Delineating the cell intrinsic mechanisms of peripheral nerve regeneration

The peripheral nervous system connects the signals from the brain and spinal cord to our skin and muscles, allowing us to move, feel and sense our surroundings. When these nerves are injured these functions are disturbed and patients usually need surgery to repair the damaged nerves. Unlike the nerves in the central nervous system of the brain and spinal cord, peripheral nerves are able to regrow after injury. Unfortunately this is never enough to provide full recovery for the patients as the regrowth process is very slow and unstable. Surgery to repair the nerves can help with recovery but these operations have remained unchanged for over 60 years and still do not lead to a complete recovery. This leaves patients with both physical and emotional impairment. Understanding how the peripheral nerves are able to self-repair is key to developing new methods of treating nerve injuries.
This research project will focus on what happens inside the individual cells that make up our peripheral nerves after injury. This will be carried out in a rat model of peripheral nerve injury. I will use fluorescent markers which attach to the proteins inside the cells to illuminate specific structures which may be involved in coordinating the process of nerve regrowth. This will be combined with the use of state of the art microscopes which are able to take high-resolution videos of the cells whilst they regrow. This will give us an understanding of what the process of nerve regrowth after injury looks like and also why it might go wrong.
There are some specific structures inside cells which are thought to be very important in allowing peripheral nerves to regrow and ultimately reconnect to the skin and muscle that they send electrical signals to. One of these structures is called the Golgi apparatus and this usually is used for carrying proteins from one end of the nerve to the other, allowing it to function properly. Another function for the Golgi apparatus is thought to be in reforming the skeleton of the cell after injury, allowing the very long extension that connects the spinal cord to the skin and muscles (known as the axon) to regrow. I will illuminate this structure and find out whether its shape and position within the cells changes during the process of regrowth. By changing the genetic code of the cells and disrupting the function of the Golgi apparatus I will be able to determine whether this structure is needed for nerves to regrow. If the Golgi apparatus is needed, this could be very important as we may be able to develop new treatments for nerve injury which can target this structure.
It is known that if surgery to repair injured nerves is delayed, this can lead to poorer recovery for patients but it is not fully known why this is the case. It could be that the Golgi apparatus responds to the 'injury signals' quicker if the nerve in repaired faster. In order to test this I will use my rat model of peripheral nerve injury. Repair of this injury will be carried out immediately, after one week or after two months. I will then take fluorescent images of the nerve to assess how much the nerve has regrown and also what the structure and position of the Golgi apparatus is. Again, I will alter the genetic code of the cells within the nerve and disrupt the function of the Golgi apparatus. It is expected that this will lead to disordered nerve regrowth.
All of these experiments aim to find out which structures inside our nerve cells might allow for regrowth to occur. We think that the Golgi apparatus may be important and will focus on this. Finding out what makes nerves regrow and also why early surgical repair is needed will be important for two reasons: 1.) it will enable specific structures in nerve cells to be targeted when thinking about making new treatments for patients with nerve injuries. 2.) It may tell us why nerves in the brain and spinal cord are not able to regenerate and how we might overcome this.

Peripheral nerve injury (PNI) is common and can be physically, psychologically and socioeconomically challenging. Microsurgical nerve repair remains the only clinical intervention yet seldom leads to adequate recovery.

Aims: To assess the underlying neurobiology driving regeneration by determining the sites of microtubule nucleation in in vitro, ex vivo and in vivo models of PNI, thus enabling the development of centrally acting therapeutics targeted to the intrinsic regenerative machinery.

Objectives:
1. Determine the spatio-temporal control of regeneration using high-resolution live-cell imaging.
2. Identify sub-cellular structures involved in axonal elongation following PNI.
3. Examine the intracellular response to nerve repair in an in vivo model.

Methods: Adult rat dorsal root ganglia in vitro and ex vivo tissue cultures will undergo genetic transfection via electroporation. Fluorescent DNA constructs or siRNAs will be delivered to the nucleus allowing for visualisation and manipulation of specific sub-cellular structures within the neurons. Combining this with high-resolution fixed and live-cell imaging will allow for in depth assessment of the spatial and temporal dynamics of peripheral nerve regeneration at the cellular and sub-cellular level. An in vivo rat model of PNI with microsurgical repair will be used to examine the intracellular response to repair and, in particular, response to delayed intervention.

Impact: This work will enhance understanding of the neurobiological processes of peripheral nerve regeneration as well as identify targets for the development of novel therapeutics and eventual clinical translation. My models provide an ideal system on which to test therapeutics in their early stages and may provide knowledge on why nerves of the central nervous system are unable to regenerate. Thus, this project will impact basic and translational scientists, clinicians and also those in fields outside of peripheral nerve repair.

This research is anticipated to have far-reaching benefits for a number of individuals/groups including, academics, clinicians, industry and the wider public; however, our primary motivation is to impact injured patients. Peripheral nerve injury (PNI) is a major unmet medical need, with >1 million new cases worldwide each year. Whilst microsurgical innovation has improved outcomes for some PNI patients, the principles have not much changed in the last 60 years and clinical outcomes remain poor. A limb following PNI will never regain its former function; almost 1 in 4 of these injuries will impact function of the hand such that less than 60% of these patients return to work within a year; whilst 1 in 20 lose entire arm function resulting in little prospect of a return to previous employment. This functional morbidity typically fosters psychosocial dysfunction alongside significant socio-economic impact. It is now apparent that a purely microsurgical approach to PNI will fail to address complex neurobiology following injury. It is therefore critical that novel therapies are sought to enhance quality of life of injured patients. Our approach focuses on critical cell-biological mechanisms controlling axonal regeneration that may be targeted therapeutically, thus having the potential for direct clinical benefits in the future. This is in line with the MRC health focus theme of 'advanced therapies'.

In the short term, the impact of this project will be most evident for academics working to enhance understanding of neurobiology and disease, particularly those in peripheral nerve science but additionally central nervous system regeneration and peripheral neuropathy. Focus will be placed on the role of the Golgi apparatus in peripheral nerve regeneration. Since the Golgi is involved in such diverse cellular functions, the relevance of this project will be far-reaching and may influence research in a wide range of disciplines relating to cell biology. Moreover, the ability to image neurons whilst they are living will enable testing of novel therapeutics including drugs, biomaterials and stem cells and could facilitate the early biocompatibility testing of these. Thus, it is anticipated that cross-disciplinary collaborations will form. This will benefit the wider research society and also stimulate the biotechnology industry into a new potential market.

Improving long-term outcomes in PNI would provide the NHS and wider society significant economic benefit, both from costs directly associated with healthcare and indirectly from unemployment. PNI can be particularly devastating for those without reliable access to healthcare; therefore research outputs will be shared through existing collaborations in developing countries (Zimbabwe/Uganda/Bangladesh) via the BFIRST charity.

Social responsibility is one of the UoM's three core goals in the Manchester 2020 strategy. We will embed engagement with patients and the public into this research program, and build upon existing patient involvement groups. Outreach events will plan to inform the whole spectrum of the population. We will produce visually stimulating content with fluorescent super-resolution images and live-cell movies, which would be ideal on display at outreach events in galleries/museums. This will generate cultural interest and also inform the wider population of the impact of PNI on patients and the search for new therapies. These movies will be utilised for education purposes for students, clinicians and researchers to aid in learning the basic biological mechanisms of peripheral nervous system's response to injury.

Personally, being awarded this fellowship would be pivotal in my progression towards becoming an independent clinical researcher. The support from the MRC and the training provided at the UoM would equip me with essential skills to apply for an MRC clinician scientist fellowship, which would be pivotal in establishing my career as a clinical academic.