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Recommendations for measuring and standardizing light for laboratory mammals to improve welfare and reproducibility in animal research [1]

['Robert J. Lucas', 'Centre For Biological Timing', 'School Of Biological Sciences', 'Faculty Of Biology Medicine', 'Health', 'University Of Manchester', 'Manchester', 'United Kingdom', 'Annette E. Allen', 'George C. Brainard']

Date: 2024-03

Having considered how light may be quantified, we turn our attention to direct advice on how this new metrology could be used to improve animal husbandry and experimentation [50]. Our advice is summarized in Box 3 and elucidated below.

The resources available (see above) mean that reporting light in 4 dimensions need not be onerous. Nevertheless, we also considered the additional problem of this quantification when it comes to recreating experimental conditions, as it is all but impossible to simultaneously match intensity across 4 α-opic dimensions. This complexity is unavoidable when applying light as an experimental parameter and should be accounted for in study design. For more general applications, however, it would be very helpful to have a single target metric when standardizing measurements or replicating experimental conditions. The answer to the question of which α-opic quantity to adopt for this purpose may differ according to the nature of the experiment, but as a rule of thumb we suggest using melanopic EDI. This choice is partly to retain consistency with the guidance for husbandry (see below). Furthermore, the similarity in spectral sensitivity between melanopsin and rods means that melanopic and rhodopic irradiance are strongly correlated across light sources. Consequently, under most circumstances melanopic irradiance will represent a good approximation of effective intensity for the retinal photoreceptors with lowest (rod) and highest (melanopsin) activation thresholds [ 51 ]. Matching melanopic irradiance may not always be sufficient to normalize experimental conditions (for example, when using lights of very divergent spectral power distribution), but melanopic irradiance will have much greater tolerance than the current practice of matching photopic lux.

We were aware that quantifying α-opic irradiances lacks the simplicity of a single metric (c.f. photopic lux). For most lab mammals, 4 α-opic values would be required. This complexity reflects biology, as not only do light-evoked responses typically reflect a weighted output from all photoreceptive systems, but these weightings may differ across physiological outputs, or indeed between species. Applying the α-opic methodology to quantify light as experienced by individual photoreceptors removes those uncertainties and is the only way to capture the animal’s full experience. Moreover, our view is that this approach will itself provide a framework to better describe the photoreceptor origins of the myriad biological effects of light, in an approach that is transferable and comparable between species (and has already happened for humans) [ 2 , 32 ]. Finally, reporting all α-opic measures provides information about both effective irradiance and color.

The most complete description of experimental conditions would encompass a complete quantification of light as experienced by the animal. This can be achieved by reporting species-specific α-opic irradiance (or EDI) for each photoreceptor. Ideally, this would be provided in methods sections both for general housing and, where appropriate, experimental conditions.

Animal husbandry

In the case of laboratory rodent husbandry, we feel that there is sufficient information to go beyond recommending that light is appropriately quantified and documented, and to provide some quantitative recommendations for light exposure. Many factors were considered in determining these, including circadian biology, light preference and aversion, human health and safety, and the animal’s species-specific experience. The guidance we provide is for light as experienced by the animal, and it is important to note that this will be determined not just by the nature of room lighting, but also by rack orientation, cage location within the rack, and cage color [52–54]. For this reason, the figures we give relate to in-cage light measurements, with the detector pointing towards the major light source (and the cage in its position in the rack if appropriate). The guidelines in Box 3 are based on available information, but this evidence base is certainly incomplete, and guidelines may evolve as new data are presented.

The first decision in defining healthy levels of lighting is which metric to provide targets for. As mentioned above, a complete description of the animal experience requires quantification in all α-opic irradiances. We note that there is good evidence that circadian and related neurophysiological responses can be engaged by all photoreceptors in laboratory rodents [9,55–61], and hope that the α-opic metrology will facilitate studies aimed at resolving their contribution to factors relevant for husbandry. Nevertheless, given the substantial practical advantages to using a single metric, we provide guidance here in terms of melanopic irradiance. Several factors persuaded us that this quantity could be applied to achieve a reasonable approximation of the animal experience. First, melanopsin-expressing ipRGCs are responsible for important determinants of animal welfare, including circadian photoentrainment and light-induced changes in physiological and behavioral states [6,8]. Secondly, as melanopsin cells have lower sensitivity than rods and comparable sensitivity to cones [51], as well as a spectral sensitivity in the short to middle wavelength portion of the visible range, any light sufficient to engage melanopsin will also be sufficient to support vision. Furthermore, as outlined above, the similarity in spectral sensitivity between melanopsin and rods means that melanopic irradiance would quantify light with sufficient accuracy across the full range of intensities to which mammals respond.

