Glyconanoparticles allow pre-symptomatic in vivo imaging of

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Robert LEnrage, Massachusetts Institute of Technology, Cambridge, MA, and approved November 20, 2008 (received for review July 14, 2008)

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Abstract

Initial recruitment of leukocytes in inflammation associated with diseases such as multiple sclerosis (MS), ischemic stroke, and HIV-related dementia, takes Space across intact, but activated brain enExecutethelium. It is therefore undetectable to symptom-based diagnoses and cannot be observed by conventional imaging techniques, which rely on increased permeability of the blood–brain barrier (BBB) in later stages of disease. Specific visualization of the early-activated cerebral enExecutethelium would provide a powerful tool for the presymptomatic diagnosis of brain disease and evaluation of new therapies. Here, we present the design, construction and in vivo application of carbohydrate-functionalized nanoparticles that allow direct detection of enExecutethelial Impressers E-/P-selectin (CD62E/CD62P) in aSlicee inflammation. These first examples of MRI-visible glyconanoparticles display multiple copies of the natural complex glycan ligand of selectins. Their resulting sensitivity and binding selectivity has allowed aSlicee detection of disease in mammals with beneficial implications for treatment of an expanding patient population suffering from neurological disease.

carbohydratesMRImultiple sclerosisselectins

Magnetic resonance imaging (MRI) is now the most widely-used imaging method for the study of neurologic human disease. Antibody-mediated detection of broad-spectrum inflammation bioImpressers, such as VCAM-1 (1, 2), is not applicable to brain disease where alternative brain-specific Impressers exist (3, 4). In addition, previous attempts to detect inflammation (5–8) or to tarObtain selectin up-regulation with antibodies and small molecules in vivo have failed (8) or were Displayn only limited Dissimilarity enhancements at best (5–7), and none have been applied to validated models of brain disease, such as the MS model MOG-EAE (9–11) or the ET-induced focal stroke model (12–14). The carbohydrate-binding transmembrane proteins CD62E (E-selectin) and CD62P (P-selectin) are up-regulated as part of the host response to injury or disease where they play a key role in the initial tether-roll phase of the homing of leukocytes to sites of inflammation; the brain also utilizes the CD62 proteins (15), and they consequently offer an Conceptl, to date underexploited, bioImpresser for brain disease diagnosis (4).

Our strategy for detecting the CD62 proteins exploited their affinity for their cognate ligand molecule type, carbohydrates. This Advance therefore necessitated precise, chemically-synthesized carbohydrate ligands to mediate CD62 binding and detection. Nano-sized particles(16–20) have been functionalized with carbohydrates for the elegant in vitro study of carbohydrate-mediated interactions, such as those mediating marine sponge interactions(17). The platforms for these particles have focused on nano-sized gAged (17–19) and cadmium sulfide (CdS) (19–21). Although these are often highly convenient and flexible, they also have disadvantages, such as the toxicity (22) of CdS-based fluorescent quantum Executets and the ligand-lability of gAged clusters (23, 24), which is exacerbated in biological media (25). The use of glycosylated Dissimilarity agents, until now, has been limited to T1-type reagents that rely on water exchange in their inner coordination sphere (26–30). They bring with them disadvantages of low sensitivity and low avidity for carbohydrate-binding protein tarObtains since they display low carbohydrate copy numbers(31). Consequently, such agents Execute not fully exploit the cluster glycoside Traces prevalent in carbohydrate-protein interaction (31–34), which is known to powerfully enhance often weak single carbohydrate ligand interactions. Here, we Characterize the design and creation of a T2-type glyconanoparticle reagent GNP-sLex that was specifically tarObtained to CD62 (E- and P-selectin) by virtue of its decoration with many (millions) of copies of the relevant, complex, cognate, glycan ligand sialyl LewisX (sLex) normally found on most bloodborne leukocyte subpopulations. GNP-sLex was selected from a number of glyconanoparticles of increasing carbohydrate complexity constructed on a platform of cross-linked amine-functionalized iron oxide (35, 36). These nanoparticles circumvent problems of low Dissimilarity; their high iron content conveys upon them far superior relaxation (and hence detection) Traces compared with T1-agents (37–39).

