Rapid development in nanomaterial synthesis and functionalization has led to advanced studies in actuation and manipulation of cellular functions for biomedical applications. Often these actuation techniques employ externally applied magnetic fields to manipulate magnetic nanomaterials inside cell bodies in order to drive or trigger desired effects. While cellular interactions with low-frequency magnetic fields and nanoparticles have been extensively studied, the fundamental mechanisms behind these interactions remain poorly understood. Additionally, modern investigations on these concurrent exposure conditions have been limited in scope, and difficult to reproduce. This study presents an easily reproducible method of investigating the biological impact of concurrent magnetic field and nanoparticle exposure conditions using an in-vitro CHO-K1 cell line model, with the purpose of establishing grounds for in-depth fundamental studies of the mechanisms driving cellular-level interactions. Cells were cultured under various nanoparticle and magnetic field exposure conditions from 0 to 500 μg/ml nanoparticle concentrations, and DC, 50 Hz, or 100 Hz magnetic fields with 2.0 mT flux density. Cells were then observed by confocal fluorescence microscopy, and subject to biological assays to determine the effects of concurrent extreme-low frequency magnetic field and nanoparticle exposures on cellnanoparticle interactions, such as particle uptake and cell viability by MTT assay. Current results indicate little to no variation in effect on cell cultures based on magnetic field parameters alone; however, it is clear that deleterious synergistic effects of concurrent exposure conditions exist based on a significant decrease in cell viability when exposed to high concentrations of nanoparticles and concurrent magnetic field.
Phosphatidylinositol4,5-biphosphate (PIP2) is a membrane phospholipid of particular importance in cell-signaling pathways. Hydrolysis of PIP2 releases inositol-1,4,5-triphosphate (IP3) from the membrane, activating IP3 receptors on the smooth endoplasmic reticulum (ER) and facilitating a release of intracellular calcium stores and activation of protein kinase C (PKC). Recent studies suggest that nanosecond pulsed electric fields (nsPEF) cause depletion of PIP2 in the cellular membrane, activating the IP3 signaling pathway. However, the exact mechanism(s) causing this observed depletion of PIP2 are unknown. Complicating the matter, nsPEF create nanopores in the plasma membrane, allowing calcium to enter the cell and thus causing an increase in intracellular calcium. While elevated intracellular calcium can cause activation of phospholipase C (PLC) (a known catalyst of PIP2 hydrolysis), PIP2 depletion has been shown to occur in the absence of both extracellular and intracellular calcium. These observations have led to the hypothesis that the high electric field itself may be playing a direct role in the hydrolysis of PIP2 from the plasma membrane. To support this hypothesis, we used edelfosine to block PLC and prevent activation of the IP3/DAG pathway in Chinese Hamster Ovarian (CHO) cells prior to applying nsPEF. Fluorescence microscopy was used to monitor intracellular calcium bursts during nsPEF, while MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) survivability assays were utilized to determine whether edelfosine improved cell survival during nsPEF exposure. This work is critical to refine the role of PIP2 in the cellular response to nsPEF, and also to determine the fundamental biological effects of high electric field exposures.
A temperature detection system using a micropipette thermocouple sensor was developed for use within mammalian cells during laser exposure with an 8.6-μm beam at 532 nm. We have demonstrated the capability of measuring temperatures at a single-cell level in the microscale range by inserting micropipette-based thermal sensors of size ranging from 2 to 4 μm into the membrane of a live retinal pigment epithelium (RPE) cell subjected to a laser beam. We setup the treatment groups of 532-nm laser-irradiated single RPE cell and in situ temperature recordings were made over time. Thermal profiles are given for representative cells experiencing damage resulting from exposures of 0.2 to 2 s. The measured maximum temperature rise for each cell ranges from 39 to 73°C; the RPE cells showed a signature of death for all the cases reported herein. In order to check the cell viability, real-time fluorescence microscopy was used to identify the transition of pigmented RPE cells between viable and damaged states due to laser exposure.
