Addressing the shift from classical animal testing to high-throughput in vitro and/or simplified in vivo proxy models has been defined as one of the upcoming challenges in ecotoxicology and neurotoxicology.
In our research we are focusing on non-invasive and minimally invasive as well as alternative bioanalytical approaches in line with 3Rs and Animal Welfare principles.
We are also actively pursuing alternative animal and non-animal models that enable ethical and high-throughput approaches outlined in the Toxicity Testing in the 21st Century: A Vision and a Strategy (TT21C, U.S. National Academy of Sciences, 2007)
Small aquatic organisms such as diverse invertebrates as well as fish and frog embryos and eletheuro-embryos at very early developmental stages are convenient alternative models in aquatic ecotoxicity testing, behavioural ecotoxicology, predictive and developmental neurotoxicology as well as neuroactive drug discovery.
In eco-neurotoxicology they are commonly used in a plethora of tests ranging from simple locomotory activities to elucidation of mating, feeding traits and cognitive responses under laboratory as well as micro-and mesocosm studies.
Due to relative simplicity of their central nervous systems, many small aquatic organisms are also particularly well suited to explore molecular mechanisms of neurotoxicity.
Planarian neurotoxicity model
Immunohistochemistry labelling of cephalic ganglions and ventral nerve cords (VNC) in a planarian Schimdtea mediterranea using anti-synapsin antibodies.
Zebrafish transgenic Fli1:EGFP model
Five days post fertilisation zebrafish larval stage expressing EGFP in vasculature as well as central nervous system.
Cells and cell lines
Cell lines and primary cells are very convenient and inexpensive models to study cellular and molecular mechanisms of cytotoxicity as well as rapidly evaluate cytotoxic effects of chemicals and drugs in very high throughput fashion.
We specialise in applications of diverse cell-based assays using time-lapse fluorescence microscopy, imaging cytometry, flow cytometry and laser scanning cytometry.
In particular we have expert know-how in multi-parameter bioassays for analysis of programmed cell death, mitochondrial toxicity, cell proliferation and cell cycle.
We employ diverse methods that allow for implementation of apoptotic assays on live suspension and adherent cells as well as fixed specimens such as cell and tissue samples.
Time-resolved analysis of apoptosis using cell lines
Time-lapse epifluorescence microscopy of U937 cells exposed to an investigative chemical was performed using fluorescent probes such as Annexin V (AV-APC, detecting externalisation of phosphatidyl serine in early apoptotic cells) and propidium iodide (PI, detecting late stages of apoptotic membrane permeabilisation).
Freshwater rotifers are sensitive models for ecotoxicity screening of investigational chemicals, effluents and wastewater as well as assessment of the quality of surface and groundwater.
Planktonic rotifers are commonly used in standard acute toxicity testing with calculation of the 24h LC50.
However, we have demonstrated that neuro-behavioural analysis of their swimming behaviour is a much more sensitive toxicological endpoints that enable analysis of sub-lethal concentrations of chemicals.
We have also developed new systems and biotests to enable behavioral analysis in rapidly swimming planktonic micro-invertebrates such as rotifers using high-definition video-microscopy.
Freshwater rotifers Brachionus calyciflorus
Planktonic rotifers are commonly used in standard acute toxicity testing but the video-microscopic analysis of their swimming behaviour enables sensitive sub-lethal biotests.
Planarians are the most primitive organisms that exhibit bilateral symmetry and cephalization, with a fully centralized nervous system that features true synaptic transmission. Their CNS, despite its higher level of organizational and functional complexity, remains tractable on the cellular level with approximately 10000 identified neurons. Interestingly, similar to vertebrates the planarian brain acts as a central processing and decision-making unit.
The relative complexity of the CNS, combined with a complex sensory system gives planarians a rich repertoire of stereotyped behavioural patterns. These behaviours range with sensitivity of response to thermotaxis,chemotaxis, thigmotaxis, electric fields (electrotaxis) and magnetic fields (magnetotaxis)
In recent years freshwater planarians have emerged as very effective new model species in higher throughput neurotoxicity and ecotoxicity testing with diverse behavioural tests that can be performed on this model.
Planarian models in ecotoxicology and neurotoxicology
Planarians are recently re-discovered neoclassical models in toxicology and neuroscience. They offer significant benefits for neuro-behavioural studies, neurotoxicology as well as in situ analysis of programmed cell death, cell proliferation and tissue regeneration.
Diverse small freshwater and marine crustaceans and their larval stages are popular models in conventional ecotoxicity testing.
We are using standard freshwater models such as cladocerans (Daphnia magna, Daphnia pulex), ostracods (Heterocypris incongruens), phyllopodous branchiopods (Thamnocephalus platyurus). We are also commonly using amphipods (Allorchestes compressa) and brine shrimp (Artemia franciscana) in marine aquatic toxicology projects.
