Introduction

Secondary lymphedema is a debilitating chronic disease whereby lymphatic insufficiency due to lymph node removal causes fluid accumulation and limb swelling, and eventual soft tissue changes¹. Prolonged lymph stasis in advanced disease induces a chronic inflammatory state leading to a cyclical progression of fibrosis, adipose expansion, and further lymphatic impairment^2,3.

Much research on adipose tissue pathology in lymphedema has been performed in animal models or clinical tissue biopsies⁴. Noninvasive MRI could be an important in vivo tool for probing fibroadipose tissue pathology and potentially to guide therapy development and implementation⁵. Fibrosis is discernible on T₁-weighted MRI, yet such anatomical imaging is largely insensitive to inflammation which may adjust before and after treatments. Sodium imaging (²³Na-MRI) has gained recent attention and has sensitivity to many inflammatory disease processes⁶. Combining ¹H/²³Na-MRI to study lymphedema could help localize fibrosis and interrogate the inflammatory tissue environment associated with disease progression.

We apply proton and sodium ¹H/²³Na-MRI in a preliminary cohort of patients with advanced secondary lower extremity lymphedema (LEL) with unilateral disease. We test the hypotheses that (1) subcutaneous adipose tissue (SAT) volume is elevated in affected compared to contralateral legs, forming distinct patterns of fibrosis, and (2) fibroadipose tissue has elevated sodium content compared to surrounding non-fibrosed tissue.

Methods

All participants provided informed consent in accordance with the local Institutional Review Board. Patients with secondary LEL were enrolled from a lymphedema therapy clinic who met inclusion criteria of i) pelvic or inguinal lymph node removal for cancer therapy or hysterectomy procedures, ii) unilateral disease, and iii) no history of kidney disease or conditions affecting sodium handling. The affected limb LEL stage was evaluated by a certified physical therapist according to International Society of Lymphology criteria².

MRI Acquisitions.
Participants were scanned supine at 3T (Philips Healthcare; Best, The Netherlands) using the body coil for radiofrequency transmission/reception in ¹H-MRI acquisitions and a single-tuned receive-only quadrature sodium coil (Rapid Biomedical GmbH; Rimpar, Germany) for ²³Na-MRI acquisitions. Both legs were scanned independently, with the mid-gastrocnemius calf muscle at the isocenter (Figure 1a).

We utilized a multi-nuclear MRI protocol^7,8 in the lower extremities. The two-point Dixon water-fat method (TR=100ms, TE₁/TE₂=1.19/2.37ms, in-plane resolution=1x1mm², slice thickness=6mm) provided separate water- and fat-weighted contrasts in the transverse plane (Figure 1b). ²³Na-MRI was acquired in the same field-of-view as Dixon MRI, using a 3D gradient-echo sequence with minimal echo time (TR=130ms, TE=0.99ms, in-plane resolution=3x3mm², slab thickness=30mm, number signal acquisitions=26) (Figure 1c).

Imaging and statistical analyses.
Affected legs with lymphatic impairment were determined to have radiologically-evident fibrosis; contralateral limbs without evidence of fibrosis are considered an internal control.

To evaluate hypothesis 1, SAT was segmented manually from fat-weighted Dixon images of both limbs. Within the SAT, a Gaussian mixture model was used to identify hyperintense regions on water-weighted images corresponding to SAT fibrosis. Cross-sectional slices were partitioned into equiangular quadrants around the centroid of the SAT mask, approximately corresponding to the following anatomical regions: anterior, posterior, lateral, and medial (Figure 1d). SAT volume (mL) in each quadrant was compared between affected and contralateral legs using a Wilcoxon signed-rank test. Fibrosis extent (fibrosis volume/SAT volume, %) was evaluated between quadrants in the affected leg of patients with lymphedema using the Kruskal-Wallis test with Dunn’s post-hoc test.

Tissue sodium content (TSC) maps were calculated by calibrating signal intensity of ²³Na-MRI from standard sodium concentrations using linear interpolation⁷. Mean TSC in each quadrant was compared in the affected vs. contralateral legs using the Kruskal-Wallis test. To evaluate hypothesis 2, TSC in fibroadipose tissue, surrounding non-fibrosed adipose tissue, and contralateral SAT were compared pairwise by Wilcoxon signed-rank tests.

All statistical tests were considered significant at the two-sided P≤0.05 level.

Results

The cohort consisted of seven participants with unilateral secondary leg lymphedema (age=56.4±8.6 years, BMI=28.3±6.1 kg/m², LEL stage=2 or 3). SAT volume was significantly higher in affected limbs compared to contralateral limbs (Table 1). Fibrosis extent was highest in the anterior calf (Table 2) and significantly elevated in the anterior compared to posterior region (P=0.009).

TSC was also significantly higher in the anterior compared to the posterior region of the affected limb (P=0.014, Table 2). Fibroadipose tissue segmentations has elevated TSC (mean ± standard deviation=54.11±13 mmol/L) compared to surrounding non-fibrosed adipose tissue (25.36±11 mmol/L, P=0.016) and contralateral leg SAT (14.21±4 mmol/L, P=0.016, Figure 2).

Fibrosis and sodium deposition patterns prominent in the anterior SAT in the calf are visualized in a representative patient with LEL (Figure 3).

Discussion

This work expanded upon existing MRI studies of secondary lymphedema fat and fibrosis pathology, by utilizing regional analysis and incorporating multi-nuclear ¹H/²³Na MRI^5,9. Together, quantitative imaging revealed remarkably high tissue sodium in specific regions of fibrosis within the lower-extremity SAT expansion in participants with advanced LEL. Locoregional fibrosis formation and sodium deposition, with prominence to the anterior region, appear consistent with anterior lymphatic drainage pathways ^3,10. Further multi-modal imaging using MR lymphangiography could help interrogate these lymphatic pathways potentially corresponding to tissue sodium and fibrosis.^5,11.

Conclusion

Multi-nuclear proton and sodium MRI demonstrated SAT fibrosis and high tissue sodium deposition in advanced stage LEL. Findings are useful for understanding lymphatic disease pathophysiology, but also may provide a much-needed imaging biomarker of lymphedema.

Acknowledgements

Funding was provided by the National Institutes of Health (NIH)/NINR 1R01NR015079, the NIH/NHLBI 1R01HL157378, NIH/NHLBI 1R01HL155523. Imaging experiments were performed at the Vanderbilt Human Imaging Core, using research resources supported by the NIH grant 1S10OD021771-01. We are grateful for Philips support from Charles Nockowski and Ryan Robison, and to Human Imaging Core MRI technologists. Recruitment through www.ResearchMatch.org and services at the Clinical Research Center are supported by the National Center for Advancing Translational Sciences (NCATS) Clinical Translational Science Award (CTSA) Program, award number 5UL1TR002243-03. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.