Alterations of the synapse of the inner retinal layers after chronic intraocular pressure elevation in glaucoma animal model

Abstract

Background: Dendrites of retinal ganglion cells (RGCs) synapse with axon terminals of bipolar cells in the inner plexiform layer (IPL). Changes in RGC dendrites and synapses between bipolar cells in the inner retinal layer may critically alter the function of RGCs in glaucoma. Recently, synaptic plasticity has been observed in the adult central nervous system, including the outer retinal layers. However, few studies have focused on changes in the synapses between RGCs and bipolar cells in glaucoma. In the present study, we used a rat model of ocular hypertension induced by episcleral vein cauterization to investigate changes in synaptic structure and protein expression in the inner retinal layer at various time points after moderate intraocular pressure (IOP) elevation.

Results: Synaptophysin, a presynaptic vesicle protein, increased throughout the IPL, outer plexiform layer, and outer nuclear layer after IOP elevation. Increased synaptophysin after IOP elevation was expressed in bipolar cells in the innermost IPL. The RGC marker, SMI-32, co-localized with synaptophysin in RGC dendrites and were significantly increased at 1 week and 4 weeks after IOP elevation. Both synaptophysin and postsynaptic vesicle protein, PSD-95, were increased after IOP elevation by western blot analysis. Ribbon synapses in the IPL were quantified and structurally evaluated in retinal sections by transmission electron microscopy. After IOP elevation the total number of ribbon synapses decreased. There were increases in synapse diameter and synaptic vesicle number and decreases in active zone length and the number of docked vesicles after IOP elevation.

Conclusions: Although the total number of synapses decreased as RGCs were lost after IOP elevation, there are attempts to increase synaptic vesicle proteins and immature synapse formation between RGCs and bipolar cells in the inner retinal layers after glaucoma induction.

Figure 1

Verifying intraocular pressure (IOP) elevation and reduction of retinal ganglion cells (RGCs) in a chronic hypertension glaucoma model.(A) The change of IOP after cauterization in shown. The IOP of control and cauterized eyes was measured 0, 1, 4, and 8 weeks after cauterization. The IOP of the cauterized eyes was elevated to 30.4 ± 2.10 mmHg 1 week after cauterization and remained elevated during the 8 week experimental period (31.2 ± 2.26 mmHg). (B) Quantification of RGCs after elevation of IOP. The number of RGCs labeled with Fluorogold in control and cauterized eyes after 1, 4, and 8 weeks are shown. The loss of RGCs in the cauterized retinas relative to control (set at 100%) was approximately 97.0%, 84.6, and 67.1% at 1, 4, and 8 weeks after cauterization, respectively. Bar represents mean ± SD. Student’s t-test was used for statistical evaluation. *P < 0.05 compared to the control.

Figure 2

Confocal micrographs of retinal sections double-stained for synaptophysin and PKCα, a bipolar cell marker. In the control, synaptophysin was expressed in the innermost IPL and OPL (A). After intraocular pressure elevation, expression of synaptophysin was increased throughout the IPL, OPL, and innermost ONL (B and C, left panel). Co-staining with PKCα revealed that upregulation of synaptophysin occurs in bipolar cells in the innermost IPL, where they synapse with retinal ganglion cells. Expression of synaptophysin and co-staining between synaptophysin and PKCα increased in the INL and OPL after 8 weeks of IOP elevation (C, middle panel) compared with 4 weeks (B, middle panel). Magnified confocal micrographs of the innermost inner plexiform layer in hypertensive eyes at 4 (B, right panel) and 8 weeks (C, right panel) post-surgery. Compared to the control (set to 1.0), the co-stained area significantly increased at 1, 4, and 8 weeks after IOP elevation (D). *P < 0.05 compared with the control. Scale bars = 50 μm.

Figure 3

Confocal micrographs of retinal sections double-stained for synaptophysin and parvalbumin, an amacrine cell marker. At 4 weeks after intraocular pressure elevation, immunoreactivity for synaptophysin increased (A). Little co-staining with parvalbumin was seen (B). For cross-sectional immunohistochemical staining, n = 6 for control and n = 6 for glaucoma retinas at each time period; total n = 24. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bars = 50 μm.

Figure 4

Confocal micrographs of flat-mounted retinas double-stained for synaptophysin and calretinin, an amacrine cell and retinal ganglion cell marker, was focused on the border between the ganglion cell layer and the inner plexiform layer. Celretinin stained the soma of the displaced amacrine cells and retinal ganglion cells in the ganglion cell layer. Immunoreactivity for synaptophysin increased at 1 (B), 4 (C), and 8 weeks (D) after intraocular pressure (IOP) elevation compared with the controls (A). Magnification at 4 weeks after IOP elevation (E). The area with both synaptophysin and calretinin co-staining did not change after IOP elevation (control set as 1.0, F). For flat mount immunohistochemical staining, n = 6 for control and n = 6 for glaucoma retinas at each time period; total n = 24. Scale bars = 50 μm.

Figure 5

Confocal micrographs of flat-mounted retinas double-stained for synaptophysin and SMI-32, a retinal ganglion cell (RGC) marker, focused on the border of the ganglion cell layer and the inner plexiform layer. SMI-32 staining was seen in both the soma and dendrites of RGCs. Synaptophysin immunoreactivity increased at 1 (B), 4 (C), and 8 weeks (D) after intraocular pressure (IOP) elevation compared with controls (A). SMI-32 immunoreactivity revealed increased and thickened RGC dendrites at 1 (B) and 4 weeks (C), and rounded somas and decreased, beaded dendrites at 8 weeks after IOP elevation (D). Magnification of flat mounts at 4 weeks after IOP elevation (E). Co-staining between synaptophysin and SMI-32 showed that synaptophysin was expressed in the dendrites of RGCs. Co-stained areas significantly increased at 1 and 4 weeks after IOP elevation compared with the control (set to 1.0, F). For flat mount immunohistochemical staining, n = 6 for control and n = 6 for glaucoma retinas at each time period; total n = 24. Scale bars = 50 μm.

Figure 6

The results of western blot analysis of synaptic vesicle proteins. Synaptophysin, a presynaptic vesicle protein, was significantly increased in the retina at 4 and 8 weeks after intraocular pressure (IOP) elevation compared to controls (A). PSD-95, a postsynaptic vesicle protein, was significantly increased in the retina at 4 and 8 weeks after IOP elevation compared to controls (B). For western blot analysis, n = 6 for control and n = 6 for glaucoma retinas at each time period; total n = 48. Bar represents mean ± SD. Student’s t-test was used for statistical evaluation. *P < 0.05 compared to the control.

Figure 7

Transmission electron micrographs of ribbon synapses between RGCs and bipolar cells in the inner plexiform layer. After 8 weeks of intraocular pressure elevation, synapse diameter (A) and the number of synaptic vesicles (C) increased, and active zone length (B) and the number of docked vesicles (D) decreased. For electron microscopy, n = 6 for control and n = 6 for glaucoma retinas at 8 weeks, total n = 12. *P < 0.05 compared with the control. Scale bars = 0.5 μm.