Development and Application of Radiation-Induced Skin Injury Animal Models Based on Strontium-90 β-rays

Summary

Here, we present a protocol to establish reproducible animal models of radiation-induced skin injury using strontium-90 (⁹⁰Sr) β-radiation. This protocol enables the investigation of dose effects and inflammatory responses in superficial radiation damage, providing a foundation for future mechanistic studies.

Abstract

Radiation-induced skin injury is a severe complication frequently encountered in nuclear accident emergencies or radiation-related occupations, necessitating the establishment of standardized, specificity-focused injury models for mechanistic research. Conventional gamma or high-energy X-rays, with strong penetrating ability, induce skin damage but inevitably trigger complex deep-tissue reactions, interfering with investigations of superficial skin effects. Given the risk of pronounced superficial tissue damage from β-radiation in scenarios such as nuclear fallout, we developed a strontium-90 (⁹⁰Sr) β-rays source-based animal model of radiation-induced skin injury to address this challenge. This model leverages the physical property of β-rays, which deposit energy predominantly in superficial tissues, enabling precise simulation of localized biological effects caused by radiation material contact with the skin while avoiding the potential systemic responses triggered by deep-penetrating radiation. This study systematically outlines the entire workflow, including ⁹⁰Sr radiation source parameterization, animal irradiation protocols, and pathological specimen collection and evaluation methods. This model provides a reliable animal tool for elucidating the pathophysiological mechanisms of radiation-induced skin injury and stimulating the development of mechanistic investigations or therapeutic strategies.

Introduction

Ionizing radiation is widely used across multiple fields, including energy, medicine, industry, and agriculture. While it brings significant benefits, it also poses direct and potential threats to human health, particularly for occupational workers and cancer patients^1,2. Given that the skin is the body's largest organ and forms its outer covering, it inevitably serves as the primary target upon exposure to ionizing radiation. Composed of the epidermis, dermis, and subcutaneous tissue, the skin performs indispensable physiological functions. However, the mechanisms underlying radiation-induced skin injury and effective strategies for its prevention and treatment remain insufficiently elucidated. Therefore, there is a critical need for a standardized and reproducible experimental model that specifically targets radiation-induced skin injury in the superficial layer and is suitable for mechanistic and interventional studies. Consequently, to advance research in this field, we have successfully established a novel animal model of radiation skin injury.

In conventional studies, high-energy radiation, such as X-rays or γ-rays, is commonly used to induce skin injury models^3,4. These types of radiation possess high penetrating ability, allowing them to interact easily with subcutaneous and deeper tissues and organs. Consequently, while inducing skin damage, they also trigger extensive responses in deeper tissues, which compromise the specificity of the model for skin-focused research and significantly complicate the interpretation of results⁵. This lack of tissue specificity represents a major limitation when the primary research objective is to investigate skin-restricted radiation injury rather than systemic radiation effects.

In contrast, we have developed a more specific and refined skin injury model, leveraging the characteristics of strontium-90 (⁹⁰Sr) β-radiation, namely its low energy and limited tissue penetration. Research has shown that the damage caused by β-rays is predominantly confined to the epidermal and dermal layers of the skin⁶, thereby effectively minimizing interference from deep tissue response. Compared to high-penetration radiation models, this approach allows for precise localization of injury, reduced involvement of visceral organs, and improved interpretability of skin-specific pathological and inflammatory changes. In addition, utilizing β-rays to establish a radiodermatitis model is also justified by critical considerations of potential real-world threats. Lessons from the Chernobyl nuclear accident demonstrate that high-dose external exposure to β particles can lead to severe β burns and even fatal injuries⁷. Furthermore, potential long-term health risks posed by β-radiation from radiation contaminants also exist^8,9.

To explore these mechanisms, selecting an appropriate animal to be subjected to β-ray irradiation is also crucial. While minipigs have been successfully used to establish β-rays induced skin injury models^10,11, their general applicability in research is more limited compared to mice. Furthermore, a key characteristic of mouse skin is its high density of hair follicles, which makes it a suitable model for exploring the effects of radiation on skin appendages in the study of radiation skin injury. Thus, using a mouse model has notable advantages. In addition, mouse models offer practical advantages, including lower cost, higher throughput, genetic manipulability, and broader compatibility with molecular and immunological analyses, making them particularly suitable for mechanistic studies and intervention screening.

