Secondary Lymphedema Patients Research Articles

Improved lymphangiogenesis around vascularized lymph node flaps by periodic injection of hyaluronidase in a rodent model

Vascularized lymph node transfer (VLNT) is an advanced surgical approach for secondary lymphedema (SLE) treatment, but tissue fibrosis around the lymph node flap (VLNF) inhibiting lymphangiogenesis is the biggest challenge undermining its therapeutic efficacy. This study explored the effects of periodic hyaluronidase (HLD) injection in reducing fibrosis and promoting lymphangiogenesis in 52 Sprague–Dawley rats with a VLNF over 13 weeks. The results demonstrated that HLD administration significantly enhanced swelling reduction, lymphatic drainage efficiency, and lymphatic vessel regeneration, with up to a 26% decrease in tissue fibrosis around the VLNF. These findings suggest that combining VLNT with periodic injections of HLD could substantially improve SLE treatment outcomes in clinical settings. It offers a promising direction for future therapeutic strategies and drug development aimed at increasing the efficacy of surgical treatment for SLE patients.

148. Extravasated Lymph from Secondary Lymphedema Patients Increases Cellular Permeability and Decreases Lymphatic Endothelial Cell VEGFR3 Expression

148. Extravasated Lymph from Secondary Lymphedema Patients Increases Cellular Permeability and Decreases Lymphatic Endothelial Cell VEGFR3 Expression

Serum Cathepsin D was Elevated in Patients with Limb Lymphedema.

Lymphedema (LE) is a chronic disease that can lead to disability. Currently, the pathogenesis of LE remains unclear, and there is a lack of serum proteins applicable for diagnosis in clinical practice. This study aimed to screen and identify the differentially expressed proteins in serum samples of limb lymphedema and normal subjects and to further explore their value in the diagnosis of LE. Nano-flow reverse phase liquid chromatography-tandem mass spectrometry (Nano RPLC-MS/MS) was used to establish the serum protein profiles of primary lymphedema (PLE), secondary lymphedema (SLE), and normal controls (NC). Differentially expressed serum proteins were screened and identified. Subsequently, enrichment analysis was performed for proteins that were upregulated in the LE group compared to the NC group. The target protein was validated by western blot (WB) and enzyme-linked immunosorbent assay (ELISA). Both the receiver operating characteristic (ROC) curve and Spearman's correlation test were employed to evaluate the diagnostic performance of the protein and its relationship with disease severity. A total of 362 serum proteins were identified, among which 241 proteins were differentially expressed among PLE, SLE, and NC subjects (p < 0.05, fold change > 1.2). The enriched pathway correlated with cornified envelope formation was selected for further analysis. Cathepsin D (CTSD), a target protein involved in the selected pathway, was found to be up-regulated in the serum of PLE and SLE patients compared to NC. The AUCs of CTSD were 0.849 and 0.880 for patients with PLE and SLE, respectively. There was a significant positive correlation between the levels of serum CTSD and disease severity in the PLE group. Proteomic analysis found that the levels of serum proteins related to cornified envelope formation were elevated in patients with limb lymphedema. Serum CTSD was highly expressed in patients with limb lymphedema and showed good diagnostic value.

Lymphaticovenular Anastomosis for Advanced-Stage Peripheral Lymphedema: Expanding Indication and Introducing the Hand/Foot Sign

BackgroundEffective lymphaticovenular anastomosis (LVA) requires identification of functioning lymphatics, which are not always visible with contrast-based imaging in advanced-stage lymphedema patients. Ultrasound (US) allows to identify preoperatively functioning lymphatic vessels even in limbs severely affected by lymphedema. Moreover, in our experience, we observed an interesting clinical sentry in advanced-stage lymphedema patients, the hand/foot sign that is analyzed in this paper. Patients and MethodsFrom January 2016 to January 2019, 76 consecutive advanced-stage secondary lymphedema patients underwent LVA. Preoperative planning included lymphoscintigraphy, indocyanine-green lymphography (ICG-L) and US. Patients’ features, the hand/foot sign (preservation of more normal skin on the dorsum of the hand or foot), lymphatic degeneration, quantitative, qualitative, and composite outcomes at 1-year follow-up were evaluated. ResultsAn average number of 3±0.1 LVA was performed in upper limb lymphedema (ULL) (range 2–5, 47 patients) and of 4±1.08 LVAs in lower limb lymphedema (LLL) cases (range 4–7, 29 patients). The composite outcome was positive in 45 cases (59.7%). The “negative” hand /foot sign was significantly associated with presence of functioning lymphatic channels. The incidence of adverse outcomes was significantly higher in patients with positive hand/foot sign. ConclusionPatients with no functioning lymphatic vessels detectable by lymphoscintigraphy and ICG-L may still have functioning lymphatic channels that can be identified preoperatively by ultra-high-frequency ultrasound and salvaged by LVA. The “hand/foot sign” is a simple clinical sentry that appears to be correlated with higher probability of being able to localize functional lymphatics for potential lymphovenous bypass surgery.

