 mA intensityand 5 s duration in the early life span of rats (14th day and 21st day)has been used to study the deleterious effects of early life stress onspontaneous locomotor activity Q16at maturity (Kim and Seo, 2013). Inour study, application of mild electric foot shock stress of 0.15 mAintensity with duration 0.5 s for 4 days was used to investigate effectsof mild stress on memory improvement (Bali et al., 2013). A mildextrinsic foot shock stressor of 0.1 or 0.3 mA for 1-s for 7 days hasbeen used to reactivate the memory of a discriminative avoidancetask in mice (Takatsu-Coleman et al., 2013). An exposure of 3 electricfoot shocks trials/day for 7 consecutive days with each trialcomprising of 0.2 mA intensity, 6 s duration for 10 times with a30 s interval is shown to induce post-traumatic stress disorder in rats

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(Kim and Seo, 2013). Ten foot shock shocks of 0.6 mA intensity andeach of 2 s duration within a period of 5 min for 7, 14 or 21consecutive days has been employed to study the effects of stressconditions on the transcription and translation of actin-relatedcytoskeletal genes in the rat brain (Santha et al., 2013). Scientistshave also used this stressor to examine the effects of stress in alteringthe humoral and cellular immune function that include application ofshocks of 1 mA of 0.5 s duration every 5 s for 30 min/day for5 consecutive days (Zhang et al., 2005); 2 mA of 10 s duration withinterval of 50 s for 2 h/day (Yamamoto et al., 2009); 120 electric footshocks, each of 0.15 mA intensity, 5 s duration over a period of 1 h(every 30 s) for 5 days (Brevet et al., 2010) (Table 2).

2.3.2. Electric foot shock as psychological stressorElectric foot shock has also been used to produce psychological

stress in rats indirectly by visual, olfactory and auditory sensationfrom the foot shock subjected rats. In a typical model, 1 foot shock/s of 1 mA intensity for 1 h/day are provided during acute (1 day)and chronic (15–30 day) stress studies (Greisen et al., 2005;Rostamkhani et al., 2012). Other protocols include delivery of 10unpredictable shocks of 0.5 mA of 1 s duration at random for 1 day(acute) and 5 days (chronicQ17 ) (Daniels et al., 2008); 1 mA, once/s for1 h/day for 5 weeks have also been used to induce psychologicalstress (Li et al., 2013). However, this type of indirect stressor isrelatively mild as compared to direct physical stress.

2.4. Social isolation stress

Early life events have profound consequences on subsequentquality of life (Ladd et al., 2004). It has been shown that the earlylife stress of neonatal isolation in rats produces immediate andlong lasting neural and behavioral effects (Kuhn et al., 1990).Rearing rats in isolation (post-weaning) is an animal model ofsocial deprivation and rodents reared in social deprivation exhibit

an abnormal behavior that includes hyper-locomotion in responseto a novel environment, and disrupted exploratory Q18behaviors(Varty et al., 2000).The postnatal period of mice from postnatalday 1 to postnatal day 12 is characterized by stress hypo-responsive period during which pups exhibit decreased respon-siveness to a variety of stressors due to low basal corticosteronelevels (Cirulli et al., 1992; Schmidt et al., 2003).A close contactbetween the dam (mother) and the litter is essential for thenormal development of the HPA axis in rodents. Maternal signalsincluding licking and feeding are important in sustaining the HPAaxis of the pups in a hypo-responsive state. However, prolongeddisruption of this mother-pup interaction in the form of maternaldeprivation for about 24 h increases the sensitivity of otherwisequiescent HPA axis to ACTH and other mild stressors (Schmidtet al., 2004). Furthermore, the lack of the protection during theabsence of the dam makes the pups more vulnerable to stressors.Although isolation of pup from its mother does not grossly effectthe growth in neonatal stage or in adult stage (Hamm et al., 1983;Kehoe and Bronzino, 1999), yet it induces stress characterized bystimulation of HPA axis and morphological changes in the hippo-campus including neurodegeneration (Kosten et al., 2007). There-fore, this stressor has been very useful in evaluating the effect ofstress on cognition and memory development. Furthermore, earlylifetime stress also increases the vulnerability to addiction and ithas been employed for those particular studies (Kosten and Kehoe,2005). Other significant changes due to maternal deprivationduring neonatal period include development of tentative Q19behavior(Kuhn et al., 1990), exacerbation of the severity of trinitrobenzenesulfonic acid (TNBS)-induced colitis (Barreau et al., 2004) andgastric ulcer susceptibility in the adult (Ackerman et al., 1978).

