Evaluating rearing behaviour as a model-specific pain indicator in mouse osteotomy models.

To assess pain in mouse models of bone fractures, currently applied assessment batteries use combinations of clinical signs with spontaneous behaviours and model-specific behaviours, including walking and weight-bearing behaviour. Rearing behaviour - an upright position on the hindlimbs - has a motivational and an ambulatory component. Thus, rearing behaviour might have the potential to be an indicator for model-specific pain in mouse fracture models. To date, the assessment of rearing behaviour in bone fracture models using mice is only scarcely described. In this study, we aimed to determine whether the duration of rearing behaviour is affected by osteotomy of the femur in male and female C57BL/6N mice with external fixation (rigid vs. flexible) and could be an additional sign for model-specific pain, such as the presence of limping. Rearing duration was significantly decreased after osteotomy in male and female mice at 24 h, 48 h and 72 h, but was not affected by anaesthesia/analgesia alone. In male mice, the relative rearing duration increased over 72 h (both fixations) and at 10 days in the rigid fixation group but remained significantly lower in the flexible fixation group. In contrast, in female mice, no increase in rearing duration was observed within 72 h and at 10 days post-osteotomy, independent of the fixation. We did not identify any association between relative rearing time and presence or absence of limping. In summary, our results do not provide sufficient evidence that altered rearing behaviour might be an indicative sign for pain in this model.

Anesthesia and analgesia for experimental craniotomy in mice and rats: a systematic scoping review comparing the years 2009 and 2019.

Experimental craniotomies are a common surgical procedure in neuroscience. Because inadequate analgesia appears to be a problem in animal-based research, we conducted this review and collected information on management of craniotomy-associated pain in laboratory mice and rats. A comprehensive search and screening resulted in the identification of 2235 studies, published in 2009 and 2019, describing craniotomy in mice and/or rats. While key features were extracted from all studies, detailed information was extracted from a random subset of 100 studies/year. Reporting of perioperative analgesia increased from 2009 to 2019. However, the majority of studies from both years did not report pharmacologic pain management. Moreover, reporting of multimodal treatments remained at a low level, and monotherapeutic approaches were more common. Among drug groups, reporting of pre- and postoperative administration of non-steroidal anti-inflammatory drugs, opioids, and local anesthetics in 2019 exceeded that of 2009. In summary, these results suggest that inadequate analgesia and oligoanalgesia are persistent issues associated with experimental intracranial surgery. This underscores the need for intensified training of those working with laboratory rodents subjected to craniotomies. Systematic review registration: https://osf.io/7d4qe.

A buprenorphine depot formulation provides effective sustained post-surgical analgesia for 72 h in mouse femoral fracture models.

Adequate pain management is essential for ethical and scientific reasons in animal experiments and should completely cover the period of expected pain without the need for frequent re-application. However, current depot formulations of Buprenorphine are only available in the USA and have limited duration of action. Recently, a new microparticulate Buprenorphine formulation (BUP-Depot) for sustained release has been developed as a potential future alternative to standard formulations available in Europe. Pharmacokinetics indicate a possible effectiveness for about 72 h. Here, we investigated whether the administration of the BUP-Depot ensures continuous and sufficient analgesia in two mouse fracture models (femoral osteotomy) and could, therefore, serve as a potent alternative to the application of Tramadol via the drinking water. Both protocols were examined for analgesic effectiveness, side effects on experimental readout, and effects on fracture healing outcomes in male and female C57BL/6N mice. The BUP-Depot provided effective analgesia for 72 h, comparable to the effectiveness of Tramadol in the drinking water. Fracture healing outcome was not different between analgesic regimes. The availability of a Buprenorphine depot formulation for rodents in Europe would be a beneficial addition for extended pain relief in mice, thereby increasing animal welfare.

Evaluation of the effect of tramadol, paracetamol and metamizole on the severity of experimental colitis.

Application of dextran sodium sulfate (DSS) is often used to induce experimental colitis. Current state of the art is to refrain from the use of analgesics due to their possible interaction with the model. However, the use of analgesics would be beneficial to reduce the overall constraint imposed on the animals. Here, we analyzed the effect of the analgesics Dafalgan (paracetamol), Tramal (tramadol) and Novalgin (metamizole) on DSS-induced colitis. To study the effect of those analgesics in colitis mouse models, acute and chronic colitis was induced in female C57BL6 mice by DSS administration in the drinking water. Analgesics were added to the drinking water on days four to seven (acute colitis) or on days six to nine of each DSS cycle (chronic colitis). Tramadol and paracetamol had minor effects on colitis severity. Tramadol reduced water uptake and activity levels slightly, while mice receiving paracetamol presented with a better overall appearance. Metamizole, however, significantly reduced water uptake, resulting in pronounced weight loss. In conclusion, our experiments show that tramadol and paracetamol are viable options for the use in DSS-induced colitis models. However, paracetamol seems to be slightly more favorable since it promoted the overall wellbeing of the animals upon DSS administration without interfering with typical readouts of colitis severity.

