Sylvia Mechsner, MD - Neurogenic inflammation in peritoneal lesions and clinical consequences

I'm so happy to be here. And when I saw the program, thank you so much for this program because I love the story of the nerve fibers and I agree with all of these points with you. So we started many years ago also with the research on the topic of the nerve fibers because one of the main points in endometriosis is a pain and we need to understand the pain in more detail. So I picked out this idea to explore in more detail, the pathways of the cyclic and more cyclical pain due to peritoneal lesions, because it is really intriguing that such a small lesion, puritinal lesion, endometriosis lesion is able to induce so strong pain. Whereas in patients with ovarian cancer, it's still without any symptoms. That is really, really interesting. So endometrial lesion, peritoneal lesions, it's a proliferative disease, but not only this, and I think we miss a lot of information because of the HE staining because there's nothing to see.

But when we are doing stainings for pain mediators, inflammatory mediators, growth factors, we see that peritoneal lesions are highly active. But we have also this inflammation in immune cell infiltrates are affecting the peritoneum. And then we have the neuro, the nerves inside of the lesions. We explored also that they are not together with blood vessels that are without blood vessels. So we thought, okay, there are newly grown nerve fibers into the endometritic lesions. We have also the angiogenesis and lymphungiogenesis present in this tissue. And we have also, that is also often forgotten that we have also smooth muscle cells around the lesions. And this smooth muscle cells or myofibroblasts are able to release collagen. And so we have also problem with fibrosis in this tissue. So we have a lot of topics which could be involved in pain generation. And at the beginning for me, of course, it's a hormonal dependent disease.

The release of pain mediators is it's beginning cyclical. So we have this situation that we started with the cyclical pelvic pain in patients with endometriosis that is often reported by the patients. But then there is one point that the cyclical pain shifted also to an acyclical pain or better. There is an additional acyclical pain and was on top cyclical pain then. So there is something different. And my question was then to explore if a neuroimmune modulation could be the cause for the acyclical lesional pain. What is the meaning of neurogenic inflammation? That means that activated nerve fibers release neuropeptides and lead also to inflammation in the tissue extravasation and edema in this tissue. And this is a complex process of peripheral and central sensitization, and that is better understood in other diseases, for example, migraine. So in migraine, we already have an antibody against migraine for the prophylaxis.

CGRP is one of these released neuropeptides from the sensory nerves. Also involved in the complex regional pain syndrome, it's also investigated in this area and also postoperative pain syndrome are well known that there's neuroimmune modulation and neurogenic inflammation involved. And we have also very good data from the arthritis research because they were the first who explored this neurogenic inflammation. We see here that the number of sensory nerveables is increased in this inflamed tissue, but surprisingly, the sympathetic nerve fibers are decreased in this tissue. And also the simple ... So the sensory nerve fibers release substance P that is also pro- inflammatory neurotransmitter. And the no-andrelein and adrenaline released by the sympathetic nerve fibers is more anti-inflammatory. And they postulated this shift, this inverse correlation of this sensory nerve fabric and the loss of the sympathetic nerve abuse is one of the main pathways in this inflammation and pain generation in arthritis.

So that was the reason that we started also to look in more detail. We found the nerve fibers before. My first publication about the nerve fibers present was in 2007. I was one of the first who thought and who looked for the nerve fibers, but then we started to count them. So we did staining with PGPs, a pan marker for nerve fibers, looked in endometriosis and healthy tissue and healthy peritoneal tissue. And we found also a higher number of sensory nerve fibers in the endometritic tissue and a loss or a lower number of sympathetic nervous in the endometriosis in comparison to the healthy controls. So we have an invarious correlation in this tissue. Of course, the next step was then to look for substance BNGRP is the release of this neurotransmitters in the lesions, and we did also staining for this. And of course, also here we can found a higher level of this neurotransmitters in the lesions direct in the direct microenvironment of the lesions and a lower level in the periphery of the lesions and in healthy controls.

We have no relevant elevation of these neurotransmitters. And of course, the next step was to look for the NK1A receptor. That is a receptor for substance B, and that is also increased in immune cells around the lesions. So we started to think about how can we explore it in a in vivo in vitro model, and then we decided to use sensory dose root ganglia from embryo chicken that is easy to prepare them and to use it in the cell culture model. And we incubated lumbus peritoneal fluid of patients with and without endometriosis. And as you can see here on the left side, there's an outgrowth of nerve fibers when we incubated the CRGs with peritoneal fluid from patients with endometriosis. And that is not the case when we do it with peritoneal fluid from healthy controls. We used also sympathetic ganglia and here it's contrary present.

We have no induction of sympathetic outgrowth due to the peritoneal fluid from patients with endometriosis, but the healthy peritoneal fluid is bringing or is induced the outgrowth of this sympathetic nerve fabric. So we were able to confirm our finding from the immune histochemistry also in this in vitro model. Yes, what's going on with this sympathetic nerve fiber? So why is there a deletion of the sympathetic nerve fibers? Also here, we reviewed the literature and found also there is a protein family zmaphyrines, which are involved in development of brain and nerve fibers. And also the researcher, the rheumatoid arthritis researcher looked for the cymophrenes, especially cimophrine 3C and 3F seems to be involved in a damage of sympathetic nerve fibers. So they found it also in the rheumatoid arthritis. So we looked for cimaphorin 3C and 3F, also imperitoneal lesions in comparison to healthy controls. We did double labeling.

