BJA/RCoA non-clinical PhD Studentships

Background
Malignant hyperthermia (MH) is a genetic condition that affects the control of calcium within skeletal muscle tissue. The body normally compensates for this, but when a patient with the genetic risk for MH is given one of the commonly used anaesthetic gases, the muscle cells lose control of calcium regulation in a dramatic and potentially fatal way. The currently used definitive diagnostic test for MH requires a muscle biopsy through a 2-3 inch incision. Genetic (DNA) diagnosis is available for some families but the complex genetics of MH precludes making this more widely available. The genetic model that best explains our current knowledge is one where individual gene changes sensitise the calcium release mechanism but not enough for a reaction to be triggered during anaesthesia. If there are one or more additional gene changes that can amplify this calcium release MH is likely to be triggered.

The gene most commonly implicated in MH is called RYR1 but changes in unidentified genes are likely to play a role in at least 45% of cases. Using the latest DNA sequencing technology we have identified changes in a series of additional genes that have the potential to amplify a sensitized calcium release mechanism. We now need to show that they have this effect and we will look at changes in two genes, PYGM and CPT2.

Aims
To assess the effect of newly identified gene changes on calcium release in muscle cells that carry a change in the RYR1 gene associated with malignant hyperthermia.

Methodology
This is a laboratory-based project suitable for a PhD student. In order to assess the effects of a gene change we need to compare the function of cells that differ only in that specific gene change. Our ability to do this has been greatly enhanced by the development of gene editing technology known as CRISPR-Cas. The muscle cells we will use for gene editing are from a genetically engineered mouse model of MH in which we have introduced the RYR1 gene change most commonly found in UK MH patients. The mouse cell model has advantages over human cells because the mouse cells can be maintained for longer in the laboratory which is vital for gene editing. Calcium release in muscle cells (gene edited compared with non-edited) will be measured in response to inhalation anaesthetic using calcium indicator dyes that fluoresce in the presence of calcium.

Expected outcomes
We expect to find that some of the newly identified changes in the PYGM and CPT2 genes contribute to the calcium dysregulation responsible for MH. This training project will enable the student to acquire a range of state-of-the-art molecular and cellular biology techniques.

Implications
The results of this project are likely ultimately to lead to the availability of DNA testing instead of a muscle biopsy in further patients at risk of MH.

Background
People who have a spinal cord injury usually lose movement and sensation below the injury site, but about half of patients also have severe nerve pain, known as neuropathic pain, which occurs due to inflammation of the nervous system, is hard to control. Studies using rats have shown giving either a fatty acid called omega-3 docosahexaenoic acid (DHA) or melatonin can reduce and/or prevent neuropathic pain after spinal cord injury.

Aim
Each of the treatments work in different ways and the proposed PhD studies will investigate whether giving both drugs together will provide even better pain prevention after spinal cord injury in a rat model, or even reduce pain after it has developed. The rat studies will be complemented by experiments using cells to investigate the mechanisms which may be involved.

Study design
There will be two complementary experimental approaches: (i) using cells in the laboratory and (ii) using a rat model of spinal cord injury. Two types of cells involved in neuropathic pain and inflammation will be cultured with inflammatory stimuli and the effect of different concentration combinations of DHA and melatonin on cell activation and function, along with measurement of key proteins which may offer insight into possible mechanisms of the effects, will be studied. For the rat model, a spinal cord injury will be performed under general anaesthesia and animals will be randomly allocated to received DHA alone, melatonin alone or DHA/melatonin combined. Natural behavioural responses to pain, such as burrowing behaviour will be combined with the usual pain response tests such as reaction to prodding or heat. This approach is novel and more realistically reflects pain measures in humans. At the end of the studies, rats will be anaesthetized and samples of brain and spinal cord tissue will be used to identify changes in cell activation and proteins which may be involved. The numbers of rats have been carefully considered to use as few as possible and rescue protocols are in place to minimise any animal suffering.

The student will gain expertise in pre-clinical (animal) research and a range of laboratory assay techniques. He or she will join an established group which is actively working on projects investigating treatments for pain which can be rolled out to human studies in future, since both DHA and melatonin have excellent safety profiles and can be used in clinical trials.

Impact
This is a straightforward study with the potential to contribute to treatment for neuropathic pain as a result of spinal cord injury. However, pain is a common denominator across all medical specialities and this study may identify new therapeutic approaches relevant to other types of pain.