Retinal ganglion cell apoptosis in glaucoma is related to intraocular pressure and IOP-induced effects on extracellular matrix

Abstract

Purpose: To investigate the effect of IOP on retinal ganglion cell (RGC) apoptosis and correlate the effects with IOP-induced changes in extracellular matrix (ECM) in the retina and optic nerve head (ONH) in glaucomatous rat eyes.

Methods: Thirty-seven Dark Agouti rats had elevated IOP induced in the left eye by hypertonic saline episcleral vein injections. Eyes were examined at 3 months histologically for RGC apoptosis and expression of specific ECM components.

Results: RGC apoptosis was significantly related to IOP exposure (integral DeltaIOP P <0.001; peak IOP P <0.01). In the RGC layer, elevated IOP correlated positively to a significant increase in MMP-9 activity (P <0.001), tissue inhibitor of matrix metalloproteinase (TIMP-1) (P <0.05), and collagen I (P <0.01), and negatively correlated to deposition of laminin (P <0.05) and TGF-beta2 (P <0.05). There was a significant correlation between MMP-9 activity and both RGC apoptosis (P <0.001) and loss of laminin (P <0.01). IOP exposure was also associated with increased deposition of TGF-beta2 and collagen I at the ONH (P <0.01).

Conclusions: The results demonstrated that RGC apoptosis in glaucoma correlates strongly with elevated IOP and is significantly associated with IOP-induced changes in specific ECM components in the RGC layer. The study shows for the first time a link between MMP-9, laminin degradation, RGC apoptosis, and IOP exposure in glaucoma. The findings suggest that abnormal ECM remodeling in the glaucomatous retina may relate to RGC death and support the notion that the retina is a primary site of injury in glaucoma.

Figure 1

RGC apoptosis was detected in all rat eyes, although it was considerably greater in glaucomatous retinas. Immunohistochemistry confirmed that apoptotic cells were localized to the RGC layer (RGCL). Typical fluorescence micrographs from the control eye (A) and the contralateral, IOP-elevated eye (B–D) show apoptotic cells stained by Alexa-488 –labeled annexin V (green) and RGCs labeled by DiAsp (A, B, red), with DAPI as a nuclear stain (C, D, blue), both in retinal wholemounts (A–C) and cross sections (D). INL, inner nuclear layer. Scale bars, 15 μm.

Figure 2

RGC apoptosis correlated positively with both ΔIOP integral (A) and peak IOP (B) (Pearson's r = 0.766 and 0.533, P < 0.001 and < 0.01, respectively). Similarly, there was a significant correlation between the percentage loss of RGCs and ΔIOP integral (C) and peak IOP (D) (Pearson's r = 0.777 and 0.612, P < 0.005 and < 0.05, respectively).

Figure 3

Immunohistochemistry on paraffin-embedded cross sections shows typical staining in control (A, B) and glaucomatous (C, D) eyes of laminin (A, C) and TIMP-1 (B, D). Insets: Same cross section of retina at low magnification. Immunostaining demonstrated a decrease in laminin (C) and an increase in TIMP-1 (D) in all glaucomatous eyes compared with the control. On analysis, loss of laminin (E) and increased TIMP-1 expression (F) correlated significantly with ΔIOP integral (Pearson's r = −0.661 and +0.605, respectively, P < 0.05). Scale bars, 10 μm.

Figure 4

Immunohistochemistry of frozen cross sections illustrated MMP-9 (A, blue) colocalizing to apoptotic cells in the RGC layer stained with Alexa-488 –labeled annexin V (B, green) and RGCs labeled with NF-200 (C, red). (D) Composite image and low-magnification (inset) demonstrated discontinuity in MMP-9 immunostaining with areas of skip lesions. Bar, 10 μm.

Figure 5

Increase in MMP-9 immunoreactivity in glaucomatous eyes was strongly positively correlated with ΔIOP integral (A, Pearson's r = 0.898, P < 0.001) and peak IOP (B, Pearson's r = 0.721, P < 0.01). A correlation between MMP-9 upregulation and a decrease in laminin immunostaining in the RGC layer was also demonstrated (C, Pearson's r = −0.726, P < 0.01), as also a direct relationship between MMP-9 and the number of apoptotic RGCs (D, Pearson's r = 0.898, P < 0.001).

Figure 6

Immunohistochemistry confirmed changes in ECM components in the rat ONH in glaucomatous eyes (A), including increased expression of TGF-β2 (red) compared with control (B) in the transition region. TGF-β2 (C) and collagen I (D) deposition were positively associated with an increase in ΔIOP integral (Pearson's r = 0.688 and 0.686, respectively, P < 0.01). Histochemistry (hematoxylin and eosin) confirmed scleral canal expansion and cupping with loss of nerve fibers in a typical glaucomatous eye (E) compared with a control eye (F).