This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.
Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker — a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.
One in 6 people will have a stroke at some point in their lifetime. Ischemic stroke is by far the most common stroke type, and accounts for about 80 percent of all stroke cases. Because thromboemboli cause the majority of these ischemic strokes, there is an increasing interest in direct thrombus imaging.
It was estimated that about 2 million brain cells die during every minute of middle cerebral artery occlusion1, leading to the slogan "Time is Brain". Computed tomography (CT) studies can be done rapidly, and are widely available; for this reason, CT remains the imaging of choice for the initial diagnosis and treatment of hyperacute ischemic stroke. CT is particularly valuable for informing the critical early decisions: administering tissue plasminogen activator (tPA) for thrombolysis and/or triaging to endovascular clot-retrieval2. Current CT-based thrombus imaging, however, cannot serially track cerebral thromboemboli in vivo, because it uses indirect methods to demonstrate thrombi: after opacification of the blood pool by iodinated contrast, the thrombi are demonstrated as filling defects in the vessels. There are dose limits and risks associated with the repeated administration of iodinated contrast, which preclude repeated imaging of thrombi in this manner.
Thus, there is a critical need for a direct imaging methodology for cerebral thrombi in stroke patients, to allow faster and better treatment decisions to be made. We propose to accomplish this by enhancing the value of CT, the currently used frontline imaging modality for stroke, with the use of a thrombus-seeking nanoparticular molecular imaging agent.
We have demonstrated the use of this agent using micro-computed tomography (microCT), a high-resolution ex vivo or in vivo (small animal) imaging version of CT that allows rapid data acquisition3,4. Even with the relatively poor soft tissue contrast available for small animal microCT (much worse than available from human sized scanners), the imaging agent was able to seek and mark thrombi by making them hyperdense on CT, a 'dense vessel sign' enhanced by molecular imaging.
Complementing the CT technique, our group has previously developed an optical direct thrombus imaging technique using Cy5.5 near-infrared fluorescent (NIRF) probe to visualize cerebral thrombus burden5. This is an ex vivo technique on post mortem brains, but is highly sensitive, and serves to confirm in vivo data in the research setting.
Having both CT and NIRF based thrombus-seeking imaging techniques allows us to compare and contrast these techniques to achieve highly informative data on the role of thrombus and thrombus imaging in the process of ischemic stroke development.
Here, we describe a detailed protocol of a combined technique of in vivo microCT and ex vivo NIRF imaging to directly visualize thromboemboli in a mouse model of embolic stroke. These simple and robust methods are useful to advance our understanding of thrombotic diseases by enabling the accurate in vivo assessment of thrombus burden / distribution and characterization of dynamic thrombus evolution in a prompt and quantitative manner in vivo during therapy, followed by ex vivo data that serves as a control and reference standard for the confirmation of in vivo imaging findings.
All animal procedures demonstrated in this protocol have been reviewed and approved by the Dongguk University Ilsan Hospital Animal Care and Use Committee and conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Animals.
1. Preparation of Exogenously Formed Clot Labeled with Fluorescence Marker (Figure 1)
2. Modeling a Mouse Model of Thromboembolic Stroke (Figure 2)
3. In Vivo MicroCT Imaging of Cerebral Thrombus (Figure 3)
4. Thrombolysis and In Vivo MicroCT Imaging of Cerebral Thrombus (Figure 3)
5. Ex Vivo NIRF Thrombus Imaging and Triphenyl Tetrazolium Chloride (TTC) Staining of the Brain Tissue (Figure 4)
6. Quantification of Thrombus Area Using MicroCT Images and ImageJ (1.49d) Software (Figure 5)
Baseline microCT images, obtained in vivo after administering fib-GC-AuNP (10 mg/ml, 300 µl) at 1 hr after embolic stroke, clearly visualized cerebral thrombus in the MCA – ACA bifurcation area of the distal internal carotid artery (Figure 6). Follow-up microCT imaging showed no change in the COW thrombus with saline treatment. However, treatment with tPA showed a gradual dissolution of the COW thrombus (blue arrowheads in Figure 6). This finding demonstrates that microCT thrombus imaging can allow therapeutic monitoring of thrombolysis. There is an excellent correspondence between in vivo CT density and ex vivo NIRF for residual thromboemboli at 24 hr. Note that hyperdense microCT areas due to endogenous thrombus-marking by the fibrin-targeting AuNPs nicely co-localized with NIRF signal areas due to exogenous thrombus-marking, which was performed before embolic stroke by using a Cy5.5 fluorescent probe sensitive to the coagulation factor XIIIa-mediated fibrin-crosslinking activity in the process of the maturation of the preformed clot (Figure 6). The combined technique of direct thrombus imaging to demonstrate the tPA-mediated thrombolytic effect reflects histological stroke outcome as measured by TTC staining. TTC staining showed a clear reduction in the amount of ischemic brain damage (whitish areas in Figure 6) for the animal receiving tPA thrombolysis.
