Table 2.

Characteristics of blood-brain barrier markers described in this review.

Marker	Size		Binding1		Visualization			Quantification3,4	Toxic5	Renal clearance6	Cost
	Da	Radius nm	Protein	Tissue2	Macro	LM	EM
Biotin ethylenediamine	286a	NR	NR	No	No	Yes	Yes	Qualitative only	NR	Yes	Med
Radio-sucrose	342.3b	0.51i	No	No	No	No	No	Accurate	No	Yes	High
Na fluorescein	376c	NR	Weak	NR	No	Yes	No	Unreliable	No	Yes	Low
Evans blue	960d	NR	Yes	Yes	Yes	Yes	No	Unreliable	Yes	No*	Low
Trypan blue	961e	NR	No	Yes	Yes	Yes	No	Unreliable	Yes	No*	Low
Radio-inulin	≈7000b	1.3i	No	No	No	No	No	Accurate	No	Yes	High
Horseradish peroxidase	≈44,000f	3.0j	NR	NR	No	Yes	Yes	Unreliable	Yes	No	Low
Albumin (unlabeled)	69,000g	3.5i	No	No	No	IHC	Yes	Unreliable	No	No	Low
Radio-albumin	69,000g	3.5i	No	No	No	IHC	No	Accurate	No	No	High
IgG	≈155,000g	5.3i	No	No	No	IHC	No	Qualitative only	No	No	Low
Fibrinogen	340,000g	11.0k	No	No	No	IHC	No	Qualitative only	No	No	Low
Dextrans	1500 to 2,000,000h	0.8–38.2l	NR	NR	No	Yes	Yes	Qualitative in tissue	No	Only low MW	High

MW, molecular weight; Macro, visible to unaided eye; LM, light microscopy; EM, electron microscopy; NR, not reported; IHC, immunohistochemistry; Med, medium cost;

^*unless protein binding capacity in plasma exceeded.

^aEk et al. (2006),

^bWeast (1986),

^cMalmgren and Olsson (1980),

^dAllen and Orahovats (1950),

^eT1826, isomer of Evans blue, Allen and Orahovats (1950),

^fwww.sigmaaldrich.com,

^gThompson (2005),

^hhttp://www.thermofisher.com,

ⁱDziegielewska et al. (1979),

^jRennke et al. (1978),

^kBoyd et al. (1969),

^lArmstrong et al. (2004), Grznárová et al. (2005).

¹Dyes are generally problematic because they bind to proteins in plasma and to tissues in a reversible equilibrium, so when visualized it is unclear whether they are free dye or bound dye and how they became located at a particular site.

²Many markers may be taken up by a variety of cells, including choroid plexus epithelial cells. Also neurons and glia, but only if the blood-brain barrier is breached.

³With currently available methods only markers with radiolabels (e.g., ³H, ¹⁴C, ¹²⁵I) can be reliably quantitated. For reliable measurements this requires checking on the stability of the label and absence of contaminating labeled products (Evans et al., 1974). For liquid scintillation counting of ³H and ¹⁴C particular care is required to allow for differential quenching from variations in protein content of samples; internal quench correction is usually inadequate. Colorimetric and spectrophotometric methods are generally inaccurate because of spectral shifts produced by different composition of brain tissue and standards.

⁴Many groups attempt to deal with the problem of blood contamination by perfusing the cerebral circulation with “physiological” solutions at the termination of the experiment; this is probably variably effective, particularly in the case of any local disturbance to cerebral circulation e.g., brain trauma, as local intravascular coagulation will limit entry of both marker and of washout fluid.

⁵At concentrations used in marker experiments.

⁶Depends on molecular diameter, charge and protein binding. Dyes that bind to proteins in plasma would not be expected to appear in urine unless the concentration in plasma exceeded the binding capacity of the proteins. Neutral substances with effective molecular diameters of < 4 nm are freely filtered. Above 8 nm filtration is negligible. Between these values the amount filtered depends on molecular diameter and charge (Barrett et al., 2012).