Iron Overload contributes to General Anesthesia-induced Neurotoxicity and Cognitive Deficits

doi:10.21203/rs.2.18898/v1

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Iron Overload contributes to General Anesthesia-induced Neurotoxicity and Cognitive Deficits

https://doi.org/10.21203/rs.2.18898/v1

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Journal Publication

published 11 Apr, 2020

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Background

Increasing evidence suggests that exposure to general anesthesia (GA) could be detrimental to cognitive development in young subjects, and might also contribute to accelerated neurodegeneration in the elderly. Iron is essential for normal neuronal function and excess iron in brain is a hallmark of neuroinflammation and is implicated in several neurodegenerative diseases. However, the role of iron in GA-induced neurotoxicity and cognitive deficits has not been studied.

Methods

We used the primary hippocampal neurons and rodents including young rats and aged mice to examine whether GA impacts iron metabolism and whether the impact contributed to neuronal outcomes. In addition, a pharmacological suppression of iron metabolism was performed to explore the molecular mechanism underlying GAs-mediated iron overload in the brain.

Results

Our results demonstrated that GA, induced by intravenous ketamine or inhalational sevoflurane, disturbed iron homeostasis and caused iron overload in both in vitro hippocampal neuron culture and in vivo hippocampus. Interestingly, ketamine or sevoflurane-induced cognitive deficits, very likely, result from a novel regulated iron-dependent cell death, ferroptosis. Notably, iron chelator deferiprone attenuated the GA-induced mitochondrial dysfunctions, ferroptosis, and further cognitive deficits. Moreover, we found that GA-induced iron overload was activated by NMDAR-RASD1 signalling via DMT1 action in the brain.

Conclusion

We conclude that disturbed iron metabolism may be involved in the pathogenesis of GA-induced neurotoxicity and cognitive deficits. Our study provides new insights into a potential novel therapy for prevention in GA-associated neurological disorders.

Neurobiology of Disease

Iron

Ferroptosis

General anesthesia

Neurotoxicity

Cognition

Millions of paediatric and elderly patients undergoing surgery require general anaesthesia (GA) each year worldwide. Accumulating evidence is forcing the anaesthesia community to question the safety of GA at the extremes of age[1, 2]. A large body of preclinical as well as some retrospective clinical studies suggest that general anaesthetics (GAs) can have profound and long-lasting effects in young subjects by increasing neuronal death and reducing neurogenesis[3]. On the other hand, elderly patients are recognized to take an increased risk of postoperative cognitive dysfunction (POCD) after GA and surgery[4]. Although GAs have been used clinically for over a century, both their targets of action and the nature of any potentially neurotoxic side effects remain incompletely understood.

GAs can be broadly classified as either volatile inhaled compounds (such as nitrous oxide, sevoflurane, isoflurane and desflurane), or intravenously administered compounds (such as propofol and ketamine). Two main targets have been extensively described to the GAs: the excitatory N-methyl-D-aspartate (NMDA) glutamate receptors and the inhibitory gama-aminobutyric acid (GABA_A) receptors. By binding glutamate, NMDA receptors (NMDAR) gate an influx of Ca²⁺ and Na⁺ to cause membrane depolarization, while GABA binds to GABA_A receptors, an influx of Cl⁻ results in hyperpolarization[5]. Most GAs, such as ketamine and sevoflurane, activate the inhibitory ion channels and/or inhibit the excitatory ion channels, thereby reduce neuronal excitability and lead to GA[5]. There are other potential targets of anaesthetic relevance including glycine receptors and K⁺ channels[6, 7]. This diversity of potential targets increases the probability of both positive and negative non-anaesthetic effects. NMDA neurotoxicity can be mediated through an iron uptake signalling cascade, i.e., NMDAR stimulation activates neuronal nitric oxide synthase, leading to S-nitrosylation and activation of RASD1 (also called Dexras1, a novel GTPase), which physiologically induces iron uptake via DMT1 (divalent metal transporter 1) and selective iron chelation prevents NMDA neurotoxicity in cortical cultures^[8]. Therefore, we question whether iron is involved in GA-induced neurotoxicity.

