Degeneration and dysfunction of retinal neurons in acute ocular hypertensive rats: involvement of calpains

Abstract

Purpose: Retinal ischemic diseases primarily lead to damage of the inner retinal neurons. Electrophysiological studies also suggest impairment of the inner retinal neurons. Our recent studies with acute ocular hypertensive rats confirmed damage predominantly in the inner retinal layer along with the ganglion cell layer, changes that are ameliorated by the calpain inhibitor SNJ-1945. However, we do not know which specific neuronal cells in the inner retinal layer are damaged by calpains. Thus, the purpose of the present study was to identify specific calpain-damaged neuronal cells in the inner retina from acute ocular hypertensive rats.

Methods: Intraocular pressure was elevated to 110 mm Hg for 40 min. One hour after ocular hypertension (OH), SNJ-1945 was administrated as a single oral dose of 50 mg/kg. Retinal function was assessed by scotopic electroretinography (ERG). Histological degeneration was evaluated by hematoxylin and eosin, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL), and immunostaining in thin sections and flat mounts of the retina. Calpain activation was determined by proteolysis of the calpain substrate α-spectrin.

Results: OH caused calpain activation, increased TUNEL-positive staining, decreased thickness of the inner nuclear layer (INL), and decreased amplitudes of the ERG a- and b-waves and oscillatory potentials (OPs). SNJ-1945 significantly inhibited calpain activation and the decrease in ERG values. Interestingly, the changes in the b-wave and OPs amplitudes were significantly correlated to changes in the thickness of the INL. In the inner retinal layer, the numbers of rod bipolar, cone-ON bipolar, and amacrine cells were decreased after OH. SNJ-1945 suppressed the loss of cone-ON bipolar and amacrine cells, but did not inhibit the loss of rod bipolar cells. We also observed increased glial fibrillary acid protein-positive staining in the Müller cells after OH and the treatment with SNJ-1945.

Conclusions: Calpains may contribute to ischemic retinal dysfunction by causing the loss of cone-ON bipolar and amacrine cells and causing the activation of Müller cells. Calpain inhibitor SNJ-1945 may be a candidate compound for treatment of retinal ischemic disease.

FIG. 1.

Representative ERGs (A) and quantitative measurement of a-waves (B), b-waves (C), and OPs amplitudes (D), showing significant decrease in amplitudes of a- and b-waves and OPs 7 days after OH, and the protective effect of oral 50 mg/kg SNJ-1945. Data are expressed as mean±SEM (n=16 each). ^#P<0.05 relative to normal (paired Wilcoxon's rank sum test). *P<0.05 relative to vehicle (Student's t-test). ERGs, electroretinographies; OPs, oscillatory potentials.

FIG. 2.

Degeneration in retina after OH in rats. Representative retinal sections stained with H&E from normal rats (A) and from rats 1 day (B) or 7 days (C) after OH at 110 mm Hg for 40 min. Representative TUNEL staining in normal retina (D) and 1 day after OH (E), showing TUNEL-positive nuclei (arrows) in INL and ONL. Cell nuclei were stained with TOPRO-3 (blue). Retinal sections stained with PKCα (F), Goα (G), and TUNEL (H) were merged (I). The area with an asterisk in (I) was magnified (J). Rod bipolar cells (J, arrows) staining Goα-positive (purple) and PKCα-positive (green), stained positive for TUNEL. Cone-ON bipolar cells (arrowheads), staining Goα-positive and PKCα-negative, likewise stained positive for TUNEL (J). Retinal section stained with calretinin (K) or with calbindin (N) were merged with TUNEL staining (L, O). AII amacrine cells (arrows), staining positive for calretinin (green), stained positive for TUNEL (M, merged K, L). Horizontal cells (N), staining positive for calbindin (green) were TUNEL-negative (O), are shown merged in (P). Scale bar=50 μm (A), 20 μm (D), and 10 μm (F, K, N). OH, ocular hypertension; H&E, hematoxylin and eosin; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling.

FIG. 3.

Photomicrographs of sections from rat retina stained with H&E (A) and measurement of thickness in IPL (B) and INL (C), showing significant a decrease in thickness of the IPL and INL 7 days after OH and the protective effect of a single oral dose of 50 mg SNJ-1945/kg. Scale bar=50 μm. Data are mean±SEM (n=16 each). ^#P<0.05 relative to normal and *P<0.05 relative to Vehicle (Tukey's test). Correlation between relative b-wave (D) or OPs (E) amplitudes and thickness of the INL in individual eyes from vehicle-treated (closed circles) and 50 mg/kg SNJ-1945-treated (open circles) rats 7 days after OH. When using all data, statistically significant, positive correlations were observed (D): % b-wave=3.86×(μm INL)−49.40, Pearson's linear correlation coefficient r=0.54, P<0.05 (n=16); and (E): % OPs=2.44×(μm IPL)−17.41, Pearson's linear correlation coefficient r=0.45, P<0.05 (n=16).

FIG. 4.

(A) Representative immunoblot of lysate from whole retina showing intact α-spectrin at 280 kDa (closed arrowhead) and SBDPs at 145 and 150 kDa (open arrowheads) 4 hrs after OH and inhibition of these changes by SNJ-1945. (B) Densitometric image analysis showing increased calpain-specific SBDP at 145 kDa and inhibition by oral SNJ-1945 at 50 mg/kg. Data are expressed as mean±SEM (n=8–10). ^#P<0.05 relative to normal and *P<0.05 relative to vehicle (Tukey's test). Dose-dependent inhibition of decreased cell numbers in the GCL (C) and decreased ganglion cells (D) by administration of oral SNJ-1945 after OH. Data are expressed as mean±SEM (n=27–29 (C), n=11–12 (D)). ^#P<0.05 relative to normal and *P<0.05 relative to vehicle (Tukey's test). SBDPs, α-spectrin breakdown products.

FIG. 5.

Immunohistochemistry for rod bipolar cells (arrows), staining Goα-positive (purple) (A, D, G) and PKCα-positive (green) (B, E, H); and cone-ON bipolar cells (arrowheads), staining Goα-positive (purple) and PKCα-negative. Quantitative measurement of cell numbers (J, K), showed loss of bipolar cells 7 days after OH (D–F) and protection of cone ON-bipolar cells by 50 mg/kg SNJ-1945 (G–I). Area with an asterisk in normal retina was magnified and inserted in panel (C). Scale bar=20 μm. Data are expressed as mean±SEM (n=3 each). *P<0.05 (Tukey's test). PKCα, protein kinase Cα.

FIG. 6.

Immunohistochemistry of AII amacrine cells, staining (green) for calretinin-positive (arrows) (A), and quantitative measurement of cell numbers (B). AII amacrine cells were lost after 7 days of OH and protected by 50 mg/kg SNJ-1945. Scale bar=20 μm. Data are expressed as mean±SEM (n=3 each). *P<0.05 (Tukey's test).

FIG. 7.

Immunohistochemistry of astrocytes and Müller cells, staining (green) for glial fibrillary acid protein.