Department of Pathology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo, 060, Japan
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ABSTRACT |
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It is well known that
the blood-brain barrier (BBB) matures at ~2 wk after birth in the
rat. Recently, we showed that glial cell line-derived neurotrophic
factor (GDNF) enhances the barrier function of porcine endothelial
cells forming the BBB in culture. In the present study, we examined the
relation between permeability of the BBB, using Evans blue as a tracer,
and expression of the GDNF family receptor (GFR-1) during postnatal
development of the BBB. Morphometric analysis showed that exudation of
Evans blue from capillaries of the cerebral cortex progressively
decreased until postnatal day 21. Inversely,
immunohistochemical examinations showed expression of GFR
-1 in the
capillaries at postnatal day 3 and expression that reached
the same levels as observed in adult rats by postnatal day
10. However, c-ret, which is thought to mediate a
signal evoked by binding of GDNF to GFR
-1, was not expressed in the
capillaries of the brain cortex in 3-mo-old rats. On the other hand,
the tight junction proteins occludin and ZO-1 appeared to be fully
expressed at birth. The reciprocal relation between GFR
-1 expression
and the permeability of the BBB strongly suggests active participation
of GDNF in postnatal development of the BBB, although the mechanism(s)
involved is still veiled.
blood-brain barrier; maturation; glial cell line-derived
neurotrophic factor; glial cell line-derived neurotrophic factor family
receptor -1; Evans blue
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INTRODUCTION |
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THE CAPILLARY ENDOTHELIUM in the brain forms a highly impermeable structure between the blood and the central nervous system (CNS) brain interstitium, called the blood-brain barrier (BBB), and plays an essential role in maintaining homeostasis of the CNS. In the BBB, highly impermeable tight junctions between endothelial cells are the most important cellular apparatus for paracellular barrier function as well as limited transcytosis of endothelial cells (23). It is well known that the BBB of the rat fully matures in postnatal weeks 3-4 (2, 3) and that exudation of tracers from the BBB ceases at ~2 wk after birth (18, 32). However, none of the molecular mechanisms of development of the BBB have yet been fully clarified.
Several proteins associated with tight junctions have been disclosed (5). Of these proteins, occludin and the claudins are considered to be essential for tight junctions because they are integral membrane proteins comprising tight junction strands (29). Occludin is known to be much more highly expressed in brain endothelial cells than in endothelial cells of nonneural tissue (12), whereas the expression of claudins in various tissues and cell types has not been fully clarified (19). To date, the roles of these proteins in regulation of tight junctions are mostly unknown.
In the brain, astrocytes have been suggested to contribute to
development of the BBB because the cells have vascular feet ensheathing
the brain capillaries. In addition to this anatomic observation, it has
been reported that astrocytes presumably secrete unknown factors
differentiating capillaries to the BBB-type capillary, in terms of
forming impermeable tight junctions (1, 6,
15, 20, 23,
26). In this context, it was reported that
interleukin-6 secreted by astrocytes contributes to induction of BBB
properties such as activities of alkaline phosphatase and the
Na+-K+-Cl cotransporter
(24, 25).
The rat glial cell line B49 secretes glial cell-derived neurotrophic
factor (GDNF) to maintain dopaminergic (17) and motor neurons (11) in vivo. GDNF is a member of the transforming
growth factor- family and binds to receptor GFR
-1 on the cell
surface of neurons. GFR
-1 then interacts with c-ret, a
receptor kinase (7, 16, 27,
28). Recently, we demonstrated that GFR
-1 was expressed
in endothelial cells of the cerebral cortex in adult rats and that GDNF
activated the barrier function of endothelial cells isolated from the
porcine brain in vitro (13), suggesting that GDNF is a
potent differentiating factor of the BBB. Thus in the present study we
examined the expression of GFR
-1 and tight junction-associated
proteins during postnatal maturation of the BBB in the rat as compared
with leakage of Evans blue from the BBB as a marker of permeability. We
show here a reciprocal relationship between leakage of Evans blue and
expression of GFR
-1, strongly suggesting that GDNF is a potent
differentiating factor for the BBB.
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MATERIALS AND METHODS |
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Animals. F344/Jcl rats were used on postnatal days 1, 3, 5, 7, 10, and 14 and as adults (postnatal month 6) for immunohistochemical detection. Moreover, postnatal day 4 (n = 3), 7 (n = 3), 10 (n = 3), 14 (n = 3), and 21 (n = 3) rats as well as adult rats (n = 3, postnatal month 3) were used for evaluation of Evans blue leakage. Three 4-wk-old Crj:CD-1 (ICR) mice were used for preparation of total RNAs from the brain, spinal cord, and thymus, because the complete sequence of c-ret is only available for the mouse and human.
