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Direct Imaging of Cerebral Thromboemboli Using Computed Tomography and Fibrin-targeted Gold Nanoparticles.

Kim JY, Ryu JH, Schellingerhout D, Sun IC, Lee SK, Jeon S, Kim J, Kwon IC, Nahrendorf M, Ahn CH, Kim K, Kim DE - Theranostics (2015)

Bottom Line: Glycol-chitosan-coated gold nanoparticles (GC-AuNPs) were synthesized and conjugated to fibrin-targeting peptides, forming fib-GC-AuNP.This targeted imaging agent and non-targeted control agent were characterized in vitro and in vivo in C57Bl/6 mice (n = 107) with FeCl3-induced carotid thrombosis and/or embolic ischemic stroke.Fibrin-binding capacity was superior with fib-GC-AuNPs compared to GC-AuNPs, with thrombi visualized as high density on microCT (mCT). mCT imaging using fib-GC-AuNP allowed the prompt detection and quantification of cerebral thrombi, and monitoring of tPA-mediated thrombolytic effect, which reflected histological stroke outcome.

View Article: PubMed Central - PubMed

Affiliation: 1. Molecular Imaging and Neurovascular Research Laboratory, Dongguk University College of Medicine, Goyang, South Korea;

ABSTRACT
Computed tomography (CT) is the current standard for time-critical decision-making in stroke patients, informing decisions on thrombolytic therapy with tissue plasminogen activator (tPA), which has a narrow therapeutic index. We aimed to develop a CT-based method to directly visualize cerebrovascular thrombi and guide thrombolytic therapy. Glycol-chitosan-coated gold nanoparticles (GC-AuNPs) were synthesized and conjugated to fibrin-targeting peptides, forming fib-GC-AuNP. This targeted imaging agent and non-targeted control agent were characterized in vitro and in vivo in C57Bl/6 mice (n = 107) with FeCl3-induced carotid thrombosis and/or embolic ischemic stroke. Fibrin-binding capacity was superior with fib-GC-AuNPs compared to GC-AuNPs, with thrombi visualized as high density on microCT (mCT). mCT imaging using fib-GC-AuNP allowed the prompt detection and quantification of cerebral thrombi, and monitoring of tPA-mediated thrombolytic effect, which reflected histological stroke outcome. Furthermore, recurrent thrombosis could be diagnosed by mCT without further nanoparticle administration for up to 3 weeks. fib-GC-AuNP-based direct cerebral thrombus imaging greatly enhance the value and information obtainable by regular CT, has multiple uses in basic / translational vascular research, and will likely allow personalized thrombolytic therapy in clinic by a) optimizing tPA-dosing to match thrombus burden, b) enabling the rational triage of patients to more radical therapies such as endovascular clot-retrieval, and c) potentially serving as a theranostic platform for targeted delivery of concurrent thrombolysis.

No MeSH data available.


