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Animal use protocol assistance

Small Animal MR Facility, Last Updated 6/1/2010 Documentation to assist in developing an Animal Use Protocol for Small Animal MR.
Investigators intending to use the UGA small animal imaging facilities for in vivo studies are
required to have an approved Animal Use Protocol (AUP) that includes the use of MR prior to
initiating research. Information below is to assist in addressing some of the questions related to
the use of MR at UGA. Details related to specific studies performed need to be described in
detail. The information provided is to assist with study design and generation of an AUP. It is
recommended strongly that if you plan to use the MR facility that you contact the Manager (Dr.
Khan Hekmatyar, hekmat@uga.edu) to assist with developing studies.

Section 2
2.1 Personnel and Qualifications: (example, list all involved)

Position:
Procedures:
Imaging, training associated with MRI/MRS Facility note, all investigators are required to have personnel who are on an approved AUP to
assist with MR. Dr. Khan Hekmatyar is the MRI Manager and can assist with training and/or
operation of MR.

Section 3
3.1 Objective:
(general MRI information; actual abstract needs project specifics)
MR imaging and spectroscopy is a non-invasive technique that permits visualization of internal
organs and anatomic structures or quantification of contrast agents and/or metabolic substances
within intact living subjects (humans and animals). It can provide an investigator with serial data
sets, e.g., individual animal tumor burden, tumor stage and/or vascular status that are unavailable
from other means. Using the combination of magnetic field and radio waves, MRI creates three-
dimension images and spectra of normal and diseased tissues (e.g., tumors). This technique can
be applied to temporal imaging of normal growth or pathological conditions, such as the growth
of tumors. This allows researchers to rapidly and serially track the size and location of tumors (or
other organs of interest) without euthanizing the animal.
3.2 Significance (project specific)

3.3 Please provide a justification of use of animals
MR procedures are not considered to be painful or to have lasting health consequences. Without
MR imaging methods, the only way obtain similar data is to euthanize large groups of mice/rats
at different time points to compare results from different populations within an experiment.
Therefore, the use of MR imaging can substantially decrease the numbers of animals needed
to obtain scientifically relevant information concerning basic science questions about cancer.
Small Animal MR Facility, Last Updated 6/1/2010 Section 4

4.1 Overall Animal Use Category Information (project specific)
4.1.2, animals can be imaged for prolonged periods under anesthesia. This is typically not
considered prolonged physical restraint.
4.1.4, MRI is generally considered pain free and has no lasting effects on the animal.
4.1.5, animals will need to be transported to MRI facility. If serially imaging is to be performed
it is recommended that the animals be housed at Coverdell (rm 033) that is reserved for animals
being imaged.
4.1.8, there should be no evidence of stress or signs of illness following MRI. Any project
specific stresses should be noted.
4.2 Animal Use Category Classification (this is project specific)

Section 6
6.3.1 Justification of numbers of animals:
(example)

□ Other (Explain Below):
For any new imaging sequence additional control animals should be
included to optimize instrument parameters. For MRI, image contrast results from a broad range
of factors, including but not limited to: field strength, tissue properties, scanner hardware, and
manufacturer pulse program. The number of animals would be dependent on the animal model
and techniques being performed. It is recommended that you consult with the MR Director and
UGA Veterinarian to estimate appropriately the number of animals needed.
Section 7
7.1 Experimental Design and Procedures
(project specific)

7.2 Summary of Procedures
Provide a chronological description of procedures performed on animals, preferably in the
format of a brief timeline:


