3Rs Principle Research Articles

#4145 ACLARA: AN ON-LINE CALCULATOR TO ESTIMATE CREATININE CLEARANCE IN RATS

Abstract Background and Aims Creatinine clearance (ClCr) is a standard method for the measurement of glomerular filtration rate in rats. Recently, we developed ACLARA, an open and freely available neural network (NN)-based calculator of ClCr in Wistar rats (http://idal.uv.es/aclara). By working solely on plasma creatinine concentration and body weight, ACLARA instantly and easily provides accurate ClCr estimations with an identical concept to that underlying human glomerular filtration rate (GFR)-estimating formulae utility in the clinical setting. In addition to saving costs and time, ACLARA aligns with the 3Rs principles by providing a methodological refinement tool that reduces experimental animal stress derived from avoiding confinement in metabolic cages for individual, 24-hour urine collection. As a potential limitation, ACLARA was developed with data from Wistar rats. Accordingly, the aim of this study is to study ACLARA's accuracy and utility in a different rat strain, namely the spontaneous hypertensive rat (SHR). Method In order to further validate the calculator, we used a dataset with 1,458 entries from unpublished experiments of AKI induced in Wistar and SHR male rats. Measured ClCr (mClCr) was determined with a standard procedure, as we previously described [1], and estimated ClCr (eClCr) was calculated using ACLARA. Calculator performance was assessed with the mean absolute error (MAE) and the Pearson product-moment correlation coefficient calculated as previously described [2]. Results of eCLCr were compared with their corresponding mClCr values in the context of individual experiments. Results Our study reveals that ACLARA performs with reasonably high accuracy at ClCr estimation in both Wistar and SHR rats as indicated by Pearson's product-moment correlation coefficient (Corr) and MAE. The global Corr for the 1,458 measured versus estimated data values reaches 0.9, and a MAE of 0.18 mL/min. In general, estimated data provide identical results to measured data for individual experiments considered in the whole and, if anything, depict more congruent behavior. We interpret this improvement as the consequence of bypassing experimental errors introduced by the well-known discrepancies between the real and measured urinary flow in metabolic cages. Interestingly, ACLARA performs with accuracy through the range of ages and for all experimental conditions tested, including pharmacological treatments. Figure 1 shows a representative experiment in which measured and estimated data are compared holistically. Conclusion ACLARA is an easy-to-use and reliable tool to estimate creatinine clearance in SHR and Wistar rats from plasma creatinine concentration and body weight data. Validation for a second rat strain suggests a potential, wider application of ACLARA through laboratory rats, which needs to be further studied. In this sense, collaboration and use by independent investigators and recruitment of additional data sets from other laboratories is strongly sought to further adjust the calculator in order to enhance its accuracy, extend its applicability and improve its utility to the scientific community. This study was supported by Project PI21/01226, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, and a grant from the Consejería de Educación, Junta de Castilla y León (IES160P20), Spain, co-funded by FEDER funds; and a grant from the Valencian Government with reference number CIAICO/2021/184. Noelia Diaz-Morales is recipient of a Juan de la Cierva-Formación postdoctoral contract (FJC2020-043205-I) funded by MCIN/AEI/10.13039/501100011033 and European Union “NextGenerationEU/PRTR”.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.

Caenorhabditis elegans for research on cancer hallmarks.

After decades of research, our knowledge of the complexity of cancer mechanisms, elegantly summarized as 'hallmarks of cancer', is expanding, as are the therapeutic opportunities that this knowledge brings. However, cancer still needs intense research to diminish its tremendous impact. In this context, the use of simple model organisms such as Caenorhabditis elegans, in which the genetics of the apoptotic pathway was discovered, can facilitate the investigation of several cancer hallmarks. Amenable for genetic and drug screens, convenient for fast and efficient genome editing, and aligned with the 3Rs ('Replacement, Reduction and Refinement') principles for ethical animal research, C. elegans plays a significant role in unravelling the intricate network of cancer mechanisms and presents a promising option in clinical diagnosis and drug discovery.

Front Cover: Polystyrene‐Supported Aminocatalyst Derived from Diarylprolinol for Asymmetric α‐Amination of Aldehydes (Eur. J. Org. Chem. 21/2023)

The Front Cover illustrates the natural environment that needs to be protected by following the principles of 3Rs (Reduce, Reuse, and Recycle). The background of the picture shows our institute's riverfront, where a reusable polymer-supported aminocatalyst was developed for the sustainable synthesis of chiral amino acid derivatives. Cover design by Ms. Gayathri Chandrakumar. More information can be found in the Research Article by C. Appayee et al.

