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Abstract Secondary lymphedema is a common sequel of oncologic surgery and presents a global health burden still lacking pharmacological treatment. The infiltration of the lymphedematous extremities with CD4+T cells influences lymphedema onset and emerges as a promising therapy target. Here, we show that the modulation of CD4+FOXP3+CD25+regulatory T (Treg) cells upon anti-CTLA4 treatment protects against lymphedema development in patients with melanoma and in a mouse lymphedema model. A retrospective evaluation of a melanoma patient registry reveals that anti-CTLA4 reduces lymphedema risk; in parallel, anti-CTLA4 reduces edema and improves lymphatic function in a mouse-tail lymphedema model. This protective effect of anti-CTLA4 correlates with a systemic expansion of Tregs, both in the animal model and in patients with melanoma. Our data thus show that anti-CTLA4 with its lymphedema-protective and anti-tumor properties is a promising candidate for more diverse application in the clinics.

Background:. Recent advances in robotic microsurgery have enabled the application of robotic technology in central lymphatic reconstruction. Although the use of microsurgical robots demands careful consideration of associated costs and potentially prolonged operating times, it may offer improved surgical approaches and enhanced accessibility to deeper anatomical structures such as the thoracic duct (TD). Methods:. We report on successful reconstruction of the central lymphatic system using the Symani Surgical System in four patients with lesions of the central lymphatic system. The patients were of different age (range: 8 mo–60 y) and had variable conditions, including central conducting lymphatic anomaly and other rare anomalies of the central lymphatic pathways. Results:. Depending on the underlying pathology, a cervical access (n = 1) or median laparotomy (n = 3) was chosen to access the TD and perform anastomosis with a nearby vein. In all patients, anastomoses were patent, and chyle leakage decreased postoperatively. From a surgical perspective, the Symani Surgical System improved the precision of the microsurgeon and accessibility to the deep-lying TD. Conclusion:. Considering the high morbidity and rarity of pathologies of the central lymphatic system, robotic-assisted microsurgery holds substantial promise in expanding and improving the microsurgical treatment for central lymphatic anomalies.

Facial paralysis is a highly burdening condition, resulting in a patient's inability to move his mimic musculature on one or both sides of his face. This condition compromises the patient's communication and facial expressions, and thus dramatically reduces his quality of life. The current treatment for chronic facial paralysis relies on a complex reconstructive surgery. This publication proposes a novel, less invasive approach for dynamic facial reanimation. The use of a smart material, namely a Dielectric Elastomer Actuator (DEA) is proposed for facial motion restoration, thus avoiding the traditional two-stage free muscle transfer procedure and allowing for a faster recovery of the patient. DEAs are a type of electroactive polymers, showing promising properties similar to natural muscles such as the fact that they are soft, lightweight and allow for large displacements. As a result, a study of the facial muscles and neural interfaces, notably the ones responsible for mouth movement, was performed, in order to implement a realistic setup. In this paper, a non-invasive neural interface based on myoelectric signal is used in order to establish a real-time control of the actuator. Visible motion of a skin model is produced in real time, by synchronizing the actuator to the activity of a healthy muscle, with a maximal delay of 108 ​ms resulting from the signal processing and a delay of less than 30 ​ms related to the actuation of the DEA. This shows that the usage of DEA combined with a neural interface presents a promising approach for treatment of facial paralysis.

Background:. Lower extremity trauma can have a significant impact on a patient’s quality of life. The LIMB-Q is a recently developed and validated patient-reported outcome measure that assesses patient-specific outcomes and experience of health care. The aim of this study was to translate and linguistically validate the LIMB-Q from English to German. Methods:. The translation was performed by combining World Health Organization and Professional Society for Health Economics and Outcomes Research guidelines. The process consisted of forward translations, a backward translation, expert panel meetings, cognitive debriefing interviews with patients, and several rounds of discussion and reconciliation with the creators of LIMB-Q. The goal was to obtain a culturally and conceptually accurate translation of LIMB-Q into German for use in Switzerland. Results:. From the two forward translations, there was one primary discrepancy between the two translators that was discussed to determine the most conceptually accurate translation. From the backward translations, there were 63 items that required discussion and re-translation. Nine patients participated in the cognitive debriefing interviews, which led to three items being modified. The translation process led to a linguistically validated and conceptually equivalent German version of the LIMB-Q. Conclusions:. The German (Switzerland) version of LIMB-Q is now available. This will offer a valuable tool for lower extremity trauma research and clinical care in German-speaking populations.

