Studying Chronic Exposure of Mice to Ultraviolet B Radiation

Summary

The protocol describes a study of the systemic and local skin effects of Ultraviolet B exposure in mice.

Abstract

Ultraviolet B (UVB) radiation (280-320 nm) has been recognized as a carcinogen since 1928, leading to sun exposure minimization. However, epidemiological studies suggest that sun exposure correlates with increased life expectancy and reduced incidence of cardiovascular diseases and certain cancers such as colon and endometrial cancer. UVB exposure also influences liver metabolism, protects against hepatocellular lipotoxicity, and affects metabolic health. These effects are partly due to vitamin D synthesis, though some are independent of vitamin D. Moreover, phototherapy with UVB has immunomodulatory properties and is used to treat skin conditions such as vitiligo and psoriasis while also affecting the systemic immune system. Given UVB's broad physiological effects, there are gaps in understanding the mechanism behind most of these effects. We showed that daily low-level UVB exposure enhances food-seeking behavior in male mice but not in females due to increased ghrelin secretion from skin adipocytes. In females, estrogen inhibits adipocytes' p53 activity, blocking ghrelin elevation and demonstrating a sex-dependent response to UVB radiation. Additionally, UVB-induced p53 activation in epidermal keratinocytes activates the hypothalamus-pituitary-gonadal axis, inducing sexual behavior and ovarian pro-fertility. This paper outlines a protocol for studying local and systemic effects of low UVB radiation using a C57BL/6J mouse model. Key elements include precise calibration of UVB doses, housing conditions, and careful hair removal to ensure consistent exposure. Mice are subjected to a graded UVB exposure regime over several weeks, with monitoring for health and behavioral changes. Methods for validation of radiation efficiency through pigmentation measurements are also detailed. By emphasizing calibration of UVB doses, rigorous animal care, and methodological controls, this protocol will facilitate the study of the comprehensive effects of UVB exposure, contributing to a nuanced understanding of its therapeutic benefits and risks.

Introduction

The frequencies of solar radiation that reach the earth's surface include ultraviolet (UV), UVA radiation (320-400 nm), and UVB radiation (280-320 nm). UVB is a mutation inducer. UV was recognized as a carcinogen in 1928¹, and from the mid-1900s, reducing sun exposure was recommended as a precautionary measure to avoid skin cancer². Although UVB is clearly an environmental trigger for skin cancer, research in the past decade³ with recent studies from our group^4,5,6 has shown that UVB has broad effects on physiological systems. A study of a cohort of women followed over 20 years revealed that life expectancy was higher by 2 years in women who did not avoid the sun compared to women who did⁷. Additional studies demonstrated that sun exposure was correlated with a decrease in cardiovascular disease and overall mortality^8,9,10. Inverse correlations have also been reported between sunlight and colon cancer and endometrial cancer⁸. UVB exposure also induces liver metabolism¹¹, protects against hepatocellular lipotoxicity and metabolic diseases, and has positive effects on endorphins and sexual-related behaviors^3,6. These lines of evidence indicate the detrimental effects of avoidance of sun exposure on human health. The beneficial effects of sun exposure have been principally attributed to the induction of vitamin D production however, at least some of the health benefits of sunlight are independent of vitamin D levels^4,6,12

Skin is the largest organ of the body, and it is the first line of defense against environmental stressors. The epidermal and dermal layers of skin are separated by a basal epidermal layer composed of undifferentiated, self-renewing keratinocytes and melanocytes¹⁰. Keratinocytes at increasing stages of terminal differentiation are found in the upper epidermal layer. The dermis includes fibroblasts and blood vessels, and the hypodermis mainly consists of adipocytes. The skin is a dermato-endocrine organ since multiple receptors of hormones, such as androgen receptor and corticotropin-releasing hormone receptor, are expressed in the skin, suggesting that hormones influence processes in the skin by paracrine and autocrine routes^13,14,15. The skin is also a producer of hormones. With a few exceptions, however, hormone production in the skin and release into the blood has not been documented, nor are the triggers for expression known (review in¹⁶).

