My name is Julian Clark, I'm a medical student at Washington University School of Medicine, and I work in the Zipfel Cerebrovascular Lab. Today I'm presenting my work on functional connectivity deficits after experimental subarachnoid hemorrhage. So to give a bit of background first, aneurysmal subarachnoid hemorrhage has the capability of being a very devastating injury, a lot of poor patient outcome, high mortality. The drivers of these poor outcomes are split into initial and secondary brain injury, secondary consisting of both early brain injury or EBI and delayed cerebral ischemia or DCI. There are various sequelae of each that contribute to poor patient outcome, and my focus of this research is primarily within the paradigm of DCI after SAH. So in our lab we've been looking at conditioning-based therapy for subarachnoid hemorrhage, specifically hypoxic conditioning after SAH injury, or otherwise known as hypoxic post-conditioning. Some of our work has previously shown that hypoxic post-conditioning induces neurovascular protection against SAH. If you look at the image on the right, this is our neurobehavioral outcome, and you can see that SAH mice with hypoxic post-conditioning at 8% oxygen for two hours had a significant improvement in their neurological score in comparison to those without. Other figures here, the second and third figure, are looking at vessel diameter for vasospasm as well as percent area with microthrombosis. Both of these are also decreased or protected against with hypoxic post-conditioning. So in addition to hypoxic post-conditioning therapy providing protection, we've also looked at the mechanism of what exactly is mediating this protective process and discovered that SIRT2 and 1, or SIRT1, is the primary mediator of this protective effect. So using EX527, a SIRT1 inhibitor, we also removed the protective effect of hypoxic post-conditioning on vasospasm, microthrombosis, and neuroscore. My work is taking us away from looking at the different sequelae of DCI, like vasospasm and microvascular thrombosis, and looking more globally at the effects of SAH on the brain. And I'm doing this through analysis of functional connectivity, which refers to the zero-lag Pearson correlation analysis performed across various cortical regions in mice. The optical intrinsic signaling imaging system that I use measures functional connectivity by quantifying fluctuations in oxygenated and deoxygenated hemoglobin, which gives us a surrogate readout of neural activity due to the close coupling of neurovascular activity in this process. So this is just a sample of our readout from the FCOIS imaging system. You can visualize a lot of different areas of the mouse cortex using this system. The map on the right there is just a sample map of what the readout looks like, with the red corresponding to a higher level of connectivity or a higher correlation between the two hemispheres. So you'll see more of this in my data as well. So for my experiments, cranial windows were installed on mouse brains prior to subretinoid hemorrhage induction, which allows me to visualize that cortical surface and to image through them as well. This is done at least seven days or so prior to subretinoid hemorrhage induction. Baseline neural score is taken, and then the mice were separated into surgery groups, either sham surgery or subretinoid hemorrhage surgery using the endovascular perforation model. In my first experiment, mice were either left as is, sham versus subretinoid hemorrhage, or they were subjected to hypoxic post-conditioning with or without the SIRT1 inhibitor EX527, so a total of four groups. And in the second experiment, rather than using the hypoxic post-conditioning paradigm, I used the SIRT1 activator drug resveratrol to pharmacologically activate SIRT1 rather than going through the hypoxic chamber. All of these mice were imaged on day three, which corresponds with the timeframe of DCI in mice. This slide right here shows the first major finding of my study, which is that subretinoid hemorrhage induces major deficits in functional connectivity, both globally and regionally. So going through these figures, top left to start, this is just my neural score figure, and this is really just showing that the mice in the sham group versus the subretinoid hemorrhage group have significant differences in their neurological outcome. These subretinoid hemorrhage mice have much poorer neurological outcome. Top right now, this is a bilateral functional connectivity map, and so this is taking the map I showed you a little bit earlier as an example and just expanding on it. So it's all of the functional connectivity scores of all of the sham mice averaged and compared to the same thing for the group of subretinoid hemorrhage mice. And what you really see here is, remember, red is good. So the more red, the stronger connectivity, and the more yellow to green to blue, that's weaker. And so these subretinoid hemorrhage mice have this big wiped out area of this connectivity map that's corresponding to lower connectivity in comparison to these sham mice. And really, that's just a really strong qualitative look at what we're seeing. Getting more quantitative now, these histograms on the bottom row. The bottom left first, basically the closer you get to one on the x-axis of these, there's a stronger connectivity correlation, and the bottom left