Previous Awardees – Vacation Studentships

2010

Identification of the Molecular Interactions of Xenon with the NMDA Receptor Glycine Site
Dr Robert Dickinson, Imperial College, London
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Abstract: The general anaesthetic inert gas xenon inhibits NMDA receptors and has been shown to beneuroprotective in in vitro and in vivo models. Xenon is currently undergoing early clinical trials for neuroprotection in neonatal ischemia & cardiopulmonary bypass. However, the molecular mechanism of xenon's neuroprotection remains to be elucidated. We have recently shown that xenon inhibits the NMDA receptor by competing with the co–agonist glycine. This is the first time that a specific anaesthetic binding –site has been identified on the NMDA–receptor. This mode of NMDA–receptor inhibition may explain why xenon has beneficial clinical properties (e.g neuroprotection, profound analgesia) while certain other anaesthetics do not. Certain drugs that inhibit NMDA receptors have neuroprotective properties. However, many of these NMDA receptor antagonists are also neurotoxic (e.g.ketamine, MK801, nitrous oxide). Our recent observation that xenon competes at the glycine site of the NMDA receptor may have important implications for general anaesthesia and neuroprotection. Neuroprotective NMDA receptor glycine site antagonists (e.g. Gavestinel) are well tolerated in patients and devoid of psychotomimetic side effects common with many NMDA receptor antagonists.
Our molecular modelling identified 12 aminoacids in the glycine binding site of the NMDA receptor likely to interact with xenon. The project aims to determine which of these
amino acids are involved in inhibition by xenon and isoflurane. We have mutated these amino acids using site–directed mutagenesis. As a first stage we have already determined glycine–affinity of 4 of the mutant receptors. The summer project student will determine the sensitivity of these mutant receptors to xenon. Using patch–clamp electrophysiology he will perform inhibition measurements at different glycine concentrations. We will use a Lineweaver–Burk analysis to determine the effect of the mutations on the competitive inhibition at the glycine site. Our hypothesis is that the mutations will reduce or eliminate the xenon's competitive inhibition.
The mutated NMDA receptors will be expressed in HEK 293 cells. Patch–clamp recordings will be ma de of NMDA–activated currents at different glycine concentrations in the presence & absence of xenon. Lineweaver–Burk analysis will be used to quantify the degree of competitive inhibition which will be compared with our published data in wild–type receptors.

Potential of iron chelators in protecting against mitochondrial damage under conditions of sepsis
Dr Damon Lowes, Academic Unit of Anaesthesia & Intensive Care, University of Aberdeen.  
Student:

Abstract: Sepsis is a common clinical condition in the intensive therapy unit and still carries an unacceptably high mortality rate from multiple organ dysfunction syndrome. Excessive production of inflammatory mediators, including reactive oxygen and nitrogen species, commonly leads to immune dysregulation, organ failure and death.  There is much evidence that links the pathogenesis of organ failure in sepsis to mitochondrial injury in both animal models and in patients, due to oxidative stress. Excessive oxidative stress has been shown to damage the mitochondrial membrane, inactivate mitochondrial enzymes and oxidise mitochondrial DNA, disrupting respiratory chain activity, causing loss of cytochrome c and initiation of apoptosis.
Within mitochondria superoxide production increases during sepsis and does not readily cross the mitochondrial membrane.  This molecule is relatively un–reactive and is converted within the mitochondrion by manganese containing superoxide dismutase (MnSOD) to hydrogen peroxide which is removed by several enzyme systems. However, MnSOD slowly becomes inactivated by reactive nitrogen species allowing build up of superoxide.. Superoxide has been shown to displace iron atoms from several mitochondrial iron containing enzymes such as aconitase and can react with hydrogen peroxide to form the very damaging hydroxyl radical via Fenton chemistry. 
Iron release from mitochondrial enzymes during sepsis, in addition to free iron that exists between intracellular cellular uptake and binding to iron storage proteins, can be redox reactive and has been implicated in oxidative stress and tissue injury. However, this free iron has been found to be amenable to chelation and several intracellular and intramitochondrial iron chelators have been developed.
We have undertaken several studies using endothelial cells cultured in an environment designed to mimic sepsis, which mirror the mitochondrial dysfunction seen during sepsis. The aim of this project will be to expose human umbilical endothelial cells to a septic environment (lipopolysacchraide and peptidoglycan G) in the presence of intracellular/intramitochondrial iron chelators to determine any potential protective role of these in terms of mitochondrial function and oxidative stress. 
The role of free iron in mitochondria has not been defined in detail during the cellular events seen in sepsis and has not been studied in with regard to mitochondrial function.

