Photobiology Presentation of a blue light hazard in vivo experiment - - PDF document

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/342702678

Photobiology – Presentation of a blue light hazard in vivo experiment on the rat

Article in Light and Engineering · January 2015

CITATIONS READS

6

6 authors, including: Some of the authors of this publication are also working on these related projects: Inverse Problems and Parameter Estimation in Engineering View project baseline estimation View project Christophe Martinsons Centre Scientifique et Technique du Bâtiment

82 PUBLICATIONS 408 CITATIONS

SEE PROFILE

Samuel Carré Centre Scientifique et Technique du Bâtiment

33 PUBLICATIONS 189 CITATIONS

SEE PROFILE

All content following this page was uploaded by Christophe Martinsons on 20 March 2020.

The user has requested enhancement of the downloaded file.

Boulenguez, P. et al. PHOTOBIOLOGY – PRESENTATION OF A BLUE LIGHT HAZARD IN VIVO EXPERIMENT ON THE RAT

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

Boulenguez P.1, Jaadane I.2, Martinsons C.1, Carré S.1, Chahory S.3, Torriglia A.2

1Centre Scientifique et Technique du Bâtiment, Grenoble, FRANCE, 2Institut National de la Santé et de la Recherche Médicale, Paris, FRANCE, 3Ecole Nationale Vétérinaire d’Alfort, Alfort, FRANCE,

Pierre.Boulenguez@cstb.fr

Abstract

This paper presents an in vivo experiment designed to gain a better understanding of the mechanisms underlying blue-light retinal toxicity. Groups of Wistar rats were exposed to light from four types of LEDs. The eyes were analyzed using Western Blot, immunofluorescence, Terminal transferase dUTP nick end labelling (TUNEL), and transmission electron microscopy. Dosimetry and illumination device are discussed in detail. Keywords: Blue Light Hazard, Exposure Limit Values, Wistar Rat, Dosimetry.

1 Introduction Blue Light Hazard

Blue light hazard (BLH) is an incompletely understood phenomenon by which radiations in the blue-end of the visible spectrum induce lesions in the retinal pigment epithelium (RPE) and photoreceptors layer. Effects of rapid onset following high-dose exposure (retinal dose > 20 J/cm2) have long been

• bserved, but low-dose chronic exposition is also suspected to play a role in age-related

macular degeneration (AMD) since seminal studies [Ham76]. No epidemiological study demonstrated it so far yet; it is now known that A2E (a constituent of lipofuscin, which is involved in the formation of drusen, the hallmark of AMD) is an initiator of blue light-induced apoptosis of RPE cells.

Solid State Lighting

Figure 1 InGaN LED and BLH action spectra. The regain of interest for the BLH owes to the emergence of light-emitting diodes (LEDs) as general lighting service (GLS) sources. LEDs have excellent luminous efficacy and durability,

Boulenguez, P. et al. PHOTOBIOLOGY – PRESENTATION OF A BLUE LIGHT HAZARD IN VIVO EXPERIMENT ON THE RAT

but the ubiquitous indium gallium nitride (InGaN) “white-light” LED emits significantly more blue- light per lumen than the lamps it intends to replace (cf. Figure 1).

Exposure Limit Values

The BLH action spectrum in Figure 1 was established through animal experiment. In a recent survey [NORREN11], the following animal models were identified: rats (9), macaques (7), rabbits (2), and squirrel (1). These studies moreover form the basis of the International Commission on Non-Ionizing Radiation Protection (ICNIRP) exposure limit values (ELVs) [ICNIRP13]. It is noteworthy that a threshold safe-dose of BLH-weighted retinal irradiance was taken at 2,2 J/cm2 (considering that deleterious effects were observed for doses of the order of 20 to 30 J/cm2 [ICNIRP05]). This dose was converted to spatially-averaged BLH-weighted radiance (of sources) by accounting, notably, for human vision physiology. These ELVs are the foundation of the classification of lamps and lamp systems into photobiological risk groups by the Illuminating Engineering Society of North America (IESNA), the International Commission on Illumination (CIE), and the International Electrotechnical Commission (IEC) [CIES009].

