Irradiation of Rodents

A. Animals exposed to irradiation must be monitored and findings documented daily for the first 14 days. However, after protocols have been established and the principal investigator (PIPrincipal Investigator View Boston University's policy on...) has experience with the particular strain of mice and dose, three/week monitoring and documentation is acceptable. If animals experience morbidity or mortality, daily checks are required. Procedures for care of irradiated rodents must be observed.

B. Irradiation must be scientifically justified in the IACUC protocol.

C. The planned dose of irradiation, or dose range, must be specified.

D. Fractionated doses may be useful in certain studies if practical, since animal morbidity and mortality will be lower.

E. Unless literature references are available, a pilot study to determine the best dose is recommended if the PI is starting a new study or using a new strain of mice.

F. If control animals (animals that are lethally irradiated but not reconstituted with bone marrow) will be used, the PI must justify the use of these animals, use as few animals as possible, use historical controls as much as possible, and comply with the Humane Endpoints Policy.

G. The IACUC will place non-reconstituted, lethally irradiated control rodents with death as an endpoint in USDAUnited States Department of Agriculture Category E. Scientific justification of unrelieved pain and distress or potential pain or distress of lethal radiation doses is required.

H. Prior to starting an irradiation experiment, the PI is required to submit a form to Boston University Animal Science Center (BU ASCThe Animal Science Center manages and oversees animal-relate...) (request for supplies needed for post-irradiation animal care).

Ionizing irradiation causes breaks in the DNA double-strand, thus it mostly affects mitotically active cells. This damage leads to cell death through either necrosis or apoptosis, primarily affecting the cells of the hematopoietic and gastrointestinal tract ² (Duran-Struuck, R. and Dysko, R, 2009).

A. Weight loss

Lethargy, inappetence, diarrhea resulting in anorexia, and body weight loss, which peaks at about seven days post-irradiation. Depending on the dose and whether immune reconstitution had been provided, recovery will usually occur in 2–3 weeks. Mice may never regain the body weight they had before irradiation.

B. Anemia

C. Infection

Severe bacteremia may occur as a result of translocation of bacteria from the GI tract ⁶ (Duran-Struuck, R. et.al., 2008).

D. Intestinal bleeding

E. Transplant failure

Successful survival of a bone marrow graft requires suppression of the host’s immune system. If the irradiation dose has been too low, Graft-versus-host Disease (GVHD) will ensue. As in humans, older mice are more prone to develop GVHD.

F. Graying of hair coat

Black mice, such as C57BL, will frequently turn gray after irradiation.

G. Development of secondary neoplasias

The development of neoplasia after irradiation has been reported in humans and many large animal species. This may occur in mice on long-term studies as well.

H. Incisor damage

One non-neoplastic illness reported in mice is incisor damage and subsequent difficulty in eating. Giving softened food during the recovery phase is required.

Animals to be lethally irradiated should be healthy and free from infectious agents, including Pseudomonas aeruginosa.¹ Mice are tested for P. aeruginosa at the vendor and at the annual sentinel surveillance at BU ASC. The drinking water is tested for P. aeruginosa annually as well. Immunocompetent mice are not made sick even though they may carry commensal microbial agents. After exposure to TBI, mice are immunocompromised and subject to infections not previously clinically important. If post-irradiation infections occur and interfere with recovery of the animals, this possibility should be taken into consideration.

A. Purchasing the pie cage

Each investigator is required to purchase their own pie cage. The type recommended is a pie cage with filters which holds up to 20 mice and which can be autoclaved between uses. Pie cages must be sanitized between uses.

• These may be obtained from Braintree Scientific. The dimensions of the irradiator slot is circular with a diameter of 12 inches and a depth of 4 inches. There is plenty of space for either type of pie cage used for mice or rats.

B. Scheduling

The PI must call the Radiation Protection office (RPO) at 617-638-7052 to schedule a time. If the dose is to be fractionated, this must be specified and time must be scheduled for three hours later as well.

C. The approved IACUC protocol number is given to the RPO staff.

D. The RPO staff sets and runs the irradiator.

E. Dose

The PI must provide the RPO staff with a suitable dose required for their study.

F. Time of exposure

Animals are generally irradiated for a short period of time (less than 15 minutes). The amount of time spent inside the irradiator varies depending upon the dose and the radioisotope decay charts.

