MODEL TO STUDY THE STREES ACTIVITY BOTH IN-VIVO AND IN-VITRO

1. 1
Screening of anti stress agents
By,
Mayur Patil.
Dattatrya Sirsat,
Manik Bainwad,
Mohanlal,

2. 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.
2

3. 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
3

4. 4
Different Physiological And Pathological Changes In The Body In Response To Stressor

5. Drugs used for treatment of stress
Antianxiety drugs
Benzodiazepine, Buspirone,
Antidepressants
Duloxetine, Escitalopram, Fluoxetine
Beta blockers
Propranolol, Atenolol
5

6.  Immobilization/restrain-induced stress
 Cold-water restraint stress
 Forced swimming induced stress
 Anoxic stress tolerance test
 Food-deprived activity stress
 Immersion in cold water
6
 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 agents
Physical stress models Psychological stress models

7. 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.
7

8. 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.
8

9.  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
drug
 The 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.
9
Tail Suspension Test (TST):

10. Immobilization/restrain-induced stress
When 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.
Procedure
Animals
Albino mice (20-25 g) of either sex are used
Mice 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
10

11. All the treatment continuously given for 12 days
On 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.
11

12.  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
12

13.  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
13

14. 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
14

15. Procedure
 Animals
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.
15

16. 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
16

17.  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.
17
Continue…

18. Psychological stress models
18

19. 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.
19

20. Procedure
 Chronic 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
20

21. 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.
21

22. 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 .
22
Assessment of Memory and Retention by Object Recognition Test

23.  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.
23

24. LEARNED “HELPLESSNESS” TEST
24
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.

25. 25
 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.

26. 26
 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

27. 27
Thank you…