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Sandip R Final Synopsis
Sandip R Final Synopsis
Sandip R Final Synopsis
For approval of topic of the thesis to be submitted in the partial fulfillment of the
requirement for the degree of Master of Pharmacy in Pharmacology.
Sandip R. Rahangdale
B. Pharm.
Guide
Co-guide
Prof. C. T. Chopde
M. Pharm., Ph.D., F.I.C.
Dr. R. R. Ugale
M. Pharm., Ph.D.
s
SMT. KISHORITAI BHOYAR COLLEGE OF PHARMACY,
New Kamptee, Nagpur, Maharashtra, 441 002
Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur.
2010-2011
administration
of
agmatine
produced
several
effects
including
antinociceptive (Onal et al., 2003), neuroprotective (Olmos et al., 1999), anxiolytic (Lavinsky et
al., 2003), anticonvulsant (Bence et al., 2003), mood elevator (Zomkowski et al., 2002; Halaris et
al., 1999) and antidepressant-like effect in the forced swimming test (FST) and tail suspension
test (TST) in mice, through a mechanism that seems to involve an interaction with NMDA
receptors, the L-argininenitric oxide pathway, and/or 2-adrenoreceptors (Zomkowski et al.,
2002). A clinical study has shown an increase in plasma agmatine level in patients with major
depression compared with healthy controls (Halaris et al., 1999). It was also found that brain
agmatine was depleted in depressive patients (Piletz et al., 1994).
Allopregnanolone (3, 5 THP), dehydroepiandrosterone (DHEA) and its sulfated moiety
DHEA(S) in particular are of significance in depression. Both the indirect genomic and nongenomic mediated effects of 3, 5 THP are involved in depression, while DHEA(S) only exerts
non-genomic effect. In patients suffering from major depression, disequilibrium of neurosteroid
concentrations in plasma and cerebrospinal fluid (CSF) from control subjects and normalize after
successful treatment with antidepressants (Romeo et al., 1998; Uzunova et al., 1998; Strohle et
al., 1999, 2000).
The neurosteroid (3, 5 THP) is synthesized from progesterone in the brain (Baulieu,
1981). It alters rapidly (milliseconds to minutes) neuronal excitability through positive allosteric
interaction with steroid recognition site on gamma-aminobutyric acid type A (GABA A) receptorion channel (GRC) (Paul and Purdy, 1992). At lower (nanomolar) concentrations 3, 5 THP
increases the frequency or duration of openings, or both of the GRC (Lambert et al., 1995).
However, at very high (micromolar) concentrations, it exerts a certain intrinsic agonistic activity
at GABAA receptor in the absence of GABA (Puia et al., 1990). It also modulate ligand gated ion
channels including the voltage gated calcium ion channels (Ffrench-Mullen et al., 1994),
nicotinic acetylcholine receptors (Bullock et al., 1997) and 5-HT3 receptors (Wetzel et al., 1998).
Neurosteroids regulate the neuronal function through their concurrent influence on
neuronal excitability and gene expression (Rupprecht and Holsboer, 1999). Exogenous
administration of 3, 5 THP or drugs that increase its endogenous brain content exhibit
antidepressant-like activity in mouse FST (Khisti et al., 2000), behavioral effect such as antistress (Zimmerberg and Blaskey, 1998), anxiolytic (Akwa et al., 1999), anticonvulsant (Frye,
1995), cataleptic (Khisti et al., 1998; Mandhane et al., 1999).
Serotonin (5-HT) system is strongly implicated in the neural regulation of mood.
Dysregulation in 5-HT neurotransmission underlies pathophysiology of depression. Reduced
level of 5-HT and 5-HIAA is reported in CSF of depressed patients (Risch and Nemeroff, 1992).
In addition, enhancement of serotonergic transmission underlies the therapeutic response to
various types of antidepressant treatments (Blier and Montigny, 1994).
The olfactory bulbectomized rat model of depression is a well-characterized animal
model of depression. Bilateral removal of the olfactory bulbs of rats results in a constellation of
behavioral, neurochemical, neuroimmune and neuroendocrine alterations, similar changes
observed in major depressive disorder (Cryan et al., 2002). The most widely studied behavioral
deficit produced by bulbectomy is a response to a novel and brightly lit environment and reduced
gain of body weight (Kelly et al., 1997). It is sensitive almost exclusively to chronic
antidepressant treatment (Cryan et al., 1998). It exhibits ventricular enlargement as well as
altered signal intensity in cortical, caudate, hippocampal and amygdaloid regions as measured by
magnetic resonance imaging (Wrynn et al., 2000).
Considering the fact that i) Agmatine and serotonergic agents produces an antidepressant
effect as well as a well established role of neurosteroids (3, 5 THP) in FST and TST.
ii)Serotonergic agents modulate antidepressant-like effect of 3, 5 THP in mice. iii) Fluoxetine
increases the level of neurosteroids (3, 5 THP) in brain. iv) Agmatine and fluoxetine produce
synergistic effect in sub-effective doses (Zomkowski et al., 2004). Present study sought to
investigate: i) Antidepressant-like effect of agmatine in olfactory bulbectomized rats. ii) Role of
3, 5 THP in antidepressant-like effect of agmatine by altering endogenous neurosteroids using
neusteroidogenic agents or its biosynthesis blocker in olfactory bulbectomized rats.
Objectives:
1) To investigate the antidepressant-like effect of agmatine in olfactory bulbectomized
rats.
2) To investigate the role of 3, 5 THP in antidepressant-like effect of agmatine in
olfactory bulbectomized rats.
Plan of work:
1) The rats will undergo surgery for removal of olfactory bulb, after recovery period of not less
than 14 days.
