An In Vivo Murine Simulated Seaside System for Assessing Sunscreen Efficacy and Durability

Summary

This study presents a simulated seaside system using a mouse model to quantitatively evaluate the retention rate and photoprotective efficacy of sunscreens under complex environmental stressors, including ultraviolet irradiation, seawater immersion, wave washout, and wind drying. It serves as a preclinical screening tool and does not replace standardized clinical trials.

Abstract

Ultraviolet (UV) radiation is a primary environmental factor causing skin photoaging, sunburn, and acute UV-induced skin injury. Sunscreen is the most common method of photoprotection; however, current evaluation methods fail to accurately simulate complex seaside environments or pose high costs and ethical limitations. This study presents a novel simulated seaside testing system combined with a live murine model to quantitatively evaluate the efficacy and durability of sunscreens under complex environmental stressors. The custom-designed chamber integrates controllable UV irradiation, seawater immersion with simulated waves, and wind-drying mechanisms. Depilated BALB/c mice were treated with different sunscreens and exposed to a standardized 120-minute simulated seaside cycle. The retention rate of the sunscreens was quantified using fluorescence imaging, while the photoprotective efficacy was evaluated by Hematoxylin and Eosin (H&E) staining for epidermal thickness and inflammatory infiltration. The results demonstrated that this system could effectively differentiate the performance of various sunscreens. Specifically, a high-protection water-resistant sunscreen exhibited the highest retention rate and provided the best protection against UV and environmental damage. This protocol offers a cost-effective, highly reproducible, and ethical preclinical screening alternative for exploring skin protection products under simulated extreme environments, serving as an exploratory bridge rather than a validated replacement for mandatory human SPF testing.

Introduction

Ultraviolet (UV) radiation, particularly UVA and UVB, is the leading cause of skin photoaging, sunburn, and acute UV-induced skin injury^1,2,3. The use of sunscreen is the most widely adopted strategy to mitigate these adverse effects^4,5. Therefore, the accurate evaluation of sunscreen efficacy is of critical importance to both dermatological research and the cosmetic industry.

Currently, the primary methods for testing sunscreen efficacy include in vitro instrumental analyses and human clinical trials^6,7. In vitro methods, such as UV spectrophotometry on quartz plates or artificial skin, are cost-effective and highly reproducible^8,9. However, they fail to simulate the complex dynamics of sunscreen film-forming, water resistance, and washout resistance on living skin. Conversely, human clinical trials, which measure the Minimal Erythema Dose (MED) on volunteers' backs, are considered the gold standard for determining the Sun Protection Factor (SPF) in accordance with International Organization for Standardization (ISO) (SPF: ISO 24444, water resistance: ISO 16217) and FDA guidelines^{10,11,12,13,14}. Despite their reliability, clinical trials are expensive, time-consuming, subject to significant individual variation, and heavily restricted by ethical considerations, especially when testing under extreme or potentially harmful environmental conditions.

A significant limitation of existing evaluation techniques is the inability to comprehensively simulate practical seaside environments. During seaside activities, sunscreens undergo a synergistic degradation process involving high-salinity seawater immersion and wave washout^15,16. These factors drastically compromise the adhesion stability and protective duration of the applied products. To overcome these limitations, this study introduces a novel simulated seaside testing system utilizing a BALB/c mouse model. This system integrates multiple key environmental parameters—including UV intensity, seawater salinity, water temperature, wave force, and wind speed—into a single, highly controllable platform. By combining this hardware with quantitative fluorescence imaging and histological analysis, the protocol enables precise evaluation of both sunscreen retention rate and physiological protective efficacy. This method provides a reliable, low-cost, and efficient solution for assessing sunscreen performance in complex, practical marine scenarios.

Protocol

All methods involving vertebrate animals were performed in compliance with the institutional guidelines and approved by the laboratory animal care and ethics committee of Guangdong Laidi Biomedical Research Institute Co., Ltd., under approval No. LDSW2025050-4.

