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Screening of anti stress agents

By,Mayur Patil.Dattatrya Sirsat,Manik Bainwad,Mohanlal,

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What is stress?Stress is a state of threatened homeostasis that produces

different physiological as well as pathological changes depending on severity, type and duration of stress.

The physiological changes associated with stress are mobilization of energy to maintain brain and muscle function; sharpened and focused attention of the perceived threat, enhanced cardiovascular output and respiration.

Prolonged stress play an important role in depression and neurodegenerative disorders.

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Stress begins with a stimulus of external or internal origin that activates the hypothalamic–pituitary–adrenal axis (HPA) and the sympathetic nervous system (SNS)

HPA and SNS activation leads to generation of glucocorticoids and catecholamine

Causes of stress :Stress came from various factors like problems at work, difficult

relationships, worrying about money, or dealing with an ongoing illness. Traumas such as war, illness, or natural disaster may lead to severe stress disorders

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Different Physiological And Pathological Changes In The Body In Response To Stressor

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Drugs used for treatment of stress

Antianxiety drugs Benzodiazepine, Buspirone,

AntidepressantsDuloxetine, Escitalopram, Fluoxetine 

Beta blockersPropranolol, Atenolol

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Immobilization/restrain-induced stress Cold-water restraint stress Forced swimming induced stress Anoxic stress tolerance test Food-deprived activity stress Immersion in cold water

Repeated social defeat stress Neonatal isolation stress Predatory stress Day–night light change-induced stress (sleep deprivation-induced stress) Noise-induced stress Post-traumatic stress disorder

Animal models for screening of anti stress agentsPhysical stress models Psychological stress models

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Forced swimming induced stress

It is the tendency of the living being to escape a noxious condition.

If the animal is not able to escape the stressful stimuli or it feels threatened, the animal will show stress response.

This principle is used for developing forced swimming model for inducing stress in laboratory animals

Procedure Animals Adult albino rats (200- 250g) of either sex The animals are divided into 4 groups of six rats in each group. Group I - received saline served as vehicle control Group II-received saline and stress, served as negative control.

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Group III-received standard drug, diazepam (2 mg/kg, i.p) and stress, served as positive control.

Group IV- treated with test compound and Stress. Treatment is given to rats, once daily for period of 7 days. On 8th day the rats

are subjected to swimming stress by keeping them in tank of dimension (37X37X30 cm), filled with water to a height of 25cm

The endpoint is taken when the animal started drowning and the mean swimming time for each groups is calculated

After induction of stress, blood is collected, serum is separated and biochemical parameters like serum glucose, triglycerides, cholesterol, BUN(Blood,Urea,Nitrogen), cortisol and blood cell count are estimated.

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All the rats of either sex were divided in six different groups. The first group assigned as control receiving only vehicle (Nacl

5ml/kg). The other four groups received acute dose of aqueous, extract of

drugThe sixth group received standard drug (30mg/kg).The total duration of immobility induced by tail suspension was

measured by placing the mice were suspended 50cm above the floor by adhesive tape placed approximately 1cm from the tip of tail.

Immobility time was recorded during a 6min period. Mice were considered immobile only when they hung passively and

were motionless.

Tail Suspension Test (TST):

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Immobilization/restrain-induced stressWhen the animals are kept with its limbs stretched on a board and

immobilized, it produces stress in animals and increase in concentrations of blood constituents like glucose, cholesterol, triglycerides, blood urea nitrogen (BUN), and plasma cortisole.

ProcedureAnimals Albino mice (20-25 g) of either sex are usedMice are randomly divided into 4 group of 6 animals in each.Group 1 and 2 receives distilled water, group 3 test compound and group 4

receives standard drug

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All the treatment continuously given for 12 daysOn 12 one hour after treatment forelimb and hind limb of the mice are tied

by adhesive tape there by immobilizing them.After 2 hour tapes are removed blood is collected and mice are sacrified,

brain adrenal gland and spleen are removed. Blood used for estimation of glucose, cholesterol, triglyceride, cortisone etc,

and compared with standard.

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In this method, the rats are placed individually in a tank (25 35 40 cm) of cold water (depth 15.5 cm; temperature 15–20C) for 15 min, where they either swim or remain in an upright position, keeping their heads above the water level .

Immersion in cold water (ICW)

A modification has been made in the method by subjecting the animals to coldwater immersion stress for 5 min at 4c and this situation lasts for 15 min unless the rats sink.

In that event, rats are removed before the cut-off time and are not included in the experiments.

Stressors are applied, both acutely (5–15 min) and chronically (during 4, 12 and 20 days) at the onset of the light phase as well as at the onset of the dark phase of the light/dark cycle .

Procedure

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Immersion in cold water elicits a clear increase in plasmatic corticosterone levels , regardless of the time cycle of the stressors.

For acute stress, rats are killed 30 min after the stress exposure. For chronic stress, animals are exposed to this stressor for 7–10 days and

thereafter, the rats are killed 1 h after the last stress session.

The major advantage of this type of stressor is that acute stress can be achieved in a relatively short period of time .

However, the major drawback of this model is that the body adapts to change in temperature on chronic exposure to low temperature and hence, stress response gets highly diminished with repeated episodes of stress .

Evaluation

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Cold-water restraint stress

It is combination of both immobilization and cold stress.

