We studied the efficacy of mild hyperthermia as a protective measure against subsequent laser-induced thermal damage. Using a well established in vitro retinal model for laser bioeffects, consisting of an artificially pigmented human retinal pigment epithelial (RPE) cell culture (hTERT-RPE1), we found both protection and sensitization to laser damage that depended upon the location of pigment granules during the hyperthermia preconditioning (PC). Photothermal challenge of cell monolayers consisted of 16 independent replicate exposures of 65 W/cm 2 at 514 nm and post laser damage was assessed using fluorescence indicator dyes. Untreated cells had 44% damage, but when melanosome particles (MPs) were intracellular or extracellular during the hyperthermia treatment, laser-induced cell damage occurred 94% or 25% of the time, respectively. Using a recently published method called microthermography, we found that the hyperthermia pretreatment did not alter the threshold temperature for cell death, indicating an alteration in absorption or localization of heat as the mechanism for sensitization and protection. Raman microspectroscopy revealed significant chemical changes in MPs when they were preconditioned within the cytoplasm of cells. Our results suggest intracellular pigment granules undergo chemical modifications during mild hyperthermia that can profoundly affect absorption or heat dissipation.
We measured threshold temperatures for cell death resulting from short (0.1-1.0 s) 514-nm laser exposures using an in vitro retinal model. Real-time thermal imaging at sub-cellular resolution provides temperature information that is spatially correlated with cells at the boundary of cell death, as indicate by post-exposure fluorescence images. Our measurements indicate markedly similar temperatures, not only around individual boundaries (single exposure), but among all exposures of the same duration in a laser irradiance-independent fashion. Two different methods yield similar threshold temperatures with low variance. Considering the experimental uncertainties associated with the thermal camera, an average peak temperature of 53 ± 2 °C is found for laser exposures of 0.1, 0.25, and 1.0 s. Additionally, we find a linear relationship between laser exposure duration and time-averaged integrated temperature. The mean thermal profiles for cells at the boundary of death were assessed using the Arrhenius rate law using parameter sets (frequency factor and energy of activation) found in three different articles.
We use laser damage thresholds in an in-vitro retinal model, and computational simulations to examine the laser exposure durations at which damage transitions from photothermal to photochemical at 413 nm. Our results indicate a dramatic shift in 1-h damage thresholds between exposure durations of 60 and 100 s. The trend in our in-vitro results is similar to a trend found in a recent study where retinal lesions were assessed 1-h post laser exposure in the rhesus eye Our data suggest that nonthermal mechanisms did not significantly contribute to cell death, even for exposures of 60 s. Knowledge of the transition point, and lack of concurrent thermal and nonthermal damage processes, are significant for those wishing to devise a comprehensive computational damage model.
Even though laser exposures of 1 s or less are non-isothermal events, researchers have had to rely upon the
isothermal treatise of Arrhenius to describe the laser damage rate processes. To fully understand and model
thermal damage from short exposure to laser irradiation we need to experimentally obtain the temperature
history of exposed cells and correlate it with the cellular damage outcomes. We have recorded the thermal
response of cultured retinal pigment epithelial cells in real-time with laser exposure using infrared imaging
(thermography). These images were then overlaid with fluorescence images indicating cell death taken 1 hr
post laser exposure. The image overlays allowed us to define the thermal history of cells at the boundary
(threshold) of laser-induced death. We have found a correlation between the onset of cell death and the
average temperature over the course of the laser exposure.
KEYWORDS: In vitro testing, Animal model studies, Data modeling, Laser damage threshold, Eye models, In vivo imaging, Retina, Mathematical modeling, Beam shaping, Laser induced damage
Without effective in vitro damage models, advances in our understanding of the physics and biology of laser-tissue interaction would be hampered due to cost and ethical limitations placed on the use of nonhuman primates. We extend our characterization of laser-induced cell death in an existing in vitro retinal model to include damage thresholds at 514 and 413 nm. The new data, when combined with data previously reported for 532 and 458 nm exposures, provide a sufficiently broad range of wavelengths and exposure durations (0.1 to 100 s) to make comparisons with minimum visible lesion (in vivo) data in the literature. Based on similarities between in vivo and in vitro action spectra and temporal action profiles, the cell culture model is found to respond to laser irradiation in a fundamentally similar fashion as the retina of the rhesus animal model. We further show that this response depends on the amount of intracellular melanin pigmentation.
Thresholds for photochemical damage were performed in RPE cell lines (artificially pigmented) taken from either human (hTERT-RPE1), wild type (wt) mouse, or transgenic mice deficient (+/-) in either superoxide dismutase 1 (SOD1) or SOD2. The four cell lines were characterized by immunohistochemical and immunoblot analyses to
determine relative abundance of the SOD proteins. There was no difference in sensitivity between the human, murine wt and murine SOD1-deficient cells, whereas there was a dramatic (2 fold) increase in threshold irradiance value for the murine SOD2-deficient cells. Possible explanations for the unexpected result are provided.
Corneal organotypic cultures were generated as per existing methods, which included growth on polycarbonate inserts and air-lifting for one week. The corneal simulant cultures were exposed, with real-time IR imaging, to the 2-μm wavelength output of a thulium fiber laser with 4 mm beam diameter for 0.25 seconds in a thermally controlled
environment and then assayed for damage. The in vitro threshold (ED50 value of 12.5 W/cm2) and peak temperature (74.5 °C) at threshold irradiance are compared with rabbit corneal data in the literature.
