Research ArticleRETINAL DEGENERATION

Inhibition of the alternative complement pathway preserves photoreceptors after retinal injury

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Science Translational Medicine  22 Jul 2015:
Vol. 7, Issue 297, pp. 297ra116
DOI: 10.1126/scitranslmed.aab1482
  • Fig. 1. Activity of the alternative complement pathway during RD.

    (A) ELISA analysis of factor B production in human vitreous from patients with RD (n = 10) compared to nondetached retina control patients with a macular hole or epiretinal membrane (n = 4). (B) Image of RD in a mouse model. The dotted yellow line outlines the region of retina that is detached (about 60%). (C) Time course of gene expression for Fb (encoding factor B), Cd55, and Cd59 in the retina of mice after RD. The red line tracks Fb gene expression, the blue line tracks Cd55 expression, and the green line tracks Cd59 expression at intervals over a period of 48 hours. Color of asterisks corresponds to the gene (n = 4 for each time point). (D) Representative 3,3′-diaminobenzidine labeling (brown) for Cd55 mRNA expression in the attached outer nuclear layer (left panel) and the detached outer nuclear layer (right panel) of the same eye (n = 4). (E) 3,3′-Diaminobenzidine labeling (brown) for Cd59 mRNA expression in the attached outer nuclear layer (left panel) and the detached outer nuclear layer (right panel) of the same eye (n = 4). ns, not significant. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; unpaired Student’s t test. Scale bar, 20 μm.

  • Fig. 2. Apoptosis in complement-deficient mice after RD.

    (A) Representative TUNEL labeling in the outer nuclear layer (ONL) of C3−/− mice and wild-type (WT) control (C57Bl6) mice 24 hours after RD. DAPI, 4′,6-diamidino-2-phenylindole. (B) Quantitation of TUNEL cells in the outer nuclear layer of C3−/− mice (n = 8) and WT control mice (n = 8). (C) Representative TUNEL labeling in the outer nuclear layer of mice injected with an Ab against C3 compared to injection with an immunoglobulin G (IgG) isotype control Ab. (D) Quantitation of TUNEL cells in the outer nuclear layer of mice injected with an Ab against C3 (n = 4) compared to injection with an IgG isotype control Ab (n = 10). (E) Representative TUNEL labeling in the outer nuclear layer of C3−/− mice injected with PBS (control) or cobra venom factor (CVF) to activate the complement system. (F) Quantitation of TUNEL-positive cells in the outer nuclear layer of C3−/− mice injected with PBS control (n = 5) or cobra venom factor to activate complement in C3−/− mice (n = 8). **P ≤ 0.01, ****P ≤ 0.0001; unpaired Student’s t test. Scale bars, 50 μm.

  • Fig. 3. Apoptosis in alternative pathway–deficient mice after RD.

    (A) Representative TUNEL labeling in the outer nuclear layer 24 hours after RD in mice lacking factor B (Fb−/−) and WT control mice. (B) Quantitation of TUNEL-positive cells from Fb−/− mice (n = 6) and WT control mice (n = 6) 24 hours after RD. (C) Representative TUNEL labeling 24 hours after RD in mice injected with an anti–factor D (Fd) Ab and IgG isotype control Ab. (D) Quantitation of TUNEL-positive cells from mice injected with an anti–factor D Ab (n = 9) and IgG isotype control Ab (n = 9) 24 hours after RD. ****P ≤ 0.0001, unpaired Student’s t test. Scale bars, 50 μm.

  • Fig. 4. Hypoxia in the retina after RD.

    (A) Representative immunohistochemistry images of the outer nuclear layer labeled with Hypoxyprobe (brown staining) comparing the detached portion of a mouse retina (right panel) to the attached portion of the same retina (left panel) 24 hours after RD. Both sides of the retina were stained with toluidine blue stain (n = 7). (B) In vivo oxygen concentrations taken in the retina 24 hours after RD in an attached retina (right eye) compared to the detached retina (left eye) (n = 7). (C) Representative TUNEL labeling in the outer nuclear layer 24 hours after RD in mice kept in room air (left panel) compared to mice kept in 75% oxygen (right panel). (D) Quantitation of TUNEL-positive cells in mice kept in room air (n = 5) for 24 hours after RD compared to mice kept in 75% oxygen (n = 6). (E) RT-PCR showing gene expression for Fb (encoding factor B) in the attached or detached retina 24 hours after RD; comparing mice kept in room air to mice kept in 75% oxygen (attached, n = 3; room air, n = 3; 75% O2, n = 4). (F) Under hypoxic conditions (1% O2), increasing mouse serum concentrations (used as a source of complement) led to an increased percentage of cell death in 661W cells assessed using a live/dead assay (n = 4). *P ≤ 0.05, ****P ≤ 0.0001; unpaired Student’s t test. Scale bars, 50 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/297/297ra116/DC1

    Materials and Methods

    Table S1. Human patient data for ELISA samples.

    Fig. S1. Human samples of RD.

    Fig. S2. Mouse model of RD.

    Fig. S3. Complement expression in the mouse RD model.

    Fig. S4. Cd55 and Cd59 are down-regulated in the detached retina.

    Fig. S5. Outer nuclear layer expression of complement regulator Cd55.

    Fig. S6. Expression of several key complement proteins after RD.

    Fig. S7. The role of the lectin and classical complement pathways in outer nuclear layer cell death after RD.

    Fig. S8. Cd55 and Cd59 are down-regulated in a photoreceptor cell line during hypoxia, and increasing serum concentrations cause MAC formation.

    Fig. S9. Hypoxia makes photoreceptors more susceptible to complement-mediated cell death.

    References (59, 60)

  • Supplementary Material for:

    Inhibition of the alternative complement pathway preserves photoreceptors after retinal injury

    J. Harry Sweigard, Hidetaka Matsumoto, Kaylee E. Smith, Leo A. Kim, Eleftherios I. Paschalis, Yoko Okonuki, Alexandra Castillejos, Keiko Kataoka, Eiichi Hasegawa, Ryoji Yanai, Deeba Husain, John D. Lambris, Demetrios Vavvas, Joan W. Miller, Kip M. Connor*

    *Corresponding author. E-mail: kip_connor{at}meei.harvard.edu

    Published 22 July 2015, Sci. Transl. Med. 7, 297ra116 (2015)
    DOI: 10.1126/scitranslmed.aab1482

    This PDF file includes:

    • Materials and Methods
    • Table S1. Human patient data for ELISA samples.
    • Fig. S1. Human samples of RD.
    • Fig. S2. Mouse model of RD.
    • Fig. S3. Complement expression in the mouse RD model.
    • Fig. S4. Cd55 and Cd59 are down-regulated in the detached retina.
    • Fig. S5. Outer nuclear layer expression of complement regulator Cd55.
    • Fig. S6. Expression of several key complement proteins after RD.
    • Fig. S7. The role of the lectin and classical complement pathways in outer nuclear layer cell death after RD.
    • Fig. S8. Cd55 and Cd59 are down-regulated in a photoreceptor cell line during hypoxia, and increasing serum concentrations cause MAC formation.
    • Fig. S9. Hypoxia makes photoreceptors more susceptible to complementmediated cell death.
    • References (59, 60)

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