Jerry J. Wilmink,

Research Summary

Laser induced surgical wounds are superior to scalpel excisions because they can provide more precise tissue ablation without direct contact. Effective medical laser procedures are achieved by selecting laser parameters that minimize undesirable tissue damage. In this study, heat shock protein 70 (Hsp70) was employed as a surrogate marker for laser induced thermal damage. Hsp70 was used to compare the characteristics of excisional wound repair after surgery with the free electron laser (FEL) as a function of wavelength, fluence, and exposure time. Wound repair and Hsp70 expression was compared at 2.94, 6.1, 6.45 μm using fluences of 7.96, 9.95, 11.94, 15.92, 17.91, and 19.89 J/cm2. The FEL was operated in 5 μs micropulses, with a 30 Hz repetition rate, and the beam was delivered in a computer-controlled raster pattern to produce a 6.5 mm square. Using a transgenic mouse strain with the hsp70 promoter driving luciferase expression, we assessed wound repair by ! observing hsp70 expression, neovascularization, biomechanical properties, and wound histology for up to 62 d. Bioluminescence (BLI) was monitored non-invasively in real time with the Xenogen IVIS 200 System, blood velocity and neovascularization was measured with three-dimensional Doppler ultrasound and two-dimensional laser Doppler techniques, and tensile properties were measured with the BTC-2000 tensiometer.

The spatial and temporal kinetics of hsp70 expression allowed for adequate intra and inter-wound assessments. Comparing FEL ablations using λ = 6.45 μm and 6.1 μm, the BLI data revealed margin differences in gene expression between the wavelengths. Hsp70 expression was higher (4x) and more confined (3x steeper slope) to the treated tissue region at 6.45 μm than at 6.1 μm. For the first time, these findings demonstrate at a gene expression level, the mechanisms that are responsible for the superiority of laser ablation seen at 6.45 μm. The BLI data also served to illustrate, for the first time, the wavelength, fluence, and exposure time dependence of hsp70 expression. BLI indicated that peak hsp70 expression occurred 6-12 hours after exposure to laser irradiation. Increasing the FEL pulse energy/fluence resulted in non-linear increases in hsp70 expression and more rapid induction of response. Increases in fluence resulted in more robust ne! ovascularization. We are currently working examining the histological correlates of Hsp70 expression. These results indicate that noninvasive, quantitative BLI and pulsed wave Doppler can be used as a high throughput screening platform for laser-tissue interaction studies.

Research Keywords

» 1. Wilmink GJ, Jeffrey M. Davidson, E. Duco Jansen. Wavelength-Dependent Dynamics of Heat Shock Protein 70 Expression in Free Electron Laser Wounds. SPIE Proceedings. Paper number: 6440-2. (volume in preparation)

» 2. Wilmink GJ, Susan R. Opalenik, Joshua T. Beckham, Jeffrey M. Davidson, E. Duco Jansen. Assessing laser-tissue damage with bioluminescent imaging. Journal of Biomedical Optics, 2006. 11(4): p. 041114.

» 3. Wilmink GJ and E. Duco Jansen. Using bioluminescent reporter genes in a novel skin model to temporally and noninvasively monitor gene expression. Lasers in Surgery and Medicine. Volume 36, Issue S17, p4, 2005.

» 4. Wilmink GJ and E. Duco Jansen. Quantification of gene expression in organotypic raft cultures during wound healing using bioluminescent reporter genes. Lasers in Surgery and Medicine- Volume 34, Issue S16, p. 9, 2004.