Veterans Affairs Medical Center and Tulane University School of Medicine, New Orleans, Louisiana 70146
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ABSTRACT |
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Diffusion from brain regions lacking a
blood-brain barrier (BBB) and saturable transport across capillaries
are possible pathways for the entry of blood-borne interleukin-1
into the central nervous system (CNS). To assess the involvement of
these putative routes, mice received intravenous injections of
radioiodinated interleukin-1
, and their brains were subjected to
emulsion autoradiography. The resulting patterns of silver grain
distribution showed that diffusion of interleukin-1
from the choroid
plexus and the subfornical organ was greatly restricted. These
restrictive properties were quantified by the determination of
D1/2 values, the
distances needed for the concentration of silver grains to decrease by
one-half. Within several brain regions, a subset of the
microvasculature indicated transport of interleukin-1
across the
BBB. Individual microvessels showed different patterns of transport
ranging from robust to absent. The high degree of containment of
blood-borne interleukin-1
within the regions lacking a BBB indicates
that these sites cannot account for total delivery of the cytokine into
the brain and suggests instead that the microvascular network may serve
as the major route of entry into the CNS.
cytokine; transport; mouse; subfornical organ; blood-brain barrier; interleukin-1; autoradiography; circumventricular organ
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INTRODUCTION |
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NEUROIMMUNOLOGICAL communication between the brain and the body is mediated in part by numerous cytokines, signaling agents produced by both the immune and central nervous systems (CNS). Disease or infection can stimulate the release of several cytokines into the bloodstream. These blood-borne proteins may then penetrate the blood-brain barrier (BBB) to deliver information to the brain.
Our laboratory has characterized endogenous saturable transport systems
that carry several of these compounds, including interleukin-1 (IL-1
), interleukin-1
, interleukin-1 receptor antagonist, and tumor necrosis factor-
, across the murine BBB into the parenchymal space of the brain (2, 4, 11, 12). Analysis of the pharmacokinetic profile of IL-1
has shown a biphasic disappearance of the cytokine from the blood, with a half-time of 131 min for phase
1 and a half-time of 2.06 min for
phase 2 (4). HPLC analysis showed 100% of the radioactivity to be intact radioiodinated IL-1
in mouse
serum 30 min after intravenous administration (4), indicating that
intact IL-1
is available for transport across the BBB after injection into the blood.
Within the brain, transported IL-1 also displays a striking degree
of structural integrity. HPLC analysis in two separate studies has
demonstrated 125I-labeled IL-1
to be 100% intact in mouse brain tissue 10 min after intravenous
injection (1) and over 40% intact 30 min later (4). These observations
show that ample intact IL-1
is present in the CNS after transport
and available for interactions with central tissues.
For IL-1, transport of the cytokine into the posterior division of
the septum (PDS) in the mouse brain is particularly high, as previously
demonstrated by quantitative film autoradiography (14). Transport into
both whole brain and the PDS is saturable (4, 14). A possible site for
such regulated transport of cytokines into the CNS is the capillary
bed.
Another postulated route of entry for IL-1 and other cytokines into
the central environment, which may not be saturable, is through
circumventricular organs (CVOs) such as the subfornical organ (SFO).
Many CVOs have capillaries that lack a BBB. It has been proposed that
cytokines might leak from the CVOs to enter areas of the brain with a
BBB.
To investigate the possible involvement of the CVOs and the
microvasculature in the delivery of blood-borne cytokines to the CNS,
we applied emulsion autoradiography to demonstrate anatomical and
cellular localization of radioiodinated IL-1 within the brain after
intravenous administration. We measured the concentration of exposed
silver grains, which reflect transport into the CNS, in several
regions, including the SFO, choroid plexus (CP), ventricular space, and
other tissues both proximal and distal to the ventricular network. We
also analyzed silver grain concentrations at defined increments within
and around the SFO to determine whether the patterns of distribution
could implicate the CVO as a site for the entry into the CNS of
blood-borne IL-1
. Finally, we studied the transport of IL-1
at
individual capillaries to determine the possible involvement of the
microvascular bed in the delivery of IL-1
to the brain.
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METHODS |
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Animals and tissue preparation. Adult
male ICR mice (Charles River Laboratories, Wilmington, MA), 20-25
g, were used in this study. The mice were anesthetized with
intraperitoneal urethan (4.0 g/kg). The left and right jugular veins
were exposed. Mice received an injection into the left jugular vein of
~5 × 107 counts/min of
human recombinant IL-1, labeled with
125I, in a volume of 200 µl of
1% bovine serum albumin in lactated Ringer's (LR) solution.
