healing after endovascular treatments. To begin with, despite enormous advancements for endovascular aneurysm treatment in recent years, the recurrence rate after coiling remains much, much higher than this with clipping. And this can at least partially be explained by the direct physical endothelial-to-endothelial contact immediately after clipping, whereas in coiling, the coil introduces thrombus inside the aneurysm, which then needs to become organized into fibrous scar tissue and new cells need to be recruited to form a neointima. So in other words, once the aneurysm is thrombosed, it needs cell migration for cells that promote aneurysm healing. However, in reality, previous studies have shown that a lot of growing aneurysms actually have a lack of cells in their aneurysm wall. And as a consequence, the thrombus is steadily built, remodeled, built, remodeled, but it doesn't mature into mature scar tissue. And as a result, there is some remaining residual perfusion or recurrence in these aneurysms. So the question we wanted to address with this study was, where do the cells that mediate aneurysm healing origin from? Are they coming from the aneurysm wall? Are they derived from the parent artery or rather just from circulating progenitor cells? To answer that question, we went into the lab. We are using the Helsinki rat aneurysm model. In this model, a standardized piece of the thoracic aorta is ligated at the far end and sutured end-to-side on the abdominal aorta of a recipient animal, which then forms a highly standardized aneurysm. Now, for the purpose of that study, we used transgenic rats, which express green fluorescent protein, so-called GFP, in a mosaic pattern. And that allowed us to suture a green aneurysm on a wild-type animal and vice versa. These surgically created aneurysms were then treated either by coil or by stent. And upon a predefined follow-up of 28 days max, they were examined for residual flow. As you can see in the fluorescence angiography, there was substantial residual perfusion in this aneurysm with no treatment. By contrast, this aneurysm with stent treatment did not show any signs of residual perfusion, but strong blood flow into underlying parent artery. Aneurysms were then harvested and macroscopically inspected. And again, we see central residual perfusion to a large extent in this aneurysm, but a very strong neoentema on the level of the stent in that one. As a next step, we did histological staining. Here we have the aneurysm on top of the parent artery. Here you see some artifact from the stent. And we have already quite good healing, a strong neoentema with some residual hematoma on top of that aneurysm. Now let's put that into a timeline. This is muscle staining, so green for collagen fibers and red is blood. And you can see that already after three days, there is a small but existing neoentema, which steadily grows from the periphery towards the center and from the basis of the aneurysm upwards in the dome. And at the same time, the hematoma is continuously organized and matures into scar tissue with only some minor hematoma left on the very top of the aneurysm after 28 days. So if I may sum up for the first time, light microscopy showed us that in biological aneurysm healing, there is a pattern of organization from the periphery towards the center and from the basis upwards into the dome of the aneurysm. As a next step, we did fluorescence microscopy. This is the same aneurysm in DARPA staining, so all the nuclei stain as bright dots. And again, the same aneurysm with a green filter. You see that the image goes all the way down here. This is just artifact, but we have a green signal on the level of the aneurysm, but not in the parent artery. So that's the first positive control, because we have a GFP-positive aneurysm on a wild-type animal. These two images can be merged together, which allows us then to see GFP positivity for every single cell, if present. So we went on to study the proportion of GFP-positive cells in stent-treated aneurysms and compared that to the proportion in coiled aneurysms, specifically for three regions of interest. In the aneurysm wall, every other cell or so shows a GFP-positive signal, which makes sense because, again, remember, GFP is expressed in a mosaic pattern, and that's exactly the same for stent treatment. In the thrombus, we see some GFP-positive cells, but the majority of all cells is GFP-negative. And again, that's very similar, some single GFP-positive cells, but the majority is negative. Lastly, in the neointima, we see some GFP-positive cells in coiled treatment, but hardly any, actually very few, in stent treatment. So we performed a cell count in all aneurysms, specifically for all three regions of interest, and that reveals about 50% of cells in the aneurysm wall for either treatment were GFP-positive, and about one-fifth of cells in the thrombus. However, quite the difference in the neointima, about one-third of cells with coiled treatment, but less than 10% with stent treatment. And we think that's because the stent acts as a scaffold. It helps cells to migrate along the stent and settle down on the surface and build that neointima, whereas there is no surface in coiled treatment, which means more cells need to migrate also from the aneurysm wall. Now, as a last question, one is wondering what kind of cells we see, and this is why we did immunostaining. But first for your orientation, this is the parent arteries lumen. There is some artefact from stent or coils, and in the top row, we have a coiled treated aneurysm with substantial residual central perfusion, whereas we see a very strong and thick neointima of excellent healing in this stent-treated animal. Now, very clearly, cells in the thrombus and in the neointima, they express smooth muscle acting, so they may be myofibroblasts or myoblasts. And with von Willebrand staining, we see endothelial lining towards the lumen, but also around foreign bodies such as stents or coils. That brings me to the conclusion. We were able to show that aneurysm healing after endovascular treatments depends on cell migration from the parent artery and from the aneurysm wall. Cell migration from the aneurysm plays more important role in coiling than in stent treatment, and in complete healed aneurysm, there is a continuous endothelial lining along the parent artery. Taking these things together, this may be a reason for better healing in stent-assisted coiling than in coiling alone. I would like to thank all the members of our lab group, and thank you very much indeed for your attention.

healing process

endovascular treatments

aneurysms

coiling

clipping