Intraoperative localization of insulinoma and normal pancreas using invisible near-infrared fluorescent light

Abstract

Background: Neuroendocrine tumors of the pancreas, such as insulinoma, are difficult to localize, and complete resection is essential for cure. Our hypothesis is that a near-infrared (NIR) fluorophore exhibiting uptake in insulinoma could provide high-sensitivity detection intraoperatively.

Materials and methods: The optical properties of methylene blue (MB) were measured in vitro in 100% serum at 37 degrees C and in vivo after tissue uptake. MB was injected as a rapid intravenous bolus at doses ranging from 0.25 to 2 mg/kg into wildtype rats and pigs, and into insulinoma-bearing transgenic mice. The FLARE imaging system was used to acquire color video and NIR fluorescence images simultaneously and in real-time. The signal-to-background ratios (SBR) of tissues and tumors were quantified using FLARE software.

Results: When appropriately diluted, MB exhibits moderate NIR fluorescence emission peaking at 688 nm. At doses > or =1 mg/kg, certain normal tissues, such as pancreas, accumulate MB and remain NIR fluorescent for up to 1 h with an SBR > or = 1.6. MB spectral properties are maintained after uptake into tissue. Interestingly, insulinoma exhibits even higher MB signal than normal pancreas, resulting in insulinoma-to-pancreas ratios of 3.7 and insulinoma-to-muscle ratios of 16.2. MB permitted high-sensitivity, real-time localization of primary, multicentric, and metastatic insulinoma and permitted differentiation among tumor, normal pancreas, and other abdominal structures.

Conclusion: A single intravenous injection of a clinically available, commonly used NIR fluorophore provides prolonged intraoperative localization of normal pancreas and insulinoma using invisible NIR fluorescent light.

Keywords: Intraoperative Imaging; Methylene Blue; Near-Infrared Fluorescence; Neuroendocrine Tumors; Pancreas.

Figure 1. NIR Fluorescent Contrast Agent and Imaging System

A. Methylene blue (MB) chemical structure and molecular weight (MW). B. Absorbance (solid curve; left axis) and fluorescence emission (dotted curve; λ_Exc = 655 nm; right axis) spectra of 10 μM MB in 100% serum at 37°C. C. Schematic of the FLARE^™ (Fluorescence-Assisted Resection and Exploration) intraoperative NIR fluorescence imaging system, showing excitation and emission wavelengths, and dichroic mirrors D1 and D2. Camera images are acquired and displayed simultaneously, and in real-time.

Figure 2. NIR Fluorescence Imaging of Normal Rat Pancreas Using Intravenously Injected MB

A. Representative images (n = 3 animals) of the abdominal cavity of rat pre-injection (top row) and 15 min post-injection (bottom row) of 1.5 mg/kg MB given as an IV bolus. Shown are color video (left), 700 nm NIR fluorescence (middle), and a pseudo-color (lime green) merge of the two (right). Arrows = pancreas. Arrowheads = food particles in lumen of small bowel. NIR fluorescence images were acquired with a 150 msec camera exposure time and displayed with identical normalizations. Du = duodenum; Je = jejunum; Li = liver; Pa-H = head of pancreas; Pa-T = tail of pancreas; Re = rectum; Sp = spleen. B. Signal-to-background ratio (SBR; mean ± SEM) over time in normal rat pancreas after IV bolus injection of MB at the doses shown and a 150 msec camera exposure time. N = 3 rats per group.

Figure 3. NIR Fluorescence Imaging of Normal Pig Pancreas Using Intravenously Injected MB

A. Representative images (n = 4 animals) of the abdominal cavity of pig pre-injection (top row) and 15 min post-injection (bottom row) of 1.5 mg/kg MB given as an IV bolus. Shown are color video (left), 700 nm NIR fluorescence (middle), and a pseudo-color (lime green) merge of the two (right). NIR fluorescence images were acquired with a 250 msec exposure time and displayed with identical normalizations. Du = duodenum; Je = jejunum; Ki = kidney; Li = Liver; Pa = pancreas. B. Reflectance absorbance and fluorescence spectra of pig pancreas 1 h post-injection of 1.5 mg/kg MB. Fluorescence excitation was 655 nm.

Figure 4. NIR Fluorescence Imaging of Insulinoma Using Intravenously Injected MB

Insulinoma-bearing NOD/ShiLt-Tg(RipTAg)1Lt/J mice were imaged 15 min after IV bolus injection of 1.5 mg/kg MB. Shown are color video (left), 700 nm NIR fluorescence (middle), and a pseudo-colored (lime green) merge of the two (right). NIR fluorescence images were acquired with a 150 msec exposure time and displayed with identical normalizations. Shown are animals bearing a single primary tumor (arrow) in the pancreas (top row), multi-centric pancreatic tumors (arrows; middle row), and metastatic tumors (arrowheads; bottom row).

Figure 5. Histological Analysis of Normal and Tumor-Bearing Pancreas

A. Consecutive sections from normal rat pancreas stained with H&E (left) or unstained and imaged at 700 nm using a NIR fluorescent microscope (right). Rats were either uninjected (top row) or injected IV with 1.5 mg/kg MB and sacrificed at 15 min post-injection (bottom row). NIR fluorescence images have identical exposure times (5 sec) and normalizations. Results are representative of n = 3 independent experiments. I = pancreatic islets; E = exocrine pancreas. B. Consecutive sections from insulinoma-bearing pancreas of NOD/ShiLt-Tg(RipTAg)1Lt/J mice stained with H&E (left) or unstained and imaged at 700 nm using an NIR fluorescent microscope (right). Mice were either uninjected (top row) or injected IV with 1.5 mg/kg MB and sacrificed at 15 min post-injection (bottom row). NIR fluorescence images have identical exposure times (5 sec) and normalizations. Results are representative of n = 3 independent experiments. Insulinoma is indicated; E = exocrine pancreas.