Browsing by Author "Mohan, Ganesh"
Now showing 1 - 10 of 13
Results Per Page
Sort Options
Item 142. Optimization of the Murine Hindlimb Lymphedema Model(Wolters Kluwer, 2025-04-24) Ahmed, Shahnur; Mohan, Ganesh; Sullivan, Steven J.; Jorge, Miguel; Sinha, Mithun; Hassanein, Aladdin H.; Surgery, School of MedicinePURPOSE: Secondary lymphedema is limb swelling from lymphatic injury. It frequently occurs following lymph node dissection and radiation during the treatment of malignancies such as breast cancer or melanoma. The murine tail is the most commonly used model to study secondary lymphedema and involves full thickness tail skin excision and lymphatic vessel disruption. The murine hindlimb model, which has been less frequently used in the literature, offers a more clinically translatable method. However, there is inconsistency and variability, including the benefit of radiation, which have contributed to the model being less widely adapted than the tail model. The purpose of this study is to 1) optimize the murine hindlimb lymphedema to achieve consistent results and 2) assess the effect of radiation on outcome in the murine hindlimb model. METHODS: C57BL/6 mice either underwent 20 Gy irradiation of one hindlimb seven days prior to surgery (n=11) or no preoperative radiation (n=9). For all mice, a circumferential skin incision was created at the proximal hindlimb exposing the subcutaneous soft tissues. Lymphatics were identified with isosulfan blue dye injection into the paw and disrupted. Popliteal lymph nodes were excised. The skin was sutured leaving a 3 mm gap. The contralateral hindlimb served as the control. Paw thickness and calf thickness measurements were obtained at weekly intervals and indocyanine green (ICG) near-infrared laser lymphangiography was used to assess lymphatic function. RESULTS: For the irradiated mice, the average paw thickness of the operated hindlimb on postoperative day (POD) 14 was 3.5±0.3 cm compared to 2.1±0.05 cm on the contralateral limb (p=0.0001). At POD-90, the average paw thickness of the irradiated, operated hindlimb was 2.4±0.1 cm compared to 2.1±0.1 cm for the contralateral limb (p=0.01). ICG lymphangiography at 24-hours postinjection on POD-42 demonstrated an average signal intensity of 97.7±28.5 arbitrary fluorescent units (AFU) in the operated hindlimb compared to 33.6±6.2 AFU in the non-operated hindlimb (p=0.003). In the mice that did not undergo radiation, the average paw thickness was 2.5±0.2 cm on POD-42 was greater than the contralateral limb (2.1±0.1 cm) (p=0.0002) but smaller than hindlimbs that underwent radiation (3.2±0.1 cm) (p=0.0002). The nonradiated mice had greater paw thickness than the contralateral control until POD-56 whereas the radiated mice sustained significant paw thickness until Day 90. CONCLUSION: Radiation of the murine hindlimb model results in sustained lymphedema compared to non-irradiated mice. The murine hindlimb lymphedema model is clinically more translatable than the murine tail model and includes limb lymphatic vessel disruption, and popliteal lymphadenectomy and ideally radiation for consistent results with lymphedema sustained for 90 days.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 97. Focal Gene Delivery in the Murine Lymphedema Tail Model Using Tissue Nanotransfection Technology (TNT)(Wolters Kluwer, 2022) Mohan, Ganesh; Khan, Imran; Sinha, Mithun; Gordillo, Gayle M.; Sen, Chandan K.; Hassanein, Aladdin H.; Surgery, School of MedicineBackground: Lymphedema is chronic limb swelling resulting from lymphatic dysfunction. It affects an estimated 5 million Americans. There is no cure for this disease. Experimental gene-based therapeutic approaches (e.g., using viral vectors) have had limited translational applicability. Tissue nanotransfection technology (TNT) utilizes a direct, transcutaneous non-viral vector, gene delivery using a chip with nanochannel poration in a rapid (<100ms) focused electric field. This platform technology has been used for various applications in tissue reprogramming. The ability to deliver genetic cargo at a focal, non-global site would have practical clinical potential in lymphedema treatment. The purpose of this study is to experimentally evaluate the applicability of TNT for lymphedema. 