Dramatic Effect of Oral CSF-1R Kinase Inhibitor on Retinal Microglia Revealed by In Vivo Scanning Laser Ophthalmoscopy

Ebneter, Andreas; Kokona, Despina; Jovanovic, Joël; Zinkernagel, Martin

doi:10.1167/tvst.6.2.10

Cited by 13 publications

(16 citation statements)

References 10 publications

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“…At D3-7, microglial cell bodies and processes got thicker and showed a bipolar shape associated with an axon. Scale bars: 100 μm and 25 μm in (a) and (b) [Color figure can be viewed at wileyonlinelibrary.com] (Figure 4c), similar to the previous report (Ebneter, Kokona, Jovanovic, & Zinkernagel, 2017). In our condition, PLX did not change the number of RGCs in the absence of NMDA.…”

Section: Pharmacological Ablation Of Microglia Protects Rgcssupporting

confidence: 89%

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Microglia mediate non‐cell‐autonomous cell death of retinal ganglion cells

Takeda

Shinozaki

Kashiwagi

et al. 2018

Glia

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Excitotoxicity is well known in the neuronal death in the brain and is also linked to neuronal damages in the retina. Recent accumulating evidence show that microglia greatly affect excitotoxicity in the brain, but their roles in retina have received only limited attention. Here, we report that retinal excitotoxicity is mediated by microglia. To this end, we employed three discrete methods, that is, pharmacological inhibition of microglia by minocycline, pharmacological ablation by an antagonist for colony stimulating factor 1 receptor (PLX5622), and genetic ablation of microglia using Iba1‐tTA::DTAtetO/tetO mice. Intravitreal injection of NMDA increased the number of apoptotic retinal ganglion cells (RGCs) followed by reduction in the number of RGCs. Although microglia did not respond to NMDA directly, they became reactive earlier than RGC damages. Inhibition or ablation of microglia protected RGCs against NMDA. We found up‐regulation of proinflammatory cytokine genes including Il1b, Il6 and Tnfa, among which Tnfa was selectively blocked by minocycline. PLX5622 also suppressed Tnfa expression. Tumor necrosis factor α (TNFα) signals were restricted in microglia at very early followed by spreading into other cell types. TNFα up‐regulation in microglia and other cells were significantly attenuated by minocycline and PLX5622, suggesting a central role of microglia for TNFα induction. Both inhibition of TNFα and knockdown of TNF receptor type 1 by siRNA protected RGCs against NMDA. Taken together, our data demonstrate that a phenotypic change of microglia into a neurotoxic one is a critical event for the NMDA‐induced degeneration of RGCs, suggesting an importance of non‐cell‐autonomous mechanism in the retinal neuronal excitotoxicity.

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Section: Pharmacological Ablation Of Microglia Protects Rgcssupporting

confidence: 89%

“…and (b) [Color figure can be viewed at wileyonlinelibrary.com] (Figure 4c), similar to the previous report(Ebneter, Kokona, Jovanovic, & Zinkernagel, 2017). In our condition, PLX did not change the number of RGCs in the absence of NMDA.…”

supporting

confidence: 90%

Microglia mediate non‐cell‐autonomous cell death of retinal ganglion cells

Takeda

Shinozaki

Kashiwagi

et al. 2018

Glia

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“…To investigate the role of resident microglia on the LPS-induced phenotype, we employed the highly selective CSF-1R inhibitor PLX5622, which has been previously shown to deplete microglia in the CNS [24, 56]. Accumulation of sub-retinal fluid and retinal swelling were completely abrogated by PLX5622 (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, a single LPS injection triggered the production of a plethora of inflammatory mediators in the retina and this phenomenon was abolished after repeated injections. In addition, the use of a colony-stimulating factor 1-receptor (CSF1-R) inhibitor, which we have previously shown to dramatically deplete retinal microglia [24], provides evidence that the breakdown of the BRB can be abrogated by suppression of the innate immune response. These data suggest that resident retinal microglia in combination with monocytes invading from the circulation are responsible for the breakdown of the BRB and that BRB integrity can be preserved by inhibition of the CSF-1R.…”