Inflammatory cytokines are biologically active small molecules or proteins secreted by both immune and non-immune cells, broadly classified as pro-inflammatory or anti-inflammatory. In skin damage induced by ionizing radiation, these cytokines are key drivers. In fact, such damage is fundamentally an uncontrolled inflammatory disease, primarily orchestrated by a network of cytokines and chemokines¹². IL-6 is a key pro-inflammatory mediator and one of the critical cytokines involved in acute inflammatory responses to radiation and infection^13,14,15. Recent studies have shown that radiation activates the IL-6/STAT3 signaling pathway and cellular senescence, thereby promoting immune cell infiltration and contributing to radiation dermatitis, skin inflammation, and hair loss¹⁶. Interestingly, it also exhibits radioprotective effects under specific contexts^17,18. In contrast, IL-10 is an important anti-inflammatory regulator. Its functions include limiting excessive inflammatory responses, enhancing innate immunity, promoting tissue repair mechanisms, and maintaining tissue homeostasis during infection and inflammation^19,20. IL-10 often acts in concert with IL-6, demonstrating beneficial effects in promoting dermal wound healing, reducing scar formation, and ameliorating certain dermatological conditions^21,22,23. While existing research has demonstrated that IL-6 and IL-10 respond to radiation and ultraviolet exposure, respectively^16,24,25, their responses to β-radiation remain unclear.

In summary, this study aims to establish a stable and reproducible mouse model of radiation-induced skin injury by ⁹⁰Sr β-radiation. This model is especially appropriate for studies focusing on cutaneous radiation injury, β-radiation exposure scenarios, inflammatory regulation, skin appendage damage, and long-term tissue remodeling. However, it is not intended to replicate deep-tissue or solid tumor radiotherapy, which typically requires highly penetrating radiation such as X-rays or γ-rays. We present the procedures for model establishment and for systematically validating its feasibility through multi-level assessments, including radiation injury score, histological examination, and molecular expression of key cytokines (IL-6 and IL-10). Dynamic analysis of these parameters will help clarify the regulatory roles of IL-6 and IL-10 in the injury process, thereby providing a solid experimental basis and theoretical reference for the development of targeted intervention strategies in the future.

Protocol

All experimental procedures involving animals were reviewed and approved by the Animal Experimentation Ethics Committee of the West China School of Basic Medical Sciences & Forensic Medicine (Chengdu, China).

1. Animal preparation

1. Animal housing and grouping
   1. Assign twelve SPF-grade female C57BL/6J mice (weight range: 18–25 g, age: 3 months) to four standard ventilated cages and randomly allocate them into four groups (n=3) for exposure to varying doses of external irradiation (a non-irradiated control group and 3 experimental irradiation groups).
   2. Maintain all animals on a 12-hour light/dark cycle with ad libitum access to standard rodent chow and water.
   3. Use numbered ear tags to uniquely identify each mouse and distinguish between experimental groups.
   4. Allow the experimental animals to acclimate to the new environment for 3 days prior to subsequent procedures.
   5. Place one Sprague Dawley (SD) rat (age: 2 months; weight: around 160 g) under identical conditions.
2. Anesthesia
   1. Anesthetize the mice via intraperitoneal injection of 1.25% tribromoethanol solution. To ensure safe anesthesia, calculate the injection volume based on body weight, administering 15–20 mL/kg of body weight, in accordance with established reference standards.
3. Preparation of the irradiation site
   1. Following anesthesia, remove fur over the intended dorsal irradiation site using electric clippers.
   2. Using a red marker pen and a pre-prepared rigid template of approximately 3 cm in diameter, mark a circular area for irradiation on the shaved dorsal skin.
      NOTE: Depilatory creams are not used, as they may cause skin irritation and potentially alter histological architecture, thereby confounding experimental results.

2. Dose calculation for irradiation

NOTE: ⁹⁰Sr emits high-energy β-rays through its decay to ⁹⁰Y (Figure 1A). β-rays have poor penetration performance, typically affecting only the epidermis. Precise determination of irradiation time for mice requires computation of absorbed dose rate (D, cGy/s).