Summary: Micro- and supermicrosurgeries have become standard techniques for lymphatic reconstruction. As increasingly smaller vessels are being targeted, robotic-assisted surgery has emerged as a new approach to push reconstructive limits owing to its ability of motion scaling and providing better accessibility of deep anatomical regions. The precision of the robot is achieved at the expense of operating speed among other variables; therefore, the surgeon must weigh the enhanced dexterity against the additional operating time and cost required for the robotic surgical system itself to ensure optimal resource utilization.Here we present a case series of 8 patients who underwent robot-assisted lymphatic microsurgery for omental flap transfer to the axilla and lympho-venous anastomosis. The Symani® Surgical System was used with a conventional microscope or 3D exoscope. The use of 3D exoscope provided clear benefits in terms of surgeon positioning. Moreover, access to the recipient vessels near the thoracic wall was significantly improved with the robotic setup. In addition, suture precision was excellent, resulting in patent anastomoses. Operating time for anastomosis was comparable to that for manual anastomosis and demonstrated a steep learning curve.The benefits of robotic systems in operating fields with good exposure require further evaluation. However, owing to longer instruments, additional stability, dexterity, and motion precision, robotic systems offer a marked advantage for operating in deep anatomical planes and on small structures. A potentially new field for the implementation of robotic surgery is central lymphatic reconstruction. Progress in terms of operating time and cost is crucial, and future research should validate the effectiveness of robotic-assisted surgery in larger clinical studies.

IntroductionIn recent years advances have been made in the microsurgical treatment of congenital or acquired central lymphatic lesions. While acquired lesions can result from any surgery or trauma of the central lymphatic system, congenital lymphatic lesions can have a variety of manifestations, ranging from singular thoracic duct abnormalities to complex multifocal malformations. Both conditions may cause recurrent chylous effusions and downstream lymphatic congestion depending on the anatomical location of the thoracic duct lesion and are associated with an increased mortality due to the permanent loss of protein and fluid.MethodsWe present a case series of eleven patients undergoing central lymphatic reconstruction, consisting of one patient with a cervical iatrogenic thoracic duct lesion and eleven patients with different congenital thoracic duct lesions or thrombotic occlusions.ResultsAnastomosis of the thoracic duct and a nearby vein was performed on different anatomical levels depending on the underlying central lymphatic pathology. Cervical (n = 4), thoracic (n = 1) or abdominal access (n = 5) was used for central lymphatic reconstruction with promising results. In 9 patients a postoperative benefit with varying degrees of symptom regression was reported.ConclusionThe presented case series illustrates the current rapid advances in the field of central microsurgical reconstruction of lymphatic lesions alongside the relevant literature.

Background: Burn wounds pose significant challenges in medical treatment due to their devastating nature and resource-intensive requirements. Temporary coverage of burn wounds using synthetic or biological dressings allows for reepithelization before definitive skin grafting. Allogenic skin grafts have been widely used but come with drawbacks such as rejection and disease transmission. The use of amniotic membranes (AMs) offers a promising alternative for temporary coverage, as they possess biological properties that promote faster healing and improved scar quality. The various components of the amniotic membrane, including pluripotent stem cells, extracellular matrix proteins, and regenerative factors, contribute to cell growth, migration, and differentiation, as well as preservation of the original epithelial phenotype. Objective: Reliable information on the treatment of burn wounds with AM is needed. The knowledge gained in this project may help to include this advantageous modern concept of biological dressings in clinical practice. The purpose of this study is to use human amniotic membranes from our in hospital laboratory, as an allogenic biological dressing after enzymatic debridement in superficial partial thickness, deep partial thickness or full thickness burn wounds. Methods: We will include 30 patients in a randomized-controlled trial with each patient receiving the study intervention and the control intervention. Two 7 × 7 cm burn wound areas will be compared regarding percentage of skin graft take, healing time, healing percentage value and total healing time. Human amniotic membranes will be compared to allogenic skin grafts.

