SMAFIRA: a literature-based web tool to assist researchers with retrieval of 3R-relevant information.

The search for 3R-relevant information is a prerequisite for any planned experimental approach considering animal use. Such a literature search includes all methods to replace, reduce and refine (3Rs) animal testing with the aim of improving animal welfare, and requires an intensive screening of literature databases reflecting the current state of knowledge in experimental biomedicine. We developed SMAFIRA, a freely available online tool to facilitate the screening of PubMed/MEDLINE for possible alternatives to animal testing. SMAFIRA employs state-of-the-art language models from the field of deep learning, and provides relevant literature citations in a ranked order, classified according to the experimental model used. By using this classification, the search for alternative methods in the biomedical literature will become much more efficient. The tool is available at https://smafira.bf3r.de.

Automatic classification of experimental models in biomedical literature to support searching for alternative methods to animal experiments.

Current animal protection laws require replacement of animal experiments with alternative methods, whenever such methods are suitable to reach the intended scientific objective. However, searching for alternative methods in the scientific literature is a time-consuming task that requires careful screening of an enormously large number of experimental biomedical publications. The identification of potentially relevant methods, e.g. organ or cell culture models, or computer simulations, can be supported with text mining tools specifically built for this purpose. Such tools are trained (or fine tuned) on relevant data sets labeled by human experts. We developed the GoldHamster corpus, composed of 1,600 PubMed (Medline) articles (titles and abstracts), in which we manually identified the used experimental model according to a set of eight labels, namely: "in vivo", "organs", "primary cells", "immortal cell lines", "invertebrates", "humans", "in silico" and "other" (models). We recruited 13 annotators with expertise in the biomedical domain and assigned each article to two individuals. Four additional rounds of annotation aimed at improving the quality of the annotations with disagreements in the first round. Furthermore, we conducted various machine learning experiments based on supervised learning to evaluate the corpus for our classification task. We obtained more than 7,000 document-level annotations for the above labels. After the first round of annotation, the inter-annotator agreement (kappa coefficient) varied among labels, and ranged from 0.42 (for "others") to 0.82 (for "invertebrates"), with an overall score of 0.62. All disagreements were resolved in the subsequent rounds of annotation. The best-performing machine learning experiment used the PubMedBERT pre-trained model with fine-tuning to our corpus, which gained an overall f-score of 0.83. We obtained a corpus with high agreement for all labels, and our evaluation demonstrated that our corpus is suitable for training reliable predictive models for automatic classification of biomedical literature according to the used experimental models. Our SMAFIRA - "Smart feature-based interactive" - search tool ( https://smafira.bf3r.de ) will employ this classifier for supporting the retrieval of alternative methods to animal experiments. The corpus is available for download ( https://doi.org/10.5281/zenodo.7152295 ), as well as the source code ( https://github.com/mariananeves/goldhamster ) and the model ( https://huggingface.co/SMAFIRA/goldhamster ).

The fate of surplus laboratory animals: Minimizing the production of surplus animals has the greatest potential to reduce the number of laboratory animals: Minimizing the production of surplus animals has greatest potential to reduce the number of laboratory animals.

To meet regulatory requirements and the political pressure to minimize the number of animals used in research, it is critical to reduce the production of surplus animals.

A guide to open science practices for animal research.

Translational biomedical research relies on animal experiments and provides the underlying proof of practice for clinical trials, which places an increased duty of care on translational researchers to derive the maximum possible output from every experiment performed. The implementation of open science practices has the potential to initiate a change in research culture that could improve the transparency and quality of translational research in general, as well as increasing the audience and scientific reach of published research. However, open science has become a buzzword in the scientific community that can often miss mark when it comes to practical implementation. In this Essay, we provide a guide to open science practices that can be applied throughout the research process, from study design, through data collection and analysis, to publication and dissemination, to help scientists improve the transparency and quality of their work. As open science practices continue to evolve, we also provide an online toolbox of resources that we will update continually.

Declaration of common standards for the preregistration of animal research-speeding up the scientific progress.