Evaluation of Evans blue leakage. Evans blue (4% wt/vol) was intraperitoneally injected into the rats, and systematic distribution of the dye was confirmed by a change in skin color 2 h after injection. After decapitation, the brains were frozen in nitrogen, sectioned 5 µm thick, and mounted on glass slides. The extent of the autofluorescence of Evans blue in or over the capillaries was noted with a Nikon FX epifluorescence photomicroscope with the use of Photoshop (Adobe Photoshop 4.0J) under the same luminance conditions. Five micrographs were randomly taken from one rat, and then each area stained by Evans blue was measured by using Photoshop under the same luminance conditions. The area ratios of Evans blue leakage were determined by dividing the mean area of the postnatal rats by the mean area of adult rats. The mean ± SD of one area stained with Evans blue was calculated from the values of three rats.
RNA extraction and RT-PCR. RNA preparation and RT-PCR were performed according to standard protocols (21). Briefly, total RNA was isolated from the cerebral cortices and spinal cords of two male Crj:CD-1 (ICR) mice with the use of the single-step thiocyanate-phenol-chloroform extraction method as modified by Xie and Rothblum (30). RT-PCR was performed using an RT-PCR kit supplied by PerkinElmer (Branchburg, NJ) according to the manufacturer's recommendations. To confirm the finding that no c-ret was present in the cerebral cortex, total RNA was extracted from the various tissues of 3- to 4-wk-old mice followed by RT-PCR using 1 µg of the total RNA as a template. The primers for mouse c-ret (14) were 5'-GGATGCCCCTGGAGAAGTGCC-3' [nucleotides (nt) 171 to 191] and 5'-CATTCCTCACACTCGGGGCGC-3' (nt 1,598 to 1,578). PCR was performed for 40 cycles. Aliquots of PCR products (10 µl) were loaded on a 1% agarose gel containing ethidium bromide.
Immunohistochemistry.
About 1 mm3 of the brain cortex tissue was put
between glass slides. The slides were pressed against each other,
pulled apart, and immediately immersed in a cold mixture of acetone and
ethanol (1:1) for 5 min. This procedure is suitable for observation of brain tissues, particularly vessels, because the cell shape and tissue
are well preserved by avoiding freezing. Five 6-µm-thick frozen
sections of the brain cortex and spinal cord, as well as cultured cells
on polycarbonate filters, were also dipped in the mixture for 5 min.
After the slides were rinsed with phosphate-buffered saline (PBS), they
were incubated overnight with the primary antibody at 4°C. The
tissues were then incubated with appropriate secondary antibodies
(DAKO, Glostrup, Denmark) labeled with FITC for 1 h at room
temperature. Primary antibodies used included goat polyclonal anti-rat
GFR-1 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal
anti ZO-1 (Zymed, San Francisco, CA), rabbit polyclonal anti-c-ret (Santa Cruz Biotechnology), and rabbit polyclonal
anti-occludin (Zymed). Actin was visualized by using
rhodamine-phalloidin. All samples were examined with a Nikon FX
epifluorescence photomicroscope (Nikon, Tokyo, Japan) and/or a confocal
laser scanning fluorescence imaging system (MRC-500J, Bio-Rad).
Primary cultures of porcine brain capillary endothelial cells. Porcine brain capillary endothelial cells were purified as described previously (13). Briefly, cortical gray matter of brains obtained from miniature pigs weighing ~20 kg was minced with scissors into small pieces and digested in 0.25% dispase (Godo Shusei, Tokyo, Japan) and 0.12% collagenase (Yakult, Tokyo) in Ca2+-, Mg2+-free Hanks' balanced saline solution (HBSS) at 39°C for 60 min. During the enzyme digestion, the solution containing the tissues was bubbled with a mixture of 95% O2-5% CO2. After extensive pipetting, the capillaries were separated from the remaining slurry by centrifugation at 1,000 g for 15 min in PBS containing 25% bovine serum albumin. After several rinses by centrifugation, fragments of capillaries were seeded onto 12-well tissue culture plates coated with type IV collagen (Nitta Gelatin, Osaka, Japan) in Dulbecco's modified Eagle's medium 1:1 with Ham's F-12 nutrient (D/F12) mixture (Kyokuto), supplemented with 15% heat-inactivated fetal bovine serum (Moregate), 75 µg/ml endothelial growth supplement (Sigma, St. Louis, MO), 80 µg/ml heparin (Sigma), 5 µg/ml insulin (Collaborative Biomedical, Bedford, MA), 5 µg/ml transferrin (Collaborative Biomedical), 5 ng/ml selenous acid (Collaborative Biomedical), 100 U/ml penicillin, and 0.1 mg/ml streptomycin. At day 1 after plating, 60 nM vincristin (Sigma) was added to the cultures to eliminate undesirable cells until the cell density reached subconfluence (4). The medium was renewed every other day. When the endothelial cells reached subconfluence, they were released by 0.25% trypsin-EDTA (GIBCO, Grand Island, NY) and seeded at 25, 000 cells/filter on 0.33-cm2 rat-tail collagen-coated polycarbonate Costar Transwell filters (0.4-µm pore size; Costar, Cambridge, MA) for transcellular electrical resistance (TER) measurements and immunocytochemical examinations. After 4 days of cultivation without vincristin after passage, the medium of the endothelial cells was changed to medium containing 0.1 ng GDNF/ml, 125 µM 8-(4-chlorophenylthio)-cAMP (CPT-cAMP; Sigma), and 17.5 µM phosphodiesterase inhibitor (PDE-I) RO20-1724 (RBI, Natick, MA). The cells were then cultured for 8 h.
