Determining Occlusion Pressure in Lymphatic Vessels

Injection of contrast agent Indocyanine Green (ICG) is a tricarbocyanine dye that is commonly used for hepatic, cardiovascular, plastic surgery and ophthalmology applications. It is usually injected intravenously at a dose comprised between 0.1 and 0.5 mg/kg. After administration, ICG binds to plasmatic proteins, lipoproteins and lipids, and is thereafter rapidly and completely cleared from blood by the liver. ICG is excited and emits fluorescence in the near-infrared (around 760 nm).

In this study, we will suspend ICG in 25 ml pure water and subsequently diluted with pure water to reach a final concentration of 1 mg/ml. Each subject will receive a subcutaneous injection of 0.2 ml of diluted ICG (e.i. 200µg), using a hypodermic needle. We will standardize the injection point and inject ICG subcutaneously in the first interdigital space, according to the injection method used for lymphoscintigraphy. In this way, we highlight systematically the same area of the superficial lymphatic network.

We know that in the upper limbs, lateralization or handedness coincides with functional (and/or anatomical) asymmetries. To avoid any bias, the injected arm in healthy patients will be randomized tossing heads or tails.

Fluorescence images acquisition After injection, the injection sites will be covered with tape to avoid camera oversaturation, and to avoid spreading of ICG on the skin and undesirable fluorescent background on the images. To obtain fluorescence imaging of arm lymphatic flow, a near-infrared fluorescence imaging system will be used (Photo Dynamic Eye, or PDE camera from Hammamatsu Photonics - Japan). The camera is maintained in a fix position, 15 cm above the investigation field, by a holding system from Noga Tools. The amplified analogic signal is transformed into a digital signal by a converter from Terratec (Model Grabster AV 450 MX) and transferred to a monitor, which allows to visualize lymphatic network and lymph flow in real-time. Images will be recorded on a dedicated external hard disk drive Iomega, model MDHDU.

The session will take place in a dark room, to allow NIR fluorescence images acquisition.

Specific manual lymphatic drainage, developed by the first author and his team under NIRF feedback, is performed immediately after the injection, during the whole experimental session in order to be sure that the examined lymphatic vessels permanently contain lymph. The lymph is propelled with the hand of the operator from the injection point to a limit line drawn on the skin at 5 cm before the distal extremity of the transparent cuff. This optimized manual lymphatic drainage will be performed for 3 minutes in order to easily detect the superficial lymphatic vessels at the volar region of the arm and to mark the area of interest. The MLD is standardized by the first author for this experiment, and composed by a continuous and repeated sequence, executed from distal to proximal, of 3 "fill up" maneuvers realized on the injection point, succeeded by 2 "wash out" maneuvers realized from the injection point to the limit line before the cuff. Then, two fluorescent lines are drawn on the skin in a distance of 50 mm in order to limit the space of reference.

The cuff of the transparent sphygmomanometer is placed in the middle of the arm.

The sphygmomanometer is connected to a pressure meter, indicating mm of Mercury (Testo® 510 Digital Manometer precision 0.1mmHg.).

The PDE camera will be maintained perpendicularly on the major axis of the arm, at the level of the middle of the cuff.

In order to make sure that observed lymphatic vessels contain lymph, the fluorescent lymph is propelled during all the experience with the hand of the operator from the injection point to a limit line drawn on the skin at 5 cm before the distal extremity of the transparent cuff.

• First step: as baseline we record and observe the lymphatic flow under the un-inflated cuff.
• Second step: the cuff is inflated at 30mmHg, images are recorded during three lymph flow passages.

Three independent observers are present. They control the three passages of lymph in order to give the consent to go over to the next pressure step.

• Third step: We increase the inflated pressure in steps of 10 mmHg recording images at each increase (waiting for three passages of lymph at each step).

The experiment is completed as soon as we visually note the complete stop of lymph progression under the transparent cuff. At that moment, the pressure is reduced by 10mmHg in order to validate the return of the lymph flow. After this step, we observe and record images of the lymphatic vessel from the injection point up to the root of the limb.