Iron is one of the most abundant trace elements in the human body. Iron plays a critical role in brain development and physiology, neurotransmitter synthesis (including monoamine transmitters and GABA), cytoplasmic protein function, and mitochondrial reactions[9]. Non-transferrin-bound iron (NTBI) in the central nervous system appears to be the main physiologically available iron source and DMT1 is a very important iron transporter for iron uptake[10, 11]. Excess iron in the brain causes neuroinflammation and significant cognitive impairments, which has been implicated in the pathogenesis of several neurological disorders including hypoxic ischemic brain injury and periventricular white matter injury in neonates[12]; and neurodegenerative disorders in elders[9, 13]. We propose that disruption of iron homeostasis involves GA-induced neurotoxicity.

In this study, we examined the effects of intravenous ketamine and inhalational sevoflurane on iron homeostasis, mitochondrial function, and neuronal outcomes in vitro and in vivo. Our hypothesis was confirmed by the finding that iron chelation by deferiprone (DFP) reverses the neurotoxicity. In addition, we found that the GA-induced iron overload and neurotoxicity was regulated by NMDAR-RASD1-DMT1 signalling.

Animals

Sprague-Dawley rat pups at postnatal day (PND) 6 and fifteen-month-old male C57BL/6 mice were used in the present study. All experimental procedures and protocols were reviewed and approved by the Animal Investigation Ethics Committee of Nanjing University and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals from the National Institutes of Health, USA. The animals were housed in a room maintained under standard environmental condition (temperature 22–24 °C, a 12 h light/dark cycle, and 50 ± 10% humidity) with free access to food and water. The rat pups were housed with their mothers till PND 20. At PND 21, the pups were weaned and housed 4–5 per cage in standard condition.

General anesthesia

GA was performed based on our previous optimization [14, 15], in which ketamine (75 mg/kg) intraperitoneally or 3% sevoflurane 2 h inhalation daily for three consecutive days can induce neurotoxicity and cognitive impairments. For ketamine GA, rat pups at PND 6 or fifteen-month-old mice received ketamine (75 mg/kg) intraperitoneally daily for three consecutive days. For sevoflurane GA, animals were put in an anesthetizing chamber delivered with 3% sevoflurane plus 30% oxygen (O₂) for 2 h daily for three consecutive days. For control experiments, 30% O₂ was delivered at the same flow rate. For drug treatment, DFP (75 mg/kg, intraperitoneally, synthesized in China Peptides Co., Ltd., Shanghai, China) or DMT1i (50 mg/kg, orally, MedChemExpress, China)[16, 17] was administered to the animals 1 hour before GA daily for three consecutive days.

The composition of the chamber gas was continuously monitored using a DatexTM infrared analyzer (Capnomac, Helsinki, Finland). Animals were kept normothermic throughout the experiment. Animals were kept warm on a plate heated to 37 ± 1 °C and returned to the cage until recovery of the right reflex after GA exposure.

Rat hippocampal neuronal culture and anesthetic exposure

Primary neuronal cultures were prepared from embryonic day 16–17 (E16-17) embryos of Sprague-Dawley rats as previously described[18]. Neurons were dissociated and seeded on Poly-Lysine pre-treated culture dishes with neurobasal medium, supplemented with B-27, glutamine, antibiotics and 5% FBS (Invitrogen GIBCO Life Technologies, Carlsbad, CA, USA). After 2 h incubation, primary cultures were maintained in neurobasal medium without FBS in 5% CO₂ incubator at 37 °C. Half of the culture medium was refreshed every 3 d. After 9 d of culture (DIV 9), the neurons were treated with ketamine (10 µM) in 5% CO₂ incubator or 3% sevoflurane plus 5% CO₂ for six hours at 37 °C, whereas the control group was maintained in same amount of culture medium. For drug treatment, DFP (100 µM) or DMT1i (3 µM) was added to the culture medium 30 min before GAs exposure.

