CD163+ macrophages infiltrate axon bundles of postmortem optic nerves with glaucoma

  • Milica A. MargetaEmail author
  • Eleonora M. Lad
  • Alan D. Proia



Prior research in animal models has shown that macrophages and microglia play an important role in pathogenesis of glaucoma, but the phenotype and distribution of macrophages in human glaucomatous tissue have not been sufficiently characterized.


We analyzed H&E, CD68-, and CD163-immunostained slides from 25 formaldehyde-fixed, paraffin-embedded autopsy eyes: 12 control eyes and 13 eyes with glaucoma. The diagnosis of glaucoma was made based on a history of glaucoma as reported in the medical record and histological changes characteristic of glaucoma. Glaucoma cases and controls were matched in terms of age, sex, and race.


Qualitative analysis of the conventional outflow pathway and the optic nerve revealed that all eyes contained CD163+ cells but a negligible number of CD68+ cells. CD163+ macrophages infiltrated the trabecular meshwork and surrounded Schlemm’s canal of normal eyes and eyes with glaucoma, but the pattern was variable and qualitatively similar between groups. In optic nerves of control eyes, CD163+ macrophages were present at low levels and restricted to septa between axon bundles. In glaucomatous optic nerves, the number of CD163+ cells was increased both qualitatively and quantitatively (glaucoma 5.1 ± 0.6 CD163+ cells/mm2, control 2.5 ± 0.3 CD163+ cells/mm2, p < 0.001), with CD163+ cells infiltrating axon bundles in cases of both mild and severe diseases.


The increase in CD163+ cell number in eyes with mild and severe glaucoma is the first demonstration of macrophage infiltration in glaucomatous human optic nerves. This finding supports a role for macrophages in glaucoma pathogenesis and progression.


Glaucoma Optic nerve Macrophages Microglia Neurodegeneration Neuroprotection 



NIH/NEI provided financial support in the form of salary/research support [NIH/NEI K12 EY016335 (MAM); NIH/NEI K23-EY026988 (EML)]. The sponsors had no role in the design or conduct of this research.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee at Duke and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. For this type of study formal consent is not required.

Supplementary material

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Supplementary Table 1 (DOCX 27 kb)
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Supplementary Figure 1

(PNG 11705 kb)

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High resolution image (TIF 51568 kb)