Measurement of TER. The TER of the endothelial cells on the filters was measured using an Epithelial Voltohmmeter (World Precision Instruments, Sarasota, FL) equipped with STX-2 Ag-AgCl electrodes (Endohm, World Precision Instruments). The measurements of TER were performed at 37°C on a thermal plate (Fine, Tokyo, Japan). TER was expressed in standard units of ohm · cm2. For calculation of the resistance of endothelial cell monolayers, resistance of blank filters was subtracted from that of filters covered with cells. Each value was calculated from five or six cultures.
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RESULTS |
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Evaluation of Evans blue leakage.
First, we evaluated the function of the BBB by using leakage of Evans
blue from capillaries of the cerebral cortex. Extensive exudation of
Evans blue in the surrounding capillaries was observed widely at
postnatal day 4. With time, the leakage of this dye decreased (Fig. 1). Five micrographs
randomly taken under the same luminance conditions per rat were
analyzed to calculate the mean value of the Evans blue-stained area,
and then the mean value and SD were calculated by using three rats per
age group. Thus area ratios of Evans blue leakage (postnatal rats/adult
rats) were determined (Fig. 2). The
ratios at postnatal days 4, 7, and 14 were
~4.5, 2.3, and 1.9 times those of adults, respectively. At
postnatal day 21, the degree of dye leakage was not
significantly different from that observed in adult rats. The diameter
of the capillaries did not significantly change during the observation period (data not shown).
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Expression of GFR-1 and c-ret protein in the cerebral cortex
capillaries during postnatal development of the BBB.
We immunohistochemically investigated expression of GFR
-1 during
postnatal development of the BBB to clarify the relationship between
GDNF and the postnatal development. At birth, expression of GFR
-1
was not detected. GFR
-1 was initially expressed in the capillaries
at postnatal day 3. Its signals became stronger with time.
By postnatal day 10, GFR
-1 appeared to be fully expressed as detected in adult rats.
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Expression of tight junction-associated proteins in the
capillaries during postnatal development of the BBB.
Tight junctions are an important cellular apparatus for
regulating the paracellular pathway of the BBB. Thus, as an initial approach to investigate the involvement of tight junction-associated proteins in development of the BBB, two well-known proteins, occludin and ZO-1, were studied by using an immunohistochemical technique. It
was reported that ZO-1 is expressed at postnatal days 8 and 70, whereas occludin is expressed at postnatal day
70 but hardly detectable at postnatal day 8 (12). In the present experiments, even at birth, these
proteins appeared to be expressed at cell-cell contacts of endothelial
cells of the cerebral capillaries. The intensity of the
junctional staining of the proteins in the brain capillaries at birth
was comparable to that in adult rats (Fig. 6). Figure 6 is representative of 10 separate experiments for staining of ZO-1 and occludin.
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Effects of GDNF on occludin of cultured porcine endothelial cells
forming the BBB.
To examine the relationship between the expression of occludin and ZO-1
and the permeability of tight junctions, endothelial cells of the
porcine cerebral cortex were cultured and treated with GDNF or agents
elevating the intracellular cAMP level (CPT-cAMP and PDE-I) for 8 h. The results are summarized in Fig.
7A. The TER of the cells
treated with GDNF and the cAMP-elevating agents was significantly
higher than those of the cells treated with the agents alone. The TER
of the cells treated with GDNF alone, however, did not increase. These
results are consistent with our previous report that GDNF enhances
barrier function (13). Under these conditions, we
immunocytochemically examined the localization and staining intensity
of occludin, showing no marked alteration of occludin by GDNF or the
cAMP-elevating agents. On the other hand, the distribution of actin was
altered from a stress-fiber dominant to a circumferential pattern by
addition of the cAMP-elevating agents, whereas GDNF had no effect on
actin organization (Fig. 7B). Figure 7B is
representative of five separate experiments for staining of occludin
and actin.
