Skip to main content

Advertisement

SpringerLink
Log in

Spatial distribution of CD115+ and CD11b+ cells and their temporal activation during oxygen-induced retinopathy in mice

  • Basic Science
  • Published:
Graefe's Archive for Clinical and Experimental Ophthalmology Aims and scope Submit manuscript

Abstract

Purpose

The model of oxygen-induced retinopathy (OIR) is widely used to analyze pathomechanisms in retinal neovascularization. Previous studies have shown that macrophages (MP) play a key role in vessel formation in OIR, the influence of microglia (MG) having been discussed. The aim of our study was to analyze the spatial and temporal distribution and activation of MP/MG expressing CD115 and CD11b during the process of neovascularization in OIR.

Methods

We used MacGreen mice expressing the green fluorescence protein (GFP) under the promoter for CD115. CD115+ cells were investigated in vivo by scanning laser ophthalmoscopy at postnatal days (P) 17 and 21 in MacGreen mice with OIR (75% oxygen from P7 to P12), and were compared to MacGreen room-air controls. In addition MP/MG were examined ex vivo using immunohistochemistry for CD11b+ detection on retinal flatmounts at P14, P17, and P21 of wild type mice with OIR.

Results

In-vivo imaging revealed the highest density of activated MP/MG in tuft areas at P17 of MacGreen mice with OIR. Tufts and regions with a high density of CD115+ cells were detected close to veins, rather to arteries. In peripheral, fully vascularized areas, the distribution of CD115+ cells in MacGreen mice with OIR was similar to MacGreen room-air controls. Correspondingly, immunohistochemical analyses of retinal flatmounts from wild type mice with OIR induction revealed that the number of CD11b+ cells significantly varies between vascular, avascular, and tuft areas as well as between the retinal layers. Activated CD11b+ cells were almost exclusively found in avascular areas and tufts of wild type mice with OIR induction; here, the proportion of activated cells related to the total number of CD11b+ cells remained stable over the course of time.

Conclusions

Using two different approaches to monitor MP/MG cells, our findings demonstrated that MP/MG concentrate within pathologically vascularized areas during OIR. We were able to clarify that reactive changes of CD11b+ cell distribution to OIR primarily occur in the deep retinal layers. Furthermore, we found the highest proportion of activated CD11b+ cells in regions with pathologic neovascularization processes. Our findings support previous reports about activated MP/MG guiding revascularization in avascular areas and playing a key role in the formation and regression of neovascular tufts.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Smith LE, Wesolowski E, McLellan A, Kostyk SK, D’Amato R, Sullivan R, D’Amore PA (1994) Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci 35:101–111

    CAS  PubMed  Google Scholar 

  2. Stahl A, Connor KM, Sapieha P, Chen J, Dennison RJ, Krah NM, Seaward MR, Willett KL, Aderman CM, Guerin KI, Hua J, Löfqvist C, Hellström A, Smith LE (2010) The mouse retina as an angiogenesis model. Invest Ophthalmol Vis Sci 51:2813–2826. https://doi.org/10.1167/iovs.10-5176

    Article  PubMed  PubMed Central  Google Scholar 

  3. Hartnett ME, Penn JS (2012) Mechanisms and management of retinopathy of prematurity. N Engl J Med 367:2515–2526. https://doi.org/10.1056/NEJMra1208129

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Caprara C, Grimm C (2012) From oxygen to erythropoietin: relevance of hypoxia for retinal development, health and disease. Prog Retin Eye Res 31:89–119. https://doi.org/10.1016/j.preteyeres.2011.11.003

    Article  CAS  PubMed  Google Scholar 

  5. Uemura A, Kusuhara S, Katsuta H, Nishikawa S (2006) Angiogenesis in the mouse retina: a model system for experimental manipulation. Exp Cell Res 312:676–683. https://doi.org/10.1016/j.yexcr.2005.10.030

    Article  CAS  PubMed  Google Scholar 

  6. Naug HL, Browning J, Gole GA, Gobé G (2000) Vitreal macrophages express vascular endothelial growth factor in oxygen-induced retinopathy. Clin Experiment Ophthalmol 28:48–52

    Article  CAS  PubMed  Google Scholar 

  7. Kataoka K, Nishiguchi KM, Kaneko H, van Rooijen N, Kachi S, Terasaki H (2011) The roles of vitreal macrophages and circulating leukocytes in retinal neovascularization. Invest Ophthalmol Vis Sci 52:1431–1438. https://doi.org/10.1167/iovs.10-5798