UVB has been shown to have therapeutic potential in skin diseases. Phototherapy, including psoralen-UVA and narrowband UVB therapy, is the principal treatment modality for generalized vitiligo¹⁷. Response to phototherapy occurred in 74% of patients at 6 months and 75% of patients at 12 months after initiation of treatment¹⁸. The mechanism of action of phototherapy in vitiligo is not well understood. Its therapeutic action involves a combination of effects on cell-cycle kinetics, alterations in cytokine expression, effects on melanocytes, and immunomodulation. Numerous studies have examined the effects of phototherapy on skin immunity^19,20,21; however, the systemic effects are poorly understood, especially in the context of vitiligo. In contrast to psoriasis, where a study showed a decrease in proinflammatory cytokines and an increase in regulatory T cells after phototherapy treatment, in vitiligo, the number of regulatory T cells decreased with UV phototherapy²¹.

Although UVB has effects on the brain^14,15, adrenal glands²², ovaries^5,6, testicles⁶, liver¹¹, and lymph nodes²³, the mechanisms by which UVB modulates systemic systems are mostly not known. This research protocol describes a mouse model for studying the systemic and local (skin) effects of UVB exposure that will facilitate the study of the comprehensive effects of UVB exposure. The UVB mouse model is of broad interest in skin physiology, dermatology, and skin cancer, especially melanoma. The overall goal of this method is to provide a simple yet robust, effective protocol to develop the UVB mouse model for skin-related scientific research questions. The protocol describes how to overcome the challenges of skin erythema associated with hair removal and UVB exposure under physiological conditions and is effective in studying the local and systemic effects of UVB on skin.

Protocol

All the experimental procedures described were performed in accordance with the guidelines of the Tel Aviv University Institutional Animal Care and Use Committee (IACUC permit: 01-20-043).

1. Habituation of the mice for the experiment

1. Obtain 7- to 8-week-old C57BL/6J mice by either purchasing from a commercial vendor or rearing in-house, depending on the IACUC protocol of the experimenter. This protocol tests the 15-week-old mice in C57BL/6J background, but the protocol must be calibrated on other mice strains and for younger ages (< 7 weeks old).
   NOTE: The protocol to use the different age-grouped mice should be approved by the IACUC of the institution where the experiment will take place. Details on the health status of the animals are provided (Supplementary Table 1).
2. Habituate the mice for at least 7 days before experiment initiation. The habituation is critical for the mice after transportation and logistics.

2. Housing the mice

1. House the animals in home cages with standard bedding ad libitum access to standard chow and water in a standard dark/light cycle of 12:00 h (light hours: 07:00 - 19:00 h, and dark hours: 19:00-07:00 h) at standard temperature 22 ± 1°C and 32%-35% humidity.
   NOTE: We have tested this protocol on restricted dietary access, and it works fine under those experimental conditions as well.
2. Ensure the number of mice per cage is in accordance with their body weight, litter size, and experimental hypothesis.

3. Shaving and hair removal

1. Prior to shaving, place mice in the isoflurane chamber connected to an anesthesia vaporizer set on 5% isoflurane and 1 L/min oxygen.
   NOTE: Female experimenters should be cautious while using isoflurane, as exposure might cause serious health hazards, and pregnant women must avoid it in any form.
2. Ensure the mice are immobile and consistently breathing. Use ocular eye ointment to prevent dryness.
3. Turn ON the shaver machine (blade dimensions LxWxH: 204 x 50 x 41 mm) and hold it parallel to the mouse skin. Gently move the shaver machine in the shaver machine in the forward direction. Do not push the machine into mouse skin; a couple of rounds of shaving should sufficiently shave the mice from the dorsal side. Shave the mice dorsally so that 50%-60% of the dorsal region is shaved (Figure 1) prior to the start of radiation exposure. Re-shave the mice whenever there are patches of fur regrowth which is typically around 0.5 cm.
   NOTE: Carefully use the shaver so as not to hurt or tear up the skin of the animals; the experimenter must keep the shaver as parallel as possible with the skin as possible. The frequency of the hair shaving depends on the strain and the type of experimental treatment given to the mice.
4. Anesthetize mice from one cage at a time and then shave them. After shaving, apply depilatory cream (enough to cover the cotton pad applicator) to the shaved area using cotton pads. Be careful not to expose the depilatory cream closer to the eyes of the animals. Doing otherwise can cause serious complications and itching.
5. After 1-2 min, wash the area gently with tissues dipped in sterile phosphate buffer saline (PBS) to remove traces of the depilatory cream from the mouse body to remove the traces of the stuck hair.
   NOTE: Prolong timing (> 5 min) with the depilatory cream might harm the skin of the mice and should be avoided. Please make sure that the temperature of the PBS is room-temperature ambient and not too cold.
6. Dry the shaved area with soft tissues, followed by placing the mice in the clean, dry recovery cage with cage bedding. Closely watch the mice until they regain consciousness.
   NOTE: The infrared light lamp can ensure that the mice's body temperature does not drop drastically after the wash steps.
7. Let the mice rest in their home cage with ab libitum food and water and recover from the shaving procedure for at least 10 days before the first UVB exposure.
   NOTE: If the mice are exposed to UVB before 10 days after shaving, it might lead to skin erythema.

