We'll go ahead and get started. Thank you all for joining us at 5 p.m. on a Friday. That's an impressive turnout for sure, so I appreciate your attention and your time. My name is David Brogan. I'm from Washington University in St. Louis, and I'll be helping to moderate this session with Dr. Moore, and the title is Back to the Future, Translational Techniques that Will Change Peripheral Nerve Repair. And so our hope, we've really assembled a pretty austere faculty that's very impressed with their accomplishments, and they're going to enlighten all of us with different techniques and hopeful promises of things that may change what we do in the future. So how do we kind of break through our current results, and what can we do to improve them? So I'm looking forward to it. We'll have these topics, and leading off, Adam Reed is going to help talk to us about the basic science of peripheral nerve injury and repair. And Adam, if you can come on up here, please. Fantastic. And thank you very much for the invitation. So I'm Adam Reed. I'm a plastic surgeon and senior clinical lecturer at the University of Manchester in England. I'm really delighted to be in Boston. I heard so much nerve talks already today, just a few more, and a little bit about the basic science that I've been involved in. First of all, disclosures. I don't receive any personal payments from any company. Research funding is government or association charities, British Society, Royal College of Surgeons, BAPRAS, and a small amount of industrial funding for a PhD project, which I will talk about. So this is Manchester, a beautiful place, if you've never been. We have a fantastic university, really fantastic basic science expertise in there, and the hospital right next door, one of the largest trusts in the UK, with a major trauma centre built in. As part of the university, I lead the Blon-McIndoe Laboratories, which is surgeons and scientists working together on things like peripheral nerve injury, wound healing, are the things that we're interested in. It being Manchester, this is the view from the labs. So today, I'm going to present data that's been generated by, mostly by my PhD fellows. Ralph, who you may have heard from this morning, on ultrasound, he also did some work to work out how common is nerve injury, and we'll have a look at the UK overview data. What is the focus of basic science in nerve injury? We'll have a look at that, and the different sites. In particular, the repair site, and a very quick slide on the distal stump, but the neuronal cell body, because we've got some quite exciting work going on, looking at that just now in a collaboration within Manchester. So Ralph presented ultrasound data this morning, but what we realised in trying to generate funding for some of these projects, is they all want to know the epidemiology, and if you read the papers, most of the epidemiological papers on peripheral nerve injury are very, very poor. So whilst the NHS has its flaws, it has many good things as well, in that we domineer the market in trauma, so we have almost all trauma cases recorded through the NHS. So this is really good data, national data, we've not published this yet, hopefully later this year, but we've demonstrated that in the UK there's a mean instance of somewhere between 11 and 12 events per 100,000 population per year. And then that equates to somewhere between 9 and 10,000 nerve injuries per year. And as you might know already, the vast majority of these are wrist and distal, with some in the forearm, about a sixth of the number in the forearm, and then a few less frequently plexus injuries. And then all the other nerve injuries are way below that. Typically, men, three times more common than women, and we know the age distribution, these are predominantly young working males. But I think for the first time, Ralph will be able to show with this data the true national level epidemiology. So this is why nerve injury is important, and we've heard a lot clinically today about the impact upon the patient, so I'm not going to go over old ground. In treating nerve injury, there is complex and really counterproductive neurobiology, which I'll touch a bit upon. But current therapies really revolve around a few stitches and some glue, and nothing really has been a game changer in terms of getting the nerve to regenerate. Now, there have been advances in surgical techniques, sure, and nerve transfers and tendon transfers, and that's been great for the patient. But in terms of nerve regeneration, there isn't an awful lot that we can do differently over the last hundred years or so, since Tenel described repairing nerves using nerve grafts. So the focus of basic science has, in the surgical domain, predominantly been at the site of nerve injury. However, we know from Goran Lundberg's work that there is an immediate and longstanding change in the cortical neurons in the central nervous system. We know that in the cell body, there's changes, in particular in the sensory system. So the sensory cells in the dorsal root ganglion die in great numbers after an injury, and this is particularly important for sensory recovery, which is determined by the number of the quantity of reinnervation is directly proportional to the recovery of sensation. So if we lose 40% of our neurons, then we're probably getting less across the repair site and less to the final destination in skin. And I'll talk a little bit about what we're looking at in the neuronal cell body. The site of repair, now this is where all the work has been done, and I'll talk just a little bit about what we're doing in Manchester. And I'm interested to hear the other speakers later. I want to know, I've just felt this nerve tape. Still feels like it's stuck on my finger, but it looks like an exciting thing to repair a nerve. PEG fusion sounds like a very exciting paradigm shift in nerve regeneration. Electrical stimulation, we're going to hear about today, and conduits in cells. So I'll talk a little bit about what Manchester's doing. Medical stump, just a slide, and today the end organ reinnervation. So there's changes across the whole of the nerve, from the central nervous system all the way to the end organ, that we need to be thinking about. Liam is another PhD fellow. He is partially funded by Manchester Biogel, who are a start-up in Manchester, and together with them, we've generated a fairly straightforward self-assembling peptide made up of three essential amino acids. It's entirely synthetic and reproducible, and we think can kind of replicate the extracellular matrix. Their predominant market is going to be in cell culture, and removing the necessity for something like Matrigel. If you work in a lab, you'll know. But our goal really is to see if we can use this as a scaffold to carry cells, which we think are necessary for long-gap nerve repair. So Liam, with this work, has shown that the nerve can grow through and regenerate through this hydrogel. He's shown that, compared to collagen, that it can support stromovascular fraction from fat, so this is stem cells and a mixture of other cell components, and can keep them alive and proliferate. So we've put them into animal models and looked at regeneration, compared against the autograft, and we seem reasonably confident that this, in a 12mm nerve gap, produces a really excellent result comparable to autograft. This is animal models, we've all seen lots of data previously, with lots of different types of conduits and fillers and everything else, but this is the area that we're working in at the repair site in Manchester. Strom measures, motor recovery, sensory recovery, I won't go too much more into detail on them. But suffice to say, cells are required. In almost every in vivo experiment we've done, even in a 12mm nerve gap in the animal, the addition of cells makes a difference to outcomes. These are some of the other outcomes that we're looking at, micro-CT and whole mount staining, but I won't go too much into detail. One slide on the distal stump. This is yet another unpublished project, god they all are, finding the time, eh? But this was a serial block face, SEM, and we got a medical student to draw around each of these structures and to generate in 3D response. We did this in distal stumps of sciatic nerves of rats that had been repaired, and we compared aged animals against young animals, with the hypothesis being, let's look at the impediments to nerve regeneration. We found these big red blobs which are myelin which have not been taken up by the Schwann or the macrophages, and this we found, particularly in aged animals, was a big problem. So there's retained myelin isn't there, there's these inhibitory proteoglycans that prevent regeneration, and I think if you don't have efficient valerian degeneration, and this tends to happen in aged nerves, then there are significant problems for nerve regeneration. So I'm interested to hear David talk later about SARM1 and how that's going to feed in to this paradigm where we need efficient valerian degeneration not to prevent it from happening. Okay, so in the neuron now, Alice is another PhD student, she's funded by the Medical