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.

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.

A Proof-of-Concept Preclinical Study Using a Novel Thermal Insulation Device in a Porcine Kidney Auto-Transplantation Model.

Ischemia-reperfusion injury remains a fundamental problem during organ transplantation logistics. One key technical factor is the rapid allograft rewarming during the time of vascular reconstruction in the recipient. In this pilot study, a new thermal insulation bag (TIB) for organ transplantation was used. Insulation capacity, tissue compatibility, and usability were tested initially ex vivo on porcine kidneys (n = 24) followed by the first in vivo usage. Fourteen female German landrace pigs underwent kidney auto-transplantation after 24 h cold storage (4 °C). During the implantation process the kidney was either insulated with the new TIB, or it was not thermo-protected at all, which represents the clinical standard. In this proof-of-concept study, the usability (knife-to-skin-time) and the general thermal capacity (30 min warm storage at 38 °C ex vivo p < 0.001) was shown. The clinical outcome showed significant differences in the determination of CRP and pi-GST levels. Syndecan-1 Antibody staining showed clear significant higher counts in the control group (p < 0.01) indicating epithelial damage. However, the effect on renal outcomes in not severely pre-damaged kidneys does not appear to be conclusively significant. A close follow-up study is warranted, especially in the context of marginal organs or in cases where anastomosis-times are prolonged due to surgical complexity (e.g., multiple vessels and complex reconstructions).

[Physical long-term consequences of cancer]. / Körperliche Langzeitfolgen von Krebserkrankungen.

The number of long-term survivors of malignant diseases is steadily increasing, which is due to the further development and optimization of multimodal therapy strategies and the mechanisms of new substance classes. These can now be combined with classical treatment methods or used sequentially. At the same time the number of patients who suffer from physical and psychosocial long-term consequences of cancer therapies or have to live with chronic side effects under the long-term therapies increases. Every therapy, whether radiation, chemotherapy, targeted therapy, or operation, has undesirable long-term side effects that contribute to the decrease of one's quality of life. These affect all parts of the body. As a result, patients can be heavily burdened. In oncology and in other disciplines involved in aftercare, these consequences must therefore be increasingly addressed and clarified and treatment strategies further developed. Unfortunately, there is still a considerable need for research in this area; moreover, there is a lack of clinical studies examining the evidence of a wide variety of holistic therapy methods.

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.

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.

A New Percutaneous Approach to Treat Combined Right Ventricular and Respiratory Failure: The "Aachen Cannula".

INTRODUCTION: Right ventricular failure (RVF) on its own is a life-threatening condition. Often it manifests as a two-organ failure in the final phase of several lung diseases. Mechanical circulatory support is a proven treatment of RVF but remains challenging. Our objective is to develop a novel, simplified, and minimally invasive cannula approach to treat both RVF and respiratory failure. METHODS: We conceptualized a dual lumen cannula approach to allow oxygenated right-to-left shunting at an atrial level to decompress right-sided circulation. A minimally invasive approach through percutaneous, transjugular insertion and transseptal placement should enable patients to be non-sedated and even ambulatory. In an iterative design, pre-prototyping, prototyping, and anatomic fitting process, such a cannula was generated and tested in both cadaveric and fluid dynamic studies. RESULTS: After various modifications and improvements, a 27-Fr 255-mm-long double-lumen cannula with an inner line (oxygenated blood return to patient into the left atrium) of 18 Fr and an inflatable balloon (with a volume of approximately 1 mL) at the outflow tip was produced - one version with a straight head and another one with a curved head. In our anatomic studies, the "Aachen Cannula" allowed an easy transjugular introduction and advancement into the right atrium by Seldinger technique. Transseptal placement was achieved by puncture (Brockenbrough needle) in combination with dilatation and was then secured in place with the stabilizing balloon, even under slight tension. The cannula prototype enabled a flow of up to 3.5 L/min, at which common pressure drops were observed. CONCLUSION: In conclusion, we successfully conceptualized, designed, and verified a minimally invasive one-cannula approach for the treatment of either isolated right heart failure and even combined RVF and respiratory failure through our transseptal Aachen Cannula. This concept may also be carried out in ambulatory conditions. Moreover, this approach completely avoids recirculation issues and ensures reliable oxygenated coronary as well as cerebral perfusion.

