Localization and quantification of platelet-rich thrombi in large blood vessels with near-infrared fluorescence imaging.

BACKGROUND: Imaging of thrombus formation in vivo has been limited by the inability to directly visualize and measure thrombi in large blood vessels in real time. Near-infrared light, with its superior tissue penetration and reduced scatter, could potentially solve this problem. METHODS AND RESULTS: Platelets were labeled with the near-infrared fluorophore IR-786. Optimal total fluorescence yield occurred at 6 attomoles of IR-786 per platelet. IR-786-labeled platelets were tested for their ability to detect thrombus formation in large animal model systems relevant to common human vascular procedures. Invisible near-infrared light did not distort the surgical field in any way, and even after optimization of per-platelet fluorescent yield, platelets remained fully functional. Intravenous infusion of just 3.6x10(10) labeled platelets into a 35-kg Yorkshire pig permitted thrombus visualization, with a signal-to-background ratio > or = 2, for at least 2 hours in coronary, carotid, and femoral vessels. Platelet-rich, actively growing clots were monitored in real time and quantified with respect to size and kinetics after injury to vessels, cutaneous incisions, intravascular stent insertion, or introduction of embolic coils. Similarly, formed clots were monitored in real time during thrombolysis with streptokinase and heparin. Vessel patency was assessed independently with a second near-infrared fluorescent blood pool agent. CONCLUSIONS: IR-786-labeled platelets provide sensitive, specific, and real-time visualization of thrombi in thick-walled blood vessels. In addition to immediate application in cardiac, transplant, and vascular surgery, the mechanisms that underlie thrombus formation in large blood vessels can now be investigated.

Measuring noncalcified coronary atherosclerotic plaque using voxel analysis with MDCT angiography: phantom validation.

OBJECTIVE: This purpose of this study was to evaluate the accuracy and reproducibility of a voxel analysis technique for measuring noncalcified plaque in the coronary arteries. MATERIALS AND METHODS: Polyethylene phantoms representing noncalcified plaque were scanned in both MDCT and micro-CT scanners and inter- and intrareader variability of volume calculation was performed. RESULTS: Volume measurements by both MDCT and micro-CT were comparable to the true volume as measured by micrometry (< 3%, p = 0.05). CONCLUSION: There appears to be no significant difference (< 3%) between MDCT and micro-CT measurements.

Optical imaging of hydroxyapatite in the calcified vasculature of transgenic animals.

OBJECTIVE: To detect the hydroxyapatite component of vascular calcification in vivo so that the process of calcium deposition can be studied in transgenic model systems. METHODS AND RESULTS: We have previously developed a near-infrared fluorescent bisphosphonate derivative that binds with high affinity and specificity to hydroxyapatite, and an intraoperative near-infrared fluorescence imaging system for small animals. Using these tools, and a transgenic mouse strain with homozygous deletion of the matrix GLA protein (Mgp(-/-)), we demonstrate that the hydroxyapatite component of vascular calcification can be detected in vivo with high sensitivity, specificity, and resolution. CONCLUSIONS: The hydroxyapatite component of vascular calcification can be detected optically, in real-time, without sacrifice of the animal. It is now possible to study the earliest events associated with vascular mineralization, at the cell and organ level, and to monitor the process in living animals.

Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping.

The use of near-infrared or infrared photons is a promising approach for biomedical imaging in living tissue. This technology often requires exogenous contrast agents with combinations of hydrodynamic diameter, absorption, quantum yield and stability that are not possible with conventional organic fluorophores. Here we show that the fluorescence emission of type II quantum dots can be tuned into the near infrared while preserving absorption cross-section, and that a polydentate phosphine coating renders them soluble, disperse and stable in serum. We then demonstrate that these quantum dots allow a major cancer surgery, sentinel lymph node mapping, to be performed in large animals under complete image guidance. Injection of only 400 pmol of near-infrared quantum dots permits sentinel lymph nodes 1 cm deep to be imaged easily in real time using excitation fluence rates of only 5 mW/cm(2). Taken together, the chemical, optical and in vivo data presented in this study demonstrate the potential of near-infrared quantum dots for biomedical imaging.

Image-guided quantification of cardioplegia delivery during cardiac surgery.

