Free radicals, such as reactive oxygen species (ROS) which are released abruptly after deflation of an ischaemic tourniquet, cause reperfusion injuries. Ischaemic precondition (IPC), however, can reduce the injury. In clinical practice, the sequential application and release of tourniquets is often used in bilateral total knee replacement (TKR) to obtain a clearer operative field, but the effects on the production of free radicals and lipid peroxidation have not been studied. In this study, we have observed the production of free radicals and the subsequent lipid peroxidation in bilateral TKR with sequential application of a tourniquet to examine the effect of IPC. Patients undergoing elective TKR under intrathecal anaesthesia were studied. Blood samples were obtained after spinal anaesthesia, one minute before and five and 20 minutes after release of each tourniquet. We used the lucigenin chemiluminescence analysis and the phosphatidylcholine hydroperoxide (PCOOH) assay to measure the production of ROS and lipid peroxidation. Our results showed that production of ROS significantly increased at five and 20 minutes after release of the first tourniquet and at five minutes after release of the second tourniquet, but returned to normal at 20 minutes after the second reperfusion. The peak production of ROS was at 20 minutes after the first reperfusion; lipid peroxidation did not change significantly. We conclude that in spite of significant production of ROS after the release of tourniquet, the IPC phenomenon occurs during bilateral TKR with sequential application of a tourniquet

Aim. This aim of this study was to investigate apoptosis, reactive oxygen species (ROS), and their upstream markers in Anteromedial Gonarthrosis (AMG). Methods. Ten resection specimens, from patients undergoing unicompartmental knee replacement for AMG, and ten control specimens, collected from vascular disease patients undergoing above knee amputation, were used. Routine histology and immunohistochemical studies were conducted for Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Active Caspase 3, Cytochrome C, Active Bax, Bim, 3-Nitrotyrosine and Forkhead box O3A (FOXO 3A). Results. Cell death was shown predominantly in the surface layer of chondrocytes of damaged cartilage (p<0.001). There was a significant difference in TUNEL staining between regions (p=0.001). This ranged from 26% (most damaged) to 4% (undamaged) and was significantly higher (p<0.001) in AMG compared to the control samples which showed an average of 2% TUNEL overall. Upstream markers of apoptosis (Active Caspase 3, Cytochrome C, Active Bax), assessed qualitatively, were present in a similar distribution to that of TUNEL staining. 3-Nitrotyrosine, an indicator of ROS mediated damage, was also shown to be a predominantly surface phenomenon. There was a significant difference (p<0.001) between regions, ranging from 58% (most damaged) to 10% (undamaged). Again, this was significantly higher that the control samples (p<0.001). In line with indicators of ROS mediated damage, Bim and FOXO3A were also detected. Discussion. In AMG, apoptosis and ROS appear to be a part of the biological process leading to cartilage degeneration. Such cellular responses in ‘stressed’ chondrocytes provide possible targets for disease modification, thus delaying or preventing the need for joint arthroplasty. Further work is required to demonstrate these pathways and the effects of intervention