Research Council, and we thought it would be neat to live image neurons to understand regeneration, what's happening to the sub-cellar organelles in regeneration. We wanted a live imaging assay, we wanted to know where, when and how the neuronal cytoskeleton is formed. The neuronal cytoskeleton is made up largely by microtubules, stained in red here. They're really important for the structure of the cell and for the axon. They have some control over the signalling in the cell, transport up and down the axon, and polarity of the cell, and the microtubules need to be polarised in order to head in the right direction and to traffic up and down. So microtubules are critical. If we think we're asking when there's a nerve injury, we're asking for regeneration to take place maybe up to a metre beyond. So they've got a tall ask after an injury, and we don't know where they come from after an injury. So we're using, again, an in vivo model, transfecting with fluorescent probes to look at various proteins. Now, it's known in other adult cells, outwith the neuron, that microtubules can be nucleated so they can be formed and they can be polarised by the Golgi apparatus. In development, this is a centrosome, which you might have heard of, and together they're the microtubule organising centres, or mTOCs, but we don't know what happens in a neuron. So our hypothesis is that this happens in the Golgi apparatus because it has the necessary components, GM130, which is the cis end of the Golgi, and it needs binding with ACAP9 and gamma tubulin in order. These are prerequisites for microtubules to form. One of our early images of this demonstrated that the microtubules in the Golgi apparatus were maybe in some way related. You can see the Golgi in red and the microtubules in blue emanating out of here, and we wanted to see what was happening in live and real time. So this is the picture on the left, it's just a very simple cell membrane tracker, and you can see really beautiful neuronal regeneration. So this is a rat dorsal ganglion neuron, and it's clear that at 24 hours, in this condition, this is the critical time point where axons start to sprout, and we saw that across all the dishes. So it's happening at 24 hours, and then we look at what's happening at one week, and then another beautiful neuron with an extension. And so our challenge from here was, so what's happening at 24 hours to push out this neurite, and can we image and do things further downstream to look at what makes the growth cone head in a particular direction? Okay, we then did some live tracking of these microtubules, and we could also stain, transfecting with two different fluorescent probes, the Golgi. So the Golgi are in red, the microtubules are in blue, and we saw that these microtubules are emanating from the Golgi, and they're heading down the axon. So this was another bit of evidence. The lines that you see on there are the start of some tracing, which we can use mathematical models to work out where they've come from and where they're going to. So that's all work in progress. And again, we need gamma tubulin ACAP9 and GM130. So if we look at the overlay, we can see that ACAP9 is involved at the critical 24-hour time point, and importantly, the gamma tubulin is as well, and we've looked at the overlay and at different time points and found that 24 hours is the critical time point in this system where it's all happening. So all the proteins are in place, the Golgi are there, and they're producing the microtubules. So our theory is that there's disorganisation immediately after injury. If you look at the two-hour one, this is fairly disorganised. The Golgi apparatus are all over the place. And then it starts to polarise, and it starts to build the necessary components required to push out a new right. And this starts to happen from 24 hours, and then it can extend a new right. And then interestingly, as we followed down at 72 hours, we can see in the growth cone, so this image here in red shows the growth cone at 72 hours. There's microtubules there, all swimming about, doing their thing, starting to elongate. But there's Golgi as well, and we think what's happening, we've got a few more experiments to do to demonstrate this, but we think what's happening is that there are outposts for further microtubule nucleation or formation, for polarisation, going on down the new right at different time points as we go. So this is interesting because it lends itself well, perhaps, to interventions. There are drugs that deal with microtubules. So Alice's future work is to validate this finding in human cells. This is quite tricky, but we've started to do this in fixed human cells, injured after certain time points. We're going to perform functional knockouts and look at all the different protein components that I spoke to you about, and see whether they're necessary for the time points that we've seen. Okay. A quick shout out to Jay Pras, of which I'm an editor. We take all kinds of papers, including from orthopaedic surgeons, David. And thank you for inviting us to Boston. Adam, thank you for that whirlwind tour, and sets us up perfectly for a discussion of kind of novel and exciting things. Thanks, David and Amy, for putting together an outstanding program. Adam, I did visit Manchester some years ago to see Giorgio Terenzi, learn about Schwann cells. It's an amazing place. So my charge is to talk about advanced imaging. Just a show of hands, who uses ultrasound frequently in nerve injury? I'm going to give you a scenario. A humerus fracture, plated, comes in to see you, they came from somewhere else, radial nerve palsy. Who would get an ultrasound for that? So it's just good to know, like, I'm just going to walk you down this path. Five clinical scenarios I'm going to talk about with advanced imaging. One is atrogenic nerve injury. Two, Parsonage-Turner syndrome. Three, painful neuromas. Four, assessing post-op recovery. Five, brachial plexus injury. Number one, atrogenic nerve injury. We published this in 2000, or actually this year, using ultrasound. So that's kind of giving you that background there. The controversy. Somebody comes in, humerus fracture, somewhere else, whatever, palsy, and many times they're just told from the original surgeon, just wait. Or, you know, is it swelling or something else, right? And we'll just see. And I think there's been previous problems with imaging. People get MRI, and there's just metal everywhere, you can't see anything, and they just go, well, you know, from what we can tell, it looks intact. So we looked at this, and we compared MRIs and ultrasound, had a smattering of different kind of injuries, humeral shaft fractures, even radius, fractured disc radius, biceps, and we characterized them on the ultrasounds. Basically, I was telling my radiologist, tell me, is it cut? Is it cut in half? Is it impinged? Is there metal on it? Is there a plate on it? Is there a screw through it? Is it entrapped in the fracture? Is it a neuroma in continuity? Or is it intact and it's scarred? Okay, tell me all these things. And what we found was that, and this, we correlated what they found in ultrasound pre-op to what I found when I did surgery. And all 13 of these were, she called it correctly. And I would, like, you know, it's interesting, because reports were always a little bit, somewhat vague, and this is like, becomes a very, like in the United States, a very medical legal issue. You know, somebody plates a humerus, and they've, you know, injured a nerve, maybe. So I'd always have to call her, you know, tell me the real deal. MRI, only about half. And we just had a recent case where the MRI was red, you know, it was okay. And then later when I went, I put them in TAS, and they said, well, we actually couldn't, you know, you couldn't actually see in certain areas the nerves, because of the metal scatter. So I basically do MRIs and ultrasound for most things, you know, mainly for study. But for some scenarios, ultrasound is way better. You need somebody who's very skilled in it, but I think it's very valuable. Versus just saying, oh, just wait. You know, we don't know. And, you know, it seems like, you know, it's not the way to go. I don't think in 2022, that's the way to go for nerves. You got to, you should know. There's ways to know better. I'm going to give you some case examples. Patient was treated in, I work in New York, hospital surgeries. Patient comes from Virginia. Fixed, both bones, forearm fracture, terrible pain that goes down the dorsal forearm. Can't move. It hurts really badly. Not only was the plate on the, that radius plate impinging the ulna so she couldn't rotate, but they put the plate on the nerve. So when I see an incision that's like this big, and a plate that's this big, a surgeon does not put a plate on a nerve they're looking at, you hope, right? What they do is they do little ones, and the trauma surgeon's like, I'm not, who are orthopods here? Who are plastics? Plastics? Okay. The trauma orthopods, they like little, some of them like little incisions to slide a plate in. You know, in some areas it's okay, you know, femur or something, but you don't do that