CMGI SOP for ImagingMice and Rats 8/30/10
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The Center for Molecular and Genomic Imaging (CMGI) provides small animal imaging services for preclinical research. Animal handling procedures for the several imaging modalities offered by the facility are described below.
General anesthesia procedures
Animals are anesthetized (described below)for the duration of the imaging study to prevent motion. A heated animal bed, heating pads and, if necessary, a heating lamp, will be used to ensure thatbody temperature is maintained both pre-imaging and during the procedure. For non–terminal studies animals are monitored during recovery. After recovery, animalsare returned to the vivarium. Special handling applies to animals injected with short-lived radioactive contrast agents asdescribed below.
Anesthesia protocols
Mice and rats will be anesthetized with an inhalation anesthetic (isoflurane). Anesthesia will be induced in an induction chamber (2-5% isoflurane), after which the animal will be placed in the imaging instrument and fitted with a nose cone connected to a vaporizer tomaintain isoflurane (1.0-2.5%) during the procedure. We have found this range of concentrations produces a level ofanesthesia that prevents animal movement during scanning. If respiratory rate begins to accelerate or slow down, theisoflurane concentration will be increased or decreased, respectively. If theanimal only needs to be anesthetized for a relatively short period of time (e.g, 30 min or less), we may use an injectableanesthetic (see Injectable Anesthetics below).
Injections
Animals may be injected with a contrast agent for some imaging procedures, or with an injectable anesthetic. Injection volumes and needle sizes for mice: iv: <0.25 ml/20g (25-30 ga); ip: <0.4 ml/20g (25-27 ga); ); sc: <0.4 ml/20g (23-25 ga); im: <0.05 ml/injection site (25-27 ga). Injection volumes and needle sizes for rats: iv: <5 ml/kg (22-25 ga); ip: <10 ml/kg (25 ga); sc: <10 ml/kg (23-25 ga); im: <0.1 ml/site (25 ga). In most cases injections will be iv or ip. Intramuscular injection will be avoided if possible.
Monitoring heart rate and respiratory rate
For PET, CT, SPECT and MRI imaging, ECG and respiration rate may be electronically monitored to provide a trigger signal for gated data acquisition. For ECG monitoring, neonatal ECG pads may be applied to the shaved limbs, or fine gauge needles (approximately 28-gauge) may be inserted subcutaneously on three limbs of the animal while it is anesthetized. For monitoring respiration rate, a respiration pad may be taped onto the animal at the level of the thorax while it is anesthetized. These pads are very sensitive and do not need to be taped so tight as to restrict breathing.
mPET, mCT, mSPECT imaging procedures
High quality injections of contrast agents are a prerequisite for scientifically useful imaging studies. Contrast agents include iodine-based compounds for x-ray computed tomography (CT) and radioactive tracers for positive emission tomography (PET) and single photon emission computed tomography (SPECT). For some radiotracers, mice or rats will be fasted (water unrestricted) for up to 20 hours to improve radiotracer uptake. Various radiotracers (see Radiotracer list below) are available for micro-PET (mPET) imaging in small animals. Trace amounts (<1 mCi, mice; <4 mCi, rats) of a short-lived radioisotope will be administered by iv,ipor scinjection prior to imaging. Only micro- to nanogram doses of radiotracerwill be utilized, which will not cause any pharmacologic effect. In some cases, a small catheter (mouse or rat: 28 ga or 30 ga needle on PE10 or MicroRenathane tubing; rat: commercially available 22 or 24 ga iv catheter) will be temporarily inserted into the tail vein of the conscious or anesthetized animalfor isotope injection. For conscious animal injection, animals are briefly restrained in a commercial restrainer for catheter placement or tail vein injection of radiotracer. The animal’s tail may be immersed in warm water for 30-60 seconds to increase vasodilation of the tail vein. Prior to catheter insertion, the tail is cleaned with an alcohol wipe and the catheter, filled with heparinized saline, is inserted into the tail vein. A needle/syringe or catheter plug is inserted into the end of the catheter until the radioisotracer is ready to be injected. The isotope syringe is held in a syringe shield whenever possible to minimize radiation dose to personnel. The radiotracer uptake period prior to imaging is typically 0.5 hr, but may last from 0-2 hr depending on the protocol. During this time the animal may be continuously anesthetized or conscious. If the animal is anesthetized during this time, it is kept warm by a heat lamp or other heating device (e.g., Deltaphase pads).
For image acquisition, the animal will be placed on plastic-backed absorbent paper to contain any excreted urine, and imaged by mPET or mSPECT scanner (typically for 10 min to 2 hr) or mCT (typically for 10-30 min). In some longitudinal studies, animals may be rescanned several times per week for several weeks. For some studies, two animals may be imaged simultaneously, each with its own nose cone for anesthetic delivery. For mCT imaging, x-ray contrast agents (DOUG – what is the agent we’re using now? e.g., Fenestra; iv or ip) may be utilized to highlight certain soft tissue features, or to visualize the vascular system. The typical dose for Fenestra is 0.2 ml/20 g in the mouse, and 5-10 ml/kg in the rat.
