Special Section on Pioneers in Biomedical Optics: A.J. Welch

Assessing laser-tissue damage with bioluminescent imaging

[+] Author Affiliations
Gerald J. Wilmink

Vanderbilt University, Department of Biomedical Engineering, 5824 Stevenson Center, Nashville, Tennessee 37235

Susan R. Opalenik

Vanderbilt University, Department of Pathology and Department of Veterans Affairs Medical Center, Nashville, Tennessee 37212

Joshua T. Beckham

Vanderbilt University, Department of Biomedical Engineering, 5824 Stevenson Center, Nashville, Tennessee 37235

Jeffrey M. Davidson

Vanderbilt University, Department of Pathology and Department of Veterans Affairs Medical Center, Nashville, Tennessee 37212

E. Duco Jansen

Vanderbilt University, Department of Biomedical Engineering, VU Station B #351631, Nashville, Tennessee 37235

J. Biomed. Opt. 11(4), 041114 (August 31, 2006). doi:10.1117/1.2339012
History: Received October 07, 2005; Revised February 28, 2006; Accepted February 28, 2006; Published August 31, 2006; Online August 31, 2006
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Effective medical laser procedures are achieved by selecting laser parameters that minimize undesirable tissue damage. Traditionally, human subjects, animal models, and monolayer cell cultures have been used to study wound healing, tissue damage, and cellular effects of laser radiation. Each of these models has significant limitations, and consequently, a novel skin model is needed. To this end, a highly reproducible human skin model that enables noninvasive and longitudinal studies of gene expression was sought. In this study, we present an organotypic raft model (engineered skin) used in combination with bioluminescent imaging (BLI) techniques. The efficacy of the raft model was validated and characterized by investigating the role of heat shock protein 70 (hsp70) as a sensitive marker of thermal damage. The raft model consists of human cells incorporated into an extracellular matrix. The raft cultures were transfected with an adenovirus containing a murine hsp70 promoter driving transcription of luciferase. The model enables quantitative analysis of spatiotemporal expression of proteins using BLI. Thermal stress was induced on the raft cultures by means of a constant temperature water bath or with a carbon dioxide (CO2) laser (λ=10.6μm, 0.679 to 2.262Wcm2, cw, unfocused Gaussian beam, ωL=4.5mm, 1min exposure). The bioluminescence was monitored noninvasively with an IVIS 100 Bioluminescent Imaging System. BLI indicated that peak hsp70 expression occurs 4 to 12h after exposure to thermal stress. A minimum irradiance of 0.679Wcm2 activated the hsp70 response, and a higher irradiance of 2.262Wcm2 was associated with a severe reduction in hsp70 response due to tissue ablation. Reverse transcription polymerase chain reaction demonstrated that hsp70 mRNA levels increased with prolonged heating exposures. Enzyme-linked immunosorbent protein assays confirmed that luciferase was an accurate surrogate for hsp70 intracellular protein levels. Hematoxylin and eosin stains verified the presence of the thermally denatured tissue regions. Immunohistochemical analyses confirmed that maximal hsp70 expression occurred at a depth of 150μm. Bioluminescent microscopy was employed to corroborate these findings. These results indicate that quantitative BLI in engineered tissue equivalents provides a powerful model that enables sequential gene expression studies. Such a model can be used as a high throughput screening platform for laser-tissue interaction studies.

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© 2006 Society of Photo-Optical Instrumentation Engineers

Citation

Gerald J. Wilmink ; Susan R. Opalenik ; Joshua T. Beckham ; Jeffrey M. Davidson and E. Duco Jansen
"Assessing laser-tissue damage with bioluminescent imaging", J. Biomed. Opt. 11(4), 041114 (August 31, 2006). ; http://dx.doi.org/10.1117/1.2339012


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