Ischaemic preconditioning protected skeletal myotubes against the effects of ischaemia-reperfusion in vitro. This protection was associated with increased Nrf2 signalling. Ischaemic preconditioning (IPC) is a well recognised and powerful phenomenon where a tissue becomes more tolerant to a period of prolonged ischaemia when it is first subjected to short bursts of ischaemia/reperfusion. While much is known about the ability of ischaemic preconditioning to protect myocardial tissue against ischaemia-reperfusion injury, its potential to confer benefit in an orthopaedic setting by protecting skeletal muscle remains relatively unexplored to date. One mechanism by which ischaemic preconditioning may induce protection is through a reduction in oxidative stress. Reactive oxygen species (ROS) are generated both during prolonged ischaemia and also upon reperfusion by infiltrating neutrophils, thereby leading to an increase in oxidative stress. The transcription factor, NF-E2-related factor 2 (Nrf2), is a key regulator of the cells response to oxidative stress as it regulates the expression of a network of anti-oxidant/detoxifying enzymes. Nrf2 signalling has recently been shown to protect against ischaemia-reperfusion injury in both a kidney cell line and in liver biopsies, indicating that this transcription factor may play a key role in the protection provided by ischaemic preconditioning. To date, the involvement of Nrf2 in the response of skeletal muscle to ischaemia-reperfusion has not been investigated. Thus, the aims of this study were to investigate the ability of ischaemic preconditioning to protect skeletal myotubes against ischaemia-reperfusion and to determine the role of Nrf2 signalling in this protection.Summary Statement
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. 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.Introduction
Methods
Local anaesthetic has been reported to have a potentially detrimental effect on human chondrocytes both in vitro and in vivo. Due to chondroproliferative effects, magnesium may be an alternative intra-articular analgesic agent following arthroscopy. We aimed to examine the dose response effect of commonly used local anaesthetics on chondrocyte viability and also to report on the effect of adding magnesium to the local anesthetic agent. Human chondrocytes were grown under standard culture conditions. Cells were exposed to either lignocaine (0.5, 1, 2%), levobupivacaine (0.125, 0.25, 0.5%), bupivacaine (0.125, 0.25, 0.5%) or ropivacaine (0.1875, 0.375, 0.75%) for 15 minutes. Cells were also exposed to a local anesthetic agent with the addition of magnesium (10, 20, or 50%). Cells exposed to culture media or saline served as controls. The MTS assay was used to assess cell viability 24 hours after exposure. One-way ANOVA were used to test for statistical significance.Introduction
Methods
