A novel pilot animal model for the surgical prevention of lymphedema: the power of optical imaging.

BACKGROUND: Breast cancer-related lymphedema affects more than 400,000 survivors in the United States. In 2009, lymphatic microsurgical preventive healing approach (LYMPHA) was first described as a surgical technique to prevent lymphedema by bypassing divided arm lymphatics into adjacent veins at the time of an axillary lymph node dissection. We describe the first animal model of LYMPHA. METHODS: In Yorkshire pigs, each distal hind limb lymphatic system was cannulated and injected with a different fluorophore (human serum albumin-conjugated indocyanine green or Evans Blue). Fluorescence-assisted resection and exploration imaging system was used to map the respective lymphangiosomes to the groin. Baseline lymphatic clearance of each hind limb lymphangiosome was obtained by measuring the fluorescence of each dye from centrally obtained blood samples. A lymphadenectomy versus lymphadenectomy with LYMPHA was then performed. The injections were then repeated to obtain clearance rates that were compared against baseline values. RESULTS: Human serum albumin-conjugated indocyanine green and Evans Blue allowed for precise lymphatic mapping of each respective hind limb using fluorescence-assisted resection and exploration imaging. Lymphatic clearance from the distal hind limb dropped 68% when comparing baseline clearance versus after a groin lymphadenectomy. In comparison, lymphatic clearance dropped only 21% when comparing baseline clearance versus a lymphadenectomy with LYMPHA. CONCLUSIONS: We describe the first animal model for LYMPHA, which will enable future studies to further evaluate the efficacy and potential limitations of this technique. Of equal importance, we demonstrate the power of optical imaging to provide real-time lymphatic clearance rates for each hind limb.

Quantitative assessment of nipple perfusion with near-infrared fluorescence imaging.

Preserving the nipple-areolar complex with a nipple-sparing mastectomy improves cosmesis compared with skin-sparing mastectomy. However, complications such as necrosis of the nipple-areolar complex significantly affect cosmetic outcome. Many factors influence nipple-areolar perfusion, and no consensus currently exists on optimal incisional choice. This study evaluates 2 nipple-sparing mastectomy incision models using near-infrared fluorescence to assess perfusion quantitatively. The periareolar and radial incisions were compared with 2 control models in Yorkshire pigs (N = 6). Methylene blue and indocyanine green were injected intravenously, and near-infrared fluorescence images were recorded at 3 time points: before surgery, immediately after (0 hour), and 3 days postoperatively. Contrast-to-background ratio was used to assess perfusion. At 72 hours, radial incisions showed a statistically significantly higher perfusion compared with periareolar incisions (P < 0.05). Based on our findings, radial incisions for nipple-sparing mastectomy may be preferable due to higher perfusion; however, clinical trials are necessary for further assessment.

Intraoperative prediction of postoperative flap outcome using the near-infrared fluorophore methylene blue.

Methylene blue (MB) is a near-infrared fluorophore that provides a stable visual map of skin perfusion after intravenous injection. We explored the capability of MB to predict submental flap postoperative outcome using a single intraoperative measurement. Submental flaps were created in N = 15 pigs and imaged using the FLARE imaging system immediately after surgery and at 72 hours. Using the first 3 pigs, optimal MB dosing was found to be 2.0 mg/kg. Training and validation sets of 6 pigs each were then used for receiver operating characteristic analysis. In the training set, a contrast-to-background ratio (CBR) threshold of 1.24 provided the highest sensitivity and specificity to predict tissue necrosis at 72 hours. In the validation set, this threshold provided a prediction sensitivity of 95.3% and a specificity of 98.0%. We demonstrate that a single intraoperative near-infrared measurement can predict submental flap outcome at 72 hours.

Real-time simultaneous near-infrared fluorescence imaging of bile duct and arterial anatomy.

