Protocol No.

APPLICATION FOR PERMISSION FOR ANIMAL EXPERIMENTS

SUBMITTED TO

INSTITUTIONAL ANIMAL ETHICS COMMITTEE (IAEC)

ON

Date, 2022

BY

University Address

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 Form B (per rule 8(a)* for Submission of Research Protocol (s) Application for
Permission for Animal Experiments
Application to be submitted to the CPCSEA, New Delhi after approval of Institutional Animal Ethics
Committee (IAEC)

Section -I

Siddhartha Institute of Pharmacy, Dobachi near IT

1. Name and address of establishment Park Sahastradhara Road, Dehradun Uttrakhand India,
248001

Regd. No. 1435/PO/Re/S/11/CPCSEA Date:

2. Registration number and date of registration. 20/04/2011

Shri Guru Ram Rai Institute of Medical & Health

Name, address and registration number of Sciences, Dehradun, Uttrakhand India,
3. breeder from which animals acquired (or to be 248001
acquired) for experiments mentioned in parts
B&C

Siddhartha Institute of Pharmacy, Dobachi near IT

Place where the animals are presently kept (or Park Sahastradhara Road, Dehradun
4. proposed to be kept). Uttrakhand India, 248001

Siddhartha Institute of Pharmacy, Dobachi near IT

Place where the experiment is to be performed Park Sahastradhara Road, Dehradun
5. (Please provide CPCSEA Reg. Number) Uttrakhand India, 248001
Regd. No. 1435/PO/Re/S/11/CPCSEA

Date of commencement (Tentative): Jan 2023

6. Date and Duration of experiment. Duration (Tentative): 6 months

Educational Research
Type of research involved (Basic
7. Research / Educational/ Regulatory/
Contract Research)

Signature
Name and Designation of Investigator
Dr. Balvinder Singh
Professor
Department of Pharmaceutical Sciences, BMU,
Rohtak
Date: 23/12/2022

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Place: Dehradun

Section -II

Protocol form for research proposals to be submitted to the Institutional Animal Ethics
Committee/ CPCSEA, for new experiments or extensions of ongoing experiments using animals.

1. Project / Dissertation / Thesis Title: Investigation of anti-depressant activity of NSAIDs in mice.

2. Principal Investigator / Research Guide / Advisor:

a. Name:
b. Designation:
c. Dept / Div/ Lab:
d. Telephone No.:
e. E-mail Id:
f. Experience in Lab animal experimentation:

3. List of all individuals authorized to conduct procedures under this proposal.

(i)
a. Name:
b. Designation:
c. Department:
d. Telephone No. :
e. E-mail Id:
f. Experience in Lab animal experimentation:
(ii)
a. Name:
b. Designation:
c. Department:
d. Telephone No.:
e. E-mail Id:
f. Experience in Lab animal experimentation:

4. Funding Source / Proposed Funding Source with complete address (Please attach the proof)

5. Duration of the animal experiment: 1 Year

a. Date of initiation (Proposed):

b. Date of completion (Proposed):

6. Describe details of study plan to justify the use of animals (Enclose Annexure-i)
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 S. Grou No. of Treatm Dose Route Observation
N p Animal ent of /duration
O. name s (n) Admini
stration
1. Contro 6 Vehicle 1-1.5 Oral 21 days
l (Normal ml
Saline)
2. Standa 6 Chronic - - 21 days
rd mild
stress
(CMS)
3. Standa 6 Venlafaxi 10 Oral 21 days
rd drug ne mg/kg
4. Test 1 6 Etoricoxi 5 Oral 21 days
b mg/kg
5. Test 2 6 Indometh 5 Oral 21 days
acin mg/kg

7. Animals required

a. Species and Strain: Swiss Albino mice

b. Weight: 25-30 g
c. Gender: Female
d. Number to be used (Year-wise breakups and total figures needed to be given in tabular form):
30
e. Number of days each animal will be housed: 43 days
8. Rationale for animal usage
a. Why is animal usage necessary for these studies?
The model animals manifest depression-like symptoms that basically cover the onset of
depression in humans, and most acute symptoms can be reversed by antidepressants within 3–5
days. Moreover, the correlation between cognitive impairment and behavior caused by autonomic
nervous system dysfunction in rats is obvious, which provides a model for investigating nerve
circuits in the course of depression onset.
b. Whether similar study has been conducted on in vitro models? If yes, describe the
leading points to justify the requirement of animal experiment.
No
c. Why are the particular species selected?
The work related to the present study is reported from world-wide on this species and as well
as due to the availability of species and they also have physiology similar to that of humans.

d. Why is the estimated number of animals essential?

