Introduction: Strict maintenance of normoglycaemia with intensive insulin therapy in the critically ill surgical patients has helped to reduce morbidity and mortality by almost 50%. The notion that insulin may act independently of glucose as an anti inflammatory agent is of interest. Orthopaedist manipulate the inflammatory cascade through the practice of Damage Control Surgery. By delaying surgery they prevent a second hit in the Systemic Inflammatory Response Syndrome (SIRS) and attenuate excessive inflammation which may lead to Multiorgan Failure (MOF). An insulin infusion is a novel method of modulating the inflammatory cascade through the strict control of hyperglycaemia.

The role of neutrophils and endothelium are an integral part of the inflammatory cascade. Our aim was to investigate whether insulin had an independent effect on endothelial cell activation.

Aim: We hypothesise that insulin, independent of glucose, has a cytoprotective effect on the endothelium as an anti inflammatory agent.

Methods: We subjected human umbilical vein endothelial cells (HUVEC) to normoxia, hypoxia and hypoxia reoxygenation to simulate trauma. These 3 groups were incubated with insulin at 0uU/ml, 10uU/ml, 50uU/ml and 100uU/ml for 24h and 48h normoxia and hypoxia. For the hypoxia reoxygenation study HUVEC were exposed to 24h of hypoxia and then 24h of reoxygenation. Proliferation of endothelial cells was measured using an MTT study.

Results: Our experiment shows that hypoxia reduces HUVEC proliferation. Results show that treatment with 50uU/ml insulin for 24 hours attenuates the effect of hypoxia. This suggests that insulin at post prandial, physiological levels, in non diabetics has a cytoprotective effect on endothelial cells. This was significant in hypoxic conditions in a dose dependent manner.

Conclusions: Hypoxia simulates injury and when injury occurs it activates an inflammatory response which could lead to SIRS. It has not previously been investigated how insulin acts as an anti inflammatory mediator in the control of hyperglycaemia. We can conclude that insulin may act to protect the endothelium, independent of glucose, under hypoxia and hypoxia reoxygenation conditions.

Introduction: The beneficial effects of insulin in the maintenance of normoglycaemia in non-diabetic myocardial infarct and intensive care patients have recently been reported. Hyperglycaemia and neutrophilia have been shown to be independent prognostic indicators of poor outcome in the traumatised patient. The role of insulin and the maintenance of normoglycaemia in the trauma patient have as yet not been explored. We hypothesised that through the already described anti-inflammatory effects of insulin and the maintenance of normoglycaemia, that the systemic inflammatory response would be attenuated, in the injured patient. This might result in less adult respiratory distress syndrome (ARDS) and multi-organ dysfunction and therefore less morbidity and mortality in trauma patients.

Materials and Methods: We used a previously validated rodent trauma model. There were 3 groups, two groups underwent bilateral femur fracture and 15% blood loss via cannulation and aspiration of the external jugular vein. The third group were anaesthetised only. The treatment group immediately receive subcutaneous insulin according to a recently identified sliding scale, and thereafter subcutaneous boluses, dependent on ½ hourly blood sugar estimations. The control groups received the same volume of normal saline ½ hourly, subcutaneously. The animals were maintained under anaesthetic for 4 hours from injury via inhaled isoflurane and oxygen. Core temperature and O2 saturations were recorded throughout. At 4 hours, each animal underwent midline laparotomy and cannulation of the IVC for blood sampling for full blood counts and lactate levels. Serum was also taken for flow cytometric analysis of neutrophil activation via respiratoy burst and CD11b levels. Broncho-alveolar lavage (BAL) was performed for neutrophil content and total protein estimation. The left lower lobe was harvested for wet-dry lung weight ratios.

Results: While O2 saturations were equal throughout in both groups, respiratory rates were persistently elevated in the controls. Wet:Dry lung weight ratios (p< 0.05) and lactate levels were reduced in the insulin treated animals compared to controls. There were similiarly fewer neutrophils in the BAL specimens of the insuliln treated animals compared to injured controls (p< 0.05).

Conclusions: Insulin reduces leukocyte lung sequestration in the injured animal model. This work confirms that insulin may have a role in reducing ARDS in the trauma patient, be that as an anti-inflammatory agent or anti-hyperglycaemic agent, or both, indicating that outcomes might be improved by treating hyperglycaemic trauma patients with insulin. Further work needs to done to elucidate its exact mechanism of action and role in the injured patient.

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.

Methods: For the tendon repair study, a previously validated rabbit tendo-achilles tenotomy model was chosen. Animals underwent a transverse tenotomy of the FDL and TA tendons. These were immediately repaired using 3/0 ethibond sutures using the modified Kessler technique, prior to local application of either a phenytoin or buffer gel formulation. At 21 days post-op, the animals were euthanased and the TA harvested for tensiometry testing and collagen content estimation, and the FDL was harvested for histological analysis.

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.

Results: There was no difference in tendon diameter, gross adhesion formation, ultimate tensile strength or collagen content between the groups. Histologically, however, there were a significantly greater number of inflammatory cells (p< 0.05) and blood vessels (p< 0.05) in the phenytoin treated tendons compared to controls.

At both 2 and 4 weeks there was no statistical difference in stiffness or strength of the phenytoin treated fractures compared to controls.

Conclusions: The study phenytoin formulations whilst apparently promoting neovascularisation in the healing tendon, did not augment healing strength in either tissue suggesting that at these doses and dosing schedules the role of phenytoin is limited in these tissues.