1. Setup of the simulated seaside chamber

1. Assemble the test chamber, which consists of a transparent, airtight metal box (dimensions: 40 cm length × 30 cm width × 30 cm height) with access ports for the animals.
2. Integrate the UV Simulation Unit: Install UV lamps (6W) at the top of the chamber, providing an approximate intensity of 2 mW/cm². This simulates the average strong midday seaside sunlight.
   CAUTION: UV radiation is hazardous to the eyes and skin. Ensure the chamber is fully sealed and wear appropriate safety goggles and protective clothing when operating the UV unit.
3. Integrate the water environment unit. Fill the built-in water tank with artificial seawater.
4. Prepare the seawater by dissolving commercial sea salt in purified water at a ratio of 1 kg (± 0.05 kg) salt to 30 L (± 0.5 L) water. Set the water temperature controller to maintain a constant temperature of 25 °C (± 1 °C).
5. Activate the wave-maker at a flow rate of approximately 10 L/min to simulate the mechanical friction of moderate shore waves.
6. Position the blower fan to direct airflow across the animal holding area. Calibrate airflow to 35 cubic feet per minute (CFM) to simulate a seaside breeze.
7. Connect all components to a central control interface. Prior to testing, input the target environmental parameters into the control system and calibrate them using the corresponding measurement instruments.
   NOTE: Execute this initial verification by utilizing an external handheld digital UV radiometer, a waterproof thermometer, and an anemometer, ensuring that the UV intensity (2 mW/cm²), water temperature (25 °C), and wind speed (35 CFM) match the intended setpoints prior to initiating the biological experiments.
8. During the experiment, toggle the UV lamps, wave-maker, and water drainage on or off via the interface to precisely execute the alternating immersion and emersion phases.

2. Preparation of the animal model

1. House adult BALB/c mice (weighing approximately 20 g to 25 g) in standard conditions and allow them to acclimatize for one week prior to the experiment.
2. Briefly anesthetize the mice using 1.5% isoflurane via inhalation to minimize stress. Pre-shave the fur on the backs of the mice using an animal hair removal device.
3. Next, apply a commercial chemical depilatory cream evenly to the dorsal skin of the mice. After 3 to 5 min, gently wipe away the hair using warm, damp gauze.
   CAUTION: Chemical depilatory creams and paraformaldehyde used in histology are toxic and irritants. Handle them in a well-ventilated area while wearing protective gloves. Disposal: Dispose of all chemical waste (paraformaldehyde) and biological waste (animal carcasses and tissues) in designated biohazard and chemical waste containers strictly in accordance with institutional and local regulatory requirements.
4. House the depilated mice for an additional 2 days to allow recovery. Before the experiment, visually inspect the dorsal skin to confirm the complete absence of erythema or irritation from the depilatory process.
5. Draw a cross on the exposed dorsal skin using a black marker to divide the area into four distinct quadrants (Quadrants 1 to 4).

3. Sunscreen application and pre-treatment

1. Prepare the test sunscreens. Randomly select three commercial sunscreens: A, B, and C.
2. Apply the sunscreen to the designated areas using a 20 μL micropipette, and spread the formulation evenly with a sterile gloved fingertip in a gentle circular motion for 10 s to ensure the formation of a uniform film as follows:
   NOTE: Ensure the pressure applied by the fingertip remains consistent across all quadrants. Uneven spreading or excessive pressure can lead to irregular film thickness, which will directly compromise the reproducibility of the subsequent retention evaluation.
3. Quadrant 1 (Top-Left): Apply 20 mg/cm² of Sunscreen A.
4. Quadrant 2 (Top-Right): Apply 20 mg/cm² of Sunscreen B.
5. Quadrant 3 (Bottom-Left): Apply 20 mg/cm² of Sunscreen C.
6. Quadrant 4 (Bottom-Right): Leave untreated (Unprotected control).
7. Place the mice in a resting position for 5 min to ensure that the sunscreen is evenly and stably distributed on the dorsal skin.

