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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Arterioscler Thromb Vasc Biol. Author manuscript; available in PMC Nov 1, 2013.
Published in final edited form as:
PMCID: PMC3516622
NIHMSID: NIHMS414555

Inflammation Modulates Murine Venous Thrombosis Resolution In Vivo: Assessment by Multimodal Fluorescence Molecular Imaging

Crystal M. Ripplinger, Ph.D.,*,1,2 Chase W. Kessinger, Ph.D.,*,1 Chunqiang Li, Ph.D.,3 Jin Won Kim, M.D., Ph.D.,1,4 Jason R. McCarthy, Ph.D.,5,6 Ralph Weissleder, M.D., Ph.D.,5,6 Peter K. Henke, M.D.,7 Charles P. Lin, Ph.D.,3,6 and Farouc A. Jaffer, M.D., Ph.D.1,3,5

Abstract

Objective

Assessment of thrombus inflammation in vivo could provide new insights into deep vein thrombosis (DVT) resolution. Here we develop and evaluate two integrated fluorescence molecular-structural imaging strategies to quantify DVT-related inflammation and architecture, and to assess the effect of thrombus inflammation on subsequent DVT resolution in vivo.

Methods and Results

Murine DVT were created with topical 5% FeCl3 application to thigh or jugular veins (n=35). On day 3, mice received macrophage and matrix metalloproteinase (MMP) activity fluorescence imaging agents. On day 4, integrated assessment of DVT inflammation and architecture was performed using confocal fluorescence intravital microscopy (IVM). Day 4 analyses showed robust relationships among in vivo thrombus macrophages, MMP activity, and FITC-dextran deposition (r>0.70, p<0.01). In a serial two-timepoint study, mice with DVT underwent IVM at day 4 and at day 6. Analyses revealed that the intensity of thrombus inflammation at day 4 predicted the magnitude of DVT resolution at day 6 (p<0.05). In a second approach, noninvasive fluorescence molecular tomography-computed tomography (FMT-CT) was employed, and detected macrophages within jugular DVT (p<0.05 vs. sham-controls).

Conclusions

Integrated fluorescence molecular-structural imaging demonstrates that the DVT-induced inflammatory response can be readily assessed in vivo, and can inform the magnitude of thrombus resolution.

Keywords: deep vein thrombosis, inflammation, molecular imaging, intravital microscopy, post-thrombotic syndrome

INTRODUCTION

Deep vein thrombosis (DVT) occurs in over 350,000 patients annually in the United States.1 In addition to the acute risk of pulmonary embolism, up to 50% of all DVT patients will develop the post-thrombotic syndrome (PTS), despite appropriate anti-coagulant therapy.2 PTS symptoms include pain, heaviness, swelling and cramping in the affected limb that are frequently aggravated by standing and walking. In advanced cases extensive varicose vein and acute venous ulcers can form. PTS occurs after incomplete DVT resolution and DVT-induced vein wall and valve damage, leading to venous hypertension, valvular incompetence, and chronic inflammation.

It is now established that venous thrombosis is an inflammatory process,3-5 with accumulating evidence demonstrating that inflammation orchestrates DVT resolution.6-11 At present however, minimal data are available regarding in vivo spatiotemporal measures of leukocyte influx and inflammatory mediators, and their subsequent effects on DVT resolution. Furthermore, it is not known whether the extent of inflammation can predict the magnitude of subsequent DVT resolution. Therefore the development and application of in vivo inflammation molecular imaging approaches12-14 could provide new insights into DVT resolution, and inform the future development of PTS.

In this experimental murine investigation, we harness multispectral and multimodal fluorescence molecular imaging to quantify the in vivo inflammatory response elicited by subacute DVT, and then assess the impact of DVT inflammation on subsequent DVT resolution. We first validate and then utilize multi-wavelength intravital microscopy (IVM) to simultaneously assess thrombus macrophages, matrix metalloproteinase (MMP) activity, and thrombus architecture in resolving venous thrombi. We then apply serial, two-timepoint IVM to investigate whether the initial intensity of the inflammatory response in FeCl3-induced DVT predicts the magnitude of DVT resolution in vivo. In a translational study, we assess whether noninvasive integrated fluorescence molecular tomography-computed tomography (FMT-CT) can detect inflammatory macrophages in subacute jugular DVT.

