- Has anybody irradiated C57/BL6 mice at 400 Rad for adoptive transfer of EAE? In the first attempt, I irradiated C57/BL6 mice at 350 Rad and I could not observe successful induction of disease (mice being normal). In the second attempt, I irradiated mice at 400 Rad and after only 5 days, I could see the drastic weakness in mice. Day by day they reduced in weight with the hair on their body looking very moist. As such I cannot see any symptom of EAE like hind limb paralysis but there seems to be the weakness in their hind limbs. They are drastically loosing wight day by day. Can any body tell me if these are the symptoms of high radiation dose or some other thing is going on?
Dear Tabasum,
I think its fatal irradiation. Didi you complement your mice with intact bone marrow from wt or from eae mice?
All my best
Marc
Dear Tabasum Sidiq
I did only active EAE, but if you ask people from Prof. Dr. Britta Engelhardt (Theodor Kocher Institute from University of Bern) you will get the right answer.
Faik
You do not have to irradiate mice in order to adoptively transfer EAE. First, induce the disease actively in the first group of mice. After about 11 days, harvest lymph nodes and re-stimulate the lymph node culture with MOG peptide. Then isolate CD4+ T cells from the cultures and then inject those (preferably via tail vein) into the second group of recipient mice. Then, you should begin observing symptoms of EAE.
Yes, there is no need for irradiation of the mice. Attached are our papers that detail the methods for EAE induction.
Hi Tabasum,
I did adoptive transfer EAE in C57BL/6 mice with irradiation (400rad) of the recipient mice at the day befor transfer (of MOG restimulated CD4 T cells from EAE mice). I could not observe any weight loss as you described. - mice slightly gained weight within 14 days post transfer. Subsequent to EAE onset (d13) mice lost weight.
Just check the Nat Protocol Publication from Stromnes & Goverman : PMID:
17487182
Good luck! Sylvia
Justin is correct. You do not need to irradiate mice in this case. I'd only add that you can re-stimulate your cells with MOG in the presence of IL-12 or IL-23 to drive a Th1 or Th17 response. Also, it isn't strictly necessary to isolate CD4+ T cells at the end of the culture.
An alternate approach, if you have access to them, is to culture myelin-specific TcR transgenic 2d2 T cells ex vivo (with plate-bound CD3+CD28), prior to transfer to immunologically naive animals. See: http://www.ncbi.nlm.nih.gov/pubmed/19890056
For induction of passive EAE, the encephalogenecity and health state of T cells can influence the outcome of EAE. Usually, people use activated T cells ((re)-stimulated with autoantigen in vitro on Day 3). A few millions (from 1 to 10) of such cells can sucessfully induce overt passive EAE. Too few cells or too many cells can not induce overt passive EAE. One need to do a titration to decide the optimal cell number to be injected. It should be noted that the encephalogenicity of T cells generated from different experiments may not be the same. Therefore, to do samll pilot experiments to establish the optimal conditions is required.
The 400 rad irradiation is not fatal for B6 mice. However, if you use pMOG35-55-specific T cells for inducing passive EAE, the irradiation is not needed.
One minor thing to comment: Before the onset of overt clinical EAE, mice may lose the body weughts; and after the onset the EAE, they may further lose body weight.
Dear Ming-Chao Zhong,
I could inject only 3 million cells per mouse due to less number of cells after Lympholyte-M step. After only 5 days of injecting CD4 T cells which were MOG35-55 re-stimulated in vitro, I could see the reduction in body weight of mice. Moreover, the mice were not active at all. They stayed in the cage clinging to each other closely, with one of the mouse having swelling in face. On day 10, two of the mice died with extreme reduction in weight and their hair looked moist and sticky all over the body. So, I don't know exactly what the reason can be. Is this the irradiation but it was not fatal or something else?
If you can check histology of brain and spinal cord of the dead mice and you can see many invaded T cells in CNS, then the mice may die from EAE. Otherwise, the mice died from other cause or combined causes. 400 rad irradiation alone can not led to the death of mice. However, if the mice were under stress for prolonged time (combined with side-effects of irradition, the transferred autoimmune T cells, and other stresses unidentified), they might die.
For passive EAE, if the encephalogenicity of injected T cells is very strong and the cell number injected is too high, the recipient mice may die before they showed any overt clinical signs of EAE (paralysis of tail and limbs). In this case, histology examination can real many invaded T cells in CNS.
Thank you all. Your valuable suggestions have shown me the directions to follow.
Tabasum
Thank you Ming-Chao Zhong . You are right. I appreciate your valuable suggestion and will follow it.
Tabasum
Dear Tabasum Sidiq,
Recently I have same problem like you when doing adoptive EAE (7 million CD4 T cells/recipient i.v). Most of the recipient showed reduction in body weight, swelling in their face, being unactive at day 4-5 after injection, and died within 1 week. For some reason, I couldn't do the histology examination. So far, have you found any reason why this kind of thing happened ? And how did you solve this phenomenon? Your comment will be precious for me. Thank you !!!
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