In Vivo Dynamic and Static Analysis of Lymphatic Dysfunction in Lymphedema Using Near-Infrared Fluorescence Indocyanine Green Lymphangiography

Abstract

Background: Near-infrared fluorescence indocyanine green lymphangiography, a primary modality for detecting lymphedema, which is a disease due to lymphatic obstruction, enables real-time observations of lymphatics and reveals not only the spatial distribution of drainage (static analysis) but also information on the lymphatic contraction (dynamic analysis). Methods: We have produced total lymphatic obstruction in the upper limbs of 18 Sprague-Dawley rats through the dissection of proximal (brachial and axillary) lymph nodes and 20-Gy radiation (dissection limbs). After the model formation for 1 week, 9 animal models were observed for 6 weeks using near-infrared fluorescence indocyanine green lymphangiography by injecting 6-μL ICG-BSA solution of 20-μg/mL concentration. The drainage pattern and leakage of lymph fluid were evaluated and time-domain signals of lymphatic contraction were observed in the distal lymphatic vessels. The obtained signals were converted to frequency-domain spectrums using signal processing. Results: The results of both static and dynamic analyses proved to be effective in accurately identifying the extent of lymphatic disruption in the dissection limbs. The static analysis showed abnormal drainage patterns and increased leakage of lymph fluid to the periphery of the vessels compared with the control (normal) limbs. Meanwhile, the waveforms were changed and the contractile signal frequency increased by 58% in the dynamic analysis. Specifically, our findings revealed that regular lymphatic contractions, observed at a frequency range of 0.08 to 0.13 Hz in the control limbs, were absent in the dissection limbs. The contractile regularity was not fully restored for the follow-up period, indicating a persistent lymphatic obstruction. Conclusions: The dynamic analysis could detect the abnormalities of lymphatic circulation by observing the characteristics of signals, and it provided additional evaluation indicators that cannot be provided by the static analysis. Our findings may be useful for the early detection of the circulation problem as a functional evaluation indicator of the lymphatic system.

Impact of Cervical Lymphatic Obstruction on Brain Pathophysiology in Cervical Lymphedema Animal Models

“…Furthermore, abnormal waveforms of lymphatic contraction were observed in the CLN dissection group. A previous study showed a change in lymphatic contraction induced by lymphatic obstruction in upper limb lymphedema models 18 . Figure 2C demonstrates a disruption of the cervical lymphatic circulation.…”

“…In addition, we measured lymphatic contraction signals in the same collective lymphatic vessels in both groups. In a previous study, we examined a method for obtaining lymphatic contraction signals through signal processing and identified variations in lymphatic contraction signals caused by lymphatic obstruction 18 .…”

“…Therefore, we obtained 15-min real-time videos to identify these variations in lymphatic contraction and extracted lymphatic contraction signals in both groups using ImageJ software. FFT signal processing was performed using Origin Pro 9 software (Origin 9.0; OriginLab, Northampton, MA, USA) for numerical calculations and visualization based on previous research 18 .…”

“…Lateral and ventral images of the neck were consistently measured and analyzed from the same posture, and the crosssectional area was calculated according based on the neck diameter using ImageJ software (ImageJ 1.48 v, http://rsbweb.nih.gov/ij/; NIH, Bethesda, MD, USA). After anesthesia, the animals were flexed on a plane in a completely relaxed state, and the diameter of the neck was measured using a customized near-infrared fluorescence (NIRF) imager (Figure S3) 18,61,67,68 .…”