Hello, my name is Devin Bitzell, and I'm a medical student working in Dr. Brian Ho's lab at the University of Florida. Today I'll be discussing our project entitled neutrophils contribute to intracranial aneurysm pathogenesis in an estrogen-dependent manner. I have no financial disclosures, so for some background, the prevalence of intracranial aneurysms is between 3.6 and 6%. When an aneurysm ruptures, this is often a life-threatening condition with a 30-day mortality between 36 and 38%, and up to 50% of patients actually develop a permanent disability. Now the mechanism of aneurysm rupture is currently unknown. It's thought that inflammatory pathways play a key role, and from epidemiological studies, we know that females are typically more affected than males, with postmenopausal women having the greatest risk of rupture. And this suggests a role for sex hormones, although this role is currently unclear. So to go over what we know about estrogen and neutrophils, first for estrogen, we know that it protects against aneurysm formation and rupture in mice. And some work from our lab has studied the role of inflammatory pathways in the context of estrogen deficiency, and we've shown that in mice that are estrogen deficient, there's upregulation of T helper 17 cells and increased interleukin 17. There's also increased interleukin 6, which increases macrophage infiltration, and both of these inflammatory pathways contribute to aneurysm rupture. The role of neutrophils, however, is currently unknown. It's been shown that estrogen attenuates neutrophil chemotaxis and models the vascular injury, and in a mice model of abdominal aortic aneurysms, it's been shown that neutrophil depletion prevents AAA formation. So putting this together, we hypothesized that neutrophils contribute to aneurysm pathogenesis in an estrogen-dependent manner. For our methods, we used our marine model for aneurysm formation and rupture, which combines a variety of factors, including hypertension, hemodynamic changes, and vessel wall damage. To study the role of neutrophils, we used a rabbit antipolymorphonuclear antibody to induce neutrophil depletion, or anti-PMN, and then the control group received rabbit serum control. We studied the role of neutrophil depletion in three distinct cohorts. We had estrogen-intact females, estrogen-deficient females, in which we performed bilateral overectomy, and in males. For the first experiment, we induced aneurysms in a cohort of mice and used immunofluorescence for neutrophil elastase to assess neutrophil infiltration, and what we found is that there was significantly increased neutrophil staining in aneurysm tissue compared to normal vasculature. And here's some representative immunohistochemistry images where we can see in aneurysm tissue that there are increased neutrophils, depicted here by the white arrows, compared to normal vasculature. For the neutrophil depletion experiments, we analyzed four outcomes, aneurysm formation, rupture, symptom-free survival, and we also measured blood pressure at different points throughout the experimental model to see if the antipolymorphonuclear antibody affected blood pressure in mice. So for the first cohort, which is estrogen-intact females, we saw that neutrophil depletion had no effect on aneurysm formation rates or on aneurysm rupture rates. Similarly, symptom-free survival was not affected by neutrophil depletion, and with regards to blood pressure, we saw that in mice treated with the antipolymorphonuclear antibody, there was no change in blood pressure compared to those that received the rabbit serum control. In estrogen-deficient females, neutrophil depletion had no effect on aneurysm formation rates. However, mice that were treated with the neutrophil depletion antibody, there was significantly fewer aneurysm ruptures compared to the control group. Similarly, in symptom-free survival, mice that were depleted of neutrophils had a higher symptom-free survival compared to the control group, although this difference was not statistically significant, and there was no change in blood pressure, suggesting that the antipolymorphonuclear antibody does not affect blood pressure in mice. And finally, in the cohort of males, we see that in mice treated with neutrophil depletion, there was significantly fewer aneurysms that formed compared to the control group, and there was also fewer aneurysm ruptures, although this difference was not statistically significant. With regards to symptom-free survival, mice that were treated with neutrophil depletion had a higher percent survival compared to the control group, and once again, we see that our antipolymorphonuclear antibody does not affect blood pressure. So in conclusion, we can say that aneurysms have increased neutrophil infiltration compared to normal vessels. In estrogen-intact females, neutrophils do not affect aneurysm formation or rupture. In estrogen-deficient females, however, neutrophils contribute to aneurysm rupture. And in males, neutrophils promote aneurysm formation and may even affect aneurysm rupture. And further studies are needed to elucidate the underlying mechanisms by which neutrophils contribute to aneurysm pathogenesis. In closing, I'd like to acknowledge our various sources of funding, and I'd also like to say thank you to all the members of our lab who make this work possible. Thank you for your attention.

neutrophils

intracranial aneurysms

estrogen-dependent

inflammatory pathways

rabbit model