Experiment design is made to utilize the minimal number of animals by which a relevant and
statistically significant result could be obtained, the number suggested here is least possible.

e. Are similar experiments conducted in the past in your establishment?

Yes

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 f. If yes, justify why new experiment is required?
Previous experiments were conducted for other drugs and the present study comprised of
different formulation for treatment of depression.
g. Have similar experiments been conducted by any other organization in same or other in
vivo models? If yes, enclose the reference.
Yes, references are included in below section.
9. Describe the procedures in detail:

a. Describe all invasive and potentially stressful non-invasive procedures that

animals will be subjected to in the course of the experiments)
Chronic mild stress
b. Furnish details of injections schedule Substances:
(a) Doses: Venlafaxine
Sites: Oral
Volumes: 10 mg/kg
Prepared formulation
Etoricoxib and Indomethacin by oral route
Volumes: Dose-5mg/kg (Etoricoxib), Indomethacin (5 mg/kg)

c. Blood withdrawal Details

Volumes: 1 ml
Sites : Marginal vein
d. Radiation (dosage and schedules): N/A
Nature of compound/Broad Classification of drug/NCE: Anti-depressant activity of
NSAIDs
10. Does the protocol prohibit use of anesthetic or analgesic for the conduct of painful
procedures? If yes, justify.
No
11. Will survival surgery be done?
No

If yes, the following to be described.

a. List and describe all surgical procedures (including methods of asepsis)
b. Names, qualifications and experience levels of personnel’s involved.
c. Describe post-operative care
d. Justify if major survival surgery is to be performed more than once on a single animal.

12. Describe post-experimentation procedures.

a. Scope for Reuse : No

b. Rehabilitation (Name and Address, where the animals are proposed to be rehabilitated):
N/A
c. Describe method of Euthanasia (If required in the protocol): Spinal dislocation
d. Method of carcass disposal after euthanasia: N/A

13. Describe animal transportation methods if extra-institutional transport is envisaged.

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 By Vehicle as per the CPCSEA Norms.

Swiss Albino Mice

Maximum No. of Animal per Cage 1-2
Material used in transport Box Metal cardboard, synthetic material
Space per Animal (cm. sq) 80-100
Minimum height of box (cm) 14

14. Use of hazardous agents (use of recombinant DNA-based agents or potential human
pathogens requires documented approval of the Institutional Biosafety Committee (IBC).
For each category, the agents and the biosafety level required, appropriate therapeutic
measures and the mode of disposal of contaminated food, animal wastes and carcasses must
be identified).
If, your project involved use of any of the below mentioned agent, attach copy of the
approval
certificates of the respective agencies: Not required

(a) Radionucleotides (AERB)

No
(b) Microorganisms / Biological infectious Agents (IBSC)
No
(c) Recombinant DNA (RCGM)
No
(d) Any other Hazardous Chemical / Drugs
No

Investigator’s declaration.

1. I certify that the research proposal submitted is not unnecessarily duplicative of

previously reported research.

2. I certify that, I am qualified and have experience in the experimentation on animals.

3. For procedures listed under item 10, I certify that I have reviewed the pertinent
scientific literature and have found no valid alternative to any procedure described
herein which may cause less pain or distress.

4. I will obtain approval from the IAEC/ CPCSEA before initiating any changes in this
study.

5. I certify that performance of experiment will be initiated only upon review and approval
of scientific intent by appropriate expert body (Institutional Scientific Advisory
Committee / funding agency / other body).

6. I certify that I will submit appropriate certification of review and concurrence for
studies mentioned in point 14.

7. I shall maintain all the records as per format (Form D) and submit to Institutional
Animal Ethics Committee (IAEC).
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 8. I certify that, I will not initiate the study before approval from IAEC/ CPCSEA received
in writing. Further, I certify that I will follow the recommendations of IAEC/ CPCSEA.
9. I certify that I will ensure the rehabilitation policies are adopted (wherever required).

Signature

Name of Investigator Date:

Certificate
This is to certify that the project proposal no. ..................................entitled ………………………
……………………… submitted by Dr./ Mr. / Ms. ………………………………………………………….
has been approved/recommended by the IAEC of……………(Organization) in its meeting held
on…………. (date) and ……………… (Number and Species of animals) have been sanctioned under this.

Authorized by Name Signature Date

Chairman: ……………………. ……………… ………………..

Member Secretary: ……………………. ……………… ………………..

Main Nominee of ……………………. ……………… ………………..

CPCSEA:

(Kindly make sure that minutes of the meeting duly signed by all the participants are maintained by
Office)

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 Annexure-1
Investigation of anti-depressant activity of NSAIDs in mice
Study Title

 To explore the anti-depressant activity of NSAIDs in Mice

Objectives
 To study the effect of selected drugs on behavior of animals
 To carry out the biochemical analysis

Swiss Albino mice, Weight: 25-30 gm

Species

Housing Animals are kept in plastic cage in an air-conditioned animal room.

