A Novel Approach to Subcutaneous Collecting Lymph Ducts Using a Small Diameter Wire in Animal Experiments and Clinical Trials
Abstract
Background: While performing microsurgery, including lymphaticovenous anastomosis (LVA) for chronic limb lymphedema, it is a common procedure to identify the subcutaneous collecting lymph ducts with near-infrared fluorescence lymphangiography (NIR) using indocyanine green. However, due to limitations such as minimum observable depth, only a few lymphatic ducts can be identified with this procedure. Hence, we developed a new smaller-diameter ‘‘lymphatic wire’’ (LW) that could be inserted directly into lymphatic collecting ducts of the limbs, enabling accurate identification and localization.
Methods and Results: First, used the LW on the hind limbs of 6 swine, and 36 porcine lymphatic collecting ducts were identified, the outer diameter of which varied from 0.3–0.7 mm (mean 0.41 – 0.11 mm). We could insert the LW after creating a side opening in 30 of these ducts. We encountered no difficulties during the procedure. In the pathological examination, adverse events such as valve dysfunction and perforation were not identified. Based on the results, a clinical evaluation of the LW was performed in two patients with lower extremity lymphedema, and the LW helped us identify lymphatic ducts in the subcutaneous layer, even at the sites where the NIR had proved ineffective.
Conclusion: Based on our results, we suggest that the procedure for identifying lymphatic vessels using the newly developed LW is a useful technique that can be utilized before performing a LVA for lymphedema. However, further clinical study is required to develop this device and technique, for wider clinical application in the future.
Keywords: lymphedema, wire, collecting lymph duct, animal experiment
Introduction
HE MAINsTAY of TREATMENT for lymphedema until re- cently was conservative management, which centered on performing manual drainage massage of the affected limb along with compression therapy.1 In recent years, advances in medical technology and surgical procedures have led to an upsurge in the utilization of microsurgery for lymphedema, and various techniques such as lymphaticovenous anasto- mosis (LVA),2–6 vascularized lymph node transplantation,7–9 and lymph vessel transplantation10,11 have been used. Of these, a LVA is widely used across the world due to its minimally invasive nature, and its usefulness has been reported by several researchers. However, some drawbacks of this technique have also been noted. We have reported that the anastomotic site gets obstructed over time and that the long-term efficacy of LVA alone in reducing lymphedema is limited when the standard operative method is used.12–14 Hence, it is a clinical challenge to develop a technique that can be applied to construct a ‘‘high quality anastomosis,’’ having long-term patency, and which leads to effective drainage over a larger area.
Therefore, we focused on developing a method of identi- fication of the deeper subcutaneous lymphatic ducts. When a procedure such as LVA is performed, the subcutaneous lymphatic ducts are commonly identified using near-infrared fluorescence lymphangiography (NIR). In this method, a fluorescent dye is injected into the affected limb, and the dynamic lymphatic flow is observed in real time.15–18 Al- though it is a highly useful method of duct localization, the observable depth with this technique is only *10 mm, as fluorescence of only the near-infrared wavelength is ob- servable through the camera. In a lymphedematous limb, the lymph taken up by the collecting lymphatic duct flows back into the subcutaneous layer or the dermis, causing a lym- phatic dermal back flow (DBF). While the site of DBF leakage in the dermal layer fluoresces on NIR, we cannot observe the ducts or tissues deeper to this level. Conse- quently, when there could be several latent functional lym- phatic vessels available for a better anastomosis, only a limited number of superficial collecting ducts can be identi- fied and utilized, using NIR alone.
Therefore, inspired by the guidewire used in endovascular treatment for peripheral arterial disease, we developed a novel, moderately flexible wire with a smaller diameter that could be directly inserted into a lymphatic duct. In this arti- cle, we report the preclinical evaluation of this wire in animal experiments and the outcome of its use in human subjects.
