Combined use of multiparametric high-content-screening and in vitro circadian reporter assays in neurotoxicity evaluation.

Accumulating evidence indicates that chronic circadian rhythm disruption is associated with the development of neurodegenerative diseases induced by exposure to neurotoxic chemicals. Herein, we examined the relationship between cellular circadian rhythm disruption and cytotoxicity in neural cells. Moreover, we evaluated the potential application of an in vitro cellular circadian rhythm assay in determining circadian rhythm disruption as a sensitive and early marker of neurotoxicant-induced adverse effects. To explore these objectives, we established an in vitro cellular circadian rhythm assay using human glioblastoma (U87 MG) cells stably transfected with a circadian reporter vector (PER2-dLuc) and determined the lowest-observed-adverse-effect levels (LOAELs) of several common neurotoxicants. Additionally, we determined the LOAEL of each compound on multiple cytotoxicity endpoints (nuclear size [NC], mitochondrial membrane potential [MMP], calcium ions, or lipid peroxidation) using a multiparametric high-content screening (HCS) assay using transfected U87 MG cells treated with the same neurotoxicants for 24 and 72 h. Based on our findings, the LOAEL for cellular circadian rhythm disruption for most chemicals was slightly higher than that for most cytotoxicity indicators detected using HCS, and the LOAEL for MMP in the first 24 h was the closest to that for cellular circadian rhythm disruption. Dietary antioxidants (methylselenocysteine and N-acetyl-l-cysteine) prevented or restored neurotoxicant-induced cellular circadian rhythm disruption. Our results suggest that cellular circadian rhythm disruption is as sensitive as cytotoxicity indicators and occurs early as much as cytotoxic events during disease development. Moreover, the in vitro cellular circadian rhythm assay warrants further evaluation as an early screening tool for neurotoxicants.

Intranasal administration enhances size-dependent pulmonary phagocytic uptake of poly(lactic-co-glycolic acid) nanoparticles.

BACKGROUND: Nanoparticles exhibit distinct behaviours within the body, depending on their physicochemical properties and administration routes. However, in vivo behaviour of poly(lactic-co-glycolic acid) (PLGA) nanoparticles, especially when administered nasally, remains unexplored; furthermore, there is a lack of comparative analysis of uptake efficiency among different administration routes. Therefore, here, we aimed to comprehensively investigate the real-time in vivo behaviour of PLGA nanoparticles across various administration routes. PLGA-NH2 nanoparticles of three sizes were synthesised using an oil-in-water single-emulsion method. We assessed their uptake by murine macrophage RAW264.7 cells using fluorescence microscopy. To enable real-time tracking, we conjugated p-SCN-Bn-deferoxamine to PLGA-NH2 nanoparticles and further radiolabelled them with 89Zr-oxalate before administration to mice via different routes. Nanoparticle internalisation by lung immune cells was monitored using fluorescence-activated cell sorting analysis. RESULTS: The nanoparticle sizes were 294 ± 2.1 (small), 522.5 ± 5.58 (intermediate), and 850 ± 18.52 nm (large). Fluorescent labelling did not significantly alter the nanoparticle size and charge. The level of uptake of small and large nanoparticles by RAW264.7 cells was similar, with phagocytosis inhibition primarily reducing the internalisation of large particles. Positron emission tomography revealed that intranasal delivery resulted in the highest and most targeted pulmonary uptake, whereas intravenous administration led to accumulation mainly in the liver and spleen. Nasal delivery of large nanoparticles resulted in enhanced uptake by myeloid immune cells relative to lymphoid cells, whereas dendritic cell uptake initially peaked but declined over time. CONCLUSIONS: Our study provides valuable insights into advancing nanomedicine and drug delivery, with the potential for expanding the clinical applications of nanoparticles.

Histopathological pulmonary lesions in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques experimentally infected with wild-type severe acute respiratory syndrome coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a viral pneumonia characterized by acute interstitial pneumonia and diffuse alveolar damage in humans. Non-human primates (NHPs) are widely used as preclinical animal models for vaccine development against SARS-CoV-2. However, the pathological changes in NHPs have been described only in selected facets and inconsistent nomenclature is used, making it difficult to interpret and compare the outcomes between studies. Here, we present a standardized methodology for histopathological evaluation of experimental infection outcomes in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques. Evaluation criteria for vascular and epithelial changes in the early (3 days post infection [dpi]) and late (21 dpi) phases of the infection were developed, and a four-grade classification encompassing all the histopathological lung lesions was established. The grades of histopathological lung lesions were higher at 3 dpi compared with 21 dpi in both species of macaques, and there were no statistically significant differences in the grades between the two species at 3 dpi and 21 dpi. This study contextualized the pathological SARS-CoV-2 presentation and standardized the terminology and grading scale for lesion severity to facilitate histopathological examination in the macaque model. By referring to the standardized histopathological criteria and grades proposed here, comparable results with high reproducibility can be obtained in future studies of pathogenicity.