Cell viability assays

The neurons were seeded in 96-well plates for 9 d. Cell Counting Kit-8 (Beyotime Biotechnology) was used to assess cell viability according to the manufacturer’s instructions. Results were expressed as the percentage of reduction of absorbance at 450 nm by calibration with the absorbance of the control cells.

Lactate dehydrogenase (LDH) assays

LDH is a stable cytoplasmic enzyme present in all types of cells and released into the cell culture medium through damaged plasma membrane. The neuron cells were plated into 12-well plates for 9 d and LDH release was measured by LDH Assay Kit according to the manufacturer’s instructions (Sigma-Aldrich, US) following the treatment as needs.

Mitochondria isolation

Mitochondria from hippocampus or primary hippocampal neurons were isolated using Qproteome Mitochondria isolation kit (Qiagen, Cat. No. 37612, USA). In brief, tissue or cells were homogenized in ice-chilled Dounce homogenizers using isolation buffer and centrifuged at 1,000 g for 10 min at 4 °C. The supernatant was then centrifuged at 6,000 g for 10 min to separate the mitochondria and cytoplasm. The later mitochondria-enriched pellets were gently resuspended and washed with isolation buffer, then pelleted by centrifugation at 6,000 g for 10 min. The later supernatant was transferred into new EP tubes and centrifuged at 1,2000 g for 10 min at 4 °C. This supernatant was considered as cytosolic fraction. The protein was determined by the Micro BCA protein assay kit (Beyotime Institute of Biotechnology).

Ferrozine iron assays

Iron content in the hippocampus was measured using a colorimetric ferrozine-based assay with some modifications[19]. Briefly, 22 µl concentrated HCl (11.6 mol/L) was added to 100 µl cell lysate (~ 500 µg total protein). The mixed sample was heated at 95 °C for 20 min, then centrifuged at 12,000 g for 10 min. Supernatant was transferred very gently into fresh tubes. Ascorbate was added to reduce the Fe (III) into Fe (II). After 2 min of incubation at room temperature, ferrozine and saturate ammonium acetate (NH4Ac) were sequentially added to each tube and the absorbance was measured at 570 nm (BioTek EL x 800, Shanghai, China) within 30 min.

Determination of mitochondrial swelling

To initiate mitochondrial swelling by Ca²⁺ uptake, freshly isolated mitochondria were suspended in mitochondrial suspension buffer [120 mM KCl, 25 mM sucrose, 5 mM KH2PO4, 0.1 mM EGTA, 20 MOPS (pH 7.2)] in the presence of 5 mM malate and 5 mM pyruvate as substrates. Swelling was recorded as the decrease of the density of the mitochondrial matrix at 540 nm with a UV/Vis spectrophotometer after adding 0.5 mM Ca²⁺ into the medium.

Determination of mitochondrial membrane potential (MMP) levels and ATP contents

Levels of MMP and ATP contents were determined in hippocampal neurons using a 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) mitochondrial membrane potential detection kit (Solarbio Science & Technology Co. Ltd.) and ATP content assay kit (Beyotime Biotech) according to the manufacturer’s instructions.

Mitochondrial morphology imaging

Mitochondrial morphology was observed under the confocal microscopy following staining with the mitochondria targeting dye MitoTracker Red CMXRos (Beyotime Biotech). After GA treatment for 6 h, the neurons were washed and stained with 20 nM MitoTracker Red CMXRos in neurobasal medium for 30 min at 37˚C in dark in a 5% CO₂ incubator. The cells were then washed with HBSS and immersed in neurobasal medium to prevent cell damage. Images were obtained with confocal microscopy (Fluoview FV 10i, Olympus) and analysed using FV10-ASW 2.1 Viewer software.