Materials and Methods
All animal experiments were approved by the Executing Agency under the jurisdiction of the Ministry of Health, Labor and Welfare. The experimental operation was con- ducted in strict accordance with the guidelines of the animal laboratory facilities and as per ‘‘Animal Welfare and Man- agement Law,’’ ‘‘Standards for Relieving and Storing La- boratory Animals and Relief of Pain,’’ and the Ministry of Health, Labor and Welfare Basic Guidelines on the Im- plementation of Animal Experiments. We obtained six swine (specific pathogen free, livestock pigs, female sex, 3 months old) weighing 38.0–41.6 kg. Following the animal study, we applied the guidewire clinically, during microsurgery for lymphedema, in two patients, according to a research plan approved by the ethics committee of our university.
Development of lymphatic wire
In cooperation with the TRS Company Ltd. (Okayama, Japan), we developed a smaller-diameter ‘‘lymphatic wire (LW),’’ the basic structure of which was the same as the existing guidewire used for blood vessels. The material used was nitinol, a nickel and titanium composite. The maximum outer diameter of the wire was 250 lm (0.099 inches) and it tapered toward the tip. In a part of the tip, gold was wound in a coiled shape, which made it moderately mobile. The whole wire was then coated with a hydrophilic polymer.
Animal experimental study for evaluation of operability
After induction of general anesthesia in the swine, we conducted experiments with the animals in the supine posi- tion. First, we injected indocyanine green (ICG) intrader- mally between the toes of the hind limbs and performed NIR. The subcutaneous lymphatic flow of the hind limb was ob- served using a fluorescence imager (PDE-neo®; Hamamatsu Photonics K.K., Hamamatsu, Japan), which traced the flow of the dye by detecting the near-infrared radiation in the tissue up to a depth of 10 mm from the skin surface. To improve the visibility of the subcutaneous lymphatic vessels after the skin incision, a 5% blue dye (Patent Blue®) was also injected intradermally (Fig. 1).
Based on the findings of the NIR, we made skin incisions in the distal part of the hind limb, corresponding to the visualized positions of the lymphatic ducts, using a microscope ( M320 F12®; Leica Microsystems, Germany) to magnify the operative field. The subcutaneous lymphatic collecting ducts were identified, and their outer diameter was measured. We made a side opening in the lymphatic duct, 0.5 mm in size, using a micro scalpel and inserted the LW. If the LW could be inserted without resistance, we used the wire as a guide to insert and position *1–2 cm of the smaller diameter sheath and introduced Iopamidol (Bystage®; Takeda Teva—Pharma ª, Japan) into the duct. We then used an X-ray fluoroscope (OEC 9800®; GE Yokogawa Medical Systems) to perform antegrade imaging of the lymphatic duct.
FIG. 1. NIR for the hind limbs of the swine. (A) Skin markings of NIR pattern in the hind limb of the swine. ICG was injected into the dermis between the toes (arrow head). (B) The NIR pattern was visualized using a near-infrared camera and demarcated. Several linear patterns were iden- tified, which show the pathways of collecting lymphatic ducts. In the groin area, a faintly-stained oval-shaped area is seen on fluoroscopy, which indicated the presence of a lymph node (arrow). ICG, indocyanine green; NIR, near- infrared fluorescence lymphangiography.
Thereafter, the wire was slowly advanced under fluoro- scopic guidance, and the directional pathways of the lym- phatic ducts were identified. A skin incision was made in the vicinity of the inguinal region, and the LW and the collecting duct were identified directly at that site. We also inserted the LW from a side opening to a site considered to be a lymph node and reciprocated the wire five times with an intention to damage the lymphatic duct.
To check whether the wire had caused a functional ab- normality, we made another side opening in the damaged duct and performed retrograde lymphangiography using the sheath and the LW as a guide. To confirm the occurrence of any mechanical damage, the injured lymphatic vessels and lymph nodes were extracted and histologically analyzed us- ing hematoxylin–eosin and Masson trichrome staining.
Clinical experimental study
Of the patients diagnosed with lymphedema using lym- phoscintigraphy, for whom LVA was indicated, two patients agreed to participate in this study.
FIG. 2. Identification of collecting lymphatic ducts and insertion of LW. (A) An incision had been made in the below- knee region, and several collecting lymph ducts were detected. To avoid damage, the lymph duct had been ablated with the surrounding soft tissue. (B) Measurement of the outer diameter of the lymphatic ducts. (C) Pinhole made in the lateral side of the lymph duct with a scalpel followed by insertion of the LW. (D) The LW insertion had been smooth due to its superior flexibility and its hydrophilic coating. LW, lymphatic wire.