In some cases, 2-3 small, low-radioactivity fiducial markers may be affixed to the fur or skin of the animal. These markers are visible on both the PET and CT scan, and allow coregistration of the PET and CT images, thereby providing the anatomical information afforded by CT imaging to better interpret the PET image. If attached to the skin, the animal will be shaved in the regions where the markers will be attached, and a commercial hair removal cream may be applied briefly to remove stubble if necessary. The markers will either be taped in place or, if the animal is to be euthanized after the scan, may be glued to the skin.
MRI imaging procedures
Mice or rats will be anesthetized, placed inside the MRI scanner and imaged typically for aperiod of up to 1 hour. Animals are warmed by the imaging bed to maintain body temperature. Contrast agents (e.g., paramagnetic gadolinium-containing liposomes, superparamagnetic iron oxide nanoparticles) may beutilized to highlight certain soft-tissue features, and these would be typically be injected iv unless there isdifficulty with iv injection and it is known that the agent can be absorbed by ip injection.
Optical imaging procedures
For optical imaging, anesthetized animals may be imaged in a Xenogen IVIS system, typically for bioluminescence imaging, or a Maestro 2 system, typically for fluorescence imaging. In some cases, animals will be shaved in the regions where signal is anticipated and a commercial hair removal cream may be applied briefly to remove stubblein order to optimize transmission of the light signal emitted from within the animal’s body. For bioluminescence imaging, anesthetized animals carrying a bioluminescent reporter gene are injected ip or iv with the substrate for luciferase (luciferin, 150 mg/kg iv/ip or coelenterazine, 3 mg/kg iv/ip). Animals are placed ona warmed surface in a light-tight box (IVIS imaging chamber) and imaged with a sensitive CCD camera for typically 20 min or less. For fluorescence imaging, anesthetized animals are injected (iv, ip, sc, or im) with a small quantity
of fluorescently-labeled murine antibody, fluorescently-labeled microspheres, or red/near-infrared emitting optical contrast agent. Animals are placed on a warmed surface in a light-tight box and the fluorescent light is imaged with a sensitive CCD camera for periods typically ranging from seconds up to sixty minutes.
Animal Housing
Following the scan, the animals may be euthanized (see below) or returned to the vivarium. To minimize the risk of contamination of the vivarium, animal cages will be sprayed with Cidex or other approved disinfectant before they are returned to the vivarium. If animals have received a radioactive tracer, a radioactive label will be placed on the cage to indicate the date, isotope, dose, and probable date that animals will no longer be radioactive. Radioactive animals will be transferred to a designated ‘hot’ room (0658) in the GBSF vivarium (for conventional clean animals), or in Tupper Hall (for possible pathogen-carrying animals). Radioactivity levels of animals will be checked with a survey meter by CMGI staff, who will remove the radioactivity label when animals are no longer radioactive.
Blood Sampling
In some studies, blood samples (typically 5-50 uL) will be collected to determine radioactivity levels in the blood, or to provide serum or plasma for chemical analysis of specific substances in the blood, e.g., the injected radiotracer. In mice and rats, this will be performed by any of three techniques: catheter (typically in the tail vein), tail vein nick, or tail tip. For all techniques, the site will be cleaned before the procedure, and following collection of the sample, blood flow will be stopped by applying pressure before returning the animal to its cage. Tail vein nick: performed by inserting a sterile hypodermic needle into the lateral tail vein. Tail tip: performed by cutting off approximately 3 mm of the tail tip with a sharp scissors or scalpel blade. For repeated sampling, the clot can be removed to obtain another sample, but the tail tip will be clipped off no more than twice. The maximum blood volume that will be drawn in a 2-week period is 1% of the animal’s body weight.
Euthanization
In some cases animals will be euthanized at the end of their imaging studies. Euthanization will be by cervical dislocation under anesthesia (mice); or by CO2 (mice or rats); or by sodium pentobarbital (150-200 mg/kg, ip or iv) overdose; or by 3x the initial dose of ketamine/xylazine or ketamine/xylazine/acepromazine.
Radiotracers:
PET: 18F-fluorodeoxyglucose (FDG),18F-fluorothymidine (FLT),18F-fluoromisonidazole (FMISO),18F-penciclovir analog (FHBG),18F-paclitaxel,or 18F-labeled peptides, antibodies, microspheres, acoustically active liposomes, microbubbles or cells; 64Cu-PTSM, 64Cu-ATSM, or 64Cu-labeled microspheres, microbubbles, liposomes, peptides, antibodies or cells; 11C-raclopride, 11C-SCH23390, 11C-PK11195, or other 11C-labeled compounds; 89Zr-labeled proteins, peptides, antibodies, microbubbles, liposomes or cells; 90Y-labeled compounds, microbubbles, liposomes or cells.
SPECT: 99mTc, 111In, 123I, 125I and compounds labeled with these radioisotopes may be utilized.
Injectable Anesthetics:
Ketamine/Xylazine: Possible mouse doses: 80/20; 100/10; 100/5; 200/10 mg/kg,IP (1/3 original dose of ketamine for maintenance)
Ketamine/Xylazine: Possible rat doses: 65-80/10-12.5 mg/kg, IP (1/3 original dose of ketamine for maintenance)
Ketamine/Xylazine/Acepromazine: Mouse or rat: 50/5/0.5 mg/kg, IP (1/3 original dose of ketamine for maintenance)
Pentobarbital: Mouse: 40-70 mg/kg, IP; Rat: 30-45 mg/kg, IP (1/3 original dose for maintenance)