Real-time intraoperative ureteral guidance using invisible near-infrared fluorescence

Abstract

Purpose: Invisible near-infrared light is safe and it penetrates relatively deeply through tissue and blood without altering the surgical field. Our hypothesis was that near-infrared fluorescence imaging would enable visualization of the ureteral anatomy and flow intraoperatively and in real time.

Materials and methods: CW800-CA (LI-COR, Lincoln, Nebraska), the carboxylic acid form of near-infrared fluorophore IRDye 800CW, was injected intravenously, and its renal clearance kinetics and imaging performance were quantified in 350 gm rats and 35 kg pigs. High performance liquid chromatography and electrospray time-of-flight mass spectrometry were used to characterize CW800-CA metabolism in urine. The clinically available near-infrared fluorophore indocyanine green was also used via retrograde injection into the ureter. Using the 2 near-infrared fluorophores the ureters were imaged under the conditions of steady state, intraluminal foreign bodies and injury.

Results: In rat models the highest signal-to-background ratio for visualization occurred after intravenous injection of 7.5 microg/kg CW800-CA with values of 4.0 or greater and 2.3 or greater at 10 and 30 minutes, respectively. In pig models 7.5 microg/kg CW800-CA clearly visualized the normal ureter and intraluminal foreign bodies as small as 2.5 mm in diameter. Retrograde injection of 10 microM indocyanine green also permitted the detection of normal ureter and pinpointed urine leakage caused by injury. Electrospray time-of-flight mass spectrometry, and absorbance and fluorescence spectral analysis confirmed that the fluorescent material in urine was chemically identical to CW800-CA.

Conclusions: A convenient intravenous injection of CW800-CA or direct injection of indocyanine green permits high sensitivity visualization of the ureters under steady state and abnormal conditions using invisible light.

Figure 1. Real-time visualization of the ureters using invisible light

Minutes after IV injection of A) 7.5 μg/kg CW800-CA into rat and B) 7.5 μg/kg into pig, the ureters become highly NIR fluorescent (arrows). Shown are the color video (left), NIR fluorescence (middle), and merged images of the two (right). NIR fluorescence images have identical exposure times (100 msec) and normalizations. Data are representative of N = 4 independent experiments in rat and N = 6 independent experiments in pig.

Figure 2. Quantification of SBR kinetics for ureteral NIR fluorescence emission

The SBR (mean ± SEM) of the ureter relative to A) abdominal wall and B) kidney was quantified over time after IV injection of 1.5 μg/kg, 3.0 μg/kg, 7.5 μg/kg, and 15 μg/kg CW800-CA in N = 3 rats each.

Figure 3. Assessment of ureteral patency and injury in large animals approaching the size of humans

1. After IV injection of 7.5 μg/kg CW800-CA in the pig, the ureters could be visualized for the next 60 min. Shown is the identification of two foreign bodies (2.5 mm diameter beads; arrows) within the lumen of the ureter. NIR fluorescence exposure times were 100 msec. Data are representative of results from N = 4 independent experiments.

2. Retrograde injection of 10 ml of 10 μM ICG in saline provided immediate visualization of the ureters. NIR fluorescence exposure times were 60 msec. Data are representative of N = 2 independent experiments.

3. A site of injury to the ureter made during tissue dissection, in this case from a scalpel, can be pinpointed immediately (red arrow) and repaired. NIR fluorescence exposure times were 60 msec. Data are representative of N = 2 independent experiments.

Figure 4. Mass spectroscopic analysis of urine after IV injection of CW800-CA

A) 700 nm absorbance (top) and 800 nm fluorescence (bottom) of urine (left) and pure CW800-CA (right) after separation on a C18 column. B) The single non-void volume, NIR fluorescent peak from each chromatography column was subjected to mass spectroscopic analysis, with the obtained fragmentation pattern shown. Data are representative of N = 4 independent experiments.