1. Calculate the decay constant (λ) from the physical half-life (T½) using the formula:
   T½ ≈ 0.693/λ
   Given that the half-life of ⁹⁰Sr is approximately 28.6 years, λ ≈ 0.02423 year^-1.
2. Calculate the number of radionuclides (N) using the following formula:
   A = λ N
   Where:
   A: Radioactivity (mCi)
   N: Number of radionuclides after time t (years)
   With an initial radioactivity A₀ = 30 mCi (1 Ci = 3.7x10¹⁰ Bq) and λ ≈ 0.02423,
   N₀ = A₀/λ = 1238.13454
   Then, apply the general radiation decay law, i.e., the number of radionuclides decreases exponentially over time:
   dN/dt = -λ N
   Integrating yields:
   N = N₀е^-λt
   Where:
   N₀: Initial number of radionuclides at t = 0
   N: Number of radionuclides remaining after time t
3. Finally, calculate the absorbed dose rate, D, in cGy/s using the following formula:
   Conversion formula for the absorbed dose rate of a ⁹⁰Sr-⁹⁰Y applicator:
   D = [(A×1770/S) ×(E⁹⁰_Y/E³²_P) ×Fm] /3600 = (A/S) ×0.6347
   Where
   D: Absorbed dose rate (cGy/s)
   A: Millicurie (mCi)
   S: Area (cm²)
   1770: Ionization constant for ³²P
   E⁹⁰_Y: Average energy = 0.93 MeV
   E³²_P: Average energy = 0.693 MeV
   Fm: Exposure-to-dose conversion factor (0.962)
   3600: Hour-to-second conversion factor
   NOTE: The calculations of the ⁹⁰Sr parameters are summarized in Table 1. An example calculation is shown below:
   On March 3, 2023:
   Applicator outer diameter = 3 cm, inner diameter = 2.47 cm, radius = 1.235 cm, Radioactivity A = 30 mCi
   S = π ×1.235²
   D=(A/S) ×0.6347 ≈ 3.9738 cGy/s
   On November 3, 2023 (t = 0.583 years later):
   N(t) = N₀е^-λt = 1238.13454×e^{-0.02423×0.583} ≈ 1220.7675
   A(t) = λN(t) = 0.02423×1220.7675 ≈ 29.579 mCi
   D = (A/S) ×0.6347 ≈ 3.918 cGy/s
   After t = 2.25 years:
   N = N₀е^-λt ≈ 1238.13454×e^{-0.02423×2.25} ≈ 1172.4391
   A = λN ≈ 0.02423×1172.4391 ≈ 28.408 mCi
   D = (A/S) ×0.6347 ≈ 3.7629 cGy/s
4. Calculate the irradiation time of each dose group based on the data obtained (see Table 2).

3. Irradiation

NOTE: Although β-rays emitted by ⁹⁰Sr have limited penetration, typically affecting only the epidermal layer (Figure 1D), they still pose potential risks to human health. Ensure that all procedures involving ⁹⁰Sr sources follow appropriate protective measures to minimize radiation exposure to operators and the environment. Operators must complete specialized training on radiation source handling. The procedures for handling and using radiation sources strictly adhered to the international standards outlined in The 2007 Recommendations of the International Commission on Radiological Protection (ICRP Publication 103).

1. Pre-use preparation: Submit an application for ⁹⁰Sr source usage. After approval by the supervisor, collect the laboratory key and the alarm system disarm code.
2. Prepare all required materials, including mice with shaved and marked dorsal areas, 1 mL syringes, tribromoethanol solution, food-grade plastic wrap, timers, tape, and foam boards.
3. Mouse pretreatment
   1. Anesthetize mice via intraperitoneal injection of tribromoethanol solution (see step 1.2).
   2. Once fully sedated, place the mouse dorsum-up on a rigid board, cover its dorsal surface with plastic wrap, and secure the wrap with tape.
4. Irradiation procedure
   1. Enter the radiation source room, disarm the alarm system, don protective gear (protective suit, cap, goggles, and personal dosimeter), and then retrieve the ⁹⁰Sr source from the lead shield.
   2. Attach a metal rod to the top knob of the source to lift and move the applicator.
   3. To simulate β-radiation-induced skin injury relevant to nuclear accidents or occupational exposure, position the emitting surface (Figure 1B) of the ⁹⁰Sr applicator over the marked shaved dorsal area of the anesthetized mouse (Figure 1C). Start the timer immediately upon placement. Precisely after the preset duration, retract the applicator and temporarily store it in its original container.
      CAUTION: Never face the applicator's bottom toward personnel.
   4. Return the ⁹⁰Sr source to the lead shield after use.
      NOTE: For the 10 Gy×5 group, irradiate once daily for 5 consecutive days to achieve a total dose of 50 Gy.
5. Post-irradiation steps
   1. Complete the Radiation Source Inspection Record and Radiation Source Usage Log, reactivate the alarm system, lock the door, return the key, and report the source status.
   2. Collect all radioactive waste generated during the procedure and dispose of it as per institutional guidelines.

