Speaking of Science: Interrogating the physiology of the human vagus nerve with Professor Vaughan Macefield

For our April edition of Speaking of Science in acknowledgement of World Creativity and Innovation Week, we were joined by Professor of Neuroscience at Monash University, Professor Vaughan Macefield.

As the first researcher to record electrical signals from the human vagus nerve in awake humans in 2020, Professor Macefield is interrogating how our largest parasympathetic nerve controls organs affected in certain disease states such as cardiovascular disease.

Watch and listen as Professor Macefield discusses his groundbreaking (and inspiring) innovative research and what it means for the future of neuroscience.

Recorded on Tuesday 15 April from 11:00AM – 12:00PM AEST.

Video transcript

0:16 Dr Julie Glover
Good morning, all.

I'm just waiting for people to flow in from the waiting room. I'll just give it another minute because I can see it's a little bit of a lag as people come in.

OK, Welcome everyone. Before we begin this morning, I would like to acknowledge the traditional owners of the lands on which we're meeting. I'm here on Ngunnawal country and pay my respects to elders past, present and emerging.

I recognise and respect their cultural heritage, their beliefs and relationship with the land, which continue to be important to the Ngunnawal people today. I extend this respect to Aboriginal and Torres Strait Islander people who are present today and acknowledge that we're meeting on lots of lands around Australia and also internationally.

My name is Julie Glover, I'm the Executive Director of the Research Foundations branch here at NHMRC and I'm your host here this morning.

Beginning with housekeeping. You would have seen that we are recording this meeting.

I just also remind people that the Australian government is in Caretaker and we're currently operating in accordance with the Caretaker Conventions. NHMRC is unable to answer policy type questions, but that's OK because we're here to hear from Vaughan and also there will be a chance to ask him questions at the end of his presentation.

If you do have questions, you can enter them into the chat, and we will get through as many of those as we can. One last reminder you, you will have seen that we are recording, there are past, all of our past recordings for these.

This series are available on our website, and today's discussion will also be added to our website in the coming days.

Welcome to a very special edition of Speaking of Science and one in which I hope that you walk away from feeling empowered to use new ideas, make new decisions, and make the world a better place with your creativity. This month's talk is timed with the start of World Creativity and Innovation Week, which is a United Nations global day of observance that is dedicated to celebrating all forms of creativity.

Collectively speaking, NHMRC funded research is a prime example of creativity, of the outstanding breakthroughs that can happen when innovation and creativity are supported to improve human health.

In particular, the overarching objective of the Ideas Grant scheme supports innovative and creative projects in any area of research from discovery right through to implementation science, and so the Ideas Grant scheme is part of our theme today.

That brings me to our guest speaker who is very familiar with our Ideas Grant scheme and that is Professor Vaughan Macefield, who's on screen here. Vaughan is a Professor of Neuroscience in the Department of Neuroscience at Monash University, and he specialises in recording single nerve fibres via microelectrodes inserted into the peripheral nerves of awake human participants. Vaughan is here because he holds the rare distinction of receiving both the 2022 NHMRC Marshall and Warren Ideas Grant Award and the NHMRC Marshall and Warren Innovation Award for his Ideas Grant titled Microelectrode Recordings from the Vagus Nerve in Awake Humans.

Just to explain what this means, this means that Vaughan's application not only had the highest overall score in around with over 2,100 applications, it also received the highest innovation and creativity score. Vaughan is the only Ideas Grant recipient since the inception of the scheme in 2019 to achieve this sort of joint award. That's a great accomplishment.

Today we'll be hearing from Vaughan on his research and how he is looking at the Physiology of the human vagus nerve. I will now hand over to you Vaughan.

Just a reminder, we'll take questions at the end, but you can also drop them in the chat as we go through.

Thanks Vaughan.

5:07 Professor Vaughan Macefield
Thank you very much, Julie and thank you NHMRC for that great honour of collecting these two awards.

I should say that this it was the third time I put that grant in. So, third time lucky, yeah.

I'm delighted to have been invited to speak to you all today and it's wonderful to see so many people online.

As you heard, I've been recording from peripheral nerves for a long time and I'm just going to give a bit of a background there. So if, let me just, OK.

The technique that I'll be talking about is called microneurography, and it was developed in Sweden in the mid 60s by Karl-Erik Hagbarth and Åke Vallbo. I've worked with both of those, and I feel very privileged to have done so.

As you can see, it's contributed a lot of information on our sense of touch proprioception, how we know where our limbs are in space and it has given us information on how we process pain, how we control the hand for instance, or the legs, and in particular how we control the sympathetic nervous system.

