Animal Monitoring in the Center for Biomedical Imaging

Introduction and Purpose

These guidelines form the basis of institutional policy, as approved by the Boston University Institutional Animal Care and Use Committee (BU IACUC), Center for Biomedical Imaging (CBI), and Boston University Animal Science Center (BU ASC). These guidelines apply to all animal imaging conducted at CBI (X-building B01) using imaging facilities, equipment and other resources also used for imaging human patients. These guidelines apply to both BU research staff as well as investigators bringing animals from other institutions for imaging sessions at CBI.
Contents:

I.    Study Requirements
II.  Animal Transportation To and From Imaging Suites
III. Animal Preparation Prior to Entering the Imaging Facility
IV. Hygiene
V.   Animal Monitoring
VI. Termination of Scan
VII. Contact Information
VIII. Certification

I. Study Requirements

All animal imaging studies and related activities must be performed according to established procedures of the BU IACUC and in accordance with applicable federal laws and regulations, which include:

Public Health Service Policy on Humane Care and Use of Laboratory Animals
USDA-APHIS Animal Welfare Act
Guide for the Care and Use of Laboratory Animals – “The Guide”

1. IACUC Protocol

All protocols and any amendments must be pre-approved by both the BU IACUC and CBI, in accord with the Policy and Procedure for Animal Transfer to/from BU Biomedical Imaging. If a non-BU animal or investigator is involved, the BU IACUC must review and approve the off-site IACUC protocol before imaging is permitted. Imaging and related activities must adhere to the approved version in the IACUC protocol(s). Evidence that the research staff are qualified to perform the study will be demonstrated by means of his/her having obtained approvals for the IACUC protocol and by completing IACUC and CBI training or another institution’s equivalent. The primary responsibility for the conduct of the experiment, for the care of each animal subject during the experiment and the safety of research staff resides with the PI.

2. MRI Safety Training

All personnel must have received MR safety training from the CBI before imaging studies commence. During this training all personnel who will be entering an MR facility will be screened for contraindications to entering a magnetic field (e.g. whether the staff members have pacemakers, cochlear implants, aneurysm clips).

3. BU ASC meeting

A meeting must be scheduled with BU ASC Veterinary Services (617-638-4086) prior to the first imaging session to address specific issues of safety, animal transportation, anesthesia, monitoring of vital functions, general animal care while in the facility, and cleanup procedures upon completion of the scan. The investigator must provide a clearly written experimental outline to BU ASC staff prior to the meeting.

All personnel participating in the imaging activity must have documented training in transporting and handling the pertinent species, the potential hazards associated with such animal handling, and must be current in their occupational health assessment (including evaluation for tuberculosis every six months for non-human primate protocols). Guidelines for such training and safeguards are provided in a CDC publication, Guidelines for Environmental Infection Control in Health-Care Facilities.

4. Imaging studies

Imaging studies at the CBI will be coordinated through the CBI Director or his designee. The principal investigator is responsible for ensuring that BU ASC is informed well in advance of any scans (7 days, if possible). In addition, the CBI Director will inform BU ASC monthly of animal scans.

II. Animal Transportation to and from Imaging Suites

See also: Policy and Procedure for Animal Transfer to/from BU Biomedical Imaging

Animals must be accompanied at all times by research staff from the institution of origin or an LASC designee. Research staff must have the approved IACUC protocol number readily accessible. Recent individual medical records, including the most recent tuberculin tests for non-human primates, will also accompany animals.

Animal transport to and from CBI will be in vehicles that comply with federal laws and regulations and The Guide. Animals will be transported within BU buildings in appropriate, secure containers that limit exposure between animals and humans and ensure safety of the animal.

Animals will be fully anesthetized prior to imaging (unless a specific exemption was obtained). Animals already at LASC will be transported in an enclosed escape-proof cage or carrying box that is opaque or completely covered by a sheet or drape, and lined with an absorbable pad for the collection of bodily fluids (e.g., urine). Standard operating procedures for transportation within BU must be approved by the IACUC.