The determination of safe exposure levels for lasers has come from damage assessment experiments in live animals, which typically involve correlating visually identifiable damage with laser dosimetry. Studying basic mechanisms of laser damage in animal retinal systems often requires tissue sampling (animal sacrifice), making justification and animal availability problematic. We determined laser damage thresholds in cultured monolayers of a human retinal pigment epithelial (RPE) cell line. By varying exposure duration and laser wavelength, we identified conditions leading to damage by presumed photochemical or thermal mechanisms. A comparison with literature values for ocular damage thresholds validates the in vitro model. The in vitro system described will facilitate molecular and cellular approaches for understanding laser-tissue interaction.
KEYWORDS: Animal model studies, In vitro testing, Data modeling, Laser induced damage, Laser damage threshold, Eye models, In vivo imaging, Systems modeling, Beam shaping, Tissues
Ocular laser exposures resulting in damage at the retina typically involve cellular alterations in the retinal pigment epithelial (RPE) layer. To provide guidelines for eye-safe exposure to lasers, the laser safety community has relied on damage assessment in nonhuman primate studies. Simple and reliable model systems for laser bioeffects that use cultured RPE cells, rather than animals, are thus desirable. We have characterized our artificially pigmented hTERT-RPE1 model by identifying ED50 thresholds over a wide
range of laser parameters and cell culture conditions. When summarized as action spectra and temporal action profiles (log threshold fluence versus log exposure duration), trends (pigment-dependent) in our cell model data are strikingly similar to the threshold trends reported for animal models (literature). In addition,
the rapidity and flexibility (laser delivery) with which studies are performed in our culture model has benefited computational modeling efforts.
Artificially pigmented hTERT-RPE1 cells were exposed to a mode-locked or continuous wave (CW) laser at 458 nm for one hour in a modified culture incubator. Exposure conditions were selected to give greatest likelihood of damage due to a photochemical mechanism, with interest in possible differences between CW and mode-locked damage thresholds. After post-exposure-recovery (PER) for either 1-hour or 24-hour, cells were concurrently stained with annexin V and 6-CFDA to determine if they had undergone necrosis or apoptosis. Alternatively, cells were stained with Ethidium Homodimer (EthD-1) and Calcein AM to determine if they had undergone necrosis following 1-hour and 24-hours PER. Preliminary results indicate that laser exposure induced some apoptosis following 1-hour PER, with irradiance required for apoptosis being lower than that for necrosis with mode-locked exposure conditions. Probit analysis yielded necrosis thresholds for cell culture following 1-hour PER using data compiled from both dye sets. CW exposures resulted in a lower threshold than mode-locked exposures for necrosis following 1-hour PER. A thermal model provided the predicted temperature rise in cell culture due to laser exposure. The thermal model validates our choice of laser parameters to obtain photochemical damage. Data following 24-hours PER were inconclusive. Considerations of cell migration are included in the interpretation of data and further improvements to methods when using live cell assays are recommended.
KEYWORDS: Luminescence, In vivo imaging, Laser damage threshold, Gaussian beams, In vitro testing, Microscopes, Amplifiers, Sensors, Eye, Argon ion lasers
We performed measurements to validate damage threshold trends in minimum visible lesion (MVL) studies as a function of spot size for nanosecond laser pulses. At threshold levels, nanosecond pulses produce microcavitation bubbles that expand and collapse around individual melanosomes. This microcavitation process damages the membranes of retinal pigment epithelium (RPE) cells. A spot size study on retinal explants found cell damage fluence (energy/area) thresholds were independent of spot size when microcavitation caused the damage, contradicting past in vivo retinal spot size experiments. The explant study (ex vivo) used a top-hat beam profile, whereas the in vivo studies used Gaussian beams. The difference in spot size trends for damage in vivo versus ex vivo may be attributed to the optics of the eye but this has not been validated. In this study, we exposed artificially pigmented human RPE cells (hTERT-RPE1)-in vitro-to 7 ns pulsed irradiation from a Ti:Sa TSA-02 regenerative amplifier (1055 nm) with beam diameters of 44, 86, and 273 μm (Gaussian beam profiles). We detected the microcavitation event with strobe illumination and time-resolved imaging. We used the fluorescent indicator dye calcein-AM, with excitation by an Argon laser (488 nm), to assess cell damage. Our current results follow trends found in the in vivo studies.
A thermal model was used to calculate the skin temperature rise in porcine skin and predict the damage thresholds in terms of laser power for various wavelengths, pulse durations, skin parameters and laser spot sizes. Laser exposures of 1.54 μm, 0.60 ms in duration and using a 0.7 mm spot size were applied to the porcine skin. The damage thresholds were determined at 1-hour and 24-hour post exposures using probit analysis. Only one subject was exposed giving adequate fiducial limits at the 95% confidence level. The ED50 for these 72 exposures was determined to be 58 mJ, giving a radiant exposure of 15 Jcm-2. The damage threshold is compared with model predictions, with work previously published in the literature and with the ANSI Standard’s MPE for 1540 nm lasers at 0.60 ms.
With the advent of future weapons systems that employ high energy lasers, the 1315 nm wavelength will present a new laser safety hazard to the armed forces. Experiments in non-human primates using this wavelength have demonstrated a range of ocular injuries, including corneal, lenticular and retinal lesions, as a function of pulse duration and spot size at the cornea. To improve our understanding of this phenomena, there is a need for a mathematical model that properly
predicts these injuries and their dependence on appropriate exposure parameters. This paper describes the use of a finite difference model of laser thermal injury in the cornea and retina. The model was originally developed for use with shorter wavelength laser irradiation, and as such, requires estimation of several key parameters used in the computations. The predictions from the model are compared to the experimental data, and conclusions are drawn
regarding the ability of the model to properly follow the published observations at this wavelength.
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