What it involves is inserting a Tungsten microelectrode, a very, very fine microelectrode with respect 200 microns in diameter, electrolytically sharpened to a very fine tip, insulated right down to the very tip, so only two to five microns are exposed. A very focal recording area.

This can be inserted into any accessible peripheral nerve, such as the median, all nerves of the wrist, median all nerves in the upper arm, the radial nerve in the upper arm or behind the knee, the tibial nerve or the side of the knee, the common perineal nerve, or at the ankle, the serial or the posterior tibial nerve.

There have also been a handful of studies in which people have put these microelectrodes into branches of cranial nerves, such as branches of the trigeminal nerve, such as the infraorbital nerve below the eye, the super orbital above the facial nerve and I have a dentist colleague in Sweden who put some microelectrode in the mouth and in the same nerve that the dentist will block with local anaesthetic, he can record from single sensory endings in the teeth.

There is a bit of a background to applying this to the cranial nerves. But I've been wanting to record from the vagus nerve for years. I submitted an ethics application in late 2019 and surprisingly it got through and here we are.

Just some background. This is what the microneurography setup looks like. We have the, you can just see the head stage, this is a participant's knee. The silver lead is the ground lead, which is just a surface lead.

We mark out the course of the nerve by giving a weak electrical stimulation over the skin with a little surface probe and if we're over the nerve, then the muscles of the foot will twitch, and the participant will report radiating pins and needles.

We then insert this active microelectrode into the skin. We have a reference electrode just under the skin and we electrically stimulate through the microelectrode. We know that we're inside the nerve and particular inside a single fascicle of the nerve if we're getting twitches of a particular muscle or radiating pins and needles into a particular area of the skin at 20 microamps and then we can listen and we can hear signals such as these.

Here's a cross section of a peripheral nerve. I use this image all the time because it took a long time to make, but basically we can record from the very large diameter axons, the myelinated axons in this case. Example here is a muscle spindle firing spontaneously and related to muscle stretch, and we can also recall from the very, very small axons.

These are the unmyelinated axons or the C fibres. These large, myelinated axons have a diameter of about 11.50 microns, 1/5 diameter of the human hair, whereas these small ones have a diameter of a micron.

Importantly for this talk, when it gets the vagus nerve is the fact that the tungsten microelectrode being a monopolar electrode, when it records from myelinated axons those nerve action potentials, the spikes shown here are positive going.

Conversely, if we record from C fibres on myelinated axons such as this example, which is a muscle based constricted neuron, a sympathetic post ganglionic axon, these generate negative going action potentials.

With this, when we record from the vagus, we can determine whether we're recording from myelinated axons or unmyelinated axons. We can also record from individual C fibres, and this is something I did with Gunavelin and Okavalvo over 30 years ago.

Just like any single unit recording, whether we are recording from one and only one axon really depends on the principle that the action potentials. The spikes, note these are negative going spikes, when they're superimposed as shown down here, we'll have a uniform spike morphology. We can conclude beyond reasonable doubt that this firing represents the activity of one and only one axon.

Now this leads to the topic of the talk, can we record from the vagus nerve? Well, I was thinking, well, it should be possible. Just by way of background, most of you will know that it's the largest and longest cranial nerve. It supplies structures in the neck, the chest, the abdomen. But it's also the only cranial nerve in which the majority of the innovation territory resides outside the head, the chest and the abdomen, for instance, and the neck.

But just like all the peripheral nerves that I've been recording from for many, many years, the vagus nerve is surround by thick fibrous sheath. My PhD was all neurophysiology and experimental animals. I was recording from the vagus nerve in rabbits, for instance, and we know that the sheath is very, very tough and this is called the epineurium.

The human vagus nerve is composed of about 6 to 8 separate fascals and each of those is surrounded by their own sheath. Basically, knowing that the structure of the nerve is very similar to the structure of peripheral nerves, it should be possible to impale the cervical vagus nerve and that was the starting position. It should be possible.

I wanted to do it after, of course, obtaining ethics approval. In early 2020, a postdoc from Pisa, Italy, and I'd been visiting Italy, collaborating over there. He was recording from the vagus nerve in the pigs, and they were developing novel interfascicular electrodes. He said, oh, please, please, please can I come and work in your lab? This is Matteo Ottaviani shown down here and so he was the first author.

Basically, we performed the first recordings on ourselves, just three of the authors and we used ultrasound to guide the microelectrode into the nerve and I'll show you that shortly. Unfortunately, if you all recall, 2020 was our annus horribilis, the year in which COVID broke out and after China, you will all recall that Italy was the first country to report cases. Matteo had to return home.

