Detection of alternative subpleural lymph flow pathways using indocyanine green fluorescence.

PURPOSE: Pulmonary lymphatic fluid predominately flows along the bronchi. However, there are reports suggesting that an alternative lymphatic pathway exist, which may result in skip metastases. The aim of this study was to evaluate the subpleural lymph flow in vivo using indocyanine green (ICG) fluorescence. METHODS: One hundred cases were enrolled. ICG was injected into the macroscopically healthy subpleural space. Intraoperative fluorescence images were then observed in real time. RESULTS: ICG fluorescence was observed moving through subpleural channels in 58/100 cases. ICG flowed into adjacent lobes over interlobar lines in 18 cases and flowed from the visceral pleura directly into the mediastinum in 5 cases. The frequency of mediastinal detection without hilar lymph node detection was significantly higher in the left lung compared to the right (p < 0.05). The subpleural lymph flow detection rates were significantly lower in patients with smoking pack-years ≥ 40 than those with < 40 (p < 0.05). CONCLUSIONS: The flow of lymphatic fluid directly into the mediastinum suggests one mechanism of skip metastasis. In addition, the reduction of the subpleural lymph flows in smokers with ≥ 40 pack-years suggests that smoking might modify lymph flow patterns. These findings may assist in selecting the optimal therapy for patients with possible skip metastasis.

Breast Cancer-Related Lymphedema: Differentiating Fat from Fluid Using Magnetic Resonance Imaging Segmentation.

BACKGROUND: Lymphedema is an iatrogenic complication after breast cancer treatment in which lymph fluid in the affected limb progresses to fat deposition and fibrosis that are amenable to liposuction treatment. Magnetic resonance imaging (MRI) for lymphedema can differentiate fat tissue from fluid, but estimating relative volumes remains problematic. METHODS AND RESULTS: Patients underwent routine bilateral arm MRI both before and after liposuction for advanced lymphedema. The threshold-based level set (TLS) segmentation method was applied to segment the geometric image data and to measure volumes of soft tissue (fat, muscle, and lymph fluid) and bone. Bioimpedance testing (L-Dex®) to detect extracellular fluid was also used. Volumes derived by using TLS or girth measurement were evaluated and showed consistent agreement, whereas L-Dex showed no significant reduction between pre- and postoperative measures. The percentage median volume difference between the affected and unaffected sides was 132.4% for girth measures compared with 137.2% for TLS (p = 0.175) preoperatively, and 99.8% and 98.5%, respectively (p = 0.600), postoperatively. MRI segmentation detected reductions in fat (median 52.6%, p = 0.0163) and lymph fluid (median 66%, p = 0.094), but the volumes of muscle and bone were relatively constant. CONCLUSIONS: MRI imaging with TLS technology may be a useful tool to quantitatively measure fat tissue and fluid for patients with advanced lymphedema and may assist in the selection of eligible liposuction candidates at initial assessment and follow-up of patients who proceed with surgery.

Simple Estimation of the Endolymphatic Volume Ratio after Intravenous Administration of a Single-dose of Gadolinium Contrast.

