RICK MUNSCHAUER, MD: Hello, and welcome to MS Conversations. My name is Rick Munschauer. I'm a neurologist and Chairman of the Department of Neurology at the University of New York at Buffalo, and Chief of the Baird Multiple Sclerosis Center there.

JEFFREY GREENSTEIN, MD: Hello, and welcome to MS Conversations. I'm Dr. Jeff Greenstein from the Multiple Sclerosis Institute in Philadelphia, and I will be your host for tonight's MS Conversation. This month's conversation is titled "Neuronal Degeneration in Multiple Sclerosis." Joining me to discuss this topic are Dr. Doug Arnold and Dr. Bruce Trapp.

Dr. Arnold is the James McGill Professor in the Department of Neurology and Neurosurgery at McGill University and Director of the Magnetic Resonance Spectroscopy Unit and the Clinical Research Unit of the Montreal Neurological Institute.

Dr. Trapp is Chairman of the Department of Neurosciences at the Lerner Research Institute Cleveland Clinic Foundation and a Professor of Neurosciences at Case Western Reserve University and the Ohio State University. He was also the recent recipient of the Dystel Award for MS Research, which is awarded by the American Academy of Neurology and the National Multiple Sclerosis Society. So welcome to both of you tonight to MS Conversations, and thank you very much for joining us.

First of all, Bruce, let me ask you the question of what the evidence is for the presence of external pathology in multiple sclerosis?

BRUCE TRAPP, MD: Well, Jeff, I think, you know, the evidence now comes from multiple research areas. The one I've been most actively involved in is actually looking at the brains of MS patients and to examine them for axonal loss, and certainly that evidence is unequivocal that there is axonal transection, axonal degeneration and atrophy of the brain. Certainly, brain imaging studies that Dr. Arnold has spearheaded in many ways also point in that direction, and I think I'd let him respond to that part, because that data is just as convincing as that that we've collected.

JEFFREY GREENSTEIN, MD: Before we get to Doug answering some of the questions, let me ask you just an extension of the question as to whether or not you think there's one kind of pathology that's actually going pathologically, or whether in fact there may be more than one event that's going on that affects axons.

BRUCE TRAPP, MD: Well, I think at the present time we believe that there is more than one way to transect an axon or to have neuronal degeneration, and that may be dependent on the stage of the disease. Certainly, we know axons are transected during inflammatory demyelination, and that those numbers can be quite high, over 11,000 per cubic millimeter in an active lesion.

I think there is increasing evidence now, although somewhat harder to prove or demonstrate, that chronic demyelination causes axonal loss, and in fact, we believe that this may be the major cause of axonal loss in MS patients. And that is that the axons degenerate because they're missing trophic support from the myelin sheaths, and this is a very chronic event that occurs over not just years but over decades.

JEFFREY GREENSTEIN, MD: Yeah. Doug, could you perhaps address the issue of MR evidence for axonal pathology, what the measures are, and perhaps also a little about how this might relate to clinical progression of disease.

DOUGLAS ARNOLD, MD: Well, the main evidence, I think, comes from spectroscopy studies. This is a specialized form of magnetic resonance imaging which allows us to look specifically at the integrity of the axons and the neurons. And these studies show that there is in fact a significant amount of pathology involving both the axons and the neurons that is not necessarily related to the lesions, and that seems to occur quite early in the course of MS. This problem also gets progressively worse, and may well be a substrate for the secondary progressive phase of the disease when it eventually appears, as it does in the majority of patients.

For more conventional imaging techniques, the kind of MRI that most people are familiar with, the evidence for neurodegeneration is also there, and it's strong, but it's a little less direct, and it's related to the fact that there is a significant amount of abnormality that involves the parts of the brain that don't include the MS lesions, so the so-called normal-appearing white matter. And there's also the development of atrophy, which, again, seems not necessarily related to the development of lesions. It seems to occur early in the disease and not necessarily in patients who have a large lesion volume. So this is suggestive -- atrophy is suggestive of a degenerative process in many neurologic disorders, and it may well be suggested of the degenerative process in MS.

JEFFREY GREENSTEIN, MD: Yeah. One of the things that strikes me about axonal transections is that there are some recent reports suggesting that they occur relatively early in the disease, and yet clearly progression is something which occurs relatively late. And I guess my question for either of you would be, how can we relate the pathology and perhaps the NAA [N-AcetylAspartate] changes to what we see in the clinical evolution of the disease.

BRUCE TRAPP, MD: Well, you know, I think, Jeff, that the reason we have lots of transection early in the disease and very little clinical readout of that is that the brain has a remarkable ability to compensate for neuronal and axonal loss, and I think one has to reach a threshold of neuronal or axonal loss for clinical manifestations to come.