In the case of an escaped animal, the following guidelines must be followed:

• Contain the animal as best as possible (e.g. within the room).
• Contact BU ASC immediately. Contact information is listed in Section VII.

III. Animal Preparation Prior to Entering the Imaging Facility

Animal preparation, including hair shaving, catheter placement, anesthesia induction and intubation are to be performed in the prep room by investigators prior to entering the scanner. Research personnel will bring their own routine medical supplies that may be necessary for animal restraint and for the imaging procedure. Research personnel will monitor and document animal anesthesia and recovery in accordance with federal laws and regulations and The Guide. If the investigator prefers veterinary technical assistance to perform these tasks or is interested in receiving training, contact BU ASC. All pertinent entries must note the date and time, and be initialed by the person making the entry in the individual animal health record

Animals must have completed the required acclimation period or been released from quarantine after review by the designated facility veterinarian. Animals known to have common zoonotic diseases may not enter the suite unless all personnel involved have been properly trained and the risk of infection or cross-contamination can be minimized to an acceptable level. The scanning of any known or suspected infectious animal must be explicitly approved by LASC and by CBI, and research staff must inform all personnel who are present in the MRI suite when an infected animal is present.

All personnel (including research staff, CBI staff, BU ASC staff, etc) will be instructed in and wear appropriate PPE during the entire prep and imaging session. This includes but may not be limited to gowns, face shields or safety glasses, masks, shoe covers and gloves when working with non-human primates. Research staff are responsible for notifying CBI staff and visitors of special PPE requirements for their animals. All staff who are present when primates are being scanned must have a negative TB test within the past 6 months.

All primate users will ensure the availability in the prep room of monkey bite/scratch kits and post-exposure instructions at each scan, and will notify Occupational Health and LASC Veterinary Services promptly in the event of an exposure.

IV. Hygiene

Disposable absorbent pads (“chucks”) will be used whenever possible to minimize direct contact of animals to surfaces which come into contact with human patients. The animal will be placed on top of the chucks for the duration of the study. No one other than persons actively involved in the animal research project will be allowed in the room. Personnel must wear proper PPE (which may include gowns or scrubs, gloves, masks, safety glasses, and shoe covers) when handling animals. Gloves must be removed before touching control panels, video equipment, telephones, doorknobs, elevator buttons, or other objects in shared spaces, including the control room.

If surgical manipulation or blood withdrawal is necessary, areas that may come in contact with blood, body fluids, and animal dander must also be draped. Sanitization supplies must be available for immediate use. At completion of the scan, the room will be cleaned thoroughly. The table, floor, and any instruments in contact with the animals such as surface coils must be cleaned with disinfectant solution, available in the MRI facility, allowing sufficient contact time for agent effectiveness. Sharps containers are readily accessible in the MRI suite and prep area. No human patients will be permitted to enter the area until it has been cleaned. Post-scan sanitization should be arranged immediately after the scan. The research staff will dispose of all biological waste and disposable items in red biohazard bags; CBI will make arrangements with building services for disposal of red bag waste. When possible, human patient care equipment which comes in direct contact with the animal must be sterilized before re-use. Steam, gas, or chemical sterilization should be used, as appropriate for each item. Ideally this equipment should be duplicated whenever possible and labeled and stored separately from patient equipment.

No food or drink is permitted in the suite or the control room during the study. The designated investigator listed on the IACUC protocol is responsible for adequately cleaning and disinfecting the MR Suite after the animal is removed from the room. All surfaces which have come into contact with animals or have been touched by animal handlers must be disinfected using Quatricide, MB-10, or other approved disinfectant. Personnel handling the animals or sample material must wash their hands prior to leaving the MR suite.

All waste material generated during the study will be considered medical-pathological waste and disposed of according to current guidelines from the Safety Office. The PI or designee will be responsible for ensuring that medical-pathological waste is disposed of properly. All animal associated waste will be red-bagged for disposal.