4. Radiation injury scores

1. Assess the skin damage progression in mice by examining the irradiation site every 2–3 days during the initial phase. Upon observation of any abnormal skin appearance, increase the assessment frequency to daily. Once lesion progression has stabilized, return the assessment interval to every 2–3 days.
   NOTE: Throughout the entire process, the unirradiated control group underwent identical procedures as the irradiated groups, including subsequent scoring and tissue collection. Sampling timepoints for the control groups are scheduled to coincide with the progression phase of skin injury in the irradiated groups.
2. Photograph the irradiated skin areas periodically and assess the status of radiation-induced dermatitis on the mouse dorsum.
3. Score skin reactions within the irradiated field using a validated scale (see Table 3).
   NOTE: When scoring the injuries, have three investigators independently perform blinded assessments without knowledge of the irradiation dose. Base the scoring on the predominant wound characteristics according to the established criteria. Assign a final score by consensus, acknowledging that some wounds might not perfectly match every descriptor in the scoring system but still reflect the essential pathology.

5. Sampling and staining

1. Obtaining skin tissue: On days 13, 19, 27, and 32 post-irradiation, collect serial full-thickness skin biopsies from mice in each treatment group, within the pre-marked circular irradiation field.
   1. Anesthetize the mice by intraperitoneal injection of tribromoethanol solution (15–20 mL/kg) as in step 1.2.
   2. Disinfect the surgical area with 75% alcohol. Then, use sterile fine forceps to stabilize the target skin.
   3. Excise a full-thickness skin sample of approximately 0.5 cm² vertically using surgical scissors along the transition zone between damaged tissue, ensuring inclusion of the central injured area. Hold the scissors perpendicular to the skin surface and extend the cut down to the subcutaneous fascial layer.
   4. Place the harvested tissue immediately in 4% paraformaldehyde for fixation.
   5. Apply gentle pressure to the wound for hemostasis. For each sampling session, follow a strategy of moving from one of four directions toward the center of the irradiation field.
2. Histological and immunofluorescence staining
   NOTE: H&E and immunofluorescence staining were performed with the assistance of a commercial service provider (Servicebio Biotechnology, Wuhan, China) in accordance with their standard optimized protocols.
   1. Perform hematoxylin and eosin (H&E) staining on the skin samples collected at all four time points (days 13, 19, 27, and 32 post-irradiation).
      1. First, embed the fixed tissue samples in paraffin and create tissue sections.
      2. Perform dewaxing and hydration by immersing the sections sequentially in Environmental Friendly Dewaxing Transparent Liquid I (20 min), Environmental Friendly Dewaxing Transparent Liquid II (20 min), anhydrous ethanol I (5 min), anhydrous ethanol II (5 min), and 75% ethanol (5 min), then rinse with tap water.
      3. Treat all sections with the HD constant staining pretreatment solution for 1 min. Then, stain with the hematoxylin solution for 3–5 min, followed by rinsing with tap water.
      4. Next, immerse the sections in hematoxylin differentiation solution and rinse again with tap water. Then, treat them with hematoxylin bluing solution followed by another rinse.
      5. Subsequently, place sections in 95% ethanol for 1 min and counterstain with eosin dye for 15 s.
      6. Next, dehydrate the sections using a series of absolute ethanol (three changes, 2 min each), normal butanol (two changes, 2 min each), and xylene (two changes, 2 min each), and finally seal them with neutral gum.
   2. Perform immunofluorescence staining to visualize the inflammatory cytokines IL-6 and IL-10.
      NOTE: Based on the overall progression of injury, select tissues obtained on day 13, when visible lesions began to appear, and day 27, when injury progression stabilizes and shows signs of recovery, for immunofluorescence staining to visualize the inflammatory cytokines IL-6 and IL-10.
      1. Deparaffinize and rehydrate the sections using xylene (three changes, 10 min each) and graded ethanol (three changes of pure ethanol, 5 min each), then rinse them in distilled water.
      2. For antigen retrieval, heat the slides in 1× citric acid antigen repair solution (pH 6.0) at 70°C for 20 min. Allow the slides to cool naturally, then wash them in PBS (pH 7.4) on a decolorizing shaker (three washes, 5 min each).
      3. Next, apply 3% BSA to block nonspecific binding at room temperature for 30 min.
      4. Prepare and apply a mixed primary antibody solution containing anti-IL-6 (1:3000) and anti-IL-10 (1:2000), then incubate the sections at 4°C overnight in a humidified chamber.
      5. The following day, wash the slides in PBS (three times, 5 min each) and incubate with appropriate secondary antibodies at room temperature for 50 min in the dark.
      6. After washing in PBS (three times, 5 min each), counterstain the nuclei with DAPI for 10 min in the dark. Then, wash again in PBS (three times, 5 min each), treat with the autofluorescence quencher for 5 min, and rinse under running water for 10 min.
      7. Finally, mount coverslips using anti-fade mounting medium.
3. Quantitative analysis
   1. Use Image J software to quantify epidermal thickness from H&E-stained sections.
      1. Calibrate the image scale in Image J by tracing the scale bar with the straight-line tool and setting the known length in Analyze > Set Scale.
      2. Then, go to Analyze > Tools > ROI Manager to select the regions of interest and add them to the ROI Manager.
      3. Finally, click Measure in the ROI Manager to obtain the actual lengths.
   2. Use Image J software to measure the mean immunofluorescence intensity of IL-6 and IL-10.
      1. Import the image and split channels via Image > Color > Split Channels. Then select the channel corresponding to the target fluorescence (e.g., red for IL-6, green for IL-10).
      2. Apply auto-thresholding under Image > Adjust > Auto-Threshold > Try All. In the Threshold window, select the optimal algorithm and manually adjust the slider to match the actual fluorescence.
      3. Set measurements under Analyze > Set Measurements by checking Mean Gray Value and enabling Limit to threshold.
      4. Finally, execute Analyze > Measure to obtain intensity data.