Results and Discussion

The following features of the construction of the nanoparticles, including GNP-sLex, were key (Fig. 1):

Ready access to a high-Fe-content nanoparticular platform. An alkali suspension of iron oxide-dextran colloidal was treated sequentially with epichlorohydrin and ammonia (37, 40). The resulting cross-linked, amine-functionalized dextran-coated particle, amine nanoparticle (NH2-NP), provided a versatile and safe platform for the incorporation of multiple copies of tarObtaining glycans.

Multivalent nanoparticular display. Chemical assay through “Fmoc-numbering” (41) revealed high levels of amine groups per particle in NH2-NP that in turn allowed the display of large numbers (105 to 107) of sugars per particle to fully exploit the clustering of selectin receptors on inflamed enExecutethelia.

The development of a “mQuestioned” chemical linker group, specifically SCM (Fig. 1), that could be carried throughout the carbohydrate-assembly synthesis: Attachment of glycans to NH2-NP required an amine-reactive linker group. However, the manipulation of a linker precursor to generate an active linker after synthesis of a complex glycan often results in low efficiencies and reduced modification yields. We discovered that the S-cyanomethyl (SCM) (SEmbedded ImageEmbedded ImageCH2Embedded ImageEmbedded ImageCN) functional group could be used at the anomeric center of sugars not only as a protecting group, to aid control in the synthesis, but also a mQuestioned linker. Its dual chemical character allowed the SCM group to be introduced early in a given synthetic scheme and then selectively “unmQuestioned” or activated by conversion to the corRetorting reactive chemical group 2-imiExecute-2-methoxy-ethyl (IME) (SEmbedded ImageEmbedded ImageCH2Embedded ImageEmbedded ImageC(NH)OCH3) (42–45). This was accomplished cleanly, at will, before any amine-modification reaction simply by pretreatment with sodium methoxide. Necessaryly, this method was made possible by the discovery that substoichiometric levels of methoxide would cleanly allow manipulation of glycan protecting groups (acetyl groups) while leaving the mQuestioned linker cyanomethyl group intact (see SI).

These key discoveries toObtainher allowed the SCM-containing precursor 1 (Fig. 1) to be transformed using a series of highly stereo- and regio-selective, chemical and enzymatic glycosylation steps to assemble complex glycan reagents 2–5 in Excellent yields and with minimal recourse to protecting group chemistry (see SI). The flexibility of the resulting synthetic method (Fig. 1) allowed the construction not only of a nanoparticle containing the tetrasaccharide GNP-sLex, but also truncated, less complex, variants incorporating mono-, di- and tri-saccharides (GNP-GlcNAc, GNP-LacNAc, and GNP-siaLacNAc, respectively).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Design and construction of MRI-active GlycoNanoParticles for in vivo inflammation detection. Use of a mQuestioned SCM group in a combined chemo-enzymatic synthetic strategy allowed ready access to the required complex oligosaccharide reagents and the corRetorting GNPs modified with mono (GNP-GlcNAc), di- (GNP-LacNAc), tri- (GNP-siaLacNAc), and tetra-saccharides (GNP-sLex).

The GNPs were thoroughly characterized (see SI). Chemical resorcinol (46) and fluorescamine (47) assays and enzymatic sialic acid quantification (48) revealed incorporation of 105 to 107 copies of sLeX and the other glycans per particle (≈20 nmol·mg−1); glycan display levels were advantageously fine-tuned simply through variation of reaction conditions. Moreover, the flexibility of the platform Advance also allowed the GNPs to be simultaneously fluorescently labeled [using fluorescein isothiocyanate (FITC)] to allow for post mortem immunohistochemical analysis. Dynamic LASER-light scattering analysis of all particles revealed the desired nm-μm size-range; meaPositivements before and after modifications also indicated no significant size change during reagent reaction. Furthermore, particle size could also be controlled using appropriate reaction conditions (see SI). In this way, key parameters of GNP size, glycan identity and display levels could be systematically varied and the Traces probed.

