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Deferoxamine Treatment Decreases Levels Of Reactive Oxygen Species And Cellular Apoptosis In Irradiated Skin
Abra H. Shen, SB, Sandeep Adem, MS, Nestor M. Diaz Deleon, Mimi R. Borrelli, MBBS, MSc, Harsh N. Shah, MPH, Ankit Salhotra, BS, Michael T. Longaker, MD, MBA, Geoffrey C. Gurtner, MD, Derrick C. Wan, MD.
Department of Surgery, Division of Plastic Surgery, Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.

PURPOSE: Radiation therapy is a cornerstone of oncologic treatment. Unfortunately, radiation can lead to pain and ulceration in the acute phase, as well as stiffness and fibrosis in the chronic phase. Deferoxamine (DFO) is an FDA approved iron chelator that has been shown to increase skin vascularization by stabilizing hypoxia-inducible factor 1-alpha levels and improve radiation-induced fibrosis. However, it is unclear whether there are other mechanisms underlying the therapeutic effects that DFO exhibits on the skin. Previous work has shown that DFO delivered transdermally improves healing in diabetic ulcer wounds by decreasing oxidative stress and cellular apoptosis. This study evaluates the effects of transdermal DFO on reactive oxygen species and cell death in the context of irradiation (IR) to skin.

METHODS: CD-1 nude immunodeficient mice (n=8) were randomized to one of two conditions: 1. Control patch (IR only) (n=4) and 2. DFO patch (n=4) (Figure 1A). The study period consisted of a 14-day pre-IR period, followed by a 12-day course of fractionated IR which delivered a total of 30 Gray to all mice. The control group received patches without DFO and the DFO group received patches containing DFO throughout the experiment with patches changed daily (Figure 1B). Mouse scalp skin was harvested one day after the completion of IR. Tissue samples were either fixed in 4% PFA, snap frozen in OCT, or directly stored at -80C. Fixed samples were embedded in paraffin, sectioned, and stained for iron with Prussian blue. Snap frozen samples were cryosectioned at 10um, mounted, stained with dihydroethidium (DHE), and imaged with confocal microscopy. DHE detects reactive oxygen species. Stored samples were mechanically homogenized and enzyme-linked immunosorbent assays (ELISAs) were performed to measure levels of human Bax and Cleaved Caspase-3, markers of apoptosis.

RESULTS: Treatment with DFO was associated with decreased levels of iron in the skin, confirming DFOs role as an iron chelator. DHE fluorescence was more intense in the control patch group than in the DFO patch group (***p<0.0001), indicating that ROS generation decreased with DFO treatment (Figure 1C). Similarly, apoptosis was highest in the control group and lowest in the DFO group as shown by higher human Bax protein (*p=0.028, Figure 1D, left) and Cleaved Caspase-3 (**p=0.0038) levels (Figure 1D, right).

CONCLUSIONS: DFO delivered transdermally leads to a dose-dependent decrease in reactive oxygen species and apoptotic markers. These results provide further insight into the mechanisms underlying its restorative effects on irradiated skin. Future work will investigate the relationship of reactive oxygen species and fibroblast subpopulations in the skin.

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