Iron overload resulting from the chronic oral administration of ferric citrate induces parkinsonism phenotypes in middle-aged mice

Chronic oral administration of ferric citrate induces selective iron overload in the brain

To evaluate the effects of the chronic supplementation of ferric citrate on the brain functions of middle-aged subjects, 9-month C57BL/6 mice were intragastrically administered ferric citrate (2.5 mg or 10 mg) daily for 16 weeks. Weekly body weight and food intake, as well as brain weight, were measured, and no significant differences among the different groups were observed during the experimental period (Figure 1A–1C). The accumulation of iron in the body was analyzed after the mice were killed. The absorption of ferric citrate led to a robust increase in the serum iron level in the ferric citrate groups (Figure 1D), and the accumulation of iron was also observed in the heart, liver, spleen and kidney, especially in the 5% ferric citrate group (Figure 1E). In the brain, the iron level was quantified by flame atomic absorption analysis. We found that the accumulation of iron was dramatically increased in the substantia nigra (SN), caudate putamen (CPu), olfactory bulb (OB) and thalamus (THA) after ferric citrate administration, and the hippocampal (Hip) iron level moderately increased in the high-dose ferric citrate group, but no such changes were detected in the cortex (Ctx), cerebellum (CB) and hypothalamus (HYP) (Figure 1F). The accumulation of iron in the SN and CPu, further confirmed by Prussian blue staining, indicated that there was a marked dose-dependent increase in the positive signals in the ferric citrate groups (Figure 1G and 1H). Increased iron transport, as indicated by the upregulated expression of the major iron uptake transporter TFR1, may be responsible for the accumulation of iron in the brain after ferric citrate supplementation (Figure 1I). Excessive iron is excreted by the protective exporter mechanism of the brain, and FPN1 functions as an iron efflux transporter in the brain [35]. A robust increase in FPN1 expression was detected in the 1.25% ferric citrate group, while a dramatic decrease was observed in the 5% ferric citrate group (Figure 1J), suggesting the dose- and time-dependent destruction of the balance between iron uptake and export with ferric citrate supplementation. These data demonstrated that the chronic oral supplementation of ferric citrate, especially at a high dose, could lead to an accumulation of iron in the brain with selective regional differences. This finding is consistent with previous reports that the concentration of iron varies greatly among different regions of the brain, and more iron tends to accumulate in the regions associated with motor functions than nonmotor-related regions [36, 37].

Figure 1. Chronic oral administration of ferric citrate induces selective iron overload in the brain. (A to C) Quantifications show no differences in the body weight, daily food intake and brain weight of mice supplemented with ferric citrate. Error bars indicate SD. (D) Quantification shows the increased serum iron levels of mice supplemented with ferric citrate (N=10). Error bars indicate SEM. (E) Quantification shows the increased peripheral tissue iron levels of mice supplemented with ferric citrate (N=10). Error bars indicate SEM. (F) Quantification shows the selective iron overload in the brains of mice supplemented with ferric citrate (N=10). Error bars indicate SEM. (G and H) Representative images from Prussian blue staining show the excessive iron accumulation in the Cpu and SN of mice supplemented with ferric citrate. Bars, 100 μm. (I) qRT-PCR shows the increased mRNA levels of TFR1 in the Cpu and SN of mice supplemented with ferric citrate (N=5). Error bars indicate SEM. (J) qRT-PCR shows that the mRNA levels of FPN1 increased in the Cpu and SN of mice from the 1.25% ferric citrate group but decreased in those of mice from the 5% ferric citrate group (N=5). Error bars indicate SEM. Compared with the Ctr group, *p<0.05 and **p<0.01. Compared with the 1.25% ferric citrate group, ^#p<0.05 and ^##p<0.01.

Motor and cognitive defects are associated with iron accumulation in ferric citrate-supplemented mice

