Kanako Hayashi, PhD. - Central sensitization driven by neuroinflammation in endometriosis

Thank you for the introduction. Thank you for the invitation. I'm first time in New York City. Very excited to be here. Yeah, it's a little overwhelming, all buildings. Anyway. So I'm not going to talk about endometriosis. Everyone knows the endometrials except a few experts in different field, but just want to remind one thing. Yes, so far it has been widely accepted virtual grade menstruation is a source of endometriosis. I understand some other theories exist, but let's just focus on this lateral grade menstruation. I'm going to talk about neural inflammation. So neuroinflammation is an inflammatory response within the brain and spinal cord characterized by the infiltration of the leukocyte, mainly macrophages. Activation of glio cells based basically brain macrophages. And then production of pro- inflammatory cytokines and chemical, this is also in the brain. Microglia IV1, he also mentioned that mycoglia microglia and osteocytes GFAP are key regulator of inflammatory responses within this central nervous system.

So as I mentioned, retrograde menstruation occur. This actually happened repeatably. Menstruation generally happen every month if you have normal period. So this level is coming into the pelvic cavity. This is Dr. Celebrin's review paper captured menstruation and definitely has a bra study endometriotic lesions. Once come to there, we think it's centralized peripheral centralization, centralized sensitization, and probably neuroinflammations involve to further centralize the brain. That's why causing the endometriosis associated chronicles to the entire body everywhere. So to understand this mechanism, yes, it's very hard to study in humans. So yes, we need to have a model and it's very hard to do non-animal model because of the entire body. So we use, yes, it's a mouse model. This is the limitation, but what we try to do is to mimic the situation of the menstruation, which means we repeatably induce the region multiple times, not just the one times.

We decided to do six times. I don't like mouse model regions are definitely different from the humans, but we just want to use this model to understand the entire central sensitization model. So that's what I want to say. What's the limitation once induced the lesion mice they develop? This is definitely different than humanitation. Yes, I understand retrograde menstruation over 90% of the women. About 10% women develop the endometriosis. This is very high, but majority of the women are actually healthy. We can understand. We cannot understand why majority of women actually do not develop the endometriosis. We just want to establish the model, then try to understand. So once induced, that's why once induced six times, there are many regions there. The systemize again. When you look at the lesion, there are more macrophages, more the vascularization and geogenesis are going, as well as the neuroangiogenesis.

We have been listening whole story. This is a quality quantified data. We also obtain the resource MAC endometriosis. Non-human primate measures spontaneously develop the endometriosis. These mark examples are spontaneously develop in advanced stages because these markers suffer the pain eventually needed to be euthanized. So that's why we think this is advanced stage of the endometriosis, not by stages, but as a pain. So when you look at these mark example, we have a eutopic endometrium without the endometriosis with endometriosis, and this is the lesions. You see more macrophages definitely in the region. When you look at the neuropylament of the innovation, you see more neurons are there. We also found the neurons, they make the fiber bundling. Those things exist in the neurons as well. So going back to the mouse model, we performed them fly analysis, mechanical sensitivity. We did two area. One is abdominal.

We six is this is more direct because very skin is a very thin. Also, the hind pole withdrawal. We think this is more like a systemic pain because needed to go the brain, central sensitize, and then go down to the different places, which is the hind pole. So once we compare one-time induction and the six-time induction, what's the difference is that here, the six-time induction six week after the last induction, they're more sensitive. And then hindpoint, this is more clear. This is a systemic at least we consider. At the six weeks time point, they return to almost the baseline, which is before the lesion induction. But if you look at the six times induced group, they're still very sensitive. So we think it's definitely chronic sustained pains actually happen in the evening muscle model here repeatably induced the regions. So that's why we went to the brains.

So we look at the IBA1 as marker of microglia, GFAP, marker of gastrocytes. You see more staining, basically. So we quantify where we chose the region cortex, hippocampus, thalamus, and hypothalamus. The lesions are related for pain processing, pain streamers, also anxiety and depression. If you see, first of all, you see the soma cells. The soma cemetery means the activation of the disglion cells. We see the hippocampus salamis, especially six times in use group, more larger soma cells, as well as the number of the IBA-1, as well as the area. Most of the region tended to have more activated gliosis in the brain. So we also had marker samples. And this is, again, the spontaneously developed in other study that we could receive the control markers. Anyway, this is most likely peripheral context, and then we examined the white matter as well as the gray matter.

