Near-infrared fluorescent nanoparticles as combined MR/optical imaging probes

A number of quantitative three-dimensional tomographic near-infrared fluorescence imaging techniques have recently been developed and combined with MR imaging to yield highly detailed anatomic and molecular information in living organisms (1, 2). Here we describe magnetic nanoparticle based MR contrast agents that have a near-infrared fluorescence (NIRF) that is activated by certain enzymes. The probes are prepared by conjugation of arginyl peptides to cross-linked iron oxide amine (amino-CLIO), either by a disulfide linkage or a thioether linker, followed by the attachment of the indocyanine dye Cy5.5. The NIRF of disulfide-linked conjugate was activated by DTT, while the NIRF of thioether-linked conjugate was activated by trypsin. Fluorescent quenching of the attached fluorochrome occurs in part due to the interaction with iron oxide, as evident by the activation of fluorescence with DTT when nanoparticles that have less than one dye attached per particle. With a SC injection of the probe, axillary and brachial lymph nodes were darkened on MR images and easily delineated by NIRF imaging. The probes may provide the basis for a new class of so-called smart nanoparticles, capable of pinpointing their position through their magnetic properties, while providing information on their environment by optical imaging techniques.     

Ratio imaging of enzyme activity using dual wavelength optical reporters

The design of near-infrared fluorescent (NIRF) probes that are activated by specific proteases has, for the first time, allowed enzyme activity to be imaged in vivo. In the current study, we report on a method of imaging enzyme activity using two fluorescent probes that, together, provide improved quantitation of enzymatic activity. The method employs two chemically similar probes that differ in their degradability by cathepsin B. One probe consists of the NIRF dye Cy5.5 attached to a particulate carrier, a crosslinked iron oxide nanoparticle (CLIO), through cathepsin B cleavable L-arginyl peptides. A second probe consists of Cy3.5 attached to a CLIO through proteolytically resistant D-arginyl peptides. Using mixtures of the two probes, we have shown that the ratio of Cy5.5 to Cy3.5 fluorescence can be used to determine levels of cathepsin B in the environment of nanoparticles with macrophages in suspension. After intravenous injection, tissue fluorescence from the nondegradable Cy3.5-D-arginyl probe reflected nanoparticle accumulation, while fluorescence of the Cy5.5-L-arginyl probe was dependent on both accumulation and activation by cathepsin B. Dual wavelength ratio imaging can be used for the quantitative imaging of a variety of enzymes in clinically important settings, while the magnetic properties of the probes allow their detection by MR imaging.     

In vivo imaging of tumors with protease-activated near-infrared fluorescent probes

We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model in vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous injection, the NIRF probe carrier accumulated in solid tumors due to its long circulation time and leakage through tumor neovasculature. Intratumoral NIRF signal was generated by lysosomal proteases in tumor cells that cleave the macromolecule, thereby releasing previously quenched fluorochrome. In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters. This strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.     

In vivo NIRF imaging-guided delivery of a novel NGR–VEGI fusion protein for targeting tumor vasculature

Pathological angiogenesis is crucial in tumor growth, invasion and metastasis. Previous studies demonstrated that the vascular endothelial growth inhibitor (VEGI), a member of the tumor necrosis factor superfamily, can be used as a potent endogenous inhibitor of tumor angiogenesis. Molecular probes containing the asparagine–glycine–arginine (NGR) sequence can specifically bind to CD13 receptor which is overexpressed on neovasculature and several tumor cells. Near-infrared fluorescence (NIRF) optical imaging for targeting tumor vasculature offers a noninvasive method for early detection of tumor angiogenesis and efficient monitoring of response to anti-tumor vasculature therapy. The aim of this study was to develop a new NIRF imaging probe on the basis of an NGR–VEGI protein for the visualization of tumor vasculature. The NGR–VEGI fusion protein was prepared from prokaryotic expression, and its function was characterized in vitro. The NGR–VEGI protein was then labeled with a Cy5.5 fluorophore to afford Cy5.5-NGR–VEGI probe. Using the NIRF imaging technique, we visualized and quantified the specific delivery of Cy5.5-NGR–VEGI protein to subcutaneous HT-1080 fibrosarcoma tumors in mouse xenografts. The Cy5.5-NGR–VEGI probe exhibited rapid HT-1080 tumor targeting, and highest tumor-to-background contrast at 8 h post-injection (pi). Tumor specificity of Cy5.5-NGR–VEGI was confirmed by effective blocking of tumor uptake in the presence of unlabeled NGR–VEGI (20 mg/kg). Ex vivo NIRF imaging further confirmed in vivo imaging findings, demonstrating that Cy5.5-NGR–VEGI displayed an excellent tumor-to-muscle ratio (18.93 ± 2.88) at 8 h pi for the non-blocking group and significantly reduced ratio (4.92 ± 0.75) for the blocking group. In conclusion, Cy5.5-NGR–VEGI provided highly sensitive, target-specific, and longitudinal imaging of HT-1080 tumors. As a novel theranostic protein, Cy5.5-NGR–VEGI has the potential to improve cancer treatment by targeting tumor vasculature.     

