Retrospective Assessment of Animals Experimentation Projects in Romania – A Critical Analysis of Non-Technical Summaries

The aim of the present inquiry was to design an overview about the non-technical summaries available on the web page of the National Sanitary Veterinary and Food Safety Agency (Romania). We analyzed the compliance to the requirement of replacement, reduction and refinement (the 3R’s). 56 projects were found, the most of them (48), were assigned as “basic research”, 2 as “translational and applied research”, 2 as “regulatory use and routine analysis”, while 4 projects targeted “higher education or training”. The most common animals species used were rats, and mice, followed by rabbits, pigs, hamsters, guinea pigs and frogs (Rana sp.). According to cumulative severity of the project, 20 projects were classified as “severe”, six as “moderate”, three “mild”, and three as “non-recovery”. 24 projects had nor a severity assessment, neither a proper description of the level of severity. In our opinion, the number of projects classified as “severe” is too high; at least for some of them, the level of severity could be reduced using appropriate pain control techniques and / or human end-points. Overall, our recommendation is further improving the knowledge and skills of the personal involved in authorization and execution of the projects and of the authority inspectors that authorized the projects

Long-term treatment with chloroquine increases lifespan in middle-aged male mice possibly via autophagy modulation, proteasome inhibition and glycogen metabolism

Previous studies have shown that the polyamine spermidine increased the maximum life span in C. elegans and the median life span in mice. Since spermidine increases autophagy, we asked if treatment with chloroquine, an inhibitor of autophagy, would shorten the lifespan of mice. Recently, chloroquine has intensively been discussed as a treatment option for COVID-19 patients. To rule out unfavorable long-term effects on longevity, we examined the effect of chronic treatment with chloroquine given in the drinking water on the lifespan and organ pathology of male middle-aged NMRI mice. We report that, surprisingly, daily treatment with chloroquine extended the median life span by 11.4% and the maximum life span of the middle-aged male NMRI mice by 11.8%. Subsequent experiments show that the chloroquine-induced lifespan elevation is associated with dose-dependent increase in LC3B-II, a marker of autophagosomes, in the liver and heart that was confirmed by transmission electron microscopy. Quite intriguingly, chloroquine treatment was also associated with a decrease in glycogenolysis in the liver suggesting a compensatory mechanism to provide energy to the cell. Accumulation of autophagosomes was paralleled by an inhibition of proteasome-dependent proteolysis in the liver and the heart as well as with decreased serum levels of insulin growth factor binding protein-3 (IGFBP3), a protein associated with longevity. We propose that inhibition of proteasome activity in conjunction with an increased number of autophagosomes and decreased levels of IGFBP3 might play a central role in lifespan extension by chloroquine in male NMRI mice.UEFISCDI (EU Horizon 2020 Research and Innovation Programme), Consiliul National al Cercetarii Stiintifice (CNCS), Unitatea Executiva pentru Finantarea Invatamantului Superior, a Cercetarii, Dezvoltarii si Inovarii (UEFISCDI

Development of Innovative Biocomposites with Collagen, Keratin and Hydroxyapatite for Bone Tissue Engineering

This study follows the process for the development of an innovative biomimetic composite derived from bovine collagen with keratin, with hydroxyapatite being hybridized into its architecture, and it builds a comprehensive evaluation of the composite’s characteristics. The novel biomimetic materials are tailored with special traits to be achieved for the repair of osteochondral defects (OCDs). The purpose of the present research is to create a reliable effective alternative to existing bone graft materials while leveraging the intrinsic properties of the components for enhanced osteoinduction and integration. The composites were characterized based on their morphological properties, including water absorption, through scanning electron microscopy (SEM), and their structural properties were characterized by Fourier-Transform Infrared Spectroscopy (FTIR). Biological performance was assessed in vitro using human bone marrow mesenchymal stem cells (BMSCs), focusing on cytotoxicity, cell viability, and the ability to support cell colonization with forthcoming results. This in vivo study illustrates the real potential that this class of novel composites exhibits in regard to bone and cartilage tissue engineering and encourages further exploration and development for future clinical applications

Developing a Novel Murine Meningococcal Meningitis Model Using a Capsule-Null Bacterial Strain

