Abstract Body: Introduction: Iron is the most abundant metal in the brain and plays an important role in cellular processes. Thus, an imbalance of iron metabolism in the brain negatively affects brain function. Iron accumulates in the brain with aging, which contributes to the progression of different neurological disorders. In two previous independent studies in our lab using an iron overload model and a chronic cerebral hypoperfusion (CCH) model (bilateral carotid artery stenosis, BCAS) in aged mice, we observed that mice of both models showed enhanced brain iron deposition and cognitive deficits, compared with control mice. Here, we investigate if these two models share the same molecular mechanisms in brain iron metabolism, and whether the combination of iron overload and CCH exacerbates the phenotype.

Methodology: 18-month-old C57BL/6 mice of both sexes were subjected to survival surgery to implant 0.18 mm diameter micro-coils in both common carotid arteries to induce CCH (BCAS mice). Sham mice underwent the same procedure without coil implantation. Two weeks after surgery, mice were administered 5% ferric citrate (FC, 3 alternate days/week for 6 weeks) by oral gavage as a model of iron overload or were given saline solution. Two months after surgery, mice were tested for open field, elevated plus maze, and fear conditioning. We analyzed their brains to determine the expression of genes involved in iron metabolism by qPCR.

Results: Iron overload and/or BCAS did not affect body weight, nor velocity and distance moved of mice. However, FC mice showed reduced freezing time (p=0.002) in fear conditioning, and BCAS mice spent more time in the open arms (p=0.04) in the elevated plus maze, compared with control mice. We did not find differences between BCAS+FC mice and control mice. Interestingly, CCH reduced the levels of ferrous iron (p=0.049) in the serum of BCAS mice. We also found that BCAS, but not iron overload, increased the expression of both Gss (glutathione synthetase, an antioxidant enzyme; p=0.01) and Ncoa4 (nuclear coactivator 4, a mediator of ferritinophagy; p=0.005).

Conclusions: BCAS and FC affect brain function via different mechanisms, and the combination of both does not worsen the phenotype. BCAS, but not FC, increased the expression of Gss and Ncoa4. Next, we will determine whether ferritinophagy is activated in the brains of BCAS mice and whether the higher expression of Gss is a response to mitigate the oxidative stress induced by CCH.