Immunohistochemical changes in rat retinas at various time periods of elevated intraocular pressure

Abstract

Purpose: To study alterations in different retinal cell types associated with retinal ganglion cell (RGC) death after elevation of intraocular pressure (IOP) in rats.

Methods: IOP was elevated by episcleral vein cauterization of the rat left eye. The right unoperated eye was kept as the control. IOP was measured when rats were awake. The animals were euthanized after one week (n=4) and five weeks (n=4). Their eyes were enucleated, postfixed, cryoprotected, and embedded in optimal cutting temperature (OCT) medium. Cryosections of the retina were cut at 14 microm thickness and processed for immunocytochemistry with 15 antibodies that specifically stain different retinal cell types. The distribution and intensity of the label was analyzed by comparing sections of control and glaucomatous retinas obtained from identical locations.

Results: The amount of amacrine cells identified by calcium binding proteins and choline acetyltransferase antibodies decreased after five weeks of elevated IOP. By using the anti-protein kinase C-alpha antibody, we were able to label a subpopulation of rod bipolar cells in control retinas but not in retinas that had elevated IOP. No changes were found in RGCs labeled with brain derived neurotrophic factor when comparing control and glaucomatous retinas. Glial fibrillary acidic protein and vimentin expression in glial cells increased after one week of elevated IOP.

Conclusions: After one week of elevated IOP and before the onset of RGC death, it was evident that inner retinal cells showed remarkable changes in their molecular expression.

Figure 1

Time-course changes in intraocular pressure (IOP) after experimental glaucoma. Panel A shows the changes in IOP (mmHg) in group 1 (1 week of elevated IOP). Panel B shows the changes in IOP (mmHg) in group 2 (5 weeks of elevated IOP). Blue dots represent control values measured in the right eye, and purple dots represent experimental values measured in the left operated eye. The results of the IOP reading were accepted if the confidence interval was greater than or equal to 95%. The mean values of the IOP measurements were eventually averaged, and results were expressed as mean IOP±SEM. Five such measurements were made. Statistically significant differences were assessed according to Student’s t-test. Asterisks denote the level of significance as follows: *p≤0.05 and **p≤0.01.

Figure 2

M/L and S cone opsin immunohistochemical localization. Control (A, D, and E) and glaucomatous retinas (B, C, F, and G) were labeled with M/L and S cone opsin. Photographs B and F represent retinas after 1 week of elevated IOP and photographs C and G show retinas after 5 weeks of elevated IOP. Panel H represents the number of cone OS/mm in control and experimental groups as indicated (n=40 in all experimental groups). Abbreviations: inner nuclear layer (INL), intraocular pressure (IOP), inner segment (IS), outer nuclear layer (ONL), outer plexiform layer (OPL), outer segment (OS), Differential Interference Contrast (DIC), 4,6-diaminodiphenyl-2-phenylindole (DAPI). Scale bar equals 20 μm.

Figure 3

Calretinin, calbindin, parvalbumin, Chat, and TH inmunolocalization. Panels A-C show caretinin (green) and calbindin (red). Panels D-F show parvalbumin (green) and DAPI (blue). Panels G-I show ChAT (green) and DAPI (blue). Panels J-L show TH (green) and DAPI (blue). Pictures A-J represent control retinas, pictures B, E, H, and K represent glaucomatous retinas from experimental group 1 and pictures C, F, I, and L show glaucomatous retinas from experimental group 2. Abbreviations: choline acetyltransferase (ChAT), ganglion cell layer (GCL), inner nuclear layer (INL), intraocular pressure (IOP), inner plexiform layer (IPL), outer nuclear layer (ONL), outer plexiform layer (OPL), parvalbumin (PV), tyrosine hydroxylase (TH). Scale bar equals 20 μm.

Figure 4

Number of cells/mm labeled with parvalbumin and ChAT antibodies. Panel A depicts the number of amacrine cells, in the inner nuclear layer, labeled with parvalbumin. Panel B shows the number of amacrine cells, in the inner nuclear layer, labeled with ChAT. Panel C shows the number of ChAT positive cells localized in the ganglion cell layer (n=40 in all experimental groups). Statistically significant differences were assessed according to Student’s t-test. Asterisks denote the level of significance as follows: *p≤0.05 and **p≤0.01. Abbreviations: choline acetyltransferase (ChAT), ganglion cell layer (GCL), inner nuclear layer (INL).

Figure 5

Confocal images of PKC-α, calretinin, bassoon, and Goα immunolocalization. Panels A-D show PKC-α (green), calretinin (red) and DAPI (blue). Panels E-H show PKC-α (green), bassoon (red), and DAPI (blue). Panels I-Q show PKC-α (green), Goα (red), and DAPI (blue). Arrows in B, C, F, G, and I-Q indicate a subpopulation of PKC-α-positive cells, located in the innermost INL, that may be affected by glaucoma. Abbreviations: ganglion cell layer (GCL), inner nuclear layer (INL), intraocular pressure (IOP), inner plexiform layer (IPL), outer nuclear layer (ONL), outer plexiform layer (OPL), protein kinase C-α (PKC-α). Scale bar equals 20 μm.

Figure 6

Number of cells per mm located in the INL labeled with PKC-α and Goα antibodies. (A) corresponds to one week of elevated IOP, and (B) to five weeks of elevated IOP (n=40 in all experimental groups). Results are expressed as mean±SEM. Statistically significant differences were assessed according to Student’s t-test. The two asterisks denote the level of significance, p≤0.01. Abbreviations: intraocular pressure (IOP), protein kinase C-α (PKC-α).

Figure 7

Fluorescent immunoreactivity of BDNF in control and glaucomatous retinas. Control retinas (A and B), and experimental glaucomatous retinas after one week (C) and five weeks (D) of elevated IOP. Abbreviations: brain derived neurotrophic factor (BDNF), ganglion cell layer (GCL), inner nuclear layer (INL), intraocular pressure (IOP), inner plexiform layer (IPL), outer nuclear layer (ONL), outer plexiform layer (OPL). Scale bar equals 20 μm.

Figure 8

Fluorescent immunoreactivity of vimentin and GFAP in control and glaucomatous retinas. Panels A-D depict vimentin (red) antibody expression in control (A-B) and glaucomatous retinas after one week (C), and five weeks (D) of elevated IOP. Panels E-H indicate GFAP (green) antibody expression in astrocytes in control (E-F), one week (G), and five weeks of elevated IOP (H). Nuclei of the cells were marked with DAPI (blue). Abbreviations: ganglion cell layer (GCL), green fibrillary acidic protein (GFAP), inner nuclear layer (INL), intraocular pressure (IOP), inner plexiform layer (IPL), outer nuclear layer (ONL), outer plexiform layer (OPL). Scale bar represents 20 μm.