And I think this not only happens in MS, but it happens in other neurodegenerative diseases, so it's been estimated that Parkinson's patients don't have clinical symptoms until they've lost as much as 80% of the dopaminergic neurons, or that ALS patients don't have clinical symptoms until they've lost 50% of their lower motor neurons, these both being the primary targets of those diseases. So I think that similar things are happening in MS, and that is that you have to lose a substantial number of axons before you have clinical manifestation.

DOUGLAS ARNOLD, MD: There's actually MR evidence to support what Dr. Trapp has just said. One can do a kind of MR called functional MRI which shows areas of brain activation that are required to perform a certain task. And if you do that in patients with MS, you can see that at a time when they may not be showing much clinical evidence of disability, they will show increased activation of areas of brain that would normally be activated to do a certain task, and in fact evidence of reorganization and activation of parts of the brain that normally wouldn't be required to perform a certain task may well be activated.

JEFFREY GREENSTEIN, MD: So that really, I think, would support the notion that there is certainly a great degree of compensation in the brain to really overcome the loss of critical pathways.

One of the other things that I was interested in, Doug, was work that you had done looking at reductions in NAA, both acutely and longitudinally in lesions, and also looking at the distribution of NAA decrease in normal-appearing white matter. And do you think that this, again, would fit with the evolution of the disease in its natural history from acute attacks to a more chronic and progressive disorder?

DOUGLAS ARNOLD, MD: Well, that's actually a somewhat complicated question with, I think, two separate issues. So the spectroscopy of the acute lesions shows that there is, in fact, axonal injury that occurs essentially at the very early stage of lesion formation. So there's an old concept that axonal damage is a late phenomenon in MS, and I think this has now clearly been shown to be wrong by MR evidence and evidence such as, you know, Dr. Trapp has provided from pathology. So this -- It's interesting that some of the acute injury to the axons is reversible early on, and that, I think, is a positive aspect of lesion evolution that may be able to be influenced so that one can have better recovery of lesions, if one understood exactly what was determining the extent of recovery of NAA or axonal marker in those acute lesions.

The diffuse decreases of NAA, that seems to involve all the white matter and the gray matter, as well, actually -- are probably not related to the lesions that we see. You know, the evidence for that is indirect, but it's too great to be explained by the small volume of lesions that are evident on MRI, and they can be seen very early in patients with very low lesion volume, so it suggests that there is another mechanism that's producing this axonal and neuronal dysfunction. You know, very possibly degenerative, although there may be other mechanisms, such as a low-grade, diffuse inflammation that could be contributing to this that we're not really imaging very well at the moment.

JEFFREY GREENSTEIN, MD: I guess one of the key questions -- and this, I think, probably is going to ask you to speculate a little -- is what the actual pathogenesis of the external injury really is, what kinds of cytokines or other processes might go on that might actually cause axonal injury in a disease which, at least conceptually, was thought of as being a disease that's been primarily demyelinating. Perhaps you might, either of you, speculate a little as to what mechanisms you think might be involved.

BRUCE TRAPP, MD: Well, go ahead, Doug.

DOUGLAS ARNOLD, MD: Well, you know, in an acute -- I mean, we have to, I think, talk separately about the lesions and the non-lesional tissue or the normal-appearing tissue, at least on MRI. So when one has the formation of an acute MS lesion, there is a very aggressive inflammatory response which is probably directed against myelin. But there is this so-called bystander damage that occurs because of the -- the inflammation is not completely direct. It's a very angry environment with a lot of proteolytic enzymes and cytokines, as you've mentioned, and nonspecific immunologic attack mechanisms. So I think to a large extent, the axons are damaged secondarily in the acute focal lesions of MS.

The mechanism for possible axonal injury outside of lesions, that's not related to the lesions, is, I think, less clear and less well understood. There's some evidence for increased inflammation throughout the brain of patients with MS, and if you think about it, the oligoclonal bands that are found in the spinal fluid had to get there somehow, and they probably came from abnormal inflammatory cells that are in the brain tissue. So these antibodies are, in fact, diffusing throughout the brain substance. There's pathologic evidence, I think, for diffuse abnormal inflammation throughout the brain that Bruce might be -- might want to comment about.

JEFFREY GREENSTEIN, MD: Yeah. Perhaps, Bruce, we can bring you into this with your thoughts on this issue.

BRUCE TRAPP, MD: Well, yeah. I mean, I agree with Doug that there could be this diffuse inflammatory event going on throughout the brain, and I think one of the critical questions is what is the target of that. And I'd like to raise the possibility -- again, I think we're still collecting evidence -- that one of the potential targets in this disease might be mitochondria in the axon. And I raise this for a few reasons.

