Three-Dimensional Tracking of Intracellular Calcium and Redox State during Real-Time Control in a Hypoxic Gradient in Microglia Culture: Comparison of the Channel Blocker and Reoxygenation under Ischemic Shock

Jana, Soumya and Giri, Lopamudra (2023) Three-Dimensional Tracking of Intracellular Calcium and Redox State during Real-Time Control in a Hypoxic Gradient in Microglia Culture: Comparison of the Channel Blocker and Reoxygenation under Ischemic Shock. ACS Chemical Neuroscience, 14 (10). pp. 1810-1825. ISSN 1948-7193

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Abstract

Real-time three-dimensional (3-D) imaging is crucial for quantifying correlations among various molecules under acute ischemic stroke. Insights into such correlations may be decisive in selecting molecules capable of providing a protective effect within a shorter period. The major bottleneck is maintaining the cultures under severely hypoxic conditions while simultaneously 3-D imaging intracellular organelles with a microscope. Moreover, comparing the protective effect of drugs and reoxygenation remains challenging. To address this, we propose a novel workflow for the induction of gas-environment-based hypoxia in the HMC-3 cells along with 3-D imaging using laser-scanning-confocal microscopy. The imaging framework is complemented with a pipeline for quantifying time-lapse videos and cell-state classification. First, we show an imaging-based assessment of the in vitro model for hypoxia using a steep gradient in O2 with time. Second, we demonstrate the correlation between mitochondrial superoxide production and cytosolic calcium under acute hypoxia. We then test the efficacy of an L-type calcium channel blocker, compare the results with reoxygenation, and show that the blocker alleviates hypoxic conditions in terms of cytosolic calcium and viability within an acute window of one hour. Furthermore, we show that the drug reduces the expression of oxidative stress markers (HIF1A and OXR1) within the same time window. In the future, this model can also be used to investigate drug toxicity and efficacy under ischemic conditions.

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IITH Creators:
IITH CreatorsORCiD
Jana, Soumyahttp://www.orcid.org/0000-0002-2049-2239
Giri, Lopamudrahttp://orcid.org/0000-0002-2352-7919
Item Type: Article
Uncontrolled Keywords: calcium; Fluo-4AM; HIF1A; HMC-3; Hoechst 33342; hypoxia; microglia; MitoSOX Red; OXR1; Rhod-2AM; human; hypoxia; ischemic stroke; metabolism; microglia; oxidation reduction reaction; Calcium; Humans; Hypoxia; Ischemic Stroke; Microglia; Oxidation-Reduction; Oxygen
Subjects: Electrical Engineering
Chemical Engineering
Divisions: Department of Chemical Engineering
Department of Electrical Engineering
Depositing User: Mr Nigam Prasad Bisoyi
Date Deposited: 28 Sep 2023 11:00
Last Modified: 28 Sep 2023 11:02
URI: http://raiith.iith.ac.in/id/eprint/11711
Publisher URL: https://doi.org/10.1021/acschemneuro.2c00807
OA policy: https://v2.sherpa.ac.uk/id/publication/7763
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