Aya Matsui

Class 1A PI3K regulates vessel integrity during development and tumorigenesis

PI3K is important in the regulation of growth, proliferation, and survival of tumor cells. We show that class 1A PI3K is also critical in the tumor microenvironment by regulating the integrity of the tumor vasculature. Using Tie2Cre-mediated deletion of the PI3K regulatory subunits (p85α, p55α, p50α, and p85β), we generated mice with endothelial cell-specific loss of class 1A PI3K. Complete loss of all subunits caused acute embryonic lethality at E11.5 due to hemorrhaging, whereas retention of a single p85α allele yielded viable mice that survived to adulthood. These heterozygous mice exhibited no vascular defects until challenged with a pathological insult, such as tumor cells or high levels of VEGF. Under these pathological conditions, heterozygous mice exhibited localized vascular abnormalities, including vessel leakage and the inability to maintain large vessels, which caused a deceleration of tumorigenesis. Furthermore, we show that a PI3K inhibitor can mimic the effects of class 1A PI3K loss, which suggests that targeting class 1A PI3K may be a promising therapy for blocking tumor angiogenesis.

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, near-infrared, fluorescence-guided identification of the ureters using methylene blue

BACKGROUND The aim of this study was to determine whether the invisible near-infrared (NIR) fluorescence properties of methylene blue (MB), a dye already approved by the U.S. Food and Drug Administration for other indications, could be exploited for real-time, intra-operative identification of the ureters. METHODS The optical properties of MB were quantified in vitro. Open surgery and laparoscopic NIR fluorescence imaging systems were employed. Yorkshire pigs were injected intravenously with 0.1-mg/kg MB (n = 8), 10-mg furosemide followed by 0.1-mg/kg MB (n = 6), or 0.5-mg/kg MB (n = 6). The contrast-to-background ratio (CBR) of the kidney and ureters, and the MB concentration in the urine, were quantified. RESULTS Peak MB absorbance, emission, and intensity in urine occurred at 668 nm, 688 nm, and 20 mumol/L, respectively. After intravenous injection, doses as low as 0.1-mg/kg MB provided prolonged imaging of the ureters, and a dose of 0.5 mg/kg provided statistically significant improvement of CBR. The preinjection of furosemide increased urine volume but did not improve CBR. Laparoscopic identification of the ureter using MB NIR fluorescence was demonstrated. CONCLUSION Ureteral imaging using MB NIR fluorescence provides sensitive, real-time, intra-operative identification of the ureters during open and laparoscopic surgeries.

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.

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.

Image-Guided Perforator Flap Design Using Invisible Near- Infrared Light and Validation with X-Ray Angiography

Although perforator flaps mark an important conceptual change in reconstructive surgery, individual perforator vessels show a high degree of variability with respect to anatomic landmarks. We have developed an intraoperative imaging system that simultaneously displays surgical anatomy and otherwise invisible near-infrared images. In 22 adult pigs, perforating vessels were identified within seconds using this optical imaging system and systemic injection of indocyanine green. Perforator flaps were then designed based on these results, and vessel location confirmed by direct visualization and anatomic dissection. Since x-ray angiography remains the gold standard for identification of underlying vessels, conventional x-ray angiography was also performed in 8 pigs to verify the location of perforators. There was full correlation of all the perforators identified among near-infrared fluorescence angiography, x-ray angiography, and anatomic dissection. The technology we describe provides high-sensitivity real-time image guidance throughout perforator dissection, and permits patient-specific flap design.

Predicting the survival of experimental ischaemic small bowel using intraoperative near-infrared fluorescence angiography

Predicting the long‐term viability of ischaemic bowel during surgery is challenging. The aim was to determine whether intraoperative near‐infrared angiography (NIR‐AG) of ischaemic bowel might provide metrics that were predictive of long‐term outcome.

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.

Preclinical study of near-infrared-guided sentinel lymph node mapping of the porcine lung.

BACKGROUND The presence of lymph node metastasis is the most important prognostic factor in early non-small cell lung cancer. Our objective was to develop a rapid, simple, and reliable method for thoracic sentinel lymph node (SLN) identification using near-infrared fluorescence imaging and clinically available contrast agents. METHODS Indocyanine green (ICG) reconstituted in saline, human serum albumin, human fresh frozen plasma, and autologous porcine plasma was evaluated for optimal formulation and dosing for SLN within porcine lungs. Animals were imaged using the fluorescence-assisted resection and exploration for surgery imaging system. The SLN identification rate, time to identification and fluorescence intensity of the SLN, bronchus, and background were measured. RESULTS The SLN identification rates varied widely, ranging from 33% to 100% as a function of the carrier used for ICG reconstitution. No significant difference was noted in SLN fluorescence intensity; however, bronchial intensity was significantly higher with ICG: albumin, which resulted in the lowest rate of SLN identification. Subsequent evaluation with 125 μM and 250 μM ICG:porcine plasma resulted in identification of strongly fluorescent SLNs, with identification rates of 93% and 100% and median signal-to-background ratios of 8.5 and 12.15, respectively, in less than 2 minutes in situ. CONCLUSIONS Near-infrared fluorescence imaging with ICG is a reliable method for SLN mapping in the lung with high sensitivity. Mixing of ICG with plasma resulted in strong SLN fluorescence signal with reliable identification rates.

MRI contrast using solid-state, B1-distorting, microelectromechanical systems (MEMS) microresonant devices (MRDs)

Presently, signal generation in MRI depends on the concentration and relaxivity of protons or other MR‐active nuclei, and contrast depends on local differences in signal. In this proof‐of‐principle study, we explore the use of nonchemical, solid‐state devices for generating detectable signal and/or contrast in vitro and in vivo. We introduce the concept of microresonant devices (MRDs), which are micron‐sized resonators fabricated using microelectromechanical systems (MEMS) technology. Fifteen‐micrometer (15‐μm)‐thick, coil MRDs were designed to resonate at the 3T Larmor frequency of protons (127.7 MHz) and were fabricated using tantalum (Ta) oxide thin‐film capacitors and copper‐plated spiral inductors. The performance of MRDs having final diameters of 300, 500, and 1000 μm were characterized in saline using a radio frequency (RF) scanning microscope and a clinical 3T MR scanner. The measured B1 fields of 300 μm to 1000 μm MRDs ranged from 3.25 μT to 3.98 μT, and their quality factors (Q) ranged from 3.9 to 7.2. When implanted subcutaneously in the flank of a mouse, only MRDs tuned to the resonant frequency of protons generated a measurable in vivo B1 field. This study lays the foundation for a new class of solid‐state contrast agents for MRI. Magn Reson Med, 2009.