Browsing by Author "Maredia, Navin"
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Item Burn Serum Stimulated Mitochondrial Fission Was Decreased with IL-6 Antibody Treatment(2017-01-17) Sehat, Alvand; Song, Juquan; Kulangara, Rohan; Maredia, Navin; Maxwell, Christian; Panwar, Kunal; Huebinger, Ryan M.; Carlson, Deborah L.; Zang, Qun S.; Wolf, Steven E.INTRODUCTION: Burn patients suffer muscle mass loss associated with hyperinflammation and hypercatabolism. We previously observed that burn serum resulted in cell death with elevated mitochondrial fragmentation in C2C12 myoblast. IL-6 as the key cytokine response to thermal injury has been showed to increase mitochondrial fragmentation. We thus posit that inhibition of IL-6 expression in burn serum will alleviate mitochondrial fragmentation. The aim of this study is to investigate the neutralization effect of IL-6 antibody in burn serum stimulated myoblasts. METHODS: Murine myoblasts C2C12 cells were cultured with recombinant IL-6 protein from 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, and 100 ng/ml. Cells were labeled with MitoTracker Green dye and live cell images were captured with Confocal microscopy. Next, C2C12 cells were exposed to medium containing 1) 10% serum from control rat, 2) 10% serum from burn rat, and 3) 10% serum from burn rat and 0.5 ug/ml of IL-6 antibody. All cells were labeled as the first experiment and live cell images were recorded. The caspase 3 activity was further examined from cell protein lysate. RESULTS: The 1 ng/ml dose of r-IL6 showed a fourfold increase in mitochondrial volume (μm3) at 24 hours post challenge. The intensity signal of Mitotracker was significantly increased in the 1 ng/ml IL-6 dose group at 48 hours. The 1 ng/ml dose of r-IL6 showed an increase in mitochondrial fragmentation. In cells cultured with 10% serum from control rats, mitochondrial morphology maintained the elongated linear shape during the 48 hours. In cells cultured with 10% serum from burned rats, a reduction in mitochondrial sizes was significant. In cells cultured with 10% serum and IL-6 antibody treatment this effect was reversed. Further, the intensity signals increased in response to the burn serum challenge. However, with addition of IL-6 antibody treatment the intensity signals decreased. In addition, burn serum increased the total volume of mitochondria about 1.4 fold, while with IL-6 antibody treatment the total volume was decreased. Consistently, a significant decrease in the expression of caspase 3 in the IL-6 antibody treatment group was observed. CONCLUSION: IL-6 stimulates an increase in mitochondria fragmentation in myoblasts, while IL-6 antibody treatment decreases mitochondrial fragmentation and cell death in burn serum stimulated myoblasts. This project has shown that targeting cytokine levels may be an effective treatment strategy in the management of burn patients.Item Metastatic Penile Melanoma(2018-04-08) Maredia, Navin; Goenka, AnamikaMelanoma, the most serious skin cancer and the sixth most common cancer in North America, is associated with UV light radiation from sun exposure. The majority of melanomas develop on sun-exposed areas, but melanoma can also occur on non-sun exposed areas. This case Illustrates the importance of doing a thorough physical exam, formulating an extensive differential diagnosis, and identifying possible cognitive biases that may lead to misdiagnosis.Item Muscle Function Improvement in Injured Mice with Combination Treatment(2017-01-17) Kulangara, Rohan G.; Sehat, Alvand, J.; Maredia, Navin; Maxwell, Christian; Liu, Ming-Mei; DeSpain, Kevin; Wolf, Steven E.; Song, JuquanINTRODUCTION: Loss of skeletal muscle from direct injury presents debilitating effects to an individual. Current treatments addressing muscle loss are limited by insufficient reconstitution of functioning muscle. Novel regenerative medicine technologies include the application of Urinary Bladder Matrix (UBM) and mesenchymal stem cells (MSCs) to restore functional muscle tissue. In our previous studies, we found that UBM increased muscle myoblast cell proliferation. Therefore, we examined whether co-treatment with MSCs