BACKGROUND: We hypothesized that two independent wavelengths of near-infrared (NIR) fluorescent light could be used to identify bile ducts and hepatic arteries simultaneously, and intraoperatively. MATERIALS AND METHODS: Three different combinations of 700 and 800 nm fluorescent contrast agents specific for bile ducts and arteries were injected into N = 10 35-kg female Yorkshire pigs intravenously. Combination 1 (C-1) was methylene blue (MB) for arterial imaging and indocyanine green (ICG) for bile duct imaging. Combination 2 (C-2) was ICG for arterial imaging and MB for bile duct imaging. Combination 3 (C-3) was a newly developed, zwitterionic NIR fluorophore ZW800-1 for arterial imaging and MB for bile duct imaging. Open and minimally invasive surgeries were imaged using the fluorescence-assisted resection and exploration (FLARE) surgical imaging system and minimally invasive FLARE (m-FLARE) imaging systems, respectively. RESULTS: Although the desired bile duct and arterial anatomy could be imaged with contrast-to-background ratios (CBRs) ≥ 6 using all three combinations, each one differed significantly in terms of repetition and prolonged imaging. ICG injection resulted in high CBR of the liver and common bile duct (CBD) and prolonged imaging time (120 min) of the CBD (C-1). However, because ICG also resulted in high background of liver and CBD relative to arteries, ICG produced a lower arterial CBR (C-2) at some time points. C-3 provided the best overall performance, although C-2, which is clinically available, did enable effective laparoscopy. CONCLUSIONS: We demonstrate that dual-channel NIR fluorescence imaging provides simultaneous, real-time, and high resolution identification of bile ducts and hepatic arteries during biliary tract surgery.

Intraoperative near-infrared fluorescence imaging in perforator flap reconstruction: current research and early clinical experience.

Despite recent advances in perforator flap reconstruction, there can be significant variability in vessel size and location. Although preoperative evaluation may provide valuable information, real-time intraoperative methods have the potential to provide the greatest benefit. Our laboratory has developed the Fluorescence-Assisted Resection and Exploration (FLARE) near-infrared (NIR) fluorescence imaging system for intraoperative visualization of details of the underlying vasculature. The FLARE system uses indocyanine green, a safe and reliable NIR fluorophore already FDA-approved for other indications. The system has been optimized in large-animal models for the identification of perforator size, location, and perfusion and has also been translated to the clinic for use during breast reconstruction after mastectomy. In this article, we review our preclinical and clinical data, as well as literature describing the use of similar NIR fluorescence imaging systems in plastic and reconstructive surgery.

Cardioscopic tricuspid valve repair in a beating ovine heart.

BACKGROUND: Open heart surgery is commonly associated with cardiopulmonary bypass and cardioplegic arrest. The attendant risks of cardiopulmonary bypass may be prohibitive in high-risk patients. We present a novel endoscopic technique of performing tricuspid valve repair without cardiopulmonary bypass in a beating ovine heart. METHODS: Six sheep underwent sternotomy and creation of a right heart shunt to eliminate right atrial and right ventricular blood for clear visualization. The superior vena cava, inferior vena cava, pulmonary artery, and coronary sinus were cannulated, and the blood flow from these vessels was shunted into the pulmonary artery via a roller pump. The posterior leaflet of the tricuspid valve was partially excised to create tricuspid regurgitation, which was confirmed by Doppler echocardiography. A 7.0-mm fiberoptic videoscope was inserted into the right atrium to visualize the tricuspid valve. Under cardioscopic vision, an endoscopic needle driver was inserted into the right atrium, and a concentric stitch was placed along the posterior annulus to bicuspidize the tricuspid valve. Doppler echocardiography confirmed reduction of tricuspid regurgitation. RESULTS: All animals successfully underwent and tolerated the surgical procedure. The right heart shunt generated a bloodless field, facilitating cardioscopic tricuspid valve visualization. The endoscopic stitch resulted in annular plication and functional tricuspid valve bicuspidization, significantly reducing the degree of tricuspid regurgitation. CONCLUSION: Cardioscopy enables less invasive, beating-heart tricuspid valve surgery in an ovine model. This technique may be useful in performing right heart surgery without cardiopulmonary bypass in high-risk patients.

Adjustable, physiological ventricular restraint improves left ventricular mechanics and reduces dilatation in an ovine model of chronic heart failure.