in an upper extremity generally. But anyway, so it was on the nerve. I ended up taking out the plate, doing a nerve allograft. There was improvement, but you know, once you do that to a nerve, it's never like great, right? But improved. Here's another case. This patient was another country, you know, came from another country, lives in the United States, was fixed in Europe somewhere, I'm not going to say where, but anyway, so she had a palsy after, and it looks pretty good. I mean, from the orthopod's perspective, it's a very good, you know, it looks great. And so she's told, you know, it's okay. I get an ultrasound, and it's an earlier, it's a newer radial, it's a real story, and she's kind of hemmed and hawed. I was like, I think there might be something going on. So I had my senior radiologist do it. There's a screw pushing on the nerve there. The nerve isn't cut, but the nerve is pushed on there. And this went through like a good couple weeks of going through this. Go back, get another ultrasound, see another person. You know, it gets kind of a little sticky. But my radiologist was like, there's definitely a screw on that nerve, and I was like, all right. Well, then I talked to the patient. I was like, you know, there's something on that nerve. I'll go, and I'll take that screw out. I had to go from the other side to take the screw out, and then neuralyzer from the other side because, you know. So no little thing. And afterwards, she had resolution. She came out with a palsy, serotonin palsy. Such a happy patient. You know, she comes in, was told by a number of other people. She came from another state and was told, it looks fine, x-rays look fine, you know, there's nothing going on there. So ultrasound, very helpful. And so like in these cases, these are radial nerve. That's what we published. But you can really, I mean, I extrapolate that for all, you know, all situations. If there's metal in the way, get a good radiologist. Some people here probably do their own ultrasounds. Anybody do their own for these things? I do them for simpler things. I haven't gone to the level of this, but very helpful. Scenario number two, Parsonage-Turner syndrome. We published a few things. You might have seen some of this stuff. Big shout out to my senior partner, Scott Wolfe, who a lot of you know, Daryl Sneeg, an amazing MR radiologist, and Kenny Nowaka, who is the whiz at ultrasound nerve. There, that's, I mean, to find a group who's gonna do that, not easy. We published in a number of areas, or a number of journals, but you can see these hourglass constrictions on advanced imaging inside the epineurium. So if you look at the nerve, you've dissected it, it looks like a regular nerve. You have to open the epineurium, and you see this. This is a case I just recently did. So this is, if you look on like the right and the left of the nerve, that was before the epineurium was taken off. It just looked like a regular nerve. So when they find the hourglass, I have to tell them exactly, tell me how many centimeters from a bony landmark. Because when you open it, it doesn't, you can't tell. And you don't wanna be like, you know, flaying it open and, you know, going all over the nerve. You wanna just go right at it, and they're always right on. So it looks like this. So this is from one of our publications. If you look at that left, that's an MRI, MR neurography, actually. And you see that hourglassing. Not, I mean, you need a really dedicated neurologist, or a radiologist. But on the right side is that hourglassing. This is one of Scott Wolfe's cases. So we've gone into, for some cases, doing a micro neurolysis. I know this is controversial, but, you know, because like the old way of teaching was parts in his turn, or they just get better. Well, they don't all get better. And some of them, if you study them, they have these hourglass, and if you open those up, they actually do get better after that. And these are people who have had it for 12, 16, 18 months, and they haven't gotten better. And we get serial EMGs, there's no change, and then we do it. So we don't do it early. But we, in this series, we had 24 patients, 11 out of 24 had surgery, 13 were non-op. Once again, those are the indications. So we wait a long time. And you can see, not everybody gets better, but those numbers are pretty, you know, I mean, we're collecting more and more to try to show it. But, you know, you do the operation, and just a few weeks later, they're getting some function back. So I'm pretty certain. And the other thing is when you open it up and you take the bands off, you actually see it start plumping up at the hourglass. Number three, painful neuromas. There's a lot of neuroma papers nowadays, and it's interesting, because there's tons of patients with neuroma pain, and they're just wallowing in pain clinics. But end neuroma, neuroma in continuity, scar to the nerves. And if you look at, you know, past literature, ultrasound is useful for seeing the neuromas, and then giving an anesthetic and seeing relief. We published this year 12 patients who we noticed, we saw the neuromas on ultrasound, gave an anesthetic, gave a good relief, and we reconstructed or did translocation, and there was improvement. Some of these quick case examples, flowerpot on nerve, you know, standard, right? Superficial radial nerve hurts, and we got Tonell, the whole situation, you get it. And over here, you can see a neuroma, anesthetic, pain relief. I give Marcane, because it lasts longer, and they can leave the clinic and test it. But here she is, the neuroma, and that's removed and reconstructed. You know, I just wanted to give the nerve, like if there are two good nerve ends, I'd try to reconstruct it. You know, nerve wants to grow back. Case number two is a patient who had severe leg pain, polytrauma, you know, reoperation, this compartment syndrome, the whole deal. Tonell, pain in the superficial perineal, that's a really, that's a very common nerve to get neuromas in. End neuroma in an ultrasound, relief with the anesthetic. Here it was, removed the neuroma, and so versus just kind of saying, oh, that hurts, and I don't know. You know, you can see it, and you give the injection, and they say that really was a lot of pain relief. I think it's very helpful. So number four for imaging, assessing post-op recovery. Here's a case of an 18-year-old man who had median ulnar nerves reconstructed 14 months prior. Ulnar nerve was doing fine, median nerve painful at the repair sites, and there was no recovery. If you look at the top panels, the upper left is the ulnar nerve proximal. Doesn't look totally normal, but you can see that hint of the fascicles going all the way through. I asked the radiologist, do you see fascicular continuity? That's what I'm asking for. And if you see that, that's the proximal, and the distal is, you know, straight on through. Usually the distal looks better. If you look at the median on the bottom, bottom left, see how that's bulbous, but you don't see those lines going all the way through and connecting. And that's when we say there's not fascicular continuity. If somebody's doing poorly post-op, this is very helpful. Like for me, if they're not getting recovery, they hurt at the repair site, you know, like a really painful situation there, and the imaging shows that, that's a reason for me to revise that. So post-op recovery, when it's not going well, instead of just going, wait longer, wait longer, get an EMG and you can't tell, like this, you can see it if it's happening. And I have more cases of doing post-op, you know, checks like this. Last one quickly is plexus injuries. I'm not talking about normal plexus injuries that follow the standard traction, supercubicular. I'm talking about ones that are weird. You know, you're like, why is that pattern like this? I don't quite understand it. Usually they have clavicle fractures, scapular fractures, proximal humerus, you know, a direct blow. And what we do is we get imaging to see where is it all happening. A lot of times it's like a, you know, there's like double crushed cervical, and there's a double plexus injury. Sometimes they have a traction and an injury here. And it really helps to know where it's happening for pre-op planning. So this is a study I'm doing with Neil Basha. We have 170 infraclavicular injuries. And you can see where they happen. This is just, it's too short to go into the whole details, but here's just an example, a man who had a gunshot wound, did an ultrasound. And this is one where, you know, gunshot wounds are often thought, okay, it's just, you know, a blast, and it'll just, you know, neuropraxy will get better. But if you see this, you see that tract, look at the upper right, the tract of the gun, of the bullet going through the median and all the nerves. So instead of just saying, wait, like some of them you wait. If you get the imaging and the nerves are continuous, that's a neuropraxia. If they're not continuous, then you might, you know, do something for that. Early reconstruction, if you know it's like that, can be helpful. Like in this series we've had, for the use of the infra, a good recovery from surgery. So