Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis

Summary

Here we present a protocol to induce radiation-induced skin fibrosis in the hind limb of mice and perform post-irradiation measurements of chronic impairment via limb excursion and gait index analyses to evaluate the functional outcome. The model elucidates radiation-related skin fibrosis mechanisms and is useful in subclinical therapeutic studies.

Abstract

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. Radioactive beams can penetrate through the skin and affect the structures in their path including skin, muscles, and internal organs. Skin is the first structure to get exposed to radiation and is susceptible to develop chronic fibrosis, which is challenging to treat. Currently, limited treatment options show moderate efficacy in mitigating radiation-related skin fibrosis. A key factor hindering the development of effective countermeasures is the absence of a convenient and robust model that could allow for translation of the experimental findings to humans. Here, a robust and reproducible murine hind limb skin fibrosis model has been established for prophylactic and therapeutic evaluation of possible agents for functional and molecular recovery.

The right hind limb was irradiated using a single dose of 40 (Gray) Gy to induce skin fibrosis. Subjects developed edema and dermatitis in the early stages proceeded by visible skin constriction. Irradiated limbs showed a significantly reduced limb range of motion in the following weeks. In late stages, acute side effects subsided, yet chronic fibrosis persisted. A gait index was performed as an additional functional assay, which demonstrated the development of functional impairment. These non-invasive methods demonstrated reliable measurements for tracing fibrosis progression, which is supported by histological analyses. The radiation dose, application, and post-irradiation analyses employed in this model offer a vigorous and reproducible method for studying radiation-induced skin fibrosis and testing the efficacy of therapeutical agents.

Introduction

The skin is the largest organ of the body, covering and protecting the body from hazards. It has three distinct layers: epidermis, dermis, and hypodermis. Each layer has its unique functions: the epidermis prevents dehydration and microbial invasion; the dermis has a rich network of cells, and an extracellular matrix that provides tensile strength and elasticity¹; the dermal layer contains the sensory receptors, hair follicles, glands, and vessels for lymphatic and capillary networks. The hypodermis or subcutaneous tissue, with its abundance of adipose tissue, contours the body and distributes mechanical stress^2,

Protocol

Ethical animal use was approved by the Institutional Animal Care and Use Committee (IACUC), which acts in compliance with the Animal Welfare Act. Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) approved facility and treated according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

1. Anesthesia

1. Place mice in the box of a small animal anesthesia system (

Discussion

Skin injury is a likely outcome of accidental or medical treatment-related exposure to radiation. Nuclear reactors possess an accidental breach risk due to human error or natural disasters like Chernobyl and Fukushima^26,27. Therapeutical dosing for cancer treatment is the most common exposure, which uses fractionated repeated dose regimens that risk causing radiation-related fibrosis in the treated areas. This common chronic adverse reaction can be prevalent in u.......

Materials

Name	Company	Catalog Number	Comments
10% Formalin	Fischer Scientific	23-427098
Bolus	Orfit	8333.SO1/R
Clipper	Kent Scientific Corp.	CL8787-KIT
CO2	Various
CO2 Chamber	E-Z Systems Inc.	E-22000
Depilatory Cream	Church & Dwight Co., Inc.		Nair
Digital Camera	Wolfang	GA100
Eppendrof Tubes	Eppendorf	22364111
Eye Lubricant	Dechra		Puralube Ophthalmic Ointment
Gauze	Covidien	682252
Image Processing Program	NIH		Image J
Isoflurane	Dechra		USP Inhalation Anesthetic
Linear Accelaerator	Varian Medical Systems, Inc.		23EX
PBS	Cytiva	SH30256.LS
Pentobarbital	Akorn Pharmaceuticals		Nembutal
Protractor	Westcott	550-1120
Small Animal Anesthesia System	E-Z Systems Inc.	EZ-SA800	Single animal system
Spreadsheet Software	Microsoft		Excel
Surgical Scissors	Medline	MDS0834111
Surgical Tape	3M	1538-1
Tape	3M	H-1113