BACKGROUND: Ventricular restraint is a nontransplantation surgical treatment for heart failure. The effect of varying restraint level on left ventricular (LV) mechanics and remodeling is not known. We hypothesized that restraint level may affect therapy efficacy. METHODS AND RESULTS: We studied the immediate effect of varying restraint levels in an ovine heart failure model. We then studied the long-term effect of restraint applied over a 2-month period. Restraint level was quantified by use of fluid-filled epicardial balloons placed around the ventricles and measurement of balloon luminal pressure at end diastole. At 4 different restraint levels (0, 3, 5, and 8 mm Hg), transmural myocardial pressure (P(tm)) and indices of myocardial oxygen consumption (MVO2) were determined in control (n=5) and ovine heart failure (n=5). Ventricular restraint therapy decreased P(tm) and MVO2, and improved mechanical efficiency. An optimal physiological restraint level of 3 mm Hg was identified to maximize improvement without an adverse affect on systemic hemodynamics. At this optimal level, end-diastolic P(tm) and MVO2 indices decreased by 27% and 20%, respectively. The serial longitudinal effects of optimized ventricular restraint were then evaluated in ovine heart failure with (n=3) and without (n=3) restraint over 2 months. Optimized ventricular restraint prevented and reversed pathological LV dilatation (130+/-22 mL to 91+/-18 mL) and improved LV ejection fraction (27+/-3% to 43+/-5%). Measured restraint level decreased over time as the LV became smaller, and reverse remodeling slowed. CONCLUSIONS: Ventricular restraint level affects the degree of decrease in P(tm), the degree of decrease in MVO2, and the rate of LV reverse remodeling. Periodic physiological adjustments of restraint level may be required for optimal restraint therapy efficacy.

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.

Real-time visualization and quantification of retrograde cardioplegia delivery using near infrared fluorescent imaging.

BACKGROUND AND AIM: Homogeneous delivery of cardioplegia is essential for myocardial protection during cardiac surgery. Presently, there exist no established methods to quantitatively assess cardioplegia distribution intraoperatively and determine when retrograde cardioplegia is required. In this study, we evaluate the feasibility of near infrared (NIR) imaging for real-time visualization of cardioplegia distribution in a porcine model. METHODS: A portable, intraoperative, real-time NIR imaging system was utilized. NIR fluorescent cardioplegia solution was developed by incorporating indocyanine green (ICG) into crystalloid cardioplegia solution. Real-time NIR imaging was performed while the fluorescent cardioplegia solution was infused via the retrograde route in five ex vivo normal porcine hearts and in five ex vivo porcine hearts status post left anterior descending (LAD) coronary artery ligation. Horizontal cross-sections of the hearts were obtained at proximal, middle, and distal LAD levels. Videodensitometry was performed to quantify distribution of fluorophore content. RESULTS: The progressive distribution of cardioplegia was clearly visualized with NIR imaging. Complete visualization of retrograde distribution occurred within 4 minutes of infusion. Videodensitometry revealed retrograde cardioplegia, primarily distributed to the left ventricle (LV) and anterior septum. In hearts with LAD ligation, antegrade cardioplegia did not distribute to the anterior LV. This deficiency was compensated for with retrograde cardioplegia supplementation. CONCLUSIONS: Incorporation of ICG into cardioplegia allows real-time visualization of cardioplegia delivery via NIR imaging. This technology may prove useful in guiding intraoperative decisions pertaining to when retrograde cardioplegia is mandated.

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.

Intraoperative identification of esophageal sentinel lymph nodes with near-infrared fluorescence imaging.

OBJECTIVE: In esophageal cancer, selective removal of involved lymph nodes could improve survival and limit complications from extended lymphadenectomy. Mapping with vital blue dyes or technetium Tc-99m often fails to identify intrathoracic sentinel lymph nodes. Our purpose was to develop an intraoperative method for identifying sentinel lymph nodes of the esophagus with high-sensitivity near-infrared fluorescence imaging. METHODS: Six Yorkshire pigs underwent thoracotomy and received submucosal, esophageal injection of quantum dots, a novel near-infrared fluorescent lymph tracer designed for retention in sentinel lymph nodes. Six additional pigs underwent thoracotomy and received submucosal esophageal injection of CW800 conjugated to human serum albumin, another novel lymph tracer designed for uptake into distant lymph nodes. Finally, 6 pigs received submucosal injection of the fluorophore-conjugated albumin with an endoscopic needle through an esophagascope. These lymph tracers fluoresce in the near-infrared, permitting visualization of migration to sentinel lymph nodes with a custom intraoperative imaging system. RESULTS: Injection of the near-infrared fluorescent lymph tracers into the esophagus revealed communicating lymph nodes within 5 minutes of injection. In all 6 pigs that received quantum dot injection, only a single sentinel lymph node was identified. Among pigs that received fluorophore-conjugated albumin injection, in 5 of 12 a single sentinel lymph node was revealed, but in 7 of 12 two sentinel lymph nodes were identified. There was no dominant pattern in the appearance of the sentinel lymph nodes either cranial or caudal to the injection site. CONCLUSION: Near-infrared fluorescence imaging of sentinel lymph nodes is a novel and reliable intraoperative technique with the power to assist with identification and resection of esophageal sentinel lymph nodes.

