Relative contributions of a CVO and the microvascular bed to delivery of blood-borne IL-1alpha to the brain

Lawrence M. Maness, Abba J. Kastin, and William A. Banks

Veterans Affairs Medical Center and Tulane University School of Medicine, New Orleans, Louisiana 70146

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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-1alpha into the central nervous system (CNS). To assess the involvement of these putative routes, mice received intravenous injections of radioiodinated interleukin-1alpha , and their brains were subjected to emulsion autoradiography. The resulting patterns of silver grain distribution showed that diffusion of interleukin-1alpha 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-1alpha across the BBB. Individual microvessels showed different patterns of transport ranging from robust to absent. The high degree of containment of blood-borne interleukin-1alpha 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-1alpha ; autoradiography; circumventricular organ

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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-1alpha (IL-1alpha ), interleukin-1beta , interleukin-1 receptor antagonist, and tumor necrosis factor-alpha , across the murine BBB into the parenchymal space of the brain (2, 4, 11, 12). Analysis of the pharmacokinetic profile of IL-1alpha 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-1alpha in mouse serum 30 min after intravenous administration (4), indicating that intact IL-1alpha is available for transport across the BBB after injection into the blood.

Within the brain, transported IL-1alpha also displays a striking degree of structural integrity. HPLC analysis in two separate studies has demonstrated 125I-labeled IL-1alpha 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-1alpha is present in the CNS after transport and available for interactions with central tissues.

For IL-1alpha , 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-1alpha 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-1alpha 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-1alpha . Finally, we studied the transport of IL-1alpha at individual capillaries to determine the possible involvement of the microvascular bed in the delivery of IL-1alpha to the brain.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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-1alpha , 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-1alpha 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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Fig. 1.   Example of sampling ladder for image analysis. This approach was used to determine concentrations of silver grains at 10-µm intervals within and away from the subfornical organ (SFO). Scale bar, 50 µm.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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-1alpha 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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Fig. 2.   Sigmoidal distribution of silver grains around the ventral and dorsal borders of the SFO. , Measurements taken from the ventral SFO to the dorsal thalamus; and down-triangle, measurements from the dorsal SFO to the ventral hippocampal commissure (VHC); values are reported as grain counts ± SE. Dotted line represents SFO-tissue interface.

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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Fig. 3.   Percent values of maximal SFO grain concentrations at SFO-tissue interface. Values found on the linear decline phase of the sigmoidal curve were plotted relative to their distances from either the SFO-thalamus interface (, ) or the SFO-VHC interface (bullet , open circle ). Slopes of the resulting lines were significantly different (P = 0.0057). Distances needed for concentration of silver grains to decrease by one-half (D1/2 values) for the ventral and dorsal aspects of the SFO were calculated from these slopes. Silver grain concentrations were found to be one-half the maximal SFO concentration at 22.72 µm ventrally and 37.93 µm dorsally.

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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Fig. 4.   Cerebral microvessels within the striatum (A-C) and the anterior hypothalamus (D) seen transporting radioiodinated interleukin-1alpha (IL-1alpha ) into the brain parenchyma, as demonstrated by silver grain labeling on and laterally from microvessels. Additional microvessels within the septum and cortex (not shown) also displayed blood-borne IL-1alpha transport properties. Scale bar, 50 µm.


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Fig. 5.   Degree of IL-1alpha transport by individual microvessels. A: representative transporting capillary showing different IL-1alpha transport rates along the length of the microvessel; B: box B from A, showing a region of high transport, with extensive grain scatter away from the capillary. C: box C from A, showing minimal IL-1alpha transport activity and grain scatter comparable to background levels; D: image showing 2 capillaries only 300 µm apart, one showing vigorous transport of IL-1alpha (open arrow) and the other displaying no transport activity (solid arrow). Scale bars: A and D, 50 µm; B and C, 20 µm.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

These results provide evidence that blood-borne IL-1alpha 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-1alpha 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-1alpha out of the SFO and into the bordering neuropil.

An absence of unimpeded diffusion of IL-1alpha 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-1alpha from the CVO.

We did not investigate the movement of radioiodinated IL-1alpha 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-1alpha , from the CVO.

The degree of inhibition of passage of IL-1alpha 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-1alpha , 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-1alpha from the dorsal SFO.

The high degree of containment of blood-derived IL-1alpha 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-1alpha 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-1alpha , 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-1alpha 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-1alpha . 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-1alpha into the PDS (14). Mapping studies of IL-1alpha receptors are consistent with the possibility that the CNS may influence the IL-1alpha transporter. Several of the regions we have shown to contain IL-1alpha -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-1alpha 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-1alpha transport, such delivery would affect equally the entire microvascular bed. Because only a subset of the capillaries appear to transport IL-1alpha 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-1alpha 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-1alpha into the brain was observed.

    ACKNOWLEDGEMENTS

We thank Melita Fasold for assistance in the preparation of the manuscript.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Endocrinol Metab 275(2):E207-E212
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