Results

Physical examination findings
Mice were exposed to β-radiation from a ⁹⁰Sr source at three different dosage regimens: 0 Gy, 25 Gy, 10 Gy×5 fractions, and 50 Gy. Concurrent on-site evaluations were performed to document the progression of skin damage until injury progression had stabilized. The experimental results demonstrated that ⁹⁰Sr β-radiation induced radiation-induced skin injury across all groups, and the degree of injury depended on the irradiation dose. Regardless of th...

Discussion

In establishing an animal model of radiation skin injury, we utilized long-lived radionuclide ⁹⁰Sr as a source of β-radiation. Precise calculation of the radiation dose rate and irradiation time constituted a critical step in the successful development of this model. The clinical application of ⁹⁰Sr in tumor therapy has been documented, with its reliability receiving some partial validation^27,28. While other β-ray radionuclides (e....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-IL-6 Rabbit pAb	Servicebio	GB11117	Immunofluorescence staining
Anti-IL-10 Rabbit pAb	Servicebio	GB11108	Immunofluorescence staining
Citric acid antigen repair solution (PH6.0)	Servicebio	G1202	Immunofluorescence staining
Dehydrator	DIAPATH	Donatello	H&E and Immunofluorescence  staining
Digital camera	Canon	EOS 70D	Document the progression of skin injury during the experiment
Disposable sterilized syringe	Professional Manufacturer Of Disposable Sterile Medical Devices.	GB20163140107	For intraperitoneal injection
Disposable Latex Glove	Medicom	1186	Protection
Environmental Friendly Dewaxing Transparent Liquid	Servicebio	G1128-1L	H&E and Immunofluorescence  staining
Frozen platform	Wuhan Junjie Electronics Co., Ltd	JB-L5	H&E  staining
Hematoxylin-eosin (H&E) HD constant dye kit	Servicebio	G1076	H&E  staining
Image J	National Institutes	Image processing	Enable precise quantification of histological staining data
Lead apron	Shandong Oureixin Radiation Protection Engineering Co., Ltd.	N/A	Protect personnel from radiation exposure
Liquid Blocker PAP Pen	Servicebio	G6100	Immunofluorescence staining
Medical film	3M	10163337409520	Prevent contamination of the radiation source by biological surfaces
Neutral gum	SCRC	10004160	H&E  staining
Nuclear radiation detector	FNIRSI	100128262296	Real-time monitoring of ambient radiation levels around the operator to ensure personnel safety
Paraformaldehyde Fixative (4%)	Biosharp	BL539A	Fix skin tissue samples
Pathology slicer	Shanghai Leica Instrument Co., Ltd	RM2016	H&E and Immunofluorescence  staining
Paraffin embedding machine	Wuhan Junjie electronics	JB-P5	H&E and Immunofluorescence  staining
Strontium-90	Khlopin Radium Institute Joint-Stock Company (Radium Institute JSC,Russia)	N/A	Provide β-rays
Tribromoethanol	Nanjing Aibei Biotechnology Co.,Ltd	M2910	Anesthetize mice
Water bath-slide drier	ZHEJIANG JINHUA KEDI INSTRUMENTAL EQUIPMENT	KD-P	H&E and Immunofluorescence  staining