Oxygen gradient generator to improve in vitro modeling of ischemic stroke

Abstract

Introduction: In the core of a brain infarct, perfusion is severely impeded, and neuronal death occurs within minutes. In the penumbra, an area near the core with more remaining perfusion, cells initially remain viable, but activity is significantly reduced. In principle, the penumbra can be saved if reperfusion is established on time, making it a promising target for treatment. In vitro models with cultured neurons on microelectrode arrays (MEAs) provide a useful tool to investigate how ischemic stroke affects neuronal functioning. These models tend to be uniform, focusing on the isolated penumbra, and typically lack adjacent regions such as a core and unaffected regions (normal perfusion). However, processes in these regions may affect neuronal functioning and survival in the penumbra.

Materials and methods: Here, we designed, fabricated, and characterized a cytocompatible device that generates an oxygen gradient across in vitro neuronal cultures to expose cells to hypoxia of various depths from near anoxia to near normoxia. This marks a step in the path to mimic core, penumbra, and healthy tissue, and will facilitate better in vitro modeling of ischemic stroke.

Results: The generator forms a stable and reproducible gradient within 30 min. Oxygen concentrations at the extremes are adjustable in a physiologically relevant range. Application of the generator did not negatively affect electrophysiological recordings or the viability of cultures, thus confirming the cytocompatibility of the device.

Discussion: The developed device is able to impose an oxygen gradient on neuronal cultures and may enrich in vitro stroke models.

Keywords: hypoxia; in vitro model; ischemic penumbra; ischemic stroke; microelectrode arrays (MEAs); neuronal cultures; oxygen gradient.

FIGURE 1

Various schematics of the oxygen gradient generator and the setup. (A) 3D model of the device. After closing the top of the culture well (MEA with the culture chamber) with the gradient lid, it is sealed with the lid lock (orange ring). The lid assembly (on the right) consist of a top holder, two inserts and 19G stainless-steel connectors. The inserts are facing each other and each one has attached a gas permeable silicone membrane. (B) Top view of the device sealed with the ring, indicating the dimensions of the device and the placement of the oxygen sensors grid. Membranes through which gas exchange takes place (7.2 × 3 mm) are indicated in yellow. The lower border of the membranes was ∼0.2 mm above the surface of the MEA. (C) Representation of the complete setup for calibration with the calibration cage (smaller and darkened), the grid of sensors which was exposed to gas mixtures of known O₂ concentrations, and a cross-sectional view of the glass plate, silicone culture chamber and lid assembly. (D) Representation of the complete setup for cell experiments in the recording cage (bigger and not darkened), the gas mixtures supplied to the generator (green: compressed air with CO₂; red: N₂ with CO₂), and a cross-sectional view of the MEA, silicone culture chamber and lid assembly. (E) Representation of the grid of sensors and the definition of the ROIs for the calculation of the red phosphorescence intensity and estimation of oxygen concentration.

FIGURE 2

Calibration of the oxygen sensors. (A) Example of stepwise phosphorescence intensity changes observed at one of the sensors when supplying both channels of the gradient generator with the six known gas mixtures, indicated in each of the steps that were considered stable (marked as green). (B) At all oxygen concentrations, phosphorescence (median ± 25–75% quartiles) was slightly but significantly lower when measured in culture medium than in water (*p < 0.001 for a sample of n = 35 electrodes). (C) When exposing all sensors to N₂ through the inserts while the gas supplied to the calibration cage was switched from N₂ (red line period) to air (blue line period), changes were less than 1% O₂ during a period of 45 min (n = 40 sensors; mean ± SD). (D) Example of Stern-Volmer equation derived calibration for one sensor with R² = 0.997.

FIGURE 3

Establishment of the oxygen gradient. (A) Example of time course of establishment of the oxygen gradient in one illustrative experiment, showing stability and linearity of the gradient after 30 min. Error bars indicate SD and refer to differences between sensors in the same column perpendicular to the gradient (n = 4 sensors per column). For clarity error bars are shown only for t = 0, t = 30 min, and t = 150 min, other curves had error bars of comparable magnitude. (B) Changing the direction of flow through the inserts did not significantly change the readout values of sensors in lines of the grid (direction of the gradient), showing that there was no transverse component. (C) Real gradients observed in three experiments (shown for the measurable length using the grid of sensors in each experiment) which were averaged and extrapolated for the full gap between the inserts (Average Gradient, traced in black) to obtain the mean slope and offset for the generalization of the oxygen gradient produced by the generator.

FIGURE 4

Electrophysiological recording (A) example of recorded action potentials during the various phases in one experiment, where 21 of the 60 electrodes recorded activity. Position in the figure represents the position of recording electrodes in the Microelectrode Array. Mean waveform of action potentials (shape and amplitude) did not significantly change between phases. (B) Close up of a representative electrode showing the similarities in mean waveform between the phases “Conventional Lid” and “Generator Lid-off.” (C) Close up of a representative electrode showing the similarities in mean waveform between the phases “Generator Lid -off” and “Generator Normoxia.” (D) Example of a raster plot (only active electrodes), with instances of network bursts. (E) Evolution of the mean firing rates per minute during the recordings, normalized to “Conventional Lid” values, showing that there is a decrease in the “Generator Lid-off” and “Generator Gradient” phases, both compensated when oxygen flow through the channels is restored (error bars indicate 25–75% quartiles; n = 41 active electrodes from both experiments combined). All phases lasted 1 h, except “Generator Gradient,” which lasted 2 h.