Immunohistochemical analysis of the cellular localization of Hb expression in the human retina and optic nerve head. Consistent with proteomic findings, histologic sections of the retina and optic nerve head from human donor eyes exhibited immunolabeling with a specific Hb antibody. (
A,
B) Immunoperoxidase labeling of retina sections for Hb in glaucomatous donor eyes with moderate (
A) or more advanced (
B) damage and age-matched controls. (
C) Immunoperoxidase labeling of optic nerve head sections in a glaucomatous donor with moderate damage and the age-matched control. The level of glaucomatous damage in these donor eyes was assessed based on the available clinical data, including optic disc assessments and visual field tests.
19 An increase was detectable in the extent of Hb immunolabeling and the number of immunolabeled cells in most (31/38 eyes with glaucoma) but not all glaucomatous eyes, which also exhibited regional differences with an indistinct pattern. In addition to erythrocytes within blood vessels (v), intracellular Hb immunolabeling was most prominent in macroglial cells. Based on known morphologic features and retinal distributions of macroglial cell types, retinal Hb immunolabeling mainly corresponded to astrocytes (
black arrow) characterized by smaller and oval shape nuclei and close localization to retinal vasculature in the RGC (
white arrow) layer. In addition, some cells in the inner nuclear layer, likely corresponding to Müller cells, exhibited Hb immunolabeling. (
C) Optic nerve head astrocytes also exhibited immunolabeling for Hb. (
D) Double immunofluorescence labeling of the glaucomatous human retina using specific antibodies to Hb (
green) and GFAP or Brn-3 (
red) as marker for retinal macroglia or RGCs. In merged images, most of the Hb immunolabeling (
yellow) was localized to GFAP-positive macroglial cells. However, some Brn-3-positive RGCs also exhibited Hb immunolabeling. rgc, retinal ganglion cell layer; in, inner nuclear layer; on, outer nuclear layer. Scale bar: (
A,
B) 100 μm; (
C) 250 μm; (
D) 150 μm.