Evaluation of the Genetic Response of U937 and Jurkat Cells to 10-Nanosecond Electrical Pulses (nsEP).

Nanosecond electrical pulse (nsEP) exposure activates signaling pathways, produces oxidative stress, stimulates hormone secretion, causes cell swelling and induces apoptotic and necrotic death. The underlying biophysical connection(s) between these diverse cellular reactions and nsEP has yet to be elucidated. Using global genetic analysis, we evaluated how two commonly studied cell types, U937 and Jurkat, respond to nsEP exposure. We hypothesized that by studying the genetic response of the cells following exposure, we would gain direct insight into the stresses experienced by the cell and in turn better understand the biophysical interaction taking place during the exposure. Using Ingenuity Systems software, we found genes associated with cell growth, movement and development to be significantly up-regulated in both cell types 4 h post exposure to nsEP. In agreement with our hypothesis, we also found that both cell lines exhibit significant biological changes consistent with mechanical stress induction. These results advance nsEP research by providing strong evidence that the interaction of nsEPs with cells involves mechanical stress.

Nanosecond pulsed electric fields modulate the expression of Fas/CD95 death receptor pathway regulators in U937 and Jurkat Cells.

In this publication, we demonstrate that exposure of Jurkat and U937 cells to nanosecond pulsed electrical fields (nsPEF) can modulate the extrinsic-mediated apoptotic pathway via the Fas/CD95 death receptor. An inherent difference in survival between these two cell lines in response to 10 ns exposures has been previously reported (Jurkat being more sensitive to nsPEF than U937), but the reason for this sensitivity difference remains unknown. We found that exposure of each cell line to 100, 10 ns pulses at 50 kV/cm caused a marked increase in expression of cFLIP (extrinsic apoptosis inhibitor) in U937 and FasL (extrinsic apoptosis activator) in Jurkat, respectively. Measurement of basal expression levels revealed an inherent difference between U937 cells, having a higher expression of cFLIP, and Jurkat cells, having a higher expression of FasL. From these data, we hypothesize that the sensitivity difference between the cells to nsPEF exposure may be directly related to expression of extrinsic apoptotic regulators. To validate this hypothesis, we used siRNA to knockdown cFLAR (coding for cFLIP protein) expression in U937, and FasL expression in Jurkat and challenged them to 100, 10 ns pulses at 150 kV/cm, a typical lethal dose. We observed that U937 survival was reduced nearly 60% in the knockdown population while Jurkat survival improved ~40%. These findings support the hypothesis that cell survival following 10 ns pulse exposures depends on extrinsic apoptotic regulators. Interestingly, pretreatment of U937 with a 100-pulse, 50 kV/cm exposure (to amplify cFLAR expression) significantly reduced the lethality of a 150 kV/cm, 100-pulse exposure applied 24 h later. From these data, we conclude that the observed survival differences between cells, exposed to 10 ns pulsed electric fields, is due to inherent cell biochemistry rather than the biophysics of the exposure itself. Understanding cell sensitivity to nsPEF may provide researchers/clinicians with a predicable way to control or avoid unintended cell death during nsPEF exposure.

Thermal evaluation of laser exposures in an in vitro retinal model by microthermal sensing.

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 micropipettebased 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.

No effect of femtosecond laser pulses on M13, E. coli, DNA, or protein.

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.

Hyperthermia sensitizes pigmented cells to laser damage without changing threshold damage temperature.

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/cm2 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.

Spatially correlated microthermography maps threshold temperature in laser-induced damage.

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.

In-vitro retinal model reveals a sharp transition between laser damage mechanisms.

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.

In vitro model that approximates retinal damage threshold trends.

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.

Damage thresholds for cultured retinal pigment epithelial cells exposed to lasers at 532 nm and 458 nm.

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.

Damage Thresholds for Exposure to NIR and Blue Lasers in an In Vitro RPE Cell System.

PURPOSE: Until reliable nonanimal systems of analysis are available, animal models will be necessary for ocular laser hazard analysis and for evaluating clinical applications. The purpose of this work was to demonstrate the utility of an in vitro system for laser bioeffects by identifying photothermal and photochemical cytotoxicity thresholds for continuous-wave (cw) and mode-locked (ml) laser exposures. METHODS: Exogenous melanosomes were added to hTERT-RPE1 cells in exposure wells 1 day before laser exposure. Thermal or photochemical laser exposures were delivered to artificially pigmented retinal pigment epithelial (RPE) cultures, with subsequent assay for viability 1 hour after exposure. Beam delivery for the 1-hour photochemical exposures was via a modified culture incubator. The cytoprotective effect of pretreatment with two antioxidants was investigated. RESULTS: Phagocytosis of melanosomes by the RPE cells was efficient, yielding cultures of uniform pigmentation. The damage threshold for the thermal exposure was consistent with published in vivo results. Thresholds for both blue exposures (cw and ml) were identical. Overnight treatment of cells with ascorbic acid (AA) minimized cell death from both cw and ml blue laser exposure, whereas similar treatment with N-acetyl-L-cysteine (NAC) was less effective. CONCLUSIONS: The in vitro system described is suitable for measuring meaningful thermal and photochemical laser damage thresholds. The system is also useful in comparative laser bioeffects studies, such as comparisons between cw and ml laser exposures, cells with various degrees of pigmentation, and studies determining the efficacy and mechanisms of treatments altering the response of cells to lasers.