Research ArticleCELLULAR NEUROSCIENCE

Analysis of the bystander effect in cone photoreceptors via a guided neural network platform

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Science Advances  09 May 2018:
Vol. 4, no. 5, eaas9274
DOI: 10.1126/sciadv.aas9274
  • Fig. 1 Dissecting the mechanisms of photoreceptor degeneration via the bystander effect using the NN-Chip platform.

    (A) Excessive light irradiation induces apoptosis in photoreceptors, which are highly connected throughout the photoreceptor layer by random synapses. (B) To dissect photoreceptor communication, which is normally highly coupled in tissue and in dish culture, we loaded the 661W cells individually onto the NN-Chip. Each microwell was connected by microchannels to allow synapse formation between each photoreceptor, generating a highly guided neural network. (C) To study the bystander killing effect between cones, we selectively induced apoptosis using blue light sources. (D) To quantitatively analyze the role of Cx36 in bystander killing between cones on the NN-Chip, we used CRISPR (clustered regularly interspaced short palindromic repeats)–based gene-edited cells in conjunction with a microinjection unit.

  • Fig. 2 Operation and design of the NN-Chip platform.

    (A) Schematic of the cell loading process. (B) Representative image of loading efficiency. The 661W cells were incubated in the medium with calcein for 20 min before loading. (C) Cell morphology after incubation for 1 hour on chip after loading. Scale bar, 40 μm. (D) Results of large-area loading by using the MDA-MB-231 cells with optimized parameters. Scale bar, 40 μm. (E) A scanning electron microscopy (SEM) image shows the neural network structure, including the microwells and their connecting microchannels, of the NN-Chip at a 30° tilt angle. (F) Representative images show that the 661W cells formed neural network on the NN-Chip. The actin (green) was labeled by phalloidin, and the nuclei (blue) was labeled with 4′,6-diamidino-2-phenylindole (DAPI). (G) A representative SEM image shows a 661W cell with synapses extending into the surrounding chambers along with the microchannel. (H) Neural network created by iCell neurons. Cells were stained by calcein after culturing on chip for 12 hours. Scale bar, 20 μm.

  • Fig. 3 Maintaining the blue light–sensitive characters of 661W cells after loading and culturing on the NN-Chip.

    (A) ROS production accumulation, as measured by the mean florescence intensity (MFI) of H2DCFDA, was assessed following blue or white light irradiation for various times (n = 10). (B) Representative images show JC-1–stained 661W cells after blue light irradiation. The healthy cells with high mmp appeared red (aggregates), the apoptotic cells with low mmp appeared green (monomers), and the pre-apoptotic cells appeared red and green. Scale bar, 20 μm. (C) Number of the SYTOX Green–labeled cells under blue light irradiation for different time intervals. (D) Percentage of cells in each JC-1–stained group, which were counted in a 100 × 100 cell array (n = 10). (E) Representative immunostaining images show S-opsin aggregation in 661W cells on the NN-Chip after blue light irradiation for 9 and 18 hours. The cells were stained for actin [green fluorescent protein (GFP)] and S-opsin (Alexa Flour 647), and the nuclei were stained with DAPI. Scale bar, 20 μm. (F) Quantitative analysis showed that the percentage of S-opsin–aggregated cells was increased by blue light irradiation compared with that by white light irradiation.

  • Fig. 4 Evidence of the gap junction–mediated bystander killing effect in the 661W cells.

    (A) Representative bright field (BF) and fluorescence images show apoptosis distribution, detected by SYTOX staining, in different gap junction inhibitors (octanol, 1 mM; quinine, 50 μM; meclofenamic, 100 μM) after 6 hours of blue light irradiation on the 661W-containing NN-Chip. White circles represent the initial irradiated area. Scale bar, 1 mm. (B) Schematic of the 405-μm laser spot diameter irradiation of the NN-Chip. Apoptosis was counted every hour for 24 hours after blue light irradiation (n = 10).