Intraluminal coronary shunting preserves regional myocardial perfusion and function.

BACKGROUND: Coronary artery hemostasis during offpump coronary artery bypass (OPCAB) may be achieved with extraluminal coronary occlusion or intraluminal coronary shunting. We sought to determine with a normal porcine beating-heart model whether coronary shunting preserves regional myocardial perfusion and function compared with coronary occlusion. METHODS: Six pigs (50-60 kg) underwent sternotomy and instrumentation. Two pairs of ultrasonic crystals were placed in the distribution of t h e left anterior descending (LAD) and left circumflex (LCx) arteries for measurement of fractional change in area (FCA), an index of regional contractility. Regional myocardial blood flow (RMBF) was determined with radiolabeled microspheres. Data were recorded for each animal at baseline and after LAD arteriotomy and vascular control with (1) a 1.5-mm intraluminal shunt, (2) proximal occlusion, and (3) proximal and distal occlusion. RESULTS: One pig experienced ventricular fibrillation during LAD manipulation and was excluded from the study. Data were summarized for the remaining 5 animals. Coronary shunting maintained RMBF and function (FCA) compared with baseline. Proximal occlusion led to 50% (P =.05) and 47% (P =.04) decreases in RMBF and FCA, respectively, in the LAD region. Proximal and distal occlusion led to 55% (P =.03) and 51% (P = 02) decreases in RMBF and FCA, respectively, in the LAD region. There were no significant changes in RMBF or FCA in the LCx (control) region. CONCLUSION: Intraluminal coronary shunting is capable of preserving distal myocardial perfusion and function in a normal porcine heart. Coronary occlusion, in contrast, significantly reduces regional perfusion and function. More frequent use of intracoronary shunting may facilitate OPCAB by minimizing ischemia and hemodynamic compromise.

Restraint to the left ventricle alone is superior to standard restraint.

OBJECTIVE: In standard ventricular restraint therapy, a single level of restraint is applied to the entire ventricular surface. We showed previously that at high restraint levels, cardiac tamponade develops because of the thin-walled right ventricle, even while the left ventricle remains unaffected. We now hypothesize that applying restraint exclusively to the left ventricle permits higher levels of restraint, resulting in increased benefit to the left ventricle. METHODS: The acute effect of restraint applied to the left ventricle alone was analyzed in healthy and cardiomyopathic sheep hearts. Restraint therapy was applied by fluid-filled epicardial balloons placed solely around the left ventricle. Restraint level was defined by the measured balloon luminal pressure at end diastole. At incrementally higher restraint levels (0, 3, 5, 8, 10, 12, and 14 mm Hg), transmural myocardial left ventricular pressure (P(tm) = Left ventricle pressure - Balloon pressure) and indices of myocardial oxygen consumption were measured in healthy sheep (n = 5) and in sheep with heart failure (n = 6). RESULTS: Increasing restraint from 0 to 14 mm Hg decreased transmural myocardial pressure by 48.8% (P ≤ .02) and the left ventricle tension-time index by 39.1% (P ≤ .01), and the pressure-volume area decreased by 58.4% (P ≤ .01). Similarly, stroke work decreased by 57.9% (P ≤ .03). Systemic hemodynamics were unchanged. There was no difference in the trend for all indices between animals that were healthy and those with heart failure. CONCLUSIONS: We showed previously that, with standard restraint, right ventricle tamponade develops at high restraint levels, limiting restraint therapy. We now show that restraint applied to the left ventricle alone permits increased restraint levels, without causing right ventricle or left ventricle tamponade, for greater therapeutic benefit. We conclude that partial left ventricle restraint may be more effective than standard restraint.