Measurement of cellular ROS, MDA and GSH levels

Intracellular ROS levels were estimated using a fluorescence-labelled probe DCFH-DA (Beyotime Biotech). Cellular MDA and GSH levels were measured following the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Western blot analysis

Proteins were obtained from hippocampal tissue or cultured neurons or isolated mitochondria. For western blotting, 35–50 µg protein was added per lane of 12% SDS-PAGE. Primary antibodies were diluted in primary antibody dilution buffer (Beyotime Biotech., Shanghai, China). Antibodies used included: anti-GAPDH (Abgent Biotech. Co. Ltd., Suzhou, China), anti-ferritin, SOD2, Mitoferrin1, Drp1, Mfn2, RASD1, NMDAR1, and NMDAR2A (Abcam, Cambridge, MA, USA), anti-TfR1 (Zymed, San Francisco, CA, USA), anti-IRP2 (polyclonal, raised from rabbit), and anti-DMT1 (Alpha Diagnostic International, San Antonio, TX, USA). Detection was performed using peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, UK). Quantification of the density of the western bands was done with programme ImageJ (http://rsb.info.nih.gov/ij/).

Morris Water Maze (MWM) Tests

To investigate spatial learning and memory function, we subjected the rats (n = 12 for each group) to the MWM tests (XR-XM101; Shanghai Xinruan Information Technology Co., Ltd., Shanghai, China) at PND 60. The MWM was a black metal tank (120 cm in diameter, 60 cm in depth) equipped with a platform (10 cm in diameter) 1–2 cm below the surface of the water. The MWM task was performed according to our previous study [15]. Briefly, it consists of two phases, training phase for five consecutive days and probe trial phase on day 6. In the training phase, the rat was allowed to face to the pool wall in four random places (N, S, E, W) in the pool to find the fixed platform. Release positions were randomly predetermined. The trial was terminated once the rat reached the platform. If the rat failed to reach the platform within 60 s, it would be guided to the platform and allowed to stay for 15 s, then the latency was recorded for 60 s. In the probe test, single-probe trial was conducted with the original platform removed 24 h after the last training session. The rat was released at a random start position and allowed to swim for 60 s in the pool.

Fear Conditioning Tests

To measure the abilities of learning and memory, we employed the fear conditioning paradigm (30 cm long × 26 cm wide × 22 cm high, XR-XC404, Shanghai Softmaze Information Technology Co. Ltd.) to the aged mice 24 hours after GA. Each mouse was exposed in the conditioning chamber for 3 min of accommodation then one tone-foot-shock pairing (tone, 30 s, 65 dB, 3 kHz; foot-shock, 3 s, 0.75 mA) was delivered. The contextual fear conditioning test was performed 24 h later by placing mice back to the same test chamber for 5 min without any stimulation. Two hours later, each mouse was placed in a novel chamber altered in shape, color, and smell, and the same tone was presented for 3 min without the foot shock to evaluate tone fear conditioning. Cognitive deficits in the test were assessed by measuring the length of time of “freezing behavior”, which is defined as a completely immobile posture except for respiratory efforts. Freezing behaviors were automatically recorded by the video tracking system.

Statistics analysis

Data were presented as mean ± SEM and analysed by the Statistical Product for Social Sciences (SPSS; version 18.0, IL, United States). The difference between the groups was determined by one-way analysis of variance followed by the Bonferroni test. Comparisons for the spatial training sessions of MWM were performed by repeated two-way ANOVA followed by the LSD test. A p-value < 0.05 was considered statistically significant.