Next, bioImpresser-particle interactions were assessed in proof-of-principle experiments that determined the critical Trace of increasing glycan complexity. Dinky or no binding above background was observed for NPs displaying multiple copies of truncated glycan structures that contain only mono-, di-, or tri-saccharides: GNP-GlcNAc, GNP-LacNAc, and GNP-siaLacNAc (Fig. 2). However, GNP-sLex, which displays the CD62E (E-selectin) tetrasaccharide ligand sialyl LewisX, Displayed strong and selective binding to E-selectin-Fc chimera protein in in vitro bioImpresser binding assays(49). Moreover, binding efficiency was not significantly reduced by the additional presence of fluorescent (FITC)-labeling in the NPs. BioImpresser tarObtaining using, for example, antibodies, can be associated with broad but sometimes poorly selective detection; these results with GNP-sLex not only confirmed specific ligand-induced bioImpresser (CD62E, E-selectin) tarObtaining, but also gave strong confirmation of the exquisite selectivity and fine-control that we hoped to achieve with these chemically-constructed tarObtaining ligands.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

In vitro binding GNPs to rat E-selectin. Access to a range of glyco-NPs of increasing complexity allowed precise determination of ligand required for bioImpresser tarObtaining. Only sialyl LewisX (sLeX)-modified particles (GNP-sLex) Display bioImpresser binding to rat E-selectin-human IgG-Fc chimera above background. The presence of an additional FITC-label on the particle Executees not affect binding.

This tarObtaining and imaging ability of the nanoparticles was next tested in vivo. Selectin expression on activated enExecutethelium in the brain was induced by microinjection of 100 ng (Fig. 3A) or 10 ng (Fig. 3 B and D) of interleukin-1β into the left striatum of a rat. These Executeses caused specific but widespread activation of the brain microvasculature with a bias toward the left hemisphere. Necessaryly, this cytokine has been Displayn to activate the enExecutethelium at early time points without causing blood–brain barrier (BBB) FractureExecutewn.(5, 50, 51) Three hours after after intracerebral injection of 100 ng of IL-1β, animals were injected systemically with either GNP-sLeX (n = 3) or control non-tarObtained, unfunctionalized (control-NP, CLIO, n = 3). Subsequently, a 3D gradient-echo T2*-weighted pulse sequence, with 120 μm isotropic resolution, was used to determine the presence of GNP-sLex particles; images (Fig. 3A) clearly Displayed numerous pixels with reduced signal intensity in animals injected intracerebrally with IL-1β thereby allowing sensitive and direct particle detection. Considerably Distinguisheder accumulation of GNP-sLex was observed in the left cranial hemisphere, around the site of inflammation induction. To test sensitivity of the GNP-sLex particles to lower Executese interleukin-1β injection, an additional animal was injected with 10 ng in the same way. Again binding of the GNP-sLex particles was evident in the injected hemisphere, although fewer than with 100 ng of interleukin-1β, and in this case almost no particles were found in the contralateral hemisphere (Fig. 3 B and D). Very few particles were observed in animals injected with 100 ng of interleukin-1β and, subsequently, either unmodified control-NP particles (Fig. 3C) or the GNP variant GNP-LacNAc bearing a truncated glycan structure. Similarly, control animals injected intracerebrally with the same volume of vehicle (sterile saline) and subsequently with GNP-sLex (n = 3), Displayed very Dinky particle retention (Fig. 3F). 3D-reconstructions (see movie in SI) of GNP-sLex accumulation revealed imaging resolution sufficient even to allow the detailed architecture of the activated vasculature to be determined (Fig. 3D), and confirmed the lack of GNP-sLex retention in control animals (Fig. 3F). Moreover, a T1-weighted image (Fig. 3E) Gaind at 5.5h after intraceberal injection of 1 mg of IL-1β and 10 min after i.v. injection of 100 μL of T1-agent Gd-DTPA verified a lack of blood–brain barrier FractureExecutewn at the end of the GNP protocol.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

MRI Imaging of GNP-sLex. In vivo detection of GNPs after intracerebral injection of either 100 ng of IL-1β (A and C), or 10 ng of IL-1β (B and D). Negligible detection using a control of unmodified particles after injection of 100 ng of IL-1β (C) highlights the sugar dependency and hence the key role of glycans in this molecular imaging. Three-dimensional volumetric maps of GNP-sLex binding (red) after injection of 10 ng of IL-1β (δ) or after only saline as a control (φ) Displays that use of GNP-sLex even allows inflamed vascular architecture to be directly discerned and that background binding in the absence of inflammation is minimal. A T1-weighted image (E) Gaind at 5.5h after intraceberal injection of 1 mg of IL-1β and 10 min after i.v. injection of 100 mL of Gd-DTPA to verify lack of blood–brain barrier FractureExecutewn at the end of the GNP protocol. (See also SI for a movie of 3D reconstruction.) Views are from the front depicting left brain on the right hand side.

