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Cited by 111Retinal Development and DisordersNeuroinflammation and Neurodegeneration MechanismsGlaucoma and retinal disorders

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Targeting translocator protein (18 kDa) (TSPO) dampens pro-inflammatory microglia reactivity in the retina and protects from degeneration

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Scholz, Rebecca, Caramoy, Albert, Bhuckory, Mohajeet B., Rashid, Khalid, Chen, Mei, Xu, Heping, Grimm, Christian, Langmann, Thomas·2015·Journal of Neuroinflammation
DOI 10.1186/s12974-015-0422-5PMC PMC4630900
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Abstract

BACKGROUND: Reactive microglia are commonly seen in retinal degenerative diseases, and neurotoxic microglia responses can contribute to photoreceptor cell death. We and others have previously shown that translocator protein (18 kDa) (TSPO) is highly induced in retinal degenerations and that the selective TSPO ligand XBD173 (AC-5216, emapunil) exerts strong anti-inflammatory effects on microglia in vitro and ex vivo. Here, we investigated whether targeting TSPO with XBD173 has immuno-modulatory and neuroprotective functions in two mouse models of acute retinal degeneration using bright white light exposure. METHODS: BALB/cJ and Cx3cr1(GFP/+) mice received intraperitoneal injections of 10 mg/kg XBD173 or vehicle for five consecutive days, starting 1 day prior to exposure to either 15,000 lux white light for 1 h or 50,000 lux focal light for 10 min, respectively. The effects of XBD173 treatment on microglia and Müller cell reactivity were analyzed by immuno-stainings of retinal sections and flat mounts, fluorescence-activated cell sorting (FACS) analysis, and mRNA expression of microglia markers using quantitative real-time PCR (qRT-PCR). Optical coherence tomography (OCT), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stainings, and morphometric analyses were used to quantify the extent of retinal degeneration and photoreceptor apoptosis. RESULTS: Four days after the mice were challenged with bright white light, a large number of amoeboid-shaped alerted microglia appeared in the degenerating outer retina, which was nearly completely prevented by treatment with XBD173. This treatment also down-regulated the expression of TSPO protein in microglia but did not change the TSPO levels in the retinal pigment epithelium (RPE). RT-PCR analysis showed that the microglia/macrophage markers Cd68 and activated microglia/macrophage whey acidic protein (Amwap) as well as the pro-inflammatory genes Ccl2 and Il6 were reduced after XBD173 treatment. Light-induced degeneration of the outer retina was nearly fully blocked by XBD173 treatment. We further confirmed these findings in an independent mouse model of focal light damage. Retinas of animals receiving XBD173 therapy displayed significantly more ramified non-reactive microglia and more viable arrestin-positive cone photoreceptors than vehicle controls. CONCLUSIONS: Targeting TSPO with XBD173 effectively counter-regulates microgliosis and ameliorates light-induced retinal damage, highlighting a new pharmacological concept for the treatment of retinal degenerations.

