Date of Award
Fall 8-22-2025
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Neuroscience
First Advisor
Meharvan Singh
Abstract
Ischemic stroke is a leading cause of death worldwide and often results in permanent disabilities that can greatly hinder quality of life in surviving patients. Treatment options for ischemic stroke are extremely limited, emphasizing a critical need for the development of novel treatment modalities. Progesterone (P4) is a gonadal steroid hormone which has been shown to be protective in experimental models of stroke. However, the underlying protective mechanisms remain unclear. Previously, our lab has shown that inhibition of the microRNA let-7i enhances the protective effects of P4 in an animal model of stroke. In this study, we sought to evaluate the differential responses between astrocytes and neurons to P4 + let-7i antagomir treatment and assess the protective effects when administered as an intervention in vitro after ischemic insult. Furthermore, we explore a minimally invasive method of antagomir delivery using exosomes expressing the rabies virus glycoprotein (RVG), a brain-targeting peptide. Using cultures of primary cortical astrocytes, neurons, and mixed cultures (containing neurons and glia) exposed to conditions of oxygen-glucose deprivation (OGD), we report that there are cell-specific changes in expression of let-7i, as well as two key mediators of P4’s protective effects, progesterone receptor membrane component 1 (PGRMC1) and brain derived neurotrophic factor (BDNF). Our data also demonstrates that treatment with P4 + the let-7i antagomir (let-7i inhibitor), when administered 3 hours after ischemic insult, directly protects astrocytes, and indirectly protect neurons through astrocyte mediated mechanisms. Additionally, P4 + let-7i antagomir treatment does not affect BDNF protein levels in astrocytes and neurons cultured separately, but significantly increases BDNF protein levels in mixed cell cultures. Initial characterization of our exosome-mediated delivery of the let-7i antagomir revealed that RVG-expressing exosomes were non-toxic to astrocytes. However, upon evaluating the delivery of the antagomir in astrocytes exposed to OGD, we found negligible changes in expression of let-7i, PGRMC1, and BDNF. Additionally, RVG-expressing exosomes failed to produce the same protective effect seen when the let-7i antagomir was directly transfected into astrocytes, suggesting further development and/or optimization of the exosome-mediated delivery approach is warranted. In summary, we report that the protective effects of P4 and the let-7i antagomir are primarily mediated by glia, and overall, require the presence of both astrocytes and neurons to elicit a maximal protective response. These data highlight the therapeutic potential of P4 + let-7i antagomir treatment as a delayed intervention following stroke and emphasize the role of glia in mediating its protective effects. Additionally, since glia are the primary target for this intervention, our results invite further studies on effective and safe delivery methods for the let-7i antagomir, specifically to astrocytes, following ischemic stroke.
Recommended Citation
Krishnamoorthy, Vignesh Raghu, "Let-7I Mirna Antagomir as a Novel Adjuvant Intervention for the Treatment of Ischemic Stroke" (2025). Dissertations. 4267.
https://ecommons.luc.edu/luc_diss/4267
