Assessing laser-tissue damage with bioluminescent imaging

Abstract

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 (lambda=10.6 microm, 0.679 to 2.262 Wcm2, cw, unfocused Gaussian beam, omegaL=4.5 mm, 1 min exposure). The bioluminescence was monitored noninvasively with an IVIS 100 Bioluminescent Imaging System. BLI indicated that peak hsp70 expression occurs 4 to 12 h after exposure to thermal stress. A minimum irradiance of 0.679 Wcm2 activated the hsp70 response, and a higher irradiance of 2.262 Wcm2 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 150 microm. 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.

Spatially correlated microthermography maps threshold temperature in laser-induced damage

“…4,21 We were surprised that our data do not follow the common single-process rate model (Arrhenius) when used to estimate thermal damage from laser exposures of 0.1-1.0 s. Additional studies, characterizing the kinetics of the damage response to short photothermal exposures, are needed to formulate new hypotheses and computational models that describe the damage rate processes evident in our in vitro retinal model. Although cells receiving threshold thermal doses and greater are destined to die, cells outside of the boundary of death are expected to have temperature-dependent heat shock responses 5,6,21,22 that may trigger delayed death, 23 oncogenesis, 24 or even protective adaptive responses. 25 In addition, microthermography in cell cultures could provide thermal dose-response assessments for efficient killing in selective photothermal therapy regimes using aptamer-conjugated nanoparticles, 26 reducing the number of research animals required in preclinical trials.…”

Wound healing from a cellular stress response perspective

“…Based on a review of the cytoprotection literature as it relates to thermal induction of HSP70, the authors hypothesized that laser pretreatment of skin to induce HSP70 would lead to improved dermal wound healing as measured by wound burst strength. Prior work from this group describes the laser thermal doses used to obtain dermal HSP70 expression (Wilmink et al 2006). A single treatment (43°C×15 min), 12 h before wounding resulted in significantly stronger tissue repair compared to the untreated animals.…”

Identification of microRNAs associated with hyperthermia-induced cellular stress response

“…Cell culture conditions and hyperthermia stress protocol Normal adult human dermal fibroblasts (HDF) were cultured as described previously (Wilmink et al 2006). In brief, HDFs were plated in 60-mm dishes (5,000 cells/cm 2 ) and were incubated overnight.…”

“…RNA isolation and normalization RNA was harvested from samples 4 h post-exposure, a time point shown to have maximum HSPA1A mRNA levels, using RNeasy Mini-Kit (Qiagen) (Wilmink et al 2006). The RNA concentration was assessed on a NanoDrop Spectrophotometer (NanoDrop Technologies), and the quality was measured on a 2100 Bioanalyzer™ (Agilent Technologies).…”

“…Calibrator RNA was used as control (Cell Applications Inc). PCR was conducted using a threeprogram LightCycler® protocol and analyzed as previously described (Wilmink et al 2006). miRNA microarrays and PCR miRNA microarrays were used to identify stress-responsive miRNAs, and qPCR was conducted to verify their expression.…”