would further augment regeneration as compared to individual treatments. METHODS: Twenty C57BL/6 male adult mice received bilateral laceration injuries on the gastrocnemius muscle under anesthesia, and were randomly grouped to a designed treatment applied 14 days after injury. Treatment groups were 1) DMEM culture medium, 2) UBM only (150μg), 3) MSCs only (1 million mouse derived cells), and 4) UBM+MSCs. 4 additional mice served as a control baseline not receiving injury. Efficacy of treatment was analyzed through isometric muscle force testing as well as histomorphologic examination at 50 days after injury. Two-way ANOVA was applied for statistical analysis. RESULTS: Isometric muscle force was measured, including twitch (Pt), tetanic (Po), and fatigue isometric functions with the muscle stretched to optimal length (Lo). Muscle twitch (Pt) significantly decreased in the DMEM group compared to the non-injured group at day 50 (p < 0.05). Furthermore, twitch significantly increased with UBM treatment, but not with MSC treatment. Regenerating myofiber nuclei were counted and myofiber cross sectional area was measured with histology. New myotubes were identified as having centrally located nuclei. Further, Ki-67 nuclear immunofluorescence staining was performed to demonstrate proliferating satellite cells. The myofiber cross sectional area and the number of Ki-67/DAPI overlapping stained nuclei significantly increased in the DMEM group compared to the non-injured group (p < 0.05). No differences were observed with other treatments in injured mice at day 50. CONCLUSION: We observed a significant improvement in muscle function with combination treatment and single UBM treatment applied 50 days post-injury. The current animal model provides a tool to study muscle regeneration, and is feasible for clinical translation to address impairment in skeletal muscle function after burn injury.Item Myokine Musclin Expression Is Elevated in Rats after Burn(2017-01-17) Maredia, Navin; Song, Juquan; Sehat, Alvand; Maxwell, Christian; Panwar, Kunal; Kulangara, Rohan; Carlson, Deborah; Huebinger, Ryan; Wolf, StevenINTRODUCTION: Annually, over 2 million people in the US experience severe burns, a condition marked by a hypercatabolic state with significant muscle loss. Muscle is necessary for glucose and lipid metabolism. Previous studies have shown detrimental effects of insulin resistance and hyperglycemia associated with muscle loss due to burns. Recently, the novel skeletal muscle myokine musclin has been found to regulate glucose in vitro. Thus, we attempted to better understand the effects of burns on musclin levels. We aimed to investigate the effects of burns on 1.) musclin levels systemically and 2.) musclin mRNA expression in vitro. METHODS: Thirty-one adult male Sprague-Dawley rats received 40% total body surface area (TBSA) burns. Rat serum was collected from 6 hours to 14 days after burn. Nine animals without injury served as control. Musclin levels in serum were measured by ELISA. Mouse C2C12 myoblasts were stimulated with 10% rat burn serum for 24 hours. Cells were stimulated with non-burn serum, 6-hour post burn serum, 72-hour post burn serum, and 14-day post burn serum. Following stimulation for 24 hours, C2C12 cells were collected and musclin expression was quantified by real time PCR analysis. RESULTS: Circulating musclin levels were 59.3 ± 3.3 ng/mL in non burned control rats. Musclin levels in the serum significantly increased to 76.7 ± 6.0 ng/mL at 6 hours and 76.7 ± 1.9 ng/mL at 24 hours after burn (p<0.05). Musclin levels in the serum returned to baseline until 14 days. Normalized to GAPDH mRNA level, musclin mRNA expression was 7.87 ± 0.56 fold in C2C12 myoblasts with 10% non-burn serum stimulation. Musclin mRNA expression significantly increased with the addition of the following burn rat serums: 22.66 ± 5.18 fold with 6-hour post-burn serum, 15.00 ± 1.93 fold with 72-hour post-burn serum, and 12.17 ± 0.82 fold with 14-day post-burn serum (p<0.05). CONCLUSIONS: Musclin levels increase in rat serum following 40% TBSA burn injury. In vitro stimulation of muscle cells with burn serum increases musclin expression.