The left and right jugular veins were severed 15 or 30 min after
injection, and the brains were washed free of their vascular contents
by intracardiac perfusion of 20 ml of cold LR into the left ventricle
of the heart while the thoracic aorta was occluded. Mice were then
quickly decapitated, and the blood-free brains were carefully removed.
The intact brains were immediately dipped in 30°C isopentane
for 12 s and moved to a bed of crushed dry ice for 10 min, being turned
once for even freezing. The brains then were wrapped in plastic and
stored at
70°C for 24 h.
Frozen brains were warmed to 15°C, coronally sliced at 20 µm, and mounted on gelatin-coated slides. The sections were
desiccated for 24 h at 4°C and defatted through a series of
alcohols and xylenes.
Autoradiographic image analysis. After drying, the slides were dipped in a solution of 50% NTB3 autoradiographic emulsion (Kodak) at 45°C. Once the emulsion was completely dry, the slides were placed in light-tight slide boxes that contained desiccant-filled capsules and stored at 4°C. After 48 wk, the emulsion was developed with D-19 developer (Kodak), and the individual cells were counterstained with the nuclear stain cresyl violet. The distribution of the developed silver grains was analyzed with an MCID image analysis system (Imaging Research, St. Catharines, Ontario, Canada).
Micrographic images of various regions of the brain with the overlying
silver grains were captured with the imaging system. A
400-µm2 sampling tool (20 µm × 20 µm) was used to take multiple measurements within each of
the areas examined to determine mean concentrations of grains. These
means were then used to statistically compare the degrees of
penetration of blood-borne IL-1 into different regions of the brain.
Another image sampling box of 400 µm2 (40 µm wide × 10 µm high) was used to more closely monitor the changes in the concentration of grains at distances away from the SFO. The sampling box was placed 80 µm from and parallel to a ventral border of the SFO within the dorsal thalamus. The number of exposed grains within the box was determined, the sampling box was moved toward the SFO by 10 µm, and the process was repeated. The sampling "ladder" extended 80 µm into the SFO (Fig. 1). Measurements were made at midline and at both lateral aspects of the SFO, and the mean distribution profiles were determined for each image analyzed. The same approach also was used to determine the distribution of silver grains at the SFO-ventral hippocampal commissure (VHC) interface.
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RESULTS |
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Labeling at CP and SFO. Analysis of the emulsion-coated tissue sections was performed under both light- and dark-field illumination. Two-way analysis of variance (ANOVA) showed no significant differences in grain concentrations between the 15- and 30-min circulation times. Given the previously observed slow rates of passive diffusion within the brain parenchyma (3, 15), the lack of statistical significance between the two circulation times was not unexpected. Therefore, the results were combined across time and analyzed by regions. Examination of these tissues revealed different concentrations of developed silver grains at various sites.
Within the cerebral ventricular system, numerous grains were associated with the CP (18.40 ± 0.84 grains/400 µm2). The dense grain pattern closely followed the folds of the CP. Much lower levels (1.86 ± 0.40) were detected in the cerebrospinal fluid (CSF) that bathed the CP.
A high degree of labeling also was observed at the SFO (14.6 ± 1.33). Increased concentrations of grains were detected within the CVO at the transitional, central, and caudal subregions. At the ventral interface of the SFO and the third ventricle/dorsal thalamus, the number of grains abruptly decreased (1.99 ± 0.50). Similarly, at the dorsal border of the SFO, another sharp decline in the concentration of developed grains was observed where the SFO edge contacted the VHC (4.03 ± 1.24). Within both of these peri-SFO tissues, grain counts were more comparable to those seen in the neocortex (1.49 ± 0.30), a region of the brain distant from both the CVOs and the ventricular system.
ANOVA applied to the silver grain counts obtained by computer-assisted
image analysis of the various regions revealed a statistically significant relationship (F5,6 = 84.97, P < 0.0001). The results of a
Newman-Keuls multiple comparison test identified differences among the
defined regions. The SFO had significantly more autoradiographic labeling than the cortex, thalamus, VHC, and the ventricles
(P < 0.001). The grain density
within the CP was significantly greater than in the cortex, thalamus,
VHC, ventricles (P < 0.001), and SFO
(P 0.05). All other posttest
comparisons were nonsignificant (P > 0.05).
