Protocol Review - Lab Animal 30(3), 2001

Protocol Review

Jerald Silverman, DVM, Column Coordinator

A Pain in the Rat

Dr. Peter Paul Price came to Great Eastern University from the Southern Cape A&M School of Medicine. He came with money, people, his Southern Cape-approved IACUC protocol, and a problem for the Great Eastern IACUC.

It was Great Easterns policy to give new investigators a 60-day window to rewrite their existing IACUC protocols on Great Eastern forms. The protocols then went through the regular IACUC approval process. During those 60 days, investigators could perform their research, as long as they had an IACUC-approved protocol from their previous institution; the previous institution was NIH-Assured; and, if applicable, the institution was USDA-registered. In the past, the Great Eastern IACUC had occasionally requested some small changes to protocols, but nothing with which the investigator had trouble complying. Prices protocol was different. Southern Cape had an NIH Assurance, and the protocol was properly approved, but the approval did not include the use of postoperative analgesia, and Price was performing some rather extensive cranial and brain surgery. Price had justified this exclusion to Southern Cape, and now to Great Eastern, on two grounds. First, he said that in his many years of experience, his rats always recovered uneventfully from surgery and were eating and acting normally within a day. Second, since he subsequently would record brain electrical activity from induced pain (using a tail flick test), and his surgical procedure caused the tail flick to be most sensitive 10 days after surgery, he could not risk any residual effects of postoperative analgesics.

After some consideration, the IACUC asked Price to do a pilot study using analgesia to determine if his recordings would be affected. Price wanted to know who would pay for the study; how many analgesics he would have to try if the first had an adverse effect on his research; what would be a reasonable number of animals to use; and how many studies that he performed in past years would have to be repeated, since he tested different analgesics and psychoactive drugs as part of his standard research protocol.

Should the Great Eastern IACUC agree with the Southern Cape IACUC, or is there another way of approaching the issue of postoperative pain relief?

Wheres the Pilot?

Robin Minkel and Karen Krueger, DVM

It is unclear whether Price completed a search of the literature for alternatives. Under USDA guidelines, pain and distress must be alleviated, unless scientifically justified. The defense given by Pricethat he has always performed his studies without analgesiadoes not alone carry enough scientific justification. The Attending Veterinarian (AV) should be consulted to suggest analgesics that have less residual effects and to assist Price with the alternative search by suggesting key words. It is possible that the alternative search will generate newer methods or data that support the use of analgesia for postoperative pain management. It is also possible that the search will provide further support to the investigators claim that analgesics will affect the study, or that the surgical procedure does not produce any pain or distress, resulting in further justification for withholding analgesia.

If the literature does not provide any clear alternatives to the use of analgesics nor demonstrates that the procedure does not cause pain or distress, it is appropriate for the IACUC to request a pilot study. A pilot study that repeats previous experimental situations with the addition of analgesia, with close monitoring by the AV, would carry more weight and also demonstrate good scientific practices. By working together, the AV, investigator, and IACUC should establish clear criteria for monitoring the animals with specific limits and guidelines that can be easily interpreted. These guidelines should be used to evaluate all animals in each test group. The pilot study would need, at minimum, two test groups: one that undergoes the surgery without the benefit of postoperative analgesia, and one that receives analgesia minimally the day of surgery. The results of the tail flick test conducted on day 10 can then be a direct comparison to determine if the analgesia had any effect. These results should be well documented and sent to the IACUC for review.

As the IACUC is requesting the pilot study, ideally, they should be the group to fund the project. However, very few IACUCs have their own allocation of resources to allow this. As such, the burden falls back onto Price. As the number of animals needed would be minimal, the burden is not likely to be great. Perhaps a grant search could be conducted that would allow the pilot study to proceed (with the results then made available to other research facilities) and be incorporated as a refinement or serve as justification for withholding analgesics for these types of studies. Another question that Price raises deals directly with the nature of his research. If the pilot study concludes that analgesics, in fact, do not have residual effects, would that compromise the integrity of his previous work? A short answer should be, "No." It is unclear if the surgical procedure caused any pain or distress, as indicated by Prices account that the animals were eating and acting normally within a day of surgery. The animals involved in the previous experiments may have experienced pain during surgery or immediately postoperatively that could have been alleviated, but during the testing phase there would have been no changes in the results. If the pilot study indicates that the use of analgesics do have a significant residual effect, then Price was justified in withholding pain relief.

