Estrogen deficiency modulates the local tissue response after stroke in a photothrombotic stroke model

Introduction: : In women, stroke risk increases post-menopause and stroke severity correlates with estrogen (E2) deficiency, but hormone replacement therapy does not mitigate this and may be associated with increased stroke risk1. Concomittantly, recovery after stroke declines with age due to the brain’s reduced regenerative capacity, decreased blood flow, slower metabolism, and increased inflammation2. Currently, no post-stroke therapies address the role of E2 in neuroprotection and wound healing. The stroke infarct is characterized by a necrotic core filled with inflammatory signals and cells and degraded ECM, surrounded by a glial barrier. While the area surrounding the core has increased angiogenesis, the core itself does not remodel. E2 supports wound healing through lowering inflammation and supporting pro-regenerative cell phenotypes in skin2 and stroke wounds4. We have previously shown stroke core remodeling and functional improvement through an angiogenic biomaterial delivery into the stroke core, but this therapy failed to promote tissue regeneration in female mice5. We hypothesize that an estrogenic biomaterial that provides a physical scaffold for cell infiltration, attenuates inflammation, and promotes angiogenesis may improve functional recovery after stroke in female mice by addressing the critical role of E2 in tissue regeneration. Towards the development of this translational biomaterial, we have established an E2-deficient photothrombotic stroke model for investigating E2-mediated neuroprotection and the therapeutic potential of E2 for regeneration and functional recovery post-stroke.

Materials and

Methods: : C57BL6 female mice were ovariectomized (OVX) at 13 weeks of age, then after two weeks subjected to daily vaginal cytology smears to monitor cyclicity and E2 washout. Once persistent diestrus was observed (~18 days post-OVX), E2-deficient mice and age-matched controls underwent photothrombosis to induce stroke as previously described3. Briefly, anesthetized mice were placed on a stereotactic apparatus, where a midline incision was made to expose the skull and connective tissue was removed. A laser was positioned 1.5 mm lateral from the bregma, and the mouse was injected interperitoneally with Rose Bengal, a photosensitive dye, in sterile PBS at 0.1 µg per gram of bodyweight. After 7 minutes of circulation time, the brain was illuminated at 42 mW through the intact skull for 13 minutes to induce photothrombosis. The incision was then closed using Vetbond. Two weeks after stroke, mice were perfused and sacrificed for harvesting brains and reproductive tissues. The study design is summarized in Figure A. For immunofluorescent staining, anti-GFAP (Abcam ab33922) was used to visualize astrocytes and the glial scar, alongside anti-estrogen receptor β (Santa Cruz sc-53494), and DAPI to visualize nuclei. 30 µm whole brain slices were imaged at 6 µm intervals to make maximum intensity projections representing the entire 30 µm section. PIPseq will be implemented to characterize the cellular landscape in the peri-infarct with and without OVX, as well as flow cytometry to understand the inflammatory milieu post-stroke in both conditions.

Results, Conclusions, and Discussions:: All mice showed normal estrus cycle patterns in a screening period prior to OVX; OVX mice (n=3) showed persistent diestrus when vaginal cytology was resumed 18 days after OVX (Figure C). Harvested uteri showed normal morphology in intact mice, while OVX mice displayed uterine atrophy, as expected with estrogen deficiency. Whole brain sections from the animals sacrificed 14 days after photothrombotic stroke were stained against Iba1 (yellow) to visualize microglia and GFAP (red) to visualize astrocytes (Figure D), along with DAPI (blue). We observed a dramatic difference in infarct size between groups, where the glial scar is clearly visible in the OVX group but injuries were small in intact mice and thus difficult to quantify (Figure D). In both conditions we observed localization of microglia to the site of injury. We next investigated whether OVX and/or photothrombotic stroke modulate the expression of estrogen receptor (ER) β in the infarct area. ERβ is a classical nuclear membrane receptor that has been shown to play critical roles in modulating cutaneous wound healing4, but has not previously been studied in the context of ischemic stroke. Thus its elevated expression in the peri-infarct makes it an interesting target for future therapies (Figure E). Importantly, ERβ expression was observed in both intact and OVX conditions, showing that these receptors persist regardless of circulating E2 levels. These results together point to the role of estrogen as a neuroprotective factor in the central nervous system and set the stage future work in understanding how estrogen modulates the local and systemic responses to injury in the brain. We will investigate these factors further via flow cytometry to understand the inflammatory milieu in intact versus OVX mice, and PIPseq to understand phenotypic changes in cells in the peri-infarct. Here we have established an estrogen-deficient model of ischemic stroke to elucidate the neuroprotective and regenerative capacities of estrogen signaling in the brain. Future work in this area should focus on uncovering the mechanisms by which estrogen mediates neuroprotection and which cell types most prominently drive this effect towards the development of estrogenic therapies to support functional recovery after stroke.

Acknowledgements and/or References (Optional):: 1 Johansson, T. et al. Oral Contraceptives, Hormone Replacement Therapy, and Stroke Risk. Stroke 53, 3107-3115 (2022). https://doi.org/10.1161/STROKEAHA.121.038659
2 Tsao, C. W. et al. Heart Disease and Stroke Statistics-2023 Update: A Report From the American Heart Association. Circulation 147, e93-e621 (2023). https://doi.org/10.1161/CIR.0000000000001123
3 Wilson, K. L. et al. SDF-1 Bound Heparin Nanoparticles Recruit Progenitor Cells for Their Differentiation and Promotion of Angiogenesis after Stroke. Adv Healthc Mater 13, e2302081 (2024). https://doi.org/10.1002/adhm.202302081
4 Campbell, L. et al. Estrogen promotes cutaneous wound healing via estrogen receptor beta independent of its antiinflammatory activities. J Exp Med 207, 1825-1833 (2010). https://doi.org/10.1084/jem.20100500