Vascularized lymph node transplantation successfully reverses lymphedema and maintains immunity in a rat lymphedema model.

Vascularized lymph node transplantation (VLNT) has shown inspiring results for the treatment of lymphedema. Nevertheless, it remains unclear how VLNT restores lymphatic drainage and whether or not immunity recovers after surgery. Hindlimb lymphedema model was created using rats with extensive groin and popliteal lymph node removable following with radiotherapy, and the lymphedema was confirmed using indocyanine green (ICG) lymphangiography and micro-computer tomography for volume measurement. VLNT was performed 1 month later. Volume measurement, ICG lymphangiography, histology, and immune reaction were done 1 month after surgery. VLNT successfully reduced the volume of the lymphedema hindlimb, restored lymphatic drainage function with proven lymphatic channel, and reduced lymphedema-related inflammation and fibrosis. It promotes lymphangiogenesis shown from ICG lymphangiography, histology, and enhanced lymphangiogenesis gene expression. Dendritic cell trafficking via the regenerated lymphatic channels was successfully restored, and maintained systemic immune response was proved using dinitrofluorobenzene sensitization and challenge. VLNT effectively reduces lymphedema and promotes lymphatic regeneration in the capillary lymphatic but not the collecting lymphatic vessels. Along with the re-established lymphatic system was the restoration of immune function locally and systemically. This correlated to clinical experience regarding the reduction of swelling and infection episodes after VLNT in lymphedema patients.

Enhancing lymphangiogenesis and lymphatic drainage to vascularized lymph nodes with nanofibrillar collagen scaffolds.

BACKGROUND: This study investigated the effect of nanofibrillar collagen scaffold (BioBridge) implantation from the affected limb to the unaffected contralateral femoral vein or lymph node in a rat model. METHODS: Hind limb lymphedema in Lewis rats was created with lymphadenectomy and inguinal circumcision without radiation. The volumetric difference (greater than 5%) using computed tomography and indocyanine green fluorescence evaluated the progress of lymphedema at 4 weeks. The lymphedema rats have separated into Group I: Controls; Group II: implanted BioBridge to the contralateral femoral vein; and Group III: implanted BioBridge to the contralateral inguinal lymph node. RESULTS: A total of 14 of 30 (46.7%) rats developed hind limb lymphedema with a mean volume difference of 5.83 ± 0.99% and showed diffuse dermal backflow at 4 weeks postlymphadenectomy. Four weeks postimplantation of BioBridge, the mean volumetric difference was 5.62 ± 2.11%, 4.97 ± 0.59%, and -2.47 ± 2.37% in Group I, II, and III, respectively (p < 0.05). The dermal backflow on the affected limb increased in Groups I and II but decreased in Group III. CONCLUSIONS: Implantation of BioBridge from the affected limb to the contralateral inguinal lymph node significantly reduced the hind limb lymphedema at 4 weeks.

Long-term outcomes of arterial ischemia or venous occlusion on vascularized groin lymph nodes in a rat model.

BACKGROUND: This study investigated the long-term effects of arterial ischemia and venous occlusion on lymph node drainage function in a rat model. METHODS: Bilateral groin lymph node flaps of 18 Lewis rats were dissected. The pedicle artery was clamped for 4, 5, and 6 h (A4, A5, and A6 groups), and the vein for 3, 4, and 5 h (V3, V4, and V5 groups) in six flaps. At 4 weeks, the evaluations included gross morphomics, indocyanine green (ICG) lymphography, histological section, immunofluorescence of terminal deoxynucleotidyl transferase assay, and heme oxygenase-1 (HO-1) stain. RESULTS: The lymph node flaps developed shrinkage and partial necrosis in A5, A6, V4, and V5 groups. Hemorrhage in the lymph node cortex and medulla was observed histologically in A5, A6, and V5 groups. ICG lymphography showed loss of lymphatic drainage function in 2 of 6 flaps in A6 and V5 groups. Cell death was shown partly in cortical follicles in A5 and V4 groups and completely in A6 and V5 groups. The HO-1 expression was statistically increased in A5 and V5 groups, respectively (p < 0.05). CONCLUSIONS: The critical arterial ischemia and venous occlusion time were 4 h at 4 weeks of follow-up.

Impacts of arterial ischemia or venous occlusion on vascularized groin lymph nodes in a rat model.