V. Veterinary Oversight

LASC Veterinary Services must be notified in advance of any animal MRI scans. LASC veterinary staff or designee (e.g. consulting veterinarian) will be present for the animal preparation and scan, or will be available on call for consultation and attendant care in the event of animal escapes or veterinary emergencies. LASC contact information is listed in Section VII.

VI. Animal Monitoring

Anesthesia monitoring and supportive care is required for all animals that enter the CBI. A complete medical record of all procedures and the administration of fluids and drugs will be generated along with an anesthesia monitoring sheet for periodically recording the animal’s vital signs (heart and respiratory rates, etc.) and other vitals where appropriate. Documentation of supportive care (which may include fluid therapy, supplemental heat, etc.) is also required. These records will be kept in the animal’s individual health record. A summary or copy of these records will be made and accompany animals that return to the LASC animal facility. In case of a veterinary medical emergency, the investigative staff must have extra anesthetics and emergency drugs available during the scan. A crash kit and defibrillator are available in CBI for veterinary emergencies.

The following general guidelines for animal care should be considered and addressed as part of the protocol for each study. Individual protocols might vary from these guidelines as required by the particular research study, as approved by the IACUC.

1. Pre-anesthetic evaluation

All animals should be in good health prior to the administration of general anesthesia. If the animal has any pre-existing medical conditions or diseases (anemia, high blood pressure, diabetes, kidney problems, etc.), please consult LASC prior to the administration of any drugs.

2. Handling & Restraint

Personal protection equipment should always be worn when handling animal to help prevent the transmission of allergens and zoonotic diseases, and to protect the health status of the animal. The amount of restraint (generally chemical) and its duration should be kept to the minimum necessary to complete the procedure. All necessary equipment and reagents for the procedure should be ready prior to restraint. The use of pre-anesthetic sedatives/tranquilizers will help reduce anxiety and the subsequent doses of other agents.

3. Ophthalmic ointment

Anesthetized animals should have ophthalmic ointment applied to the eyes to prevent drying of the eyes and corneal trauma during manipulation.

4. Vascular access

Placement of an intravenous catheter allows for easy and rapid administration of fluids and drugs during the procedure. This may be a lifesaving factor if an emergency arises. For long procedures (> 30 min.), an intravenous catheter should be placed. The cephalic, saphenous, or femoral veins are most commonly employed for vascular access. For arterial sampling or direct blood pressure monitoring, the femoral artery is most commonly employed. Intramuscular injections are generally given in the thigh or back muscles (parallel to the spine). The smallest possible injection volume should be employed.

5. Hydration

Fluid support should be provided for procedures lasting longer than 30 minutes. Intravenous fluids should be given at an initial rate of 10ml/kg/hr for most procedures. Alternatively, a bolus of subcutaneous (SQ) fluids may be administered at the end of the procedure. For species commonly used in the CBI (2 kg – 12 kg body weight), a range of 50 – 100 ml fluids may be given subcutaneously.

6. Hypothermia

Mammals are prone to hypothermia during anesthesia. Animals that weigh less than 5 kg, those undergoing long procedures and those given Telazol® and/or xylazine are most at risk. Hypothermia can cause bradycardia, low cardiac output, and delay recovery from anesthesia. Serial evaluations of body temperature during anesthesia and recovery should be performed. Warm surgical scrub and warm sterile saline can be used when necessary to prepare the animal. A circulating water blanket covered with a drape should be used as an external heat source during anesthesia. Electric heating pads and hot water bottles may be used during transport and recovery, provided that close monitoring is utilized to prevent thermal burns and/or hyperthermia; exposed skin should be covered or if a heat lamp is used it should be placed such as to indirectly warm the inside of a cage. The lamp should be kept 3-4 feet away from the cage and the cage surface temperature should be checked with your hand periodically. The surface should be comfortably warm to the touch and the formation of hot spots on the cage surface must be avoided. Warm IV or SQ fluids may also be helpful.

7. Endotracheal intubation

Airway control via endotracheal intubation is highly desirable during anesthesia and required in some situations where controlled ventilation is mandatory, e.g. use of paralytic agents. Administration of inhalation anesthetics is best accomplished via an endotracheal tube. Intubation requires some skill and practice and is best accomplished using a laryngoscope. Personnel must demonstrate proficiency in this technique per the approved IACUC protocol.