METHODS

IVM imaging system and image analysis, IVM FITC-dextran time course experiments, histological image analysis, correlative histopathology, and fluorescence microscopy are detailed in the Supplemental data file.

Experimental Venous Thrombosis

The Subcommittee on Research Animal Care at Massachusetts General Hospital approved all animal studies. Male C57Bl/6 mice (n=35 total, 26 for thigh IVM studies, 9 for jugular FMT-CT/FRI studies) were obtained from Jackson Laboratory (Bar Harbor, ME) and were 12-16 weeks of age at the time of study (Figure 1). DVT were created using topical ferric chloride (FeCl3)15,16 to induce vessel wall injury and venous thrombosis in the thigh containing the distal femoral vein and the proximal saphenous vein. At Day 0, mice were anesthetized with an intraperitoneal (IP) injection of ketamine and xylazine (80/12 mg/kg). For the thigh vein thrombosis model, a midline skin incision was made and vessels were gently exposed by blunt dissection. A 1x2 mm strip of #1 filter paper (Whatman, Inc.) was soaked in 5% FeCl3 and applied to the anterior surface of the femoral/saphenous vein for 5 minutes. After removal of the filter paper, the area was thoroughly rinsed with sterile saline and the surgical incision was sutured closed.

Figure 1
Experimental model of venous thrombosis and study protocol. (A) Diagram of mouse femoral/saphenous bundle with example images of vein thrombus induction using 5% FeCl3 soaked filter paper. Images shown are at 15 minutes post-induction at Day 0, then at ...

A similar procedure was followed to create jugular vein thrombi. After blunt dissection of the jugular vein, a 1 mm wide strip of filter paper soaked in 7.5% FeCl3 was placed on the anterior surface of the vein for 3 minutes. The area was then rinsed with sterile saline and the surgical incision was sutured closed. Sham surgery with topical saline was also performed on the contralateral jugular vein. In all mice, buprenorphine analgesia (0.05-0.1 mg/kg IP) was administered 30 minutes prior to induction of anesthesia and every 12 hours post-procedure.

Fluorescent Imaging Agents

Crosslinked dextran-coated magnetofluorescent nanoparticles (MFNP) were harnessed for imaging of macrophages.17, 18 Briefly, crosslinked iron oxide (CLIO) nanoparticles were obtained from the Center for Systems Biology Chemistry Core at Massachusetts General Hospital. To 20 mg of CLIO (9.98 mg Fe/mL) was added 1 mg of AlexaFluor 555 (λmax absorption = 555 nm, λmax emission = 565 nm, AF555, Invitrogen, Carlsbad, CA) or, for FMT studies, 1 mg of VivoTag 680 (λmax absorption = 670 nm, λmax emission = 688 nm, VT680, PerkinElmer, Waltham, MA) in 200 μL dimethylsulfoxide. The reaction was allowed to proceed for 16 hours, at which time it was filtered through Sephadex G25 to yield the MFNP (CLIO-AF555, 3.6 mg Fe/mL, 1.8×10−04 M AF555, CLIO-VT680, 4.7 mg Fe/mL, 1.0×10−04 M VT680).

MMP activity within the thrombus was imaged using a protease-activatable near-infrared fluorescence reporter (MMPSense680, ex/em 680/700 nm, PerkinElmer). MMPSense is optically silent upon injection and becomes highly fluorescent following gelatinase MMP-mediated activation.19, 20

Fluorescein isothiocyanate (FITC)-modified dextran (FITC-dextran, 0.5 mg in 100 μL PBS, λmax absorption = 490 nm, λmax emission = 520 nm, MW: 2,000,000, Sigma Chemical, St. Louis, MO), injected immediately prior to IVM, enabled vessel angiography and thrombus anatomical imaging. Tetramethylrhodamine (TMR)-modified dextran (TMR-dextran, 0.5 mg in 100 μL PBS, λmax absorption = 557 nm, λmax emission = 576 nm, MW: 2,000,000, Sigma Chemical, St. Louis, MO) was also utilized in IVM validation experiments.

