(T0930-04-25) ISMICAP: A Model for Delving into Blood-Brain Barrier Mysteries

Purpose: The blood-brain barrier (BBB) is a dynamic anatomical and metabolic interface regulating the traffic to and from the brain. The endothelial cells lining the cerebral microvasculature have a complex network of transporters and they are interconnected by tight junctional proteins, restricting the paracellular diffusion of most molecules. As most neurotherapeutics are unable to reach the brain, targeting strategies have been developed to overcome the BBB. Hundreds of delivery vectors appear promising in preclinical studies, yet poorly perform clinically. Culture-based models fail to replicate many native BBB properties that result from the interaction of endothelial cells with other cells. On the other hand, the in vivo approaches maintain the complexity of the BBB but it is often hard to define how and where the systemically applied vehicles enter the brain parenchyma, especially in the presence of altered perfusion or peripheral metabolism. Many open questions remain unanswered such as knowing whether BBB properties fluctuate across different brain regions, delivery vehicles behave differently while crossing the embryonic BBB, and how the activity of transporters changes in pathological states.

Methods: We developed a BBB model combining in situ microperfusion of capillaries (ISMICAP) in acute brain slices with high-resolution multiphoton microscopic imaging. ISMICAP allows to quantitively assess BBB permeability to molecules at cellular and subcellular levels in real-time. In our set-up, the vasculature is perfused with test molecules under controlled pressure, while the entire slice is submerged in an oxygenated buffer simulating the extracellular brain milieu. After proper validation, we used ISMICAP to assess the integrity of tight junctions as well as the activity of two ABC transporters (ABCB1 and ABCC1). In addition, hyperosmotic mannitol perfusion, an established BBB temporary opening technique that is used clinically, was tested with the presented model.

Results: ISMICAP overcomes many limitations of previous models and offers additional advantages. The preparation of acute brain slices for electrophysiological recordings is a long-established technique, where neurons remain viable for hours. Our results demonstrate that the viability of neurons, their electrophysiological properties and the functionality of ABC-transporters are preserved. Three substrates for ABC transporters (Calcein-AM, rhodamine and Hoechst) were found to be potently extruded from the endothelial cells throughout the entire experimental window over a period of at least 60 min. The substrates became detectable only after blocking ABC transporters. As ABC transporters are ATP-dependent, the endothelial cells in the ISMICAP set-up can essentially generate ATP for extended periods of time. Therefore, ISMICAP can significantly reduce the risk of underestimating efflux phenomena in drug discovery research. In addition, perfusing mannitol solution into the vasculature along with fluorescent bovine serum albumin resulted in their extravasation and accumulation in perivascular macrophages.

Conclusion: Asssessing native BBB properties with our newly developed ISMICAP model confirms its translational values. Our set-up delivers versatility and universality as the slices can be prepared from almost any brain region, at any developmental stage, from animal models or human tissue samples, and they can be used for up to 8 hours. However, a proper choice of the BBB model to suit the target read-out is the bottle neck to ultimately result in better chances of moving drug delivery to the brain ahead in clinical settings.