Preregistration of studies is a recognized tool in clinical research to improve the quality and reporting of all gained results. In preclinical research, preregistration could boost the translation of published results into clinical breakthroughs. When studies rely on animal testing or form the basis of clinical trials, maximizing the validity and reliability of research outcomes becomes in addition an ethical obligation. Nevertheless, the implementation of preregistration in animal research is still slow. However, research institutions, funders, and publishers start valuing preregistration, and thereby level the way for its broader acceptance in the future. A total of 3 public registries, the OSF registry, preclinicaltrials.eu, and animalstudyregistry.org already encourage the preregistration of research involving animals. Here, they jointly declare common standards to make preregistration a valuable tool for better science. Registries should meet the following criteria: public accessibility, transparency in their financial sources, tracking of changes, and warranty and sustainability of data. Furthermore, registration templates should cover a minimum set of mandatory information and studies have to be uniquely identifiable. Finally, preregistered studies should be linked to any published outcome. To ensure that preregistration becomes a powerful instrument, publishers, funders, and institutions should refer to registries that fulfill these minimum standards.

Moderately aged OFA rats as a novel model for mild age-related alterations in learning and memory.

Aged rodents have been used as preclinical models of age-associated cognitive decline. Most of those models displayed substantial impairments in learning and memory. The initial, more subtle changes that precede more severe losses in cognitive abilities have not been well characterized. Here, we established a model detecting initial subtle cognitive changes by comparing the performance of moderately aged Oncins France Strain A Sprague Dawley rats with young rats in the Morris water maze (MWM) and the Open Field (OF) test. Both age groups improved their performance during the training period at a similar rate; however, the older rats performed worse in several parameters measured in the MWM. Our results suggest that already at the age of 18-20 months rats show changes in their approach to solve the spatial memory task while their ability to learn is not yet diminished. The disparate spatial information processing of the moderately aged rats provides a novel animal model for early age-related cognitive alterations that could be useful to test the effect of early intervention strategies. Moreover, our results suggest that the sensitivity of cognitive tests in the elderly could be substantially enhanced if they assess both the improvement after several trials, and the strategy used to solve a certain task.

How many animals are used for SARS-CoV-2 research?: An overview on animal experimentation in pre-clinical and basic research.

Non-technical summaries of research projects allow tracking the numbers and purpose of animal experiments related to SARS-CoV2 research so as to provide greater transparency on animal use.

Analgesic treatment with buprenorphine should be adapted to the mouse strain.

Buprenorphine is a commonly used opioid to treat moderate to severe pain in mice. Although strain differences regarding basal pain sensitivity and the analgesic effect of other opioids have been described for mice, the data for buprenorphine is incomplete. Hence, we investigated basal pain sensitivity and the analgesic effect of buprenorphine (0.42, 4.0 mg·kg-1) in male C57BL/6J, Balb/cJ and 129S1/SvImJ mice using the incremental hot plate. Additionally, we verified single nucleotide polymorphisms in Cytochrome P450 3a (Cyp3a) genes, which encode for enzymes that are relevant for buprenorphine metabolism, and analyzed serum and brain concentrations of buprenorphine and its metabolites. Finally, in a pilot survey we determined µ-opioid receptor (MOR) protein expression in whole brain lysates. Basal pain sensitivity differed significantly between the mouse strains (Balb/cJ > C57BL/6J > 129S1/SvImJ). Additionally, buprenorphine showed a dose- and strain-dependent effect: at a higher dose it led to increased antinociception in C57BL/6J and Balb/cJ mice, whereas in 129S1/SvImJ mice this effect was diminished. Serum and brain concentrations of buprenorphine and its metabolites dose-dependently increased and differed slightly between the strains at the high dose. However, these slight strain differences did not correlate with pain behavior. Furthermore, serum buprenorphine metabolic ratio and distribution of buprenorphine and its metabolites between brain and blood showed no dose- and only some strain-dependent differences independent from nociceptive behavior. Western blot analysis revealed no strain difference in the basal MOR protein expression in brain lysates. Our results indicate that buprenorphine dosing should be determined in a pilot study for the respective mouse strain to optimize pain treatment and to avoid unwanted side effects. The present pharmacokinetic data and the coarse determination of MOR expression do not explain the strain differences in the analgesic effect of buprenorphine. However, follow-up studies focusing on more specific pharmacodynamic factors could further elucidate the reasons.