Summary:. Advances in the development of robotic systems have recently enabled the use of robotic technology in reconstructive lymphatic surgery. Although the advantages of microsurgical robots must be weighed carefully against the costs, their use may allow for smaller surgical approaches and easier access to anatomically deeper structures or even smaller vessels. We report on a case of a patient with central lymphatic dilation causing abdominal pain and severely reduced physical capacity. Sonography-assisted intranodal injection of indocyanine green allowed for localization of the lymphatic cyst and anastomosis with the left ovarian vein, applying robotic-assisted microsurgery for the first time on the central lymphatic system. Following the successful reconstruction of lymphatic drainage and decompression of the cyst, the patient reported a complete regression of her preoperative symptoms. From a surgical point of view, the Symani Surgical System improved precision and allowed significantly smaller surgical access. Considering the high morbidity and rarity of pathologies of the central lymphatic system, central lymphatic surgery is to date rarely performed. With improved precision and significantly smaller surgical access, robotic-assisted microsurgery has great potential to expand the treatment options for central lymphatic lesions.

Summary: Robotic systems have recently been introduced into micro- and supermicrosurgery showing potential benefits for reconstructive surgery. After showing the feasibility and safety of using the Symani Surgical System® for lymphatic microsurgical procedures in humans, we present the results of the first twenty-two patients operated with the robot. The main goal of the study was to determine the learning curve using the Symani Surgical System® . In addition, we aimed at exploring the potential of robot-assisted anastomosis for lymphatic, free flap, and nerve reconstruction and defining the advantages and drawbacks of implementing the system into our daily routine. The operating times were compared between robotic-assisted and hand-sewn anastomoses. Moreover, outcomes and complications were recorded. In this first patient series, anastomotic times were significantly faster with the hand-sewn technique (14.1±4.3 min) when compared with the robot-assisted technique (25.3±12.3 min; p

Dendritic cell (DC) migration from peripheral tissues via afferent lymphatic vessels to draining lymph nodes (dLNs) is important for the organism’s immune regulation and immune protection. Several lymphatic endothelial cell (LEC)-expressed adhesion molecules have thus far been found to support transmigration and movement within the lymphatic vasculature. In this study, we investigated the contribution of CD112, an adhesion molecule that we recently found to be highly expressed in murine LECs, to this process. Performing in vitro assays in the murine system, we found that transmigration of bone marrow-derived dendritic cells (BM-DCs) across or adhesion to murine LEC monolayers was reduced when CD112 was absent on LECs, DCs, or both cell types, suggesting the involvement of homophilic CD112–CD112 interactions. While CD112 was highly expressed in murine dermal LECs, CD112 levels were low in endogenous murine dermal DCs and BM-DCs. This might explain why we observed no defect in the in vivo lymphatic migration of adoptively transferred BM-DCs or endogenous DCs from the skin to dLNs. Compared to murine DCs, human monocyte-derived DCs expressed higher CD112 levels, and their migration across human CD112-expressing LECs was significantly reduced upon CD112 blockade. CD112 expression was also readily detected in endogenous human dermal DCs and LECs by flow cytometry and immunofluorescence. Upon incubating human skin punch biopsies in the presence of CD112-blocking antibodies, DC emigration from the tissue into the culture medium was significantly reduced, indicating impaired lymphatic migration. Overall, our data reveal a contribution of CD112 to human DC migration.

Background:. Patients with upper limb lymphedema and lower limb lymphedema experience a wide range of physical and psychological symptoms that affect quality of life. The benefits of lymphatic reconstructive surgery for patients with lymphedema are undisputed. However, recording volume reduction alone may be insufficient with regard to postoperative outcome because measurements are often inadequate, depend on many factors, and do not reflect improvement in quality of life. Methods:. We conducted a prospective single center study patients receiving lymphatic reconstructive surgery. Patients received volume measurements preoperatively and at standardized postoperative intervals. To evaluate patient-reported outcomes, patients completed the following questionnaires: LYMPH-Q Upper Extremity Module, quickDASH, SF 36, Lymphoedema Functioning, Disability and Health Questionnaire for Lower Limb Lymphoedema, and Lower Extremity Functional Scale at the aforementioned intervals. Results:. We included 55 patients with upper limb lymphedema (24%) and lower limb lymphedema (73%) of lymphedema grades I–III. Patients received lymphovenous anastomosis only (23%), free vascularized lymph node transfer (35%) or a combination of both (42%). Analysis of patient-reported outcome measurements revealed improvements with respect to a broad range of complaints, particularly physical function, symptoms, and psychological well-being. There was no correlation between the extent of volume reduction and improvement in quality of life (Pearson correlation coefficient below ±0.7; P > 0.05). Conclusions:. Based on a broad range of outcome measurements, we observed an improved quality of life in almost all patients, even in those without measurable volume loss of the extremity operated on, which emphasizes the need for a standardized use of patient-reported outcome measures to evaluate the benefits of lymphatic reconstructive surgery.

