Real-time impedance-based cell analyzer as a tool to delineate molecular pathways involved in neurotoxicity and
Zoya Marinova1, Susanne Walitza2, Edna Grünblatt2
1University Clinics for Child and Adolescent Psychiatry, University of Zürich; zoya.marinova@kjpdzh.ch.
Abstract:
Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations - most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.
Insights
This study introduces a real-time impedance assay for assessing neuroprotection and neurotoxicity in neuronal cells. The method enables label-free, kinetic analysis and evaluation of downstream signaling pathways for drug development.
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- Neuronal cell death is central to many brain disorders.
- Existing in vitro assays lack real-time kinetic and downstream pathway analysis.
- Developing advanced models is crucial for understanding neuroprotection/neurotoxicity and drug development.
Purpose of the Study:
- To present a real-time, label-free impedance-based assay for assessing neuroprotection in neuronal cells.
- To demonstrate the utility of this assay in identifying downstream signaling pathways involved in neuroprotection.
- To evaluate the contribution of cell proliferation to observed neuroprotective effects.
Main Methods:
- Utilized a real-time impedance-based cell analyzer with microelectronic plates (E-Plates).
- Measured impedance changes reflecting cell status (adherence, viability, morphology).
- Derived a Cell Index from impedance measurements for quantitative analysis.
Main Results:
- Successfully determined neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in real-time.
- Delineated downstream signaling molecules using second messenger pathway inhibitors.
- Assessed the impact of cell proliferation on neuroprotection.
Conclusions:
- Real-time impedance analysis offers a label-free, high-throughput method for evaluating neuroprotective compounds.
- This technique provides detailed kinetic and mechanistic insights into neuroprotection/neurotoxicity.
- Facilitates the selection of promising therapeutic candidates for neurological disorders.
