BJA/RCoA Project Grants

Up to 70% of patients undergoing standard cancer chemotherapy treatment will develop chemotherapy-induced peripheral neuropathy (CIPN, a type of nerve damage) causing numbness, tingling and pain in their hands and feet. As well as the debilitating effect of the pain, patients often find CIPN significantly impacts their daily activities, for example difficulty in buttoning up their clothing due to lack of fine touch sensation, difficulties in walking and driving and increased risk of burns to hands and feet because of numbness. Currently, there is no treatment for CIPN and no method to detect which patients will be affected, burdening a large and growing population of cancer patients and cancer survivors with chronic pain following chemotherapy. CIPN can persist for months, even years following the end of chemotherapy treatment. If we could identify those patients who are likely to develop CIPN before it appears it may be possible to prevent CIPN and its symptoms by modifying chemotherapy treatment or providing early analgesic treatment. In addition, if we can understand what causes CIPN, we can identify or develop novel treatments for CIPN. This project will address both these aims.

Mitochondria are commonly known as the 'powerhouse' of the cell and are found throughout the body providing many vital functions. In blood and tissues from rats with CIPN, we have found increases in number of mitochondria and impaired function of mitochondria. We think this happens because chemotherapy damages mitochondria and inhibits their functions, therefore the body tries to repair this damage by producing more mitochondria. Based on these results from animal studies, we believe that measurements of the number or function of mitochondria will change in blood samples from patients. This project will measure changes in blood mitochondria at three different stages to answer the following questions:

1) Can the number or function of mitochondria in a patient's blood before starting
chemotherapy, predict if he/she will develop CIPN?
2) After the first chemotherapy treatment cycle, does the number or function of mitochondria in a patient's blood predict if he/she will develop CIPN?
3) Once the patient is receiving chemotherapy and CIPN has occurred, can the number or function of mitochondria in a patient's blood help us understand what causes CIPN?

Research into CIPN is greatly under-funded, as symptom-control research is often considered lower priority by funding bodies. Given the current lack of CIPN treatments, there is substantial unmet clinical need to alleviate pain in this large patient population. This project will provide vital research into a potential blood biomarker and help us understand what is causing CIPN. Such research will potentially have a substantial impact on survivorship and quality of life in the millions of patients affected each year by CIPN, worldwide.

What are we looking at?
Cardiac arrest is the term used to describe sudden cessation of heart function. After cardiac arrest blood stops being circulated to the vital organs and consciousness is lost within seconds. Each year about 30,000 people receive resuscitation for an Out of Hospital Cardiac Arrest (OHCA) in the United Kingdom (UK). Only one in every twenty people that have an OHCA survive to go home from hospital. The reason for this in 2 out of 3 patients is brain damage caused by lack of blood flow to the brain during the cardiac arrest.

Why are we looking into this?
Following cardiac arrest, we would like to be able to predict accurately which patients are likely to recover and which will suffer untreatable brain damage. This will help guide doctors in updating family members about relatives at an earlier stage of their illness. This information will also help us to decide when complex and highly invasive supportive techniques are more likely to make a difference to survival.

How do we plan to do this?
There are several ways of predicting brain damage following cardiac arrest including examination of the patient, x-ray images of the brain (CT or MRI scan) as well as tests of brain electrical activity. These are not very accurate, are impossible to do quickly at the patient's bedside and are influenced by sedative medications. Accurate measurement of how the pupils respond to a bright light (pupillometry) is not affected by drugs used to induce coma, can be done rapidly by a hand held device and may provide valuable information about patients' prospects of recovery. This project will take place at Barts Health Heart Attack Centre. Research nurses will test the response of the eyes to light at specific time points over the first 2 days, in patients who are admitted following OHCA. In patients who survive to go home from hospital, the research nurses will also make an assessment of how well their brain is functioning.

How will the project help?
The information will be used to obtain a better understanding of the possible role of pupillometry in working out which patients who survive OHCA will recover brain function. This may allow us to provide better care to patients likely to survive with good brain function and also give early information to families about patients who are unlikely to recover brain function or who may die as a result of brain damage.

Background
Recovery from major surgery can be hampered by many diverse complications, which strike at different systems within the body. They include infections, poor wound healing, heart disease and stroke, and may result in severe illness, prolonged hospital stay, reduced lifespan and long-term quality of life. Postoperative complications thus inflict a significant physical and psychological cost on the individual, as well as a crippling financial burden on the NHS. Preventing such suffering is a key priority for perioperative medicine. Modern innovations have ameliorated postoperative complications to a certain extent, but their impact remains unacceptably high. We have much to learn about the biology that leads to these complications if we are to effectively target and prevent them in the future. One important avenue for exploration is the effect of the "stress" of surgery on the cells of the body, and we are interested in a process called "oxidative stress." Most of the energy that cells require to survive and function properly is produced by tiny 'power stations' that exist within them: the mitochondria. Surgery may cause a disruption of the mitochondrial environment, impairing their ability to produce energy, and even leading to their destruction. Impairment of mitochondrial function has been linked to worse outcomes in other serious conditions and we propose that it may also lead to worse outcomes following surgery.

