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Electronic Supplementary MaterialJournal: Psychopharmacology

Electronic Supplementary Material

Journal: Psychopharmacology

In-vivo and In-vitro attenuation of naloxone precipitated experimental opioid withdrawal

syndrome by insulin and selective KATP channel modulator

Prabhat Singh a, Bhupesh Sharma b, c, *, Surbhi Gupta d and B. M. Sharma e

Neuropharmacology Lab, Department of Pharmacology, School of Pharmacy, BIT, India

Conscience Research, Delhi, India

a Research Student, Neuropharmacology Lab., Department of Pharmacology, School of

Pharmacy, Bharat Institute of Technology, Partapur Bypass, Meerut, Pin- 250103, Uttar Pradesh,

India. +91-954-8116443; [email protected]

b Associate Professor and Head, Department of Pharmacology, School of Pharmacy, Bharat

Institute of Technology, Partapur Bypass, Meerut, Pin- 250103, Uttar Pradesh, India

+91-995-8219190, +91-879-1636281; [email protected]

c Chief Consultant, CNS Pharmacology, Conscience Research, Pocket F-233, B, Dilshad Garden,

Delhi-110095 India. +91-995-8219190; [email protected]

d Research Student, Neuropharmacology Lab., Department of Pharmacology, School of

Pharmacy, Bharat Institute of Technology, Partapur Bypass, Meerut, Pin- 250103, Uttar Pradesh,

India. +91-992-7932746; [email protected]

e Assistant Professor, Department of Pharmacology, School of Pharmacy, Bharat Institute of

Technology, Partapur Bypass, Meerut, Uttar Pradesh, India

* Corresponding Author: Dr. Bhupesh Sharma

Category of paper: Original Investigation

1

mailto:[email protected]





Material and Method

Animals

Swiss albino mice (25±2g) and Wistar rats (250±20g), of either sex, were maintained on

a standard laboratory diet and having free access to tap water. To control for sex-related

variations in the results, male and female mice were divided equally among all treatment groups.

Animals were housed in the departmental animal house and were exposed to 12 hour light and 12

hour dark cycle. The Experiments were conducted in a semi sound-proof laboratory. The

experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC),

whereas the animals were taken care by following the guidelines of the Committee for the

Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of

Environment and Forests, Government of India (Reg. No. 25/230/2011/AWD/CPCSEA). Mice

were used for in-vivo studies and Wistar rats (ileum) were used for in-vitro studies.

Drugs and chemicals

Morphine sulphate and naloxone were obtained from Troikaa pharmaceuticals Ltd.,

India. Gliclazide was obtained from Morepen laboratories Ltd., India. Insulin (Huminsulin,

biphasic isophane insulin injection IP) was obtained from Gland Pharma Ltd., India. 5,5`-

dithiobis(2-nitrobenzoic acid) (DTNB) and N-naphthylethylenediamine were purchased from

Sigma Aldrich, USA.

Morphine, naloxone and insulin were dissolved in 0.9% saline solution, prepared in

triple distilled water and gliclazide was suspended in 0.5% carboxy methyl cellulose (CMC). All

drug solutions were freshly prepared before use. Selection of doses and the dosing schedule were

based on previously published reports from other labs.

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For in-vivo study, morphine (5mg/kg; intraperitoneally), naloxone (8mg/kg;

intraperitoneally), insulin (4 and 8 I.U/kg/day; intraperitoneally) and gliclazide (3 and

6mg/kg/day; orally) were administered to mice (Jafari et al. 2004; Satyanarayana and Kilari

2006; Rehni and Singh 2012). For in-vitro study the concentration of morphine (10−5 M),

naloxone (10−5 M), gliclazide (5×10-6 M and 5×10-5 M) and insulin (1x10-6 M and 1x10-5 M) were

used (Thomson et al. 1997; Rehni and Singh 2012; Sliwinska et al. 2012). The duration of action

of insulin and gliclazide are 18-24 hours and 12-18 hours, respectively, so the treatment drugs

were administered once daily for 6 days after 1 hour of morphine injection.

