Computational models predicting cell damage responses to transient temperature rises generated by exposure to lasers have implemented the damage integral (Ω), which time integrates the chemical reaction rate constant described by Arrhenius. However, few published reports of empirical temperature histories (thermal profiles) correlated with damage outcomes at the cellular level are available to validate the breadth of applicability of the damage integral. In our study, an analysis of photothermal damage rate processes in cultured retinal pigment epithelium cells indicated good agreement between temperature rise, exposure duration (τ), and threshold cellular damage. Full-frame thermograms recorded at high magnification during laser exposures were overlaid with fluorescence damage images taken 1 h postexposure. From the image overlays, pixels of the thermogram correlated with the boundary of cell death were used to extract threshold thermal profiles. Assessing photothermal responses at these boundaries standardized all data points, irrespective of laser irradiance, damage size, or optical and thermal properties of the cells. These results support the hypothesis that data from boundaries of cell death were equivalent to a minimum visible lesion, where the damage integral approached unity (Ω = 1) at the end of the exposure duration. Empirically resolved Arrhenius coefficients for use in the damage integral determined from exposures at wavelengths of 2 μm and 532 nm and durations of 0.05–20 s were consistent with literature values. Varying ambient temperature (Tamb) between 20°C and 40°C during laser exposure did not change the τ-dependent threshold peak temperature (Tp). We also show that, although threshold laser irradiance varied due to pigmentation differences, threshold temperatures were irradiance independent.
Photobiomodulation (PBM) is a biological outcome of exposure to low-level light in the red and near-infrared (NIR) wavelengths. Current literature has attributed beneficial effects to PBM, to include improved wound healing, enhanced mitochondrial function, functional enhancements in patients suffering from stroke, and improved cognitive function in a murine model for traumatic brain injury. Cytochrome c oxidase, also named complex IV (C-IV) in the electron transport chain (ETC), is the expected primary chromophore for the red and NIR exposures. The direct evidence that PBM is a consequence of absorption by C-IV is incomplete. Recently, our lab has found metabolic perturbations in cells and isolated mitochondria from low-level exposures to blue and green light as well. To study the immediate and early events of PBM we used a combination of fluorescence microscopy, resonance Raman spectroscopy, Fourier transformed IR (FTIR) spectroscopy, and ultrafast transient absorption spectroscopy (TAS) on cells, isolated mitochondria, and purified ETC enzymes. In this paper, we show that FTIR spectroscopy is useful in determining substrate-dependent, steady-state rates of CO2 production by the tricarboxylic acid (TCA) cycle. The method allows for determinations of wavelength-specific changes in metabolic rate in real time with low-level light exposures. These data will help determine if any mitochondrial components have absorption spectra that correlate with the global PBM response in the literature.
Photobiomodulation, also known as low level laser therapy (LLLT), is a technique that uses light in the red and near infrared (NIR) range (600-900 nm) to elicit a clinically beneficial physiological change in tissue. This physiological change is thought to begin in the mitochondria by altering the metabolic rate for the electron transport chain (ETC). Resonance Raman spectroscopy at 532 nm was used to determine the reduction/oxidation (redox) state of cytochrome c in isolated mitochondria after undergoing LLLT. Mitochondria from hTERT-RPE1 cells were isolated and placed in glutamate buffer and then exposed to violet (405 nm) or red (635 nm) light. The resonance Raman spectrum of the cytochrome c redox state before and after light illumination was measured. This gives us an insight into the types of metabolic changes that occurs within the mitochondria while being illuminated by light during photobiomodulation.
Photobiomodulation therapy has been around for several decades with a number of applications increasing every year. However, the exact mechanisms of energy/signal transduction, which lead to the observed changes, and the lack of those in living cells, is still poorly understood. We propose and experimentally implement a novel research platform, which uses a microfluidic chip and controllable excitation condition to precisely initiate the process of photobiomodulation. Resonance Raman spectroscopy is employed to assess the structural transformational stages on different time-scales. We will present our first experimental results and discuss those results with respect to potential therapeutic applications.