We do this by having the participant's neck rotated to the side and we insert the microelectrode just posterior to the sternocleidomastoid muscle, this big muscle here, and having used ultrasound to image the carotid arteries, which we can see here.

This is now the internal carotid artery and the internal jugular vein. This was when we went up quite high, but usually we come further down the common carotid artery, and this is the vagus nerve. We can see this on ultrasound. The microelectrode is highly echogenic, being metal, and we just have to have the ultrasound in the right plane so we can image it.

This is what it looks like when we're going in. Let me just show this video. What you can see is I'm just moving the electrode in and out and I'm trying to get just the right trajectory.

Here is the pulsating carotid artery, here is the vagus nerve, and here is the internal jugular vein, which is very compliant and squashy. But we can see that the microelectrode is aiming towards the vagus nerve, and once we are in and here we are in, we can just move to the next slide, we can just record, sorry, slide's not progressing, we can record activity.

I should point out that the microelectrode, you can clearly see that the vagus nerve is pulsing with a carotid artery when we let go of the microelectrode and the microelectrode tip is insitu within the nerve, the nerve and the microelectrode and the carotid artery are pulsating as one. The microelectrode moves with the nerve.

It's the same principle of how we obtain stable recordings from the median or ulnar nerve in the upper arm, both of which are located above the brachial artery, which is not, doesn't have the diameter of which is not so dissimilar to that of the carotid artery.

We can get very stable recordings on the brachial artery from the median or on nerves rather despite the pulsations.
Now here's an example. We have respiration on the top, we've got ECG, heart rate, continuous blood pressure recorded from the finger at the bottom. We can see some far field activity related to inspiration. There's just a smooth version shown in the RMS, the root mean squared process signal.

But here there was evidently an increase in the depth or the rate of inspiration, which wasn't really reflected in this respiration band around the chest. But nevertheless, we can see there's a recruitment of an axon here during inspiration.

Now we know that during inspiration several things occur, one of which is the larynx opens to allow air flow. This could be a neuron supplying a laryngeal abductor, a muscle that opens the posterior cricoarytenoid muscle that opens the larynx, or it could be a stretch receptor in the tracheobronchial tree such as a pulmonary stretch receptor. These are the largest myelinated axons within the vagus nerve.

The vagus nerve is full of respiratory related activity by the way. Or we could ask the participants to take a deep breath hold if it was a layer and we can see the same neuron is firing tonically as the participant holds his breath. I think it was he. When we hold our breath, we are holding our breath against the closed glottis, so we go, and the larynx constricts. It couldn't be a laryngeal dilator. It could not be a motor axon to the posterior cricoarytenoid muscle because otherwise this activity would go down.

The most parsimonious explanation here is that this is indeed a stretch receptor in the tracheobronchial tree.

It's not a laryngeal constrictor, otherwise it wouldn't be active during inspiration, and it also was not active during humming. Which we generate humming by passing airflow through the partially constricted larynx.

We also find sites; this is a multi-unit site in which the dominant activity is not during inspiration but during expiration. We can see here that there was a spontaneous swallow that the participant made. This didn't dislodge the recording. Participants can talk and they can swallow. What they can't do is move their neck, obviously.

We can see during the swallow there's an increase in heart rate and there's another burst of activity. We conclude again the most parsimonious explanation because we can't actually cut open the chest in these living humans to poke around and see where we are. We have to try and interpret these signals on basis of behavioural criteria. We assume that this is a laryngeal set of motor neurons going to the laryngeal constrictors.

Subsequent paper. This is Mikaela Patros who did her honours with me, and this is from her honours project, which is a very ambitious project to say the least, where we wanted to quantify the activity within the vagus.

We're looking for the low hanging fruit here. We're looking for signals that are correlated to the ECG, to the heart rhythm or to the respiratory rhythm. We know that the vagus nerve supplies the gut, most of the gut apart from the distal part of the colon, but we don't have any way of correlating activity in the gut at this stage. We're looking for activity that's related to the cardiac rhythm or the respiratory rhythm.

Here's an example where we've got an increase in signal intensity during inspiration, just as you saw previously. But also we've got these little bursts occurring at the asterisks, single spikes here at the asterisks, that are occurring with what appears to be a cardiac rhythmicity, and we can quantify that by performing cross correlation analysis.

Now there'll be a few figures like this, I'll just take you through it.