Near-infrared imaging of flare-up arthritis with native fluorochrome Cy5.5 and albumin-bound Cy5.5

The aim of the study was to visualize chronic experimental arthritis with near-infrared fluorescence imaging (NIRF) in a murine experimental arthritis model of rheumatoid arthritis (RA) (flare-up arthritis). The flare-up arthritis model is a modification of the primary antigen-induced arthritis (AIA) model. NIRF was done for two preparations of the fluorochrome Cy5.5, one native and the other albumin conjugated. Histological features of flare-up arthritis were evaluated.AIA was induced in 16 mice (strain C57/Bl6); flare-up arthritis was induced in a subgroup of eight. On day 7 after induction of flare-up arthritis, four mice received 50 nmol/kg native dye and four mice equimolar concentrations of the dye as albumin-dye conjugate intravenously. NIRF imaging was performed immediately before injection (baseline) and until 72 h thereafter. Arthritis severity was evaluated histologically for primary AIA and flare-up arthritis mice.NIRF imaging revealed higher fluorochrome uptake in all inflamed knees compared to contralateral ones. The signal intensities induced by native Cy5.5 were higher than those generated by albumin-Cy5.5 conjugate. Histological evaluation of arthritic joints showed similar abnormalities in flare-up arthritis and in primary AIA joints.Imaging of flare-up arthritis in the near-infrared range was successful for both fluorochrome preparations, but albumin conjugation prior to injection does not improve the uptake of dye in arthritic joints. Flare-up arthritis is a feasible model of chronic relapse of arthritis in human RA.     

Developing a peptide-based near-infrared molecular probe for protease sensing

Recently near-infrared (NIR) molecular probes have become important reporter molecules for a number of types of in vivo biomedical imaging. A peptide-based NIR fluorescence probe consisting of a NIR fluorescence emitter (Cy5.5), a NIR fluorescence absorber (NIRQ820), and a protease selective peptide sequence was designed to sense protease activity. Using a MMP-7 model, we showed that NIRQ820 efficiently absorbs the emission energy of Cy5.5 resulting in a low initial signal. Upon reacting with its target, MMP-7, the fluorescence signal of the designed probe was increased by 7-fold with a K(cat)/K(m) of 100 000 M(-)(1) s(-)(1). The described synthetic strategy should have wide application for other NIR probe preparations.     

Preparation of a cathepsin D sensitive near-infrared fluorescence probe for imaging.

A variety of proteases are overexpressed or activated during pathogenesis and represent important targets for therapeutic drugs. We have previously shown that optical imaging probes sensitive in the near-infrared fluorescence (NIRF) spectrum can be used for in vivo imaging of enzyme activity. In the current study, we show that these probes can be designed with specificity for specific enzymes, for example, cathepsin D which is known to be overexpressed in many tumors. A NIR cyanine fluorochrome served as the optical reporter and was attached to the amino terminal of an 11 amino acid peptide sequence with specificity for cathepsin D. The peptides were subsequently attached to a synthetic graft copolymer for efficient tumoral delivery. The close spatial proximity of the multiple fluorochromes resulted in quenching of fluorescence in the bound state. A 350-fold signal amplification was observed post cleavage during in vitro testing. Cell culture experiments using a rodent tumor cell line stably transfected with human cathepsin D confirmed enzyme specific activation within cells. This sequence but not a scrambled control sequence showed enzyme specificity in vitro. We conclude that activatable NIRF optical probes can be synthesized to potentially probe for specific enzymes in living organisms.     