One is, this is the source of NAA. Mitochondria contain the enzymes that synthesize NAA, which Doug and others have shown to be diffusely changed, and there's a number of mechanisms whereby inflammatory reactions of different types could affect mitochondrial function, one being nitric oxide. And we know that inducible nitric oxide synthetase is dramatically increased in the inflammatory lesions of MS being expressed, we believe, primarily by astrocytes. So that's certainly one mechanism.

I think in the more chronic stages of the brain, one could have what Steve Waxman has called an axonopathy, where you have a redistribution of sodium channels, and this low level of chronic inflammation could be affecting mitochondria through a different mechanism, particularly if you redistribute the sodium channels along the axon, the energy requirements go dramatically up for firing the action potential through the demyelinated axons, and if the mitochondria are not sufficient to supply that energy, you get an influx of calcium into the axon, which, again, could cause dysfunction of the mitochondria, potentially upregulate calcium-activated proteases and cause axonal dysfunction, if not transfection. Transection, that is.

So I think, again, that may depend on the environment, and there may be more mechanisms. Certainly, another specific mechanism, potentially, is glutamate, which there is significant evidence in the literature suggesting that it may play a role, also.

JEFFREY GREENSTEIN, MD: Yeah. And we actually recently have just shown that there is increased glutamate in the brain in MS, particularly in active lesions and areas where there has been significant inflammation.

But to change slightly, I guess one of the issues that might explain some of the NAA change of white matter is -- in normal-appearing white matter is wallerian degeneration. And you certainly, Bruce, have shown that very clearly in projection pathways, and I presume, Doug, that that might explain some of the NAA change which you're measuring in an MR spectroscopy.

DOUGLAS ARNOLD, MD: Yes, I absolutely agree with that. And I don't think we should ignore it. Perhaps I didn't emphasize it enough. I think that is a contributing factor. It's one that I think is, in a sense, obvious, and the reason I focused on the others is that it seems to me that most of the decrease of NAA must be due to something else because it occurs early in patients with low lesion volume. So I don't think that the wallerian degeneration that one would expect from patients who have relatively small burden of disease can explain those widespread decreases of NAA, although they're certainly contributing to it.

This actually raises another issue with respect to the NAA measurements in patients with MS, and that is that they tend -- they basically reflect a density measure. So when we do spectroscopy, we measure the amount of NAA in a volume, be it one element of a spectroscopic examination, or even if it's in the whole brain, normalized to brain volume. It's basically a density measure, and so if an axon in the myelin is lost and the damaged tissue is cleaned up and the brain shrinks, then the density is actually not affected by that, by that process. I think this actually attenuates the amount of decrease of NAA that we see due to the acute lesions.

BRUCE TRAPP, MD: And I should say that I agree with what Doug is saying, and we have looked at this in postmortem tissue of MS brain by quantifying NAA biochemically, and correlating that with axonal volumes within various lesions. And certainly a significant amount of the NAA loss is due to wallerian degeneration and axonal loss. But what we also demonstrated is that normally myelinated axons in these MS spinal cords also have reduced NAA per unit area of axon, so this is a different confirmation of what Doug has been talking about, Doug's theory that in fact NAA is reduced in normal myelinated axons in MS patients.

JEFFREY GREENSTEIN, MD: I guess one other aspect of neurodegeneration that we haven't covered is the issue of cortical involvement of the brain in MS. And again, Bruce, perhaps you could speak a little to some of the pathologic studies that have been done, and Doug, perhaps consideration of measurement of cortical size and NAA content from an MR perspective.

BRUCE TRAPP, MD: Yeah, well, let me start, Jeff, by stating that the cerebral cortex is myelinated and the major fibers coming in and out of the cortex are myelinated, and some of the intracortical fibers also are myelinated. So there is myelin. It's less than what we see in white matter, but we and others now have characterized demyelination of the cerebral cortex, and although it's early days in this, it's possible, based on our early studies, that cortical demyelination may be equal to or even greater than white matter demyelination. And along with this comes similar pathologies, as we described in the white matter, such as neuritic transection. That would be transection of both the axon and the dendrite, neuronal apoptosis.

I think one of the big challenges right now for the MS community is to really characterize the dynamics of that. We've looked at end-stage disease at autopsy. We need to know why -- or how often these lesions occur, and when do they occur during the process of the disease? We need to get total lesion load, which is difficult to do from a morphological perspective. A big challenge is to be able to image these lesions, and we do not see them on routine MRI imaging for two reasons. One, these are not hypercellular lesions. That is, there are not a lot of immune cells in these lesions. And two, as far as we can tell, there's no dramatic breakdown in the blood-brain barrier, and many of our MRI measurements are dependent on alterations in extracellular/intracellular water due to that.

JEFFREY GREENSTEIN, MD: Yeah. Doug, perhaps you could try to address the issue from the MR perspective as to how one could characterize the gray matter involvement in MS and what techniques potentially are available at this point.