take-home points, advance imaging and help with diagnosis, treatment plans. Just gave you five scenarios where it's helped me. And you really need a team, though. You need a team of really dedicated, you know, these radiologists, they get paid the same for like an ACL MRI read that takes them five minutes, and one of these that takes them an hour. So they have to be dedicated, and a lot of times it's ones that, you know, I have an interest academically, too. So I'm fortunate that I have that. I've talked to other people, and they're like, my radiologist won't do that. And I don't know, I'm not sure what the answer is. With ultrasound, I know a lot of surgeons are doing it themselves, and a lot of people who do nerve surgery do it themselves. I do it, I'm getting into it more, haven't gone to that sophisticated level yet, but that's another solution. Thank you. to augment our nerve practice. And I think, Dr. Moore, do you want to come up next? And we'll have you do, oh, I'm sorry. Yeah, we'll do Amy. Let's do it. You'll be more interesting. I'll do mine at the end, while everybody's asleep. Well, good evening, everyone. I just want to correct the status. Dave was very kind to make me a co-moderator, but I did zero work. And so as much as people have said he is the hero tonight, and it's awesome seeing you here, I'm going to talk about electrical stimulation. And my disclosure is important, because I am a research collaborator with Checkpoint. So I refer to it, and make sure you're aware when I'm using that. And these slides have been approved by the Hand Society's team already. So facts of nerve generation, we just heard some great talks to tell us that. But what we can say for pretty simplified is that once a nerve's injured, there's a series of events that happen. Our rate is slow, one to three millimeters faster if you're younger, slower if you're older. And it's inefficient, so that if you have a coaptation, you can see that as the neuron extends its axons, the axons taste the world as they sprout, and they're very inefficient on it. And so brief electrical stimulation has been around for quite a while, and there's great research from really smart people discussing about if an hour of duration at 20 hertz can show that it accelerates growth and helps with that misdirection. So it's not that it speeds the regeneration up so that it wins the race, it's more that more across the finish line. And so that's where it becomes effective, so you can see in this image. So a mechanism has been brought out that once you stimulate directly on the nerve, this isn't the stimulation of the muscle, this isn't stimulation transcutaneously, and this isn't stimulation transcortically, it is that when you're touching the nerve, they found with these great different studies that it is happening at the proximal aspect at the neuron level. And so different mechanisms have been borne out, but really it's at the neuron level and the regenerative cascade of associated genes. And so that's where it's exciting to see, okay, where can we go with nerve if we have a way to prime at the neuron and something to do distally, and if we hear about, okay, we're gonna protect the muscle distally, these are all avenues that we can to try to augment and get the best and understanding our why, which is to make our patients with nerve injuries better. So again, looking at what the question was, okay, if there's all this great science, why hasn't it been clinically made available to us? What has been the resistance either, it's not because it wasn't known, and I'm sure that it could be, it's more that we didn't have the tool. We had these big clunky machines, and the great work that's been done clinically thus far has been out of Edmonton, and showing using this machine and putting wires in that then stay in the patient in the post-operative area and then get pooled, and so I think that that's been part of our limitation. And so we thought to say, well, if we can provide the same parameters with the device, and this is what's so great about being a surgeon scientist and I'm sure that I can echo that with all of my peers on the panel, is that we can take these questions and we can answer them in the laboratory, and so that's what we did here. This, again, is funded by Checkpoint, but we controlled all of the data and how we presented it. The tibial nerve transection model, we did no treatment, and then compared it to what we have studied in the laboratory at Washington University, used to be my laboratory, but this is all the work by Susan McKinnon, looking at FK-506 or tacrolimus, and tacrolimus is the only drug that we've shown to increase consistently in all animal models of about 15% the speed of regeneration. And so we took that against one hour of e-STEM. And what we found is that there was no statistical differences between the brief electrical stimulation and FK, but both were statistically better results on multiple different outcome metrics than no therapy alone. So this was very exciting for us. I get asked all the time, did we put them together? No, we didn't. I'm sure they're probably working on that now. But then we said, okay, well, there's the problem. We can do pretty good recovery if we do a direct repair and the right timing, but we all know that no matter what the length of the graft is, we've made a more difficult time for the nerve to recover, and therefore we try to see, can we still see, in this more serious injury, can we get effectiveness? And again, we were able to show that, that with brief electrical stimulation, you can get growth. But then I tried to get a clinical trial funded, and an hour of stimulation in the OR is very expensive in the US system. And the idea for paying for anesthesia, paying for the patient OR time was cost prohibitive. And so then it became, okay, well, can we do this for shorter periods of time? Because there are studies that showed doing it for longer periods of time. And so we said, okay, well, let's figure it out. And so again, with looking at multiple different outcome metrics, different time points, they showed that 10 minutes versus an hour versus no stimulation, 10 minutes had no statistical differences than 60 minutes. And so this gave us our window to say, okay, can we now study using this handheld device in patients? And so we also showed with footfall, with functional outcome metrics, so I'm sorry for the double block, but the first one was looking at all of the counts, and then here is the function. And so we were able to, by changing this, we were able to, to 10 minutes, get it funded with collaborators and are currently enrolling two arms. The first arm is looking at it in a compression model of owner compression at the elbow. And then distally, we're looking at, the second arm is going to be looking at it in the more severe contransections, which we're just getting started. So the clinical studies to show this, that again, using the antiquated or the more difficult big machines, and was there was Tessa Gordon, again, one of my heroes in neuroscience. Carpal tunnel release showed with these patients, one hour stimulation increased the number of motor units they were able to see. And then another study with Alston Wong showed that with the digital nerve transection, again, 36 patients, six months follow-up, again, can improve regeneration in these clinical studies. And then last but not least, Holly Power, a previous fellow and superstar, did a double-blinded randomized controlled trial. They looked at 31 patients. She followed them for over three years and showed that they could increase pinch grip and improvement on electrical studies. So I think that there's something to be said. Stay tuned on if we can do it for a shorter duration, but I'm hopeful that we can augment all of the great work and by coming out of this segment today, that we'll be able to push it forward. So final thoughts, nerve regeneration is fixed, and now maybe have brief electrostimulation help us. Thank you. Thank you, Dr. Moore. That's exciting. I look forward to seeing what your results are from that trial. So now we have Dr. Isaacs, who's going to show us a novel repair technique called nerve tape. Assuming we can get your PowerPoint up. Come on, baby. Come on, PowerPoint. I don't know. I don't know. I've been told the system was overloaded today, and that's been the issue for – sometimes it likes – there we go. I can act it out. Go ahead. Oh, close. That was too complicated for me. All right, well, thank you, David and Amy. I can't help but give you credit for being the moderator. Thank you both for including me. I showed David this a few, many months ago and got myself an invitation to participate in this really cool panel. So, but this is something I've been working on, actually, for like 10 or 12 years. Some of my colleagues are probably sick of me talking about it, but important to note that this is something that is generating royalties, and I have stock options, and hopefully I'll get more royalties down the road. I shamelessly say. But I've spent much of my career looking at this question on why do repairers do so poorly. And it's really, it's a multifaceted