Noninvasive near-infrared imaging of fluorochromes within the brain of live mice: an in vivo phantom study

Near-infrared fluorescence (NIRF) imaging has great potential for studying physiological and pathophysiological processes noninvasively in several locations of the body. In this study, we evaluated the feasibility of NIRF imaging to visualize fluorescent compounds within the brains of live mice commonly used in brain research. To simulate the presence of a molecular NIRF reporter agent at the site of a lesion, we developed a new in vivo phantom model wherein capsules containing different amounts of an NIRF dye (Cy5.5) were stereotactically implanted deep into the left hemispheres of living mice. To precisely locate the implanted capsules, magnetic resonance imaging (MRI) was performed. Fluorescence reflectance imaging (FRI) and transillumination fluorescence imaging (TFI) were conducted to analyze and compare sensitivity and target-to-background ratios of the two methods. The sensitivities of FRI and TFI to background fluorescence from circulating dye was tested by imaging fluorescent capsules in mice intravenously injected with increasing amounts of long-circulating Cy5.5-dextran. The results show that capsules containing dye amounts as low as 10(-12) mol can be detected. TFI yielded significantly higher target-to-background ratios than FRI at 10(-11) mol (p < .05). Comparatively low amounts of fluorescence in the blood vessels can extinguish the signal. We conclude that keeping the signal from circulating NIRF dye low, NIRF imaging offers high sensitivity in detecting fluorochromes noninvasively within brains of mice, especially by using TFI. This encourages the application of NIRF for molecular imaging in the mouse brain using NIRF reporters.     

Oleyl-chitosan nanoparticles based on a dual probe for optical/MR imaging in vivo

Oleic acid-conjugated chitosan (oleyl-chitosan) is a powerful platform for encapsulating oleic acid-decorated iron oxide nanoparticles (ION), resulting in a good magnetic resonance imaging (MRI) probe. Oleyl-chitosan could self-assemble into core-shell structures in aqueous solution and provide the effective core compartment for loading ION. ION-loaded oleyl-chitosan nanoparticles showed good enhanced MRI sensitivity in a MR scanner. Cy5.5 dye was accessed to the oleyl-chitosan conjugate for near-infrared (NIR) in vivo optical imaging. After intravenous injection of ION-loaded Cy5.5-conjugated oleyl-chitosan (ION-Cy5.5-oleyl-chitosan) nanoparticles in tumor-bearing mice, both NIRF and MR imaging showed the detectable signal intensity and enhancement in tumor tissues via enhanced permeability and retention (EPR) effect. Tumor accumulation of the nanoparticles was confirmed through ex vivo fluorescence images and Prussian blue staining images in tumor tissues. It is concluded that ION-Cy5.5-oleyl-chitosan nanoparticle is highly an effective imaging probe for detecting tumor in vivo.     

Magneto/optical annexin V, a multimodal protein

Multimodal proteins, or proteins labeled with both fluorescent and magnetic reporter groups, can be used in a wide range of applications including FACS or fluorescence microscopy, MRI and or near-infrared based optical imaging, or to fractionate cells by magnetic cell sorting. A problem with multimodal proteins, however, is the need to maximize bioactivity, often achieved by minimizing the number of modification points of the protein, while attaching fluorescent and magnetic labels. Here we describe the synthesis of a magneto/optical form of annexin V, achieved by reacting the amino-CLIO nanoparticle with Cy5.5 and SPDP, to produce a fluorescent, sulfhydryl reactive nanoparticle. A single reactive sulfhydryl group was added to annexin V by reaction with SATA that preserved the protein's ability to bind apoptotic Jurkat T cells. Reacting SATAylated annexin V with an SPDP activated nanoparticle yielded Anx-CLIO-Cy5.5, a magneto/optical form of annexin V. The binding of Anx-CLIO-Cy5.5 was specific for apoptotic Jurkat T cells and had an EC(50) of 3.66 nM. This was comparable to the strength of the interaction of unmodified annexin V with apoptotic cells, measured as the displacement of FITC-annexin by annexin V (2.4 nM). Our conjugation strategy preserves the strength of the interaction between annexin V and apoptotic cells, while yielding a probe, Anx-CLIO-Cy5.5, that is readily detectable by standard MR imaging or NIRF optical methods.     

