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

Noninvasive measurement of intraocular pressure in rats with the iCare TonoVet rebound tonometer

Rebound tonometry is an accurate method of measuring intraocular pressure (IOP) that is easy to perform and does not involve the use of anesthesia. This, together with biological and genetic advances contributes to the use of ocular etiopathogenetic data of rats. The aim of the study was to evaluate the applicability, reproducibility and accuracy of a rebound tonometer in measuring IOP in rats. IOP was measured three times, at different time intervals, in 40 male and female Sprague Dawley rats, 10 weeks old. The animals studied were kept in normal living conditions, not subject to external stress. The mean IOP expressed consecutively of six successive measurements for each eye was determined with the iCare TonoVet rebound tonometer. The readings generated gave IOP values between 13.3 - 14.5 mmHg in males and 12.6-16.1 mmHg in females. There were no significant differences between the eyes in terms of the values obtained in a measurement so the variability was 0.02 mmHg. Rebound tonometry is convenient, can be used without topical anesthesia and provides fast and accurate results. These can be useful to the clinician, when we talk about the rat as a pet or to scientists, when the rat is chosen as an animal model for various biomedical research

The rat and the sheep, animal models for the study of periodontitis and induced periimplantitis of bacterial strains specific to human oral microbiote

Periodontitis and periimplantitis are two diseases that have as a common element the progressive loss of alveolar bone, eventually leading to the loss of teeth and dental implants. The causes of the two diseases are multiple but the composition of the local bacterial biofilm is one of the important triggers. The aim of this review was to establish the main bacterial strains that can induce experimental periimplantitis and periodontitis as well as the techniques by which diseases can reproduce. The rat and the sheep are commonly used animal models in this branch of research because it reflects the main characteristics of human periodontitis or periimplantitis. The results obtained from the recent literature show that Porfiromonas gingivalis, Aggregatibacter actinomicemcomitans, Streptococcus oralis or Fusobacterium nucleatum (bacterial species commonly found in the human oral microbiota) are among the bacteria that can easily reproduce the two diseases of the oral cavity. Induction techniques include oral gavage, ligation technique, lipopolysaccharide injection, or the use of preinfected implant devices. The data accumulated in this review will be useful for research on the pathology of periodontal or periimplant diseases but also the approach of innovative therapies

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

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

Table_2.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

Technology transfer of oil-in-water emulsion adjuvant manufacturing for pandemic influenza vaccine production in Romania: preclinical evaluation of split virion inactivated H5N1 vaccine with adjuvant

Millions of seasonal and pandemic influenza vaccine doses containing oil-in-water emulsion adjuvant have been administered in order to enhance and broaden immune responses and to facilitate antigen sparing. Despite the enactment of a Global Action Plan for Influenza Vaccines and a multi-fold increase in production capabilities over the past 10 years, worldwide capacity for pandemic influenza vaccine production is still limited. In developing countries, where routine influenza vaccination is not fully established, additional measures are needed to ensure adequate supply of pandemic influenza vaccines without dependence on the shipment of aid from other, potentially impacted first-world countries. Adaptation of influenza vaccine and adjuvant technologies by developing country influenza vaccine manufacturers may enable antigen sparing and corresponding increases in global influenza vaccine coverage capacity. Following on previously described work involving the technology transfer of oil-in-water emulsion adjuvant manufacturing to a Romanian vaccine manufacturing institute, we herein describe the preclinical evaluation of inactivated split virion H5N1 influenza vaccine with emulsion adjuvant, including immunogenicity, protection from virus challenge, antigen sparing capacity, and safety. In parallel with the evaluation of the bioactivity of the tech-transferred adjuvant, we also describe the impact of concurrent antigen manufacturing optimization activities. Depending on the vaccine antigen source and manufacturing process, inclusion of adjuvant was shown to enhance and broaden functional antibody titers in mouse and rabbit models, promote protection from homologous virus challenge in ferrets, and facilitate antigen sparing. Besides scientific findings, the operational lessons learned are delineated in order to facilitate adaptation of adjuvant technologies by other developing country institutes to enhance global pandemic influenza preparedness