Lymphedema causes local fluid retention and edema in subcutaneous tissue as a result of dysfunction in the circulating interstitial fluid between the thoracic duct and blood-stream due to damage or flaws in lymphatic systems [1, 2]. Penetration of inflammatory cells in lymphedema tissues damages dermal connective tissue elements, ground substances, microfilaments, collagen, and elastic fibers. As a result, angiogenic factors are generated by vascular endothelial and dermal inflammatory cells to cause vascular proliferation and angiosarcomas, leading to edema, chronic inflammation, fibrosis of tissue, and other conditions [3-6].

Tabibiazar et al. induced lymphedema in an animal model using a mouse tail, and observed an increase in fibroblast cells, hyperkeratosis, edema in epidermis, and expanded lymphatics in the dermis and subdermis in mouse tail histological samples [7].

Many progressive lymphedema patients can be diagnosed through distinguishing medical history, clinical features, and different imaging studies. Current diagnosis of lymphedema is done through the volume changes of affected limbs [8], and the tests that can diagnose lymphedema include isotopic lymphoscintigraphy, indirect and direct lymphography, lymphatic capillaroscopy, magnetic resonance imaging (MRI), axial tomography and ultrasonography [9]. However early diagnosis may be difficult because these volume and tissue changes are not clearly observed in early stages of the disease. Various studies have attempted to diagnose lymphedema through the volume changes, but the definition of volume change varies among studies [10-14]. In addition, diagnosis is not easy in early stages of mild or intermittent edema [9, 15]. Diagnosis of early stage lymphedema requires an invasive histologic assessment. However, this may cause complications such as infection or worsening of edema, so it is limited in clinical application.

Optical coherence tomography (OCT) is a new optical diagnostic technology which was first introduced as an imaging assessment method of the retina by Huang et al. in 1991 [16]. This technology scans and radiates near-infrared light to the body, then measures the reflected light intensity in each tissue in different positions and obtains images of the tissue through computer signal processing. It is similar to ultrasonography, but uses light instead of sound to obtain near-histological resolution.

In the 1990s when OCT was first introduced, it was shown to be effective in obtaining a non-invasive, contactless high resolution image used to study structural changes in the retina and optic nerve head [17]. Studies using OCT allow the visualization of changes in muscle microstructure in Duchenne muscular dystrophy [18, 19] and observation of microarchitecture by comparing the OCT images of axillary lymph node and histologic findings of breast cancer patients [20, 21].

However, studies on OCT imaging of tissues in cases of lymphedema have not been reported. Therefore, we constructed a mouse tail model of lymphedema and compared the OCT image to histologic findings to determine the utility of OCT in imaging of lymphedema.

Two male mice from the Institute of Cancer Research were used in this study. The mouse body weights were approximately 170~220 g. The mice were acclimated for one week before the experiments. The room temperature was maintained at 22~23°C with a humidity of 50~55% and lighting was on a 12-hour light-dark cycle. A diet of commercial rodent pellets and water was supplied ad libitum. We conducted surgery to yield a lymphedema on mouse tails. All animal procedures were conducted in accordance with the guidelines published in the Guide for the Care and Use of Laboratory Animals (DHEW publication NIH 85-23, revised 2010, Office of Science and Health Reports, DRR/NIH, Bethesda, MD). The study protocol was approved by the Committee on Animal Research of the College of Medicine at Kosin University.

Six-week-old mice were obtained to create a model of acute lymphedema. An additional group of unmanipulated normal mice was used for comparison. Before the surgery, the mice were anesthetized with Zoletil (0.04 ml/100 g body weight, intraperitoneal inj.) (Virbac, Carros, France) and Xylazine (0.01 ml/100 g body weight, intraperitoneal inj.) (Rompun, Bayer, Germany). To create a mouse tail model of acute secondary lymphedema, skin and subcutaneous tissues 5-10 mm from the tail base were removed without injury to the blood vessels on either side of the tail.