A survey of the impact of COVID-19 on the management of animal experiments and laboratory animal facilities in Korea.

The pandemic has affected the lives of people all over the world. The effects of the pandemic on laboratory animal facilities and their operations through this unusual global event are poorly understood. Here, we have applied a methodological framework of qualitative approach including semi-structured interviews to investigate laboratory animal operations in Korea and how it has shaped the on-going management and laboratory operations of such facilities. A total of fifty-two individuals, including members and administrators of the Institutional Animal Care and Use Committee (IACUC), researchers, and animal facility managers and staff, were surveyed through purposeful sampling. Survey questions explored how the pandemic impacted the IACUC and the functioning of animal facilities, and what steps to take in preparation of a future pandemic-like crisis. Our survey found evidence of an increase in animal experiments in Korea during the pandemic that correlated with increases in research funding during that period, such as for vaccine development. Also, operational challenges due to pandemic-related health issues in personnel were resolved through overtime, rather than by reducing facility operations. Moreover, a refinement of post-approval monitoring (PAM) practices was also discussed by respondents. Taken together, our study offers insights into animal facility operations during the pandemic and outlines recommendations for safeguarding operations in such future scenarios.

Spatial transcriptome atlas reveals pulmonary microstructure-specific COVID-19 gene signatures in cynomolgus macaques.

Characterizing the host response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the molecular level is necessary to understand viral pathogenesis and identify clinically relevant biomarkers. However, in humans, the pulmonary host response during disease onset remains poorly understood. Herein, we utilized a spatial transcriptome atlas to identify pulmonary microstructure-specific COVID-19 gene signatures during the acute phase of lung infection in cynomolgus macaques. The innate immune response to virus-induced cell death was primarily active in the alveolar regions involving activated macrophage infiltration. Inflamed vascular regions exhibited prominent upregulation of interferon and complement pathway genes that mediate antiviral activity and tissue damage response. Furthermore, known biomarker genes were significantly expressed in specific microstructures, and some of them were universally expressed across all microstructures. These findings underscore the importance of identifying key drivers of disease progression and clinically applicable biomarkers by focusing on pulmonary microstructures appearing during SARS-CoV-2 infection.

Flubendazole exposure disrupts neural development and function of zebrafish embryos (Danio rerio).

Flubendazole (FBZ) is a benzimidazole anthelmintic drug widely used for treating parasitic infections by disrupting microtubule formation and function through tubulin binding. Recently, its use has extended to include anticancer applications, leading to increased environmental exposure to benzimidazole drugs. However, the impact of FBZ on neural development in aquatic organisms, particularly in aquatic vertebrates, remains poorly understood. This study aimed to investigate the potential developmental toxicity of FBZ during neural development using zebrafish model. Various assessments, including analysis of overall developmental changes, morphological abnormalities, apoptosis, gene expression alterations, axon length measurements, and electrophysiological neural function, were performed. FBZ exposure resulted in concentration-dependent effects on survival rate, hatching rate, heartbeat, and the occurrence of developmental abnormalities. Notably, FBZ-induced changes included reductions in body length, head size, and eye size, as well as the detection of apoptotic cells in the central nervous system. Gene expression analysis revealed upregulation of apoptosis-related genes (p53, casp3, and casp8), downregulation of neural differentiation-related genes (shha, nrd, ngn1, and elavl3), and alterations in neural maturation and axon growth-related genes (gap43, mbp, and syn2a). Additionally, shortened motor neuron axon length and impaired electrophysiological neural function were observed. These findings provide novel insights into the potential risks of FBZ on the neural development of zebrafish embryos, emphasizing the need for risk prevention strategies and therapeutic approaches to address the environmental toxicity of benzimidazole anthelmintics.

Comparative spatial transcriptomic profiling of severe acute respiratory syndrome coronavirus 2 Delta and Omicron variants infections in the lungs of cynomolgus macaques.