8. Monitoring and Recovery

Anesthetic monitoring is of vital importance for proper patient management during the MRI procedure. The goal of monitoring is to maintain adequate anesthetic depth while preserving the normal function of organ systems. Anesthetic depth may be assessed via palpebral and corneal reflexes, jaw tone, and the absence of movement in response to painful stimuli (such as a toe pinch). Reduction and/or loss of these reflexes and responses is associated with an adequate surgical depth of anesthesia. Increased respiratory rate in response to surgical stimuli is a sensitive indication of inadequate depth of anesthesia. Increases in heart rate may be due to pain perception or from increased blood level of carbon dioxide. Blood pressure is a valuable marker for anesthetic depth; for example the anesthetist could become confused by the paradoxical effect of increased heart rate in response to very low blood pressure, thus making the determination of anesthetic depth (i.e. too deep versus too light) more challenging to decipher. Finally, the desired anesthesia depth for MRI studies may differ from that of surgical procedures (e.g. lack of limb movement may be the only requirement, however the lack of retinal movement may necessitate the use of neuromuscular blocking agents). Animals must be monitored continuously until fully recovered from anesthesia (ambulatory) in their home cage. Vital signs need to be checked and recorded periodically in unconscious or tractable animals until recovery is complete.

Cardiovascular monitoring: Heart rate and rhythm, pulse quality, mucus membrane color and capillary refill time should be evaluated at regular intervals to access cardiovascular status. The use of an esophageal stethoscope, lead II ECG, and blood pressure monitor can help facilitate cardiovascular monitoring.

Respiratory monitoring: Proper monitoring of this system involves quantitation of oxygenation and ventilation. For evaluating oxygenation, mucus membrane color is most commonly used, it is however a very insensitive technique. Patients may become dangerously hypoxic by the time a change in mucus membrane color (pink to blue) is noted. Increases in respiratory rate may also indicate low blood oxygen levels; however, this parameter is primarily regulated by blood levels of carbon dioxide. Pulse oximetry is a non-invasive method for continuous measurement of oxygen saturation and heart rate. Other methods include arterial blood gas sampling and end-tidal CO2 monitoring.

9. Adjunctive drugs

Anticholinergics: (Atropine, Glycopyrrolate) These drugs are used to decrease salivation and bronchial secretions during anesthesia. Atropine may also be used to treat bradycardia (low heart rate) induced by some drugs ( e.g. opioids and xylazine) or by reflex responses. These agents should be used with care as they may cause very rapid heart rates or potentially fatal abnormal electrical activity (arrhythmias) in the heart. Anticholinergics should be administered prior to the use of xylazine and are recommended to help facilitate endotracheal intubation.

Neuromuscular Blocking Agents: (Pancuronium, Vecuronium, Atracurium, and others) Neuromuscular blocking agents (paralytics) are used in conjunction with general anesthetics to achieve greater control of muscle activity. These drugs produce skeletal muscle relaxation and are very useful when control of ventilation or muscle movement is needed (e.g. ophthalmic scans). These agents do not have any anesthetic or analgesic properties; furthermore, even if animals are not subjected to painful procedures, the inability to move spontaneously when conscious produces unacceptable distress to the animal. Therefore, neuromuscular blocking agents can only be used in anesthetized animals. Extreme care must be taken to ensure that the patient is at a proper level of anesthesia when using these agents. Since the ability of the animal to move is eliminated, parameters such as heart rate and blood pressure must be used to monitor anesthetic depth. These agents paralyze the diaphragm and cause respiratory arrest; therefore, the animal must be intubated and mechanically ventilated. Pancuronium lasts longer than vecuronium and produces mild increases in blood pressure and heart rate. Special attention to recovery from neuromuscular blocking agents must be given: the ability to breathe consistently is regained after the first spontaneous breathe/cough/sigh is observed, so continued ventilatory support is required until certain that the animal can support itself. For more detailed information, see the BU IACUC Policy on the Use of Neuromuscular Blocking Agents.