Rethinking the incentive system in science: animal study registries: Preregistering experiments using animals could greatly improve transparency and reliability of biomedical studies and improve animal welfare.

The Animal Study Registry offers scientists a range of benefits by preregistering their studies. Wider adoption could address the reproducibility problem in biomedical research and enhance animal welfare.

Refining animal research: The Animal Study Registry.

The Animal Study Registry (ASR; www.animalstudyregistry.org) was launched in January 2019 for preregistration of animal studies in order to increase transparency and reproducibility of bioscience research and to promote animal welfare. The registry is free of charge and is designed for exploratory and confirmatory studies within applied science as well as basic and preclinical research. The registration form helps scientists plan their study thoroughly by asking detailed questions concerning study design, methods, and statistics. With registration, the study automatically receives a digital object identifier (DOI) that marks it as intellectual property of the researcher. To accommodate the researchers concerns about theft of ideas, users can restrict the visibility of their registered studies for up to 5 years. The full content of the study becomes publicly accessible at the end of the embargo period. Because the platform is embedded in the infrastructure of the German Federal Government, continuity and data security are provided. By registering a study in the ASR, researchers can show their commitment to transparency and data quality to reviewers and editors, to third-party donors, and to the general public.

A mouse model for intellectual disability caused by mutations in the X-linked 2'­O­methyltransferase Ftsj1 gene.

Mutations in the X chromosomal tRNA 2'­O­methyltransferase FTSJ1 cause intellectual disability (ID). Although the gene is ubiquitously expressed affected individuals present no consistent clinical features beyond ID. In order to study the pathological mechanism involved in the aetiology of FTSJ1 deficiency-related cognitive impairment, we generated and characterized an Ftsj1 deficient mouse line based on the gene trapped stem cell line RRD143. Apart from an impaired learning capacity these mice presented with several statistically significantly altered features related to behaviour, pain sensing, bone and energy metabolism, the immune and the hormone system as well as gene expression. These findings show that Ftsj1 deficiency in mammals is not phenotypically restricted to the brain but affects various organ systems. Re-examination of ID patients with FTSJ1 mutations from two previously reported families showed that several features observed in the mouse model were recapitulated in some of the patients. Though the clinical spectrum related to Ftsj1 deficiency in mouse and man is variable, we suggest that an increased pain threshold may be more common in patients with FTSJ1 deficiency. Our findings demonstrate novel roles for Ftsj1 in maintaining proper cellular and tissue functions in a mammalian organism.

Impact of repeated anesthesia with ketamine and xylazine on the well-being of C57BL/6JRj mice.

Within the scope of the 3Rs of Russel and Burch, the number of laboratory animals can be reduced by repeated use of an animal. This strategy only becomes relevant, if the total amount of pain, distress or harm the individual animal experiences does not exceed the severity of a single manipulation. For example, when using imaging techniques, an animal can be examined several times during a study, but it has to be anesthetized each time imaging is performed. The severity of anesthesia is thought to be mild according to the Directive 2010/63/EU. However, the Directive does not differentiate between single and repeated anesthesia, although repeated anesthesia may have a greater impact on well-being. Hence, we compared the impact of single and repeated anesthesia (six times at an interval of three to four days) by injection of ketamine and xylazine (KX) on the well-being of adult female and male C57BL/6JRj mice. After anesthesia, well-being of mice was assessed according to a protocol for systematic assessment of well-being including nesting, the Mouse Grimace Scale (MGS), a test for trait anxiety, home cage activity, and the rotarod test for motor activity, food intake, and body weight, as well as corticosterone (metabolite) analysis. Repeated anesthesia increased the MGS in mice of both sexes and caused short-term effects on well-being of female mice in the immediate post-anesthetic period, indicated by longer lasting effects on trait anxiety-related behavior. However, corticosterone metabolite concentrations suggested that mice habituated to the stress induced by repeated KX administration. Hence, the mildly negative effects on well-being of repeated KX anesthesia do not seem to accumulate over time using the respective regimen. However, further observations for severity classification are warranted in order to more specifically determine the duration of mild distress and trait anxiety.