Hold your breath! How to build little tracheas in the combat against COVID-19. SARS-CoV-2 research with regard to the 3Rs principle

Hold your breath! How to build little tracheas in the combat against COVID-19. SARS-CoV-2 research with regard to the 3Rs principle

Swine intestinal segment perfusion model for the evaluation of nutrients bioaccessibility.

Nutrition science requires more science-based evidences for the development of effective functional diets. To reduce animals for experimental purposes innovative reliable and informative models, simulating the complex intestinal physiology, are needed. The aim of this study was to develop a swine duodenum segment perfusion model for the evaluation of nutrient bioaccessibility and functionality across time. At the slaughterhouse, one sow intestine was harvested following Maastricht criteria for organ donation after circulatory death (DCD) for transplantation purposes. Duodenum tract was isolated and perfused in sub-normothermic conditions with heterologous blood after cold ischemia induction. Duodenum segment perfusion model was maintained under controlled pressure conditions through extracorporeal circulation for 3 hours. Blood samples from extracorporeal circulation and luminal content samples were collected at regular intervals for the evaluation of glucose concentration by glucometer, minerals (Na+, Ca2+, Mg2+, K+) by ICP-OES, lactate-dehydrogenase and nitrite oxide by spectrophotometric methods. Dacroscopic observation showed peristaltic activity caused by intrinsic nerves. Glycemia decreased over time (from 44.00±1.20 mg/dL to 27.50±0.41; p < 0.01), suggesting glucose utilization by the tissue confirming the organ viability in line with histological examinations. At the end of the experimental period, intestinal mineral concentrations were lower than their level in blood plasma suggesting their bioaccessibility (p < 0.001). A progressive increase of LDH concentration over time was observed in the luminal content probably related to a loss of viability (from 0.32±0.02 to 1.36±0.02 OD; p < 0.05) confirmed by histological findings that revealed a de-epithelization of the distal portion of duodenum. Isolated swine duodenum perfusion model satisfied the criteria for studying bioaccessibility of nutrients, offering a variety of experimental possibilities in line with 3Rs principle.

Использование приматов в доклинических исследованиях препаратов моноклональных антител

The emergence of new biopharmaceuticals, including therapeutic monoclonal antibodies (MABs), required a revision of the preclinical development strategy. To provide human risk assessment data, preclinical in vivo efficacy and safety studies of biotechnology-derived pharmaceuticals should be conducted in pharmacologically relevant species. A relevant species is one in which the test material is pharmacologically active due to the expression of the epitope and is one which demonstrate a similar tissue cross-reactivity profile as for human tissues. Often, the only relevant species for preclinical safety assessment of therapeutic MABs are non-human primates (NHPs). If there are no other alternatives (homologous proteins, genetically modified animals), all measures should be taken to reduce the number of animals involved in preclinical studies in accordance with the 3Rs principle. There is not a single solution for therapeutic MABs preclinical development program. When planning research, a scientifically-based approach should always be applied, taking into account all available data. While NHPs are currently necessary to provide risk assessment data for many therapeutic MABs, the very characteristics of MABs biology such as high target specificity, predictable metabolic pathways, and low toxicity, also provide opportunities to minimize the use of NHPs in safety testing and to increase the efficiency of MABs development. The opportunities to adapt development program for MAB at low risk of toxicity include reducing the number of chronic studies, dose groups and recovery group animals. If rodents are also a pharmacologically relevant species, the use of NSPs may be reduced. The paper discusses examples of preclinical programs for therapeutic MABs and possible strategies aimed to minimize the number of NHPs during preclinical safety studies without compromising human safety.

The chick embryo chorioallantoic membrane patient-derived xenograft (PDX) model.

The chick embryo chorioallantoic membrane (CAM) CAM is an extraembryonic membrane generated by the fusion of the chorion with the vascularized allantoic membrane. It performs multiple functions during embryonic development, including respiration, calcium transport from the eggshell, acid-base homeostasis, and ion/water reabsorption from the allantoic fluid. The CAM is a widely used model for the study of angiogenesis, anti-angiogenesis, tumor growth, and metastasis as well as drug efficacy. Ethical approval is omitted if experiments are terminated at embryonic day 14 in most countries, facilitating screenings of pharmacological or physics-based therapies with high reproducibility at large scales supporting the 3Rs principle. Being naturally immunodeficient, the chick embryo accepts transplantation from various tissues and species without immune response. This review article is focused on the analysis of the literature and personal data concerning the effects of patient-derived xenografts (PDX) on the CAM.

