Victor Song: So, our project is to develop a method to image optic nerve function and pathology directly from mouse to human. Optic nerve is a vital tissue transmitting visual information from the retina to the brain. Optic nerve injury usually leads to impaired visual function and eventually vision loss. And it's commonly affected in various diseases and injury, for example, glaucoma and optic neuritis, two diseases that commonly affect the optic nerve. A clinical assessment of visual function is convenient and very useful, but even though you can locate the site of injury, you can detect the impairment of vision, but this cannot determine whether the injury is inflammation, demyelination, or axonal injury. And then it's really difficult to determine if this axonal injury is irreversible axonal loss. So imaging the optic nerve is needed, and is very important to determine in patient management. There are many different methods to imaging optic nerve. And one of the most commonly used technique is OCT. It's a light based imaging technique, but unfortunately you can only image everything intraocular so doesn't reach out to the later part of the optic nerve. To image, ordinarily the most effective method would be MRI because it has no limitations to penetration. Conventional T1W or T2W image can detect lesions, for example, but unfortunately would not be able to tell you the difference of underlying pathology. So the diffusion tensor imaging was developed, that would allow, one to assess demyelination and axonal injury. Unfortunately, these measurements also are confounded by the presence of axonal loss or inflammations. The goal for the current study is to develop Diffusion Basis Spectral Imaging to image optic nerve function and pathology. So what's Diffusion Basis Spectral Imaging? See, here is a cartoon of an image voxel representing image containing the optic nerve. So where we can see, you know, it consists of demyelinated axons, the intact axons and the injured axons and then you have a lot infirmative cells present and the intra-axonal  space here. So if we do Diffusion Basis Spectrum Imaging, basically what we trying to do is, we will detect the compose or order signal contribution from each component, and it gave you a specific image, you know, DBSI model to signaling patterns. For example, we can model intact axon with demyelinated axon which increase the actual diffusivity, gives you much fatter here. And then when with the injured axon we can do a shorter actual diffusivity, and then the cell to the less diffusion distribution. And then within that edema or extracellular intra-axonal space will be isotropic diffusivities. For the DTI model basically you have to take an average of all these effects to show you know the presentation, the sheer size of this is tensor, to guess what happens. So it's insufficient. That's why DBSI is a much better method and I will show you how it works. But this is a the first occasion we do an EAE mouse model, is a multiple sclerosis model. So, how we have to detect an axonal injury and the demyeliation in this model. So what you can see it is the same animal, the same nerve, we do it before immunization, and then onset and then your two days after. You can see that the actual diffusivity of DTI, to look at, you can see a significant decrease actual diffusivity, but in contrast to a DBSI, it's a more accurate reflection of an injury is you can see that the injury was not prominent until a second time point. Similarly to the DTI, radial diffusivity, you know, the increase in demyelination, and then your DBSI can you tell you it's not as significant but also it's present. And then DBSI was able to show you what happened in fundamental component. Look at cellularity, you can see slight increase over time. And the edema has a significant increase in a second time point at edema. And you will take a look at what's in the group, And once again you can see here, the DTI suggests axonal injury early at its first time point at onset and then it gets worse, and demyelination at the second time point and they look at FA overall and the compound effect will get accelerated in specific and see is more significantly affected in time two. But look at the DBSI, you know, axial diffusivity that is generated by axonal injury is the same at the onset, but significant injury a second or two days later. And then you can see here on the radial diffusivity demyeliation, there's no demyeliation at this time point and even a second point is not significant. You look at an overall fiber for axonal anisotropy and again you can see, again indicating the second time point more significant damage to the axons. And then you can see this inflammatory component can see here if we see, you know, significant vasoginity at at onset and get worse at the second timepoint. And you can see cellularity increase. It's insignificant at first time point but there's a trend, but the second time point its a more significant increase. And then you, and I think very important we wanted to do with a DBSI is, you know, we want to see if we can quantify the axonal loss, as we can see in the EAE model, the interferometry, it causes the the optic nerve to swell. You can see it, the swelling of the optic nerve, but it was, it is swelling and we are able to use DBSI to see that you tend towards the axonal loss, axonal volume, basically it's the equivalent of the histology axonal count. So you can see we have the And it is axonal volume that is linearly correlated with the visual acuity in mice. Okay, so, so this is just, you know, the DBSI does can look at the imagery optic nerve pathology. How about function. So we can do similarly, diffusion imagery to see the functional changes. So this is the control and the EAE mice. The center of the image shows this. At the baseline the radial diffusivity is low. But once it turns on the visual stimulation it decreased even more further. So you're even lower, but then you stop, it will go back to normal. You can see here. Okay. You can you drop a 25% significant decrease and then return to normal as it stops stimulation. Its good. In contrast, in the EAE, the baseline is higher because you have demyelination and inflammation, and with stimulation, you do have, you know, decreasing radial diffusivity and it does increase, returning to normal. Not entirely to normal. But you look at