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Behrad Garmsiri, Jennifer V. Robertson, Alexander K. Ball, Judy A. West-Mays; Retinal Ganglion Cell Loss Correlates With Increased IOP in MMP-9 Knockout Mice. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6942.
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Matrix metalloproteinases (MMPs) are endopeptidases that degrade the retinal ganglion cell (RGC) layer extracellular matrix after injury to RGCs. Retinal MMP-9 is upregulated in ischemia, NMDA excitotoxicity, and elevated intraocular pressure (IOP) associated RGC death. Abnormal expression of MMP-9 has recently been implicated in the etiology of glaucoma. We have previously reported that IOP is elevated in MMP-9 knock out (KO) mice. The purpose of this study was to examine RGC survival in MMP-9 KO mice after several weeks of elevated IOP.
IOP measurements were made in anaesthetized (Avertin) 3-4 month old MMP-9 KO mice on a C57BL/6 background and age-matched wild type (WT) littermates using rebound tonometry (TonoLab). Full-field Electroretinograms (ERGs) were recorded to evaluate the amplitude of the a-wave, b-wave (distal retina), and scotopic threshold response (STR; proximal retina) in both KO and WT mice. The mice were euthanized and their eyes fixed in phosphate buffered 4% formaldehyde, washed, and retinas removed. Retinas were incubated in goat∝Brn3 (c-13; Santa Cruz; 1:500) to label RGCs and rabbit∝PGP9.5 (UltraClone; 1:500) to label amacrine cells and RGCs in the RGC layer. RGCs and all neurons in the midperiphery of each quadrant of the RGC layer were counted in flatmounted retinas.
It has been reported that Brn3 antisera labels 85% of retrograded labeled RGCs from the superior colliculus in mice and is not downregulated in surviving RGCs after injury. We counted 2066±413 Brn3 immunoreactive (-IR) cells/mm² in WT retinas, and only 1283±257 Brn3 cells/mm² in the MMP-9 KO mice, which had a sustained 20% increase in IOP sustained over several weeks. There was a significant (t-test) 38% decrease in RGCs, which is equivalent to the loss seen in mice 7 days after optic nerve transection. The retina was otherwise normal. There was an insignificant (ANOVA), but expected decrease in PGP9.5-IR neurons in the RGC layer (WT: 4612±333 vs KO: 4291±393 cells/mm²) indicating that the only cells affected were Brn3-IR RGCs. There was also no significant change in a- or b-wave ERG amplitudes, suggesting that the distal retinal neurons were functionally normal. There was no significant difference in the STR due to high variability in this response. A Pearson correlation analysis showed that there was a strong correlation between the number of surviving Brn3-IR RGCs and the increase in IOP (0.749; P=0.05).
Our results demonstrate that MMP-9 KO mice have elevated IOPs that correlate with a specific loss of RGCs. An impaired ability to remodel the ECM by eliminating MMP-9 activity did not ameliorate RGC loss in these animals. These findings implicate MMP-9 in the development of high IOP and RGC loss in a mouse model of glaucoma.
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