Related in: MedlinePlus

Correlation between imaging and histology: targeted fib-GC-AuNPs vs. non-targeted GC-AuNPs. A-C, mCT thrombus images in representative C57Bl/6 mice with embolic stroke that was induced by injecting Cy5.5-fluorescently labeled clot into the bifurcation area of the distal internal carotid artery. One hour later, and 5 minutes after intravenous injection of 300 μL (120 mg/kg) fib-GC-AuNPs, cerebral thrombus is visualized on the in vivo mCT images: a reconstructed 2 mm thick horizontal view at the level of the circle of Willis (COW) shows the thrombus in the bifurcation as a Y-shaped hyperdensity (arrow) (A). A 2 mm thick coronal view that corresponds to the green-colored brain region in A also shows the thrombus as dotted hyperdensity in the vessel (arrow) (B), which is corroborated by 50 μm thick coronal view that corresponds to the middle of the green-colored brain region of A (inset of B with a red-square containing the thrombus-related hyperdense lesion). Ex vivo mCT imaging of a 2 mm thick brain section (C), corresponding to the location for the in vivo mCT image displayed in the inset of B, also shows the cerebral thrombus (red-square). D, An ex vivo optical image of the excised whole brain: visualization of thrombus-related fluorescent signal in the COW (arrow), corresponding to a similar viewpoint as in A (D). E, An ex vivo optical image of a 2 mm thick coronal section with thrombus (arrow), which corresponds to the viewpoint in B and C (= coronal views of the green-colored brain region of A and D). Please note that fluorescent and mCT visualizations of the thrombus are nearly equivalent. F, Hematoxylin-eosin staining of a thin section (10 μm thickness) from the 2 mm tissue slab used in C. This shows the cerebral thrombus (arrow in the inset) that co-localizes with the hyperdense mCT lesion observed earlier (inset of B as well as in C). G-I, Microscopic fluorescent images. A magnified view of the thrombosed site (arrow) in F and near-infrared fluorescent microscopy image of the same area (G) demonstrate that Cy5.5 fluorescent thrombus is located within the cerebral artery. Fibrin immunostaining of the same section (H, FITC channel) and Cy5.5 - FITC merged image (I) shows that the major thrombus component is fibrin, which is the target for fib-GC-AuNPs. A'-I' shows the same data for an animal injected with non-targeted GC-AuNPs. Please note that there is no mCT density (A'-C') associated with the clot (D'-I') for the GC-AuNP compound, although a thrombus is clearly present on histology and fluorescent imaging, indicating the specificity of fib-GC-AuNP over non-targeted GC-AuNP. Concentrations of nanoparticles are reported as 'mg Au / kg of animal'. Scale Bars = 2 mm (A-F, inset of B); 500 μm (inset of F); 200 μm (G).
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Figure 3: Correlation between imaging and histology: targeted fib-GC-AuNPs vs. non-targeted GC-AuNPs. A-C, mCT thrombus images in representative C57Bl/6 mice with embolic stroke that was induced by injecting Cy5.5-fluorescently labeled clot into the bifurcation area of the distal internal carotid artery. One hour later, and 5 minutes after intravenous injection of 300 μL (120 mg/kg) fib-GC-AuNPs, cerebral thrombus is visualized on the in vivo mCT images: a reconstructed 2 mm thick horizontal view at the level of the circle of Willis (COW) shows the thrombus in the bifurcation as a Y-shaped hyperdensity (arrow) (A). A 2 mm thick coronal view that corresponds to the green-colored brain region in A also shows the thrombus as dotted hyperdensity in the vessel (arrow) (B), which is corroborated by 50 μm thick coronal view that corresponds to the middle of the green-colored brain region of A (inset of B with a red-square containing the thrombus-related hyperdense lesion). Ex vivo mCT imaging of a 2 mm thick brain section (C), corresponding to the location for the in vivo mCT image displayed in the inset of B, also shows the cerebral thrombus (red-square). D, An ex vivo optical image of the excised whole brain: visualization of thrombus-related fluorescent signal in the COW (arrow), corresponding to a similar viewpoint as in A (D). E, An ex vivo optical image of a 2 mm thick coronal section with thrombus (arrow), which corresponds to the viewpoint in B and C (= coronal views of the green-colored brain region of A and D). Please note that fluorescent and mCT visualizations of the thrombus are nearly equivalent. F, Hematoxylin-eosin staining of a thin section (10 μm thickness) from the 2 mm tissue slab used in C. This shows the cerebral thrombus (arrow in the inset) that co-localizes with the hyperdense mCT lesion observed earlier (inset of B as well as in C). G-I, Microscopic fluorescent images. A magnified view of the thrombosed site (arrow) in F and near-infrared fluorescent microscopy image of the same area (G) demonstrate that Cy5.5 fluorescent thrombus is located within the cerebral artery. Fibrin immunostaining of the same section (H, FITC channel) and Cy5.5 - FITC merged image (I) shows that the major thrombus component is fibrin, which is the target for fib-GC-AuNPs. A'-I' shows the same data for an animal injected with non-targeted GC-AuNPs. Please note that there is no mCT density (A'-C') associated with the clot (D'-I') for the GC-AuNP compound, although a thrombus is clearly present on histology and fluorescent imaging, indicating the specificity of fib-GC-AuNP over non-targeted GC-AuNP. Concentrations of nanoparticles are reported as 'mg Au / kg of animal'. Scale Bars = 2 mm (A-F, inset of B); 500 μm (inset of F); 200 μm (G).