1. GA causes hippocampal iron overload

As stated above, GAs may produce profound and long-lasting effects in extremes of lifetime by increasing neuronal death and reducing neurogenesis. Iron deposition in hippocampus, cortical areas, and basal ganglia has been revealed to impair the spatial memory, aversive memory, and recognition memory[9]. To test our hypothesis that GA would induce iron overload, which could be involved in GA-induced neurotoxicity in brain, we first measured the iron content in cultured hippocampal neurons and in the hippocampus of developing rats and aged mice after exposure to ketamine or sevoflurane. The results showed that both ketamine and sevoflurane caused cytosolic and mitochondrial iron accumulation in cultured hippocampal neurons (Fig. 1A, B) and in the hippocampi of developing rats (Fig. 1C, D) and aged mice (Fig. 1D, E).

Deferiprone (DFP) is a low-molecular-weight iron chelator, which has been clinically used to treat iron overload, particularly for patients with hemosiderosis. DFP can freely cross the blood-brain barrier and bind iron in multiple subcellular and extracellular locations[20, 21]. To see if the DFP would reverse the GA-induced overload, we treated the neurons or animals 30 min or 1 h before GA exposure. The results showed that the effect of ketamine or sevoflurane on the content of cytosolic and mitochondrial iron was prevented (Fig. 1). These findings suggested that both intravenous and inhalational GAs could cause iron overload in hippocampal neurons and in developing and aged brains.

2. GA disturbs neuronal iron metabolism

Cells maintain optimal cytosolic iron levels by iron regulatory proteins (IRP1 and IRP2), which can posttranscriptionally regulate the expression of a set of genes such as transferrin receptors 1 (TfR1) for iron uptake and ferritin for iron storage. Therefore, we detected the expression levels of these proteins after ketamine or sevoflurane exposure. Our results showed that both ketamine and sevoflurane reduced the expression of IRP2 and TfR1 and increased the expression of ferritin in the cultured hippocampal neurons (Fig. 2A) and in the hippocampi of developing rats (Fig. 2B) and aged mice (Fig. 2C), respectively. Moreover, depletion of iron with DFP successfully revered the iron status (Fig. 2).

Mitochondria play a vital role in cellular iron metabolism. These organelles are major hubs of iron utilization, and the sole site of heme synthesis and the major site for biogenesis of iron-sulfur clusters, which are vital for cell functions[22–24]. The mechanisms of mitochondrial iron transport have not been well revealed. The best-known pathway is inward iron transport mediated by mitoferrin (Mfrn), a protein located in the inner mitochondrial membrane[22, 25]. In this study, we found that Mfrn1 level was significantly increased in the GAs-treated groups when compared to the control group in the cultured hippocampal neurons (Fig. 3A) and in the hippocampi of developing rats (Fig. 3B) and aged mice (Fig. 3C). Taken together, it is demonstrated that both ketamine and sevoflurane intensify the import of iron into cytoplasm and mitochondria, resulting in the accumulation of iron in the cells or tissues.

3. GA-induced iron overload causes mitochondrial dysfunction

Neurons are very sensitive to iron overload, which toxicity may lead to neuronal mitochondrial dysfunction[22–24]. We have previously demonstrated that mitochondrial dysfunction is involved in the earliest pathogenesis of GA-induced cognitive impairments in developing or aged rodent brain[14, 26]. Whether the GA-induced iron overload elicits mitochondrial dysfunction is not understood. To address this question, we investigate the iron involvement in between.

Mitochondria are highly dynamic organelles that continuously divide and fuse through the processes of fission and fusion, respectively. First, we measured the protein levels of Drp1 and Mfn2, which are fission and fusion-related proteins, respectively. The result showed that the levels of Drp1 elevated and no significant variation of Mfn2 levels was observed after ketamine or sevoflurane stimulation, whereas iron chelation by DFP reversed the expression of Drp1 (Fig. 3A-C), suggesting that iron overload induced by GAs exposure led to the mitochondrial fragmentation. Then, we examined the mitochondrial morphology after incubation of the hippocampal neurons with mito-tracker with the confocal microscopy following GAs exposure. Consistent with the changes of mitochondrial dynamic proteins, mitochondria became fragmented and condensed after 6 h of ketamine or sevoflurane treatment (Fig. 3D). Finally, we evaluated the mitochondrial function by determining the levels of mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential (MMP), and ATP production, three important parameters of mitochondrial function-related indexes. The results showed that the opening of mPTP (Fig. 3E) was enhanced and MMP (Fig. 3F) and ATP production (Fig. 3G) were reduced. However, all the GAs-induced effects were prevented by pretreatment with DFP as shown in Fig. 3.

