Therapeutic Potential of the Prolyl Hydroxylase Inhibitor Roxadustat in a Mouse Hindlimb Lymphedema Model.

Background: Lymphedema is an intractable disease with no curative treatment available. Conservative treatment is the mainstay, and new drug treatment options are strongly needed. The purpose of this study was to investigate the effect of roxadustat, a prolyl-4-hydroxylase inhibitor, on lymphangiogenesis and its therapeutic effect on lymphedema in a radiation-free mouse hindlimb lymphedema model. Methods and Results: Male C57BL/6N mice (8-10 weeks old) were used for the lymphedema model. Mice were randomized to an experimental group receiving roxadustat or a control group. The circumferential ratio of the hindlimbs was evaluated, and lymphatic flow of the hindlimbs was compared by fluorescent lymphography up to 28 days postoperatively. The roxadustat group showed an early improvement in hindlimb circumference and stasis of lymphatic flow. The number and area of lymphatic vessels on postoperative day 7 were significantly larger and smaller, respectively, in the roxadustat group compared with the control group. Skin thickness and macrophage infiltration on postoperative day 7 were significantly reduced in the roxadustat group compared with the control group. The relative mRNA expression of hypoxia-inducible factor-1α (Hif-1α), vascular endothelial growth factor receptor-3 (VEGFR-3), vascular endothelial growth factor-C (VEGF-C), and Prospero homeobox 1 (Prox1) on postoperative day 4 was significantly higher in the roxadustat group compared with the control group. Conclusions: Roxadustat demonstrated a therapeutic effect in a murine model of hindlimb lymphedema through promotion of lymphangiogenesis through the activation of HIF-1α, VEGF-C, VEGFR-3, and Prox1, suggesting the potential of roxadustat as a therapeutic option in lymphedema.

Sentinel node restoration by vascularized lymph node transfer in mice.

BACKGROUND: Recent reports have indicated that vascularized lymph node transfer (VLNT) may improve the impaired immunity in lymphedema but there has been no report concerning anti-cancer immunity. In the early tumor immune response, dendritic cells (DCs) participate in tumor recognition and antigen presentation in local lymphatics. Here, we investigated the impact of VLNT on DC dynamics against cancer in mouse models. METHODS: Forty-seven 8-week-old C57BL/6 N male mice were divided into three surgical groups: a VLNT model in which a vascularized inguinal lymph node (LN) flap was transferred into the ipsilateral fossa after a popliteal LN was removed; a LN dissection (LND) model in which the popliteal LN was dissected; and a control model in which a skin incision was made at the popliteal fossa and an ipsilateral inguinal LN was removed. Postoperative lymphatic flows were observed by indocyanine green lymphography and B16-F10-luc2 mouse melanoma were implanted into the ipsilateral footpad. The proportion of DCs in the transplanted nodes was measured by CD11c immunohistochemistry using digital imaging analysis 4 days after cancer implantation. Metastases to the lungs and LNs were quantitatively evaluated by luciferase assay 4 weeks after cancer implantation. RESULTS: After VLNT, lymphatic reconnection was observed in 59.2% of mice. The proportion of DCs was significantly higher in the VLNT group with lymphatic reconnection (8.6% ± 1.0%) than in the naïve LN (4.3% ± 0.4%) (p < .001). The tumor burden of lung metastases was significantly less in the VLNT group with lymphatic reconnection compared with the LND group (p = .049). CONCLUSIONS: Metastasis decreased in mice with reconnected lymphatics after VLNT. A possible explanation was that lymphatic restoration may have contributed to the tumor immune response by allowing DC migration to LNs.

Novel Surgical Technique for Correction of Incomplete Median Cleft Lip Deformity in Oral-Facial-Digital Syndrome Type II.

