In vivo Blood-brain Barrier Permeability Assays Using Clostridium perfringens Epsilon Toxin

Background

To maintain the intricate homeostasis required for proper brain function, the central nervous system (CNS) has developed a selective barrier that stringently regulates movement of blood-borne material into the brain. This is called the blood-brain barrier (BBB). This barrier is generated by highly specialized brain endothelial cells (BEC) whose unique properties are influenced by the close contact of CNS pericytes and astrocytes (Balabanov and Dore-Duffy, 1998; Engelhardt, 2003; Abbott et al., 2006; Abbott and Friedman, 2012; Alvarez et al., 2013). This increased barrier activity is achieved using two main mechanisms in BEC. The first is reduced paracellular permeability via formation of tight junctions at cell-to-cell contacts and the second is decreased transcellular permeability through reduced pinocytic activity (Gloor et al., 2001; Wolburg and Lippoldt, 2002; Preston et al., 2014; Tietz and Engelhardt 2015; De Bock et al., 2016). An increase in BBB permeability can have devastating neuropathological outcomes and can result in death. Because of the destructive consequences that BBB permeability can have on normal brain function, it is important to establish methods that reliably measure BBB permeability to help identify both the causes of and treatments for BBB dysfunction.

To measure BBB permeability in vivo in mice, animals are typically injected intravenously (IV) with a tracer or dye normally excluded from the brain by an intact BBB. The most commonly used tracer is Evans Blue (Saunders et al., 2015). However, it has been argued that Evans Blue has significant limitations as a tracer for BBB permeability assays and has been extensively reviewed in Saunders et al. (2015). Because Evan Blue binds to plasma albumin, extraversion of Evans Blue into the CNS is reasoned to be a measurement of albumin permeability. However, it has been demonstrated that Evans Blue binds to several other plasma proteins and may even be present as a free dye, suggesting that Evans Blue is an unsuitable tracer for BBB permeability. To help reduce the amount of uncertainty of accessing BBB permeability with Evan’s Blue, we sought to identify tracers that were better characterized and would therefore allow us to better define the specific aspects involved in BBB dysfunction. For our purposes, we wished to identify the size of molecules a damaged BBB had become permeable to, indicating if certain blood proteins could cross the BBB. Extravasation of certain blood-borne proteins such as complement, fibrinogen, and immunoglobulins may have lasting pathological effects on the CNS even after a permeable BBB has been repaired. Based on these criteria, we selected a panel of easily detectable tracers including exogenous 376Da fluorescein salt (FITC-Na), 66.5 kDa Alexa fluor 594 conjugated bovine serum albumin (BSA-594), and 70 kDa fluorescein isothiocyanate conjugated dextran (FITC-dextran); and the endogenous ~155 kDa tracer, mouse IgG.

In this paper, we outline procedures on how to use FITC-Na, BSA-594, -FITC-dextran, and endogenous IgG as tracers for BBB permeability and their acceptable downstream applications. In addition, we also describe a method to induce BBB permeability in mice using Clostridium perfringens epsilon toxin (ETX) (Linden et al., 2019). ETX-induced BBB permeability opens the BBB to all the tracers described here, specifically through caveolae dependent-transcytosis using both receptor-mediated and fluid-phase mechanisms (Linden et al., 2019). This panel allows us to access permeability to substances with varied molecular sizes and well as different molecular characteristics. FITC-Na can be used as a measure of solute and ion permeability, while BSA, 70 kDa dextran, and IgG can be used as measures of protein permeability (Nag, 2003). Influx of specific markers may also help identify what paracellular or transcellular mechanisms are involved in BBB dysfunction. For example, albumin transport is mediated via caveolae dependent-transcytosis (Schnitzer et al., 1994; Schubert et al., 2001; Frank et al., 2003). However, specific mechanisms of BBB permeability need to be confirmed using additional experimental methods. We strongly suggest ultrastructure examination via electron microscopy to determine if permeability is a result of increased paracellular permeability via tight junction dysfunction or increased transcellular permeability because of elevated endocytic activity. We also describe the best methods to use to evaluate extravasation for each tracer, as some techniques are not compatible with certain tracers, and can result in significant reduction of signal (Buxton, 1978; Hoffmann et al., 2011, Saunders et al., 2015). Finally, we also describe some of the advantages and disadvantages for each of the methods and tracer combinations (Figure 1). These methods may be used to measure BBB permeability in numerous disease models, transgenic mouse models, and CNS drug delivery evaluations.