Research reportPermeability of the blood–brain barrier to neurotrophins
Introduction
Neurotrophins are secretory proteins that can affect cell survival and activity in the CNS 13, 15. For instance, neurotrophins can block neuronal cell death in lesioned animals and increase the expression of cholinergic and dopaminergic neuronal phenotypes, thereby indicating a possible therapeutic potential in degenerative diseases [15]. The family includes NGF, BDNF, NT3, NT4/5, and NT6. Neurotrophins can be produced within the CNS with regional differences in distribution [11]and can enter the CNS by retrograde axonal transport [7].
The mammalian CNS is known to have poor regenerative capacity after insults such as anoxia, ischemia, or trauma because of inhibitory factors and insufficient neurotrophic support. In recent years, there have been several studies indicating that locally applied neurotrophins can enhance survival and regrowth of the damaged CNS, including delivery through retroviral vectors, application of gel foams, and cellulose fibers soaked with neurotrophins 6, 27, 28.
Peripheral administration of neurotrophins also has been explored, but penetration into the CNS is limited by enzymatic degradation and by the BBB 19, 21, 22. There are differences in previous reports about the extent to which these problems limit CNS penetration. Most investigators have assumed that parental delivery of neurotrophins is an impractical route to exert effects in the degenerating CNS. More recently, however, the ability of the blood-borne neurotrophin NGF [8]and its subunit βNGF [14]to enter the CNS was demonstrated by autoradiography. One route for the entry of blood-borne neurotrophins into the CNS could be by direct permeation through the BBB at the brain and spinal cord [22].
The BBB has low permeability to large proteins and many other hydrophilic molecules, but still shows a selective permeability to many such substances [2]. Regional differences in the permeability of the BBB could be an important issue in the delivery of neurotrophins from the periphery, because individual neurotrophins have various effects on selected neuronal populations 5, 12.
In this report, we compared the rate of penetration and volume of distribution in different regions of the brain and spinal cord for NGF, NT3, and NT5 and the differential entry of 7S NGF vs. βNGF. We also examined neurotrophin integrity, capillary association, and saturability of transport after intravenous administration.
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Radiolabeling and purification
Human βNGF [27 kDa dimer, 120 amino acid residues, recombinant and expressed in the NSO cell line (R&D System, Minneapolis, MN] was reconstituted in 0.25 M PBS (pH 7.4) at a concentration of 0.5 μg/μl. Aliquots were stored for up to 3 months at −70°C until use. Natural human NT3 (0.25 μg/μl) and NT5 (0.43 μg/μl), kindly supplied by Genentech (San Francisco, CA), were stored in 10 mM acetate. Natural human NGF (7s) from Genentech was stored at 1.4 μg/μl in 20 mM succinate. The enzymobead method
Lipid solubility determined by octanol/PBS partition coefficients
Table 1 shows the means and standard errors of octanol/PBS partition coefficients for the iodinated neurotrophins (n=3 for each compound). No significant difference was found among the compounds [F3,9=1.03, p>0.05] as analyzed by ANOVA. When the tissue/serum ratio of radioactivity was plotted against octanol/PBS partition coefficients, no correlation was present.
Acid precipitation for relative stability
The acid precipitability for iodinated neurotrophins in buffer was greater than 95% for each compound. Table 2 shows the percentage of
Discussion
In this study, we showed that iodinated neurotrophins are relatively stable in the blood and brain, interact with the cerebral vasculature, and have a variable permeability across the BBB. The passage of iodinated NGF and NT3 was inhibitable by unlabeled neurotrophins, indicating the presence of saturable transport systems.
The high influx rates of the neurotrophins suggest that entry cannot be easily explained by simple diffusion. Simple diffusion of peptides across the BBB usually correlates
Acknowledgements
Supported by VA Rehab. The authors thank Ms. Melita B. Fasold for editorial help.
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