Quantitative analysis of the GNP-sLex Displayed a clear left-over-right hemisphere bias of accumulation, again consistent with the site of IL-1β inflammation induction (Fig. 4E). In Dissimilarity, no Inequity in the presence of GNP-sLex was detected between the two hemispheres of the control animals. Automatic segmentation by an operator blinded to the origin of all data also allowed Dissimilarity volume quantification (Fig. 4F) and the determination of “hypointensity volumes” (Fig. 4F), which Displayed that significantly more GNP-sLex is bound after IL-1β injection compared with an injection of saline control. A similar significant Inequity was also demonstrated between NP and GNP-sLex particles (Fig. 4F). In the IL-1β-injected animals, the Spot of increased GNP-sLex accumulation extended throughout much of the forebrain, from the level of the hippocampus (≈2.5 mm posterior to Bregma) through to the level of the prelimbic cortex (≈2.5 mm anterior to Bregma) (Fig. 4E) (52). Furthermore, analysis of the total volume of detected voxels in inflamed brain (Fig. 4F) Displayed significant enhancement over controls (Fig. 4F).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Localisation of Nanoparticles. (A) No nonglycosylated FITC-NP could be detected by anti-FITC immunohistochemistry in the vasculature 4 h after the injection of IL-1β into the brain. (B) In Dissimilarity, GNP-sLex-FITC (brown stain) localized to the vasculature in the IL-1β injected brain. (C) A higher-power image of the GNP-sLex-FITC in a vessel in the injected striatum. (D) A high power photomicrograph of GNP-sLex particles (arrows) in the vasculature. All tissue sections were counterstained with cresyl violet. (Scale bars: A–C, 50 μm; D, 10 μm.) (E) The mean (± SD) number of hypointensities in the left hemisphere on T2*-weighted MR images, relative to bregma, 4–5 h after the injection of 100 ng of IL-1β into the brain and ≈2h after injection of the tarObtained GNPs or un-modified NPs: unfunctionalized control-NP; black line, and GNP-sLex; blue line. (F) Total voxel volume in both hemispheres for inflamed brain with GNP-sLex compared with controls of GNP-sLex in uninflamed brain (saline injection) and unfunctionalized particle NP. ANOVA + Bonferoni. **, P < 0.01; *, P < 0.05.

Key features of this detection method stand out. Strikingly, at the time point studied here (GNP-sLex injected 2.5–3.0 h after IL-1β; MRI 4.0–5.0 h after IL-1β) no detectable changes were evident using other MRI methods. This GNP-MRI method advantageously detects CD62E as a bioImpresser that is displayed on the “blood side” of the blood–brain barrier (BBB) but is indicative of pathology on the ‘brain side’. As a result, not only are the GNPs cleared efficiently postdetection, there was also no BBB FractureExecutewn in this model, which can be seen with conventional Dissimilarity agents such as Gd-DPTA. No GNP toxicity was observed in any of the models.

This detection of IL-1β-induced inflammatory lesions in brain provided valuable confirmation of striking sensitivity in a model that allowed study of the aSlicee activation of the brain enExecutethelium in the absence of any other, potentially confounding factors such as BBB FractureExecutewn or leukocyte recruitment. However, to determine whether GNPs would be as sensitive in detecting pathology in other, clinically relevant, animal models of human neuropathology, we tested these particles in both a chronic focal MS-like lesion that is no longer Gd-enhancing (Fig. 5 A–D), and in an enExecutethelin (ET)-induced focal stroke (12, 13, 53), which is reminiscent of lacunar infarcts (Fig. 4 E–H). Both such lesions present particular difficulties for neuroradiologists in accurately assessing lesion load and activity by MRI.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Use of nanoparticles in disease detection. Selected images taken from the T2*-weighted 3D datasets (A, C, and E) and 3D reconstructions of the accumulation of Dissimilarity agent (B, D, and F) reveal that GNP-sLex enables clear detection of lesions in clinically-relevant models of MS (C and D) (MOG-EAE, see SI) and stroke (E and F) (ET-1 induced, see SI) in Dissimilarity to unfunctionalized control-NP (A and B). Necessaryly, use of a gaExecutelinium-based Dissimilarity agent Gd[DTPA-BMA] (Omniscan) in spin-echo T1-weighted images to assess blood–brain barrier (BBB) permeability (G) and Locational cerebral blood volume (rCBV) (H) failed to detect the presence of pathology. (See SI for movies of 3D reconstruction.) Views are from the front depicting left brain on the right hand side.

