Pharmacological preconditioning to improve outcome in free tissue transfer
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Introduction. Free tissue transfer is the 'gold standard' of surgical care for patients requiring composite tissue reconstruction when local options are unavailable or unsuitable. It is a form of autologous transplant wherein composite tissue is harvested from a distant site and used to reconstruct the primary defect. The flap is rendered ischaemic following transection of its vascular pedicle until successful anastomosis with the recipient vessels is completed. Ischaemia depletes cellular ATP, lowers pH and strains cellular homeostatic mechanisms. The only way to halt the inevitable progression to cell death is by reperfusion. However, reperfusion per se initially worsens the injury through the influx of inflammatory cells and mediators. This biphasic injury is named ischaemia reperfusion injury (IRI) and is characterized by microcirculatory dysfunction primarily mediated by oxidative stress. This can lead to inadequate perfusion and ultimately tissue necrosis. IRI occurs in all transplants, is unavoidable and has no treatment. Preconditioning is an intervention performed before a known event that improves the outcome of that event. The elective nature of transplants permits such interventions to be executed. Haem-oxygenase 1 (HO-1) is a cytoprotective enzyme that is up-regulated in response to diverse stressors including oxidative stress. Haem arginate (HA) is a potent inducer of this enzyme. Pharmacological preconditioning with HA has been shown to reduce IRI and improve clinical outcome in models of visceral IRI. Aim: to investigate whether HA could be used to improve outcome in myocutaneous flaps. Objectives: (1) to establish a reliable model of myocutaneous IRI (2) to assess the effects of pharmacological preconditioning with HA on clinical outcome measures and (3) to investigate the mechanisms underlying the effects of HA preconditioning demonstrated in the in vivo model by in vitro work. Methods. An in situ transverse rectus abdominis myocutaneous (TRAM) flap was developed. Forty male, Lewis rats were randomly assigned to receive IV: Control (NaCl); HA; HA + tin mesoporphyrin (SnMP, an HO-1 inhibitor) and SnMP alone. Laser Doppler imaging (LDI) scans were performed to assess perfusion. Clinical outcome was assessed by percentage area flap necrosis and perfusion. In vitro adult human epidermal keratinocytes (HEKa) were treated in: Control medium; HA; SnMP and Desferrioxamine (DF) or combinations thereof. MTT and VialightTM plus ATP assays were used to assess cytotoxicity. Intracellular reactive oxygen species (ROS) concentration was determined by flow cytometry (CMH2DCFDA assay). Statistical analysis was performed by one-way analysis if variance (ANOVA) followed by Tukey's test. Results. In vivo preconditioning with HA increased HO-1 protein expression and level of bioactivity. This bioactivity was successfully inhibited by SnMP. In the skin, HO-1 up-regulation occurred in macrophages. HA based treatments resulted in significantly worse necrosis at 48 h: Control vs HA (p = 0.01). HA based treatments significantly decreased perfusion at: 24 h (Control vs HA, p = 0.0002) and 48 h (Control vs HA, p = 0.04). By contrast, SnMP did not affect either clinical outcome measure. In vitro preconditioning with HA was cytotoxic and increased intracellular ROS: both were reversed by co-administration of DF but not SnMP. Conclusion. In contrast to data from visceral models, HA preconditioning proved deleterious in myocutaneous flaps. This is most likely due to the generation of ROS by free haem independent of HO-1 up-regulation.