overall is out here so you can see about 7% drop but does not reach statistical significance. But then condition was normal. There was a bit of a trend. You can see the functional responses affected in this EAE mice. And then, what does this diffusion fMRI signal change means. And then we so we performed perfused isolated Frog Sciatic Nerve to see the extent of decreasing radial diffusivity upon the electrical stimulation is linearly proportional to the number of impulse we give. Suggesting this is indeed a marker of nerve activation. So then we try to do this and you'll see how we can translate to a human. Here we can see in a human, you have an anatomic image you can identify where the optic nerve is. And then to ensure we see a cross section perpendicular to the nerve every time, repeatable. And we do this careful planning, and then we can reduce the field of view to see focused into where optic nerve is. The reason for this is because when we do diffusion fMRI we need to do echo planar imaging, which is really susceptible to multiple artifacts so we needed to a different field of view to see where the optic nerve is. You can see once we do a field of view here, we reduce it and then we can see these nerves are right here. And based on this image, we can perform the DBSI here is a DBSI result on one example on multiple sclerosis. We can see intracellularity, okay DBSI can show it doesn't really increase in MS in this particular patient, a little decrease. Look at edema you do see the optic nerve has a significant amount of edema and axonal densities, you can see you have decrease axonal density, such as maybe you have some axonal loss. And then axonal injury. But the DTI, the DBSI and can show-- the DTI doesn't show it. Okay, obvious, but a DBSI you can see axonal injury in this particular mass it look like the peripheral region is decreased, to look for increased, so in averaging it may not actually change. So this needs further investigation. And look at the demyelination, you can see you have increase in radial diffusivity in DBSI, but even more decrease in DTI. So that suggests you may have demyelination and may or may not have axonal injury in this patient. So we look at this, group averaging you can see here, compare MS patient and the Glaucoma patient here. DTI radial diffusivity is increasing rate in MS patients. And then DBSI shows in your the increase in both Glaucoma and MS patients. The most interesting thing here is you know the axial diffusivity did not change among these two group. It means the axons may not be injured. They look like normal. And then the DTI showed it more variable. But show a similar trend. So how I can do here is look at the inflammation and axonal loss. And so you can see that there's no cellularity change and there's slight increase in vasogenic edema or maybe tissue void and then you see a decrease in axonal volume. So axon count is lower in MS and in Glaucoma. Okay, so one of the things that we are interested to do is look at the natural history of disease progressions. This is an example of MS patients undergoing, you know, their individual, respective treatment. And how we have here can see we have a patient imaging three times. After the first imaging, with six months later, we do a second image and then a year later, we do third image. Okay, so this is a healthy control we're doing DBSI -- actual diffusivity can see basically, which we see this axonal injury progress in this three time point. And the most interesting thing here is, first time point that we see radial diffusivity is high, significant demyelination. But then, since this patient is undergoing treatment, this could reflect the treatment effect or re-myelination, but we're not sure. Okay, but more side are needed, but we can see this, the vasogenic edema is increasing over time. And the most interesting thing here is we do see a very significant axonal loss over time. The first time point is the lower than normal, but then it progressively decreases. So this suggests you know You know, maybe the axonal loss is -- could contribute to the progressive disease, second to progressive MS. A similar look at a functional MRI in a human is also doable. We can see it, it will compare DBSI and the DTI you go to the same data. So you can enter it by the two different models. We consider do a DTI you really just didn't see any changes here. The DBSI can see some changes. Okay. And we look overall at all the numbers you can see radial diffusivity decease upon turning on the visual stimulation and then you know did not recover, you know, as expected. Okay, maybe we take a longer because this stimulation is pretty long and pretty stressful. And then an interesting thing here we did not see in the animal, but we do see in a human is actual diffusivity decreased. Again, although it did not recover when he turned it off, it's in a control. And then we apply this in MS patient, MS is more compliant because they are younger And in Glaucoma this paradigm doesn't work. So, how we have can see in the actual diffusivity, upon stimulation, control decreased and then you know did not return to normal, but MS decreased the response, and they come back. Similarly radial diffusivity, you also see the response and established in the control. Now MS sure you know similar trend as we see in the healthy controls. So, just so we could use to the MS and the most interesting thing here is that DTI doesn't work. So such. So this really needed to look at DBSI if you have the isolated all the surrounding signal, so you know the account confounding factors to affect your signal changes. So after this study. You know, we have some things for the the future study, we wanted to do is want to develop a more friendly protocol to to do functional measurements. So, you know, hopefully we can include more patients. And then we want to refine the DBSI models or so we can improve sensitivity, including some intra-axonal models. Another thing very important here is we realized recently if it's axonal loss, it's unlikely we can regenerate. Because regeneration, that the progress is slow and a very short distance. So maybe we should focus on preserving the residual axons. So we are trying to see if we can remodel DBSI to allow it to estimate the extent of injured and non injured axons in the optic nerve. So this is that the project. Thank you very much.