Now I'd like to introduce Raj Mehta, who will be presenting the Kline Research Awardee, the talk on Neuroanatomical Analysis of Distal Supercharged Endocide Nerve Repair for Incontinuity Nerve Injuries in Rodents. Thanks, Mark. Eric, that was a great talk. Rob, wonderful talk. So Sudheesh was supposed to have given this talk, but I couldn't make it, so I borrowed a picture of him. So that's Sudheesh in a 10-gallon hat in Calgary. So if you have a tradition in Calgary, we give out these 10—this started when Ralph Kline was our mayor a number of years ago, and we had this thing called a stampede in Calgary, and whenever we have a visitor to Calgary, Mayor Kline used to give them this white 10-gallon hat as part of the stampede tradition. So anyway, I gave one to Sudheesh, that's Sudheesh's. Sudheesh and Mustafa, two fellows, sequential fellows, did all the work that I'm going to present here, and Joanne is my research assistant. And this is a work related to a neuroanatomical study that tries to understand some aspects of a so-called endocide transfer. So thanks to the Kline—to Integra, who supported the Kline Award, and we were able to get this small grant a couple of years ago to do this work, and the paper's actually in press in Journal of Neurosurgery, so if you don't—if you are interested in reading further about this, it'll be coming out, I think, soon. So I think we all know this. We recognize this classification of nerve injury, and an easy way to think about it is a stunned nerve. When I translate it to an animal model, it's a stunned nerve, a biochemical injury, a simple crush injury causes in a rodent, an axiomatic injury, and then the more severe injuries called internal disruption, and then, of course, if you cut the nerve, it's a laceration. And I want to bring this particular clinical problem to this attention because this is a form of nerve injury that was one of the commonest forms of nerve injury we see. So we have many patients with severe ulnar neuropathy at the elbow, some of them who present end-stage, and as many of you know, one of the treatments that's being recommended, and it's being done without much literature, I would say without much validity so far, is a so-called supercharge end-to-side transfer where the end of the pronator quadratus branch is taken to the side of the ulnar motor nerve branch at the wrist forearm level to hope to get back better functional outcome, even though the injury is more proximal. And the reason to do that would be because you want to spare the anatomical pathway of the ulnar nerve. You don't want to cut the motor, the ulnar motor nerve, and do an end-to-end transfer. So you try to kind of, as David used to say, belt and suspenders, right, do a little bit of both. So that idea led to the formulation of this study and this hypothesis that I'll talk about in a moment. Now, before I get to that, I think we all know that with great suite of four neuroendocrine injury, the standard approach, of course, is to, if the patient is spontaneously showing clinical electrophysiological function, is to approach the lesion electrophysiologically evaluated, and Dave's done immense work in this area, and of course, depending on what the findings are, consider doing a nerve graft repair or doing a neuralysis. And we know that results from neuralysis are excellent because that indicates that the nerve is actually spontaneously regenerating, and of course, that means it's a spontaneous recovery. A less severe injury, the outcomes are going to be excellent. But then we end up often needing to do a graft or an end-to-end repair depending on the gap length, and of course, the newer, I would say newer in terms of the popularization of nerve transfers, which I think, as Rob pointed out, really revolutionized the field. And so most of the transfers that we're doing right now are end-to-end transfers. So this is a situation of a very proximal nerve injury such as for a brachial plexus where you're essentially converting a very proximal nerve injury into a distal nerve injury where you're taking a donor such as a branch of the fascicle of the ulnar nerve to the end of the distal stump of the biceps mansion of the musculocutaneous nerve, so that's an end-to-end transfer. Now, recognize that with an end-to-end transfer, you're eliminating the possibility of any spontaneous regeneration in the native pathway. So a second approach then, therefore, is that paradigm I told you about, which is the so-called reverse end-to-side transfer where you actually take the donor nerve and you plug it into the side of a recipient nerve so you still preserve this pathway. And why has this been postulated? Susan McKinnon coined the term supercharge. Susan's really, you know, very clever, amazing, innovative person, and she's also very, you know, I think she liked the idea of supercharge because she thought, well, you're actually like supercharging the end organ. You're babysitting it, you're kind of providing extra axons, motor axons, and perhaps, you know, you may get some incremental increase in recovery. So the idea would be not only positively impact the muscle to babysit it, but also potentially positively impact the denovated pathway to condition it to perhaps be a stump for improved regeneration. So this is the theory. We don't actually know that this actually works in reality. And of course, the results are modest for this. When you actually look at the clinical series, it's all level three to four evidence. It's anecdotal, small case theories. So it's not proven yet for these injuries, and we thought we'd look at an animal model. So what we did was we used the peroneal tibial animal model system in the rodent, which is anatomically a nice, easy model system to work with. So the idea is the peroneal nerve, tibial nerve come off the sciatic in the proximal thigh. And if you create a, and I'll talk about the neuroendocrine, you can create a NIC injury in the tibial system and then use the distal tibial nerve