Animal models in lymph node transfer surgery: A systematic review

Lymph node transfer surgery (LNTS) is indicated in secondary lymphedema (LE) patients who do not respond to conservative therapy. Animal models are the spearhead of LE research and were used to pioneer most of the surgical interventions currently in practice. We conducted a systematic review of the literature to explore animal models dedicated to LNTS to compare different species, techniques, and outcomes. Four databases were searched: PubMed, Cumulative Index of Nursing and Allied Health Literature, Scopus, and Web of Science. We used the Preferred Reporting Items for Systematic Reviews and Meta-Analysis as our basis of organization. Avascular lymph node graft (ALNG) and vascularized lymph node transfer (VLNT) effectively treated LE and lead to better outcomes than controls. Whole ALNGs are superior to fragmented ALNGs. Larger fragments are more likely to be reintegrated into the lymphatic system than small fragments. VLNT was superior to whole and fragmented ALNG. Increasing the number of VLNT resulted in better outcomes. Adipose-derived stem cells improved outcomes of VLNT; vascular endothelial growth factor C and D and platelet-rich plasma improved outcomes for ALNG. Cryopreservation of lymph nodes (LNs) did not affect outcomes for ALNG. The critical ischemia and venous occlusion time for LN flaps were 4-5 and 4 h, respectively. The critical time for reperfusion injury was 2 h. Some of the novel models included venous LNT, and cervical adipocutaneous flap to groin. Current evidence from animals favors VLNT over other surgical interventions. Several pharmacological therapies significantly improved outcomes of ALNG and VLNT. LE is a chronic condition affecting millions of patients worldwide. LNTS is becoming more popular as a LE treatment. Animal models have led the LE research for decades and developing new models for LE are essential for LE research. This systematic review aims to summarize the existing animal models dedicated to LNTS. We believe that this review is critical to guide researchers in the selection of the model that is best fit for their hypothesis-driven experiments.

The pig as a model for immunology research.

The pig is an omnivorous, monogastric species with many advantages to serve as an animal model for human diseases. There are very high similarities to humans in anatomy and functions of the immune system, e g., the presence of tonsils, which are absent in rodents. The porcine immune system resembles man for more than 80% of analyzed parameters in contrast to the mouse with only about 10%. The pig can easily be bred, and there are less emotional problems to use them as experimental animals than dogs or monkeys. Indwelling cannulas in a vein or lymphatic vessel enable repetitive stress-free sampling. Meanwhile, there are many markers available to characterize immune cells. Lymphoid organs, their function, and their role in lymphocyte kinetics (proliferation and migration) are reviewed. For long-term experiments, minipigs (e.g., Göttingen minipig) are available. Pigs can be kept under gnotobiotic (germfree) conditions for some time after birth to study the effects of microbiota. The effects of probiotics can be tested on the gut immune system. The lung has been used for extracorporeal preservation and immune engineering. After genetic modifications are established, the pig is the best animal model for future xenotransplantation to reduce the problem of organ shortage for organ transplantation. Autotransplantation of particles of lymphnodes regenerates in the subcutaneous tissue. This is a model to treat secondary lymphedema patients. There are pigs with cystic fibrosis and severe combined immune deficiency available.

Rationale for Study of the Deep Subfascial Lymphatic Vessels During Lymphoscintigraphy for the Diagnosis of Peripheral Lymphedema.

The current study aimed to determine the utility of including the study of deep subfascial lymphatic vessels in a 2-compartment lymphoscintigraphy for the diagnosis of lymphedema in patients with limb swelling. Lymphoscintigraphy is a valuable imaging tool for the timely diagnosis of peripheral lymphedema. However, there is a lack of standardization in its application, especially regarding which type of lymphatic vessels to examine (superficial, deep, or both). Two hundred fifty-eight patients with lymphedema underwent segmental lymphoscintigraphy. The transport index (TI) was calculated to categorize the flow of the superficial and deep vessels as normal (<10) or pathological (≥10). The scores from 248 patients (48 unilateral arm, 86 unilateral leg, 114 bilateral leg) were tested with a 3-way analysis of variance to examine the relationship between affected limb, deep or superficial pathways, and primary or secondary lymphedema. The relationship between clinical presentation and TI was also investigated. In general, primary and secondary lymphedema patients had similar patterns of lymphoscintigraphic lymphatic abnormalities. Patients with unilateral clinical presentation can have bilateral TI abnormalities. The vast majority of patients (88%-98%) had either the deep subfascial vessels alone, or both the superficial and deep vessels, with a pathological TI. A 2-compartment lymphoscintigraphy is able to accurately detect lymphatic flow abnormalities in patients with limb swelling. Given that the vast majority of patients had deep lymphatic vessels abnormalities, inclusion of these vessels in the lymphoscintigraphic diagnostic protocol is recommended.

