1

Association between central neurotransmitters and inflammatory

mediators in depressive state- An experimental study

Ph.D. Synopsis

Submitted To

Gujarat Technological University

For The Degree

Of

Doctor of Philosophy

in

Pharmacy

By

Mr. Shailendra Bhatt

Batch: 2011

Enrollment No: 119997290049

Supervisor Dr. Sunita Goswami

Associate Professor

Dept.of Pharmacology

L. M. College of Pharmacy

2

Title of the thesis

Association between central neurotransmitters and inflammatory mediators in depressive

state- An experimental study.

Abstract

In the present study, a preliminary behavioural study using single dose of anti-inflammatory

and antidepressant was done in SD rats and albino mice. The drugs used were aspirin (10

mg/kg, p.o.) dexamethasone (1mg/kg p.o.) and amitriptyline (10 mg/kg p.o., reference

standard) respectively. Amitriptyline was also used in combination with aspirin and

dexamethasone to inspect any synergistic effects. Tests performed were FST in rats and TST,

EPM and Light dark box. This was followed by studies using the above drugs in interferon -

α- 2b model (21 days) of depression and chronic mild stress (CMS) model (28 days) in male

Sprague–Dawley rats. Tests performed towards the end of the studies included Sucrose

preference test, behavioural tests like forced swim test, elevated plus maze, light dark box,

locomotor activity, and biochemical estimations like serum cortisol and brain

neurotransmitters. Disease control group (CMS treated) and interferon produced significant

depressive behaviour in rats. The animals treated with aspirin showed increased sucrose

preference, decreased immobility time in forced swim test, decreased serum cortisol and

increased brain monoamines levels signifying antidepressant action in both the models. There

was aggravation of depressive behaviour in rats treated with dexamethasone in contrast to the

single dose study in which dexamethasone significant antidepressant behaviour. Together,

these findings suggest that aspirin can serve as a potential antidepressant both individually

and as adjunctive agent in the treatment of depression. Inhibition of the inflammatory

mediators during stress procedures or any other potential physiological and biochemical

mechanisms may be involved in its antidepressant effect.

Brief description on the state of the art of the research topic:

Major depression is one of the most commonly diagnosed neuropsychiatric disorders, with a

worldwide lifetime prevalence of 17%. It is estimated that by 2020 major depression will be

second most disabling condition in the world 1,2. Historically, treatment options for

depression and associated disorders have focused on the medications that modify the activity

of monoamine neurotransmitter systems 3.

Several studies support the role of inflammation and immune system deregulation in

pathophysiology of depression 4, 5. Patients with major depression have been found to exhibit

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all of the vital features of inflammation, including elevation of inflammatory cytokines, acute

phase proteins, chemokines, adhesion molecules, and inflammatory mediators such as

prostaglandins. Repeated administration of IL-1, TNF-α and IL-6 elicits depressive like

behaviour in animals 6-8. The mechanisms contributing towards pathogenesis of depression

incorporates abnormal neurotransmitter metabolism, altered neuro-endocrine functions and

distorted neural plasticity 9, 10.

Studies show that systemic administration of IL-1, IL-2 and IL-6 causes pronounced effect on

dopamine and serotonin activity in the brain 11, 12. At very low concentrations, IL-2 increases

dopamine release, but inhibits dopamine release at high concentration 13. Low level of

dopamine in brain is associated with depression. The cytokines also alter norepinephrine

activity in the locus coeruleus, hippocampus and the hypothalamus leading to depressive

symptoms 14, 15. Elevated level of cytokines activate HPA axis and cause activation of

hypothalamus corticotropin releasing Factor (CRF). Pituitary gland release ACTH (Adreno-

corticotropin-releasing hormone) that stimulate adrenal cortex and release cortisol in blood 16,

17. In normal conditions, high level of ACTH exerts negative feedback control on release of

CRF however, there is failure of suppression of cortisol secretion in the patients with major

depression 18, 19.