First, we identified the subcutaneous collecting lymphatics using NIR intraoperatively, according to the usual method. We created side openings in the lymphatic duct, under the microscope and inserted the LW. We let the LW enter the duct precisely through the opening we had created, while moni- toring the advancement of the wire through the duct, using X-ray fluoroscopy. Wherever the surgeon encountered resis- tance, indicating that the LW tip was caught or obstructed, the overlying skin was carefully marked. A skin incision was made under the microscope over the demarcated site, and the LW and subcutaneous collecting lymphatics were located.
Results
Porcine experiment
The subcutaneous collecting lymphatic ducts in the hind limbs of the swine were located and identified in the manner described. We demarcated 36 collecting ducts, with outer di- ameters ranging from 0.3 to 0.7 mm (mean 0.41 – 0.11 mm). The side openings were created, and the LWs were inserted into 30 of these ducts (0.3–0.7 mm, mean 0.41 – 0.11 mm). We were able to perform the insertion smoothly, without any issues (Fig. 2).
The collecting lymphatic ducts of swine differed from those in humans, as they were not as confluent and had fewer branches. During lymphangiography, the contrast agent could be injected into the ducts without resistance, and a valve at intervals of about 1 cm was seen on contrast imaging, which gave the duct a segmented appearance. The LW was also easily visualized using X-ray fluoroscopy. When a sheath was placed retrogradely at a damaged site where the LW insertion had been repeated, retrograde imaging could not be con- ducted, as we met strong resistance while attempting to inject the dye into the duct (Fig. 3).
In the histological analysis, a slight separation of the en- dothelium and inflammatory cell infiltrates was observed on the examination of a cross-section of the collecting lym- phatic ducts (Fig. 4; Table 1). Findings suggestive of bleeding were observed in the damaged groin lymph node. Although accumulated inflammatory cells centered on eosinophils were observed in the histopathological examination of the lymph node, similar findings were also seen in the normal lymph nodes (Fig. 4; Table 2).
Limited human trial
In both clinical cases, the collecting lymphatic ducts were identified in the lower thigh near the knee joint. The LW was inserted and positioned using an X-ray fluoroscope. The wire entered the proximal thigh smoothly without encountering any resistance. Thereafter, the overlying skin was incised, the subcutaneous tissue dissected, and the point of entry of the LW into the subcutaneous collecting lymph duct was iden- tified. A side opening was created under a microscope, and a lymphaticovenous side-to-end anastomosis procedure was performed by anastomosing an adjacent subcutaneous vein to the side opening into which the LW was inserted. In one of the two patients, an enlargement of the lymphatic duct side pore at the site of the LW insertion was observed, but it was sutured and repaired under the microscope (Fig. 5).
FIG. 3. Findings of X-ray fluoroscopy and lymphangiography. (A) Anterograde lymphangiography: The authors could inject contrast agent smoothly with no resistance. (B) LW was inserted and examined using X-ray fluoroscopy. The LW was easily detected and inserted without resistance. (C) Retrograde lymphangiography: A retrograde sheath was installed as a port for the LW to an injured lymph duct followed by lymphangiography. It was very difficult to introduce the contrast agent, which could not be injected due to resistance.
FIG. 4. Histological findings. (A, D) The microscopic appearance of a cross-section taken from a control lymph duct. (B, E) The microscopic appearance of a cross-section taken from a lymph duct treated with a LW. Dissections of endothelium (arrow) were observed in a part of the collecting lymphatic duct. (C, F) Findings suggestive of bleeding (arrow head) were observed in the groin lymph node cross-section. HE, hematoxylin eosin staning; MT, masson trichrome staining.
Discussion
In this study, we confirmed that the newly developed small-diameter LW could be utilized effectively as a guide- wire to identify subcutaneous lymphatic ducts in animal ex- periments and in limited clinical application.
