LOW INTENSITY LASER THERAPY SPEEDS WOUND HEALING IN HEMOPHILIA B MICE Maureane Hoffman, Anna G. McDonald, Dougald M. Monroe Durham VA Medical Center, Duke University and The University of North Carolina North Carolina, USA Abstract Introduction: Wound healing is delayed and histologically abnormal in FIX knockout (HB) mice [1]. The current work tests the hypothesis that low intensity laser therapy (LILT) can speed healing in HB mice. Methods: A 3 mm punch biopsy was placed on the dorsal skin of each HB mouse as described [1]. The mice were given LILT or sham LILT treatment daily for 3 days, starting immediately after wound placement (day 0). LILT parameters were chosen based on published studies on the effects of LILT in animal models and cell culture. In LILT the wound was illuminated at 650 nm to deliver an energy "dose" of 4 J/sq cm. The Sham treatment was carried out as for LILT, but power to the laser was not activated. Results: The skin defects were significantly smaller in LILT-treated than Sham-treated mice on days 1-4 after wounding. LILT also significantly shortened the time to epithelial closure - from 13 days in Sham- to 11 days in LILT-treated mice. LILT did not normalize all histologic parameters of healing. However, there was notably less hemorrhage in the skin and subcutaneous tissues of LILT- compared to Sham-treated HB mice. Conclusions: LILT treatments enhanced wound healing in HB mice. LILT shortened the time to epithelial closure, and, surprisingly, also appeared to reduce hemorrhage in HB mice. Results As shown in the top panel, 3 daily LILT treatments decreased the expansion in wound size seen in untreated or sham-laser-treated HB mice to a point that the laser-treated HB wounds were not significantly larger than wounds placed on WT mice. As shown in the second panel, LILT also shortened the time to closure of the surface epithelial defect in HB mice. It reduced the time for closure of 100% of the wounds from 12 days in sham-treated HB wounds to 10 days in LILT-treated wounds. The LILT-treated HB wounds had the same time to closure as control WT mice and were significantly different (p<0.05) from sham-treated HB by Fisher’s exact test. However, as shown in the third panel, the influx of macrophages into the wound site was not normalized by LILT treatment. While WT mice exhibit some bleeding around the biopsies, they never develop subcutaneous hematomas, which are common in the HB mice [1]. Much to our surprise, hemorrhage was impressively reduced by LILT. The proportion of sham-treated HB mice that developed hematomas was 50% (6 of 12 mice). However, only 18% (3 of 17) of LILT-treated HB mice developed hematomas. This difference was significantly significant by Fisher’s exact test. We do not know the mechanism by which LILT reduced hemorrhage, but we are actively pursuing this unexpected effect. Introduction We have recently reported that wound healing is delayed in hemophilia B mice [1]. In addition, the influx of macrophages is delayed, likely due to the reduced level of thrombin generated in hemophilia. Recurrent hemorrhage occurs, even after surface healing resulting in iron accumulation near the wound site. Low-intensity laser therapy (LILT) has been reported to enhance wound healing and reduce pain and inflammation. LILT utilizes light with wavelengths in the red and infrared range (600 to 940 nm). In contrast to high-energy (up to 300 W) lasers used in surgery, LI lasers have power in the milliwatt range. They do not heat or damage tissue and have been classified as nonsignificant risk devices by the FDA Many studies (of variable quality) have been published on the effects of LILT on healing. A recent meta-analysis, including only studies of high quality, found a significant effect of LILT on wound healing in animal and human subjects, with strongest positive effects on the time to healing, inflammation and reduction of dermal necrosis [2]. The current work was designed to determine whether LILT could improve the time course and/or tissue features of healing in hemostatically impaired mice. Conclusions Conclusions LILT treatment of HB mice reduced: wound size extent of hemorrhage and time to healing Methods Wound healing studies on hemophilia B (HB) and wild type (WT) mice were conducted under a protocol approved by the Animal Care and Use Committee of the University of North Carolina. Biopsy placement, tissue harvesting, processing and histologic evaluation was carried out as previously described [1]. Laser instrumentation for these studies was calibrated and provided by Dr. Melyni Worth and Peter Jenkins of Thor International, Inc. The laser probe emits light at 650 nm and has a spacer ring of 6 mm in diameter around the tip of the probe to keep the lens a defined distance from the skin surface. The laser probe has been calibrated to deliver an energy dose to the wound surface of 4 J/sq cm in a treatment time of 32 seconds. The wavelength and treatment schedule were selected based on conditions found to be effective in a meta- analysis of LILT effects on wound healing [2]. The wound sites were treated for three successive days starting immediately after wound placement. Sham-treated animals were restrained and the laser probe applied to the wound site in a manner identical to the LILT- treated mice, but the laser was not activated. References Hoffman M, Harger A, Lenkowski A, Hedner U, Roberts HR, Monroe DM. Cutaneous wound healing is impaired in hemophilia B. Blood 2006; 108, 3053-60. Woodruff, L. D., Bounkeo, J. M., Brannon, W. M., Dawes, K. S., Barham, C. D., Waddell, D. L., and Enwemeka, C. S. 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