Induction of morphine withdrawal syndrome (In-vivo)

To develop dependence, morphine (5 mg/kg, intraperitoneally) was administered twice

daily (at 08: 00 and 20: 00 h) for 6 days. However, the test drug/vehicle was administered daily

(at 10:00 am) after one hour injection of morphine (Rehni and Singh 2012). The withdrawal

signs were precipitated by injecting naloxone (8 mg/kg, intraperitoneally), 2 hours after the final

injection of morphine on the 6th day. This in-vivo model has been extensively used for induction

of morphine withdrawal syndrome in various laboratories (Way et al. 1969; Rehni and Singh

2012). Immediately after a naloxone injection, mice were individually placed in an observation

chamber and behavioural observations were made in initial 30 min. The observations were

carried out in a transparent Perspex observation chamber with dimensions of 30cm×30cm×30

cm. Two observers, which were blind to the treatment schedule, simultaneously observed each

animal for all of the withdrawal measures and the mean value of both observations were

recorded. The withdrawal symptoms are reported as a summary of all of the signs that were seen.

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Induction of experimental morphine withdrawal response in isolated rat ileum (In-vitro)

Morphine withdrawal by naloxone can be induced in-vitro (rat ileum), which has been

utilized previously for screening of agents as well as for assessing morphine withdrawal

syndrome. Naloxone contracture (basal and treatment) was measured in terms of tension ratio,

using isolated rat ileum preparation on student organ bath assembly. Briefly, overnight fasted

adult rats (250±20 g), were killed by injecting high doses of thiopental sodium followed by

carotid bleeding. A small 2-3 cm section of ileum was isolated from the intestine. Rat ileum

preparation was prepared by tying a loop on one end of the tissue and tying another thread on a

diagonally opposite aspect in order to ensure the complete opening up of the linen of the tissue

while the same was mounted on the tissue bath. The tissue was then suspended in an organ bath

containing 20 ml of Krebs solution (NaCl 118, KCl 4.75, K2HPO4 1.2, CaCl2 1.26, MgSO4 1.2,

NaHCO3 25 and glucose 11.1 mM) at 37°C, aerated with 95% O2 and 5% CO2. A resting tension

of 1 g was applied to the tissue. The tissue was allowed to equilibrate for 40-60 min, and three

responses to acetylcholine (ACh, 10−6 M) were obtained so that the withdrawal response could be

expressed as the percentage of a particular mean ACh response. The experimental procedure was

similar to that described for guinea pig ileum previously (Valeri et al. 1995; Capasso and

Sorrentino 1997; Rehni and Singh 2012). Morphine (10−5 M) was added to the bath and the tissue

was exposed to the opioid agonist for a period of 4 min. Naloxone (10−5 M) was then added in

the bath to elicit a strong opioid withdrawal contracture in the morphine withdrawn rat ileum

(Rehni and Singh 2012). After a washout, another ACh response was obtained (to verify whether

the ileum’s response was modified after withdrawal contracture). After a 10 min resting period,

test drug/vehicle (varying concentrations as per the protocol) was added in the bath along with a

4 min exposure of the ileum then added to elicit a response. Following a washout, ACh response

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was repeated to affirm the functional ability of the tissue. Moreover, in order to avoid the

development of tolerance to repeated morphine exposures, each preparation was exposed to three

challenges with morphine and naloxone. Naloxone per se did not produce any effect on naive

preparations or those washed after morphine contact.

Naloxone contracture- The size of the contracture produced by naloxone challenge in all the

groups was expressed as a fraction of the mean contraction obtained with ACh in the isolated

morphine treated rat ileum preparation, calculated in terms of tension ratio as follows:

Tension Ratio = (Response to naloxone/Mean acetylcholine response) × 100

Assessment of withdrawal syndrome and overall morphine withdrawal severity (OMWS)

Naloxone, an opioid receptor antagonist, is commonly used for induction of withdrawal

syndrome (WS) in morphine withdrawal animals. Various behavioural parameters such as: Fore

paw tremors (FPT), wet dog shake (WDS), straightening, sneezing, jumping frequency (JF),

rearing frequency (RF), fore paw licking (FPL) and circling behaviour (CB) in mice were

assessed to evaluate the overall severity of WS in all the groups.

Animals were observed for each of the behavioural parameters for a period of 30 min.

Forepaw tremors (shakes of paws unrelated to grooming), wet dog shake (full body shakes

unrelated to grooming or lateral rotatory movements of the trunk to shake excessive water

present on the body hair away), straightening [the state of animal when, during locomotion, the

anterior aspect of the body (supported by forepaws) pulls the remaining body forward while its

hind paws remain stationary] and sneezing (an involuntary reflex behaviors that forcibly expels

air out of the respiratory tract) (Rabbani et al. 2012; Enquist et al. 2012), jumping frequency (A

spring or leap off the surface by a muscular effort of the legs and feet or escape attempts, all four

paws off the floor were defined as a jumping response) (Rehni and Singh 2012; Enquist et al.