Accumulating evidence suggests that the pro-inflammatory cytokines TNF-α, IL-1, and IL-6,

as well as interferons and their receptors, are constitutively expressed in various brain

regions. The inflammatory cytokines have a significant role in regulating synaptic plasticity

in major depressive disorder in rodent models of depression 20. Increased IL-1, IL-6 and TNF

activity in brain is associated with reduced hippocampus neurogenesis 21.

Most of the antidepressants used clinically have anti-inflammatory properties. Selective

serotonin reuptake inhibitors (SSRI) Like fluoxetine and citalopram, tricyclic

antidepressants (TCA) like amitryptilline and desipramine 22,23, mono amine oxidase -B

inhibitor like moclobamide 24 and hyperforin (chemical constituent from herbal plant St

John’s wart) 25, and other herbal antidepressants have been reported to possess anti-

inflammatory properties 26,27. Conversely, there has been use of antidepressant amitriptyline

in therapy of chronic tension headache 28. Recently, it was shown that Aspirin when added to

fluoxetine led to improvement in treatment resistant depressive rats 29. Further, use of

selective COX -2 inhibitor Celecoxib enhanced the effect of reboxetine and fluoxetine on

cortical noradrenaline and serotonin output in rats 30.

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Definition of the Problem:

Given the above-described distinct pathologic signatures of inflammatory mechanism

in depression, it is reasonable to assume that use of anti-inflammatory drugs may have a

different impact on the treatment of depression. In this context, the aim of the present study

was to investigate the effects of aspirin, dexamethasone and the prototypical antidepressant,

amitriptyline, in single dose studies in albino mice and SD rats and with repeated treatments

in the Interferon model and CMS model of depression in SD rats.

Objective and Scope of work:

The primary objective of our study is to investigate the antidepressant activity of anti

inflammatory drugs using experimental models of depression.

The secondary objective is to study association between central neurotransmitters and

inflammatory mediators in depressive state.

Only 50 percent of the currently used antidepressants are clinically effective when compared

with the effectiveness of placebo (30%). In this context, the use of the anti-inflammatory

drugs in the treatment of depression can be an optimum and effective strategy.

Original contribution by the thesis:

The work is original in terms of use of aspirin and dexamethasone in the current experimental

models of depression. The studies conducted have been published in peer reviewed journals.

Methodology of Research, Results / Comparisons

Study protocol:

The study was divided into four phases in which the first two phases focused on single dose

effect and the last 2 phases focused on effects on the established animal model of depression

like interferon induced and CMS induced model of depression.

Study 1 – Preliminary single dose study using Diclofenac, Dexamethasone and Amitriptyline

in SD rats with FST.

Study 2- Single dose study using Aspirin, Dexamethasone and Amitriptyline in albino mice

and tests performed included TST, EPM and LD box.

Study 3:-

Study using interferon - α- 2b model of depression in Sprague Dawley rats. The animals in

different groups were treated for 21 days with aspirin (10 mg/kg, po) dexamethasone (1mg/kg

5

po) and amitriptyline (10 mg/kg po). Amitriptyline was used as reference standard, and was

also used in combination with aspirin and dexamethasone to examine any synergy.

Interferon-α-2b (6000 I.U./ k.g, ip) was administered in above groups daily, except normal

control. Tests performed included Sucrose preference test, behavioural tests like forced swim

test, elevated plus maze, light dark box and locomotor activity and biochemical estimations

like serum cortisol and brain neurotransmitters.

Study 4:

Study using chronic mild stress (CMS) model of male Sprague–Dawley rats. All the animals

in different groups, except the normal control group were exposed to CMS procedure for 28

days and concurrently treated with aspirin (10 mg/kg, p.o.) dexamethasone (1mg/kg p.o.) and

amitriptyline (10 mg/kg p.o., reference standard) respectively. Amitriptyline was also used in

combination with aspirin and dexamethasone to inspect any synergistic effects. Tests

performed towards the end of the study included Sucrose preference test, behavioural tests

like forced swim test, elevated plus maze, light dark box, locomotor activity, and biochemical

estimations like serum cortisol and brain neurotransmitters.