Ultra High-frequency Ultrasonographic Imaging with 70 MHz Scanner for Visualization of the Lymphatic Vessels.

Identification and localization of functional lymphatic vessels are important for lymphaticovenular anastomosis. Conventional high-frequency ultrasound (CHFUS) has been reported to be useful for them, but it has some disadvantages. In this article, we present new capabilities of ultra high-frequency ultrasound (UHFUS) for imaging of the lymphatic vessels, which may overcome the weakness of CHFUS. Thirty unaffected extremities in 30 unilateral secondary lymphedema patients (13 upper limbs and 17 lower limbs) were examined. Identification of the lymphatic vessels using UHFUS and CHFUS were performed at 3 sites in each unaffected extremity. Number and diameter of the detected lymphatic vessels were compared between UHFUS and CHFUS groups. At the same time, new characteristics of the lymphatic vessels seen with UHFUS were investigated. One hundred sixty-nine lymphatic vessels were detected with UHFUS, and 118 lymphatic vessels with CHFUS. The number of lymphatic vessels found in upper and lower extremities was significantly larger with UHFUS than with CHFUS. The diameter of lymphatic vessels found in upper and lower extremities was significantly smaller with UHFUS than with CHFUS. All lymphatic vessels that were detected in UFHUS were less likely to collapse when the transducer was against the skin of the examined sites. Detection rate of the lymphatic vessels in nonlymphedematous extremities with UHFUS was higher than that with CHFUS. UHFUS provides images with extremely high resolution, demonstrating new characteristics of the lymphatic vessels.

Site Specific Evaluation of Lymphatic Vessel Sclerosis in Lower Limb Lymphedema Patients.

Histological changes in the collecting lymphatics in patients with lymphedema are classified as Normal type, Ectasis type, Contraction type, and Sclerosis type (NECST) classification. In this study, we investigated the condition of the lymphatic vessels in different sites of the legs. We prospectively investigated the lymphatic vessels of patients with lymphedema who underwent lymphaticovenous anastomosis (LVA) from August 8, 2014 to August 4, 2015 based on the NECST classification. Lymphedema was diagnosed using lymphoscintigraphy, indocyanine green (ICG) lymphography, and the International Society of Lymphology (ISL) Classification. The affected limbs were divided into four sites: proximal thigh (Site 1), distal thigh (Site 2), proximal crus (Site 3), and distal crus (Site 4). A total of 109 patients (205 limbs and 1028 lymphatics) were included in this study. Of the 109 patients, there were 100 women and 9 men with an average age of 61 years. The ratio of Ectasis type vessels increased toward the distal end of the limb with the highest occurrence rate being 54% at Site 4. As ISL stage, ICG stage, and lymphoscintigraphy stage advanced, so too did the ratio of Sclerosis type. In secondary lymphedema patients with lymphedema, the ratio of Ectasis type was more predominant in the distal end of the limb, whereas this tendency was not observed in primary lymphedema patients. Sclerotic lymphatics are more predominantly found in the proximal limb whereas nonsclerotic vessels are more often found toward the distal end. These findings help lymphatic surgeon determine incision sites.

Real-Time Direct Evidence of the Superficial Lymphatic Drainage Effect of Intermittent Pneumatic Compression Treatment for Lower Limb Lymphedema.

Intermittent pneumatic compression (IPC) is a widely used lymph drainage therapy that can be performed at home. However, the effectiveness of IPC for lymph drainage remains unclear. The aim of this study is to investigate the real-time change in the lymph flow velocity during IPC and consider which mode and pressure are best for treating lower limb lymphedema. Eight lower left limbs of 8 healthy volunteers and 17 lower limbs of 15 secondary lymphedema patients were investigated. Indocyanine green lymphography was performed with the subject covered with a transparent six-chambered IPC garment. The IPC treatment was administered in several modes (sequential or nonsequential inflation mode, sequential or interrupted deflation mode, and under high or low pressure). Using a brightness intensity analysis software program, the real-time change in the fluorescence intensity during the treatment was recorded and graphed. The maximum inclination of the graph between 2 seconds in the inflation phase (SLOPE) and the mean SLOPE value of all subjects (average SLOPE) were calculated. The average SLOPEs of each mode of treatment were then compared. The average SLOPEs were also compared between patients with mild and moderate lymphedema. There were no significant differences among the SLOPEs in the healthy group. However, in the patient group, the average SLOPE was significantly higher in the sequential inflation mode as well as under high pressure than in the nonsequential inflation mode. On comparing the mild and moderate lymphedema groups, the average SLOPE tended to be higher in the mild group; however, the difference was not statistically significant. Sequential programs and high pressure resulted in a faster lymphatic flow than other modes. These results suggest that a greater treatment effect could be obtained by adjusting the mode of treatment and the pressure of IPC.

