8-week-old C57BL/6 mice were irradiated from 7 to 10Gy with a new X-ray irradiator in an attempt to decide optimal X-ray dose for bone marrow chimera experiment. The goal was to find the dose of X-ray that completely eradicate recipient BM cells and achieve >99% donor cells. It has been 4 weeks since mice were irradiated and BM cells from CD45.1 B6 were transferred. Now irradiated mice show lesions on their dorsal skin and it seems mice with higher dose are severer. 7 and 8Gy mice show just hair loss on small area but 10Gy mice have scabs all around their back. I collected and analyzed blood from mice with concern that I might have over-dosed it, but CD45.1+ donor cells were only found less than 20%. It does not make sense because I heard that BM-derived cells are the least resistant to irradiation, and I think if X-ray dose was strong enough that there is skin damage, then recipient BM cells should be long gone. 9-10Gy is also routine dose in other publications. What do you think went wrong? I would appreciate inputs from experienced colleagues. Thanks.
edited: there was a mistake at irradiation unit. error corrected.
You don't mention the energy of your x-rays. If you used 250kVp with inherent filtration, the beam contains a large low energy component and the dose at mouse midline is much less than at the skin. This may be OK for cell cultures being irradiated from the top with only a few mm of medium for the beam to traverse. For mice you should use a Thoraeus filter in the beam, then you would have a much harder beam with almost uniform dose throughout the mouse. Check the half value layer (HVL) of the beam you are using to see how much dropoff you have with your current irradiation conditions and select conditions where the mouse exit dose is no less than 95% of the entry dose for uniform irradiation throughout.
The beam energy should be high enough for Compton scattering as the mode of energy deposition rather than lower energy photoelectric effect . The bone is transparent foe the former, while the marrow is relatively protected for the latter.
Fully agree with Lange. In addition you may use maximal backscatter (5 cm perspex or hard board at the beam exit to improve dorsal to abdominal dose homgeneity and make sure you have a homogeneous radiation field in which the mice are positioned. Best is to recruit the assistance of a radiation physicist.
Thank you Dr. Lange and Dr. Bekkum for your advice. Now I clearly understand what happened. I should consult with the manufacturer of the irradiator for animal use.
Fortunately there was a publication using the same X-ray irradiator and I followed their settings, which includes 0.5mm aluminium filter. I was able to acheive >80% donor cell population after 6 weeks of irradiation. Although it is a huge improvement from ~20%, it is still not enough. And burns on the back of mice are still there. I guess the x-ray need more hardening, but my options are limited. I have 0.5mm and 1mm Al, and 0.1mm and 0,3mm Cu filters. The problem is that the irradiator can take only one filter at a time, while Thoraeus filter is made of tin, copper and aluminium. So now I am wondering which one I should use for the best result.
One more question, I may have made a mistake by building an animal cage for x-ray irradiation with 2mm acrylic. At that time I did not know that acrylics can interfere with the irradiation, but apparently it blocks x-ray in some way. What exact properties does acrylic have on x-ray?
I agree with Lange , Bekkum and Lee. I think the burning and skin damage are mainly due very high dose. To my knowledge the permissible dose for the bone marrow irradiation is about 0.5 Gy. Again, a small suggestion, just replace the 2mm acrylics cage by 1 mm Al cage, carryout your experiment (if we suspect the Compton scattered low energy photons, by changing the cage we can reduce the same)
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