A systematic review on reporting of refinement measures in mouse ECG telemetry implantation surgery.

Telemetric monitoring is used in many scientific fields, such as cardiovascular research, neurology, endocrinology, as well as animal welfare research. Nowadays, implanted electrocardiogram (ECG) radiotelemetry units are the gold standard for monitoring ECG traces, heart rate and heart rate variability in freely moving mice. Telemetry technology can be a valuable tool when studies utilize it adequately, while prioritizing animal welfare. Recently, concerns have been raised in many research fields, including animal research, regarding the reproducibility of research findings, with insufficient reporting being one of the underlying causes.A systematic review was performed by making use of three literature databases, in order to include all publications until 31.12.2019, where the surgical placing of ECG recording telemetry devices in adult mice was involved. Data extracted from the publications included selected items recommended by the ARRIVE guidelines. We focused on aspects related to the refinement of the surgery and experimental conditions that aim to improve animal welfare. In general, the quality of reporting was low in the analyzed 234 publications. Based on our analyses, we assume there has been no improvement in this field's reporting quality since 2010 when the ARRIVE guidelines on reporting were introduced. Additionally, even though expert recommendations on telemetry surgery refinement have been available since many years now, no increase in uptake (or reporting) of these measures prior (e.g., acclimatization), during (e.g., asepsis) or after (e.g., social housing) the surgery could be observed.

RELSA-A multidimensional procedure for the comparative assessment of well-being and the quantitative determination of severity in experimental procedures.

Good science in translational research requires good animal welfare according to the principles of 3Rs. In many countries, determining animal welfare is a mandatory legal requirement, implying a categorization of animal suffering, traditionally dominated by subjective scorings. However, how such methods can be objectified and refined to compare impairments between animals, subgroups, and animal models remained unclear. Therefore, we developed the RELative Severity Assessment (RELSA) procedure to establish an evidence-based method based on quantitative outcome measures such as body weight, burrowing behavior, heart rate, heart rate variability, temperature, and activity to obtain a relative metric for severity comparisons. The RELSA procedure provided the necessary framework to get severity gradings in TM-implanted mice, yielding four distinct RELSA thresholds L1<0.27, L2<0.59, L3<0.79, and L4<3.45. We show further that severity patterns in the contributing variables are time and model-specific and use this information to obtain contextualized between animal-model and subgroup comparisons with the severity of sepsis > surgery > restraint stress > colitis. The bootstrapped 95% confidence intervals reliably show that RELSA estimates are conditionally invariant against missing information but precise in ranking the quantitative severity information to the moderate context of the transmitter-implantation model. In conclusion, we propose the RELSA as a validated tool for an objective, computational approach to comparative and quantitative severity assessment and grading. The RELSA procedure will fundamentally improve animal welfare, data quality, and reproducibility. It is also the first step toward translational risk assessment in biomedical research.

Grimace scale, burrowing, and nest building for the assessment of post-surgical pain in mice and rats-A systematic review.

Several studies suggested an informative value of behavioral and grimace scale parameters for the detection of pain. However, the robustness and reliability of the parameters as well as the current extent of implementation are still largely unknown. In this study, we aimed to systematically analyze the current evidence-base of grimace scale, burrowing, and nest building for the assessment of post-surgical pain in mice and rats. The following platforms were searched for relevant articles: PubMed, Embase via Ovid, and Web of Science. Only full peer-reviewed studies that describe the grimace scale, burrowing, and/or nest building as pain parameters in the post-surgical phase in mice and/or rats were included. Information about the study design, animal characteristics, intervention characteristics, and outcome measures was extracted from identified publications. In total, 74 papers were included in this review. The majority of studies have been conducted in young adult C57BL/6J mice and Sprague Dawley and Wistar rats. While there is an apparent lack of information about young animals, some studies that analyzed the grimace scale in aged rats were identified. The majority of studies focused on laparotomy-associated pain. Only limited information is available about other types of surgical interventions. While an impact of surgery and an influence of analgesia were rather consistently reported in studies focusing on grimace scales, the number of studies that assessed respective effects was rather low for nest building and burrowing. Moreover, controversial findings were evident for the impact of analgesics on post-surgical nest building activity. Regarding analgesia, a monotherapeutic approach was identified in the vast majority of studies with non-steroidal anti-inflammatory (NSAID) drugs and opioids being most commonly used. In conclusion, most evidence exists for grimace scales, which were more frequently used to assess post-surgical pain in rodents than the other behavioral parameters. However, our findings also point to relevant knowledge gaps concerning the post-surgical application in different strains, age levels, and following different surgical procedures. Future efforts are also necessary to directly compare the sensitivity and robustness of different readout parameters applied for the assessment of nest building and burrowing activities.