Professor DG Lambert and Dr JP Thompson, Department of Cardiovascular Sciences, Division of Anaesthesia, University of Leicester
Student: Mr Gareth Jones

Abstract: Cannabis–like molecules (cannabinoids) are involved in a range of processes within the body and
have a role in the perception of pain. However, cannabinoids also produce effects on the heart and
blood pressure. Cannabinoid receptors are currently classified as CB1, CB2 and GRP55 and the
pharmaceutical industry is interested in producing drugs which act at these receptors. The presence
of these receptors in the human heart is controversial. The research that we carried out specifically
looked at whether there was genetic message for these receptors and also for an enzyme
responsible for the breakdown of the body's ‘natural cannabis’.
Genetic code for cannabinoid receptors was measured using a technique known as polymerase chainreaction. We studied one of the chambers of the heart, the right atrium, in patients with varying degrees of heart failure. Tissue was obtained at the time of routine coronary artery bypass surgery. The primary finding was that the genetic message for CB1, CB2, GPR55 and the breakdown enzyme were found in human right atrial tissue. This is the first time that GPR55 has been measured in the heart. There was more enzyme and CB1 than CB2 and GPR55, but no relationship between receptor or enzyme and degree of heart failure.

Is pentraxin 3 regulated by antioxidants under conditions of sepsis?
Dr D Lowes and Professor H Galley, Academic Unit of Anaesthesia and Intensive Care, University of Aberdeen.
Student: Miss Amy Hill

Abstract: Pentraxins are a group of acute phase proteins which are produced in response to inflammatory conditions in vivo and which play a key role in the humoral innate immune system. Routinely used in the diagnosis and monitoring of inflammation and infection, C reactive protein is a classical short pentraxin.CRP is produced in the liver in response to inflammation and is the main inducer of interleukin 6, together with serum amyloid P component (SAP), another member of the short pentraxin protein group. Recently discovered pentraxin 3 (PTX3) is the first member of the long pentraxin family group. Unlike CRP and SAP, PTX3 is expressed in the tissues following exposure to pro–inflammatory stimuli including specific microbial constituents, TNFα and IL–1β. Studies have revealed that PTX3 levels correlate well with disease severity in the septic patient.5, 6 However, the validity of using PTX3 as a routine marker in intensive care patients has yet to be elucidated. Oxidative stress has consistently been demonstrated in patients with severe sepsis and acts as a trigger for PTX3 up–regulation in endothelial cells, fibroblasts and macrophages. During oxidative stress, loss of essential protective antioxidants occurs, free radicals are formed and overwhelming degrees of tissue injury result. A greater understanding of oxygen radicalmediated
mechanisms will hopefully enable the development of new therapies for use in critical care patients with the aim of improving clinical outcomes in this group. Consequently, the aim of this project was to treat human endothelial cells with a range of antioxidants with and without bacterial cell components or proinflammatory cytokines and to determine the subsequent expression of PTX3 using enzyme linked immunosorbent assay (ELISA). To date, the regulation of PTX3 within a generated model of sepsis has yet to be defined

Publications Arising:
Lowes DA, Hill AL, Sheth CC, Webster NR, Gow NAR, Galley HF. Relative expression of pentraxin–3 by endothelial cells in response to lipopolysaccharide, cytokines or candida albicans. Br J Anaesth 2009; 103: 320P–321P. Hill AL, Lowes DA, Webster NR, Sheth CC, Gow NAR, Galley HF. Regulation of pentraxin–3 expression by antioxidants. Br J Anaesth 2009; 103: 833–9.

Intrathecal stability of clinical admixtures of morphine, bupivacaine, clonidine and baclofen.
Professor R Sneyd, Peninsula Medical School, Anaesthesia Research, Plymouth.
Student: Mr Mark Ferrigan

The purpose of this study was to determine the long term (3 month) stability of 8 intrathecal drug
admixtures currently used clinically. The admixtures studied were:
1) Morphine sulphate (40 mg / ml) 3.75 ml + clonidine (1000mg) 0.5 ml + bupivicaine (3%) 13.75
ml
2) Morphine sulphate (40 mg / ml) 4.5 ml + clonidine (6500 mg) 3.25 ml + bupivicaine (3%) 10.25
ml
3) Morphine sulphate (40 mg / ml) 4.5 ml + clonidine (3000 mg) 1.5 ml + bupivicaine (3%) 12 ml
4) Morphine sulphate (40 mg / ml) 4.5 ml + bupivicaine (3%) 14.5 ml
5) Morphine sulphate (40 mg / ml) 4.5 ml + bupivicaine (3%) 6.5 ml + NaCl (0.9%) 7 ml
6) Morphine sulphate (40 mg / ml) 4.5 ml + baclofen (2000 mg) 1 ml + bupivicaine (3%) 12.5 ml
7) Morphine sulphate (40 mg / ml) 5.0 ml + clonidine (1000 mg) 0.5 ml + NaCl (0.9%) 14.4 ml
8) Morphine sulphate (40 mg / ml) 12.25 ml + bupivicaine (3%) 22.75 ml
Admixtures were checked at regular intervals over the 6 weeks I was involved with the project (and
thereafter for a further 6 weeks). The preparations were inspected for any colour changes,
precipitation, pH changes and then analysed for drug concentration using liquid
chromatography/mass spectrometry.
The project at this stage is incomplete as the stability study continued beyond the end of the
studentship. In addition, certain methodological difficulties caused problems with drug
concentration determination; in particular the over one hundred–fold differences in drug
concentrations in the admixtures caused difficulties with the bupivacaine quantitation. All samples
have been frozen for bupivacaine re–analysis using an alternative technique.
Once complete, it is envisaged that the work will have a direct bearing on improving the safety of
current clinical prescribing practices for intrathecal admixtures.