Rodent Tests Research Articles

Determination of the LD50 with the chick embryo chorioallantoic membrane (CAM) assay as a promising alternative in nanotoxicological evaluation

Toxicity tests in rodents are still considered a controversial topic concerning their ethical justifiability. The chick embryo chorioallantoic membrane (CAM) assay may offer a simple and inexpensive alternative. The CAM assay is easy to perform and has low bureaucratic hurdles. At the same time, the CAM assay allows the application of a broad variety of analytical methods in the field of nanotoxicological research. We evaluated the CAM assay as a methodology for the determination of nanotoxicity. Therefore we calculated the median lethal dose (LD50), performed in vivo microscopy and immunohistochemistry to identify organ-specific accumulation profiles, potential organ damage, and the kinetics of the in vivo circulation of the nanoparticles. Zinc oxide nanoparticles were intravascularly injected on day 10 of the egg development and showed an LD50 of 17.5 µM (1.4 µg/mLeggcontent). In comparison, the LD50 of equivalent amounts of Zn2+ was 4.6 µM (0.6 µg/mLeggcontent). Silica encapsulated ZnO@SiO2 nanoparticles conjugated with fluorescein circulated in the bloodstream for at least 24 h. Particles accumulated mostly in the liver and kidney. In immunohistochemical staining, organ damage was detected only in liver tissue after intravascular injection of zinc oxide nanoparticles in very high concentrations. Zinc oxide nanoparticles showed a different pharmacokinetic profile compared to Zn2+ ions. In conclusion, the CAM assay has proven to be a promising methodology for evaluating nanotoxicity and for the assessment of the in vivo accumulation profiles of nanoparticles. These findings may qualify the methodology for risk assessment of innovative nanotherapeutics in the future.

Behavior Testing in Rodents: Highlighting Potential Confounds Affecting Variability and Reproducibility.

Rodent models of brain disorders including neurodevelopmental, neuropsychiatric, and neurodegenerative diseases are essential for increasing our understanding of underlying pathology and for preclinical testing of potential treatments. Some of the most important outcome measures in such studies are behavioral. Unfortunately, reports from different labs are often conflicting, and preclinical studies in rodent models are not often corroborated in human trials. There are many well-established tests for assessing various behavioral readouts, but subtle aspects can influence measurements. Features such as housing conditions, conditions of testing, and the sex and strain of the animals can all have effects on tests of behavior. In the conduct of behavior testing, it is important to keep these features in mind to ensure the reliability and reproducibility of results. In this review, we highlight factors that we and others have encountered that can influence behavioral measures. Our goal is to increase awareness of factors that can affect behavior in rodents and to emphasize the need for detailed reporting of methods.

Novel 1,3,4-Oxadiazole Derivatives of Pyrrolo[3,4-d]pyridazinone Exert Antinociceptive Activity in the Tail-Flick and Formalin Test in Rodents and Reveal Reduced Gastrotoxicity.

Despite the availability of the current drug arsenal for pain management, there is still a clinical need to identify new, more effective, and safer analgesics. Based on our earlier study, newly synthesized 1,3,4-oxadiazole derivatives of pyrrolo[3,4-d]pyridazinone, especially 10b and 13b, seem to be promising as potential analgesics. The current study was designed to investigate whether novel derivatives attenuate nociceptive response in animals subjected to thermal or chemical noxious stimulus, and to compare this effect to reference drugs. The antinociceptive effect of novel compounds was studied using the tail-flick and formalin test. Pretreatment with novel compounds at all studied doses increased the latency time in the tail-flick test and decreased the licking time during the early phase of the formalin test. New derivatives given at the medium and high doses also reduced the late phase of the formalin test. The achieved results indicate that new derivatives dose-dependently attenuate nociceptive response in both models of pain and exert a lack of gastrotoxicity. Both studied compounds act more efficiently than indomethacin, but not morphine. Compound 13b at the high dose exerts the greatest antinociceptive effect. It may be due to the reduction of nociceptor sensitization via prostaglandin E2 and myeloperoxidase levels decrease.