4. Sunscreen retention assessment

1. Mix a safe fluorescent marker (0.01% w/w Sulfo Cy7) into Sunscreen A, Sunscreen B, and Sunscreen C.
2. Apply the fluorescently labeled sunscreens to the dorsal skin of the mice as described in section 3.
3. Capture the initial baseline fluorescence image (F0) of the dorsal skin using an in vivo fluorescence imaging system.
   NOTE: Verify that the imaging parameters, including exposure time and focal plane, remain strictly locked for each animal. An inaccurate baseline (F0) capture will invalidate the final calculation of the sunscreen retention rate.
4. Place the mice into the testing chamber. Turn off the UV lamps and set the water temperature to 25 °C.
5. Raise the water level and activate the wave-maker so that the mice undergo a 30-minute immersion phase (swimming), ensuring their heads safely remain above the water surface.
6. Next, turn off the wave-maker and lower the water level to expose a stepped metal platform inside the chamber, allowing the mice to rest safely on the platform for 30 min (emersion phase).
7. Repeat the 30-minute immersion phase and the 30-minute emersion phase for a second cycle (120 min in total).
8. Capture the post-exposure fluorescence image (F1) using the in vivo imaging system.
9. Calculate the retention level using the formula:
   Retention level = (F1 / F0) × 100%      (1)
10. Calculate the relative fluorescence intensity using the formula:
    Fluorescence intensity = (F1(X) / F1(A)) × 100%      (2)
    where F0 is the baseline fluorescence intensity, F1 is the post-experiment fluorescence intensity, F1(A) is the post-experiment fluorescence intensity of the area applied with Sunscreen A, and F1(X) is the post-experiment fluorescence intensity of the area applied with Sunscreen B or C.
    NOTE: Ensure the UV lamps remain off during the fluorescence monitoring experiment to prevent UV-induced quenching of fluorescence. The total exposure time is 120 min, and ensure constant monitoring of the animals' respiratory rate and activity status throughout the immersion phase to confirm the absence of signs of distress or exhaustion.

5. Photoprotective efficacy detection

1. Apply the standard (non-fluorescent) sunscreens to the backs of the mice as described in section 3.
2. Apply a piece of black, UV-blocking tape to the upper neck area of the mouse to serve as a UV-shielded internal control. Record the baseline skin conditions via macroscopic photography.
   NOTE: Press the edges of the tape firmly to prevent water seepage or accidental detachment during the wave wash phase. If the tape detaches, the internal control area will be compromised by exposure to UV and water, confounding the histological baseline.
3. Expose the mice to the same water cycling program described in section 4, with the UV simulation unit activated: raise the water level for 30 min (immersion), lower the water level for 30 min (emersion phase), and repeat for a second cycle (total 120 min).
4. Remove the mice from the chamber and record the presence of erythema, peeling, or hardening on the skin using macroscopic photography.
   NOTE: Based on the chamber dimensions (40 cm × 30 cm × 30 cm) and the animals' activity space relative to the light source, the cumulative UV dose delivered during the 120-minute cycle is estimated at approximately 2.5 J/cm². Dosages around this level effectively induce measurable epidermal thickening and acute inflammation while ensuring the safety of the mice and strictly maintaining animal welfare limits³.
5. At the experimental endpoint, euthanize the mice humanely via cervical dislocation under deep isoflurane anesthesia, strictly following the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals, to harvest skin tissues for the subsequent histological evaluation. Excise the dorsal skin tissues from the four quadrants and the tape-protected area.
6. Fix the tissues in 4% paraformaldehyde for 24 h, followed by standard dehydration, paraffin embedding, and sectioning.
7. Stain the sections with hematoxylin and eosin (H&E). Briefly, deparaffinize the sections in xylene, rehydrate through graded ethanol solutions, stain with hematoxylin for 5 min, rinse in running tap water, counterstain with eosin for 1 min, dehydrate, and mount with a resinous mounting medium.
8. Observe the stained sections under a light microscope. Measure the thickness of the epidermis and assess the level of skin inflammation by counting the infiltrating inflammatory cells.
   NOTE: The UV dose delivered during the cycle is sufficient to induce acute erythema for efficacy evaluation but remains well within the safe, non-lethal threshold, as evidenced by the mice showing no signs of severe systemic damage and maintaining normal physical activity after the testing cycle.

6. Statistical analysis

1. Perform statistical analysis using GraphPad Prism 9.5. Express quantitative variables as mean ± standard deviation. Use one-way ANOVA followed by Tukey’s post-hoc test for comparisons among multiple groups. Statistical significance was defined as *p < 0.05, **p < 0.01, and ns indicates no significance (p > 0.05).

Results

Environmental characterization of the testing system
Before the in vivo evaluation, the physical parameters of the simulated seaside chamber were calibrated to ensure high environmental fidelity. The UV simulation unit utilized a 6 W lamp emitting a broad spectrum ranging from 300 to 420 nm with a peak wavelength at 340 nm. This specific spectral distribution ensures a dominant UVA emission accompanied by a biologically relevant portion of UVB, which effectively mirrors the typical ultraviolet spectrum of natural midday sunlight reaching the Earth's surface¹⁷. The wave maker operated at a flow rate of 10 L/min, which generates a mild fluid shear stress comparable to gentle coastal wave washout. Additionally, the airflow unit provided a constant wind speed of 35 CFM, translating to a wind velocity of approximately 3 to 4 m/s over the test area. This correlates with a gentle breeze (Beaufort scale 3) typical of seaside meteorological conditions¹⁸. These combined parameters reliably replicate the cyclic mechanical and environmental stressors experienced during typical seaside activities.