Near-infrared fluorescence imaging of CD13 receptor expression using a novel Cy5.5-labeled dimeric NGR peptide

In this study, we synthesized a novel Cy5.5-labeled dimeric NGR peptide (Cy5.5-NGR2) via bioorthogonal click chemistry, and evaluated the utility of Cy5.5-NGR2 for near-infrared fluorescence imaging of CD13 receptor expression in vivo. The dimeric NGR peptide (NGR2) was conjugated with an alkyne-containing PEG unit followed by mixing with an azide-terminated Cy5.5 fluorophore (Cy5.5-N₃) to afford Cy5.5-NGR2. The probe was subject to in vitro and in vivo evaluations. The bioorthogonal click chemistry provided a rapid conjugation of the alkyne-containing NGR2 with Cy5.5-N₃ in a quantitative yield within 15 min. The laser confocal microscopy revealed that binding of Cy5.5-NGR2 to CD13 receptor is target-specific as demonstrated in CD13-positive HT-1080 cells, CD13-negative MCF-7 cells, and a blocking study in HT-1080 cells. For in vivo optical imaging, Cy5.5-NGR2 exhibited rapid HT-1080 tumor targeting at 0.5 h postinjection (pi), and highest tumor-to-background contrast at 2 h pi. The CD13-specific tumor accumulation of Cy5.5-NGR2 was accomplished by a blocking study with unlabeled NGR peptide in HT-1080 tumor bearing mice. The tumor-to-muscle ratio of Cy5.5-NGR2 at 2 h pi reached 2.65 ± 0.13 in the non-blocking group vs. 1.05 ± 0.06 in the blocking group. The results from ex vivo imaging were consistent with the in vivo findings. We concluded that Cy5.5-NGR2 constructed by bioorthogonal click chemistry is a promising molecular probe, not only allowing the NIR optical imaging of CD13 overexpressed tumors, but also having the potential to facilitate noninvasive monitoring of CD13-targeted tumor therapy.     

In vivo time domain optical imaging of renal ischemia-reperfusion injury: discrimination based on fluorescence lifetime

Fluorescence lifetime is an intrinsic parameter of the fluorescent probe, independent of the probe concentration but sensitive to changes in the surrounding microenvironment. Therefore, fluorescence lifetime imaging could potentially be applied to in vivo diagnostic assessment of changes in the tissue microenvironment caused by disease, such as ischemia. The aim of this study was to evaluate the utility of noninvasive fluorescence lifetime imaging in distinguishing between normal and ischemic kidney tissue in vivo. Mice were subjected to 60-minute unilateral kidney ischemia followed by 6-hour reperfusion. Animals were then injected with the near-infrared fluorescence probe Cy5.5 or saline and imaged using a time-domain small-animal optical imaging system. Both fluorescence intensity and lifetime were acquired. The fluorescence intensity of Cy5.5 was clearly reduced in the ischemic compared with the contralateral kidney, and the fluorescence lifetime of Cy5.5 was not detected in the ischemic kidney, suggesting reduced kidney clearance. Interestingly, the two-component lifetime analysis of endogenous fluorescence at 700 nm distinguished renal ischemia in vivo without the need for Cy5.5 injection for contrast enhancement. The average fluorescence lifetime of endogenous tissue fluorophores was a sensitive indicator of kidney ischemia ex vivo. The study suggests that fluorescence lifetime analysis of endogenous tissue fluorophores could be used to discriminate ischemic or necrotic tissues by noninvasive in vivo or ex vivo organ imaging.     

Spectroscopically well-characterized RGD optical probe as a prerequisite for lifetime-gated tumor imaging

Labeling of RGD peptides with near-infrared fluorophores yields optical probes for noninvasive imaging of tumors overexpressing ανβ3 integrins. An important prerequisite for optimum detection sensitivity in vivo is strongly absorbing and highly emissive probes with a known fluorescence lifetime. The RGD-Cy5.5 optical probe was derived by coupling Cy5.5 to a cyclic arginine-glycine-aspartic acid-d-phenylalanine-lysine (RGDfK) peptide via an aminohexanoic acid spacer. Spectroscopic properties of the probe were studied in different matrices in comparison to Cy5.5. For in vivo imaging, human glioblastoma cells were subcutaneously implanted into nude mice, and in vivo fluorescence intensity and lifetime were measured. The fluorescence quantum yield and lifetime of Cy5.5 were found to be barely affected on RGD conjugation but dramatically changed in the presence of proteins. By time domain fluorescence imaging, we demonstrated specific binding of RGD-Cy5.5 to glioblastoma xenografts in nude mice. Discrimination of unspecific fluorescence by lifetime-gated analysis further enhanced the detection sensitivity of RGD-Cy5.5-derived signals. We characterized RGD-Cy5.5 as a strongly emissive and stable probe adequate for selective targeting of ανβ3 integrins. The specificity and thus the overall detection sensitivity in vivo were optimized with lifetime gating, based on the previous determination of the probes fluorescence lifetime under application-relevant conditions.     