The unique cellular response to nanosecond pulsed electric field (nsPEF) exposure, as compared to longer pulse exposure, has been theorized to be due to permeabilization of intracellular organelles including the mitochondria. In this investigation, we utilized a high-throughput oxygen and pH sensing system (Seahorse® XF24 extracellular flux analyzer) to assess the mitochondrial activity of Jurkat and U937 cells after nsPEF. The XF Analyzer uses a transient micro-chamber of only a few μL in specialized cell culture micro-plates to enable oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to be monitored in real-time. We found that for nsPEF exposures of 10 pulses at 10-ns pulse width and at 50 kV/cm e-field, we were able to cause an increase in OCR in both U937 and Jurkat cells. We also found that high pulse numbers (>100) caused a significant decrease in OCR. Higher amplitude 150 kV/cm exposures had no effect on U937 cells and yet they had a deleterious effect on Jurkat cells, matching previously published 24 hour survival data. These results suggest that the exposures were modulating metabolic activity in cells possibly due to direct effects on the mitochondria themselves. To validate this hypothesis, we isolated mitochondria from U937 cells and exposed them similarly and found no significant change in metabolic activity for any pulse number. In a final experiment, we removed calcium from the buffer solution that the cells were exposed in and found that no significant enhancement in metabolic activity was observed. These results suggest that direct permeabilization of the mitochondria is unlikely a primary effect of nsPEF exposure and calcium-mediated intracellular pathway activation is likely responsible for observed pulse-induced mitochondrial effects.
Data showing what appears to be nonthermal inactivation of M13 bacteriophage (M13), Tobacco mosaic virus, Escherichia coli (E. coli), and Jurkatt T-cells following exposure to 80-fs pulses of laser radiation have been published. Interest in the mechanism led to attempts to reproduce the results for M13 and E. coli. Bacteriophage plaque-forming and bacteria colony-forming assays showed no inactivation of the microorganisms; therefore, model systems were used to see what, if any, damage might be occurring to biologically important molecules. Purified plasmid DNA (pUC19) and bovine serum albumin were exposed to and analyzed by agarose gel electrophoresis (AGE) and polyacrylamide gel electrophoresis (PAGE), respectively, and no effect was found. DNA and coat proteins extracted from laser-exposed M13 and analyzed by AGE or PAGE found no effect. Raman scattering by M13 in phosphate buffered saline was measured to determine if there was any physical interaction between M13 and femtosecond laser pulses, and none was found. Positive controls for the endpoints measured produced the expected results with the relevant assays. Using the published methods, we were unable to reproduce the inactivation results or to show any interaction between ultrashort laser pulses and buffer/water, DNA, protein, M13 bacteriophage, or E. coli.
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.
The goal of this project is to explore the role of nitric oxide (NO) in regulating the response of hTERT-RPE to low-level exposures to red light. Exposure to low-level red light has been shown to positively affect wound healing, reduce pain, and encourage cell proliferation. The current explanation for this effect is described as an interaction between the photons and cytochrome c oxidase (Cco), which plays a role in regulation of intracellular NO levels in addition to being the mitochondrial protein complex where reduction of oxygen occurs in the process of oxidative phosphorylation. Exposure to 2.88 J/cm2 of 671-nm and 637-nm light shows a two-fold increase in NO immediately after exposure, and a 56% increase in ATP measured at ~5 h post exposure. Levels of NF-κB mRNA and protein were measured at six and 24 h, respectively, and found to increase six fold, correlating with increases in NO levels. Light-stimulated increased levels of NO also correlated with an 11-fold increase in Bcl-2 and a 70% decrease in Bax mRNA levels, relative to controls. NF-κB promotes cell growth and Bcl-2 is an apoptosis suppressor protein. Bax is a positive apoptotic effector protein. These results support the hypothesis that light-induced changes in the intracellular levels of NO play a role in the beneficial effects of low-level light photobiomodulation
The objective of this study was to elucidate cellular mechanisms of protection against laser-induced
thermal killing utilizing an in vitro retina model. When exposed to a 1-sec pulse of 2-μm laser
radiation 24 hr after illuminating hTERT-RPE cells with red light (preconditioning), the cells are
more resistant to thermal challenge than unilluminated controls (adaptive response). Results of
efforts to understand the physiology of this effect led us to two genes: Vascular Endothelial Growth
Factor C (VEGF-C) and Micro RNA 146a (miR-146a). Transfecting wild type (WT) cells with
siRNA for VEGF-C and miR-146a mRNA resulted in knockdown strains (VEGF-C(KD) and miR-
146a(-)) with 10% and 30% (respectively) of the constitutive levels expressed in the WT cells.