BACKGROUND: Cosmetic surgery tourism has increased in popularity in recent years, with arising complications and post-operative follow-up care often managed in the client’s home country, thereby burdening the Swiss health care system. METHODS: We retrospectively reviewed patients with complications after cosmetic surgeries abroad and in Switzerland who were treated at the University Hospital Zurich between 2015 and 2019. Data were collected from medical records and reviewed for patient characteristics, procedures, complications, and treatment modalities. RESULTS: A total of 228 patients (207 females and 21 males) were identified with female mean age of 40.9±12.0 years and male mean age of 34.3±8.9 years. Most complications were observed for procedures performed in Europe (69%) with only thirty-six patients (16%) experiencing complications due to a procedure undertaken in Switzerland. Breast surgery was the most frequently performed procedure (60%), followed by body contouring (17%) and facial surgery (12%). The most common complications occurring after surgeries abroad were pain and discomfort (19%) as well as aesthetic dissatisfaction (18%), followed by wound breakdown (14%) and infection (11%). Most patients (76%) were treated as outpatients and the treatment of all patients over the observed period cost the healthcare system $ 795,574. CONCLUSIONS: There is an ongoing trend of cosmetic surgery tourism leading to an increasing number of patients with complications requiring aftercare in Switzerland. In contrast to previous research, more men are seeking cosmetic surgery abroad and the most common complications, such as wound healing disorders and infection, descreased in favor of aethetic dissatisfaction, possibly indicating ameliorated patient aftercare abroad.

The application of minimal invasive mastectomy has allowed surgeons to perform nipplesparing mastectomy via a shorter, inconspicuous incision under clear vision and with more precise hemostasis. However, it poses new challenges in microsurgical breast reconstruction, such as vascular anastomosis and flap insetting, which are considerably more difficult to perform through the shorter incision on the lateral breast border. We propose an innovative technique of transcutaneous medial fixation sutures to help in flap insetting and creating and maintaining the medial breast border. The sutures are placed after mastectomy and before flap transfer. Three 4-0 nylon suture loops are placed transcutaneously and into the pocket at the markings of the preferred lower medial border of the reconstructed breast. After microvascular anastomosis and temporary shaping of the flap on top of the mastectomy skin, the three corresponding points for the sutures are identified. The three nylon loops are then sutured to the dermis of the corresponding medial point of the flap. The flap is placed into the pocket by a simultaneous gentle pull on the three sutures and a combined lateral push. The stitches are then tied and buried after completion of flap inset.

Lipedema is a chronic and progressive adipose tissue disorder, characterized by the painful and disproportionate increase of the subcutaneous fat in the lower and/or upper extremities. While distinct immune cell infiltration is a known hallmark of the disease, its role in the onset and development of lipedema remains unclear. To analyze the macrophage composition and involved signaling pathways, anatomically matched lipedema and control tissue samples were collected intra-operatively from gender- and BMI-matched patients, and the Stromal Vascular Fraction (SVF) was used for Cytometry by Time-of-Flight (CyTOF) and RNA sequencing. The phenotypic characterization of the immune component of lipedema versus control SVF using CyTOF revealed significantly increased numbers of CD163 macrophages. To gain further insight into this macrophage composition and molecular pathways, RNA sequencing of isolated CD11b+ cells was performed. The analysis suggested a significant modification of distinct gene ontology clusters in lipedema, including cytokine-mediated signaling activity, interleukin-1 receptor activity, extracellular matrix organization, and regulation of androgen receptor signaling. As distinct macrophage populations are known to affect adipose tissue differentiation and metabolism, we evaluated the effect of M2 to M1 macrophage polarization in lipedema using the selective PI3Kγ inhibitor IPI-549. Surprisingly, the differentiation of adipose tissue-derived stem cells with conditioned medium from IPI-549 treated SVF resulted in a significant decreased accumulation of lipids in lipedema versus control SVF. In conclusion, our results indicate that CD163+ macrophages are a critical component in lipedema and re-polarization of lipedema macrophages can normalize the differentiation of adipose-derived stem cells in vitro evaluated by the cellular lipid accumulation. These data open a new chapter in understanding lipedema pathophysiology and may indicate potential treatment options.

