Irradiation Conditions Necessary for Murine Bone Marrow Ablation Utilizing an X-Ray-Based Irradiator.
Authors: Kennedy Patrick J. , Wang Lei , Burke Michael J. , Sullivan Glenn , Hernandez Jose M. , Tse William T. 
Published: April 2020

Abstract
In mouse models of bone marrow transplantation, irradiation of recipient animals to ablate endogenous hematopoietic tissues is conventionally given by radioisotope-based irradiators. Recently, cabinet-size, X-ray-based irradiators have been advertised as a safe and cost-effective alternative source of radiation. Whereas 137Cesium-generated gamma-rays have an energy of 662 kV, X-rays generated from cabinet irradiators typically have a peak energy of only 130 kV, thus potentially limiting their ability to penetrate tissues. In this study, we performed dosimetry studies of a Faxitron RX650 irradiator operating at peak voltage and tested its effectiveness in ablating murine bone marrow. Thermo-luminescence dosimetry (TLD) chips were placed on and under the skin of freshly sacrificed mice or embedded in the tissue next to the pelvis and long bones. The mice were placed inside a Plexiglas cage at a distance of 16 inches from the X-ray tube and irradiated in a fashion that simulated live mouse irradiation. The TLD chips recorded radiation doses of 150 cGy/min to the skin, 73 cGy/min to the pelvis and 47 cGy/min to the femur, which were dramatically lower than the nominal dose of 527 cGy/min suggested by the manufacturer. This result demonstrated that there was a marked attenuation of radiation by intervening materials and a significant difference in doses delivered to superficial and deep tissues of irradiated mice. Preliminary studies performed on live mice showed that irradiation at a dose high enough to ablate bone marrow cells caused extensive ulceration of back skin, necrosis of ear cartilage and increased mortality rates post-irradiation. To overcome these problems, a 4-point-rotation irradiation procedure was subsequently adopted, whereby the mice to be irradiated were first anesthetized and then placed inside a confinement cage to receive equal fractions of radiation in the supine, prone, left lateral decubitus and right lateral decubitus positions, with four hours between the first and last two fractions. Dosimetry analysis showed that this irradiation protocol gave an effective average dose of 55 cGy/min to hematopoietic tissues of the irradiated mice. To confirm the biological validity of this protocol, four cohorts of mice were given effective doses of either 200, 600, 900 or 1100 cGy and then transplanted with 80,000 bone marrow cells. Twelve days later, prominent spleen colonies (CFU-S) derived from transplanted hematopoietic progenitors were seen in the 900 and 1100 cGy cohorts, whereas no colonies were observed in the 200 and 600 cGy cohorts, indicating successful endogenous bone marrow ablation using the higher radiation doses. Engraftment studies were then performed in which four cohorts of C57BL/6 (B6) mice were given effective doses of either 200, 600, 900 or 1100 cGy, respectively, and transplanted with 5x106 B6.SJL bone marrow cells per mouse. After four weeks, peripheral blood analysis showed donor engraftment rates of &lt;10% in mice irradiated with 200 or 600 cGy but &gt;90% in mice irradiated with 900 or 1100 cGy. Our studies showed that X-ray-based irradiators can be used effectively for bone marrow ablation in mice, but careful dosimetry calibration and a 4-point-rotation irradiation procedure is necessary to prevent radiation-associated tissue damage.