DOUGLAS ARNOLD, MD: So Bruce has, I think, rather nicely indicated why the conventional imaging methods don't show the focal lesions. So we've basically been reduced to methods that look at gray matter as a whole, but don't necessarily have the resolution to distinguish between the lesions and the surrounding tissue but do provide quantitative measures of gray matter pathology. And so this consists of measuring the cortical thickness so that we can actually assess cortical atrophy. And in fact these measures do show loss of thickness in patients with MS, and this may occur, although the data are rather preliminary here, this may occur rather early in the disease, and it may be particularly prominent in patients with disability who don't have much in the way of MR physical white matter lesion load to explain that disability.

Other quantitative techniques also show evidence of gray matter pathology. If you look carefully enough, you can see it with magnetization transfer ratio imaging, which is presumable sensitive to pathology in the myelin that's present in the gray matter. Those changes are small and hard to detect because of the relatively low level of myelin in the gray matter compared to the white matter.

You can also see changes in the NAA in gray matter in patients with MS. The exact time and extent of these changes, I think, is not entirely clear. There are some reports that suggest they may be very great and very early in the disease, and there are other observations that suggest they may be -- they may occur somewhat later. I think this is still an area that needs more work before it will be worked out.

JEFFREY GREENSTEIN, MD: How do we tie in studies with measurement of brain atrophy with some of the processes that we've been talking about, both pathologically and on MR with NAA reductions? Doug, you might perhaps want to address that.

DOUGLAS ARNOLD, MD: Well, from a point of view of pathology, I think the cortical atrophy could represent a number of things. It can represent -- it can be secondary to lesions and loss of tissue in those lesions. It can be secondary to more subtle mechanisms of secondary neurodegeneration, which I think Bruce should probably speak to in a couple of minutes. But these mechanisms could lead to, let's say, retrograde changes in neurons from injury or -- injury to axons within the white matter, resulting, for example, in dendritic atrophy and decrease in the size of the neurons. So both these mechanisms way well be active, and I think the atrophy that we see may reflect both lesional and nonlesional pathology in the gray matter.

JEFFREY GREENSTEIN, MD: In the last few minutes what I'd like to do is, perhaps, just turn a little bit to the implications of all this work to therapy in MS, and perhaps address the issue of whether or not the therapies that we currently have available and approved by regulatory agencies are really doing what we might hope them to do as far as reducing the development of neurodegeneration in MS, and whether or not we ought to be looking at other strategies that might complement the therapies that we already have. Again, Bruce, perhaps I can sort of throw it out to you first from the pathologic side, and then Doug to talk about the MR and clinical side.

BRUCE TRAPP, MD: Well, you know, first I want to say, I'm not a clinician, so I don't treat patients, but certainly I know the literature and follow it well. I think from the pathological perspective, we've had a major advance in recent years in the trend to treat MS patients early and to treat them continuously. And I think that that is very important with current therapies. But I think we all know and understand that these therapies do not stop the disease. They slow the disease. So I think the hopes of the future is that additional therapies will be developed.

I think a critical question for understanding the pathogenesis of this disease is if we can stop all the inflammatory lesions, do we stop the disease. And I think that raises a very important question that we ask ourselves all the time, and that is, do we fully understand the cause of this disease, and is that cause inflammatory demyelination, or is there another, or an additional pathological process that is going on.

I would hope that in the future we're going to see neuroprotective therapies, and I think just to follow up on a comment I made earlier about the Parkinson's patients and the ALS patients, these folks don't get to the clinician until significant amounts of their neuronal targets have degenerated. The inflammatory lesions in MS bring the patient over the clinical threshold very early in the disease, and I think we can argue that MS may be the disease of choice for testing neuroprotective therapies, because we can get at the patient when there is still probably 95 or greater percent of the target in the brain.

JEFFREY GREENSTEIN, MD: Doug, your thoughts on the issue of therapy?

DOUGLAS ARNOLD, MD: I basically agree with Bruce. It's important to realize that the current therapies are directed pretty much against the focal inflammatory component of MS, and they're not really primarily directed against preventing neural degeneration. So as Bruce said, if the degeneration is, in fact, secondary to the focal inflammation, either acutely or with some delay because of the loss of trophic support from the myelin, then the antiinflammatory therapies may, in fact, work for both components of the disease, but it's not clear that the degenerative component is in fact secondary to the focal inflammation, and if it is in fact an independent process it will require, I think, other approaches to support the axons that are there.

JEFFREY GREENSTEIN, MD: Well, unfortunately, we've really come to the end of our allotted time, and I really would like to thank you both very much for having participated in tonight's discussion. I hope it's been informative for everybody and, again, thank you very much for joining us. Good night.