The Aachen Minipig: Phenotype, Genotype, Hematological and Biochemical Characterization, and Comparison to the Göttingen Minipig.

BACKGROUND: The pig is one of the most frequently used large animal models for biomedical research, especially in the field of translational research and surgical models. While standard livestock breeds are used in short-term and acute studies, minipig breeds are the preferred breeds in long-term and chronic studies due to their limited growth and body weight. OBJECTIVE: In consideration of the 3R principle (refinement, reduction, replacement) and the increasing demand, the aim of this study was to generate a new, robust, non-specific-pathogen-free minipig breed, the Aachen minipig. METHODS: Phenotype, genotype, and hematological as well as clinical chemistry parameters were characterized, and reference values of the Aachen minipig were generated and compared to the values in the commonly used Göttingen minipig. Organ weights of the heart, kidney, liver, lung, spleen, and brain were determined using a laboratory balance. Blood samples were collected for hematology and clinical chemistry. Assessment of genetic diversity was performed by microsatellite markers. Nasal swabs were collected from 11 individual minipigs representing 6 races for DNA extraction. DNA was quantified and the identity and origin of the Aachen minipigs at the genomic level was determined by microsatellites. RESULTS: The Aachen minipig established here is based on the Mini-LEWE breed and consists of the Vietnamese potbelly pig, the Schwäbisch Hällisch Landpig, the German Landrace, and the Minnesota minipig. Relative organ weights (lung, heart, kidneys, brain), hematology (hemoglobin, hematocrit, platelet count, mean corpuscular hemoglobin concentration, segmented neutrophils, lymphocytes, eosinophils, basophils), and clinical chemistry parameters (sodium, calcium, chloride, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, lactate dehydrogenase, triglycerides, blood urea nitrogen, creatinine, total bilirubin, total protein, creatine kinase) of the Aachen minipigs and the Göttingen minipigs were not significantly different. Significant differences where only seen in relative organ weights (liver, spleen), hematology (red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, white blood cell count, banded neutrophils, monocytes), and clinical chemistry parameters (inorganic phosphorus, potassium, glucose, cholesterol, albumin, amylase). CONCLUSION: The Aachen minipig is a suitable model for research due to its similarity to other minipig breeds, especially the Göttingen minipig. The reference values established in this study may be used for the comparison of scientific data and encourage the use of the Aachen minipig as an animal model for biomedical research.

Machine perfusion of the liver and in vivo animal models: A systematic review of the preclinical research landscape.

Machine perfusion (MP) is often referred to as one of the most promising advancements in liver transplantation research of the last few decades, with various techniques and modalities being evaluated in preclinical studies using animal models. However, low scientific rigor and subpar reporting standards lead to limited reproducibility and translational potential, hindering progress. This pre-registered systematic review (PROSPERO: CRD42021234667) aimed to provide a thematic overview of the preclinical research landscape on MP in liver transplantation using in vivo transplantation models and to explore methodological and reporting standards, using the ARRIVE (Animal Research: Reporting of In Vivo Experiments) score. In total 56 articles were included. Studies were evenly distributed across Asia, Europe, and the Americas. Porcine models were used in 57.1% of the studies, followed by rats (39.3%) and dogs (3.6%). In terms of graft type, 55.4% of the studies used donation after cardiac death grafts, while donation after brain death grafts accounted for 37.5%. Regarding MP modalities, the distribution was as follows: 41.5% of articles utilized hypothermic MP, 21.5% normothermic MP, 13.8% subnormothermic MP, and 16.9% utilized hypothermic oxygenated MP. The stringent documentation of ARRIVE elements concerning precise experimental execution, group size and selection, the choice of statistical methods, as well as adherence to the principles of the 3Rs, was notably lacking in the majority of publications, with less than 30% providing comprehensive details. Postoperative analgesia and antibiotics treatment were not documented in 82.1% of all included studies. None of the analyzed studies fully adhered to the ARRIVE Guidelines. In conclusion, the present study emphasizes the importance of adhering to reporting standards to promote reproducibility and adequate animal welfare in preclinical studies in machine perfusion. At the same time, it highlights a clear deficiency in this field, underscoring the need for further investigations into animal welfare-related topics.