Collectively, these data support that mitochondrial dynamics and function are impaired by ketamine or sevoflurane exposure and iron chelation may ameliorate the injury.

4. GA triggers iron-mediated ferroptosis

Recently, it was reported that ferroptosis, an iron-dependent regulated cell death process, is an important cell death pathway for a number of diseases, including cancer and neurological disorders[27]. Moreover, ferroptosis can be prevented by iron chelators such as DFP, deferoxamine (DFO), ciclopirox (CPX), and 2,2-bipyridyl (2,2-BP)[28, 29]. Here we hypothesize that ferroptosis may play an important role in the GA-induced neurotoxicity.

Although the characteristics of ferroptosis have not been perfectly understood, the key biomarkers include iron dependence and the stress levels of lipid peroxidation[30]. Here we showed that GAs treatment induced cells to exhibit condensed and ruptured mitochondria (Fig. 3D), which also belongs to the morphological characteristics of ferroptotic cells[31]. Not only the cell morphology, viability, and cytotoxicity (Fig. 4A, B, C), but also other biomarkers (Fig. 4D-G) all matched the phenotypes which combination represents the process of ferroptosis and they were all rescued by iron chelation. For instance, the general reactive oxygen species (ROS, Fig. 4D), the final lipid peroxidation product malondialdehyde (MDA, Fig. 4E), glutathione (GSH, Fig. 4F) content, and even the scavenger superoxide dismutase 2 (SOD2, Fig. 4G) altered after GAs exposure and reversed after DFP treatment. These results suggest that ferroptosis might be derived from GA-induced neurotoxicity.

5. Chelating neurotoxic iron ameliorates GA-induced cognitive deficits

To estimate the extent to which GA-induced cognitive outcomes were attributed to iron overload, the adolescent rats were engaged in MWM tests after GAs exposure with DFP pretreatment. The MWM tests showed long escape latency in training tests (Fig. 5A) and decreased target quadrant time and crossing-platform times (Fig. 5B, C, D) in probe trial in adolescent rats after exposure to either ketamine or sevoflurane. For aged mice, fear conditioning tests were performed and showed, comparing to the control, a decreased percentage of freezing time in the 24 h-context test (Fig. 5E and F), which reflects the impairment of hippocampus-dependent memory, after ketamine or sevoflurane-treatment. Beyond our expectation, DFP pretreatment significantly improved the performance in the behavioural tests (Fig. 5). These results indicate that chelating neurotoxic iron with DFP ameliorated GA-induced cognitive deficits and suggest that iron restriction might provide a preventative effect for paediatric patients undertaking GA and might have a therapeutic effect for POCD treatment.

6. NMDAR-RASD1-DMT1 pathway is involved in GA-induced iron overload

Based on the above results, we further explored the molecular mechanism underlying GAs-mediated iron overload in the brain. NMDAR is one of the two main targets to the GAs. Previous experiments revealed that ketamine or sevoflurane induced a compensatory upregulation of NMDAR expression[32, 33], which promoted iron uptake through DMT1 action[8, 34]. Here we found increased expression of NMDAR1 (NR1) and NMDAR2A (NR2A), as well as RASD1 and DMT1, after 6 h ketamine or sevoflurane treatment (Fig. 6A). To reveal whether GA-induced iron accumulation could be via the NMDAR-RASD1-DMT1 pathway, a DMT1-specific inhibitor (DMT1i)[16, 17] was utilized. Intriguingly, DMT1i pretreatment before ketamine or sevoflurane exposure significantly supressed the expression of all the tested components in NMDAR-RASD1-DMT1 complex (Fig. 6A) and iron contents in cytosol and mitochondria also maintained evenly (Fig. 6B) in rat hippocampal neuronal culture. Amazingly, DMT1i ameliorated the GA-induced cognitive impairments in vivo in all the tested assays including escape latency (Fig. 6C), target quadrant time (Fig. 6D) and crossing-platform times (Fig. 6E) in MWM tests of the rats and freezing time (Fig. 6F) in fear conditioning tests of the aged mice. The freezing time to tone tests did not show the difference after DMT1i treatment. Our data, overall, confirm the effect of GA-induced iron neurotoxicity and suggest that NMDAR-RASD1-DMT1 pathway is crucial in the process leading to cognitive impairments in the rodents at extreme ages of life.