as the recipient of potentially of endocyte transfer. So what we did was we employed the model that Jacob Alland had developed in the lab a number of years ago. And Jacob had discovered that when you apply a traction force at the same time as you compress the nerve quite severely with a modified malleus nipper in this case, you're able to actually create graded injuries in the nerve, which, based on some very nice neural assays of behavioral function, you could actually determine not only, you know, histologically where you could see the, in this case of this NIC injury, where you could see very aberrant regeneration and axons leaving the peroneal interface and going through the internal epineurium. So really a histological model of our NIC, so our grade three to four injury. But in fact, if you did behavioral function, you could see very nicely when you did a simple crush injury, which is a grade two injury, by eight weeks you were back to baseline, which is about what you'd expect in these two different assays. But you could see the NIC injuries, they stayed poor. So the animals made a slip half the time, so every second step was a slip. So, you know, these animals stayed severely impaired in terms of behavioral performance. So we had an assay to look at this. So this is the model we used. And then this is the experimental paradigm of this study that I'll show you. So the idea here is we're going to be creating, this is the main experimental group, so you've got the neurone and continuity injury here. And at the same surgical time when you create the NIC injury, you do a distal end-to-side transfer from the perineal nerve to the side of the tibial nerve. And you have the appropriate control group. So you have control groups where you, the positive control, of course, you don't injure anything, you just do a sham operation. And then you have all the other controls, including cutting both the nerves, cutting just the tibial nerve end-to-side, just to see what the perineal nerve contribution does. And then in group three, which is the main, sorry, group two, which is the main control for the NIC condition, basically doing the NIC injury, but not doing the end-to-side repair. So we had all the appropriate control groups. So this is phase one of the study. And this was really to answer the functional question of whether this actually made a difference in outcome. But then to ask a more important anatomical question of what is the contribution of the neuron, whether it comes from the native versus the foreign pathway, we did a sequential double-labeling study. So this is the model here. Here's the side nerve coming down. There's the NIC injury. This is the 15 millimeters distal to the NIC injury. And we have this perineal nerve end-to-side repair with an epineural window. So that's important. It's an epineural window. We make a small slit in the epineurium. We plug it in. We put one microsuture. We used standard techniques that I think all of you are aware of. If you look at the histology results, you can see the negative control. There's effectively no regeneration distally into the tibial nerve. This is a normal nerve control with the NIC injury approximately and the end-to-side repair. You can see there's pretty good regeneration. We don't know what those axons are from, but very good regeneration down here distally, similar to this group. And this is probably the important slide from the first phase of the study. So first of all, if you look at neuroanatomical regeneration, which is best done by looking at spinal cord counts, you can see that end-to-side actually does improve the number of motor neurons that regenerate into the distal tibial nerve. There's almost a 50% increase as compared to group G2, which is the main control group. So that end-to-side does bring more motor neurons into the end organ, no question. However, the function is not improved. The function is not improved. Now, why is the function not improved? Well, it's an antagonistic donor. You're plugging in a perineal nerve into the side of a tibial nerve, and you could say, well, the hind paw is not working properly. Interestingly, even the CMAP didn't improve, which suggests that even the number of functional motor units is not actually increasing with the end-to-side repair. But you know, so that was kind of unexpected to see that to be improved because, you know, you've got a lot more motor neurons. So now we ask the question is, what is the source? So again, we use sequential double labeling. We used DyeI as the initial tracer. It fills up the motor neurons that were originally in the tibial motor neuron pool. You come back with fast blue, distillate into the tibial nerve, and then you can go back and count the number of single and double-labeled neurons in the spinal cord. And this is a critical data slide. So first, let me just take you through this because it's a little bit complex. So you know, basically, we're looking at the spinal cord. We're counting. The person counting is blinded to experimental conditions. You can count the number of DyeI-labeled cells, number of fast blue, and the number of double-labeled cells. So first of all, the model works. So if you just put DyeI in and you have the control group, you can see there's around 1,500 motor neurons labeled in the spinal cord, which is similar in all three conditions, which you expect. So that's the baseline. So that just tells us that we can effectively, retrogradedly fill the motor neuron pool with DyeI. This data point here, this bar graph's important, panel B, because this tells us what's the fast blue counts at termination of the experiment. And you can see, again, we've got the same results as the first experiment. It's about 50% more, so this confirms the first experiment. There's 50% more motor neurons when you have the supercharged. So it's supercharged. Does supercharge work? Yes, it works. You bring 50% more motor neurons into the end organ. But these two slides are actually the