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Fig. 1 XBD173 treatment of light-exposed mice prevents microglia reactivity. a Light exposure regimen and mode of XBD173 administration. Representative photomicrographs show retinal sections (b–d) and flat mounts (e–j) stained with IBA1 (green) and DAPI (blue). In control retinas, microglial cells were located in the OPL, IPL, and GCL (b, e, h). Light-exposed retinas injected with vehicle control showed a massive thinning of the ONL and many amoeboid-shaped, reactive microglia in the ONL and the subretinal space (c, f, i). Compared to vehicle controls, the ONL of XBD173-treated retinas appeared markedly preserved and less amoeboid microglia were detectable in the ONL and the subretinal space (d, g, j). The total number of Iba1-positive microglial cells in the ONL (k) and the subretinal area (l) after light damage was significantly reduced in the XBD173 therapy group. Data show mean ± SEM (control n = 3, light exposure and vehicle treatment n = 6, light exposure and XBD173 treatment n = 12 sections). The percentage of CD11b-positive cells in the retina as determined by flow cytometry was strongly reduced by XBD173 treatment in representative FACS plots (m–o) and quantitative analyses of n = 3–4 retinas (p). ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer. *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar 50 μm
Fig. 1 XBD173 treatment of light-exposed mice prevents microglia reactivity. a Light exposure regimen and mode of XBD173 administration. Representative photomicrographs show retinal sections (b–d) and flat mounts (e–j) stained with IBA1 (green) and DAPI (blue). In control retinas, microglial cells were located in the OPL, IPL, and GCL (b, e, h). Light-exposed retinas injected with vehicle control showed a massive thinning of the ONL and many amoeboid-shaped, reactive microglia in the ONL and the subretinal space (c, f, i). Compared to vehicle controls, the ONL of XBD173-treated retinas appeared markedly preserved and less amoeboid microglia were detectable in the ONL and the subretinal space (d, g, j). The total number of Iba1-positive microglial cells in the ONL (k) and the subretinal area (l) after light damage was significantly reduced in the XBD173 therapy group. Data show mean ± SEM (control n = 3, light exposure and vehicle treatment n = 6, light exposure and XBD173 treatment n = 12 sections). The percentage of CD11b-positive cells in the retina as determined by flow cytometry was strongly reduced by XBD173 treatment in representative FACS plots (m–o) and quantitative analyses of n = 3–4 retinas (p). ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer. *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar 50 μmCC BY · PubMed Central
Fig. 2 XBD173 treatment of light-exposed mice down-regulates TSPO in microglia and prevents gliosis. Representative photomicrographs of retinal sections and flat mounts from mice 4 days after light exposure. Control retinas show weak TSPO labeling (a), and strong up-regulation of TSPO expression confined to microglia was present upon light exposure and vehicle treatment (b). XBD173-treated mice displayed only constitutive expression of TSPO in the RPE, but no strong signal in the retina was found (c). Number of analyzed photomicrographs: control n = 5 sections from two individual mice, light exposure and vehicle treatment n = 7 sections from five individual mice, light exposure and XBD173 treatment n = 11 sections from five individual mice. The expression of TSPO in the RPE was relatively unaffected in the different conditions as shown in flat mount images stained with anti-TSPO antibody and phalloidin (d–f). Glutamine synthetase (GS), a constitutive Müller cell protein, and glial fibrillary acidic protein (GFAP) as markers for gliosis were analyzed (g–n). GS expression did not differ in the various treatment groups (g–i, m) whereas GFAP expression was changed significantly (j–l, n). Data show mean ± SEM (control n = 5 sections from five individual mice, light exposure and vehicle treatment n = 5 sections from three individual mice, light exposure and XBD173 treatment n = 5 sections from four individual mice). **p < 0.01. ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer. Scale bar 50 μm
Fig. 2 XBD173 treatment of light-exposed mice down-regulates TSPO in microglia and prevents gliosis. Representative photomicrographs of retinal sections and flat mounts from mice 4 days after light exposure. Control retinas show weak TSPO labeling (a), and strong up-regulation of TSPO expression confined to microglia was present upon light exposure and vehicle treatment (b). XBD173-treated mice displayed only constitutive expression of TSPO in the RPE, but no strong signal in the retina was found (c). Number of analyzed photomicrographs: control n = 5 sections from two individual mice, light exposure and vehicle treatment n = 7 sections from five individual mice, light exposure and XBD173 treatment n = 11 sections from five individual mice. The expression of TSPO in the RPE was relatively unaffected in the different conditions as shown in flat mount images stained with anti-TSPO antibody and phalloidin (d–f). Glutamine synthetase (GS), a constitutive Müller cell protein, and glial fibrillary acidic protein (GFAP) as markers for gliosis were analyzed (g–n). GS expression did not differ in the various treatment groups (g–i, m) whereas GFAP expression was changed significantly (j–l, n). Data show mean ± SEM (control n = 5 sections from five individual mice, light exposure and vehicle treatment n = 5 sections from three individual mice, light exposure and XBD173 treatment n = 5 sections from four individual mice). **p < 0.01. ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer. Scale bar 50 μmCC BY · PubMed Central
Fig. 3 XBD173 treatment dampens microglia-related pro-inflammatory marker expression after light exposure. To determine the mRNA expression of inflammation-associated genes in the retina, qRT-PCR analysis was performed 4 days after light exposure. a–c The microglia markers Cd68 and Amwap were up-regulated after light exposure, and TSPO levels were also increased. In contrast, the retinas from XBD173-treated mice expressed significantly less Cd68, Amwap, and Tspo transcripts after light exposure. d, e XBD173 treatment reduced the expression of Ccl2 and Il6 that were elevated by light exposure. f iNOS expression was not altered. Data show mean ± SEM out of three independent experiments (control n = 4–8, light exposure plus vehicle treatment n = 8–15, light exposure plus XBD173 treatment n = 8–15 per group, measured in duplicates) with *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 3 XBD173 treatment dampens microglia-related pro-inflammatory marker expression after light exposure. To determine the mRNA expression of inflammation-associated genes in the retina, qRT-PCR analysis was performed 4 days after light exposure. a–c The microglia markers Cd68 and Amwap were up-regulated after light exposure, and TSPO levels were also increased. In contrast, the retinas from XBD173-treated mice expressed significantly less Cd68, Amwap, and Tspo transcripts after light exposure. d, e XBD173 treatment reduced the expression of Ccl2 and Il6 that were elevated by light exposure. f iNOS expression was not altered. Data show mean ± SEM out of three independent experiments (control n = 4–8, light exposure plus vehicle treatment n = 8–15, light exposure plus XBD173 treatment n = 8–15 per group, measured in duplicates) with *p < 0.05; **p < 0.01; ***p < 0.001CC BY · PubMed Central