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). TNT was applied with square wave pulse electric stimulation (10x10ms pulses, 250 V, 10 mA). The efficiency of gene delivery was assessed through qRT-PCR using primers with SYBR Green fluorescence quantification and immunostaining with anti-Prox1 antibody. Results: The experimental Group II exhibited four-fold increased expression of Prox1 using qRT-PCR compared to control Group I at the site of TNT treatment (P<0.05). Increased expression of Prox1 was also observed with immunohistochemistry 3 days post-TNT application. Intensity quantification of immunohistochemistry revealed greater expression of Prox1 in Group II when compared to Group I (P<0.05). Conclusion: This study demonstrates a novel, focal nanotechnology approach for local genetic cargo delivery in murine tail lymphedema model without the use of viral vectors for transfection. TNT can rapidly and effectively be applied for potentially therapeutic delivery of factors locally at the site of lymphedema.Item Biofilm-derived oxylipin 10-HOME–mediated immune response in women with breast implants(ASCI, 2024-02) Khan, Imran; Minto, Robert E.; Kelley-Patteson, Christine; Singh, Kanhaiya; Timsina, Lava; Suh, Lily J.; Rinne, Ethan; Van Natta, Bruce W.; Neumann, Colby R.; Mohan, Ganesh; Lester, Mary; VonDerHaar, R. Jason; German, Rana; Marino, Natascia; Hassanein, Aladdin H.; Gordillo, Gayle M.; Kaplan, Mark H.; Sen, Chandan K.; Kadin, Marshall E.; Sinha, Mithun; Chemistry and Chemical Biology, School of ScienceThis study investigates a mechanistic link of bacterial biofilm–mediated host-pathogen interaction leading to immunological complications associated with breast implant illness (BII). Over 10 million women worldwide have breast implants. In recent years, women have described a constellation of immunological symptoms believed to be related to their breast implants. We report that periprosthetic breast tissue of participants with symptoms associated with BII had increased abundance of biofilm and biofilm-derived oxylipin 10-HOME compared with participants with implants who are without symptoms (non-BII) and participants without implants. S. epidermidis biofilm was observed to be higher in the BII group compared with the non-BII group and the normal tissue group. Oxylipin 10-HOME was found to be immunogenically capable of polarizing naive CD4+ T cells with a resulting Th1 subtype in vitro and in vivo. Consistently, an abundance of CD4+Th1 subtype was observed in the periprosthetic breast tissue and blood of people in the BII group. Mice injected with 10-HOME also had increased Th1 subtype in their blood, akin to patients with BII, and demonstrated fatigue-like symptoms. The identification of an oxylipin-mediated mechanism of immune activation induced by local bacterial biofilm provides insight into the possible pathogenesis of the implant-associated immune symptoms of BII.Item Biofilm-derived oxylipin 10-HOME–mediated immune response in women with breast implants(The American Society for Clinical Investigation, 2023-11-30) Khan, Imran; Minto, Robert E.; Kelley-Patteson, Christine; Singh, Kanhaiya; Timsina, Lava; Suh, Lily J.; Rinne, Ethan; Van Natta, Bruce W.; Neumann, Colby R.; Mohan, Ganesh; Lester, Mary; VonDerHaar, R. Jason; German, Rana; Marino, Natascia; Hassanein, Aladdin H.; Gordillo, Gayle M.; Kaplan, Mark H.; Sen, Chandan K.; Kadin, Marshall E.; Sinha, Mithun; Surgery, School of MedicineThis study investigates a mechanistic link of bacterial biofilm–mediated host-pathogen interaction leading to immunological complications associated with breast implant illness (BII). Over 10 million women worldwide have breast implants. In recent years, women have described a constellation of immunological symptoms believed to be related to their breast implants. We report that periprosthetic breast tissue of participants with symptoms associated with BII had increased abundance of biofilm and biofilm-derived oxylipin 10-HOME compared with participants with implants who are without symptoms (non-BII) and participants without implants. S. epidermidis biofilm was observed to be higher in the BII group compared with the non-BII group and the normal tissue group. Oxylipin 10-HOME was found to be immunogenically capable of polarizing naive CD4+ T cells with a resulting Th1 subtype in vitro and in vivo. Consistently, an abundance of CD4+Th1 subtype was observed in the periprosthetic breast tissue and blood of people in the BII group. Mice injected with 10-HOME also had increased Th1 subtype in their blood, akin to patients with BII, and demonstrated fatigue-like symptoms. The identification of an oxylipin-mediated mechanism of immune activation induced by local bacterial biofilm provides insight into the possible pathogenesis of the implant-associated immune symptoms of BII.Item Breast Implant-Associated Immunological Disorders(Hindawi, 2022-05-04) Suh, Lily J.; Khan, Imran; Kelley-Patteson, Christine; Mohan, Ganesh; Hassanein, Aladdin H.; Sinha, Mithun; Surgery, School of MedicineBackground: Breast implants are commonly placed postbreast cancer reconstruction, cosmetic augmentation, and