OCT provides cross-sectional images of micro-tissue morphology with a high resolution (2-10 μm) in a non-invasive manner. OCT acquires images of tissue by illuminating a biological sample with a focused beam of near-infrared light, then detecting the light that is backscattered. Backscattering from different depths within the tissue is separated through a process referred to as interferometry which can achieve a narrow, one-dimensional depth scan into the tissue at a specific location. Different tissues have different scattering and absorption properties, allowing OCT to provide high-resolution morphologic imaging.

OCT was applied to image the detailed structure of the lymphedema lesion in the mouse tail in the second week. After in vivo OCT imaging was done, the mouse tail was cut off (5-10 mm from tail base) and immediately fixed in formalin solution. Paraffin blocks were made using the fixed mouse tail samples. The paraffin-embedded mouse tail samples were stained with hematoxylin-eosin to examine histological changes. Images of the stained slides were captured by a digital camera (Digital sight DS-U1, Nikon, Tokyo, Japan) attached to a microscope (Eclipse 80i, Nikon, Tokyo, Japan).

We constructed a spectrometer-based OCT system using a fiber-optic Michelson interferometer, as shown in Fig. 1. A broadband light was delivered to a fiber based 2×2 beam splitter through optical fiber and the split beams were directed to reference and sample arm. The beams were reflected by a reference mirror and sample surfaces in tissue under a two-axis scanner, respectively. Two beams, then, were interfered at the beam splitter and directed to the detection arm. We used a broadband light source (SLED Butterfly, EXALOS, Schlieren, Switzerland) with a center wavelength of 1310 nm and a full width at half maximum of 80 nm. The two-axis scanner was customized using two galvanometers (6220H, Cambridge Technology, MA, USA) and had a maximum scanning area of 10.24×10.24 mm². The detection arm was composed of a grating (1145 lines/mm, Wasatch Photonics, NC, USA), a prism, and a line scan InGaAs camera with 1024 pixels and a line rate of 92 kHz (SU1024-LDH2, Goodrich, Princeton NJ, USA). We acquired volume images of each mouse tail with 1024×512×2048 voxels which corresponded to 5.12×2.3×10.24 mm³. To obtain specifications of the OCT system, we measured the point spread function. The OCT system had a depth resolution of 11.629 μm in air (measured at a depth of 130 μm), an entire imaging depth of 2.3 mm, a roll-off distance of 1 mm at 5.65 dB power drop, a dynamic range of 106 dB, and a frame rate of 70 fps with 1024 A-lines.

Based on observation with the naked eye, the volume of the mouse tail gradually increased in lymphedema samples, reaching a maximum on the 14th day after the operation, and displaying twice as much volume as prior to the operation (Fig. 2).

In the OCT image of the epithelial layer of the mouse tail, the highest and second highest signal peaks provided information on the thickness of the epidermis, and the second and third highest signal peaks provided information on the thickness of the dermis. In the normal tail, an even surface and regular epidermal layer thickness were observed, and the distinctions between epidermis and dermis, and dermis and subcutaneous layer were relatively clear. However, in the lymphedema model, an uneven surface and irregular epidermal layer thickness were observed, and the boundaries between epidermis and dermis, and dermis and subcutaneous layer became unclear (Fig. 3).

Also, skin inflammation in the lymphedema model accompanied edema and displayed an increased light attenuation in the dermis and subcutaneous tissue (Fig. 3). Similar to OCT image findings, histological biopsy also showed an inflammatory response involving edema, more neutrophils in the epidermis and subdermis, and lymphatic microvascular dilatation (Fig. 4). Furthermore, the lymphedema model showed an increase in thickness of the dermis using both methods.

In this study, we created a mouse tail model of lymphedema to visualize using OCT, a real-time noninvasive high-resolution video imaging technology, and compared images to findings from histologic analysis. The usefulness of OCT as an imaging technology to diagnose and observe progress of lymphedema treatment was assessed through the results of this study.

The study of lymphedema requires an understanding through pathophysiologic analysis, but there are few experimental studies due to insufficient experimental subjects. As a result, treatment methods are applied to patients based on theoretical analyses. While there has been a long history of studying lymphedema, the mechanism behind its cause and histologic changes is still not clear, so it is often treated with a non-pharmacological method called complex decongestive physical therapy rather than medication. Therefore, development of a proper experimental model would greatly help the study and treatment of lymphedema.