BACKGROUND: Reported ischemia time of vascularized lymph nodes was 5 hours. This study investigated the effects of arterial ischemia and venous occlusion on vascularized lymph node function in rats. METHODS: Bilateral pedicled groin lymph node flaps were raised in 27 Lewis rats. Femoral artery and vein were separated and clamped for 1, 3, 4, or 5 hour(s). Lymph node flap perfusion and drainage were assessed by laser Doppler flowmetry and indocyanine green lymphography. Histologic changes were assessed using hematoxylin and eosin stain, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), and glutathione assays. RESULTS: Perfusion units of 2.84 ± 1.41, 2.46 ± 0.64, 2.42 ± 0.37, and 2.01 ± 0.90 were measured in arterial ischemia groups, and 1.71 ± 0.45, 2.20 ± 0.98, 1.49 ± 0.35, and 0.81 ± 0.20 in venous occlusion groups after 1, 3, 4, and 5 hours of clamping, respectively. Lymphatic drainage showed mean latency periods of 5.33 ± 0.88, 9.00 ± 3.21, 10.00 ± 2.08, and 24.50 ± 11.50 seconds in arterial clamping groups, and 25.00 ± 3.61, 26.00 ± 3.06, 23.33 ± 4.41, and 152.00 ± 0 seconds in venous clamping groups, respectively. Severe medullary and cortical congestion and hemorrhage on histology and cell damage by glutathione levels and TUNEL assay were found after 4 hours of venous clamping. CONCLUSIONS: Arterial ischemia and venous occlusion impact the function and viability of vascularized lymph node flaps differently. The critical venous occlusion time was 4 hours.

Efficacy validation of a lymphatic drainage device for lymphedema drainage in a rat model.

BACKGROUND: Vascularized lymph node transfer (VLNT) is an effective surgery for extremity lymphedema. This study evaluated a lymphatic drainage device (LDD) for the drainage of accumulated fluid into the venous system. METHODS: Micropore filtering membranes with pore sizes of 5, 0.65, and 0.22 µm polyvinylidene difluoride, and 0.8 µm Nylon Net Filter were evaluated to determine the in vitro efficiency of drainage flow of an LDD. The two superior membranes were further used for the evaluation of the inflow and outflow of the LDD in vivo using 5% albumin. RESULTS: At 5 minutes, the volumes drained with 5, 0.65, and 0.22 µm polyvinylidene difluoride and 0.8 µm nylon membranes were 15.2, 2.77, 2.37, and 0.59 mL, respectively (P < .01). At 10 minutes, the collected volumes of 5 and 0.65 µm polyvinylidene difluoride were 1788 and 1051 µL (P = .3). The indocyanine green fluorescence was detected at 50 seconds for the 5 µm polyvinylidene difluoride membrane but not for the 0.65 µm membrane. CONCLUSIONS: The study successfully demonstrated the proof-of-concept of the LDD prototype that mimicked VLNT with drainage of 5% albumin into the venous system in a rat model.

Critical Ischemia Time, Perfusion, and Drainage Function of Vascularized Lymph Nodes.

BACKGROUND: Vascularized lymph node transfer is a promising surgical treatment for lymphedema. This study investigated the effect of ischemia on the lymphatic drainage efficiency of vascularized lymph node flaps and the critical ischemia time of lymph nodes. METHODS: Twenty-four lymph nodes containing groin flaps in 12 Sprague-Dawley rats were dissected. Clamping of the vascular pedicle was performed for 0, 1, 3, 5, 6, or 7 hours; then, each was allowed to reperfuse by means of the vascular pedicle for 1 hour. Perfusion and ischemic changes were assessed using indocyanine green lymphography; laser Doppler flowmetry; and histologic studies with associated lymphatic vessel endothelial hyaluronan receptor-1, CD68, 4',6-diamidino-2-phenylindole, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, and glutathione assay stains. RESULTS: The mean latency period of the groin lymph node flaps was 247 ± 67, 83 ± 15, 72 ± 42, 30 ± 18, and 245 ± 85 seconds in the 0-, 1-, 3-, 5-, and 6-hour groups, respectively. Perfusion detected by laser Doppler was 85.2 ± 14.5, 87.2 ± 36.7, 129.8 ± 33.7, 140.4 ± 148.5, 156.1 ± 91.4, and 41.2 ± 34.8 perfusion units at ischemia times of 0, 1, 3, 5, 6, and 7 hours, respectively. Cell damage measured by glutathione was 46.8 ± 10.2, 67.7 ± 14.2, 62.8 ± 15.4, 126.6 ± 5.9, 259.0 ± 70.3, and 109.1 ± 27.5 at ischemia times of 0, 1, 3, 5, 6, and 7 hours, respectively. Histologically, as ischemia time increased, hemorrhage and congestion became more severe. CONCLUSIONS: The critical ischemia time of vascularized lymph nodes is 5 hours in the rodent animal model, verified by indocyanine green lymphatic fluid uptake, laser Doppler perfusion, and histologic assessments. Interestingly, lymphatic drainage and perfusion of vascularized lymph nodes were improved with an increased ischemia time before the critical 5 hours was reached.