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2012), frequency of rearing (repetitive raising of the frontal aspect of the mouse body while

balancing the body on the hind paws), fore paw licking (the mouse licking both of its forepaws at

least once) and circling (completions of a complete circle of the entire observation chamber were

recorded as a unit of circling behavior) were measured that were used to quantify the WS in mice

(Rehni and Singh 2012). Further, an overall morphine withdrawal severity was also measured in

all the groups, where mean frequencies of all the parameters of respective groups were summed

up. This suggests that overall morphine withdrawal severity is in proportional to every

withdrawal parameters.

Overall morphine withdrawal severity was calculated as:

OMWS = JF + RF+ FPT + CB + WDS + straightening + sneezing + FPL

Whereas, OMWS– Overall morphine withdrawal severity; JF– mean jumping frequencies; RF–

mean frequencies of rearing; FPT– mean frequencies of fore paw tremors; CB– mean

frequencies of circling behaviour; WDS– mean frequencies of wet dog shake; Sneezing– mean

sneezing frequencies; FPL– mean frequencies of fore paw licking

Biochemical assessment

Dissection and homogenization

Immediately after the estimation of behavioural parameters, the animals were sacrificed

by decapitation. Brain of each animal was separated by putting on ice and weighed individually.

10% (w/v) tissue homogenate was prepared in 0.1 M phosphate buffer (pH 7.4). The homogenate

was centrifuged at 10,000 g at 4°C for 15 min. An aliquot of supernatant was separated and used

for biochemical estimations.

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Assessment of serum glucose level

Morphine administration or its removal has been found to induce alterations in glucose

levels (Simpkins et al. 1990). Due to this reason the blood sample was collected by retro-orbital

bleeding for estimation of serum glucose. The blood was kept at room temperature for 30 min

and then centrifuged at 4000 rpm for 15 min to separate serum which was then used for

estimation of serum glucose level. The fasting blood glucose level was estimated

spectrophotometerically (UV-1800 ENG 240V; Shimadzu Coorporation, Japan) at 550 nm by

glucose per-oxidase (GOD-POD) method using commercially available kit (Sharma and Singh

2010).

Assessment of brain malondialdehyde (MDA) levels

Morphine dependent animals have been shown to increase the levels of malondialdehyde

(MDA) in the brain (Abdel-Zaher et al. 2011). Brain thiobarbituric acid reactive substances

(TBARS), was measured spectrophotometerically (UV-1800 ENG 240V; Shimadzu

Coorporation, Japan) at 532 nm (Sharma and Singh 2008a; Sharma and Singh 2010; Sharma and

Singh 2012c). The quantitative measurement of TBARS, an index of lipid peroxidation in brain

was performed. 0.2 ml of supernatant of the homogenate was pipetted out in a test tube, followed

by the addition of 0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 30% acetic acid (pH 3.5),

1.5 ml of 0.8% of thiobarbituric acid and the volume was made up to 4 ml with distilled water.

The test tubes were incubated for 1 h at 950C, then cooled and added 1 ml of distilled water,

followed by the addition of 5 ml of n-butanol-pyridine mixture (15:1 v/v). The tubes were

centrifuged at 4000 g for 10 min. The absorbance of developing pink colour was measured

spectrophotometerically at 532 nm. A standard calibration curve was prepared using 1-10 nM of

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1,1,3,3-tetra methoxy propane. The TBARS value was expressed as nanomoles per gram wet

weight of the brain (Sharma and Singh 2008b).

Assessment of brain glutathione (GSH) level

It has been reported that naloxone in morphine dependent animals, reduces the levels of

glutathione in the brain (Abdel-Zaher et al. 2011). Because of this the different glutathione

content in the brain was estimated according to the different methods such as reduced

glutathione. The reduced GSH content in the brain was estimated spectrophotometerically (UV-

1800 ENG 240V; Shimadzu Coorporation, Japan) at 412 nm. Briefly, the supernatant of the

homogenate was mixed with trichloroacetic acid (10% w/v) in 1:1 ratio. The tubes were

centrifuged at 1000 g for 10 min at 40C. The supernatant obtained (0.5 ml) was mixed with 2 ml

of 0.3 M disodium hydrogen phosphate. Then 0.25 ml of 0.001 M freshly prepared DTNB [5,5`-

dithiobis (2-nitro benzoic acid) dissolved in 1% w/v sodium citrate] was added and absorbance

was noted spectrophotometerically at 412 nm. A standard curve was plotted using 10-100 μM of

the reduced form of glutathione and results were expressed as micromoles of reduced glutathione

per gram wet weight of the brain (Sharma and Singh 2008a; Sharma and Singh 2013).

