2.3. Acute hypoxia/reoxygenation imaging on cultured neurons

To perform imaging of neurons in a state of hypoxia/reoxygenation, we used a Nikon Eclipse Ti2 inverted fluorescence microscope. For HyPer7, SypHer3s, SoNar excitation LED-DA/FI/TR/Cy5-B (DAPI/FITC/TRICTC/Cy5- Full Multiband Quad filters were used (ex = 352–404 nm, 461–488 nm), alongside with LED-DA/FI/TR/Cy5-B (DAPI/FITC/TRICTC/Cy5- Full Multiband Quad filters for emission (Em = 500–530 nm). Most of measurements were carried out using a 20X (S PLAN FL LWD 20x/0.70) and 40X (Plan Apo λ 40x/0.95 ∞/0.11–0.23, WD 0.25–0.17 mm) objectives.

Before starting imaging experiments, the growth medium was changed to a KRS solution (120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 25 mM NaНCO₃, 5.5 mM HEPES, 10 mM d-glucose, pH 7.4), containing no serum, then cells were incubated at 37 °C in 5% CO₂ for 20 min.

The effect of acute hypoxia/reoxygenation on cultured neurons was investigated using a perfusion chamber. The cell dish was placed in the stage top incubator (Miniature Incubator TC-MWP, Bioscience Tools). Two 1L bottles with KRS solution were used for perfusion. The deoxygenated solution was prepared by bubbling 95% N2/5% CO₂ gas mixture through the solution, the control solution was bubbled with 95% air/5% CO₂ (gas mixtures were produced by CO₂–O₂ controller CO₂–O₂-MI, Bioscience Tools, and CO₂ controller Tokai Hit). The solutions were supplied into the cell dish via tygon tubes by peristaltic pumps. The atmosphere in the incubator was 95% N2/5% CO₂ under hypoxic conditions, 95% air/5% CO₂ under control and re-oxygenation conditions.

The oxygen content of the solutions was monitored using a multichannel oximeter (Multi-Channel Oxygen Meter OXY-4 SMA (G3), PreSens Precision Sensing GmbH) connected to optical fibers recording the signal from a fluorescent oxygen sensors FTC-PSt3; PreSens Measurement Studio 2 software was used for data acquisition. Oxygen sensors were installed in both source bottles and inside the perfusion chamber – one in the deoxygenated solution inlet and one in the outlet tubes. With this setup we were able to induce hypoxia/reoxygenation in neurons in course of seconds and control the absence of oxygen directly in the cell dish.

The protocol of the experiment was as follows. First, the baseline fluorescence was recorded under control conditions for 15–20 min: a control solution was fed to the cell dish, the perfusion chamber was filled with 95% air/5% CO₂. Then fluorescence under hypoxia conditions was recorded for 35 min: deoxygenated solution was fed into the chamber; the perfusion chamber was filled with 95% N₂/5% CO₂. Then fluorescence was recorded under reperfusion conditions (same as in the control) for 15–20 min. At the end of the experiment, perfusion was stopped and H₂O₂ (200 μM) or l-Lactate (20 mM) was added into the dish to record the maximal ratiometric response of the biosensors.