# Photobiology - Presentation of a Blue Light Hazard In Vivo Experiment on The Rat

Pierre Boulenguez, Imene Jaadane, Christophe Martinsons, Samuel Carré, Sabine Charory, Alicia Torriglia

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Pierre Boulenguez, Imene Jaadane, Christophe Martinsons, Samuel Carré, Sabine Charory, et al. Photobiology - Presentation of a Blue Light Hazard In Vivo Experiment on The Rat. Light and Engineering (Svetotechnika), 2015, 23 (4), pp.41-46. hal-04695428

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# PHOTOBIOLOGY – PRESENTATION OF A BLUE LIGHT HAZARD IN VIVO EXPERIMENT ON THE RAT

Boulenguez P.<fnref n="1" />, Jaadane I.<fnref n="2" />, Martinsons C.<fnref n="1" />, Carré S.<fnref n="1" />, Chahory S.<fnref n="3" />, Torriglia A.<fnref n="2" />

Pierre.Boulenguez@cstb.fr

## Abstract

An experiment was conducted aiming at a better understanding of blue-light retinal toxicity. Freely moving Wistar rats were exposed to light from four types of light-emitting diodes (LEDs) for up to eighteen hours. Their retinas were analyzed using transmission electron microscopy, Western Blot, immunofluorescence, and Terminal transferase dUTP nick end labelling (TUNEL). In this paper, the biological results are summarized and the theoretical aspects of the dosimetry are detailed.

**Keywords:** blue light hazard, animal experiment, Wistar rat, white-light LED.

## 1 Introduction

### 1.1 Blue Light Hazard

Blue light hazard (BLH) is an incompletely understood phenomenon by which radiations in the blue-end of the spectrum induce lesions in the layer of photoreceptors and retinal pigment epithelium (RPE).

Damages of rapid onset following high-dose exposure (retinal dose > 20 J/cm<fnref n="2" />) have long been observed [Ham 76]. Since seminal studies, low-dose chronic exposure has also been suspected to play a role in age-related macular degeneration (AMD).

While no epidemiological study demonstrated this link so far, it is now known that a protein found in drusen of lipofuscin -the hallmark of AMD- called N-retinylidene-N-retinylethanolamine (A2E) reacts with blue light [ARNAULT13].

### 1.2 Solid State Lighting

<figure><img src="image_2.png" /><figcaption>Figure 1. InGaN LED spectrum and BLH action spectrum.</figcaption></figure>

<footnote n="1">¹Centre Scientifique et Technique du Bâtiment, Grenoble, FRANCE,</footnote>

<footnote n="2">²Institut National de la Santé et de la Recherche Médicale, Paris, FRANCE,</footnote>

<footnote n="3">³Ecole Nationale Vétérinaire d'Alfort, Alfort, FRANCE,</footnote>
Concerns over the BLH have recently intensified because of the advent of "white-light" LEDs. A typical Indium gallium nitride (InGaN) LED indeed emits significantly more blue-light per lumen than the sources it intends to replace (cf. Figure 1).

## 1.3 Exposure Limit Values

The BLH action spectrum of Figure 1 was determined by animal experiment. Nineteen such studies were surveyed in [NORREN11], relying on the following model organisms: rat (9), macaque (7), rabbit (2), and squirrel (1).

The International Commission on Non-Ionizing Radiation Protection (ICNIRP) used these works (and mostly [HAM76]) to estimate that toxicity starts with a retinal dose of BLH-weighted radiation of 20-30 J/cm² [ICNIRP05]. Taking a safety factor into account, a safe-dose threshold was set at 2.2 J/cm², defining the guideline exposure limit value (ELV) for the BLH [ICNIRP13].

A so-called spatially-averaged BLH-weighted radiance (SABLHWR) is generally employed to assert a source would not induce an excess of this ELV for the user. It is obtained by weighting spectral radiance with the BLH action spectrum, and averaging the obtained quantity over a finite solid angle (of which the angular extent increases with exposure time) [CIES009].

SABLHWR accounts for physiological effects such as eye movements and pupillary constriction. Accuracy in its assessment is essential for a correct classification of a source into a photobiological risk group [BOULENGUEZ13].