Minkel is IACUC Administrator and Krueger is a member of the veterinary staff at Genetics Institute, Andover and Cambridge, MA.

Pass the Baton

Kerry Taylor, DVM, MSc, DACLAM, and Axel Wolff, MS, DVM

Unfortunately, this scenario did not identify a funding source. If we assume that Prices project is supported by a Public Health Service (PHS) agency, such as the National Institutes of Health (NIH), this scenario would not play out; Great Easterns policy to allow new investigators a 60-day window for processing paperwork through their IACUC would be noncompliant with the PHS/NIH grants policy as well as the PHS Policy and the Great Eastern Animal Welfare Assurance on file with OLAW.

Requests for transfer of PHS/NIH-supported research must follow specific procedures. First, a transfer requires the prior approval of the PHS/NIH funding component which would trigger a number of additional steps to ensure full compliance with grants policy prior to funding, such as determining that Great Eastern has an Assurance, that the IACUC approved the protocol, and that Great Eastern has all the resources necessary to fulfill the goals of the grant. In addition, the institution relinquishing the grant must do so prior to the issuance of a new award. If the request to transfer the grant was approved, the new grantee, with an approved Assurance, must assure that an IACUC is in place and certify that the protocol has been reviewed and approved. These PHS/NIH grants policies would have challengedif not eliminatedGreat Easterns policy and would have forced a Great Eastern IACUC review in accordance with the PHS Policy. The issue of reducing the burden of dual review is not pertinent; once Southern Cape A&M relinquishes the grant, and NIH issues a new award, all accountability transfers to Great Eastern. The Great Eastern IACUC must conduct a review that satisfies the PHS Policy for the transfer of the grant to be effective, and for Price to be able to continue his work at the gaining institution. Regarding the evaluation of pain in the rats, the previous IACUC may have agreed to an earlier accepted interpretation concerning pain alleviation in rodents, an issue frequently debated in todays research climate. As science advances and more information is available about animal pain, protocols must necessarily be evaluated (or re-evaluated) against a higher standard, using more complete criteria.

As an Assured institution, Great Eastern must follow PHS Policy, which specifically states that, "Procedures that may cause more than momentary or slight pain or distress to the animals will be performed with appropriate sedation, analgesia, or anesthesia, unless the procedure is justified for scientific reasons in writing by the investigator." The burden of proof is on Price to convince the new IACUC that analgesia is not necessary or must be withheld.

In addition, the US Government Principles states, "Proper use of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with sound scientific practices, is imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in other animals." Prices study may fit this category, but if exceptions are to be made, they must be IACUC-approved.

If the IACUC has concerns about the study design, well-executed pilot studies are certainly a reasonable request, and can often provide useful information. Pilot studies are generally funded by the investigator, and the numbers necessary (both animals and experimental agents) depend on the quantity of data needed for an informed IACUC decision. Price and his colleagues would be expected to have sufficient expertise for a pilot design, but other experts in the field could be consulted. Price would not likely need to repeat prior studies if the scientific information was acceptable. If he has already tested analgesics as part of the protocol, he is in a good position to recommend an effective postoperative drug for the animals under study. The AV must be intimately involved in oversight of the protocol, to advise, monitor, and assess the level of postoperative pain experienced by the animals, rather than accepting the investigators assertion that the animals were "normal." In addition, studies designed to characterize behavioral and physiological parameters for the purpose of pain assessment have demonstrated that an unbiased, medical assessment of the animals condition can readily validate that analgesics either are not necessary or must be withheld.

Taylor is Deputy Director, Office of Animal Care and Use, and Wolff is Senior Assurance Officer, Division of Animal Welfare, NIH, Bethesda, MD.

