Near-infrared Navigation System for Real-time Visualization of Blood Flow in Vascular Grafts

Summary

We describe the procedure for real-time monitoring of blood flow in vascular grafts using indocyanine green (ICG), a near-infrared (NIR) dye, and a portable near-infrared navigation (NAVI) detectible camera system. The flow of dye in vascular grafts and the camera efficiency have been compared with Doppler and cine-angiography procedures.

Abstract

Vascular grafting failures are often attributed to inadequate anastomotic perfusion assessments. If successful, vascular anastomosis can be rapidly confirmed through the visualization of continuous blood flow upon completion of the grafting process. Surgeons can then minimize graft failures, thus decreasing morbidity in a cost-effective manner. Fluorescence image-guided surgery using near-infrared (NIR) dye is one of the methods that can be performed to monitor grafting success. To address the current logistical challenges and costs of these systems, a compact camera system was used for intraoperative fluorescence real-time NIR imaging. Combined with benchtop experiments, a swine model was used to demonstrate the procedure of using a near-infrared navigation system (NAVI) to visualize grafted vessels in vivo. This was done by performing bilateral arteriovenous grafts and imaging intravenously injected ICG as it circulated through the grafted vessels. The fluorescent images obtained by NAVI were corroborated with Doppler flow measurements and cine-angiography, the current gold standard for the evaluation of vascular grafts.

Introduction

Intraoperative assessments of blood flow following arterial or venous grafting, as well as direct visualization methods evaluating anastomosis, allow surgeons to make immediate decisions on the likelihood of success or failure, allowing for repair, if needed^1,2. Techniques frequently employed are fluoroscopic angiography³, direct visualization, palpation of pulsatile flow, pulse oximetry², Doppler ultrasound⁴, transit time flowmetry^5,6,7, computed tomography angiography⁸, and visible light and near-infrared spectrophotometry⁹. As the evaluation of surgical outcomes continues, improvements in equipment and an increasing availability of technology allow for the more widespread use of these methods. Challenges exist in many of these techniques; while they are excellent at evaluation, some limitations include cumbersome equipment, a lack of proper anatomical definition, and exposure to radiation¹⁰. One technique that avoids many of these drawbacks and that has been tested across a wide variety of surgical repairs is the use of near-infrared (NIR) fluorescence imaging, specifically with FDA-approved indocyanine green (ICG)¹¹. ICG fluoresces brightly at low concentrations and is well confined to the vasculature due to its high plasma protein binding capabilities¹². Additionally, the liver clears it from blood circulation within minutes, allowing for multiple injections^13,14.

Fluorescence image-guided surgery is an optical imaging procedure that allows surgeons to visualize real-time tissue images and to evaluate circulation and perfusion intraoperatively. Capturing real-time images improves surgical outcomes by allowing the rapid identification and repair of graft failures¹⁵. ICG is injected intravenously and visualized under an NIR wavelength range (700-900 nm). ICG visualization in this wavelength range results in high contrast against a black background due to minimal tissue absorbance and scattering,meaning minimal auto-fluorescence. While frequently used to measure hepatic function and ophthalmic angiography¹¹, this dye has shown promise for several procedures that include but are not limited to: lymphatic mapping^16,17,18,19, hepatobiliary surgery²⁰, dermal vasculature mapping for burn wound severity²¹, cerebral aneurysm repair^22,23, gastrointestinal anastomosis verification²⁴, and venous and arterial grafting¹². A recent, encompassing review of the usage of ICG in surgical procedures revealed the need for improvements in the image processing, portability of the instrumentation, and economic viability¹².

A portable and economical fluorescence imaging system that could serve as an alternative to the costly and unwieldy existing fluorescence imaging modality is desired to assist surgeons in intraoperatively evaluating the success of the vascular grafting process. We used a portable NIR navigation system (NAVI), which has the capability of visualizing ICG in deep tissues and recording real-time images with high sensitivity. The ability of the NAVI system to visualize selected regions of interest, confirm blood flow, and detect leaks from sites of anastomosis has been demonstrated by in vivo arteriovenous grafting procedures in a swine study.