Reversal Agents: (Nalaxone, Yohimbine, Neostigmine) Anesthesia in general and some anesthetics in particular are prone to cause hypothermia, cardiovascular and respiratory depression. Therefore, reversal agents are recommended to speed recovery from anesthesia following MRI procedures (which are considered non-invasive and non-painful).

1) Naloxone (Narcan®) is an opioid antagonist that is used to reverse the effects of morphine, oxymorphone, or fentanyl. It rapidly and effectively reverses opioid sedation, respiratory depression, and bradycardia. Naloxone can be given IV or IM. It should be noted that naloxone will also eliminate the analgesic effects of opioids and its duration of action may be shorter than the opioid it is being used to reverse. Careful monitoring and re-dosing may be needed if the animal begins to relapse as a result of this difference in duration of action.

2) Yohimbine is used to reverse the action of xylazine under similar circumstances as indicated for naloxone. In general, only one dose of yohimbine is needed to reverse the effects of xylazine.

3) Neostigmine is used to reverse the effects of neuromuscular blocking agents. It is best administered IV in this indication, although IM and SQ are also acceptable. To avoid cholinergic toxicity, the use of neostigmine should be titrated based on the waning effects of the paralytic agent used. Assurance that the animal has the ability for unaided respiratory function is necessary before discontinuing ventilatory support.

10. Emergency Resuscitation

In the event of cardiopulmonary complications, contact LASC Veterinary Services at the numbers listed below. For emergencies after hours (7 pm – 7 am, and Saturdays/Sundays), call the Control Center (x8-4144) who will notify LASC. These instructions are provided as a guideline for emergency treatment prior to the arrival of veterinary staff. Follow the “ABCs” (airway, breathing, cardiovascular support, drugs) for basic CPR:

The most important procedure is to ventilate the animal. Establishing control of the airway and breathing should be the first priority, do nothing else first!!!

A. Tracheal access. If the patient is not already intubated place an endotracheal tube. If an endotracheal tube is not available use a face mask to ventilate.

B. Ventilation. Ventilate with 100% O2 at a rate of 1 breath every 5 seconds. If using inhalant agents make sure the vaporizer is turned off. An Ambu-bag is available in the crash kit for manual ventilation if the ventilator machine is not easily accessible.

C. Chest compressions. After establishing control of ventilation, start regular strong chest compressions (at least 1 per second). It is not necessary to coordinate compressions with ventilation. Rapid infusion of IV fluids should be started to support perfusion.

D. Emergency drugs. If no heart beat can be heard or no pulses felt, then epinephrine should be given IV. Reversal drugs should be given if opioids, xylazine, or paralytics have been used. Dopram® may be given to stimulate respiration if spontaneous breathing is not noted. Have emergency drugs and instruments in the suite ready for use.

VII. Termination of Scan

A scan may be terminated by CBI or LASC if the procedures outlined in these guidelines and the approved IACUC protocol are not followed or if the animal is considered to be at unnecessary risk by the MR staff or LASC staff. In addition, the BU IACUC may suspend protocol activities after a convened IACUC meeting if there are concerns of compliance and/or animal welfare.

If euthanasia is performed, the methods must be in accordance with guidelines established by the BU IACUC and the American Veterinary Medical Association:

IACUC Euthanasia Policy
AVMA Guidelines on Euthanasia

VIII. Contact Information

BU Control Center (617) 638-4144

LASC Office (617) 638-4086

CBI: (617) 414-2370
Dr. Ron Killiany, Director

IACUC:
Dr. William Cruikshank, Chair (617) 638-5295
Kate Wiklanski, Director (617) 638-7198

Applicable Standard Operating Procedures and Policies:
• Policy and Procedure for Animal Transfer to/from BU Biomedical Imaging
• 3 T Facility (3TF) Application Review Process
• IACUC Policy for Animal Monitoring in CBI
• IACUC Policy for the Use of Neuromuscular Blocking Agents