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. 2020 Aug 28;37(1):55–69. doi: 10.1007/s12264-020-00563-x

Chronic Oral Administration of Magnesium-L-Threonate Prevents Oxaliplatin-Induced Memory and Emotional Deficits by Normalization of TNF-α/NF-κB Signaling in Rats

Xin Zhou 1,#, Zhuo Huang 1,#, Jun Zhang 1,#, Jia-Liang Chen 3, Pei-Wen Yao 1, Chun-Lin Mai 1, Jie-Zhen Mai 1, Hui Zhang 2,✉, Xian-Guo Liu 1,4,✉
PMCID: PMC7811972  PMID: 32857294

Abstract

Antineoplastic drugs such as oxaliplatin (OXA) often induce memory and emotional deficits. At present, the mechanisms underlying these side-effects are not fully understood, and no effective treatment is available. Here, we show that the short-term memory deficits and anxiety-like and depression-like behaviors induced by intraperitoneal injections of OXA (4 mg/kg per day for 5 consecutive days) were accompanied by synaptic dysfunction and downregulation of the NR2B subunit of N-methyl-D-aspartate receptors in the hippocampus, which is critically involved in memory and emotion. The OXA-induced behavioral and synaptic changes were prevented by chronic oral administration of magnesium-L-threonate (L-TAMS, 604 mg/kg per day, from 2 days before until the end of experiments). We found that OXA injections significantly reduced the free Mg2+ in serum and cerebrospinal fluid (from ~ 0.8 mmol/L to ~ 0.6 mmol/L). The Mg2+ deficiency (0.6 mmol/L) upregulated tumor necrosis factor (TNF-α) and phospho-p65 (p-p65), an active form of nuclear factor-kappaB (NF-κB), and downregulated the NR2B subunit in cultured hippocampal slices. Oral L-TAMS prevented the OXA-induced upregulation of TNF-α and p-p65, as well as microglial activation in the hippocampus and the medial prefrontal cortex. Finally, similar to oral L-TAMS, intracerebroventricular injection of PDTC, an NF-κB inhibitor, also prevented the OXA-induced memory/emotional deficits and the changes in TNF-α, p-p65, and microglia. Taken together, the activation of TNF–α/NF–κB signaling resulting from reduced brain Mg2+ is responsible for the memory/emotional deficits induced by OXA. Chronic oral L-TAMS may be a novel approach to treating chemotherapy-induced memory/emotional deficits.

Electronic supplementary material

The online version of this article (10.1007/s12264-020-00563-x) contains supplementary material, which is available to authorized users.

Keywords: Magnesium-L-threonate, Oxaliplatin, Tumor necrosis factor-alpha, Nuclear factor-kappaB, Cognitive deficit, Hippocampus, Medial prefrontal cortex

Introduction

Chemotherapy-induced cognitive dysfunction and chemotherapy-induced mood disorders are common side-effects in cancer patients. The former is manifested as a decline in attention and visuospatial skills [1], and the latter as anxiety and depression [2]. These disorders often persist for a long period after the termination of chemotherapy, seriously reducing the quality of life and the ability of cancer survivors to work [3]. At present, the treatment of these side-effects is a major challenge in clinical practice.

To date, it is still unclear how anticancer agents with different chemical structures and anticancer mechanisms, such as oxaliplatin (OXA) [4], paclitaxel [5, 6], and vincristine [7, 8], have common side-effects – memory/emotional deficits. It has been shown that many anticancer agents, including OXA [9], paclitaxel [10], vinblastine [11], cisplatin [12], and cetuximab [13], result in hypomagnesemia in patients. Magnesium deficiency is associated with high levels of serum pro-inflammatory cytokines including TNF-α in humans [14]. These cytokines play important roles in the initiation of chemotherapy-induced memory [15] and emotional deficits [16]. NF-κB, a potent transcription factor for many inflammatory cytokines and chemokines, is involved in cancer genesis and treatment [17]. However, currently little is known about the role of NF-κB in chemotherapy-induced memory and emotional deficits. The causal link between Mg2+ deficiency and the activation of TNF-α/NF-κB signaling in the central nervous system after chemotherapy has not been clarified.