question that we're not going to get into, though. So certainly we're hearing some novel ideas on how to approach some of these issues, but I particularly have focused on misdirection and loss of axons at the coaptation site. So that's what I have focused on quite a bit. And I think everybody would agree that these are well accepted principles that you have to resect out of the zone of injury, you have to avoid excessive tension, and you have to have a well aligned repair. And while I say these are well accepted principles, I don't think that they're always realized in practice. And so we developed this goal that we were going to try to improve coaptations. The first thing we felt we had to do was we had to get away from sutures. And sutures, I believe, or microsutures, are what make these repairs technically challenging, which means that there's a limited number of people that can do them and that they become time and subsequently resource expensive. And then sutures also will generate at least some scar tissue. Our second overall goal was to obviously better align repairs. And again, this ties a little bit into the microsutures because when we look at microsuture repairs, and we've done this in the cadaver lab, Bernstein did a similar study where they had very similar results. If you look at that scale there, yes, that's just the technical appearance of the repair, but about 40% of suture repairs, at least in cadaver nerves, look something like what you're seeing up on the screen there. That's a median nerve. Those are fascicles pooching out of the side. Yeah, it's a cadaver nerve. But you want your median nerve repair to look like that? I don't think any of us would. Well, none of us want a median nerve repair, but if you had one, you certainly want it to look like that. I'm not taking credit for coming up with this idea that intubating repairs can improve the alignment, but I certainly believe it and have supported it and tried to develop it. And in fact, from the same study that I just showed you with the fascicles pooching out of the side, if we did sutures and then intubated the repair, we had a much higher percentage of at least technically well-aligned repairs. So we came up with this tool. We're going to get away from sutures. So to hold the tissue, we came up with this idea of using micro hooks. And when I say micro, I mean really small, less than half a millimeter. So you'll see in a minute about 400 microns long. And then to incorporate this into a backing that could intubate and align the repair. P.S., maybe some little extra benefits, theoretically at least, some tension distribution that's been clearly shown to improve axon regeneration, maybe a protected micro environment. You get a little concentration of neurotrophic factors, block some axons from escaping, block scar tissue from invading. And so we've got these little extra potential benefits. So we looked at a couple of backings, but settled pretty quickly on this process laminated porcine intestinal submucosa. It is very similar to products that are already used around nerves, which is nice. But it's also really flexible. It's bioinert. It's resorbable. And anybody who's played with it can attest that it's quite resilient. The micro hooks, though, we played around with a little bit more. Our first prototypes were all made of stainless steel. And stainless steel is really nice because it's cheap and it's really easy to work with. But the problem, and I think you can kind of see that on the screen, it can crimp. And by crimping, sometimes it meant that it didn't form to the surface of the nerve very well. And at other points, it meant that it actually could indent and actually pinch the nerve. I think everybody would agree that that's not a good solution. So we switched over to nitinol. Now, nitinol is nice because it's elastic, semi-elastic, which means that it has some flexibility and it doesn't kink. And it conforms really nicely to the nerve surface. And it's really nice because it's used in the body all the time. And we felt really comfortable with that. The downside of it, of course, is that it's super expensive and it's much harder to work with. So here you can see one of our early prototypes. So you can see that's the SIS material. You can see the micro hooks are embedded in that. Again, micro hook's about 400 microns long. You can see that's my thumb in the background to give some context and comparison. And you can see my thumb is a normal-sized thumb, as I sit up here. So here's how we would envision this being used or how we certainly have used it in our own animal models. So obviously, this is a cadaver nerve. But if you imagine that this is a tissue bed, so we would lay the nerve tape down. These are the opposing micro hooks are now obviously on the surface. And we're going to lay the nerve on there, give a little back tug to get it to engage those micro hooks. Here, we're purposely over-approximating it. And you want to over-approximate a little bit. Here, I did it a little extra on purpose just so I could reposition it. Some people had questioned how hard it would be to reposition. Obviously, it's not hard at all. And again, even when I'm putting it in a more correct position, I still, you saw, I over-imposed just a little bit so that when we do that backward tug, it'll kind of let everything settle into place. And now we're going to take the other side with the micro hooks in it. I think you can see the micro hooks nicely there as I'm wrapping around. And I'll milk that down just a little bit. And then take the other leaf without the micro hooks and we're going to pass this around. And we spent a long time thinking about how we wanted to close this. And ultimately, we decided we don't want to close it. We're going to let the surface tension hold it closed. It seems to work really well. And also should allow the nerve to swell, which is certainly something that we were worried about. So you can see a little irrigation just to create a little surface tension there. And you can see really a rapid, very nice repair showing that it has some stability. And then obviously, this next move, you wouldn't do this in the operating room, but just to show how strong the repair is. And that's me pulling it. And those of you who know me, I'm very, very strong. So if I'm pulling it this hard and it's staying together, you could count that it's a very solid repair. We have a lot of validation work already done. This is our biomechanical testing. You could see the nerve tape obviously on the left of the screen. We did small and large nerves for the small nerves, about 2 millimeters. We used three 9-0-9-1 sutures for the larger nerve, which I think would be on the order of a median nerve, something around 6-millimeter diameter. I used eight 8-0-9-1 sutures. I didn't want anybody to come back and say that I set ourselves up for success, put it into a custom testing stage and showed that the nerve tape was either stronger or at least as strong as the sutures. We also have significant work out of animal models. This is a rabbit tibial nerve. You're losing the micro hooks in the glare there a little bit, but the micro hooks are holding the nerve there. You could see the completed repair. And we harvest these at four months. We looked at scar tissue where we could see that the scar tissue was, with the nerve tape, it was actually less than with the suture groups. A big question obviously is, well, what's going to happen to all those micro hooks? Are they going to damage the inside of the nerve? Here's some histology showing a micro hook path that you could see is not penetrating into the fascicles, but that wasn't enough. And we actually ended up, it's hard to even admit it. It gives me some post-traumatic stress when I remember it. We actually looked at 3,600 digital images. It was so important to us to verify what these micro hooks did inside the nerves. So these are the comparative, these are sutures and not infrequently we found sutures inside the fascicles. We never found a micro hook that had penetrated into the fascicle. You could see here that they did sometimes abut or even indent the fascicle, but they never penetrated through the perineurium. And I think this is translated into our efficacy data where we showed that the nerve conduction was actually a little bit better in the nerve tape group compared to sutures. And we also had a group where we just intubated or a connector assisted repair. And our axon counts are very solid, stable across the three groups. So no difference including G ratios. So, you know, I feel really good about where we are. I think it's an intuitive design. I think it's intuitively appealing. Our data so far is really promising as I think you could see. I want to end up being able to call it, say it's better, faster, easier. I envision that as kind of our catch line, but I think we need some more validation before we could comfortably say that. Clinical implants are pending. We have received our 510K clearance. And I really feel that we have developed a product and will continue to develop a product that will really help improve nerve repair outcomes. And with that, thank