Assessment of brain nitrite/nitrate level

Naloxone in morphine dependent animals has been reported to increase the levels of

nitrite/nitrate in the brain (Abdel-Zaher et al. 2011). Brain nitrite concentration was measured

spectrophotometerically (UV-1800 ENG 240V; Shimadzu Coorporation, Japan) at 545 nm

(Sharma and Singh 2011a; Sharma and Singh 2012a; Sharma and Singh 2013). Briefly, 400 μl of

carbonate buffer (pH 9.0) was added to 100 μl of brain or standard sample followed by the

addition of small amounts (0.15 g) of copper-cadmium alloy. The tubes were incubated at room

temperature for 1 hour to reduce nitrate to nitrite. The reaction was stopped by adding 100 μl of

8


0.35 M sodium hydroxide. Following this, 400 μl of zinc sulphate solution (120 mM) was added

to deproteinate the brain samples. The samples were allowed to stand for 10 min and then

centrifuged at 4000 g for 10 min. Greiss reagent (250 μl of 1.0% sulphanilamide prepared in 3 N

HCl and 250 μl of 0.1% N-naphthylethylenediamine (prepared with water) was added to aliquots

(500 μl) of clear supernatant and brain nitrite was measured spectrophotometerically at 545 nm.

The standard curve of sodium nitrite (5 to 50 μM) was plotted to calculate the concentration of

serum nitrite (Sharma and Singh 2011b; Sharma and Singh 2012b).

Assessment of brain calcium (Ca+2) levels

The administration of opiates modifies the content and movement of Ca+2 in the brain.

Morphine dependence is associated with a large increase in the content of Ca+2 in the brain of

mice (Bongianni et al. 1986). The increased Ca+2 levels in the brain were measured by using an

Auto analyzer (RMSBCA 201). The test procedure was performed according to method of Carl

et al. (1996).

Experimental protocol

In the present investigation, for in-vivo studies total twelve groups were employed with

each group comprising of eight Swiss albino mice of either sex and for in-vitro studies eight

groups were employed, with each group comprising of six Wistar rats of either sex (Figure 1).

Protocol for morphine withdrawal syndrome in mice (In-vivo):

Group I and II-- Vehicle control group (0.9% w/v saline and 0.5% CMC): Animals were

administered with saline (10 ml/kg/day; intraperitoneally) and CMC (10 ml/kg/day, orally) for 6

days respectively.

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Group III, IV, V and VI-- Insulin (dose 1 and dose 2) and gliclazide (dose 1 and dose 2) per

se: Animals were administered with insulin (4 and 8 I.U/kg/day; intraperitoneally) and gliclazide

(3 and 6 mg/kg/day; orally) for 6 days.

Group VII-- Morphine treatment group: Animals were administered with morphine (5

mg/kg/day; intraperitoneally) for 6 days.

Group VIII-- Morphine + Naloxone treatment group: All animals were administered with

morphine (5 mg/kg/day; intraperitoneally) for 6 days. On 6th day these animals were

administered with naloxone (8 mg/kg; intraperitoneally) after 2 hours of morphine treatment.

Group IX-- Morphine + Insulin (dose 1) + Naloxone treatment group: All animals were

administered with morphine (5 mg/kg/day; intraperitoneally) and simultaneously 1 hour later

with insulin (4 I.U/kg/day; intraperitoneally) for a period of 6 days. On the 6th day, naloxone (8

mg/kg, intraperitoneally) was administered to the animals after 2 hours of morphine treatment.

Group X-- Morphine + Insulin (dose 2) + Naloxone treatment group: All animals were

administered with morphine (5 mg/kg/day; intraperitoneally) and simultaneously 1 hour later

with insulin (8 I.U/kg/day; intraperitoneally) for a period of 6 days. On the 6th day, naloxone (8

mg/kg; intraperitoneally) was administered to the animals after 2 hours of morphine treatment.

