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4. MATERIALS AND METHODS

Age matched Wistar rats of either sex, weighing 180-300g housed in animal house

and provided 12h light and 12h dark cycle were used. They were fed on standard

chow diet (Ashirwad Industries Ltd., Ropar, India) and provided water ad libitum. The

experimental protocol was approved by the Institutional Animal Ethics Committee in

accordance with the National Guidelines on the Use of Laboratory animals.

4.1 Induction of Experimental Diabetes Mellitus and Hyperlipidaemia

The rats were administered streptozotocin (50 mg/kg, i.p., once) dissolved in 0.1M

citrate buffer (prepared by dissolving 49.5 ml of 0.1M Citric acid and 50.5ml of 0.1M

Trisodium Citrate, pH 4.4) to induce experimental diabetes mellitus (Ozansoy and

Akin, 2004). Serum glucose level >200 mg/dl were considered to be hyperglycaemic.

Experimental hyperlipidaemia was produced by feeding high fat diet (corn starch

44.74g, casein 14g, sucrose 10g, butter 20g, fiber 5g, mineral mix 3.5g, vitamin mix

1g, choline 0.25g, ter-butylhydroquinone 0.0008g, cholesterol 1g, cholic acid 0.5g)

for 6 weeks (Reeves, 1997; Lorkowska, et al., 2006). Hyperlipidaemia was

documented by estimating the level of total cholesterol (TC) and triglycerides (TG) in

serum using commercially available kits (Vital Diagnostics (P) Ltd., Mumbai, India).

Blood samples were collected before and after 1 week of administration of

streptozotocin for analysis of serum glucose and after 6 weeks of high fat diet for

measurement of total serum total cholesterol (TC) and triglycerides (TG), in clean

centrifuge tubes from 12h fasted rats and were allowed to clot for 15min at room

temperature. Serum was separated by centrifugation at 3000 rpm for 20 min, aliquoted

and then stored at -20°c for analysis of serum glucose and total cholesterol (TC) and

triglycerides (TG).

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4.2 Estimation of Serum Glucose

Serum glucose was estimated spectrophotometrically at 505nm by glucose

oxidase/pyruvate oxidase (GOD-POD) method (Trinder, 1969; Lott and Turner, 1975)

using an enzymatic kit (Kamineni Life Sciences Pvt. Ltd. Hyderabad, India)10µl of

serum was added to 1000µl of glucose reagent to prepare test sample. 10µl of glucose

standard (100 mg/dl) was added to 1000µl to prepare standard sample. 10µl of

distilled water was added to 1000µl of glucose reagent to prepare blank. The samples

were mixed, incubated for 30 min. at room temperature. The absorbance of test and

standard were measured against blank spectrophotometrically (UV-1700

Spectrophotometer, Shimadzu, Japan) at 505nm. The amount of serum glucose was

calculated using following formula:

4.3 Estimation of Serum Total Cholesterol

The total cholesterol was estimated by cholesterol oxidase peroxidase

(CHOD-PAP) method (Allain et al., 1974) using commercially available kit (Vital

Diagnostics (P) Ltd. Mumbai India). 1000 µl of cholesterol reagent was added to 10

µl of serum, 10 µl of standard cholesterol (200 mg/dl) and 10 µl of purified water to

prepare test, standard and blank, respectively. All the test tubes were incubated at

room temperature for 15 min. The absorbances of test and standard samples were

noted against blank at 505 nm spectophotometrically.

Absorbance of Test X 100

Absorbance of Standard

Cholesterol esters + H2O Cholesterol esterase

Cholesterol + Fatty acids

Concentration of glucose (mg/dl) =

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Cholesterol esterase hydrolyses esterified cholesterols to free cholesterol. The free

Cholesterol is oxidized to form hydrogen peroxide which further reacts with phenol

and 4-aminoantipyrine by the catalytic action of peroxidase to form a red coloured

quinoeimine dye complex. Intensity of colour formed is directly proportional to the

amount of cholesterol present in sample.