Periosteal Osteogenic Capacity Depends on Tissue Source.

Periosteal osteogenic capacity can be exploited to enhance bone formation in the fields of tissue engineering and regenerative medicine. Despite this importance, there have been no studies examining the composition, structure, and osteogenic capacity of periostea from different bone sources. In this study, structure and osteogenic factor content were compared among periostea from rib, calvarial, femoral, and tibial bones, in which the native bones of these four regions were harvested and subjected to histological analysis. The osteogenic capacity of grafted periosteum was evaluated using an in vivo vascularized pedicle model of bone tissue engineering. Poly(ethylene glycol)-poly(l-lactic acid) (PEG-PLLA) copolymer hydrogels were seeded with bone marrow mesenchymal stem cells and implanted with grafted periosteum harvested from either calvarial or tibial bone, which were representative of thin and thick native periostea, respectively. The cambium layer thickness of periostea from the femoral and tibial bones (36.9% ± 2.5% and 36.8% ± 2.6%) was greater than that from the calvarial and rib bones (26.8% ± 2.4% and 25.5% ± 1.9%). The osteocalcin and alkaline phosphatase levels were comparatively higher in the femoral and tibial periostea than those in periostea harvested from the calvarial and rib bones. The construct implanted with grafted tibial periosteum resulted in greater neo-bone regeneration and higher osteocalcin and alkaline phosphatase expression. This study is the first investigation of the osteogenic capacity of periostea from diverse sources. The results can be used to guide clinical strategies that exploit periostea for tissue engineering and clinical applications.

Promoting chondrocyte cell clustering through tuning of a poly(ethylene glycol)-poly(peptide) thermosensitive hydrogel with distinctive microarchitecture.

Hydrogels are considered to be attractive cell-matrix for chondrocytes due to their similarity in properties to the natural cartilage. However, the formation of chondrocyte cell clusters in hydrogels has been mostly limited to naturally-derived or relatively fast degrading materials. In this study, a series of diblock copolymer poly(ethylene glycol)-poly(alanine) (mPEG-PA) was synthesized and investigated as injectable biomimic hydrogels for the culturing of chondrocytes. Depending on the poly(alanine) chain length, afforded hydrogels exhibited variable mechanical property and microarchitecture due to difference in secondary structure arrangement. After 21days of culture, cell clusters were observed in all hydrogels with longer PA chains and these hydrogels supported more homogenous and established clustering as well as significantly higher glycosaminoglycan and collagen deposition. Interestingly, scanning electron microscopy revealed a distinct micron range fibrillar-like microarchitecture that may be responsible for maintaining chondrocyte phenotype and matrix production. In addition, micrographs revealed the presence of collagen fibrils and an extensive extracellular matrix network. Therefore, it is reasonable to conclude that mPEG-PA hydrogels possess the desirable properties for chondrocyte cluster formation and serve as potential candidate in cartilage tissue engineering.

Proposed pathway and mechanism of vascularized lymph node flaps.

OBJECTIVE: To investigate the pump mechanism and pathway of lymph transit in vascularized lymph node flaps. BACKGROUND: Microsurgical treatment of lymphedema with vascularized lymph node transfer can improve signs and symptoms of disease, but the pathways and mechanisms of these flaps warrant further exploration. METHODS: (Animal model) 72 flaps were raised in 18 rats: 36 groin flaps contained lymph nodes (LN), 36 deep inferior epigastric artery perforator flaps did not (non-LN). Indocyanine green (ICG) was added into normal saline (NS), 1%, 3%, 5%, 7% and 10% albumin. Three rats were assigned to each group. LN and non-LN flaps were submerged in solution and surveyed for venous fluorescence. In the 7% albumin and NS groups, volumetric change of solution was measured. (Human model) A similar experiment was performed in humans using five submental LN flaps. RESULTS: (Animal model) Fluorescence was detected in the venous pedicle of LN flaps submerged in 5%, 7% and 10% albumin, and half of flaps submerged in 3% albumin. Fluorescence was not detected in LN node flaps submerged in ICG-containing NS or 1% albumin solution. Fluorescence was not detected in non-LN flaps. There was greater volume reduction with LN flaps than non-LN flaps (p<0.001). (Human model) Fluorescence was detected in the venous pedicle of all flaps immersed in lymph. CONCLUSIONS: ICG fluorescence was detected in the venous pedicle of rat and human LN flaps submerged in lymph or albumin when the concentration was greater than 3%. Based on these results, a pathway for lymphatic uptake is presented.