Aim
We hope to determine whether a relationship exists between the oxidative stress induced by major surgery, the function of mitochondria and the development of postoperative complications. We also hope to identify whether preoperative fitness, as defined by exercise testing, is related to the degree of susceptibility to this stress, or to the function of the mitochondria at the beginning and end of surgery.

Study design
We plan to conduct an observational study in patients classified as 'high-risk' for developing complications following major surgery. Prior to the day of surgery, participants will undergo a standard fitness test on an exercise bicycle. Immediately prior to the surgical procedure, we shall take a sample of blood and small piece of thigh muscle from each patient, while they are under general anaesthetic. Both tests will be repeated at the end of the operation. Samples will be analysed in our laboratory to quantify the degree of oxidative stress and mitochondrial function at baseline and following the surgical insult. Patients will be followed up to determine whether or not they develop postoperative complications. The experimental measures will then be compared in the group of patients who develop complications and the group of patients who do not, in attempt to discover whether adverse outcomes are related to oxidative stress and mitochondrial function. Potential correlation of these measures with preoperative fitness will also be investigated.

Potential impact
The results of this study may identify whether oxidative stress leads to mitochondrial dysfunction during major surgery, and whether this contributes to adverse outcomes. It could highlight important processes that are amenable to drugs, which may allow us to reduce harm to high risk patients undergoing operations in the future.

Many frail and elderly patients undergo surgery for hip fracture every year. Many of these patients have other health problems including heart disease and anaemia (low haemoglobin or "low blood count") either from chronic illness, from bleeding at the time of their injury or during subsequent surgery. The vast majority (more than 95%) of these patients will go on to have surgery. This surgery is often high risk. Patients with this type of injury may already be frail, may be in hospital for a long time and will need rehabilitation. Many of them will develop complications, including heart attacks and some will die.

Doctors looking after these patients commonly prescribe a blood transfusion around the time of surgery. These patients often have anaemia before surgery and lose more blood during their operations. A benefit of blood transfusion is that it may increase the amount of oxygen the blood can carry. One of the main reasons that anaesthetists prescribe blood around the time of surgery is to prevent heart attacks, which can occur if the heart doesn't receive enough oxygen. Another possible benefit of blood transfusion is that it may help patients get out of bed more quickly after surgery. This is another important aspect of their recovery.

However, blood transfusions can have side effects such as causing heart failure or increasing infections after surgery. These can delay patient recovery too. Although some research has been done in this area, anaesthetists and surgeons are still unsure of when to prescribe blood transfusions to these patients. In particular, we are not sure about how low the blood count should be before a blood transfusion is ordered. Some doctors prescribe blood when the haemoglobin count is less than 9 and some at a lower level of 7. Current guidelines suggest that prescribing at a lower haemoglobin count is better, but there is research which suggests that this level is too low if the patient has a history of heart disease.

We wish to see if it is possible to undertake a study comparing blood transfusion at two different levels of anaemia to see which is best for patients. All patients that present to our hospital with a broken hip and cardiac disease will be able to take part in this study. If they become anaemic during their treatment they will be allocated to either be transfused when their blood count is less than 9 or less than 7. In all patients we will measure heart damage with a blood test that is very sensitive. We will also collect data on the incidence of heart attacks and other complications.

If we are able to carry out a small study of blood transfusion in these patients in our hospital, then we will use this data to plan a bigger study involving more hospitals and more patients.

Risk assessment for major surgery currently focuses on the risk of complications and death in the 30 days immediately following surgery (the perioperative period). From the patient's perspective long-term survival and indeed recovery to the health that they enjoyed before surgery are at least as important. At present preoperative risk prediction models are based on data obtained in hospital. This looks at only one part of the individual's journey through the healthcare system. Somebody undergoing elective surgery will usually have visited their general practitioner before being sent to hospital and will return to the care of their GP following surgery. Primary care in the UK generally uses high-quality electronic patient records. It is likely that the primary care patient record contains information that would be of value in judging the patient's fitness for surgery. By the same token, complications and disability that manifest themselves after discharge from hospital may well be recorded in the general practice record but not in the hospital notes. In the ideal world there would be a seamless connection between the primary and secondary care medical records. In practice the systems generally do not talk to each other. The proposed project seeks to go some way towards closing this gap by using a large anonymised database of primary care consultation records to test the feasibility of using information recorded in general practice to support preoperative risk assessment, to identify complications following surgery, and produce predictive models for such complications.