The serum total cholesterol was calculated using the following formula:

4.4 Estimation of Serum Triglycerides

The serum triglyceride was estimated by glycerophosphate oxidase peroxidase (GOD-

PAP) method (Trinder, 1969, Bucolo and David, 1973) using commercially available

kit (Vital Diagnostics (P) Ltd. Mumbai India). 1000 µl of enzyme reagent was added

to 10 µl of serum, 10 µl of standard (200 mg/dl) and 10 µl of purified water to prepare

test, standard and blank, respectively. All the test tubes were incubated at room

temperature for 15 min. The absorbances of test and standard samples were noted

against blank at 505 nm spectrophotometrically.

Triglycerides Lipoprotein Lipase

Glycerol +Free Fatty acids

Absorbance of Test X 200

Absorbance of Standard

Peroxidase H2O2 + 4-Aminoantipyrine+ Phenol

Cholesterol + O2

Cholesterol Oxidase Cholestenone + H2O2

Red Quinoneimine dye + H2O

Total cholesterol (mg/dl) =

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Lipoprotein lipase hydrolyses triglycerides to glycerol and free fatty acids. The

glycerol formed with ATP in the presence of glycerol kinase forms glycerol-3-

phosphate which is oxidized by enzyme glycerol phosphate oxidase to form hydrogen

peroxide. The hydrogen peroxide further reacts with phenolic compound and 4-

aminoantipyrine by the catalytic action of peroxidase to form a red coloured

quinoneimine dye complex. Intensity of the colour formed is directly proportional to

the amount of triglycerides present in the sample.

The serum triglyceride was calculated using the following formula:

4.5 Assessment of Myocardial Injury

The extent of myocardial injury was assessed by the estimation of Lactate

Dehydrogenase and Creatine Kinase in the coronary effluent. The assessment of

myocardial infarct size was done by using triphenyltetrazolium chloride (TTC)

staining method while LDH and CK-MB were estimated by using commercial kits.

Values of LDH and CK-MB were expressed in international units per litre (IU/L).

Triglycerides (mg/dl) = Absorbance of Test

Absorbance of Standard X 200

H2O2 + 4-Aminoantipyrine + Phenol Red Quinoneimine dye + H2O Peroxidase

Glycerol-3-Phosphate + O2

Glycerol-3-PO Dihydroxyacetone phosphate + H2O2

Glycerol + ATP Glycerol Kinase

Glycerol-3-Phosphate + ADP

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4.6 Assessment of Myocardial Infarct Size

The heart was removed from the Langendorff apparatus. Both the atria and root of

aorta were excised and ventricles were kept overnight at -40C. Frozen ventricles were

sliced into uniform sections of 1-2 mm thickness. The slices were in-cubated in 1%

triphenyl tetrazolium chloride (TTC) for 30 min at 370C in 0.2M Tris-chloride buffer

(CDH Pvt. Ltd., New Delhi) (prepared by dissolving 7.27g of tris-(hydroxymethyl)-

methyamine and 5.27g of sodium chloride in water, adjusting pH to 7.4, finally

diluting upto 1000ml with distilled water) (Fishbein et al., 1981). TTC is converted to

red formazone pigment by NADH and dehydrogenase enzyme and therefore, the

viable cells were stained brick red (Nachlas and Schnitka, 1963). The infracted cells

had lost the enzyme and cofactor and thus remain unstained or dull yellow. The

ventricular slices were placed between two glass plates. A transparent plastic grid with

100 squares in 1cm2

was placed above it. Average area of ventricular slice was

calculated by counting the number of squares on either side. Similarly, numbers of

square falling over non-stained dull yellow area were counted. Infarct size was

expressed as percentage of average ventricular volume (Klein et al., 2000; Singh and

Chopra, 2004).

4.7 Estimation of lactate Dehydrogenase (LDH) Release

LDH was estimated in samples of coronary effluent collected after stabilization and

immediately and 30 min. after reperfusion by King (1965), method using

commercially available kit (Vital Diagnostics (P) Ltd. Mumbai India)

Spectrophotometrically (UV-1700 Spectrophotometer, Shimadzu, Japan) at 340nm.

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate and

NADH to NAD. LDH activity is directly proportional to the rate of decrease in the

absorbance of NADH at 340nm.

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LDH catalyzes the following reaction:

Pyruvate + NADH + H+ LDH

L-Lactate + NAD +

1000µl of LDH working reagent (containing buffer and starter reagent) was pipetted

into a clean and dry test tube and incubated for 1 min. at room temperature. After that

50µl coronary effluent was pipetted to same test tube. The contents were mixed well

and again incubated at 370C for 1 min. Initial absorbance was then immediately read

spectrophotometrically (UV-1700 Spectrophotometer, Shimadzu, Japan) at 340nm

against a blank sample (Distilled Water). Subsequently, absorbance readings were

taken exactly at 60, 120 and 180 seconds.