Restricted diffusion of IL-1 from
ventral and dorsal SFO. Quantification of silver grain
distributions around the ventral and dorsal borders of the SFO with the
sampling ladder resulted in comparable diffusion profiles. Sigmoidal
distributions were seen at both sites when the grain counts were
plotted over distance (Fig. 2). In each
case, a basal level of grains was detected within the peri-SFO tissue,
and steadily increasing concentrations of grains that eventually
reached plateau values were observed within the CVO. The grain counts
in each region were converted to percentages of the upper plateau value
of the best-fit sigmoidal curve for each region.
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The percent values corresponding to the region of the SFO-tissue
interface were then plotted against their distribution relative to the
dorsal or ventral SFO interface on a semilog plot and subjected to
linear regression (Fig. 3). The values for
the slopes of the resulting lines were compared by statistical analysis
and revealed significant differences between the slopes
(F1,10 = 12.26, P = 0.0057). The slopes of the lines
then were used to determine the D1/2 values, the distances needed
for the number of silver grains to decrease by one-half, an approach
used to analyze images on autoradiographic films (15). The
D1/2 values were calculated for
both the dorsal and ventral SFO-tissue interfaces by multiplication of
the inverse log values of the slopes by 0.301. The
D1/2 value for the ventral surface
of the SFO was 22.72 µm, and the
D1/2 value for the dorsal surface
of the SFO was 37.93 µm.
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Labeling of microvasculature. In addition to labeling at the SFO and CP, heavy silver grain accumulations also were observed in association with microvascular components (Fig. 4). Appropriate lengths and branching morphologies were used to identify these structures as capillaries. Labeled microvessels were detected in multiple regions of the brain, including the septal forebrain, striatum, hypothalamus, and neocortex. The vessels displayed concentrations of silver grains directly on the chains of endothelial cells as well as laterally from the capillaries. However, a large degree of variation in grain density was observed along the lengths of the labeled capillaries and at their side branches (Fig. 5). Furthermore, other microvessels were observed lacking any radioactive labeling.
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DISCUSSION |
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These results provide evidence that blood-borne IL-1 entering the
CNS at the SFO is greatly restricted to the SFO and does not freely
penetrate into the surrounding regions. We have also shown that
blood-borne IL-1
localizing to the CP of the cerebral ventricles
does not readily spill into the CSF bathing the CP or into the tissues
surrounding the ventricular system. Although the microvasculatures of
both the SFO and CP lack a BBB, a formidable challenge to unobstructed
diffusion into surrounding areas exists at both the CP and the ventral
border of the SFO.
The restricted dispersion observed at these sites can be attributed to
the ependymal cells that line both the ventral SFO and the CP. Tight
junctions have been demonstrated between these cells that impede the
passage of materials from the CVO into the CSF and surrounding
parenchyma (5, 9, 17). Furthermore, ZO-1, a protein associated with the
tight junctions between cerebral vascular endothelial cells, has
recently been demonstrated within the tight junctions of the
nonvascular ependymal cells lining the SFO and other CVOs (16). The
presence of ZO-1 further suggests that the ependymal lining functions
as a true barrier to the passage of IL-1 out of the SFO and into the
bordering neuropil.
An absence of unimpeded diffusion of IL-1 from the dorsal aspect of
the SFO into the region of the VHC also was observed. This, however,
does not appear to be the result of an obvious physical barrier, such
as tightly joined ependymal cells. Instead, this lack of movement may
reflect the restrictive nature of the parenchymal space within the
brain, a phenomenon that has previously been described and detailed for
several other compounds (3, 6, 15).
A statistically significant difference was seen between the slopes of
the regression lines generated from the linear phase of the sigmoidal
curve plots. This contrast indicates that the ventral lining of
ependymal cells establishes a more restrictive environment than the VHC
at the dorsal aspect of the SFO. However, even in the absence of a
"classic" barrier, the dorsal interface still appears to greatly
hinder free diffusion of IL-1 from the CVO.
We did not investigate the movement of radioiodinated IL-1 away from
other CVOs. However, ependymal cells possessing tight junctions and
ZO-1 also are located at the ventricular interface of the median
eminence, area postrema, and organum vasculosum laminae terminalis (13,
16). Therefore, it is likely that the properties that impede
penetration from the SFO also may restrict free diffusion from other
central regions lacking a BBB.