All experiments were conducted after the approval from Institutional Animal Care Ethical

Committee. (Protocol number-LJIP/IAEC/12-13/75).

Material and methods

Study 1:

1a: Animals: Male Sprague-Dawley rats (200–250g) and albino mice (30-35 g) were

obtained from the Department of Laboratory Animal Science, Zydus Research Centre

(Ahmedabad, India).

1b: Pharmacological treatments and experimental protocol: The animals were divided into six

groups (6 in each group) and were treated with drugs specified in Table 1, which included

Diclofenac sodium 31, dexamethasone 32, 33 and amitriptyline 34 were given orally one hour

before conducting behavioural tests.

Table 1: Treatment of drugs and their doses in different groups.

Group 1 Normal Saline

Group 2 Amitryptilline 10 mg/kg p.o.

Group 3 Dexamethasone 1 mg/kg p.o.

Group 4 Diclofenac sodium 10 mg/kg p.o.

Group 5 Amitryptilline Dexamethasone

10 mg/kg p.o. 1mg/kg p.o.

Group 6 Amitryptilline

Diclofenac

10 mg/kg p.o.

10 mg/kg p.o.

6

Results

Study 1 Preliminary study: Single dose study: Forced swim test.

Figure 1.1: Immobility time after single dose treatment in various groups as observed in forced swim test. Bar diagram represents

total immobility time out of 300 seconds swim test. Results are represented as mean + SEM with n=6 rats in each group, # p˂0.05

when compared with the Amitriptyline treated group. * p < 0.05 ,** p< 0.01 when compared with the normal group.

Study 2:

Male albino mice (30-35 g) were obtained from the Department of Laboratory Animal

Science, Zydus Research Centre (Ahmedabad, India). They were housed under standard

conditions (23_1 °C; relative humidity, 55 ± 5%) and ad libitum food and water. They were

allowed to acclimatize to the colony for at least 7days before any experimentation. The

animals were divided into six groups (6 in each group) and were treated with drugs specified

in Table 2, which included Aspirin 36 dexamethasone and amitriptyline were given orally one

hour before conducting behavioural tests.31-34, 36

Table 2: Treatment of drugs and their doses in different groups.

Group 1 Normal Saline

Group 2 Amitriptyline 10 mg/kg p.o.

Group 3 Dexamethasone 1 mg/kg p.o.

Group 4 Aspirin 10 mg/kg p.o.

Group 5 Amitriptyline

Dexamethasone

10 mg/kg p.o.

1mg/kg p.o.

Group 6 Amitryptilline

Aspirin

10 mg/kg p.o.

10 mg/kg p.o.

2a: Tail suspension test

The total time of immobility induced by tail suspension was measured according to method

described in previous studies [37].

2b: Light/dark box - choice paradigm

The light/dark test is based on the initial model described in 38.

2c: Elevated plus-maze

The Elevated Plus-Maze apparatus as described in 39 was used for the study.

7

Results:

Effect of single dose treatment on immobility time

T a i l s u s p e n s i o n t e s t

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

N o r m a l

A m i t r y p t i l l i n e

D e x a m e t h a s o n e

A s p i r i n

A m i+ D e x a

A m i + A s p i r i n

*

* *

#

T r e a t m e n t G r o u p s

Imm

ob

ilit

y t

ime

(s

ec

)

*

Fig.2 Immobility time on single dose treatment as observed in Tail suspension test. Bar diagram represents total immobility time out of 300

seconds of Tail suspension test. Results are represented as mean + SEM with n=6 mice in each group, * p< 0.05, ** p< 0.01, when compared with the normal group. # p˂0.05 when compared with Amitriptyline treated group.

