In-vivo optical imaging of hsp70 expression to assess collateral tissue damage associated with infrared laser ablation of skin

Abstract

Laser surgical ablation is achieved by selecting laser parameters that remove confined volumes of target tissue and cause minimal collateral damage. Previous studies have measured the effects of wavelength on ablation, but neglected to measure the cellular impact of ablation on cells outside the lethal zone. In this study, we use optical imaging in addition to conventional assessment techniques to evaluate lethal and sublethal collateral damage after ablative surgery with a free-electron laser (FEL). Heat shock protein (HSP) expression is used as a sensitive quantitative marker of sublethal damage in a transgenic mouse strain, with the hsp70 promoter driving luciferase and green fluorescent protein (GFP) expression (hsp70A1-L2G). To examine the wavelength dependence in the mid-IR, laser surgery is conducted on the hsp70A1-L2G mouse using wavelengths targeting water (OH stretch mode, 2.94 microm), protein (amide-II band, 6.45 microm), and both water and protein (amide-I band, 6.10 microm). For all wavelengths tested, the magnitude of hsp70 expression is dose-dependent and maximal 5 to 12 h after surgery. Tissues treated at 6.45 microm have approximately 4x higher hsp70 expression than 6.10 microm. Histology shows that under comparable fluences, tissue injury at the 2.94-microm wavelength was 2x and 3x deeper than 6.45 and 6.10 microm, respectively. The 6.10-microm wavelength generates the least amount of epidermal hyperplasia. Taken together, this data suggests that the 6.10-microm wavelength is a superior wavelength for laser ablation of skin.

Fig. 1

Schematic representation of experimental methodology. (a) Free electron laser (FEL) optical positioning setup and parameters (λ: 2.94, 6.10, 6.45 μm; H: 1.59, 3.17, 4.76, 6.35, 7.96, 9.95, 11.94, 15.92, 17.91, 19.89 J/cm²; ω_r: 200 μm ± 10 μm; τ_p: 5 μs, repetition rate 30 Hz. A 10 week old hsp70A1-L2G transgenic mouse was placed and treated on a 20 cm diameter holding plate. (b) Computer-assisted-surgical-technique (CAST) guided FEL wounding.

Fig. 2

Visualization of hsp70 promoter activity on laser-treated mouse skin. Bioluminescent imaging time course of a FEL wounded hsp70-luc mouse dermis. (a) Time= 0 h. (b) Time= 5-h postsurgery. (c) Time= 7-h postsurgery. (d) Time= 12-h postsurgery. The FEL parameters used to make the wounds were the following: λ=6.10 μm, ω_r = 200 μm, τ_p = 5 μs, 30 Hz; H: 7.96, 11.94, 15.92, and 19.89 J/cm². Laser-treated tissue has ~18× higher bioluminescence intensities (scale bar= 6 mm).

Fig. 3

Quantification of hsp70 expression using bioluminescent imaging (BLI). Hsp70 fold-induction (BLI relative to control) plotted versus time. Bioluminescent images were integrated over 30 s. Values are reported as mean± SEM. FEL at (a) λ= 6.45 μm, (b) λ= 6.10 μm, and (c) λ= 2.94 μm.

Fig. 4

Morphological methods to measure depth of damage and fluorescent imaging to measure hsp70 expression. Histology and confocal fluorescence imaging of sections of laser-irradiated dermal tissue. For a consistent comparison, exposures that induced comparable (12-fold) increases in hsp70 expression are shown. Radiant exposures of 11.94, 15.92, and 1.59 J/cm² were used for the wavelengths of 6.45, 6.10, and 2.94 μm, respectively. Tissue was harvested 12-h postinjury. (a), (b), and (c) Fluorescence imaging of hsp70 induced GFP expression (scale bar = 100 μm, epidermis= epi, dermis= d, a = adipose tissue, and pc= panniculus carnosus). (d), (e), and (f) HE stained sections (scale bar= 75 μm). (g), (h), and (i) Gomori’s (green) trichrome stain is used to visualize the depth of damage of laser-treated tissue (scale bar= 75 μm).

Fig. 5

Quantification of fluorescence signal. (a) Fluorescent integrated intensity versus depth of tissue (μm). Fluorescent intensity was integrated within rectangular regions of interest (13×100 μm) going from the skin surface (z = 0 μm) to the muscle (z = 250 μm). Mean fluorescence intensity is plotted and the SD <5% (SD not plotted for clarity). (b) Average cell number per area versus depth of tissue. (c) The fluorescent intensity values plotted in (a) were normalized to the cell numbers in (b) and plotted as function of tissue depth.

Fig. 6

Collateral thermal damage. (a) The max depth of damage at 12 h plotted versus radiant exposure (J/cm²). Higher FEL radiant exposures cause deeper tissue damage. At a given radiant exposure the 2.94-μm treatment damages ~2× deeper than 6.45 μm and ~3× deeper than 6.10 μm. Depth of damage was measured from tissue sections using histological stains (Gomori’s trichrome). The max depth of damage is expressed as mean± SD, n = 90. (b) Peak hsp70 fold-induction is plotted versus radiant exposure (J/cm²) for 2.94, 6.10, and 6.45 μm. (c) Peak hsp70 fold-induction plotted versus depth of tissue damage (μm). The data for 6.45 and 6.10 μm lie on the same line (black dotted line). The 2.94-μm data are shifted (i.e., more hsp70 expression for a given depth of damage) for radiant exposures up to 4.76 J/cm² (gray dotted line), and then shows a decrease in hsp70 expression for higher radiant exposures (and deeper depths) that is not seen in either 6.10- or 6.45-μm irradiated tissues.

Fig. 7

Epidermal hyperplasia: an early wound repair response in laser-treated tissues. (a–d) Sample representation of Gomori trichrome stains of tissues evaluated 120-h postsurgery (scale bar= 50 μm). (a) Untreated tissue, (b) λ= 6.10 μm, (c) λ= 6.45 μm, and (d) λ = 2.94 μm. All tissues were treated with a radiant exposure of 11.94 J/cm². The 6.10 μm generated the least amount of epidermal thickening. (Note: arrow demarcates the zone of epidermal hyperplasia.) (e) The magnitude of epidermal thickening 120 h after FEL exposure is plotted versus the radiant exposure for each wavelength. (Note: untreated tissue has an average thickness of 20 μm). The magnitude of epidermal thickening is expressed as the mean± SEM. Samples were compared using a student’s t-test (* = P <0.01, ** = P <0.005, *** = P <0.001, and n = 25).