When animal is immobilized and subjected to cold condition it produces more stress in animal than either of the two method

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ProcedureAnimals

Albino rats of wistar strain (weighing 200-250 g) of either sex.Stress is induced by subjecting the animals to cold restrain stress by immobilizing

them at 4C for 4 hours daily in cylindrical cage over a period of 7 days. Standard(Diazepam) and test drugs administered to the respective groups  for  all  7days.   Animals  are  sacrificed  on  the day 7 using  ether.  Blood is withdrawn and serum is separated to study various biochemical

parameters like  Glucose  (GLU),  Triglycerides  (TG),  Cholesterol  (CHOL), and corticosterone.Adrenal glands are removed  aseptically and  relative adrenal gland weight is

measured and compared with standard.

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Anoxic stress tolerance test Stress Can be produced in animal by subjecting them to anoxia, when animals are

placed in airtight container they show typical convulsions due to stress.

Procedure Swiss albino mice (25 ± 2 g) of either sex are used. Animals are divided into 4 groups of six animals in each group Group 1 receive vehicle serves as negative control Group 2 receive vehicle and stressed serves as positive control Group 3 receive standard drug Group 4 receive test compound

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Conical flasks of 250 mL capacity are used for the study. These flasks are made airtight using rubber cork before beginning the experiment On day 14th and 21st, 1 hour after the treatment, each animal is placed in the airtight

vessel and time is observed using a stopwatch. The moment animal showed first convulsion, it is removed immediately from the

vessel. The time duration from the entry of the animal in the hermetic (conical flask) vessel

to the appearance of the first convulsion is taken as the time of “Anoxic stress tolerance”.

The data obtained are subjected to statistical analysis.

Continue…

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Psychological stress models

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Noise-induced stress

Noise exposure of any kind above 90 dB, is a stressor.Noise stress in laboratory rats can be produced by loudspeakers (15 W), driven

by a white noise generator (0–26 kHz), installed 30 cm above the cage.A noise level can be set at 100 dB or above uniformly throughout the cage and

can be monitored using a sound level meter.Noise stress has a depletory effect on free radical scavenging enzymes in the

brain leading to moderate to severe oxidative stress.Noise stress alters the biogenic amine levels in brain.

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ProcedureChronic model-Each animal to be treated is exposed to noise stress for 4 h/day

for 15 days.An acute model -exposure of rats to noise stressor of 10 kHz, 100 dB stress for

30 min.

Control group rats are kept in the cage during the corresponding period of time,

without noise stimulation to avoid the influence of handling stress on evaluation

of effects due to noise exposure.

The effect of noise stress exposure can be determined by estimating the brain

biogenic amine level

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Day–night light change-induced stress(sleep deprivation-induced stress)

Changes in the circadian rhythm have profound effect on physical and psychological well being of an individual.

Changes in circadian rhythms are regulated by pineal gland through the secretion of melatonin.

Melatonin is released from the pineal gland in response to dark or dim light whereas its functional antagonist serotonin is secreted in response to bright light.

The serotonin–melatonin cycle is responsible for regulation of sleep–awake state of the body.

There is an increase in the hypothalamic and thalamic oxidative stress level following sleep deprivation , which in turn is responsible for the cognitive impairment.

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Procedure Inbred Swiss albino male mice (20-25 gm).Female mice are not considered because their changes in the concentration of

estrogen and progesterone may influence in the cognitive behavior of the animal.On the 1st day of the experiment, the animals are divided randomly into two

groups of six animals in each. Group I: Normal control place in normal laboratory condition. Group II: Subjected for 5 days sleep deprivation and they receive normal food

and water.The objects to be discriminated were placed at diagonally opposite corners of the

wooden box (70 X 60 X 30 cm) and were in two different shapes: pyramid side and cylinder .

Assessment of Memory and Retention by Object Recognition Test

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On day 0, animals were allowed to explore the box without any object for 2 minutes.

On first trail (T1), two identical objects were presented in two opposite corners of the box, and the time taken by each mouse to complete 20 seconds exploration was measured.

Exploration meant directing the nose at a distance less than 2 cm to an object

and / or touching with the nose.

During the second trail (T2, 90 minutes after T1), a new object replaced one of

the objects present in T1, and mice were left in the box for 5 minutes.

The time spent for exploring new (N) and familiar (F) objects were recorded

separately.

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LEARNED “HELPLESSNESS” TEST

PURPOSE AND RATIONALE Animal is exposed to inescapable & unavoidable electric shock in one

situation later fail to escape shock in a different situation when escape is possible

METHOD Male Sprague –Drawly rats(300g) is used. Learned helplessness is produced by exposure to electric shock 0.7mA; 10s of

shock/min repeatedly given for 1 hr. A box with grid floor is taken. At 20cm height above the floor ,a platform can be inserted through one side

wall to allow jump up escape.

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The platform is not available during training At the beginning of trial, after the appropriate treatment with the drug, the platform is pushed in the box and a 0.4mA shock initiated.

If an escape response occur, animal is allowed to be on the platform for 10 sec and then returned to the grid floor.

10 trials with an interval for 20 sec are given.

EVALUATION: A drug is considered to be effective if the animal helplessness is

reduced and the number of failures to escape is decreased.

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A review on animal models for screening potential anti-stress agents Amteshwar Singh Jaggi, Nitish Bhatia, Naresh Kumar,Nirmal Singh, Preet Anand ,Ravi Dhawan.

Reference

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Thank you…