So you see here, these are the glio cell IBM positive and GFP positive. Quantitatively, the IVL1 showing the larger thrombocytes in the white matter, and then the FAP also showing the vigor. You can see how more activated glyosis in the brain. And also this is more GFAP for positive ostracizing white matter. Look at the DLG. This we consider peripheral. We examined especially DLG L4-6 related to pelvic organs. We look at the TRP1 SPGRP. We had so many times, I'm not going to repeat what's what, but elevated and sustained all expression, especially in the system induced groups as well. So then go back to the peritoneal cavity. So pretonial capital human and even animals, there's so many immune cells there. So we look at, especially focused on macrophages. We have a whole other data T-cells and B-cell, but let's just focus on microphages for the today.

When we examine the CD11B, this is a total macrophages. We didn't see any differences the total microphages. However, then when we look at the neutrophil marker LY6C, I'm sure KD is going to talk about more this afternoon, but what's the important neutrophils fast immune cell, whatever the stimulus come. So they come and go and they die. So two weeks after the lesion induction, we don't see any more difference in general, but the six time induction, however, two weeks is still high, which means that someulator keep going never go away. So that's why we think neutrophils are still high in two weeks after six time induced group. And then TM4 high MHT2 law these are most likely residential macrophages, which they need to be there. They are important for the homeostasis. Once tumors come, what's happening, they disappear, macrophage disappearance reaction occur. So that's why two weeks after they disappear.

But the six weeks after one-time induction, they replenish going back to the normal stages. However, in the six-time induced group, they further disappear and even six weeks after they are still remains lower. On the other hand, the TM4 low image to high macrophages, these are the mainly pro- inflammatory microphages circulating monocytes recruited monocytes differentiated from inflammatory microphages in the pelvic cavity, so they're high in the pelvic cavity. So we definitely go in pro- inflammation if we induce more times. So for a bit, this is a unique microphages we're very into about this population. They have a character, yes, these are tissue regulational macrophages, but they are usually differentiated from pro- inflammatory macrophages. So what's happening is the ones induced six times, they're still high and they're still high in the six weeks after. When we followated this population, we see a higher MHT2 expression, which means these are not likely tissue regination more likely differentiated from the monocyte and keep as a pro inflammatory and they actually reduce a lot cytokines.

So in fact, when we examine the TNFR fire beta and IL-6, those cytokines are high and sustained in the six-time-induced group. So what we did is just confirm the idea we treated DNOGES synthetic progestin and also fingolimod. This is the immunosuppressor or especially the relapse multiple sclerosis, just the proof of concept. I understand the energies is now approved and FDF4 endometriosis associated pain is for the pill in the US. But most of the other country, the endogenous available endometriosis associated pain. This is also FDA approved, but not for endometriosis purpose. But we treated this one, these two drugs three weeks after regions are developed. When we treated three weeks and waited one week just away from the handling stress mice are definitely stress when we treated the drug. So unfortunately, we didn't see any lesion number or volume differences treated three weeks on both drug.

However, when we examined the behavior study, one time induced one didn't see any differences. However, six times induced group, both treatment improved, especially the day 49. Well, with six days 21 such too early, they are just still too sensitive, but the day 49, both treatments improved for the abdominal sensitivity. When we examine the brain that's more clear, treatment of the both drugs is likely improve most of the group, especially in the six times as well. DLG, same way if you treated this drug and they are more likely in a six time induced group, they are much better expressions with PSPS and TP1 as well. So the current idea is that yes, retrograde menstruations are repeatedly okay in women and so far the most of the research will just induce one time and maybe not capture the situation. Again, this is the most model. There is definitely tons of limitation.

However, try to understand once they induce that inflammatory situation is definitely elevated in the pelvic cavity. If you induce again, they cannot clear the existing inflammation. That's why the sensitized peripheral sensitization happened. Central sensitization happened. They were associated to chronic pain. Neuroinflammation definitely helped sensitize, but also based on our results, they can be probably directly contributed to the anxiety depression based on the brain. The stimulation so far we have observed. They can do the top-down things. We don't have data, but probably once neuroinflammation is activated, they will do top down to further the sensitized and peripheral sensitization. The drugs, the NLGS finger mode so far work to inhibit the endometriosis associated pain. Understand the finger mode. Yes, it's approved by FDA, but the side effect is quite high. Directly use this drug systemically treated long-term for endometrial specialty, probably not going to work. However, we are mostly trying to do targeting specific cellotypes in the endometriosis.

So we see how that work. I think we need to do some kind of targeting using nanotherapy and nano drug to target the specific population that so far we found. Then lastly, the acknowledgement members, collaborators, also finding sources. Thank you very much.