In a typical neonatal isolation procedure, the pup is removedfrom the cage on the second day after its birth and placedindividually in an opaque plastic container (9 cm diameter and8 cm deep) with no bedding for 1 h, which in turn is placed in atemperature and humidity-controlled chamber with white noise

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Table 2Different acute and chronic electric foot shock stress protocols in preclinical studies to study different aspects of stress.

S.no.

Duration Species Objective of study Reference

Electric foot shock

1. Acute stress(a) Four un-signaled electric shocks (2 s, 0.8 mA, pulsed) with interval of

1 minRat To trigger anhedonia-like behavior and depression Enkels et al.

(2010)(b) Shocks of 3 mA of 200 ms and a frequency of 1 per second over a 5-min

periodRat To investigate the effect of stress on stress hormones Retana-

Márquezet al. (2003)

(c) 0.5 mA foot shock for 1 h for moderate shock (FS-medium) or 1.5 mAfor severe shock (FS-high), one shock every 60 s in a regular schedule

Mice To investigate adaptive response of the HPA axis Rabasa et al.(2011)

(d) 120 electric foot shocks each of 0.15 mA intensity of 5 s duration over aperiod of 1-h session (every 30 s)

Mice To investigate the effects of stress in altering the humoral andcellular immune function

Brevet et al.(2010)

(e) Six foot shocks of 1 mA intensity of 750 ms duration within a period of2 min

Rat To study transcriptional changes in brain Santha et al.(2013)

2. Chronic stress(a) Mild electric foot shock stress of 0.15 mA intensity with duration 0.5 s for

4 daysMice To investigate effects of mild stress on memory improvement Bali et al.

(2013)(b) A mild extrinsic foot shock Mice To evaluate the effects of stress on memory Takatsu-

Coleman et al.(2013)

(c) 60 unpredictable shocks of 0.8 mA intensity and 5 s duration in the earlylife span of rats (14th day & 21st day)

Rat To study the deleterious effects of early life stress onspontaneous locomotor activity at maturity

Kim and Seo(2013)

(d) Eposure of 3 electric foot shocks trials/day for 7 consecutive days witheach trial comprising of 0.2 mA intensity, 6 s duration for 10 times with a30 s interval

Rat To investigate stress induced post-traumatic stress disorder inrats

Kim and Seo(2013)

(e) 6 shocks, of 1 mA for 750 ms in 2 min, for 3 consecutive days and 10shocks of 0.6 mA intensity for 2 s in 5 min for 7, 14 or 21 day

Rat To study the effects of stress conditions on the transcriptionand translation of actin-related cytoskeletal genes in the ratbrain

Santha et al.(2013)






to mask the calls of other pups. Thereafter, the litters are placedback in home cage with mother. This isolation procedure isrepeated for 8 days to induce chronic stress (Knuth and Etgen,2007; Kosten et al., 2003, 2004, 2007).

3. Stress assessment in preclinical studies

3.1. Behavioral tests

Apart from well-defined stress evaluating stress procedures forbehavioral testing, an initial battery of behavioral tests may beconducted in mice in their home cages to provide important clues.These mouse behavior including eye blink, whisker, ear twitching,locomotion, rearing, visual cliff, righting reflex, hanging, toe pinchand, acoustic startle that may be used to assess the neurologicalstatus. The following behavioral tests are more commonlyemployed to assess stress-induced behavioral changes:

3.1.1. Open field exploration testHall et al. (1998) originallyQ20 described the open field test for the

study of emotionality in rodents. The open field is a paradigm tosystematically assess novel environment exploration, generallocomotor activity, and screen stress-related behavior in rodents(Prut and Belzung, 2003; Weiss and Greenberg, 1996). Highlyanxious mice hug the walls (thigmotaxis) and non-anxious micerun in the center of the open field. This test simultaneousmeasures the spontaneous locomotion (line crossing), exploration(rearing), and fear or anxiety (peripheral square entries, defeca-tion) (Manchanda et al., 2011; McFadyen et al., 2002). A standardopen field arena used for mice is generally square and mostappropriate size is 44�44 cm2 and 30 cm height. However,numerous research studies have employed open field arena ofdifferent sizes including 45.5�45.5�39.5 cm3 (Zimprich et al.,2014); 20�30�33 cm3 (Saitoh et al., 2014) and 30�30�20 cm3

to assess anxiety-like behaviors in mice (Kulesskaya and Voikar,2014; Wang et al., 2014). The size of this field is relatively large forrats and different sizes employed in stress-related studies include90�90�38 cm3 (Manchanda et al., 2011) 80�80�50 cm3