Fig. 4 XBD173 preserves retinal thickness in light-exposed mice. SD-OCT was performed 4 days after light exposure to analyze changes in retinal structures. a–c Light-exposed mice show an altered reflectance in the ONL, which was not present in XBD173-treated mice. d–f Representative heat maps show the average retinal thickness of control and light-exposed mice after vehicle or XBD173 treatment, respectively. Light-exposed mice show a significant thinning of the retina, in the central (g) and more peripheral area (h), which was preserved by XBD173 treatment. g, h One data point represents the average thickness of the central retina, calculated from four different areas around the optic nerve head in circle diameters of 3 mm (g) and 6 mm (h), respectively. Data show mean ± SEM out of two independent experiments (control n = 6, light exposure plus vehicle treatment n = 10, light exposure plus XBD173 treatment n = 10/group) with ***p < 0.001
Fig. 4 XBD173 preserves retinal thickness in light-exposed mice. SD-OCT was performed 4 days after light exposure to analyze changes in retinal structures. a–c Light-exposed mice show an altered reflectance in the ONL, which was not present in XBD173-treated mice. d–f Representative heat maps show the average retinal thickness of control and light-exposed mice after vehicle or XBD173 treatment, respectively. Light-exposed mice show a significant thinning of the retina, in the central (g) and more peripheral area (h), which was preserved by XBD173 treatment. g, h One data point represents the average thickness of the central retina, calculated from four different areas around the optic nerve head in circle diameters of 3 mm (g) and 6 mm (h), respectively. Data show mean ± SEM out of two independent experiments (control n = 6, light exposure plus vehicle treatment n = 10, light exposure plus XBD173 treatment n = 10/group) with ***p < 0.001CC BY · PubMed Central
Fig. 5 XBD173 protects the light-exposed retina from cell death. a–c Representative panorama sections of the retina were stained with DAPI to further characterize structural changes. Light exposure caused severe degeneration particularly of the ONL. XBD173 administration reduced degeneration, represented by a clearly thicker photoreceptor layer, scale bar 200 μM. d–f Representative photomicrographs of TUNEL-stained retinal sections show the amount of apoptotic cells 4 days after light exposure. Light exposure caused a strong increase of apoptotic cells, especially in the ONL. Only very few TUNEL-positive cells were visible after XBD173 administration. Scale bar 50 μm (g). For quantification of retinal thickness, anterior and posterior areas were divided into ten sections with the optic nerve as reference and rows of photoreceptor nuclei were counted. Data show mean ± SEM (control n = 4, light exposure plus vehicle treatment n = 8, light exposure plus XBD173 treatment n = 10/group) with *p < 0.05; **p < 0.01; ***p < 0.001 light exposed + vehicle versus control (red color) and light exposed + XBD173 versus light exposed + vehicle (blue color). h mRNA analyses revealed a significantly lower expression of caspase 8 in light-exposed XBD173-treated mice compared to controls and vehicle-injected retinas. Data show mean ± SEM out of two independent experiments (control n = 4, light exposure plus vehicle treatment n = 8, light exposure plus XBD173 treatment n = 8/group, measured in triplicates) with **p < 0.01; ***p < 0.001
Fig. 5 XBD173 protects the light-exposed retina from cell death. a–c Representative panorama sections of the retina were stained with DAPI to further characterize structural changes. Light exposure caused severe degeneration particularly of the ONL. XBD173 administration reduced degeneration, represented by a clearly thicker photoreceptor layer, scale bar 200 μM. d–f Representative photomicrographs of TUNEL-stained retinal sections show the amount of apoptotic cells 4 days after light exposure. Light exposure caused a strong increase of apoptotic cells, especially in the ONL. Only very few TUNEL-positive cells were visible after XBD173 administration. Scale bar 50 μm (g). For quantification of retinal thickness, anterior and posterior areas were divided into ten sections with the optic nerve as reference and rows of photoreceptor nuclei were counted. Data show mean ± SEM (control n = 4, light exposure plus vehicle treatment n = 8, light exposure plus XBD173 treatment n = 10/group) with *p < 0.05; **p < 0.01; ***p < 0.001 light exposed + vehicle versus control (red color) and light exposed + XBD173 versus light exposed + vehicle (blue color). h mRNA analyses revealed a significantly lower expression of caspase 8 in light-exposed XBD173-treated mice compared to controls and vehicle-injected retinas. Data show mean ± SEM out of two independent experiments (control n = 4, light exposure plus vehicle treatment n = 8, light exposure plus XBD173 treatment n = 8/group, measured in triplicates) with **p < 0.01; ***p < 0.001CC BY · PubMed Central
Fig. 6 XBD173 treatment prevents microglia reactivity and rescues cone photoreceptors in focal light-induced damage. a Light exposure regimen and mode of XBD173 administration. b Representative fundus image showing the focal light lesion 4 days after Cx3cr1 GFP/+ mice were exposed to 50,000 lux for 10 min. c–e Representative confocal images of GFP-positive retinal microglia in flat mounts from control mice (c) and lesion areas of light-exposed mice (d, e). The average size of microglial cells was assessed using the grid cross point system (f). Data show mean ± SEM (n = 40–180 cells per group) with ***p < 0.001. g–i Retinal flat mounts were processed for cone arrestin staining and examined by confocal microscopy. Representative confocal images of cone arrestin-positive cells in flat mounts from control mice (g) and lesion areas of light-exposed mice (h, i). j Average density of cone photoreceptor cells. Data show mean ± SEM (n = 6–8 retinas per group) with *p < 0.05; ***p < 0.001. Scale bar 50 μm
Fig. 6 XBD173 treatment prevents microglia reactivity and rescues cone photoreceptors in focal light-induced damage. a Light exposure regimen and mode of XBD173 administration. b Representative fundus image showing the focal light lesion 4 days after Cx3cr1 GFP/+ mice were exposed to 50,000 lux for 10 min. c–e Representative confocal images of GFP-positive retinal microglia in flat mounts from control mice (c) and lesion areas of light-exposed mice (d, e). The average size of microglial cells was assessed using the grid cross point system (f). Data show mean ± SEM (n = 40–180 cells per group) with ***p < 0.001. g–i Retinal flat mounts were processed for cone arrestin staining and examined by confocal microscopy. Representative confocal images of cone arrestin-positive cells in flat mounts from control mice (g) and lesion areas of light-exposed mice (h, i). j Average density of cone photoreceptor cells. Data show mean ± SEM (n = 6–8 retinas per group) with *p < 0.05; ***p < 0.001. Scale bar 50 μmCC BY · PubMed Central