ABSTRACT: Oral-facial-digital syndromes (OFDSs) represent a heterogenous group of embryonic development disorders characterized by malformations of the face, oral cavity, and extremities. Oral-facial-digital syndrome type II is an autosomal recessive disease characterized by median cleft lip, gingival frenula, cleft lobulated tongue, and polydactyly. There are few reports on surgical techniques for correction of incomplete median cleft lip. Here we describe a novel surgical method that we used to correct an incomplete median cleft lip in a 5-year-old girl with oral-facial-digital syndrome type II. She had previously undergone surgery for congenital heart disease, oral anomalies, and polydactyly. Cheiloplasty was performed at 5 years and 8 months using a surgical approach that focused on repair of the median tubercle using lateral labial elements. A reasonably good Cupid's bow and median tubercle were achieved. Our technique for surgical correction of moderate incomplete median cleft lip provides adequate philtral height, vermillion fullness, and a good-shaped Cupid's bow.

Topical Application of Conditioned Medium from Hypoxically Cultured Amnion-Derived Mesenchymal Stem Cells Promotes Wound Healing in Diabetic Mice.

BACKGROUND: Mesenchymal stem cells or their conditioned medium improve chronic wound healing, and their effect is enhanced by hypoxia. Diabetic foot ulcers are chronic wounds characterized by abnormal and delayed healing, which frequently require amputation. The authors evaluated the effect of topical application of conditioned medium from hypoxically cultured amnion-derived mesenchymal stem cells on wound healing in diabetic mice. METHODS: Amnion-derived mesenchymal stem cells were cultured under 21% oxygen to prepare normoxic conditioned medium and under 1% oxygen to prepare hypoxic conditioned medium. Hydrogels containing standard medium, normoxic conditioned medium, or hypoxic conditioned medium were topically applied to excisional wounds of mice with streptozotocin-induced diabetes. Ulcer tissues were harvested on day 9; immunohistochemical and quantitative polymerase chain reaction analyses were performed to analyze angiogenesis, inflammatory cell infiltration, and expression levels of inflammation-related genes. RESULTS: Hypoxic conditioned medium significantly enhanced wound closure, increased capillary density and epithelization, and reduced macrophage infiltration. It also tended to reduce the infiltration of neutrophils and enhance the infiltration of regulatory T cells; it showed a tendency to downregulate the expression of the inflammation-related genes interleukin-1ß, interleukin-6, chemokine ligand 1, and chemokine ligand 2. Normoxic conditioned medium exhibited similar effects, although they were of lesser magnitude than those of hypoxic conditioned medium. CONCLUSIONS: Hydrogels containing hypoxically cultured, amnion-derived mesenchymal stem cell conditioned medium accelerated wound healing in diabetic mice by enhancing angiogenesis, accelerating epithelization, and suppressing inflammation. Therefore, topical application of amnion mesenchymal stem cell-derived hypoxic conditioned medium could be a novel treatment for diabetic foot ulcers.

Combined internal and external negative pressure wound therapy: breakthrough treatment for lymphocutaneous intractable fistula.

PURPOSE: Lymphocutaneous fistula after lymph node dissection is intractable, yet there is no established treatment strategy. This study demonstrates the wound closure time achieved by a new method of combined internal and external negative pressure wound therapy (CIEN) in patients with lymphocutaneous fistula. METHODS: The subjects of this study were six consecutive patients with lymphocutaneous fistula after lymphatic surgery, who were treated with CIEN between 2018 and 2020. The CIEN technique can be summarized as follows: first, internal foam is inserted into the fistula from the opening of the fenestration. Next, a slightly larger area of external foam is applied above the fistula flap outside the external margin of the foam-filled fistula. After bridging the internal foam and external foam, negative-pressure wound therapy is carried out on this bridging foam block. RESULTS: CIEN led to rapid and complete wound healing in all six patients. Fistula flap margin ischemia developed in one patient, but adjusting the mode and pressure settings resulted in improvement. Three patients suffered contact dermatitis. There were no signs of tumor or fistula recurrence in any patients after at least 3 months of follow-up. CONCLUSION: CIEN is an effective and less invasive treatment modality than the conventional method of managing lymphocutaneous fistula.

A Comparison of Outcomes After Palatoplasty in Patients With Non-Syndromic Pierre Robin Sequence Versus Patients With Non-Syndromic Isolated Cleft Palate.