Confocal Intravital Fluorescence Microscopy (IVM)

For intravital fluorescence microscopy (IVM) studies, N=16 mice were injected on day 3 with the macrophage sensor CLIO-AF555 (10 mg Fe/kg). Twelve of the sixteen CLIO-AF555 injected mice were additionally injected with MMPSense680 (150 nmol/kg) dissolved in sterile phosphate buffered saline. Macrophage and MMP activity agents were injected 24 hours prior to imaging. Additional uninjected mice with DVT (N=3) served as IVM controls.

All mice were injected with the angiographic agent FITC-dextran as described above. To determine whether FITC-dextran would deposit into thrombi in vivo, a set of serial IVM time course experiments were performed (see Supplemental methods).

In the first IVM imaging study, mice underwent IVM once at day 4 (N=16, 10 injected with inflammation imaging agents, 6 uninjected), followed by sacrifice and histological assessment. In a second IVM imaging experiment, mice underwent serial IVM once on day 4 and then once on day 6 (N=10, N=6 for two timepoint inflammation imaging, and N=4 for FITC-dextran time course imaging).

During the IVM procedure, the thigh vessels of anesthetized mice were exposed and the thrombus region was visually identified in the femoral/saphenous vein. The area was then bathed in sterile saline at room temperature, a coverglass was placed on the vessel surface, and the mouse was placed on the stage of a custom-built laser scanning intravital confocal microscope.21

See supplement for details on the IVM imaging system and IVM image analysis methodology.

Fluorescence reflectance imaging (FRI)

To image macroscopic DVT inflammation, N=9 mice with subacute jugular DVT underwent ex vivo FRI studies. At day 3 post-DVT, mice were injected via tail vein with the near-infrared macrophage reporter CLIO-VT680 (10 mg Fe/kg, MGH-CSB, N=6) or phosphate buffered saline (N=3). On day 4, mice were sacrificed and both jugular veins were carefully dissected, rinsed in sterile phosphate buffered saline, and placed on the stage of a FRI system (Kodak Image Station 4000MMPro, Carestream Health Inc, New Haven, CT). Optical images at 630/700 nm excitation/emission were collected with 42x42 micron spatial resolution and 8 second exposure time. For these images, the target-to-background ratio (TBR) was calculated as mean thrombus signal/mean sham signal, and therefore represents number-fold change over sham-operated veins.

Fluorescence Molecular Tomography-Computed Tomography (FMT-CT)

Prior to FRI, the six mice that received CLIO-VT680 underwent noninvasive in vivo FMT-CT imaging of jugular DVT on day 4. FMT was performed at 680nm/700nm excitation/emission wavelengths with 1 mm isotropic resolution using an FMT 2500 system (PerkinElmer) as previously described.22 Briefly, anesthetized mice were placed in a multi-modal imaging cartridge that allows for co-registration of FMT and CT data through the use of multi-modal fiducial markers. FMT imaging took approximately 5-8 minutes. Immediately following FMT, CT angiography was performed to map the jugular veins (Inveon PET-CT, Siemens). The 3D CT spatial resolution was 80 μm. To visualize the lumen of jugular veins, mice were continuously infused with the radiopaque contrast agent Isovue-370 (Bracco Diagnostics) at 50 μL/min via tail vein. CT imaging took approximately 10 minutes. Reconstructed FMT and CT images were imported into OsiriX software to co-register and create fusion images. After FMT-CT, resected jugular veins were imaged by ex vivo FRI, as described above.

Statistical Analysis

All results are reported as mean±SD. Correlation coefficients were determined using linear regression and calculating Pearson’s correlation coefficient (r). Differences between thrombus area and length at day 4 and day 6 were assessed using the paired Students’ t-test. Differences in thrombus length and area measurements in the FITC-dextran time course experiments were assessed using ANOVA. A p-value <0.05 was considered statistically significant. To assess rate agreement for the IVM measurement of thrombus length, the intraclass correlation coefficient (ICC, one-way random effect model) was calculated using IBM SPSS Statistics version 21. All other statistical analyses were performed using Prism (v 5.0c, GraphPad, La Jolla, CA).

