γ-H2AX as a marker for dose deposition in the brain of wistar rats after synchrotron microbeam radiation

Abstract

Objective: Synchrotron radiation has shown high therapeutic potential in small animal models of malignant brain tumours. However, more studies are needed to understand the radiobiological effects caused by the delivery of high doses of spatially fractionated x-rays in tissue. The purpose of this study was to explore the use of the γ-H2AX antibody as a marker for dose deposition in the brain of rats after synchrotron microbeam radiation therapy (MRT).

Methods: Normal and tumour-bearing Wistar rats were exposed to 35, 70 or 350 Gy of MRT to their right cerebral hemisphere. The brains were extracted either at 4 or 8 hours after irradiation and immediately placed in formalin. Sections of paraffin-embedded tissue were incubated with anti γ-H2AX primary antibody.

Results: While the presence of the C6 glioma does not seem to modulate the formation of γ-H2AX in normal tissue, the irradiation dose and the recovery versus time are the most important factors affecting the development of γ-H2AX foci. Our results also suggest that doses of 350 Gy can trigger the release of bystander signals that significantly amplify the DNA damage caused by radiation and that the γ-H2AX biomarker does not only represent DNA damage produced by radiation, but also damage caused by bystander effects.

Conclusion: In conclusion, we suggest that the γ-H2AX foci should be used as biomarker for targeted and non-targeted DNA damage after synchrotron radiation rather than a tool to measure the actual physical doses.

Fig 1. Schematic representation of a broad beam and a microbeam array.

Fig 2. γ-H2AX stain comparison between micro- and broad beam configurations.

Horizontal sections of the irradiated right cerebral hemisphere and the cerebellum of Wistar rats. Images A—D were obtained from the irradiated right cerebral hemisphere and cerebellum of animals exposed to 350 Gy of either microbeam or broad beam; dissected 8 hours after irradiation. For the MRT array, the center-to-center distance was 200 μm. The position and intensity of the γ-H2AX marker (green) correlate with the deposition of the peak synchrotron doses. A) Radiation tracks of the microbeams in the right cerebral hemisphere. B) Right cerebral hemisphere after broad beam irradiation C) Radiation tracks of the microbeams in the cerebellum. D) Cerebellum after broad beam irradiation. E) Horizontal section through the whole cerebellum after exposure of the right hemisphere to microbeams of 350 Gy; dissected at 4 hours after irradiation.

Fig 3. Comparison of radiation tracks produced by the same intended microbeam configuration (center-to-center distance of 200 μm).

By design the collimator spatially fractionates the microbeams with a center-to-center distance of 400 μm; to generate a center-to-center distance of 200 μm the collimator moves laterally followed by a second passage of the animal through the beam. A) Variable center-to-center distance of the microbeams due to inaccurate lateral translation of the collimator (35 Gy, 4 hours after irradiation). B) Accurate delivery of the microbeams. (350 Gy, 8 hours after irradiation).

Fig 4. Mean thickness of the radiation tracks in the cerebellum.

Microbeam irradiation was given to both normal (A) and tumour-bearing rats (B). The width of the microbeams was 25 μm, which is represented by the dotted line. Animals were exposed to 35, 70, or 350 Gy to their right cerebral hemisphere. Four and 8 hours indicate the two dissection times after irradiation. Different letters and different letter cases indicate significant differences between groups and within each group respectively. Error bars show SD.

Fig 5. Intensity of the fluorescence measured through a 100 μm profile line traced perpendicular to the direction of the microbeams peak radiation path.

A) Intensity profile of normal rats. B) Intensity profile in tumour-bearing animals.

Fig 6. Different width of the radiation paths.

A) Microbeam tracks as outlined by γ-H2AX stain at 4 and 8 hours after a 350 Gy irradiation. The high dose delivered resulted in an increase in the width of the microbeam track over time. B) Intensity of the fluorescence of the areas depicted in A.

Fig 7. Number of γ-H2AX positive cells per unit area.

The measurements were performed in the granular layer of the cerebellum. A) Shows the number of cells in normal rats while B) shows the number of cells in tumour-bearing animals. Stars show significant differences between 4 and 8 hours. Error bars correspond to SD.