The membrane-active tri-block copolymer pluronic F-68 profoundly rescues rat hippocampal neurons from oxygen-glucose deprivation-induced death through early inhibition of apoptosis

Abstract

Pluronic F-68, an 80% hydrophilic member of the Pluronic family of polyethylene-polypropylene-polyethylene tri-block copolymers, protects non-neuronal cells from traumatic injuries and rescues hippocampal neurons from excitotoxic and oxidative insults. F-68 interacts directly with lipid membranes and restores membrane function after direct membrane damage. Here, we demonstrate the efficacy of Pluronic F-68 in rescuing rat hippocampal neurons from apoptosis after oxygen-glucose deprivation (OGD). OGD progressively decreased neuronal survival over 48 h in a severity-dependent manner, the majority of cell death occurring after 12 h after OGD. Administration of F-68 for 48 h after OGD rescued neurons from death in a dose-dependent manner. At its optimal concentration (30 μm), F-68 rescued all neurons that would have died after the first hour after OGD. This level of rescue persisted when F-68 administration was delayed 12 h after OGD. F-68 did not alter electrophysiological parameters controlling excitability, NMDA receptor-activated currents, or NMDA-induced increases in cytosolic calcium concentrations. However, F-68 treatment prevented phosphatidylserine externalization, caspase activation, loss of mitochondrial membrane potential, and BAX translocation to mitochondria, indicating that F-68 alters apoptotic mechanisms early in the intrinsic pathway of apoptosis. The profound neuronal rescue provided by F-68 after OGD and the high level of efficacy with delayed administration indicate that Pluronic copolymers may provide a novel, membrane-targeted approach to rescuing neurons after brain ischemia. The ability of membrane-active agents to block apoptosis suggests that membranes or their lipid components play prominent roles in injury-induced apoptosis.

Figure 1.

F-68 rescues hippocampal neurons from OGD insult. A, Structure of the tri-block copolymer F-68. B, Left, Pseudocolored photomontage of contiguous 20× fields obtained by high-content imaging of cultured hippocampal neurons over an entire 15-mm-diameter coverslip. Images are overlays of calcein (green) and DRAQ5 (red) images. Squares indicate the location of the 42 fields on the coverslip used for automated cell counting. Scale bar, 500 μm. Top right, Overlaid calcein and DRAQ5 images from the indicated site in the montage. Scale bar, 10 μm. Bottom right, Result of automated image segmentation performed on the image above. Arrows show identified dead (red) and living (green) cells. C–F, Each experimental unit (n) consists of six coverslips per condition, with high-throughput imaging counting ∼1300 cells per coverslip. * indicates significantly different from control (Ctrl); # indicates significantly different from OGD. C, Changes in mean ± SD neuronal survival over time after 30, 45, and 60 min of OGD. n = 3 per condition. D, Changes in mean ± SD percentage neuronal survival induced by F-68 treatment as a function of OGD duration, measured 48 h after OGD (n = 3 per condition per OGD duration). E, Concentration dependence of F-68-induced rescue of neurons after 45 min OGD, measured 48 h later (n = 3 per condition). F, Changes in mean ± SD percentage neuronal survival with increasing delay in F-68 addition after OGD, measured 48 h after OGD (n = 3 per condition). Neuronal rescue from OGD persists when F-68 addition is delayed as much as 12–15 h after OGD. G, Raw, time-dependent [Ca²⁺]_i changes in response to NMDA or potassium-induced membrane depolarization in control neurons (top left) and in neurons 5 d after rescue from 45 min OGD with F-68 (30 μm; bottom right). Right, Mean ± SD peak fura-2 ratios obtained in each condition (n = 3, each study of at least 5 neurons). H, Voltage responses to current injection are similar between control neurons and neurons, 5 d after rescue from OGD-induced death with F-68 (30 μm). I, Action potential morphology is identical between control (orange) neurons and neurons 5 d after rescue from OGD with F-68 (30 μm; black). Traces are offset for clarity. J, Representative NMDA-activated currents in hippocampal neurons studied before (orange) and after 3 min exposure to F-68 (30 μm; black). Mean ± SD current densities were −24.3 ± 2 pA/pF before and −21.4 ± 2 pA/pF after exposure of nine cells to F-68 (30 μm). Mean ± SD times to recovery to half-maximal amplitude were 246 ± 9.5 and 246 ± 8 ms, respectively.

Figure 2.