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Metadata

Authors
Scholz, R., Caramoy, A., Bhuckory, M. B., Rashid, K., Chen, M., Xu, H., Grimm, C., & Langmann, T.
Affiliations
  • University of Cologne (DE)
  • Queen's University Belfast (GB)
  • University of Zurich (CH)
Corresponding author
Langmann, Thomas
ORCID
Venue
Journal of Neuroinflammation
Publisher
Springer Science and Business Media LLC
Published
2015-11-02
Volume
12
Issue
1
Pages
201-201
ISSN
1742-2094 (ISSN-L)
DOI
10.1186/s12974-015-0422-5
PMC
PMC4630900
OpenAlex
W1944096616
Cited by
111
References
60
Citations by year
2026: 5 · 2025: 5 · 2024: 11 · 2023: 5 · 2022: 12 · 2021: 18 · 2020: 10 · 2019: 11 · 2018: 13 · 2017: 13 · 2016: 7 · 2015: 1
FWCI
4.92
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Journal of Neuroinflammation (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Kölner Universitäts PublikationsServer (Universität zu Köln) (publishedVersion)
  • Zurich Open Repository and Archive (University of Zurich) (acceptedVersion, cc-by)
  • Research Portal (Queen's University Belfast) (submittedVersion, cc-by)
  • Zurich Open Repository and Archive (University of Zurich) (submittedVersion, cc-by)
  • Zurich Open Repository and Archive (University of Zurich)
Funders
  • Deutsche Forschungsgemeinschaft (LA1203/6-2, LA1203/9-1, LA1203/10-1, FOR2240)
  • Velux Stiftung (Project 967)
  • Hans und Marlies Stoock Foundation (S 061-10.013)
  • Pro Retina Foundation (ProRetinaStip LA)
  • Bayer (Bayer Graduate Program in Pharmacology)
  • Fight for Sight UK (1425/1426)
Topics
  • Health Sciences › Medicine › Ophthalmology › Retinal Development and Disorders
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Ophthalmology › Glaucoma and retinal disorders
Keywords
translocator protein (TSPO), microglia, retinal degeneration, light-induced retinal damage, photoreceptor apoptosis, neuroprotection, anti-inflammatory activity, Müller cells, CX3CR1, MCP-1, interleukin-6, retinal pigment epithelium, choroidal neovascularization
MeSH terms
Animals, Disease Models, Animal, Female, Flow Cytometry, Immunohistochemistry, Inflammation, Male, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Transgenic, Purines, Retinal Degeneration, Microglia, Receptors, GABA, Reverse Transcriptase Polymerase Chain Reaction, In Situ Nick-End Labeling, Tomography, Optical Coherence, Mice, Real-Time Polymerase Chain Reaction

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