Effect of single dose treatment on number of transitions

L i g h t D a r k B o x

0

5

1 0

1 5

2 0

N o r m a l

A m i t r y p t i l l i n e

D e x a m e t h a s o n e

A s p i r i n

A m i+ D e x a

A m i + A s p i r i n

*

T r e a t m e n t G r o u p s

Nu

mb

er

of T

ra

ns

itio

ns

#

Fig 3. Number of transitions observed in light dark box after single dose treatment s in different groups. Bar diagram represents total number

of transitions in the light dark box. Results are represented as mean + SEM with n=6 mice in each group, * p˂0.05 when compared with the normal group. # p < 00.05 when compared with the aspirin treated group.

Effect of single dose treatment on time spent in open arm

E l e v a t e d p l u s m a z e

0

2 0

4 0

6 0

8 0

N o r m a l

A m i t r y p t i l l i n e

D e x a m e t h a s o n e

A s p i r i n

A m i+ D e x a

A m i + A s p i r i n

T r e a t m e n t G r o u p s

Tim

e s

pe

nt i

n o

pe

n a

rm

(s

ec

)

Figure 2.3: Time spent in open arm observed after single dose treatment in various groups. Bar diagram represents total time spent in

seconds in open arm of 300 seconds duration. Results are represented as mean + SEM with n=6 mice in each group.

Study 3: Interferon model of depression

3a: Animals and experimental methology

Healthy Male Sprague Dawley Rats (200- 250 grams) were divided into seven groups

(6 in each group) and were treated with approximately 6000 international units (I.U.)/kg 40

reliferon (Human recombinant interferon alpha-α 2b, Reliance life science ) ip and normal

8

control rats were treated with vehicle 0.2 ml (0.9% w/v NaCl) ip once daily for 21 days.

Other drug treatments were given orally half an hour before interferon treatments (Table 3)

After 14 days of treatment; sucrose preference test was done to measure anhedonia.

Behavioral tests were performed on 18th, 19th 20th day and observations were video

recorded by use of the Video-Track system. On 21st day, the blood samples were collected

for cortisol measurements and the rats were sacrificed to collect brain for neurotransmitter

study.

Table 3: Treatment of drugs and their doses in different groups in the interferon-α-2b model

of Sprague Dawley rats

Group 1 Normal Saline po

Group 2 Interferon-α-2b 6000 (I.U.)/kg ip

Group 3 Amitryptilline

Interferon-α-2b

10 mg/kg po

6000 (I.U.)/kg ip

Group 4 Dexamethsone Interferon-α-2b

1 mg/kg po 6000 (I.U.)/kg ip

Group 5 Aspirin

Interferon-α-2b

10 mg/kg po

6000 (I.U.)/kg ip

Group 6 Amitryptilline Dexamethasone

Interferon-α-2b

10 mg/kg po 1mg/kg po

6000 (I.U.)/kg ip

Group 7 Amitryptilline Aspirin

Interferon-α-2b

10 mg/kg po 10 mg/kg po

6000 (I.U.)/kg ip

po :per oral, ip: intra peritoneal, I.U. : -α-2b International Units

Study 3:

3a. Sucrose preference test: In order to measure the anhedonia which directly correlates with

the degree of depression, sucrose preference test was performed 41.

3b. Forced swim test: Forced swim test was carried out as explained in 1a.

3c. Light/dark box - choice paradigm : The light/dark test was done as explained in 2b

4c. Elevated plus-maze :The Elevated Plus-Maze test was done as explained in 2c

5c. Locomotor activity: Locomotor activity was done as described in the method. 42

6c. Serum cortisol measurements: Cortisol levels were measured in serum using a cortisol

assay 43 on the Diagnova Elisa reader.

7c. Neurotransmitter estimation: Tissue extracts were prepared by the method of Ciarlone 44

for the estimation of the serotonin, norepinephrine, and dopamine neurotransmitters by

spectroflourimetry method.