RESULTS

In Vivo Assessment of Inflammation in Subacute DVT

Inflammatory macrophages and MMP activity in day 4 murine DVT were visualized via IVM and the spectrally distinct fluorescence imaging reporters CLIO-AF555 and MMPSense-680, respectively. Prior experimental work suggests the day 4 timepoint as the transition point of polymorphonuclear leukocyte to monocyte/macrophage influx.3, 23 Vessel angiograms, obtained with FITC-dextran, provided high-resolution images of dark thrombi surrounded by bright luminal signal intensity (Figure 2). The average thrombus length was 1.09±0.27 mm. Macrophage and MMP activity IVM signals were detectable to a depth of approximately 90 μm from the top of the vessel (Supplemental Figure 1), with heterogeneous signal intensity noted throughout the thrombus. Elevated inflammatory signals were noted often along the thrombus edges (Figures 2,,33).

Figure 2
Representative multi-wavelength IVM of FeCl3-induced murine thigh venous thrombosis. (A) FITC-dextran angiogram (ANGIO, grayscale) alone and then overlaid onto thrombus macrophages illuminated by CLIO-AF555 uptake (MAC, green), or onto MMP activity generated ...
Figure 3
Representative histological and fluorescence microscopy of targeted imaging agents in venous thrombosis. Adjacent axial tissue sections of venous thrombus were stained with H&E (left column), evaluated for fluorescence microscopy (center column), ...

The average macrophage and MMP activity target-to-background ratios (TBR), calculated by encompassing the entire thrombus area, were 2.07±0.51, and 1.21±0.27, respectively. The CLIO-AF555 macrophage signal reflected the signal above the circulating background, which is typically low 24 hours after injection of CLIO 10 mg/kg.20 Although the MMP TBR was just slightly greater than 1, it was significantly greater than the TBR of uninjected mice in the 680nm channel (TBR uninjected: 0.70±0.18, p=0.009), with a TBR<1.0 due to stronger light absorbance of thrombi.

Hematoxylin and eosin histopathology of the femoral/saphenous bundle demonstrated partially occlusive venous thrombi at the site of FeCl3 treatment in all animals (Supplemental Figure 2). Fluorescence microscopy of CLIO-AF555 and MMPSense-680 signals corresponded with immunoreactive macrophages and MMP-9, consistent with prior investigations of CLIO18 and the MMP activity sensor19, 20 (Figure 3A,B).

In Vivo Relationships Among Inflammation, FITC-dextran, and Area in Resolving DVT

Substantial topographical heterogeneity of thrombus macrophages, and MMP activity was noted along the thrombus length (Figures 2A). Quantitative IVM analyses revealed that whole-thrombus macrophage and MMP activity associated closely (r=0.84, p=0.001, Figure 4A). At day 4, measures of whole-thrombus inflammation and FITC-dextran deposition did not correlate with day 4 thrombus area (p>0.05; Figures 4D, E, and F).

Figure 4
In vivo relationships between whole thrombus inflammation measures 4 days post thrombus induction. (A) Macrophage vs. MMP activity. (B) Macrophage vs. FITC-dextran (FD) thrombus enhancement. (C) MMP activity vs. FD deposition. (D-F) Macrophage, FD thrombus ...

Interestingly, forty-five minutes after FITC-dextran injection, IVM images demonstrated augmented FITC-dextran signal within thrombus zones, often including thrombus-lumen edge interfaces (Figure 2A). To further understand whether the FITC signal within thrombi was due time-dependent uptake of FITC-dextran (as opposed to pores within the thrombus), a serial IVM time course experiment was performed. Serial IVM images (Supplemental Figure 3) demonstrated that thrombus regions enhanced over the 60 minute imaging period, with minimal thrombus interior signal noted at the 10 minute timepoint, in contrast to the 30, 45, and 60 minute timepoints.

