Determination of Microcirculatory Changes and Angiogenesis in a Model of Frostbite Injury In Vivo

Abstract

The breakdown of skin microcirculation and the leukocyte-endothelium interaction are assumed to play a key role in the pathophysiology of frostbite injuries. However, little is known as yet. The aim was to develop an in vivo frostbite model to monitor microcirculatory changes and angiogenesis after frostbite injury. Deep partial thickness frostbite injuries were inflicted with a no-touch-technique to the ears of hairless mice (n=9). To this end, a gas jet of nitrogen vapor (T=-195,8±2.7°C) was delivered onto an area of 1.9 mm(2) for 1,5 s. Intravital fluorescent microscopy in combination with FITC-dextran and Rhodamin 6G as fluorescent dyes was used to assess microcirculatory changes, leukocyte behavior, and angiogenesis during the 14 d of wound healing. The area of no perfusion decreased significantly over the observed period, and perfusion was almost completely restored due to angiogenesis by d 14 (day 1: 1.89 [mm(2)]±0.44SEM, d 14: 0.02±0.01). No post-traumatic extension of the trauma could be observed. Edema formation increased significantly up to d 7. The number of adherent leukocytes showed a significant increase during the first 7 d. Functional vessel density showed a significant post-frostbite decrease to 60% of the baseline value. This novel frostbite model provides a simple and nonetheless highly effective technique of creating locally limited reproducible frostbite injuries using a no touch technique. Tissue damage can be fully attributed to the thermal trauma, and the model allows repetitive intravital fluorescent microscopy of the microcirculation, leukocyte-endothelium interaction, and angiogenesis.