Utilisation of in vitro and in vivo model systems to investigate complex physiological responses of endothelial cells to stress and stimuli

The blood-brain barrier (BBB) continues to present one of the most significant challenges to the successful development of therapies for central nervous system (CNS) diseases. Mechanical modulation of the BBB using focused ultrasound (FUS) and microbubbles (MB) has shown considerable promise in enhancing therapeutic delivery to the brain. However, concerns remain regarding the possible long-term effects of such forced disruption. These include questions such as: (1) Are there significant differences in the BBB between homeostatic and disease states that should be considered when designing BBB-modulating drug delivery techniques? (2) Is there a clinical pipeline in place to determine the appropriate use of alternative drug delivery techniques, if BBB integrity is compromised in patients? (3) What pre- or post-operative measures should be put in place to minimise or monitor the adverse physiological effects methodically?
To address these concerns, the work described in this thesis initially focused on understanding brain endothelial cell behaviour in vitro when stimulated ultrasonically with microbubbles. Both 2- and 3-dimensional BBB models were developed and validated with various orthogonal parameters such as trans-endothelial electrical resistance, confocal microscopy, and image-based permeability assays. The 2D set-up was used to observe microbubble-cell interactions in vitro. Ultra-high-speed optical imaging (106 frames per second) was used to capture microbubble dynamics, whilst fluorescence imaging was used simultaneously to monitor the GFP signals from the vascular endothelial cadherin (VE-Cad) proteins in the brain microvascular endothelial cells (BMVEC).
Based on the recorded images, we identified gaps between cells that formed during exposure to ultrasound. To quantify these gaps between different conditions, the regions of interest were (1) masked, (2) binarised and (3) normalised for quantification. The resulting analyses showed that there were paracellular changes occurring during exposure, indicating that these openings can be recorded in real-time with the relevant biological model system.
In parallel to the in vitro work, an Niemann-Pick type C (NPC) mouse model was also utilised to evaluate whether FUS provided an effective means to disrupt the tight junctions between the brain endothelial cells in vivo and allow extravasation of the non-BBB permeable, 2-hydroxypropyl-β-cyclodextrin (HPβCD) into the brain parenchyma. Furthermore, the treated mice were monitored for possible long-term effects of BBB modulation in NPC disease. We first performed a proof-of-concept study on healthy animals and showed a 7- to 9-fold increase of HPβCD uptake with FUS+MB compared with controls. Based on the preliminary results, NPC-affected mice were treated with HPβCD with or without FUS+MB and their responses were monitored over 4 weeks. The results showed modest improvement in the behavioural phenotype of the treated mice compared with controls.
There are inevitably important questions that remain unanswered, such as whether pre-existing neurovascular pathology in CNS diseases can exacerbate, both in vitro and in vivo, the adverse effects of FUS+MB-mediated BBB disruption. Future work could include quantification of vascular patterns, specifically for complex in vitro models (CIVM), that can be used as a reliable metric to identify homeostatic and pathological vascular development. A more robust method of vascular analyses will allow for more accurate pre- and post-treatment evaluation of therapeutic modulation – such as FUS+MB – and its possible adverse effects on the vasculature.

Keywords: blood-brain barrier
endothelial heterogeneity
organ-on-chip
focused ultrasound
Advisor: Stride, E