In vivo measures of whole-thrombus inflammation (macrophage, MMP activity) were found to significantly associate with in vivo FITC-deposition (r=0.75, p=0.001 and r=0.74, p=0.006, respectively, Figures 4B,C). This finding further suggested that FITC-dextran was depositing into the thrombus. This observation was confirmed on fluorescence microscopy (Figure 3C, Supplemental Figures 4 and 5).

To further understand mechanisms of FITC-dextran deposition into thrombi in vivo, a line-by-line segmental analysis of thrombus macrophages and FITC-dextran signal was performed across thrombi (Supplemental Figure 4A). One-dimensional IVM line-by-line plots of the average signal intensity demonstrated a close relationship between thrombus macrophages and FITC-dextran deposition in vivo (average r=0.58±0.25, p<0.0001 for 8 thrombi analyzed). On ex vivo fluorescence microscopy analyses, FITC-dextran deposition and macrophage signals were also strongly correlated (average r=0.93±0.04, p<0.0001 for 3 thrombi analyzed, 103426±59229 pixels analyzed per thrombus, Supplemental Figure 4B). In addition, we evaluated the association between FITC-dextran deposition and von Willebrand Factor (vWF) thrombus staining, as vWF-positive areas may reflect thrombus neovessels and indicate thrombus resolution, as demonstrated previously.9, 24, 25 Analyses of microscopic sections (Figure 3C, Supplemental Figure 5) demonstrated a good correlation between FITC-dextran deposition and vWF-positive area in thrombi (r=0.86, p=0.003). Fluorescence microscopy revealed that the FITC-dextran deposited into deeper thrombus zones (Figure 3C, Supplemental Figures 4 and 5), suggesting that diffusion did not exclusively govern FITC-dextran deposition into murine DVT.

Thrombus inflammation and subsequent DVT resolution

To investigate whether the intensity of the DVT-associated inflammatory response could inform the future extent of DVT resolution, we performed a serial IVM two-timepoint study in a separate group of mice (N=6). IVM was performed on day 4 for thrombus inflammation and architecture, and then again on day 6 for thrombus architecture. DVT resolution was then evaluated by quantifying the change in thrombus area and thrombus length from day 4 to day 6.

First, to confirm the above IVM methodology could accurately measure thrombus length and area over both imaging sessions, IVM data from the time course studies (N=4 mice) and additional mice (N=3) were analyzed. The time-course studies revealed that thrombus length and area measurements derived from FITC-dextran images were stable over the 60 minute IVM imaging session (Supplemental Figures 3D and 3E). To evaluate the agreement of thrombus length measurement among three readers, the intraclass correlation coefficient (ICC) was calculated from 12 measurements (4 per reader) of the coronal and sagittal IVM datasets (Supplemental Figure 3F-3G), and was found to be 0.915.

IVM of day 4 femoral/saphenous DVT (Figure 5) demonstrated a range of whole-thrombus inflammation levels in both macrophages (TBR range, 1.47-3.18) and MMP activity (TBR range, 0.87-1.83). Serial IVM allowed calculation of DVT resolution, specifically the reduction in thrombus area and thrombus length over a 48 hour period from day 4 to day 6. Time-course experiments demonstrated that FITC-dextran from day 4 did not remain in the thrombus at day 6 (Supplemental Figure 3C), and therefore did not produce a day 6 thrombus measurement error. Quantification of day 4 to day 6 DVT resolution across all animals (Figure 6A, 6D) demonstrated a reduction in mean DVT length (1.21 mm ± 0.22 to 0.941 mm ± 0.27, p = 0.002) and reduction in DVT area (0.18 ± 0.07mm2 to 0.11 ± 0.04mm2, p = 0.002).

Figure 5
Serial IVM micrographs day 4 and day 6 of two DVT subjects. (A) In a thrombus with relatively high inflammatory macrophage and MMP activity at day 4, the thrombus length and area were reduced by 37.0% and 50.5% respectively from day 4 to 6. (B) In a thrombus ...
Figure 6
Intensity of thrombus inflammation and the magnitude of subsequent DVT resolution 48 hours later. (A) Thrombus length and day 4 and day 6, measured from IVM FITC-dextran micrographs (p=0.002). Day 4 (B) macrophage accumulation and (C) MMP activity vs. ...