Time 0 is the first R wave of the ECG. These black columns represent the time at which the next R wave of the ECG occurs, and then the one after that, one after that, and then to the left, the ones back in time. We’ve just analysed hundreds of R waves of the ECG shown in black, and we've analysed the same number of sweeps of the vagus nerve activity in grey.

I think it's very clear to see, and this smooth curve shows this, that there is indeed cardiac modulation in the vagus nerve. We can quantify that by measuring the amplitude of this modulation. We get a modulation index which is basically the peak minus the trough, divided by the peak.

This is activity from two participants, so clearly the modulation in panel A, this participant is higher than that in panel B because it's a lot more background activity in this participant in the lower panel that is not related to cardiac rhythm. Nevertheless, with a multi-unit recording, with so many things going on, so much activity related respiration, etcetera, we can still find activity related to the cardiac rhythm.

Likewise, we can find activity related to the respiratory rhythm. So here time 0 is the first in spiritual peak and then we got the subsequent breaths to the right, previous breaths to the left and the vagus activity in grey. Again, there are peaks that have clear respiratory rhythmicity. Two participants are shown here.

If we ask them to take to undertake slow deep breathing, then no surprise that activity is augmented. We can measure the magnitude of the modulation and basically respiratory modulation is always dominant because there is just so much more activity related to respiration in the vagus nerve. Sensory endings coming from the trachea, the bronchi and the bronchioles as well as inspiratory related activity to the larynx and also expiratory activity related to the larynx.

However, we do find some sites in which we have very marked cardiac locked burst. Here's the ECG and this is the smooth version, and I think you can see and over the loudspeaker would hear, with each heartbeat. This is very strong activity.

When we look at the modulation in this site, we've got very clear cardiac modulation. If we compare that to muscle sympathetic nerve activity which we record from the leg, that has very marked cardiac rhythmicity as you can see in the bottom trace.

What we see in the vagus nerve is not as strong as what we see in sympathetic basal constricted neurons going to skeletal muscle recorded from the leg or any other peripheral nerve. But we do see nonetheless very marked cardiac modulation.

Here are some recent examples where again we've got marked cardiac modulation. In this case there's negative going spikes. The C fibres and this each of these bursts is occurring during the cardiac rhythm and it's higher when blood pressure is increasing and during slow deep breathing.

We can see this here that during slow deep breathing, it becomes constrained to occur during the expiratory phase when we've got an increase in, well after we've got an increase in, atrial filling during inspiration. Because these are C fibres and conduct slowly, we see this activity in expiration. But we presume given the fact that we're recording from the right vagus nerve that these indeed originate in the right atrium.

We also found sites in which, this is the end of a maximum spiritual breath hold, so you can see the respiratory signal here. What we've got here are two bursts of cardiac intervals. So instead of going with each heartbeat, it's going, which was very, very interesting.

If we show this on an expanded time base, I should point out that the blood pressure recording we obtain is from the finger, and so it takes quite some time for the blood pressure wave to travel down to the finger. If you shift this blood pressure signal back in time, I think you'd be convinced that the first burst is related to systole and the second burst to just after the dichroitic notch.

What could this be? Well, perhaps it is something related to the opening and closing of these valves. The tricuspid or the pulmonary valves, these are obviously opening and closing during systole and so maybe the first burst is related to opening and the second to closing. Again, this is just conjecture because we can't go in and poke around, but these are based on the most parsimonious interpretation based on the signals we've got and what we know from animal work.

Now, in 2022 I was invited over to Croatia. They'd heard about my vagus nerve recording, and how could I refuse going to Split, Croatia where they studied breath hold divers? They've been diving for the sponges for generations and it's a family of thing, but they've got some world champions who can hold their breath for over 11 minutes. Very, very remarkable. But I had to walk along a pebbly beach, and we were not used to that in Australia.

But here we have a, a happy customer. She has a microelectrode in her nerve and clearly it's well tolerated. Here we have a participant, one of the divers. This was a young male, and you can see we've got the microelectrode here. Here's some ultrasound gel, the head stage and he's been holding his breath at this stage for about 5 minutes and he's clearly very good at it and he could hold his breath for 7 minutes.

When we record the activity in the vagus nerve in these participants, we've done this from 11 of these so far. It's hard to see it in the left panel, but if we do the cross-correlation analysis, which I'll show you shortly, we can actually see some low level cardiac locked activity.

