Assessment of zebrafish embryo photomotor response sensitivity and phase-specific patterns following acute- and long-duration exposure to neurotoxic chemicals and chemical weapon precursors.

Zebrafish are an attractive model for chemical screening due to their adaptability to high-throughput platforms and ability to display complex phenotypes in response to chemical exposure. The photomotor response (PMR) is an established and reproducible phenotype of the zebrafish embryo, observed 24 h post-fertilization in response to a predefined sequence of light stimuli. In an effort to evaluate the sensitivity and effectiveness of the zebrafish embryo PMR assay for toxicity screening, we analyzed chemicals known to cause both neurological effects and developmental abnormalities, following both short (1 h) and long (16 h+) duration exposures. These include chemicals that inhibit aerobic respiration (eg, cyanide), acetyl cholinesterase inhibitors (organophosphates pesticides) and several chemical weapon precursor compounds with variable toxicity profiles and poorly understood mechanisms of toxicity. We observed notable concentration-responsive, phase-specific effects in the PMR after exposure to chemicals with a known mechanism of action. Chemicals with a more general toxicity profile (toxic chemical weapon precursors) appeared to reduce all phases of the PMR without a notable phase-specific effect. Overall, 10 of 20 chemicals evaluated elicited an effect on the PMR response and eight of those 10 chemicals were picked up in both the short- and long-duration assays. In addition, the patterns of response uniquely differentiated chemical weapon precursor effects from those elicited by inhibitors of aerobic respiration and organophosphates. By providing a rapid screening test for neurobehavioral effects, the zebrafish PMR test could help identify potential mechanisms of action and target compounds for more detailed follow-on toxicological evaluations. Approved for public release: distribution unlimited.

Comparison of Tricaine Methanesulfonate (MS-222) and Alfaxalone Anesthesia in Zebrafish (Danio rerio).

The research use of zebrafish has risen exponentially over the past decade while anesthetic options have remained largely unchanged.6 ricaine methanesulfonate (MS-222) is widely accepted as an anesthetic for routine husbandry procedures, however it has limitations and safety concerns. 11 A greater variety of effective anesthetic options for surgical procedures would be advantageous for the research community. Adult zebrafish were randomly assigned to one of the following groups (n = 10, 5 males and 5 females): 200 mg/L MS-222; 6-, 10-, 13-, and 16-mg/L alfaxalone, and control. All zebrafish in the MS-222 group reached a surgical plane of anesthesia within 95 ± 32 s. By contrast, only 2 of 10, 1 of 10, 0 of 10, and 0 of 4 of the 6, 10, 13, and 16 mg/L alfaxalone groups, respectively, reached a surgical plane of anesthesia within the allotted 10-min period. Recovery time was also significantly slower in the alfaxalone groups as compared with MS-222, with some fish taking greater than 10 min to recover. In addition, 33 of 34 zebrafish (the 16 mg/L group was not completed due to safety concerns) in the alfaxalone groups lost opercular movements for greater than one minute during their anesthetic event and had to be removed to the recovery tank. The results demonstrated that alfaxalone was unable to provide a reliable and safe surgical plane of anesthesia at any of the drug doses tested. Therefore, we recommend alfaxalone not be used as an anesthetic for painful procedures on zebrafish and conclude that MS-222 remains a more viable anesthetic for immersion anesthesia in zebrafish.

High throughput embryonic zebrafish test with automated dechorionation to evaluate nanomaterial toxicity.

Engineered nanomaterials pose occupational health and environmental concerns as they possess unique physical and chemical properties that can contribute to toxicity. High throughput toxicity screening methods are needed to address the increasing number of nanomaterials in production. Here we used a zebrafish photomotor response (PMR) test to evaluate a set of fifteen nanomaterials with military relevance. Automated dechorionation of zebrafish embryos was used to enhance nanomaterials bioavailability. Optimal PMR activity in zebrafish embryos was found at 30-31 hours post-fertilization (hpf). Behavioral and toxicological responses were measured at 30 and 120 hpf; behavioral responses were found for thirteen of the fifteen nanomaterials and acute toxicity (LC50) levels for nine of the fifteen nanomaterials below the maximum test concentration of 500 µg/ml. Physico-chemical characterization of the nanomaterials detected endotoxin and bacterial contamination in two of the tested samples, which may have contributed to observed toxicity and reinforces the need for physical and chemical characterization of nanomaterials use in toxicity testing. The zebrafish PMR test, together with automated dechorionation, provides an initial rapid assessment of the behavioral effects and toxicity of engineered nanomaterials that can be followed up by physico-chemical characterization if toxicity is detected, reducing the amount of time and monetary constraints of physico-chemical testing.

Mid-Tibiofibular Amputation as a Method of Terminal Blood Collection in Xenopus Laevis.

The African clawed frog, Xenopus laevis, is a widely used model for biomedical research. X. laevis could be more useful as a model with a better method for collection and analysis of its blood and serum. However, blood collection in X. laevis can be challenging due to their small size, lack of peripheral vascular access, and species-specific hematology variables. The goal of this study was to compare cardiocentesis, the current gold standard terminal blood collection method, with a leg amputation technique. Blood samples were collected from 24 laboratory-reared X. laevis, randomized to either the cardiocentesis or leg amputation method, with 6 males and 6 females in each group. Hematology and serum biochemistry were also conducted to identify any lymph contamination in the samples. The leg amputation method produced significantly higher blood volumes in shorter times and showed no significant differences in clinical pathology parameters as compared with cardiocentesis. These results indicate that blood collection by leg amputation may be a valuable approach for increasing the utility of an already valuable biomedical research model.