Irradiation of the zebrafish caudal fin.

(A) Schematic representation of currently used beam types for preclinical radiotherapy studies. (B) Overview of a semi-amputated zebrafish caudal fin. Five to seven days post amputation, a single beam with a dose of 5000 Gy and a width of 25, 50, 100, 200, 400 or 800 µm was applied through both mature and immature parts of the fin.

Early cellular response: local adhesion of neutrophils and thrombocytes

(A,A′) Stereomicroscopic image showing that fli1a⁺ blood cells adhered to the venous wall inside the beam path (white arrowheads) in vivo. (B) Electron micrograph depicting an accumulation of neutrophil (Ne). (C) Scatter plot of peripheral blood with back-gated fli1a⁺ cells (green) clustering in two distinct populations. Red = lymphocytes and thrombocytes, blue = precursor cells. (D) Blood smear of untreated zebrafish whole blood. Fli1a⁺ cells were identified by fluorescence microscopy and relocated after staining with Giemsa-May Grünwald. (E) Fraction of fli1a⁺ cells in the whole blood (n = 40 [number of animals]). (F,G) Pseudo-color plots showing the absence of fli1a-expression in CD4⁺ and CD8⁺ leukocytes. (H) Example of a thrombocyte expressing CD41 and fli1a in 5 dpf old fish in vivo. (I) Fraction of CD41 and fli1a expressing blood cells in the posterior caudal vein in vivo (n = 10 [number of animals]).

Width-dependent effects of IR on the immature caudal fin vasculature in vivo and as schematic representations.

Dashed lines depict the beam path. Titles (left grey column) refer to the applied beam width. (A-F) Overviews. (G) Non-irradiated vein, 5 days post amputation. (H) 25 µm-wide beams induced adhesion of few fli1a⁺ thrombocytes in the beam path. (I) 50 µm-wide beams: adhesion of fli1a⁺ thrombocytes to the venous wall in the beam path. (J) Vein hit by a 100 µm-wide beam: About 1 µm sized spots of clumped, intensely fluorescent cells. (K) 200 µm-wide beams: Disrupted arteries and veins. (L) 800 µm-wide beams induced massive cell death inside the beam path. (M-R) Schematic representation of the beamlet width-dependent effects integrated from in vivo- (fli1a⁺ blood cells) and electron microscopy (neutrophils). Black arrows indicate the presence or absence (in case the arrow is crossed) of blood flow in arteries or veins. Accumulations of fli1a⁺ thrombocytes and neutrophils are indicated by green and black icons, respectively.

Effects of micro- (50 µm) and minibeam (800 µm) irradiation on the mature and immature vasculature.

(A-H) Stereomicroscopic image of the vascular network in vivo at the site of irradiation. At 6 hpi (hours post irradiation), the mature vasculature was not affected by any type of irradiation. In contrast to microbeam-irradiated fish, massive vascular damage was visible in the mature fin of minibeam-irradiated fish at 48 hpi. (I,J) Brightfield images of whole caudal fins at 96 hpi.

Structural alteration after micro- and minibeam irradiation.

(A-D′) Semithin sections of the immature fins stained with toluidine blue. Shown are two neighboring bony fin rays (FR) and the loose connective tissue with blood vessels (BV). The site of irradiation is indicated by dashed lines and arrows. The cells in the beam path were characterized by solitary dark intracellular bodies and inclusions. Their number increased in the path of 200 µm wide beamlets. (A′′-D′′) Transmission electron microscopy demonstrated normal tissue appearance within the control fins with fibroblasts (F) of normal appearance, embedded in loose extracellular matrix and endothelial cells (EC). Electron-dense intracellular apoptotic bodies (ApB). Macrophages (Mφ) containing cellular debris and autophagic vacuoles (arrowheads). (D) The inter-ray tissue was disrupted and infiltrated by neutrophils (Ne).

Attaching and detaching fli1a⁺ thrombocytes in the beam path previously irradiated with one 50 µm-wide beam. Real-time movie of a vein in the immature caudal fin at 6 hpi. Fli1a⁺ thrombocytes (white arrowheads) continuously attach and detach to the site of irradiation (orange encircled area).

Extravasated and tissue-resident neutrophils (Ne) in the loose connective tissue. Neutrophils (Ne), fibroblast (F), blood vessel (BV).

Neutrophils did not express fli1a. (A) Blood smear of zebrafish blood 3h post LPS-injection. (B) Neutrophils (red arrowheads) did not express fli1⁺.

Microbeam IR with 100 µm-wide beams intersects arteries (6 hpi). Vasculature of fin rays irradiated with a 100 µm-wide beam. The yellow dashed line indicates the site of irradiation. Arteries are disrupted, whereas fli1a⁺ blood cells (white arrowheads) adhere to the vessel wall in veins sited in the beam path.

Blood perfusion of the mature and immature fin. In the mature fin, the blood flows past the irradiation site in both directions. In the immature fin, the blood vessels are discontinued, causing a defective blood perfusion distal to the site of irradiation.

Movie of CD41⁺/fli1a^- blood cells rolling on endothelium in 5 dpf old fish

Effects of microbeam (25, 100 µm) irradiation on the mature and immature vasculature at 6, 48, and 96 hpi.

Effects of microbeam (200, 400 µm) irradiation on the mature and immature vasculature at 6, 48, and 96 hpi.