2 In Vivo Experiment

This paper presents an animal experiment aiming at a better understanding of the biological mechanisms underlying blue-light retinal toxicity.

Animal Model

Figure 2 Outbred albino Wistar rat. Wistar rat (cf. Figure 2) is widely considered as the golden standard general multipurpose model

• rganism. The strain has been used in ophthalmological studies since its inception [1] yet the

translation of the model to human being remains to this day a matter of debate. All procedures were conducted in compliance with the animal use and care committee of the veterinary school of Maison Alfort (ENVA).

Boulenguez, P. et al. PHOTOBIOLOGY – PRESENTATION OF A BLUE LIGHT HAZARD IN VIVO EXPERIMENT ON THE RAT

Experimental Set-Up

Figure 3 Illumination device: the cage was placed below a plane emitting diffuse light. Six weeks old males moving freely in a cage were exposed to blue-light for eighteen hours. The illumination device, presented on Figure 3 and 4, was designed to maximize irradiance uniformity in the plane of the eyes. Figure 4 Illumination device: assembling of sources. Four different sources were used (cf. Table 1) in order to study the impact of spectral distribution (a group of rats was exposed to a single source). Table 1 Four types of LEDs were used.

XP-E Blue XP-E Royal Blue NCSE119A NCSB119 Type LED LED LED LED Brand Cree Cree Nichia Nichia Dominant wavelength (nm) 473 449 507 473

Boulenguez, P. et al. PHOTOBIOLOGY – PRESENTATION OF A BLUE LIGHT HAZARD IN VIVO EXPERIMENT ON THE RAT

Dosimetry

Figure 5 Dosimetry software (Python). Dosimetry was based on in situ measurements (section 2.3.1), and on a theoretical model linking retinal spectral irradiance (section 2.3.2) to retinal dose (section 2.3.3). A software was developed to ease interpretation of results (cf. Figure 5).

Spectrophotometric Measurements

Spectral irradiance was measured within the cages using a fibre spectrophotometer (with a diffusing head). Irradiance uniformity (given by the ratio

𝐹𝑛𝑗𝑜 𝐹𝑏𝑤𝑓) was about 0,7 for all sources.

Retinal Spectral Irradiance

Table 2 Parameters used for the Wistar rat eye model. Pupil diameter 𝒆𝒒 Focal length 𝒈 Ocular transmission 𝝊 5mm 2,4mm 100% Retinal spectral irradiance of a rat was dependent upon his head glaze. For overall dose estimation, it was considered that, on average, a rat keeps his head aligned with his body. Following the arguments in [NORREN11], the eye was approached as a globe. As only the upper plane was emitting light (cf. Figure 3), average corneal spectral irradiance was estimated as: 𝐹𝜇,𝑑𝑝𝑠 =

𝐹𝜇 2 [W/nm.m2],

and the average retinal spectral irradiance was approached as: 𝐹𝜇,𝑠𝑓𝑢 = 𝜐𝐹𝜇,𝑑𝑝𝑠

𝐵𝑑𝑝𝑠 𝐵𝑠𝑓𝑢 [W/nm.m2],

where 𝜐 is the transmittance of the ocular media, 𝐵𝑑𝑝𝑠 is the effective area of the illuminated cornea, and 𝐵𝑠𝑓𝑢 the area of the illuminated retina. The corneal area 𝐵𝑑𝑝𝑠 was estimated as: 𝐵𝑑𝑝𝑠 =

𝜌𝑒𝑞

2

4 [m2],

where 𝑒𝑞 is the diameter of the pupil. The retinal area 𝐵𝑠𝑓𝑢 was approached as half that of the

• cular globe:

𝐵𝑠𝑓𝑢 = 2𝜌𝑔2 [m2], where 𝑔 is the focal length (the diameter of the ocular globe for an eye focused at infinity). The average spectral retinal irradiance was thus: 𝐹𝜇,𝑠𝑓𝑢 = 𝜐𝐹𝜇

𝑒𝑞

2

16𝑔2 [W/nm.m2].