Evaluation of Non-Invasive Sampling Methods for Detection of Hepatitis E Virus Infected Pigs in Pens

Pigs are a reservoir of hepatitis E virus (HEV), which causes hepatitis in humans. To study the epidemiology of HEV in pig farms, sampling methods are currently used that cause discomfort to pigs, such as rectal sampling. In line with the 3Rs principle, we aimed to evaluate non-invasive methods to detect pens with HEV-shedding pigs. Twenty-eight pens of one farm were sampled cross-sectionally. Individual rectal swabs (IRS) were collected to determine prevalence within pens. Four pen-level samples were compared: a pool of IRS per pen (P), boot socks (BS), oral fluid (OF) and pooled faecal droppings (FD). Each sample was tested by RT-PCR and the sensitivity and specificity of each method was determined by Bayesian latent class analysis. According to IRS, 19/28 pens were HEV positive. BS had a sensitivity of 95% and detected HEV in pens with 10% of pigs shedding; however, specificity was below 30%. FD were comparably accurate to P, with a sensitivity and specificity of 94% and 86%, respectively. BS sampling is thus advised to detect early shedding of HEV or pen contamination, and FD to determine the duration of shedding. This study demonstrates that non-invasive sampling can replace rectal swabs in research on HEV in pigs.

Evaluation of the EPR Effect in the CAM-Model by Molecular Imaging with MRI and PET Using 89Zr-Labeled HSA

Mouse models are commonly used to study the biodistribution of novel radioligands, but alternative models corresponding to the 3Rs principles, such as the chorioallantoic membrane (CAM) model, are highly required. While there are promising data from the CAM model regarding target-specific radiolabeled compounds, its utility for assessing macromolecule biodistribution and analyzing the EPR effect remains to demonstrated. Using 89Zr-labeled human serum albumin, the accumulation of nontarget-specific macromolecules in CAM and mouse xenograft models was studied using PET and MRI. Therefore, the radioligand [89Zr]Zr-DFO-HSA was analyzed in both chicken embryos (n = 5) and SCID mice (n = 4), each with TZM-bl and PC-3 tumor entities. Dynamic PET and anatomical MRI, as well as ex vivo biodistribution analyses, were performed to assess ligand distribution over 24 h. Histological staining and autoradiography verified the intratumoral accumulation. The tumors were successfully visualized for CAM and mouse models by PET, and the albumin influx from the blood into the respective tumors did not differ significantly. The accumulation and retention of HSA in tumors due to the EPR effect was demonstrated for both models. These results highlight that the CAM model is a potential alternative to the mouse model for initial studies with novel radiolabeled macromolecules with respect to the 3Rs principles.

When less is more: Experimental Bishop-Koop technique for reduction in the use of laboratory animals for intestinal pathophysiological studies.

The use of animals to gain knowledge and understanding of diseases needs to be reduced and refined. In the field of intestinal research, because of the complexity of the gut immune system, living models testing is mandatory. Based on the 3Rs (replacement, reduction and refinement) principles, we aimed to developed and apply the derived-intestinal surgical procedure described by Bishop and Koop (BK) in rats to refine experimental gastrointestinal procedures and reduce the number of animals used for research employing two models of intestinal inflammation: intestinal ischemia-reperfusion injury and chemical-induced colitis. Our results show the feasibility of the application of the BK technique in rodents, with good success after surgical procedure in both small and large intestine (100% survival, clinical recovery and weight regain). A considerable reduction in the use of the number of rats in both intestinal inflammation models (80% in case of intestinal ischemia-reperfusion damage and 66.6% in chemical-induced colitis in our experimental design) was achieved. Compared with conventional experimental models described by various research groups, we report excellent reproducibility of intestinal damage and functionality, survival rate and clinical status of the animals when BK is applied.

Organ-on-a-chip technologies for biomedical research and drug development: A focus on the vasculature.

Current biomedical models fail to replicate the complexity of human biology. Consequently, almost 90% of drug candidates fail during clinical trials after decades of research and billions of investments in drug development. Despite their physiological similarities, animal models often misrepresent human responses, and instead, trigger ethical and societal debates regarding their use. The overall aim across regulatory entities worldwide is to replace, reduce, and refine the use of animal experimentation, a concept known as the Three Rs principle. In response, researchers develop experimental alternatives to improve the biological relevance of in vitro models through interdisciplinary approaches. This article highlights the emerging organ-on-a-chip technologies, also known as microphysiological systems, with a focus on models of the vasculature. The cardiovascular system transports all necessary substances, including drugs, throughout the body while in charge of thermal regulation and communication between other organ systems. In addition, we discuss the benefits, limitations, and challenges in the widespread use of new biomedical models. Coupled with patient-derived induced pluripotent stem cells, organ-on-a-chip technologies are the future of drug discovery, development, and personalized medicine.

