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Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_IV | Pages 133 - 133
1 Mar 2012
Murphy C Chen G Winter D Bouchier-Hayes D
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Introduction

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.

Aims

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.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_III | Pages 53 - 53
1 Feb 2012
Kearns S Daly A Murray P Kelly C Bouchier-Hayes D
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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.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_I | Pages 127 - 127
1 Mar 2008
Kearns S Daly A Murray P Bouchier-Hayes D
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Purpose: Compartment syndrome (CS) is a unique form of skeletal muscle ischaemia. N-acetyl cysteine (NAC) is an anti-oxidant with beneficial microcirculatory effects. We aim to assess the effect of NAC administration on CS induced muscle injury.

Methods: 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 were assessed at24-hours.

Results: 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 24hrs 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).

Conclusions: |NAC provides extended protection to skeletal muscle against compartment syndrome induced injury by both direct reducing neutrophil mediated tissue toxicity and by reducing neutrophil recruitment to the site of injury.


Orthopaedic Proceedings
Vol. 88-B, Issue SUPP_III | Pages 393 - 393
1 Oct 2006
Donnelly M Timlim M Kiely P Condron C Murray P Bouchier-Hayes D
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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.


Orthopaedic Proceedings
Vol. 88-B, Issue SUPP_II | Pages 319 - 319
1 May 2006
O’Grady P Watson R Redmond H Bouchier-Hayes D
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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) in vitro but this has not as yet been demonstrated in vivo.

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.


Orthopaedic Proceedings
Vol. 88-B, Issue SUPP_II | Pages 283 - 283
1 May 2006
Donnell M Nelligan M Condron C Murray P Bouchier-Hayes D
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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.


Orthopaedic Proceedings
Vol. 87-B, Issue SUPP_III | Pages 267 - 267
1 Sep 2005
Thornes B Murray P Bouchier-Hayes D
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Introduction: Histamine is an integral mediator following traumatic injury. Histamine-2 receptors have previously been identified on lymphocytes and monocytes.

Materials and methods: Two rodent models (1) Bilateral femoral fracture and intramedullary nailing, with resulting indirect lung injury (n=30). (2) In vivo model of orthopaedic implant contaminated by Staphylococcus epidermidis (n=36). Animals were randomised to receive ranitidine or placebo (saline).

Results: Markers of lung injury (MPO activity, BAL proteins and wet:dry ratios) increased 24 hours following bilateral femoral fracture, but were reduced if ranitidine was administered systemically after the injury. Production of Th-1 cytokines was blocked by ranitidine, whilst Th-2 cytokine production remained unaffected by ranitidine. These suggest an anti-inflammatory effect of ranitidine, blocking the early (Th-1) pro-inflammatory response following major injury.

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.

Conclusions: The findings highlight the complex balance in vivo, a double-edged sword: the risk of increasing implant infection versus reducing indirect lung injury following major injury. The administration of ranitidine in major trauma patients with severe pro-inflammatory responses may block and reduce early multi-organ dysfunction and improve survival. However, owing to infection, the peri-operative administration of ranitidine should be avoided in elective cases.


The Journal of Bone & Joint Surgery British Volume
Vol. 86-B, Issue 6 | Pages 906 - 911
1 Aug 2004
Kearns SR Daly AF Sheehan K Murray P Kelly C Bouchier-Hayes D

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.


Orthopaedic Proceedings
Vol. 85-B, Issue SUPP_II | Pages 140 - 140
1 Feb 2003
Timlin M Toomey D Condron C Power C Street J Bouchier-Hayes D Murray P
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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.


The Journal of Bone & Joint Surgery British Volume
Vol. 84-B, Issue 5 | Pages 758 - 760
1 Jul 2002
Thornes B Murray P Bouchier-Hayes D

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 Staphylococcus epidermidis. We randomised 44 Sprague-Dawley rats to have bone cement implanted subcutaneously containing either gentamicin or saline (control). The wound was inoculated with a dilute solution of gentamicin-sensitive Staphylococcus epidermidis. At two weeks the cement was retrieved and microbiologically tested. A lower overall rate of infection was seen in the gentamicin-loaded cement group, but there was a significantly higher rate of gentamicin-resistant infection in this group (Fisher’s exact test, p < 0.01). Antibiotic-impregnated cement has an optimum surface for colonisation and prolonged exposure to antibiotic allows mutational resistance to occur. Gentamicin-loaded cement may not be appropriate for revision surgery if it has been used already in previous surgery.


Orthopaedic Proceedings
Vol. 84-B, Issue SUPP_I | Pages - 7
1 Mar 2002
Kearns S Moneley D Murray P Kelly C Bouchier-Hayes D
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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.


Orthopaedic Proceedings
Vol. 84-B, Issue SUPP_I | Pages 6 - 6
1 Mar 2002
Kiely PJ Condron C Monley D Murray P Bouchier-Hayes D
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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.