  1. 1.
    Wax MB, Tezel G (2009) Immunoregulation of retinal ganglion cell fate in glaucoma. Exp Eye Res 88(4):825–830. CrossRefPubMedGoogle Scholar
  2. 2.
    Soto I, Howell GR (2014) The complex role of neuroinflammation in glaucoma. Cold Spring Harb Perspect Med 4(8). CrossRefGoogle Scholar
  3. 3.
    Williams PA, Marsh-Armstrong N, Howell GR (2017) Lasker IIoA, Glaucomatous neurodegeneration P neuroinflammation in glaucoma: a new opportunity. Exp Eye Res. CrossRefGoogle Scholar
  4. 4.
    Evangelho K, Mogilevskaya M, Losada-Barragan M, Vargas-Sanchez JK (2017) Pathophysiology of primary open-angle glaucoma from a neuroinflammatory and neurotoxicity perspective: a review of the literature. Int Ophthalmol.
  5. 5.
    Zeng HL, Shi JM (2018) The role of microglia in the progression of glaucomatous neurodegeneration—a review. Int J Ophthalmol 11(1):143–149. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Alvarado JA, Katz LJ, Trivedi S, Shifera AS (2010) Monocyte modulation of aqueous outflow and recruitment to the trabecular meshwork following selective laser trabeculoplasty. Arch Ophthalmol 128(6):731–737. CrossRefPubMedGoogle Scholar
  7. 7.
    Neufeld AH (1999) Microglia in the optic nerve head and the region of parapapillary chorioretinal atrophy in glaucoma. Arch Ophthalmol 117(8):1050–1056CrossRefGoogle Scholar
  8. 8.
    Yuan L, Neufeld AH (2001) Activated microglia in the human glaucomatous optic nerve head. J Neurosci Res 64(5):523–532. CrossRefPubMedGoogle Scholar
  9. 9.
    Bosco A, Steele MR, Vetter ML (2011) Early microglia activation in a mouse model of chronic glaucoma. J Comp Neurol 519(4):599–620. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Howell GR, Soto I, Zhu X, Ryan M, Macalinao DG, Sousa GL, Caddle LB, MacNicoll KH, Barbay JM, Porciatti V, Anderson MG, Smith RS, Clark AF, Libby RT, John SW (2012) Radiation treatment inhibits monocyte entry into the optic nerve head and prevents neuronal damage in a mouse model of glaucoma. J Clin Invest 122(4):1246–1261. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bosco A, Romero CO, Breen KT, Chagovetz AA, Steele MR, Ambati BK, Vetter ML (2015) Neurodegeneration severity can be predicted from early microglia alterations monitored in vivo in a mouse model of chronic glaucoma. Dis Model Mech 8(5):443–455. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Chidlow G, Ebneter A, Wood JP, Casson RJ (2016) Evidence supporting an association between expression of major histocompatibility complex II by microglia and optic nerve degeneration during experimental glaucoma. J Glaucoma 25(8):681–691. CrossRefPubMedGoogle Scholar
  13. 13.
    Bosco A, Inman DM, Steele MR, Wu G, Soto I, Marsh-Armstrong N, Hubbard WC, Calkins DJ, Horner PJ, Vetter ML (2008) Reduced retina microglial activation and improved optic nerve integrity with minocycline treatment in the DBA/2J mouse model of glaucoma. Invest Ophthalmol Vis Sci 49(4):1437–1446. CrossRefPubMedGoogle Scholar
  14. 14.
    Wang K, Peng B, Lin B (2014) Fractalkine receptor regulates microglial neurotoxicity in an experimental mouse glaucoma model. Glia 62(12):1943–1954. CrossRefPubMedGoogle Scholar
  15. 15.
    Nakazawa T, Nakazawa C, Matsubara A, Noda K, Hisatomi T, She H, Michaud N, Hafezi-Moghadam A, Miller JW, Benowitz LI (2006) Tumor necrosis factor-alpha mediates oligodendrocyte death and delayed retinal ganglion cell loss in a mouse model of glaucoma. J Neurosci 26(49):12633–12641. CrossRefPubMedGoogle Scholar
  16. 16.
    Roh M, Zhang Y, Murakami Y, Thanos A, Lee SC, Vavvas DG, Benowitz LI, Miller JW (2012) Etanercept, a widely used inhibitor of tumor necrosis factor-alpha (TNF-alpha), prevents retinal ganglion cell loss in a rat model of glaucoma. PLoS One 7(7):e40065. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Cueva Vargas JL, Belforte N, Di Polo A (2016) The glial cell modulator ibudilast attenuates neuroinflammation and enhances retinal ganglion cell viability in glaucoma through protein kinase A signaling. Neurobiol Dis 93:156–171. CrossRefPubMedGoogle Scholar
  18. 18.
    Liu X, Huang P, Wang J, Yang Z, Huang S, Luo X, Qi J, Shen X, Zhong Y (2016) The effect of A2A receptor antagonist on microglial activation in experimental glaucoma. Invest Ophthalmol Vis Sci 57(3):776–786. CrossRefPubMedGoogle Scholar
  19. 19.