Mentions: There was a clear difference between targeted and non-targeted nanoparticles on cerebral CT imaging (Figure 3), with excellent thrombus demonstration on CT with targeted nanoparticles, and excellent matching to corresponding histological findings, while CT demonstration failed for non-targeted nanoparticles, even though thrombi were clearly present on histology. After intravenous administration of fib-GC-AuNPs in mice with embolic stroke, in vivo mCT imaging detected hyperdense lesions in the brain, and these lesions co-localized with Cy5.5 fluorescent thromboemboli on ex vivo NIRF images of the whole brain tissue and 2 mm thick sections (Figure 3 and Supplementary Material: Figure S4). NIRF microscopic imaging of 10 μm thick cryosections of the tissue confirmed thrombi filling in cerebral arteries (Figure 3). However, non-targeted GC-AuNPs could not always visualize cerebral thrombus in the COW, even when these thrombi could be clearly imaged by ex vivo NIRF imaging of the brain tissues and NIRF microscopic imaging of the brain sections. Histological findings clearly showed that mCT signal corresponded to cerebral arterial thrombi on H&E-stained or fibrin immune-stained brain sections. Electron microscopy studies confirmed that numerous fib-GC-AuNPs were scattered within the thrombi, whereas GC-AuNPs were only sparsely observed (Figure 4). Target-specific and localized accumulation of fib-GC-AuNPs in millimeter-sized carotid or cerebral thrombi may have allowed successful mCT demonstration of the thrombi even at the low doses administered (2 or 12 mg/kg vs. 20 or 120 mg/kg, respectively).


Direct Imaging of Cerebral Thromboemboli Using Computed Tomography and Fibrin-targeted Gold Nanoparticles.

Kim JY, Ryu JH, Schellingerhout D, Sun IC, Lee SK, Jeon S, Kim J, Kwon IC, Nahrendorf M, Ahn CH, Kim K, Kim DE - Theranostics (2015)

Correlation between imaging and histology: targeted fib-GC-AuNPs vs. non-targeted GC-AuNPs. A-C, mCT thrombus images in representative C57Bl/6 mice with embolic stroke that was induced by injecting Cy5.5-fluorescently labeled clot into the bifurcation area of the distal internal carotid artery. One hour later, and 5 minutes after intravenous injection of 300 μL (120 mg/kg) fib-GC-AuNPs, cerebral thrombus is visualized on the in vivo mCT images: a reconstructed 2 mm thick horizontal view at the level of the circle of Willis (COW) shows the thrombus in the bifurcation as a Y-shaped hyperdensity (arrow) (A). A 2 mm thick coronal view that corresponds to the green-colored brain region in A also shows the thrombus as dotted hyperdensity in the vessel (arrow) (B), which is corroborated by 50 μm thick coronal view that corresponds to the middle of the green-colored brain region of A (inset of B with a red-square containing the thrombus-related hyperdense lesion). Ex vivo mCT imaging of a 2 mm thick brain section (C), corresponding to the location for the in vivo mCT image displayed in the inset of B, also shows the cerebral thrombus (red-square). D, An ex vivo optical image of the excised whole brain: visualization of thrombus-related fluorescent signal in the COW (arrow), corresponding to a similar viewpoint as in A (D). E, An ex vivo optical image of a 2 mm thick coronal section with thrombus (arrow), which corresponds to the viewpoint in B and C (= coronal views of the green-colored brain region of A and D). Please note that fluorescent and mCT visualizations of the thrombus are nearly equivalent. F, Hematoxylin-eosin staining of a thin section (10 μm thickness) from the 2 mm tissue slab used in C. This shows the cerebral thrombus (arrow in the inset) that co-localizes with the hyperdense mCT lesion observed earlier (inset of B as well as in C). G-I, Microscopic fluorescent images. A magnified view of the thrombosed site (arrow) in F and near-infrared fluorescent microscopy image of the same area (G) demonstrate that Cy5.5 fluorescent thrombus is located within the cerebral artery. Fibrin immunostaining of the same section (H, FITC channel) and Cy5.5 - FITC merged image (I) shows that the major thrombus component is fibrin, which is the target for fib-GC-AuNPs. A'-I' shows the same data for an animal injected with non-targeted GC-AuNPs. Please note that there is no mCT density (A'-C') associated with the clot (D'-I') for the GC-AuNP compound, although a thrombus is clearly present on histology and fluorescent imaging, indicating the specificity of fib-GC-AuNP over non-targeted GC-AuNP. Concentrations of nanoparticles are reported as 'mg Au / kg of animal'. Scale Bars = 2 mm (A-F, inset of B); 500 μm (inset of F); 200 μm (G).
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Related In: Results  -  Collection