Neutrophil activation by nanomaterials in vitro: comparing strengths and limitations of primary human cells with those of an immortalized (HL-60) cell line

Assessment of nanomaterial (NM) induced inflammatory responses has largely relied on rodent testing via measurement of leukocyte accumulation in target organs. Despite observations that NMs activate neutrophil driven inflammatory responses in vivo, a limited number of studies have investigated neutrophil responses to NMs in vitro. We compared responses between the human neutrophil-like HL-60 cell line and human primary neutrophils following exposure to silver (Ag), zinc oxide (ZnO), copper oxide (CuO) and titanium dioxide (TiO2) NMs. NM cytotoxicity and neutrophil activation were assessed by measuring cellular metabolic activity, cytokine production, respiratory burst, and release of neutrophil extracellular traps. We observed a similar pattern of response between HL-60 cells and primary neutrophils, however we report that some neutrophil functions are compromised in the cell line. Ag NMs were consistently observed to stimulate neutrophil activation, with CuO NMs inducing similar though weaker responses. TiO2 NMs did not induce a neutrophil response in either cell type. Interestingly, ZnO NMs readily induced activation of HL-60 cells but did not appear to activate primary cells. Our findings are relevant to the development of a tiered testing strategy for NM hazard assessment which promotes the use of non-rodent models. Whilst we acknowledge that HL-60 cells may not be a perfect substitute for primary cells and require further investigation regarding their ability to predict neutrophil activation, we recommend their use for initial screening of NM-induced inflammation. Primary human neutrophils can then be used for more focused assessments of neutrophil activation before progressing to in vivo models where necessary.

Sequential reversal learning: a new touchscreen schedule for assessing cognitive flexibility in mice.

The widespread deficits in cognitive flexibility observed across psychiatric disorders call for improved rodent tests to understand the biology of cognitive flexibility and development of better psychotherapeutics. Current reversal learning paradigms have a forced-choice setup that challenges the interpretation of results. We aimed at developing a free-choice reversal learning test, where images are presented sequentially and animals are free to move, to enable investigation of the cognitive sub-processes that occur during reversal. Behavior in female C57BL/6JOlaHsd mice was characterized using chronic fluoxetine as a reference compound. Additional tests were included to support the interpretation of results and exclude confounding pharmacological effects. Behaviors in vehicle-treated mice were furthermore analyzed for relatedness to deepen the understanding of parameters measured. We found that exploitation of the previously rewarded image was independent of exploration and acquisition of the new reward contingency and could be differentially modulated by fluoxetine, supporting recent theories that these processes are not mutually exclusive. Specifically, fluoxetine reduced mistake rate, premature and perseverative responses, and promoted conservative strategies during reversal without affecting hit rate. These effects appeared to be most prominent during the late stage of reversal learning, where accuracy was above chance level. Analysis of behaviors in vehicle-treated mice suggested that exploitation was related to an impulsive-like deficit in response inhibition, while exploration was more related to motivation. This new schedule was feasible, easy to implement, and can provide a deeper understanding of the cognitive sub-processes during reversal.

Suppressing Anterior Cingulate Cortex Modulates Default Mode Network and Behavior in Awake Rats.

The default mode network (DMN) is a principal brain network in the mammalian brain. Although the DMN in humans has been extensively studied with respect to network structure, function, and clinical implications, our knowledge of DMN in animals remains limited. In particular, the functional role of DMN nodes, and how DMN organization relates to DMN-relevant behavior are still elusive. Here we investigated the causal relationship of inactivating a pivotal node of DMN (i.e., dorsal anterior cingulate cortex [dACC]) on DMN function, network organization, and behavior by combining chemogenetics, resting-state functional magnetic resonance imaging (rsfMRI) and behavioral tests in awake rodents. We found that suppressing dACC activity profoundly changed the activity and connectivity of DMN, and these changes were associated with altered DMN-related behavior in animals. The chemo-rsfMRI-behavior approach opens an avenue to mechanistically dissecting the relationships between a specific node, brain network function, and behavior. Our data suggest that, like in humans, DMN in rodents is a functional network with coordinated activity that mediates behavior.