Quantity of lymph nodes correlates with improvement in lymphatic drainage in treatment of hind limb lymphedema with lymph node flap transfer in rats.

PURPOSE: This study was conducted to investigate the correlation between the number of vascularized lymph nodes (LN) transferred and resolution of hind limb lymphedema in a rat model. METHODS: Unilateral hind limb lymphedema was created in 18 male Sprague-Dawley rats following inguinal and popliteal LN resection and radiation. A para-aortic LN flap based on the celiac artery was subsequently transferred to the affected groin. The three study groups consisted of Group A (no LN transfer), Group B (transfer of a single vascularized LN), and Group C (transfer of three vascularized LNs). Volumetric analysis of bilateral hind limbs was performed using micro-CT imaging at 1, 2, and 3 months postoperatively. Lymphatic drainage was assessed with Tc(99) lymphoscintigraphy preoperatively and at 3 months postoperatively. RESULTS: A statistically significant volume reduction was seen in Groups B and C compared to Group A at all time points. Volume reduction of Group A vs.Group B at 1 month (8.6% ± 2.0% vs. 2.7% ± 2.6%, P < 0.05), 2 months (9.3% ± 2.2% vs. -4.3% ± 2.7%, P < 0.05), and 3 months (7.6% ± 3.3% vs. -8.9% ± 5.2%, P < 0.05). Volume reduction of Group A vs. Group C at 1 month (8.6% ± 2.0% vs. -6.6% ± 3.1%, P < 0.05), 2 months (9.3% ± 2.2% vs. -10.2% ± 4.6%, P < 0.05), and 3 months (7.6% ± 3.3% vs. -9.1% ± 3.1%, P < 0.05). Of note, comparison of Groups B and C demonstrated greater volume reduction in Group C at 1 (P < 0.02) and 2 (P = 0.07) months postoperatively. CONCLUSIONS: LN flap transfer is an effective procedure for the treatment of lymphedema. The number of vascularized LNs transferred correlates positively with the degree of volume reduction.

Developing a Lower Limb Lymphedema Animal Model with Combined Lymphadenectomy and Low-dose Radiation.

BACKGROUND: This study was aimed to establish a consistent lower limb lymphedema animal model for further investigation of the mechanism and treatment of lymphedema. METHODS: Lymphedema in the lower extremity was created by removing unilateral inguinal lymph nodes followed by 20, 30, and 40 Gy (groups IA, IB, and IC, respectively) radiation or by removing both inguinal lymph nodes and popliteal lymph nodes followed by 20 Gy (group II) radiation in Sprague-Dawley rats (350-400 g). Tc(99) lymphoscintigraphy was used to monitor lymphatic flow patterns. Volume differentiation was assessed by microcomputed tomography and defined as the percentage change of the lesioned limb compared to the healthy limb. RESULTS: At 4 weeks postoperatively, 0% in group IA (n = 3), 37.5% in group IB (n = 16), and 50% in group IC (n = 26) developed lymphedema in the lower limb with total mortality and morbidity rate of 0%, 56.3%, and 50%, respectively. As a result of the high morbidity and mortality rates, 20 Gy was selected, and the success rate for development of lymphedema in the lower limb in group II was 81.5% (n = 27). The mean volume differentiation of the lymphedematous limb compared to the health limb was 7.76% ± 1.94% in group II, which was statistically significant compared to group I (P < 0.01). CONCLUSIONS: Removal of both inguinal and popliteal lymph nodes followed by radiation of 20 Gy can successfully develop lymphedema in the lower limb with minimal morbidity in 4 months.

The mechanism of vascularized lymph node transfer for lymphedema: natural lymphaticovenous drainage.

