Abstract
Introduction
Ischaemic preconditioning (IPC) is a phenomenon whereby a tissue is more tolerant to an insult if it is first subjected to short bursts of sublethal ischaemia and reperfusion. The potential of this powerful mechanism has been realised in many branches of medicine where there is an abundance of ongoing research. However, there has been a notable lack of development of the concept in Orthopaedic surgery. The routine use of tourniquet-controlled limb surgery and traumatic soft tissue damage are just two examples of where IPC could be utilised to beneficial effect in Orthopaedic surgery.
Methods
We conducted a randomized controlled clinical trial looking at the role of a delayed remote IPC stimulus on a cohort of patients undergoing a total knee arthroplasty (TKA). We measured the effect of IPC by analysing gene expression in skeletal muscle samples from these patients. Specifically we looked at the expression of Heat shock protein-90 (HSP-90), Catalase and Cyclo-oxygenase-2 (COX-2) at the start of surgery and at one hour into surgery. Gene analysis was performed using real time polymerase chain reaction amplification. As a second arm to the project we developed an in-vitro model of IPC using a human skeletal muscle cell line. A model was developed, tested and subsequently used to produce a simulated IPC stimulus prior to a simulated ischaemia-reperfusion (IR) injury. The effect of this on cell viability was investigated using crystal violet staining.
Results
In the clinical arm of the study 4 patients were randomized to a control group and 4 randomized to IPC. Operative and post-operative periods were without any adverse incident. For each gene in question there was a different pattern in expression. COX-2 showed an initial up-regulation of 1.43 (p=0.83) at the start of surgery and a subsequent down-regulation of 0.07 (p=0.01) at one hour into surgery. Catalase expression was lower than control at the start of surgery (0.62, p= 0.46) and at one hour into surgery (0.5, p=0.1). HSP-90 expression was initially lower than control at the start of surgery (0.59, p= 0.07) then up-regulated at one hour into surgery (1.13, p=0.62).
In the in-vitro section of the study we found that 15 hours of simulated ischaemia was required for a cell death of approximately 50 % (p=0.00001). The introduction of a simulated IPC stimulus increased cell death at a 1 hour reperfusion time-point (IPC group had 18% more cell death than IR group, p=0.003) and at a 24 hour reperfusion time-point (IPC group had 19% more cell death than IR group, p= 0.00001). At a 72 hours reperfusion time-point the IPC group had a 30% greater survival than the IR group (p=0.000006)
Conclusion
Our clinical study was subject to small sample size. Despite this it suggests a particular importance of COX-2 in the IPC mechanism. The in-vitro model we developed is an essential resource for further studies into IPC in Orthopaedic Surgery. Preliminary results from this model point towards the ‘second window of protection’ of IPC as a stronger phenomenon than immediate preconditioning.
