Virtual Reality in Biomedical Education in the sense of the 3Rs.

Article 23(2) of EU Directive 2010/63 on the protection of animals used for scientific purposes requires staff involved in the care and use of animals to be adequately educated and trained before carrying out procedures. Therefore, the 3Rs (refinement, reduction, and replacement) and knowledge of alternative methods should be part of the education and training itself. For this purpose, the digital learning concept "Virtual Reality (VR) in Biomedical Education" evolved, which successfully combines VR components with classical learning content. Procedures, such as anesthesia induction, substance application, and blood sampling in rats, as well as aspects of the laboratory environment were recorded in 360° videos. The generated VR teaching/learning modules (VR modules) were used to better prepare participants for hands-on training (refinement) or as a complete replacement for a live demonstration; thus, reducing the number of animals used for hands-on skills training (reduction). The current study evaluated users' experience of the VR modules. Despite little previous VR experience, participants strongly appreciated the VR modules and indicated that they believed VR has the potential to enhance delivery of procedures and demonstrations. Interestingly, participants with previous experience of laboratory animal science were more convinced about VR's potential to support the 3Rs principle, and endorsed its use for further educational purposes. In conclusion, VR appeared to be highly accepted as a learning/teaching method, indicating its great potential to further replace and reduce the use of animals in experimental animal courses.

The Open Field Test as a Tool for Behaviour Analysis in Pigs: Recommendations for Set-Up Standardization - A Systematic Review.

INTRODUCTION: The open field test (OFT) is a common tool to assess anxiety and behavioural changes in rodents. It has been adapted to pigs with no systematic investigation of how environmental changes may alter the performance of pigs. Currently, the number of published studies including the OFT in domestic pig models is increasing without standardization. METHODS: Our review aimed to investigate the open field (OF) set-ups in published studies and the similarities between performance and published parameters. RESULTS: Following the PRISMA guidelines for reviews, we selected 69 studies for inclusion in this systematic review. We determined the specific set-up conditions such as dimensions, duration, and time of day for most of the included studies; we found high variability across studies with respect to these test specifics. DISCUSSION: Our results indicate the inconsistent implementation of the set-up, including dimensions, timing, parameters, and additional combined tests (e.g., new object tests). Based on our findings, we have made recommendations for the performance of the OFT, according to the current literature.

Implementation of the Surgical Apgar Score in Laboratory Animal Science: A Showcase Pilot Study in a Porcine Model and a Review of the Literature.

INTRODUCTION: In an attempt to further improve surgical outcomes, a variety of outcome prediction and risk-assessment tools have been developed for the clinical setting. Risk scores such as the surgical Apgar score (SAS) hold promise to facilitate the objective assessment of perioperative risk related to comorbidities of the patients or the individual characteristics of the surgical procedure itself. Despite the large number of scoring models in clinical surgery, only very few of these models have ever been utilized in the setting of laboratory animal science. The SAS has been validated in various clinical surgical procedures and shown to be strongly associated with postoperative morbidity. In the present study, we aimed to review the clinical evidence supporting the use of the SAS system and performed a showcase pilot trial in a large animal model as the first implementation of a porcine-adapted SAS (pSAS) in an in vivo laboratory animal science setting. METHODS: A literature review was performed in the PubMed and Embase databases. Study characteristics and results using the SAS were reported. For the in vivo study, 21 female German landrace pigs have been used either to study bleeding analogy (n = 9) or to apply pSAS after abdominal surgery in a kidney transplant model (n = 12). The SAS was calculated using 3 criteria: (1) estimated blood loss during surgery; (2) lowest mean arterial blood pressure; and (3) lowest heart rate. RESULTS: The SAS has been verified to be an effective tool in numerous clinical studies of abdominal surgery, regardless of specialization confirming independence on the type of surgical field or the choice of surgery. Thresholds for blood loss assessment were species specifically adjusted to >700 mL = score 0; 700-400 mL = score 1; 400-55 mL score 2; and <55 mL = score 3 resulting in a species-specific pSAS for a more precise classification. CONCLUSION: Our literature review demonstrates the feasibility and excellent performance of the SAS in various clinical settings. Within this pilot study, we could demonstrate the usefulness of the modified SAS (pSAS) in a porcine kidney transplantation model. The SAS has a potential to facilitate early veterinary intervention and drive the perioperative care in large animal models exemplified in a case study using pigs. Further larger studies are warranted to validate our findings.