shows a left shift of the subretinoid hemorrhage mice indicating weaker overall connectivity. But also this second histogram beside it looks specifically at what turns out to be the somatosensory and parietal regions of the mouse cortex, shown in that little box there with the blacked out area. And you can see a very clear distinction between the subretinoid hemorrhage and the sham mice here too, with the subretinoid hemorrhage mice, again, shifted left in comparison to the sham mice. And then this last graph here is just showing that the neurological scoring corresponds to the functional connectivity deficits. So the sham mice have a better neurological outcome, and they also have better functional connectivity scores. And this is just showing that there's a significant correlation between the two. And these subretinoid hemorrhage mice that have weaker functional connectivity scores are also showing poor neurological outcome, and the two are correlated. And this next slide here really ties it all together. You know, the last slide, we talked about those bilateral maps, and we saw the difference between the sham and the subretinoid hemorrhage mice in their connectivity and their strength of connectivity. But this takes it one step further. And so now these subretinoid hemorrhage mice, this is the same map as previously with that wiped out area in that somatosensory and parietal mouse cortical region. And if you look beside the subretinoid hemorrhage map to the hypoxia map, you'll see that there's a lot more red there. It's just corresponding to a lot more connectivity. It's just stronger, and it looks more like that sham mouse from the previous slide. And then in comparison, when you administer EX-527 to these mice and subject them to the hypoxic chamber, you're not really seeing the same result. And so this, you know, they look more like they're leaning towards the subretinoid hemorrhage results in comparison to the hypoxic conditioning results. And when you look over at the histogram, you see the same thing, is those sham mice really have the strongest connectivity still, and the subretinoid hemorrhage mice have the weakest connectivity. But in between, you see that the hypoxia mice are really moving towards the sham, while the hypoxia mice that have been subjected to EX-527 are not. And so what this is telling us here is that not only does hypoxic post-conditioning protect against subretinoid hemorrhage-induced functional connectivity deficits, which is very important, but also that it's doing it in a SIRT1-mediated fashion. And then solidifying the data from the previous slide that the protective effects are SIRT1-dependent are these next group of experiments. So this resveratrol experiment here, looking at the NeuroScore, there's a clear difference between each group, with the resveratrol group improving over the subretinoid hemorrhage mice, but not quite reaching the same outcome as the sham group. This is the same pattern we've seen previously. You also see in the bilateral maps improved functional connectivity qualitatively, and then the histogram shows those respective shifts to the left of subretinoid hemorrhage in comparison to the sham, and then the resveratrol group starts to shift back towards the sham group. So this is just showing that pharmacologic SIRT1 activation via resveratrol also provides significant protection against functional connectivity deficits after subretinoid hemorrhage. So from here, I think this research can take us in a number of different directions. You know, I think using the same experimental model as this research, but instead of using hypoxic post-conditioning or resveratrol, using genetics to our advantage, so genetically knocking out SIRT1 or genetically up-regulating over-expressing SIRT1 would make an even stronger case that SIRT1 is the primary mediator of this protective effect if we were to see the same things genetically. And then excitingly, I think this could eventually be an option for assessing viability of the therapeutic options in humans. There's already data that suggests that cognitively impaired SAH patients have significant differences in functional connectivity across multiple networks. This is shown both in fMRI and in MEG. And I think that this could potentially be a viable option for testing and understanding novel therapies such as SIRT1 activation on subretinoid hemorrhage patients. So in conclusion, this study shows that experimental subretinoid hemorrhage causes significant deficits in functional connectivity and that these deficits are protected against both by hypoxic post-conditioning and resveratrol in a SIRT1-mediated fashion. This is further evidence that the delay between subretinoid hemorrhage and delayed cerebral ischemia is a window of opportunity for a SIRT1-based conditioning strategy. And then also, this shows that functional connectivity analysis might be an important imaging application for further understanding and testing of novel therapeutics in subretinoid hemorrhage patients. And I just want to take the opportunity here to thank Dr. Ziffel for his mentorship and guidance and thank everybody else that participated in this project. I appreciate everybody's hard work throughout this process. Thank you.

functional connectivity deficits

subarachnoid hemorrhage

poor patient outcomes

delayed cerebral ischemia

hypoxic post-conditioning