The "WWHow" Concept for Prospective Categorization of Post-operative Severity Assessment in Mice and Rats.

The prospective severity assessment in animal experiments in the categories' non-recovery, mild, moderate, and severe is part of each approval process and serves to estimate the harm/benefit. Harms are essential for evaluating ethical justifiability, and on the other hand, they may represent confounders and effect modifiers within an experiment. Catalogs and guidelines provide a way to assess the experimental severity prospectively but are limited in adaptation due to their nature of representing particular examples without clear explanations of the assessment strategies. To provide more flexibility for current and future practices, we developed the modular Where-What-How (WWHow) concept, which applies findings from pre-clinical studies using surgical-induced pain models in mice and rats to provide a prospective severity assessment. The WWHow concept integrates intra-operative characteristics for predicting the maximum expected severity of surgical procedures. The assessed severity categorization is mainly congruent with examples in established catalogs; however, because the WWHow concept is based on anatomical location, detailed analysis of the tissue trauma and other intra-operative characteristics, it enables refinement actions, provides the basis for a fact-based dialogue with authority officials and other stakeholders, and helps to identify confounder factors of study findings.

A systematic review on the reporting quality in mouse telemetry implantation surgery using electrocardiogram recording devices.

Telemetric monitoring is used in many scientific fields, such as cardiovascular research, neurology, endocrinology, animal welfare research and many more. Nowadays, implanted electrocardiogram (ECG) radiotelemetry units are the gold standard for monitoring ECG traces, heart rate and heart rate variability in freely moving mice. This technology can be a valuable tool when studies utilise it adequately, while also prioritizing animal welfare. Recently, concerns about the reproducibility of research findings have been raised in many scientific fields with insufficient reporting being one of the underlying causes. A systematic review was performed in three literature databases to include all published studies until 31.12.2019 using surgery that involves the placing of ECG recording telemetry devices in adult mice. Data extracted from the publications included selected items recommended by the ARRIVE guidelines and SYRCLE`s tool for assessing risk of bias. We focused on aspects related to quality of reporting, risk of bias reduction measures and ECG measurements characteristics. In general, the quality of reporting was low to moderate in the 234 analyzed publications regarding the animal, husbandry, statistics, and risk of bias related items, but good for more specific telemetry study characteristics. Based on our analyses we assume that there is no or only slight improvement in the reporting quality since 2010, when the ARRIVE guidelines were published.

Analysis of Pain and Analgesia Protocols in Acute Cerulein-Induced Pancreatitis in Male C57BL/6 Mice.

Pancreatitis is known to be painful in humans and companion animals. However, the extent of pain in experimental mouse models of acute pancreatitis is unknown. Consequently, the severity classification of acute pancreatitis in mice is controversially discussed and standardized pain management is missing. In this study, we investigated acute Cerulein-induced pancreatitis with pain-specific and well-being orientated parameters to detect its impact on mice. Male C57BL/6J male mice were injected with Cerulein; animals that received saline injections served as control group. The animals were observed for weight change and water intake. To assess pain, behaviors like stretch-and-press and reduced rearing, the Mouse Grimace Scale, and von Frey hypersensitivity were assessed. Fecal corticosterone metabolites and burrowing behavior were assessed to detect changes in the animal's well-being. Pancreatitis severity was evaluated with amylase and lipase in the blood and pancreas histology. To investigate whether different analgesics can alleviate signs of pain, and if they influence pancreas inflammation, animals received Buprenorphine, Paracetamol in combination with Tramadol, or Metamizole in the drinking water. The calculated intake of these analgesics via drinking reached values stated to be efficient for pain alleviation. While pancreatitis did not seem to be painful, we detected acute pain from Cerulein injections that could not be alleviated by analgesics. The number of inflammatory cells in the pancreas did not differ with the analgesic administered. In conclusion: (1) Cerulein injections appear to be acutely painful but pain could not be alleviated by the tested analgesics, (2) acute pancreatitis induced by our protocol did not induce obvious signs of pain, (3) analgesic substances had no detectable influence on inflammation. Nevertheless, protocols inducing more severe or even chronic pancreatitis might evoke more pain and analgesic treatment might become imperative. Considering our results, we recommend the use of Buprenorphine via drinking water in these protocols. Further studies to search for efficient analgesics that can alleviate the acute pain induced by Cerulein injections are needed.