    Article  CAS  PubMed  Google Scholar 

  8. Yang Y, Liu F, Tang M, Yuan M, Hu A, Zhan Z, Li Z, Li J, Ding X, Lu L (2016) Macrophage polarization in experimental and clinical choroidal neovascularization. Sci Rep 6:30933. https://doi.org/10.1038/srep30933

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. He L, Marneros AG (2014) Doxycycline inhibits polarization of macrophages to the proangiogenic M2-type and subsequent neovascularization. J Biol Chem 289:8019–8028. https://doi.org/10.1074/jbc.M113.535765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Fischer F, Martin G, Agostini HT (2011) Activation of retinal microglia rather than microglial cell density correlates with retinal neovascularization in the mouse model of oxygen-induced retinopathy. J Neuroinflammation 8:120. https://doi.org/10.1186/1742-2094-8-120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dorrell MI, Aguilar E, Jacobson R, Trauger SA, Friedlander J, Siuzdak G, Friedlander M (2010) Maintaining retinal astrocytes normalizes revascularization and prevents vascular pathology associated with oxygen-induced retinopathy. Glia 58:43–54. https://doi.org/10.1002/glia.20900

    Article  PubMed  PubMed Central  Google Scholar 

  12. Ritter MR, Banin E, Moreno SK, Aguilar E, Dorrell MI, Friedlander M (2006) Myeloid progenitors differentiate into microglia and promote vascular repair in a model of ischemic retinopathy. J Clin Invest 116:3266–3276. https://doi.org/10.1172/JCI29683

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Vessey KA, Wilkinson-Berka JL, Fletcher EL (2011) Characterization of retinal function and glial cell response in a mouse model of oxygen-induced retinopathy. J Comp Neurol 519:506–527. https://doi.org/10.1002/cne.22530

    Article  CAS  PubMed  Google Scholar 

  14. Checchin D, Sennlaub F, Levavasseur E, Leduc M, Chemtob S (2006) Potential role of microglia in retinal blood vessel formation. Invest Ophthalmol Vis Sci 47:3595–3602. https://doi.org/10.1167/iovs.05-1522

    Article  PubMed  Google Scholar 

  15. Chen L, Yang P, Kijlstra A (2002) Distribution, markers, and functions of retinal microglia. Ocul Immunol Inflamm 10:27–39

    Article  PubMed  Google Scholar 

  16. Egensperger R, Maslim J, Bisti S, Holländer H, Stone J (1996) Fate of DNA from retinal cells dying during development: uptake by microglia and macroglia (Müller cells). Brain Res Dev Brain Res 97:1–8

    Article  CAS  PubMed  Google Scholar 

  17. Yang P, de Vos AF, Kijlstra A (1996) Macrophages in the retina of normal Lewis rats and their dynamics after injection of lipopolysaccharide. Invest Ophthalmol Vis Sci 37:77–85

    CAS  PubMed  Google Scholar 

  18. Alt C, Runnels JM, Mortensen LJ, Zaher W, Lin CP (2014) In vivo imaging of microglia turnover in the mouse retina after ionizing radiation and dexamethasone treatment. Invest Ophthalmol Vis Sci 55:5314–5319. https://doi.org/10.1167/iovs.14-14254

    Article  PubMed  Google Scholar 

  19. Jolivel V, Bicker F, Binamé F, Ploen R, Keller S, Gollan R, Jurek B, Birkenstock J, Poisa-Beiro L, Bruttger J, Opitz V, Thal SC, Waisman A, Bäuerle T, Schäfer MK, Zipp F, Schmidt MH (2015) Perivascular microglia promote blood vessel disintegration in the ischemic penumbra. Acta Neuropathol 129:279–295. https://doi.org/10.1007/s00401-014-1372-1

    Article  PubMed  Google Scholar 

  20. Crespo-Garcia S, Reichhart N, Hernandez-Matas C, Zabulis X, Kociok N, Brockmann C, Joussen AM, Strauß O (2015) In vivo analysis of the time and spatial activation pattern of microglia in the retina following laser-induced choroidal neovascularization. Exp Eye Res 139:13–21. https://doi.org/10.1016/j.exer.2015.07.012