BACKGROUND: Homogenous distribution of cardioplegia delivered to the myocardium has been identified as an important predictor of post-cardiopulmonary bypass ventricular recovery and function. Presently, a method to determine adequate distribution of cardioplegia in patients during cardiac surgery does not exist. The goal of this study was to evaluate the feasibility of quantifying cardioplegia delivery using a novel, noninvasive optical method. Such a system would permit instantaneous imaging of jeopardized myocardium and allow immediate, intraoperative corrective measures. METHODS: We have previously developed a portable, intraoperative near-infrared (NIR) fluorescence imaging system for use in large animal cardiac surgery that simultaneously displays color video and NIR fluorescent images of the surgical field. By introducing exogenous, NIR fluorophores, specific cardiac functions can be visualized in real-time. RESULTS: In a porcine cardiopulmonary bypass model, we demonstrate that the FDA-approved intravascular fluorophore indocyanine green (ICG) permits real-time assessment of cardioplegia delivery. ICG was injected into an aortic root and/or transatrial coronary sinus catheter during delivery of crystalloid cardioplegia solution. Segmental distribution was immediately noted at the time of injection. In a subset of animals, simulated coronary occlusions resulted in imaging defects consistent with poor cardioplegia delivery and jeopardized myocardium. Videodensitometric analysis was performed on-line to quantify distribution to the right ventricle and left ventricle. CONCLUSION: We report the development of a novel, noninvasive, intraoperative technique that can easily and safely provide a visual assessment of cardioplegia delivery (antegrade and/or retrograde) and that offers the potential to quantify the relative segmental distribution during cardiac surgical procedures.

Tissue-like phantoms for near-infrared fluorescence imaging system assessment and the training of surgeons.

We demonstrate how to construct calibrated, stable, and inexpensive tissue-like phantoms for near-IR (NIR) fluorescence imaging applications. The bulk phantom material is composed of gelatin, intralipid, hemoglobin, and indocyanine green (ICG). Absorbance, scatter, background fluorescence, and texture can be tuned as desired. NIR fluorescent inclusions are comprised of ICG-labeled polystyrene divinylbenzene beads and Pam78-labeled hydroxyapatite crystals. The former mimic tumor masses of controllable size and contrast agent concentration, and the latter mimic microcalcifications in breast cancer. NIR-fluorescent inclusions can be positioned precisely in phantoms, with one or more regions having different optical properties, and their position can be verified independently using microcomputed tomography. We demonstrate how these phantoms can be used to calibrate and compare imaging systems, and to train surgeons to operate under NIR fluorescence image guidance.

Humoral bone morphogenetic protein 2 is sufficient for inducing breast cancer microcalcification.

Microcalcifications are an important diagnostic marker for breast cancer on mammograms, yet the mechanism of their formation is poorly understood. Indeed, there is presently no short-latency, high-yield, syngeneic rodent model of the process. Bone morphogenetic protein 2 (BMP-2) is a key mediator of physiologic bone formation and pathologic vasculature calcification, but its role in breast cancer microcalcification is unknown. In this study, R3230 rat breast tumors were adapted to cell culture, transduced with adenoviral BMP-2, and inoculated into a syngeneic host. Tumor growth and calcium salt deposition were quantified in living animals over time using micro-computed tomography and probed chemically using near-infrared fluorescence. Plasma BMP-2 levels were quantified over time by enzyme-linked immunosorbent assay. Within 3 weeks, 100% of the breast tumors developed microcalcifications, which were absent from all normal tissues. Importantly, when two tumors were initiated in a single host, the ipsilateral tumor expressing BMP-2 was able to induce microcalcification in the contralateral tumor that was not expressing BMP-2, suggesting that BMP-2 can act humorally. Taken together, we describe the first reproducible rodent model of breast cancer microcalcification, prove that BMP-2 expression is sufficient for initiating the process, and lay the foundation for a new generation of targeted diagnostic agents.

Sentinel lymph node mapping of the gastrointestinal tract by using invisible light.

