Media 1: Dual-mode laparoscopic fluorescence image-guided surgery using a single camera

Originally published in Biomedical Optics Express on 01 August 2012 (boe-3-8-1880

Improved Intraoperative Visualization of Nerves through a Myelin-Binding Fluorophore and Dual-Mode Laparoscopic Imaging

<div><p>The ability to visualize and spare nerves during surgery is critical for avoiding chronic morbidity, pain, and loss of function. Visualization of such critical anatomic structures is even more challenging during minimal access procedures because the small incisions limit visibility. In this study, we focus on improving imaging of nerves through the use of a new small molecule fluorophore, GE3126, used in conjunction with our dual-mode (color and fluorescence) laparoscopic imaging instrument. GE3126 has higher aqueous solubility, improved pharmacokinetics, and reduced non-specific adipose tissue fluorescence compared to previous myelin-binding fluorophores. Dosing and kinetics were initially optimized in mice. A non-clinical modified Irwin study in rats, performed to assess the potential of GE3126 to induce nervous system injuries, showed the absence of major adverse reactions. Real-time intraoperative imaging was performed in a porcine model. Compared to white light imaging, nerve visibility was enhanced under fluorescence guidance, especially for small diameter nerves obscured by fascia, blood vessels, or adipose tissue. In the porcine model, nerve visualization was observed rapidly, within 5 to 10 minutes post-intravenous injection and the nerve fluorescence signal was maintained for up to 80 minutes. The use of GE3126, coupled with practical implementation of an imaging instrument may be an important step forward in preventing nerve damage in the operating room.</p></div

Optical properties of GE3126 in different solvents.

<p>Ɛ = molar extinction coefficient; Abs Max = absorbance maximum wavelength; Em Max = emission maximum wavelength; QY = quantum yield.</p><p>Optical properties of GE3126 in different solvents.</p

Kinetics and dosing in mice following IV administration of GE3126 formulated with 80% distilled/deionized water, 10% 2- HPβCD, and 10% propylene glycol.

<p>Imaging was performed using a commercial fluorescence stereomicroscope with multispectral detection. Excitation was achieved using a filter centered at 406 nm with a 15 nm bandwidth. Numerical data represented the area under the curve for emission wavelengths ranging from 550 nm to 720 nm acquired in sciatic nerves, adjacent muscle and adipose tissue, with n = 3 mice per group. (A) For kinetics, each mouse was given 16 mg/kg of GE3126 and imaging was performed at 0.5, 1, 2, 3, or 4 h post-injection. (B) In the dosing study, mice were given 1.6, 3.3, 6.6., 10.0, 13.3, 16.6, 20.0, 23.3, or 46.6 mg/kg of GE3126 and imaging was performed 1 h post-injection. Control mice were given a single injection of IV formulation (vehicle only) and imaged to determine background fluorescence. (C) Representative fluorescence multispectral image of a mouse injected with 16.6 mg/kg of GE3126. The sciatic nerve (reddish-orange) is indicated by an arrow. Note fluorescence labeling of adjacent adipose tissue in green. The control animal tissue appeared dark. Scale bar ~0.5 mm. (D) Emission spectra of nerve, muscle, and adipose tissue are shown to illustrate spectral separations between tissue types in both GE3126-treated mouse and control mouse.</p

Dual-mode laparoscopic imaging in the porcine model.

<p>A dose of 0.74 mg/kg GE3126 was injected into the pig. An incision was made into the left brachial plexus and video was recorded just prior to GE3126 injection and at defined time points up to 100 min post-injection. (A) Individual frames were extracted and the fluorescence intensities of nerve, adjacent muscle and adipose tissue were plotted over time. (B) Representative white light and fluorescence images extracted from the video show the brachial plexus (top) and the retroperitoneal region (bottom) at ~80 min post-injection. Nerves are shown with white arrows. (C) Fluorescence microscopy images of a pig nerve tissue section taken prior to injection of GE3126, and at ~90 min post-injection. Scale bar in B ~1 mm; scale bar in C = 20 μm.</p

Dual-mode laparoscopic imaging in a porcine model and corresponding histology confirmation.

<p>(A) White light imaging (top left) showing a vessel, marked “a”, and a harder-to-visualize nerve, in the vena cava, marked “b”. The corresponding fluorescence image (top right) shows the fluorescently labeled nerve “b” more clearly than white light imaging. (B) H&E staining of tissue sections of “a” and “b” confirmed the identity of the non-labeled blood vessel (left) and labeled nerve (right). Scale bar in A~ 1 mm; scale bar in B = 100 μm.</p

Modified Irwin screening and supplemental battery tests in rats to assess potential adverse effects on the nervous system.

<p>Four Sprague-Dawley rats were injected with 35 mg/kg GE3126 and observed at defined time intervals, up to 24 h post-injection for gross signs of reactions. The number of rats (of the 4 injected showing a reaction was reported for each adverse effect. Note that the same rat exhibited the transient effect in respiration, startle and sniffing. Four control rats (not shown) were injected with formulation buffer alone and none exhibited any reactions.</p

<i>In vitro</i> properties of the fluorophores.

<p>^a Measured maximal solubility when dissolved in 58.5% distilled water, 30% 2- HPβCD, 10% propylene glycol, 1% polyethylene glycol-300, and 0.5% DMSO</p><p>^b From Reference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130276#pone.0130276.ref031" target="_blank">31</a>]</p><p><i>In vitro</i> properties of the fluorophores.</p

Individual frames extracted from real-time video recorded during minimal access (A, B) and open (C) surgery of a rat injected with 12.2 mg/kg of GE3126 using a custom dual-mode laparoscopic imaging instrument.

<p>Arrows are pointing to nerves. A small incision was made in the diaphragm allowing access of the laparoscope to the thoracic cavity highlighting the areas around the phrenic nerve (A) and the vagus nerve (B). White light images (left column) identify anatomical markers (such as lung and blood vessels), and fluorescence images (right column) show small diameter nerves easily obscured by surrounding fascia. (C) Open incision in the brachial plexus. The medial and lateral cords (arrowheads) are more easily distinguished in fluorescence mode. Smaller nerves that are more visible under fluorescence, such as the suprascapular, anterior thoracic, and thoracodorsal nerves are shown with white arrows. Asterisk in (B) is a gloved finger of the operator. Scale bar ~1 mm.</p