Male Sprague Dawley rats will be housed in animal conditions by maintaining standard conditions of 25 degree Celsius room temperature, 12hour light/day cycle and 40-50% humidity.The rats will be provided with free access to food and water ad Libitum.The rats will be made unconscious using intra peritoneal injections of 80 mg/kg Ketamine and 10 mg/kg Xylazine. The anaesthesia will be sustained using supplementary injections.
The procedure of tracheotomy will be performed followed by endotracheal intubation using a 16 gauge catheter. The entire procedure will be carried out on a heating pad to maintain the body temperature at 37 degree Celsius . A rectal thermometer will be used to monitor the body temperature.Mechanical ventilation will be supplied to the rats by room air supplemented with oxygen such that a stroke volume of 1-1.5 ml/100g of rat weight and 52 strokes per minute. This will enable in sustaining the normal range of arterial blood pressure and blood pH. A catheter which is filled with 10IU/ml heparinised saline will be introduced into the right carotid artery.
This will assist in collecting blood samples and measuring blood pressure at different times if follow up. ECG waves and arterial blood pressure will be assessed through data acquisition system.A cannula will be inserted into the tail vein to dispense Evans blue dye.The heart of the rat is exposed by performing left thoracotomy between third and fourth inter coastal spaces.The incision made will be expanded using blunt ended retractors.
The pericardial sac around the heart will be slit in order to identify the left descending coronary artery. After the determination of the position of left coronary descending artery, a 6-0 silk ligature will be passed around the artery a little distant from the origin site. The two ends if the ligature will be tied together.Partial closure of the chest will be executed. The animals will be given a period of 30 minutes to recover from the effects during which heart relate and blood pressure will also become stabilised.
After blood pressure and heart rate will become stable hemodynamic parameters will be measured for a time period of 15 minutes.The experimental protocol will then be initiated.The division of the rats will be done into 9 groups.
The rats will undergo ischemia for 30 minutes by occlusion of coronary artery that occurs when tension is applied to the silk ligature.The study consists of three protocols. Protocol 1 includes control group animals. Protocol 2 includes oxytocin group animals.
Protocol 3 involves Atosiban (ATO) group animals. 30 minutes before the occlusion of left anterior descending coronary artery intraperitoneal administration of : saline in control group animals, different doses of Oxytocin (OT) (in 0.015,0.03,0.
06 and 1.2 ?g/rat) in Oxytocin group and Atosiban which is an antagonist of Oxytocin receptor will be used in three groups (in ATO+OT 0.015, ATO+OT 0.
03 and ATO+OT 1.2 ?g). The Atosiban will be administered 10 minutes earlier to Oxytocin.Following this, the Electrocardiogram will be analysed to determine the changes in the ejection fraction. The recording of the echocardiographic parameters will be completed by following American society if echocardiography guidelines.The changes in number and duration of arrhythmias, if any will also be determined in accordance with Lambeth conventions.The statistical analysis will be performed using parametric as well as non parametric tests.The assessment of the morphological changes in the myocytes will be conducted.
Following the completion of experimental protocols blood samples will be collected and coronary artery reocclusion will take place. The Evans blue dye will then be injected through the cannula into the tail vein inorder to detect the area at risk.The heart will then be removed and washed rapidly in cold water.
After freezing the hearts for 60 minutes at -20 degree Celsius, the hearts will be cut into transverse sections if 2mm measurement. These slices will be incubated in Triphenyltetrazolium Chloride (TTC) stain which will be pre-warmed in phosphate buffered saline for a time of 15 minutes at 37 degree Celsius. These slices will be fixed in 10% formalin for two days to enhance the contrast of the applied stains. The transverse sections will be observed under a microscope to identify the morphological changes and the area at risk.
The blood samples collected after the completion of every experiment will be used for the biochemical analysis of myocardial specific enzymes : Creatine-kinase MB isoenzyme and lactate dehydrogenase. The activity if these enzymes will be measure using a commercial kit.The hemodynamic parameters like blood pressure and Heart rate will be measured and recorded at all the various steps of the experiment.