Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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).
Conclusions: :
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.
Keywords: ganglion cells • intraocular pressure • electroretinography: non-clinical