8c. Statistical Analysis

Data were expressed as mean + SEM. Analysis was performed with Prism version 3.0

software using one-way analysis of variance (ANOVA) followed by Tukey`s comparison test

where results showing p< 0.05 were considered statistically significant.

9

Results:

Fig. 4: Time spent in open arm observed on 19th day. Bar diagram

represents total time spent in seconds in open arm of 300 seconds.

Results are represented as mean + SEM with n=6 rats in each group

10

STUDY 4:

In the present study, chronic mild stress (CMS) model of male Sprague–Dawley rats was

used to evaluate the antidepressant effect of aspirin and dexamethasone.

Drug treatments

Drug treatments were given every morning between 10 am and 11 am. The doses of aspirin

(10 mg/kg) p.o. and dexamethasone (1 mg/kg) p.o. were selected on the basis of methods

described in earlier studies31-34, 36. Amitriptyline (10 mg/kg) p.o. was used as a reference

standard The animals were divided into 7 groups and the drug treatments were done as per

(table 4).

4.3 CMS procedure

The unpredictable chronic mild stress was applied for 4 weeks. The stress regime used in this

study was a modified version of models previously described in 45-46. The stress sequence

was changed every week in order to make the stress procedure unpredictable.

Sucrose preference test was done at the end of first, second and third week of the study. The

observations of sucrose preference test were utilized as a measure of anhedonia. (Indicator

for depressive behaviour) Behavioral tests were performed on 25th, 26th 27th day and

observations were video recorded by use of the Video-Track system. On 28st day, the blood

11

samples were collected for cortisol measurements and the rats were sacrificed to collect brain

for neurotransmitter study.

Table.4 Treatment of drugs and their doses in different groups in the chronic mild stress

model of rats.

S u c r o s e P r e fe r e n c e

0

2 0

4 0

6 0

8 0

1 0 0

N o rm a l

C M S

C M S + D e x a m e th a s o n e

C M S + A s p ir in

C M S + A m itry p till in e

C M S + A m i + D e x a

#

# # #

***

C M S + A m i + A s p ir in

******

T re a tm e n t G ro u p s

%

of

Su

cro

se

Pre

fere

nc

e # # #

+

Fig. 1: Percentage of sucrose preference on 21st day of CMS exposure. Bar diagram

represents percentage of sucrose preference (%). Results are represented as mean + SEM

with n=6 rats in each group, ***p˂0.001 when compared with the Normal control group.

# p< 0.05, ### p< 0.001 when compared with the CMS group. + p<0.05 when compared

with CMS group.

F o rc e d S w im T e s t

0

5 0

1 0 0

1 5 0

2 0 0

N o rm a l

C M S

C M S + D e x a m e th a s o n e

C M S + A s p ir in

C M S + A m itry p till in e

C M S + A m i + D e x a

# # ## # #

# # #

***

C M S + A m i + A s p ir in

*

T re a tm e n t G ro u p s

Imm

ob

ilit

y t

ime

(se

c)

+

L ig h t D a rk B o x

0

5

1 0

1 5

2 0

N o rm a l

C M S

C M S -D e x a m e th a s o n e

C M S -A S P IR IN

C M S -A M IT R Y P T IL IN E

C M S - A M I+ D E X A

**

C M S - A M I+ A S P IR IN

T re a tm e n t G ro u p s

Nu

mb

er o

f T

ra

ns

itio

ns

**

Fig. 2: Immobility time as observed in forced swim test. Bar diagram represents total immobility time out of 300 seconds swim test. Results

Fig. 3: Number of transitions observed in light dark box. Bar diagram represents total number of transitions in the light dark

Group Stress procedure Drugs/Dose

Group 1 Nil Saline p.o.

Group 2 Chronic mild stress (CMS) Saline p.o.

Group 3 CMS Amitriptyline (10 mg/kg) p.o.

Group 4 CMS Dexamethasone (1 mg/kg) p.o.

Group 5 CMS Aspirin (10 mg/kg) p.o.