G. Rodents are restrained in a rotating pie-shaped holder (to limit mobility and ensure equal doses of irradiation).

This may be done in the PI’s laboratory in a biosafety cabinet (BSC), or in the rodent housing room in a BSC, and the rodents then transported to the irradiator inside the pie cage. Alternatively, rodents are transported to the irradiator in their home cage and transferred to the pie cage once inside the irradiator room. This may be necessary if more rodents are irradiated than can be contained inside one pie cage.

H. Rodents may or may not be anesthetized for irradiation.

I. Transporting the mice to the RPO

The IACUC guidelines for Transportation of Rodents must be followed with the proviso that mice may be transported inside the pie cage.

J. Rodents can only stay in the pie cage for a maximum of one hour.

A. The PI is responsible for bringing the following items:

• Pie cage and rodents

B. The following items are available in the irradiator room:

1. Chucks (diapers) to cover the work counter
2. Paper towels
3. MB-10
4. Trash bags
5. PPE as needed. If mice are handled, the minimum required PPE include:
   • Disposable gown
   • Surgical mask
   • Gloves
   • Bonnet

1. If animals are transferred from home cage to pie cage in the irradiator room, the PI or research staff are required to first don PPE.
2. After the irradiation is completed, the PI or research staff are required to sanitize the counter and place all trash in a trash bag and remove the trash bag from the irradiator room, take it back to the BU ASC, and dispose of it according to BU ASC policy.
3. If animals are transported in the pie cage, it is not necessary to don PPE or sanitize the room.

1. After the pie cage is loaded, it is placed in the irradiator.
2. Mice must be returned to the animal housing room or laboratory as soon as the irradiation is completed.

After irradiator exposure, mice must be placed in a cage and kept clean and quiet. Sterilized caging, food, and water is recommended. The PI must order these from BU ASC prior to the start of irradiation.

The PI or designated research staff are responsible for caring for the irradiated animals. BU ASC staff will provide special care in an emergency and in such a case the PI will be charged accordingly. For the first 14 days, animals must be checked daily, or three times a week, their condition and care documented on the rodent post-procedure monitoring sheet. Care, especially during the first week, must be dedicated to making animals as comfortable as possible, keeping them clean and quiet, and making sure that they stay hydrated and have ready access to moistened food and Napa Nectar if indicated. Please refer to the section Policies for Animals on Studies Exposed to Irradiation above. Post-Procedure Monitoring Form

The PI is responsible for placing rodents on SulfaTrim antibiotic water a few days before the planned irradiation in order for the animals to get used to the taste. Rodents are kept on the SulfaTrim water for at least 14 days and up to 28 days post-irradiation.

Irradiated mice will suffer from irradiation sickness and will not feel well for the first 7–14 days. They may lose up to 25% of their body weight, which in the case of a successful bone marrow transplant will be mostly regained by day 14–21 post-irradiation. It is important to provide easy access to water.

Napa Nectar must be provided on the bottom of the cage during the first 14 days if morbidity or mortality is seen. Napa Nectar is available in the animal room free of charge. Placing a new Napa Nectar in the cage daily can be done by the PI or research staff or can be done by BU ASC for fee for service. The Napa Nectar becomes contaminated with fecal material and must be replaced daily.

Giving softened food during the recovery phase is required. Powdered chow is available in the rodent housing rooms and should be mixed with water and served in a small petri dish on the cage floor. Pellets moistened with water dry up easily and are not recommended. Placing a new moistened food dish in the cage daily must be done by the PI or research staff or can be done by BU ASC for fee for service.

It is important to realize that even after bone marrow transplantation, lethally irradiated mice are severely immunosuppressed for the first two weeks and providing a totally sterile housing environment is recommended^{1, 3} (Fox et al. 2007, & Duran-Struuck, R and Dysko, R., 2009). Sterile caging, food, and water is strongly recommended and is required if post-irradiation complications occur.

• 3.5ml stock → 250ml water = _3.5 x 240mg __ = 0.6720mg/ml drinking soln.
• 5ml x 250ml
• Mouse dose = 0.6720mg x 150mg = 100.8mg/kg ~ 100mg/kg/24 hours POml x kg x 24 hours

• 2.78ml stock → 250ml water = _2.78 x 240mg __ = 0.5358mg/ml drinking soln.
• 5ml x 250ml
• Rat dose = 0.5358mg x 100mg = 53.58mg/kg ~ 54mg/kg/24 hours POml x kg x 24 hours