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Shanu Markand, Alan Saul, Rima Rozen, Arul Shanmugam, Vadivel Ganapathy, Sylvia B Smith; Mild hyperhomocysteinemia (Hhcy) leads to modest retinal ganglion cell (RGC) dysfunction and focal vascular leakiness in rd8-free methylenetetrahydrofolate reductase (Mthfr) deficient mice.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4262.
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© ARVO (1962-2015); The Authors (2016-present)
We hypothesize that endogenous, mild Hhcy alters retinal neurovasculature. Hhcy is implicated in diabetic retinopathy, glaucoma, CRVO & AMD, but its role is unclear. At ARVO 2014, we reported a fulminant retinal neurovasculopathy in mice deficient in Mthfr; a key enzyme in homocysteine-methionine metabolism. Further studies, revealed the Crb1 (rd8) mutation in Mthfr+/- mice, confounding our interpretation of HHcy effects on retina. To test our hypothesis, it was critical to evaluate HHcy effects due to Mthfr mutation independent of the rd8 mutation.
Mthfr+/- mice were backcrossed with C57BL6/J mice for several generations to eliminate the rd8 mutation (confirmed by genotyping).Wildtype (WT) mice (n=18) & Mthfr+/- mice (n=16) were subjected to comprehensive retinal evaluation using ERG, fundoscopy, fluorescein angiography (FA), SD-OCT, morphometric & immunohistochemical (IHC) analyses of isolectin-B4 & GFAP at 8, 12, 16, 24 wks. Two way ANOVA was used to determine whether there were significant differences between mouse groups (p < 0.05).
Assessment of retinal function revealed a significant decrease in positive scotopic threshold response (pSTR) from retinas of Mthfr+/- mice at 24 wks. pSTR reflects RGC function. Fundoscopic evaluation revealed a normal appearing fundus in both groups at all ages studied. FA revealed areas of focal vascular leakage in 20% of Mthfr+/- mice at 12-16 wks, which increased to 60% by 24 wks; no leakage was observed in WT mice. OCT revealed a significant decrease in nerve fiber layer (NFL/GCL) thickness at 24 wks in Mthfr+/- mice (10.11 ± 0.87 µM) compared to WT (14 ± 0.72 µM). Morphometric & IHC analyses at 24 wks revealed ~20% reduction in number of cells in GCL of Mthfr+/- mice, no differences in isolectin-B4 staining, but a significant increase in GFAP labeling in Mthfr+/- mice, particularly in Müller cells.
Mild HHcy (especially due to Mthfr mutations) is quite prevalent in humans. Earlier studies of the role of HHcy on retina were confounded because Mthfr+/- mice harbored the rd8 mutation. Elimination of rd8 has now permitted direct assessment of mild HHcy and revealed altered RGC function, thinner NFL & mild vasculopathy by 24 wks. The data support our hypothesis that mild Hhcy has moderate deleterious effects on retinal neurovasculature.
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