Abstract
Purpose :
The conventional Slc4a11 knock out shows significant corneal edema at eye opening. This complicates the study of the initial events that lead to corneal edema in absence of Slc4a11. We have previously reported that Slc4a11 is an NH3-activated mitochondrial uncoupler that facilitates glutamine catabolism and suppresses mitochondrial superoxide (O2-) production. Here, we tested the hypothesis that inducible knock out of Slc4a11 in the adult mouse leads to progressive corneal edema and oxidative stress in the corneal endothelium.
Methods :
Slc4a11 Flox /Flox mice were obtained from Ozgene. Mice expressing the estrogen receptor –Cre Recombinase fusion protein: Cre-ERT2 were obtained from Jackson Laboratories. Slc4a11 Flox/Flox//CreERT2/Cre-ERT2 mice at 8 weeks of age were fed Tamoxifen (Tam) enriched chow (0.4 g/Kg) for 2 weeks, followed by 2 weeks of normal chow. Controls of the same genotype were fed normal chow throughout. Corneal thickness (CT) was measured before and after treatment by Optical Coherence Tomography (OCT). Mitochondrial O2- was quantified by MitoSOX staining. Tight junction integrity was qualitatively observed by ZO-1 staining. Cell density was estimated by DAPI staining. Slc4a11 expression on corneas was tested by QPCR.
Results :
Tam treatment induced almost complete Slc4a11 knock out in corneal tissue (Relative Quantity, Control: 1.00 ± 0.11 vs Tam: 0.02 ± 0.005, p= 3.07 x 10-8, n=4). Tam produced mild but significant edema at 4 weeks after initiation of treatment (CT, Control: 103.89 ± 5.67 μm vs Tam: 132.21 ± 13.26, p<0.0001, n=4) consistent with gradual development of the phenotype. Corneal endothelial density was not changed (Control: 2350 ± 28 cells/mm2 vs Tam: 2321 ± 42, p=0.59, n=4). Mitochondrial O2- was increased in corneal endothelium of Tam fed mice (Control: 30.33±4.01 Mean Fluorescence Intensity/Cell vs Tam: 71.16±5.53, p=0.001, n=4). Tight junction appearance of Tam treated mice was not altered.
Conclusions :
Inducible Slc4a11 KO in the corneal endothelium of adult mice induces a mild corneal edema phenotype at 4 weeks of induction. Longer studies will be needed to study the progression of corneal edema and attempt to dissect the mechanism. Mitochondrial O2- is induced early in Slc4a11 knock out, consistent with a role of Slc4a11 in oxidative stress control.
This is a 2020 ARVO Annual Meeting abstract.