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.

Design and in vivo evaluation of a microparticulate depot formulation of buprenorphine for veterinary use.

Buprenorphine is a frequently used analgetic agent in veterinary medicine. A major drawback, however, is the short duration of action requiring several daily administrations. We therefore designed a poly-lactic-co-glycolic acid (PLGA) based microparticulate drug formulation for sustained parenteral drug release. Particles were designed to allow for a fast onset of action and a duration of the analgesic effect of at least two days in laboratory mice. Microparticles were produced using a solvent evaporation technique. Release rate was dependent on polymer type and particle size. Spherical particles used for subsequent animal studies had a mean size of 50 µm and contained 4.5% of buprenorphine. Drug release was characterized by an initial burst release of 30% followed by complete release over seven days. In vivo pharmacokinetic experiments in female C57BL/6 J mice confirmed prolonged exposure in plasma and brain tissue and correlated with the pharmacological effect in the hot plate assay or after minor abdominal surgery. No adverse side effects with respect to food and water intake, body weight, local tolerability, or nesting behavior were observed. Our formulation is an attractive alternative to established immediate release formulations. A use for prolonged pain management in laboratory animals is proposed.

Semi-automated generation of pictures for the Mouse Grimace Scale: A multi-laboratory analysis (Part 2).

The Mouse Grimace Scale (MGS) is an established method for estimating pain in mice during animal studies. Recently, an improved and standardized MGS set-up and an algorithm for automated and blinded output of images for MGS evaluation were introduced. The present study evaluated the application of this standardized set-up and the robustness of the associated algorithm at four facilities in different locations and as part of varied experimental projects. Experiments using the MGS performed at four facilities (F1-F4) were included in the study; 200 pictures per facility (100 pictures each rated as positive and negative by the algorithm) were evaluated by three raters for image quality and reliability of the algorithm. In three of the four facilities, sufficient image quality and consistency were demonstrated. Intraclass correlation coefficient, calculated to demonstrate the correlation among raters at the three facilities (F1-F3), showed excellent correlation. The specificity and sensitivity of the results obtained by different raters and the algorithm were analysed using Fisher's exact test (p < 0.05). The analysis indicated a sensitivity of 77% and a specificity of 64%. The results of our study showed that the algorithm demonstrated robust performance at facilities in different locations in accordance with the strict application of our MGS setup.

Measurement of corticosterone in mice: a protocol for a mapping review.

Severity assessment for experiments conducted with laboratory animals is still based mainly on subjective evaluations; evidence-based methods are scarce. Objective measures, amongst which determination of the concentrations of stress hormones, can be used to aid severity assessment. Short-term increases in glucocorticoid concentrations generally reflect healthy responses to stressors, but prolonged increases may indicate impaired welfare. As mice are the most commonly used laboratory animal species, we performed a systematic mapping review of corticosterone measurements in Mus musculus, to provide a full overview of specimen types (e.g. blood, urine, hair, saliva, and milk) and analysis techniques. In this publication, we share our protocol and search strategy, and our rationale for performing this systematic analysis to advance severity assessment. So far, we have screened 13,520 references, and included 5337 on primary studies with measurements of endogenous corticosterone in M. musculus. Data extraction is currently in progress. When finished, this mapping review will be a valuable resource for scientists interested in corticosterone measurements to aid severity assessment. We plan to present the data in a publication and a searchable database, which will allow for even easier retrieval of the relevant literature. These resources will aid implementation of objective measures into severity assessment.

A safe bet? Inter-laboratory variability in behaviour-based severity assessment.

Evidence-based severity assessment is essential as a basis for ethical evaluation in animal experimentation to ensure animal welfare, legal compliance and scientific quality. To fulfil these tasks scientists, animal care and veterinary personnel need assessment tools that provide species-relevant measurements of the animals' physical and affective state. In a three-centre study inter-laboratory robustness of body weight monitoring, mouse grimace scale (MGS) and burrowing test were evaluated. The parameters were assessed in naïve and tramadol treated female C57BL/6J mice. During tramadol treatment a body weight loss followed by an increase, when treatment was terminated, was observed in all laboratories. Tramadol treatment did not affect the MGS or burrowing performance. Results were qualitatively comparable between the laboratories, but quantitatively significantly different (inter-laboratory analysis). Burrowing behaviour seems to be highly sensitive to inter-laboratory differences in testing protocol. All locations obtained comparable information regarding the qualitative effect of tramadol treatment in C57BL/6J mice, however, datasets differed as a result of differences in test and housing conditions. In conclusion, our study confirms that results of behavioural testing can be affected by many factors and may differ between laboratories. Nevertheless, the evaluated parameters appeared relatively robust even when conditions were not harmonized extensively and present useful tools for severity assessment. However, analgesia-related side effects on parameters have to be considered carefully.

Administration of Tramadol or Buprenorphine via the drinking water for post-operative analgesia in a mouse-osteotomy model.

Adequate analgesia is essential whenever pain might occur in animal experiments. Unfortunately, the selection of suitable analgesics for mice in bone-linked models is limited. Here, we evaluated two analgesics - Tramadol [0.1 mg/ml (Tlow) vs. 1 mg/ml (Thigh)] and Buprenorphine (Bup; 0.009 mg/ml) - after a pre-surgical injection of Buprenorphine, in a mouse-osteotomy model. The aim of this study was to verify the efficacy of these opioids in alleviating pain-related behaviors, to provide evidence for adequate dosages and to examine potential side effects. High concentrations of Tramadol affected water intake, drinking frequency, food intake and body weight negatively in the first 2-3 days post-osteotomy, while home cage activity was comparable between all groups. General wellbeing parameters were strongly influenced by anesthesia and analgesics. Model-specific pain parameters did not indicate more effective pain relief at high concentrations of Tramadol. In addition, ex vivo high-resolution micro computed tomography (µCT) analysis and histology analyzing bone healing outcomes showed no differences between analgesic groups with respect to newly formed mineralized bone, cartilage and vessels. Our results show that high concentrations of Tramadol do not improve pain relief compared to low dosage Tramadol and Buprenorphine, but rather negatively affect animal wellbeing.

Collagen I-based scaffolds negatively impact fracture healing in a mouse-osteotomy-model although used routinely in research and clinical application.

Although several biomaterials for bone regeneration have been developed in the last decades, clinical application of bone morphogenetic protein 2 is clinically only approved when applied on an absorbable bovine collagen I scaffold (ACS) (Helistat; ACS-H). In research, another ACS, namely Lyostypt (ACS-L) is frequently used as a scaffold in bone-linked studies. Nevertheless, until today, the influence of ACS alone on bone healing remains unknown. Unexpectedly, in vitro studies using ASC-H revealed a suppression of osteogenic differentiation and a significant reduction of cell vitality when compared to ASC-L. In mice, we observed a significant delay in bone healing when applying ACS-L in the fracture gap during femoral osteotomy. The results of our study show for the first time a negative influence of both ACS-H and ACS-L on bone formation demonstrating a substantial need for more sophisticated delivery systems for local stimulation of bone healing in both clinical application and research. STATEMENT OF SIGNIFICANCE: Our study provides evidence-based justification to promote the development and approval of more suitable and sophisticated delivery systems in bone healing research. Additionally, we stimulate researchers of the field to consider that the application of those scaffolds as a delivery system for new substances represents a delayed healing approach rather than a normal bone healing which could greatly impact the outcome of those studies and play a pivotal role in the translation to the clinics. Moreover, we provide impulses on underlying mechanism involving the roles of small-leucine rich proteoglycans (SLRP) for further detailed investigations.