BACKGROUND: Because many gastrointestinal (GI) tumors spread by way of lymphatics, histological assessment of the first draining lymph nodes has both prognostic and therapeutic significance. However, sentinel lymph node mapping of the GI tract by using available techniques is limited by unpredictable drainage patterns, high background signal, and the inability to image lymphatic tracers relative to surgical anatomy in real time. Our goal was to develop a method for patient-specific intraoperative sentinel lymph node mapping of the GI tract by using invisible near-infrared light. METHODS: We developed an intraoperative near-infrared fluorescence imaging system that simultaneously displays surgical anatomy and otherwise invisible near-infrared fluorescence images of the surgical field. Near-infrared fluorescent quantum dots were injected intraparenchymally into the stomach, small bowel, and colon, and draining lymphatic channels and sentinel lymph nodes were visualized. Dissection was performed under real-time image guidance. RESULTS: In 10 adult pigs, we demonstrated that 200 pmol of quantum dots quickly and accurately map lymphatic drainage and sentinel lymph nodes. Injection into the mid jejunum and colon results in fluorescence of a single lymph node at the root of the bowel mesentery. Injection into the stomach resulted in identification of a retrogastric node. Histological analysis in all cases confirmed the presence of nodal tissue. CONCLUSIONS: We report the use of invisible near-infrared light for intraoperative sentinel lymph node mapping of the GI tract. This technology overcomes the limitations of currently available methods, permits patient-specific imaging of lymphatic flow and sentinel nodes, and provides highly sensitive, real-time image-guided dissection.

Catheter-based antegrade intracoronary viral gene delivery with coronary venous blockade.

The purpose of this study is to evaluate the feasibility of percutaneous antegrade myocardial gene transfer (PAMGT). A consistent and safe technique for in vivo gene transfer is required for clinical application of myocardial gene therapy. PAMGT with concomitant coronary venous blockade was performed in 12 swine. The myocardium was preconditioned with 1 min of occlusion of the left anterior descending and left circumflex arteries. The anterior interventricular vein was occluded during left anterior descending artery delivery, and the great cardiac vein at the entrance of the middle cardiac vein was occluded during left circumflex artery delivery. With arterial and venous balloons inflated (3 min) and after adenosine (25 mug) injection, PAMGT was performed by antegrade injection of an adenoviral solution (1 ml of 10(11) plaque-forming units in each coronary artery) carrying beta-galactosidase or saline through the center lumen of the angioplasty balloon. In one set of animals, PAMGT was performed with selective coronary vein blockade (n = 9); in another set of animals, PAMGT was performed without coronary vein blockade (n = 5). At 1 wk after gene delivery, the animals were killed. Quantitative beta-galactosidase analysis was performed in the left and right ventricular walls. PAMGT was successfully performed in all animals with and without concomitant occlusion of the coronary veins. Quantitative beta-galactosidase analysis showed that PAMGT with coronary blockade was superior to PAMGT without coronary blockade. beta-Galactosidase activity increased significantly in the beta-galactosidase group compared with the saline group: 1.34 +/- 0.18 vs. 0.81 +/- 0.1 ng (P

High-affinity near-infrared fluorescent small-molecule contrast agents for in vivo imaging of prostate-specific membrane antigen.

Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR) fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM), single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.

In vivo optical imaging of pleural space drainage to lymph nodes of prognostic significance.

BACKGROUND: Understanding the spatial and temporal drainage patterns of the pleural space could have profound impact on the treatment of lung cancer and mesothelioma. The purpose of this study was to identify the in vivo pattern of drainage from the pleural space to prognostic lymph node stations. METHODS: Fifty-six rats underwent pleural space injection of a novel lymph tracer composed of recombinant human serum albumin (HSA) covalently conjugated to the near-infrared (NIR) fluorophore IRDye78 via an amide bond (HSA-78). Nodal uptake was imaged at 10, 20, 30, and 60 minutes and 4, 12, and 24 hours after injection with a custom system that simultaneously acquires color video, NIR fluorescence of HSA-78, and a merged picture of the two. Six pigs underwent the same procedure with imaging at 30 minutes, 1 hour, and 24 hours. RESULTS: In both the rat model and the pig model, HSA-78 drained from the pleural space to superior mediastinal lymph nodes first, followed by other intrathoracic and then extrathoracic lymph nodes over the course of 24 hours. CONCLUSION: NIR fluorescence imaging in two species shows that the superior mediastinal lymph nodes are the first to drain the pleural space. Over the course of 24 hours, the pleural space also communicates with other intrathoracic and then extrathoracic lymph nodes. This study also demonstrates an intraoperative method for identifying nodes communicating with the pleural space, with potential utility in the staging and/or resection of lung cancer and mesothelioma.