Group 6 CMS

Amitriptyline (10 mg/kg) p.o.

Dexamethasone (1mg/kg) p.o.

Group 7 CMS

Amitriptyline (10 mg/kg) p.o.

Aspirin ( 10 mg/kg) p.o.

12

are represented as mean + SEM with n=6 rats in each group.* p< 0.05,

*** p< 0.001 when compared with the normal group. ### p< 0.001, when compared with the CMS group. +p<0.05 when compared with

CMS group

box for four minutes. Results are represented as mean + SEM

with n=6 rats in each group, * p< 0.5, ** p< 0.01 when compared with the normal control group.

E L E V A T E D P L U S M A Z E

0

2 0

4 0

6 0

8 0

N o rm a l

C M S

C M S + D e x a m e th a s o n e

C M S + A s p ir in

C M S + A m itry p t ilin e

C M S + A m i+ D e x a

C M S + A m i+ A s p ir in

T re a tm e n t G ro u p s

Tim

e s

pe

nt

in o

pe

n a

rm(s

ec

)

L o c o m o to r A c tiv ity

0

2 0

4 0

6 0

N o rm a l

C M S

C M S + D e x a m e th a s o n e

C M S + A s p ir in

C M S + A m itry p till in e

C M S + A m i + D e x a

C M S + A m i + A s p ir in

T re a tm e n t G ro u p s

No

. o

f C

uto

ff ** *

Fig. 4: Time spent in open arm observed on 19th day. Bar diagram represents total time spent in seconds in open arm of 300 seconds.

Results are represented as mean + SEM with n=6 rats in each group

Fig. 5: Number of cut-offs observed in photoactometer in various groups. Bar diagram represents number of cut-offs in

photoactometer in five minutes. Results are represented as mean

+ SEM with n=6 rats in each group. *p<0.05 when compared with the normal group.

S e ru m C o r t is o l

0

5

1 0

1 5

2 0

N o rm a l

C M S

C M S + D e x a m e th a s o n e

C M S + A s p ir in

C M S + A m itry p tillin e

C M S + A m i + D e x a

***

## # #

***

C M S + A m i + A s p ir in

# # #

***

T re a tm e n t G ro u p s

se

ru

m c

orti

so

l (n

g/m

l)

+ +

B ra in S e r o to n in

0

2 0 0

4 0 0

6 0 0

N o rm a l

C M S

C M S + D e x a m e th a s o n e

C M S + A s p ir in

C M S + A m itry p tillin e

C M S + A m i + D e x a

C M S + A m i + A s p ir in

**

# # # #

Se

ro

to

nin

(n

g/g

m)

#

Fig. 6: Serum Cortisol levels on 28th day of treatment of CMS. Bar

diagram represents Cortisol in ng/ml. Results are represented as mean

+ SEM with n=6 rats in each group, *** p˂0.001 when compared with the normal group. #p< 0.05, ### p< 0.001 when compared with

the CMS group. ++ p< 0.01 when compared with the CMS group.

Fig. 7: Estimation of Serotonin in brain homogenate on 28th day

of administration of drugs in various groups. Bar diagram

represents serotonin in ng/ gm of brain tissue. Results are represented as mean + SEM with n=6 rats in each group. *p

<0.05 when compared with the normal group. # p<0.05 and ##p<

0.01 when compared with CMS group.

B ra in N o re p in e p h r in e le v e ls

0

2 0 0

4 0 0

6 0 0

**

***

T re a tm e n t G ro u p s

No

re

pin

ep

hrin

e

(n

g/g

m)

***

N o rm a l

C M S

C M S + D e x a m e th a s o n e

C M S + A s p ir in

C M S + A m itry p tillin e

C M S + A m i + D e x a

C M S + A m i + A s p ir in

B ra in D o p a m in e le v e ls

0

2 0 0

4 0 0

6 0 0

N o rm a l

C M S

C M S + D e x a m e th a s o n e

C M S + A s p ir in

C M S + A m itry p tillin e

C M S + A m i + D e x a

C M S + A m i + A s p ir in

**

T re a tm e n t G ro u p s

Do

pa

min

e (

ng

/gm

)