PURPOSE: To evaluate the feasibility of a simple estimation for the endolymphatic volume ratio (endolymph volume/total lymph volume = %ELvolume) from an area ratio obtained from only one slice (%EL1slice) or from three slices (%EL3slices). The %ELvolume, calculated from a time-consuming measurement on all magnetic resonance (MR) slices, was compared to the %EL1slice and the %EL3slices. METHODS: In 40 ears of 20 patients with a clinical suspicion of endolymphatic hydrops, MR imaging was performed 4 hours after intravenous administration of a single dose of gadolinium-based contrast material (IV-SD-GBCM). Using previously reported HYDROPS2-Mi2 MR imaging, the %ELvolume values in the cochlea and the vestibule were measured separately by two observers. The correlations between the %EL1slice or the %EL3slices and the %ELvolume values were evaluated. RESULTS: A strong linear correlation was observed between the %ELvolume and the %EL3slices or the %EL1slice in the cochlea. The Pearson correlation coefficient (r) was 0.968 (3 slices) and 0.965 (1 slice) for observer A, and 0.968 (3 slices) and 0.964 (1 slice) for observer B (P < 0.001, for all). A strong linear correlation was also observed between the %ELvolume and the %EL3slices or the %EL1slice in the vestibule. The Pearson correlation coefficient (r) was 0.980 (3 slices) and 0.953 (1 slice) for observer A, and 0.979 (3 slices) and 0.952 (1 slice) for observer B (P < 0.001, for all). The high intra-class correlation coefficients (0.991-0.997) between the endolymph volume ratios by two observers were observed in both the cochlea and the vestibule for values of the %ELvolume, the %EL3slices and the %EL1slice. CONCLUSION: The %ELvolume might be easily estimated from the %EL3slices or the %EL1slice.

Lymphatic transit rate as a novel predictive parameter for nodal metastasis in primary truncal skin cancers.

Prediction of nodal metastasis in skin cancer before sentinel lymph node (SLN) biopsies is ideal to avoid unnecessary SLN biopsy performance. Primary truncal skin cancers are characterized by the lymphatic flow that drains from the primary lesion, occasionally to plural nodal basins. The scintigraphic appearance time (SAT), defined as the time between radionuclide injection and first SLN visualization, can potentially predict nodal metastasis, and a short SAT is a predictive parameter for metastasis. We recently introduced a novel method to measure the lymphatic flow rate using dynamic lymphoscintigraphy exhibiting a time-activity curve in the SLN. The time at which the count reaches a plateau in the SLN is termed the scintigraphic saturation time (SST) and can be a good alternative to the SAT. Moreover, the value obtained by division of the distance between the primary lesion and the SLN by the SST was termed the lymphatic transit rate (LTR), which represents the scintigraphic saturation velocity. In the present study, we evaluated LTR as a predictive parameter for nodal metastasis. Data for 22 lymph nodes from 18 patients with primary truncal skin cancers were used. Histopathologically, nodal metastasis was determined in nine nodes of eight patients. Because the mean LTR were 1.84 cm/min in non-metastatic SLN and 2.38 cm/min in metastatic SLN, the LTR was significantly higher in metastatic SLN than in non-metastatic SLN. All SLN with LTR of less than 1.8 cm/min were histopathologically evaluated as non-metastatic. The LTR may be a predictive indicator for nodal metastasis.

In vivo flow cytometry: a horizon of opportunities.

Flow cytometry (FCM) has been a fundamental tool of biological discovery for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents studying cells in their native environment. These problems can be overcome by use of in vivo FCM, which provides detection and imaging of circulating normal and abnormal cells directly in blood or lymph flow. The goal of this review is to provide a brief history, features, and challenges of this new generation of FCM methods and instruments. Spectrum of possibilities of in vivo FCM in biological science (e.g., cell metabolism, immune function, or apoptosis) and medical fields (e.g., cancer, infection, and cardiovascular disorder) including integrated photoacoustic-photothermal theranostics of circulating abnormal cells are discussed with focus on recent advances of this new platform.

Effects of autotransplanted lymph node fragments on the lymphatic system in the pig model.

