Skip to main content
main-content

01.12.2017 | Research | Ausgabe 1/2017 Open Access

Journal of Neuroinflammation 1/2017

Microglia activation is essential for BMP7-mediated retinal reactive gliosis

Zeitschrift:
Journal of Neuroinflammation > Ausgabe 1/2017
Autoren:
Subramanian Dharmarajan, Debra L. Fisk, Christine M. Sorenson, Nader Sheibani, Teri L. Belecky-Adams
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12974-017-0855-0) contains supplementary material, which is available to authorized users.

Abstract

Background

Our previous studies have shown that BMP7 is able to trigger activation of retinal macroglia. However, these studies showed the responsiveness of Müller glial cells and retinal astrocytes in vitro was attenuated in comparison to those in vivo, indicating other retinal cell types may be mediating the response of the macroglial cells to BMP7. In this study, we test the hypothesis that BMP7-mediated gliosis is the result of inflammatory signaling from retinal microglia.

Methods

Adult mice were injected intravitreally with BMP7 and eyes harvested 1, 3, or 7 days postinjection. Some mice were treated with PLX5622 (PLX) to ablate microglia and were subsequently injected with control or BMP7. Processed tissue was analyzed via immunofluorescence, RT-qPCR, or ELISA. In addition, cultures of retinal microglia were treated with vehicle, lipopolysaccharide, or BMP7 to determine the effects of BMP7-isolated cells.

Results

Mice injected with BMP7 showed regulation of various inflammatory markers at the RNA level, as well as changes in microglial morphology. Isolated retinal microglia also showed an upregulation of BMP-signaling components following treatment. In vitro treatment of retinal astrocytes with conditioned media from activated microglia upregulated RNA levels of gliosis markers. In the absence of microglia, the mouse retina showed a subdued gliosis and inflammatory response when exposed to BMP7.

Conclusions

Gliosis resulting from BMP7 is mediated through an inflammatory response from retinal microglia.
Zusatzmaterial
Additional file 1: Figure S1. PU.1 localizes with retinal microglia. Co-label of PU.1 antibody with antibody against GFP that cross-reacts with YFP in a retinal section from P30 mice which have YFP tag on vascular endothelial cadherin (VE-YFP), a marker expressed in endothelial cells (A-D). No co-label of PU.1 was observed with VE-YFP. PU.1 was also co-labeled with microglia marker IBA1 to show localization was restricted to microglial cells (E-H). Hoechst merged with the images of green and red channels are shown in D and H. Magnification bar in E = 50 μm, for images A–H. (TIF 857 kb)
12974_2017_855_MOESM1_ESM.tif
Additional file 2: Figure S2. Expression of BMP signaling molecules in microglia in vehicle and BMP7-injected retinas. Retinal sections from P30 mouse injected with vehicle or BMP7 24 h postinjection were double-labeled with antibodies that labels microglia cytoplasm (IBA1) and phospho SMAD 1/5/9 (pSMAD; A–F) or phospho TAK1 (pTAK1; G–L). Thin plane confocal microscopy images with y,z (strips to right of the panel) and x,z planes (strips at the bottom of the panels) shown in C, F, I and L. pSMAD-labeled cells were primarily found in the GCL in the vehicle-treated retina, with some co-localization with the cytoplasmic microglial marker IBA1 (A-C). The BMP7-injected retina had an increase in pSMAD expression in the INL as well as substantial co-localization with IBA1 (D–F). Vehicle-injected retina showed pTAK1 expression in the GCL with little to no IBA1 co-localization (G–I), while the BMP7-injected retinas showed increased levels of pTAK1 levels in the INL, as well as significant co-localization with IBA1 (J–L). Magnification bar in A = 50 μm, for images A–L. (TIF 688 kb)
12974_2017_855_MOESM2_ESM.tif
Additional file 4: Figure S4. IF label of retinas for GFAP, S-100-β, and NCAN in P30 uninjected and 3 and 7 days vehicle-injected retinas. Retinal sections from uninjected P30 mouse, vehicle-injected P30 mouse, obtained 3 and 7 days postinjection, labeled for GFAP (A, D, G), S100-β (B, E, H), and NCAN (C, F, I). Label for all three markers appears to be similar in the uninjected and the vehicle-injected retinas. Magnification bar in A = 50 μm, for images A–I. (TIF 5611 kb)
12974_2017_855_MOESM4_ESM.tif
Additional file 5: Figure S5. Protein levels in PLX-treated mice. Protein isolated from control and PLX-treated mice injected with vehicle or BMP7 changes in protein levels of gliosis markers GFAP, S100-β, and TXNIP, with β-Tubulin used as a loading control. GFAP showed elevated levels in the BMP7-injected control mice, while PLX mice had GFAP levels similar to the vehicle injection. S100-β was elevated in the 3 and 7 days BMP7-injected PLX mice as well as in the 7 days BMP7-injected control mice, compared to the respective vehicle controls. TXNIP levels did not change in the control and PLX mice injected with vehicle or BMP7 3 days postinjection. Seven days postinjection, TXNIP levels did increase in the control BMP-injected mice, while no such change was observed in the PLX mice. No statistical significance was observed in the densitometric analysis (B) of blots from (A). (TIF 472 kb)
12974_2017_855_MOESM5_ESM.tif
Literatur
Über diesen Artikel

Weitere Artikel der Ausgabe 1/2017

Journal of Neuroinflammation 1/2017 Zur Ausgabe

Neu in den Fachgebieten Neurologie und Psychiatrie

Meistgelesene Bücher in der Neurologie & Psychiatrie

  • 2016 | Buch

    Neurologie

    Das Lehrbuch vermittelt Ihnen das gesamte Neurologie-Prüfungswissen für Ihr Medizinstudium und bereitet auch junge Assistenzärzte durch detailliertes Fachwissen optimal auf die Praxis vor. Die komplett überarbeitete Auflage enthält sechs neue, interdisziplinäre Kapitel.

    Herausgeber:
    Werner Hacke
  • 2016 | Buch

    Komplikationen in der Neurologie

    Das Buch schildert Ereignisse im Rahmen der Neuromedizin, die während der Diagnostik und Therapie neurologischer Erkrankungen und Symptome auftreten können. Die Fallbeispiele sensibilisieren Sie für mögliche Risikofaktoren, um das Auftreten solcher Komplikationen zu vermeiden.

    Herausgeber:
    Frank Block
  • 2017 | Buch

    Facharztwissen Psychiatrie, Psychosomatik und Psychotherapie

    Leitsymptome, Untersuchungsmethoden, Krankheitsbilder, Notfälle & Co. – mit der Neuauflage des "Facharztwissens" sind Sie auf die Facharztprüfung in Psychiatrie und Psychotherapie optimal vorbereitet. In dieser 2. Auflage sind die Kapitel zu psychosomatischen Störungen deutlich ausgebaut.

    Herausgeber:
    Prof. Dr. Dr. Frank Schneider
  • 2019 | Buch

    Kompendium der Psychotherapie

    Für Ärzte und Psychologen

    Dieses Werk wendet sich an Ärzte und Psychologen, die an psychiatrischen und psychosomatischen Kliniken oder an Psychotherapeutischen Ausbildungsinstituten arbeiten und in den vorhandenen Lehrbüchern der Psychotherapie den Brückenschlag zur …

    Herausgeber:
    Tilo Kircher