8-24 weeks
Age

Environmental
conditions 22±5˚C temperature and 60±5% humidity

Light cycle 12 h light / 12 h dark cycle

Study Plan The experiment will be carried out as follows:

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STUDY PLAN

S.N Group No. of Treatment Dose Observation

O. name Animals Route of /duration
(n) Administratio
n

1. Control 6 Vehicle (Normal 1-1.5 ml Oral 21 days

Saline)

2. Standard 6 Chronic mild stress - - 21 days

(CMS)

3. Standard 6 Venlafaxine 10 mg/kg Oral 21 days

drug

4. Test 1 6 Etoricoxib 5 mg/kg Oral 21 days

5. Test 2 6 Indomethacin 5mg/kg Oral 21 days

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Annexure-2

Aim- Investigation of anti-depressant activity of NSAIDs in mice

Objectives-
 To explore the anti-depressant activity of NSAIDs in Mice
 To study the effect of selected drugs on behavior of animals
 To carry out the biochemical analysis Requirements-

Plant/Drug required-

o Venlafaxine

o Etoricoxib, Indomethacin

Chemicals required-

● Distilled water
· Dosing schedule-

 After chronic mild stress induction

 Assessment of physical parameters such as body weight, dietary and water intake etc.

Anti-depressant activity procedure-

Material & Method: 

 Material:
 Standard drug (Venlafaxine) and Test Formulation (Etoricoxib and Indomethacin)
Method:
In-Vivo Anti-depressant Activity

Experimental protocol
In the present study, a chronic mild stress model of Swiss albino mice will be used to evaluate the
antidepressant effect of analgesic and anti-inflammatory drugs (Etoricoxib and Indomethacin) in
individual and concomitantly with established antidepressant drug (venlafaxine) using chronic mild
stress model. The whole protocol was as per OECD guidelines [1-2].

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Drug treatments
The dose of Etoricoxib (5mg/kg) p.o. and Indomethacin (5mg/kg) p.o. were selected on the basis of
previous studies [3-4]. The test drugs will be prepared in 0.5% carboxymethyl cellulose solution and
would be administered orally daily using oral gavage. Venlafaxine (10 mg/kg) p.o. via oral gavage
will be used as a reference standard (dissolved in distilled water), and its dose was selected as per the
earlier study [5]. The animals will be divided into six groups.

Chronic mild stress (CMS) procedure

Depression may result from neuronal toxicity brought on by persistent stress. According to a
previously documented approach, the mice will be subjected to mild chronic stress [6-7]. Mice will be
subjected to each type of stress pattern once per day for three weeks. Thirty minutes prior to the
introduction of the stress pattern, the medications will be administered to the appropriate groups.
Chronic stress can cause neuronal toxicity, which can lead to depression. The mice will be given
chronic mild stress in accordance with a previously described procedure [7]. The different stress
patterns will be induced in mice once a day for three weeks. The drugs would be given to the
respective groups 30 min before the induction of the stress pattern. In the first week of CMS, animals
will be immobilised for 2 h on day 1, introduced to empty water bottles on day 2 for 1 h, subjected to
foreign bodies for 24 h on day 3, kept under nocturnal illumination on day 4, tilted cage at 45◦ for 7 h
on day 6, and tail pinch (30 s) on day 7. The stress patterns’ order will be altered throughout weeks
two and three.

Behavioral Parameters
Sucrose preference test

Animals were trained to consume sucrose solution while fasted for two days prior to exposing them to
persistent mild stress. Three days later, after a 23-h fast, the animals were introduced to two bottles,
one containing regular water and the other containing sucrose solution. The baseline percentage of
sucrose solution preference was computed [6]. The test was repeated after 21 days of therapy to
ascertain the impact of therapy on the subjects’ preference for sucrose solution as a percentage, which
will serve as an indicator for depression brought on by stress.

Forced swim test

Forced swim test will be carried out as explained earlier [8]. The mice will be taken to a separate
room and immediately placed in a cylinder (45 cm high, 20 cm diameter) filled to 30 cm depth and
maintained at 25 ± 1°C. Mice will be examined for the duration of 5 minutes. The oral treatments in

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the various groups will be carried out 1 hour prior to forced swim test. Clean water will be used for
each behavioral trial.