The IVM data demonstrated greater reductions in DVT burden in mice with higher degrees of DVT inflammation at day 4 (Figures 5 and and6).6). The intensity of the macrophage response (whole thrombus macrophage TBR) correlated strongly with reductions in thrombus length and area (r=0.92 and r=0.93, Figure 6B and 6E, respectively, p<0.05). The intensity of the MMP activity (whole thrombus MMP activity TBR) also correlated strongly with reductions in thrombus length (r=0.92, Figure 6C, p<0.05), and showed a trend with reduced thrombus area (r=0.54, Figure 6F, respectively, p>0.05).

Noninvasive FMT-CT Imaging of Thrombus Inflammation

Noninvasive imaging of thrombus macrophages may offer a translational approach to assess inflammation in DVT resolution and the future development of PTS. Therefore, integrated noninvasive FMT-CT22, 26 was performed on day 4 mice with jugular DVT (Figure 7). To enable in vivo noninvasive fluorescence imaging, the CLIO-based macrophage sensor (CLIO-VT680, ex/em 670/688nm) was red-shifted into the near-infrared (NIR) window.14 Noninvasive FMT-CT of the CLIO-VT680 injected mice demonstrated focal signal enhancement within thrombosed jugular veins, as localized by fiducial co-registered CT venograms showing the absence of intravascular contrast material within the thrombosed vein (Figure 7A). After FMT-CT, mice underwent macroscopic ex vivo fluorescence reflectance imaging (FRI). FRI revealed that the near infrared fluorescence (NIRF) signal from CLIO-VT680 in the DVT was significantly higher in the CLIO treated group compared to a saline treated group (CLIO-VT680 group N=6, thrombus TBR=1.65±0.09 vs. saline group N=3, thrombus TBR=1.04±0.14, p<0.006; Figure 7B and C).

Figure 7
Noninvasive FMT-CT imaging of thrombus macrophages in day 4 jugular DVT. At day 3, mice received either the near-infrared fluorescence (NIRF) macrophage sensor (CLIO-VT680) or saline. Mice were then imaged on day 4. (A) Three representative axial FMT-CT ...

DISCUSSION

In this experimental murine venous thrombosis study, we investigated the in vivo spatiotemporal relationships of inflammatory cells and proteinase factors that mediate DVT resolution. We found that the inflammatory response can predict the extent of DVT resolution in a ferric chloride DVT model. Intravital microscopy (IVM) and fluorescence molecular tomography (FMT), in conjunction with specialized macrophage and MMP activity fluorescent imaging reporters, enabled assessment of the inflammatory response in murine thigh and jugular DVT. Compared to histological studies of DVT resolution, multi-target IVM provided in vivo, whole-thrombus insights into venous thrombus inflammation, architecture, and resolution.

Recruited thrombus macrophages play a pivotal role in mediating DVT resolution.3 Landmark histological-based experimental studies demonstrated that deficiency of macrophages impaired DVT resolution,8-10 and that heightened macrophage responses improved DVT resolution.6, 27, 28 In day 4 ferric chloride-induced DVT, the present IVM data revealed substantial thrombus infiltration by macrophages that were heterogeneously distributed along the length of the thrombi. Architecturally, the in vivo thrombus resolution process, as measured by macrophage and MMP activity, demonstrated an outward-in pattern (Figures 2, ,33 and and5),5), from the edges of the thrombus towards the middle, extending prior ex vivo observations.9 These findings suggest an active interface between peri-thrombus leukocytes and the thrombus itself.8, 9 In addition, the developed IVM imaging method shows great potential for evaluating therapies that might increase monocyte entrance into the thrombus to accelerate resolution.9

Thrombus MMP activity was simultaneously imaged using a spectrally distinct MMP-activatable imaging agent that reports on MMP activity, in particular MMP-9 and MMP-2, in vivo.19, 20, 29 MMPs, predominantly gelatinases MMP-2 and MMP-9, likely play an important role in thrombus resolution by promoting collagenolysis and neoangiongenesis, fibrinolysis,30 and facilitating leukocyte influx into thrombi.31 A robust correlation of whole-thrombus MMP activity and macrophages was found (r=0.84, p=0.001), supporting that macrophages are a predominant source of active gelatinases in day 4 subacute DVT.