The purpose of this retrospective study was to compare outcomes of cleft palate repair in patients with non-syndromic Pierre Robin sequence (NS-PRS) versus those with non-syndromic isolated cleft palate (NS-ICP). Pierre Robin sequence (PRS) was defined as a diagnosis of the triad of microretrognathia, glossoptosis, and cleft palate, and the severity of PRS was assessed based on the presence of respiratory and feeding problems. All patients underwent palatoplasty between January 2000 and December 2011. The authors examined age at palatoplasty, type of cleft palate, fistula rate, velopharyngeal (VP) function, nasal emission, hypernasality, and need for secondary speech surgery, in addition to PRS severity in the NS-PRS patients. A total of 15 NS-PRS patients and 40 NS-ICP patients were reviewed. The incidence of VP dysfunction, nasal emission, hypernasality, and secondary speech surgery was not significantly different between the NS-PRS patients and NS-ICP patients. Age at palatoplasty was significantly different between the 2 groups (P = 0.012) but type of CP was not (P = 1.00). Only 2 NS-PRS patients were classified as category III (severe), and all of the NS-PRS patients who had VP insufficiency were classified as PRS severity category I (not severe). The findings of this study indicate that NS-PRS patients may not have worse outcomes than NS-ICP patients.

Ulcerated Infantile Hemangioma of the Hard Palate: Diagnostic Treatment With Oral Propranolol.

Infantile hemangiomas arising in the palate are rare. The authors describe a case of ulcerated infantile hemangioma of the hard palate with feeding difficulty. To our knowledge, this is the first reported case of immunohistochemically diagnosed palatal infantile hemangioma successfully treated using oral propranolol.

Oculo-Auriculo-Fronto-Nasal Syndrome With Duane Retraction Syndrome and Dysplastic Bony Structure in the Midline of Nose.

Oculo-auriculo-fronto-nasal syndrome (OAFNS) is a rare anomaly characterized by features overlapping those of frontonasal dysplasia (FND) and the oculo-auriculo-vertebral spectrum (OAVS). The FND features malformation of frontonasal process-derived structures, characterized by anomalies in the central portion of the face. The OAVS is characterized by developmental anomalies of the first and second pharyngeal arches. The OAFNS is a condition with clinical features of both FND and OAVS.Here, the authors present the case of a male with OAFNS who not only exhibited typical OAFNS symptoms but also a dysplastic bony structure that bridged the anterior nasal spine and inferior nasal bones, and unilateral type 3 Duane retraction syndrome (absence of right-eye abduction). Abnormal nasal bones are characteristic of OAFNS; such abnormalities are absent from FND and OAVS. The authors reduced the dysplastic nasal bony structure via open external rhinoplasty, followed by lateral nasal osteotomy when he was 16 years of age. The nasal dorsum appeared natural after surgery and he was satisfied with the result.

Advancement Flap Using Excess Skin for Upper Eyelid Full-Thickness Defects.

Reconstruction of upper eyelid defects should aim for a moveable lid with perfect corneal protection and good aesthetic quality. Numerous procedures to reconstruct large upper eyelid defects have been reported, but these methods require 2-stage procedures. A new method for reconstructing full-thickness upper eyelid defects after tumor excision in a single stage was presented in this study. The preferred technique uses excess skin as an advancement flap together with an ear cartilage graft for the lining. Reconstruction was performed with an advancement flap using excess skin and ear cartilage for full-thickness defects after upper eyelid tumor excision. The rectangular flap was outlined on the excess skin of the upper eyelid. After tumor resection of the full-thickness defect, the ear cartilage was sutured to the remaining tarsus. The residual levator aponeurosis and posterior lamellar were connected to the transplanted ear cartilage. The outer layer was reconstructed with an advancement rectangular flap. The authors performed this technique for 4 patients, aged 62 to 88 years, for upper eyelid reconstruction. Good functional and aesthetic results were achieved for all patients. Our method involves a single-stage reconstruction, which is simpler and less invasive than other techniques.

Changes in high endothelial venules in lymph nodes after vascularized and nonvascularized lymph node transfer in a murine autograft model.