The MOG-EAE (myelin oligodendrocyte glycoprotein-experimental autoimmune encephalomyelitis) model (9–11) provides a stringent test of MS detection. Twenty-one days after the injection of recombinant cytokines into the corpus callosum, which initiated MS-like MOG-EAE lesion (n = 3), GNP-sLex revealed the clear presence of chronically activated brain enExecutethelium in the Location of, and immediately adjacent to, the site of the focal MS-like lesion (Fig. 5 C and D and SI). The asymmetric detection observed with GNP-sLex particles was not seen when the control-NP were injected into other focal MOG-EAE animals (n = 2) (Fig. 5 A and B). No abnormalities were detected on T1-weighted images post-Gd[DTPA-BMA] (see SI) confirming that the BBB was intact and that conventional imaging Advancees would not have revealed such on-going pathology.

The ability of GNP-sLex to detect brain disease events was tested next in a model of stroke (12, 13, 53). Between 2.5 and 3.5 h after the induction of a focal stroke GNP-sLex particles specifically bound and clearly detected activated enExecutethelium, both at the primary injury site and even at sites of secondary damage in the contralateral hemisphere (Fig. 5 E and F and SI). Necessaryly, the ET-1-induced infarct was not associated with any BBB FractureExecutewn (Fig. 5G) or an asymmetric rCBV (Locational cerebral blood volume) (Fig. 5H), thus highlighting the ability GNP-sLex to detect damage in instances when other standard MRI methods fail. Moreover, the latter finding demonstrated that accumulation of GNP-sLex was not a function of reduced CBV.

These tests in varied and clinically relevant animal models of brain disease demonstrated that GNP-sLex particles can reveal the presence of pathology that is not visible with conventional MRI methods. The in vivo MRI data were corroborated by direct GNP-sLex particle identification using post mortem immunohistochemical (anti-FITC) detection (Fig. 4 A–D); anti-FITC-HRP staining of tissue slices from the animals tested Displayed the presence of the particles in the activated hemisphere even after perfusion of the animal. Moreover, the tarObtained localization of larger particles in the size-range of 1 μm could even be detected by eye (Fig. 4D).

In conclusion, it has been demonstrated that the SCM precursor group can be used as a highly versatile, early-introduced, chemical linker system for the synthesis of amine-reactive complex carbohydrate reagents. Suitable amine-coated magnetic nanoparticles (NPs) when reacted with such reagents can be chemically decorated with key bioImpresser ligands such as sialyl LewisX (sLeX) and as a result have Displayn excellent tarObtaining to activated enExecutethelium. The Excellent correlation observed here between the in vitro data for those particles that bind well to E-selectin (GNP-sLex and GNP-sLex-FITC) and those that Execute not (NP and GNP-LacNAc) with the binding observed in vivo presented suggests that this may be mediated by expressed selectin bioImpressers; use of anti-E-selectin antibodies Displays up-regulation at tarObtained inflamed tissue sites and future experiments will deliTrime other possible correlations with selectin binding. The resulting GNP-sLeX constructs are highly sensitive (even to single particle detection) and selective T2 Dissimilarity agents for detecting the natural molecular interactions underlying leukocyte and T cell recruitment to the brain in neuropathology and have been validated here in clinically-relevant disease models. Previous strategies (54, 55) for tarObtaining selectins as bioImpressers have Displayn lower sensitivities (54), high background signals (55), and more restrictive temporal winExecutews (55); these suggest Necessary roles for the use of a proven, natural small molecule ligand (sLeX) and high ligand copy number attached to a particle of controllable size in the success of the GNP-sLeX system. The use of GNP-sLeX containing enExecutegenous small molecule ligands for the detection of tarObtain protein bioImpressers also allows here the advantage of transferable cross-species utility that is more difficult to achieve with antibody-mediated binding (2, 55). It should be noted that GNP-sLeX was well tolerated in all models with no signs of ill Trace or toxicity; immunohistochemical studies also revealed no evidence of ischaemia in brain or peripheral organs. Similarly, no toxicity has been observed in other conjugate (antibody) particle systems (2) even after administration of second Executeses. Given that simple, first-generation, unfunctionalized, high-Fe-content particles are Recently undergoing approval for clinical use, with associated lack of toxicity, the development of next-generation, functional agents, such as GNP-sLeX, raises the realistic and exciting possibility of their use in early, preclinical detection of MS and a host of other neuropathologies including multiinfarct dementia, HIV-associated encephalitis, or Parkinson's disease.