Secondary lymphedema often develops after removal of lymph nodes in combination with radiation therapy, in particular in patients with breast cancer, inguinal cancer, cervical cancer and melanoma. No convincing treatment for the prevention and therapy of acquired lymphedema exists so far, therefore we wanted to show the reintegration of transplanted avascular lymph node fragments in the lymphatic system and positive effects of the transplanted fragments on the restoration of the lymphatic flow in this study. A total of 26 minipigs underwent lymphadenectomy of both groins. A minimum of one lymph node was retransplanted. The lymph nodes were cut into small pieces and retransplanted in the left groin (n = 17) or in both groins (n = 9). Different retransplantation techniques were investigated, transplantation of large versus small fragments, with and without capsule. The lymph flow was evaluated 5 and 8 months after surgery, using SPECT/CT and Berlin Blue. The results were confirmed by dissection. The lymph node transplants were assessed histologically. In contrast to the lymph flow in the transplanted groin, the lymph flow in the non-transplanted groin was often malfunctioning. Large lymph node fragments were found reintegrated in the lymphatic system more often than small slices of lymph node fragments. About 5 months after surgery impairment of lymph flow was seen especially after retransplantation of small slices of lymph node fragments. In seven out of eight minipigs a dermal backflow developed in the non-transplanted groin, 8 months after surgery. Only one minipig of these groups developed dermal backflow in both groins. All lymph node fragments showed an organized structure histologically. Autologous lymph node transplantation has positive effects on the regeneration of lymph vessels and restoration of lymph flow after lymphadenectomy.

Nanoradioliposomes molecularly modulated to study the lung deep lymphatic drainage.

UNLABELLED: Lung deep lymphatic drainage (LDLD) plays an important role in the removal of foreign materials from lungs being alveolar macrophages the first line of phagocytic defence with high affinity for pathogenic microorganisms. Bacillus subtilis is a well-known genome- decoded saprophyte of the human respiratory tract used in research and in the biotechnology industry. Lung deep lymphatic chains (LDLC) constitute one of the first sites of lung tumours' dissemination. In this work we intended to develop and validate a non-invasive method for assessing LDLC by nanoradioliposomes aerosolised modulated on the Bacillus subtilis spore wall. The final goal was to produce a nanoradioliposome formulation that can mimics the dynamics of preferential removal of spores by LDLD and present the ideal properties as a tracer for molecular imaging studies. Seven different liposomal formulations were tested, and the formulation-F demonstrated physicochemical and radiopharmaceutical properties that make it an ideal candidate as an in vivo probe for molecular imaging studies of the LDLC. Nanoradioliposomes of the formulation-F after labelling with 99mTc-HMPAO were administered as aerosols to 20 Sus scrofa. Hilar and interpulmonary communications were visualized in first 5 minutes post-inhalation, infradiaphragmatic chains between 10 and 20 minutes, the ganglia of the aortic chain at 20 minutes and those of the renal hilar region at 30 minutes. CONCLUSION: the proposed method enables visualization of deep lymphatic lung network and lymph nodes. Besides, this technique involving the modulation of nanoradioliposomes targeting specific organs or tissues may be an important tool for diagnostic or even for therapeutic purposes.

Topography of accumulation of stagnant lymph and tissue fluid in soft tissues of human lymphedematous lower limbs.

BACKGROUND: The knowledge of where does excess tissue fluid accumulate in obstructive lymphedema is indispensable for rational physical therapy. However, it has so far been limited to that obtained from lymphoscintigraphic, ultrasonographic, and MR images. None of these modalities provide composite pictures of dilated lymphatics and expanded tissue space in dermis, subcutis, and muscles. So far, only anatomical dissection and histological processing of biopsy material can visualize the tissue lymphatic network and the sites of accumulation of the excess of mobile tissue fluid. METHODS AND RESULTS: We visualized the "tissue fluid and lymph" space in skin and subcutaneous tissue of foot, calf, and thigh in various stages of lymphedema in specimens obtained during lymphatic microsurgical procedures or tissue debulking, using special staining techniques. The volume of accumulated fluid was calculated from the densitometric data of stained tissue sections. We found that lymph was present only in the subepidermal lymphatics, whereas the collecting trunks were obliterated in most cases. Mobile tissue fluid accumulated in the spontaneously formed spaces in the subcutaneous tissue, around small veins and above and underneath muscular fascia. Deformation of subcutaneous tissue by free fluid led to formation of interconnecting channels. The volume of subcutaneous free fluid ranged around 50% of total tissue volume and there were no significant differences in various stages of lymphedema. This could be explained by the presence of thick layers of subcutaneous fat tissue even in the most advanced stage of lymphedema. CONCLUSIONS: In lymphedema caused by obliteration of collecting trunks, lymph is present only in the subepidermal lymphatics, whereas the bulk of stagnant tissue fluid accumulates in the subcutaneous tissue and above and beneath muscular fascia. These findings should be useful for designing pneumatic devices for limb massage as well as for rational manual lymphatic drainage in terms of sites of massage and level of applied external pressures.