Increasing evidence has demonstrated that excessive iron accumulation in selective brain regions may induce oxidative stress-related damage and thereby cause neurobehavioral dysfunctions that are widely implicated in NDs [38, 39]. Considering the potential accumulation of iron in the brain after ferric citrate supplementation, multiple behavioral tests were performed during the experiment. First, locomotor functions were assessed by an open field test. Representative maps of mouse activities showed that the oral administration of ferric citrate could reduce the mobility of mice (Figure 2A). Further statistical results found that the total travel distance and the speed, frequency, distance and time spent in the center zone were decreased in the ferric citrate groups in a time- and dose-dependent manner (Figure 2B–2F). Second, the accelerated rotarod test and pole test were performed to measure the gross motor skill and motor coordination of these mice [40, 41]. Quantification showed that compared with the mice in the other groups, the mice supplemented with 5% ferric citrate displayed a significant time-related decrease in fall latency (Figure 2G), while the times required for the mice to turn around and descend to the floor in the pole test were remarkably increased (Figure 2H and 2I). Then, in the last experimental week, the grip strength of these mice was measured with a traction test [42], and the results showed that the mice from the 5% ferric citrate supplementation group spent much less time on the rope than those from the other two groups (Figure 2J). In addition, as mentioned above, the iron concentration in the hippocampus was also increased in the 5% ferric citrate-supplemented mice; thus, we also performed a Y-maze test to assess the cognitive function of these mice [43]. As shown in Figure 2K, the frequency that mice entered the novel arm of the Y-maze was lower in the 5% ferric citrate group than in the control group. These results showed the effects of the chronic oral intake of ferric citrate on impairing the motor and cognitive functions of middle-aged mice, and these behavioral defects are known to be indicative of experimental parkinsonism [44]. Therefore, we consider these middle-aged ferric citrate over-supplemented mice to be a potential PD animal model, which will be a powerful tool for research on PD mechanisms and drugs.

Figure 2. Motor and cognitive defects are associated with iron accumulation in ferric citrate-supplemented mice. (A) Representative maps of mouse activities in the open field test. (B) Effect of ferric citrate on the distance traveled by mice. (C) Effect of ferric citrate on the speed of mice. (D) Effect of ferric citrate on the time mice spent in the center zone. (E) Effect of ferric citrate on the frequency mice moved into the center zone. (F) Effect of ferric citrate on the distance mice traveled in the center zone. (G) Effect of ferric citrate on the fall latency of mice. (H and I) Effect of ferric citrate on the performance of mice in the pole test. (J) Effect of ferric citrate on the time to fall of mice in the traction test. (K) Effect of ferric citrate on the cognitive functions of mice, as evidenced by the quantification of their frequency to enter the novel arm in the Y-maze test. Error bars indicate SD. Compared with the Ctr group, *p<0.05 and **p<0.01. Compared with the 1.25% ferric citrate group ^#p<0.05 and ^##p<0.01.

Iron overload induced by ferric citrate supplementation causes neurotoxicity in SN and CPu

Given that the brain iron accumulation resulting from the chronic oral uptake of ferric citrate caused motional and cognitive defects, we further explored the underlying histopathological damage. As shown by H&E staining, nerve cell swelling was present in the SN, while white matter edema and vasodilatation were observed in the CPu of the mice supplemented with 5% ferric citrate (Figure 3A and 3B), but no observable pathological changes were found in the 1.25% ferric citrate and control groups. Moreover, cell swelling or white matter edema was also found in the globus pallidus, thalamic and red nuclei (Supplementary Figure 1). These histopathological findings suggested the occurrence of neuroinflammation after ferric citrate supplementation, which was further evidenced by detecting the expression of inflammatory factors. As shown in Figure 3E, the expression levels of the proinflammatory factors TNF-α and IL-6 were increased, while the expression of the anti-inflammatory factor IL-4 was suppressed in the 5% ferric citrate group (Figure 3E). Nissl staining was performed to quantify the numbers of neurons in the SN and CPu and displayed a remarkable neuronal loss in the 5% ferric citrate group (Figure 3C, 3D and 3F). Specifically, the neurons lost in the SN were dopaminergic neurons, as indicated by tyrosine hydroxylase (TH) staining and qRT-PCR (Figure 3G–3I), which further resulted in the depletion of dopamine (DA) and its metabolite (dihydroxyphenylacetic acid, DOPAC) in the CPu (Figure 3J and 3K). Besides, the expression of dopamine transporter (DAT) in striatum was also reduced in 5% ferric citrate group (Figure 3L). Moreover, our study further demonstrated that cellular apoptosis was responsible for the neuronal loss in the SN and CPu, as many more positive signals were observed in the subjects in the 5% ferric citrate group by TUNEL and cleaved caspase-3 staining (Figure 3M to 3O). Dopaminergic neurons constitute a major source of dopamine, which is one of the most important neurotransmitters involved in the nigrostriatal pathway that controls voluntary motor movement [45]. Therefore, the neurotoxicity to SN dopaminergic neurons after the chronic oral uptake of ferric citrate may be the cause for the behavioral defects previously observed. Lewy bodies are important clinical manifestation in PD patients, but in our model, even we have detected increased expression of alpha synuclein (a-syn) in the CPu from 5% ferric citrate group (Figure 3L), but we didn’t observe any Lewy body both in SN and CPu (Data not shown).