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Figure 3: Correlation between imaging and histology: targeted fib-GC-AuNPs vs. non-targeted GC-AuNPs. A-C, mCT thrombus images in representative C57Bl/6 mice with embolic stroke that was induced by injecting Cy5.5-fluorescently labeled clot into the bifurcation area of the distal internal carotid artery. One hour later, and 5 minutes after intravenous injection of 300 μL (120 mg/kg) fib-GC-AuNPs, cerebral thrombus is visualized on the in vivo mCT images: a reconstructed 2 mm thick horizontal view at the level of the circle of Willis (COW) shows the thrombus in the bifurcation as a Y-shaped hyperdensity (arrow) (A). A 2 mm thick coronal view that corresponds to the green-colored brain region in A also shows the thrombus as dotted hyperdensity in the vessel (arrow) (B), which is corroborated by 50 μm thick coronal view that corresponds to the middle of the green-colored brain region of A (inset of B with a red-square containing the thrombus-related hyperdense lesion). Ex vivo mCT imaging of a 2 mm thick brain section (C), corresponding to the location for the in vivo mCT image displayed in the inset of B, also shows the cerebral thrombus (red-square). D, An ex vivo optical image of the excised whole brain: visualization of thrombus-related fluorescent signal in the COW (arrow), corresponding to a similar viewpoint as in A (D). E, An ex vivo optical image of a 2 mm thick coronal section with thrombus (arrow), which corresponds to the viewpoint in B and C (= coronal views of the green-colored brain region of A and D). Please note that fluorescent and mCT visualizations of the thrombus are nearly equivalent. F, Hematoxylin-eosin staining of a thin section (10 μm thickness) from the 2 mm tissue slab used in C. This shows the cerebral thrombus (arrow in the inset) that co-localizes with the hyperdense mCT lesion observed earlier (inset of B as well as in C). G-I, Microscopic fluorescent images. A magnified view of the thrombosed site (arrow) in F and near-infrared fluorescent microscopy image of the same area (G) demonstrate that Cy5.5 fluorescent thrombus is located within the cerebral artery. Fibrin immunostaining of the same section (H, FITC channel) and Cy5.5 - FITC merged image (I) shows that the major thrombus component is fibrin, which is the target for fib-GC-AuNPs. A'-I' shows the same data for an animal injected with non-targeted GC-AuNPs. Please note that there is no mCT density (A'-C') associated with the clot (D'-I') for the GC-AuNP compound, although a thrombus is clearly present on histology and fluorescent imaging, indicating the specificity of fib-GC-AuNP over non-targeted GC-AuNP. Concentrations of nanoparticles are reported as 'mg Au / kg of animal'. Scale Bars = 2 mm (A-F, inset of B); 500 μm (inset of F); 200 μm (G).
Mentions: There was a clear difference between targeted and non-targeted nanoparticles on cerebral CT imaging (Figure 3), with excellent thrombus demonstration on CT with targeted nanoparticles, and excellent matching to corresponding histological findings, while CT demonstration failed for non-targeted nanoparticles, even though thrombi were clearly present on histology. After intravenous administration of fib-GC-AuNPs in mice with embolic stroke, in vivo mCT imaging detected hyperdense lesions in the brain, and these lesions co-localized with Cy5.5 fluorescent thromboemboli on ex vivo NIRF images of the whole brain tissue and 2 mm thick sections (Figure 3 and Supplementary Material: Figure S4). NIRF microscopic imaging of 10 μm thick cryosections of the tissue confirmed thrombi filling in cerebral arteries (Figure 3). However, non-targeted GC-AuNPs could not always visualize cerebral thrombus in the COW, even when these thrombi could be clearly imaged by ex vivo NIRF imaging of the brain tissues and NIRF microscopic imaging of the brain sections. Histological findings clearly showed that mCT signal corresponded to cerebral arterial thrombi on H&E-stained or fibrin immune-stained brain sections. Electron microscopy studies confirmed that numerous fib-GC-AuNPs were scattered within the thrombi, whereas GC-AuNPs were only sparsely observed (Figure 4). Target-specific and localized accumulation of fib-GC-AuNPs in millimeter-sized carotid or cerebral thrombi may have allowed successful mCT demonstration of the thrombi even at the low doses administered (2 or 12 mg/kg vs. 20 or 120 mg/kg, respectively).

