IACUC GUIDELINESFOR THE USE OF NEUROMUSCULAR BLOCKING DRUGS (NMBD)

Guidelines for the Use of Neuromuscular Blocking Drugs (NMBD)

NMBD can be administered only when the animal is adequately anesthetized and an appropriate means of mechanical ventilation is provided. Federal Animal Welfare Regulations give the PI responsibility to ensure that anesthesia is adequate throughout the period of time NMBD are used. The level of anesthesia must be sufficient to induce unconsciousness and to provide adequate pain relief.

One complication of using NMBD is that it is more difficult to determine if the level of anesthesia is adequate. Changes in skeletal muscle tone (especially jaw tone), changes in the rate and effort of respiration, and gross purposeful movement are all valuable tools used clinically to determine the level of anesthesia. When NMBD are administered to animals, these useful parameters are lost.

NMBD must therefore be used only in conjunction with an anesthetic protocol known from clinical veterinary experience to produce a stable and adequate level of general anesthesia for the entire period the animal will be under chemical paralysis. The investigator must take appropriate measures to insure that the correct amount of anesthetic agent(s) is being administered at all times.

Guidelines for the submission of an Animal Use Protocol that proposes the use of NMBD

1. The need for NMBD must be scientifically justified.
2. The anesthetic protocol, NMBD regimen, and method of ventilation must be specified.
3. A description of the method(s) that will be used to monitor anesthetic depth during NMBD paralysis must be included. A thorough anesthetic monitoring record must be kept for all animals. Physiological parameters appropriate for the species being studied must be monitored and documented periodically. Details of the specific parameters to be monitored and frequency of documentation must be included as part of the Animal Use Protocol.
   
   Some examples of adequate anesthetic and monitoring procedures to be used in conjunction with NMBD are included in the Appendix.

Appendix

There are several methods used to determine that the level of anesthesia is appropriate for a protocol that utilizes NMBD. One method is to either perform a sham procedure or carry out the entire procedure in a subset of animals without administering NMBD, thereby determining the anesthetic requirement. Subsequent animals would all receive that predetermined anesthetic dose (or a higher dose if indicated by clinical signs in an individual animal). A second method is to determine the anesthetic requirement for each animal, prior to administering the NMBD. With this method, once NMBD are administered, the amount of anesthesia is never decreased. With a third method, the NMBD is periodically withdrawn and the anesthetic requirement determined when skeletal muscle function has returned. The most suitable method used to determine that an appropriate level of anesthesia is maintained during NMBD administration may depend on the type of investigation. These methods are described in more detail below. In animals receiving NMBD, the following parameters may be considered for monitoring: electrocardiogram, electroencephalogram, body temperature, respiratory rate, respiratory volume, oxygen saturation, end-tidal CO2. If a volatile anesthetic agent is used, the inspired and endtidal 02 concentration, N20 concentration, and agent concentration should be continuously monitored. Regardless of which method(s) will be used for a study, it must be described in detail in the IACUC protocol.

Sham procedure or withholding NMBD

Performing sham procedures or complete experiments where the NMBD is withheld are sometimes used to determine the anesthetic requirement for a particular study. With sham studies, the animals are anesthetized using the proposed anesthetic technique; however, the entire study is not performed. A noxious stimulus is applied (e.g., toe-pinch or tail-pinch) to determine if the depth of anesthesia is adequate. Once the anesthetic dose requirement is determined, all subsequent animals will receive at least that dose of anesthetic (higher doses should be administered if clinical signs of inadequate anesthesia are observed in individual animals). If the noxious stimulus is greater in the actual experiment (e.g., a thoracotomy has higher anesthetic requirements than a toe-pinch), this would not be the best method of determining the anesthetic requirement. In addition, if the dose range of anesthetic is not consistent between multiple sham animals, a good measure of anesthetic requirements for all subsequent animals is not determined. Due to biologic variation between individuals, the more sham animals that are used to determine the anesthetic dose requirement, the more accurate the minimum anesthetic determination will be. The specific details of conditions and number of “shams” necessary should be determined in consultation with LARC veterinarians.

