3.10 CARDIOTONIC EXPERIMENTS
3.11.1 Chemically induced models of arrhythmia
To test the anti-arrhythmic effect of our test compounds on the chemically induced arrhythmia, six anaesthetised rats were used per compound. The rats were anaesthetised by intraperitoneal injection of sodium thiopentone (40 mg/kg body weight). All the chemicals used to induce the arrhythmias were administered through the cannulated right jugular vein.
The arrhythmias were induced 20 minutes after intraperitoneal administration of a test compound. This was done like this to check whether the test compounds had any prophylactic potential on the occurrence of the chemically induced-arrhythmias. Before the application of the test compound, control ECG of each arrhythmogenic chemical was registered.
3.11.1.1 BaCh -induced arrhythmia
BaCh-induced arrhythmias were used to investigate the antiarrhythmic action of our test compound on the rhythmicity of the myocardium and prolongation of the action potential by decreasing the efflux of potassium. Unlike the dosage of 2 mg/kg as was used by
Petkov and Manolov (1972), a dosage of 1 mg/kg was used in this study because it was found in preliminary experiments that the former dose killed the rats. BaCh (1 mg/kg) was administered intravenously 20 minutes after intraperitoneal administration of the test compounds. Control ECG of BaCh alone was recorded so as to compare it with the ECG obtained from the BaCh applied on the background of the test compound.
3.11.1.2 eaCh - induced arrhythmia
CaCh-induced arrhythmia was used to evaluate possible antiarrhythmic calcium antagonistic effects of the test compounds. The arrhythmia was initiated by administering a 2.5 % CaCh solution (140 mg/kg) (Papp et al., 1966) on the background of the test compound that was administered 20 minutes before. Changes in ECG were recorded after the application of CaCh before and on the background of the test compounds
3.11.1.3 Adrenaline-induced arrhythmia
Adrenaline-arrhythmia in rats were induced through intravenous injection of adrenaline in a dose of 10 Ilg/kg for the evaluation of ~-blocking activity (Barrett and Cullum, 1968). Just like above, changes in ECG were recorded after the application of CaCh before and on the background of the test compounds.
3.11.2- Ischemia-reperfusion arrhythmia
To evaluate the effect of the test compounds on the ischaemia-reperfusion model of arrhythmia, six anaesthetised rats were used per each compound. The rats were
anaesthetised by intraperitoneal injection of sodium thiopentone (40 mg/kg body weight).
The trachea was cannulated and artificial respiration was achieved by a positive-pressure rodent respirator (Phipps and Bird Inc., USA). This artificial respiration used atmospheric air at a tidal volume of approximately 5 ml at a rate of 25 breaths per minute. Throughout the whole experiment, changes in ECG (ECG-Cl, ESAOT Biomedica, Italy) were monitored from standard limb lead II with needle electrodes. Haemodynamic parameters (mean arterial pressure and heart rate) were monitored from a cannulated carotid artery with a catheter connected to a Statham MLT 0380 pressure transducer (Ad instruments), compatible with the PowerLab System ML410/W, Australia.
Regional myocardial ischaemia model of arrhythmia and the reperfusion model of arrhythmia were used since as has been stated in chapter two, these two arrhythmias are initiated by different mechanisms (Curtis and Hearse, 1989). Ischaemia-reperfusion arrhythmias were induced according to the method described by Kane et al., (1984) with slight modifications. In anaesthetised and artificially ventilated rats, the chest was opened using a left thoracotomy, followed by sectioning of ribs 4 and 5, approximately 2 mm to the left of the sternum. Positive pressure artificial respiration was started immediately to maintain normal
peo
l, POl and pH parameters. After incising the pericardium, the heart was exposed out of the chest using gentle pressure on the rib cage. A 6/0 silk non- traumatic suture was passed through the epicardial layer under the left main coronary artery, about 2 mm from the origin. A small plastic button was threaded through the ligature and placed in contact with the heart. The end of the ligature was passed through a small vinyl tube and exteriorised. The heart was massaged before being replaced in the chest, and then thorax was closed after removing the residual air to avoid pneumothorax.The animal was left to recover for 15 minutes. Any animal in which this procedure
produced arrhythmias or a sustained fall in blood pressure to less than 70 rrunHg was discarded. Applying tension to the ligature could then occlude the artery and reperfusion was achieved by releasing the tension
Two types of studies were undertaken for the antiarrhythmic actions of our test compounds.
In the first type of experiments, the artery was occluded for 30 minutes to induce experimental ischaemia, and then the tension was released for 20 minutes to produce reperfusion. In this study, any heart that was not in sinus rhythm during 2 seconds just before and at the moment of reperfusion was excluded from the reperfusion study and was immediately replaced (Walker et ai., 1988; Curtis and Hearse, 1989). The ischaemia were done either 20 minutes after intraperitoneal administration of the test compound or 10 minutes after intravenous injection of the antiarrhythmic drugs. In the second set of experiments, the drug pre-treatment was replaced by ischaemic preconditioning produced by three 3-minutes occlusion periods interspaced with 3-mnutes reperfusion period. After the last 3-mute reperfusion, the artery was occluded for 30 minutes (to produce ischaemia) followed by 20 minutes reperfusion.
To determine whether reperfusion was in fact occurring, 0.1 ml 100-1 (body weight) of a 10
% solution of fluorescein (Sigma) was administered through the jugular vein immediately before sacrificing the animals. The heart was rapidly removed from the chest and placed in ice cold KCI solution (10 % weight/volume) for 1 min to cause cardioplegia. A gross examination of the heart was made under ultraviolet light to determine whether areas of non-perfused myocardium were present. These appeared dark blue under ultraviolet light whereas perfused were stained green. The hearts were then photographed under ultraviolet light to give a permanent record. As a comparison, this procedure was also carried out in
hearts with a permanent ligation, after 30 minutes of ischaemia, and in control non- ischaernic hearts.
3.12- STATISTICAL ANALYSIS
All results were evaluated originally as means ± SEM. After statistical analysis, the results were presented, when indicated, as % of the baseline value. For all analyses, INSTAT V2.04 program was used, including one-way and two-way ANOVA, (-test and Chi-square test. A p-value less than 0.05 was considered to be statistically significant.