High-resolution 3D volumetry versus conventional measuring techniques for the assessment of experimental lymphedema in the mouse hindlimb

www.nature.com/scientificreports OPEN received: 14 July 2016 accepted: 16 September 2016 Published: 04 October 2016 High-resolution 3D volumetry versus conventional measuring techniques for the assessment of experimental lymphedema in the mouse hindlimb Florian S. Frueh1,2,*, Christina Körbel1,*, Laura Gassert1, Andreas Müller3, Epameinondas Gousopoulos4, Nicole Lindenblatt2, Pietro Giovanoli2, Matthias W. Laschke1 & Michael D. Menger1 Secondary lymphedema is a common complication of cancer treatment characterized by chronic limb swelling with interstitial inflammation. The rodent hindlimb is a widely used model for the evaluation of novel lymphedema treatments. However, the assessment of limb volume in small animals is challenging. Recently, high-resolution three-dimensional (3D) imaging modalities have been introduced for rodent limb volumetry. In the present study we evaluated the validity of microcomputed tomography (μCT), magnetic resonance imaging (MRI) and ultrasound in comparison to conventional measuring techniques. For this purpose, acute lymphedema was induced in the mouse hindlimb by a modified popliteal lymphadenectomy. The 4-week course of this type of lymphedema was first assessed in 6 animals. In additional 12 animals, limb volumes were analyzed by μCT, 9.4 T MRI and 30 MHz ultrasound as well as by planimetry, circumferential length and paw thickness measurements. Interobserver correlation was high for all modalities, in particular for μCT analysis (r = 0.975, p < 0.001). Importantly, caliper-measured paw thickness correlated well with μCT (r = 0.861), MRI (r = 0.821) and ultrasound (r = 0.800). Because the assessment of paw thickness represents a time- and cost-effective approach, it may be ideally suited for the quantification of rodent hindlimb lymphedema. The lymphatic system is important for the regulation of fundamental biological processes such as immune response, intestinal lipid absorption and tissue fluid homeostasis1,2. The cardinal manifestation of lymphatic dysfunction is lymphedema, a condition which is characterized by limb swelling, chronic interstitial inflammation and connective or fat tissue deposition3,4. Based on the triggering cause it can be classified into primary and secondary lymphedema5. Primary lymphedema has a genetic background with a dysfunctional lymphatic system, either already symptomatic after birth or later in life6. In contrast, acquired damage of collecting lymphatic vessels causes secondary lymphedema. In the United States, more than 5 million people suffer from cancer-related lymphedema7, the most common form in developed countries. Lymph node dissection and irradiation result in the formation of scar tissue, which is a key inhibitor of lymphatic regeneration8. In particular, breast cancer and melanoma are associated with high rates of secondary lymphedema9. Despite reduced surgical invasiveness, recent data indicate that 20% of female breast cancer patients undergoing axillary lymph node dissection will develop arm lymphedema10. Given the livelong course of the disease, lymphedema has a highly relevant socio-economic burden. Many animal models have been established for the analysis of lymphedema pathophysiology and the development of novel approaches for its treatment. However, the induction of chronic lymphedema in animals is 1 Institute for Clinical and Experimental Surgery, Saarland University, 66421 Homburg/Saar, Germany. 2Division of Plastic Surgery and Hand Surgery, University Hospital Zurich, 8091 Zurich, Switzerland. 3Clinic of Diagnostic and Interventional Radiology, Saarland University Medical Center, Homburg/Saar, Germany. 4Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH Zurich, Zurich, Switzerland. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to F.S.F. (email: ) Scientific Reports | 6:34673 | DOI: 10.1038/srep34673 1 www.nature.com/scientificreports/ challenging and requires lymphatic interruption by means of surgery and irradiation11. The rodent limb is a frequently used model to study lymphatic regeneration after lymph node resection12,13 and emerging lymphedema treatments such as vascularized lymph node transfer14–18 or stem cell transplantation19,20. For this purpose, the valid assessment of limb volume is a major prerequisite. Recently, high-resolution three-dimensional (3D) imaging techniques such as magnetic resonance imaging (MRI) or microcomputed tomography (μCT) have been introduced for rodent limb volumetry21,22. However, the validity and reliability of these complex 3D techniques for limb volume assessment compared to conventional measuring techniques have not been systematically evaluated so far. Therefore, in the present study we assessed the hindlimb volumes in an acute lymphedema mouse model by means of μCT, 9.4 T MRI and high-resolution 30 MHz ultrasound (hrUS) as well as planimetry, circumferential length and paw thickness measurements. Subsequently, we calculated interobserver variability and performed a correlation analysis for the comparison of the different techniques. Results Acute hindlimb lymphedema model. Acute lymphedema was induced in C57BL/6 mice by means of popliteal lymphadenectomy, circular skin incision and cautery (Fig. 1a–c). In pilot experiments, we first assessed the course of this type of acute lymphedema during 28 days. Limb swelling peaked 1 to 3 days after surgery (maximal ratio operated/non-operated leg: 1.7) and rapidly decreased throughout the following observation period (Fig. 1d–f). Noteworthy, there was still significant paw swelling after 28 days (ratio: 1.1, p <  0.05) (Fig. 1g). Based on these results the evaluation of different volumetric techniques was performed in groups of 3 animals between day 3 and 10 after surgery, which guaranteed a well-distributed data set for correlation analyses (Fig. 1g). Additional qualitative histological and immunohistochemical analyses revealed markedly increased paw swelling and dilated lymphatic vessels on day 3, reflecting acute lymph stasis (Fig. 1h,j). In contrast, 10 days after surgery, paw volume had decreased and lymphatic vessels exhibited normal configuration (Fig. 1i,k). 3D hindlimb volumetry. The volumes of operated and contralateral non-operated hindlimbs were assessed by means of μCT, 9.4 T MRI and hrUS (Fig. 2a–l). The analysis included the determination of the overall volume (in mm3) of each hindlimb. For this purpose, boundaries were manually outlined in parallel slices separated by 1 mm step size in the 3D modality images and volumes were calculated by integrating the outlined areas. Volumes were assessed with the gluteal skinfold as a landmark (Fig. 2e), because circumferential hindlimb boundaries could be outlined up to this point. All 3D techniques resulted in high-resolution hindlimb images. In addition, due to high soft-tissue contrast, MRI and hrUS allowed visualization of edematous and t (...truncated)