(Ennaceur et al., 2006); 122�122�46 cm3 (Desikan et al.,2014); 99Q5 �99�45 cm3 (Lio et al., 2014); 100�100�30 cm3

(Bai et al., 2014).

3.1.2. Hole board testHole board test is also commonly used to study stress-related

changes in exploratory behavior of animals. The test is based onthe assumption that head-dipping activity of the animals isinversely proportional to their anxiety state. The low level of headdipping reflects high anxiety state level in animals. During 10 mintest, numberQ21 of head dips is noted as a parameter of stress-associated anxiety (Agrawal et al., 2011; Brown and Nemes, 2008).Different hole board design used to study the behavioral disordersin rats include 68�68�40 cm3 with equidistance holes in baseboard (Agrawal et al., 2011; Manchanda et al., 2011; Lee et al.,2013); 60�60�35 cm3 (Garabadu and Krishnamurthy, 2014;Masood et al., 2009); 50�50�30 cm3 (Valdés-Cruz et al., 2012)and the hole board is placed 28 cm above the ground on a stand. Incase of mice, the size range include 40�40�15 cm3 (Gupta et al.,2014; Kurhe et al., 2014); 50�50�50 cm3 (Tsuji et al., 2014);31.5�31.5�20.5 cm3 (Mesa-Gresa et al., 2013) and the hole boardis placed 15 cm above the ground on a stand.

3.1.3. Social interaction testThe social interaction is also one of the commonly employed tests

to assess stress-related behavioral changes in which the behavior of amouse with its social partner is observed during a specified test

period (Berardi et al., 2014; Ko et al., 2014; Kumar et al., 2012;Manchanda et al., 2011; Zotti et al., 2013). The social behavior test isperformed in an open-field apparatus. The main principle of this testis based on the free choice by an experimental mouse to spend timean unfamiliar mouse during experimental sessions. During a 10 mintest period, the social behavior parameters of experimental mouse inrelation to unfamiliar mouse in the test arena are observed includingtotal number of contacts, total duration of contacts, total number andduration of other behaviors such as freezing, self-grooming andwalking (Toth et al., 2008). The deficits in social interactive behaviorinclude object interaction (represents interaction with an inanimateobject) such as sniffing and attempt of climbing over box.

3.2. Biochemical parameters

In preclinical studies, the levels of corticosterone and ACTH(product of HPA axis) are taken as biochemical indices of stress.

3.2.1. Plasma corticosteroneSimilar to cortisol in humans as biological marker of stress,

plasma corticosterone levels are taken as an index of stress in non-human animals. During variety of stress exposure, includingelectric foot shock, immobilization, social isolation and restrainstress, significant release of corticosterone from the adrenal glandtakes place in response to rise in plasma ACTH levels (Bhatia et al.,2011; Kumar et al., 2012). The plasma corticosterone levels aretaken as an external marker of central nervous system activity(particularly limbic-hypothalamic activity) because HPA axis iscontrolled by long and short feedback loops operating through thelimbic system (Katz, 1981). However, corticosterone is a goodmarker of stress in rodents only for low to middle intensitystressors because of the ceiling effect of circulating ACTH on theadrenal responsiveness. Repeated exposure of stressors is gener-ally associated with reduced corticosterone levels due to adapta-tion of HPA axis. The reduced corticosterone levels are particularlyevident in response to repeated exposures of low to middleintensity stressors than with middle to high intensity stressors(Chaouloff et al., 1989; De Boer et al., 1990; Kant et al., 1983;Pitman et al., 1988). Plasma corticosterone is generally measuredby radioimmunoassay (RIA) (Lahmame et al., 1996). However,corticosterone has also been estimated with the spectrofluoro-metric method using sulfuric acid which yield fluorescent moietyby forming a complex with the corticosterone (Bhatia et al., 2011;Glick et al., 1964; Mattingly et al., 1989).