Mean absorbance change per minute (�A/min) was calculated. LDH activity was

calculated by the following formula:-

LDH activity in IU/L = �A/min × 3333

4.8 Estimation of Creatine Kinase -MB (CK-MB) Release

CK-MB release was estimated in samples of coronary effluent after stabilization and

5min after reperfusion by Hughes, (1962) method using commercially available

kit (Vital Diagnostics (P) Ltd. Mumbai India) spectrophotometrically (UV-1700

Spectrophotometer, Shimadzu, Japan) at 340nm.

Creatine kinase catalyzes the following reaction:

Creatine phosphate + ADP creatine kinase Creatine + ATP

Glucose + ATP hexokinase Glucose-6-phosphate + ADP

Glucose-6-phosphate + NADH G-6 PDH Gluconate-6-phosphate + NADPH + H+

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1000µl of CK-MB working reagent (containing enzyme and starter reagent) was

pipetted into a clean and dry test tube and incubated for 1 min. at room temperature.

After that 50µl coronary effluent was pipetted to same test tube. The contents were

mixed well and again incubated at 370C for 5 min. Initial absorbance was then

immediately read spectrophotometrically (UV-1700 Spectrophotometer, Shimadzu,

Japan) at 340nm against a blank sample (Distilled Water). Subsequently, absorbance

readings were taken exactly at 60, 120 and 180 seconds.

Mean absorbance change per minute (�A/min) was calculated.

CK-MB activity : CK-MB activity in IU/L = �A/min × 6666.

4.9 Isolated Perfused Rat Heart

After 6 weeks of administration of STZ and feeding of HFD rat hearts were harvested.

Rats were administered heparin (500 IU/L, i.p.) 20 min. prior to sacrificing the animal

by cervical dislocation. Heart was rapidly excised and immediately mounted on

Langendorff’s apparatus (Digital Langendorff system, Radnoti LLC, Monrovia, USA)

(Langendorff’s, 1895). The heart was enclosed by a double walled jacket, the

temperature of which was maintained by circulating water heated to 37.8°C. The

preparation was retrogradely perfused at constant pressure (By using peristaltic pump)

with Kreb’s-Henseleit (K-H) buffer (NaCl 118 mM; KCl 4.7 mM; CaCl2 2.5 mM;

MgSO4.7H20 1.2 mM; KH2PO4 1.2 mM; C6H12O6 11 mM), PH 7.4, bubbled with

95% O2 and 5%CO2 . Global ischemia was produced for 30 min. by closing the

inflow of Kreb’s Henseleit solution which was followed by 120 min. of reperfusion.

Coronary effluent was collected before ischemia, immediately, 5 min. and 30 min.

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after reperfusion for estimation of Lactate Dehydrogenase (LDH) and Creatine Kinase

(CK-MB).

4.10 Preconditioning of Rat Heart

a- Classical ischemic preconditioning

Isolated rat heart was subjected to four brief episodes of ischemia and reperfusion

(each episode comprised of 5 min. ischemia followed by 5 min. of reperfusion) after a

stabilization period of 10 min. Then, the heart was subjected to 30 min. ischemia

followed by 120 min. of reperfusion.

b- Delayed cardioprotection

Late cardioprotection in the rat was induced by administration of GSK-3� inhibitor

i.e. Lithium chloride (200 mg/kg i.p., Selenica et al., 2007), SB 216763 (0.6 mg/kg

i.p., Lai et al., 2006; Fu et al., 2007) and Indirubin-3 monooxime (0.4 mg/kg i.p.,

Sugihara et al., 2004; Wang et al., 2007) , 24 h before the isolation of heart.

4.11 Experimental Protocol

The animals were divided into following subgroups, each group comprised of six rats.

4.11.1 Classical ischemic preconditioning

a- Normal and diabetic rat heart.

Group 1: Sham control; n=6; Isolated normal rat heart preparation was stabilized for

10 min. and then perfused continuously with K-H buffer solution for 190 min. without

subjecting them to global ischemia and reperfusion.