Recently emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants are generally less pathogenic than previous strains. However, elucidating the molecular basis for pulmonary immune response alterations is challenging owing to the virus's heterogeneous distribution within complex tissue structure. Here, we revealed the spatial transcriptomic profiles of pulmonary microstructures at the SARS-CoV-2 infection site in the nine cynomolgus macaques upon inoculation with the Delta and Omicron variants. Delta- and Omicron-infected lungs had upregulation of genes involved in inflammation, cytokine response, complement, cell damage, proliferation, and differentiation pathways. Depending on the tissue microstructures (alveoli, bronchioles, and blood vessels), there were differences in the types of significantly upregulated genes in each pathway. Notably, a limited number of genes involved in cytokine and cell damage response were differentially expressed between bronchioles of the Delta- and Omicron-infection groups. These results indicated that despite a significant antigenic shift in SARS-CoV-2, the host immune response mechanisms induced by the variants were relatively consistent, with limited transcriptional alterations observed only in large airways. This study may aid in understanding the pathogenesis of SARS-CoV-2 and developing a clinical strategy for addressing immune dysregulation by identifying potential transcriptional biomarkers within pulmonary microstructures during infection with emerging variants.

Postnatal exposure to trimethyltin chloride induces retinal developmental neurotoxicity in mice via glutamate and its transporter related changes.

Exposure to toxic substances during postnatal period is one of the major factors causing retinal developmental defects. The developmental toxicity of trimethyltin chloride (TMT), a byproduct of an organotin compound widely used in agriculture and industrial fields, has been reported; however, the effect on the mammalian retina during postnatal development and the mechanism have not been elucidated to date. We exposed 0.75 and 1.5 mg/kg of TMT to neonatal ICR mice (1:1 ratio of male and female) up to postnatal day 14 and performed analysis of the retina: histopathology, apoptosis, electrophysiological function, glutamate concentration, gene expression, and fluorescence immunostaining. Exposure to TMT caused delayed eye opening, eye growth defect and thinning of retinal layer. In addition, apoptosis occurred in the retina along with b-wave and spiking activity changes in the micro-electroretinogram. These changes were accompanied by an increase in the concentration of glutamate, upregulation of astrocyte-related genes, and increased expression of glial excitatory amino acid transporter (EAAT) 1 and 2. Conversely, EAAT 3, 4, and 5, mainly located in the neurons, were decreased. Our results are the first to prove postnatal retinal developmental neurotoxicity of TMT at the mammalian model and analyze the molecular, functional as well as morphological aspects to elucidate possible mechanisms: glutamate toxicity with EAAT expression changes. These mechanisms may suggest not only a strategy to treat but also a clue to prevent postnatal retina developmental toxicity of toxic substances.

Use of laboratory animals and issues regarding the procurement of animals for research in Korea.

BACKGROUND: Laboratory animals remain critical to biomedical research, despite the increasing availability of alternative approaches. Indeed, scientists strive to reduce and refine and replace the use of laboratory animals, even in the face of public calls for ever-more stringent regulation for the protection and care of animals in research. This report outlines the current status and legal regulatory issues with regard to the procurement and use of animals for research in Korea. RESULTS: The number of animals used for education and research purposes was increased nationwide, from 2.5 to 4.9 million in 2015 and 2021, respectively. When compared with figures from the UK, institutions in Korea were found to use more mammals such as mice and dogs. In our research, we identified three major issues concerning recent animal supply in Korea, particularly: (1) Purchase of dogs from unregistered animal supplier for a dog cloning project; (2) Purchase of dogs from an unclear source for veterinary education and training; (3) Illegal cat experiments using cats obtained from unauthorized routes. CONCLUSIONS: Our findings support the notion that alternatives to laboratory animal research should be implemented. We conclude that improvements in the regulations and guidelines for animal suppliers, together with the recent introduction of legislation will improve animal safety and wellbeing of animals in laboratory research in Korea.

Evaluation of exhaust air dust polymerase chain reaction as a supplement method for soiled bedding sentinel monitoring in specific pathogen free mouse facility using two different individually ventilated cage racks.