It Doesn't Have to Hurt

Tracy Peace, DVM, MS, DACLAM, and Laurie Goodchild, DVM

Prices assertion that since his rats are "eating and acting normally within a day," they therefore do not experience postoperative pain is akin to the recently disproved notion that rats are resistant to postoperative infection, since they recover from nonsterile surgical procedures. Rats are indeed susceptible to wound infection, as evidenced by measurable changes in physiology and behavior1. Prices rats eat and act normally within a day of surgery, but this does not prove that they did not experience postoperative pain. Postoperative pain likely peaks within the first 24 hours after the procedure, since associated behavioral changes typically peak at that time2.

Simply stating that the use of anesthetics, analgesics, or tranquilizers may interfere with research results is not adequate justification to withhold them. Additionally, according to animal welfare regulations, procedures that cause pain and distress in humans are assumed to cause pain and distress in animals unless proven otherwise. Spinal and cranial surgery in the rat may result in mild to severe pain, depending on site and amount of trauma3. Lack of clear scientific justification or proof (i.e., objective, measurable data) that the animals are not experiencing pain, and the use of invasive techniques, should be red flags to any IACUC.

The Guide4 endorses the use of pilot studies conducted under IACUC oversight when dealing with procedures that have the potential to cause pain or distress that cannot be reliably controlled. Although conducting a pilot study to evaluate the use of analgesics, anesthetics, or tranquilizers is a valid option, the optimal first step is to examine objective and easily measured pain assessment parameters.

Food and water consumption, body weight, and behavioral/locomotory assessments can be performed in rats. Decreases in food and water consumption are considered useful indicators of postoperative pain in rodents5 and are easy to measure. Furthermore, the administration of analgesics (e.g., opiates, nonsteroidal anti-inflammatories) has been proven to reduce the drop in food and water consumption seen post-surgery, and, indeed, "If the depression in food and water consumption is related to the presence of postoperative pain, then these findings suggest that analgesics should be administered to rats following surgical procedures6."

The IACUC should ask Price to establish a list of measurable pain assessment parameters and a schedule for data collection, allowing his work to continue without disruption while simultaneously obtaining very valuable information. At the conclusion of the data collection, the information should be reviewed by the IACUC. If food and water consumption are significantly reduced by the surgery and resultant postoperative pain, the IACUC should then mandate the performance of a pilot study with pain-relieving drugs. It should also be noted that good surgical technique minimizes tissue trauma, reduces the length of the surgical procedure, and has a direct impact on postoperative pain and distress. The IACUC should verify the surgeons expertise. If a pilot study using pain-relieving medications is warranted, Price should be enlightened that the field of pain relief has evolved from postoperative, to intra-operative, to preemptive administration. Drugs administered prior to the painful procedure can prevent sensitization of pain receptors. Also, since Price has concerns regarding residual effects of analgesics, he should undertake a careful review of the literature to reveal information related to the potential interactive effects of various drugs.


1. Bradfield, J.F., et al. Behavioral and physiologic effects of inapparent wound infection in rats. Lab. Animal Sci.; 42(6):572-578, 1992.
2. Hansen, B. Through a glass darkly: using behavior to assess pain. Semin. Vet. Med. Surg. (Small Animal) 12(2):61-74, 1997.

3. Hobbs, B.A. Assessment of painful procedures. In: Guttman, H.N., ed. Guidelines for the Well-Being of Rodents in Research. Scientists Center for Animal Welfare, Bethesda, MD. pp. 70-79, 1990.
4. Guide for the Care and Use of Laboratory Animals. National Academy Press. Washington, DC, 1996.
5. Flecknell, P.A. and Silverman, J. Pain and distress. In: Silverman, J., Suckow, M.A., and Murthy, S., eds. The IACUC Handbook. CRC Press, Washington, DC, pp. 221-249, 2000.
6. Liles, J.H. and Flecknell, P.A. A comparison of the effects of buprenorphine, carprofen and flunixin following laparotomy in rats. J. Vet. Pharmacol. Ther.; 17(4):284-290, 1994. Peace is Attending Veterinarian and Goodchild is Training Coordinator/Clinical Veterinarian, Battelle Memorial Institute, Columbus OH.

Peace is Attending Veterinarian and Goodchild is Training Coordinator/Clinical Veterinarian, Battelle Memorial Institute, Columbus OH.

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