Several methods apart from NIR can help identify subcuta- neous collecting lymph ducts, such as lymphoscintigraphy,19–23 single photon emission computed tomography/computed to- mography lymphoscintigraphy (SPECT/CT LS),24,25 and magnetic resonance lymphography.26–28 These techniques are quite useful, because they provide us objective images having excellent spatial resolution with no limitation of ob- servable depth. However, these imaging modalities can take an accurate snapshot, but do not provide a direct real-time visualization of a lymphatic duct and its pathway. Therefore, it is difficult to use these imaging modalities intraoperatively for identification of the collecting ducts with the LW. For example, SPECT/CT LS can be used to identify the lymphatic ducts with greater accuracy, even in the thigh. However, due to the difference of positioning the body in SPECT/CT LS and surgery, sometimes a lot of intraoperative time is consumed in localizing the exact position of the lymphatic ducts, and the surgeon may even be required to perform an additional inci- sion on the skin to extend the visual field. Utilizing the LW as a guidewire addresses these difficulties.
Furthermore, using a novel LW, it was able to identify subcutaneous collecting lymph ducts on the thigh and the back of DBF. We believe that performing an LVA on such lymphatic ducts and creating a shunt can provide better drainage. This is because theoretically, if the shunt is created more proximally, drainage of the lymph outflow from a wider area is possible. In addition, the subcutaneous collecting lymph ducts, which are in the thigh or in the back of the DBF, are considered to be stagnant and not to be conductive. Chronic lymphedema, especially secondary lymphedema, causes progressive lymphatic vessel injury from proximal to the distal end of the ducts.29–32 Lymph fluid that has lost its destination due to lymph node dissection or radiation damage stagnates in the collecting lymph ducts. They remain patent, even become dilated, and collateral circulation through the subepidermal plexus develops, which is so called DBF.
Therefore, there are many lymphatic vessels with stagnant lymphatic fluid in the thigh or in the back of the DBF. However, it is difficult to identify such lymphatic vessels by the lymph flow evaluation tests such as NIR and lymphoscintigraphy, which have been frequently used in recent years. This is be- cause the administered contrast agent, fluorescent dye, or ra- dioisotope does not reach the lymphatic vessels that are ‘‘patent but not conducted.’’ Some attempts to efficiently identify the lymphatic vessels with stagnant lymphatic fluid using NIR and ultrasound have been reported.33,34 However, using LW, we can identify them directly with real-time visualization. It is therefore expected that a larger range of collecting ducts can be identified using this technique, but it does not quantitatively assess lymphatic vessel damage or stasis, internal pressure, or lymphatic degeneration. Consequently, further extensive clinical studies utilizing the LW, NIR, and ultrasound simul- taneously are needed to determine the wider effectiveness of this technique in identifying optimal anastomotic sites.
Several techniques for inserting wires directly into lymph vessels have been reported. Specifically, there are reports of retrograde imaging performed using an intravenous approach to chylothorax.35,36 In cases where it was indicated, a retro- grade cannulation of the thoracic duct was performed before embolizing the affected duct. There is also a report of a pelvic lymphocele (arising after a gynecological cancer surgery), which was punctured under computed tomography guidance and the intervention was carried out from that point of entry.37 In both cases, a guidewire was inserted first, followed by cannulation with an indwelling catheter before injecting the contrast agent and using imaging guidance to perform an in- direct embolization with a lipid soluble contrast agent. How- ever, all these approaches are only applicable to the major lymphatic ducts with a large diameter such as the lymphatic trunk, cisterna chyli, thoracic ducts, and lymphoceles. To our knowledge, there have been no reports of a guidewire utilizing technique that allows direct access to the subcutaneous col- lecting lymphatic ducts in the periphery of the limb.