The integral components and assembly of NAVI (Figure 1) for the visualization of florescence emitted from ICG are listed and briefly described below. The source is five 770-nm NIR LEDs assembled onto a battery DC power supply, with a 775-nm short pass filter. The short pass filter allows only 770-nm emitted light to reach the imaging site while reducing any possible stray radiation above 770 nm. The camera is an NIR imaging camera with an added band pass filter. The width of the pass region is 37 nm, centered at 832 nm, and is employed for the purpose of rejecting radiation collected by the NIR imaging camera that does not emanate from ICG fluorescence or from stray light. The NIR imaging camera has an RCA analog output for the image signal. The interface is the composite video image from an NIR imaging camera that is interfaced to a USB port on a computer via an analog-to-video interface designed to translate composite video to digital for the purpose of capturing, storing, processing, and providing the output of the digital signal to an image display unit. The interface system accepts the analog output. The computer is a single-board with a USB input that has the processing capabilities necessary to store and process images. The monitor is a conventional video monitor of any size and is used to display the images generated by the computer. The stands are two mechanical arms for suspending the source and NIR camera over selected tissue areas, as well as a foot control that aids in the movement of the NIR camera.

Protocol

Animal studies were performed following approval from the University of Missouri Animal Care and Use Committee. The University of Missouri is USDA-licensed and AAALAC-International accredited.

1. Pre-operation procedure

1. Obtain a domestic Yorkshire/large white pig (4 months old; 57 kg) one day prior to the study and provide water ad libitum.
2. Sedate the pig with 5 mg/kg Telazol and 2.2 mg/kg xylazine. Provide 3-4% isoflurane by nose cone until the pig can be intubated and then reduce the isoflurane to 2%.
3. Intubate the pig, attach ECG leads, and place the pig on mechanical ventilation for the entire procedure. Confirm the depth of anesthesia by performing a toe pinch and observing the loss of the pedal reflex. Throughout the procedure, monitor the heart rate, absence of limb withdrawal, and absence of palpebral reflex to ensure the maintenance of a surgical plane of anesthesia.
4. Place the pig in dorsal recumbent position on the operating table.
5. Place a 20-gauge IV catheter in the right auricular vein. Administer IV fluids (0.9% NaCl) at 5 mL/kg/h via an auricular catheter.

2. Carotid artery grafting

1. Incise approximately 12 cm of the jugular furrow with a size-10 scalpel blade. Expose the external jugular vein and the carotid arteries via blunt dissection of the sternomastoidius and sternocephalic musculature using Metzenbaum scissors.
2. Using curved Metzenbaum scissors and Adson-Brown tissue forceps, dissect an 8-cm segment of the right jugular vein. Apply proximal and distal 2-0 silk ligatures and remove it.
3. Apply vascular clamps to the carotid artery and make a small, vertical arteriotomy on the anterior wall of the vessel. Circumferentially suture one end of the jugular vein with 6-0 Prolene sutures to the arteriotomy in an end-to-side manner to complete the proximal anastomosis.
4. Likewise, perform distal anastomosis with the other end of the jugular vein to complete the graft.
5. Remove the clamps to allow the diverted blood to flow through the graft.
6. Repeat the jugular vein grafting on the contralateral side.

3. Visualization by NAVI

1. Prepare 10 mL of ICG (1 mg/mL) solution in sterile water in a sterile red-top tube and keep it protected from light. Draw up the injection amount with a sterile 20-gauge needle and syringe, as needed.
2. Focus the camera, adjust the NIR light source, and open the software preloaded in the laptop screen. Use low ambient light and turn off all room lights when the camera is functioning.
3. Rapidly inject 3.0 mL of ICG (0.05 mg/kg) in the auricular catheter and flush with 8 mL of sterile saline.
4. After the injection of ICG, press the small switch on the NIR light source to turn it on and illuminate the graft region; simultaneously press the switch to turn on the NIR imaging camera.
5. View the fluorescent images on the laptop screen. Simultaneously, click the record icon in the software to record the images.
6. Wait approximately 7 min to allow the fluorescence of the ICG to diminish.
7. Place vascular clamps in the middle of the graft to occlude anastomosis. Repeat the injection at a lower dose of 1.5 mL of ICG (0.03 mg/kg) and flush with saline.
8. Repeat steps 3.2-3.5 to record the flow of ICG into the carotid segment grafting and perform fluorescence measurements on the contralateral side.
9. Perform blood flow measurements simultaneously using Doppler flow probes, as described below in step 4.

