All the studies described herein were approved by the Ethical Committee of Hamamatsu University School of Medicine, and informed consent was obtained from all the participants.

The measurement was performed as previously reported [9]. Briefly, using a 27-gauge needle, we subcutaneously injected 0.3 mL of ICG (Diagnogreen 0.5 %; Daiichi-Sankyo Pharmaceutical, Tokyo, Japan) at the dorsum of each participant’s foot. Immediately after the injection, fluorescence images of the subcutaneous lymphatic drainage were obtained using an infrared camera system (PDE^TM, Hamamatsu Photonics K.K. Hamamatsu, Japan), which activates ICG with light emitted at a wavelength of 760 nm and filters out light at a wavelength <820 nm. The light source for the emission of ICG consisted of 760-nm light-emitting diodes, and the detector was a charge-coupled device camera. The fluorescence images were continuously observed on the monitor of a laptop (VAIO, Type T; SONY Co., Tokyo, Japan).

Before the injection of ICG, a custom-made transparent sphygmomanometer cuff was wrapped around the lower leg just below the popliteal fossa. The cuff was connected to a standard mercury sphygmomanometer. Lymphatic pumping was measured with the participants in the sitting position. Immediately after the subcutaneous injection of ICG, the transparent cuff was inflated to 70 mmHg, and then, it was gradually deflated to lower the pressure by increments of 5 mmHg every 2 min. This was performed until the fluorescence dye exceeded the upper border of the cuff, indicating that the lymphatic contraction had overcome the cuff pressure. The value of the cuff pressure at this point was used to measure P lymph pump (Fig. 42.2).

This method was confirmed by comparing data with the values obtained with a dynamic lymphoscintigram using the same cuff technique. We found that there was a significant correlation between the values with ICG fluorescence lymphography and dynamic lymphoscintigraphy [8].

Three hundred and ninety-nine participants (199 men and 200 women) were recruited from the Hamamatsu University School of Medicine. The subjects medical history was screened preliminarily, and those with no serious allergy and no history of leg injury or surgery were included. Those with an iodine allergy were excluded according to the industry’s recommendation because ICG contains iodine. In addition, subjects with varicose vein in the leg (i.e., C3, 4, and 5 according to the Clinical Etiology Anatomy Pathophysiology (CEAP) classification) were excluded. P lymph pump was measured as previously described.

Figure 42.3 shows that there was a significant correlation between the leg lymphatic pumping and age: r = −0.34 (p < 0.0001). Both sexes showed a significant correlation between leg lymphatic pumping and age (male: r = −0.32, p < 0.0001; female: r = −0.37, p < 0.0001).

Aging affects the arteries and causes arteriosclerosis. However, venous stasis syndrome causes phlebosclerosis. Based on this study’s findings, aging may affect the lymph vessels and cause lymphosclerosis. This concept was proposed by Rabinovits et al. almost half century ago [10]. They performed a histological study on the human thoracic duct in elderly humans and reported changes in the distribution of smooth muscle cells and fibrosis. Our novel method of measuring enabled to demonstrate that “lymphosclerosis” may change the lymphatic vessels functionally. The mechanisms of contraction/relaxation in lymph vessels are yet to be fully understood. Similar to blood vessels, lymphatic endothelial cells have an ability to produce nitric oxide (NO) [11]. The endothelium-dependent modulation via NO production is considered to be a key mechanism of lymph pumping. In an animal study, endothelial NO synthase expression in the thoracic duct was markedly decreased, and flow-dependent modulation of the lymph pumping was completely abolished, suggesting that aging may disturb NO-dependent regulatory mechanisms of the lymphatic vessels [12].

Our study demonstrated the effect of aging on lymphatic vessels, which may begin a new era of lymphatic research regarding antiaging, lifestyle, and new drug developments [9].