Resonance Raman spectroscopy of cytochrome c was used to follow reduction/oxidation (redox) states of isolated mitochondria in response to blue or red laser exposure. Mitochondria were isolated from hTERT-RPE1 cells and were kept in a buffer formulation known to be conducive to electron transport chain (ETC) activity. Using either pyruvate or succinate as substrates for ETC, we found differences in the redox responses of cytochrome c for different exposure laser irradiance and excitation wavelength. We anticipate that the proposed new method will be valuable in the study of metabolic processes in mitochondria in response to low level laser exposure, and thus aid in elucidating the mechanism(s) of photobiomodulation.
Thermal damage rate processes in biological tissues are usually characterized by a kinetics approach. This stems from experimental data that show how the transformation of a specified biological property of cells or biomolecule (plating efficiency for viability, change in birefringence, tensile strength, etc.) is dependent upon both time and temperature. Here, two disparate approaches were used to study thermal damage rate processes in cultured retinal pigment epithelial cells. Laser exposure (photothermal) parameters included 2-μm laser exposure of non-pigmented cells and 532-nm exposures of cells possessing a variety of melanosome particle densities. Photothermal experiments used a mid-IR camera to record temperature histories with spatial resolution of about 8 μm, while fluorescence microscopy of the cell monolayers identified threshold damage at the boundary between live and dead cells. Photothermal exposure durations ranged from 0.05-20 s, and the effects of varying ambient temperature were investigated. Temperature during heat transfer using a water-jacketed cuvette was recorded with a fast microthermister, while damage and viability of the suspended cells were determined as percentages. Exposure durations for the heat transfer experiments ranged from 50- 60 s. Empirically-determined kinetic parameters for the two heating methods were compared with each other, and with values found in the literature.
KEYWORDS: Optical character recognition, Acquisition tracking and pointing, Oxygen, In vitro testing, NOx, Electron transport, Curium, Control systems, Pulsed laser operation, Absorption
Exposure to 2.88 J/cm2 of red light induces an adaptive response against a lethal pulse of 2.0 μm laser radiation in hTERT-RPE cells in vitro, but not in a knockdown mutant for vascular endothelial growth factor c (VEGF-C). The generally accepted initiation sequence for photobiomodulation is that absorption of red light by cytochome c oxidase (CCOX) of the electron transport chain increases the binding affinity of CCOX for O2 vs. nitric oxide (NO). This results in displacement of NO by O2 in the active site of CCOX, thereby increasing cellular respiration and intracellular ATP. We've previously reported that red-light exposure induces a small, but consistently reproducible, increase in NO levels in these cells. But the relative importance of NO and oxidative phosphorylation is unclear because little is known about the relative contributions of NO and ATP to the response. However, if NO dissociation from CCOX actually increases oxidative phosphorylation, one should see a corresponding increase in oxygen consumption. A Seahorse Extracellular Flux Analyzer was used to measure oxygen consumption rates (OCR) in normal and mutant cells as a proxy for oxidative phosphorylation. Both basal respiration and maximum respiration rates in normal cells are significantly higher
than in the mutant. The normal cells have a significant amount of “excess capacity,” whereas the VEGF-C(KD) have
little or none. The OCR in exposed normal cells is lower than in unexposed cells when measured immediately after
exposure. The exposures used for these experiments had no effect on the OCR in mutant cells.
Laser damage thresholds were determined for exposure to 2.5-ms 532-nm pulses in an established in vitro retinal model. Single and multiple pulses (10, 100, 1000) were delivered to the cultured cells at three different pulse repetition frequency (PRF) values, and overt damage (membrane breach) was scored 1 hr post laser exposure. Trends in the damage data within and across the PRF range identified significant thermal additivity as PRF was increased, as evidenced by drastically reduced threshold values (< 40% of single-pulse value). Microthermography data that were collected in real time during each exposure also provided evidence of thermal additivity between successive laser pulses. Using thermal profiles simulated at high temporal resolution, damage threshold values were predicted by an in-house computational model. Our simulated ED50 value for a single 2.5-ms pulse was in very good agreement with experimental results, but ED50 predictions for multiple-pulse trains will require more refinement.