FITC-dextran, a routine angiographic agent for IVM, was found to deposit into thrombi at timepoints after 30 minutes after injection. In vivo and histological assessments demonstrated an association of the FITC-dextran signal with macrophages, MMP activity and vWF+ regions within thrombi. While vWF staining in thrombi was present, thrombus neovessels were not clearly visible, possibly due to the earlier day 4 timepoint employed here, as opposed to later timepoints.9,25 Additional studies are required to determine whether specific mechanisms are responsible for FITC-dextran deposition into venous thrombi.

To provide new insights into the inflammatory response and subsequent magnitude of DVT resolution, serial IVM of femoral/saphenous DVT was performed in mice at day 4 and at day 6 (Figures 5, ,6).6). We found that the intensity of day 4 inflammatory signals significantly predicted the magnitude of the reduction in DVT burden at day 6 (r-values, 0.54-0.92). The overall IVM findings provide new in vivo evidence that local macrophage and MMP activity facilitate favorable DVT resolution, presumably by promoting plasminogen activation and fibrinolysis, as well as neoendothelialization and possibly early collagenolysis.8,9,30 In future studies, the developed IVM methodology can be utilized to evaluate the in vivo effects of inflammation-modulating therapies (genetic and/or pharmacological) on DVT resolution.

FMT-CT imaging enabled the detection of macrophages in subacute DVT (Figure 7), and thus offers a noninvasive molecular-structural approach to track the DVT inflammatory response. In addition, as the CLIO particle can also serve as an MRI macrophage imaging agent,17, 32 noninvasive MRI of macrophages in DVT may also be possible (MRI was not employed in this study due to confounding signal changes from FeCl3 33). As CLIO-like superparamagentic nanoparticles have already been tested in patients, translational extension of this work may enable molecular imaging of inflammation in DVT at two timepoints: early after DVT (<1 month), where impaired inflammation is likely to identify poorly resolving thrombi and therefore patients at risk for PTS,34 and later after DVT (>3 months), where persistent inflammation indicates a high risk for PTS.35, 36

Limitations are present in this study. Later timepoints beyond day 6 DVT were not amenable to IVM due to superficial scar formation induced by FeCl3, which limited light penetration and thus IVM assessment of deeper vessels. Alternative murine thrombosis models such as ligation with venous stasis may overcome this limitation, and be more clinically relevant, but require greater technical development in the smaller femoral/saphenous vessels. As thrombi extended deeper than 100 μm, confocal IVM did not permit full thrombus volume measurement; multiphoton IVM approaches may allow deeper imaging. Regarding delivery of the imaging agents to the thrombus, if restricted diffusion, i.e. limited thrombus permeability, is present, it is possible that the CLIO and MMP signals may not reflect the full extent of macrophages and MMP activity, respectively, within the thrombus. Of note, MMP activity, while beneficial for thrombus resolution, may detrimentally affect the vein wall.37 The ability to resolve the vein wall from the thrombus was not feasible in the current IVM confocal imaging study, but may become feasible using higher resolution multiphoton imaging approaches coupled with motion compensation methods.

Supplementary Material

Acknowledgements

We acknowledge Yoshiko Iwamoto BS for assistance with histology; and Peter Waterman BS and Brett Marinelli BS, for assistance with FMT-CT imaging and data analyses; and Tetsuya Hara MD PhD and Jie Cui MD for assistance with area and length measurements.

Sources of Funding

American Heart Association Scientist Development Grant #0830352N (F.A.J.), Howard Hughes Medical Institute Career Development Award (F.A.J.), NIH R01 HL 108229-01A1 (F.A.J.),NIH T32 HL07208 support to C.M.R., NIH T32 HL076136 support to C.W.K., NIH U24CA092782 Small Animal Imaging Resource (R.W.).

Footnotes

Disclosures

None.

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