Euthanasia of laboratory mice: Are isoflurane and sevoflurane real alternatives to carbon dioxide?

In the European Union (EU) millions of laboratory mice are used and killed for experimental and other scientific purposes each year. Although controversially discussed, the use of carbon dioxide (CO2) is still permitted for killing rodents according to the Directive 2010/63/EU. Within the scope of refinement, our aim was to investigate if isoflurane and sevoflurane are an appropriate alternative killing method to CO2 in mice. Different concentrations of CO2 (filling rates of 20%, 60%, 100%; CO2 20, 60, 100), isoflurane (Iso 2%, 5%) and sevoflurane (Sevo 4.8%, 8%) were compared in two mouse strains (NMRI, C57Bl/6J) using a broad spectrum of behavioral parameters, including the approach-avoidance test, and analyzing blood for stress parameters (glucose, adrenaline, noradrenaline). We focused in our study on the period from the beginning of the gas inlet to loss of consciousness, as during this period animals are able to perceive pain and distress. Our results show that only higher concentrations of CO2 (CO2 60, 100) and isoflurane (5%) induced surgical tolerance within 300 s in both strains, with CO2 100 being the fastest acting inhalant anesthetic. The potency of halogenated ethers depended on the mouse strain, with C57Bl/6J being more susceptible than NMRI mice. Behavioral analysis revealed no specific signs of distress, e. g. stress-induced grooming, and pain, i. e. audible vocalizations, for all inhalant gases. However, adrenaline and noradrenaline plasma concentrations were increased, especially in NMRI mice exposed to CO2 in high concentrations, whereas we did not observe such increase in animals exposed to isoflurane or sevoflurane. Escape latencies in the approach-avoidance test using C57Bl/6J mice did not differ between the three inhalant gases, however, some animals became recumbent during isoflurane and sevoflurane but not during CO2 exposure. The rise in catecholamine concentrations suggests that CO2 exposure might be linked to a higher stress response compared to isoflurane and sevoflurane exposure, although we did not observe a behavioral correlate for that. Follow-up studies investigating other fast-acting stress hormones and central anxiety circuits are needed to confirm our findings.

Liver lobe and strain differences in the activity of murine cytochrome P450 enzymes.

The cytochrome P450 (CYP) enzyme superfamily is the most important enzyme system for phase I biotransformation. For toxico- and pharmacokinetic studies, use of liver-based microsomes, including those of mice, is state-of-the-art to study CYP-dependent metabolism. However, reproducibility and interpretation of these data is still very variable, partly because current testing guidelines do not cover details on organ sampling and potential liver lobe differences. Hence, we analyzed CYP activity, CYP protein content, mRNA expression of CYP1A, CYP2C, CYP2D and CYP3A isozymes, and cytochrome P450 reductase (CPR) activity of the four different liver lobes and processus papillaris of male C57BL/6J mice in comparison to whole liver. Additionally, we used whole liver of Balb/cJ and 129S1/SvImJ for strain comparison. Our data show significant differences in CYP activity, being most prominent in lobus sinister lateralis and lobus medialis, and lowest in processus papillaris. These differences were not caused by varying Cyp gene expression or CYP protein level, but partly correspond with lobe specific CPR activities. We also observed significant strain differences in CYP mRNA expression and activities with overall high activities in 129S1/SvImJ mice and low activities in Balb/cJ mice compared to C57BL/6J mice. In addition, strain specific differences in CYP2C and CYP2D activity seem to be reflected in strain dependent differences in CPR activity. In summary, our results indicate that in mice CYP activity and gene expression are strain dependent and may vary highly between liver lobes. To ensure reproducibility and comparability of different probes and studies, this should be taken into account when liver samples are collected for the analysis of CYP-dependent metabolism.

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.

Rethinking 3R strategies: Digging deeper into AnimalTestInfo promotes transparency in in vivo biomedical research.