Figure 3. Iron overload induced by ferric citrate supplementation causes neurotoxicity in the SN and CPu. (A and B) Representative images of H&E staining display the histopathological damage in the CPu and SN induced by ferric citrate supplementation. Red arrows show white matter edema, red stars show vasodilatation, and green arrows display nerve cell swelling. (C, D and F) Representative images and quantification of NISSL staining display the decreased numbers of neurons in ferric citrate-supplemented mice. Error bars indicate SD. (E) qRT-PCR showed increased mRNA levels of TNF-α and IL-6 and decreased expression of IL-4 in the Cpu and SN of mice supplemented with ferric citrate (N=5). Error bars indicate SEM. (G and H) Representative images and quantification of TH staining display decreased numbers of dopaminergic neurons in the SN of mice supplemented with ferric citrate. Error bars indicate SD. (I) qRT-PCR shows decreased mRNA levels of TH in the SN of mice supplemented with ferric citrate (N=5). Error bars indicate SEM. (J and K) Quantifications show the decreased levels of DA and DOPAC in mice supplemented with ferric citrate. Error bars indicate SEM. (L) qRT-PCR show the mRNA levels of DAT and a-syn in the SN of mice supplemented with ferric citrate (N=5). Error bars indicate SEM. (M to O) Representative images and quantification from TUNEL and cleaved caspase-3 staining display the increased neuronal apoptosis in the SN of mice supplemented with ferric citrate. Bars, 100 μm. Compared with the Ctr group, *p<0.05 and **p<0.01. Compared with the 1.25% ferric citrate group, ^#p<0.05 and ^##p<0.01.

Oxidative stress-induced neuronal loss is implicated in the neurotoxicity of ferric citrate supplementation

As a transition metal, iron is capable of generating hydroxyl radicals via the Fenton reaction. Consequently, elevated iron deposition induces oxidative stress and triggers the accumulation of oxidative damage and neuronal death, which is widely implicated in NDs [8, 46]. To explore whether oxidative stress was induced by chronic ferric citrate supplementation, oxidative damage was analyzed in the SN and CPu of mice. Lipid peroxidation was evaluated by 4-hydroxynonenal (4-HNE) staining, and a widespread increase in 4-HNE positive signals was observed in the SN and CPu of mice, especially in the 5% ferric citrate group (Figure 4A and 4B). This increased 4-HNE level was accompanied by an increase in malondialdehyde (MDA) (Figure 4C), another product generated from lipid peroxidation [47]. Oxidative damage to proteins and DNAs was quantified by a protein carbonylation assay kit and an 8-hydroxydeoxyguanosine (8-OHdG) assay kit, respectively [48]. The data showed that markedly higher levels of protein carbonylation (PC) and 8-OHdG were present in the SN and CPu of mice in the 5% ferric citrate supplementation group than in the control group (Figure 4D and 3E). Iron accumulation was reported to result in the depletion of reduced glutathione (GSH), resulting in decreased oxidative defense [49]. Consistent with this observation, we detected a significant decrease in GSH in the SN and CPu of mice from the 5% ferric citrate group (Figure 4F). In addition, the expression levels of multiple critical antioxidant defense genes, such as superoxide dismutase 1 (SOD1), catalase (CAT) and glutathione peroxidase (GPX), were downregulated in the SN and CPu of mice supplemented with 5% ferric citrate (Figure 4G), and the activities of SOD in these tissues were also reduced (Figure 4H). Accumulating oxidative damage triggered cellular apoptotic processes, as shown in Figure 3L and 3M. This finding suggested that the oxidative stress generated in the ferric citrate-supplemented mice was involved in dopaminergic neuronal loss and neurobehavioral defects.