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Group 2: Ischemia-reperfusion control; n=6; Isolated normal rat heart preparation

was allowed to stabilize for 10 min followed by 40 min of perfusion with K-H

solution. Then it was subjected to 30 min. global ischemia followed by 120 min. of

reperfusion.

Group 3: Ischemic preconditioning control; n=6; Isolated normal rat heart preparation

was allowed to stabilize for 10 min. and subjected to four cycles of ischemic

preconditioning, each cycle comprised of 5 min. global ischemia followed by 5 min.

reperfusion with K-H solution. Then the preparation was subjected to 30 min. global

ischemia followed by 120 min. of reperfusion.

Group 4, Ischemic Preconditioning (IPC) and atractyloside (20µM) treated normal

heart, n=6; Rat heart was reperfused with atractyloside (20µM) in the last episode of

reperfusion of IPC. Then the preparation was subjected to 30 min global ischemia

followed by 120 min of reperfusion.

Group 5: Ischemic preconditioning in diabetic rat heart; n=6; Isolated heart

preparation from diabetic rat was allowed to stabilize for 10 min. and subjected to

four cycles of ischemic preconditioning, each cycle comprised of 5 min. ischemia

followed by 5 min. reperfusion with K-H solution. Then the preparation was subjected

to 30 min. global ischemia followed by 120 min. of reperfusion.

Group 6, Lithium chloride (LiCl) (20mM) preconditioning in diabetic rat heart, n=6;

Isolated heart from diabetic rat was perfused for 5 min with K-H solution containing

LiCl (20mM) followed by 5 min perfusion with K-H solution. Four such cycles have

been employed for lithium chloride induced preconditioning. Then the preparation

was subjected to 30 min global ischemia followed by 120 min of reperfusion.

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Group 7, Indirubin-3 monooxime (1µM) preconditioning in diabetic rat heart, n=6;

Isolated heart from diabetic rat was perfused for 5 min with K-H solution containing

indirubin-3 monooxime (1µM) followed by 5 min perfusion with K-H solution. Four

such cycles have been employed for Indirubin-3 monooxime induced preconditioning.

Then the preparation was subjected to 30 min global ischemia followed by 120 min of

reperfusion.

Group 8, SB216763 (3µM) preconditioning in diabetic rat heart, n=6; Heart was

isolated from diabetic rat and was perfused for 5 min with K-H solution containing

SB216763 (3µM) followed by 5 min perfusion with K-H solution. Four such cycles

have been employed for SB216763 induced preconditioning. Then the preparation

was subjected to 30 min global ischemia followed by 120 min of reperfusion.

Group 9, Lithium chloride (LiCl) (20mM) and atractyloside (20µM) treated diabetic

rat heart, n=6; In last episode of perfusion of LiCl (20mM) preconditioning in diabetic

rat heart was perfused with atractyloside (20µM) followed by global ischaemia and

reperfusion as described in group 4.

Group 10, Indirubin-3 monooxime (1µM) and atractyloside (20µM) treated diabetic

rat heart, n=6; In last episode of perfusion of indirubin (1µM) preconditioning in

diabetic rat heart was perfused with atractyloside (20µM) followed by ischaemia and

reperfusion as described in group 4.

Group 11, SB216763 (3µM) and atractyloside (20µM) treated diabetic rat heart, n=6;

In the last episode of perfusion of SB216763 (3µM) preconditioning, heart of diabetic

rat was perfused with atractyloside (20µM). Rest of the procedure was similar to

group 4.

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b- Hyperlipidaemic rat heart.

Group 1: Ischemic preconditioning in hyperlipidaemic rat heart; n=6; Isolated heart

preparation from hyperlipidaemic rat was allowed to stabilize for 10 min. and

subjected to four cycles of ischemic preconditioning, each cycle comprised of 5 min.

ischemia followed by 5 min. reperfusion with K-H solution. Then the preparation was

subjected to 30 min. global ischemia followed by 120 min. of reperfusion.

Group 2, Lithium chloride (LiCl) (20mM) preconditioning in hyperlipidaemic rat

heart, n=6; Isolated heart from hyperlipidaemic rat was perfused for 5 min with K-H

solution containing LiCl (20mM) followed by 5 min perfusion with K-H solution.

Four such cycles have been employed for lithium chloride induced preconditioning.