# 2 In Vivo Experiment

An animal experiment was thus conducted in order to gain at a better understanding of blue-light retinal toxicity. All experimental and animal care procedures were approved by the animal use and care committee of the veterinary school of Maison Alfort (ENVA).

## 2.1 Animal Model

<figure><img src="image_3.png" /><figcaption>Figure 2. Wistar rat.</figcaption></figure>

The model organism was the Wistar rat (cf. Figure 2). The strain is sometime presented as the golden standard general multipurpose animal model. It has been used in ophthalmological studies since its inception [CLAUSE93].

In a BLH study context, the translation of the model to human context being remains a matter of discussion. Debated aspects are the facts that the animal is nocturnal, albino, deprived of macula, and its retinal repair mechanisms are less efficient than that of human. It is nonetheless considerably easier to work with than other animals (e.g. macaques).
## 2.2 Illumination Procedure

<figure><img src="image_4.png" /><figcaption>Figure 3. Illumination device. Top: the cages were placed below an emitting surface. Below left: eighteen source modules were installed above the surface. Bottom right: a source module was made of four LEDs.</figcaption></figure>

Groups of six-week-old males freely moving in their cages were exposed for up to eighteen hours, using the dedicated multispectral illumination device presented in Figure 3.

<table><caption>Table 1. Sources installed in the illumination device.</caption><thead><tr><th></th><th>XP-E Blue</th><th>XP-E Royal Blue</th><th>NCSE119A</th><th>NCSB119</th></tr></thead><tbody><tr><th>Type</th><td>LED</td><td>LED</td><td>LED</td><td>LED</td></tr><tr><th>Brand</th><td>Cree</td><td>Cree</td><td>Nichia</td><td>Nichia</td></tr><tr><th>Dominant wavelength (nm)</th><td>473</td><td>449</td><td>507</td><td>473</td></tr></tbody></table>

The device was made up of eighteen source modules, each consisting of four different LEDs (cf. Table 1), allowing the spectral distribution of the stimulus to vary. Each group of rats was exposed using a single type of LED.
## 2.3 Dosimetry

<figure><img src="image_5.png" /><figcaption>Figure 4. Dedicated dosimetry toolbox.</figcaption></figure>

Dosimetry relied on spectrophotometric measurements within the cages (section 2.3.1), and on a theoretical model relating these measurements to the retinal dose (section 2.3.2).

### 2.3.1 Measurements

Spectral irradiance within the cages was measured using a diffusing head fibre spectrophotometer. Illuminance uniformity in the plane of the eyes (given by the ratio $E_{v,min}/E_{v,ave}$) was about 0.7 for all sources. Average spectral irradiance is noted $E_λ$ in the following.

### 2.3.2 Retinal Dose

At any given moment, retinal irradiance of an animal was dependent upon its head glaze. To estimate the overall retinal dose, it was considered that: (i) on average, a rat keeps his head aligned with his body, and (ii) as in [NORREN11], the eye can be approached as half a globe (taking focal length $f$ as diameter).

As only the upper surface was emitting light (cf. Figure 3), a horizontal to corneal irradiance correction factor was applied:

$$E_{\lambda,cor} = \frac{E_{\lambda}}{2} \quad [W/nm.m^2].$$

Retinal spectral irradiance was then approached as:

$$E_{\lambda,ret} = \tau E_{\lambda,cor} \frac{A_{cor}}{A_{ret}} \quad [W/nm.m^2],$$

Where $\tau$ is the transmittance of the ocular media, $A_{cor}$ is the effective area of the illuminated cornea, and $A_{ret}$ the area of the illuminated retina. The former area was estimated as:

$$A_{cor} = \frac{\pi d_p^2}{4} \quad [m^2],$$

Where $d_p$ is the diameter of the pupil. The retinal area $A_{ret}$ was approached as half that of the ocular globe:

$$A_{ret} = 2\pi f^2 \quad [m^2],$$

The average spectral retinal irradiance was finally estimated as:

$$E_{\lambda,ret} = \tau E_{\lambda,cor} \frac{d_p^2}{8f^2} \quad [W/nm.m^2].$$

Values for $d_p$, $f$, and $\tau$ were chosen according to [NORREN11] ($d_p$ = 5mm, $f$ = 5.525mm, and $\tau$ = 0.58 at 403nm), consistent with the values found in [MEYER77] and [HUGHES79]. Other values for $d_p$ are given in [BLOCK69] (0.4mm $\le d_p$ $\le 1.2mm$) and [SLINEY84] ($d_p$ = 0.5mm). An
explanation for this discrepancy may be the difference in sizes between the entrance pupil and the real pupil.

