Long bone surgery and marrow instrumentation represent significant surgical insults, and may cause severe local and systemic sequelae following both planned and emergent surgery. Preconditioning is a highly conserved evolutionary endogenous protective mechanism, but finding a clinically safe and acceptable method of induction has proven difficult. Glutamine, a known inducer of the heat shock protein (HSP) response, offers pharmacological modulation of injury through clinically acceptable preconditioning. This effect has not been previously demonstrated in an orthopaedic model. The aim of the study was to test the hypothesis that glutamine preconditioning protects against the local and systemic effects of long bone trauma in a rodent model.Introduction
Aims
Compartment syndrome (CS) is a unique form of skeletal muscle ischaemia. N-acetyl cysteine (NAC) is an anti-oxidant in clinical use, with beneficial microcirculatory effects. Sprague-Dawley rats (n=6/group) were randomised into Control, CS and CS pre-treated with NAC (0.5g/kg i.p. 1 hr prior to induction) groups. In a post-treatment group NAC was administered upon muscle decompression. Cremasteric muscle was placed in a pressure chamber in which pressure was maintained at diastolic minus 10 mm Hg for 3 hours inducing CS, muscle was then returned to the abdominal cavity. At 24 hours and 7 days post-CS contractile function was assessed by electrical stimulation. Myeloperoxidase (MPO) activity was assessed at 24-hours. CS injury reduced twitch (50.4±7.7 vs 108.5±11.5, p<0.001; 28.1±5.5 vs. 154.7±14.1, p<0.01) and tetanic contraction (225.7±21.6 vs 455.3±23.3, p<0.001; 59.7±12.1 vs 362.9±37.2, p<0.01) compared with control at 24 hrs and 7 days respectively. NAC pre-treatment reduced CS injury at 24 hours, preserving twitch (134.3±10.4, p<0.01 vs CS) and tetanic (408.3±34.3, p<0.01 vs CS) contraction. NAC administration reduced neutrophil infiltration (MPO) at 24 hours (24.6±5.4 vs 24.6±5.4, p<0.01). NAC protection was maintained at 7 days, preserving twitch (118.2±22.9 vs 28.1±5.5, p<0.01) and tetanic contraction (256.3±37 vs 59.7±12.1, p<0.01). Administration of NAC at decompression also preserved muscle twitch (402.4±52; p<0.01 versus CS) and tetanic (402.4±52; p<0.01 versus CS) contraction, reducing neutrophil infiltration (24.6±5.4 units/g; p<0.01). These data demonstrate NAC provided effective protection to skeletal muscle from CS induced injury when given as a pre- or post-decompression treatment.
The mechanism by which cells die is important in an immune response and its resolution. The role of apoptosis in sepsis and trauma, and its regulation by cytokines is unclear. During the systemic inflammatory response, rates of human neutrophil apoptosis are decreased. Peritoneal macrophage apoptosis has been induced by nitric oxide and Lipopolysaccharide (LPS) We examined the induction and effects of macrophage apoptosis in a model of trauma and sepsis. One hundred female CD-I mice were randomised into four groups: Control, Septic model, challenged with intraperitoneal LPS (1.Img/200ul/mouse), Traumatic model, received hind limb amputation (HLA) and a Combined trauma/septic model. After 24 hrs mice were sacrificed and peritoneal macrophages were assessed for apoptosis by morphology and DNA fragmentation by flow cytometry and DNA gel electrophoresis Peritoneal lavage from septic models had a decreased percentage of macrophages in comparison to control and trauma groups. The septic model also had a significantly increased incidence of apoptosis in comparison to control and trauma levels. There was no significant difference between control and traumatic groups. These findings demonstrate that in a murine model of sepsis, lipopolysaccharide induces macrophages apoptosis. Modulation of this immune response may have important roles in the management of trauma patients.
Phenytoin has previously been shown to accelerate wound healing through upregulation of angiogenesis and promotion of collagen deposition. These reported effects led us to hypothesise that phenytoin could be used locally at the tendon repair site to increase the rate and strength of healing. Systemic treatment with phenytoin has also been shown to increase the thickness and density of calvarial and maxillary bones in humans, and promote fracture healing in rabbits, rats and mice. Based on these and similar studies we hypothesised that local percutaneous injection of phenytoin solution into a fracture site would result in improved fracture healing without the risk of the side effects of systemic administration of the drug.
For the fracture study, a rat femur fracture model was utilised. Adult male Sprague-Dawley rats were anaesthetised. Following a medial parapatellar approach, the femur was cannulated using an 18 gauge cannula. The cannula was cut flush with the distal femur and countersunk. The skin and retinaculum were closed with 5.0 monocryl. The nailed femur was then fractured using a 3 point bending technique. The femurs were xrayed to ensure each fracture was mid-diaphyseal and transverse. At 6 hours post op animals underwent either 1) Fracture site percutaneous injection with 100 μmol phenytoin solution 2) Fracture site percutaneous injection with phosphate buffer solution (PBS) 3) No percutaneous injection. This procedure was once again repeated at 72 hours. At 2 and 4 weeks post op 6 animals from each group were euthanased, their femurs were harvested for biomechanical analysis of stiffness and strength.