Sunscreen retention assessment
First, the simulated seaside apparatus was assembled. As shown in Figure 1A, the system primarily consists of a blower fan, a UV irradiation system, a simulated seawater tank, and a control system, which are respectively used for wind-drying, UV irradiation, seawater immersion, and centralized control. After placing the mice in the apparatus and starting the system, the conditions of immersion and emersion phases on the shore were successfully simulated (Figure 1B). Based on this setup, we first investigated sunscreen retention under these conditions. Initially, commercially available Sunscreens A, B, and C, labeled with a fluorescent marker, were applied to the dorsal skin of the mice. Subsequently, these mice were subjected to an alternating cycle in the simulation apparatus, consisting of 30 min of immersion (simulating swimming) and 30 min of emersion phase (simulating sunbathing on the shore). After 2 h, following a complete cycle of wave and wind, the retained fluorescence on the skin was detected using in vivo imaging. As shown in Figure 2A–B, compared to pre-simulation, all sunscreen groups exhibited a decrease in fluorescence intensity, indicating that the wash-off effect of seawater waves causes a significant amount of sunscreen to be removed. Among them, the quadrant treated with Sunscreen C retained a relatively high amount of fluorescence, Sunscreen B showed a moderate retention rate, and Sunscreen A exhibited the lowest retention rate (Figure 2C). This indicates that Sunscreens C and B possess relatively stronger retention capabilities. These data confirm that our apparatus can quantitatively differentiate the durability of different formulations under complex environmental stress.

Photoprotective efficacy
To further investigate the UV-protective effects of these sunscreens in a simulated seaside environment, macroscopic observations of the dorsal skin of mice were conducted. As shown in Figures 3A and 3B, the unprotected control exhibited UV-induced damage, characterized by pronounced erythema, redness, and sunburn. In contrast, the regions treated with sunscreens showed significantly alleviated sunburn symptoms. Furthermore, histological analysis using H&E staining was performed and was consistent with the aforementioned macroscopic observations (Figure 4A–B). When the unprotected control was exposed to the seaside simulator, significant thickening of the epidermis and marked infiltration of inflammatory cells (Figure 4C) in the dermis were observed, demonstrating that the simulated seaside testing system effectively replicates skin sunburn in a practical seaside environment. The application of sunscreens significantly mitigated these pathological changes. Notably, the epidermis thickness and inflammatory cell infiltration of the skin tissue treated with Sunscreen C were remarkably similar to those of the UV-shielded internal control subjected to the same environmental stress. This indicates that Sunscreen C provides the optimal protective effect in the simulated marine environment, likely attributable to its high retention rate and inherent photoprotective properties.

Figure 1: Overview of the simulated seaside testing system. (A) Schematic diagram of the apparatus. (B) Mice inside the testing chamber. The customized test chamber integrates a UV irradiation device, a water tank with a wave generator, and a blower fan, effectively simulating a practical seaside environment. No copyright concerns are associated with this figure as it is obtained by the authors. Please click here to view a larger version of this figure.

Figure 2: Sunscreen retention fluorescence analysis. (A) Representative fluorescence images of the mouse dorsal skin before and after the 120-minute experiment. (B) Quantitative analysis of fluorescence retention. (C) Relative fluorescence intensity analysis. Data are presented as mean ± SD (n = 5). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05, **p < 0.01. Please click here to view a larger version of this figure.

Figure 3: Macroscopic appearance of mouse skin after environmental exposure. (A) Representative macroscopic images of the mouse dorsal skin after a 120-minute exposure to the simulated seaside environment. (B) Locally enlarged views showing the occurrence of erythema and peeling in the unprotected control area compared to the sunscreen-protected areas and the UV-shielded internal control (Normal). Please click here to view a larger version of this figure.

Figure 4: Histological evaluation of photoprotective efficacy. (A) Representative H&E-stained sections of the mouse dorsal skin from different treatment areas following environmental exposure. Indicator lines identify the boundaries of the measured epidermis. Scale bar = 250 µm. (B) Quantitative analysis of epidermal thickness in each designated area. (C) Quantitative assessment of relative inflammatory cell counts (%). The UV-shielded internal control (Normal) was set as the baseline (100%). Data are presented as mean ± SD (n = 5). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. Please click here to view a larger version of this figure.