Does Sex Matter in Liver Surgery? Comparison of Severity Assessments between Female and Male Rats after Partial Hepatectomy: A Pilot Study.

INTRODUCTION: Current animal-based biomedical research, including studies on liver function and disease, is conducted almost exclusively on male animals to mitigate confounding effects of the estrous cycle. However, liver diseases afflict both men and women, so translational research findings should also be applicable to female patients. This pilot study investigated sex differences in objective and subjective severity assessment parameters in rats following 50% partial hepatectomy. MATERIALS AND METHODS: This study was performed using Wistar Han rats, in which measurements of body weight, spontaneous motor activity in the open field (OF) (movement distance, movement velocity, rearing frequency), and fecal corticosterone metabolites were conducted at baseline and at multiple times after partial hepatectomy. Subjective postsurgical severity assessments were conducted using modified score sheets. Blood parameters such as leukocyte count and serum aspartate aminotransferase, as well as estrogens and testosterone were measured from samples obtained during partial hepatectomy and at sacrifice. In addition, the amount of resected liver tissue was measured at partial hepatectomy, and the proliferated liver was weighed at sacrifice. RESULTS: Fecal corticosterone metabolite concentrations differed significantly between males and females at baseline and following hepatectomy. Also, leukocyte counts and estrogen concentrations were significantly different between sexes before partial hepatectomy. Alternatively, there were no sex differences in severity assessments, body weight changes, and behavior in the OF at any measurement time point. Liver weight was significantly different in males and females at the time point of partial hepatectomy and sacrifice. CONCLUSION: The results of this pilot study suggest that males and females respond similarly following partial hepatectomy. Examination of both sexes is very important for translation to humans, where both men and women suffer from liver disease. Furthermore, the use of both sexes in animal-based research would improve the utilization of the animal breeding in terms of the 3 Rs. However, due to some limitations, larger scale investigations including a broader spectrum of pathophysiolological, behavioral, and pharmacokinetic measures are planned.

3R Blackboard: A platform for animal and organ sharing.

Since the embedding of the principles of the 3Rs (Replacement, Reduction and Refinement) in national and international regulations on the use of animals, scientists have been challenged to find ways to reduce the number of animals in their research. Here, we present a digital platform, called '3R Backboard', linked to a laboratory animal management system, which facilitates sharing of surplus biological materials from animals (e.g. tissues, organs and cells) to other research teams. Based on information provided, such as genotype, age and sex, other animal workers were able to indicate their interest in collecting specific tissues and to communicate with the person providing the animals. A short pilot study of this approach conducted in a limited academic environment presented strong evidence of its effectiveness and resulted in a notable reduction of the number of mice used. In addition, the use of 3R Blackboard led to resource saving, knowledge exchange and even establishment of new collaboration.

Assessing the severity of laparotomy and partial hepatectomy in male rats-A multimodal approach.