Figure 1: Overview of the Preparation of Fluorescently Labeled Blood Clot. (A) Mixing of fresh whole blood with Cy5.5 near-infrared fluorescent (NIRF) probe (C15) that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. The mixed blood is injected into a polyethylene (PE)-50 tube and left for 24 hr to allow clot formation and maturation. (B) NIRF imaging to confirm the optical labeling of the thrombus with the C15 probe. Note the persistent fluorescence after washing the thrombus that was removed from the PE tube. (C) Thrombus-loaded PE-10 tube with a PE-50 tube modified to have a tapered end (200 μm diameter) for thromboembolism in the middle cerebral artery – anterior cerebral artery bifurcation area of the internal carotid artery in mice. Scale bars: 2 mm. Please click here to view a larger version of this figure.
Figure 2: Overview of Modeling a Mouse Model of Thromboembolic Cerebral Infarction. (A) Laser Doppler monitoring of cerebral blood flow (CBF) before and after occlusion of the middle cerebral artery (MCA), with the distal end of the optical fiber of the laser Doppler flowmeter glued to the left temporal bone surface (1 mm left and 4 mm below from the bregma). Note the post-occlusion decrease of Doppler CBF, which should be lowered by 30% or lower compared to baseline. (B) Insertion of the thrombus-containing catheter (i.e., the distally tapered polyethylene-50 tube in Figure 1C) into the hole in the external carotid artery (ECA), followed (eventually) by advancing the catheter about 9 mm into the MCA – anterior cerebral artery (ACA) bifurcation area of the distal internal carotid artery (ICA). CCA, common carotid artery; PPA, pterygopalatine artery. Please click here to view a larger version of this figure.
Figure 3: Overview of In vivo MicroCT-based Direct Thrombus Imaging using Fibrin-targeted Gold Nanoparticles to Visualize Cerebral Thromboemboli and Monitor Tissue Plasminogen Activator (tPA)-mediated Thrombolytic Effects. (A) Intravenous injection of fibrin-targeted glycol-chitosan gold nanoparticles (fib-GC-AuNPs) in a mouse with thromboembolic stroke. (B) The mouse is placed on the bed of a microCT imager. (C) Acquiring microCT images at 5 min after the injection of fib-GC-AuNPs. (D) Image reconstruction to transform the raw image data into Digital Imaging and Communications in Medicine (DICOM) format using a software package installed on the microCT scanner. (E) Image analysis and preparation of axial / coronal images rendered to have 2 mm thickness by using maximal intensity projection mode. (F) Intravenous tPA-mediated thrombolysis, which can be monitored and analyzed by using the microCT-based direct thrombus imaging method (C – E). Please click here to view a larger version of this figure.
Figure 4: Overview of Ex Vivo Near-Infrared Fluorescent (NIRF) Imaging of Cerebral Thromboemboli. (A and B) A representative brain tissue removed from a mouse with thromboembolic stroke. Take precautions not to lose thromboemboli (red arrow) in the middle cerebral artery (MCA) – anterior cerebral artery (ACA) bifurcation area of the distal internal carotid artery (ICA, blue arrows) when removing the brain. (C and D) Ex vivo NIRF thrombus imaging. The brain tissue is placed inside the light-tight chamber of a NIRF imager (C) for the visualization of the thrombus (red arrow in D) pre-labeled with the Cy5.5 fluorescent marker C15 before embolic occlusion of the MCA – ACA bifurcation area. Compare to Figure 5 for the CT appearance. Please click here to view a larger version of this figure.