Previous work has shown that oral administration of magnesium-L-threonate (L-TAMS, also called MgT), which can elevate brain Mg2+, improves hippocampus-dependent spatial learning in naïve rats [18] and in a mouse model of Alzheimer’s disease [19]. Oral L-TAMS also prevents and restores the memory deficits induced by peripheral nerve injury [20] and enhances fear extinction, but does not affect the amygdala-dependent delay-cued fear memory [21]. Mechanistically, L-TAMS upregulates the NR2B subunit of N-methyl-D-aspartate receptors (NMDARs) and brain-derived neurotrophic factor, as well as improving synaptic plasticity/density in the hippocampus [18] and prefrontal cortex, but not in the amygdala [21]. A recent clinical trial indicated that the global cognitive ability of older adults can be improved by oral L-TAMS [22]. Furthermore, Mg2+ deficiency results in anxiety and depression in animals and humans [23]. However, whether oral L-TAMS can prevent chemotherapy-induced memory/emotional deficits remains unknown.

The present study was designed to test the hypothesis that OXA induces memory and emotional deficits by activation of TNF-α/NF-κB signaling via reducing Mg2+ in the brain, and to determine whether supplemental Mg2+ by chronic oral L-TAMS can prevent the Mg2+ deficiency and memory/emotional deficits induced by OXA.

Materials and Methods

Animals

Adult male Sprague-Dawley rats weighing 220–250 g and Sprague–Dawley rats 5–7 days old (~ 10 g) were purchased from the Institute for Experimental Animals of Sun Yat-sen University. The rats were housed in separate cages in a temperature-controlled room (24 ± 1°C) with 50%–60% humidity under a 12:12-h light/dark cycle in a specific pathogen-free environment, and permitted free access to sterile water and standard laboratory chow. All experimental procedures were approved by the Animal Care and Use Committee of Sun Yat-sen University and carried out under the guidelines on animal care and the ethical guidelines of the National Institutes of Health. Rats were randomly assigned to different experimental and control conditions.

Drug Administration

Oxaliplatin sulfate (Jinrui Pharmaceutical Co., Ltd, Hainan, China) dissolved in 5% glucose/H2O to 1 mg/mL was intraperitoneally injected at 4 mg/kg/day for 5 consecutive days [24]. Control animals received an equivalent volume of 5% glucose/H2O. Based on previous studies [18], L-TAMS (Neurocentria Inc., Walnut Creek, CA, USA) was administered via drinking water (604 mg/kg per day; 50 mg/kg per day elemental Mg2+), initiated 2 days in advance of chemotherapy and continued until the end of the experiments. The average volume of drinking water (~ 30 mL/day) and daily food intake (containing 0.15% elemental Mg2+) were monitored. Accordingly, the concentration of L-TAMS in the drinking water was adjusted to reach the appropriate target dose.

Experimental Design

Behavioral Tests

The novel object-recognition test (NORT) was applied to assess short-term memory as previously described [25]. Briefly, rats were tested in an open field apparatus made up of a circular arena 80 cm in diameter. In the first stage, each rat was placed in the arena for 5 min with two identical objects that served as “old objects”. Ten minutes later, a new object replaced the less preferred old object. Each rat was allowed to explore the two different objects for 5 min. The recognition index was calculated as the ratio of time spent exploring the novel object over total exploration time.

The elevated plus-maze test (EPMT) was carried out in an EPM apparatus (RWD Life Science, Shenzhen, China) formed by 2 open arms and 2 closed arms surrounded by walls 30 cm high in a plus shape. Each rat was placed in the center of the maze and allowed to explore freely for 5 min. The anxiety-like behaviors were measured using TopScan3D software (Clever Sys Inc., Reston, VA, USA). The percentage of time and bouts in the open arms in different groups were compared. A reduction of time and bouts in the open arms indicated anxiety-like behavior.

The forced swimming test (FST) was based on previous studies [26]. Rats were forced to swim for 6 min in a glass cylinder (25 cm in diameter and 55 cm high) containing 30 cm of water at 25 ± 1°C. In the last 4 min, the immobility time was used as an indicator of depression-like behavior.