Discussion

In this study, a multi-factor simulated seaside testing system combined with a live murine model was successfully developed and validated. Beyond providing a novel apparatus, this integrated protocol enables the dynamic evaluation of sunscreen durability and photoprotective efficacy under synergistic environmental stressors. By bridging the gap between simplified in vitro assays and complex human clinical trials, this method establishes a comprehensive preclinical platform for understanding how skin barrier formulations perform in practical marine settings.

The primary advantage of this protocol over conventional methods is its high environmental fidelity. Standard water-resistance tests typically involve static water immersion in a laboratory tub¹⁹. In contrast, our system integrates the mechanical friction of wave action, the osmotic stress of high-salinity seawater, the evaporative effect of wind, and continuous UV irradiation. These factors act synergistically to break down the sunscreen film, providing a much more rigorous and realistic stress test. It should be noted that during the fluorescence retention assessment, UV lamps were turned off to prevent fluorophore quenching. Consequently, this specific test evaluates physical durability against mechanical washout and osmotic stress, but lacks the UV-induced chemical degradation factor. To compensate for this, the subsequent histological evaluation was conducted with the UV module activated, providing a holistic assessment of practical efficacy. The results clearly demonstrated that while all sunscreens provided some baseline protection, the retention rate (Figure 2) and the corresponding histological protection (Figure 4) varied significantly depending on the formulation's resistance to these combined dynamic forces.

The use of BALB/c mice provides an excellent in vivo platform that accounts for biological variables such as sebum secretion, transepidermal water loss, and dynamic skin movement, which artificial skin models cannot replicate. The UV dose delivered during the 120-minute cycle was strictly controlled to effectively induce acute sunburn on the exposed skin area without causing any systemic toxicity or severe damage, keeping it strictly within ethical limits for BALB/c mice. Furthermore, by dividing a single mouse's back into four quadrants, we eliminated inter-individual biological variation, allowing for highly accurate paired comparisons between different sunscreen formulations.

A critical step in this protocol is ensuring the complete recovery of the murine skin after depilation, as any baseline irritation will confound the histological readout. Furthermore, the uniform application of the sunscreen film, stable calibration of the environmental parameters prior to testing, and precise timing of the immersion and emersion phases are vital to ensuring high reproducibility between batches. Regarding troubleshooting, several foreseeable issues may arise. If uneven sunscreen spreading is observed, recalibrate the pipetting volume and ensure a gentle circular motion. If erythema resulting from depilation persists, the recovery period should be extended, or the affected animals should be excluded. To prevent animal fatigue during immersion, strictly monitor their activity and ensure the resting platform is fully accessible during the immersion phase.

A limitation of this model is the anatomical and physiological difference between murine skin and human skin (e.g., hair follicle density and epidermal thickness). Murine skin possesses higher hair follicle density and a thinner epidermis, which may alter sunscreen film formation. The abundant hair follicle invaginations might act as micro-reservoirs, potentially favoring the mechanical retention of particulate-based (inorganic) sunscreens over strictly organic formulations. While it is a robust comparative tool for screening formulations, human clinical trials remain necessary for final SPF labeling. Future applications of this system are broad. Beyond sunscreens, this apparatus can be adapted to evaluate the durability of other topical cosmetics, barrier creams, wound dressings, and even UV-protective textiles under harsh environmental conditions. The integration of this system streamlines the R&D process, offering cosmetic chemists and dermatological researchers a rapid, cost-effective, and ethical screening method before moving to human trials.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Eosin	Macklin	770427	Used for H&E staining
Ethanol	Macklin	E809059	Used for H&E staining
Hair removal cream	Veet	TMG-001	Used for removing fine hairs
Hematoxylin	Macklin	H810910	Used for H&E staining
Image J			Used for image analysis
In vivo Imaging System	Perkinelmer	IVIS Lumina III	Used for detecting fluorescence retention
Isoflurane	RWD Life Science	R510-22-10	Used for anesthetizing mice
Paraformaldehyde	Biosharp	BL539A	Used for fixing skin tissues
Shaver	Chemmole	CM700015	Used for removing most of the hair
Sodium chloride	Macklin	S805285	Used for manufacturing simulated seawater
Sulfo Cy7	Ruixibio	R-Z54515	Used for marking sunscreen
UV lamp	Kanfu Lighting Technology	T5-6W	Used for providing ultraviolet rays
Xylene	Macklin	X821391	Used for H&E staining