This study assessed the postoperative severity after three different visceral surgical interventions in rats by using objective parameters pertaining to various disciplines. The objective was to evaluate whether the degree of severity increases with the invasiveness of the intervention and whether this is in accordance with the EU Directive 2010/63. 136 adult male WistarHan rats were assigned to three groups: Sham-laparotomy (Sham) [7 days post-surgical survival time]; 50% partial hepatectomy (PH); 70% PH [PH groups with 1, 3, or 7 days post-surgical survival times]. Post-surgical severity assessment was performed via several multimodal assessment tools: I) model-specific score sheet focusing on body weight, general condition, spontaneous behavior, and the animals' willingness to move as well as on wound healing; II) Open Field tests evaluating the total distance and velocity an animal moved within 10 minutes and its rearing behavior during the test; III) telemetric data analyzing heart rate and blood pressure; and IV) analysis of blood (AST, ALT, and hemogram) and fecal samples (fecal corticosterone metabolites). Significant differences among the experimental groups and models were observed. We demonstrated that the Open Field test can detect significant changes in severity levels. Sham-laparotomy and removal of 50% of the liver mass were associated with comparable severity (mild-moderate); the severity parameters returned to baseline levels within seven days. Removal of 70% of the liver tissue seemed to be associated with a moderate severity grade and entailed a longer recovery period (>7 days) for complete regeneration. We recommend the use of Open Field tests as part of multimodal objective severity assessment.

Re-Sterilisation of Single-Use Telemetric Devices.

Implantable telemetric transponders for contactless measurement of physiological parameters are often used in animal-based research. After explantation, single-use devices cannot be re-implanted because of non-validated functionality and necessary re-sterilisation. This is disadvantageous because the battery life would enable a second implantation cycle in another animal. To save costs and time taken for the manufacturer's refurbishing process, we validated and implemented a re-sterilisation protocol for single-use transponders using hydrogen peroxide gas. The described protocol was established with models, i.e., for large (n = 7) and small (n = 3) animals, of telemetric device from 2 different manufacturers (Data Science International and EMKA). All transponders, prepared according to the protocol, were previously implanted subcutaneously in the flank of pigs or rats for a duration of 21 days. Our investigations demonstrate that disinfection only is not sufficient against bacterial contamination and that sterility can only be achieved by additional gas sterilisation with hydrogen peroxide. Furthermore, re-implantation of the re-sterilised transponders into pigs caused neither undesired tissue reactions along the transponder nor impairment of the measured values when compared to the first implantation and after necropsy in 4 cases. We were able to demonstrate that, using our protocol, re-implantation of reprocessed single-use telemetric devices can be performed without compromising transponder quality.

Best variable identification by means of data-mining and cooperative game theory.

OBJECTIVE: To develop and evaluate methods to assess single and grouped variables impact on measuring intervention severities and support a search for most expressive variables. METHODS: Datasets of cohort studies are analyzed automatically based on algorithms. For this, a metric is developed to compare measured variables in different cohorts in a data-mining process. Variables are measured in all possible combinations to detect possible synergies of certain variable constellations and allow for a ranking of the combinations' expressiveness. Such ranking serves as a basis for a wide range of algorithmic data analysis. In an exemplary application, every group member's impact on the total result is determined based on the principle of the cooperative game theory besides to the total expressiveness of the variable groups. RESULTS: For different types of interventions, the method is applied to experimental data containing multiple recorded medical lab values. The expressiveness of variable combinations to indicate severity is ranked by means of a metric. Within each combination, any variable's contribution to the total effect is determined and accumulated over whole datasets to yield local and global variable importance measures. The computed results have been successfully matched with clinical expectations to prove their plausibility. CONCLUSION: Algorithmic evaluation shows to be a promising approach in automatized quantification of variable expressiveness. It can assess descriptive power of measurements, help to improve future study designs and expose worthwhile research issues.

Severity assessment in mice subjected to carbon tetrachloride.