A similar type of determination could be carried out where all procedures of a study are performed; however, the NMBD is not administered. Similar to above, if the dose of anesthetic is not consistent between multiple animals undergoing the procedures, this would also not be a good measure of the anesthetic requirement for all subsequent animals. Again, the more animals that are used to determine the anesthetic dose requirement, the more accurate the minimum anesthetic determination will be. This method of anesthetic determination is not applicable to investigations where paralysis is a requirement of the study. The specific details of conditions and number of animals studied should be determined in consultation with LARC veterinarians.

Anesthetic requirement determined in each patient

This method determines the anesthetic requirement for each animal prior to administering the NMBD. A portion of the study is performed without paralysis when skeletal muscle tone and movement can be monitored (along with other clinical signs of anesthetic depth) and the anesthetic requirement defined. This method accounts for normal biologic diversity in anesthetic dose requirements since each animal serves as is its own control. With this method of anesthetic requirement determination, it is imperative that the maximum noxious stimulus that will be encountered during the study occurs before NMBD are administered. This method of anesthetic dose determination is particularly useful for protocols that include an initial surgical phase that is followed by a data collection phase.

Many investigators find it most useful to perform the surgical/instrumentation portion of the study without using NMBD. Except in certain types of procedures, the use of NMBD is not justified during the surgical phase. Without NMBD paralysis during this phase of the study, the anesthetist can monitor skeletal muscle tone, a valuable indicator of anesthetic depth. Many investigators find that inhalation anesthetics such as isoflurane are the safest and most easily controlled anesthetic for the surgical phase of the study. For certain protocols (e.g., many neuroscience investigations) inhalation anesthetics depress the responses being studied. Therefore, following the surgical/instrumentation phase, the anesthetic technique is often changed from isoflurane to another method for the data collection phase. In this case, a physiological steady state under the new anesthetic should be achieved before NMBD are administered. Following paralysis, the anesthetist will be dependent upon the sympathetic nervous system responses to indicate the level of anesthesia, thus it is important that the patient also be physiologically stable before NMBD are administered. The time required to achieve steady-state anesthesia with stable physiological variables can vary dramatically, depending upon the method of anesthesia. Once adequate steady-state anesthesia is achieved, NMBD can be administered. The rule of thumb is that the anesthetic level should not be decreased after the NMBD has been administered; however, it can be increased if clinical signs of an inadequate level of anesthesia are observed.

NMBD periodically withheld

If animals are anesthetized for several days, the anesthetic regimen may require adjustment during this period. To do so, NMBD should be withheld from the animal for a period adequate to allow for return of normal skeletal muscle responses prior to the anesthetic adjustment. Normal muscle responses can be assessed using an electronic twitch monitoring device (MiniStim®) that stimulates skeletal muscles to contract. The frequency of NMBD interruptions and anesthetic adjustments must be specified and justified in the Animal Use protocol for approval. An alternative to NMBD interruptions when using a volatile anesthetic is to monitor end-tidal anesthetic concentration and to maintain an appropriate level throughout the procedure. Again, the end-tidal anesthetic concentration must be defined in the Animal Use protocol for approval.

References:

1. Preparation and maintenance of higher mammals during neuroscience experiments. Report of a National Institute of Health workshop. 1991 Van Sluyters RC, Oberdorfer MD, eds. NIH Publication No. 91-3207.
2. Drummond JC, 1996. Use of neuromuscular blocking drugs in scientific investigations involving animal subjects. Anesthesiology, 85(4):607-699
3. Guidelines for the use of neuromuscular blocking drugs in anesthetized animals. Animal Care and Use Committee, University of California, Berkeley. Appendix K: Home Office guidelines on the use of neuromuscular blocking agents. Guidance on the Operation of the Animals (Scientific Procedures) Act 1986. (UK)