The beneficial effects of insulin in the maintenance of normoglycaemia in non-diabetic myocardial infarct and intensive care patients have recently been reported. Hyperglycaemia and neutrophilia have been shown to be independent prognostic indicators of poor outcome in the traumatised patient. The role of insulin and the maintenance of normoglycaemia in the trauma patient have as yet not been explored. We hypothesised that through the already described anti-inflammatory effects of insulin and the maintenance of normoglycaemia, that neutrophil activation and endothelial dysfunction would be attenuated, in the injured patient. This might result in less adult respiratory distress syndrome (ARDS) and multi-organ dysfunction and therefore less morbidity and mortality for the trauma patient.

Materials and Methods: To study this we used a previously validated rodent trauma model. There were 2 groups, both groups underwent bilateral femur fracture and 15% blood loss via cannulation and aspiration of the external jugular vein. The treatment group immediately receive subcutaneous insulin according to a recently identified sliding scale, and thereafter subcutaneous boluses, dependent on half hourly blood sugar estimations. The control group received the same volume of normal saline half hourly, subcutaneously. The animals were maintained under anaesthetic for 4 hours from injury via inhaled halothane and oxygen. Core temperature and 0₂ saturations were recorded throughout. At 4 hours, each animal underwent midline laparotomy and cannulation of the IVC for blood sampling for full blood counts, lactate levels and for flow cytometry to estimate neutrophil activation via respiratory burst and CD11b upregulation. Bronchoalveolar lavage (BAL) was performed for neutrophil content and total protein estimation. The left lower lobe was harvested for wet-dry lung weight ratios.

Results: While 0₂ saturations were equal throughout in both groups, respiratory rates were persistently elevated in the controls. Wet:Dry lung ratios and lactate levels were reduced in the insulin treated animals compared to controls. There were similarly fewer neutrophils in the BAL specimens of the insulin treated animals (p< 0.05).

Conclusions: Insulin reduces leukocyte lung sequestration in the injured animal model. This work confirms that insulin may have a role in reducing ARDS in the trauma patient, be that as an anti-inflammatory agent or anti-hyperglycaemic agent, or both, indicating that outcomes might be improved by treating hyperglycaemic trauma patients with insulin. Further work needs to be done to elucidate its exact mechanism of action and role in the injured patient.

Introduction: Patients with multiple skeletal injuries are susceptible to Systemic Inflammatory Response Syndrome (SIRS) and consequently Acute Respiratory Distress Syndrome (ARDS). Fracture haematoma contains pro-inflammatory mediators. The aim of our study was to show in vitro that fracture haematoma is implicated in neutrophil mediated injury, SIRS, ARDS and MOF.

Methods: Fracture haematoma was isolated from 10 patients at the time of surgery. Neutrophils (PMN) were isolated from 10 healthy volunteers. PMN were exposed to the fracture haematoma supernatant and PMN activation in both primed and unprimed neutrophils were examined (CD11b and CD18 adhesion receptor expression and respiratory burst). PMN phagocytosis and apoptosis were also assessed using flow cytometry. Transmigration across an endothelial barrier was also measured following exposure to fracture haematoma.

Results: Fracture haematoma had a marked effect on respiratory burst in primed PMNs (control = 100% vs 20% fracture haematoma = 1044% ± 405, p=0.04). CD11b and CD18 adhesion receptor expression were not upregulated in the fracture haematoma group. PMN phagocytosis of E coli was increased following treatment with fracture haematoma (control = 100% vs fracture haematoma = 171% ± 6SE, p=0.0001). Transendothelial migration of treated neutrophils was unaffected. Treatment of endothelial monolayers with fracture haematoma did not result in upregulated ICAM1 expression but was observed to induce significant endothelial cell death. PMN apoptosis was significantly delayed following exposure to fracture haematoma (control = 46% ± 5 vs fracture haematoma = 8% ±2, p=0.0005).

Discussion: We have shown that fracture haematoma activates neutrophils, increases phagocytosis and respiratory burst whilst delaying apoptosis. These effects, whilst beneficial at the site of injury, may cause neutrophil mediated tissue injury systemically.

Matsen in 1975 described Compartment Syndrome (CS) as a condition in which the circulation and function of tissues within a closed space are compromised by increased pressure within that space. Raised intra-compartmental pressures result in progressive venous obstruction, capillary stagnation and microvascular hypoxia.

N-acetyl cysteine (NAC) is an anti-oxidant used clinically to reduce liver injury following paracetamol overdose. NAC has been shown previously to reduce lung injury following exposure to endotoxin. Our aim was to evaluate the efficacy of n-acetyl cysteine in the prevention of CS induced acute muscle injury.

Sprague-Dawley rats (n=6/group) were randomised into Control, CS and CS pre-treated with N-Acetyl Cysteine (0.5g/kg i.p. 1 hr prior to induction). Cremasteric muscle was isolated on its neuro-vascular pedicle and CS injury was induced by placing the muscle in a specially designed pressure chamber. Arterial blood pressure was measured via a cannula placed in the carotid artery. To induce compartment syndrome chamber pressure was maintained at diastolic-10 mm Hg. After three hours pressure was released stimulating surgical fasciotomy. One hour after decompression muscle function was assessed by electrical field stimulation: peak twitch (PTV) and maximum tetanus (MTV) values were recorded. Tissue oedema was assessed by wet to dry ratio (WDR).

Compartment Syndrome (CS) resulted in a significant decrease in muscle function (PTV, MTV). CS also resulted in a significant increase in tissue oedema (WDR). Pre-Treatment with N-Acetyl Cysteine attenuated CS injury as assessed by these parameters. These data show that administration of the anti-oxidant N-Acetyl Cysteine results in significant attenuation of the muscle injury and oedema caused by Compartment Syndrome.

This work was supported by a grant from the Cappagh Trust.

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).

Results: The cohort of animals with the greatest injury severity manifested evidence of acute lung injury when compared with controls, this was associated with evidence of interstitial leucosequestration. This data suggests that neutrophils are involved in mediating an acute lung injury following musculoskeletal trauma.