4. UVB exposure set-up

1. Wear a long-sleeved shirt, pants, a lab coat, closed shoes, double gloves, and a face shield. Perform irradiation inside a biological hood with the 10 mm plexiglass cover down to prevent the experimenter's UVB exposure.
2. Place the UVB lamp on the transparent Plexi glass chamber without turning it ON, as shown in Figure 2.
3. Turn ON the UVB lamp and allow it to operate until the intensity is steady. Place the UVX radiometer by turning ON as monitored using a UVX radiometer equipped with a UVB-measuring head. Make sure to switch ON the UVX radiometer to 20 mW/cm² and read the settings on the radiometer. Allow the UVB lamp to warm up and stabilize for at least 2 min. When the lamp is ready for use, take out the UVX radiometer.
4. Use a clean cage other than the home cage for irradiation, and place it in the biological hood under the UV lamp. Transfer the mice into the irradiation cage prior to treatment with the UV lamp ON or OFF for the respective UV or mock exposures. Treat only 2 mice at a time to provide enough space.
   NOTE: The chamber had a floor area of 500 cm2 and an internal height of 14 cm to allow mice to move freely without climbing on each other during irradiation. The reason for using the exposure chamber is to avoid the mice overlapping each other during the exposure and to ensure that all mice get the same amount of UVB exposure. Introducing more than two mice in the chamber will lead to a scenario where mice climb on each other, which will create inconsistency in the UVB dose given to each mouse.
5. Calculate the amount of time required to expose the mice to the desired UVB intensity using the calculator. For example, if the UVX radiometer shows 2 mW/cm² on the display screen and the desired exposure is 50 mJ/cm² to be given to the mice, expose the mice for 25 s in the UV irradiation chamber while the UV lamp is ON.
   NOTE: Before starting make sure to put the appropriate protective gear as mentioned in step 4.1. The duration of exposure throughout the treatment ranged between 20-120 s. However, the duration could vary by radiation intensity of the UVB lamp and thus should be calculated for each treatment. Mice are not expected to be removed from the study as no signs of weight loss, pain signals, including severe skin burns, bleeding, erythema, apathy, or difficulty breathing are normally seen with this protocol.
6. Expose male and female mice separately and clean the chamber with disinfectant between each sex to avoid cross-contamination of odors for the behavioral studies. In case of exposure of different groups of mice that were treated with additional treatment or genetically transgenic or control animals, clean the chamber between exposure of each group to avoid cross-contamination of unknown factors that are different between the two groups.