2) Dose dependent study of agmatine and 3, 5 THP will be carried out by analysing locomotor
activity in open field apparatus and measure body weight. On the basis of this study the final
effective and sub-effective doses will be selected.
3) In another set of experiment the selected sub-effective dose of agmatine will be combined
with sub-effective dose of 3, 5 THP and effect will be analysed in open field test and measure
body weight.
4) In another set of experiment the positive and negative neurosteroid modulators will be
combined with sub-effective and effective doses of agmatine respectively and effects will be
analysed in open field test and measure body weight.
will not be aspirated. Postoperative analgesia will be consisted of buprenorphin (Temgesic) (0.3
mg/kg s.c.) once a day for 3 days (Uzunova et al., 2003).
After surgery, the rats will be placed in a group of 5-6 animals per cage and handled daily
throught recovery period to reduce stress and/or aggressive behavior.
Method:
Apparatus:
Experimental design:
The rats will undergo surgery for removal of olfactory bulbs and after recovery different
drug treatment will be started at 10-11 a.m. as described above. Effects on locomotor activity and
body weight will be assessed as given below:
Locomotor activity in open field and measurement of body weight of animal:
After recovery period of two week rats will be given treatment of different drugs once a
day for 14 days and accessed for the acute, sub-chronic and chronic effect of different drug
treatment on 1, 7 and 14th day of initiation of treatment respectively. Several positive and
negative modulators will be given at defferent time interval before administration of subeffective and effective dose of agmatine. Effect on locomotor activity will be accessed 30 min
after last dosing of agmatine in open field for ambulations and rearings during a period of 3 min
(as described above) and also measure body weight on same day.
Treatment groups:
1) To study the effect of agmatine:
Bulbectomized animal
A) Control group: Saline (5 ml/kg, i.p.) for 14 days.
B) Treatment group: Agmatine (5, 10 or 20 mg/kg, i.p.) for 14 days.
Sham operated animal
A) Control group: Saline (5 ml/kg, i.p.) for 14 days.
B) Treatment group: Agmatine (5, 10 or 20 mg/kg, i.p.) for 14 days.
Effect on locomotor activity and body weight will be analysed same as described above.
Bulbectomized animal
A) Control group: Vehicle (5 ml/kg, i.p.) for 14 days.
B) Treatment group: 3, 5 THP (0.5, 1 or 2 mg/kg i.p.) for 14 days.
Sham operated animal
A) Control group: Vehicle (5 ml/kg, i.p.) for 14 days.
B) Treatment group: 3, 5 THP (0.5, 1 or 2 mg/kg, i.p.) for 14 days.
Effect on locomotor activity and body weight will be analysed same as described above.
Bulbectomized animal
A) Control group: Vehicle (5 ml/kg, i.p.) 30 min later, agmatine (sub-effective dose, i.p.)
for 14 days.
B) Treatment group: 3, 5 THP (sub-effective dose, i.p.) 30 min later, agmatine (sub-
Bulbectomized animal
A) Control group: Vehicle (5 ml/kg, i.p.) 30 min later, agmatine (sub-effective dose, i.p.)
for 14 days.
B) Treatment group: Progesterone (5 mg/kg, i.p.) 30 min later, agmatine (sub-effective
Bulbectomized animal
C) Control group: Vehicle (5 ml/kg, i.p.) 30 min later, agmatine (sub-effective dose, i.p.)
for 14 days.
A) Treatment group: Metyrapone (50 mg/kg, i.p.) 30 min later, agmatine (sub-effective
Bulbectomized animal
A) Control group: Vehicle (5 ml/kg, i.p.) 30 min later, agmatine (sub-effective dose, i.p.)
for 14 days.
B) Treatment group: 4CD (15 mg/kg, i.p.) 30 min later, agmatine (sub-effective dose,
A) Control group: Vehicle (5 ml/kg, i.p.) 30 min later, agmatine (sub-effective dose, i.p.)
for 14 days.
B) Treatment group: 4CD (15 mg/kg, i.p.) 30 min later, agmatine (sub-effective dose,
i.p.) for 14 days.
Effect on locomotor activity and body weight will be analysed same as described above.
5) To study the influence of neurosteroid biosynthesis inhibitors (negative modulators of
neurosteroids) on antidepressant effect of agmatine:
i) For observing the effect of indomethacin:
Bulbectomized animal
A) Control group: Vehicle (5 ml/kg, i.p.) 20 min later, agmatine (effective dose, i.p.) for
14 days.
B) Treatment group: Indomethacin (5 mg/kg, i.p.) 20 min later, agmatine (effective
Bulbectomized animal
A) Control group: Vehicle (5 ml/kg, i.p.) 120 min later, agmatine (effective dose, i.p.) for
14 days.
B) Treatment group: Trilostane (30 mg/kg, i.p.) 120 min later, agmatine (effective
Bulbectomized animal
A) Control group: Vehicle (5 ml/kg, i.p.) 30 min later, agmatine (effective dose, i.p.) for
14 days.
B) Treatment group: PK11195 (15 mg/kg, i.p.) 30 min later, agmatine (effective dose,
Data analysis:
Data will be analysed by two-way analysis of variance (ANOVA). Significant interaction
will be assessed by post hoc Dunnett's or StudentNewmanKeuls test. The criterion for
statistical significance will be P < 0.05.
Possible outcomes:
1) Agmatine may have antidepressant-like effect in olfactory bulbectomized rats.
2) 3, 5 THP may have role in antidepressant-like effect of agmatine in olfactory
bulbectomized rats.
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Signature of Guide
Prof. C. T. Chopde
Date:
Place:
Signature of Co-guide
Dr. R. R. Ugale
Signature of Candidate
S. R. Rahangdale