Dynamic contrast-enhanced magnetic resonance imaging of tumor-induced lymph flow.

The growth of metastatic tumors in mice can result in markedly increased lymph flow through tumor-draining lymph nodes (LNs), which is associated with LN lymphangiogenesis. A dynamic magnetic resonance imaging (MRI) assay was developed, which uses low-molecular weight gadolinium contrast agent to label the lymphatic drainage, to visualize and quantify tumor-draining lymph flow in vivo in mice bearing metastatic melanomas. Tumor-bearing mice showed greatly increased lymph flow into and through draining LNs and into the bloodstream. Quantitative analysis established that both the amount and the rate of lymph flow through draining LNs are significantly increased in melanoma-bearing mice. In addition, the rate of appearance of contrast media in the bloodstream was significantly increased in mice bearing melanomas. These results indicate that gadolinium-based contrast-enhanced MRI provides a noninvasive assay for high-resolution spatial identification and mapping of lymphatic drainage and for dynamic measurement of changes in lymph flow associated with cancer or lymphatic dysfunction in mice. Low-molecular weight gadolinium contrast is already used for 1.5-T MRI scanning in humans, which should facilitate translation of this imaging assay.

Quantitative lymph imaging for assessment of lymph function using indocyanine green fluorescence lymphography.

OBJECTIVES: A new diagnostic imaging technique that can assess lymph function is needed as a screening test in daily practice. This study assessed the use of indocyanine green (ICG) fluorescence lymphography in subjects without leg oedema. METHODS: 0.3ml of ICG (0.5 %) was injected subcutaneously at the dorsum of the foot. Subsequently, the movement of ICG dye from the injection site to the groin was traced by visualizing its fluorescence signal with an infrared light camera. The time for the dye to reach the knee and groin were measured (Transit time to knee: TT(K), Transit time to groin: TT(G)). TT(G) was measured while standing, lying at a supine position, standing with massage, and sitting while using a cycle ergometer exercise at an intensity of 50W at 50rpm in ten healthy volunteers at intervals of 14 days. RESULTS: Mean TT(G) during standing was 357+/-289 and 653+/-564 seconds for the right and left legs respectively. Compared to TT(G) in the standing position, all other conditions shortened TT(G). In another seventeen subjects without leg oedema, we compared transit time obtained with ICG fluorescence lymphography to that with dynamic lymphoscintigraphy. A significant correlation between transit time measured with ICG lymphography and dynamic lymphoscintigraphy was identified (r(2)=0.64, p<0.01). CONCLUSIONS: ICG fluorescence lymphography has the potential to become an alternative lymphatic imaging technique to assess lymph function.

Multimodal nanoprobes for radionuclide and five-color near-infrared optical lymphatic imaging.

Current contrast agents generally have one function and can only be imaged in monochrome; therefore, the majority of imaging methods can only impart uniparametric information. A single nanoparticle has the potential to be loaded with multiple payloads. Such multimodality probes have the ability to be imaged by more than one imaging technique, which could compensate for the weakness or even combine the advantages of each individual modality. Furthermore, optical imaging using different optical probes enables us to achieve multicolor in vivo imaging, wherein multiple parameters can be read from a single image. To allow differentiation of multiple optical signals in vivo, each probe should have a close but different near-infrared emission. To this end, we synthesized nanoprobes with multimodal and multicolor potential, which employed a polyamidoamine dendrimer platform linked to both radionuclides and optical probes, permitting dual-modality scintigraphic and five-color near-infrared optical lymphatic imaging using a multiple-excitation spectrally resolved fluorescence imaging technique.