This study provides the first integrative evidence that intravenous and inhalational GAs, such as ketamine and sevoflurane, may induce iron overload in the hippocampus of developing and aged brains to be involved in the cognitive deficits, likely, through neuronal ferroptosis. Our study shed new light on understanding the mechanism of GAs to generate neurotoxicity, at least partly, through modulation of iron metabolism in the brain.

The developing or aged brain has several significant differences from the adult brain that provide a physiological basis for enhanced resistance to insults, including exposure to perioperative stressors[4, 5]. Recent studies have demonstrated that excess iron may be detrimental for neural development, behavioural and cognitive functioning, particularly, at the extremes of age[9, 12, 13]. In this study, we found that both ketamine and sevoflurane disrupted iron homeostasis by affecting the expression of proteins involved in iron uptake further to induce cytosolic and mitochondrial iron accumulation in the cultured hippocampal neurons and in the hippocampus of developing rats and aged mice. Interestingly, pretreatment with DFP successfully ameliorated the turbulence of iron metabolism and attenuated the ketamine or sevoflurane-induced neuronal toxicity. Previous studies have revealed that surgical trauma induces iron accumulation in the hippocampus and that iron chelator DFO, whose mode of action is still under investigation, protects against neuroinflammation and cognitive impairments in an aged rodent model of POCD[35, 36]. However, the blood-brain barrier is thought to be relatively impermeable to DFO [37]. Unlike DFO, DFP, a small positively charged molecule (only 139 Da), is neutral in the circulation and, whether free or bound to iron, readily penetrates cells[38]. Therefore, we used DFP to chelate the accumulated cellular iron in brain or ex vivo neurons, which is in line with the clinical first-line treatment for removing and/or preventing iron accumulation in the brain[39].

Mitochondria are important organelles in all cell types, particularly in the neurons. Mitochondrial dysfunction has been previously shown to link to the earliest pathogenesis of GA-induced cognitive impairments in developing or aged mammalian brain[14, 26]. On the one hand, we confirmed our previous findings including the mitochondrial dysfunction after GAs exposure[40]; on the other hand, we observed that pretreatment with DFP prior to GAs exposure significantly attenuated GA-induced effects on mitochondrial function and dynamics. This means that GA-induced impairment of mitochondria is directly associated with iron accumulation, which is consistent with the previous studies in other iron-overload models[41]. Therefore, we can draw a rough conclusion that iron toxicity is involved in the mechanism underlying GA-induced mitochondrial dysfunction.

Ferroptosis is a relatively new form of regulated cell death. Though still more to be unfolded, it involves iron-dependence and lethal lipid-based ROS generation. Emerging data suggest that ferroptosis is one, if not the main, driver of cell death in cancer and neurodegenerative diseases like Parkinson's disease, Alzheimer's disease and Friedreich's ataxia[27]. In the present study, our results indicates that ketamine or sevoflurane initiates neuronal death, presenting the combination of a few ferroptotic biomarkers including iron-dependence, increased lipid peroxidation, and decreased GSH content[30]. Among them, it is very attractive that iron restriction by iron chelation with DFP or blockage of iron uptake with DMT1i not only abolishes GAs-induced ferroptosis but also resists GA-induced neurotoxicity and cognitive deficits. Both ways to limit the iron availability are to restrict NTBI in a short time frame, suggest that the iron-dependence of ferroptosis is rather NTBI- than other sources of iron-dependence.