On the day of imaging, animals are transported from the animal facility following the URAR
SOP for transport (in closed cages, usually on the dumb waiter if they are housed in CRV) and
prepared for imaging, e.g., anesthetic is administered, contrast agent is administered (if required),
animal is positioned and mildly restrained, imaging is performed, following imaging, animals are
Small Animal MR Facility, Last Updated 6/1/2010 kept warm & monitored until animals regain consciousness and normal mobility, animals are returned to an approved animal facility. Note, once animals have been taken to the imaging facility they are not permitted to be returned to general animal facility housing. A quarantine room is available at Coverdell (rm. 033). Permission to house post-imaging animals in any other animal facility requires approval from the URAR Assistant Director in charge of the facility. As animals are returned to the animal facility, they are placed into clean cages, and the cages that went upstairs to the BIRC will be taken to the dirty cage wash prep area for sanitation. Anesthesia Animals will be anesthetized prior to imaging either by inhalation of Isoflurane (available at MR-Facility) or by injection (IP/IM) of ketamine/xylazine mixture (67-100/7-10 mg/kg) (provided by and maintained by the investigator) or other IACUC approved anesthetic. Note, ketamine is a DEA controlled substance and will not be maintained by the facility. For Isoflurane anesthesia, animals will be placed within dedicated induction chambers and connected to the Isoflurane anesthesia unit. Anesthetic chambers will be disinfected with an approved product (e.g., Vimoba or Spore-Klenz). After anesthesia any surgical clips or metal objects will be removed prior to imaging. Anesthesia during MR imaging is maintained by means of a nose bane (cone) supplying 0.75-3% Isoflurane delivered in medical air at 1 L/min as deemed necessary. Response to toe-pinch will be performed to determine the extent of anesthesia before putting placing animal into the magnet. After use nose banes will be disinfected with an approved product (e.g., Vimoba or Spore-Klenz). Waste gases are removed by low pressure vacuum and exhausted through a scrubber (carbon canisters). Canisters will be weighed prior to and after each use and their weight recorded and replaced as suggested by the manufacture. Note the facility has an Isoflurane vaporizer, other gas anesthetics cannot be used in this device. It is recommended that all PI’s consider using Isoflurane as opposed to injectable anesthesia. Animals can be sedated quickly and recover more rapidly at the completion of the experiment compared to injectable anesthetics. It is recommended that investigators purchase individual nose banes to further minimize the risk of transmission of disease and/or agents across colonies. Magnetic resonance imaging Magnetic resonance imaging (MRI) will be carried out using a horizontal 210 mm (inner diameter) bore, 7 Tesla Varian MR system. Following the induction of anesthesia, the animals’ eyes are covered with Paralube (or other approved ophthalmic lubricant) and placed within an acrylic tube, and may contain an MR-compatible pillow for monitoring respiration. Body temperature is controlled using a circulating water pump with a MR-compatible blanket connected to a temperature controlled water pump. The use of a water-pad minimizes the risk of burns sometimes observed with the use of electrical coils. The animal is positioned into the center of the scanner for imaging. A monitoring system (Small Animal Instruments Co.) that can be used to monitor physiological signals such as heart rate, respiration, and body temperature (rectus) is available if necessary. Respiration rate can be obtained from a small pneumatic pillow sensor placed next to the animal’s abdomen. Heart rate can be monitored using ECG by placing three leads with sub-dermal needle electrodes, and Temperature measured by insertion of a rectal Small Animal MR Facility, Last Updated 6/1/2010 thermometer or by fiber optic sensor. Following imaging, the animal is removed, and the holder and thermometer are disinfected and sanitized with a cold sterilant as described above.
Facility note, the tables below provide a list of biologics and experimental drugs and agents that
are commercially available and are commonly used in MR studies and their known toxicities.


Compound
FDA-approved gadolinium-based contrast agents MultiHance, Eovist) MRI Contrast Agent Gadomer-17 (synthetic dendrimer) MRI contrast MacroGd: (methoxy-PEG succinyl-poly-L-lysine- MRI Contrast Agent Iron-oxide based contrast agents (Ferex, Feridex, Molday ION) MRI Contrast Agent 3M VetBond Tissue Facility note, MR imaging contrast agents have varied sites of enhancement (global, vascular, liver, brain etc) and differing degrees of enhancement that must be characterized individually for determination of appropriate use. Small Animal MR Facility, Last Updated 6/1/2010 7.3 Use of Hazardous Substances (project specific)
Facility note, all studies using hazardous substance need to be communicated to the MR
Director prior to initiating studies. Note, many therapeutic agents, e.g., chemotherapeutic
agents, have additional handling precautions that need to be identified prior to initiating studies.

Contrast Agents: (example)
Facility note, publically available toxicity information on FDA-approved MR contrast agents is
included in the charts below. FDA-approved MR contrast agents listed in Tables 1 and 2 are
generally considered to have excellent safety profiles at their prescribed dosages.

Table 1: Acute toxicities (LD 50) occurs at 19x or more the administered doses listed above
Acute Toxicity
Brand Name
Compound Name
(LD 50) mmol/kg
Table 2: No reported sub-acute toxicity in small animals at clinical doses. Compound
Toxicity/Safety Assessment
No histo-pathologic study on rodents is available publicly. In humans, “hematologic, blood chemistry, and urinary evaluations showed no apparent drug-related effects, with the exception of a transient increase in some patients. Other safety assessments - electrocardiography, electroencephalography, neurologic examinations, and vital signs - showed no clinically significant trends in change from baseline results.”3 Transient decrease in blood pressure and heart rate. 5 No histo-pathologic studies available publicly. No effect on mortality, morbidity, body weight or food consumption. No evidence of tissue damage. 2 1 Oksendal AN, Hals PA. “Biodistribution and toxicity of MR imaging contrast media.” J Magn Reson Imaging. 2 Weissleder R et.al.“Superparamagnetic Iron Oxide: Pharmacokinetics and Toxicity” Am. J. of Roentgenology, Small Animal MR Facility, Last Updated 6/1/2010 3 Goldstein HA, et.al. “Safety Assessment of Gadopentetate Dimeglumine in U.S. Clinical Trials” Radiology. 1990 4 Harpur ES, et.al. “Preclinical safety assessment and pharmacokinetics of gadodiamide injection, a new magnetic resonance imaging contrast agent.” Invest Radiol. 1993 Mar;28 Suppl 1:S28-43. 5 Vittadini G, et. al. “Preclinical profile of Gd-BOPTA. A liver-specific MRI contrast agent.” Invest Radiol. 1990 6 Döhr O, et. al. “Preclinical safety evaluation of Gd-EOB-DTPA (Primovist).” Invest Radiol. 2007
Section 8 (project specific)