Open-source raspberry Pi-based operant box for translational behavioral testing in rodents

BackgroundRodents have been used for decades to probe neural circuits involved in behavior. Increasingly, attempts have been developed to standardize training paradigms across labs; and to use visual/auditory paradigms that can be also tested in humans. Commercially available systems are expensive and thus do not scale easily, and are not optimized for electrophysiology. New methodUsing the rich open-source technology built around Raspberry Pi, we were able to develop an inexpensive (<$1000) visual-screen based operant chamber with electrophysiological and optogenetic compatibility. The chamber is operated within MATLAB/Simulink, a commonly used scientific programming language allowing for rapid customization. ResultsHere, we describe and provide all relevant details needed to develop and produce these chambers, and show examples of behavior and electrophysiology data collected using these chambers. We also include all of the tools needed to allow readers to build and develop their own boxes (CAD models for 3D printing and laser-cutting; PCB-board design; all bill of materials for required parts and supplies, and some examples of Simulink models to operate the boxes). Comparison with existing methodsThe new boxes are far more cost-effective than commercially available environments and allow for the combination of automated behavioral testing with electrophysiological read-outs with high temporal precision. ConclusionThese open-source boxes can be used for labs interested in developing high-throughput visual/auditory behavioral assays for ∼ 10th the cost of commercial systems.

Антидепрессивная активность димерного дипептидного миметика BDNF ГСБ-106 при однократном пероральном введении на модели социального стресса у мышей

Изучено влияние низкомолекулярного миметика BDNF — соединения ГСБ-106 в таблетированной лекарственной форме на агедонию в тесте предпочтения раствора сахара и синаптогенез в гиппокампе (Вестерн-блот анализ) при однократном введении внутрь мышам линии C57Bl/6, находящимся в депрессивно-подобном состоянии, вызванном социальным стрессом, в сравнении с трициклическим антидепрессантом амитриптилином. У стрессированных мышей наблюдали статистически достоверное снижение предпочтения раствора сахара в среднем на 5 % (p = 0,04), а также уменьшение содержания синаптофизина в ткани гиппокампа на 27 % (p = 0,01), по сравнению с интактными мышами. ГСБ-106, как и амитриптилин, полностью восстанавливал предпочтение мышами раствора сахара и вызывал тенденцию (p = 0,08) к восстановлению содержания синаптофизина в гиппокаме. Таким образом, дипептидный миметик BDNF ГСБ-106 при однократном пероральном введении мышам на фоне хронического социального стресса оказывал антиагедонический эффект и улучшал нарушенную пластичность мозга.

Development of an antibody cocktail for treatment of Sudan virus infection

Antibody-based therapies are a promising treatment option for managing ebolavirus infections. Several Ebola virus (EBOV)-specific and, more recently, pan-ebolavirus antibody cocktails have been described. Here, we report the development and assessment of a Sudan virus (SUDV)-specific antibody cocktail. We produced a panel of SUDV glycoprotein (GP)-specific human chimeric monoclonal antibodies (mAbs) using both plant and mammalian expression systems and completed head-to-head in vitro and in vivo evaluations. Neutralizing activity, competitive binding groups, and epitope specificity of SUDV mAbs were defined before assessing protective efficacy of individual mAbs using a mouse model of SUDV infection. Of the mAbs tested, GP base-binding mAbs were more potent neutralizers and more protective than glycan cap- or mucin-like domain-binding mAbs. No significant difference was observed between plant and mammalian mAbs in any of our in vitro or in vivo evaluations. Based on in vitro and rodent testing, a combination of two SUDV-specific mAbs, one base binding (16F6) and one glycan cap binding (X10H2), was down-selected for assessment in a macaque model of SUDV infection. This cocktail, RIID F6-H2, provided protection from SUDV infection in rhesus macaques when administered at 50 mg/kg on days 4 and 6 postinfection. RIID F6-H2 is an effective postexposure SUDV therapy and provides a potential treatment option for managing human SUDV infection.

