Name: systematic assessment of well-being in mice for procedures using general anesthesia
Uploaded: 2018-03-20T13:00:00
Description: Watch this Scientific Journal Video about Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia at JoVE.com

Thanks to Sabine Jacobs for assisting with the sample collection, Edith Klobetz-Rassam for analysis of FCM, PD Dr. med. vet. habil. Roswitha Merle for assisting with statistical analysis, and Wiebke Gentner for proofreading the manuscript. The study is part of the Berlin-Brandenburg research platform BB3R (www.bb3r.de) and was funded by the German Federal Ministry of Education and Research (grant number: 031A262A) (www.bmbf.de/en/index.html).&#160;

The study was performed according to the guidelines laid down by the German Animal Welfare Act and was approved by the Berlin State Authority (&#34;Landesamt f&#252;r Gesundheit und Soziales&#34;, permit number: G0053/15).
NOTE: The main objective of this protocol was to investigate the effect of repeated anesthesia on glucocorticoid metabolites. A sample size calculation was performed to determine the number of animals to be used: n &#8805; 2 &#215; (s/&#181;1- &#181;2)<...

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The protocol was originally developed to assess well-being of C57BL/6JRj mice that received either a single anesthesia or repeated isoflurane anesthesia. The results confirm that tests of luxury behaviors, as well as other measures (e.g. the free exploratory paradigm, the MGS, burrowing food intake) were sensitive methods for assessing well-being. Repeated isoflurane anesthesia caused short-term effects on trait anxiety-related behavior, the MGS, and burrowing behavior. Moreover, repeated isoflurane anesthesia a...

EU Directive 2010/631 stipulates that the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch2 is to be applied whenever animal experimentation is necessary. The ultimate goal of the EU Directive is to phase out all animal testing, but the Directive acknowledges that, for the time being, some animal experiments are still needed to conduct research that will protect human and animal health. Thus, if an animal experiment cannot be replaced by any alternative method, only the minimum number of laboratory animals is to be used to obtain reliable results. In addition, the amount of pain, suffering, and distress accompanying experimental procedures should be minimized using appropriate refinement measures. EU Directive 2010/63 stipulates that the severity of a procedure must be prospectively classified as non-recovery, mild, moderate, or severe1. As severity classification is decided on a case-by-case basis, it is important to have scientifically sound tools to estimate the severity of a given procedure.
Score sheets as proposed by Morton and Griffith3 are an essential tool in detecting any deviations from normal status, including negative effects on well-being4. Score sheets are used to retrospectively determine pain, suffering, and distress caused by an experiment and focus on visible changes in the physical state of the individual animal (e.g., body weight, fur, gait). Although, Annex VIII of EU Directive 2010/63 provides examples of each severity category, researchers still lack tools to estimate the degree of severity of a given procedure using scientifically based data.
The absence of indicators showing negative well-being is not the only way to determine the state of the animal; the presence of indicators pointing to positive well-being is also important5,6,7,8. For example, animals display luxury behaviors like burrowing and nest building behavior only when all their essential needs are met. If well-being is reduced, luxury behaviors are the first to decline5,7. Protocols to be used in assessing well-being should include indicators pointing to the physical, physiological/biochemical, and psychological states of animals in order to evaluate their well-being in a detailed and comprehensive manner9.
Within the context of refinement, a protocol was developed to meet these requirements and to assess the effects of a procedure involving general anesthesia on well-being of mice10. At the same time, the goal was to minimize any additional stress to enable the easy integration of the protocol into a given experiment. The protocol considers burrowing behavior, home cage behavior such as activity, food intake, and nesting, and trait anxiety-related behavior. In addition, it includes the Mouse Grimace Scale (MGS), and the non-invasive analysis of corticosterone metabolites in feces. The protocol is designed to facilitate the assessment of well-being in a scientific and animal-centered manner and to provide information on well-being that supports the classification of the degree of severity. In addition to score sheets, it can provide useful information for the severity classification of a procedure. As the protocol is easy to carry out and does not require extensive equipment, it can be integrated into an ongoing experiment without influencing the results of a study. It should be noted that the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guideline11 is to be observed in all studies involving animal experiments, with the goal of improving design, analysis, and reporting.