The graphs that show the decreasing concentrations of silver grains at
defined intervals away from the dorsal and ventral borders of the SFO
illustrate several points. The decline in concentration of grains away
from the center of the SFO appears to actually begin within the CVO
tissue. This suggests that an environment restrictive to the diffusion
of materials exists even before the interfaces of the SFO and bordering
tissues. At the actual intersection of the SFO and the neighboring
tissues, the containment of the cytokine is further strengthened by
both physical barriers and cellular densities. Together, these
characteristics act to severely restrict the diffusion of blood-derived
materials, including IL-1, from the CVO.
The degree of inhibition of passage of IL-1 out of the SFO becomes
even more evident when the D1/2
values are compared with the movement of albumin out of the ventricular
system of the brain, an area where the ependymal lining is not joined
by tight junctions. In an earlier study, periventricular penetration of
radioiodinated albumin was measured from the base of the third
ventricle of the rat after intracerebroventricular injection (15).
Despite having a mass four times greater than IL-1
, this protein
displayed a D1/2 of 88.25 µm,
representing a distance four times greater than the penetration of the
cytokine from the ventral SFO and more than twice as far as the
penetration of IL-1
from the dorsal SFO.
The high degree of containment of blood-derived IL-1 observed within
"leaky" regions of the brain suggests that alternate routes must
provide access for the cytokine into the CNS to account for its
detected presence within other regions of the brain after peripheral
administration. In this study, we observed transport of IL-1
at
structures in multiple regions of the brain, which we identify as
cerebral vasculature. The structures are comprised of chains of cells
that form thin, irregularly branched shapes. These structures are
comparable in length and morphology to cerebral vasculature detected by
alkaline phosphatase staining in other sections of brain tissue not
subjected to emulsion autoradiography. The substantial morphological
and physiological evidence obtained by the autoradiographic approach
supports strongly the microvascular identification of these structures,
despite an absence of immunocytochemical verification, probably due to
the extensive processing of the tissues for emulsion coating.
The varying degrees of BBB penetration by microvascular transport of
IL-1, as determined by the differences in grain density observed
between individual capillaries and even along the length of a single
vessel, were striking. Notable differences in vascular and perivascular
penetration, as well as the scatter of silver grains away from the
transporting microvessels, were seen throughout the capillary bed.
Individual capillaries, often separated by only a few micrometers,
displayed vastly different patterns of grain distribution. Furthermore,
one branch of a single microvessel would show substantial delivery of
IL-1
to the parenchyma, whereas another branch of the same capillary
would display a negligible concentration of grains (Fig. 5,
A-C). Even more intriguing
was the observation that other comparable vessels within the same region as the transporting capillaries completely lacked any
silver grains, thus being classified as nontransporting vessels
(Fig. 5D).
This suggests that only a subset of endothelial cells may be able to
saturably transport blood-borne IL-1. The ability of a particular
vessel or section of a single vessel to carry the cytokine across the
BBB may reflect regional or cellular influences on vascular properties.
Induction of BBB transport mechanisms by the central environment may be
the underlying cause of regional transport into the brain, such as that
seen with IL-1
into the PDS (14). Mapping studies of IL-1
receptors are consistent with the possibility that the CNS may
influence the IL-1
transporter. Several of the regions we have shown
to contain IL-1
-transporting microvessels have been reported to
express the receptor for this cytokine, including the hypothalamus,
striatum, and septum (8, 10). This raises the possibility that CNS
cells that bind and respond to IL-1
also may regulate its entry into
the brain. Alternatively, transport might occur only at the arterioles,
capillaries, or venules, as previously reported (7, 18).
Although blood-borne compounds also may be involved in the induction of
IL-1 transport, such delivery would affect equally the entire
microvascular bed. Because only a subset of the capillaries appear to
transport IL-1
into the brain, this possibility seems less likely.
Induction of BBB transporters by the CNS also may help establish higher
degrees of communication between the brain and the periphery, and these
systems may be further regulated in response to disease or infection.
In summary, diffusion of blood-borne IL-1 from brain regions lacking
a BBB was greatly restricted as demonstrated by emulsion autoradiography. In these areas, structures such as tightly joined ependymal cells act to contain the cytokine. Communication between the
immune and central nervous systems appears instead to be maintained at
a subset of the microvascular bed, where transport of IL-1
into the
brain was observed.
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ACKNOWLEDGEMENTS |
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We thank Melita Fasold for assistance in the preparation of the manuscript.
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FOOTNOTES |
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This work is supported by the Department of Veterans Affairs and the Office of Naval Research.
Address for reprint requests: L. M. Maness, Veterans Affairs Medical Center, Research Service, 1601 Perdido St., New Orleans, LA 70146.
Received 18 December 1997; accepted in final form 16 April 1998.
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