BACKGROUND AND OBJECTIVES: Vascularized lymph node transfer (LNT) is gaining popularity in the treatment of lymphedema. However, it is unclear whether the vascularization of transferred lymph nodes (LNs) contributes to functional improvement. High endothelial venules (HEVs) are specialized vessels that allow lymphocytes to enter LNs. In this study, we compared the numbers of HEVs and lymphocytes in LNs after vascularized and nonvascularized LNT. METHODS: Fifty mice were divided into three groups (group 1, pedicled vascularized LNT; group 2, pedicled nonvascularized LNT; group 3, free nonvascularized LNT). Afferent lymphatic reconnection was confirmed by patent blue staining. The transferred LNs were harvested 4 weeks after surgery. HEVs, B-cells, and T-cells were subjected to immunohistochemical staining and quantified. RESULTS: Afferent lymphatic reconnection was observed in 13 of 20 transferred LNs in group 1, 11 of 15 in group 2, and 7 of 15 in group 3. The ratio of dilated/total HEVs in transferred LNs with afferent lymphatic reconnection was significantly higher in group 1 than in groups 2 and 3. No significant differences in numbers of B-cells and T-cells were found in the transferred LNs. CONCLUSIONS: We found that more functional HEVs were preserved in cases with successful afferent lymphatic reconnection after vascularized LNT than after nonvascularized LNT.

Feasibility of pedicled vascularized inguinal lymph node transfer in a mouse model: A preliminary study.

PURPOSE: Vascularized lymph node transfer is becoming more common in the treatment of lymphedema, but suitable small animal models for research are lacking. Here, we evaluated the feasibility of pedicled vascularized inguinal lymph node transfer in mice. METHODS: Twenty-five mice were used in the study. An inguinal lymph node-bearing flap with a vascular pedicle containing the superficial caudal epigastric vessels was transferred into the ipsilateral popliteal fossa after excision of the popliteal lymph node. Indocyanine green (ICG) angiography was used to confirm vascularity of the flap. ICG lymphography was performed to evaluate lymphatic flow at 3 and 4 weeks postoperatively. Patent blue dye was injected into the ipsilateral hind paw to observe staining of the transferred lymph node at 4 weeks postoperatively. All transferred lymph nodes were then harvested and histologically evaluated by hematoxylin and eosin staining. RESULTS: In 16 of the 25 mice, ICG lymphography showed reconnection between the transferred lymph node and the afferent lymphatic vessels, as confirmed by patent blue staining. Histologically, these transferred lymph nodes with afferent lymphatic reconnection significantly regressed in size (0.37 ± 0.24 mm2 ) and showed clear follicle formation, whereas those without afferent lymphatic reconnection showed less size regression (1.31 ± 1.17 mm2 ); the cell population was too dense to allow identification of follicles. CONCLUSIONS: We established a mouse model of vascularized lymph node transfer with predictable afferent lymphatic reconnection. Both the vascularization and reconnection might be necessary for functional regeneration of the transferred lymph node.

Comparison of Nasal Symmetry Between Presurgical Nasal Stenting and Postsurgical Nasal Retainer Placement in Unilateral Clefts.

The timing of cleft lip nose surgery remains controversial. The less invasive the procedure at the time of primary cleft lip repair, the less the growth and development of the nose is affected. Therefore, the nasal-stenting component of presurgical nasoalveolar molding has increasingly been used. However, not all cleft centers use such treatment. Conventional postsurgical placement of silicone nasal retainers remains popular. No report has yet compared presurgical nasal stenting (preNS) and postsurgical nasal retainer placement (postNR). In this study, postoperative nasal form outcomes after primary lip repair using preNS or postNR in patients with complete unilateral cleft lips, alveoli, and palates were compared. Patients in whom preNS alone was used for 6 months (group I) were compared with those receiving postNR (no preNS) for 6 months after primary nasal cartilage dissection (group II) and controls with no appliance (group III). Nasal anthropometric distances and angular relationships were measured photographically to assess nasal symmetry at 4 years of age in all groups. Compared to group III, groups I and II exhibited significantly greater nostril heights (P = 0.0075, P = 0.0015 respectively) and columellar deviation angles (P = 0.0020, P = 0.0221). Groups I and II did not differ significantly. No significant between-treatment difference in terms of nasal symmetry between preNS and postNR was observed. However, both treatments afforded significantly better results than no treatment. Since older infants tend to resist the placement of nasal devices, preNS is more feasible in this age group.