Fig. 8: Estimation of norepinephrine in brain homogenate after 28

days of administration of drugs in various groups. Bar diagram represents norepinephrine in ng/ gm of brain tissue. Results are

represented as mean + SEM with n=6 rats in each group. **p<0.01,

***p<0.001 when compared with the normal group.

Fig. 9: Estimation of Dopamine in brain homogenate after 28

days of administration of drugs in various groups. Bar diagram represents dopamine in ng/gm of brain tissue. Results are

represented as mean + SEM with n=6 rats in each group. *p

<0.05 when compared with the normal group.

Achievements with respect to objectives

In the first phase of studies 1 and 2 it has been observed that after single exposure of

dexamethasone treatment, rats under forced swim test reduced immobility time suggesting

antidepressant behaviour.

Both the forced swim test and tail suspension tests involve a procedure in which the

animals undergo stringent physical stress. During such stress, there is a release of acute

13

mediators of inflammation and activation of lipoprotein lipase pathway and COX pathway

which may be blocked by dexamethasone and aspirin respectively. In this study,

dexamethasone showed decreased immobility time significantly.

Acute effects of dexamethasone include euphoria (47) which may be attributed to

ability of corticosteroid in blocking granular uptake of noradrenaline (48) and and also its

ability to inhibit proinflammatory cytokines. (50) The elevated levels of cortisol initially

produced could be a compensatory mechanism against raised inflammatory mediators seen

during depression. There are also studies indicating antianxiety effect of dexamethasone may

be attributed to its interaction with opoidergic neurons.

Therefore, the data from the studies 1 and 2 suggests that the anti-inflammatory and

analgesic effect of drugs needs to be considered in tests like FST and TST particularly on the

immobility time and number of escape efforts. Additionally the CNS stimulant effects of

corticosteroids need to be carefully evaluated before going for behavioural test like open arm

close arm and mobility behavioiur in the photo-actometer.

In study 3 interferon treatment for 21 days in rats showed an increase (P<.05) in immobility

time in the forced swim test, along with a rise in serum cortisol and serum serotonin levels

and a reduction in sucrose preference test. The study replicated the effects of interferon on

immobility time (50, 51) and sucrose preference test. (52) The visible behavioural changes seen

in the rats treated with interferon like ptosis, piloerection, lethargy and sleep coined as

“sickness behaviour” were replicated as described in a previous study (53) The mechanism by

which IFN-α produces depressive behaviour has been explored in a recent study (54) wherein

IFN-α suppresses neuronal stem cells proliferation,

In study 4 the rats treated with chronic mild stress after 21 days produced increase

in immobility time in forced swim test, serum cortisol levels, and decreased sucrose

preference, monoamines like serotonin. The behavioural changes seen in the rats treated with

CMS were replicated as described in previous studies 55-56. Our study showed parallel results

with that of previous studies in which exposure to CMS increased immobility 57 reduced

the sucrose consumption in the rats 58.

Concomitant treatment of dexamethasone in interferon treated group and CMS model

further raised cortisol levels, decrease in sucrose preference and reduction in the brain mono

amines. Raised corticosteroids in the body cause various actions leading to depressive

behaviour 59, 60 It can also attribute to the changes in the hippocampus of the brain caused by

the long term use of corticosteroids.61, 62 The decrease in sucrose preference can be a

manifestation of behavioral changes like anhedonia, seen with long term use of

14

corticosteroids as documented in the earlier research. Further, the decrease in the brain

serotonin levels may be a result of induction of tryptophan 2, 3 dioxygenase 63 leading to

decrease in brain serotonin levels as observed in the current study. There was overall

worsening of depressive behavior by use of dexamethasone.