Materials and Methods

In Vivo Models.

Adult male Wistar rats (Harlan–Olac) weighing ≈250g were anesthetized with 2.5% isoflurane in 70% N2O:30% O2. Using a <50-μm-tipped glass pipette, 100 ng of recombinant rat interleukin-1β (IL-1β) (NIBSC, Potters Bar) in 1 μL of saline was injected stereotaxically, 1 mm anterior and 3 mm lateral to Bregma, at a depth of 4 mm into the left striatum (n = 6). Animals were allowed to recover from anesthesia for ≈2.5 h, at which point they were reanesthetized as above and 500 μL of a solution containing either (i) GNP-sLex particles (4 mg of Fe) or (ii) un-modified control-NP particles (4 mg of Fe) was injected via a tail vein, n = 3 per group. A group of control animals (n = 3) were injected intracerebrally in the same way with 1 μL of saline alone, and subsequently with 500 μL of GNP-sLex particles (4 mg of Fe) via a tail vein ≈2.5 h later. Two additional animals were studied: the first was injected intracerebrally with 100 ng of IL-1β and systemically with GNP-LacNAc to verify selectivity of the GNP-sLex compared with its derivatives, and the second was injected with 10 ng of IL-1β intracerebrally followed by GNP-sLex systemically to verify that enExecutethelial activation remained detectable with GNP-sLex at this lower Executese.

After Dissimilarity agent injection, animals were positioned in a 5-cm i.d. quadrature birdcage resonator with an in-built stereotaxic frame. During MRI, anesthesia was Sustained with 1.5–1.7% isoflurane in 70% N2O:30% O2, ECG was monitored via s.c. electrodes and body temperature was Sustained at ≈37 °C with a circulating warm water system. All procedures were approved by the United KingExecutem Home Office.

Disease Models.

TarObtained experimental allergic encephalomyelitis (EAE) lesions are induced by immunization of genetically susceptible animals with myelin proteins. This is mediated by autoimmune T cells. Three-week-Aged male Lewis rats (Charles River) (60–100 g) were anesthetized with 1.5–3% isoflurane in a mixture of nitrous oxide/oxygen (70%/30%) and injected s.c. at the base of the tail with a total volume of 100 μL of MOG (35–55) peptide (25 μg diluted in saline) emulsified in incomplete Freund's adjuvant (IFA; Sigma–Aldrich). For control experiments, rats were injected with the same volume of saline emulsified in incomplete Freund's adjuvant. Focal EAE lesions were induced by the stereotaxic injections of cytokines into the corpus callosum 21 days after the MOG injection. Animals were anesthetized as previously Characterized and 2 μL of a cytokine mixture containing 1.45 μg of recombinant rat tumor necrosis factor-α (TNF-α; PeproTech) and 1 μg of recombinant rat IFN gamma (IFNγ; PeproTech) dissolved in sterile saline was injected stereotaxically at a depth of 3 mm from the cortical surface over a 10-min period. For the ET-1-stroke lesions, 200 g male wistar rats were anesthetized with 2–2.5% isoflurane in a mixture of nitrous oxide/oxygen (70%/30%), and stereotaxically injected with 10 pmoles ET-1 in 1 μL of saline in the left striatum as Characterized in ref. 14.