But what you can see here, this is during the last stages of the breath hold. Well, he kept on going for quite some time, but here what's happening is, is inflated and it's going, is holding on, is not inhaling. But you can see there, there are these small inspiratory movements which are reflected in the transducer around the chest. There's no, there's no airflow, there's no change in lung volume, but there's clearly changes in pressure within the within the chest with each of these little efforts. You can see the vagus nerve activity is increasing there.

If we look at two participants with modulation that we saw during the hold phase before they're making these inspiratory efforts against the closed gauges. We've seen in this case the peak is occurring just before the peak of inspiration. But here in the lower example, we've got the trough around that, so the peak is on either side. There are clues there which we don't fully understand, but it may be related to the location of these receptors or the identity of these receptors. As you can see in the lower trace, when these involuntary breathing movements occur, we see this very strong cardiac locked activity, and in some instances, as shown here, the activity is very, very much strongly occurring prior to the breadth. The vagal activity is occurring just before the peak of inspiration.

I won't have much further to speak, but we've been recording from single nerve fibres because it's very hard to identify what we're recording from when we've got a multi-unit recording. We've got sensory activity, we've got motor activity, it's hard to differentiate two. But with single unit recordings we have a much better chance.

We recorded from 31 axons, single nerve fibres and we were just looking at those with cardiac rhythmicity, 10 from the left, 21 from the right, vagus nerve. This was David Farmer, who's a postdoc in the lab and has done a lot of work on the autonomic nervous system in in experimental animals.

Seventeen were defined as myelinated axons, positive going spikes and 14 as unmyelinated axons on account of their generally negative going spikes. Here's a principle, we've got a single spike here, we have a threshold and this confirms that this is indeed a recording from a single nerve fibre, single axon. As you saw previously, we got the peak of the R wave at time 0, subsequent R waves to the right, previous ones to the left. This example shows clear cardiac modulation. However, the example on the right panel did not, so not all of them exhibit cardiac rhythmicity.

Here's one that behaved where again, myelinated spikes, behaved from just like a cardio inhibitory neuron. We all know that the vagus nerve supplies the heart, and our heart rates are kept in check by the brakes exerted by the vagus nerve. Parasympathetic cardioinhibitory neuron which supplies the sinoatrial node, we conclude this because it behaved like one based on what we know from animal work. That is its peak activity is occurring just after inspiration, and it's inhibited during inspiration. This is what we would expect from a cardio inhibitory neuron, that its activity should be withdrawn. We withdraw the brakes onto the heart during inspiration, thereby allowing heart rate to increase and during expiration we put the brakes back on as shown by all these spikes in the immediate post inspiratory phase.

We can also identify, well attempt to identify other axons based on their behaviour. This one was recorded here during a maximum respiratory breath hold. Before and after the breath hold it exhibited cardiac modulation. But during the breath hold, that modulation was reduced.

Now our interpretation of that is, when you take a deep breath, hold again, the glottis is closed, intrathoracic pressure is high so the pressure across the heart from the elastic recoil of the lungs and the chest wall and the diaphragm is high and that basically compresses the atria, unloads the low-pressure barrel receptors which we believe this activity represents.

Now the final part is we've been doing some work on how vagus nerve stimulation works. Now this is a technique which has been around since the 90s. It's approved for treatment of drug-resistant epilepsy in Australia, in the US it's also used to treat drug resistant depression, and in other countries also gastrointestinal disorders and inflammatory disorders.

There's a generator just inserted under the chest. We have electrodes on the nerve, and this is just sending pulses to the nerve. We don't know how it works. One of the things we know is that we don't know much. Basically, when a patient has this implanted, the neurologist will titrate the current intensity, frequency and duty cycle so the patient can tolerate it because what it can do is activate nerve fibres going to the larynx. It can affect their voice, it can cause burping, it can cause unpleasant sensations. We don't want that, but we don't know how it all works.

We recently published a paper where we've inserted microelectrodes into the vagus nerve in patients with implanted vagus nerve stimulating devices. The whole point here is to find out which nerve fibres it activates. None of this was known before.

Here are examples just from four patients. Basically, stimulus artefact, the big spike on the left and then four different patients, different current intensities. Here on the left we can see negative going potentials, that is unmanned axons in this case being activated at half a milliamp. In this example we've got myelinated axons and unmyelinated axons, myelinated axons just here. It's early days yet, we've recorded seven so far. But this will help us to understand how it works and perhaps we'll be able to learn that, well, actually you don't need to stimulate at such high intensities that generate all these site unwanted side effects.

Now the final part is what does it do to other parts of the of the system? Because the vagus nerve doesn't just send sensory signals to the brain, it sends as said motor signals to the heart, motor signals to the gut etcetera and the lungs.