Group XI-- Morphine + Gliclazide (dose 1) + Naloxone treatment group: All animals were

administered with morphine (5mg/kg/day; intraperitoneally and simultaneously 1 hour later with

gliclazide (3 mg/kg/day; orally) for a period of 6 days. On the 6th day, naloxone (8 mg/kg;

intraperitoneally) was administered to the animals after 2 hours of morphine treatment.

Group XII-- Morphine + Gliclazide (dose 2) + Naloxone treatment group: All animals were

administered with morphine (5 mg/kg/day; intraperitoneally and simultaneously 1 hour later with

10


gliclazide (6 mg/kg/day; orally) for a period of 6 days. On the 6th day, naloxone (8 mg/kg;

intraperitoneally) was administered to the animals after 2 hours of morphine treatment.

Protocol for morphine withdrawal response in isolated rat ileum preparation (In-vitro):

Group I-- Morphine treatment: Basal contraction was obtained in the morphine dependent rat

ileum.

Group II--Insulin treatment (10x10-6 M): Basal contraction was obtained in insulin (dissolved

in 0.9% saline) treated rat ileum. The concentrations of insulin achieved in the bath employed to

assess their effect on withdrawal response was 10x10-6 M.

Group III--Gliclazide treatment (50x10-6 M): Basal contraction was obtained in gliclazide

(dissolved in 0.9% saline) treated rat ileum. The concentrations of insulin achieved in the bath

employed to assess their effect on withdrawal response was 50×10-6 M.

Group IV--Morphine-naloxone: Basal naloxone contraction was obtained in the morphine

dependent rat ileum. After 10 min resting periods, the same tissue was subjected to the vehicle

(0.9% saline) for insulin and gliclazide along with morphine treatment and the effect of naloxone

on such a tissue was then tested.

Group V--Insulin treatment (1x10-6 M) + Morphine-naloxone response: Basal naloxone

contraction was obtained in the morphine dependent rat ileum. After 10 min resting periods, the

same tissue was subjected to insulin treatment (dissolved in 0.9% saline) along with morphine

treatment and the effect of naloxone on such a tissue was then tested. The concentrations of

insulin achieved in the bath employed to assess their effect on withdrawal response was 1x10-6 M

for group V.

Group VI--Insulin treatment (10x10-6 M) + Morphine-naloxone response: Basal naloxone


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same tissue was subjected to insulin treatment (dissolved in 0.9% saline) along with morphine


insulin achieved in the bath employed to assess their effect on withdrawal response was 10x10-6

M for group VI.

Group VII--Gliclazide treatment (5×10-6 M) + Morphine-naloxone response: Basal naloxone


same tissue was subjected to gliclazide treatment (dissolved in 0.9% saline) along with morphine


gliclazide achieved in the bath employed to assess their effect on withdrawal response was 5×10-

6 M for group VII.

Group VIII--Gliclazide treatment (50×10-6 M) + Morphine-naloxone response): Basal

naloxone contraction was obtained in the morphine dependent rat ileum. After 10 min resting

periods, the same tissue was subjected to gliclazide treatment (dissolved in 0.9% saline) along

with morphine treatment and the effect of naloxone on such a tissue was then tested. The

concentrations of gliclazide achieved in the bath employed to assess their effect on withdrawal

response was 50×10-6 M for group VIII.

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References

Abdel-Zaher AO, Abdel-Rahman MS, Elwasei FM (2011) Protective effect of Nigella sativa oil

against tramadol-induced tolerance and dependence in mice: role of nitric oxide and

oxidative stress. Neurotoxicology 32:725-33

Bongianni F, Carla V, Moroni F, Pellegrini-Giampietro DE (1986) Calcium channel inhibitors

suppress the morphine-withdrawal syndrome in rats. Br J Pharmacol 88:561-7

Capasso A, Sorrentino L (1997) Differential influence of D1 and D2 dopamine receptors on

acute opiate withdrawal in guinea-pig isolated ileum. Br J Pharmacol 120:1001-6

Carl A, Burtis, Edward R Ashwood (1996) “Mineral and Bone Metabolism” in Tietz

Fundamentals of Clinical Chemistry, W.B. Saunders and company, Philadelphia, PA

685-703

Enquist J, Ferwerda M, Milan-Lobo L, Whistler JL (2012) Chronic methadone treatment shows a

better cost/benefit ratio than chronic morphine in mice. J Pharmacol Exp Ther 340:386-

92

Jafari MR, Zarrindast MR, Djahanguiri B (2004) Effects of different doses of glucose and insulin

on morphine state-dependent memory of passive avoidance in mice.