Figure 5: Overview of Quantifying Thrombus Volume using MicroCT Images and ImageJ (1.49d). (A) Importing images into a new stack file, converting the file format into the 8-bit gray scale, and changing the unit to mm. (B) Background subtraction. (C) Segmentation of the thromboembolic lesions by drawing the region of interest (ROI) around the hyperdense areas while excluding bony structures. (D) Acquiring the quantitative data for the ROI by using the menu ‘Analyze > Measure’. Please click here to view a larger version of this figure.
Figure 6: Representative Direct Cerebral Thrombus Imaging Data Acquired Before and After Treating Stroked Mice with Saline vs. Tissue Plasminogen Activator (tPA). (A) microCT (mCT) thrombus imaging to allow therapeutic monitoring of tissue plasminogen activator (tPA)-mediated thrombolysis. (B and C) An excellent correspondence between in vivo CT density (A) and ex vivo (B) near-infrared fluorescence (NIRF; C) for post-tPA residual thromboemboli at 24 hr. (D and E) Less residual thrombi (yellow arrow-heads) on coronal in vivo mCT images of the brain (D) and smaller whitish infarcts on TTC staining of the corresponding section of the removed brain (E) in the tPA-treated animal than in the saline-treated animal. Please see the main text (Representative Results) for a detailed explanation. Please click here to view a larger version of this figure.
We demonstrated the use of two complementary molecular imaging techniques for direct thrombus imaging in experimental models of embolic stroke: a fibrin targeted gold nanoparticle (fib-GC-AuNP) for in vivo microCT-based imaging, and a FXIIIa targeted optical imaging probe for ex vivo fluorescent imaging.
After intravenous administration of fib-GC-AuNPs, thrombi became visible to CT as dense structures, caused by the particles becoming entrapped in the thrombi by the action of the fibrin-targeting peptides (on the surface of the particles) after concentration-gradient-dependent diffusion into the porous structure, i.e., thrombi. This process appeared to occur in one direction only, and rapidly caused the relatively low concentrations of nanoparticle in the blood to become concentrated in the clot, and changing its CT density sufficiently to allow detection. The final in vivo microCT imaging at 24 hr nicely correlated with post-mortem NIRF imaging of the ex vivo brain, showing post-tPA residua of the initial thrombi that had been fluorescently labeled with the optical probe recognizing the fibrin-crosslinking activity of FXIIIa before being placed in the cerebral artery. In this manner, two molecular imaging probes, using different mechanisms of action and different imaging modalities, each report and cross-verify the findings of the other.
The evolution of thrombi could be followed using the same method; specifically, thrombolytic therapy with tPA could be followed in vivo, allowing a rapid and timely readout of the success of thrombolysis to be performed by CT. This has profound implications for clinical thrombolysis, and also has research applications for the development of new anti-thrombotic or thrombolytic therapies.
Previously, our group was able to detect a common complication in stroke therapy, the re-thrombosis3 often occurring around a partially treated clot, with subsequent re-occlusion and worsening outcomes. Re-thrombosis was modeled by applying 1 mm FeCl3-soaked filter papers (grade 42) sequentially on 4 different locations (left distal CCA, left proximal CCA, right distal CCA, and then right proximal CCA) at different times in the same animals. In all cases, circulating fib-GC-AuNP were demonstrated accumulating in the thrombi, and rendering these visible to imaging with CT. This duration of this effect seems to be limited only to the biological half-life of the fib-GC-AuNP, which is currently unknown, and has been observed for an extended time (up to 3 weeks) after the initial injection of fib-GC-AuNP. This is of great interest clinically, because a single dose of nanoparticle agent might prove effective for observing both the success and failures of clinical treatment regimens. Further studies are required to assess the pharmacokinetics and biodistribution of fib-GC-AuNP.
The chemical synthesis of fib-GC-AuNPs has been previously described in detail4. Briefly, fibrin-targeting peptides (tyrosine-D-glutamine-cysteine-hydroxyproline-L-3-chlorotyrosine-glycine-leucine-cysteine-tyrosine-isoleucine-glutamine) used in the EP-2104R magnetic resonance imaging (MRI) agent8 are synthesized by standard solid-phase Fmoc peptide chemistry, and conjugated to the surface of GC-coated AuNPs using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and n-hydroxy succinimide. The final products, purified fib-GC-AuNPs are collected by centrifugation.