Systematic review on the reporting accuracy of experimental details in publications using mouse femoral fracture models.

The outcomes of animal experiments can be influenced by a variety of factors. Thus, precise reporting is necessary to provide reliable and reproducible data. Initiatives such as the ARRIVE guidelines have been enrolled during the last decade to provide a road map for sufficient reporting. To understand the sophisticated process of bone regeneration and to develop new therapeutic strategies, small rodents, especially mice, are frequently used in bone healing research. Since many factors might influence the results from those studies, we performed a systematic literature search from 2010 to 2019 to identify studies involving mouse femoral fracture models (stable fixation) and evaluated the reporting of general and model-specific experimental details. 254 pre-selected publications were systematically analyzed, showing a high reporting accuracy for the used mouse strain, the age or developmental stage and sex of mice as well as model-specific information on fixation methods and fracturing procedures. However, reporting was more often insufficient in terms of mouse substrains and genetic backgrounds of genetically modified mice, body weight, hygiene monitoring/immune status of the animal, anesthesia, and analgesia. Consistent and reliable reporting of experimental variables in mouse fracture surgeries will improve scientific quality, enhance animal welfare, and foster translation into the clinic.

Lidocaine and bupivacaine as part of multimodal pain management in a C57BL/6J laparotomy mouse model.

While the use of local anesthesia as part of multimodal pain management is common practice in human and veterinarian surgery, these drugs are not applied routinely in rodent surgery. Several recommendations on the use of local anesthesia exist, but systematic studies on their efficacy and side effects are lacking. In the present study, male and female C57BL/6J mice were subjected to a sham vasectomy or a sham embryo transfer, respectively. We tested whether a mixture of subcutaneously injected Lidocaine and Bupivacaine in combination with systemic Paracetamol applied via drinking water results in superior pain relief when compared to treatment with local anesthesia or Paracetamol alone. We applied a combination of methods to assess behavioral, emotional, and physiological changes indicative of pain. Voluntary Paracetamol intake via drinking water reached the target dosage of 200 mg/kg in most animals. Local anesthesia did not lead to obvious side effects such as irregular wound healing or systemic disorders. No relevant sex differences were detected in our study. Sevoflurane anesthesia and surgery affected physiological and behavioral measurements. Surprisingly, Paracetamol treatment alone significantly increased the Mouse Grimace Scale. Taken together, mice treated with a combination of local anesthesia and systemic analgesia did not show fewer signs of post-surgical pain or improved recovery compared to animals treated with either local anesthesia or Paracetamol.

Measuring endogenous corticosterone in laboratory mice - a mapping review, meta-analysis, and open source database.

Evaluating stress in laboratory animals is a key principle in animal welfare. Measuring corticosterone is a common method to assess stress in laboratory mice. There are, however, numerous methods to measure glucocorticoids with differences in sample matrix (e.g., plasma, urine) and quantification techniques (e.g., enzyme immunoassay or radioimmunoassay). Here, the authors present a mapping review and a searchable database, giving a complete overview of all studies mea­suring endogenous corticosterone in mice up to February 2018. For each study, information was recorded regarding mouse strain and sex; corticosterone sample matrix and quantification technique; and whether the study covered the research theme animal welfare, neuroscience, stress, inflammation, or pain (the themes of specific interest in our con­sortium). Using all database entries for the year 2012, an exploratory meta-regression was performed to determine the effect of predictors on basal corticosterone concentrations. Seventy-five studies were included using the predictors sex, time-since-lights-on, sample matrix, quantification technique, age of the mice, and type of control. Sex, time-since-lights-on, and type of control significantly affected basal corticosterone concentrations. The resulting database can be used, inter alia, for preventing unnecessary duplication of experiments, identifying knowledge gaps, and standardizing or heterogenizing methodologies. These results will help plan more efficient and valid experiments in the future and can answer new questions in silico using meta-analyses.