you, David, again, for allowing me to present. exciting. So thank you, Jonathan. Next up, Dr. Schwarz is going to talk to us about PEG fusion. Thank you very much. I'm going to trust that the audience members will watch him while I give this talk. If he starts making threatening moves towards me, just tell me because I'm going to be staring at the screen. So I want to give a shout out to George Bittner, who's the guy who invented this, and he's been one of our co-investigators since I got involved with this because he taught me how to do this. Disclosures-wise that is pertinent to this, I am part of two clinical trials involving Neuraptive Therapeutics, which is the company that is commercializing PEG fusion, one DOD-funded study, and then one study funded by them that I'm an investigator on for my site. So I think we all saw the best paper talk, and we had a great summary of PEG fusion already. We use a hypotonic calcium-free solution to reopen the axillumin membrane, methylene blue. Some people say that maybe not necessary. We think as an antioxidant, stabilize the membrane, at least dyes and stains things so you can see the fascicles a little bit better, I think. The polyethylene glycol fuses the open cell membranes, we believe, and then the isotonic solution that's more calcium-enriched helps seal things and bring some calcium back in. And so that's how this works. Typically we're using a 3350 molecular weight polyethylene glycol. It's been used since the 60s or earlier to help fuse cells together. Nobel Prize has been won for this, using this technology. And it's regarded as pretty safe in humans, so we give it to our babies to make them poop so they'll stop screaming in the middle of the night. We deliver drugs by injecting, combining them with polyethylene glycol and injecting them into our circulatory system. So it's a pretty well-tolerated substance in humans. So George Bittner originally started working on this in the 1980s, using invertebrate animals, and he's been able to work up to rats, which is the standard model that all of us use. And he's been able to demonstrate that some axons in PEG fusion don't undergo valerian degeneration, and they'll demonstrate immediate recovery of behavioral function, both volitional behavioral function in the organism as well as testable function on neuroconductive testing. And so this has been kind of progressed through multiple animal models. And this is essentially a systematic review of looking at animal studies with polyethylene glycol. Most of these have been rats, but not all of them have been rats, and trying to see how successful is it. Is it just one lab that's making success, or has this been demonstrated throughout? Now there have been further studies since this 2019 study that's come out, but not all of these were successful, but the vast majority of them were. And so why do some studies show that this works and some don't? The studies that typically don't show that they work don't always do confirmatory testing, which we found in our lab to be pretty important. And so what I mean by that is demonstrating compound action potential after the PEG fusion, and even potentially compound muscle action potential. And what we'll do is we'll do our nerve repair, we'll test, we won't get a cap, and then we'll do the PEG fusion, and we'll test again. And if we get a cap, we'll call that a successful fusion. And if we don't get a cap, we'll take the nerve repair apart, and we'll start over, and we'll do it again. This technique is actually pretty difficult, and a lot of us who have microsurgical skill or practice in larger nerves will be like, I can do this, no problem. And so, you know, George keeps a log of how many times it takes people to do this before they get successful, consistent results. And so when he takes a fellow, he starts training, it may take them anywhere between 30 and 50 rats to get this. And then when he takes trained microsurgeons in there, it may take some people 10, it may take some people 20, it may take some people three. And so it has a pretty steep learning curve, and it is a technically demanding technique. But if it's unsuccessful, we take it down and we redo it. Another reason I think that some labs have had problems with this is that they're not using the right size PEG. Some labs have to make the PEG themselves, and if that's kind of a new thing for you, it can be a difficult thing, and you can definitely get bad batches. We have been able to get GMP-sourced PEG for some of our experiments. Some of it we have to make ourselves. But our approach to trying to figure out if this works for us or not has been start in the rat and then move to larger animals. And I know everybody uses the sciatic nerve in SFI, but I'm a hand surgeon. I understand the hand better than the foot. And so we use rat forelimb, and we use a model that Phil Hanright, who's one of our residents, and Sammy Tufaha, who's one of our faculty members, developed using rat forelimb with stimulated grip strength testing under anesthesia. And so we'll take the median nerve and the ulnar nerve in the forelimb, we'll cut it, we'll bury the ulnar nerve high above the elbow, and then we'll perform PEG fusion of the median nerve, and then we'll test that. We then went on to do forelimb transplants, and then we went on to do swine forelimb as well. And so here's how that looks, and we bury the ulnar nerve above, we'll do the suture repair and PEG fusion of the median nerve, all above the elbow, and then we'll stimulate above the elbow. And so we want to do confirmatory testing here to make sure it works, get caps, get C-maps. I think this is an incredibly important part of the process. And then we'll bring them back week by week by week, and we'll do stimulated testing above the elbow to see what kind of grip strength they get, then we measure this over time. Top line is what a normal arm does after just a sham exposure and closure. The two middle lines are doing this with methylene blue on the top line and doing it without methylene blue in the white line in the middle, and then the bottom line is just a standard cut repair. And, you know, you see that there's an immediate improvement over the baseline. It's certainly not normal, but you'll also see that it rises over time, which to us means that some of the axons are fusing, many of them probably aren't, and they continue to regenerate normally over time. And then this is volitional, this is letting the rat lift weights on their own, and the hand that actually hangs onto this the longest is the PEG fuse one. That may be because they don't have as good sensation, maybe, you know, they let go and it starts to hurt or something, but at any rate, this is 15 weeks out. And so our forelimb transplant, this is actually a really hard operation to do. You can't do standard micro on the vessels. We have to do this thing called a cuff technique, and then we do the nerves there as well, and this does not look all that great, but this is one of our first attempts at it, and it's the one we took the photo of, unfortunately. We do a little osteosynthesis with the hypodermic needle and the humerus. And so here's what we found there. This is using methylene blue with the PEG, and still substantially better than baseline, not as good as an arm that hasn't been transplanted, but continues to improve over time. So we think that it accelerated an augmented motor recovery in the rat forelimb PNI and forelimb transplant models, therefore, it must have implications in humans, right, for nerve injury, limb transplantation, TMR, nerve transfers, but I caution everybody, right, the road to hell isn't paved with good intentions. It's paved with rat peripheral nerve experiments. Like 99% of the stuff that we do in the lab never makes it to clinical translation, right? And so is it possible that this is one of those things? Yeah, it's possible. So we went to a larger animal, the pig, because it's got a little bit larger diameter nerves. PEG fusion is a diffusion-based technology, and so if it's a larger diameter, is it going to work as well? We worry about that. But we did find that, you know, when we looked at nerves that were seven days versus controls, there was less layer degeneration in the PEG-fused ones than in the ones that weren't. In some of the pigs that approached the success rate that we'd see in the rats, which was somewhere in a 30% to 50% range, and in some of the pigs it was a lot less, pigs developed this massive inflammatory response to nerve surgery when you go back in seven days, two weeks, three weeks, four weeks later, and so it may not be the perfect model for this, but it's a model that we're used to in our lab for some of the transplant work that we do that's unrelated to this. This is looking at motor implant innervation that was maintained as well in these PEG-fused animals versus the normal controls where it wasn't. And so we've been collaborating with the Brook Army Medical Center and Joe Alderete, who's an orthopedic oncologist there, but he has a big interest in this, and Casey Sebag and Julie Noel, who were both hand surgeons there, and Julie's now at the University of