Behavioral effects of the kappa opioid receptor partial agonist nalmefene in tests relevant to depression

Compounds with high affinity at kappa and mu opioid receptors may have clinical utility in treating major depressive disorder. Nalmefene (NMF) is a partial kappa opioid receptor agonist and potent mu opioid receptor antagonist, but there has been no preclinical evaluation of NMF in rodent tests relevant to depression and anxiety. To address this, the effects of NMF on neurochemical and behavioral endpoints in C57BL/6J mice were examined and contrasted with a structurally related analog, naltrexone (NTX). NMF exhibited kappa opioid receptor agonist activity, measured as a reduction in extracellular dopamine release in the nucleus accumbens using in vivo microdialysis following acute but not chronic administration. In the mouse forced swim test, female mice were more responsive to higher doses of NMF and NTX compared to male mice. The behavioral effects of NMF in the forced swim test were blocked in Oprk1−/− and Oprm1−/− mice. Conversely, the effects of NTX were blocked only in Oprm1−/− mice. These results indicate that both kappa and mu opioid receptors mediate the behavioral effects of NMF, but the effects of NTX in this test were modified only by mu opioid receptor engagement. Unlike NTX, NMF did not produce conditioned place aversion in either sex. Finally, NMF's activity in the marble burying test and forced swim test were retained following chronic administration. The sustained effects exerted by NMF on tests that are sensitive to antidepressant and anxiolytic compounds support further investigation of NMF as a potential therapeutic for depression.

Food/feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 59122 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/20)

Food/feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 59122 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/20)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 59122 x 1507 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/21)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 59122 x 1507 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/21)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 1507 x 59122 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/NL/2005/15)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 1507 x 59122 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/NL/2005/15)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 1507 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2004/05)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 1507 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2004/05)

A Review of the Historical Evolutionary Process of Dry and Water Maze Tests in Rodents.

This research provides an overview of the historical advances of the maze tests that are widely used to assess the cognitive impairments in rodents. Particularly, this study focuses on the issue of learning and memory behavioral tests, including dry and water mazes. Several types of mazes have been used in this setting, but their real advantages and applications depend on the type selected by the researcher. We answered some of the basic questions that any interested researcher in such studies may be faced with. The reviewed topics are as follows: the definition of maze learning, the role of the memory in the maze learning, the differences between several types of mazes, and foremost the rationale behind the maze constructions and designs.

Incorporating Laboratory Animal Science into Responsible Biomedical Research.

Biomedical research has made great strides in the past century leading to rapid advances in human life expectancy, all derived from improved understanding, prevention, and treatment of many diseases and conditions. Research involving laboratory animals has played a significant role in this medical progress. However, there continues to be controversy surrounding the use of animals in research, and animal models have been questioned regarding their relevance to human conditions. While research fraud and questionable research practices could potentially contribute to this problem, we argue that a relative ignorance of laboratory animal science has contributed to the "uncontrolled vivarium experiment" that runs parallel to the more controlled scientific experiment. Several variables are discussed, including husbandry, animal environment, social housing, and more, that can contribute to this uncontrolled experiment, and that can simultaneously decrease quality of life for rodent test subjects when ignored. An argument is put forward that laboratory animal veterinarians and scientists can and should play an important role in better controlling such variables. Similarly, the laboratory animal veterinarian and scientist should play an important role in responsible science by addressing complex interdisciplinary challenges.

Adaptation of tape removal test for measurement of sensitivity in perineal area of rat

Regeneration after spinal cord injury is a goal of many studies. Although the most obvious target is to recover motor function, restoration of sensation can also improve the quality of life after spinal cord injury. For many patients, recovery of sensation in the perineal and genital area is a high priority. Currently there is no experimental test in rodents for measuring changes in sensation in the perineal and genital area after spinal cord injury. The aim of our study was to develop a behavioural test for measuring the sensitivity of the perineal and genital area in rats. We have modified the tape removal test used routinely to test sensorimotor deficits after stroke and spinal cord injury to test the perineal area with several variations. A small piece of tape (approximately 1 cm2) was attached to the perineal area. Time to first contact and to the removal of the tape was measured. Each rat was trained for 5 consecutive days and then tested weekly. We compared different rat strains (Wistar, Sprague-Dawley, Long-Evans and Lewis), both genders, shaving and non-shaving and different types of tape. We found that the test was suitable for all tested strains, however, Lewis rats achieved the lowest contact times, but this difference was significant only for the first few days of learning the task. There were no significant differences between gender and different types of tape or shaving. After training the animals underwent dorsal column lesion at T10 and were tested at day 3, 8, 14 and 21. The test detected a sensory deficit, the average time across all animals to sense the stimulus increased from 1′32 up to 3′20. There was a strong relationship between lesion size and tape detection time, and only lesions that extended laterally to the dorsal root entry zone produced significant sensory deficits. Other standard behavioural tests (BBB, von Frey, ladder and Plantar test) were performed in the same animals. There was a correlation between lesion size and deficit for the ladder and BBB tests, but not for the von Frey and Plantar tests. We conclude that the tape removal test is suitable for testing perineal sensation in rats, can be used in different strains and is appropriate for monitoring changes in sensation after spinal cord injury.