Electrophysiological Recording

Frequency facilitation at CA3-CA1 synapses was measured in the hippocampus in vivo. After electrical stimulation of the Schaffer collateral–commissural pathway, field excitatory postsynaptic potentials (fEPSPs) were recorded from the stratum radiatum of CA1. The best placement of electrodes was based on electrophysiological criteria [27]. The recording electrode was positioned at 2.5 mm lateral to the midline, 3.4 mm posterior to bregma, and ~ 2.2 mm below the dura. The stimulating electrode was positioned at 4.2 mm posterior to bregma, 3.8 mm lateral to the midline, and ~ 4.7 mm below the dura. The baseline fEPSPs in CA1 were recorded following stimulation of CA3 with test stimuli (0.066 Hz, 0.2 ms duration). The intensity of the test stimulus was adjusted to produce 50%–55% of the maximum response. Conditioning stimuli at 2, 4, and 8 Hz at 20-min intervals were used to induce frequency facilitation in each rat. The intensity of the conditioning stimulus was identical to that of the test stimulus.

Measurement of Extracellular Free Magnesium

Blood was sampled from the left ventricle and centrifuged at 1000 g for 10 min to obtain serum. Cerebrospinal fluid (CSF) was collected from the occipital foramen. Free Mg2+ content in the fluids was measured by Calmagite chromometry [28].

Organotypic Hippocampal Slice Cultures

The method of organotypic culture of hippocampal slices from Sprague–Dawley rats 5–7 days old was as described previously [29]. Briefly, slices were cut at 300 μm and then cultured on porous (0.4 μm) insert membranes (Millipore, Billerica, MA). These membranes were transferred to 6-well culture trays in a humidified atmosphere (5% CO2, 37°C) and maintained for 7 days before use. The culture medium consisted of 50% MEM (Eagle) with Glutamax-1, 25% Earle’s balanced salt solution, 25% heat-inactivated horse serum, 6.5 mg/mL D-glucose, 50 μg/mL streptomycin, and 50 U/mL penicillin (all from Gibco, Grand Island, NY).

Intracerebroventricular (icv) Injection

Each rat, anesthetized with 10% chloral hydrate (0.4 mg/kg, i.p.), was placed in a stereotaxic frame and a cannula was implanted in a lateral cerebral ventricle at the following coordinates: 1.8 mm lateral to the midline, 1.2 mm posterior to bregma, and 3.7–4.3 mm below the skull. Seven days after the operation, pyrrolidine dithiocarbamic acid (PDTC, 10 ng/5 μL; Sigma) or artificial CSF was injected icv in a volume of 5 µL in 10 min (daily). The behavioral tests started 2 days after the last injection.

Western Blot

Under anesthesia with sodium pentobarbital (50 mg/kg, i.p.), hippocampus samples were harvested and immediately stored at –80°C until use. Total protein was extracted on ice in 15 mmol/L Tris buffer supplemented with proteinase inhibitors and phosphatase inhibitors. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane. After blocking, the blots were incubated with primary antibody against TNF-α (1:1000, rabbit; Bioworld Technology, Inc., Louis Park, MN), phosphorylated NF-κB p65 (Ser311) (1:1000, rabbit, Abcam, Cambridge, UK), NR2B (1:1000, rabbit, Abcam), and β-actin (1:1000, mouse; Cell Signaling Technology, Danvers, MA) overnight at 4°C. Horseradish peroxidase-conjugated IgG was applied for 1 h. Electrochemiluminescence was used to detect the immune complexes. Bands were quantified using a computer-assisted imaging analysis system (ImageJ; -National Institutes of Health, Bethesda, MD).

Immunohistochemistry

Rats were anesthetized and perfused through the ascending aorta with saline followed by 4% paraformaldehyde. Brains were removed and post-fixed in the same fixative for 1 h, then transferred to 30% sucrose for 3 days. Hippocampal sections (25 μm) were cut on a freezing microtome (CM3050S, Leica, Wetzlar, Germany) and processed for immunohistochemical staining. Sections were first blocked for 1 h and then incubated with primary antibody against TNF-α (1:200, Rabbit, Bioworld Technology), phosphorylated NF-κB p65 (Ser311,1:100, rabbit; Affinity Biosciences, OH), NeuN (1:400, mouse; Chemicon, Temecula, CA), GFAP (1:500, mouse, Abcam), and Iba1 (1:200, goat, Abcam) overnight at 4°C. Then the sections were incubated with Cy3-conjugated and Alexa-conjugated secondary antibodies for 1 h at room temperature, then mounted on coverslips with Fluoromount-G with DAPI (Southern Biotech, Birmingham, AL). Images of the stained sections were then captured with a Leica fluorescence microscope (Leica DFC350 FX camera, Wetzlar, Germany) or Leica fluorescence microscope (Leica DM6B camera). All parameters such as exposure, gamma, and gain were fixed to ensure standardization between slices. ImageJ software was used to analyze the fluorescence intensity.