The C15 NIRF probe for the optical labeling of preformed thrombus is a 15-amino acid peptide recognized by FXIIIa and labeled on the ε amino groups of lysine residues9,10 with Cy5.5 fluorescent dye. The optical imaging agent is covalently linked to the fibrin strands of the thrombus by the enzymatic action of the activated coagulation factor, when it crosslinks fibrin strands during the process of clot maturation11,12. The chemical synthesis of the C15 NIRF thrombus marker has also been previously described in detail4. Briefly, FXIIIa affinity peptides (glycine-asparagine-alanine-glutamate-glutamine-valine-serine-proline-leucine-threonine-leucine-leucine-lysine-tryptophan) based on the N-terminus of alph-2-antiplasmin are synthesized by solid-phase peptide synthesis and then labeled with Cy5.5 fluorochromes via the cysteine side chain. Using high-performance liquid chromatography, the product is purified and the final compound is collected.
The success of both the NIRF and gold nanoparticle agents is likely because of the strong biological design features of these agents. FXIIIa11 is a fundamentally important thrombin-activated tetrameric transglutaminase, the coagulation enzyme that mediates fibrinolytic resistance. The activity of this enzyme is regarded as a hallmark of newly formed thrombi. Both our agents interact strongly with this fundamental blood coagulation enzyme (by the FXIIIa-sensing C15 optical agent) or its substrate (by the fibrin-targeting fib-GC-AuNP CT agent).
Co-localization studies showed that there was an excellent correspondence between ex vivo fluorescence (exogenous thrombus marker) and in vivo microCT-density (endogenous thrombus marker) for cerebral thrombi imaged with the fibrin-binding nanoparticle, which is expected, because of the mutually related biological entrapment mechanisms for both the agents.
There are several things to consider when using the dual-modal direct thrombus imaging technique. First, to prevent fib-GC-AuNPs from aggregation and to achieve biocompatibility, glycol chitosan is used for surface coating13. Before intravenous injection however, the thrombus imaging agent should be resuspended in PBS, and sonicated to ensure dissolution and dispersion of the nanoparticles. Furthermore, nanoparticle stability can be assessed by using ultraviolet-visible (UV-Vis) absorption spectroscopy to compare surface plasmon resonance peaks of fresh vs. old stocks of fib-GC-AuNPs, both of which should be at about 519 nm.
Second, the resolution of microCT thrombus images can be improved by changing the scanning parameters: such as increasing the 'Scan Number' from 600 to 1,200 slices or increasing the 'Average' from 1 to 3, which will however increase the time required to complete the scan.
Third, blood and C15 NIRF imaging agent were mixed in the ratio of 7:35 for the fluorescent labeling of thrombus in this study. According to our experience, a relatively low blood volume could hamper thrombus formation, whereas a relatively high blood volume could reduce the detectability of thrombus due to the attenuation of ex vivo fluorescent signal emitting from the thrombus. In addition, it is notable that several different approaches can be used for fluorescent visualization of various components of a thrombus14.
Fourth, compared with the FeCl3-mediated in situ thrombosis model where the target artery is only partially occluded, microCT visualization of cerebral thromboemboli that completely occlude the vessel requires relatively high-doses of fib-GC-AuNPs4. Thus, there could be toxicity concerns, although gold colloids are considered to be biocompatible and fib-GC-AuNPs did not show noticeable systemic / neurobehavioral toxicity in a previous study4.
Fifth, clinical trials showed that platelet-targeted or fibrin-targeted gadolinium (Gd)-based MRI agents could increase the hyperintensity of the thrombi in the heart chambers, carotid arteries, and aortic arch on T1-weighted images15. Although MRI has an excellent soft-tissue contrast and a high spatial resolution, MRI is a time-consuming test. CT suffers from relatively poor soft-tissue contrast, however CT scans can be performed rapidly. Accordingly, CT remains the most widely used imaging technique in the acute management of stroke16. Furthermore, microCT systems for small animal imaging do not require architectural radiation shielding to be installed, and offer high-resolution images. Thus, microCT combined with ex vivo reflectance fluorescence imaging or in vivo fluorescence tomography17 is very useful for small animal research. Alternatively, MRI / CT dual-modal imaging using Au-Fe nanoparticles18-20 may also be a powerful platform for thrombus imaging in both animals and humans; MRI and CT could complement each other and, unlike optical imaging, they do not suffer from the low tissue penetration problem.