Figure 4. Oxidative stress-induced neuronal loss is implicated in the neurotoxicity of ferric citrate supplementation. (A and B) Representative images of immunohistochemical 4-HNE staining show the accumulation of lipid peroxidation in the CPu and SN induced by ferric citrate supplementation. (C–E) Quantifications show the increased peroxidation of lipids, DNAs and proteins in the CPu and SN induced by ferric citrate supplementation. (F) Quantification shows the decreased GSH levels in the CPu and SN induced by ferric citrate supplementation. (G) qRT-PCR shows the decreased mRNA levels of typical antioxidant genes in the CPu and SN of mice supplemented with ferric citrate (N=5). (H) Quantification shows the decreased activities of SOD in the CPu and SN induced by ferric citrate supplementation. Error bars indicate SEM. Bars, 100 μm. Compared with the Ctr group, *p<0.05 and **p<0.01. Compared with the 1.25% ferric citrate group, ^#p<0.05 and ^##p<0.01.

Animal care and maintenance

All animal works were performed in accordance with the requirements of “The National Institutes of Health Guide for the Care and Use of Laboratory Animal” and were approved by the Animal Welfare and Animal Ethics Committee of Sichuan Agricultural University, China. Sixty C57BL/6 male mice (9 months old) were obtained from Beijing Weitong Lihua Experimental Animal Technology Co. Ltd. and maintained in individual cages in a specific pathogen-free environment with an automatically controlled 12-hour light/dark cycle and free access to food and water for 7 days. Then, the mice were randomly divided into 3 groups, with 20 mice in each group, including the control group (Ctr), 1.25% ferric citrate group and 5% ferric citrate group. The dosages of ferric citrate refer to Toyoda’s study [31]. We used male mice to generate our model because the males do not have the physiological cycle of the females, and the hormones in males maintain a dynamic balance, which is important for the experimental stability and repeatability. Before ferric citrate administration, 5 g ferric citrate was dissolved in 100 ml physiological saline to obtain a 5% ferric citrate solution when heated to boiling. Then, a gradient dilution was performed to obtain a 1.25% ferric citrate solution. Under these conditions, the solubility of ferric citrate was good, and the solution was clear. In our study, 0.2 ml of ferric citrate solution was intragastrically administrated to the mice each day, and an equal volume of physiological saline was intragastrically administrated to the mice in the control group. Therefore, the daily chemical intake was 2.5 mg and 10 mg in the low- and high-dose ferric citrate groups, respectively (Table 1). The intragastric administration of ferric citrate was performed daily beginning at 10:00 am for 16 weeks, and the body weight and food intake were determined weekly. Behavioral tests were conducted to evaluate the effects of ferric citrate on locomotor and cognitive functions during the first week of the trial and each subsequent month. At the end of the trial, all mice from each group were randomly and evenly divided into two groups. Subjects from one group were killed by decapitation, and then the brain, heart, liver, spleen and kidney were obtained and frozen in liquid nitrogen for RNA extraction and biomedical assays. Subjects from the other group were anesthetized with 4% chloral hydrate and perfusion-fixed with 4% paraformaldehyde. The same organs were obtained and fixed in 4% paraformaldehyde and cryopreserved for subsequent histological and immunostaining.

Detection of iron

The levels of iron in serum, heart, liver, spleen and kidney were determined by a Colorimetric Assay Kit (Nanjing Built Biology, Nanjing, China) according to the manufacturer’s instructions. The iron levels in the brain were quantified by flame atomic absorption analysis. Briefly, the sample was digested in concentrated nitric acid at 180 °C for 2 h (Mars 6, Thermo Fisher Scientific Inc. Waltham, MA, USA). Then, the iron concentration was determined by flame atomic absorption spectrometry (Model PE-800, PerkinElmer, USA). Validation of the mineral analysis was conducted using green tea or bovine liver powder as a standard reference material (National Institute of Standards and Technology, Beijing, China).

Perls staining

First, paraffin slices were dewaxed, soaked in distilled water for 3 min, and then incubated in Perls solution containing 7% potassium ferrocyanide and 3% hydrochloric acid at a 1:1 ratio for 30 min, followed by three washes in PBS. Second, the slices were soaked in 1% H₂O₂ for 30 min and washed 3 times with distilled water. Finally, the slides were incubated in PBS containing 0.25 mg/mL DAB and 0.02% H₂O₂ for 10 min, counterstained for 5 min, dehydrated with gradient alcohol and mounted with xylene.

Behavioral studies

Open field test

The open field test was performed according to a previous report [67]. An open field device was provided by Jiangsu Cyrus Biotechnology Co. Ltd. and consisted of a white square arena (50×50 cm², 50 cm high) and video capture system. The test was initiated by placing the mouse at the center of the arena and allowing the mouse to explore the arena for 10 min. Then, locomotor activities were analyzed by using an ANY-maze animal behavior video analysis system (Global Biotech Inc., USA).