4. Doppler probe measurement

1. Use two 3.0-mm Doppler blood flow probes. Place one flow probe onto the carotid artery, cranial to the graft site, and apply ultrasound gel over the probe. After vascular placement, connect the flow probe to the perivascular flow module. Connect the perivascular flow module to an analog-to-digital board, which allows the Doppler signal to be measured as the blood flow in real time using data acquisition software.
2. Place the second flow probe on the bypassed segment of the carotid artery and apply ultrasound gel over the probe. After vascular placement, connect the flow probe to the perivascular flow module. Connect the perivascular flow module to an analog-to-digital board that allows the Doppler signal to be measured as blood flow in real time using data acquisition software.
3. Allow both probes to remain on the vessels for several minutes and record and compare the flow between the two flow probes.

5. Cineangiography procedure

1. Turn on the fluoroscopy unit.
2. Create a medial longitudinal skin incision in the region between the left gracilis and sartorius musculature to access the left femoral artery. Bluntly dissect with curved Metzenbaum scissors and Adson-Brown tissue forceps until the femoral artery is identified.
   1. After placing self-retaining tissue retractors, securely occlude the distal aspect of the femoral artery with 2-0 silk suture and insert a guiding catheter into the femoral artery. Pass the guide wire into the aorta via the femoral artery and external iliac artery.
3. Give a loading dose of heparin (300 U/kg) IV and continue hourly at 100 U/kg.
4. Using a 0.035 mm-diameter flexible guide wire, feed the catheter to the desired location in the carotid artery by way of the brachiocephalic trunk. Remove the guidewire.
5. Inject 8-10 mL of iohexol solution into the carotid artery (visualize each side individually).
6. Press the start key on the fluoroscopy unit to record the images.
7. View the images on a computer loaded with software for viewing the images.
8. Repeat iohexol and image acquisition for the contralateral side by moving the catheter into the contralateral carotid artery.
9. Following these procedures, sacrifice the pig with an IV pentobarbital overdose while under anesthesia.

Results

Optimization of the Concentration of ICG

The concentration of ICG required to produce optimal fluorescent images was determined using the following procedure. Different concentrations of ICG solutions ranging from 1.29 to 258 µM were prepared in microcentrifuge tubes and placed on a stand. The NIR light source and the camera were placed at a constant distance from the dye (i.e., 18 and 31 in, respectively, from the st...

Discussion

Development of NAVI for ICG Imaging Applications

NIR-based fluorescence imaging is emerging as a simple alternative procedure for intraoperative imaging, demonstrating significant benefits by: (1) eliminating the radiation exposure required by fluoroscopy and computed tomography and (2) reducing the surgical time, thus decreasing per-patient costs while maintaining efficacy and increasing safety. In this paper, we demonstrated the procedure for using NAVI to perform fluorescence imaging (using IC...

Materials

Name	Company	Catalog Number	Comments
Telazol	Zoetis		Reconstitute with sterile water prior to use
Xylazine (AnaSed)	AKORN Animal Health
0.9% NaCl	Abbott
Indocyanine green	AKRON
Heparin	Baxter
Visipaque (iodixanol)	GE Healthcare	564v
Guide catheter	Boston Scientific
LED 770-03AU, 18 mW	Roithner Laser Technik GmbH, Wiedner Hauptstrasse, Austria
Short pass filter	Edmund optics, Barrington, NJ, USA
NIR imaging  camera (Igen NV20/20-IC)	B&H optics, New york, NY, USA
Micropac USB-AVCPT interface	Sabrent, USA
CT instrument	Varian Medical System manufactured by Omega Medical Imaging
TS420 Perivascular Flow Module	Transonic Systems Inc., Ithaca, NY	TS420 & T402
PR Series Flow Probes	Transonic Systems Inc., Ithaca, NY	3PSB
Digital Board to Flow module	ADInstruments Inc., Colorado Springs, CO
Lab Chart	ADInstruments Inc., Colorado Springs, CO