BACKGROUND: Vascularized lymph node flap transfer for the treatment of upper and lower limb lymphedema has had promising results. This study was performed to investigate the mechanism of lymph drainage of a vascularized lymph node flap both experimentally and clinically. METHODS: In the experimental study, 18 Sprague-Dawley rats were used to create 36 flaps, either a groin lymph node flap or an abdominal cutaneous flap that did not contain lymph nodes. Indocyanine green dye was injected into the edge of 12 lymph node flaps, directly into a lymph node of 12 lymph node flaps, and into the edge of 12 cutaneous flaps. In the clinical study, an identical study design was used, with 24 vascularized lymph node flaps and 12 cutaneous flaps not containing lymph nodes. RESULTS: Experimentally, fluorescence was detected in the pedicle vein after a mean latency period of 153 ± 129 seconds when the edge of the lymph node flap was injected and 12.8 ± 8.1 seconds when the lymph node was directly injected. Fluorescence was not detected in the pedicle vein of the cutaneous flaps (p < 0.01). Clinically, fluorescence was detected in the pedicle vein after a mean latency period of 346 ± 249 seconds when the edge of the lymph node flap was injected and 23.5 ± 27.1 seconds when the lymph node was directly injected. Fluorescence was not detected in the pedicle vein of the cutaneous flaps (p < 0.01). CONCLUSION: The vascularized lymph node flap drains lymph into the pedicle vein, both experimentally and clinically. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Profiling lipid-protein interactions using nonquenched fluorescent liposomal nanovesicles and proteome microarrays.

Fluorescent liposomal nanovesicles (liposomes) are commonly used for lipid research and/or signal enhancement. However, the problem of self-quenching with conventional fluorescent liposomes limits their applications because these liposomes must be lysed to detect the fluorescent signals. Here, we developed a nonquenched fluorescent (NQF)1 liposome by optimizing the proportion of sulforhodamine B (SRB) encapsulant and lissamine rhodamine B-dipalmitoyl phosphatidylethanol (LRB-DPPE) on a liposomal surface for signal amplification. Our study showed that 0.3% of LRB-DPPE with 200 µm of SRB provided the maximal fluorescent signal without the need to lyse the liposomes. We also observed that the NQF liposomes largely eliminated self-quenching effects and produced greatly enhanced signals than SRB-only liposomes by 5.3-fold. To show their application in proteomics research, we constructed NQF liposomes that contained phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and profiled its protein interactome using a yeast proteome microarray. Our profiling led to the identification of 162 PI(3,5)P2-specific binding proteins (PI(3,5)P2-BPs). We not only recovered many proteins that possessed known PI(3,5)P2-binding domains, but we also found two unknown Pfam domains (Pfam-B_8509 and Pfam-B_10446) that were enriched in our dataset. The validation of many newly discovered PI(3,5)P2-BPs was performed using a bead-based affinity assay. Further bioinformatics analyses revealed that the functional roles of 22 PI(3,5)P2-BPs were similar to those associated with PI(3,5)P2, including vesicle-mediated transport, GTPase, cytoskeleton, and kinase. Among the 162 PI(3,5)P2-BPs, we found a novel motif, HRDIKP[ES]NJLL that showed statistical significance. A docking simulation showed that PI(3,5)P2 interacted primarily with lysine or arginine side chains of the newly identified PI(3,5)P2-binding kinases. Our study showed that this new tool would greatly benefit profiling lipid-protein interactions in high-throughput studies.

Elesclomol induces cancer cell apoptosis through oxidative stress.

Elesclomol (formerly STA-4783) is a novel small molecule undergoing clinical evaluation in a pivotal phase III melanoma trial (SYMMETRY). In a phase II randomized, double-blinded, controlled, multi-center trial in 81 patients with stage IV metastatic melanoma, treatment with elesclomol plus paclitaxel showed a statistically significant doubling of progression-free survival time compared with treatment with paclitaxel alone. Although elesclomol displays significant therapeutic activity in the clinic, the mechanism underlying its anticancer activity has not been defined previously. Here, we show that elesclomol induces apoptosis in cancer cells through the induction of oxidative stress. Treatment of cancer cells in vitro with elesclomol resulted in the rapid generation of reactive oxygen species (ROS) and the induction of a transcriptional gene profile characteristic of an oxidative stress response. Inhibition of oxidative stress by the antioxidant N-acetylcysteine blocked the induction of gene transcription by elesclomol. In addition, N-acetylcysteine blocked drug-induced apoptosis, indicating that ROS generation is the primary mechanism responsible for the proapoptotic activity of elesclomol. Excessive ROS production and elevated levels of oxidative stress are critical biochemical alterations that contribute to cancer cell growth. Thus, the induction of oxidative stress by elesclomol exploits this unique characteristic of cancer cells by increasing ROS levels beyond a threshold that triggers cell death.