Integrating 3Rs approaches in WHO guidelines for the batch release testing of biologicals: Responses from a survey of vaccines and biological therapeutics manufacturers.

The UK National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs) has been tasked by the World Health Organization (WHO) to review the extent to which animal-based testing methods are described in their manuals, guidelines and recommendations for vaccines and biotherapeutics. The aim is to identify and recommend where updates to these documents can lead to an increased and more harmonised adoption of 3Rs principles (i.e. Replacement, Reduction and Refinement of animal tests) in the quality control and batch release testing requirements for vaccines and biotherapeutics. Developing recommendations that are widely applicable by both the manufacturers and national regulatory authorities for vaccines and biologicals globally requires a detailed understanding of how different organisations view the opportunities and barriers to better integration of the 3Rs. To facilitate this, we developed and distributed a survey aimed at vaccine and biotherapeutics manufacturers in July 2021. In this paper, we present the key findings from this survey and how these will help inform the recommendations for wider integration of 3Rs approaches by WHO in their guidance documents applicable to the quality control and batch testing of vaccines and biotherapeutics.

Germ Cell Maintenance and Sustained Testosterone and Precursor Hormone Production in Human Prepubertal Testis Organ Culture with Tissues from Boys 7 Years+ under Conditions from Adult Testicular Tissue.

Human prepubertal testicular tissues are rare, but organ culture conditions to develop a system for human in vitro-spermatogenesis are an essential option for fertility preservation in prepubertal boys subjected to gonadotoxic therapy. To avoid animal testing in line with the 3Rs principle, organ culture conditions initially tested on human adult testis tissue were applied to prepubertal samples (n = 3; patient ages 7, 9, and 12 years). Tissues were investigated by immunostaining and transmission electron microscopy (TEM), and the collected culture medium was profiled for steroid hormones by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Culture conditions proved suitable for prepubertal organ culture since SSCs and germ cell proliferation could be maintained until the end of the 3-week-culture. Leydig cells (LCs) were shown to be competent for steroid hormone production. Three additional testis tissues from boys of the same age were examined for the number of germ cells and undifferentiated spermatogonia (SPG). Using TEM micrographs, eight tissues from patients aged 1.5 to 13 years were examined, with respect to the sizes of mitochondria (MT) in undifferentiated SPG and compared with those from two adult testicular tissues. Mitochondrial sizes were shown to be comparable between adults and prepubertal boys from approximately 7 years of age, which suggests the transition of SSCs from normoxic to hypoxic metabolism at about or before this time period.

3Rs Principle and Legislative Decrees to Achieve High Standard of Animal Research.

Animal experimentation is a vast ecosystem that tries to make different issues such as legislative, ethical and scientific coexist. Research in animal experimentation has made many strides thanks to the 3Rs principle and the attached legislative decrees, but for this very reason, it needs to be evenly implemented both among the countries that have adhered to the decrees and among the team members who design and execute the experimental practice. In this article, we emphasize the importance of the 3Rs principle's application, with a particular focus on the concept of Reduction and related key aspects that can best be handled with the contribution of experts from different fields.

Application of transgenic zebrafish for investigating inflammatory responses to nanomaterials: Recommendations for new users

Despite the increasing exploitation of nanomaterials (NMs) in an array of consumer products, there are uncertainties regarding their potential adverse impact on human health. Investigation of whether NMs activate a pro-inflammatory response is routinely used to assess their toxicity in in vitro and in vivo (rodent) studies. The use of zebrafish (Danio rerio) to investigate inflammatory responses to chemicals, pathogens and injury has increased considerably over recent years. Zebrafish have also been used to investigate the role of inflammation in disease pathogenesis and for drug discovery. Availability of transgenic strains which express fluorescent proteins in immune cells (e.g. macrophages and neutrophils) enables the visualization and quantification of immune cell accumulation in the target site(s) of interest. We therefore propose that transgenic zebrafish have great utility for screening the toxicity of NMs via investigation of inflammatory responses. Indeed, we have successfully used non-protected life stages of transgenic zebrafish with fluorescent neutrophils (Tg(mpx:EGFP114) to investigate inflammatory responses to NMs. The more widespread use of transgenic zebrafish in nanotoxicology could reduce the reliance placed on rodents and thereby enhance the implementation of the 3Rs principles. As zebrafish continue to grow in popularity it is timely to offer guidance to new users on their use. Here we will reflect on: exposure routes that can adopted to mimic human/rodent exposure, what transgenic strains and life stages are best suited to investigate inflammatory responses, selection criteria for zebrafish embryos/larvae, the inclusion of appropriate controls, the importance of dose selection and sample size, and how the (inflammatory) response can be quantified. It is hoped that our recommendations will support the development of standard protocols that can be used to assess whether NMs activate inflammatory responses. Importantly, the themes discussed are not restricted to NMs but relevant also to zebrafish application in ecotoxicology or human health focused studies.