We tested the hypothesis that chronic vagus nerve stimulation reduces muscle synthetic nerve activity. We know that this is important in the control of blood pressure, and we know that elevated muscle sympathetic nerve activity features in many diseases associated with elevated cardiovascular risk. People with heart failure, people with hypertension, their muscle sympathetic nerve activity is very, very strong. It's going with every heartbeat. Constricting the blood vessels, driving up blood pressure in the case of hypertension or trying to maintain blood pressure in the case of heart failure, where the heart clearly can't do its job of pumping out blood to the whole body. It is compromised, so the system attempts to help it by constricting the blood vessels in the periphery.

We know that epilepsy also has elevated cardiovascular risk. Sudden unexplained death in epilepsy is believed to occur due to disturbances in autonomic control. My colleagues here at Monash University, the Department of Neuroscience and the Department of Neurology have been looking at the disturbed autonomic control in epilepsy through video monitoring of ECG and respiration.

You've seen this image before. We record muscle synthetic nerve activity just through a microelectrode recording these negative going spikes. This is E print activity, motor activity, going to the blood vessels and the skeletal muscle. What we showed first was that, and this is work Mikaela Patros, my PhD student has done. After her honours she went straight into her PhD and is exploring the vagus nerve with great gusto.

Basically, patients with drug resistant epilepsy have much higher muscle sympathetic nerve activity than age match controls. This fits with them having high cardiovascular risk. Systolic blood pressure was also significantly elevated in these patients.

Here's a recording from a patient, and I should point out that these were a recording from a patient who was yet to receive the biggest nerve stimulator, we can see that the activity here is quite high.

We’ve got bursts in many a cardiac interval. This is immediately prior to implantation of the device, the cuff electrodes around the vagus nerve. This is 3 months later. Now baseline, 3 months later, we've still got a very good signal from the vagus. We've got clearly negative going spikes. We've got a very good signal, but there's very few bursts. VNS shown here, this is the green trace. This is basically we're just picking up the electrical stimulation being applied to the vagus nerve just with surface electrodes.

We can see that when the vagus nerve stimulation comes on, it doesn't do anything to the sympathetic nerve activity, but chronically it reduces the activity. Appears to be no acute effects, but chronically there is. This is a patient during the same patient tilted upright. When we tilt patients upright muscle synthetic nerve activity increases to prevent blood pooling in the lower limbs. Again, we can see that VNS is not doing anything acutely, which is a very good thing because if patients with these devices every time it came on and they were standing, it would inhibit this activity, and they'd collapse because they wouldn't be able to maintain their blood pressure.

What we've shown in a cross-sectional study, participants with epilepsy. without VNS and those with VNS, clearly you can see the activity goes down. To conclude, we can perform microelectrode recordings from the vagus nerve and it's allowing us to interrogate the physiology of this nerve for the first time in humans in what I'd like to think is in unprecedented detail.

It's well tolerated, and ultrasound guidance of the microelectrode allows us to penetrate the nerve without penetrating the carotid artery. This is clearly why nobody's attempted it before because it's tiger country in there. Again, we'd like to think that this is starting a new area of research. It's still early days, but with the funding from the Ideas Grant for which I'm incredibly grateful. I would have been out of a job if it wasn't for that are allowing us to understand how the vagus nerve is involved in normal physiology and also disturbed physiology.

We can look at things like asthma, postural hypertension, various disease states and epilepsy of course. With respect to epilepsy, we know that vagus nerve stimulation, clinical stimulation has no acute effect on muscle sympathetic nerve activity at rest, but it does show a marked reduction in people who have been stimulated with VNS for six months or more.

Because of this reduction, we feel the vagus nerve stimulation may have a protective effect on cardiovascular risk by reducing sympathetic activity, not acutely but over time through some central neuromodulation. This may contribute to the mechanism behind risk reduction for sudden unexplained death in epilepsy SUDEP in VNS treated patients.

Thanks to the National Health and Medical Research Council of Australia for funding this fundamental work and also to the National Institutes of Health for funding the epilepsy related work which we've only just started.

I'd like to thank the vagus team, Matteo, Tye, Leah, Mikaela, David and Kegan in order of involvement in the study. And to the epilepsy team for Terry O'Brien, Hugh Simpson and Shobi Sivathamboo, who has been doing a lot of the work on the SUDEP.

Thank you so much and I'm very happy to take questions.

42:40 Dr Julie Glover
Thank you very much, Vaughn. That was a fascinating talk and my mind's spinning with all the different possibilities of the next experiment, so thank you.

