A Brief Interpretation of CCAC Guide on Humane Endpoints of Animals: 2022 Edition

The 2022 Canadian Council On Animal Care (CCAC) guidelines: Identification of Scientific Endpoints, Human Intervention Points, and Cumulative Endpoints (CCAC Guide) supplements existing laboratory animal humane endpoint theory according to the latest available literature. This article summarized the main content of the 2022 CCAC Guide, and elaborated and analyzed the determination, implementation and supervision of the scientific endpoints, humane intervention points, and cumulative endpoints of animal experiments, in order to provide useful reference and information

Identification and validation of reference genes for gene expression studies in postharvest rose flower (Rosa hybrida)

AbstractOptimal reference genes are important for data normalization so that accurate and reliable gene expression measurements may be obtained in both semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR) methods. This study firstly evaluated potential reference genes in petals of rose flower (Rosa hybrida) under postharvest stress conditions and in various floral organs during flower opening and senescence, combining both RT-PCR and qRT-PCR analysis. The expression stabilities of gene members from three traditional housekeeping gene families – actin (RhACT), tubulin (RhTUB) and ubiquitin (RhUBI) – were assessed using two analysis software packages, geNorm and NormFinder. The results showed that, for cut rose flower, the optimal reference genes were RhUBI1 for dehydration treatment and receptacles; RhTUB2 for exogenous ethylene; RhACT4 for gibberellic and abscisic acid treatments, wounding and stamens; RhUBI6 for petals; RhUBI2 for sepals; and RhACT1 for gynoecia, respectively. Our results provide guidelines for reference gene(s) selection under different postharvest conditions and point the way towards more accurate and widespread use of qRT-PCR in rose flower

Loss of FMRP Impaired Hippocampal Long-Term Plasticity and Spatial Learning in Rats

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene that inactivate expression of the gene product, the fragile X mental retardation 1 protein (FMRP). In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to generate Fmr1 knockout (KO) rats by disruption of the fourth exon of the Fmr1 gene. Western blotting analysis confirmed that the FMRP was absent from the brains of the Fmr1 KO rats (Fmr1(exon4-KO)). Electrophysiological analysis revealed that the theta-burst stimulation (TBS)-induced long-term potentiation (LTP) and the low-frequency stimulus (LFS)-induced long-term depression (LTD) were decreased in the hippocampal Schaffer collateral pathway of the Fmr1(exon4-KO) rats. Short-term plasticity, measured as the paired-pulse ratio, remained normal in the KO rats. The synaptic strength mediated by the a -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) was also impaired. Consistent with previous reports, the Fmr1(exon4-KO) rats demonstrated an enhanced 3,5-dihydroxyphenylglycine (DHPG)-induced LTD in the present study, and this enhancement is insensitive to protein translation. In addition, the Fmr1(exon4-KO) rats showed deficits in the probe trial in the Morris water maze test. These results demonstrate that deletion of the Fmr1 gene in rats specifically impairs long-term synaptic plasticity and hippocampus-dependent learning in a manner resembling the key symptoms of FXS. Furthermore, the Fmr1(exon4-KO) rats displayed impaired social interaction and macroorchidism, the results consistent with those observed in patients with FXS. Thus, Fmr1exon4 KO rats constitute a novel rat model of FXS that complements existing mouse models.</p

In vivo long-term investigation of tumor bearing mKate2 by an in-house fluorescence molecular imaging system

Abstract Background Optical imaging is one of the most common, low-cost imaging tools used for investigating the tumor biological behavior in vivo. This study explores the feasibility and sensitivity of a near infrared fluorescent protein mKate2 for a long-term non-invasive tumor imaging in BALB/c nude mice, by using a low-power optical imaging system. Methods In this study, breast cancer cell line MDA-MB-435s expressing mKate2 and MDA-MB-231 expressing a dual reporter gene firefly luciferase (fLuc)-GFP were used as cell models. Tumor cells were implanted in different animal body compartments including subcutaneous, abdominal and deep tissue area and closely monitored in real-time. A simple and low-power optical imaging system was set up to image both fluorescence and bioluminescence in live animals. Results The presence of malignant tissue was further confirmed by histopathological assay. Considering its lower exposure time and no need of substrate injection, mKate2 is considered a superior choice for subcutaneous imaging compared with fLuc. On the contrary, fLuc has shown to be a better option when monitoring the tumor in a diffusive area such as abdominal cavity. Furthermore, both reporter genes have shown good stability and sensitivity for deep tissue imaging, i.e. tumor within the liver. In addition, fLuc has shown to be an excellent method for detecting tumor cells in the lung. Conclusions The combination of mKate2 and fLuc offers a superior choice for long-term non-invasive real-time investigation of tumor biological behavior in vivo

