SYNOPSIS

Effects of Imidazoline I1 receptors in fluoxetine

induced anti-hyperalgesic activity in neuropathic rats
For approval of topic of the thesis to be submitted in the partial fulfilment of the
requirement for the degree of Master of Pharmacy in Pharmacology.

By
Ku. Krutika Ramesh Sawarkar

Guide: Dr. B. G. Taksande

SMT. KISHORITAI BHOYAR COLLEGE OF PHARMACY,

New Kamptee, Nagpur-441 002, Maharashtra
Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur
2015-2016

Introduction:
Pain remains the leading factor that causes patients to live with disability
and continues to impart high health cost and economic loss to society. Currently,
clinically available analgesics are not adequate to meet all the needs and there
remains a large population with undertreated pain. Opioids remain the most
effective analgesics for many painful conditions (acute and chronic) (Jamison
and Mao, 2015). However, their clinical use is limited largely due to their
unwanted effects, such as, constipation, physical dependence, abuse and higher
doses (Nelson et al., 2015). In the past half century, pharmacotherapy of pain
has seen great progress; and prevelance of neuropathic pain has been estimated
in between 6% and 8%; thus clinical reality substantiates the urgent need to
develop novel and effective pharmacotherapies for neuropathic pain
management.
According to the international association for Study of Pain (IASP, 2011)
neuropathic pain is defined as pain caused by a lesion/disease of the somato
sensory system. Categorized as chronic pain, it is classified into hyperalgesia
and allodynia. Hyperalgesia is increased sensitivity to pain while allodynia is
increased sensitivity to pain in absence of noxious stimulus (Sandkuhler, 2009).
It may be caused by damage to nociceptors, peripheral nerve or activation of
platelet activating factor (Bonnet et al, 1981). The pain is associated with nerve
irritation or damage as in case with allodynia in neuropathic pain. In recent
years, selective serotonin reuptake inhibitors (SSRI) are used as a standard
therapy other than opioids in the management of neuropathic pain (Upadhya et
al., 2011).
Introduction of molecules:
SSRI's:
SSRI are broadly involved in management of several central diseases
such as depression (Zhau et al., 2015), epilepsy (Shiha et al., 2015), meningitis
(Liechti et al., 2015) and headache (Banzi, 2015). SSRI's are involved in
neuropathic pain (Kato et al., 2013). Recently, Saito et al. (2014) reported the
involvement of SSRI (paroxetine) in pain management.
Tricyclic antidepressants have been used for decades in the treatment of
chronic pain patients without depression (Duman, 2003).
Agmatine:
Agmatine, an amine formed by decarboxylation of L-arginine by arginine
decarboxylase (ADC), has been recently discovered in mammalian brain and
other tissues (Zhu et al., 2004) and it has many central effects like anxiolytic
(Taksande et al., 2010), anti-depressant (Moretti et al., 2015), orexigenic
(Taksande et al., 2011 and Song et al., 2014) and analgesic effect (Aggarwal et
al., 2009). Role of agmatine and imidazoline receptors in management of

neuropathic pain (Onal et al., 2003) and antidepressant activity of SSRIs

(Taksande et al., 2009) is well established. There is role of imidazoline receptor
in management of pain (Thorn et al., 2015). Moreover, it is known that
endogenous serotonin interacts with the agmatine and is involved in several
behaviors. Fluoxetine administration is reported to increase brain agmatine
levels (Taksande et al., 2009).
In view of this background, we hypothesize that imidazoline receptors
might be involved in algesic responses and that anit-hyperalgesic effect of
fluoxetine might be mediated via imidazoline I1 receptors.
Objective: To investigate the role of imidazoline I1 receptors in fluoxetine
induced anti-hyperalgesic activity in neuropathic rats.
Material and methods
Subjects:
Adult Male Sprague Dawley rats will be housed in polypropylene cages
in a temperature (252 0C) relative humidity (5070%) and maintained on a
12:12-h light/dark cycle (lights on 07:0019:00 h). Food and water will be
provided ad libitum except during specific experimental protocols. The
experiments will be performed as per the protocol approved by Institutional
animal ethical committee according to the guidelines of CPCSEA.
Drugs:
1) Fluoxetine
2) Agmatine
3) Moxonidine hydrochloride
4) Efaroxan hydrochloride
Plan of Work:
The rats will undergo surgery for neuropathic pain and implantation of
guide cannula in order to administer drugs centrally. Post-surgery
recovery will be given to the rats for 7 days.
Following 7 days, animals will be assessed for induction of neuropathic
pain using hot and cold allodynia method.
Animals will be treated with Fluoxetine, Agmatine, Moxonidine
hydrochloride or Efaroxan hydrochloride per se and their dose dependent
effects will be studied.
In order to determine the interaction between the serotonergic and
agmatinergic systems combinations studies will be performed:
1) Fluoxetine with Agmatine
2) Fluoxetine with Moxonidine hydrochloride
3) Fluoxetine with Efaroxan hydrochloride

