Histological Evaluation of In Vivo Dose Delivery in Respiration-gated Small Animal Radiotherapy

Preclinical small-animal radiotherapy is essential for advancing radiobiological research. However, respiratory motion causes significant geometric uncertainty and dose blurring, reducing treatment accuracy. This study aims to develop and validate an integrated respiration-gated irradiation technique for mice and assess its effectiveness in decreasing motion-induced targeting errors. Respiratory motion was tracked in real time using an optical displacement sensor, and a mechanical shutter was employed to gate the X-ray beam. Both ungated and gated irradiation were delivered to the liver and lung (n = 8 per group). Ex vivo γH2AX immunofluorescence staining and an image analysis pipeline were used to evaluate the radiation field. The histological dimension in the irradiated tissue was adjusted using tissue-specific shrinkage factors to account for histology-related shrinkage, and the corrected measurements were compared between the ungated and gated groups. γH2AX analysis showed that respiratory gating significantly reduced the histological dimension along the superior-inferior axis in both the liver (ungated: 3.00 ± 0.85 mm vs. gated: 1.75 ± 0.24 mm; P < 0.01) and the lung (ungated: 2.55 ± 0.64 mm vs. gated: 1.33 ± 0.19 mm; P < 0.001). This resulted in a 1.25 mm reduction in the motion-related geometric margin for the liver and a 1.22 mm reduction for the lung. This study provides direct biological evidence that respiratory gating effectively reduces motion-induced dose blurring. The proposed gating method can significantly reduce treatment margins caused by motion, thereby enhancing targeting accuracy and sparing normal tissue.