Imaging the functional anatomy of fascicles in the mammalian vagus nerve with neural tracers, electrophysiology and Electrical Impedance Tomography.

Over the past two decades, electrical stimulation of the nervous system has progressed from an academic research exercise into mainstream medical use. Implanted stimulators in the brain are now used to treat movement disorders and Parkinson's disease; electrical stimulation of the vagus nerve in the neck has been successfully used in reducing the frequency of epileptic seizures. Nerves in the body may be broadly divided into those that supply muscles and sensation in the limbs - "somatic nerves", and the "autonomic" nervous system when nerves supply bodily organs such as the heart, lungs and liver. There is currently great interest in extending the concept of electrical stimulation to treating diseases related to the autonomic nervous system such as diabetes mellitus or rheumatoid arthritis. A convenient autonomic target is the vagus nerve in the neck because it is easily accessible and a small implanted chip could be used to control about a dozen different supplied organs in the chest and abdomen. At the moment, stimulator technology is only available which would activate or suppress the entire nerve to which it is attached. For a nerve like the vagus nerve, this is unfortunate, as organs other than that intended may be stimulated. An example of this is that when patients with epilepsy have vagus nerve stimulation, they become hoarse, because nerves to the vocal cords are inadvertently stimulated.

Nerves are organised into internal bundles, termed "fascicles". The organisation of fascicles is well studied for the somatic nervous system but it is almost completely unknown for the vagus and other autonomic nerves. In order to be able to stimulate an organ selectively within the vagus nerve in the future, it is essential to understand how these fascicles are arranged within the nerve, and what their function is. For example, it is not known if any one fascicle within the vagus nerve in the neck supplies an individual organ, or whether these are all mixed together. If they are mixed together, then it will not be possible to stimulate an individual organ with a stimulator in the neck.

The purpose of this project will be to investigate the function and anatomy of the fascicles in the vagus nerve in the neck in humans. The anatomy will be studied with specialised dyes (neural tracers) which can be applied to a nerve and then travel up the nerve so that their connection all the way up to the brain is evident on inspection with a microscope. Secondly, the anatomical connection will be studied with a method in which the entire vagus nerve from the neck to its end organ will be cut into fine slices, inspected under a microscope, and a computer will be used to track all the fascicles within the nerve over its entire course. This approach will, for the first time, identify which fascicles in the cervical vagus nerve in the neck are connected to which end organs. Their electrical function may be studied using a fine array of small spike electrodes which can be inserted into the nerve or a new method, fast neural electrical impedance tomography (EIT). It enables production of images of fascicles in the nerve firing and is the only of these four techniques suitable for human use. It requires surgical placement of a flexible rubber cuff around the vagus nerve. This is ethical and practical in patients with epilepsy in whom the vagus nerve in the neck is exposed for insertion of a vagus nerve stimulator.

Studies will be initially undertaken in anaesthetised animals with all four methods. The findings from this will be used to inform a final study in human subjects using fast neural EIT. The output of this work will be, for the first time, an atlas of the anatomy and function of the vagus nerve throughout its course. This will provide a unique platform for efforts in the future where the vagus nerve will be stimulated to treat diseases and it will be beneficial to stimulate selected organs in order to avoid side effects.

