Assessment of burn wound tissue in situ by multiphoton microscopy employing fluorescence and second harmonic generation contrasts in live animals

摘要

Current assessment of burn wound depth and progression of healing for properchoice of treatment is based on time-consuming and invasive techniques that mayinterfere with the healing process. One way to overcome these problems is to usenoninvasive techniques. We use multiphoton microscopy (MPM) that employsfluorescence and second harmonic generation (SHG) contrasts to noninvasivelyfollow the burn healing process in situ as healing progresses in live animals. Healingprogression was followed in a partial thickness burn wound made on the dorsum ofanesthetized Sprague-Dawley rats with a 2.8 cm diameter brass cylinder heated at80oC for 60 seconds and pressed against the shaved rat skin for 6 seconds. Duringthe first five days, burn healing was characterized by changes in the organization ofcollagen into a compact, mat-like assembly, suggesting progressive degradation ofcollagen within the injury site.We also detected increased follicular cell damage anda strong auto-fluorescence signal from cellular debris. Deposition of new collagenwas seen after the scab fell off and gradually increased thereafter as detected withan increase in the SHG signal intensity. A great increase in the density of cells wasobserved as well. From day 21 on, a network of capillaries with blood flow wasobserved. The epidermal cell layer and the blood vessel network became progressivelymore organized. At day 29, fibrillar collagen had SHG signal levels andmorphology of the nearly preburn state. These findings were corroborated byhistology/histochemistry. In conclusion, MPM technology that employs fluorescenceand SHG contrasts is instrumental in following the healing process, in particularduring the early stages of healing. The degree and rate with which theseevents occur early after burning could help clinicians make treatment decisions. Theoutcomes of healing at later times would be indicative of the effectiveness of thetreatment applied.