Development of a Brain Vasculature Damage Model for Permeability Recovery Treatments

Introduction: The impermeability of the blood-brain barrier (BBB) presents a formidable challenge in the development of neuro-regenerative therapies. While sophisticated co-culture systems, complex microfluidic setups, and advanced machinery all offer valuable insights for closely mimicking the BBB, they often require extensive and laborious preparation and experimentation. There is a need for reliable models that can simulate BBB leakage for initial drug efficacy assessments. In this study, we aimed to establish a standardized protocol for generating such a model, determining optimal cell seeding densities and quickly assess feasibility of potential treatments. Leveraging the versatility of the 96-Transwell system, we conducted experiments to identify a damage treatment protocol that induced a leakage while maintaining cell viability. Subsequently, we utilized this simulated BBB leakage model to evaluate the efficacy of FDA-approved tri-block poloxamer (P188) as a neuro-regenerative agent for restoring a damaged endothelial monolayer. Our laboratories have previously reported the reparative effects of P188 in restoring tight junctions induced by a traumatic injury. To extend such efforts to assess the therapeutic potential of P188, we developed a brain vasculature damage model that rapidly screens therapeutic potential of the tri-block P188 in mitigating BBB leakage.

Materials and

Methods: Mouse Primary Brain Microvascular Endothelial Cells (BECs) were cultured to create a monolayer. BECs were seeded on the Corning HTS Transwell 96-well with 5 um pores to mimic the blood brain endothelium with apical and basal chambers separated by the monolayer. An optimized seeding density was determined to ensure healthy undamaged cells would minimize the flux of 70 kDa Dextran from apical to basal chamber. In all experiments, the seeding density was 9.8 x104 cells/cm2. Cell damage was induced by applying 0.01% Triton-X100 for 5 minutes at room temperature. Triton was then removed and replaced with treatment (i.e., P188) or complete media for 24 hrs. A solution containing 4 uM 70 kDa Dextran was added to the apical chamber, and the flux to the basal chamber was monitored by measuring the Dextran concentration.

Results, Conclusions, and Discussions: To ensure that a functioning endothelium was formed, the tight junction marker (ZO-1) was immunostained in control and Triton-damaged cells (Fig. 1a and 1b). As expected, the control cells demonstrated a continuous tight junction formation, whereas in the damaged cells, it showed disruption (indicated by arrows) and likely mimics a leaky endothelium. The disrupted endothelium appears to have been restored by a P188 (100 uM) treatment (Fig. 1c and 1d). The ZO-1 expression was further quantified by determining the fluorescence intensity per cell (Fig. 2). In comparison to the ZO-1 expression in the Triton-damaged cells, P188 re-established its expression by either prior or post-damage treatment, suggesting structural restoration of tight junction. Functional reparative effects of P188 were next examined by tracking the flux of Dextran to the basal chamber. In undamaged control cells, the Dextran had a basal level of transport through the endothelium (0.4 uM in the lower chamber), indicating a healthy endothelium does not allow a significant diffusion of high molecular weight molecules. Expectedly, a 0.01% Triton-X100 injury damaged the endothelium and created a leaky monolayer, allowing Dextran to diffuse to the basal chamber (0.65 uM). Interestingly, treatment of the endothelium by P188 (100 uM) prior to damage prevented the high rate of diffusion and reduced the flux of the Dextran to the same level that was observed in the control experiment. Surprisingly, treatment of the endothelium with P188 post damage restored the integrity of the endothelium and further reduced the flux of Dextran to the basal chamber ( < 0.2 uM) (Fig. 3).

In summary, by leveraging the 96-Transwell system, we have successfully established a reliable and reproducible model to study transport across the brain endothelium and demonstrated the efficacy of P188 in restoring a damaged endothelium structurally and functionally. These findings not only advance our understanding of the BBB permeability dynamics but also might lead to the development of novel therapeutic strategies aimed at addressing neurodegenerative diseases and disorders that are characterized by compromised endothelium. The model is expected to broaden the scope of investigation beyond neural engineering to exploration of compromised vasculature in other diseases.

Acknowledgements (Optional): Authors acknowledge support from the Office of Naval Research (N00014-21-1-2039, MC) and the Cancer Prevention and Research Institute of Texas grant (RR220015 to JL).