Average retinal irradiance was deduced from the average spectral retinal irradiance using numerical integration:

$$E_{ret} = \Delta_{\lambda} \sum_{i=1}^{n} E_{\lambda,ret,i} \quad [W/m^{2}],$$

with $\Delta_{\lambda}$ = 1nm. The retinal dose was finally computed, using dedicated software (cf. Figure 4), as:

$$D_{ret} = t E_{ret} \quad [J/m^{2}],$$

where t is exposure time.

While uncertainties remain in these calculations (due to assumption on the head glaze notably), it is expected that the ratios of doses between the different LEDs remain correct.

## 2.4 Biological Analysis

A fundus examination was performed immediately after exposure. The rats were then ethically sacrificed. Their eyes were enucleated, fixed in paraformaldehyde, washed, and embedded in optimal cutting-temperature for cryosection. The retinas were later analysed using Western Blot, immunofluorescence, TUNEL, and transmission electron microscopy [JAADANE15].

The fundus examination showed no bleaching, but important chemosis, indicating an oedema of the ocular tissues. This sign of irritation was probably due to exudation from abnormally permeable capillaries, and conjunctive vasodilation.

This hinted at microscopic lesions because no macroscopic damage to the retina was detected. The presence of an important oxidative damage was identified, involving proteins and nucleic acids, as well as an important amount of cell death.

The presence of necrosis was detected by staining cells with propidium iodide. A significant amount of photoreceptors were labelled, much more than photoreceptors that are TUNEL positive; indicating that permeabilization of the plasma membrane precedes degradation of deoxyribonucleic acid.

The existence of this necrosis easily explains the oedema, which was not sub-retinal but interstitial. This could also explain the presence of an early inflammatory reaction, probably due to the release of damage-associated molecular-pattern molecules.

## Conclusions

An experiment on blue-light retinal toxicity was conducted, consisting in exposing rats to blue-light rich light and then analyzing their retinas.

The main biological outcome was the observation of a wavelength dependant loss of photoreceptors and RPE cells, by mechanisms of caspase-independent apoptosis, necroptosis, and necrosis [JAADANE15].

In this paper, the illumination device and dosimetry aspects of the experiment were emphasized, as particularly relevant to the lighting science community.

A new iteration of the experiment is currently being performed, using an improved illumination device, based on new source types and alleviating the dosimetry head glaze assumption.

## Acknowledgements

The RetinaLED experiment was an interdisciplinary collaboration between biologists and ophthalmologists from the National Institute of Health and Medical Research (INSERM), veterinary ophthalmologists from the National veterinary School of Alfort (ENVA), and physicists from the Scientific and Technical Centre for Building (CSTB). It was funded by the "Agence de l'environnement et de la maîtrise de l'énergie" (ADEME).
## References

[BLOCK69] M.T. BLOCK. 1969. A note on the refraction and image formation of the rat's eye. *Vision Research* 9(11), pp. 705-711.

[HAM76] HAM W. T. Jr, MUELLER H. A., SLINEY D. H. 1976. Retinal sensitivity to damage from short wavelength light. *Nature* 260, pp. 153-155.

[HUGHES79] HUGHES A. 1979. A schematic eye for the rat. *Vision Research* 19, pp. 569-588.

[SLINEY84] SLINEY D. H. 1984. Quantifying retinal irradiance levels in light damage experiments. *Current Eye Research* 3(1), pp. 175-179.

[CLAUSE93] CLAUSE B. T. 1993. The Wistar rat as a right choice: Establishing mammalian standards and the ideal of a standardized mammal. *Journal of the History of Biology* 26, pp. 329-349.

[CIES009] CIE S 009:2002. Photobiological safety of lamps and lamp systems. European Standard 62471.