Bottom Line: Glycol-chitosan-coated gold nanoparticles (GC-AuNPs) were synthesized and conjugated to fibrin-targeting peptides, forming fib-GC-AuNP.This targeted imaging agent and non-targeted control agent were characterized in vitro and in vivo in C57Bl/6 mice (n = 107) with FeCl3-induced carotid thrombosis and/or embolic ischemic stroke.Fibrin-binding capacity was superior with fib-GC-AuNPs compared to GC-AuNPs, with thrombi visualized as high density on microCT (mCT). mCT imaging using fib-GC-AuNP allowed the prompt detection and quantification of cerebral thrombi, and monitoring of tPA-mediated thrombolytic effect, which reflected histological stroke outcome.

View Article: PubMed Central - PubMed

Affiliation: 1. Molecular Imaging and Neurovascular Research Laboratory, Dongguk University College of Medicine, Goyang, South Korea;

ABSTRACT
Computed tomography (CT) is the current standard for time-critical decision-making in stroke patients, informing decisions on thrombolytic therapy with tissue plasminogen activator (tPA), which has a narrow therapeutic index. We aimed to develop a CT-based method to directly visualize cerebrovascular thrombi and guide thrombolytic therapy. Glycol-chitosan-coated gold nanoparticles (GC-AuNPs) were synthesized and conjugated to fibrin-targeting peptides, forming fib-GC-AuNP. This targeted imaging agent and non-targeted control agent were characterized in vitro and in vivo in C57Bl/6 mice (n = 107) with FeCl3-induced carotid thrombosis and/or embolic ischemic stroke. Fibrin-binding capacity was superior with fib-GC-AuNPs compared to GC-AuNPs, with thrombi visualized as high density on microCT (mCT). mCT imaging using fib-GC-AuNP allowed the prompt detection and quantification of cerebral thrombi, and monitoring of tPA-mediated thrombolytic effect, which reflected histological stroke outcome. Furthermore, recurrent thrombosis could be diagnosed by mCT without further nanoparticle administration for up to 3 weeks. fib-GC-AuNP-based direct cerebral thrombus imaging greatly enhance the value and information obtainable by regular CT, has multiple uses in basic / translational vascular research, and will likely allow personalized thrombolytic therapy in clinic by a) optimizing tPA-dosing to match thrombus burden, b) enabling the rational triage of patients to more radical therapies such as endovascular clot-retrieval, and c) potentially serving as a theranostic platform for targeted delivery of concurrent thrombolysis.

No MeSH data available.


Related in: MedlinePlus