Diagnosis of lymphedema can be made through the circumference difference in the limb, however this also varies in definition among studies [14, 22]. Additionally, the definition of lymphedema based on volume or circumference difference does not reflect changes in fluid, muscle mass, bone, fat or other tissue compositions [8].

Most patients with progressed lymphedema can be diagnosed based on specific clinical features and physical findings. However, in the early stage of lymphedema or for intermittent edema, it becomes difficult to make a differential diagnosis between lymphedema and other edematous states. In cases where a clear diagnosis of lymphedema cannot be made based solely on a physical examination, additional tests may be needed to determine if lymphatic function is damaged. Currently, isotopic lymphoscintigraphy is most commonly used and assesses the lymphatic function by visualizing major lymphatic trunks and lymph nodes. However, the histologic changes before and after lymphedema treatments cannot be observed through lymphatic function evaluation only, so an objective assessment of treatment effect cannot be made. Due to these limitations, lymphedema evaluation has recently been done using ultrasonography which allows the observation of cutaneous, epifascial and subfascial compartment thickening in lymphedema patients and can be used in differential diagnosis and therapeutic monitoring as well [1, 9].

Until now, many areas of medicine have attempted animal model tests using OCT imaging. In the 1990s when OCT was first introduced, it was shown to be effective in obtaining a non-invasive, contactless high resolution image used to study structural changes in the retina and optic nerve head [17]. The resolution of OCT imaging is approximately 2-10 μm, which is much higher than ultrasound (200 μm), MRI (1 mm) and computed tomography (300 μ m). OCT also uses simple equipment and is less expensive than other imaging techniques. However, because of its low transmittance, it only transmits down to 1~2 mm from the surface of the body [23].

In other areas of study such as microvascular tissue and cancer, animal experimental models have recently been used to prove the clinical, diagnostic and therapeutic values of OCT [24, 25]. Also, McLaughlin et al. compared OCT imaging to histologic findings of axillary lymph nodes in breast cancer patients and found that OCT is useful for viewing microarchitecture and observing real-time node status in situ without the need for physical resection or histological processing during operation on breast cancer [20, 21].

Lymphedema typically accompanies inflammation, fibrosis, and adipose deposition, and the observation of chronic inflammation in the lymphedema area is critical for verifying treatment progress. Many cell types are involved in chronic inflammation, however macrophages are the main regulators of lymphangiogenesis and fibrosis [26]. Previous studies have visualized macrophages in coronary fibroatheroma using intravascular optical coherence tomography [27-30], so we used a mouse tail model of lymphedema and compared OCT imaging to histologic findings to examine the effectiveness of OCT in lymphedema diagnosis prior to clinically applying OCT for lymphedema verification.

In this study, OCT imaging showed that the mouse tail lymphedema model had a higher light attenuation in the dermal and subcutaneous layers compared to a normal mouse tail, presumably due to skin inflammation that accompanied the edema. Based on histological examination, more neutrophils and lymphatic microvascular dilatation were observed in the lymphedema model compared to normal tissue, which is also likely due to an inflammatory reaction from edema. In addition, increased dermis thickness was observed on the lymphedema model in both OCT and histological examination.

This study suggests that lymphedema can be effectively visualized with OCT in an animal model and OCT is a potential powerful tool that can be used to easily and non-invasively verify the increase in dermal thickness and inflammation changes in lymphedema when compared to histologic findings. In the future, high-resolution OCT imaging could be used to observe the sequence of histologic changes of lymphedema from the time of onset and non-invasively diagnose and verify treatment effects in humans. Using OCT to non-invasively verify the severity of inflammation of lymphedema territory would also be helpful in determining patients’ treatment plans and measuring their progress.

This study was limited by the fact that it did not reach the statistical approach using OCT parameters when viewing the OCT image. However, this study provides a basic reference for studying the diagnosis of lymphedema using OCT.

In the mouse tail model of lymphedema, OCT imaging showed very similar results to corresponding histological examinations. OCT provides a quick and useful diagnostic imaging technique for lymphedema and is a valuable addition or complement to other noninvasive imaging tools.