The Directive 2010/63 EU requires classifying burden and severity in all procedures using laboratory animals. This study evaluated the severity of liver fibrosis induction by intraperitoneal carbon tetrachloride (CCl4) injections in mice. 29 male C57BL/6N mice were treated three times per week for 4 weeks with an intraperitoneal injection (50 µl) of either 0.6 ml/kg body weight CCl4-vehicle solution, germ oil (vehicle-control) or handling only. Severity assessment was performed using serum analysis, behavioral tests (open field test, rotarod, burrowing and nesting behavior), fecal corticosterone metabolite (FCM) measurement, and survival. The most significant group differences were noticed in the second week of treatment when the highest AST (1463 ± 1404 vs. 123.8 ± 93 U/L, p < 0.0001) and nesting values were measured. In addition, respective animals showed lower moving distances (4622 ± 1577 vs. 6157 ± 2060 cm, p < 0.01) and velocity in the Open field, identified as main factors in principal component analysis (PCA). Overall, a 50% survival rate was observed within the treatment group, in which the open field performance was a good tracer parameter for survival. In summary, this study demonstrates the feasibility of assessing severity in mice using behavioral tests and highlight the open field test as a possible threshold parameter for risk assessment of mortality.

Severity assessment using three common behavioral or locomotor tests after laparotomy in rats: a pilot study.

The aim of this pilot study was to evaluate whether behavioral or locomotor tests (Open Field (OF), rotarod (RR), and CatWalk (CW)) can help assess the severity of laparotomy in rats.The new EU Directive (2010/63/EU) mandates severity assessment in experiments involving animals. However, validated and objective methods are needed to relate trial-specific monitoring results to the degree of distress caused to individual animals. Therefore, we focused on non-invasive or minimally invasive, simple, and convenient severity assessment methods in a surgical model.To evaluate surgical severity in this model, we compared moving velocity among three commonly used behavioral test methods (OF, RR, and CW) after midline laparotomy within postoperative 7 days.In this study, 30 adult male Wistar Han rats (n = 10 per test) were trained in their assigned test method and subsequently subjected to surgery. Severity scoring was performed daily using a modified score sheet developed previously. In addition, blood and fecal samples were collected to analyze surgical and postoperative corticosterone metabolite levels. We found significant differences among the experimental groups in terms of the analyzed parameters. In this context, the OF test was found to be the most suitable method for severity assessment after laparotomy in rats.

Defining body-weight reduction as a humane endpoint: a critical appraisal.

In many animal experiments scientists and local authorities define a body-weight reduction of 20% or more as severe suffering and thereby as a potential parameter for humane endpoint decisions. In this study, we evaluated distinct animal experiments in multiple research facilities, and assessed whether 20% body-weight reduction is a valid humane endpoint criterion in rodents. In most experiments (restraint stress, distinct models for epilepsy, pancreatic resection, liver resection, caloric restrictive feeding and a mouse model for Dravet syndrome) the animals lost less than 20% of their original body weight. In a glioma model, a fast deterioration in body weight of less than 20% was observed as a reliable predictor for clinical deterioration. In contrast, after induction of chronic diabetes or acute colitis some animals lost more than 20% of their body weight without exhibiting major signs of distress. In these two animal models an exclusive application of the 20% weight loss criterion for euthanasia might therefore result in an unnecessary loss of animals. However, we also confirmed that this criterion can be a valid parameter for defining the humane endpoint in other animal models, especially when it is combined with additional criteria for evaluating distress. In conclusion, our findings strongly suggest that experiment and model specific considerations are necessary for the rational integration of the parameter 'weight loss' in severity assessment schemes and humane endpoint criteria. A flexible implementation tailored to the experiment or intervention by scientists and authorities is therefore highly recommended.

Remote vitals monitoring in rodents using video recordings.

Laboratory animal research was always crucial for scientific breakthroughs in the fields of medicine and biology. Animal trials offer insights into various disease mechanisms, genetics, drug therapy and the effect of different external factors onto living organisms. However, conducting animal trials is highly controversial. To ensure high ethical standards, a number of directives have been adopted in the European Union, which seek to replace, reduce and refine animal trials. Hence, severity assessment plays an important role in today's laboratory animal research. Currently, severity of trials is assessed by highly rater dependent scoring systems. In this paper, we propose a method for unobtrusive, automated and contactless measurement of respiratory rate (RR) and heart rate (HR). We were able to extract RR and HR with an high agreement between our method and a contact-based reference method. The Root Mean Squared Error (RMSE) averaged 0.32 ± 0.11 breaths/min for RR and 1.28 ± 0.62 beats/min for HR in rats, respectively. In mice, the RMSE averaged 1.42 ± 0.97 breaths/min for RR and 1.36 ± 0.87 beats/min, respectively. In the future, these parameters can be used for new, objective scoring systems, which are not susceptible to inter-rater variability.