We performed the preclinical evaluation of the LW on swine hind limbs, for the detection and identification of subcutane- ous collecting lymphatic ducts, as the porcine lymphosome is similar to that of humans (except in the forelimbs).38 In fact, the sizable diameter of the collecting lymphatic ducts of the swine hind limbs at 0.3–0.5 mm was equivalent to that of humans. However, the porcine lymphatics differed from those of humans anatomically in terms of duct branching and con- fluence of the lymphatic vessels. Therefore, the wire would sometimes get caught at a branching point, but it was possible to avoid perforation by manipulating it, without applying force. Even during the clinical application in the human subjects, the ease of the operative procedure was similar to that experienced experimentally. The lymphatics were clearly visualized not only during the X-ray fluoroscopic examination but also on ultrasonography. We suggest that using the LW can help identify the collecting lymphatic ducts in the deep subcutaneous tissues, quickly and efficiently, even at the sites where the an- giography is ineffective, such as in the thigh. Consequently, a smaller skin incision would be required for locating and gaining access to the lymphatic duct.
FIG. 5. Intraoperative findings of clinical application. (A, B) Skin-mapping and NIR imaging of a leg with secondary lymphedema. DBF was visualized in the thigh region; however, no linear pattern was observed through the near-infrared camera. (C) An LW was inserted from the collecting lymph duct around the knee (arrow). Based on the identification of the LW on imaging, the proximal side of the lymph duct was marked (asterisk). (D, E) At the site of the proximal incision, we identified the collecting lymphatic duct with the inserted LW (arrow head). DBF, dermal back flow.
However, the surgeon must always beware of duct perfo- ration, as a possible complication. Guidewires have long been utilized for catheterization of coronary arteries, cerebral blood vessels, and peripheral vessels. Several types of guidewires are available. For example, there are some wires with a specific shape and structure to avoid damaging the vascular endothe- lium, and conversely, some with an improved penetrative ca- pacity. Overall, the risk of perforation of coronary vessels is very low and is reported to range from 0.1% to 0.58%, and only a part of the risk is attributed to guidewire usage.39 The char- acteristics of coronary arteries with plaque formation differ greatly from those of the subcutaneous collecting lymph ducts seen in lymphedema. However, in our study, we found lymph node damage and some findings that were indicative of injury to the lymphatic vessels. So, we concluded that frequent op- erations carry a higher risk of penetrative damage to the intima. In contrast, despite excessive manipulation of lymphatic vessels, when we tried to perform retrograde lymphangiog- raphy, we could not inject any more contrast medium. This led us to conclude that the valvular dysfunction of the duct was minimal or absent. Moreover, the small diameter wire that we developed was simply used for the purpose of iden- tifying the lymph duct and not as a traditional guidewire. Moreover, since its customized structure decreases the fre- quency of insertion and removal, the risk of failure is rela- tively low. If an auxiliary modality such as X-ray fluoroscopy is used alongside, it is possible to perform imaging and rule out or confirm a suspected perforation, intraoperatively.
There is also a risk for dilation of the side opening, through which the LW is inserted, as was observed in one of the human subjects, during clinical application. A part of the enlarged pore was sutured under microscopic view and the remaining part of the opening was anastomosed to a nearby vein. We believe that this complication can be prevented with the de- velopment and application of a sheath.
Using the basic technology of a newly-developed slimmer wire, we were able to offer a direct interventional treatment to the involved lymphatic vessels. A similar approach can be used in other instances, such as using the wire itself as a bougie or dilator or by developing a suitable catheter with a smaller diameter for the LVA, which can enable a direct measurement of the real-time, intraductal lymphatic pressure. An under- standing of the internal pressure measurements and patho- physiology of lymph flow within the affected ducts and veins is essential to predict the effectiveness of the LVA and to ensure long-term patency of the anastomosis.
However, the swine used in our experiment were not diseased and had normal lymphatic vessels. Moreover, we were able to test the clinical application of the LW in just two human subjects with lymphedema. Therefore, the risk of in- timal damage and perforation may be different, when inserting the wire into the lymphatic ducts of a wider group of patients with lymphedema. Further clinical studies are indicated to establish the ease and effectiveness of this novel approach.
Conclusion
Through animal experimentation and limited clinical ap- plication in this study, we confirmed that the newly-developed small-diameter LW is a useful tool to directly approach and identify the subcutaneous collecting lymphatic ducts in lym- phedematous limbs. Although we encountered no major ad- verse events, there is a need for further extensive LW 6 clinical study to examine the effectiveness and risk profile of this technique.