Background: Neisseria meningitidis (meningococcus) is a Gram-negative bacterium that colonises the nasopharynx of about 10% of the healthy human population. Under certain conditions, it spreads into the body, causing infections with high morbidity and mortality rates. Although the capsule is the key virulence factor, unencapsulated strains have proved to possess significant clinical implications as well. Meningococcal meningitis is a primarily human infection, with limited animal models that are dependent on a variety of parameters such as bacterial virulence and mouse strain. In this study, we aimed to develop a murine Neisseria meningitidis meningitis model to be used in the study of various antimicrobial compounds. Method: We used a capsule-deficient Neisseria meningitidis strain that was thoroughly analysed through various methods. The bacterial strain was incubated for 48 h in brain–heart infusion (BHI) broth before being concentrated and injected intracisternally to bypass the blood–brain barrier in CD-1 mice. This prolonged incubation time was a key factor in increasing the virulence of the bacterial strain. A total of three more differently prepared inoculums were tested to further solidify the importance of the protocol (a 24-h incubated inoculum, a diluted inoculum, and an inactivated inoculum). Antibiotic treatment groups were also established. The clinical parameters and number of deaths were recorded over a period of 5 days, and comatose mice with no chance of recovery were euthanised. Results: The bacterial strain was confirmed to have no capsule but was found to harbour a total of 56 genes coding virulence factors, and its antibiotic susceptibility was established. Meningitis was confirmed through positive tissue culture and histological evaluation, where specific lesions were observed, such as perivascular sheaths with inflammatory infiltrate. In the treatment groups, survival rates were significantly higher (up to 81.25% in one of the treatment groups compared to 18.75% in the control group). Conclusion: We managed to successfully develop a cost-efficient murine (using simple CD-1 mice instead of expensive transgenic mice) meningococcal meningitis model using an unencapsulated strain with a novel method of preparation

Increased Degradation Rates in the Components of the Mitochondrial Oxidative Phosphorylation Chain in the Cerebellum of Old Mice

Brain structures differ in the magnitude of age-related neuron loss with the cerebellum being more affected. An underlying cause could be an age-related decline in mitochondrial bioenergetics. Successful aging of mitochondria reflects a balanced turnover of proteins involved in mitochondrial biogenesis and mitophagy. Thus, an imbalance in mitochondrial turnover can contribute to the diminution of cellular function seen during aging. Mitochondrial biogenesis and mitophagy are mediated by a set of proteins including MFN1, MFN2, OPA1, DRP1, FIS1 as well as DMN1l and DNM1, all of which are required for mitochondrial fission. Using N15 labeling, we report that the turnover rates for DMN1l and FIS1 go in opposite directions in the cerebellum of 22-month-old C57BL6j mice as compared to 3-month-old mice. Previous studies have reported decreased turnover rates for the mitochondrial respiratory complexes of aged rodents. In contrast, we found increased turnover rates for mitochondrial proteins of the oxidative phosphorylation chain in the aged mice as compared to young mice. Furthermore, the turnover rate of the components that are most affected by aging –complex III components (ubiquinol cytochrome C oxidoreductase) and complex IV components (cytochrome C oxidase)– was significantly increased in the senescent cerebellum. However, the turnover rates of proteins involved in mitophagy (i.e., the proteasomal and lysosomal degradation of damaged mitochondria) were not significantly altered with age. Overall, our results suggest that an age-related imbalance in the turnover rates of proteins involved in mitochondrial biogenesis and mitophagy (DMN1l, FIS1) in conjunction with an age-related imbalance in the turnover rates of proteins of the complexes III and IV of the electron transfer chain, might impair cerebellar mitochondrial bioenergetics in old mice

Table_1.XLSX

<p>Brain structures differ in the magnitude of age-related neuron loss with the cerebellum being more affected. An underlying cause could be an age-related decline in mitochondrial bioenergetics. Successful aging of mitochondria reflects a balanced turnover of proteins involved in mitochondrial biogenesis and mitophagy. Thus, an imbalance in mitochondrial turnover can contribute to the diminution of cellular function seen during aging. Mitochondrial biogenesis and mitophagy are mediated by a set of proteins including MFN1, MFN2, OPA1, DRP1, FIS1 as well as DMN1l and DNM1, all of which are required for mitochondrial fission. Using N15 labeling, we report that the turnover rates for DMN1l and FIS1 go in opposite directions in the cerebellum of 22-month-old C57BL6j mice as compared to 3-month-old mice. Previous studies have reported decreased turnover rates for the mitochondrial respiratory complexes of aged rodents. In contrast, we found increased turnover rates for mitochondrial proteins of the oxidative phosphorylation chain in the aged mice as compared to young mice. Furthermore, the turnover rate of the components that are most affected by aging –complex III components (ubiquinol cytochrome C oxidoreductase) and complex IV components (cytochrome C oxidase)– was significantly increased in the senescent cerebellum. However, the turnover rates of proteins involved in mitophagy (i.e., the proteasomal and lysosomal degradation of damaged mitochondria) were not significantly altered with age. Overall, our results suggest that an age-related imbalance in the turnover rates of proteins involved in mitochondrial biogenesis and mitophagy (DMN1l, FIS1) in conjunction with an age-related imbalance in the turnover rates of proteins of the complexes III and IV of the electron transfer chain, might impair cerebellar mitochondrial bioenergetics in old mice.</p