Discussion

Rat has been employed extensively to characterise the pathophysiology of ischaemic heart disease. The rat model of myocardial ischaemia/reperfusion injury was first characterised by Johns and Olson in (1954). It has then become the model of choice (Curtis et al., 1987) to study myocardial infarction and associated arrhythmia during ischaemia and reperfusion. This model has several advantages over other animal models including simple instrumentation requited compared to the larger animals (Ytrehus, 2000). In addition, cannulation of the arterial and venous lines enables monitoring of arterial-venous differences throughout the ischaemia/reperfusion experiments, drug delivery and blood sampling compared to mouse model (Ytrehus, 2000). Rat model also affords the possibility to expose the heart either by left or mid-line thoracotomy to occlude the left coronary artery with minimum interventions compared to large animals (Hearse and Sutherland, 2000). Permanent occlusion of the rat left coronary artery has also been used to develop heart failure and study post-infarction modelling and inflammation (Qvigstad et al., 2005). Furthermore, rat heart also has a sparse collateral circulation (Maxwell et al., 1987) which is important for reproducible ischaemic area (risk zone) that determines the severity of arrhythmia and infarct size (Curtis et al., 1987). Therefore, a reperfusion phase is essential to study the infarct development and limitation (Ytrehus, 2006). Nevertheless, there are some limitations to the rat model which have to be acknowledge when compared to human. The rat considerably has a high heart rate compared to human. In addition, rat’s heart has a short action potential which means that there is a long inter-AP duration (i.e. prolonged phase 0). Taken together, these elements have a considerable impact on the incidence of

ventricular arrhythmias and the reversibility of ischaemia- and reperfusion- induced VF. Furthermore, the heart size/body size is higher in rat compared to this ration in human. Transient ligation of the proximal left coronary artery for 30 minutes followed by reperfusion leads to an infarct which is reported to be 40- 60% of the ischaemic area (are at risk) (Dillmann, 2008), which is in line with our results.

Another characteristic feature of the in vivo rat model is the pattern of ischaemia- and reperfusion-induced arrhythmias which is similar to that seen in large animals (dog and pig) compared to other small animal models (mouse and rabbit) in terms of vulnerability and reversibility. Regional ischaemia induces a first phase of arrhythmias after 4-5 minutes which lasts up to 15 minutes of ischaemia; a second phase of arrhythmia after 1.5-2.5 hours of occlusion; and a third phase occurs after 24 hours (Clark et al., 1980, Curtis et al., 1987). In our study, the first phase of ischaemia-induced arrhythmias was experienced only. Reperfusion also induces even more severe arrhythmias in comparison with ischaemia-induced arrhythmias but over a short period (first 1-5 minutes of reperfusion). However, variability in the incidence of ventricular tachycardia (VT), ventricular fibrillation (VF) and mortality emphasises that researchers need to randomise control experiments parallel to the treated group aiming to minimise that variation and to avoid any possible bias (Hearse and Sutherland, 2000).

The preliminary series of experiments were used to optimise the in vivo model of myocardial ischaemia/reperfusion injury in terms of haemodynamic parameters, successful induction of ischaemia and reperfusion, achieving a survivable infarct size, and having sufficient delineation for infarct size

determination. Regional myocardial ischaemia was induced in 60.6 ± 2.8 % of the total ventricular volume of the heart. Ischaemia was established by a transient occlusion of the left coronary artery for 30 minutes and followed by 2 hours of reperfusion. It should be acknowledged here that the AAR is relatively large which might have its implications on the resulted infarction. The incidence and pattern of VT and VF during ischaemia did not significantly vary with an onset 4-5 minutes that is consistent with others (Clark et al., 1980). Irreversible VF during ischaemia and shortly after 30 seconds of reperfusion was the main cause of death during ischaemia/reperfusion protocol. Myocardial infarction resulting from this protocol represented 50.7 ± 2.6 % of the area at risk which is consistent with other investigators using the same protocol (Wajima et al., 2006, Baker et al., 2007, De Paulis et al., 2013). Infarct size quantification was performed using a dual staining technique with Evans’ blue and triphenyltetrazolium chloride. This technique has been used routinely in myocardial infarction studies where regional myocardial ischaemia was induced. Indirect delineation of the ischaemic myocardium is performed by staining the non-ischaemic area with Evans’ blue to ascertain that the area at risk was similar between experimental groups (Black and Rodger, 1996). Infarcted tissue was then demarcated using TTC which precipitates as white particles in the dead (infarcted) tissue within the territories of the area at risk.

Ischaemic preconditioning (IPC) is the first cardioprotective mechanical manoeuvre introduced by Murry et al (1986). In the canine model, Murry et al. (1986) reported that applying a series of ischaemic episodes before the onset of long ischaemia can render the heart more resistant to ischaemia/reperfusion injury. The cardioprotective effects of IPC were then demonstrated in different

animal models including rabbit (Liu et al., 1991), rat (Li and Kloner, 1993), mouse (Suveren et al., 2012), swine (Schott et al., 1990) and in human (Kloner and Yellon, 1994). Therefore, we utilised IPC in the second phase of this study as a positive control to validate the model and ascertain that the cardioprotection of IPC can be detected. Two cycles of 3 minutes ischaemia/3 minutes reperfusion before the index ischaemia caused a significant limitation in the infarct size by 67%. It also showed a powerful antiarrhythmic effect on both ischaemia- and reperfusion-induced VT and VF. None of the preconditioned hearts suffered from VF during ischaemia in comparison to the control group. It is worth noting that this IPC protocol (2x3 min IPC cycles) is non-standard protocol and it is first time to report cardioprotection with this protocol in an in vivo rat model of myocardial ischaemia/reperfusion injury. Accordingly, it needs to be acknowledged that whether other IPC protocols could show similar cardioprotection is yet to be characterised. These results confirm that the established model can be used as a preclinical model and it is valid for screening potential cardioprotective agents.