Refinement in Post-Operative Care for Orthopaedic Models: Implementing a Sheep Walking Cast (SWC) for Effective Tibial Fracture Management.

In the healthcare system, lower leg fractures remain relevant, incurring costs related to surgical treatment, hospitalization, and rehabilitation. The duration of treatment may vary depending on the individual case and its severity. Casting as a post-surgical fracture treatment is a common method in human and experimental veterinary medicine. Despite the high importance of sheep in preclinical testing materials for osteosynthesis, there is no standardised cast system ensuring proper stabilisation and functionality of hind limbs during the healing of tibia fractures or defects. Existing treatment approaches for tibial osteosynthesis in laboratory animal science include sling hanging, external fixators, or former Achilles tendon incision. These methods restrict animal movement for 4-6 weeks, limit species-typical behaviour, and impact social interactions. Our pilot study introduces a Standardised Walking Cast (SWC) for sheep, enabling immediate physiological movement post surgery. Seven Rhone sheep (female, 63.5 kg ± 6.45 kg) each with a single tibia defect (6 mm mechanical drilled defect) underwent SWC application for 4 weeks after plate osteosynthesis. The animals bore weight on their operated leg from day one, exhibiting slight lameness (grade 1-2 out of 5). Individual step lengths showed good uniformity (average deviation: 0.89 cm). Group housing successfully started on day three after surgery. Weekly X-rays and cast changes ensured proper placement, depicting the healing process. This study demonstrates the feasibility of using an SWC for up to 72 kg of body weight without sling hanging via ceiling mounting or external fixation techniques. Allowing species-typical movement and social behaviour can significantly improve the physiological behaviour of sheep in experiments, contributing to refinement.

Long-Term Degradation Assessment of a Polyurethane-Based Surgical Adhesive-Assessment and Critical Consideration of Preclinical In Vitro and In Vivo Testing.

Tissue adhesives constitute a great possibility to improve conventional wound closure. In contrast to sutures, they enable nearly immediate hemostasis and can prevent fluid or air leaks. In the present study, a poly(ester)urethane-based adhesive was investigated which already proved to be suitable for different indications, such as reinforcing vascular anastomosis and sealing liver tissue. Using in vitro and in vivo setups, the degradation of the adhesives was monitored over a period of up to 2 years, to evaluate long-term biocompatibility and determine degradation kinetics. For the first time, the complete degradation of the adhesive was documented. In subcutaneous locations, tissue residues were found after 12 months and in intramuscular locations, tissue degradation was complete after about 6 months. A detailed histological evaluation of the local tissue reaction revealed good biocompatibility throughout the different degradation stages. After full degradation, complete remodeling to physiological tissue was observed at the implant locations. In addition, this study critically discusses common issues related to the assessment of biomaterial degradation kinetics in the context of medical device certification. This work highlighted the importance and encouraged the implementation of biologically relevant in vitro degradation models to replace animal studies or at least reduce the number of animals in preclinical testing prior to clinical studies. Moreover, the suitability of frequently used implantation studies based on ISO 10993-6 at standard locations was critically discussed, especially in light of the associated lack of reliable predictions for degradation kinetics at the clinically relevant site of implantation.

Establishment of the body condition score for adult female Xenopus laevis.

The assessment of animals' health and nutritional status using a Body Condition Score (BCS) has become a common and reliable tool in lab-animal science. It enables a simple, semi-objective, and non-invasive assessment (palpation of osteal prominences and subcutaneous fat tissue) in routine examination of an animal. In mammals, the BCS classification contains 5 levels: A low score describes a poor nutritional condition (BCS 1-2). A BCS of 3 to 4 is considered optimum, whereas a high score (BCS = 5) is associated with obesity. While BCS are published for most common laboratory mammals, these assessment criteria are not directly applicable to clawed frogs (Xenopus laevis) due to their intracoelomic fat body instead of subcutaneous fat tissue. Therefore, this assessment tool is still missing for Xenopus laevis. The present study aimed to establish a species-specific BCS for clawed frogs in terms of housing refinement in lab-animal facilities. Accordingly, 62 adult female Xenopus laevis were weighed and sized. Further, the body contour was defined, classified, and assigned to BCS groups. A BCS 5 was associated with a mean body weight of 193.3 g (± 27.6 g), whereas a BCS 4 ranged at 163.1 g (±16.0 g). Animals with a BCS = 3 had an average body weight of 114.7 g (±16.7 g). A BCS = 2 was determined in 3 animals (103 g, 110 g, and 111 g). One animal had a BCS = 1 (83 g), equivalent to a humane endpoint. In conclusion, individual examination using the presented visual BCS provides a quick and easy assessment of the nutritional status and overall health of adult female Xenopus laevis. Due to their ectothermic nature and the associated special metabolic situation, it can be assumed that a BCS ≥3 is to be preferred for female Xenopus laevis. In addition, BCS assessment may indicate underlying subclinical health problems that require further diagnostic investigation.