3.2.2. Adrenocorticotrophin hormone (ACTH)The plasma ACTH is considered as an important and reliable

marker of stress. There is a direct relationship between stress intensityand ACTH release as ACTH release response is greater in immobiliza-tion stress (more severe stress) as compared to corresponding restrainstress of same duration (less severe). Its major limitation is that theanimals are unable to sustain ACTH secretion after 2 h of continuousexposure of severe stressor such as immobilization suggesting thatprolonged exposure to a severe stressor impairs the capability of thecorticotropes to maintain a sustained ACTH release (Martı´ et al., 1999;Rivier and Vale, 1987). Furthermore, its levels are elevated not onlyduring the stress exposure, but also during post-stress period. Garcıaet al. demonstrated that during post-stress period (45 min after thetermination of stress exposure), the levels of ACTH were approxi-mately 70% of those achieved just after termination of immobilization.Since, the half-life of plasma ACTH is about 10 min in rat (Cooket al., 1972), the elevated levels in post-stress period indicates that therelease of ACTH persists beyond the actual exposure to stress. ACTH isusually determined by a double antibody RIA using 125I-ACTH as the

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tracer, rat synthetic ACTH1–39 as the standard (Orth, 1979; Iwata et al.,2012).

3.2.3. Dissociation of ACTH and glucocorticoidsThough both ACTH and corticosterone are released during stress as

a consequence of HPA axis activation and ACTH stimulates thecorticosterone release, yet, these two hormones are not alwayspositively correlated to one another and there is frequent dissociationbetween these two hormones. The dissociation of ACTH and gluco-corticoids has been described in mental disorders and besides ACTH,the release of adrenal glucocorticoid is modulated by numerous otherfactors including neuropeptides and neurotransmitters (Bornsteinet al., 2008). Furthermore, the release of glucocorticoids from theadrenal gland during the stressful state is under dual control of bothACTH and preganglionic sympathetic neurons. It has been shown thatreward dampens the adrenal glucocorticoid release independent ofACTH. Furthermore, ACTH sensitivity is also modulated by variousfactors released during chronic stress (Amann and Lembeck, 1987;Bornstein et al. 2008).

3.3. Physiological parameters

3.3.1. Food intake and body weightStress disturbs the food intake and it is considered as signifi-

cant physiological change in response to stress in both humansand animalsQ22 (Martı´ et al., 1999; Vallès et al., 2000). The moreintense the stressor and the longer the duration, the greater isreduction in food intake and body weight in the rats (Martı´ et al.,1999; Vallès et al., 2000). Females generally exhibit a largerresponse to emotional stress than males and consequently, greaterreduction in food intake has been noted in female rats (48%reduction in females vs 22% in males) (Kuriyama and Shibasaki,2004).

3.3.2. Adrenal weightThe adrenal gland is an essential stress-responsive organ and is

part of both HPA axis and the sympatho-adrenomedullary systemQ23 .Enlargement of the adrenal cortex (Aguilera et al., 1996; Tharp, 1975;Ulrich-Lai et al., 2006) and increased adrenal medullary size and/orcatecholamine content haveQ24 been demonstrated in different types ofchronic stress (Rubin et al., 1987; Wolman et al., 1993). Chronic stress-induced hypertrophy of the adrenal medulla results from repeatedactivation of the sympathetic nervous system (McCarty et al., 1988).Both cellular hypertrophy and hyperplasia in the zona fasciculatacontribute to chronic stress-induced adrenal growth in a sub-region-specific manner. On the other hand, the cells of the zona glomerulosadecrease in size in response to chronic stress. Elevated plasma levels ofACTH during chronic stress may be responsible for stimulationQ6 of zonafasciculata growth and zona glomerulosa atrophy (Gallo-Payet et al.,1996; Lehoux et al., 1998; Suwa et al., 2000).

