Abstract
Glaucoma is a common cause of blindness affecting at least 66 million people worldwide. Pigmentary glaucoma is one of the most common forms of secondary glaucoma, and its pathogenesis remains unclear. Interleukin-18 (IL-18) is an important regulator of innate and acquired immune responses and plays an important role in inflammatory/autoimmunity diseases. Using the DBA/2J mouse as an animal model of human pigmentary glaucoma, we demonstrated for the first time that the expression of the IL-18 protein and gene in the iris/ciliary body and level of IL-18 protein in the aqueous humor of DBA/2J mice are dramatically increased with age. This increase precedes the onset of clinical evidence of pigmentary glaucoma, implying a pathogenic role of inflammation/immunity in this disease. We also observed that activated NF-kappaB and phosphorylated MAPK are increased in the iris/ciliary body of DBA/2J mice, suggesting that both signaling pathways may be involved in IL-18 mediated pathogenesis of pigmentary glaucoma in the eyes of DBA/2J mice. In addition, matrix metalloproteinase-2 (MMP-2) expression in the iris/ciliary body and the activity of MMP-2 in the aqueous humor are increased whereas tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) expression in the iris/ciliary body is decreased, indicating that the degradation process is involved in this mouse model of pigmentary glaucoma. Furthermore, the expressions of apoptosis-related genes, caspase-8, Fas, FADD, FAP, and FAF, and the activity of caspase-3 are increased in the iris/ciliary body of DBA/2J mice. Elucidation of biochemical and molecular mechanisms of IL-18 participation in the pathogenesis of pigmentary glaucoma should provide approaches for developing improved and targeted treatments to ameliorate this blinding disease. The possibility that altered IL-18 expression in the eye of DBA/2J mice initiates and/or amplifies the pathogenesis of pigmentary glaucoma requires further investigation.
Highlights
Glaucoma is a common cause of blindness affecting at least 66 million people worldwide
Using the DBA/2J mouse as an animal model of human pigmentary glaucoma, we demonstrated for the first time that the expression of the IL-18 protein and gene in the iris/ ciliary body and level of IL-18 protein in the aqueous humor of DBA/2J mice are dramatically increased with age
We observed that activated NF-B and phosphorylated mitogenactivated protein kinases (MAPK) are increased in the iris/ciliary body of DBA/2J mice, suggesting that both signaling pathways may be involved in IL-18 mediated pathogenesis of pigmentary glaucoma in the eyes of DBA/2J mice
Summary
Animals—The DBA/2J and C57BL/6J mice used in this study were from the Jackson Laboratory. Retinal ganglion cell density was determined by counting the 4-di-10-ASP-labeled retinal ganglion cells (RGCs) from eight designated areas in flat-mounted retinas. Standard curves for six 2-fold dilution steps between 4 and 0.125 ng of reverse-transcribed RNA samples were run for all primer pairs in triplicate to determine the PCR efficiency for the different target genes and the housekeeping gene. The average GAPDH CT (each multiplex PCR was performed in triplicate) was subtracted from the average target gene CT; the result represents the ⌬CT This ⌬CT is specific and can be compared with the ⌬CT of a calibration sample. PCR was carried out in 50-l reaction volumes, containing 2 l of cDNA, 2 units of TaqDNA polymerase (Invitrogen), 0.2 mM dNTPs, 10 M each of forward and reverse primers, and 1.5 mM MgCl2. The products were run on 1% agarose gel containing 10 ng/ml ethidium bromide and visualized under UV light
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