A Cy5.5-labeled phage-displayed peptide probe for near-infrared fluorescence imaging of tumor vasculature in living mice

Near-infrared (NIR) fluorescence optical imaging is an emerging imaging technique for studying diseases at the molecular level. Optical imaging with a NIR emitting fluorophore for targeting tumor vasculature offers a noninvasive method for early detection of tumor angiogenesis and efficient monitoring of response to anti-tumor vasculature therapy. The previous in vitro results demonstrated that the GX1 peptide, identified by phage-display technology, is a tumor vasculature endothelium-specific ligand. In this report, Cy5.5-conjugated GX1 peptide was evaluated in a subcutaneous U87MG glioblastoma xenograft model to investigate tumor-targeting efficacy. The in vitro flow cytometry results revealed dose-dependent binding of Cy5.5-GX1 peptide to U87MG glioma cells. In vivo optical imaging with the Cy5.5-GX1 probe exhibited rapid U87MG tumor targeting at 0.5 h p.i., and high tumor-to-background contrast at 4 h p.i. Tumor specificity of Cy5.5-GX1 was confirmed by effective blocking of tumor uptake in the presence of unlabeled GX1 peptide (20 mg/kg). Ex vivo imaging further confirmed in vivo imaging findings, and demonstrated that Cy5.5-GX1 has a tumor-to-muscle ratio (15.21 ± 0.84) at 24 h p.i. for the non-blocked group and significantly decreased ratio (6.95 ± 0.75) for the blocked group. In conclusion, our studies suggest that Cy5.5-GX1 is a promising molecular probe for optical imaging of tumor vasculature.     

A novel near-infrared fluorescence imaging probe for in vivo neutrophil tracking

The development and validation of a multiscopic near-infrared fluorescence (NIRF) probe, cinnamoyl-F-(D)L-F-(D)L-F-PEG-cyanine7 (cFlFlF-PEG-Cy7), that targets formyl peptide receptor on neutrophils using a mice ear inflammation model is described. Acute inflammation was induced in mice by topical application of phorbol-12-myristate-13-acetate to left ears 24 hours before probe administration. Noninvasive NIRF imaging was longitudinally performed up to 24 hours following probe injection. The in vivo neutrophil-targeting specificity of the probe was characterized by a blocking study with preadministration of excess nonfluorescent peptide cFlFlF-PEG and by an imaging study with a scrambled peptide probe cLFFFL-PEG-Cy7. NIRF imaging of mice injected with cinnamoyl-L-F-F-F-L-PEG-cyanine7 (cFlFlF-PEG-Cy7) revealed that the fluorescence intensity for inflamed left ears was approximately fourfold higher than that of control right ears at 24 hours postinjection. In comparison, the ratios acquired with the scrambled probe and from the blocking study were 1.5- and 2-fold at 24 hours postinjection, respectively. Moreover, a microscopic immunohistologic study confirmed that the NIRF signal of cFlFlF-PEG-Cy7 was associated with activated neutrophils in the inflammatory tissue. With this probe, in vivo neutrophil chemotaxis could be correlatively imaged macroscopically in live animals and microscopically at tissue and cellular levels.     

A targeted low molecular weight near-infrared fluorescent probe for prostate cancer

Prostate-specific membrane antigen (PSMA) remains an active target for imaging and therapeutic applications for prostate cancer. Although radionuclide-based imaging is generally more sensitive and also has been deeply explored, near-infrared fluorescence imaging agents are simple to prepare and compatible with long-term storage conditions. In the present study, a near-infrared fluorescent imaging probe (Cy5.5-CTT-54.2) has been developed by chemical conjugation of Cy5.5N-hydroxysuccinimide ester (Cy5.5-NHS) with a potent PSMA inhibitor CTT-54.2 (IC(50)=144 nM). The probe displays a highly potency (IC(50)=0.55 nM) against PSMA and has demonstrated successful application for specifically labeling PSMA-positive prostate cancer cells in both two and three-dimensional cell culture conditions. These results suggest that the potent, near-infrared Cy5.5-PSMA inhibitor conjugate may be useful for the detection of prostate tumor cells by optical in vivo imaging.     