We're very happy to take questions. You can either drop your question in the chat or if you're comfortable, you can put your hand up and come off camera or come off mute to ask questions. Just not sure how I can see that. Please if you do have a question, do sing out.

I might kick off while people are thinking about that.

You mentioned a couple of times, one that these students were doing, all your postdocs were doing quite ambitious projects. How do you balance the risks of that? Because obviously the more ambitious it is, maybe the less likely it is to lead to outcomes.

43:39 Professor Vaughan Macefield
That's very true. As I said, we started this work in 2020 and we had to stop the work of this reasons.

Look, Mikaela Patros is my honour student. She approached me and very in the year, I said, look, I'm really interested in your work and is there a project you have available? I'd already assigned some honest projects to some other students. I said, well, there is something, you know, recording from the vagus nerve. It's a bit risky. It may not get good data, but we got good data and she got a paper from that, and she won a prize from the Journal of Physiology. Because of that, she got a scholarship to do a PhD and she's done very, very well.

It has been risky, but it's worth the risk to push the boundaries. I think of course, this is all under ethics approval, but yes, it's an important question.

44:48 Dr Julie Glover
Yeah, great, thanks.

There is a question in the in the chat about ethics approval. How did you convince the Ethics Committee that you could penetrate the vagus nerve without penetrating the carotid artery?

45:00 Professor Vaughan Macefield
Well, basically I put in a very large application saying I've been recording from peripheral nerves since 1887, not 1887, since 87. Some of those, as I mentioned, some of those were recordings from the median or on the nerve in the upper arm and they're just sitting over the brachial artery.

Basically, I provided all this evidence of the size of the vagus nerve, the size of the ulnar, the median nerve, the size of the carotid artery, the size of the brachial artery and the success I had in recording from the median ulnar nerve in the upper arm without penetrating the carotid artery.

Now full disclosure here, we have had in over 70 recordings from the vagus nerve, we have had five inadvertent penetrations of the carotid artery, and this happened because the sonographer just intimately lost contact, lost view of the microelectrode tip. Then we saw that it was just in the artery, and we just withdrew. There were no, the participant didn't feel it, there were no consequences.

We followed up and we reported that to the Ethics Committee and they reviewed it and they realised that, well, they know that cardiologists put very large cannula in arteries and the arteries do self-seal, and our microelectrodes are 0.2 millimetres in diameter, so 200 microns. The risk was considered acceptable.
We are very, very careful. We are much better with the ultrasound now. We've got a much higher resolution ultrasound probe. It hasn't happened again, your Honour.

46:48 Dr Julie Glover
Thanks for the detailed follow up. That's excellent.

There's a question there about whether VNS has been used for migraine treatment.

46:55 Professor Vaughan Macefield
Actually, I'm not aware of that because this of course does require surgical implantation of the electrodes and this is not approved for anything other than epilepsy in Australia. In other countries it's approved for other conditions.

There is another way of stimulating the vagus nerve. It's not as clean as this I would say, the vagus nerve supplies a little patch of skin in the ear hole and that's called the auricular branch of the vagus nerve. There are quite a few studies where they're applying electrical stimulation just to that little patch of skin. It's not as clean as this because we've got the whole nerve, not just this cutaneous branch, so when the vagus nerve is being stimulated clinically here, we're activating axons coming from the heart, the lungs, the gut, as opposed to just cutaneous input.

That is being employed for many conditions such as chronic pain, various conditions. But I'm not aware of migraine.

48:10 Dr Julie Glover
OK, thank you.

There's also a question there about auricular stimulation and whether it has similar effects to the cervical.

48:21 Professor Vaughan Macefield
Yeah, that's right.

There have been some studies by colleagues in Leeds in England and I’ve visited these and they have done electrical stimulation, the vagus nerve and the ear and they've shown some reduction in muscle sympathetic nerve activity as we showed here. We haven't been able to replicate that unfortunately.

What we do see is very robust reductions in muscle synthetic nerve activity with cervical vagus nerve stimulation and our colleagues at the University of Minnesota have also replicated that, so we know that's very robust.

But the ear stimulation, yeah, we've been playing around with that for a bit. We haven't had much success. We haven't even had success in affecting heart rate variability, which is what most people use it use that for.

49:18 Dr Julie Glover
Then there's a great question about stroke rehab and whether there are biomarkers that you would suggest as indicators to see where the is stimulating the vagal nerve, so suggested pupil dilation.