In conclusion, combined CT and fluorescent imaging for direct thrombus visualization is a powerful research tool for the investigation of ischemic stroke, promising to improve the detection of thrombus, the monitoring of therapy, and the detection of re-thrombosis complicating therapy. In addition, there is significant potential for clinical applications of these techniques.
The authors have nothing to disclose.
This work was supported by the Korea Healthcare Technology R&D Project, Ministry of Health and Welfare (HI12C1847, HI12C0066), the Bio & Medical Technology Development Program (2010-0019862) and Global Research Lab (GRL) program (NRF-2015K1A1A2028228) of the National Research Foundation, funded by the Korean government.
Machines | |||
microCT | NanoFocusRay, JeonJu, Korea | NFR Polaris-G90 | |
NIRF imaging system | Roper-scientific,Tucson, AZ | coolsnap-Ez | |
Laser Doppler flowmeter | Perimed, Stockholm, Sweden | PeriFlux System 5000 | |
Surgical microscope | Leica Microsystems, Seoul, Korea | EZ4HD | |
Inhalation anesthesia machine | PerkinElmer, Massachusetts, USA | XGI-8 | |
Software | |||
NFR control | NanoFocusRay, JeonJu, Korea | NFR Polaris-G90 | microCT control software |
Lucion | Infinitt, Seoul, Korea | Lucion | 3D render imaging software |
Lab chart 7 | ADInstruments, Colorado, USA | Lab chart 7 | rCBF |
Image J software | Wanye Rasband, NIH, USA | 1.49d | imaging analysis |
Devices/Instruments | |||
Infusion pump | Harvard, Massachusetts, USA | pump 22(55-2226) | |
Homeothermic blanket | Panlab, Barcelona, Spain | HB101 | |
Pocket cautery | Daejong, Seoul, Korea | DJE-39 | |
Brain matrice | Ted pella, CA, USA | 15003 | coronal section |
PE-50 tubing | Natsume, Tokyo, Japan | SP-45(PE-50) | I.D. 0.58 mm O.D. 0.96 mm |
PE-10 tubing | Natsume, Tokyo, Japan | SP-10(PE-10) | I.D. 0.28mm O.D. 0.61 mm |
30 gauge needle | sungshim-medical, Seoul, Korea | ||
Syringe | CPL-medical, Ansan, Korea | 1 & 3 cc | |
Gauze | Panamedic, Cheonan, Korea | ||
Tape | Scotch, Seoul, Korea | 3M-810 | |
Micro forceps | Fine Science Tools, Vancouver, Canada | 11253-27 | Dumont #L5 |
Micro scissor | Fine Science Tools, Vancouver, Canada | 15000-03 | Vannas spring |
Scissor | Fine Science Tools, Vancouver, Canada | 14084-08 | 8.5 cm |
Black silk suture | Ailee, Busan, Korea | SK6071, SK728 | 6-0 and 7-0 |
Reagents | |||
meloxicam | Yuhan, Seoul, Korea | ||
vet ointment | Novartis, Basel, Swiss | ||
10% Povidone-iodine (betadine) | Firson, Cheon-an, Korea | ||
FeCl3 | Sigma, Missouri, United States | 157740-5G | |
TTC | Amresco, Ohio, USA | 0765-100g | |
Isoflurane | Hana-Pham, Gyeonggi, Korea | Ifran | 100 mL |
PBS | Welgene, Daegu, Korea | LB001-02 | 500 mL |
Gold nanoparticles | Synthesis | ||
C15 optical agent | Synthesis | ||
Tissue plasminogen activator | Boehringer Ingelheim, Biberach, Germany | rtPA(actilyse) | 20 mg |
Normal saline | Daihan Pham, Seoul, Korea | 48N3AF3 | 20 mL |