To induce gene expression, WT or KD cells were preconditioned with 1.44 to 5.40 J/cm2, using
irradiances between 0.40 and 1.60 mW/cm2 of either 671-nm (diode) or 637-nm (laser) radiation.
Probit analysis was used to calculate threshold damage irradiance, expressed as ED50, between 10
and 100 W/cm2 for the 2-μm laser pulse. In the WT cells there is a significant increase in ED50 (p
0.05) with the maximum response occurring at 2.88 J/cm2 in the preconditioned cells. Neither KD
cell strain showed a significant increase in the ED50, although some data suggest the response may
just be decreased in the knockdown cells instead of absent. So far the response does not appear to
be dependent upon either wavelength (637 vs. 671 nm) or coherence (laser vs. LED), but there is
an irradiance dependence.
Exposure to nano-second pulsed electrical fields (nsPEFs) has been shown to cause poration of external and internal cell
membranes, DNA damage, and blebbing of the plasma membrane. Recovery from nsPEF exposure is likely dependent on
multiple factors, including exposure parameters, length of time between pulses, and extent of cellular damage. As cells
progress through the cell cycle, variations in DNA and nucleus structure, cytoskeletal arrangement, and elasticity of cell
membrane could cause nsPEFs to affect cells differently during different cell cycle phases. To better understand the
impact of nsPEF on cell cycle, we investigated CHO cell cycle progression following varying intensities of nsPEFexposures.
Cell populations were examined post exposure (10 ns pulse trains at 100, 150, or 200kV/cm) by analysis of
DNA content via propidium iodide staining and flow cytometric analysis to determine cell cycle phase. Populations
exhibited arrest in G2/M phase, but not in G1 phase at 1h post-exposure that increased in severity and duration with
increasing exposure dose. Recovery from arrest was complete after 12h, and populations did not exhibit an increase in
apoptosis as a result of exposure. Post exposure arrest in G2/M phase may indicate that nsPEF-induced damage is not
significant to cause G1 arrest or that mitotic checkpoints are more important regulators of cell cycle progression after
nsPEF exposure.
We were unable to reproduce published inactivation results, or show any interaction, between 90 femtosecond (fs) pulses
of 850 nm or 425 nm laser radiation and buffer/water, DNA, protein, M13 bacteriophage or E. coli. Using agarose
electrophoresis and polyacrylamide gel electrophoresis, we examined purified plasmid DNA (pUC19), bovine serum
albumin, and DNA and coat proteins extracted from M13 following exposures to irradiances of up to 120 MW/cm2. We
measured M13 viability using an assay for plaque-forming ability in soft agar after exposure to the same irradiances used
for the protein and DNA experiments. Exposures of up 1 GW/cm2 at 850 nm had no effect on the viability of E. coli as
measured by a colony forming assay in soft agar. Peroxynitrite, known to be toxic, to cause single strand breaks in
DNA, and fragment proteins in vitro gave positive results in all assays.
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.
Vascular endothelial growth factor (VEGF) is known for its role in neovascularization and cellular signaling pathways of
sub-threshold retinal lesions. The objective of this study was to elucidate potential protection mechanisms to laser-induced
heat stress utilizing an in vitro retinal model. The cell line was characterized to determine the relative abundance
of VEGF-C protein. Cells, preconditioned via water bath and controls, were then exposed to 2 μm laser radiation to
assess whether increases in protein production following preconditioning could confer any protection. There was no
significant increase in threshold damage irradiance (ED50) in the preconditioned cells versus control.
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.
Recent studies have determined that photochemical oxidation in cultured cells can be detected at peak irradiances as low as 8.5x108 W cm-2 (87-fs pulse). Fluorescent dyes, such as CM-H2DCFDA, enable us to quantify the oxidation response of cells to mode-locked near-infrared (NIR) laser exposure. Using a modified confocal microscope, we characterize the time-dependent 2-photon induced fluorescence generated from a given NIR laser exposure. When cultured cells were then pre-loaded with antioxidants, ascorbic acid or N-acetyl-L-cysteine (NAC), they inhibit nonlinear oxidation with different efficiencies, providing insight regarding mechanisms of damage.
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