Gareth R. Howell, Ileana Soto, Xianjun Zhu, Margaret Ryan, Danilo G. Macalinao, Gregory L. Sousa, Lura B. Caddle, Katharine H. MacNicoll, Jessica M. Barbay, Vittorio Porciatti, Michael G. Anderson, Richard S. Smith, Abbot F. Clark, Richard T. Libby, Simon W.M. John
Cells infiltrate into the ONHs of glaucoma-prone eyes prior to glaucomatous damage but were not detected in radiation-treated eyes.
(A–F) IBA1+ cell numbers were assessed from the ONH to the myelinated region (anterior to white line) of 10.5-month-old DBA/2J, Rad-D2, and D2-Gpnmb+ control eyes by immunofluorescence. (E) An example of the counted IBA1+ cells is shown. The number of IBA1+ cells increased in untreated eyes compared with that in controls but there are significantly fewer in Rad-D2 eyes (P = 0.0002). (G–K) Cell infiltration was assessed using CFDA (injected into the spleens; see Methods). CFDA+ cells had entered the optic nerves of untreated DBA/2J eyes (n = 8) but not Rad-D2 eyes (n = 6) or control eyes (n = 6). Fluorescent cells must have taken up CFDA in the spleen. H is a merged view of J (fluorescent) and K (DIC). Arrows indicate location of CFDA+ cells. (L) Flow cytometric analysis revealed that CD45hiCD11b+ monocyte numbers increased in the ONHs of untreated 10.5-month-old DBA/2J eyes compared with those in D2-Gpnmb+ controls and radiation-treated eyes (P = 0.01). CD45hiCD11b+ monocyte numbers did not increase in radiation-treated eyes compared with D2-Gpnmb+ controls (P = 0.96). CD45hi is a marker of blood-derived infiltrating cells. One-third of the CD45hiCD11b+ cells were also positive for the proinflammatory marker Ly6c+. (M) Flow cytometric analysis also shows that the CD45hi cells that infiltrate into DBA/2J eyes are CD11b+CD11c+ double-positive monocytes. This class of cell is completely absent in radiation-treated eyes (P = 0.0007, compared with untreated eyes). Scale bars: 50 μm (A–C and G–I); 20 μm (D and E and J and K).