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Type of Resource (primo): dissertations
Name: Personal
Name: Personal
Name: Personal
Name: Personal
Name: Corporate
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Note: thesis: Thesis (Sc. M.)--Brown University, 2025
Genre (aat): theses
Abstract: Objective: Despite extensive evidence of APOE4-driven vascular dysfunction, cerebral endothelial connexins that play a critical role in endothelial communication and vasodilation remain poorly characterized in Alzheimer’s disease (AD). Connexin expression changes in the hippocampal vasculature in early stages of AD may be the missing link that can bind systemic APOE dysfunction to impaired hippocampal neurovascular coupling. This study aims to (i) establish and validate a reproducible dual-label immunofluorescence and Slide Scanner pipeline to visualize endothelial Connexin-43 (Cx43) in coronal sections as a first step and (ii) develop a preliminary channel specific quantitative image analysis pipeline capable of processing high throughput whole section images in a single batch. Methods: Brain tissue was extracted from 10 ApoE ε3/ε3 and 10 ApoE ε4/ε4 mice, cryostat sectioned, immunofluorescent stained to generate 4 coronal cross sections per brain along with representative middle cerebral artery (MCA) cross section samples. These were imaged using Olympus VS200 Slide Scanner at 40×, following high-resolution validation of the protocol on Olympus FV 3000 confocal microscope at 60×. Preliminary images were analyzed using VS200 ASW, and Fiji/ImageJ. A parameter-locked Fiji/ImageJ macro was developed for all three channels to (i) compute hippocampal vessel area fraction from CD31 masks, (ii) quantify Cx43 puncta counts within atlas-anchored ROIs, and (iii) estimate nuclear density from DAPI (nuclei per mm²); the batch routine exports overlays and CSVs, with full-cohort execution deferred until validation. Results: The finalized protocol yielded continuous CD31-positive microvasculature and discrete AF594-positive puncta consistent with Cx43 along vessel walls, while secondary-only controls showed no structured signal. Slide scanning achieved complete hippocampal coverage suitable for cohort throughput, and confocal resolved submicron puncta and verified morphology. An image set of 80 hippocampal whole coronal sections with exposure records and focus-point maps was curated. The batch code produced correct overlays and tabular outputs on small ROIs of test sections. Conclusions: This study delivers a scalable staining and acquisition protocol, a modality comparison tailored to endothelial Cx43 mapping, and a ready-to-run customizable analysis pipeline. Although further refinement in image analysis pipeline are necessary for standardization, and cohort-level metrics and group comparisons are deferred, feasibility, specificity, and analysis-readiness are demonstrated, laying the groundwork to interrogate endothelial Connexin-43 as a potential mechanistic link between APOE4, disrupted neurovascular coupling, and cognitive decline.
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Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/00806532")
Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/01893632")
Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/00937740")
Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/00957258")
Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/00967947")
Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/01919344")
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