Then the preparation was subjected to 30 min global ischemia followed by 120 min of

reperfusion.

Group 3, Indirubin-3 monooxime (1µM) preconditioning in hyperlipidaemic rat heart,

n=6; Isolated heart from hyperlipidaemic rat was perfused for 5 min with K-H

solution containing indirubin-3 monooxime (1µM) followed by 5 min perfusion with

K-H solution. Four such cycles have been employed for Indirubin-3 monooxime

induced preconditioning. Then the preparation was subjected to 30 min global

ischemia followed by 120 min of reperfusion.

Group 4, SB216763 (3µM) preconditioning in hyperlipidaemic rat heart, n=6; Heart

was isolated from hyperlipidaemic rat and was perfused for 5 min with K-H solution

containing SB216763 (3µM) followed by 5 min perfusion with K-H solution. Four

such cycles have been employed for SB216763 induced preconditioning. Then the

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preparation was subjected to 30 min global ischemia followed by 120 min of

reperfusion.

Group 5, Lithium chloride (LiCl) (20mM) and atractyloside (20µM) treated

hyperlipidaemic rat heart, n=6; In last episode of perfusion of LiCl (20mM)

preconditioning in hyperlipidaemic rat heart was perfused with atractyloside (20µM).

Then the preparation was subjected to 30 min global ischemia followed by 120 min of

reperfusion.

Group 6, Indirubin-3 monooxime (1µM) and atractyloside (20µM) treated

hyperlipidaemic rat heart, n=6; In last episode of perfusion of indirubin (1µM)

preconditioning in hyperlipidaemic rat heart was perfused with atractyloside (20µM)

and followed by 30 min global ischemia followed by 120 min of reperfusion.

Group 7, SB216763 (3µM) and atractyloside (20µM) treated hyperlipidaemic rat

heart, n=6; In the last episode of perfusion of SB216763 (3µM) preconditioning, heart

of hyperlipidaemic rat was perfused with atractyloside (20µM) followed by 30 min

global ischemia followed by 120 min of reperfusion.

4.11.2 Delayed Cardioprotection or Second Window of Protection (SWOP)

a- Second window of protection (SWOP) in diabetic rat

Group 1, I/R control, n=6; Normal rat was administered DMSO, 24 h before the

isolation of heart. Isolated heart was perfused on the Langendorff apparatus, just after

the 10 min of stabilization heart was subjected to 30 min of ischemia followed by 120

min of reperfusion.

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Group 2, Vehicle control, n=6; DMSO was administered 24 h before the isolation of

heart in diabetic rat. Isolated heart was perfused on the Langendorff apparatus, just

after the 10 min of stabilization heart was subjected to 30 min of global ischemia

followed by 120 min of reperfusion.

Group 3, Lithium chloride (LiCl), 12 h before in diabetic rat, n=6; Diabetic rat was

administered LiCl (200 mg/kg, i.p.) 12 h before the isolation of heart. Isolated heart

was perfused on the Langendorff apparatus, just after the 10 min of stabilization heart

was subjected to 30 min of ischemia followed by 120 min of reperfusion.

Group 4, Indirubin-3 monooxime (Indirubin), 12 h before in diabetic rat, n=6;

Diabetic rat was administered Indirubin (0.4 mg/kg, i.p.) 12 h before the isolation of

heart. Isolated heart was perfused on the Langendorff apparatus, just after the 10 min

of stabilization heart was subjected to 30 min of ischemia followed by 120 min of

reperfusion.

Group 5, SB 216763, 12 h before in diabetic rat, n=6; Diabetic rat was administered

SB 216763 (0.6 mg/kg, i.p.) 12 h before the isolation of heart. Isolated heart was

perfused on the Langendorff apparatus, just after the 10 min of stabilization heart was

subjected to 30 min of ischemia followed by 120 min of reperfusion.

Group 6, Lithium chloride (LiCl), 24 h before in diabetic rat, n=6; Diabetic rat was

administered LiCl (200 mg/kg, i.p.) 24 h before the isolation of heart. Isolated heart

was perfused on the Langendorff apparatus, just after the 10 min of stabilization heart

was subjected to 30 min of ischemia followed by 120 min of reperfusion.

