Comprehensive characterization of retinal phenotypic changes in methylene tetrahydrofolate reductase (MTHFR) deficient mice: a model of hyperhomocysteinemia (HHcy)

Abstract

 

Purpose

 

MTHFR, a crucial enzyme in homocysteine metabolism, is mutated in 44% (heterozygous) and 12% (homozygous) of Americans. HHcy is implicated in CRVO, diabetic retinopathy, glaucoma & AMD. MTHFR mutations are the most common genetic cause of Hhcy, but there is discrepancy clinically regarding MTHFR mutations & ocular problems. Mice deficient in mthfr provide an excellent tool to address whether HHcy due to absence/lack of MTHFR manifests as retinal disruption/dysfunction.

 

Methods

 

The retinal phenotype was characterized in mthfr ^+/+ (n=24),mthfr ^+/- (n=40), mthfr ^-/- (n=20) mice (8-52 wks) using fundus photography, fluorescein angiography (FA), IOP, OCT, ERG, flatmount immunostaining, morphometry, light/electron microscopy (EM).

 

Results

 

Retinal phenotypic changes in mthfr ^-/- mice were observed as early as 8 wks (~20%); by 12 wks ~60% were affected; by 24 wks 100% manifested fundoscopic evidence of disruption including geographic atrophy; FA evidence of dilated, tortuous, beaded, leaky blood vessels (BV), which was confirmed by OCT. Morphometric analysis revealed ~20% decrease in RGCs by 8 wks; EM showed apoptotic cells in the INL and evidence of RPE atrophy adjacent to areas of RPE hypertrophy. In mthfr ^+/- (mice retinal phenotypic changes began at 24 wks (~50% were affected), while 100% showed gross disruption by 1 yr. Fundoscopy showed abnormal retinal deposits, FA revealed tortuous, leaky BV and these changes progressively worsened. Neovascularization and gliosis were observed in retinal flatmounts by isolectin -B4 and GFAP immunostaining. Disruption was confirmed by OCT. Morphometry indicated ~20% RGC loss observed by 24 wks, EM alterations were substantial by 1 yr. ERG analysis showed marked decrease in a, b, c wave amplitudes at 1 yr in ),mthfr^+/- mice. IOP was similar in all three mouse groups.

 

Conclusions

 

Absence of MTHFR manifests as marked retinal neurovasculopathy, while moderate deficiency of MTHFR shows a similar but later-onset retinal phenotype. The study provides the first comprehensive, systematic analysis of retinal function and architecture in MTHFR deficient mice. Future experiments will explore the mechanisms involved in HHcy-induced retinal neurovascular damage.

 

Keywords: 688 retina • 592 metabolism • 695 retinal degenerations: cell biology