Missouri in Columbia, but we all continue to collaborate some. And so they did the same thing in the pig, tried to get some functional outcomes after doing PEG fusion, but this is taking it a step further. We have done autografts in the animal as well, trying to see if we can get this to work across two coaptations. This is a 24-hour delay allograft model that we started working on to see if we could bank nerve and then bring it in, which may have implications later for our folks who don't maybe have a lot of great donor nerve. Could we take a larger diameter donor nerve if we had an allograft bank capable of doing that? So this is kind of the first principles for this. And so they did gait analysis, which is data that we're still trying to interpret, but they were able to generate caps and C-maps on this, and we're still waiting on the micro histo. So our animal conclusions so far, PEG fusion may stop malaria degeneration and preserve neuromuscular junctions for successfully fused axons, and again, this is random. You don't know that you got one axon diffused to the correct other axon. It substantially improved both recovery time and magnitude of functional recovery in median nerve transection models, followed by immediate neuroraphy, substantially improved both recovery time and magnitude of functional recovery in a rat forelimb allotransplant model. Early results in larger diameter swine nerves have shown similar but variable success than the smaller diameter rat nerves, but can demonstrate a similar percent fusion of the nerve in the ones that it works well in. So how about in humans? Well, there are people that have done this in humans. It's sort of sporadically, but one of the first ones was Wes Thayer, who was one of the original Hand Society members that worked with George Bittner and taught him that one of the ways that you get PEG fusion to work more long-term is you sew the nerves together when you do it. George didn't realize that there were things called microsutures, and so he'd PEG fuse these rat nerves, and their nerves would just pull them apart the next time they'd walk. So he and two other Hand Society members, Richard Trevino, and I'm spacing out on the third, I'm sorry, all contacted him independently, told him to use some microsutures, and started doing research with him. So Wes and Ravindra Bamba were able to do a small clinical trial in Thailand and then compare that to historical control digital nerve repairs in the United States, and they found that there was immediate improvement in sensation, much better than what we would consider with historical controls, but this isn't exactly, you know, a really rigorous clinical trial. So what do we do next to make it a little bit more rigorous? Well, we've been able to procure some DOD funding to do a randomized control trial, and so this is one that I'm the PI of, and I've got lots of collaborators that I'll show you, including people in this room, where we're going to be doing this for acute peripheral nerve injuries in the upper extremity, and we're going to be including nerve grafting, which is going to make this a little bit harder. We're using METRIC, the Major Extremity Trauma and Rehabilitation Consortium, which many of you may be familiar with. We've been working on a natural history prospective nerve registry with them for about five years. We're going to be able to use some of that data to help compare as an external control in addition to our inherent controls. These are our participating centers, John Isaacs, Ray Penzey at Shock Trauma, Glenn Gaston at Ortho Carolina, Rich Trevino at Wellspan in York, and then we've got Joe Alderete and Scott Tintle from our military treatment centers. Nuraptive also has their own self-funded clinical trial that I'm a member of where they're going to be looking at just simple repairs, and so just cut simple repairs, whereas my study is limited to nerves that have motor function in them. This is anything distal to the brachial plexus, including digital nerves. These are the sites that have been participating or are going to be participating with them. Their goal is 40 patients enrolled across nine sites. They'll do an interim analysis later this year, but the data is still blinded for whatever they've got so far. So we're all looking forward to seeing what both of these trials have to provide for us in terms of real human data. So possible problems. PEG fusion is technically demanding, and we don't get standard NCS testing on our intra-op patients. I just told you how important it was to do it in the lab to make sure that you've had a successful PEG fusion so that if you're going to follow these animals, you know whether it worked or not. So we're hopeful that a checkpoint stimulator for at least nerves that have some sort of motor function can be a surrogate for this. And so what we've seen in the pigs is that if we have the checkpoint on about two milliamps, that seems to give us data that is similar to what we're getting when we're testing for caps and C-maps. 0.5 may not be enough, and 20 stimulates everything, and you get some mephaptic conduction. And so we don't have rigorous data to tell us that this works, but this is still what we're going to be doing in the clinical trials. Our neurologists were uninterested in helping us with this when I approached them to try to do more rigid intraoperative neurodiagnostic testing. Intra-diameter nerves may not be as amenable due to the need for diffusion of the PEG across the nerve site. So that remains to be seen. And then enrolling patients in a clinical trial within 24 hours of the injury is going to be logistically challenging, which is what our enrollment criteria are. We want to do this as quickly as possible, and if it happens outside of the 24-hour window, then we're not going to let them be enrolled. So the name of this talk that was given to me, I didn't come up with this, was, is PEG fusion the holy grail of nerve repair? And so the answer is not yet, but it doesn't seem to interfere with normal nerve regeneration in animal models or the small number of human digital nerves that it's helped. It's well-tolerated in humans in general. And so I say if it can't hurt and it might help, then it makes sense to try. And so we hope to revisit this more conclusively in about two years' time when we'll have some final data from these clinical trials and see what we think. Thanks. Excellent. All right. Thank you very much, Jamie. That was fantastic. Thank you. And I'll round this out here. Two points, I want to point out that Dr. Schwartz is the one that compared PEG fusion to baby poop. And if Jamie's right, then we're all coming in the middle of the night for digital nerve repairs. So I just want to make that clear. So in the spirit of that, we started looking at something called SARM1 to see if we could potentially extend the window for PEG fusion repair, right? So because the trial, we're in that same interactive trial. That's an important part of my disclosures as well. So I'm participating in this. But it is logistically challenging. And so, you know, we're coming in for my partners because you have to be trained in the study. And so I'm doing nerve repairs for partners at all hours and such. And that's not fun. So it is a potential problem. So I'm going to focus today on SARM1. And it's a little bit of molecular biology. I'll try and make it as painless as possible because it's not my forte. But when we think about events after nerve injury, kind of simplify this into a few basic things. When we look at a nerve transection model, so one of the basic things that we all have is that there's a bioactive enzyme called NMNAT. That is critical for the nerve to be healthy and happy. And you need to transport that from the cell body all the way down the axon of the nerve, okay? If you have a disruption of the nerve, then you lose the transport of the NMNAT. That enzyme is no longer able to get to the distal nerve. What that happens, what happens then is that you undergo this lag phase, first calcium wave from extracellular calcium. And then you have a second phase, which is the execution phase. That's when the mitochondria become unstable. They basically expel all their contents of calcium. And that's kind of the go, no-go. It's the point of no return where the axon's going to die, the microtubules get disintegrated, all those things that we heard about in the various talks today. And so the point is you have to get PEG, if PEG works, you have to get PEG in somewhere around here, right? If you get it to here, it's probably not going to work anymore. So how can we turn this phase and make it longer so it doesn't go into this phase, basically? And the answer is, we think, SARM1 inhibition. So SARM1 is a molecule that essentially is able to help prolong and extend the life of that inhibitory phase here, this kind of phase where it's just hanging out, basically. And what SARM1 does, and why it's critical, is that your cell needs to have a ratio of NAD to NMN. It needs more NAD than NMN. NMN is the precursor to NAD. So as it breaks down, as the cell breaks down NAD, then goes to NMN, right? So they go like ATP to