    Williams PA, Braine CE, Foxworth NE, Cochran KE, John SWM (2017) GlyCAM1 negatively regulates monocyte entry into the optic nerve head and contributes to radiation-based protection in glaucoma. J Neuroinflammation 14(1):93. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Lee NY, Park HY, Park CK, Ahn MD (2012) Analysis of systemic endothelin-1, matrix metalloproteinase-9, macrophage chemoattractant protein-1, and high-sensitivity C-reactive protein in normal-tension glaucoma. Curr Eye Res 37(12):1121–1126. CrossRefPubMedGoogle Scholar
  21. 21.
    Lee NY, Kim MH, Park CK (2017) Visual field progression is associated with systemic concentration of macrophage chemoattractant protein-1 in normal-tension glaucoma. Curr Eye Res 42(7):1002–1006. CrossRefPubMedGoogle Scholar
  22. 22.
    Murray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, Gordon S, Hamilton JA, Ivashkiv LB, Lawrence T, Locati M, Mantovani A, Martinez FO, Mege JL, Mosser DM, Natoli G, Saeij JP, Schultze JL, Shirey KA, Sica A, Suttles J, Udalova I, van Ginderachter JA, Vogel SN, Wynn TA (2014) Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity 41(1):14–20. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Lichtnekert J, Kawakami T, Parks WC, Duffield JS (2013) Changes in macrophage phenotype as the immune response evolves. Curr Opin Pharmacol 13(4):555–564. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Barros MH, Hauck F, Dreyer JH, Kempkes B, Niedobitek G (2013) Macrophage polarisation: an immunohistochemical approach for identifying M1 and M2 macrophages. PLoS One 8(11):e80908. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Hogger P, Dreier J, Droste A, Buck F, Sorg C (1998) Identification of the integral membrane protein RM3/1 on human monocytes as a glucocorticoid-inducible member of the scavenger receptor cysteine-rich family (CD163). J Immunol 161(4):1883–1890PubMedGoogle Scholar
  26. 26.
    Lau SK, Chu PG, Weiss LM (2004) CD163: a specific marker of macrophages in paraffin-embedded tissue samples. Am J Clin Pathol 122(5):794–801. CrossRefGoogle Scholar
  27. 27.
    Lad EM, Cousins SW, Van Arnam JS, Proia AD (2015) Abundance of infiltrating CD163+ cells in the retina of postmortem eyes with dry and neovascular age-related macular degeneration. Graefes Arch Clin Exp Ophthalmol 253(11):1941–1945. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Wermuth PJ, Jimenez SA (2015) The significance of macrophage polarization subtypes for animal models of tissue fibrosis and human fibrotic diseases. Clin Transl Med 4:2. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Bronkhorst IH, Ly LV, Jordanova ES, Vrolijk J, Versluis M, Luyten GP, Jager MJ (2011) Detection of M2-macrophages in uveal melanoma and relation with survival. Invest Ophthalmol Vis Sci 52(2):643–650. CrossRefPubMedGoogle Scholar
  30. 30.
    Coupland SE, Penfold P, Billson F, Hoffmann F (1994) Immunohistochemistry study of the glaucomatous and normal human trabecular meshwork. Ger J Ophthalmol 3(3):168–174PubMedGoogle Scholar
  31. 31.
    Kagan DB, Gorfinkel NS, Hutnik CM (2014) Mechanisms of selective laser trabeculoplasty: a review. Clin Exp Ophthalmol 42(7):675–681. CrossRefPubMedGoogle Scholar
  32. 32.
    Durafourt BA, Moore CS, Zammit DA, Johnson TA, Zaguia F, Guiot MC, Bar-Or A, Antel JP (2012) Comparison of polarization properties of human adult microglia and blood-derived macrophages. Glia 60(5):717–727. CrossRefPubMedGoogle Scholar
  33. 33.
    Bennett ML, Bennett FC, Liddelow SA, Ajami B, Zamanian JL, Fernhoff NB, Mulinyawe SB, Bohlen CJ, Adil A, Tucker A, Weissman IL, Chang EF, Li G, Grant GA, Hayden Gephart MG, Barres BA (2016) New tools for studying microglia in the mouse and human CNS. Proc Natl Acad Sci U S A 113(12):E1738–E1746. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Satoh J, Kino Y, Asahina N, Takitani M, Miyoshi J, Ishida T, Saito Y (2016) TMEM119 marks a subset of microglia in the human brain. Neuropathology 36(1):39–49. CrossRefPubMedGoogle Scholar
  35. 35.
    Butovsky O, Jedrychowski MP, Moore CS, Cialic R, Lanser AJ, Gabriely G, Koeglsperger T, Dake B, Wu PM, Doykan CE, Fanek Z, Liu L, Chen Z, Rothstein JD, Ransohoff RM, Gygi SP, Antel JP, Weiner HL (2014) Identification of a unique TGF-beta-dependent molecular and functional signature in microglia. Nat Neurosci 17(1):131–143. CrossRefPubMedGoogle Scholar
  36. 36.
    Amici SA, Dong J, Guerau-de-Arellano M (2017) Molecular mechanisms modulating the phenotype of macrophages and microglia. Front Immunol 8:1520. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Ophthalmology, Massachusetts Eye and Ear InfirmaryHarvard Medical SchoolBostonUSA
  2. 2.Department of OphthalmologyDuke University Medical CenterDurhamUSA
  3. 3.Department of PathologyDuke University Medical CenterDurhamUSA

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