Statistical Analysis

All data are presented as the mean ± SEM. SPSS 17.0 (SPSS, Inc., Chicago, IL) was used to perform data analysis. The results from the behavioral tests, measurement of Mg2+ content, western blot, and immunohistochemistry, were analyzed with one-way or two-way ANOVA followed by Tukey’s post hoc test. The electrophysiology was analyzed with two-way ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered a significant difference.

Results

Chronic Oral Magnesium-L-Threonate Prevents the Memory and Emotional Deficits Induced by Oxaliplatin

To determine if chronic oral administration of L-TAMS is capable of preventing the memory and emotional deficits induced by OXA, rats were randomly assigned into four groups: the OXA group receiving OXA injections (4 mg/kg per day, i.p., for 5 consecutive days) and drinking normal water; the OXA + L-TAMS group receiving L-TAMS in drinking water from 2 days before OXA injection until the end of the experiments; the L-TAMS group receiving L-TAMS and vehicle injections; and the control group drinking normal water and receiving vehicle injections. Two days after the termination of the OXA or vehicle injections, behavioral tests were applied to the different groups (Fig. 1A). We found that the recognition index for short-term memory assessed with the NORT (Fig. 1B) decreased and the immobility time with the FST (Fig. 1C) increased markedly compared to controls, indicating that the OXA injections impair short-term memory and induce depression-like behavior. Furthermore, the percentage of time and bouts in the open arms measured with the EPMT were significantly lower in the OXA group than in controls (Fig. 1D, E), while the total distance traveled and the average speed did not differ among the groups (Fig. 1F, G). The data indicated that the OXA injections also cause anxiety-like behaviors without affecting motor functions. Importantly, we found that oral L-TAMS completely prevented the OXA-induced memory deficits as well as the depression-like and anxiety-like behaviors, because the index of short-term memory, the immobility time, and the percentage of time and bouts in the open arms in the L-TAMS + OXA group were significantly higher than those in the OXA group and were not different from controls (Fig. 1B–E). L-TAMS had no effect on the memory and emotion indexes in the rats treated with vehicle (Fig. 1B–E).

Fig. 1.

Fig. 1

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Magnesium-L-threonate prevents the short-term memory and emotion deficiency induced by OXA. A Experiment schedule. B Recognition index in indicated groups, accessed with the novel object recognition test (NORT) (n = 8 per group). C Immobility time in each group measured in the forced swimming test (FST). Oral L-TAMS significantly suppressed the increased immobility time induced by OXA (n = 8 per group). D, E Injections of OXA reduced the percentage of time and entries into the open arms in the elevated plus-maze test (EPMT), and the effects were prevented by oral L-TAMS (n = 8 per group). F, G Total distance traveled and average speed did not differ among the groups in the EPMT (n = 8 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs Control (Contr); #P < 0.05, ##P < 0.01, ###P < .001, vs OXA (one-way ANOVA followed by Tukey’s post hoc test).

Oral L-TAMS Prevents the Impairment of Synaptic Transmission and Downregulation of the NR2B Subunit in NMDARs in the Hippocampus Induced by OXA

An increasing number of studies have shown that the hippocampus, which is important for memory formation, also plays a role in emotion [30]. Our previous work showed that the working memory and short-term memory deficits induced by peripheral nerve injury are associated with impairment of frequency facilitation at hippocampal CA3–CA1 synapses [31], so we tested whether the same might be true in rats treated with OXA. As shown in Fig. 2, the amplitudes of fEPSPs at CA3–CA1 synapses were progressively enhanced with increased frequencies (2, 4, and 8 Hz) in controls, while the magnitudes of the facilitation induced by the same frequencies were significantly lower in the OXA group than in controls (Fig. 2B–D). Importantly, the magnitude of the facilitation in L-TAMS-treated rats was higher than that in those treated with OXA and did not differ from controls. These data indicate that OXA impairs sustained synaptic transmission, which may contribute to memory and emotional deficits, and the effects are prevented by L-TAMS.

