Dr. Margarete Arras
Improvement of Pain Therapy in Laboratory Mice
The ability of animals to feel pain is not completely understood[*] but also the expression of pain via behavioural changes differs from species to species. The alleviation of pain in regulatory and biomedical science is part of the protocols for animal experiments and inherently requires that pain is adequately recognized. In order to provide animal experimenters with better tools for pain recognition, the 3R Research Foundation is supporting projects, which elucidate this uncertainity. Support will continue in a follow-up project 96-05 which is still in an initial state and in the project 88-03 . The present results originate from project 71-00 carried out by Margarete Arras and funded partially by the 3R Research Foundation.
Pain assessment - Different types of pain
Pain assessment in laboratory mice includes the determination of symptoms and indicators of pain and correlating them to the type of pain (e.g. acute vs. chronic pain). Contemporary ways for measuring acute pain are well described for reactions (e.g. withdrawal time, pressure range) on defined, short, stimuli in analgesiometric tests (e.g. tail flick, hot plate). These tests are frequently used for substances and/or analgesics dosage testing, because the read-out is quantified according to defined endpoints.
Acute clinical pain is a different situation which occurs e.g. after surgical interventions carried out in many biomedical animal experimentations, after artificially induced inflammatory reactions (e.g. antibody production, arthritis) or experimental infections for immunological research. Clinical pain, especially in mice, is difficult to estimate, because its quality and duration is influenced by an number of variables, e.g. anaesthesia, the surgeon’s expertise, individual sensitivity, metabolic disorders, etc. In addition, in genetically modified mouse lines the impact of their phenotype on the animal`s well-being is often not known. The consequences are that i) recognizing clinical pain of a moderate degree in mice is based on assumptions and ii) the establishment of a protocol for pain relief and controlling the effectiveness can easily be guesswork.
New approaches for the assessment of clinical pain
Clinical pain is an undesirable variable in research projects. Stressors can disrupt biological functions which might be critical not only to the animal‘s welfare but also to the readout of the experiment. Accordingly one might conclude that in mice, every suspicious manipulation (as little as it might be) must be treated by a standardized pain therapy. But dosages and time course of pain therapy may be poorly adapted to the animal`s needs. The goal of the present study was to develop an approach which is suitable to recognise mild to middle grades of pain during an experiment. In the present study we used NSAIDs as analgesics. The use of opioids would induce physiological and behavioural changes which hamper the assessment of pain by an altered behaviour.
Behavioural approach: Nesting
From daily experience, we couldn’t see any changes in the appearance and behavior of the mice after laparatomy performed during embryo transfer and vasectomy with and without analgesics. Consequently, it was impossible to prove whether the pain killers that we administered routinely, were effective or even necessary to relieve pain. However, when we observed the animals in 12-h-intervals and took drafts of their cages’ appearance, e.g. condition of their nest, we found a clear difference in the animals nest building activities. The un-treated mice destroyed their nests after the operation and needed 3 days to rebuild a proper nest and structure their cage area (Fig.1). The mice that received pain killers damaged their nests negligibly during the first hours after the operation and came out with well-established nests within the first day. This disturbance of nest building behavior was not found in animals which were subjected to anesthesia only. Accordingly it was concluded that the altered behavior can be correlated to the degree of post-operative pain and could be used as a setting that provoke mice to exhibit pain-related behavior. The final goal is to establish tests that are feasible under routine laboratory conditions and could be integrated in specific pain scales and scoring systems used by investigators.
Physiological approach: Heart beat rate and variability
In order to detect a sensitive physiological marker for the immediate recognition of pain, we established a telemetry system, in which we used vasectomy as model for mild to moderate post-operative pain. Telemetric transmitters were implanted in adult, male NMRI mice. After 6 weeks of recovery, they were subjected to vasectomy (Fig. 2) followed by the application of two different analgesic regimens (NSAIDs). One group received no analgesics. Post-operatively, none of the animals exhibited overt clinical signs of pain. Locomotor activity remained stable in all groups, which confirms the absence of intolerable pain. Body core temperature showed negligible increase (below 0.5°C), suggesting that post-surgical inflammation was of no influence. Nevertheless after vasectomy in the group with no NSAIDs, the heart beat rate and variability measures increased, particularly during the daylight phase, and food consumption and body weight curves decreased by 20% respectively 2-3%.These changes were not seen in the two groups that received high doses of NSAIDs for pain therapy  or in control groups subjected to anesthesia and analgesics/vehicle administration only. We concluded that heart beat rate is a pain-related symptom which allows to detect in real-time low to middle-grade post-surgical pain and accordingly the effectiveness of the analgesic regimen in mice.
Molecular approach in the future: Pain-associated gene regulation?
Using the telemetric measurements, we were able to define time points at which the mice experienced pain. Accordingly the expression of genes involved in pain processing should be altered when compared to tissues from animals without pain. We designed a low-density microarray with over 100 selected genes involved in pain and distress. Preliminary results from hybridizations with material obtained after vasectomy indicate changes which correlate with pain management.
Benefit for mice, men and science
The combined behavioural, physiological and molecular approach in mice will provide tools for the experimenter to recognize moderate and mild clinical pain. Ideally he/she will be enabled to judge the degree of pain more precisely and to apply and control the effects of clearly targeted pain relief protocols. As a result, less side effects will be expected from pain-related physiological changes in the animal (e.g. increased stress hormone levels, complicated wound healing) and analgesics-induced adverse effects (e.g. bleeding, behavioural aberrations) will be limited to the level necessary for effective pain relief.