Functional Analysis and Immunochemical Analyses of Ca2+ Homeostasis-Related Proteins Expression of Glaucoma-Induced Retinal Degeneration in Rats

Abstract

The retinal degeneration resulting from elevated intraocular pressure was evaluated through functional and morphological analyses, for better understanding of the pathophysiology of glaucoma. Ocular hypertension was induced via unilateral episcleral venous cauterization in rats. Experimental time was set at 1 and 3 days, and 1, 2, 4, and 8 weeks post-operation. Retinal function was analyzed using electroretinography. For morphological analysis, retinal tissues were processed for immunochemistry by using antibodies against the calcium-sensing receptor and calcium-binding proteins. Apoptosis was analyzed using the TUNEL method and electron microscopy. Amplitudes of a- and b-wave in scotopic and photopic responses were found to be reduced in all glaucomatous retinas. Photopic negative response for ganglion cell function significantly reduced from 1-day and more significantly reduced in 2-week glaucoma. Calcium-sensing receptor immunoreactivity in ganglion cells remarkably reduced at 8 weeks; conversely, protein amounts increased significantly. Calcium-binding proteins immunoreactivity in amacrine cells clearly reduced at 8 weeks, despite of uneven changes in protein amounts. Apoptosis appeared in both photoreceptors and ganglion cells in 8-week glaucomatous retina. Apoptotic feature of photoreceptors was typical, whereas that of ganglion cells was necrotic in nature. These findings suggest that elevated intraocular pressure affects the sensitivity of photoreceptors and retinal ganglion cells, and leads to apoptotic death. The calcium-sensing receptor may be a useful detector for alteration of extracellular calcium levels surrounding the ganglion cells.

Keywords: Ca2+-sensing receptor; apoptosis; electroretinography; glaucoma; rats; retinal ganglion cell.

Fig. 1. IOP is temporally profiled in glaucoma and contralateral control eyes. After cauterization, IOP of glaucoma eyes is remarkably higher than that of control eyes. Data are presented as means±SE: n=10 for control and each glaucoma value.

Fig. 2. Scotopic ERGs were evaluated. In (a), representative ERGs of scotopic responses of glaucoma (G, red colored waveforms) at each time point (G1d; G 1day, G3d; G 3day, G1w; G 1week, G2w; G 2week, G4w; G 4week and G8w; G 8week) are compared with that of normal control retina (Normal, black colored waveform). In (b), a- and b-wave amplitudes of scotopic response in normal state were defined, and those in glaucoma retina at each time point were evaluated with a linear graph.

Fig. 3. Photopic ERGs were evaluated. In (a), representative ERGs of scotopic responses of glaucoma (G, red colored waveforms) at each time point (G1d; G 1day, G3d; G 3day, G1w; G 1week, G2w; G 2week, G4w; G 4week and G8w; G 8week) are compared with that of normal control retina (Normal, black colored waveform). In (b), a- and b-wave amplitudes in normal retina and glaucoma retina at each time point were evaluated with a linear graph.

Fig. 4. Photopic negative response (PhNR) was evaluated. In a, representative PhNRs of glaucoma retina (G) at each time point (G1d, G 1day; G3d, G 3day; G1w, G 1week; G2w, G 2weeks; G4w, G 4weeks; G8w, G 8weeks) are compared with that of normal control retina (N). In b, PhNR amplitudes in the retinas at normal state (Normal) and at each time point of glaucoma retinas (1d, 3d, 1w, 2w, 4w, 8w) are compared. PhNR amplitude at 2 week glaucoma shows significant reduction (p<0.01), compared to normal retina. ^*p<0.05 and ^**p<0.01, paired Student's t-test.

Fig. 5. Confocal microscopic analysis of normal and glaucoma retinas processed for CaSR and calcium-binding proteins immunohistochemistry. Immunohistochemistry for CaSR (a~d), calbindin (e~h), calretinin (i~l) and parvalbumin (m~p) was performed in normal retina (Normal: a, e, i, m), and in 1 week (G1w: b, f, j, n), 2 week (G2w: c, g, k, o), and 8 week (G8w: d, h, l, p) glaucoma retinas. CaSR immunoreactivity in the neurons in the ganglion cell layer (GCL) clearly reduced in G2w and G8w retinas. Calbindin immunoreactivity in amacrine cells in the inner nuclear layer (INL) gradually reduced in glaucoma retinas and that in the axons of ganglion cells increased. Calretinin immunoreactivity in amacrine cells showed no large change in glaucoma retinas. Parvalbumin immunoreactivity in the entire AII amacrine cell profile gradually reduced in glaucoma retinas. IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar indicates 50 µm.

Fig. 6. Evaluation of CaSR and calcium-binding proteins (CaBPs) expression levels in glaucoma retinas. Immunoreactivity bands corresponding to β-actin (42 kDa) and CaSR (130 kDa) reveal temporal expression patterns in normal and glaucoma retinas. In a, representative blots from normal and glaucoma retinas are shown. Densitometry measurements of CaSR (b) and CaBPs (c, d, e) normalized with actin as loading control for retinas are shown in accompanying graphs, n=10. ^*p<0.05, ^**p<0.01, and ^***p<0.001, paired Student's t-test (normal vs. glaucoma).

Fig. 7. Apoptotic cell death was evaluated via TUNEL. Left-side photographs (a, c, e), TUNEL+DAPI; right-side (b, d, f), TUNEL only. TUNEL-positive neurons appeared in the ganglion cell layer (GCL) (thick arrows) and in the outer nuclear layer (ONL) (thin arrows) in only 8-week glaucoma retina (G8w Rt: e, f). a & b, Normal; c & d, 8-week contralateral control retina (G8w Lt); INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer. Scale bar indicates 50 µm.

Fig. 8. Electron micrographs taken from 8-week glaucoma retina. In a, the apoptotic photoreceptor (closed arrow) appears in the outer nuclear layer (ONL). In b, the ganglion cell showing necrotic changes (opened arrow) is present. INL, inner nuclear layer; IPL, inner plexiform layer; NFL, nerve fibre layer; OPL, outer plexiform layer. Scale bars indicate 1 µm.