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.

Taurine as Antioxidant in a Novel Cell- and Oxygen Carrier-Free Perfusate for Normothermic Machine Perfusion of Porcine Kidneys.

Donor organ-shortage has resulted in the increased use of marginal grafts; however, normothermic machine perfusion (NMP) holds the potential for organ viability assessment and restoration of marginal grafts prior to transplantation. Additionally, cell-, oxygen carrier-free and antioxidants-supplemented solutions could potentially prevent adverse effects (transfusion reactions, inflammation, hemolysis), associated with the use of autologous packed red blood cell (pRBC)-based perfusates. This study compared 6 h NMP of porcine kidneys, using an established pRBC-based perfusate (pRBC, n = 7), with the novel cell- and oxygen carrier-free organ preservation solution Ecosol, containing taurine (Ecosol, n = 7). Despite the enhanced tissue edema and tubular injury in the Ecosol group, related to a suboptimal molecular mass of polyethylene glycol as colloid present in the solution, functional parameters (renal blood flow, intrarenal resistance, urinary flow, pH) and oxygenation (arterial pO2, absence of hypoxia-inducible factor 1-alpha) were similar to the pRBC group. Furthermore, taurine significantly improved the antioxidant capacity in the Ecosol group, reflected in decreased lactate dehydrogenase, urine protein and tubular vacuolization compared to pRBC. This study demonstrates the feasibility of 6 h NMP using a taurine containing, cell- and oxygen carrier-free perfusate, achieving a comparable organ quality to pRBC perfused porcine kidneys.

Diclofenac and other non-steroidal anti-inflammatory drugs (NSAIDs) are competitive antagonists of the human P2X3 receptor.

Introduction: The P2X3 receptor (P2X3R), an ATP-gated non-selective cation channel of the P2X receptor family, is expressed in sensory neurons and involved in nociception. P2X3R inhibition was shown to reduce chronic and neuropathic pain. In a previous screening of 2000 approved drugs, natural products, and bioactive substances, various non-steroidal anti-inflammatory drugs (NSAIDs) were found to inhibit P2X3R-mediated currents. Methods: To investigate whether the inhibition of P2X receptors contributes to the analgesic effect of NSAIDs, we characterized the potency and selectivity of various NSAIDs at P2X3R and other P2XR subtypes using two-electrode voltage clamp electrophysiology. Results: We identified diclofenac as a hP2X3R and hP2X2/3R antagonist with micromolar potency (with IC50 values of 138.2 and 76.7 µM, respectively). A weaker inhibition of hP2X1R, hP2X4R, and hP2X7R by diclofenac was determined. Flufenamic acid (FFA) inhibited hP2X3R, rP2X3R, and hP2X7R (IC50 values of 221 µM, 264.1 µM, and ∼900 µM, respectively), calling into question its use as a non-selective ion channel blocker, when P2XR-mediated currents are under study. Inhibition of hP2X3R or hP2X2/3R by diclofenac could be overcome by prolonged ATP application or increasing concentrations of the agonist α,ß-meATP, respectively, indicating competition of diclofenac and the agonists. Molecular dynamics simulation showed that diclofenac largely overlaps with ATP bound to the open state of the hP2X3R. Our results suggest a competitive antagonism through which diclofenac, by interacting with residues of the ATP-binding site, left flipper, and dorsal fin domains, inhibits the gating of P2X3R by conformational fixation of the left flipper and dorsal fin domains. In summary, we demonstrate the inhibition of the human P2X3 receptor by various NSAIDs. Diclofenac proved to be the most effective antagonist with a strong inhibition of hP2X3R and hP2X2/3R and a weaker inhibition of hP2X1R, hP2X4R, and hP2X7R. Discussion: Considering their involvement in nociception, inhibition of hP2X3R and hP2X2/3R by micromolar concentrations of diclofenac, which are rarely reached in the therapeutic range, may play a minor role in analgesia compared to the high-potency cyclooxygenase inhibition but may explain the known side effect of taste disturbances caused by diclofenac.

