CMGI SOP for Imaging Mice 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 that body temperature is maintained both pre-imaging
and during the procedure. For non–terminal studies animals are monitored during
recovery. After recovery, animals are returned to the vivarium. Special handling
applies to animals injected with short-lived radioactive contrast agents as described
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 to maintain isoflurane (1.0-2.5%) during the procedure. We
have found this range of concentrations produces a level of anesthesia that prevents
animal movement during scanning. If respiratory rate begins to accelerate or slow
down, the isoflurane concentration will be increased or decreased, respectively. If
the animal only needs to be anesthetized for a relatively short period of time (e.g, 30
min or less), we may use an injectable anesthetic (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, ip or sc
injection prior to imaging. Only micro- to nanogram doses of radiotracer will 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 animal for 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 a period of up to 1 hour. Animals are warmed by the imaging bed to
maintain body temperature. Contrast agents (e.g., paramagnetic gadoliniumcontaining liposomes, superparamagnetic iron oxide nanoparticles) may be utilized
to highlight certain soft-tissue features, and these would be typically be injected iv
unless there is difficulty 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 stubble in 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 on a 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), 18Ffluoromisonidazole (FMISO), 18F-penciclovir analog (FHBG), 18F-paclitaxel, or 18Flabeled 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, 11CSCH23390, 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)