Psychopharmacology (Berl) 175:457-62

Rabbani M, Hajhashemi V, Vadizadeh A (2012) Co-administration of calcium gluconate and

magnesium acetate effectively blocks the signs of morphine withdrawal in mice.

Magnes Res 25:40-8

Rehni AK, Singh N (2012) Ammonium pyrrolidine dithiocarbamate and RS 102895 attenuate

opioid withdrawal in vivo and in vitro. Psychopharmacology (Berl) 220:427-38

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Satyanarayana S, Kilari EK (2006) Influence of nicorandil on the pharmacodynamics and

pharmacokinetics of gliclazide in rats and rabbits. Mol Cell Biochem 291:101-5

Sharma B, Singh N (2010) Pitavastatin and 4'-hydroxy-3'-methoxyacetophenone (HMAP) reduce

cognitive dysfunction in vascular dementia during experimental diabetes. Curr

Neurovasc Res 7:180-91

Sharma B, Singh N (2011a) Attenuation of vascular dementia by sodium butyrate in

streptozotocin diabetic rats. Psychopharmacology (Berl) 215:677-87

Sharma B, Singh N (2011b) Behavioral and biochemical investigations to explore

pharmacological potential of PPAR-gamma agonists in vascular dementia of diabetic

rats. Pharmacol Biochem Behav 100:320-9

Sharma B, Singh N (2012a) Salutary effect of NFκB inhibitor and folacin in

hyperhomocysteinemia-hyperlipidemia induced vascular dementia. Prog

Neuropsychopharmacol Biol Psychiatry 38:207-15

Sharma B, Singh N (2012b) Experimental hypertension induced vascular dementia:

pharmacological, biochemical and behavioral recuperation by angiotensin receptor

blocker and acetylcholinesterase inhibitor. Pharmacol Biochem Behav 102:101-8

Sharma B, Singh N (2012c) Defensive effect of natrium diethyldithiocarbamate trihydrate

(NDDCT) and lisinopril in DOCA-salt hypertension-induced vascular dementia in rats.

Psychopharmacology (Berl) 223:307-17

Sharma B, Singh N (2013) Pharmacological inhibition of inducible nitric oxide synthase (iNOS)

and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, convalesce

behavior and biochemistry of hypertension induced vascular dementia in rats.

Pharmacol Biochem Behav 103:821-30

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Sharma B, Singh N, Singh M (2008a) Modulation of celecoxib- and streptozotocin-induced

experimental dementia of Alzheimer's disease by pitavastatin and donepezil. J

Psychopharmacol 22:162-71

Sharma B, Singh N, Singh M, Jaggi AS (2008b) Exploitation of HIV protease inhibitor Indinavir

as a memory restorative agent in experimental dementia. Pharmacol Biochem Behav

89:535-45

Simpkins JW, Katovich MJ, Millard WJ (1990) Glucose modulation of skin temperature

responses during morphine withdrawal in the rat. Psychopharmacology (Berl) 102:213-

20

Sliwinska A, Rogalska A, Szwed M, Kasznicki J, Jozwiak Z, Drzewoski J (2012) Gliclazide may

have an antiapoptotic effect related to its antioxidant properties in human normal and

cancer cells. Mol Biol Rep 39:5253-67

Thomson MJ, Williams MG, Frost SC (1997) Development of insulin resistance in 3T3-L1

adipocytes. J Biol Chem 272:7759-64

Valeri P, Morrone LA, Romanelli L, Amico MC (1995) Acute withdrawal after bremazocine and

the interaction between mu- and kappa-opioid receptors in isolated gut tissues. Br J

Pharmacol 114:1206-10.

Way EL, Loh HH, Shen FH (1969) Simultaneous quantitative assessment of morphine tolerance

and physical dependence. J Pharmacol Exp Ther 167:1-8

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Figure 3: Raw traces showing effect of various treatments on morphine naloxone induced contraction (during morphine withdrawal) on rat ileum.ACh: acetylcholine (10-6 M); M: Morphine (10-5 M); N: naloxone (10-5 M); I: insulin (10×10-6 M &1×10-6 M); G: gliclazide (5×10-6 M and 50×10-6 M); D1: dose 1; D2: dose 2

16

Figure 3

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