Group 7, Indirubin-3 monooxime (Indirubin), 24 h before in diabetic rat, n=6;

Diabetic rat was administered Indirubin (0.4 mg/kg, i.p.) 24 h before the isolation of

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heart. Isolated heart was perfused on the Langendorff apparatus, just after the 10 min

of stabilization heart was subjected to 30 min of ischemia followed by 120 min of

reperfusion.

Group 8, SB 216763, 24 h before in diabetic rat, n=6; Diabetic rat was administered

SB 216763 (0.6 mg/kg, i.p.) 24 h before the isolation of heart. Isolated heart was

perfused on the Langendorff apparatus, just after the 10 min of stabilization heart was

subjected to 30 min of ischemia followed by 120 min of reperfusion.

Group 9, Lithium chloride (LiCl), 24 h before in Quercetin treated diabetic rat, n=6;

Diabetic rat was administered Quercetin (4 mg/kg, i.p.) 25 h before the isolation of

heart and LiCl (200 mg/kg, i.p.) was administered 24 h before isolation of heart.

Isolated heart was perfused on the Langendorff apparatus, just after the 10 min of

stabilization heart was subjected to 30 min of ischemia followed by 120 min of

reperfusion.

Group 10, Indirubin-3 monooxime (Indirubin), 24 h before in Quercetin treated

diabetic rat, n=6; Diabetic rat was administered Quercetin (4 mg/kg, i.p.) 25 h before

the isolation of heart followed by administration of Indirubin (0.4 mg/kg, i.p.) 24 h

before the isolation of heart. Isolated heart was perfused on the Langendorff

apparatus, just after the 10 min of stabilization heart was subjected to 30 min of

ischemia followed by 120 min of reperfusion.

Group 11, SB 216763, 24 h before in Quercetin treated diabetic rat, n=6; Diabetic rat

was administered Quercetin (4 mg/kg, i.p.) 25 h before the isolation followed by

administration of SB 216763 (0.6 mg/kg, i.p.) 24 h before the isolation of heart.

Isolated heart was perfused on the Langendorff apparatus, just after the 10 min of

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stabilization heart was subjected to 30 min of ischemia followed by 120 min of

reperfusion.

b- Second window of protection (SWOP) in hyperlipidaemic rat

Group 1, Vehicle control, n=6; DMSO was administered 24 h before the isolation of

heart in hyperlipidaemic rat. Isolated heart was perfused on the Langendorff

apparatus, just after the 10 min of stabilization heart was subjected to 30 min of global

ischemia followed by 120 min of reperfusion.

Group 2, Lithium chloride (LiCl), 12 h before in hyperlipidaemic rat, n=6;

Hyperlipidaemic rat was administered LiCl (200 mg/kg, i.p.) 12 h before the isolation

of heart. Isolated heart was perfused on the Langendorff apparatus, just after the 10

min of stabilization heart was subjected to 30 min of ischemia followed by 120 min of

reperfusion.

Group 3, Indirubin-3 monooxime (Indirubin) 12 h before in hyperlipidaemic rat, n=6;

Hyperlipidaemic rat was administered Indirubin (0.4 mg/kg, i.p.) 12 h before the

isolation of heart. Isolated heart was perfused on the Langendorff apparatus, just after

the 10 min of stabilization heart was subjected to 30 min of ischemia followed by 120

min of reperfusion.

Group 4, SB 216763, 12 h before in hyperlipidaemic rat, n=6; Hyperlipidaemic rat

was administered SB 216763 (0.6 mg/kg, i.p.) 12 h before the isolation of heart.

Isolated heart was perfused on the Langendorff apparatus, just after the 10 min of

stabilization heart was subjected to 30 min of ischemia followed by 120 min of

reperfusion.

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Group 5, Lithium chloride (LiCl), 24 h before in hyperlipidaemic rat, n=6;

Hyperlipidaemic rat was administered LiCl (200 mg/kg, i.p.) 24 h before the isolation

of heart. Isolated heart was perfused on the Langendorff apparatus, just after the 10

min of stabilization heart was subjected to 30 min of ischemia followed by 120 min of

reperfusion.

Group 6, Indirubin-3 monooxime (Indirubin) 24 h before in hyperlipidaemic rat, n=6;

Hyperlipidaemic rat was administered Indirubin (0.4 mg/kg, i.p.) 24 h before the

isolation of heart. Isolated heart was perfused on the Langendorff apparatus, just after

the 10 min of stabilization heart was subjected to 30 min of ischemia followed by 120

min of reperfusion.