In the European Union (EU), animal welfare is seen as a matter of great importance. However, with respect to animal experimentation, European citizens feel quite uninformed. The European Directive 2010/63/EU for the protection of laboratory animals aims for greater transparency and requires that a comprehensible, nontechnical summary (NTS) of each authorised research project involving animals is published by the respective Member State. However, the NTSs remain sleeping beauties if their contents are not easily and systematically accessible. The German web-based NTS database AnimalTestInfo is a unique channel for scientists to communicate their work, and provides the opportunity for large-scale analyses of planned animal studies to inform researchers and the public. For an in-depth meta-analysis, we classified the duly completed NTSs submitted to AnimalTestInfo in 2014 and 2015 according to the International Classification of Diseases and Related Health Problems (ICD) system. Indexing the NTSs with ICD codes provided a fine-grained overview of the prospective uses of experimental animals. Using this approach, transparency, especially for highly controversial animal research involving, for example, nonhuman primates, is fostered, as it enables pinpointing the envisaged beneficiary down to the level of the addressed disease. Moreover, research areas with many planned projects involving animals can be specified in detail. The development of 3R (replacement, reduction, and refinement) measures in these research areas may be most efficient, as a large number of experimental animals would benefit from it. Indexing NTSs with ICD codes can support governments and funding agencies in advancing target-oriented funding of 3R research. Data drawn from NTSs can provide a basis for the development, validation, and implementation of directed 3R strategies as well as guidance for rethinking the role of animal research models.

Enhanced Stress Response in 5-HT1AR Overexpressing Mice: Altered HPA Function and Hippocampal Long-Term Potentiation.

Postsynaptic 5-HT1A receptors (5-HT1AR) play an important role in anxiety and stress, although their contribution is still controversial. Previous studies report that mice overexpressing postsynaptic 5-HT1ARs show no changes in basal anxiety, though the influence of stress conditions has not been addressed yet. In this study, we used this animal model to evaluate the role of 5-HT1ARs in anxiety response after pre-exposure to an acute stressor. Under basal conditions, 5-HT1AR overexpressing animals presented high corticosterone levels and a lower mineralocorticoid/glucocorticoid receptor ratio. After pre-exposure to a single stressor, they showed a high anxiety-like response, associated with a blunted increase in corticosterone levels and higher c-Fos activation in the prefrontal cortex. Moreover, these mice also presented a lack of downregulation of hippocampal long-term potentiation after stress exposure. Therefore, higher postsynaptic 5-HT1AR activation might predispose to a high anxious phenotype and an impaired stress coping behavior.

Severity classification of repeated isoflurane anesthesia in C57BL/6JRj mice-Assessing the degree of distress.

According to the EU Directive 2010/63, the severity of a procedure has to be classified as mild, moderate or severe. General anesthesia is thought to be mild, but the Directive does not differentiate between single and repeated anesthesia. Therefore, we investigated the impact of repeated administration of isoflurane, the most commonly used inhalation anesthetic, on the well-being of adult C57BL/6JRj mice, in comparison to single administrations and to untreated animals, when applied six times for 45 min at an interval of 3-4 days. For the animals anesthetized, excitations, phases of anesthesia, and vital parameters were monitored. Well-being after anesthesia was assessed using a behavioral test battery including luxury behavior like burrowing and nest building behavior, the Mouse Grimace Scale (MGS), the free exploratory paradigm for anxiety-related behavior, home cage activity and the rotarod test for activity, as well as food intake and body weight. Additionally, hair corticosterone and fecal corticosterone metabolites were measured. Our results show that nest building behavior, home cage activity, body weight, and corticosterone concentrations were not influenced by anesthesia, whereas changes in burrowing behavior, the MGS, food intake, and the free exploratory behavior indicated that the well-being of the mice was more affected by repeated than single isoflurane anesthesia. This effect depended on the sex of the animals, with female mice being more susceptible than male mice. However, repeated isoflurane anesthesia caused only short-term mild distress and impairment of well-being, mainly in the immediate postanesthetic period. Well-being stabilized at 8 days after the last anesthesia, at the latest. Therefore, we conclude that when using our anesthesia protocol, the severity of both single and repeated isoflurane anesthesia in C57BL/6JRj mice can be classified as mild. However, within the mild severity category, repeated isoflurane anesthesia ranks higher than single isoflurane anesthesia. Additionally, our results imply that male and female mice can differently perceive the severity of a procedure.