    Article  CAS  PubMed  Google Scholar 

  21. Sherr CJ, Roussel MF, Rettenmier CW (1988) Colony-stimulating factor-1 receptor (c-fms). J Cell Biochem 38:179–187. https://doi.org/10.1002/jcb.240380305

    Article  CAS  PubMed  Google Scholar 

  22. Mitrasinovic OM, Grattan A, Robinson CC, Lapustea NB, Poon C, Ryan H, Phong C, Murphy GM (2005) Microglia overexpressing the macrophage colony-stimulating factor receptor are neuroprotective in a microglial–hippocampal organotypic coculture system. J Neurosci 25:4442–4451. https://doi.org/10.1523/JNEUROSCI.0514-05.2005

    Article  CAS  PubMed  Google Scholar 

  23. Sasmono RT, Oceandy D, Pollard JW, Tong W, Pavli P, Wainwright BJ, Ostrowski MC, Himes SR, Hume DA (2003) A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse. Blood 101:1155–1163. https://doi.org/10.1182/blood-2002-02-0569

    Article  CAS  PubMed  Google Scholar 

  24. Sedgwick JD, Schwender S, Imrich H, Dörries R, Butcher GW, ter Meulen V (1991) Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system. Proc Natl Acad Sci U S A 88:7438–7442

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sedgwick JD, Ford AL, Foulcher E, Airriess R (1998) Central nervous system microglial cell activation and proliferation follows direct interaction with tissue-infiltrating T cell blasts. J Immunol 160:5320–5330

    CAS  PubMed  Google Scholar 

  26. Campanella M, Sciorati C, Tarozzo G, Beltramo M (2002) Flow cytometric analysis of inflammatory cells in ischemic rat brain. Stroke 33:586–592

    Article  PubMed  Google Scholar 

  27. Mattapallil MJ, Wawrousek EF, Chan CC, Zhao H, Roychoudhury J, Ferguson TA, Caspi RR (2012) The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes. Invest Ophthalmol Vis Sci 53:2921–2927. https://doi.org/10.1167/iovs.12-9662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Brockmann C, Brockmann T, Dege S, Busch C, Kociok N, Vater A, Klussmann S, Strauß O, Joussen AM (2015) Intravitreal inhibition of complement C5a reduces choroidal neovascularization in mice. Graefes Arch Clin Exp Ophthalmol 253(10):1695–1704. https://doi.org/10.1007/s00417-015-3041-z

    Article  CAS  PubMed  Google Scholar 

  29. Müther PS, Semkova I, Schmidt K, Abari E, Kuebbeler M, Beyer M, Abken H, Meyer KL, Kociok N, Joussen AM (2010) Conditions of retinal glial and inflammatory cell activation after irradiation in a GFP-chimeric mouse model. Invest Ophthalmol Vis Sci 51:4831–4839. https://doi.org/10.1167/iovs.09-4923

    Article  PubMed  Google Scholar 

  30. Kociok N, Radetzky S, Krohne TU, Gavranic C, Joussen AM (2006) Pathological but not physiological retinal neovascularization is altered in TNF-Rp55-receptor-deficient mice. Invest Ophthalmol Vis Sci 47:5057–5065. https://doi.org/10.1167/iovs.06-0407

    Article  PubMed  Google Scholar 

  31. Connor KM, Krah NM, Dennison RJ, Aderman CM, Chen J, Guerin KI, Sapieha P, Stahl A, Willett KL, Smith LE (2009) Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis. Nat Protoc 4:1565–1573. https://doi.org/10.1038/nprot.2009.187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kettenmann H (2007) Neuroscience: the brain’s garbage men. Nature 446:987–989. https://doi.org/10.1038/nature05713

    Article  CAS  PubMed  Google Scholar 

  33. Stence N, Waite M, Dailey ME (2001) Dynamics of microglial activation: a confocal time-lapse analysis in hippocampal slices. Glia 33:256–266

    Article  CAS  PubMed  Google Scholar 

  34. Davies MH, Eubanks JP, Powers MR (2006) Microglia and macrophages are increased in response to ischemia-induced retinopathy in the mouse retina. Mol Vis 12:467–477

    CAS  PubMed  Google Scholar 

  35. Jonas RA, Yuan TF, Liang YX, Jonas JB, Tay DK, Ellis-Behnke RG (2012) The spider effect: morphological and orienting classification of microglia in response to stimuli in vivo. PLoS ONE 7:e30763. https://doi.org/10.1371/journal.pone.0030763