Fig. 2.

Fig. 2

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Oral magnesium-L-threonate prevents the impairment of synaptic transmission and downregulation of NR2B induced by OXA. A Raw traces of fEPSPs at CA3–CA1 synapses evoked by the first 4 consecutive conditioning stimuli (8 Hz, 0.2 ms) in the indicated groups. B–D Facilitation of fEPSPs induced by 2, 4, and 8 Hz electrical stimuli. The magnitude of facilitation in the OXA group was significantly lower than in controls (Contr). The impairment of frequency facilitation was abolished by oral L-TAMS (n = 5–7 per group). Data are expressed as a percentage of baseline elicited by test stimuli at 0.066 Hz. The intensity of the conditioning stimulus was identical to that of the test stimulus in each experiment. E Amplitudes fEPSPs at CA1–CA3 evoked by the 20th conditioning stimulus in the different groups. F Representative blots and histogram showing the NR2B protein levels in the hippocampus in the indicated groups. Injection of OXA downregulated NR2B in the hippocampus, and the effects were prevented by oral L-TAMS (n = 5 per group). **P < 0.01, ***P < 0.001, n. s. no significant difference compared to controls (Contr) (two-way ANOVA followed by Tukey’s post hoc test).

Previous studies have shown that the expression of the NR2B but not the NR2A subunit of NMDARs in the hippocampus is important for synaptic plasticity and memory in numerous animal models [32]. Therefore, we measured the expression of NR2B in the hippocampus 5 days after the end of OXA injections and found that the NR2B levels were lower in the OXA group than in controls, while these in the L-TAMS+OXA group did not differ from controls (Fig. 2E). This NR2B down-regulation may contribute to the dysfunction of synaptic transmission. Interestingly, the effects were again prevented by the chronic administration of L-TAMS.

Oral Magnesium-L-Threonate Prevents the Mg2+ Deficiency Induced by OXA In Vivo, While Reducing Mg2+ Activates TNF-α/NF-κB Signaling and Downregulates NR2B in Cultured Hippocampal Slices

Our previous work showed that the free Mg2+ concentrations in serum and CSF are significantly reduced in vincristine-treated rats, and oral L-TAMS prevents Mg2+ deficiency [33]. In the present work, we found that injection of OXA for 5 consecutive days led to short-term memory deficits along with depression-like and anxiety-like behaviors 6–8 days after the onset of OXA treatment (1–3 days after termination of treatment). And the OXA-induced behavioral changes were prevented by supplementation with Mg2+. These data suggest that the behavioral changes might be produced by Mg2+ deficiency. To confirm this, the extracellular free Mg2+ was measured 5 and 10 days after OXA injection. Indeed, we found that the free Mg2+ in both serum and CSF were significantly lower in the OXA group than in the controls (Fig. 3A, B). But no difference between the L-TAMS + OXA group and controls was detected at any time point. These results demonstrate that OXA also induces Mg2+ deficiency, which is prevented by oral L-TAMS.

Fig. 3.

Fig. 3

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Magnesium-L-threonate prevents the Mg2+ deficiency induced by OXA in vivo, while Mg2+ deficiency upregulates NF-κB/TNF-α and downregulates NR2B in cultured hippocampal slices. A, B Histograms of the free Mg2+ concentrations in serum and CSF in the indicated groups. OXA reduced free Mg2+ and the effects were prevented by L-TAMS (n = 6–8 per group). C, D Representative blots and histograms showing the protein levels of TNF-α, p-p65, and NR2B in hippocampal slices cultured in medium containing 0.6 mmol/L or 0.8 mmol/L free Mg2+ for 24 and 48 h. *P < 0.05, **P < 0.01, ***P < 0.001 vs 0.8 mmol/L free Mg2+ (one-way ANOVA followed by Tukey’s post hoc test).