Predictive capability of near-infrared fluorescence angiography in submental perforator flap survival.

BACKGROUND: Perforator flaps have become increasingly popular in reconstructive surgery, as patients experience less donor-site morbidity than with conventional musculocutaneous flaps. Previously, the authors' laboratory described the intraoperative use of near-infrared fluorescence angiography for patient-specific perforator flap design. This study evaluates the predictive capability of near-infrared fluorescence angiography for flap survival in submental flap reconstruction. METHODS: Near-infrared angiography was performed using indocyanine green at 0, 0.5, 24, 48, and 72 hours after surgery for flap creation in 12 pigs. A single perforator artery was preserved during flap creation based on location (central or noncentral) and dominance (dominant or nondominant). Venous drainage, arterial perfusion, and perfused area as a percentage of total flap area were analyzed. Clinical assessments of perfusion were compared with those made using near-infrared imaging and histology. RESULTS: Use of near-infrared fluorescence angiography immediately after flap creation accurately predicted areas of perfusion at 72 hours (p=0.0013), compared with the initial clinical assessment (p=0.3085). Identification of necrosis by histology at 72 hours correlated with near-infrared findings of insufficient arterial perfusion immediately after flap creation. No statistically significant differences in perfusion metrics were detected based on location or dominance of the preserved perforator; however, flaps containing central perforators had a higher percentage perfused area than those with noncentral perforators. CONCLUSIONS: The use of near-infrared angiography immediately after flap creation can predict areas of perfusion at 72 hours. This predictive capability may permit intraoperative revision of compromised flaps that have a high likelihood of failure.

Real-time intra-operative near-infrared fluorescence identification of the extrahepatic bile ducts using clinically available contrast agents.

BACKGROUND: Iatrogenic bile duct injuries are serious complications with patient morbidity. We hypothesized that the invisible near-infrared (NIR) fluorescence properties of methylene blue (MB) and indocyanine green (ICG) could be exploited for real-time, intraoperative imaging of the extrahepatic bile ducts during open and laparoscopic surgeries. METHODS: In all, 2.0 mg/kg of MB and 0.05 mg/kg of ICG were injected intravenously into 35-kg female Yorkshire pigs and the extrahepatic bile ducts were imaged over time using either the Fluorescence-Assisted Resection and Exploration (FLARE) image-guided surgery system (open surgery) or a custom NIR fluorescence laparoscopy system. Surgical anatomy was confirmed using x-ray cholangiography. The contrast-to-background ratio (CBR), contrast-to-liver ratio (CLR), and chemical concentrations in the cystic duct (CD) and common bile duct (CBD) were measured, and the performance of each agent was quantified. RESULTS: Using NIR fluorescence of MB, the CD and CBD could be identified with good sensitivity (CBR and CLR > or =4), during both open and laparoscopic surgeries, from 10 to 120 min postinjection. Functional impairment of the ducts, including constriction and injury were immediately identifiable. Using NIR fluorescence of ICG, extrahepatic bile ducts did not become visible until 90 min postinjection because of strong residual liver retention; however, between 90 and 240 min, ICG provided exquisitely high sensitivity for both CD and CBD, with CBR > or =8 and CLR > or =4. CONCLUSION: We demonstrate that 2 clinically available NIR fluorophores, MB fluorescing at 700 nm and ICG fluorescing at 800 nm, provide sensitive, prolonged identification of the extrahepatic bile ducts and assessment of their functional status.

Real-time assessment of cardiac perfusion, coronary angiography, and acute intravascular thrombi using dual-channel near-infrared fluorescence imaging.

