Contributions of the glycocalyx, endothelium, and extravascular compartment to the blood–brain barrier

Abstract

The endothelial cells that form the blood–brain barrier (BBB) are coated with glycocalyx, on the luminal side, and with the basement membrane and astrocyte endfeet, on the abluminal side. However, it is unclear how exactly the glycocalyx and extravascular structures contribute to BBB properties. We used two-photon microscopy in anesthetized mice to record passive transport of four different-sized molecules—sodium fluorescein (376 Da), Alexa Fluor (643 Da), 40-kDa dextran, and 150-kDa dextran—from blood to brain, at the level of single cortical capillaries. Both fluorescein and Alexa penetrated nearly the entire glycocalyx volume, but the dextrans penetrated less than 60% of the volume. This suggested that the glycocalyx was a barrier for large but not small molecules. The estimated permeability of the endothelium was the same for fluorescein and Alexa but several-fold lower for the larger dextrans. In the extravascular compartment, co-localized with astrocyte endfeet, diffusion coefficients of the dyes were an order of magnitude lower than in the brain parenchyma. This suggested that the astrocyte endfeet and basement membrane also contributed to BBB properties. In conclusion, the passive transport of small and large hydrophilic molecules through the BBB was determined by three separate barriers: the glycocalyx, the endothelium, and the extravascular compartment. All three barriers must be taken into account in drug delivery studies and when considering BBB dysfunction in disease states.

Antibody transcytosis across brain endothelial-like cells occurs nonspecifically and independent of FcRn

“…Because of the comparable permeability of antibodies (150 kDa), 155-kDa dextran, transferrin (80 kDa) at saturation, and Fab (~50 kDa) and Fc (~32 kDa) IgG fragments (Table 1), we propose that this nominal transcytosis rate likely originates from nonspecific fluid-phase endocytosis because it is constitutive and concentration-independent. Although other studies using dextrans of varying MW have presented evidence suggesting BBB permeability is size-dependent 24,32 , our observations for the sdAb (14 kDa) and 10-kDa dextran suggest charge is a more important determinant of BEC permeability. Dextrans derived from different bacterial sources or with varying chemical modifications may vary in charge and require thorough characterizations before interpreting BEC permeability given the importance of charge on BEC transcytosis 7 .…”

“…However, previous findings have demonstrated the potential for size-dependent differences in BBB permeability in vivo 24,32 . To investigate whether nonspecific transcytosis of macromolecules across iBECs is size-dependent, we assessed the permeability of 10-kDa dextran because it is above the size limit for paracellular transport at the BBB in vivo 33 and would therefore be transported only by transcytosis.…”

The blood–brain barrier in health and disease: Important unanswered questions

“…This dense network of luminal glycoproteins prevents larger molecules from interacting with the EC. While small dyes such as fluorescein (376 daltons) and Alexa Fluor (643 daltons) permeate the glycocalyx, dextrans (40–150 kD) penetrate <60% of its volume (Kutuzov et al, 2018). In disease, glycocalyx degradation is associated with more severe BBB leakage in models of multiple sclerosis (MS) and cardiac arrest (DellaValle et al, 2018; Zhu et al, 2018).…”

Glycocalyx-Mediated Vascular Dissemination of Circulating Tumor Cells

“…33 Perfusion of tumor cells in the MVNs can be used to quantify TC extravasation events 30 . Using MVNs, 34 we dissect the process of TCs dissemination into arrest, adhesion, and trans-endothelial migration, 35 and find that the GCX plays unexpected key roles in all of these processes ultimately leading to 36 metastasis. 37 38 2.…”

“…Indeed, this increase in 51 permeability was not likely due to disrupted endothelial cell-cell junctions since ZO-1 localization 52 revealed no visible changes in tight junction morphology, and treatment with the pro-inflammatory 53 factor TNF-α, known to disrupt both the EC GCX and EC junctions 33,34 , produced a much larger 6.9- 54 fold increase in permeability (not shown). Thus, it is likely that the increase in permeability observed 55 stemmed from increased diffusion of dextran across the degraded GCX layer between ECs through 56 depletion of its solid, charged fraction, thereby increasing the GCX inter-fiber spacing and mean free 57 path of the molecules 35,36 . Consistent with this mechanism, CS exhibited a much lower expression on 58 the endothelium, and so its removal produced no measurable change in MVN permeability ( Fig.…”