We next investigated how Mg2+ deficiency in the brain causes memory/emotional deficits. A large amount of evidence has demonstrated that activation of the TNF-α/NF-κB signaling pathway plays a critical role in cognitive deficits [34], depression [35], and anxiety [36]. We therefore tested the hypothesis that Mg2+ deficiency in the brain leads to memory/emotional deficits by activation of TNF-α/NF-κB signaling. To do so, hippocampal slices were cultured in media containing normal (0.8 mmol/L) and lower (0.6 mmol/L) Mg2+, as injection of OXA decreased free Mg2+ in the CSF from ~ 0.8 to ~ 0.6 mmol/L (Fig. 3B). Western blots revealed that the levels of TNF-α and p-p65, an active form of NF-κB, were significantly higher in hippocampal slices cultured with 0.6 mmol/L Mg2+ on days 1 and 2 (Fig. 3C, D). Our previous work showed that vincristine downregulates NR2B in the hippocampus [33]. In the present work, we found that the level of NR2B was significantly reduced in hippocampal slices on day 2 but not on day 1 after culture in 0.6 mmol/L Mg2+ medium compared to that with 0.8 mmol/L medium. These data suggest that the downregulation of NR2B may result from the activation of TNF-α/NF-κB signaling.

In subsequent experiments, we used the OXA model to test whether oral L-TAMS is able to prevent the activation of TNF-α/NF-κB signaling and the resultant microglial activation in the hippocampus and medial prefrontal cortex (mPFC), which are important for memory and emotion [37, 38].

Oral Magnesium-L-Threonate Abolishes the OXA-Induced Activation of TNF-α/NF-κB and Microglial Activation in Both the Hippocampus and Medial Prefrontal Cortex In Vivo

Having shown that Mg2+ deficiency activates TNF-α/NF-κB signaling in vitro, we next investigated whether the OXA injections that reduce Mg2+ in CSF activate TNF-α-NF-κB signaling in the brain in vivo. Western blots revealed that the protein levels of both TNF-α and p-p65 in the hippocampus and mPFC increased progressively after OXA injection (Figs. 4A, B, 5A, B). The change in TNF-α reached a significant level on day 3 in the hippocampus and on day 5 in the mPFC, compared to controls (Fig. 4A, B). Of note, the TNF-α remained at these levels on days 7 and 10 after the onset of OXA injection in both regions (Fig. 4A, B). That is, TNF-α upregulation persisted for 3 and 6 days after the termination of OXA injections (see Fig, 1A for experimental schedule). Similarly, p-p65 was significantly upregulated in the hippocampus and mPFC from day 3 to day 10 after the first injection of OXA (Fig. 5A, B). These data indicate that the injection of OXA causes a long-lasting activation of TNF-α-NF-κB signaling in the hippocampus and mPFC. Importantly, we found that the levels of TNF-α and p-p65 in both regions in the L-TAMS + OXA group were significantly lower than those in the OXA group and did not differ from controls (Figs. 4A, B, 5A, B) at 10 days after OXA treatment. Similar results were obtained in the hippocampus and mPFC by immunostaining 10 days after the first OXA injection (Figs. 4C, 5C). These results indicate that oral L-TAMS normalizes the OXA-induced overexpression of TNF-α and p-p65 in the hippocampus and mPFC. Double-staining with these tissues harvested 10 days after OXA injection showed that both TNF-α and p-p65 co-localized with NeuN (a marker of the nuclei of neurons) but not with Iba1 (a microglia marker) and GFAP (an astrocyte marker) in the OXA group (Figs. 4D, 5D). These data indicate that chronic oral L-TAMS prevents the excessive activation of TNF-α–NF-κB signaling in neurons of the hippocampus and mPFC.

Fig. 4.

Fig. 4

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Oral L-TAMS blocks the OXA-induced TNF-α upregulation in both the hippocampus and mPFC. A Representative blots and histograms showing the expression of TNF-α in the hippocampus at different time points after the onset of OXA injections (n = 4 per group). The TNF-α upregulation induced by OXA is blocked by L-TAMS as measured on day 10 after the first OXA injection (n = 4 per group). B OXA injections also progressively upregulate TNF-α in the mPFC (n = 4 per group), and this overexpression is blocked by L-TAMS (n = 4 per group). C Representative immunofluorescent staining and histograms showing that L-TAMS prevents the upregulation of TNF-α induced by OXA in CA1 and the dentate gyrus (DG) of the hippocampus and the mPFC (n = 6 per group). D Double immunofluorescence staining showing that TNF-α (red) is co-localized with NeuN (green), but not with Iba1 (green) and GFAP (green) in the hippocampus and mPFC (arrowheads indicate co-localization). *P < 0.05, **P < 0.01, ***P < 0.001 vs controls (Contr) (one-way ANOVA followed by Tukey’s post hoc test).