Immune-mediated antitumor effect of a transplanted lymph node.

Lymph node (LN) transplantation is a recognized method for reconstruction of the lymphatic system and is used in the clinical setting to treat lymphedema. However, it is unclear whether transplanted LNs contribute to immune surveillance. In our study, we investigated whether a single transplanted non-vascularized LN, defined as a tumor-draining transplanted lymph node (TDTLN), could exert an immune-mediated antitumor effect. LN and lung metastases and primary tumor enlargement were evaluated in mice that were inoculated with B16-F10-luc2 melanoma cells in a hind limb footpad without (group 1) and with (group 2) popliteal lymph node (PLN) resection and in mice that underwent LN transplantation after PLN resection (group 3). The function of a TDTLN (group 3) and a tumor-draining popliteal lymph node (TDPLN; group 1) was evaluated in the context of cancer. LN and lung metastases were significantly aggravated by PLN resection but were significantly decreased by LN transplantation. Immunohistochemistry showed that the TDTLNs retained T-cells and B-cells and fluorescence-activated cell sorting analysis confirmed expansion of lymphocytes in these nodes; however, the degree of expansion in TDTLNs was different from that in TDPLNs. Expression of cytokines associated with immunostimulation was confirmed in the TDTLNs as well as in the TDPLNs. One of the differences in the immune-mediated antitumor effect of the TDPLNs and TDTLNs was ascribed to a difference in the site of lymphocyte homing to peripheral LNs through high endothelial venules. Non-vascularized LN transplantation had an immune-mediated antitumor effect.

Alternative Treatment for Residual Aortobronchial Fistula After Thoracic Endovascular Aortic Repair.

A patient with an aortobronchial fistula secondary to a thoracic endovascular aortic repair was successfully treated with a re-thoracic endovascular aortic repair with debranching technique. Five months postoperatively, the aneurysm had shrunk; however, computed tomography revealed air in the thrombo-excluded aortic aneurysmal sac without signs of infection. Because of worsening air finding at the eighth month, we performed a resection of the residual fistula and wrapped the stent graft in a pedicled muscle flap.

Microsurgical lymphaticovenous implantation targeting dermal lymphatic backflow using indocyanine green fluorescence lymphography in the treatment of postmastectomy lymphedema.

BACKGROUND: Microsurgical lymphaticovenous implantation in lymphedema is done to create a lymphaticovenous shunt by an implantation of collecting lymphatics into the small vein, as reported previously. The authors have recently introduced ultrasonograpy and indocyanine green fluorescence lymphography into this procedure. METHODS: Nine cases of postmastectomy lymphedema had received preoperative venous marking using ultrasonography and lymphatic mapping using indocyanine green fluorescence lymphography. The concept of modification is to pick up the most effective point for microsurgical lymphaticovenous implantation that involves both subcutaneous veins and the dermal backflow of excess lymphatics. Objective improvement was analyzed by the percent reduction of edema circumference at two points of the affected forearm. RESULTS: Preoperative lymphography showed a spotty image for dermal backflow in all nine extremities, a linear image on the dorsal hand in six extremities, and a linear image on the forearm in three extremities. With an average follow-up of 17 months, three patients had excellent results with the reduction of edema circumference more than 50 percent for both the distal and proximal sites of the treated forearm. Four patients had good results with the reduction of edema circumference more than 50 percent at the distal or proximal sites, two patients had fair results, and no patients had poor results. The average number of modified microsurgical lymphaticovenous implantations was 3.7 per case. CONCLUSION: Modified microsurgical lymphaticovenous implantation is expected to provide favorable results with a minimum number of these modified implantations, even though no linear lymph channel was detected by preoperative indocyanine green fluorescence lymphography.