D-Cycloserine reverses scopolamine-induced object and place memory deficits in a spontaneous recognition paradigm in rats

d-Cycloserine (DCS) is a partial agonist of the glutamatergic N-methyl-d-aspartate (NMDA) receptor-associated glycine site, and it prevents the amnesic effects of the muscarinic receptor antagonist scopolamine in various memory tests in rodents. In the present study, we tested the hypothesis that DCS has anti-amnesic effects in scopolamine-induced deficits using spontaneous object recognition and place recognition tests. In both tests, scopolamine (0.5 mg/kg, i.p.) was systemically administered 60 min prior to testing, while DCS (7.5, 15, 30 mg/kg, i.p.) was administered 30 min before testing, which consisted of a sample phase (5 min), a delay interval (15 min) and a test phase (2 min). DCS treatment reversed scopolamine-induced deficits in discriminatory behavior during the test phase. However, DCS did not affect decreased object exploration itself or increased thigmotaxis in the open-field arena induced by scopolamine. These results support our hypothesis and suggest differential contributions of glutamatergic-cholinergic system interactions to recognition memory and non-mnemonic exploratory behaviors.

Odour conditioning of positive affective states: Rats can learn to associate an odour with being tickled.

Most associative learning tests in rodents use negative stimuli, such as electric shocks. We investigated if young rats can learn to associate the presence of an odour with the experience of being tickled (i.e. using an experimenter’s hand to mimic rough-and-tumble play), shown to elicit 50 kHz ultrasonic vocalisations (USVs), which are indicative of positive affect. Male, pair-housed Wistar rats (N = 24) were all exposed to two neutral odours (A and B) presented in a perforated container on alternate days in a test arena. Following 60s of exposure, the rats were either tickled on days when odour A (n = 8) or odour B (n = 8) was present, or never tickled (n = 8). When tickled, rats produced significantly more 50 kHz USVs compared to the days when not being tickled, and compared to control rats. The level of anticipatory 50 kHz USVs in the 60s prior to tickling did not differ significantly between the tickled and control rats. As a retrieval test following the odour conditioning, rats were exposed successively in the same arena to three odours: an unknown neutral odour, extract of fox faeces, and either odours A or B. Compared to controls, 50 kHz USVs of tickled rats increased when exposed to the odour they had previously experienced when tickled, indicating that these rats had learned to associate the odour with the positive experience of being tickled. In a test with free access for 5 min to both arms of a T-maze, each containing one of the odours, rats tickled with odour A spent more time in the arm with this odour. This work is the first to test in a fully balanced design whether rats can learn to associate an odour with tickling, and indicates that positive odour conditioning has potential to be used as an alternative to negative conditioning tests.

Automated Forelimb Tasks for Rodents: Current Advantages and Limitations, and Future Promise

Rodent tests of function have advanced our understanding of movement, largely through the human training and testing and manual assessment. Tools such as reaching and grasping of a food pellet have been widely adopted because they are effective and simple to use. However, these tools are time-consuming, subjective, and often qualitative. Automation of training, testing, and assessment has the potential to increase efficiency while ensuring tasks are objective and quantitative. We detail new methods for automating rodent forelimb tests, including the use of pellet dispensers, sensors, computer vision, and home cage systems. We argue that limitations in existing forelimb tasks are driving the innovations in automated systems. We further argue that automated tasks partially address these limitations, and we outline necessary precautions and remaining challenges when adopting these types of tasks. Finally, we suggest attributes of future automated rodent assessment tools that can enable widespread adoption and help us better understand forelimb function in health and disease.