Facility note, some studies necessitate the administration (bolus or infusion) of contrast agent or
agent during study. In some instance it is desirable not to remove the animal (do not want to
reposition) so the use of a cannula (e.g., jugular vein cannulation) is needed. The following is
an example description of surgical implantation of a jugular cannulation.



A. Jugular Vein Cannulation

Justification
Serial assessment of tissue/tumor vasculature may require multi-day, dynamic infusions of MR
contrast agents during imaging. Repeated tail vein catheterization is unfeasible, and therefore
jugular vein cannulation is a more appropriate methodology.

Procedure
The animal is anesthetized by injectable anesthetic (Ketamine/Xylazine mixture). Two sites are
prepared for this procedure: a 2mm skin incision between the shoulder blades and a 0.5 cm
incision in the right ventral neck area. The surgical sites are shaved and swabbed with Betadine
and 70% alcohol. Sterile instruments, gloves and drape are used. Eyes are lubricated with
Paralube to prevent drying. For the catheter, a 10 cm length of Micro-Renathane tubing or
equivalent is used. One end of the catheter is connected to a heparin saline filled 1cc syringe and
flushed. The other end of the catheter is brought around the incision on the dorsal side, tunneling
under the skin using a probe with eye to the ventral incision in the neck. With the animal in
dorsal recumbency, the right external jugular vein is located, isolated, and cleaned by blunt
dissection. Using 5-0 silk suture, two ligatures are made to hold the vein. One ligature is tied
around the anterior aspect of the vein. The second loose ligature is wrapped around the vessel
near the clavicle. Both ligatures are clamped with forceps to produce enough tension to occlude
the flow through this portion of the vessel. A small angular cut is made on the jugular vein using
McPherson-Vannas micro scissors, the lumen is stretched using Dumont 7 forceps and the
catheter is introduced into the vein for about 0.5-1.0 cm. The loose ligature is tied around the
vessel with the catheter introduced. The catheter is flushed with saline to verify its patency. A
small drop of Vetbond is applied to anchor the catheter to the surrounding tissue. A few drops of
diluted Marcaine are dribbled in the incision line as local anesthetic and then the skin is sutured
with 5-0 nylon. The dorsal incision is also dribbled with Marcaine and sutured with 5-0 nylon.
The catheter is cut leaving approximately 1.5 cm from the skin of the animal. Using
monofilament nylon, a plug is made to seal the catheter. The animal is given 0.5-1cc saline (sub-
q) and allowed to recover under moderate warmth of heat lamp. Animals are housed individually
to prevent interference with the cannula by cage mates. Animals are given at least 2 doses of an
Small Animal MR Facility, Last Updated 6/1/2010 analgesic such as meloxicam. Meloxicam is given before surgery begins, and 24 hours later. The
dose for rats and for mice is 1.0 mg/kg, given either subcutaneously (injectable form) or orally
(oral form). If complications occur (catheter comes out; swelling, redness, or discharge indicate
infection; lethargy indicates septicemia; the animal is euthanized or the attending veterinarian or
designee will examine the animal and determine the appropriate course of action.)

Section 9
9.1 Anesthesia and Analgesia
(project specific)
Facility note, provide below is a list of common analgesics and anesthetics.

Drug/Dosage (mg/kg)
Needle Size
Isoflurane: 4% for induction, 0.75-3 % to maintain anesthesia, as determined by respiratory rate. (available at the MRI Facility) Section 10
10.1 Monitoring Interval
(project specific)
Facility note, a monitoring system (Small Animal Instruments Co.) that can be used to monitor
physiological signals such as heart rate, respiration, and body temperature (rectus) is available
if necessary. Respiration rate can be obtained from a small pneumatic pillow sensor placed next
to the animal’s abdomen. Heart rate can be monitored using ECG by placing three leads with
sub-dermal needle electrodes, and Temperature measured by insertion of a rectal thermometer
or by fiber optic sensor.

Source: http://secnmr.org/sammi/SAMMI-AnimalUseProtocolAssistance.pdf

References 2006

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