2990 Microsurgery.qxd - O'Brien Institute

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Scientific Research Bernard O’Brien Institute of Microsurgery production of highly reactive chemicals and the release of toxins from white blood cells which have moved from the blood into the tissue. Our studies have used a basic experimental model in which we apply a tourniquet to one hind limb of either a rat or a mouse for 1 to 2 hours (ischaemia), then allowing normal blood flow for 24 hours (reperfusion), before investigating the damage to the skeletal muscle. We have focused on the roles of specific factors thought to play major roles in this inflammatory process, notably nitric oxide and activated complement, chemicals made in blood vessels by white blood cells. We have also focused on the role of white blood cells known as mast cells, thought to be one of the major promoters of this inflammatory process. This investigation has also been aided by the use of genetically modified mice, also known as ‘knockout’ and ‘transgenic’ mice. In a further series of experiments we have examined the protective roles of naturally occurring chemicals within the body in ischaemia-reperfusion injury. These findings will provide new ways of reducing tissue damage in severed limbs and other forms of traumatic injury. Mast cell activation, nitric oxide and activated complement involvement in ischaemia-reperfusion injury We have been using inducible nitric oxide synthase knockout mice, which are deficient in the enzyme inducible nitric oxide synthase (iNOS). This enzyme has been implicated in many physiological and pathological processes. Its product, nitric oxide (NO), can be either beneficial or toxic – depending on the levels and sites of production. Thus, manipulating the levels of NO may be of therapeutic use in the promotion of tissue survival. It has been suggested by us that in skeletal muscle, the formation of iNOS-derived NO during ischaemia-reperfusion injury, is overall damaging. We have now tested this hypothesis by using the iNOS knockout mice. Indeed it seems that the iNOS knockout mice are a third less susceptible to muscle death following ischaemia reperfusion injury. These results suggest that iNOS is detrimental in the outcome of ischaemia-reperfusion injury and that pharmacological intervention of the iNOS pathway may be a possible therapeutic target in the treatments designed to enhance tissue survival following trauma. Further ischaemia-reperfusion experiments in rats have made the interesting observation that increased iNOS production is found almost exclusively in mast cells (a tissue-based type of white blood cell). From this information we can conclude that the inducible form of NOS is responsible for the nitric oxide formed during ischaemia-reperfusion injury to muscle and that this enzyme is located almost exclusively in mast cells. Supporting evidence that mast cells are very important in ischaemia-reperfusion injury has come from experiments with mast cell-deficient mice, which have no detectable mast cells in their skeletal muscle, and CD 46/55/59 transgenic mice, which inhibit the activation of complement in the bloodstream. In both of these mice we found a similar protective effect to that observed by iNOS knockout mice. Activated complement products have the ability to amplify the inflammatory response, for example causing mast cell activation and therefore increasing iNOS production. These results suggest that giving drugs which stabilise mast cells or which prevent complement activation, prior to reperfusion, may be protective against ischaemia-reperfusion injury. We are currently testing these drugs in our experimental model. Protective effects of the naturally occurring substances oxy-haemoglobin, uric acid and Stress Protein 70 in muscle injury Nitric oxide (NO) is a paradoxical molecule, with both beneficial and detrimental effects on cells. Normally NO is very beneficial, dilating blood vessels and preventing blood clotting. However, during ischaemia-reperfusion injury to skeletal muscle, NO can react with a ‘free radical’ known as superoxide (O - 2 ) to form peroxynitrite (ONOO - ), the latter being responsible for major cellular damage. We investigated the conditions which result in injury to skeletal muscle cells (known as myoblasts) by using a cell culture system. In Experiment 1 we found that a chemical which produces both NO and O - 2 , reduced cell survival markedly, whereas chemicals producing NO alone did not affect cell survival. In Experiment 2 we found that oxyhaemoglobin and uric acid, scavengers which neutralize NO and O - 2 respectively, prevented cell death in myoblasts exposed to injury by this toxic combination. We conclude that the reduced muscle cell survival may be due to either peroxynitrite or one of its breakdown products, eg. the potent oxidant – hydroxyl radical. Uric acid and oxyhaemoglobin are naturally occurring molecules which potentially could be used to counter ischaemia-reperfusion injury in humans. Human and animal tissues have a unique defence mechanism which can be induced to protect against trauma of many types. Stress Protein 70 is produced in response to a small degree of injury and functions to protect the tissue (eg. skeletal muscle, heart muscle, etc) from any subsequent larger degree of injury up to 12-24 hours later. This mechanism is potentially useful in protection against ischaemia-reperfusion injury to skeletal muscle. We have shown in vivo that a brief local elevation in muscle temperature to 42°C can trigger the induction of Stress protein 70, which is protective against the application of a tourniquet within 24 hours. A significant improvement in muscle survival was obtained compared with a control experimental group where Stress protein had not been induced prior to tourniquet application. In cell culture experiments where muscle was genetically engineered to synthesize Stress protein 70 continuously, we observed marked protection against a variety of toxic free radicals which are known to be formed during 17