At both 2 and 4 weeks there was no statistical difference in stiffness or strength of the phenytoin treated fractures compared to controls.
Ranitidine’s effect on implant infection rates showed higher rates (44% versus 17%, relative risk 1.8 (95% CI 1.0 to 3.3)) when systemic ranitidine was delivered peri-operatively, suggesting an immunosuppressive effect.
Compartment syndrome is a unique form of ischaemia of skeletal muscle which occurs despite patency of the large vessels. Decompression allows the influx of activated leucocytes which cause further injury. Vitamin C is a powerful antioxidant which concentrates preferentially in leucocytes and attenuates reperfusion-induced muscle injury. We have evaluated the use of pretreatment with oral vitamin C in the prevention of injury caused by compartment syndrome in a rat cremasteric muscle model. Acute and delayed effects of pretreatment with vitamin C were assessed at one and 24 hours after decompression of compartment syndrome. Muscle function was assessed electrophysiologically. Vascular, cellular and tissue inflammation was assessed by staining of intercellular adhesion molecule-1 (ICAM-1) and by determination of the activity of myeloperoxidase (MPO) in neutrophils and tissue oedema. Compartment syndrome impaired skeletal muscle function and increased the expression of ICAM-1, activity of MPO and muscle weight increased significantly. Pretreatment with vitamin C preserved muscle function and reduced the expression of ICAM-1, infiltration of the neutrophils and oedema.
We have compared the rates of infection and resistance in an animal model of an orthopaedic procedure which was contaminated with a low-dose inoculum of
Following ischaemia-reperfusion (I-R) tissues undergo a neutrophil mediated oxidant injury. Vitamin C is a water-soluble endogenous anti-oxidant, which has been shown in previous studies to abrogate neutrophil mediated endothelial injury. Our aim was to evaluate Vitamin C supplementation in the prevention of I-R induced acute muscle injury. Sprague-Dawley rats (n-6/group) were randomised into control, I-R and I-R pretreated with Vitamin C (3.3g over 5 days). Cremasteric muscle was isolated on its neuro-vascular pedicle and I-R injury induced by clamping the pedicle for 3 hours, the tissue was subsequently reperfused for 60 minutes. Following reperfusion muscle function was assessed by electrical field stimulation: peak twitch (PTV), maximum tetanus (MTV) and fatigability values were recorded. Tissue neutrophil infiltration was assessed by tissue myeloperoxidase (MPO) activity and tissue oedema by wet:dry ratio (WDR). Ischaemia-reperfusion (I-R) resulted in a significant decrease in muscle function (PTV<
MTV) there was no difference in fatigability values between groups. I-R also resulted in a significant increase in neutrophil infiltration (MPO) and tissue oedema (WDR). Pre-treatment with Vitamin C attenuated I-R injury as assessed by these parameters. This data suggests that oral Vitamin C reduce I-R induced acute muscle injury, possibly by attenuating neutrophil mediated tissue injury.
Acute respiratory distress syndrome is a long established complication and continuing cause of significant morbidity and mortality in the multiply injured patient. Systemic inflammatory response syndrome (SIRS) is classically associated with acute pulmonary dysfunction. A variety of insults including trauma, sepsis, hypoxia, ischaemia reperfusion, can trigger systemic inflammatory response and acute lung injury. In models of sepsis, endotoxaemia and ischaemia-reperfusion, acute lung injury is characterised by widespread endothelial-neutrophil interaction and neutrophil activation. Another associated finding in these models of injury, is evidence of induced diaphragm muscle dysfunction, by electrophysiological testing of muscle strips post injury. An established model of incremental increasing skeletal trauma was employed. Adult male sprague dawley rats (mean weight 476grams, 370–520g) were randomised to control, single hindlimb fracture, bilateral hindlimb fracture and bilateral hind limb fracture + 20% haemorrhage. Indices of acute lung injury studied 2 hours post injury were bronchalveolar lavage, cell counts, and protein assays. Pulmonary tissue myeloperoxidase activity was assayed as an indicator of neutrophil activation and pulmonary wet/dry weights were measured as a marker of pulmonary oedema. Diaphragmatic electrophysiological testing was also performed 2 hours post injury. Freshly harvested diaphragmatic muscle strips had peak evoked muscle twitches measured, the maximal tetanic twitch and muscle strip fatigue times were also assessed. Statistical analysis was performed by means of analysis of variance (ANOVA).