[ICNIRP05] ICNIRP. 2005. Adjustment of guidelines for exposure of the eye to optical radiation from ocular instruments: statement from a task group of the International Commission on Non-Ionizing Radiation Protection (ICNIRP). *Applied Optics* 44(11), pp. 2162-2176.

[NORREN11] VAN NORREN D., GORGELS TG. 2011. The action spectrum of photochemical damage to the retina: a review of monochromatic threshold data. *Photochemistry and Photobiology* 87(4), pp. 747-753.

[ICNIRP13] ICNIRP. 2013. Guidelines on limits of exposure to incoherent visible and infrared radiation. *Health Physics* 105(1), pp. 74-96.

[ARNAULT13] ARNAULT E., BARRAU C., NANTEAU C., GONDOUIN P., BIGOT K., VIÉNOT F., GUTMAN E., FONTAINE V., VILLETTE T., COHEN-TANNOUDJI D., SAHEL J.A., PICAUD S. 2013. Phototoxic action spectrum on a retinal pigment epithelium model of age-related macular degeneration exposed to sunlight normalized conditions. *PLoS One* 8(8).

[BOULENGUEZ13] BOULENGUEZ, P., CARRE, S., PERRAUDEAU, M., MARTINSONS, C. 2013. Blue light hazard of LEDs – Comparison of the photobiological risk groups of fifteen lamps assessed using the uniform spectrum assumption and a new hyperspectral imaging method. *Proceedings of the CIE Centenary Conference "Toward a new century of light"*, Paris.

[JAADANE15] JAADANE I., BOULENGUEZ P., CHAHORY S., CARRÉ S., SAVOLDELLI M., JONET L., BEHAR-COHEN F., MARTINSONS C., TORRIGLIA A. 2015. Retinal damage induced by commercial light emitting diodes (LEDs). *Free Radical Biology & Medicine*. S0891-5849(15)00158-6.
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Pierre Boulenguez received a master's degree in computer science in 2002 from the University of Marne-la-Vallée and a PhD degree in computer science from the University of Paris-Est in 2010. Following a Post-Doc at the CNRS/University of Poitiers in 2010, he joined the Health and Comfort Department of the Center for Building Science and Technology (CSTB) in 2012. His fields of interest include computer vision, near-field photometry, optical scattering, imaging radiometry, and photobiology.

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Imene Jaadane is a postdoctoral researcher at Institut de la Santé et de la Recherche Médicale (INSERM). She is a Doctor in molecular biology and eye physiopathology of Paris Descartes University. She works on molecular mechanisms of cell death in retinal degeneration and phototoxicity study of light emitting diodes.

<figure><img src="image_8.png" /></figure>

Christophe Martinsons is head of the lighting and electromagnetism division at the Center for Building Science and Technology (CSTB). He currently conducts research in the fields of optics and lighting in order to promote energy-efficient buildings while providing the best visual comfort for users. Christophe Martinsons has been leading several studies concerning health and environmental aspects of solid-state lighting for governmental agencies such as the
French agency for food, environmental and occupational health and safety (ANSES) and the
International Energy Agency (IEA).

<figure><img src="image_9.png" /></figure>

Samuel Carré received a master's degree in computer science in 1993 and a PhD degree in 1998 from University of Rennes 1. In 1998, he joined the lighting engineering team at the Center for Building Science and Technology (CSTB). His current research interests include physics based lighting simulation, global illumination in building environments, GPU-based rendering, and computer vision.

<figure><img src="image_10.png" /></figure>

Sabine Chahory received her DVM degree from the National Veterinary College of Alfort (Paris, France) in 1995, and pursued a residency in Veterinary Ophthalmology from 1999 to 2003. She was a Diplomate of the European College of Veterinary Ophthalmology (ECVO) since 2004. She received her PhD in Cell and Molecular Biology (Paris, France) in 2007. She currently works as an assistant professor at the Ophthalmology unit, National Veterinary College of Alfort, France. Her fields of interest are light-induced retinal degeneration.

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Alicia Torriglia is a Doctor in Medicine and PhD in Science. She is Research Director at the Institut de la Santé et de la Recherche Médicale (INSERM). Her field of expertise concerns the molecular mechanisms of cell death in the retina.