Parameters in the model are given in Table 2.

Boulenguez, P. et al. PHOTOBIOLOGY – PRESENTATION OF A BLUE LIGHT HAZARD IN VIVO EXPERIMENT ON THE RAT

Retinal Dose

Average retinal irradiance was deduced from the average spectral retinal irradiance by numerical integration: 𝐹𝑠𝑓𝑢 = 𝛦𝜇 ∑ 𝐹𝜇,𝑠𝑓𝑢,𝑗

𝑜 𝑗=1

[W/m2], with 𝛦𝜇 = 1nm. The retinal dose was finally given by: 𝐸𝑠𝑓𝑢 = 𝑢𝐹𝑠𝑓𝑢 [J/m2], where 𝑢 is the exposure time. While uncertainties remain in these doses (due to the assumption on the head glaze notably), it is expected that the ratios of doses between the different sources remain exact. A brief survey of different approaches found in the literature to estimate retinal irradiance is provided in [JAADANE15].

Biological Analysis

The rats were ethically sacrificed using sodium pentobarbital. A dilated fundus examination was performed immediately after exposition. The eyes were enucleated, fixed in paraformaldehyde, washed, and embedded in optimal cutting-temperature for cryosection. The retinas were analysed using Western Blot, immunofluorescence, Terminal transferase dUTP nick end labelling (TUNEL), and transmission electron microscopy.

3 Results and Discussion

The fundus examination showed no bleaching, but important chemosis, indicating an oedema

• f ocular tissues. This sign of eye irritation was probably due to exudation from abnormally

permeable capillaries, and conjunctival vasodilation. This hinted at microscopic photochemical lesions because no macroscopic damage to the retina was detected, which were indeed observed by the other analysis techniques. These showed the presence of an important oxidative damage, involving proteins and nucleic acids, as well as an important amount of cell death. The presence of necrosis was detected by staining cells with propodium iodide. Interestingly, a significant amount of photoreceptors were labelled, much more than photoreceptors that are TUNEL positive; indicating that permeabilization of the plasma membrane precedes degradation

• f DNA.

The existence of this necrosis easily explains the oedema, which is 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 was conducted where rats were exposed to light from four types of LEDs. Their eyes were analyzed using a broad range of ophthalmological approches. Two issues particularly relevant to the lighting community were emphasized: The design of an illumination device allowing animals to move freely in their cage; and the theoretical aspects of blue-light hazard dosimetry.

Acknowledgements

RetinaLED was an interdisciplinary collaboration between scientists from the National Institute

• f Health and Medical Research (INSERM), veterinary ophthalmologists from the National

veterinary School of Alfort (ENVA), and lighting engineers 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).

Boulenguez, P. et al. PHOTOBIOLOGY – PRESENTATION OF A BLUE LIGHT HAZARD IN VIVO EXPERIMENT ON THE RAT

References

[Ham76] HAM W. T. Jr, MUELLER H. A., SLINEY D. H. 1976. Retinal sensitivity to damage from short wavelength light. Nature 260, 153-155. [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, 329- 349. [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), 747-753. [CIES009] CIE S 009:2002. Photobiological safety of lamps and lamp systems. European Standard 62471 (CIE/IEC JTC 5 is currently working on an update). [ICNIRP13] ICNIRP 2013. Guidelines on limits of Exposure to Incoherent Visible and Infrared

• Radiation. Health Physics 105(1), 74-96.

[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), 2162-2176. [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. pii: S0891-5849(15)00158-6.

View publication stats View publication stats