Contactless Anesthesia Monitoring in Spontanously Breathing Rodents.

Laboratory animal science plays a crucial role in medical and biological research. In the last decades, stricter regulations were enforced to safeguard laboratory animals. Following the "3Rs" guiding principles, animal trials should be replaced, reduced and refined, whenever possible.A contactless modality capable of assessing the respiratory rate (RR) and additional breath related characteristics can potentially refine anesthetic interventions in rodents by continuously monitoring their anesthetic depth. This can reduce complications and thus the number of needed animals.We were able to extract the instantaneous RR in rodents with a sum squared error (SSE) of 0.26 breaths/min from color video. A correlation of 0.9781 compared to an Electrocardiography (ECG) based reference was achieved. Furthermore, additional temporal and morphological characteristics were extracted, which are sensitive for changes in the anesthetic depth.

Remote Welfare Monitoring of Rodents Using Thermal Imaging.

Animal research has always played a crucial role in various medical and scientific breakthroughs. They offer, inter alia, insights into diseases mechanisms, genetic predisposition to a disease, and drug therapy. However, the use of animals for medical research is a cause of major controversies and debates in modern science. To warrant high bioethical standards, new directives have been being adopted to replace animal research whenever possible, to reduce the number of animals, and to refine the procedures to minimize stress and pain. Here, we present two new approaches, based on thermal imaging (a remote and passive technology), to assess respiratory rate (RR) as well as exploratory behavior and general activity in rodents. In animal research, these parameters are gold standards for welfare assessment. The approaches were validated in a study conducted with both rats and mice. To test the feasibility of our algorithm to estimate RR, thermal videos from anesthetized rodents were acquired. The capability of the second approach to monitor activity was tested with videos of Open Field tests. Regarding RR, a high agreement between thermal imaging and gold standard (electrocardiography-derived RR) was achieved. The mean relative error averaged 0.50 ± 0.15 breaths/min and 4.55 ± 2.94 breaths/min for rats and mice, respectively. The second approach was capable of monitoring and tracking the activity of the rodents very well. This paper demonstrates that thermal imaging is a promising and relevant alternative for monitoring of RR and activity in rodents, thus contributing to the remote assessment of animal welfare.

Characterization of two Austrian porcine reproductive and respiratory syndrome virus (PRRSV) field isolates reveals relationship to East Asian strains.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes major problems for the swine industry worldwide. Due to Austria's central location in Europe, a large number of animals are transported through the country. However, little is known about current PRRSV strains and epidemiology. We determined full-length genome sequences of two Austrian field isolates (AUT13-883 and AUT14-440) from recent PRRSV outbreaks and of a related German isolate (GER09-613). Phylogenetic analysis revealed that the strains belong to European genotype 1 subtype 1 and form a cluster together with a South Korean strain. Remarkably, AUT14-440 infected the simian cell line MARC-145 without prior adaptation. In addition, this isolate showed exceptional deletions in nonstructural protein 2, in the overlapping region of glycoprotein 3 and 4 and in the 3' untranslated region. Both Austrian isolates caused similar lung lesions but only pigs infected with AUT14-440 developed clear clinical signs of infection. Taken together, the genetic and biological characterization of two novel Austrian PRRSV field isolates revealed similarities to East Asian strains. This stresses the necessity for a more detailed analysis of current PRRSV strains in Europe beyond the determination of short ORF5 and ORF7 sequences.