Corrigendum: Diclofenac and other non-steroidal anti-inflammatory drugs (NSAIDs) are competitive antagonists of the human P2X3 receptor.

[This corrects the article DOI: 10.3389/fphar.2023.1120360.].

A model-specific simplification of the Mouse Grimace Scale based on the pain response of intraperitoneal CCl4 injections.

Despite its long establishment and applicability in mice pain detection, the Mouse Grimace Scale still seems to be underused in acute pain detection during chronic experiments. However, broadening its applicability can identify possible refinement approaches such as cumulative severity and habituation to painful stimuli. Therefore, this study focuses on two main aspects: First, five composite MGS criteria were evaluated with two independent methods (the MoBPs algorithm and a penalized least squares regression) and ranked for their relative importance. The most important variable was used in a second analysis to specifically evaluate the context of pain after an i.p. injection (intervention) in two treatment groups (CCl4 and oil (control)) at fixed times throughout four weeks in 24 male C57BL/6 N mice. One hour before and after each intervention, video recordings were taken, and the MGS assessment was performed. In this study, the results indicate orbital tightening as the most important criterion. In this experimental setup, a highly significant difference after treatment between week 0 and 1 was found in the CCl4 group, resulting in a medium-sized effect (W = 62.5, p value < 0.0001, rCCl4 = 0.64). The oil group showed no significant difference (week 0 vs 1, W = 291.5, p value = 0.7875, rcontrol = 0.04). Therefore, the study showed that the pain caused by i.p. injections was only dependent on the applied substance, and no significant cumulation or habituation occurred due to the intervention. Further, the results indicated that the MGS system can be simplified.

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.

HBOC-301 in Porcine Kidney Normothermic Machine Perfusion and the Effect of Vitamin C on Methemoglobin Formation.

Normothermic machine perfusion (NMP) of kidneys in combination with an optimized perfusate composition may increase donor organ preservation quality, especially in the case of marginal donor grafts. Optimization of currently employed perfusates is still a subject of present research. Due to the advantages of being cell-free, easy to store, and having minimal antigenicity, hemoglobin-based oxygen carriers, such as HBOC-301 (Oxyglobin®, Hemoglobin Oxygen Therapeutics LLC, Souderton, PA, USA), offer an alternative to the commonly used perfusates based on packed red blood cells (pRBC). As previously described, using HBOC results in formation of methemoglobin (metHb) as an adverse effect, inducing hypoxic conditions during the perfusion. As a potential counterpart to metHb formation, the application of the antioxidant ascorbic acid (VitC) is of high interest. Therefore, this study was conducted in four experimental groups, to compare the effect of NMP with (1) HBOC or (3) pRBC, and additionally examine a beneficial effect of VitC in both groups (2) HBOC + VitC and (4) pRBC + VitC. All groups were subjected to NMP for 6 h at a pressure of 75 mmHg. Kidneys in the HBOC groups had a significantly lower renal blood flow and increasing intrarenal resistance, with reduced renal function in comparison to the pRBC groups, as demonstrated by significantly lower creatinine clearance and higher fractional sodium excretion rates. Clinical chemistry markers for tissue damage (LDH, lactate) were higher in the HBOC groups, whereas no significant histological differences were observed. Although the application of VitC decreased oxidative stress levels, it was not able to significantly increase the outcome parameters mentioned above in either group. This study demonstrated that HBOC-301 is inferior to pRBCs in our porcine kidney NMP model, independent of additional VitC administration. Oxidative stress and fragmentation of the hemoglobin polymers could be detected as a possible reason for these results, hence further research, focusing on the use of cell-free oxygen carriers that do not exhibit this complex of issues, is required.

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.