49:35 Professor Vaughan Macefield
Yeah, so we are very much interested in pupil dilation that is parasympathetic response. It's not via the vagus nerve, it's from the oculomotor nerve, parasympathetic axons travelling in that nerve.

With David Farmer, we're going to be looking at correlating pupil diameter with the directly recorded vagal activity to see if those two signals co-vary to see if pupil diameter changes with respiration, for instance. Because although psychologists are interested in the use of pupillometry to get a handle on the parasympathetic nervous system, the problem with these indirect measures is that both the pupil and the heart receive dual innovation from the sympathetic and parasympathetic nervous systems. So, disambiguating the two can be problematic and certainly heart rate variability is grossly over interpreted. In fact, there's no evidence that low frequency heart rate variability at rest reflects sympathetic outflow to the heart.

Where there's an increase in outflow then that's another matter. But at rest there's very little activity. I don't think vagus nerve stimulation would do anything directly to pupil diameter because that's a different nerve.

50:57 Dr Julie Glover
And then there's two questions and I think that'll be our last two questions.

But can you apply the breath holding to lowering blood pressure as a treatment for hypertension?

51:06 Professor Vaughan Macefield
That's a really interesting question.

Not breath holding, definitely slow deep breathing, slow deep breathing. There are a few studies out there. When we perform slow deep breathing, we can see in our example we get entrainment of vagal outflow. We also get entrainment of sympathetic outflow.

There have been a few studies showing that repeated episodes of slow deep breathing can reduce the total sympathetic outflow, but for a start, becomes constrained to occur during expiration and that change in pattern can indeed lead to a reduction in blood pressure.

There'll be many here on the call who may practise yoga, and the breathing techniques associated with that. I think slow deep breathing definitely has a physiological basis for lowering blood pressure and alleviating stress, of course.

52:08 Dr Julie Glover
Great. Then that final question there about the mechanism by which increased MSNA can lead to cardiorespiratory dysfunction.

52:18 Professor Vaughan Macefield
When we're recording muscle sympathetic nerve activity, it's just going to the skeletal muscle vascular bed.

But we know from recordings in the sheep for instance, and other animals that sympathetic outflow to the muscle, vascular bed, to skeletal muscle, largely parallels that to the heart, and to the kidneys, they're occurring at the same time, the phase lock to the cardiac rhythm. But there is differential control, so during stress is more sympathetic outflow to the heart.

But what we can say is that in conditions where there is elevated cardiovascular risk such as heart failure, we definitely know and this is all Murray Esler's work at the Baker Institute, who recorded from noradrenaline spillover from the heart by putting cannulas into the heart, the vessels of the heart. He showed that in patients with heart failure, there's very high levels of noradrenaline being released to the heart and there's very high levels of sympathetic nerve activity going to the muscle.

This is the basis for the use of beta blockade in heart failure. It was always thought you need to improve heart function by increasing the sympathetic activity of the heart, but we know that's actually deleterious. You want to reduce sympathetic outflow to the heart, not elsewhere. If you turned off sympathetic outflow to the muscle vascular bed, blood pressure would plummet because that's the only means by which the system is trying to maintain blood pressure.

There’s evidence from my colleagues at Monash, so Shobi Sivathamboo, Janine Liu, Terry O'Brien and so on and their colleagues showing that in animal models of epilepsy there are changes to the heart, functional and structural changes to the heart. We think that the heart is definitely receiving elevated sympathetic outflow in epilepsy. We believe that and that vagus nerve stimulation reduces that as well as reducing that to the muscle vascular bed.

We think it's having an effect on pseudo risk by reducing sympathetic output to the heart. That's my theory and I'm sticking to it.

54:43 Dr Julie Glover
That's great. Well, look, thank you. We're just coming up to time.

I wanted to say a really big thanks to our speaker for his time and efforts in putting that together for us today.

It's fabulous to hear what what's happening with such creative and innovative research.

Also, thanks to everyone for joining and this week I think emphasises just how important innovation and creativity is, helping us to shift our thinking away from current norms and to spark new ideas. This is just such a great prime example of someone thinking, oh, could we do that? I think we could do that. Can we do that?

A big thanks to everyone who joined us online. I just want to reiterate that we really appreciate and value your contributions to coming along to remember that also the Speaking of Science series is your series as just as much as it is ours. So please do continue to suggest to us topics for us to cover and reminding that the recording of this will be made available on our website shortly.

Thank you very much and enjoy the rest of your day everyone.

55:58 Professor Vaughan Macefield
Thank you.

55:58 Dr Julie Glover
Thanks again, Vaughn.

Bye.

End of transcript.