Aspirin treatment in interferon-α-2b model of depression for about 21 days, and CMS

model for 28 days showed significant antidepressant effect through the parameters such as

sucrose preference test, forced swim test, serum cortisol levels and brain monoamines. There

was increase in sucrose preference and reduction in serum cortisol in the interferon group

treated with aspirin. Further, there was noteworthy decrease in immobility in the group

treated with interferon-α-2b with aspirin. The brain serotonin levels were raised when

compared with the interferon treatment group. These findings are in line with the

observations seen in previous studies which focus on the beneficial effect of aspirin in

affective disorders. 64-67

The increase in cortisol levels might decrease serotonin levels by diminishing the

activity of tryptophan 2, 3 dioxygenase 53. Signs of an inflammatory process, in particular

increased levels of prostaglandine E2 (PGE2), have repeatedly been described in major

depression. 68 Cyclooxygenase-2 (COX-2) inhibitors 69, 70 inhibit the PGE2, suggesting

positive role in depression. Beneficial role of NSAIDS like aspirin in depression and it

neurobiological effects. 71, 72 have been reported and are replicated in the present study. It has

also been observed that lack of clinical therapeutic benefit of antidepressants is associated

with overall activation of the inflammatory system 73, which may be down regulated by use

of NSAIDS like aspirin in depression. Furthermore, one of the studies 29 showed use of

aspirin as augmentation agent in fluoxetine treatment resistant depressive rats. Increased

activity of cyclooxygenase-2 produces prostaglandin D2 74 leading to subsequent inhibition

of serotonin in the brain. Inhibition of such eicosanoids brought by aspirin may result in its

antidepressant action. Supportive evidence to this is role of hyperforin, a constituent from St.

John’s wart inhibits prostaglandin synthesis, which is an established antidepressant agent 25.

Studies show that aspirin and other non-opioid derivatives affects antinociception through

monoamines like serotonin.75 Further, latest developments suggest that aspirin decreases the

risk of depression in subjects with high plasma homocysteine 69, at cellular levels it prevents

hippocampal neuronal loss and aberrant neurogenesis 76 which can be attributed to its

antidepressant action.

Thus, our findings suggest that when targeting anti-inflammatory drugs in the

treatment of depression NSAIDS should be investigated rather than corticosteroids like anti-

15

inflammatory agents. Aspirin may serve as a potential adjunctive therapy for individuals

suffering from depression, particularly arising from use of interferon in Hepatitis B or other

forms of depression. The worsening of depressive behavior seen with long term use of

dexamethasone strengthens the use of novel antidepressants like Antalarmin CRHR-1

(Corticotropin-releasing hormone receptor-1) receptor antagonists (77) in the treatment of

depression.

Further studies are required to establish mechanism of action of aspirin for its

antidepressant action.

Conclusion

The present study investigated the role of anti-inflammatory drugs in CMS model of

depression. The anti-inflammatory drugs, particularly NSAIDs can play a significant role in

the treatment of depression, considering depression to be a psychoneuroimmunological

disorder. In contrast, use of anti-inflammatory drugs like dexamethasone for longer periods

worsens depression. The mechanism responsible for antidepressant action of aspirin is

unknown. However, it may be attributed to blocking of COX pathway, lowering of serum

cortisol levels, and neuroprotective mechanism or any other potential physiological and

biochemical mechanisms.

Papers

Bhatt S, Shukla P, Raval J, Goswami S Role of Aspirin and Dexamethasone against

Experimentally Induced Depression in Rats Basic Clin. Pharmacol. Toxicol. 2016

Jul;119(1):10-8.

Bhatt S, Kilambi P, Patel P, Patel N, Panchal A, Shah G, Goswami S Beneficial effect

of aspirin against interferon-α-2b-induced depressive behavior in Sprague Dawley rats.Clin

Exp. Pharmacol. Physiol. 2016 Aug 25. doi: 10.1111/1440-1681.12660. [Epub ahead of print]

PubMed PMID: 27561157

16

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