Similarly to rotifers we have demonstrated that neuro-behavioural analysis of crustacean swimming behaviour is a much more sensitive toxicological endpoints that enable analysis of sub-lethal concentrations of chemicals.
We have also developed new tests protocols to enable their behavioral analysis using high-definition video-microscopy.
Freshwater and marine crustaceans are popular, standard models in aquatic ecotoxicity testing.
They are also fantastic models in eco-neurotoxicology when employed in behavioural assays using the video-microscopy analysis of their swimming behaviour. (Left) freshwater cladoceran Daphnia magna, (Right) marine amphipod Allorchestes compressa.
Molluscs models in ecotoxicology and neurotoxicology can include bivalve mussels and aquatic gastropods.
Interestingly despite the fact that molluscs are second largest group of invertebrates in the world (after arthropods) they represent relatively unexplored and under-utilised biological models in eco toxicity testing.
In fact they can be used for conventional mortality tests as well as more ecologically relevant tests such embryo development, growth and reproduction as well as neuro-behavioural tests.
Historically electro-neurophysiology experiments on gastropod molluscs such as Aplysia and later also diverse mussels models paved the way for pioneering discoveries of excitability of neuronal membranes as well as neuronal communication.
The advantages of those systems for eco-neurotoxicology are high physiological relevance, low cost, as well as ability to sustain isolated neuronal preparation in microperfusion for many days. Furthermore, the results obtained using electro-physiology experiments can be easily correlated with perturbations in behavioural phenotypes.
Molluscs models in ecotoxicology and neurotoxicology can include bivalve mussels (left) and aquatic gastropods (right)
Relatively unexplored and under-utilised biological models that can be used for conventional, embryo development, growth and reproduction as well as neuro-behavioural tests.
Embryonal and early larval fish
Developmental toxicity biotests performed on embryos of zebrafish (Danio rerio, Zf), fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes) and other species have gained significant popularity as sensitive, alternative approaches to acute fish toxicity tests in chemical hazard and risk
These early developmental stages can be kept in 96-well plates and low volumes of media thus providing high-throughput screening capabilities.
In this regard the OECD TG 236 Fish Embryo Toxicity Test (FET) based on zebrafish was designed as an alternative to the Fish Acute Toxicity Test (AFT) (OECD TG 203; OECD 1992) with the purpose of determining acute toxicity of chemicals on the earliest life fish stages. It fulfils requirements postulated by implementing the postulates of “Toxicity Testing in the 21st Century (TT21C)” to develop new and simplified proxy animal models that can be deployed at higher throughputs for rapid toxicity screening. The OECD TG 236 test also aligns with postulates of the 3R framework (Replacement, Reduction and Refinement) aimed to find alternative and more ethical biological models for toxicity testing.
Importantly the zebrafish CNS shares significant structural homology with mammalian counterparts in addition to very closely related, molecular and physiological foundations of neuronal signalling. Due its relative simplicity zebrafish is particularly well suited to explore molecular mechanisms of neurotoxicity.
Embryonic and early larval stages of zebrafish can be used in high-throughput behavioural biotests in neurotoxicology, ecotoxicology as well as discovery of neuroceuticals. Embryonal stages of zebrafish exhibit for instance stereotypic photomotor responses (PMR) upon stimulation of embryos between 24-36 hpf with a light stimulus. The PMR biotests can be translated into a relatively complex biometric barcodes used as signatures for rapid classification of neuroactive or neurotoxic chemicals. The larval photomotory response assay (LPR) performed on freely swimming 5 days post fertilization stages (dpf) of zebrafish is gaining popularity in rapid assessment of freshwater aquatic pollutants with neurotoxic mode of action. The additional advantage of LPR test is the ability to study impact of toxicants on non-associative learning processes, such as habituation and sensitization because of well-developed habituation to the photic stimulus over time. The above biotests can be performed at a high throughput in 96-well plates utilizing commercially available behavioural analysis
Visualisation of neuro-developmental processes utilising a plethora of transgenic zebrafish models is enabled by the transparency of embryo and larval stages. This highlights the possibility of creating multi-dimensional data that combine molecular and cellular assessments with the analysis of behavioral alterations.
Zebrafish a modern and powerful model organism in ecotoxicity testing and drug discovery
OECD TG 236 Fish Embryo Toxicity Test (FET) is a widely accepted alternative to the Fish Acute Toxicity Test (AFT) with the purpose of determining acute toxicity of chemicals on the earliest life fish stages.
Behavioural biotests performed on early life stages of zebrafish such as embryos and larvae are convenient, high-throughput tools in ecotoxicity testing and drug discovery
(Left) zebrafish embryo photomotor responses (PMR) bioassay. (Right) larval photomotory response assay (LPR) for rapid assessment of freshwater aquatic pollutants with neurotoxic mode of action.
Advanced neuro-behavioural tests on juvenile and adult zebrafish can include sensory-motor functions such as thermotaxis and thermal preference (left) and cognitive T-maze (memory & learning, right) tests.