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DISCUSSION |
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Many studies have shown that the BBB of immature animals is more
permeable than that of adults (2, 3,
18, 32). In newborn mice, the BBB is immature
and some amino acids penetrate more freely into the brain than in
adults (22). Xu et al. (31, 32)
have demonstrated the extravasation of tracers, such as rhodamine
isothiocyanate and ferritin, into the corpus callosum in early
postnatal rats but not in rats older than 13-14 days. In the
present experiment, we consistently observed extensive exudation of
Evans blue at postnatal day 4, and the BBB became much less
permeable against Evans blue by postnatal day 21, as in
adult rats. Expression of GFR-1 progressively increased from postnatal days 3-5 to postnatal day 14 with
the postnatal development of the BBB, strongly suggesting the
participation of GDNF in the postnatal maturation of the BBB.
We immunohistochemically detected expression of GFR-1 in the
capillaries of the cerebrum but not in the endothelial cells of lung,
tongue, hypophysis, and other tissues (data not shown). These findings
are consistent with results obtained by in situ hybridization showing
that GDNF and GFR
-1mRNAs are weakly detected in the cerebral cortex
(10, 33). Thus GFR
-1 was confirmed to be
expressed in the brain capillaries forming the BBB as well as in
neurons in the brain. Our previous study revealed that GDNF induced the
barrier function of endothelial cells isolated from the porcine brain.
The present finding of a reciprocal relation between GFR
-1
expression and leakage of Evans blue during postnatal development of
the BBB provide further support for a role of GDNF in development of
the BBB.
It has been reported that the actions of GDNF are mediated by a
multicomponent receptor complex composed of c-ret and
GFR-1 (7, 16, 27,
28). Immunohistochemically, however, we failed to detect
c-ret expression in capillaries of the cerebral cortex in
the present study. Our observation is consistent with a previous report
using an in situ hybridization technique in which no signal of
c-ret was detected in brain cortex (10,
33). Thus these observations clearly indicate that
c-ret is not always necessary for the signal transduction of
GDNF, particularly in the endothelial cells of the BBB.
In endothelial cells forming the BBB, the P-face-associated particles of tight junctions observed in freeze fractures are suggested to play an important role in regulating the paracellular pathway (34). Thus the elucidation of which integral membrane proteins are crucial for tight junction formation is of importance. Of the tight junction-associated proteins (5), the integral membrane proteins occludin and the claudins are capable of forming tight junction strands, although occludin forms much shorter strands than the claudins (9). Regarding occludin expression during postnatal development of the BBB, it has been reported that occludin is expressed in the brain capillaries on postnatal day 70 but not on postnatal day 8 (12). It has also been reported that occludin expression in endothelial cells forming the BBB is not altered by treatment with astrocyte-conditioned medium and cAMP, which significantly increase TER (12). This prompted us to examine the cellular distribution of occludin in the development of the BBB. In the present experiments, however, occludin was clearly detected at cell junction areas of endothelial cells in the brain cortex, even at birth. We also observed that the localization and staining intensity of occludin were not changed by treatment with cAMP and GDNF. Thus it is suggested that the presence of occludin alone cannot account for formation of tight junctions in endothelial cells forming the BBB.
The claudin family, a newly disclosed membrane protein family of tight junctions (19), has been suggested to be a prime candidate for tight junction strands (8, 9, 29). Of the proteins of this family, it is reported that claudin 5 is highly expressed in capillaries of the brain and lung (4, 19). The tissue distribution of occludin is well correlated to the distribution of tight junctions compared with that of the claudin family; however, the role of occludin in the functioning of tight junctions remains to be clarified.
In conclusion, we demonstrated that GFR-1 was detected in rat brain
capillaries and that its expression began on postnatal days
3-5 and eventually reached the level observed in adult rats with a progressive decrease in the leakage of Evans blue. Thus it is
strongly suggested that expression of GDNF is deeply involved in the
postnatal development of the BBB.
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ACKNOWLEDGEMENTS |
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We thank Kim Barrymore for help with the manuscript.
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FOOTNOTES |
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This study was partly supported by Grants-in-Aid from the Ministry of Education, Culture, Sports and Science and the Ministry of Welfare of Japan, as well as the Akiyama Foundation, the Naitou Foundation, and the Hokkaido Geriatrics Research Institute.
Present address of H. Utsumi: Toxicology Laboratories, Yoshitomi Pharmaceutical Industries, Ltd, 214-1, Yamazaki, Fukusaki-cho, Kanzaki-gun, 679-2200, Japan.
Address for reprint requests and other correspondence: N. Sawada, Dept. of Pathology, Sapporo Medical Univ. School of Medicine, South-1, West-17, Chuo-ku, Sapporo, 060, Japan (E-mail: sawadan{at}sapmed.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 13 July 1999; accepted in final form 18 February 2000.
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