The 3Rs principle applied to laboratory experiments: from waste to potential new results

The 3Rs principle applied to laboratory experiments: from waste to potential new results

Why workshops work: Examining the efficacy of training trainers to train goats.

Experimental procedures involving farm animals are often associated with stress due to restraining. Stress can be reduced through use of positive reinforcement training, which then serves as refinement according to the 3Rs principles. Trainer skills, however, may influence the feasibility and success of animal training. The potential influence of trainer skills as well as the education of animal trainers are rarely described in literature but are necessary information for the implementation of positive reinforcement training as a refinement measure. To investigate the effect of educational programmes on animal trainers, we compared the training success of two groups of participants in training goats to elicit a behaviour that would allow simulated venipuncture. One group was educated in a two-day workshop while the other was provided with specific literature for self-instructed learning. Training success was evaluated using an assessment protocol developed for this study. A greater training success in the WORKSHOP GROUP, reflected by objective and subjective measures, was clearly supported statistically. In addition, 73 versus only 13% of the participants of the WORKSHOP GROUP and the self-instructed BOOK GROUP, respectively, stated that they could completely implement the knowledge gained in the course of this study. Our results indicate that more intensively educated trainers can train animals more successfully. In conclusion, if animal training is implemented as refinement, animal caretakers should receive instruction for positive reinforcement training.

An &lt;em&gt;ex vivo&lt;/em&gt; experimental system to track fluorescent nanoparticles inside skeletal muscle

The development of novel nanoconstructs for biomedical applications requires the assessment of their biodistribution, metabolism and clearance in single cells, organs and entire organisms in a living environment. To reduce the number of in vivo experiments performed and to refine the methods used, in accordance with the 3Rs principle, this work proposes an ex vivo experimental system to monitor, using fluorescence microscopy, the distribution of nanoparticles in explanted murine skeletal muscle maintained in a bioreactor that can preserve the structural and functional features of the organ for long periods of time. Fluorescently-labelled liposomes and poly(lactide-co-glycolide) (PLGA)-based nanoparticles were injected into the intact soleus muscle (in the distal region close to the tendon) immediately after explants, and their distribution was analysed at increasing incubation times in cross cryosections from the proximal region of the belly. Both nanocarriers were clearly recognized in the muscle and were found to enter and migrate inside the myofibres, whereas their migration in the connective tissue seemed to be limited. In addition, some fluorescent signals were observed inside the macrophages, demonstrating the physiological clearance of the nanocarriers that did not enter the myofibres. Our ex vivo system therefore provides more information than previous in vitro experiments on cultured muscle cells, highlighting the need for the appropriate functionalization of nanocarriers if myofibre targeting is to be improved.

Characterization of glioblastoma spheroid models for drug screening and phototherapy assays

Glioblastoma (GBM) spheroids present as a promising alternative testing platform for in vivo models of brain cancer, aiming at the 3Rs principle of animal use in pre-clinical trials. However, the poor characterization and irreproducibility of various GBM-based spheroids have hampered the achievement of statistically relevant data for drug screening and photodynamic therapy (PDT) assays. In a previous study (Calori et al., 2022), we defined the conditions for inducing tight compaction of U87MG, T98G, UW473, A172, and U251 GBM cell lines into reproducible 3D spheroids using type I collagen as an extracellular matrix. Herein, GBM spheroids were characterized in their growth profile, proliferation rate, cell viability, necrotic nucleus, and cell death mechanisms and applied in PDT assays. Driven by proliferation, U87MG and A172 spheroids grew with time, at least up to day 12, whereas T98G and U251 demonstrated little change. The order of cell viability of spheroids over time was U87MG > A172 > U251 > T98G. A necrotic nucleus was formed over time for all cell lines, as shown by histological assays and the inverse size-cell viability relationship. When subjected to PDT, all spheroids showed a dose-dependent response, with an excellent dose-light-cell viability correlation. The higher light dose required for PDT of GBM 3D spheroids compared with the simplest 2D monolayers and its proximity to in vivo dose response in the literature is proof of its applicability in pre-clinical tests to reduce the use of animals in research.