Biomedical Engineering Reference
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macrophages, and astrocyte endfeet (Gloor et al., 2001; Ballabh et al., 2004).
Complex tight junctions (TJs) between endothelial cells act as a barrier to the passage
of macromolecules (Engelhardt, 2003). It is known that some small molecules
can cross the BBB by passive diffusion while some are dependent on transport
systems that cross the BBB via carrier-mediated transport, receptor-mediated trans-
cytosis, or adsorption-mediated transcytosis (Greig et al., 1988; Bobo et al., 1994;
Pardridge, 2003). Three groups of transporters have been characterized in mammals:
(1) active efflux transporter (AET), which includes Pgp, a major efflux transporter,
(2) carrier-mediated transporter (CMT), and (3) receptor-mediated transporter
(RMT). These BBB transporters are localized to the luminal endothelial membrane
(on the blood side of the capillaries) and abluminal endothelial membrane (on the
brain side of the capillaries) and regulate drug influx and efflux. Moreover, proteins
and polypeptides, which represent a promising category of potential drugs for treating
various CNS disorders, cannot cross the BBB. It is widely believed that in order
to cross the BBB, a small molecule must have the following characteristics:
(1) molecular weight (MW)
400 Da, (2) lipid soluble, and (3) not a substrate for
a BBB active efflux transporter. Conventional methods for assessing compound BBB
permeability include both in silico , cell-free and cell culture system and mammalian
models. Thesemethods often involve dissection, sectioning, and histological analyses
that can be less reliable and less efficient.
In zebrafish, initial neurulation starts in the late stage of gastrulation (9hpf) and by
30hpf, four separate brain ventricles have formed and are visible throughout develop-
ment (Wilson et al., 2002), providing excellent accessibility for assessing drug perme-
ation and molecular interactions in vivo . BBB is present in all vertebrates, including
zebrafish (Cserr and Bundgaard, 1984), and during development, zebrafish exhibit a
complex angiogenic network in the brain (Lawson and Weinstein, 2002). An extensive
capillary network that exhibits angiogenic vessels has developed by 3dpf and continues
remodeling throughout development (Lawson and Weinstein, 2002). Zebrafish exhibit
comparable drug metabolism as mammals (Langheinrich et al., 2003; Parng, 2005;
Parng et al., 2006). To assess BBB formation during embryogenesis, development of
tight junctions in day 3 zebrafish was recently confirmed using ZO-1 antibody staining
(Panizzi et al., 2007;McGrath et al., 2010). Evans blue dye permeation at different stages
of development was also examined and blockage of dye diffusion from the vessels to the
brain in day 3 animals was detected, indicating that BBB functions are present in 3dpf
zebrafish (Fig. 10.6). Because the brain structure is transparent in zebrafish during early
development (days 0-22), it is possible to study drug permeation and the molecular
machinery underlying BBB formation and disruption. Drug or dye trafficking between
the blood and the brain can be easily assessed without complicated histology. Although
the BBB may not fully mature until a later stage, a functioning BBB in the transparent
embryo brain can still serve as an excellent model for studying drug permeation and
efficacy of CNS drugs. As additional data supporting the utility of zebrafish for screening
drugs that permeate the BBB, we screened 14well-characterized neuroprotectants using
a drug-induced zebrafish brain-specific apoptosis model. In this study, 11 of the 11
compounds that showed protection in other animal models caused significant protection
in zebrafish (Parng, 2005; Parng et al., 2006). Furthermore, three BBB impermeable
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