Hello, everyone. My name is William Smith. I'm a second-year medical student at the Dartmouth Geisel School of Medicine, and I'm here to present to you today my research on differential-targeted multiplex spinal cord stimulation and its enhanced reversal of neuron, glial, and plamosomes relative to high-rate and low-rate spinal cord stimulation. Before we get started, I would like to thank the Scientific Committee and the AANS for allowing me to be here and present this research, as well as my mentors, Dr. David Cedeno and Dr. Ricardo Vallejo, with whom I've worked over the last four years and who have kind of helped me get this research to where it is to present to you all today. For my disclosures, I have no disclosures, and so just getting started, neuropathic pain, it has kind of become established over the last several years that there are molecular pathways involved beyond just the neural transmission, particularly the immune and inflammatory pathways. This is something that we've highlighted in our research, looking at RNA sequencing results in rat chronic pain models and finding that, you know, immune, metabolic, and inflammatory pathways, the genes are upregulated, or at least modulated, in these chronic pain states. Similarly, we found that those genes and those pathways are able to be altered with spinal cord stimulation. We recently have begun research into differential-targeted multiplex, or DTM, spinal cord stimulation, which combines various electrical signals to target multiple cell types within the spinal cord, from neurons to different glial cells, and we were able to show in some previously published work that it was able to shift gene expression back towards pre-pain levels after they were distorted by the neuropathic pain model, and it was able to do this more effectively than high-frequency or low-frequency spinal cord stimulation, again, in those rat models, and then we were able to translate that and show benefits in a clinical feasibility study that came out just a little over a year ago. Separate from that, flow cytometry has recently emerged as a method by which one can sort cells and do individual single-cell RNA sequencing, and so that allows you to take, in any given model, be it inflammatory pain, look at specific neurons, specific microglia, specific cell types, and say, what kind of proteins, what mRNA, what are they expressing? And so that has allowed for characterization of neuron-specific inflammasomes, or microglia-specific inflammasomes, and what we focused on in this study was combining our research into these neuropathic pain models, which we suspect have this inflammatory component to them, and looking at the effect of spinal cord stimulation on genes identified as being specific to the neuron inflammasome or the microglia inflammasome. So here's our kind of materials and methods, and as you can see, we progressed through randomizing the rats either into a naive group or an SNI, which is our chronic pain injury group. They underwent behavioral testing to look at mechanical hypersensitivity via von Frey, as well as cold and warm plate testing, both at baseline, after induction of the injury, and then after they were stimulated with continuous stimulation for 48 hours with one of our three treatment groups seen down at the group on the table at the bottom right. We then biopsied the tissue subadjacent to the lead and performed RNA sequencing, which yielded around 20,000 transcripts from the spinal cord, and we cross-referenced that with those neuron and microglia inflammasomes that we identified within the literature of single-cell RNA sequencing models. And so kind of our methodology for data analysis is we took—this is kind of our previous pathway of experiments. We had, you know, we randomized the rats, we either had them injured or naive, we look at the tissue samples, we look at WGCNA, and what we'd found previously, we're now wanting to validate and look at how that compares to what we're finding in these literature databases. So first, those behavioral results, what we found was that all of the spinal cord stimulations showed a significant improvement in mechanical stimulation with von Frey filaments, compared to rats that had no spinal cord stimulation after spared nerve injury, but DTM-SCS actually had a significant improvement over both high-rate and low-rate SCS. Similarly, for cold and warm plate sensitivity testing, we found that DTM-SCS allowed the rats to sustain colder or hotter temperatures before undergoing pawl withdrawal, compared to high-rate, low-rate, or no SCS. So now looking at the transcriptomes, those molecular results, I know this slide has a lot going on, but starting over with those heat maps on the right-hand side, what we did was we said, if SNI, if the spared nerve injury chronic pain, is possibly mediated by inflammation, then the way to reduce the chronic pain would be to take these inflammasomes back to their pre-injury naive states. And so you can see we're treating naive in that leftmost column of each heat map almost as if it's a treatment. We're saying the goal then would be to have one of those spinal cord stimulations almost line up exactly with the expression values seen in the naive case. And so looking at those heat maps, we compared each of those expression profiles via Pearson correlations, and what we found was that for the microglia inflammasome, DTM-SCS and high-rate SCS both showed moderately strong positive correlations that were significant, while low-rate SCS showed a weakly negative correlation that was not significant. For the neuron inflammasome, we found that DTM-SCS showed a very strong positive correlation that was significant, while high-rate and low-rate SCS showed weak or moderately weak positive correlations that were significant. And I also want to point out on this slide, one thing that interested us here was that notice that the red represents downregulation, which would mean that the genes from the naive state were downregulated compared to where they were after the spare nerve injury. We see more of these microglia inflammasome genes are turned down compared to the neuron inflammasome genes, which are blue and turned up. So we found this to represent some kind of imbalancing of these inflammasomes between the microglia and the neurons. And so next we looked at, okay, if these are imbalanced, we want to bring them back, right? So if they start at a naive expression level and they're upregulated by spare nerve injury, then we would want spinal cord stimulation to bring them back and downregulate them. And we call that returning the gene, kind of returning it to its naive state. We found that of the genes that were returned by high-rate or low-rate SCS, DTM-SCS actually returned over 80% of those same genes in both microglia and neuron inflammasomes, while additionally modulating 63 genes that neither high-rate nor low-rate were able to return back to naive. And so going back to that data analysis methodology, as we said, we have our preview. So in conclusion, what we saw was that the spare nerve injury, this chronic pain state, creates an imbalance in the inflammatory response in these inflammasomes that are specific to neurons and microglia, as we showed earlier in the results section. Additionally, low-rate spinal cord stimulation trended towards further upregulation of microglia-based inflammasome genes. And we found this to be a particularly interesting point, given our hypothesis about bringing back towards naive. And so one area that we're exploring further now is, is there a certain subset of genes that you want to bring back towards naive that you want down or upregulated? And that's currently kind of where we're at in this inflammasome investigation. Additionally, these results are consistent with our prior RNA sequencing analyses that showed DTM had this higher impact on inflammatory processes. What we found new in this study was that it was able to rebalance, that there was an imbalancing and DTM-SCS was most effective at rebalancing these inflammasomes. And additionally, that DTM-SCS affected a population of inflammasome-specific genes in neurons and microglia that were unaffected by high-rate or low-rate commercially available SCS. And lastly, all of this that we were finding in these molecular analyses correlate with what we saw in that DTM-SCS is more effective than high-rate or low-rate in relieving mechanical hypersensitivity on those behavioral tests. So these are references, and I just want to take this opportunity to say thank you for listening to my presentation today.

spinal cord stimulation

neuropathic pain

DTM-SCS

rat models

inflammasomes