Fig 2: Deferoxamine treatment improves the iron, inflammatory and behavioral alterations observed in aged mice challenged with LPS. (A) Schematic representation of the protocol. Mice were treated i.p. with DFX (100 mg/kg) once a day for 6 consecutive days. On the last day, DFX was administered followed by LPS (0.5 mg/kg) injection. DFX, was able to reduce the LPS-dependent non-heme iron accumulation in the brain (B) and to restore to basal levels the iron-related metabolism alterations, measured as changes in DMT1, L-ferritin and FPN1 in the hippocampus (C and D). DFX also decreased the expression of pro-inflammatory proteins, such as iNOS and p65 although it was not able to restore CD68 levels (E and F). Moreover, DFX was able to decrease the expression of inflammasome-related markers: NLRP3, caspase-1 and mature IL-1ß (G and H) and reduced the mRNA levels of the pro-inflammatory cytokines TNF-a and IL-1ß (I) in hippocampus. In addition, DFX treatment was able to partially restore sickness-behavioral alterations induced by LPS, measured as social interaction (J) and locomotion (K). Data represent mean ± S.E.M. (N = 3–15, one-way ANOVA followed by Tukey post-hoc test). Significant differences were considered when: *p < 0.05 and **p < 0.01 compared to aged WT saline mice or #p < 0.05 and ##p < 0.01 compared to aged WT LPS injected mice (B–I). Data represent mean ± S.E.M. (N = 4–11, two-way ANOVA followed by Tukey post-hoc test). Significant differences were considered when: ***p < 0.001 compared to aged WT saline mice or #p < 0.05 compared to aged WT LPS injected mice (J-K).

Fig 3: Effects of liquid fructose supplementation and/or stress on copper concentration and proteins involved in copper import (CTR1 and DMT1), storage (MT), and transport to Golgi network (ATOX1). Groups: control (C), fructose-fed (F), stress (S) and fructose + stress (FS). Renal copper concentrations (a), representative Western blots (b) and protein level of CTR1 (c), DMT1 (d) MT (e) and ATOX1 (f) in renal whole cell extracts. Scatter plot with bar graphs represent the means ± SEMs for each protein normalized to ß-actin and expressed relative to controls (n = 6 animals/group). Two-way ANOVA was used to evaluate the effects of fructose and stress, and their interaction. Asterisk denotes statistically significant main effect of fructose. p < 0.05.

Fig 4: Protein Levels of DMT1 in Neurons and Glia in Four Cerebral Regions. (A–L) After treatment with 2 mg/kg/day iron dextran for 6 days, the images of DMT1 (green) costained with NeuN, GFAP, or Iba1 (red) and DAPI (blue) were visualized in FC (A–C), HP (D–F), SN (G–I), and ST (J–L). Scale bar, 50 µm. (M) The fluorescence intensity of DMT1 normalized to control for neurons, astrocytes, and microglia is presented as mean ± SD, n = 6. (N) Changes in fluorescent intensity of GFAP and Iba1 induced by treatment with 2 mg/kg/day iron dextran for 6 days. Data are presented as mean ± SD, n = 6. *P < 0.05—statistically significant difference from control group.

Fig 5: Schematic Rendition of the Effects of Excess Iron on Neural Cells. After inserting metal implants in orthopedic surgery, or after injection of iron-dextran to experimental animals the ionized Fe2+/Fe3+ enters the bloodstream. Through transportation of transferrin and DMT1, Fe2+/Fe3+ crosses BBB and blood–cerebrospinal barrier and accumulates in the brain. Facing the attack of excess iron, glial cells (astrocytes and microglia) act as protectors by increasing the uptake of ferrous by upregulating DMT1, the main iron transporter. At the same time, neurons restrict the influx of iron by degrading the DMT1 by upregulation of Ndfip1. This phenomenon has been observed in several cerebral regions, including FC, HP, SN, and ST. Pathological increase in iron levels is linked to an increased ROS production and neuronal apoptosis. Neuronal death and reactive changes in glia may instigate abnormal animal behaviors, impair memory, and cognition and contribute to the development of neurodegenerative diseases.