J Clin Invest.
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).