Intravitreous injection of a membrane depolarization agent causes retinal degeneration via matrix metalloproteinase-9.
OBJECTIVE
Membrane depolarization and subsequent synaptic release of l-glutamate have been implicated in ischemic retinal damage. However, the mechanisms that lead to ischemia-induced retinal damage are poorly understood. In this study, KCl, a classic membrane depolarizing agent, was injected into the vitreous humor, and the role of matrix metalloproteinase (MMP)-9 in KCl-induced retinal damage was investigated.


METHODS
Normal adult CD-1 mice were treated with KCl by intravitreal injection. MMP activity in retinal protein extracts was determined by gelatin zymography. Tissue localization of MMP-9 in the retina was determined by immunohistochemistry. MMP-9, MMP-2, tissue inhibitor of MMP (TIMP)-1, TIMP-2, Bax, and BCl-2 proteins in retinal extracts were determined by Western blot analysis. Apoptotic cell death in the retina was determined by TUNEL assays. Retinal damage was assessed by immunolocalization studies with antibodies against neurofilament-light (NF-L) and calretinin.


RESULTS
Depolarizing concentrations of KCl induced a dose- and time-related upregulation in MMP-9 activity and protein in the retina. KCl-mediated MMP-9 upregulation was associated with an increase in proapoptotic protein Bax and apoptotic death of cells in the ganglion cell (GCL) and inner nuclear layer (INL), and subsequent loss of NF-L-positive ganglion cells and calretinin-positive amacrine cells. Intravitreal injection of KCl along with an N-methyl-d-aspartate (NMDA)-type glutamate receptor antagonist, MK-801, and a non-NMDA-type glutamate receptor antagonist, NBQX, resulted in a reduction in KCl-mediated MMP-9 upregulation in the retina. Furthermore, a synthetic MMP inhibitor inhibited KCl-mediated MMP-9 upregulation, which led to a significant attenuation of KCl-induced retinal damage.


CONCLUSIONS
These results suggest that upregulation of MMP-9, in part, plays a causative role in KCl-induced retinal damage.
Mali
Raghuveer S
RS
Eye Research Institute of Oakland University, Rochester, MI 48309, USA.
Cheng
Mei
M
Chintala
Shravan K
SK
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D007267:Injections; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D051379:Mice; D011189:Potassium Chloride / Q000633:toxicity*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017470:Receptors, Glutamate / Q000378:metabolism; D012160:Retina / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D012162:Retinal Degeneration / Q000139:chemically induced* / Q000201:enzymology / Q000473:pathology; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D015854:Up-Regulation; D014822:Vitreous Body; D051028:bcl-2-Associated X Protein
C494827:Bax protein, mouse; D018691:Excitatory Amino Acid Antagonists; D019253:Proto-Oncogene Proteins c-bcl-2; D017470:Receptors, Glutamate; D019715:Tissue Inhibitor of Metalloproteinase-1; D051028:bcl-2-Associated X Protein; D019716:Tissue Inhibitor of Metalloproteinase-2; D011189:Potassium Chloride; D020780:Matrix Metalloproteinase 9