ADP, basically a similar correlate, right? And so SARM1 is what senses that relative ratio, right? And so when SARM1 senses that there's less NAD and too much NMN, it then starts this kind of, it basically starts the cycle of nerve degeneration. And we know that NMN increases after axonal injury. There have been a number of studies that have been done in a lot of different rats. And this all came about initially because there was this rat that just was noted a spontaneous mutation called Wollerian degeneration slow. They found out that it didn't degenerate when they, basically, Wollerian degeneration was slowed down and didn't really occur very quickly. And when they looked into this, it was because it had a mutation in an NMNAT enzyme. So there was too much NMNAT made, which basically kept the NAD higher than the NMN. All right, so this is a better graphic, but this is our happy little NMNAT going down its path. It's been blocked, right? So now NAD goes down, NMN goes up. SARM senses that, right? And then it starts this cycle of Wollerian degeneration. We know that SARM is, in part, largely responsible for that. And it happens because it's a toll-like receptor. So if you're into the biology, here's your slide. But they mutated the toll-like receptor. This is Mel Brandt and a number of people in the Genetics Department at Wash U. And what they found is that when they mutated the toll-like receptor for SARM, that the nerve wouldn't degenerate, right? That they would have this persistent axon that was okay, and the normal one, after they cut it, you'd have, basically, no nerve, and it would just kind of die out. And the reason that this has now been elucidated over the last couple of years, and really what happens is that SARM1 normally has this conformational configuration that when there's too much NMN, it preferentially binds SARM1. It changes its shape, goes from this to that. That starts cleaving, and it becomes this kind of positive feedback cycle. So it senses there's too much NMN, not enough NAD, and it starts basically destroying even more NAD over and over. And that sends it into that phase of axonal degeneration, basically. So if you stop SARM1, the nerve doesn't know that it's been cut, right? And it just stays there frozen for some period of time. So to take advantage of this, we made a mutant rat. So we did a gene therapy, and did an intrathecal injection with an AAV plasmid. And we made a SARM1 dominant negative, a mutation in that SARM1 for that rat. And what we found, when we looked at it, was that we could keep the cells alive, the axons alive, for a couple weeks at least, after a nerve injury. And we could get a pretty nice action potential, even a few days or weeks after that nerve was cut, which you wouldn't expect, and we didn't see on the other side. So that was pretty exciting. When we looked then at the muscle forces, we found kind of a bimodal distribution, where some had really good recovery early on, even at two weeks, and then some had really no recovery at two weeks. Interestingly enough, at six weeks, it didn't really seem to hold anymore. What we found later is that six weeks, the SARM kind of doesn't matter, that eventually that ratio kind of peters out, and that they'll eventually undergo bolaric degeneration. But it was pretty exciting. We saw, well, if we can get this with a partial knockout, we can get a pretty fast return of recovery with a partial. And this is what we saw when we looked at the neurofilaments. So two days, this one's beginning to degenerate, this one looks okay. This one's now, so we've stained for neurofilament, any kind of intact axons will be green here. And this looks pretty good, actually, at two weeks, right? This is our mutant. And then here at six weeks, they're kind of roughly the same. Interestingly enough, when we look at macrophages, there's less macrophages in the two weeks compared to the wild type. And at six weeks, also still fewer macrophages compared to the wild type. So at this point, I was like, oh, we got SARM1. We can essentially stop bolarian degeneration. Jamie's figuring out PEG fusion. Like, I'm gonna book my ticket to Stockholm. We got nerve problems fixed, basically, right? So then we said, okay, if we can get that with the partial knockout, basically the wild type versus the SARM1 dominant negative, what do we have if we got a complete knockout? So then we got a SARM1 complete knockout from our partners in the genetics group. And this is what I showed yesterday. And so we looked at PEG, we looked at SARM1. I won't revisit the PEG stuff. But essentially what we found is that two weeks after repair, we still had really good axons that were intact with the SARM1 knockout. In the wild type, they were already undergoing bolarian degeneration. But unfortunately, again, taking away the PEG aside, no matter what we did, suture or PEG or whatever, the SARM did worse than the wild type at six weeks. And so it didn't quite make sense. We expected that we were gonna get this great result, and it just didn't happen. And what others have figured out is that, as Adam touched on, is that when you have retained myelin and other things like that, that's inhibitory to nerves regenerating. So part of the machinery that undergoes, that basically has the cell degenerate is also critical for the cell to kind of knit together and get those two ends to come together. So we could basically freeze the cell in stasis, but the two ends wouldn't knit together because it needed that machinery to have the Schwann cells to build a bridge between them. And that's also why I thought that PEG fusion might be helpful. Unfortunately, we couldn't get it to work. But there are certainly some inhibition with the SARM1 knockout. So this is how I felt at that point, right? But other people, I think there's still more questions and more things that can be done. This has been bought, the IP for small molecule inhibitors has been bought by Eli Lilly for a substantial amount of money. Unfortunately, I was about two years too late to the party when all the genetics guys got this payout. But it is currently, there is work underway to look at small molecule inhibitors. And they've actually developed one and published about it. And so what they did for this one was that they gave basically a toxin to the nerve to cause malaria degeneration. And they call these kind of metastable axons. So axons that were in the throes of dying, they weren't completely committed to being fragmented yet, but they were on their way to certain death. And they took away the toxin and they give them the SARM1 inhibitor. And what they found is that they could actually kind of bring the cells, bring the axons back from the brink of death and have them recover with the small molecule inhibitor. And so this is essentially a nice slide of that. So untreated axons look like this. This is when they're cut. They evaluate all these are at 16 hours. They did the histology at 16 hours after the injury. But if it was cut and nothing else was done, then this is what it looked like. If they gave it two hours beforehand, then they had pretty nice retention of the axons. If they gave it three hours after the injury, right? So somebody gets their nerve cut in a lawnmower or whatever and then they come in in the ER and they get this, then it'll look, they can still maintain and rescue those metastable axons. At four hours, same thing. Five hours, it's less effective essentially, right? So timely treatment with this inhibitor can actually rescue the axons. So at this point, I have more questions than answers. I don't want to even pretend like we're even close to figuring this out. But why does the knockout do worse than the partial? I think it's probably because you need some of that molecular machinery. If it is the Schwann cells, can you maybe supplement with exogenous Schwann cells or some other factors to help pull those in or draw macrophages in, do something to augment the repair? Or could you manipulate the pathway at a different point, not just SARM1? So you get some of the inhibition, maintain the axons, but use the myelin, the Schwann cells. Or what we've anecdotally found is that when we would do the paw print testing, painting the paws for these, the ones with the SARM knockout seemed to not really mind it as much and they had less neuropathic pain. So it may be the nerve's just not recovering, but is that a possible treatment for neuroma pain? And more to come on that, we actually have some experiments in the process for that. So to take on points, this is a lot of molecular biology, but basically a normal nerve needs NMNAT to function. Loss of that leads to an increase in this NMN, which starts a cascade of insulating taxonal loss. You can freeze that for a while, a couple of weeks with inhibiting SARM, but if you knock it out completely, it will hurt your nerve in terms of recovery. We need a lot more work to fully understand this. So, all right. Thank you.

peripheral nerve repair techniques

electrical stimulation

nerve tape

nerve injury

nerve regeneration

SARM1 molecule

NMNAT enzyme

degeneration process

inhibiting SARM1

PEG fusion

molecular machinery