Laser eye protection (LEP) is a requirement anywhere lasers are used. Unfortunately, too often it is not worn because it degrades the wearer's vision—particularly in low-light situations. This is because technologies used to block the laser also obstruct the wavelengths needed to see. Therefore, the real problem with developing and fabricating LEP is not blocking the laser, but seeing through the protective equipment once the laser shield is in place. For 30 years, scientists and engineers have worked on improving technologies to accomplish this. As an alternative approach, we consider the possibility of making retinas more resistant to laser injury.
There have been reports that the illumination of mouse retinas with red light induces resistance to cell death following exposure to methanol or white light.1, 2 An induced resistance to a subsequent challenge is called an adaptive response. Because this adaptive response is induced by red light, we refer to it as photobiomodulation. Previous attempts in our laboratory to induce an adaptive response to a laser challenge by exposure to heat were unsuccessful. But, following on from this published research, we examined the effects of red light exposure.
Figure 1. Example of an experimental laser lesion in a retinal pigmented epithelial monolayer cell culture. The green cells are alive and the red cells are dead. In suspension, the cells are 10–12μm in diameter. (Image courtesy of Michael Denton).
Our work is all performed in vitro, using a line of immortalized human retinal pigmented epithelium cells (RPE).3 The experimental system is a tissue culture model for laser eye injury.4 Initial research was aimed at determining if an adaptive response occurred and, if it did, finding the precise combination of the length of exposure and the time after exposure that produced the maximum photobiomodulation effect. This is because the classical response is transient and biphasic: it occurs only within a range of doses, and does not continue increasing outside that range.5 We considered features associated with apoptosis and growth stimulation6 to determine that the optimum conditions were 24 hours after an exposure of 2.88J/cm2 to 671nm light, using irradiances between 0.40 and 1.6mW/cm2.7
Figure 2. Wild type retinal epithelium cells (WT RPE) exhibit an adaptive response after red light exposure. Key: dark blue is control; maroon is 0.40mW/cm2; green is is 0.80mW/cm2; purple is 1.00mW/cm2; light blue is 1.50mW/cm2; orange is 1.60mW/cm2. Error bars are 95% confidence intervals. The dotted lines are the range of the 95% confidence limits for the control. Probit ED50: the laser pulse energy (J/cm2)effective at producing a lesion 50% of the time.
Figure 3. Vascular endothelial growth factor-c knockdown—VEGF-C(KD)—RPE cells do not exhibit an adaptive response to red light exposure. Key: blue is WT control; red is VEGF-C(KD) control; green is VEGF-C(KD), 2.88J/cm2 at 0.40mW/cm2; purple is VEGF-C(KD), 2.88J/cm2 at 0.80mW/cm2. Error bars are 95% confidence intervals. The dotted lines are the range of the 95% confidence limits for the WT control.
To measure the resistance to cellular death following exposure to a pulse of laser radiation, we noted the formation of a ‘ lesion’ in the spot where the laser strikes the cell monolayer. In our study, a lesion is defined as a spot of red fluorescing dead cells surrounded by a field of green fluorescing live cells (see Figure 1). If we noticed this specific change, we scored the event ‘yes,’ and if not, ‘no’ CĊFrom Probit analysis8 we derived the laser pulse energy (J/cm2) effective at producing a lesion 50% of the time (ED50). We could then plot the ED50 as a function of the laser exposure to create dose-response plots for the biomodulation effect.
We conducted adaptive response experiments with two strains of RPE cells. One is the basic line obtained from American Type Culture Collection, which we call wild type cells (WT). The other is a mutant strain we created by transfecting WT cells with a silencing ribonucleic acid gene, to create vascular endothelial growth factor–c knockdown cells—VEGF-C(KD)—that constitutively express VEGF-C protein at ∼10% of the WT level. We selected these cells following previous work on how VEGF-C plays an important role in blood vessel growth for tissue healing.