3.3.3. Gastric ulcerationStress-induced gastric ulceration is a significant pathophysiological

change observed in response to chronic stress and chronic unpredict-able stress (Yadin and Thomas,1993). Chronic stress-induced excessiverelease of glucocorticoids disturbs the functional homeostasis of thebody and increases the susceptibility/vulnerability to gastric ulcera-tion. The rodent model of cold immobilization stress is a well-definedand clinically relevant experimental model for stress ulceration (Senayand Levine, 1967). Furthermore, in other chronic stress models such aswater immersion stress and restrain stress, gastric ulceration has alsobeen very well described (Guth et al., 1979). Stress stimulus rapidlyactivates the sympatho-adrenomedullary system and reduces theblood flow to the gastric mucosa leading to local hypoxia andischemia. Stress-induced gastric changes are assessed by biochemical

analyses, microscopic and macroscopic examination. For macroscopicexamination, the stomach is rapidly removed and is immersed informalin for 10 min followed by scoring the extent of hemorrhagicerosive lesion in the gastric corpus mucosa and is expressed as theulcer index (Bhatia et al., 2011; Guth et al., 1979).

4. Comparison between electric foot shock, immobilizationand cold stressor

4.1. Intensity, hormonal release and protein changes in brain

Based on the influence of chronic exposure of immobilizationand electric foot shock on the metabolic parameters such as totalfood intake and change in body weight, it has been reported thatimmobilization stress is more severe stressor than electric footshock (Rabasa et al., 2011). Both the stressors are mainly physicalin nature with some involvements of emotional components too.The different type of stressors have different actions on hormonalrelease as Palma and co-workers demonstrated that electric footshock stressor (2 mA, and 0.1 s long, 4–6 shocks/min) produces thehighest rise in plasma levels of ACTH, while 1 h immobilizationand cold stress (4 1C for 1 h)-induced rise in the plasma ACTH iscomparatively less with the order of foot shock4 immobiliza-tion4cold stress. On the other hand, cold stress produces highestrise in corticosterone levels with the order of cold4 immobiliza-tion4 foot shock (Palma et al., 2000). On the contrary, Rabasa andco-workers demonstrated that exposure of immobilization stres-sor for 1 h produces more significant rise in both corticosterone aswell as on ACTH levels as compared to medium (0.5 mA, 1 shockevery 60 s for 1 h) and high intensity (1.5 mA) foot shocks in rats.Furthermore, the post-stress recovery of HPA axis is slower inimmobilization subjected rats as compared to electric foot shocksubjected groups (Rabasa et al., 2011). Maruez and coworkersdemonstrated that electric foot shock stress (1/s; 200 ms; 3 mA for5 min)-induced increase in plasma corticosterone is similar to 2 hof immobilization Q7stress (Maruez et al., 1998).

Santha et al. compared the effect of electric foot shock, restrainstress, forced water immersion stress and psychosocial stress oncytoskeletal (beta-actin) protein translation and expression in ratbrain regions (Santha et al., 2012, 2013). β-actin is a cytoskeletalprotein and its remodeling has been shown to produce synapticdysfunction, which in turn produces behavioral and cognitive impair-ments (Kojima and Shirao, 2007; Piubelli et al., 2011). It was shownthat foot shock and retrain stress produced biphasic (U shaped)changes in beta actin in rat hippocampus region for three weeks. Theamount of β-actin in the hippocampus was increased during the firstweek of the stress, followed by decrease in second week andsubsequently increases in third week. However, there were signifi-cant differences between dynamics and regulatory mechanisms ofβ-actin transcription. In restrain stress (but not in foot shock), theexpression of β-actin regulating proteins, cofilin and MAPK-1 werealso increased suggesting that in restrain stress, dynamics of actinfilaments (actin filament assembly/disassembly) is regulated throughactin-depolymerizing factor/cofilin family and MAPK-1. In waterimmersion stress rats, there was transient rise in β-actin in hippo-campus as well in frontal cortex region during the initial stages ofstress with no changes in cofilin and MAPK-1. In contrast to otherstressors, psychosocial stress failed to induce any changes in cytos-keletal proteins in the hippocampus or frontal cortex (Santha et al.,2012, 2013).