New method for evaluation of lung lymph flow rate with intact lymphatics in anaesthetized sheep.

AIM: Lung lymph has commonly been studied using a lymph fistula created by tube cannulation into the efferent duct of the caudal mediastinal node in sheep. In this method, the tail region of the caudal mediastinal node is resected and the diaphragm is cauterized to exclude systemic lymph contamination, and cannulation is performed into one of the multiple efferent ducts originating from the caudal mediastinal node. Moreover, the pumping activity of lymphatics might be diminished by cannulation. Therefore, the purpose of the study was to evaluate the flow rate of lung lymph with maintenance of intact lymphatic networks around the caudal mediastinal node to the thoracic duct in sheep. METHODS: An ultrasound transit-time flow meter was used to measure lung lymph flow. The thoracic duct was clamped just above the diaphragm and the flow probe was attached to the thoracic duct just after the last junction with an efferent duct from the caudal mediastinal node. The lung lymph flow rate was measured at baseline and under conditions of lung-oedema formation. RESULTS: The baseline lung lymph flow rate in our model was three- to sixfold greater than values obtained with the cannulation method. With oedema-formation, the lung lymph flow rate was the same as that measured using cannulation. CONCLUSION: The lung lymph flow was unexpectedly large under the conditions of the study, and our data suggest that the drainage effect of lymphatics is significant as a safety factor against pulmonary oedema formation.

[Lymph kinetics with technetium-99m labeled radiopharmaceuticals. Animal studies]. / Lymphkinetik mit Technetium-99m-markierten Radiopharmaka: tierexperimentelle Untersuchungen.

UNLABELLED: Aim of this study was to characterize suitable technetium-99m labeled tracers for lymphoscintigraphy by comparative animal tests. ANIMALS, METHODS: To evaluate the influence of the particle size and the organ tracer-uptake on lymphtransport animal experiments were performed on six different agents (including one control group). Activity distributions were examined in Sprague-Dawley-rats by lymphoscintigraphy; the maximum uptake (count-rate) of the whole body and in lymph nodes were analyzed by regions-of-interest-technique, respectively. Additionally, for characterization of lymphatic and extralymphatic traceruptake an intra-individual relative count-rate ratio of the liver, lung, kidneys, and spleen has been calculated following organ extraction. RESULTS: Organ specific differences of distribution were clearly demonstrated. Our results indicate that the kinetics of lymphoscintigraphic (99m)Tc-bound agents substantially depends on particle size. Reliable transport from the interstitium to initial lymph vessels and lymph node uptake suggested for tracers suited for lymphoscintigraphy a median size of about <100 nm. CONCLUSION: Our data could improve standardization of diagnostic methods and lead to an objective consideration of therapeutic procedures.

[Experimental study of cranial-cervical lymph return in rabbit].

OBJECTIVE: To study the cranial-cervical lymph return and pathway in rabbit in order to provide the experimental and theoretical basis for the study of intracranial metastasis of cervical tumor and extracranial metastasis of intracranial tumor. METHOD: The distribution and clearance of tracers were observed after microinjection of lymph developer labeled by 99mTc into cerebral cortex and deep cervical lymph nodes of rabbit. RESULTS: In the cerebral cortex microinjection with 99=Tc-labeled lymph developer group, the radioactivity were detected in Willis artery, deep cervical lymph nodes and venous blood. The radioactivity curve was the same in Willis artery and deep cervical lymph nodes. The peak in the artery blood was higher than that in venous blood. In the lymph nodes microinjection with 99mTc-labeled lymph developer cervical group, the radioactivity were detected in skull base dura mater, brain, cerebrospinal fluid and venous blood. The peak in skull base dura mater showed earlier than that in cerebrospinal fluid and brain. The peak in venous blood was the last, but the radioactivity in it was the highest. CONCLUSION: The cranial-cervical lymph return in rabbit is existent. Their pathway perhaps is Willis artery, skull base dura mater and cerebrospinal fluid circulation.