We exposed the cells first to 671nm light from an LED array, then, 24 hours later, to a 1s pulse from a 2μm laser. The WT cells exposed to red light exhibited an adaptive response (see Figure 2), but the VEGF-C(KD) cells did not (see Figure 3). In the WT cells, the response was biphasic around 2.88J/cm2. The maximum response in the WT cells was an increase from ∼25J/cm2 in controls to ∼32J/cm2 in the red light-exposed cells (p ≤ 0.05). There also appears to be an irradiance dependence, with 0.80mW/cm2 being less effective than either 0.40 or 1.60mW/cm2 (p ≤ 0.05). The VEGF-C(KD) cells do not show any difference in ED50 between red light-exposed and unexposed cells. Interestingly, there is no difference in ED50 between the WT and VEGF-C(KD) controls; the difference only manifests following exposure to red light.
The aim of our future research will be to understand why the WT cells respond to photobiomodulation and the VEGF-C(KD) cells do not. We will conduct classical genetic analysis, starting with expression of apoptosis genes in WT and VEGF-C(KD), to see if we can identify key genes or pathways in this response. Then we will determine if the adaptive response can be increased (to make the ΔED50 > 7J/cm2) and/or prolonged (to possibly extend the duration of the maximum response around the 24-hour post-exposure peak). Finally, if modulating the apoptosis pathway turns out to be the key, we would need to consider the potential for negative side effects when manipulating this pathway, since its proper function is essential to homeostasis in all normal tissues in the body.
This work was funded by a grant from the United States Air Force Office of Scientific Research to Jeffrey C. Wigle.
Jeffrey C. Wigle
US Air Force Research Laboratory
San Antonio, TX
After completing his PhD in radiation biophysics and post-doctoral fellowship in genetic toxicology, Jeffrey Wigle joined the US Air Force, where he served primarily as a research program manager. After leaving active duty he worked on advanced development of laser eye protection, and has now returned to active bioscience research.
Larry E. Estlack
Conceptual MindWorks Inc.
San Antonio, TX
Larry Estlack has a BA in microbiology and worked in a variety of government, academic, and commercial laboratories before joining Conceptual MindWorks. He is a molecular biology research technician at the company.
Kurt J. Schuster
San Antonio, TX
Kurt Schuster has a BS in physics and an MS in engineering. Since receiving his undergraduate degree, he has worked in the Air Force Research Laboratory's Optical Radiation Bioeffects branch as a laser and optical engineer supporting research on laser-tissue interactions.
1. J. T. Eells, M. M. Henry, M. T. T. Summerfelt, E. V. Wong-Riley, E. V. Buchmann, M. Kane, N. T. Whelan, H. T. Whelan, Therapeutic photobiomodulation for methanol-induced retinal toxicity, Proc. National Academy of Sci. (USA) 100(6), p. 3439-3444, 2003.
2. R. Albarracin, J. Eells, K. Valter, Photobiomodulation protects the retina from light-induced photoreceptor degeneration, Investigative Ophthalmology & Visual. Sci. 52, p. 3582-3592, 2011.
3. A. G. Bodnar, M. Ouellette, M. Frolkis, S. E. Holt, C.-P. Chiu, G. B. Morin, C. B. Harley, J. W. Shay, S. Lichtsteiner, W. E. Wright, Extension of life-span by introduction of telomerase into normal human cells, Science 279, p. 349-352, 1998.
4. M. L. Denton, M. S. Foltz, K. J. Schuster, G. D. Noojin, L. E. Estlack, R. J. Thomas, In vitro model that approximates retinal damage threshold trends, J. Biomed. Opt.
13(4), p. 054014, 2008. doi:10.1117/1.2981831
5. Y-Y. Huang, A. C-H Chen, J. D. Carroll, M. R. Hamblin, Biphasic dose response in low level light therapy, Dose Response 7, p. 358-383, 2009.
6. S. Elmore, Apoptosis: a review of programmed cell death, Toxicologic Pathology 35(4), p. 495-516, 2007.
7. J. C. Wigle, L. E. Estlack, D. R. Clarkson, K. J. Schuster, A. C. Schenk, B. A. Rockwell, hTERT-RPE cells in vitro following illumination with low levels of red light, American Society for Photobiology 36th Ann. Mtg, p. 23-27, Montreal, Canada, June 2012.
8. D. J. Finney, Probit Analysis 3rd ed., Cambridge University Press, New York, 1971.