5. UVB exposure

1. Turn ON the UVB lamp and set it up from step 4.1. to 4.5.
2. Transfer the mice from the home cage to the exposure chamber for the desired time. Expose the mice to UVB once a day for the desired experimental days (here, we did 5 days/week) for 8 weeks with an XX-15 stand equipped with 15 W, 302 nm UVB bulbs.
   NOTE: The duration of exposure is subjective and dependent on the scientific question. Animals should not develop skin erythema. In case of erythema, immediately stop the exposure and report to the veterinarian. We used 5 days/week of exposure, excluding the weekends. The frequency of the UVB dose can be reduced under conditions similar to this protocol, but additional exposure will require calibration from the experimenter.
3. Expose the mice to a UVB dose of 50 mJ/cm², a sub-erythematic dose in the C57BL/6J mouse strain.
   NOTE: A UVB dose of 50 mJ/cm² is a sub-erythematic dose, and we have used this dose for 8 weeks in the experimental mice. In other experiments, we have used a single UVB exposure of 200 mJ/cm² to study DNA damage⁴.
4. Place the mock-irradiated (non-UVB exposed) shaved mice in the radiation chamber for an equal amount of time without turning the UVB lamp ON and use them as a control for UVB exposure.
   NOTE: Since mice are neophobic, the experiments involving mice should always have the control group, which undergoes the same pseudo-treatment as the experimental one.

6. UVB exposure analysis

NOTE: Mice were euthanized using CO₂ asphyxiation according to AVMA Guidelines.

1. To validate radiation efficiency, measure the ear and tail pigmentation (Figure 2) using a dermal spectrophotometer or similar instrument or by immunostaining skin biopsies followed by Fontana Masson staining, as previously described⁴.
2. Validation using dermal spectrophotometer: Calibrate the dermal spectrophotometer to set the baseline using clean, plain white paper. Once the calibration is done, place the measure sensor on the ear or tail of the mouse and note the melanin intensity readings. The melanin levels on the mock-UVB-radiated and UVB-radiated mice are shown in Figure 3.
3. Validation through Immunofluorescence: Fix mouse skin biopsies (2 cm x 2 cm) in 4% paraformaldehyde and embed in paraffin blocks. Stain tissue slices of 10 µm thickness with anti-CPD antibody (used at a concentration of 1:100)²⁴ overnight at 4 °C, followed by fluorescent-labeled secondary antibodies (Goat anti-rabbit Alexa-594-conjugated secondary antibody at a concentration of 1:1000)²⁴ for 1 h at room temperature. Counterstain the tissues with DAPI and obtain images using fluorescence microscopy.
4. Validation through Fontana Masson staining: Fix mouse skin biopsies in 4% paraformaldehyde and embed them in paraffin blocks. Stain tissue slices of 10 µm for melanin using the Fontana Masson staining protocol⁴ with silver nitrate and gold chloride solution. Counterstain tissue biopsies with Nuclear Fast Red Solution and mount them using a tissue mount, followed by bright field image capture in the microscope.

7. Animal care

1. Perform animal experiments in accordance with the Institutional Animal Care and Use Committee guidelines.
2. Weigh the mice weekly to assess their basic health state. Remove the animals with > 15% weight decreases between measurements from the experiment. Remove the animals that exhibit pain signals, including severe skin burns, bleeding, erythema, apathy, or difficulty breathing due to UVB exposure, and sacrifice according to the Institutional Animal Care and Use Committee guidelines.

Results

This method paper mentions the detailed step-by-step procedure of generating the UVB-exposure mouse model. Figure 1 clearly demonstrates the shaved area of the representative C57BL/6J mouse on the dorsal side prior to UVB exposure, demonstrating that 50%-60% of the dorsal area was shaved. The shaving protocol shows no sign of skin erythema, which is of utmost importance for dermatological studies. Figure 2 shows the set-up of the UVB exposure for the mice using ...

Discussion

UVB has long been recognized as an environmental risk factor for skin cancers; however, recent studies have illuminated a broader spectrum of UVB effects³. There is a need for studies that provide a deeper understanding of the systemic effects of UVB. The different doses used in basic research compared to clinical settings complicate our understanding of UVB effects, underscoring the importance of research that bridges these gaps.

The protocol for UVB exposure of C57BL/...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Disinfectant	Amity international	Not applicable
Exposure chamber	Made in house	Not applicable
Hair removal cream	Veet	Not applicable
Hair shaver	Heiniger	707-303 120V US
Isoflurane	Piramal Critical Care	Not applicable
Isoflurane system	Kent Scientific	VetFlo-1210S
PBS	Sigma Aldrich	TMS-012
Plexi-glass chamber	Made in house	Not applicable
UVB lamp	Ultra-Violet Products	XX-Series - 15 Watt
UVX radiometer	Ultra-Violet Products	97-0015-02