4.2. Adaptation

Rabasa et al. suggested that repeated exposure of all stressors donot lead to development of adaptive response and suggested that

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adaptive process depends upon the particular characteristics of thestressor. It was demonstrated that the repeated exposure to immobi-lization stress (1 h for 7 days) to rats produces significant adaptation ofACTH and corticosterone responses to the stressor. However, the HPAaxis adaptation was only reported with repeated exposure to footshock of medium intensity (0.5 mA) and no such adaptation wasshown in high intensity (1.5 mA) foot shock subjected rats (Rabasaet al., 2011). In contrast, Ohi et al., showed the development ofbehavioral adaptation, in terms of locomotor and freezing behavior,in male rats after repeated foot shocks of 2 mA intensity, 1 s duration,4 s inter-shock interval for 1 h daily for 10 days. The authors ascribedthe development of hypersensitivity of the 5-HT system as theprobable neuronal mechanism of stress adaptation. Moreover, it wasproposed that the 5-HT system may participate in the maintenance ofstress adaptation rather than its development. However, there were nosignificant changes in beta-adrenergic receptor density in the cortexand hippocampus regions of the brain inQ8 that study (Ohi et al., 1988).Earlier, Cohen et al. also demonstrated no changes in the beta-adrenergic receptor density in the cortex and hypothalamus regionsafter 7 days (1 h/day) of foot shock stress (0.7 mA, for 10 s) (Cohenet al., 1986). It is in contrast to studies showing that the decrease inbeta-adrenergic receptor density is associated with development ofbehavioral adaptation in response to repeated stress exposure (Stoneand Platt, 1982; Stone et al., 1985). These may be possibly due todifferent types of stressors employed in these studies. Stone et al.employed restrain stress to study stress adaptation and beta adrener-gic receptors density, while the Ohi and Cohen employed electric footshock in their studies. Therefore, it may be implied that the biochem-ical changes and associated changes in receptor density within thebrain region may be stressor specific.

Irwin et al. demonstrated the restoration of norepinephrine levelsin the hypothalamus region on repeated foot shock exposure (360shocks of 150 mA, 2 s duration at 9-s intervals on each day) for 15 daysas compared to acute foot shock. Acute inescapable shock-induceddecrease in norepinephrine levels were ascribed to its increasedutilization and restoration of norepinephrine levels after repeatedshock application was due to compensatory increase in synthesis. Theother studies have shown that foot shock exposure increases norepi-nephrine synthesis by activating tyrosine hydroxylase and dopaminebeta hydroxylase enzymes (Kramarcy et al., 1984). The authors did notevaluated behavioral response, but employed brain norepinephrinelevels to indirectly assess the effects of acute and repeated foot shockstress on adaptation (Irwin et al., 1986). In fact, there have beennumber of studies suggesting that reduced norepinephrine concen-trations in the brain render the organism less well prepared to dealwith aversive stimuli and accordingly, reduced norepinephrine isassociated with development of behavioral disturbances (Anismanet al., 1980; Weiss et al., 1981). An earlier study by Weiss et al. alsodemonstrated the restoration of acute inescapable shock-inducedimpairment in shuttle avoidance-escape task performance onrepeated application of shocks for 14 days. Moreover, repeatedapplication of shocks also elevated tyrosine hydroxylase activity andrestored acute shock-induced decrease in norepinephrine in thehypothalamus and cortex region suggesting the development of“neurochemical habituation” in response to repeated shock exposure(Weiss et al., 1975).

4.3. Sleep pattern

Immobilization stress elicits the profound changes in thesleep architecture by increasing the rapid eye movement (REM)stage of sleep (Vazquez-Palacios and Velazquez-Moctezuma,2000). It has been reported that animals submitted to 1 h ofimmobilization during the resting period demonstrate arebound increase in paradoxical sleep (REM phase of sleep)(42–65%) and slow wave sleep (15–17%) (Palma et al., 2000).