Section: Introductionmentioning

confidence: 99%

Colony-stimulating factor 1 receptor inhibition prevents disruption of the blood-retina barrier during chronic inflammation

Kokona

Ebneter

Escher

et al. 2018

J Neuroinflammation

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BackgroundMicroglia-associated inflammation is closely related to the pathogenesis of various retinal diseases such as uveitis and diabetic retinopathy, which are associated with increased vascular permeability. In this study, we investigated the effect of systemic lipopolysaccharide (LPS) exposure to activation and proliferation of retinal microglia /macrophages.MethodsBalb/c and Cx3cr1gfp/+ mice were challenged with LPS (1 mg/kg) daily for four consecutive days. For microglia depletion, mice were treated with colony-stimulating factor 1 receptor (CSF-1R) inhibitor PLX5622 1 week before the first LPS challenge and until the end of the experiment. In vivo imaging of the retina was performed on days 4 and 7 after the first LPS challenge, using optical coherence tomography and fluorescein angiography. Flow cytometry analysis, retinal whole mount, and retinal sections were used to investigate microglia and macrophage infiltration and proliferation after LPS challenge. Cytokines were analyzed in the blood as well as in the retina. Data analysis was performed using unpaired t tests, repeated measures one-way ANOVA, or ordinary one-way ANOVA followed by Tukey’s post hoc analysis. Kruskal-Wallis test followed by Dunn’s multiple comparison tests was used for the analysis of non-normally distributed data.ResultsRepeated LPS challenge led to activation and proliferation of retinal microglia, infiltration of monocyte-derived macrophages into the retina, and breakdown of the blood-retina barrier (BRB) accompanied by accumulation of sub-retinal fluid. Using in vivo imaging, we show that the breakdown of the BRB is highly reproducible but transitory. Acute but not chronic systemic exposure to LPS triggered a robust release of inflammatory mediators in the retina with minimal effects in the blood plasma. Inhibition of the CSF-1R by PLX5622 resulted in depletion of retinal microglia, suppression of cytokine production in the retina, and prevention of BRB breakdown.ConclusionsThese findings suggest that microglia/macrophages play an important role in the pathology of retinal disorders characterized by breakdown of the BRB, and suppression of their activation may be a potential therapeutic target for such retinopathies.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1373-4) contains supplementary material, which is available to authorized users.

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“…Using SLO, we could identify gfp positive microglia cells as hyperreflective signal in the auto fluorescent images, however the wide field 102 ° lens was able to cover a larger fundus area compared to the 55 ° lens. SLO can also be utilized for the monitoring of retinal microglia cells distribution and activation in reporter mice with a functional fractalkine receptor (Cx3cr1 gfp/+ mice) in normal or diseased retina 12,21,32 . The combination of SD-OCT with SLO in the same imaging session can provide information about microglia distribution and activation upon retinal degeneration and could be useful for monitoring retinal diseases in mice 21 .…”

Section: Representative Resultsmentioning

confidence: 99%

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Kokona¹,

Jovanovic²,

Ebneter³

et al. 2017

JoVE

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Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1 mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 µm and 6 µm, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate.

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Dramatic Effect of Oral CSF-1R Kinase Inhibitor on Retinal Microglia Revealed by In Vivo Scanning Laser Ophthalmoscopy

Cited by 13 publications

References 10 publications

Microglia mediate non‐cell‐autonomous cell death of retinal ganglion cells

Microglia mediate non‐cell‐autonomous cell death of retinal ganglion cells

Colony-stimulating factor 1 receptor inhibition prevents disruption of the blood-retina barrier during chronic inflammation

<em>In Vivo</em> Imaging of <em>Cx3cr1<sup>gfp/gfp</sup></em> Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

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