gender-affirming surgery. Breast implant illness (BII) is a systemic complication associated with breast implants. Patients with BII may experience autoimmune symptoms including fatigue, difficulty concentrating, hair loss, weight change, and depression. BII is poorly understood, and the etiology is unknown. The purpose of this literature review is to characterize BII autoimmune disorders and determine possible causes for its etiology. Methods: The PubMed, Google Scholar, Embase, Web of Science, and OVID databases were interrogated from 2010 to 2020 using a query strategy including search term combinations of "implants," "breast implant illness," "autoimmune," and "systemic illness." Results: BII includes a spectrum of autoimmune symptoms such as fatigue, myalgias/arthralgias, dry eyes/mouth, and rash. A review of epidemiological studies in the past ten years exhibited evidence affirming an association between breast implants and autoimmune diseases. The most commonly recognized were Sjogren's syndrome, rheumatoid arthritis, systemic sclerosis, chronic fatigue syndrome, and Raynaud's syndrome. Explantation resulted in alleviation of symptoms in over 50% of patients, strengthening the hypothesis linking breast implants to BII. Studies have shown that silicone is a biologically inert material and unlikely to be the cause of these symptoms. This is supported by the fact that increased risk of autoimmune disease was also reported in patients with other implantable biomaterials such as orthopedic implants. Recent studies shed light on a possible role of bacterial biofilm and subsequent host-pathogen interactions as a confounding factor to this problem. Conclusion: BII could be dependent on biofilm infection and the microenvironment around the implants. The true pathophysiology behind these complaints must be further investigated so that alternative treatment regimens other than explantation can be developed. Translational significance of these studies is not limited to breast implants but extends to other implants as well.Item Immunomodulatory Effects of Oxylipin 10-HOME Produced by Biofilm Results in Host-Biofilm Interaction in Breast Implant Illness(Wolters Kluwer, 2022) Khan Mohammed, Imran; Minto, Robert; Kelley-Patteson, Christine; Timsina, Lava; Singh, Kanhaiya; Van Natta, Bruce W.; Mohan, Ganesh; Chauhan, Ruvi; Lester, Mary; Hassanein, Al; Gordillo, Gayle M.; Sen, Chandan; Kadin, Marshall; Sinha, Mithun; Surgery, School of MedicinePURPOSE: The spread of biofilms on medical implants represents one of the principal triggers of persistent and chronic infections in clinical settings. Nearly 300,000 women annually have breast implant surgery in the United States, for reasons including post-mastectomy breast reconstruction, revision of prior augmentation/ reconstruction, cosmetic augmentation, and gender affirmation. There has been increased identification of patients experiencing a constellation of symptoms related to their implants termed as breast implant illness (BII). In this work, we report that bacterial biofilm associated with breast implant, metabolize fatty acid oleic acid present in the breast tissue milieu to oxylipins, one such oxylipin identified from this study is (E)-10-hydroxy-8-octadecenoic acid (10-HOME). We hypothesize that immunomodulatory effects of oxylipin 10-HOME produced by biofilm present on the implant could be a possible etiology for BII pathogenesis. METHOD: Implants, peri-prosthetic tissues and blood was collected from BII subjects (n=46) and two control groups, group I, (non-BII, n=34) patients with breast implants, no BII symptoms. Group II (normal tissue, n = 20), patients without an implant, whose breast tissue was removed due to surgical procedures. A questionnaire developed based on epidemiological studies on BII screened for the commonly reported symptoms associated with BII. Predictive variables included age, diabetes status, co-morbidities, type (smooth/textured) and duration of implant. Scanning electron microscopy (SEM), 16S rRNA (genomic) next generation sequencing (NGS) were used for bacterial biofilm identification. 