ResearchOne is a large anonymised database of primary care consultations containing some 28 million records. It is made available for non-commercial clinical research by one of the leading manufacturers of primary care electronic patient record systems (TTP). In the proposed study we will do the preliminary work required to extract data on surgical care from this database. This will require the development of a directory of codes used in the database for conditions requiring surgery and risk factors for perioperative complications. Using these codes we will identify patients who have undergone major colorectal or vascular surgery. We will undertake statistical modelling to identify factors that predict complications or long-term debility following surgery. We will also develop a directory of codes that directly or indirectly indicate that a post-operative complication has taken place. We will work with TTP to implement these statistical model as software tools within the SystemOne electronic patient record that can be used by GPs with a click of the computer mouse during the course of a consultation to generate an individual patient report on the risk of delayed or incomplete recovery after surgery. This information can then be shared with the patient and the hospital teams. We will test the functionality of this software in general practice. This will support applications for funding for larger scale studies to fully test the predictive power of the new statistical tool and to determine if interventions such as enhanced medical care in hospital or planned rehabilitation improve long term outcome following major surgery.

Control of skeletal muscle activation is a tightly controlled and highly regulated system. Regulation of muscle contraction is through the control and release of calcium ions within muscle fibres. When this control is lost, major problems can occur throughout the whole human body. Certain diseases are caused by a lack of control in calcium ion release in muscle, such as malignant hyperthermia (MH), exertional heat illness, congenital myopathies and premature muscle ageing. The causes of these diseases have not yet been fully elucidated.

MH is a potentially fatal disease triggered by inhalational anaesthetic agents as well as suxamethonium. These drugs are both commonly used during routine and emergency general anaesthesia for patients. In patients with this disease, control of calcium release is lost and the muscle cells remain constantly activated. This causes a great deal of heat to be produced from skeletal muscles, which leads to a rise in body temperature, muscle damage and a great deal of substances being released from muscle cells which can harm or damage other parts of the body.

Research into MH has shown that the ryanodine receptor, RyR1 (a skeletal muscle calcium releasing channel), is constantly activated when it has been exposed to inhalational anaesthetic agents. In 50-80% of cases, this activation is caused by a mutation in RyR1 itself. However, there are approximately 20-40% of cases where the cause of the disease has not been found, but the RyR1 still activates constantly in the presence of the inhalational agents. Work looking at the DNA from patients with MH has suggested a number of rare new variants in the RyR1 gene or in other related proteins that may cause dysregulation of calcium control in muscle cells, thereby leading to the disease. Currently, there are 6 new rare RyR1 variants that have been found in a number of families with MH where no other cause has been ascribed. In addition, we have found 3 MH families where there are no RyR1 variants, but they do have interesting rare changes in the gene of another protein related to calcium control in muscle, called Calsequestrin 1. Currently, there is no proof to suggest that any of these variant proteins are definitely causative. We therefore need to perform functional studies in cells to link these DNA variants to causing MH (either from changes within the RyR1 gene or in the genes of other related proteins, such as Calsequestrin 1). By continuing to build a comprehensive library of causative mutations in different genes, the long term goal is to create a screening panel whereby a simple blood test could diagnose MH, rather than dealing with a crisis during anaesthesia or having to perform invasive muscle biopsies as a screening test (which is what happens currently).

The understanding of opioid signalling has shifted greatly in the past few years. Previously opioids were understood to function through the binding and activation of the opioid receptor, leading to the activation of an inhibitory G-protein and the decrease of the second messengers cyclic AMP and intracellular calcium and reduced membrane excitability. The net result of these actions being a decrease in pain signal transmission. Recently, a paradigm shift has occurred in opioid signalling which has the potential for a significant impact on the development of novel opioids with reduced side-effects, such as tolerance and dependence. These have blighted opioid treatment for decades. Recent findings have demonstrated a different opioid signalling pathway, which involves rapid recruitment of a protein called β-arrestin. Importantly, in animals engineered to lack arrestin, tolerance to morphine is absent. Activation of arrestin and the effects after activation are difficult to study with high time resolution using the methods we currently have in our laboratory. In order to address this, a luciferase based system can be employed (luciferase is the firefly enzyme that glows green). The purchase of a luminometer will allow for the detection of the interaction of arrestins and their partners through the detection of transmitted light at specific frequencies, determined by the fluorophore (chemical group responsible for fluorescence) joined to our protein of interest. Recent findings by our group and others, have demonstrated that the opioid peptide, N/OFQ and its receptor NOP may play a role in inflammatory diseases. In order to determine the effect N/OFQ and NOP have on cytokine (as inflammatory markers) production, ELISA-based assays are required; these can also be performed using a luminometer. Additional uses include assessment of opioids on immune cell migration, establishment of a non-radioactive high throughput cAMP assay and a new non-radioactive receptor binding assay. This piece of equipment will significantly enhance our ability to probe old and new signaling pathways and facilitate training of clinical and non-clinical colleagues in cutting edge technologies. This will include traditional PhD and NHS based MD students.