Lipedema is a chronic disorder that mainly affects women. It is often misdiagnosed, and its etiology remains unknown. Recent research indicates an accumulation of macrophages and a shift in macrophage polarization in lipedema. One known protein superfamily that contributes to macrophage accumulation and polarization is the macrophage migration inhibitory factor (MIF) family. MIF-1 and MIF-2 are ubiquitously expressed and also regulate inflammatory processes in adipose tissue. In this study, the expression of MIF-1, MIF-2 and CD74—a common receptor for both cytokines—was analyzed in tissue samples of 11 lipedema and 11 BMI-matched, age-matched and anatomically matched control patients using qPCR and immunohistochemistry (IHC). The mRNA expression of MIF-1 (mean 1.256; SD 0.303; p = 0.0485) and CD74 (mean 1.514; SD 0.397; p = 0.0097) were significantly elevated in lipedema patients, while MIF-2 expression was unaffected (mean 1.004; SD 0.358; p = 0.9718). The IHC analysis corroborated the results for CD74 expression on a cellular level. In conclusion, our results provide first evidence for a potential involvement of the MIF family, presumably via the MIF-1-CD74 axis, in lipedema.

Summary:. Robotic microsurgery has emerged as a new technology with potential benefits for reconstructive surgery. We report the first-in-human use of the Symani surgical system to perform lympho-venous and arterial anastomosis for lymphatic reconstruction. In five patients, 10 robot-assisted anastomoses were performed. Next to lympho-venous anastomoses, two patients received a free vascularized lymph node transfer. Motion scaling was set to 10×. Visualization was either achieved with a 3D system or an optical microscope. All anastomoses were patent as confirmed by ICG. Despite a longer time to perform the first anastomoses with the robot, we observed a decline in duration of anastomosis. Among the advantages of the system were a high accuracy in placing the stitches even in very small and fragile vessels or when performing anastomoses with size mismatches. The challenges encountered included the lack of a touch sensation and the necessity to develop a “see-feel.” This could be achieved surprisingly well because the force necessary to close dilator and needle holder via the manipulators was perceived as comparable to using conventional micro instruments. Our data confirm feasibility and safety of the robotic system to perform lymphatic surgery. Larger patient cohorts and inclusion of surgeons at different training levels will be necessary to investigate the true potential of robotics in microsurgery. In addition, robot-assisted surgery shows a promising potential in opening up new frontiers in reconstructive microsurgery (eg, the reliable performance of anastomoses on even smaller blood and lymphatic vessels or on structures deeper within the body cavities—eg, the thoracic duct).

Secondary lymphedema is a chronic, debilitating disease and one of the most common side effects of oncologic surgery, substantially decreasing quality of life. Despite the progress conducted in lymphedema research, the underlying pathomechanisms remain elusive. Lymphedema is considered to be a disease affecting an isolated extremity, yet imaging studies suggest systemic changes of the lymphatic system in the affected patients. To evaluate potential systemic manifestations in lymphedema, we collected matched fat and skin tissue from the edematous and non-edematous side of the same 10 lymphedema patients as well as anatomically matched probes from control patients to evaluate whether known lymphedema manifestations are present systemically and in comparison to health controls. The lymphedematous tissue displayed various known hallmarks of lymphedema compared to the healthy controls, such as increased epidermis thickness, collagen deposition in the periadipocyte space and the distinct infiltration of CD4+ cells. Furthermore, morphological changes in the lymphatic vasculature between the affected and unaffected limb in the same lymphedema patient were visible. Surprisingly, an increased collagen deposition as well as CD4 expression were also detectable in the non-lymphedematous tissue of lymphedema patients, suggesting that lymphedema may trigger systemic changes beyond the affected extremity.