We learned from the literature they could be expressed by immune cells. So we did double labeling with CD68, which is a marker for macrophages, and another interesting cell population are activated fibroblasts. Maybe some of you know, so activated fibroblasts are used now. There is an antibody now, and we can use it for labeling for PET-CT now to detect endometriosis. When I saw that, I was a little bit upset because we found or we looked for this activated fibroblast 10 years ago, but I had no staff, no power, no manpower to go on with it. So other did it. But here, the activated fibroblasts, they are positive for zemophorine. So that is interesting. I think they have a key role also in the problem of endometriosis. Of course, we needed then to look for the expression of the recephorine receptors on the nerve fibers. So nouropiline and plexin A3 are the receptors for the cimophrines and we did double labeling tyrosine hydroxylase is a marker for sympathetic nerve fibers and in the upper lane with a peritone lesion, you can see that there's a double labeling.

So neuropeline and plexin are expressed on endometriosis associated enerves, but they are absent in healthy controls. So they seems to be upregulated in this sympathetic nerve fibers around the lesions. And that could be the reason that there is a deletion of sympathetic nerve fibers or a damage. We don't know exactly. We are going on with this research and we found some hints that there could be also a damage of this sympathetic nerve fibers in this lesions. Yes, the inflammatory immune cell infiltrates, we investigated this too, but I don't want to show you more details because of time, so I skip it. But another point is that not only the number of nerve fibers is increased in this tissue, also the nociceptor expression is changed. So we looked for the nociceptor expression of NGF-P75, TRIP as a TRPV1, which is the main activate of silent C fiber, so silent pain fibers in track A.

And of course, we again confirmed that the number of sensory nerve fibers is elevated in endometriosis, but also the expression of these nociceptors are increased. So that means that we have also a peripheral sensitization of this nerve fibers. Bringing together this idea, so we have the lesions at the beginning, and we have the release of a lot of inflammatory mediators and nerve growth factors and so on. And we found also the staff elevated in the peritoneal fluid. We have the inflammation is going on. And by the time we have an increased number of sensory nerve fibers with the release of substance P and CGRP, we were also able to found elevated concentration of substance P and CGRP in the peritoneal fluid. We have the chemotoxis of this immune cells and with the release of the simophyrine then step by step, a deletion or damage of the sympathic nerve fibers.

And then we have a higher concentration of the more pro- inflammatory substances, substance CGRP in this lesions, but lower level of the more anti-inflammatory neurotransmitters of the sympathetic nerve fibers. Yes, that is our hypothesis. And I think it has also some clinical impact because when we all try to avoid unnecessary surgeries, and when we see such a severe case, what should we do? So in my clinical day routine, we are starting hormonal treatment. We try to reach a therapeutical aminoria. So most of the people are fine, but when the pain is going on, they have no bleeding, but they started to have cyclical pain. That is for me the reason to do a surgery. We stop the hormonal treatment. And when we see this, so I know the patients suffer from pain under therapeutical aminora. I could be relative sure that the cause of the pain is then the peritoneal lesions.

So we started to remove the peritoneum completely. We did then a peritonectomy like this. And we had also, we followed more than 100 cases like this without other lesions, only the peritoneal lesions, and we had a 25% complete pain free situation later or in 50% pain improvement. So that is also very good. Of course, 25% are non-responders, maybe due to severe adenomyosis or because of central sensitization and so on, because it's complex. Yes. And when we ask our pathologist, not only for endometriosis, also for inflammation, I'm sorry, it's in German, but the red is the inflammation. Everybody can understand that it's not only endometriosis is also an inflammation present, please ask the pathologist for the inflammation and the lesions. And by accident, this lady came three years later with another problem. She developed a niche, a cesarean scarnish in the uterus, and then she had their ovular narbody present in this knee, and that was a cause for her pain, but she thought, "Dr.

Max, I'm really sure my endometriosis is back." I said, "No, I can't believe." And we did the surgery and yes, that is the same lady after the spiritonectomy three years later. I think that is, of course, it's without any endometritic lesion. So I'm summarized. So at the beginning, I think we have this typical cyclical pain, and that is a nociceptive pain. We have an acute inflammatory situation, and when the cycle is over, the pain is gone. But by the time we have the peripheral sensitization, we have the higher number of nerve fibers, and then the release of the substance B and the interaction with the blood vessels and nerve fibers and so on, blood vessels and immune cells. I'm sorry. So we have then this neurogenic inflammation, and that is a main factor in this ongoing disease. And also, I think it's under present in the understanding of the disease.

Neuroinflammation is very important. We have also to start to look for the microglia changes in the endometriosis situation, but we have some topics. We have the neuromodulation, the option of neuromodulation. Now also we started also a project with vagus nerve stimulation. We tried also spinal cord stimulation and so on. And of course we can improve the situation, the inflammatory situation with dietary supplements and so on. So there are a lot of further options to modulate this neurogenic inflammation. I'm really sure that it is a very hot topic for the future research in endometriosis. I would really like to thank my team in Berlin. I'm really proud of them. We are really close together and they are so active and brilliant. I'm really happy to have them. Yes. Thank you so much.