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Shen J, Xie B, Dong A, Swaim M, Hackett SF, Campochiaro PA (2007) In vivo immunostaining demonstrates macrophages associate with growing and regressing vessels. Invest Ophthalmol Vis Sci 48:4335–4341. https://doi.org/10.1167/iovs.07-0113

    Article  PubMed  Google Scholar 

  37. Fruttiger M (2002) Development of the mouse retinal vasculature: angiogenesis versus vasculogenesis. Invest Ophthalmol Vis Sci 43:522–527

    PubMed  Google Scholar 

  38. Ganesan P, He S, Xu H (2010) Analysis of retinal circulation using an image-based network model of retinal vasculature. Microvasc Res 80:99–109. https://doi.org/10.1016/j.mvr.2010.02.005

    Article  CAS  PubMed  Google Scholar 

  39. Lang RA, Bishop JM (1993) Macrophages are required for cell death and tissue remodeling in the developing mouse eye. Cell 74:453–462

    Article  CAS  PubMed  Google Scholar 

  40. Stephan AH, Barres BA, Stevens B (2012) The complement system: an unexpected role in synaptic pruning during development and disease. Annu Rev Neurosci 35:369–389. https://doi.org/10.1146/annurev-neuro-061010-113810

    Article  CAS  PubMed  Google Scholar 

  41. Diez-Roux G, Lang RA (1997) Macrophages induce apoptosis in normal cells in vivo. Development 124:3633–3638

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Karin Oberländer is sincerely acknowledged for her excellent technical assistance.

Funding

This work was funded by Ernst und Berta Grimmke-Stiftung (Lf.Nr. 8/13) and Deutsche Forschungsgemeinschaft (DFG, Jo 324/10-2). Claudia Brockmann and Tobias Brockmann are participants in the BIH-Charité Clinical Scientist Program funded by the Charité -Universitätsmedizin Berlin and the Berlin Institute of Health. Sabrina Dege received a scholarship from the Sonnenfeld-Stiftung. Sergio Crespo-Garcia received a Marie Curie grant from the European Commission in the framework of the REVAMMAD ITN (Initial Training Research network).

Author information

Author notes

  1. Claudia Brockmann and Sabrina Dege contributed equally to this work

Authors and Affiliations

  1. Department of Ophthalmology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Augustenburger Platz 1, 13353, Berlin, Germany

    Claudia Brockmann, Sabrina Dege, Sergio Crespo-Garcia, Norbert Kociok, Tobias Brockmann, Olaf Strauß & Antonia M. Joussen

  2. Berlin Institute of Health (BIH), Berlin, Germany

    Claudia Brockmann & Tobias Brockmann

Authors
  1. Claudia Brockmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sabrina Dege
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sergio Crespo-Garcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Norbert Kociok
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tobias Brockmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Olaf Strauß
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Antonia M. Joussen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Claudia Brockmann.

Ethics declarations

Conflict of interest

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge, or beliefs) in the subject matter or materials discussed in this manuscript.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Financial disclosure

None of the authors has any financial interests in the present work.

Electronic supplementary material

Fig. S1

Representative images showing the method of quantification of auto-fluorescent CD115+ cells in MacGreen mice. a Fluorescence angiography in an eye with oxygen-induced retinopathy at postnatal day 17 representing avascular areas and tufts. b Same image with marked regions of interest (ROIs); yellow: avascular areas; red: tufts. c Corresponding auto-fluorescence image of the same eye showing auto-fluorescent CD115+ cells. d Same image with applied ROIs. (GIF 78 kb)

High resolution Image (TIFF 3054 kb)

Fig. S2

Representative images showing the method of quantification of auto-fluorescent CD115+ cells in MacGreen mice. a Fluorescence angiography in an eye of room-air controls at postnatal day 21 representing physiological retinal vascularization. b Same image with marked regions of interest (ROIs); green: vascular area. c Corresponding auto-fluorescence image of the same eye showing auto-fluorescent CD115+ cells. d Same image with applied ROIs. (GIF 83 kb)

High resolution Image (TIFF 3165 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Brockmann, C., Dege, S., Crespo-Garcia, S. et al. Spatial distribution of CD115+ and CD11b+ cells and their temporal activation during oxygen-induced retinopathy in mice. Graefes Arch Clin Exp Ophthalmol 256, 313–323 (2018). https://doi.org/10.1007/s00417-017-3845-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00417-017-3845-0

Keywords

Search

Navigation