Background: Adipose-derived stem cells (ASCs) are multipotent mesenchymal stem cells characterized by their strong regenerative potential and low oxygen consumption. Macrophage migration inhibitory factor (MIF) is a multifunctional chemokine-like cytokine that is involved in tissue hypoxia. MIF is not only a major immunomodulator but also is highly expressed in adipose tissue such as subcutaneous adipose tissue of chronic non-healing wounds. In the present study, we investigated the effect of hypoxia on MIF in ASCs isolated from healthy versus inflamed adipose tissue.Methods: Human ASCs were harvested from 17 patients (11 healthy adipose tissue samples, six specimens from chronic non-healing wounds). ASCs were treated in a hypoxia chamber at

BACKGROUND Lymphoedema is a progressive and potentially disabling disease. A growing number of studies show promising clinical results after microsurgical reconstruction. However, this treatment is currently not supported by level 1 evidence and insurance coverage is variable. METHODS Electronic records of 55 patients with limb lymphoedema, who were eligible for lymphovenous bypass surgery and/or lymphatic tissue transfer in our department from 2017 to 2020, were reviewed. Correspondence between our department and health insurers was analysed. A web-based search and individual telephone interviews were conducted to identify health insurer policies. RESULTS We included 42 patients undergoing 46 operations and evaluated the correspondence between our department and nine different health insurers. Overall, reimbursement of costs was approved in 67% (n = 31) of all surgeries and was refused in 33% (n = 15). The mean number of applications for reconsideration sent to insurers was 1.3 ± 0.7. The time between confirmation of the indication and the final decision ranged from 6 to 300 days (mean 50 days). Reimbursement of cost coverage ranged from 0% to 100% depending on the individual insurance company. No insurance company had policies publicly available online and all stated that they determine coverage only when provided with specific patient details on a case-by-case basis. CONCLUSION Insurance companies in Switzerland do not have a uniform policy regarding cost coverage for lymphatic surgery procedures. Moreover, the decision process appeared to be rather uniform within the respective insurance company and independent of the individual case. Standardised evaluation criteria including patient reported outcome measures should be developed to underscore the beneficial effects of lymphatic surgery and facilitate insurance coverage.

Background:. Numerous approaches have been employed to treat chronic lymphocele and cutaneous lymphatic fistulas (LFs) with little success. Given a high incidence and substantial consequences for patients, there is an ongoing demand for effective therapeutic and preventive strategies. The aim of this study was to evaluate the results after microscopic lymphatic ligation (MLL) and lymphovenous anastomosis (LVA) as a therapeutic and preventive approach in this context. Methods:. Demographic data, surgical characteristics, complications, and the overall outcome of all patients undergoing surgery for postoperative LF from 2014 to 2019 were collected retrospectively. Patients were categorized in accordance with predefined inclusion/exclusion criteria and with their treatment. Statistical analysis was conducted using descriptive, summary statistics to identify a central tendency. Results:. Thirty-four patients underwent indocyanine-green-lymphangiography guided revision surgery for LF. Two patients were lost to follow-up at 6 months. LF was successfully treated in all patients (n = 32) with a multimodal approach. Only MLL was performed in 22 patients and MLL/LVA in 10 patients. LF resolved in 78% of all patients with MLL only or MLL/LVA. In the remaining 22%, LF resolved after additional sclerotherapy within 3 months. Conclusions:. Treatment of LF should follow a standardized staged surgical approach to optimize outcome. LF was treated successfully in all our patients. We therefore propose a multimodal interdisciplinary approach to this common clinical problem that includes adjunctive sclerotherapy.

Ample evidence pinpoints the phenotypic diversity of blood vessels (BVs) and site-specific functions of their lining endothelial cells (ECs). We harnessed single-cell RNA sequencing (scRNA-seq) to dissect the molecular heterogeneity of blood vascular endothelial cells (BECs) in healthy adult human skin and identified six different subpopulations, signifying arterioles, post-arterial capillaries, pre-venular capillaries, post-capillary venules, venules and collecting venules. Individual BEC subtypes exhibited distinctive transcriptomic landscapes associated with diverse biological pathways. These functionally distinct dermal BV segments were characterized by their unique compositions of conventional and novel markers (e.g., arteriole marker GJA5; arteriole capillary markers ASS1 and S100A4; pre-venular capillary markers SOX17 and PLAUR; venular markers EGR2 and LRG1), many of which have been implicated in vascular remodeling upon inflammatory responses. Immunofluorescence staining of human skin sections and whole-mount skin blocks confirmed the discrete expression of these markers along the blood vascular tree in situ, further corroborating BEC heterogeneity in human skin. Overall, our study molecularly refines individual BV compartments, whilst the identification of novel subtype-specific signatures provides more insights for future studies dissecting the responses of distinct vessel segments under pathological conditions.