The fascicular organisation of the cervical vagus nerve is almost completely unknown. The aim of this proposal is to investigate the functional anatomy of fascicles in the mammalian cervical vagus nerve which supply autonomic innervation to the stomach, heart, lungs and other visceral organs. Fascicle functional anatomy will be defined using AAV5 or other neural tracers and computerised fascicle tracing, and functional physiology with multielectrode arrays and electrophysiology, and the new method of fast neural imaging with Electrical Impedance Tomography (EIT). Initial studies will be in rat and rabbit sciatic nerve to enable technical integration of the methods. The refined methods will then be used in a defining dataset in anaesthetised pigs, and then a human study based on these findings using a non-penetrating nerve cuff with fast neural EIT; this is the only method which is non-invasive and so justifiable ethically for human studies. This will be performed for 30 minutes during an operation to insert a vagal nerve stimulator for treatment of epilepsy. It will deliver images of internal nerve activation in response to activity such as respiration or the ECG. The deliverables from this work will be : 1) A detailed anatomical atlas of the functional anatomy of fascicles in the cervical vagus nerve in the pig. 2) The first ever images of localised function in human vagus using fast neural EIT. This will be a novel resource for the autonomic community and inform the avoidance of off-target effects in "Electroceutical" autonomic nerve selective stimulation. The work will be led by an internationally leading consortium with expertise in bioengineering, computer science, autonomic physiology, large animal veterinary surgery, and a neurosurgeon who implants vagal nerve stimulators for epilepsy. The PI, David Holder, is a clinical neurophysiologist and engineer who has pioneered the development of fast neural EIT. 3) Refined method suite for future similar studies

"Electroceuticals" is a new field in which the goal is to treat a wide variety of medical diseases with electrical stimulation of autonomic nerves. A prime target for intervention is the cervical vagus nerve as it is easily surgically accessible and supplies many organs in the neck, thorax and abdomen. It would be desirable to stimulate selectively in order to avoid off target effects. This has not been tried in the past, both because of limitations in available technology but also because, surprisingly, the fascicular organisation of the cervical vagus nerve is almost completely unknown. The aim of this proposal is to investigate the functional anatomy of fascicles in the mammalian cervical vagus nerve. This will include defining innervation to the stomach, heart, lungs and, if possible, the intestines, spleen, pancreas, kidneys, adrenals, liver and gonads. We anticipate that the published Atlas will be a groundbreaking landmark resource which will be widely used in the future by academic and commercial researchers who are working on selective recording and modulation of vagus nerve activity.

The following are likely to benefit from this project.

1. Commercial companies in the Electroceuticals field.
The principal company in this field is Galvani Bioelectronics, who are a joint venture between Glaxo Smith Kline and Google Health. An improved understanding of nerve function would materially advance their applications which are planned to improve treatment in conditions including inflammatory bowel disease, arthritis, asthma, hypertension and diabetes.

2. Academics
Interest in physiology has shifted in the recent decade or two to cellular and molecular biology, and away from systems physiology. Understanding of nerve fascicle function is an unexplored area which is central to the new project of Electroceuticals. Academic groups working in this field would utilise this work in planning experiments and treatment strategies. These would include those working in inflammatory bowel disease, arthritis, asthma, hypertension and diabetes. It would also be of interest of any group working in neuroscience on how neural signals are coded in nerve. This is a leading topic in cognitive neuroscience where analysis is undertaken of neural coding in the brain. It has been less studies in peripheral nerve. Demonstration of application of these new techniques would advance this study in peripheral nerve.

3. Patients with treated conditions.
The work would lead indirectly to advances in treatment to patients with the above conditions and others in the broad range of conditions that could be managed with Electroceuticals.

4. Knowledge impact
The project contributes to the knowledge of several fields of research: Neural Anatomy, Peripheral Neural Surgery, Medical Physics. It is planned to share it with the research community through 1) releasing the project as an open access library and atlas, 2) creating community around it, 3) attending international conferences, and 4) publishing the results.

5. Follow on funding.
Autonomic systems physiology is a burgeoning field which has had relatively little attention in the past decade as interest has focused on cellular and molecular biology. This study will reawaken interest in this. We anticipate substantial international interest in the approach and findings. DH hosted an EPSRC network in the past on biomedical EIT. We will actively seek funds to establish a similar network to study the functional anatomy of the ANS and also a programme grant to extent these methods to other organs supplied by the ANS.

6. People impact
The work will contribute to the development and career progression of 3 academic staff, who will deepen their skills in science, and will learn to develop a multi-modular framework of tools. It could launch their careers in the application of interdisciplinary methods to study of the peripheral nervous system and ANS.