We propose a further simplification in order to facilitate adoption of guidelines. Although α-opic irradiances are species specific, we suggest using human melanopic irradiance as an acceptable shorthand for general animal husbandry. There is a danger of inaccuracy, but this is largely a problem when using very colored lights. Across a range of broad-spectrum lights (encompassing all commonly used room lighting), the median difference between human and mouse melanopic irradiance is only 7% (range 1% to 19%) (S3 and S4 Tables). Meanwhile, the increasing availability of light meters capable of measuring human melanopic irradiance makes it easy for any vivarium to compare their lighting against guidance specified in that measurement unit.

Turning to guidelines, we aimed for separate recommendations for the animal’s subjective daytime and night (Box 3). As complete darkness at night is neither natural nor easily achievable, we considered how much light animals might be exposed to at night in nature. The brightest natural light source at night is the moon. Although a bright super-moon can provide 0.3 photopic lux, Kyba and colleagues proposed 0.1 photopic lux as a more realistic value for moonlight [62]. We therefore suggest that light exposure during the dark phase should not exceed 0.1 lx human melanopic EDI (applying the approximation that photopic illuminance and melanopic EDI are near interchangeable for moonlight). Given the ethological basis for our decision, we believe this is a reasonable target for nighttime lighting for all mammalian species. To achieve these light levels, researchers and animal care staff will need to use dim red lighting for nighttime monitoring or welfare checks (see below).

Determining appropriate levels for daytime light is more complex. In principle, a similarly ethological approach could be taken by recommending that all animals have access to irradiances equivalent to natural daylight during the day. However, achieving such high irradiances is impractical in terms of human user experience and energy usage. We turned therefore to consider the minimum acceptable light exposure during the day. A useful starting point is the minimum intensity required for circadian entrainment. The circadian clock integrates light over long timeframes (tens of minutes [63]) and we considered a threshold here for a day (light) phase lasting at least several hours. For mice housed with dark nights, this can be very low, with entrainment reported for daytime light as low as 0.06 lx mouse melanopic EDI [64]. A more realistic target to ensure robust entrainment in all visually intact animals is 0.6 to 6 lx mouse melanopic EDI [55]. Moreover, several commonly used mouse strains have outer retinal degeneration, and the available evidence is that thresholds for entrainment are higher in animals with dysfunctional outer retina (at 6 lx mouse melanopic EDI [65]). We therefore suggest a minimum irradiance of 10 lx human melanopic EDI, which is roughly equivalent to the experience of civil twilight [5] and is much lower than the 250 lx human melanopic EDI recently recommended for humans [32].

We appreciate that 10 lx melanopic EDI is low compared to daylight and may be insufficient to fully engage the impact of light on physiological/behavioral states. The thresholds for circadian entrainment upon which it is based come from animals whose night phase is totally dark, and the impact of low light exposure in subjective night on thresholds for entrainment is not well established [66]. Moreover, the characteristics of circadian entrainment may also depend upon daytime light over a wider range [67–69]. Finally, as the threshold of 10 lx melanopic EDI is based on data from mice, it may be less appropriate for more distantly related and/or diurnal species (see e.g., data on diurnal rodents [67,70]). For these reasons, while we believe this value is supported by available evidence (and should be achievable without imposing large increases in energy usage), we stress that it should be viewed as a minimum that may be insufficient to fully normalize the animal experience and, where possible, brighter light is preferable.

We do not provide a recommended upper limit for daytime light intensity. Vivarium lighting will always be dimmer than daylight and thus fall within the range of natural light exposure. Nocturnal rodents typically avoid light when faced with a choice [71], and we recommend that cages contain a retreat space or shelter [72] and/or sufficient, suitable nesting material to allow them to do so [73]. Concerns are often raised about the potential for retinal damage under higher light intensities. For normal pigmented animals, the light intensities required to cause retinal damage are very high (>10,000 photopic lux) [74]. Where albino animals are used, current evidence suggests light levels should not exceed 20 photopic lux (corresponding to approximately 10 to 20 lx melanopic EDI, depending upon the light source) to avoid retinal damage [75].

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002535

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