Locomotor activity

Using a photoactometer, the horizontal locomotor activity ratings of control and test animals were
recorded for 5 min [6]. Each mouse was maintained in the device for five minutes. If the mouse
engaged in any exploratory behaviour, the light’s beam would interrupt, and the instrument would
automatically record the activity’s duration on its digital recorder. Digital recordings ceased recording
as soon as the animal paused its activities.
Elevated plus maze
Elevated plus maze (EPM) assesses unconditioned anxiety like behaviour in mice. EPM consisted of
two open arms (30×5 cm), two enclosed arms (30×5 cm), and a connecting central platform (5×5 cm).
The maze will be elevated 38.5 cm above the ground. At the beginning of the 5-min session, each
mouse will be placed in the central neutral zone, facing one of the close arms. Percentage time in the
open and central arms and number of head dips over the edge of open arms will be recorded by
experiments. An arm entry will be defined as a mouse having entered an arm of the maze with all four
legs [9].
Tail suspension test (TST)
TST will be performed based on the previous method that the mouse hung 25 cm above the floor by
the tip of the tail (1 cm) tied up to the level. The immobility time will be counted during a test at
period of 6 minutes (prior 1 minute to adapt and recorded the last 5 minute). The mouse may be
considered immovable if it hung passively and absolutely unmoving [10].
Biochemical analysis
Estimation of brain serotonin, noradrenaline and dopamine
The estimation of serotonin, noradrenaline and dopamine rat brain will be carried out according to the
fluorometric method [11]. Brain tissue sample will be homogenized in 10 volumes of cold acidified
N-butanol using a glass homogenizer. Duplicate internal standard tubes will be carried in parallel with
the brains homogenates. After the chemical procedure as specified in the methods section, the
monoamines will be assayed in the aqueous phase. Excitation and emission wavelengths of 295 and
355 nm, respectively, will be used for measurement of serotonin. Noradrenaline fluorescence will be
measured at excitation and emission wavelengths of 380 and 480 nm while dopamine fluorescence
will be measured at excitation and emission wavelengths of 320 and 375 nm in the same sample.
Corticosterone estimation

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For extraction of corticosterone, 0.1–0.2 ml of serum will be treated with 0.2 ml of chloroform:
methanol mixture (2:1, v/v), followed by 3 ml of chloroform. The samples will be vortexes for 30 sec
and centrifuged at 2,000 rpm for 10 minutes. The chloroform extract will be then treated with 0.1 N
NaOH by vortexes rapidly and NaOH layer will be rapidly removed. The samples will be treated with
3 ml of 30N H2SO4 by vortexes vigorously. The tubes containing H2SO4 will be kept in dark for 30-
60 min and thereafter fluorescence measurement will be carried out in fluorescence
spectrophotometer with excitation and emission wavelength set at 472 and 523.2 nm respectively [12-
13].

Data processing and statistical analysis

Results were expressed as group mean ± SEM. All results were analyzed by one-way analysis of
variance (ANOVA), followed by Tukey’s multiple comparison tests. p-Values less than 0.05 were
considered significant. The software programs used for data analyzing and making graphs were Excel
2010 and the GraphPad Prizm 6 [14].

References

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2. Vogel GH: “Drug discovery and evaluation: Pharmacological assays”, Springer Publishers,
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Baggio CH, Vital MA. 2014. Antidepressant and antioxidative effect ofibuprofen in the
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[CrossRef]
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neurotransmission in antidepressant-like activity of Emblica officinalis fruits in mice. CNS
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9. Lister RG. 1990. Ethologically-based animal models of anxiety disorders. Pharmacology &
Therapeutics 46(3):321-340.

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10. Steru L, Chermat R, Thierry B, Simon P. 1985. The tail suspension test: a new method for
screening antidepressants in mice. Psychopharmacology 85(3):367-370.
11. Ciarlone AE. 1978. Further modification of a fluorometric method for analyzing brain amines.
Microchemical Journal 23(1):9-12.
12. Grishma Patel, Sunita Goswami. Antidepressant Effect of Etoricoxib and Ibuprofen on chronic
mild stress-induced depression in mice. Asian Journal of Pharmacy and Pharmacology 2022;
8(2): 36-45.
13. Alsanie, W.F.; Alamri, A.S.; Abdulaziz, O.; Salih, M.M.; Alamri, A.; Asdaq, S.M.B.;
Alhomrani, M.H.; Alhomrani, M. Antidepressant Effect of Crocin in Mice with Chronic Mild
Stress. Molecules 2022, 27, 5462. https://doi.org/10.3390/ molecules27175462.
14. Azadeh Mesripour, Shahrzad Shahnooshi, Valiollah Hajhashemi. Celecoxib, ibuprofen, and
indomethacin alleviate depression-like behavior induced by interferon-alfa in mice. Journal of
Complementary and Integrative Medicine. 2019; 20190016.

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