Background
Infants who require surgery are exposed to pain and anaesthesia at a time when the developing brain is vulnerable to altered nervous system activity. In young adults born preterm, we found long-term alterations in pain sensitivity and brain structure, that were more marked if intensive care plus neonatal surgery was required, but which differed between males and females.

Preclinical studies in laboratory animals are necessary to: tease out the specific roles of anaesthesia, pain and surgery; identify underlying mechanisms; and test new interventions. We have shown that neonatal surgery (rat hindpaw incision) alters long-term signalling between cells in adult spinal pain pathways (neurons and microglia). Subsequent surgery unmasks more severe and prolonged sensitivity that is reduced by inhibiting microglial function (with drugs in adult clinical trials). We will now evaluate effects of neonatal surgery and/or anaesthesia in the brain, identify the cell types involved, and specifically evaluate the role of microglia. Importantly, we will see if effects differ between males and females.

Aims
1. Assess effects of neonatal incision and/or anaesthesia on the developing brain.
2. Evaluate pain response and brain structure following adult incision, and see if this is altered by prior neonatal surgery and/or anaesthesia.
3. Determine if microglial inhibitors at the time of neonatal surgery prevent longterm changes.

Methodology
Experiments will use established techniques, performed by Home Office licensed investigators, in accordance with UK Animal (Scientific Procedures) Act. Male and female neonatal mice will be randomly assigned to: anaesthesia only; anaesthesia and hindpaw incision; or no treatment.

When animals reach adulthood, long-term effects of neonatal surgery and/or anaesthesia (Aim 1) will be assessed with:
i) behavioural measures of pain sensitivity (hindlimb reflex thresholds) and motor function;
ii) magnetic resonance imaging (MRI) scans to identify brain regions with altered volume and microscopic structure.
iii) tissue analysis from affected brain regions using specific cell markers (neurons, microglia, astrocytes, oligodendrocytes).

In additional groups, adult hindpaw incision will be performed to compare groups with and without prior neonatal surgery and/or anaesthesia (Aim 2) or neonatal surgery plus perioperative microglial inhibitors (Aim 3). Effects will be compared with the same outcomes (behaviour, MRI, tissue analysis).

Expected outcomes
Effects of surgery and/or anaesthesia on the developing brain will be assessed for the first time using high-resolution MRI scans (as in clinical studies) plus specific tissue analysis. While many animal studies have evaluated anaesthesia alone, combining surgery and anaesthesia more closely mirrors clinical practice.
As neonatal surgery prolongs sensitivity after adult incision, it may represent a clinical risk factor for persistent post-operative pain. Having assessed spinal cord mechanisms, we will now evaluate brain changes, to determine if microglia also underlie altered brain structure and function, and evaluate prevention by microglial inhibitors.

Implications
Our proposal relates to UK anaesthesia perioperative care research priorities (NIAA/James Lind Alliance Partnership 2015: "effects of anaesthesia on developing brain"; "patients developing chronic pain after surgery"). Preclinical laboratory studies are essential to identify new analgesic targets and test their relative effectiveness and safety in both males and females. Results will inform future clinical trials.

Background
Sepsis (severe infection) is the main cause of death on intensive care units. Mitochondria are tiny structures inside cells where energy is made and if they are damaged they are unable to make enough energy. Such damage to mitochondria can alter the way the cell works, and contributes to organs of the body being damaged by sepsis. Melatonin, a simple molecule which is produced naturally in the body but can also be chemically manufactured, can protect these mitochondria in the laboratory under conditions designed to mimic sepsis.

We recently completed a trial where we gave different doses of melatonin to groups of healthy subjects. Despite this it is not known what happens to the extra melatonin after it is in the body and we also do not know what effects the extra melatonin may have on normal cell processes. We have over 180 samples saved from our trial that we plan to use to determine the effect of melatonin on the body's cellular reactions and also the metabolic fate of the extra melatonin.

Aim
This study will further investigate the effects of giving melatonin to healthy people. This work will give us more information into the consequences of giving extra melatonin and will contribute to our understanding when we give melatonin to patients with sepsis.

Study design
We have a large number of stored serum and urine samples from our trial which will be used to investigate the metabolic fate of the extra melatonin and also the effects of melatonin on products of the body's normal cell processes (metabolic effects of melatonin). These measurements will be done using a technique called nuclear magnetic resonance and will be undertaken at an expert centre dedicated to such work. We will work with the centre to analyse and interpret the huge amount of data which will be generated and relate the findings to the other data we already have from these subjects.The samples which we have already collected are a unique resource giving added value to our previous work and enabling increased understanding of the detailed effects of giving extra melatonin which will inform future clinical trials.