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Browsing by Author "Jorge, Miguel D."
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Item 153. Quantification of Lymphangiogenesis in Murine Lymphedema Tail Model Using Intravital Microscopy(Wolters Kluwer, 2023-05-19) Mohan, Ganesh; Khan, Imran; Diaz, Stephanie M.; Neumann, Colby R.; Jorge, Miguel D.; Sinha, Mithun; Gordillo, Gayle M.; Sen, Chandan K.; Hassanein, Aladdin H.; Surgery, School of MedicinePURPOSE: Lymphedema is limb swelling caused by lymphatic dysfunction. It occurs in 30% of patients that undergo axillary lymph node dissection in the treatment of breast cancer. There is no cure for this disease. Understanding the mechanisms of lymphatic growth will play a pivotal role in developing therapeutic strategies against these conditions. Visualization of lymphangiogenesis and functional assessment remains a challenge. Intravital two-photon microscopy (IVM) is a powerful imaging tool for investigating various biological processes in live animals. Tissue nanotransfection technology (TNT) facilitates a direct, transcutaneous non-viral vector gene delivery using a chip with nanochannel poration in a rapid (<100ms) focused electric field. TNT was used in this study to deliver the genetic cargo in the murine tail lymphedema to assess the lymphangiogenesis. The purpose of this study is to experimentally evaluate the applicability of IVM to visualize and quantify lymphatics. METHODS: The murine tail model of lymphedema was utilized. A 3 mm full thickness skin excision and lymphatic vessel disruption was performed 20 mm from the base of the tail in twelve C57BL/6 mice. TNT was applied to the murine tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: Group I (control) was given pCMV6 (expression vector backbone alone) (n=6); Group II had pCMV6-Prox1 (n=6). Post-TNT (day 10), a 3 cm segment of murine tail was deskinned distal to the site of occlusion to optimize visualization. FITC-Dextran (2000 kD) injected intradermally at the distal tail region for lymphatic uptake. Lymphatic vessels are visualized at the second skin excision site with the Leica SP8 Confocal/Multiphoton Microscope and assessed for number of branching points to determine the newly formed lymphatics. Lymphatic vessel density was also observed by immunostaining with anti-Podoplanin antibody. RESULTS: The experimental group II exhibited increased branching points (3-fold) using filamentation analysis compared to control group I at the site of TNT treatment (n=6, p<0.05). Increased lymphatic vessel density was also observed with Podoplanin immunostaining post-TNT application. Intensity quantification of immunohistochemistry revealed greater expression of Podoplanin in Group II when compared to Group I (n=6, p<0.05). CONCLUSION: This study demonstrates a novel, powerful imaging tool for investigating lymphatic vessels in live murine tail model of lymphedema. Intravital microscopy can be utilized for functional assessment of lymphatics and visualization of lymphangiogenesis following gene-based therapy.Item 80. Lymphatic Preconditioning: Novel Investigation Of A “Lymphatic Delay Phenomenon”(Wolters Kluwer, 2024-04-19) Hulsman, Luci; Mohan, Ganesh; Ahmed, Shahnur; Jorge, Miguel D.; Sullivan, Steven J.; Mohammed, Imran; Sinha, Mithun; Hassanein, Aladdin H.; Surgery, School of MedicineBackground: The vascular delay phenomenon is a well-described concept of flap physiology with many clinical applications used to increase flap viability. The approach employs a staged surgical procedure with selective partial disruption of the flap’s blood supply to increase the robustness of the remaining blood supply, followed by interval flap transfer 7-10 days later. While this vascular delay phenomenon has been thoroughly studied, no investigation has been performed to determine if a similar “delay phenomenon” exists for lymphatic vessels. Lymphedema frequently occurs following injury of lymphatics during lymph node dissection. The purpose of this study was to evaluate whether lymphatic preconditioning with staged disruption of lymphatics can be protective against lymphedema. Methods: The standard murine tail lymphedema model was utilized which involves creating a 3 mm circumferential skin excision by the base of the tail and surgically clipping two lymphatics. This standard model was used for a control (Group 1, Control A, n=6). A second control (Group 2, Control B, n=5) had 3 mm circumferential skin excision, one tail lymphatic clipped at that level, and an immediate second hemi-circumferential skin excision on the more proximal tail with disruption of the other remaining lymphatic. Group 3 (experimental lymphatic preconditioning, n=6), had a 3 mm full thickness skin excision by the base of the tail, disruption of one tail lymphatic at that level (leaving one lymphatic vessel intact) followed by staged hemi-circumferential skin excision with disruption of the remaining lymphatic vessel 7 days later. Tail volume was assessed with tail measurements using the truncated cone equation. Immunohistochemistry and histology was sent. Results: Group 3 (experimental lymphatic preconditioning) had a change in tail volume of 79.1 mm3 compared to Group 1 (Control A) 154.6 mm3 (p=0.03) and Group 2 (Control B) 126.6 mm3 (p=0.05) at 28 days post-lymphatic injury. Conclusion: Mice that underwent lymphatic preconditioning with partial lymphatic injury followed by staged completion of lymphatic disruption 7 days later exhibited less tail swelling. This study demonstrates evidence for a novel concept of “lymphatic delay phenomenon” parallel to the well-known vascular delay phenomenon. Lymphatic preconditioning has potential translational clinical applications for protective effects to minimize lymphatic dysfunction.Item Topical tissue nanotransfection of Prox1 is effective in the prophylactic management of lymphedema(Elsevier, 2024-01-18) Mohan, Ganesh; Khan, Imran; Neumann, Colby R.; Jorge, Miguel D.; Ahmed, Shahnur; Hulsman, Luci; Sinha, Mithun; Gordillo, Gayle M.; Sen, Chandan K.; Hassanein, Aladdin H.; Surgery, School of MedicineLymphedema is chronic limb swelling resulting from lymphatic dysfunction. There is no cure for the disease. Clinically, a preventive surgical approach called immediate lymphatic reconstruction (ILR) has gained traction. Experimental gene-based therapeutic approaches (e.g., using viral vectors) have had limited translational applicability. Tissue nanotransfection (TNT) technology uses a direct, transcutaneous nonviral vector, gene delivery using a chip with nanochannel poration in response to a rapid (<100 ms) focused electric field. The purpose of this study was to experimentally prevent lymphedema using focal delivery of a specific gene Prox1 (a master regulator of lymphangiogenesis). TNT was applied to the previously optimized lymphedematous mice tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: group I (sham) was given pCMV6 (expression vector backbone alone) and group II was treated with pCMV6-Prox1. Group II mice had decreased tail volume (47.8%) compared to sham and greater lymphatic clearance on lymphangiography. Immunohistochemistry showed greater lymphatic vessel density and RNA sequencing exhibited reduced inflammatory markers in group II compared to group I. Prox1 prophylactically delivered using TNT to the surgical site on the day of injury decreased the manifestations of lymphedema in the murine tail model compared to control.