10-HOME was quantitated through targeted and untargeted lipidomic analyses using LC-MS-MS. RNA-Seq analysis was performed on peri-prosthetic breast tissues. Flow cytometry and mass cytometry (CyToF) were conducted to investigate the role of immune cells. RESULTS: Bacterial biofilm was detected through SEM and 16SrRNA NGS. Bivariate analysis using cross-tabulation was performed between presence of biofilm and the study groups. Using the two-sample test of proportions with z-tests, Staphylococcus epidermidis colonization was observed to be higher in the BII group (73.33%) compared to non-BII group (16.67%, p=0.018) and the normal group (10%, p=0.036). The BII group was 2.4 times more likely to have S. epidermidis colonization compared to the non-BII group (Odds Ratio=2.4). Similarly, when comparing with normal group, the BII group was 3.4 times more likely to have S. epidermidis. Elevated levels of 10-HOME in BII compared to non-BII samples, (p<0.0001) were observed through mass spectrometry. Positive correlation was observed between bacterial abundance and concentration of 10-HOME in BII subjects (R2=0.88). RNA-Seq analysis on peri-prosthetic tissue and flow/ mass cytometry analyses from peripheral blood derived lymphocytes showed increased abundance of CD4+ Th1 cells. Th1 cells have been reported to be activated in auto-immune diseases. No significant difference was observed in the abundance of other Th subtypes (Th2, Th9 and Th22). Oxylipin 10-HOME polarized CD4+ naïve T cells to Th1 subtype in vitro. CONCLUSION: This study investigated the biofilm hypothesis of BII through a biofilm derived immunogenic metabolite. Through a systematic cause-effect based studies, the work shows activation of Th1 cells in presence of 10-HOME. The study provides the first evidence of a possible etiology of BII mediated via bacterial biofilm derived 10-HOME.Item A Murine Tail Lymphedema Model(JoVE, 2021) Hassanein, Aladdin H.; Sinha, Mithun; Neumann, Colby R.; Mohan, Ganesh; Khan, Imran; Sen, Chandan K.; Surgery, School of MedicineLymphedema is extremity swelling caused by lymphatic dysfunction. The affected limb enlarges because of accumulation of fluid, adipose, and fibrosis. There is no cure for this disease. A mouse tail model that uses a focal full thickness skin excision near the base of the tail, resulting in tail swelling, has been used to study lymphedema. However, this model may result in vascular comprise and consequent tail necrosis and early tail swelling resolution, limiting its clinical translatability. The chronic murine tail lymphedema model induces sustained lymphedema over 15 weeks and a reliable perfusion to the tail. Enhancements of the traditional murine tail lymphedema model include 1) precise full thickness excision and lymphatic clipping using a surgical microscope, 2) confirmation of post-operative arterial and venous perfusion using high resolution laser speckle, and 3) functional assessment using indocyanine green near infrared laser lymphangiography. We also use tissue nanotransfection technology (TNT) for novel non-viral, transcutaneous, focal delivery of genetic cargo to the mouse tail vasculature.Item Oxylipins in Breast Implant–Associated Systemic Symptoms(Oxford University Press, 2024) Khan, Imran; Timsina, Lava; Chauhan, Ruvi; Ingersol, Christopher; Wang, David R.; Rinne, Ethan; Muraru, Rodica; Mohan, Ganesh; Minto, Robert E.; Van Natta, Bruce W.; Hassanein, Aladdin H.; Kelley-Patteson, Christine; Sinha, Mithun; Surgery, School of MedicineBackground: A subset of females with breast implants have reported a myriad of nonspecific systemic symptoms collectively termed systemic symptoms associated with breast implants (SSBI). SSBI symptoms are similar to manifestations associated with autoimmune and connective tissue disorders. Breast tissue is rich in adipose cells, comprised of lipids. Insertion of an implant creates an oxidative environment leading to lipid oxidation. Oxylipins can influence immune responses and inflammatory processes. Objectives: In this study we explored the abundance of a spectrum of oxylipins in the periprosthetic tissue surrounding the breast implant. Because oxylipins are immunogenic, we sought to determine if they were associated with the SSBI patients. We have also attempted to determine if the common manifestations exhibited by such patients have any association with oxylipin abundance. Methods: The study included 120 patients divided into 3 cohorts. We analyzed 46 patients with breast implants exhibiting manifestations associated with SSBI; 29 patients with breast implants not exhibiting manifestations associated with SSBI (control cohort I, non-SSBI); and 45 patients without implants (control cohort II, no-implant tissue). Lipid extraction and oxylipin quantification were performed with liquid chromatography mass spectrometry (LC-MS/MS). LC-MS/MS targeted analysis of the breast adipose tissue was performed. Results: Of the 15 oxylipins analyzed, 5 exhibited increased abundance in the SSBI cohort when compared to the non-SSBI and no-implant cohorts. Conclusions: The study documents the association of the oxylipins with each manifestation reported by the patient. This study provides an objective assessment of the subjective questionnaire, highlighting which symptoms may be more relevant than the others.