However, immobilization stress of 2 h has been shown toincrease the paradoxical sleep (32%) without altering the slowwave sleep. Furthermore, longer period of immobilization (morethan 2 h) do not prolong REM sleep increasing effect (Rampin etal., 1991). The exposure of cold stressor (4 1C for 1 h) was shownto decrease the total alertness time (TAT) and increase the totalsleep time (TST) with rebound increase in slow wave sleep (14%)(Palma et al., 2000). Palma et al. (2000) delivered the electricfoot shock stressor in Wistar rats for 1 h with four to six shocksof 2 mA intensity and 0.1 s duration with a variable inter-shockinterval to study the effects of stress on the sleep pattern andreported the prolonged state of alertness in these stress sub-jected rats. In contrast, the delivery of electric foot shocks wasnot associated with any increase in paradoxical sleep or slowwave sleep. On the other hand, the delivery of electric shockswas shown to produce a prolonged state of alertness during theanimal's resting period assessed in terms of rise in total alert-ness time and decrease in total sleep time (Palma et al., 2000).Other studies have also shown the similar reports in whichdelivery of electric foot shock has been associated with increasein alertness without increase in paradoxical sleep. Vazquez-Palacios and Velazquez-Moctezuma (2000) demonstrated thatelectric foot-shocks (at intensity of 3 mA, 1/s; 200 ms; for 5 min)produces only slight and transient effect on the sleep-wakefulness cycle, in terms of increase in the number ofawakenings before the first REM sleep bout and lengthening oflatencies to SWS and REM sleep. Palma et al. (2000) attributedelectric foot shock-induced increase in alertness or wakingresponse to increase in plasma ACTH levels. Studies have shownthe alertness-inducing effects of Q25ACTH as its exogenous admin-istration is shown to increase the waking response (Chang andOpp, 1998). Alternatively, the activation of dopaminergic centralsystems following acute sessions of foot-shock may also beresponsible for alertness/wakefulness, as the neuromodulatoryrole Q26of dopamine in wakefulness/alertness has also been welldocumented (Monti, 1982; Trampus et al., 1993). On the otherhand, the increased paradoxical sleep in response to immobili-zation stress has been mainly attributed to an increased corti-cotrophin release hormone and decreased norepinephrine fromthe locus coeruleus (González andValatx, 1997).

Cui and co-workers have described differential effect of physical(foot-shock) and psychological (non-foot shock) on the sleep patternsof rats. Exposure of foot shocks of intensity 2 mA lasting for 10 s atintervals of 60 s for 1 h significantly inhibit total sleep duration, bothREM and non-REM stages, until 5 h after stress as compared to controlgroup; however psychological stress group significant increase in totalREM sleep which persisted for 4 h without any effect on non-REMsleep. The inhibition of total REM sleep by foot shock may be relatedto total number of REM episodes in the 6 h of sleep recording after thestress as reported earlier in previous findings. Other reports have alsodocumented that serotonin and dopamine were particularly increasedby physical stress and were related to inhibition of REM sleep.Moreover plasma corticosterone levels were increased significantlyafter foot shock stress exposure but not in non-foot shock group. Thus,it was suggested that sleep pattern differentially effect the sleeppatterns as in physical stress group these changes were related toHPA axis and some neurotransmitters whereas sleep patterns inpsychological stress group may not be related more to non-HPA axisfactors may participated in sleep regulation in the present physicalstress group (Cui et al., 2007).

Future prospective

The most commonly employed preclinical stress modelsincluding immobilization, restraint and foot shock are primarily

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physical stressors with some components of psychological stress.In contrast, humans experience primarily psychological stress;accordingly, these stressors do not mimic the human stressors.Therefore, there is a need of development of preclinical modelswhich simulate the human stress conditions. Furthermore, there islarge inconsistency in the procedures of stress induction particu-larly in foot shock stress models and there is a need to standardizethese models with respect to intensity of foot shocks and degree ofstress response. Another area of research in preclinical stressmodels is comparison of neurochemical, neuroendocrinologicaland behavioral responses to these commonly employed stressors.

Conclusion

Stress induction and subsequent stress assessment is of para-mount importance in stress-based studies for exploring the newtherapeutic intervention or understanding the pathophysiologicalmechanism. Most frequently employed preclinical models toinduce the response include immobilization stress, restrain stress,electric foot shock stress and social isolation stress. Stress assess-ment in animals is done at various levels i.e. physiological (foodintake), biochemical (corticosterone, ACTH) and behavioral level.These stressors act differently and changes due to these stressorsvary in terms of hormonal release, protein changes in brain,adaptation and sleep pattern.

Uncited referencesQ2

Bali and Jaggi (2014), Chiba et al. (2012), Hagbarth et al. (1972),Kosten et al. (2000), Nishimon et al. (1983), Quas et al. (2014),Wolfram et al. (2011), Kumar and Singh (Jaggi).

Acknowledgment

The authors are gratefulQ27 to the Department of PharmaceuticalSciences and Drug Research, Punjabi University, Patiala, India forsupporting this study and providing technical facilities forthe work.

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Preclinical experimental stress studies: Protocols, assessment and comparison