An artificially pigmented retinal pigment epithelial (RPE) cell model was used to study the damage rates for exposure to 1, 10, 100, and 1,000 230-μs laser pulses at 532 nm, at two different concentrations of melanosome particles (MPs) per cell. Multiple pulses were delivered at pulse repetition rates of 50 and 99 pulses per second. Standard fluorescence viability indicator dyes and the method of microthermography were used to assess damage and thermal responses, respectively. Although frame rate during microthermography was more than five times slower than the duration of laser pulses, thermal information was useful in refining the BTEC computational model for simulating high-resolution thermal responses by the pigmented cells. When we temporally sampled the thermal model output at a rate similar to our microthermography, the resulting thermal profiles for multiple pulses resembled the thermal experimental profiles. Complementary to the thermal simulations, our computer-generated thresholds were in good agreement with the in vitro data. Findings are examined within the context of common exposure limit definitions in the national and international laser safety standards.
Cells in a tissue culture model for laser eye injury exhibit increased resistance to a lethal pulse of 2.0-μm laser radiation
if the cells are first exposed to 2.88 J/cm2 of red light 24 hr prior to the lethal laser exposure. Changes in expression of
various genes associated with apoptosis have been observed, but the biochemical link between light absorption and gene
expression remains unknown. Cytochome c oxidase (CCOX), in the electron transport chain, is the currentlyhypothesized
absorber. Absorption of the red light by CCOX is thought to facilitate displacement of nitric oxide (NO)
by O2 in the active site, increasing cellular respiration and intracellular ATP. However, NO is also an important
regulator and mediator of numerous physiological processes in a variety of cell and tissue types that is synthesized from
l-arginine by NO synthases. In an effort to determine the relative NO contributions from these competing pathways, we
measured NO levels in whole cells and subcellular fractions, with and without exposure to red light, using DAF-FM, a
fluorescent dye that stoichiometrically reacts with NO. Red light induced a small, but consistently reproducible,
increase in fluorescence intensity in whole cells and some subcellular fractions. Whole cells exhibited the highest
overall fluorescence intensity followed by (in order) cytosolic proteins, microsomes, then nuclei and mitochondria.
A computer-based model has been built that simulates the response of the retinal pigmented epithelial (RPE) cell to laser
exposure in the photochemical (non-thermal) damage exposure range (≥ 100 s exposures). The modeling approach used
is knowledge-based, modular, and hierarchical, allowing the explicit modeling of the cascades of intracellular events in
response to laser application. Thus, the model can be used to both analyze existing in vitro data sets, as well as
efficiently direct sampling strategies for future in vitro and in vivo studies. This model has been validated using
laboratory data from several studies reported in the literature using blue light (413 nm and 458 nm) lasers with 100 s, 200
s, and 3600 s exposure durations. The model was able to predict the in vitro ED50 response curve from these studies, as
well as the results for which we have no in vitro data (extrapolated based on irradiance reciprocity), within 1-6% for the
shorter duration exposures. Based on exploration of this computer model using lethal vs. non-lethal laser exposure
scenarios, the RPE cell’s oxidative stress response differs quantitatively very little with respect to typical oxidative stress
sources such as superoxide and hydrogen peroxide. However, in the lethal exposure scenarios the model points to a
potential tipping point in the oxidative stress response of the mitochondrial-based cellular energetics. Further studies are
underway to explore issues related to the levels of ATP/ADP and GSH/GSSG that are predicted by the model in these
lethal vs. non-lethal exposure scenarios.
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