Pathological Changes of Lymphedematous Skin: Increased Mast Cells, Related Proteases, and Activated Transforming Growth Factor-β1.

Skin fibrosis is a clinically serious pathological process of secondary lymphedema (SLE). Previous studies have shown that mast cells (MCs) are involved in lymphedema (LE) and play a key role in the pathological process of skin fibrosis. However, the role of the protease chymase and transforming growth factor-β1 (TGF-β1) secreted by MCs in the fibrotic skins of patients with secondary lower limb LE has not been explored. In this study, full-thickness skin biopsies of lymphedematous limbs from seven SLE patients and control samples from seven healthy controls were harvested. The skin samples were assayed by Masson, immunohistochemical, and immunofluorescence staining and were analyzed by western blot and enzyme-linked immunosorbent assay. The number of MCs and the expression of proteases, TGF-β1, and latency-associated peptide TGF-β1 (LAP TGF-β1) were analyzed. The number of MCs and the expression of chymase, TGF-β1, and LAP TGF-β1 were increased in fibrotic skin compared with normal skin. The increased expression of TGF-β1 on lymphatic vessels, endothelial cells, and in skin interstitial tissues overlapped with chymase expression. Our results demonstrate that chymase and TGF-β1 expression was significantly increased in the fibrotic skin of secondary lower limb LE. The increased expression of chymase in the skin may play an important role in the development fibrosis in the lymphedematous skin. We speculate that chymase may facilitate the release of LAP TGF-β1 to generate activated TGF-β1, and the upregulation of active TGF-β1 can promote fibrosis in the SLE skin.

Development of functionally patent lymphatic meshes in postsurgical long-term resolution ofperipheral secondary lymphedema.

In oncologic surgery, secondary lymphedema of male external genital organs and upper or lower limbs frequently develops as a result of excision or mechanical obstruction of collecting lymphatic trunks. We evaluated whether the short-term and long-term outcomes of microsurgical treatment of limb and genital organs improves tissue drainage in patients with secondary lymphedema by restoring the pre-existing lymphatic networks or through new lymphangiogenesis. Of 110 secondary lymphedema patients, microsurgery was performed in 45 hospitalized patients. Patients were aged 25 to 75 years, had at least third-degree lymphedema, no satisfactory results from previous physical or pharmacologic therapy, without primitive neoplasia, at least 1 year since the last postsurgical adjuvant oncological treatment, and<15 years since the previous primary oncologic lymphedema development. A microsurgical lymphovenous shunt of the spermatic cord (n= 7), a lymphovenous shunt of the lower limbs (n= 32), or lymphatic grafting of the upper limbs (n=6) was performed. The male external genitals were treated through an innovative lymphovenous shunt of the lymphatic collectors in the pampiniform plexus of the spermatic cord. For lower limb lymphedema, the lymphatics were shunted to the collaterals or saphenous vein. For upper limb lymphedema, a shunt was performed between the lymph vessels of the jugular-supraclavicular area and those in close continuity with the axillary region. The patency of the new lymphatic pathways was assessed using Photodynamic Eye (Hamamatsu Photonic K.K., Tokyo, Japan) lymphography. Six months postoperatively, 36 responding patients showed an almost complete recovery from secondary lymphedema. Lymphatic meshes, consisting of several lymphatic vessels merging into well-canalized and complex networks developing in the perianastomotic area or between the adjacent proximal anastomotic lymphatic collectors, were commonly observed in patients who positively responded tomicrosurgery. These complexes were never encountered in nonresponding patients or in normal, nonedematous tissue. Long-term postsurgical recovery from severe secondary lymphedema requires canalizing the lymphatic collectors along their original flow pattern and developing perianastomotic meshes. Because this phenomenon can be observed with the same characteristics in different tissues, such as the spermatic cord and the inguinocrural, inguinoscrotal, inguinotesticular, and brachial regions, the development of meshes seems to reflect a generalized phenomenon oflocal lymphangiogenesis triggered by the microsurgical procedure.