OBJECTIVES: We have developed an image-guided surgical system based on invisible near-infrared fluorescent light. Presently, the only clinically available near-infrared fluorophore is indocyanine green, which fluoresces at approximately 800 nm and is used for coronary angiography. Our objective was to determine whether methylene blue, already US Food and Drug Administration approved for other indications, has useful near-infrared fluorescence properties for image-guided cardiac surgery. METHODS: The optical properties of methylene blue were measured after dissolution in 100% serum. Biodistribution and clearance were quantified in organs and tissue after intravenous bolus injection of 2 mg/kg methylene blue in 3 rats. Coronary arteriography and cardiac perfusion were imaged in real time after intravenous bolus injection of 1 mg/kg methylene blue in 5 pigs with coronary obstructions. Coronary angiography and acute thrombi were assessed by using 800-nm fluorophores, indocyanine green, and IR-786-labeled platelets, respectively. RESULTS: The peak absorbance and emission of methylene blue as a near-infrared fluorophore occur at 667 nm and 686 nm, respectively. After intravenous injection, methylene blue provides highly sensitive coronary angiography. A lipophilic cation, methylene blue is extracted rapidly into tissue, with myocardium displaying unusually high uptake. Methylene blue permits real-time visualization and quantitative assessment of myocardial perfusion. Because of absent spectral overlap, use of 2 independent fluorophores in our imaging system permits simultaneous quantification of perfusion, venous drainage, and/or intravascular thrombi. CONCLUSIONS: Methylene blue is an effective near-infrared fluorophore that provides direct visualization of coronary arteriography and cardiac perfusion. In conjunction with approximately 800-nm near-infrared fluorophores, important functional assessments during cardiac surgery are also possible.

Submental perforator flap design with a near-infrared fluorescence imaging system: the relationship among number of perforators, flap perfusion, and venous drainage.

BACKGROUND: The submental flap is a reliable alternative to microsurgical reconstruction of facial deformities, providing an excellent cosmetic match with the contour and color of the face. In this study, the authors evaluated submental flap design by using near-infrared fluorescence angiography to identify perforator arteries. The impact of the number of preserved perforator arteries on flap perfusion and venous drainage was quantified. METHODS: Indocyanine green was injected intravenously into 18 pigs. Three groups of six animals each had one, two, or three perforator arteries preserved. The fluorescence-assisted resection and exploration near-infrared fluorescence imaging system was used for image acquisition. Images were recorded before and after flap creation, and every hour, for 6 hours. The time to maximum perfusion, the drainage ratio (an indicator of venous drainage), and the percentage of perfused flap area were analyzed statistically at each time point. RESULTS: Flaps with a single dominant perforator artery had an initial mean perfused area of 80 percent, which improved to 97 percent at 6 hours. For flaps with two and three preserved perforator arteries, perfused area at 6 hours was 99.8 percent and 100 percent, respectively. A significant increase was observed in all three metrics as more vessels were preserved. Regardless of the number of perforator arteries preserved, though, all three metrics improved over 6 hours. CONCLUSIONS: Near-infrared fluorescence angiography can reliably identify submental perforator arteries for flap design and can be used to assess flap perfusion and venous drainage in real time. Flap metrics at 6 hours were equivalent when either one or multiple perforator arteries were preserved.

Quantitative assessment of perfusion and vascular compromise in perforator flaps using a near-infrared fluorescence-guided imaging system.

BACKGROUND: Techniques currently used to determine flap perfusion are mainly subjective, with the majority of reconstructive surgeons still relying on clinical examination. In this study, the authors demonstrate the use of near-infrared fluorescence angiography to directly quantify normal and abnormal perfusion in perforator flaps. METHODS: Indocyanine green was injected intravenously into anesthetized adult pigs (n = 38). A custom near-infrared fluorescence imaging system was used for image acquisition and quantitation. Thirty-nine flaps were designed based on identified perforators, and postoperative imaging was performed for comparison. In select flaps, isolated occlusion of the arterial and venous pedicle was performed. In select flaps, vascular spasm was induced by local irrigation of the vessels with epinephrine. The fluorescence intensities of select regions of interest were quantified. From these data, the authors defined two indices for abnormal perfusion: the Tmax ratio and the drainage ratio. RESULTS: The authors identified a normal pattern of perfusion before flap elevation, composed of a distinct fluorescence intensity peak at maximal arterial inflow followed by a smooth drop representing venous drainage. Delay of this peak after flap elevation, as indicated by the Tmax ratio, identified vascular spasm and arterial occlusion (p < 0.0001). Abnormal fall of fluorescence intensities after this peak, as indicated by the drainage ratio, identified venous occlusion (p < 0.0001). CONCLUSIONS: Quantitation of fluorescence intensities by near-infrared angiography accurately characterizes arterial and venous compromise. The authors' technique can assess perfusion characteristics during the intraoperative and postoperative periods and therefore complements clinically based subjective criteria now used for flap assessment.