Group 7, SB 216763, 24 h before in hyperlipidaemic rat, n=6; Hyperlipidaemic rat

was administered SB 216763 (0.6 mg/kg, i.p.) 24 h before the isolation of heart.

Isolated heart was perfused on the Langendorff apparatus, just after the 10 min of

stabilization heart was subjected to 30 min of ischemia followed by 120 min of

reperfusion.

Group 8, Lithium chloride (LiCl), 24 h before in Quercetin treated hyperlipidaemic

rat, n=6; Hyperlipidaemic rat was administered Quercetin (4 mg/kg, ip) 25 h before

the isolation of heart followed by administration of LiCl (200 mg/kg, i.p.) 24 h before

isolation of heart. Isolated heart was perfused on the Langendorff apparatus, just after

the 10 min of stabilization heart was subjected to 30 min of ischemia followed by 120

min of reperfusion.

Group 9, Indirubin-3 monooxime (Indirubin), 24 h before in Quercetin treated

hyperlipidaemic rat, n=6; Hyperlipidaemic rat was administered Quercetin (4 mg/kg,

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i.p.) 25 h before the isolation of heart followed by administration of Indirubin (0.4

mg/kg, i.p.) 24 h before the isolation of heart. Isolated heart was perfused on the

Langendorff apparatus, just after the 10 min of stabilization heart was subjected to 30

min of ischemia followed by 120 min of reperfusion.

Group 10, SB 216763 24 h before in Quercetin treated hyperlipidaemic rat, n=6;

Hyperlipidaemic rat was administered Quercetin (4 mg/kg, i.p.) 25 h before the

isolation of heart followed by administration of SB 216763 (0.6 mg/kg, i.p.) 24 h

before the isolation of heart. Isolated heart was perfused on the Langendorff

apparatus, just after the 10 min of stabilization heart was subjected to 30 min of

ischemia followed by 120 min of reperfusion.

4.12 Reagents and Chemicals

Streptozotocin (50 mg/kg i.p.) (Sigma Chemicals, St. Louis, USA) was used to induce

diabetes mellitus where as cholesterol (CDH Pvt. Ltd., New Delhi) and cholic acid

(Himedia Pvt. Ltd., Mumbai) were used for induction of experimental

hyperlipidaemia in rats. Lithium chloride (LiCl) (Central Drug House (P) Ltd.

Mumbai India) and atractyloside potassium (Sigma Chemicals, St. Louis, MO, USA)

were dissolved in minimum quantity of distilled water and added to Kreb’s Henseleit

(K-H) solution. 3-(2, 4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2, 5-

dione (SB216763) (3µM), and Indirubin-3 monooxime (Sigma Chemicals, St. Louis,

MO, USA) were dissolved in DMSO; the final concentration of DMSO in K-H

solution was 0.02%. For injection LiCl was dissolved into saline, Indirubin-3

monooxime and SB 216763 and Quercetin were dissolved in minimum quantity of

DMSO as a vehicle. The 1% w/v solution of TTC Stain (CDH Pvt. Ltd., New Delhi)

was prepared in Tris-chloride buffer (CDH Pvt. Ltd., New Delhi) was used to measure

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infarct size. The LDH enzymatic estimation kit and CK-MB enzymatic estimation kit

was purchased from Vital Diagnostics (P) Ltd., Mumbai, India. For the estimation of

All other reagents used in this study were of analytical grade and always freshly

prepared before use.

4.13 Statistical Analysis

All values were expressed as mean ± standard error of maen (SEM). Statistical

analysis was performed using Sigmastat and Graphpad Prism Software. The data

obtained from the various groups were statistically analysed using Student’s t-test,

one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison

test. P < 0.05 was considered to be statistically significant.

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Diagrammatic Representation of Experimental Protocol

a. Classical Ischemic Preconditioning

Fig. 1 Normal and diabetic rat heart.

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Fig. 2 Hyperlipidaemic rat heart

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b. Delayed Cardioprotection or Second Window of Protection (SWOP)

Fig. 3- Second window of protection (SWOP) in diabetic rat heart

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Fig. 4- Second window of protection (SWOP) in hyperlipidaemic rat