Health monitoring is essential for ensuring animal health and reliable research results. Each animal facility should establish adequate health monitoring methods, and microbiological quality control should be implemented through regular health surveillance. Recently, specific pathogen free (SPF) mice have been housed in individually ventilated cage (IVC) racks in the majority of mouse facilities globally, and health monitoring is implemented using a soiled bedding sentinel (SBS). Even though SBS monitoring is a standard method, it has a limitation in that some pathogens are not sufficiently transmitted to the sentinel housed in the IVC. The exhaust air dust polymerase chain reaction (EAD PCR) method has been reported to be a reliable complementary method to SBS monitoring based on research findings. In Korea, health monitoring programs using EAD PCR have not yet been applied to laboratory animal facilities. The microbiological status of mouse colonies housed in the two IVC racks was compared using SBS and EAD PCR monitoring in our SPF mouse facility. Except for Helicobacter spp. and Staphylococcus aureus, the detection of 16 pathogens did not differ between the two methods. In the detection of Helicobacter spp., EAD PCR was found to be more sensitive than SBS. Helicobacter spp. were not found by SBS, whereas four S. aureus positive samples were detected by either SBS or EAD PCR test. According to our findings, EAD PCR can be used as a supplement to SBS monitoring. Moreover, EAD PCR can reduce the number of animals used, making it a 3R (Replacement, Reduction, Refinement)-consistent method.

Improvement plans on the operation of the Institutional Animal Care and Use Committee: focusing on the case of Seoul National University.

BACKGROUND: The Institutional Animal Care and Use Committee (IACUC) became compulsory in 2008 by the Animal Protection Act in Korea. Seoul National University (SNU), which conducts 5% of Korea's total animal protocol reviews and uses 10% of national laboratory animal usage, has been influential in the review of animal protocols and management of animal facilities. This study was undertaken to suggest the operational improvement of the IACUC. It focused on the case of SNU. RESULTS: The methodological framework consists of a qualitative approach. In particular, this study is focused on the grounded theory approach and sixty people were surveyed through purposeful sampling. Through this study, we found that various practical educations are necessary such as: (1) education for researchers on how to write a protocol, (2) standardization of screening criteria for various animal experiments by presenting various cases, (3) training on a detailed understanding of relevant laws and policies. In particular, an integrated management system, making it possible to share information among the related committees, would be essential for smoother operation of the IACUC. CONCLUSIONS: If various levels of education and the integrated management system are established, it will be possible to enhance the excellence of researchers and to better manage the operation of the IACUC.

Operational issues of the Institutional Animal Care and Use Committee in Korea.

Korean Institutional Animal Care and Use Committee (IACUC) is currently facing some operational pressing issues. 1) Review of the animal protocol containing controversial technology. 2) Review of the multi-institution animal protocol. 3) Review of veterinary clinical trials for client-owned animals. 4) Delay the review process in large institutions with a single IACUC. Here, the following three solutions are proposed to address the above issues. 1) Establishment of public IACUC. 2) Establishment of the Veterinary Clinical Study Committee as an advisory body to the IACUC. 3) Operating multiple committees rather than increasing the number of committee members on a single committee.

Digital selective transformation and patterning of highly conductive hydrogel bioelectronics by laser-induced phase separation.

The patterning of poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) hydrogels with excellent electrical property and spatial resolution is a challenge for bioelectronic applications. However, most PEDOT:PSS hydrogels are fabricated by conventional manufacturing processes such as photolithography, inkjet printing, and screen printing with complex fabrication steps or low spatial resolution. Moreover, the additives used for fabricating PEDOT:PSS hydrogels are mostly cytotoxic, thus requiring days of detoxification. Here, we developed a previously unexplored ultrafast and biocompatible digital patterning process for PEDOT:PSS hydrogel via phase separation induced by a laser. We enhanced the electrical properties and aqueous stability of PEDOT:PSS by selective laser scanning, which allowed the transformation of PEDOT:PSS into water-stable hydrogels. PEDOT:PSS hydrogels showed high electrical conductivity of 670 S/cm with 6-µm resolution in water. Furthermore, electrochemical properties were maintained even after 6 months in a physiological environment. We further demonstrated stable neural signal recording and stimulation with hydrogel electrodes fabricated by laser.

Eliminating murine norovirus, Helicobacter hepaticus, and intestinal protozoa by embryo transfer for an entire mouse barrier facility.

Pathogens can affect physiological and immunological reactions in immunocompromised animals and genetically engineered mice. Specifically, murine norovirus (MNV), Helicobacter, and intestinal protozoa are prevalent in rodent laboratory facilities worldwide. In this study, microbiological test results of the soiled bedding of sentinel mice showed the prevalence of MNV (50.9%, 28/55), Helicobacter hepaticus (29.1%, 16/55), Trichomonas spp. (14.5%, 8/55), and Entamoeba spp. (32.7%, 18/55). No single infections were detected as all cases were confirmed to have complex infections with two or four pathogens. In previous studies, the success rate of the cross-fostering method was not perfect; therefore, in this study, the entire mouse strain of the SPF rodent facility was rederived using embryo transfer. For up to three years, we confirmed that the results were negative with regular health surveillance tests. Embryo transfer was, thus, determined to be an effective method for the rederivation of specific pathogen free (SPF) barrier mouse facilities. This is the report for the effectiveness of embryo transfer as an example of successful microbiological clean-up of a mouse colony with multiple infections in an entire SPF mouse facility and embryo transfer may be useful for rederiving.