more interesting data because the question is, where are those motor neurons from? Well, it turns out that if you look in panel C, the vast majority of the motor neurons are from the foreign perineal nerve pool. In fact, when you look at double-label neurons in panel D, which means that they were from the original tibial motor neuron pool, there is four or five-fold more motor neurons in group G2, the control group, as compared with the group that got the end-to-side repair. So that's telling us that there's a competition. And in fact, the tibial motor neurons can't grow as well through this pathway because they've been out-competed, because the perineal motor neurons are filling up the pathway. So the epineural window, with this secondary repair, actually, in a sense, kind of prevented reinnervation from the own motor neuron pool. So these are the conclusions then. So does this work as an effective strategy? Yeah, you can definitely supercharge. No question. We've demonstrated here. However, in this particular model with an epineural window, at least in a rodent, it seems that most of the regeneration is from the donor nerve, an expense to the native nerve. And at least in the Heimler model in the rat, what that means is your function is actually no better. It might actually be worse. So can SETS reverse end-to-side work? Yeah, I think it can. I think it can be used cautiously, but you have to recognize that you could actually downgrade function unless you use an agonistic donor. So I think the idea of using the EIN, perineural quadratus branch, is not a bad one, because it is agonistic donor function for motor function. So I think it's reasonable, but I think it does bring a little bit of caution. So maybe I'll stop there. Thanks. Do we have any questions? Yeah. It's a great talk, two things. Yeah, no, those are great questions. And I think corticoplasty probably doesn't work in the hind limb, because the rat's kind of a quadruped and has those very hardwired things in the spinal cord. We haven't, but other people have done similar experiments, and they've looked up to 20 weeks, and it looks like the hind limb isn't a good model in the rat for looking at, yeah, because it's too hardwired at the spinal cord level. But I think it may be different for humans. But your idea of, the first idea of actually going in and kind of taking away the babysitter after a temporal period of time, is that it's something that people have actually thought about. And I think there's actually a couple of small clinical studies where they've done that, when they've done the cross facial nerve things, for example, where they actually used that as a babysitter. And Julia Terzis talked about doing that, right? So the concept of the babysitter thing, and use that to kind of occupy those pathways and the muscle, and then later on to cut them. So it's actually been done clinically. And I'm not sure if it's been totally proven, but people have thought about it. Just a question related, but not exactly. Is there any body of evidence that's solid clinically about side-to-side anastomosis with a test or two relations? Is any of that data believable, or is it really solid? So there's only a couple of, I think there was one case series of four patients that I recall reviewing a while ago, and there's been one or two case reports. But there is a body of experimental literature in rodents where people have done side-to-side. And one study that Tessa did was she did multiple windows from one nerve to the other, and she found the more transfer points you created, so she did an experiment where she did one, two, three, and four of these end-to-side kinds of things, very far distally, and looked to try to answer that question. So in a sense, when you have an infinite number of transfer points, that's a side-to-side in a sense, right? I mean, that's exactly what it is. It seems that that actually does work, again, anatomically, whether it changes clinical outcome is uncertain. Yes, I think the criticism was there weren't enough fibers. Yeah. It's not a question you got some transfer of fibers, it's a question of whether they were robust in number enough to make a difference in outcome. I have a question for you on the CMAP project. Yes. Are you able to stimulate distal to the transfer site before it becomes muscle, or are you having to stimulate proximally? Yeah. So that's... So that's a very good criticism of the technique. So this was a standard CMAP, so we're stimulating very proximally on the sciatic nerve and recording from the muscle. So in a sense, you should be ameliorating all of the regeneration into the muscle. So I was actually surprised why G1, that group, didn't have a better CMAP. There was a trend early on... Because you're up on the sciatic nerve, so you're not counting on the perineal contribution. Well, you are, because it's got the perineal contribution also, because you're actually stimulating proximally. You're getting the full thing. Yeah. So that should actually have been higher. So that was not... Why that didn't bear out, I'm not sure. I'm not sure. Yeah. Yeah. If the rat locomotive pattern is too difficult to do, and the paradigm is for humans, upper limb, long deterioration segments, where would you suspect that a good research model... Yeah. So there was actually a... You find out that someone's actually done a better study than you after you do your study, right? So Barbara Lutz, I think. So there was a study from Austria a number of years ago where... That study was actually done from her lab, where they actually did a median to ulnar transfer in a four-limb model of rodents. And they looked at grasping task behavior. And in that model, they could actually show a functional improvement with the distal transfer as compared with the non-transfer group. So there was actually a model. Yeah. Yeah. Thank you. All right.

neuroanatomical analysis

supercharged end-to-side nerve repair

incontinuity nerve injuries

rodents

surgical technique