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Title: Imaging biological water permeability barriers using CARS microscopy
Author: Patel, Keval Dipan
ISNI:       0000 0004 7968 5216
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Alterations in the water permeability of the vasculature are attributed to several disease processes including atherosclerosis, reperfusion injury, diabetes mellitus, aging, chronic inflammation, and cancer. While vascular permeability has been extensively studied throughout the years, many of these studies have been limited by their invasiveness, specificity and spatial resolution. Coherent anti-Stokes Raman scattering (CARS) microscopy, offers a means to visualize the water permeability barrier of biological structures at high resolution within intact systems. In this thesis, water CARS imaging was characterized for determining water permeability in both dynamic and steady state conditions in model hydrogel systems. Computational models of water transport were used to validate the accuracy of water-CARS permeability mapping within these systems. This technique was then applied to the first CARS examination of water permeability in the cerebral artery and blood brain barrier. The vessels of mice deficient in the water channel protein aquaporin 1 (AQP1) were examined to evaluate the role of AQP1 in vascular water permeability. A layered hydrogel imaging phantom was constructed from poly(ethylene glycol) diacrylate (PEGDA) to validate ability of water-CARS microscopy to determine the relative permeability differences of adjacent material layers. By imaging the dynamic and steady-state H2O and D2O exchange across the uniform and layered composite hydrogel structures, it was indeed possible to measure the relative permeability differences between individual material layers and to determine the location of material interfaces from analysis of the water-CARS image profile. CARS imaging of intact biological samples poses many significant challenges. Microvessel microfluidic systems have the possibility to facilitate microscopy studies of vascular barrier function due to their flexibility of design, relative ease of preparation, and relative simplicity. In this thesis, the fabrication process of a human umbilical vein endothelial cell (HUVEC) microvessel microfluidic was investigated and optimized for future studies of vascular permeability and physiology. Several factors in the device assembly process were identified and optimized to improve the repeatability and reliability of the microvessel microfluidic fabrication protocol. CARS imaging of the mouse cerebral artery water transport produced higher quality images than what had previously been achieved in the rat mesenteric artery, likely due to their reduced wall thickness. Permeability mapping of these arteries localized the water permeability barrier to the endothelial basolateral membrane, a result consistent with previous measurements performed in the rat mesenteric arteries. Previous work by the Laboratory of Cardiac Energetics has suggested that the exclusively apical expression of the channel protein aquaporin 1 (AQP1) may account for the increase in observed permeability of the apical endothelial cell membrane. To test this hypothesis, water-CARS imaging of H2O/D2O exchange across cerebral arteries of wild type and AQP1 knockout (KO) mice was performed. No significant difference in the location of the water permeability barrier was observed. These findings indicate that AQP1 may not, in fact, be rate limiting for water transport across the apical endothelial membrane, and that it may play some other role in the physiology of the endothelial cell.
Supervisor: Huang, Shery ; Balaban, Robert Sponsor: NIH Oxford Cambridge Scholars Program
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: CARS microscopy ; engineering ; biomedical engineering ; hydrogels ; diffusion ; permeability ; blood brain barrier