Scientific Research Bernard O’Brien Institute of Microsurgery ischaemia-reperfusion injury. These experiments show the potential of utilizing this naturally inducible protein as a protective measure in microsurgical procedures. Cold preservation of skin and skeletal muscle Flushing grafts of skin and skeletal muscle with a chemically-defined solution prior to cold storage helps to preserve them, although eventually some cells die. We have commenced experiments in rats which aim to elucidate how and when cells die in cold-stored skin and muscle. For example, do these cells swell and release their contents into surrounding tissue (necrosis), or do they shrink and die by genetically programmed cell death (apoptosis)? The answer(s) to this question will assist us in the development of better preservation solutions. Future clinical application could involve the cold preservation of tissues for transplantation, for example storage of tissues/limbs of accident victims until surgery is possible. Apoptosis of cells following the cold storage of skin flaps ‘Skin flaps’, commonly used in reconstructive surgery, are composites of skin, fat and blood vessels. It has long been known that storing tissues (severed fingers, limbs, etc) on ice prior to microsurgical replacement will give the surgeon more time to complete a successful operation. In this study we found that rat skin flaps can withstand 3-4 days in the cold and still be successfully replanted. However, there is sometimes partial loss of the flap tissue. In this project we have explored the possibility that the tissue loss might be due to apoptosis (programmed cell death) as well as necrosis. The average survival of rat skin flaps subjected to cold ischaemia for 24 hours, 2, 3, 4, or 5 days (followed by 7 days of reperfusion, ie. normal blood flow) was 80, 74, 60, 47 and 12% respectively. Thus, cold storage of skin flaps for up to 4 days represents significant but potentially reversible tissue damage. A further series of skin flaps was subjected to 4 days cold storage. When specimens were harvested during the cold storage phase, no apoptotic cells were detected by histology. Apoptotic cells were only observed in the period 8-24 hours reperfusion, with increased incidence at the later reperfusion times. The major cell types involved were at the base of the dermis, in hair follicles and in blood vessels (endothelial cells, smooth muscle cells and leucocytes). Apoptotic cells in blood vessels will expose the collagen surface underneath, causing increased blood clotting. This may partly account for microvascular failure in discrete zones of the skin flap. More recently we have started to investigate the protective properties of a commercially available preservation solution known as University of Wisconsin (UW) solution. The effect of this flush solution on graft survival is currently under investigation. A new skeletal muscle free flap model In order to investigate the effect of cold storage on skeletal muscle, a new experimental model was established. This muscle flap was based on the medial gastrocnemius muscle with the femoral artery and vein at the origin of the pedicle. A survival curve was established with variable periods of cold storage (1, 2, 3 or 4 days) and 24 hours normothermic reperfusion. Viability of the flaps, determined by a histochemical stain, was estimated to be 40 to 60% after 1 day cold storage but there was little survival for longer periods. As with the skin flap model we propose to investigate the effect of preservation with UW solution and to assess the fate of cell death within the muscle tissue. Macrophages and inflammation Macrophages are derived from white blood cells called monocytes. Macrophages are dynamic cells involved in many important processes in the body, making them an exciting cell type to study. Following surgery a variety of processes may occur at the repaired site, including wound healing, inflammation, fighting infection, new blood vessel formation (angiogenesis) and blood vessel thickening (atherosclerosis). Macrophages are known to play central roles in all of these processes. Our studies aim to discover the molecular mechanisms underlying macrophage functions. Mechanisms of macrophage activation A key class of molecules essential for cell division and growth (proliferation) are the cyclins. We have previously shown that agents which block macrophage proliferation inhibit expression of D-type cyclins. However, we were surprised to find that some anti-mitogens (agents which inhibit cell proliferation) actually raised levels of cyclin D2, the opposite of the expected result. The agents which raised cyclin D2, ie. lipopolysaccharide (LPS) and interferon alpha, also activated macrophages, indicating that cyclin D2 may have a role in macrophage activation, and not just proliferation. Therefore, we believe that in cyclin D2 we have identified a new player involved in macrophage activation. We are currently trying to better understand (a) the molecular mechanisms underlying cyclin D2 induction (eg. cellular signalling pathways and transcription factors involved), and (b) the role of cyclin D2 in activated macrophages (by using macrophages from cyclin D2 ‘knockout’ mice). Type 1 interferons activate macrophages and protect them from viral infection. We wish to understand the mechanisms of these effects by using macrophages from mice with non-functional, type 1 interferon receptors (IFNARKO). In particular we are looking at the role of type 1 interferons in mediating the effects of LPS. We are examining the effect of LPS on cell survival, nitric oxide production and superoxide production in IFNARKO macrophages. 18
Page 1 and 2: Bernard O’Brien Institute of Micr
Page 3 and 4: Patrons PATRON-IN-CHIEF His Excelle
Page 5 and 6: Chairman’s Report Microsurgery Fo
Page 7 and 8: Overview of Research Bernard O’Br
Page 9 and 10: Director’s Report Bernard O’Bri
Page 11 and 12: Director’s Report Bernard O’Bri
Page 13 and 14: Scientific Research Bernard O’Bri
Page 17: Scientific Research Bernard O’Bri
Page 27 and 28: Full and Part-Time Staff Bernard O
Page 29 and 30: Publications Bernard O’Brien Inst
Page 31 and 32: Collaborations Bernard O’Brien In
Page 33 and 34: Presentations Bernard O’Brien Ins
Page 35 and 36: Visiting Lecturers Bernard O’Brie
Page 37 and 38: Named Fellowships Bernard O’Brien
Page 39 and 40: Financial Statements PROFIT AND LOS
Page 41: Donations Bernard O’Brien Institu

2990 Microsurgery.qxd - O'Brien Institute

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