Cynomolgus Macaque Model for COVID-19 Delta Variant.

With the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, which are randomly mutated, the dominant strains in regions are changing globally. The development of preclinical animal models is imperative to validate vaccines and therapeutics against SARS-CoV-2 variants. The objective of this study was to develop a non-human primate (NHP) model for SARS-CoV-2 Delta variant infection. Cynomolgus macaques infected with Delta variants showed infectious viruses and viral RNA in the upper (nasal and throat) and lower respiratory (lung) tracts during the acute phase of infection. After 3 days of infection, lesions consistent with diffuse alveolar damage were observed in the lungs. For cellular immune responses, all macaques displayed transient lymphopenia and neutrophilia in the early stages of infection. SARS-CoV-2 Delta variant spike protein-specific IgM, IgG, and IgA levels were significantly increased in the plasma of these animals 14 days after infection. This new NHP Delta variant infection model can be used for comparative analysis of the difference in severity between SARS-CoV-2 variants of concern and may be useful in the efficacy evaluation of vaccines and universal therapeutic drugs for mutations.

Germinal Center-Induced Immunity Is Correlated With Protection Against SARS-CoV-2 Reinfection But Not Lung Damage.

Germinal centers (GCs) elicit protective humoral immunity through a combination of antibody-secreting cells and memory B cells, following pathogen invasion or vaccination. However, the possibility of a GC response inducing protective immunity against reinfection following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unknown. We found GC activity was consistent with seroconversion observed in recovered macaques and humans. Rechallenge with a different clade of virus resulted in significant reduction in replicating virus titers in respiratory tracts in macaques with high GC activity. However, diffuse alveolar damage and increased fibrotic tissue were observed in lungs of reinfected macaques. Our study highlights the importance of GCs developed during natural SARS-CoV-2 infection in managing viral loads in subsequent infections. However, their ability to alleviate lung damage remains to be determined. These results may improve understanding of SARS-CoV-2-induced immune responses, resulting in better coronavirus disease 2019 (COVID-19) diagnosis, treatment, and vaccine development.

Next-Generation Intestinal Toxicity Model of Human Embryonic Stem Cell-Derived Enterocyte-Like Cells.

The gastrointestinal tract is the most common exposure route of xenobiotics, and intestinal toxicity can result in systemic toxicity in most cases. It is important to develop intestinal toxicity assays mimicking the human system; thus, stem cells are rapidly being developed as new paradigms of toxicity assessment. In this study, we established human embryonic stem cell (hESC)-derived enterocyte-like cells (ELCs) and compared them to existing in vivo and in vitro models. We found that hESC-ELCs and the in vivo model showed transcriptomically similar expression patterns of a total of 10,020 genes than the commercialized cell lines. Besides, we treated the hESC-ELCs, in vivo rats, Caco-2 cells, and Hutu-80 cells with quarter log units of lethal dose 50 or lethal concentration 50 of eight drugs-chloramphenicol, cycloheximide, cytarabine, diclofenac, fluorouracil, indomethacin, methotrexate, and oxytetracycline-and then subsequently analyzed the biomolecular markers and morphological changes. While the four models showed similar tendencies in general toxicological reaction, hESC-ELCs showed a stronger correlation with the in vivo model than the immortalized cell lines. These results indicate that hESC-ELCs can serve as a next-generation intestinal toxicity model.

The status and issues of the Institutional Animal Care and Use Committee of Seoul National University: from its establishment to the present day.

The Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU) plays a key role in monitoring and managing the humane use of animals in scientific research. Here, as one of the pioneers of the IACUC in Korea, we reported SNU-IACUC operations and activities including committee establishment and legal formulation, protocol review, and post-approval monitoring of protocols, which the IACUC has undertaken in the last decade. In addition, legal regulations and improvements were also discussed, and encompassed the limited number of committee members and the single IACUC policy in Korea. As of December, 2020, amendments are on the table at the National Assembly. We also emphasized the independent nature of the IACUC in protecting activities, including approval and monitoring animal experiments, and its public role in narrowing the knowledge gap between society and scientists. Thus, the aim of this report is to help society and scientists understand the operations of the SNU-IACUC and its role in animal welfare.