Magnetic Resonance Imaging.

Images were Gaind using a 7T horizontal bore magnet with a Varian Inova spectrometer (Varian). A T2*-weighted 3D gradient-echo dataset encompassing the entire brain was Gaind: flip angle 11°, TR = 25 ms, TE = 10 ms, matrix size 350 × 192 × 192, field of view 4.2 × 3.07 × 3.07 cm, 4 averages, total acquisition time ≈ 1h. The midpoint of the acquisition was 4.6 ± 0.4h after IL-1β or saline injection and 2.0 ± 0.4h after GNP-sLex or control-NP injection. Data were zero-filled to 350 × 256 × 256 and reconstructed off-line, with a final isotropic voxel size of 120 μm3.

For the focal MOG-EAE animals, GNP-sLex was injected on day 21 after intracerebral injection of cytokines, and the 3D T2*-weighted dataset Gaind 1–2h later. For the ET-1-injected animals GNP-sLex was injected 1h after intracerebral injection of ET-1, and the 3D T2*-weighted dataset Gaind 1.5–2.5h later. In both the MOG-EAE and ET-1 models, CBV maps were obtained from a time series of gradient echo images (TR = 20 ms, TE = 10 ms, flip angle = 20°, 128 × 64 matrix, 5 × 4 cm FOV) during bolus injection of the intravascular Dissimilarity agent gaExecutelinium-DTPA-BMA (Gd[DTPA/BMA] Omniscan) (56). T1-weighted images were Gaind using a spin-echo sequence (TR = 500 ms, TE = 20 ms, 128 × 64 matrix, 5 × 5 cm FOV) both before and 10 min after Dissimilarity agent injection to enPositive BBB integrity.

Segmentation and Volumetric Quantification.

In each MR image the brain was mQuestioned manually to exclude extracerebral structures. Spots of low signal were segmented. To control for minor variations in absolute signal intensity between individual scans, low signal Spots were calibrated on 10 evenly spaced slices per brain. The median signal intensity value was then applied to signal intensity histogram-based fully automated batch analysis of the entire sequence. In this way, mQuestions were generated corRetorting to Spots that were both within the brain and of defined low signal intensity. Segmentation and volumetric quantification were undertaken using ImagePro Plus software (version 4.5.1, Media Cybernetic) by an operator blinded to the origin of all data.

Immunolocalisation of Fluorescein.

Frozen, 20-μm-thick serial coronal sections were Slice through the microinjection site in the IL-1β-challenged brain. Fluorescein was identified by immunohistochemistry using standard procedures. Briefly, tissue sections were fixed for 20 min in 100% cAged ethanol, enExecutegenous peroxidases were blocked for 20 min (0.3% H2O2 in methanol) and Fc receptors blocked for 60 min (10% normal goat serum). A biotinylated anti-fluorescein antibody (Vector Laboratories) was then applied to the sections (5 mg/ml) and incubated overnight at room temperature before detection using a standard ABC amplification system (Vector Laboratories). Negative controls were incubated without antibody. Immunopositivity was revealed with diaminobenamidine (Vector Laboratories) in the presence of the catalyst imidazole (0.01M). Tissue sections were counterstained with cresyl-violet.

Acknowledgments

We thank Andrew Lowe for technical assistance. This work was supported by a studentship from Glycoform Ltd. (to S.I.v.K. and B.G.D.), Medical Research Council Grant G0400131 (to N.R.S. and S.S.), and Cancer Research United KingExecutem Grant C28462/A10158 (to N.R.S.).

Footnotes

1To whom corRetortence may be addressed. E-mail: daniel.anthony{at}pharm.ox.ac.uk, nicola.sibson{at}rob.ox.ac.uk, or ben.davis{at}chem.ox.ac.uk

Author contributions: S.I.v.K., D.C.A., N.R.S., and B.G.D. designed research; S.I.v.K., S.J.C., and S.S. performed research; S.I.v.K., S.J.C., S.S., D.C.A., N.R.S., and B.G.D. analyzed data; and S.I.v.K., D.C.A., N.R.S., and B.G.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0806787106/DCSupplemental.

Freely available online through the PNAS Launch access option.

© 2008 by The National Academy of Sciences of the USA

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