Accelerated rotarod test

An accelerated rotarod test was performed according to a previous study [68]. An accelerated rotarod experimental device was provided by Jiangsu Cyrus Biotechnology Co. Ltd. The mice were placed in a uniform rotating rod (rotation speed 5 r/min) with a 9 cm wide lane and a 3 cm diameter rotating rod. When the speeds of the mice were stable, they underwent a uniform acceleration process (maximum time of 5 min, speed increases every 8 s) three times. The average retention time on the revolving rod was determined. The day before the test, all the mice were pretrained on the rotarod three times (1 h interval).

Pole test

The pole test was performed as previously described [41]. Briefly, mice were placed vertically on a 50 cm tall pole with a 1 cm diameter, after which the mice make a 180° turn and return to the base of the pole. The day before the test, the mice were habituated to the pole 5 times. During the test, the amount of time was recorded for the mouse to turn toward the ground (time to turn) and to reach the ground (time to climb). Each mouse underwent five trials, and the average times were quantified.

Traction test

Both forelimbs of the mice were hung on a wire with a diameter of 1.5 mm, 30 cm above the ground, and a cap was placed 1 cm above the rod to prevent the mice from turning over. The time before landing was recorded, and each test interval was 1 min. A total of 5 tests were conducted to average.

Y-maze test

A food reward type of Y-maze test was performed as previously described [69]. Briefly, all mice were subjected to a 2 Y-maze test trials separated by a 1-h intertrial interval to assess spatial recognition memory. The mice were fasted the day before the test. The first stage was the training period. The new arm was blocked by the partition, and the mouse was placed in the labyrinth and allowed to freely move from the starting arm for 10 min. The second stage was the detection period. Food was placed at the new arm, and the mouse was allowed to freely move in the maze for 5 min. Data are expressed as the percentage of novel arm entries made during the 5-min trial.

Histopathologic analysis

H&E and Nissl staining

Organs were fixed in 4% paraformaldehyde before paraffin sectioning. Then, hematoxylin and eosin (H&E) and Nissl staining were performed according to the instructions provided by the manufacturer (Beyotime, Shanghai, China). The number of neurons was quantified by Image Pro Plus (MEDIA CYBERNETICS, USA).

Immunohistochemical staining

Organs were fixed in 4% paraformaldehyde before paraffin sectioning. Then, the paraffin slices were dewaxed and subjected to immunohistochemical staining with standard methods [70] and primary antibodies, including tyrosine hydroxylase (TH) (ENZO, USA, 1:1000), 4-hydroxynonenal (4-HNE) (Abcam, USA, 1:1000) and cleaved caspase-3 (ZEN BIO, China, 1:100). Positive signals were visualized using colorimetric detection with diaminobenzidine (DAB), and the hematoxylin indicated the nucleus. Finally, the images were photographed with a microscope (BX63, Olympus).

Enzyme-Linked Immunosorbent (ELISA) and Biochemical Reaction assay

The levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), 8-hydroxy-2- deoxyguanosine (8-OHdG) and protein carbonylation (PC) were measured by ELISA assay kits (Shanghai Enzyme Linked Organisms, Shanghai, China) according to the manufacturer’s instructions. The activity of superoxide dismutase (SOD) and the levels of glutathione (GSH) and malondialdehyde (MDA) were determined by a biochemical reaction assay kit (Nanjing Built Biology, Nanjing, China) according to the manufacturer’s instructions.

Quantitative real-time PCR

Total RNA was extracted from the sample using RNAiso Plus (TaKaRa, Dalian, China). Total RNA was subjected to reverse transcription using the PrimeScript RT reagent kit with gDNA Eraser (Perfect Real Time) (TaKaRa). Quantitative real-time PCR was performed using the Bio-Rad^® CFX96 PCR System (Bio-Rad, CA, USA), and the relative gene expression was normalized to β-actin as the internal control. The primer sequences of the target genes are described in Table 2.

Statistical analysis

The regions of the mouse brain were located according to an anatomical map by Pingyu Wang [71]. A one-way ANOVA with LSD correction was used to compare different groups. Data are expressed as the mean ± standard deviation (X±SD) for bodyweight, food uptake, behavioral test and staining, while the quantifications for iron concentration, qRT-PCR, and oxidative damage are presented as the mean ± SEM (X ± SEM). Analyses were performed using SPSS 20.0 software (IBM Corp, USA) for Windows, with the level of significance set at 0.05.