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			<td height="63" style="height:63px;width:216px;">Isofluran</td>
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			<td height="21" style="height:21px;width:216px;">InfraMot - Sensore Units</td>
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systematic assessment of well-being in mice for procedures using general anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

This protocol was originally developed to assess well-being of C57BL/6JRj mice following a single experience of isoflurane anesthesia (one 45-min anesthesia session, n = 13 females) or repeated isoflurane anesthesia (six 45-min anesthesia sessions with 3 - 4 days between the anesthesia sessions, n = 13 females) compared with the well-being of control mice (n = 6 females)10, which received no anesthesia but were tested according to the same measures. We assessed the...

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Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

The overall goal of this protocol is to assess the well-being of mice after they underwent procedures including general anesthesia and to provide scientifically sound data to support the severity classification according to EU directive 2010/63. This protocol can help answer key questions on animal welfare in the field of laboratory animal science such as the degree of distress caused by procedures involving general anesthesia. The main advantage of this protocol is that well-being is evaluated of each individual mouse in an animal-based scientific manner and this protocol can easily be adapted and integrated into other planned studies.We first had the idea for this protocol when we speculated that if repeated anesthesia cause more stress in laboratory mice than a single exposure, scientifically based data would be missing. In this step, prepare a glass box with a floor area of approximately 220 millimeters by 290 millimeters and a height of 390 millimeters. Cover the floor with approximately 0.5 centimeters of bedding material.Then scatter a handful of used bedding material from the home cage on top of the new bedding material to reduce distress caused by the new environment. Provide with food and water. Gently transfer the mouse into the observation cage and allow the mouse to habituate to the new environment for at least 30 minutes.For the mouse grimace scale, use a high-definition camera for photography. Continuously take about 30 to 40 photographs for each time point within one to two minutes. For testing burrowing behavior according to Deacon and Jirkof, prepare burrows by placing 140 grams food pellets in a standard opaque plastic water bottle.Next, place the plastic water bottle filled with food pellets parallel to the back wall of the observation cage. After two hours, weigh the food pellets remaining in the burrow. Carry out the test twice two days prior to the procedure as baseline.In this step, prepare a cage with new bedding material for the 24-hour observation period in which mice are housed individually. The 24-hour observation period begins after mouse grimace scale and burrowing behavior. The duration of single housing is kept to minimum.In the mean time, home cage activity, food intake, nest building behavior, and the levels of fecal corticosterone metabolites are measured. Next, scatter a handful of used bedding material without feces from the home cage on top of the new material in order to reduce distress. Provide a standardized square cotton nestlet of a defined weight as environmental enrichment only.After that, mount the infrared sensor on top of the cage for measuring home cage activity. Provide standard food pellets and water ad libitum and place the mouse in the cage. On the following morning, score the nest according to the five-point scale by Deacon approximately two hours after the light turns on.In addition, weigh any untorn nestlet piece that accounts for at least 5%of the initial nestlet weight. Next, place a gridded cage top in the cage at an angle of 45 degrees to the longer side of the cage. Start the timer and monitor or video record the mice for 10 minutes from a distance of approximately 1.5 meters.Note all times when the mouse climbs onto the cage top with all four paws on the cage top or leaves the cage top with one or more paws on the cage floor followed by analysis. Single experience of anesthesia as well as repeated anesthesia increased the mouse grimace scale difference scores compared to the control 30 minutes after exposure. At 150 minutes after the last anesthesia, mouse grimace scale difference scores of all mice returned to control levels.For burrowing behavior, only repeated anesthesia significantly reduced the percentage of weight of food pellets the mice removed from the burrow versus the control. For nest building behavior, there were no significant differences in the nest scores between a single experience of anesthesia, repeated anesthesia, and the control. In the free exploratory paradigm, repeated anesthesia significantly reduced the number of explorations compared to control and the total duration of exploration versus a single anesthesia one day after the last anesthesia.Eight days after the last anesthesia, there were no longer differences between the study groups. While attempting this protocol, it's important to remember to handle mice by tunnel and/or cupping methods. Picking up mice by the tail can induce stress and anxiety which in turn affects well-being and can also influence the results of the protocol.This protocol can be easily adapted to and integrated into planned studies to assess the impact of a procedure on the well-being of mice. Depending on the procedure and study design, the most suitable test of this protocol should be chosen. After watching this video, you will have a good understanding of how to assess well-being of mice underwent the procedure of general anesthesia.The protocol helps to classify the severity of the procedure in an animal-based scientific manner.

Research

JoVE Journal

Behavior

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