Recently, it has been demonstrated that increase of DMT1 expression, not increased TfR1 or decreased Fpn1, may be mainly responsible for aging or disease-dependent increase of iron in brain[42], which may also be the reason of GA-induced iron overload in this study. Accompanying with that, both ketamine and sevoflurane induced upregulation of NR1, NR2A, and RASD1. RASD1 (also called dexRas), a novel GTPase that acts at a confluence of signalling mechanisms associated with psychiatric and neurological disease, mediates iron trafficking and NMDA-dependent neurodegeneration by forming a ternary complex with DMT1, thus enhances the ability of NMDAR to activate DMT1^{[8, 34],}. The activation was inhibited by administration of DMT1 inhibitor in this study to prevent iron accumulation in neurons. Interestingly, DFP treatment reduced the protein levels of NR1, NR2A, RASD1, and DMT1, suggesting that blocking DMT1 elicits a feedback regulation. Therefore, it is possible that modulation of iron level is an effective strategy for protection against NMDAR-induced neurotoxicity.

This is the first time, at least to our knowledge, that iron overload is involved in GA-induced neurotoxicity and cognitive deficits. This work suggests a new strategy that local depletion of iron or restriction of iron influx might be beneficial for the treatment of GA or iron overload-induced neurodevelopmental toxicity.

GA:general anaesthesia; Gas:general anaesthetics; POCD:postoperative cognitive dysfunction; NMDA:N-methyl-D-aspartate; GABA:gama-aminobutyric acid; DMT1:divalent metal transporter 1; DFP:deferiprone; IRP:iron regulatory proteins; TfR1:transferrin receptors 1; Mfrn:mitoferrin; mPTP:mitochondrial permeability transition pore; MMP:mitochondrial membrane potential.

Acknowledgements

We really appreciate all the members in Dr. Li’s and Dr. Yang’s laboratories for their valuable comments to the current study.

Funding

This study was supported by the National Natural Science Foundation of China (Grants 31571218, 31871201, 81700423, 81873526 and 81901944) and the National Basic Research Program of China (Grant 2015CB856300).

Authors’ contributions

JW and KL conceived and designed the experiments. JW, SY, CY, HL, HZ and JJY performed the experiments and analyzed the data. JW and KL wrote the manuscript. All authors discussed, edited, and approved the final version of the manuscript.

Availability of data and materials

All data generated in this study are included in this manuscript.

Ethics approval

All experimental procedures and protocols were reviewed and approved by the Animal Investigation Ethics Committee of Nanjing University and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals from the National Institutes of Health, USA.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing financial interests.

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Iron Overload contributes to General Anesthesia-induced Neurotoxicity and Cognitive Deficits

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Animals

General anesthesia

Rat hippocampal neuronal culture and anesthetic exposure

Cell viability assays

Lactate dehydrogenase (LDH) assays

Mitochondria isolation

Ferrozine iron assays

Determination of mitochondrial swelling

Determination of mitochondrial membrane potential (MMP) levels and ATP contents

Mitochondrial morphology imaging

Measurement of cellular ROS, MDA and GSH levels

Western blot analysis

Morris Water Maze (MWM) Tests

Fear Conditioning Tests

Statistics analysis

Results

1. GA causes hippocampal iron overload

2. GA disturbs neuronal iron metabolism

3. GA-induced iron overload causes mitochondrial dysfunction

4. GA triggers iron-mediated ferroptosis

5. Chelating neurotoxic iron ameliorates GA-induced cognitive deficits

6. NMDAR-RASD1-DMT1 pathway is involved in GA-induced iron overload

Discussion

Conclusion

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1