  • Fig. 5 Analysis of the bystander killing phenomenon at the single-cell level.

    (A) Representative time-course images of the bystander effect at the single-cell level in SYTOX-stained 661W cells on the NN-Chip. A single cell was irradiated to blue light, and two adjacent cells eventually undergo apoptosis. Scale bar, 40 μm. (B) Schematic of the 5-μm laser spot irradiation of the NN-Chip. The number of post-apoptotic cells induced by each pretreated cell was quantified. The whiskers go down to the smallest value and up to the largest. P < 0.0001 by one-way analysis of variance (ANOVA) analysis compared with zero- and four-cell group; n = 10.

  • Fig. 6 Assessing the effects of Cx36 on apoptosis spreading in the 661W cells.

    (A) Western blot analysis shows loss of Cx36 in Cx36-KO cells relative to that in WT cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) A cell was being injected with a Femtotip (inner diameter, 0.5 μm; outer diameter, 1 μm; Eppendorf) on the NN-Chip. (C and D) Dye distribution in 15 min after injection of 100 mM Lucifer yellow (457 Da) in (C) or of 100 mM FITC-dextran (10 kDa) in (D). (E and F) Dye distribution in 15 min after the injection of blue dextran (3 kDa) (E) and Lucifer yellow (F) and into the same cell. (G and H) Representative images show blue dextran (G) and SYTOX (H) distribution 6 hours after cytochrome c (10 mg/ml) and blue dextran (20 mg/ml) co-injection into a single WT cell. (I and J) Representative images show blue dextran (I) and SYTOX (J) distribution 6 hours after cytochrome c (10 mg/ml) and blue dextran (20 mg/ml) co-injection into a single Cx36-KO cell. (K) Quantitative analysis of apoptosis in WT and Cx36-KO groups. The results are calculated from 50 single-cell injections per group. Scale bars, 20 μm (B to J).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/5/eaas9274/DC1

    fig. S1. Optimization of the parameters and conditions for high-efficiency 661W cell loading.

    fig. S2. A schematic shows the microwell BME-coating process on the NN-Chip.

    fig. S3. ROS production, mmp measurements, and SYTOX function test in the blue light–treated 661W cells.

    fig. S4. Representative immunostaining images of S-opsin from 0 to 18 hours under blue light irradiation.

    fig. S5. Existence of tight junctions in synapses and function validation of the SYTOX.

    fig. S6. Apoptotic cells were quantified with or without adjacent apoptotic cells (n = 10).

    fig. S7. Generation and verification of Cx36-KO 661W cells.

    fig. S8. Calibration of the microinjection volume.

    movie S1. Operation of NN-Chip.

    movie S2. Three-dimensional structural view of 661W cells cultured onto NN-Chip after 6 hours.

    movie S3. Time lapse of the gap junction–mediated bystander killing effect in the 661W cells.

    movie S4. Apoptosis propagation from single cell.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Optimization of the parameters and conditions for high-efficiency 661W cell loading.
    • fig. S2. A schematic shows the microwell BME-coating process on the NN-Chip.
    • fig. S3. ROS production, mmp measurements, and SYTOX function test in the blue light–treated 661W cells.
    • fig. S4. Representative immunostaining images of S-opsin from 0 to 18 hours under blue light irradiation.
    • fig. S5. Existence of tight junctions in synapses and function validation of the SYTOX.
    • fig. S6. Apoptotic cells were quantified with or without adjacent apoptotic cells (n = 10).
    • fig. S7. Generation and verification of Cx36-KO 661W cells.
    • fig. S8. Calibration of the microinjection volume.
    • Legends for movies S1 to S4

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.avi format). Operation of NN-Chip.
    • movie S2 (.mp4 format). Three-dimensional structural view of 661W cells cultured onto NN-Chip after 6 hours.
    • movie S3 (.mov format). Time lapse of the gap junction–mediated bystander killing effect in the 661W cells.
    • movie S4 (.mov format). Apoptosis propagation from single cell.

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