Spatial population distribution dynamics of big cats and ungulates with seasonal and disturbance changes in temperate natural forest

Wildlife conservation and management in human-dominated landscapes are major concerns for wildlife ecologists and managers. The dynamics of human disturbance, combined with seasonal limitations in the availability of nutritious foods, may restrict wildlife population growth and recovery. However, understanding how large mammal species adjust their population distribution in forest habitats with seasonal changes in food and disturbances requires a deeper and more extensive analysis. In this study, we found that three ungulate species, roe deer (Capreolus pygargus), sika deer (Cervus nippon), and wild boar (Sus scrofa), employ robust, conservative, and flexible distribution strategies, respectively, to adapt to the effects of seasonal changes and human disturbances. Moreover, croplands, villages, and grazing have some negative effects on the distribution of roe deer and sika deer, while wild boar can be highly abundant near human land use. Additionally, roe deer, sika deer, and wild boar are also affected by the abundance of shrub species they consume. During the cold season, the populations of the Amur tiger (Panthera tigris altaica) and Amur leopard (P. pardus orientalis) were primarily located near roads and dense forests, respectively. In the warm season, the distribution of both big cats was influenced by prey abundance, and Amur tigers also avoided grazing livestock. Nevertheless, the negative effects of human land use on Amur tigers and wild boars increased during the warm season, which was attributed to more frequent human activities during that time. Consequently, it is crucial to implement season-specific habitat management, particularly by regulating human disturbances during the warm season, in order to promote the recovery and expansion of populations of big cats and ungulates

Multistimuli-Regulated Photochemothermal Cancer Therapy Remotely Controlled <i>via</i> Fe₅C₂ Nanoparticles

Stimuli-controlled drug delivery and release is of great significance in cancer therapy, making a stimuli-responsive drug carrier highly demanded. Herein, a multistimuli-controlled drug carrier was developed by coating bovine serum albumin on Fe₅C₂ nanoparticles (NPs). With a high loading of the anticancer drug doxorubicin, the nanoplatform provides a burst drug release when exposed to near-infrared (NIR) light or acidic conditions. <i>In vitro</i> experiment demonstrated a NIR-regulated cell inhibition that is ascribed from cellular uptake of the carrier and the combination of photothermal therapy and enhanced drug release. The carrier is also magnetic-field-responsive, which enables targeted drug delivery under the guidance of a magnetic field and monitors the theranostic effect by magnetic resonance imaging. <i>In vivo</i> synergistic effect demonstrates that the magnetic-driven accumulation of NPs can induce a complete tumor inhibition without appreciable side effects to the treated mice by NIR irradiation, due to the combined photochemotherapy. Our results highlight the great potential of Fe₅C₂ NPs as a remote-controlled platform for photochemothermal cancer therapy

An Excitatory Neural Assembly Encodes Short-Term Memory in the Prefrontal Cortex

Summary: Short-term memory (STM) is crucial for animals to hold information for a small period of time. Persistent or recurrent neural activity, together with neural oscillations, is known to encode the STM at the cellular level. However, the coding mechanisms at the microcircuitry level remain a mystery. Here, we performed two-photon imaging on behaving mice to monitor the activity of neuronal microcircuitry. We discovered a neuronal subpopulation in the medial prefrontal cortex (mPFC) that exhibited emergent properties in a context-dependent manner underlying a STM-like behavior paradigm. These neuronal subpopulations exclusively comprise excitatory neurons and mainly represent a group of neurons with stronger functional connections. Microcircuitry plasticity was maintained for minutes and was absent in an animal model of Alzheimer’s disease (AD). Thus, these results point to a functional coding mechanism that relies on the emergent behavior of a functionally defined neuronal assembly to encode STM