DEVELOPMENT OF A HUMAN IN VITRO BLOOD-NERVE BARRIER MODEL
EROBOGHENE E. UBOGU, M.D., NEJLA YOSEF, M.S., ROBIN H. XIA, M.D., Ph.D.
NEUROMUSCULAR IMMUNOPATHOLOGY RESEARCH LABORATORY, DEPARTMENT OF NEUROLOGY, BAYLOR COLLEGE OF MEDICINE,
HOUSTON, TEXAS, UNITED STATES OF AMERICA.
ABSTRACT
Primary Human Endoneurial Endothelial Cells (pHEndECs)
Cellular Adhesion Molecules
Polymerase Chain Reaction
Phenotypic and functional differences exist
between vascular endothelium from different
tissues and between microvascular and
macrovascular endothelial cells from the same
tissue. Little is known about cellular interactions
at the blood-nerve barrier (BNB).
These features are consistent with the BNB. This
model provides an avenue to study mechanisms
of leukocyte entry into peripheral nerves.
p-gp
Phenotypic Characterization of pHEndECs
VCAM-1
FN CS-1
UEA-1
ICAM-1
FN CS-1
VCAM-1
GAPDH
vWF
PBMC
P3
P9
P10
Primary HEndEC
Transendothelial Electrical Resistance
180
170
DiI-Ac-LDL
160
150
ALP
140
*
130
120
110
TRANSMISSION ELECTRON MICROSCOPY SHOWING INTERCELLULAR
TIGHT JUNCTIONS AND PINOCYTIC VESICLES IN VITRO.
100
90
γ-GT
80
0
2
4
6
8
10
12
14
16
18
20
Time (Days)
0.1
18
0.09
16
0.08
P e rm e a b ility C o e ffic ie n t (c m /m in )
GLUT-1
20
P e r c e n t a g e o f In p u t
14
12
10
8
6
4
p-gp
2
Drug transport studies
Toxin/ xenobiotic influx/ efflux studies
0.07
0.06
Mechanisms of leukocyte transmigration
0.05
0.04
Microbial invasion processes
0.03
0.02
Chemokine translocation/ presentation mechanisms
0.01
0
POTENTIAL APPLICATIONS OF THE IVBNB
Endogenous solute/ macromolecular transport
studies
Permeability Coefficient: Dextran-70-FITC
Solute Permeability: Dextran-70-FITC
0
2
Human Sciatic Nerve and Endoneurial bundles
vWF
2
pHEndECs are spindle shaped, contact inhibited,
differentiate to form capillary-like tubes, are Ulex
Europaeus Agglutinin 1 and von Willebrand
Factor positive and endocytose acetylated low
density lipoprotein. They also express alkaline
phosphatase, γ-glutamyl transpeptidase, glucose
transporter-1, p-glycoprotein and low levels of
cellular adhesion molecules. Culturing pHEndECs
on collagen coated transwell inserts was used to
develop the IVBNB. High transendothelial
electrical resistances (~160 Ω.cm2; maximal 12
days after seeding) and low solute permeability to
fluoresceinated high molecular weight (70 kDa)
dextran (~0.7%) were seen. Electron microscopy
demonstrated intercellular tight junctions and 50100 nm-diameter pinocytic vesicles.
γ-GT
GLUT-1
TEER (Ohms.cm )
An in vitro BNB (IVBNB) model was developed
using primary human endoneurial endothelial
cells (pHEndECs) freshly isolated and purified
from decedent sciatic nerves via endoneurial
stripping, connective tissue enzymatic digestion
and density centrifugation.
ALP
ICAM-1
3
4
5
6
7
8
Days
RTC-coated inserts= 35-40%
9
10
11
2
3
4
5
6
7
8
9
Days
RTC-coated inserts= 0.14-0.16 cm/min
10
11
Funded by the Baylor College of Medicine New Investigator Start-Up
Program and the Guillain-Barré Syndrome/ Chronic Inflammatory
Demyelinating Polyradiculoneuropathy Foundation International.