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Melatonin inhibits thermal injury–induced hyperpermeability in microvascular endothelial cells

Wiggins-Dohlvik, Katie MD; Han, Min Suk; Stagg, Hayden W. MD; Alluri, Himakarnika MS; Shaji, Chinchusha Anasooya B. Tech; Oakley, Ryan P.; Davis, Matthew L. MD; Tharakan, Binu PhD

Journal of Trauma and Acute Care Surgery: December 2014 - Volume 77 - Issue 6 - p 899–905
doi: 10.1097/TA.0000000000000346
WTA 2014 Plenary Papers

BACKGROUND: Burns induce systemic inflammatory reactions and vascular hyperpermeability. Breakdown of endothelial cell adherens junctions is integral in this process, and reactive oxygen species (ROS) and proteolytic enzymes such as matrix metalloproteinase-9 (MMP-9) play pivotal roles therein. Outside trauma, melatonin has shown to exhibit anti-MMP activity and to be a powerful antioxidant. Consequently, we hypothesized that burn-induced junctional damage and hyperpermeability could be attenuated with melatonin.

METHODS: Sprague-Dawley rats were assigned to sham or burn groups. Fluorescein isothiocyanate–bovine albumin was administered intravenously. Venules were examined with intravital microscopy; fluorescence intensities were measured intravascularly and extravascularly. Serum was collected. Rat lung microvascular endothelial cells were grown as monolayers and divided into four groups: sham serum and burn serum with and without melatonin pretreatment. Fluorescein isothiocyanate–bovine albumin flux was measured. Immunofluorescence for adherens junction proteins and staining for actin were performed, and images were captured. Cells were grown on 96 well plates, and ROS species generation following application of burn and sham serum was analyzed with and without melatonin. Statistical analysis was conducted with the Student’s t test.

RESULTS: Intravital microscopy data revealed an increase in vascular hyperpermeability following burn (p < 0.05). Monolayer permeability was increased with burn serum (p < 0.05); this was attenuated with melatonin (p < 0.05). Immunofluorescence showed damage of rat lung microvascular endothelial cell adherens junctions with burn serum exposure, and melatonin restored integrity. Rhodamine phalloidin staining showed filamentous actin stress fiber formation after burn serum application, and melatonin decreased this. Burn serum significantly increased ROS species generation (p < 0.05), and melatonin negated this (p < 0.05).

CONCLUSION: Burns damage endothelial adherens junctions and induce microvascular hyperpermeability; melatonin attenuates this process. This insight into the mechanisms of burn-induced fluid leak suggests the role of ROS and MMP-9 but more importantly hints at the possibility of new treatments to combat vascular hyperpermeability in burns.

From the Department of Surgery, Scott and White Memorial Hospital, Baylor Scott and White Health; and College of Medicine, Texas A&M University Health Science Center, Temple, Texas.

Submitted: January 15, 2014, Revised: March 19, 2014, Accepted: March 31, 2014, Published online: July 21, 2014.

This study was presented at the 44th Annual Meeting of the Western Trauma Association, March 2–7, 2014, in Steamboat, Colorado.

Address for reprints: Binu Tharakan, PhD, Department of Surgery, Scott and White Memorial Hospital/College of Medicine, Texas A&M University Health Science Center, 702 SW H.K. Dodgen Loop, Temple, TX 76504; email:

© 2014 Lippincott Williams & Wilkins, Inc.