F-68 prevents OGD-induced apoptosis. A, C, Each experimental unit (n) consists of six coverslips per condition, with high-throughput imaging counting ∼1300 cells per coverslip. * indicates significantly different from control; # indicates significantly different from OGD. A, Mean ± SD percentage neuronal survival, 48 h after OGD (45 min) or control, the former in the presence and absence of Z-VAD-FMK (50 μm; n = 3 per condition). B, Photomontage of representative micrographs of neurons labeled with TUNEL (green) for apoptotic neurons and DAPI (red) for all cells. Images obtained 48 h after 45 min of exposure to control or OGD buffers with and without immediate treatment thereafter with F-68 (30 μm). Scale bar, 10 μm. C, Mean ± SD percentage of TUNEL-positive neurons, 48 h after exposure to control buffer, or 45 min OGD with and without immediate treatment thereafter with F-68 (30 μm; n = 3). D, Photomontage of Annexin V labeling (red) of viable neurons (green), obtained 6 h after 45 min exposure to control buffer or OGD buffers with and without immediate treatment thereafter with F-68 (30 μm). Scale bar, 10 μm. E, Mean ± SD percentage of Annexin V-positive neurons by condition (n = 5; for each study, 12 randomly selected fields of 5–10 neurons each, counted on each of 2 coverslips per condition).

Figure 3.

F-68 prevents OGD-induced caspase activation. A, Using calcein (green) to label living neurons, DAPI (cyan) to label all neurons, and FLICA (red) to label caspase-positive neurons allows identification of neurons as living/dead and caspase activation as positive/negative. Images obtained 6 h after 45 min exposure to control buffer or OGD, with and without immediate treatment thereafter with F-68 (30 μm). Scale bar, 10 μm. Arrow, Living neuron, FLICA negative; filled arrowhead, living neuron, FLICA positive; open arrowhead, dead neuron, FLICA positive; double arrowhead, dead neuron, FLICA negative. B, C, Each experimental unit (n) consists of six coverslips per condition, with high-throughput imaging counting ∼1300 cells per coverslip. * indicates significantly different from control; # indicates significantly different from OGD. B, Mean ± SD percentage of total cells at 6 h after OGD or control (n = 3). C, Mean ± SD percentage of FLICA-positive cells over time after OGD or control (n as in B). * indicates significantly different from the paired bar.

Figure 4.

F-68 prevents OGD-induced release of mitochondrial cytochrome c and dissipation of ΔΨ_m. A, Cellular distribution of cytochrome c immunoreactivity 6 h after staurosporine (1 nm) or 45 min exposure to OGD with or without F-68 or control buffer. Scale bar, 10 μm. B, Mean ± SD intensity of cytochrome c immunoreactivity over the nuclear volume 6 h after OGD; n = 6 coverslips per condition. ^#p < 0.0001; *p < 0.01. C, Histogram of individual cell TMRM intensities (red bars) 6 h after OGD or control and in the same cells after FCCP to dissipate ΔΨ_m (blue bars; measured by high-throughput imaging; n = 6 coverslips per condition).

Figure 5.

F-68 prevents OGD-induced BAX translocation from cytosol to mitochondria. A, Confocal images of hippocampal neurons expressing mitochondrially targeted GFP (green) and stained for BAX immunoreactivity (red). Scale bar, 10 μm. Note the greater overlap of BAX immunoreactivity with mitochondria after OGD but not OGD followed by F-68. B, Mean ± SD Manders coefficients, unbiased intensity correlation analyses, between BAX and mito-GFP immunoreactivity, demonstrating increased colocalization of BAX with mitochondria after OGD but not after OGD followed by F-68 (n = 7 per group). C, Representative Western blots of BAX immunoreactivity in heavy membrane and cytosolic fractions of neurons subjected to control or OGD 6 h previously. Blots were stripped and blotted with the mitochondrial marker COX IV and cytosolic marker GAPDH to identify fractions and normalize intensities for protein loading. D, E, Quantification of BAX immunoreactivity in heavy membrane (D) and cytosolic (E) fractions. Data are expressed as means ± SD of ratios of BAX/COX IV or BAX/GAPDH (n = 3 experiments, each experiment using 4 × 10⁶ cells per condition). * indicates significantly different from control; # indicates significantly different from OGD.

Figure 6.

F-68-induced inhibition of apoptosis persists when F-68 treatment is delayed for 12 h after OGD. F-68 (30 μm) or media control were administered 12 h after OGD (45 min), and measurements were made 12 h after F-68 administration. A, Mean ± SD percentage of live/dead and FLICA-positive/negative neurons. Each experimental unit (n) consists of six coverslips per condition, with high-throughput imaging counting ∼1300 cells per coverslip (n = 3). B, Mean ± SD intensity of cytochrome c immunoreactivity over the nuclear volume; n = 6 coverslips per condition. C, Representative Western blots of BAX immunoreactivity in heavy membrane and cytosolic fractions of neurons treated with F-68. Blots were stripped and blotted with the mitochondrial marker COX IV and the cytosolic marker GAPDH to identify fractions and normalize intensities for protein loading. D, E, Quantification of BAX immunoreactivity in heavy membrane (D) and cytosolic (E) fractions. Data are expressed as means ± SD of ratios of BAX/COX IV or BAX/GAPDH (n = 3 experiments, each experiment using 4 × 10⁶ cells per condition). * indicates significantly different from control; # indicates significantly different from OGD.