Mechanically processed stromal vascular fraction (mSVF) is a promising source for regenerative purposes. To study the in vivo fate of the mSVF, we herein used a vascularized tissue engineering chamber that insulates the target mSVF from the surrounding environment. In contrast to previous models, we propose an arteriovenous (AV) shunt between saphenous vessels in rats without a venous graft. Mechanical SVF was processed from the fat pads of male Sprague Dawley rats, mixed with a fibrin hydrogel and implanted into an inguinal tissue engineering chamber. An arteriovenous shunt was established between saphenous artery and vein. On the contralateral side, an mSVF-fibrin hydrogel mix without vascular axis served as a non-vascularized control. After two and six weeks, rats were sacrificed for further analysis. Mechanical SVF showed significant numbers of mesenchymal stromal cells. Vascularized mSVF explants gained weight over time. Perilipin and CD31 expression were significantly higher in the mSVF explants after six weeks while no difference in DAPI positive cells, collagen deposition and FABP4 expression was observed. Morphologically, no differentiated adipocytes but a dense cell-rich tissue with perilipin-positive cells was found after six weeks. The phosphorylation of ERK1/2 was significantly enhanced after six weeks while Akt activation remained unaltered. Finally, mSVF explants stably expressed and released VEGF, bFGF and TGFb. Vascularized mSVF is able to proliferate and express adipocyte-specific markers. The AV shunt model is a valuable refinement of currently existing AV loop models in the rat which contributes to the fundamental 3R principles of animal research.

Current diagnostic methods for evaluating the functionality of the lymphatic vascular system usually do not provide quantitative data and suffer from many limitations including high costs, complexity, and the need to perform them in hospital settings. In this work, we present a quantitative, simple outpatient technology named LymphMonitor to quantitatively assess lymphatic function. This method is based on the painless injection of the lymphatic-specific near-infrared fluorescent tracer indocyanine green complexed with human serum albumin, using MicronJet600TM microneedles, and monitoring the disappearance of the fluorescence signal at the injection site over time using a portable detection device named LymphMeter. This technology was investigated in 10 patients with unilateral leg or arm lymphedema. After injection of a tracer solution into each limb, the signal was measured over 3 h and the area under the normalized clearance curve was calculated to quantify the lymphatic function. A statistically significant difference in lymphatic clearance in the healthy versus the lymphedema extremities was found, based on the obtained area under curves of the normalized clearance curves. This study provides the first evidence that the LymphMonitor technology has the potential to diagnose and monitor the lymphatic function in patients.

Vascularized lymph node (VLN) transfer is an emerging strategy to re-establish lymphatic drainage in chronic lymphedema. However, the biological processes underlying lymph node integration remain elusive. This study introduces an experimental approach facilitating the analysis of short-term molecular and cellular effects of ischemia/reperfusion on VLN flaps. Lymph node flaps were dissected pedicled on the lateral thoracic vessels in 44 Lewis rats. VLN flaps were exposed to 45 or 120 minutes ischemia by in situ clamping of the vascular pedicle with subsequent reperfusion for 24 hours. Flaps not exposed to ischemia/reperfusion served as controls. Lymph nodes and the perinodal adipose tissue were separately analyzed by Western blot for the expression of lymphangiogenic and angiogenic growth factors. Moreover, morphology, microvessel density, proliferation, apoptosis and immune cell infiltration of VLN flaps were further assessed by histology and immunohistochemistry. Ischemia for 120 minutes was associated with a markedly reduced cellularity of lymph nodes but not of the perinodal adipose tissue. In line with this, ischemic lymph nodes exhibited a significantly lower microvessel density and an increased expression of VEGF-D and VEGF-A. However, VEGF-C expression was not upregulated. In contrast, analyses of the perinodal adipose tissue revealed a more subtle decrease of microvessel density, while only the expression of VEGF-D was increased. Moreover, after 120 minutes ischemia, lymph nodes but not the perinodal adipose tissue exhibited significantly higher numbers of proliferating and apoptotic cells as well as infiltrated macrophages and neutrophilic granulocytes compared with non-ischemic flaps. Taken together, lymph nodes of VLN flaps are highly susceptible to ischemia/reperfusion injury. In contrast, the perinodal adipose tissue is less prone to ischemia/reperfusion injury.