 Animals will be evaluated for cold allodynia using acetone test and hot
allodynia will be assessed using thermal paw withdrawal test.
Surgery for cannulation and neuropathic pain:
Procedure of cannulation, drug administration or post-surgical care has
been described (Goyal et al., 2006; Upadhya et al., 2009, 2011). Briefly, rats
will be weighed and anaesthetized with thiopental sodium (45 mg/kg, ip)
(Abbott Pharmaceuticals, Mumbai, MS, India). Hair depletor (Anne French
Creme Hair Remover, Wyeth Limited, Mumbai) will be applied to head and
scrubbed to remove hair. Each rat will be placed in a stereotaxic instrument
(David Kopf Instruments, Tujunga, CA, USA) and will be kept warm during
surgical intervention. Midsagital incision will be given on the skull and the skin
will be retracted. The soft tissues overlying on the skull will be then removed.
The landmarks of the skull, bregma and lambda, will be identified and the skull
will be oriented such that both points will be positioned at the same horizontal
level. After clearing the underlying fascia, a burr-hole will be drilled through the
skull over coordinates corresponding to the site of interest. The coordinates will
be estimated from the rat brain atlas (Paxinos and Watson, 1998). A stainless
steel guide cannula prepared in house (Kokare et al., 2011) will be implanted
into the right lateral cerebral ventricle using the stereotaxic coordinates, 0.8
mm posterior, +1.3 mm midline to lateral and 3.5 mm ventral with respect to
bregma. Guide cannula will be secured to the skull with the help of stainless
steel mounting screws and fixed by applying rapidly polymerizing dental acrylic
cement (DPI-RR cold cure, acrylic powder, Dental product of India, Mumbai) to
the surface of the skull, and after acrylic get hardened, each animal will be
removed from the stereotaxic frame. A dummy cannula (C316DC/Spc, wire
o.d., 0.25 mm) will be placed into the guide cannula to allow the guide cannula
to maintain patency. Further the animal will be subjected to surgery table for
chronic constriction injury to induce neuropathic pain.
The chronic constriction injury for neuropathic pain will be performed
according to Upadhya et al. (2009, 2011). Briefly, sciatic nerve of right hind
paw will be located and four loose ligatures tied around the nerve using chromic
catgut (4-0; Sutures India Pvt. Ltd., India) at 1 mm spacing. The incision will
then be sutured layer to layer using silk threads. In another group of rats, the
right sciatic nerve will be exposed but not ligated and same will be considered
as sham-control..
After surgery, rats will be given 3,00,000 units of penicillin G (Wyeth
Limited, Mumbai), subcutaneously to prevent infection. The animals will be
housed individually and allowed to recover from both the surgeries for 7 days.
During recovery period, animals will be habituated to the testing environment
and wound sites will be inspected and cleaned on regular basis. Rats showing
neurological and motor deficits like impairment in locomotion, grooming, social

interaction or occurrence of aggressiveness, handling induced hyper-excitability

and stereotype behaviour will be excluded from the study.
The placement of the cannula for icv injection will be tested for accuracy
by injecting dilute India ink and post-mortem examination of the distribution of
ink in the ventricles (Upadhya et al., 2009) and the animals with correct
placement will be used for statistical analysis.
Behavioural Paradigm:
Hot Allodynia using Hot Plate Test: (Aggarwal et al., 2009)
The paws of rodents are highly sensitive to heat at temperature which do
not damage their skin. They usually respond by jumping, withdrawing of paw
and licking them.
Commercially available Eddy's hot plate consists of an electrically heated
surface. The temperature is controlled at 55-56C. The animals will be placed
on the hot plate and the time until either licking or jumping occurs will be
recorded.
Cold allodynia using Acetone Test : (Boyce-Rustay et al., 2010)
The rats will be moved from the housing room to the testing room at least
60 min before pretreatment or testing. 1520 min before the onset of testing, 5
rats will be moved to acclimate in individual small plastic cages. Then inverted
on an elevated mesh floor to acclimate to their new environment. A drop of
cold acetone (100 l) will be applied using an Eppendorf repipettor to the
plantar surface of the right hind (ipsilateral) paw and response of the rat will be
recorded.( the pipettor must not come in contact with the skin).
Acetone will produce a distinct cooling sensation as it evaporates.
Normal rats will not respond to this stimulus or with a very small response (in
amplitude and duration) while sensitized animals (e.g. neuropathic animals) will
almost always respond with an exaggerated response. Responses will be
measured as the number of shakes/licking of hindpaw and it will be counted for
a set period of time or number of paw withdrawals. This will be repeated 5
times at 5 min intervals to obtain a baseline for each rat.
Statistical Analysis:
The data obtained will be statistically analyzed using either one way or two way
ANNOVA with appropriate post-hoc test. The value P<0.05 will be considered
significant.

Table: Number of animals used.

Sr. No

Particulars

1
2
3
4
5
6
7
8
9

Sham control (n=5)

acsf (n=5)
acsf + Fluoxetine (n=35)
acsf + Agmatine (n=35)
acsf + Moxonidine hydrochloride (n=35)
acsf + Efaroxan hydrochloride (n=35)
Fluoxetine + Agmatine (n=25)
Fluoxetine + Moxonidine hydrochloride (n=25)
Fluoxetine + Efaroxan hydrochloride (n=25)

No.of
animal
s (n)
5
5
15
15
15
15
10
10
10

Total number of animals required

100

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Signature of guide
Dr. B. G. Taksande
Date: 23/7/2015
Place: Kamptee

Signature of Candidate
Krutika R Sawarkar