A Mouse Ear Model for Bystander Studies Induced by Microbeam Irradiation

Abstract

Radiation-induced bystander effects have been observed in vitro and in cell and tissue culture models, however, there are few reported studies showing these effects in vivo. To our knowledge, this is the first reported study on bystander effects induced by microbeam irradiation in an intact living mammal. The mouse ear was used to investigate radiation-induced bystander effects in keratinocytes, utilizing a 3 MeV proton microbeam (LET 13.1 keV/μm) with a range in skin of about 135 μm. Using a custom-designed holder, the ear of an anesthetized C57BL/6J mouse was flattened by gentle suction and placed over the microbeam port to irradiate cells along a 35 μm wide, 6 mm long path. Immunohistochemical analysis of γ-H2AX foci formation in tissue sections revealed, compared to control tissue, proton-induced γ-H2AX foci formation in one of the two epidermal layers of the mouse ear. Strikingly, a higher number of cells than expected showed foci from direct irradiation effects. Although the proton-irradiated line was ~35 μm wide, the average width spanned by γ-H2AX-positive cells exceeded 150 μm. Cells adjacent to or in the epidermal layer opposite the γ-H2AX-positive region did not exhibit foci. These findings validate this mammalian model as a viable system for investigating radiation-induced bystander effects in an intact living organism.

FIG. 1

Panel A: The custom-designed mouse ear holder. The Plexiglas 13.9 × 5.4 cm holder consists of a mouse cradle to hold anesthetized mice in a supine position. Panel B: The left ear of the mouse is gently pulled toward the 3.2 mm circular opening where suction is applied by a vacuum handling system. The entire holder, containing the mouse with its flattened ear under vacuum, is then placed over the microbeam port and irradiated. Panel C: Thirty minutes after irradiation, the mouse was euthanized and a punch of the ear taken. Sections were cut perpendicularly to the direction of the charged particle beam, fixed and stained for biological effects as a function of the distance from the irradiated line.

FIG. 2

Panel A: Section of the ear of a sham-irradiated mouse (10× view). Panels B and C: No foci are visible in the color-outlined regions (60× view). Blue = 4′,6-diamidino-2-phenylindole (DAPI); green = Alexa Fluor 555 conjugated to anti-γ-H2AX.

FIG. 3

Panel A: Section of the ear of a proton microbeam-irradiated mouse (10×view). The 3 MeV proton microbeam with a range of 135 µm can traverse at least the stratum corneum and the first epidermal layer of the mouse ear. Panel B (purple box): γ-H2AX foci-positive keratinocytes are observed only in one area of the tissue. Cells adjacent to (panel C, red box) or opposite (panel D, orange box) the γ-H2AX-positive region do not exhibit foci (60× view). Blue = DAPI; green = Alexa Fluor 555 conjugated to anti-γ-H2AX.

FIG. 4

Panel A: Section of mouse ear exposed to whole-body 2 Gy X ray. Panels B and C: Essentially every cell in the tissue exhibits γ-H2AX foci (60× view). Blue=DAPI; green=Alexa Fluor 555 conjugated to anti-γ-H2AX.