Big data and its impact on the 3Rs: a home cage monitoring oriented review.

Undisturbed home cage recording of mouse activity and behavior has received increasing attention in recent years. In parallel, several technologies have been developed in a bid to automate data collection and interpretation. Thanks to these expanding technologies, massive datasets can be recorded and saved in the long term, providing a wealth of information concerning animal wellbeing, clinical status, baseline activity, and subsequent deviations in case of experimental interventions. Such large datasets can also serve as a long-term reservoir of scientific data that can be reanalyzed and repurposed upon need. In this review, we present how the impact of Big Data deriving from home cage monitoring (HCM) data acquisition, particularly through Digital Ventilated Cages (DVCs), can support the application of the 3Rs by enhancing Refinement, Reduction, and even Replacement of research in animals.

Data repurposing from digital home cage monitoring enlightens new perspectives on mouse motor behaviour and reduction principle.

In this longitudinal study we compare between and within-strain variation in the home-cage spatial preference of three widely used and commercially available mice strains-C57BL/6NCrl, BALB/cAnNCrl and CRL:CD1(ICR)-starting from the first hour post cage-change until the next cage-change, for three consecutive intervals, to further profile the circadian home-cage behavioural phenotypes. Cage-change can be a stressful moment in the life of laboratory mice, since animals are disturbed during the sleeping hours and must then rapidly re-adapt to a pristine environment, leading to disruptions in normal motor patterns. The novelty of this study resides in characterizing new strain-specific biological phenomena, such as activity along the cage walls and frontality, using the vast data reserves generated by previous experimental data, thus introducing the potential and exploring the applicability of data repurposing to enhance Reduction principle when running in vivo studies. Our results, entirely obtained without the use of new animals, demonstrate that also when referring to space preference within the cage, C57BL/6NCrl has a high variability in the behavioural phenotypes from pre-puberty until early adulthood compared to BALB/cAnNCrl, which is confirmed to be socially disaggregated, and CRL:CD1(ICR) which is conversely highly active and socially aggregated. Our data also suggest that a strain-oriented approach is needed when defining frequency of cage-change as well as maximum allowed animal density, which should be revised, ideally under the EU regulatory framework as well, according to the physiological peculiarities of the strains, and always avoiding the "one size fits all" approach.

Disease Animal Models for Cancer Research.

Despite nonanimal methods (NAMs) are more and more exploited and new NAMs are developed and validated, animal models are still used in cancer research. Animals are used at multiple levels, from understanding molecular traits and pathways, to mimicking clinical aspects of tumor progression, to drug testing. In vivo approaches are not trivial and involve cross-disciplinary knowledge: animal biology and physiology, genetics, pathology, and animal welfare.The aim of this chapter is not to list and address all animal models used in cancer research. Instead, the authors would like to guide experimenters in the strategies to adopt in both planning and performing in vivo experimental procedures, including the choice of cancer animal models.

Development and in vivo validation of phospholipid-based depots for the sustained release of bupivacaine.

By direct deposition of the drug at the local site of action, injectable depot formulations - intended for treatment of a local disease or for local intervention - are designed to limit the immediate exposure of the active principle at a systemic level and to reduce the frequency of administration. To overcome known drawbacks in the production of some marketed phospholipid-based depots, here we propose to manufacture drug-loaded negatively charged liposomes through conventional technologies and to control their aggregation mixing a solution of divalent cations prior to administration. We identified phosphatidylglycerol (PG) as the most suitable phospholipid for controlled aggregation of the liposomes and to modulate the release of the anesthetic bupivacaine (BUP) from liposomal depots. In vivo imaging of the fluorescently-labelled liposomes showed a significantly higher retention of the PG liposomes at the injection site with respect to zwitterionic ones. In situ mixing of PG liposomes with calcium salts significantly extended the area under the curve of BUP in plasma compared to the non-depot system. Overall, controlling the aggregation of negatively charged liposomes with divalent cations not only modulated the particle clearance from the injection site but also the release in vivo of a small amphipathic drug such as BUP.

Puberty onset curve in CD (Sprague Dawley) and Long Evans outbred male rats.

The exact timing of puberty is fundamental in preclinical studies. In male rats, the age at sexual maturity varies considerably between 40 and 60 days of age. Here, we summarize pubertal onset evaluation of two outbred rat strains (Crl:CD(SD) and Crl:LE), relying on the balano-preputial separation test. Evaluation was carried out on animals under standard barrier conditions, from four to nine weeks of age. In the Crl:CD(SD) population, 90% of males gained puberty at week 6, and 100% in the following weeks, whereas 75% of Crl:LE reached puberty at week 6, 90% at week 7 and 100% from week 8. Remarkably, in both strains, puberty onset was gained at the average weight of 200 g, suggesting that weight range, not only age range, can be considered a biomarker of puberty onset in these two strains. On the contrary, descended testes cannot be considered an additional factor to identify full puberty onset either in Crl:CD(SD) or Crl:LE rats.

Phenotyping spontaneous locomotor activity in inbred and outbred mouse strains by using Digital Ventilated Cages.

Mouse strains differ markedly in all behaviors, independently of their genetic background. We undertook this study to disentangle the diurnal activity and feature key aspects of three non-genetically altered mouse strains widely used in research, C57BL/6NCrl (inbred), BALB/cAnNCrl (inbred) and CRL:CD1(ICR) (outbred). With this aim, we conducted a longitudinal analysis of the spontaneous locomotor activity of the mice during a 24-h period for 2 months, in two different periods of the year to reduce the seasonality effect. Mice (males and females) were group-housed in Digital Ventilated Cages (Tecniplast), mimicking standard housing conditions in research settings and avoiding the potential bias provided in terms of locomotor activity by single housing. The recorded locomotor activity was analyzed by relying on different and commonly used circadian metrics (i.e., day and night activity, diurnal activity, responses to lights-on and lights-off phases, acrophase and activity onset and regularity disruption index) to capture key behavioral responses for each strain. Our results clearly demonstrate significant differences in the circadian activity of the three selected strains, when comparing inbred versus outbred as well as inbred strains (C57BL/6NCrl versus BALB/cAnNCrl). Conversely, males and females of the same strain displayed similar motor phenotypes; significant differences were recorded only for C57BL/6NCrl and CRL:CD1(ICR) females, which displayed higher average locomotor activity from prepuberty to adulthood. All strain-specific differences were further confirmed by an unsupervised machine learning approach. Altogether, our data corroborate the concept that each strain behaves under characteristic patterns, which needs to be taken into consideration in the study design to ensure experimental reproducibility and comply with essential animal welfare principles.