X-irradiation of mice

11.12 X-irradiation of mice

Mice may be immunosuppressed by 6.0–8.0 Gy whole body irradiation, the precise dose being dictated by the degree of immunosuppression required and the ‘cleanliness’ of the environment. Under these conditions there is only the occasional death by 7 days post irradiation without bone marrow therapy.

Early X-ray death is usually due to gut damage; accordingly, X-ray resistance may be increased by starving the mice 24 h before irradiation. If infection-associated deaths are noted after routine immunosuppression the following antibiotic regime may be used: in drinking water, ad libitum

1 g neomycin and 400 mg polymixin B to 10 l distilled water. Again it must be emphasized that this technique must be performed by experienced person- nel only, and under the strict guidelines of your local animal research facility. Figure 11.3 shows the variation in numbers of peripheral blood mononuclear cells, neu- trophils and platelets measured at various times after irradiation, in C57Bl mice given 6.0 Gy whole body irradiation. See below for details of a protocol for the dose–response curve of X-ray- induced immunosuppression.

mononuclear cells ( )/

Number of neutrophilis ( ) or

30 30 Number of platelets ( )

10 3 4 5 6 7 8 9 10 10 Days after irradiation

Fig. 11.3 Response of peripheral blood cells to whole body irradiation. Male C57B1 mice were given 6.0 Gy whole body irradiation and the numbers of peripheral blood cells determined at various days thereafter. Initial values of cells: neutrophils 1300 ± 340/µl; mononuclear cells 8900 ± 1500/µl; and platelets 1200 ± 180 × 10 –3 /µl. Each population showed marked variation in the response to irradiation. At 4–5 days maximum leucocyte suppression was seen, with only minimal depression of platelet numbers. Lower doses of irradiation produced much less dramatic effects on cell numbers; for example, the 4-day neutrophil count in response to 4.0 and 5.0 Gy was 170 ± 46 and 180 ± 31/µl, respectively. Whole body irradiation has no acute effects on the plasma levels of the complement components.

C H A P T E R 1 1: Immunological manipulations in vivo

11.12.1 Suppression of the immune response by X-irradiation

The dose of X-rays required to kill most types of non-dividing cells is much greater than that required to kill actively dividing cells. One of its major effects is to induce chromosomal breaks so the cells are unable to complete mitosis. Lymphocytes, however, are unusual among mammalian cells in

being susceptible to X-ray-induced death in G0. They have a D 37 of only 1.0 Gy. The dose-dependent immunosuppressive effect of X-irradiation may be demonstrated in a primary immune response against sheep erythrocytes. This experiment may also be designed to determine the lethal and immunosuppressive X-ray doses of recipient animals for other experiments (see Technical notes).

MATERIALS AND EQUIPMENT Mice (preferably inbred)

Sheep erythrocytes (SRBC) Irradiation source Materials for haemolytic plaque assay (see Section 9.1)

METHOD

Irradiate and prime the mice according to the Protocol.

Protocol.

Group (3 –5 animals per group)

Irradiation dose (Gy)

0 0 2 4 6 8 8.5 9 Immunising antigen aSRBC intravenously

1 Challenge the animals with antigen immediately before or after X-irradiation.

2 Assay the anti-SRBC haemolytic plaque response 5 days after antigen challenge.

3 Calculate the total number of plaques per spleen for each animal and the geometric mean for each group.

4 Plot a graph of the X-ray dose against the log mean plaque response. TECHNICAL NOTES

• See above for changes in blood leucocytes following whole body irradiation. • For most laboratory strains of mice there should be very few deaths during the period of assay

due to X-irradiation over the range indicated. For long-term survival, however, it is necessary to reconstitute the mice with bone marrow as X-irradiation suppresses not only lymphoprolif- eration, but also proliferation of the haemopoietic system.

11.12.2 Radioresistance

Experiments suggest that T and B cells show a differential radioresistance in the whole animal. Thus, if an animal is primed with sheep erythrocytes and X-irradiated 4 days later, functional T cells remain, whereas B-cell activity is suppressed.

11.12X-IRRADIATION OF MICE

MATERIALS AND EQUIPMENT Inbred mice Sheep erythrocytes (SRBC) Materials for haemolytic plaque assay (Section 9.1)

METHOD

1 Prime recipient mice with 2 × 10 7 sheep erythrocytes intravenously. X-irradiate (8–8.5 Gy)

4 days after priming.

2 Prepare spleen and thymocyte suspensions from normal donor mice.

3 Treat aliquots of the spleen suspension with anti-Thy-1 and complement or an irrelevant monoclonal and complement (see Section 7.8).

4 Reconstitute the X-irradiated mice according to the Protocol.

Protocol. Recipients

SRBC Group

Previous

Cells

per group

2 × 10 7 SRBC + 8 Gy*

2 × 10 7 SRBC + 8 Gy*

2 × 10 7 SRBC + 8 Gy*

Spleen treated

2 × 10 7 with anti-Thy-1 + complement

2 × 10 7 SRBC + 8 Gy*

Spleen treated with

2 × 10 7 irrelevant monoclonal + complement

* 8.0 Gy 4 days after SRBC challenge.

5 Assay all mice for anti-SRBC haemolytic plaques 8 days after cell transfer.

6 Calculate the total plaque-forming cells per spleen for each individual and the geometric mean for each group.

Knowing the cell phenotype specificity of the anti-Thy-1 serum, you should be able to identify the radiosensitive and radioresistant lymphocyte populations. This is an extremely useful method by which to prepare helper cells.

TECHNICAL NOTES • We have assayed at only one time point; both T and B cells have phases of relative radioresist-

ance after antigen stimulation. • Differential radioresistance is not shown by T and B cells in vitro.