Pan and sentinel lymph node visualization using a near-infrared fluorescent probe

A number of different types of agents have been employed to aid in the visualization of lymph nodes, particularly the sentinel lymph node, and to decrease the tissue destruction associated with the diagnosis of nodal metastases. The current study was performed to see if a novel macromolecular near-infrared fluorescent (NIRF) probe could be used to visualize lymph nodes after intravenous administration (pan-node visualization) or subcutaneous administration (sentinel node visualization), and serve as method for guiding dissection with interventional radiologic and surgical procedures. Cy5.5-PGC, the near-infrared dye Cy5.5 coupled to a protected graft copolymer (PGC), was injected (i.v. or s.c.) into nude mice. Twenty-four hours later white light and NIRF images were obtained on (i) the live animal, (ii) a partially dissected animal, and (iii) tissue specimens. With Cy5.5-PGC administered intravenously, axillary nodes were visualized from outside a living mouse. With partial dissection, iliac and aortic nodes were visible as concentrated foci of high-intensity NIRF signals. With subcutaneous injection in the front extremity, axillary and brachial nodes draining the injection site were easily visualized. NIRF imaging provides a nonradioactive method of visualizing lymph nodes through layers of tissue that can be employed with intravenous or subcutaneous injection.     

Activatable Near-Infrared Fluorescent Probe for In Vivo Imaging of Fibroblast Activation Protein-alpha

Fibroblast activation protein-alpha (FAPα) is a cell surface glycoprotein which is selectively expressed by tumor-associated fibroblasts in malignant tumors but rarely on normal tissues. FAPα has also been reported to promote tumor growth and invasion and therefore has been of increasing interest as a promising target for designing tumor-targeted drugs and imaging agents. Although medicinal study on FAPα inhibitors has led to the discovery of many FAPα-targeting inhibitors including a drug candidate in a phase II clinical trial, the development of imaging probes to monitor the expression and activity of FAPα in vivo has largely lagged behind. Herein, we report an activatable near-infrared (NIR) fluorescent probe (ANP(FAP)) for in vivo optical imaging of FAPα. The ANP(FAP) consists of a NIR dye (Cy5.5) and a quencher dye (QSY21) which are linked together by a short peptide sequence (KGPGPNQC) specific for FAPα cleavage. Because of the efficient fluorescence resonance energy transfer (FRET) between Cy5.5 and QSY21 in ANP(FAP), high contrast on the NIR fluorescence signal can be achieved after the cleavage of the peptide sequence by FAPα both in vitro and in vivo. In vitro assay on ANP(FAP) indicated the specificity of the probe to FAPα. The in vivo optical imaging using ANP(FAP) showed fast tumor uptake as well as high tumor to background contrast on U87MG tumor models with FAPα expression, while much lower signal and tumor contrast were observed in the C6 tumor without FAPα expression, demonstrating the in vivo targeting specificity of the ANP(FAP). Ex vivo imaging also demonstrated ANP(FAP) had high tumor uptake at 4 h post injection. Collectively, these results indicated that ANP(FAP) could serve as a useful NIR optical probe for early detection of FAPα expressing tumors.     

In vivo near-infrared imaging of fibrin deposition in thromboembolic stroke in mice

Objectives

Thrombus and secondary thrombosis plays a key role in stroke. Recent molecular imaging provides in vivo imaging of activated factor XIII (FXIIIa), an important mediator of thrombosis or fibrinolytic resistance. The present study was to investigate the fibrin deposition in a thromboembolic stroke mice model by FXIIIa–targeted near-infrared fluorescence (NIRF) imaging.

Materials and Methods

The experimental protocol was approved by our institutional animal use committee. Seventy-six C57B/6J mice were subjected to thromboembolic middle cerebral artery occlusion or sham operation. Mice were either intravenously injected with the FXIIIa-targeted probe or control probe. In vivo and ex vivo NIRF imaging were performed thereafter. Probe distribution was assessed with fluorescence microscopy by spectral imaging and quantification system. MR scans were performed to measure lesion volumes in vivo, which were correlated with histology after animal euthanasia.

Results

In vivo significant higher fluorescence intensity over the ischemia-affected hemisphere, compared to the contralateral side, was detected in mice that received FXIIIa-targeted probe, but not in the controlled mice. Significantly NIRF signals showed time-dependent processes from 8 to 96 hours after injection of FXIIIa-targeted probes. Ex vivo NIRF image showed an intense fluorescence within the ischemic territory only in mice injected with FXIIIa-targeted probe. The fluorescence microscopy demonstrated distribution of FXIIIa-targeted probe in the ischemic region and nearby micro-vessels, and FXIIIa-targeted probe signals showed good overlap with immune-fluorescent fibrin staining images. There was a significant correlation between total targeted signal from in vivo or ex vivo NIRF images and lesion volume.

Conclusion

Non-invasive detection of fibrin deposition in ischemic mouse brain using NIRF imaging is feasible and this technique may provide an in vivo experimental tool in studying the role of fibrin in stroke.