Fig. 5.

Fig. 5

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L-TAMS prevents p-p65 upregulation in the hippocampus and mPFC induced by OXA. A, B Representative blots and histograms showing the levels of p-p65 in the hippocampus (A) and mPFC (B) at different time points after the onset of OXA injection in the indicated groups (n = 4 per group). The OXA-induced overexpression of p-p65 is blocked by L-TAMS in both regions at 10 days after the onset of OXA (n = 4 per group). C Representative immunofluorescent staining and histograms showing the OXA-induced p-p65 upregulation is completely blocked by co-administration of L-TAMS (n = 6 per group) in both regions (n = 6 per group). D Double immunofluorescence staining reveals that p-p65 (red) is co-localized with NeuN (green), but not with Iba1 (green) and GFAP (green) in the hippocampus and mPFC (arrowheads indicate co-immunostaining of p-p65 and cell markers). *P < 0.05, **P < 0.01, ***P < 0.001 vs controls (Contr) (one-way ANOVA followed by Tukey’s post hoc test).

As microglial activation is critically involved in chemotherapy-induced cognitive impairment [39], we tested whether L-TAMS is also able to prevent OXA-induced microglial activation. Immunostaining showed that the number of microglia in the OXA group was markedly higher in CA1 and the DG of the hippocampus and in the mPFC than that in controls, while the control and L-TAM + OXA groups did not differ (Fig. 6A, B). Therefore, the microglial activation induced by OXA in the hippocampus and mPFC can be normalized by oral L-TAMS.

Fig. 6.

Fig. 6

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Oral L-TAMS prevents the OXA-induced microglial activation in CA1 and the DG of the hippocampus and in mPFC. A Representative images of the immunofluorescence staining of Iba1 (a microglia marker) and DAPI in different groups as indicated. B Histograms showing summary data of Iba1 expression (n = 6 per group). **P < 0.01, ***P < 0.001 vs controls (Contr) (one-way ANOVA followed by Tukey’s post hoc test).

OXA-Induced Memory and Emotional Deficiency are Prevented by Inhibition of NF-κB

Thus far, we showed that OXA injections led to memory/emotional deficits and activation of TNF-α/NF-κB signaling in the hippocampus and mPFC by reducing brain Mg2+, but causal links between the TNF-α/NF-κB activation and the abnormal behaviors in OXA-treated rats have not been established. To clarify this, the NF-κB inhibitor pyrrolidine dithiocarbamic acid (PDTC, 10 ng/5 μL) was injected icv 30 min before each OXA injection (Fig. 7A). The results showed that PDTC substantially prevented the short-term memory deficits and the anxiety-like and depression-like behaviors induced by OXA (Fig. 7B). Following the behavioral tests, the expression of p-p65 and TNF-α in the hippocampus and mPFC were assessed with western blots, which showed that the upregulation of p-p65 and TNF-α in both regions induced by OXA was completely prevented by PDTC (Fig. 8A, B). Furthermore, PDTC also prevented the OXA-induced microglial activation in the hippocampus and mPFC (Fig. S1). These data indicate that activation of TNF-α/NF-κB signaling is essential for OXA-induced memory, emotional deficiency, and microglial activation in the mPFC and hippocampus.

Fig. 7.

Fig. 7

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The OXA-induced deficiency in memory and emotion is prevented by intracerebroventricular injection of the NF-κB inhibitor PDTC. A Experimental schedule. B A decrease in recognition index for short-term memory assessed with the NORT (a), an increase in immobility time with the FST (b), and a reduced time in open arms and bouts in the open arms with the EPMT (c, d) induced by i.p. injection of OXA were abolished by PDTC injection 30 min before each OXA injection (n = 6 per group). There was no difference in total distance traveled and average speed between groups in the EPMT (e, f). **P < 0.01, ***P < 0.001 vs vehicle (Veh) group (one-way ANOVA followed by Tukey’s post hoc test).

Fig. 8.

Fig. 8

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OXA-induced upregulation of TNF-α and p-p65 in the hippocampus and mPFC are abolished by icv injection of PDTC. A, B Western blots and histograms showing the levels of p-p65 and TNF-α in the hippocampus (A, n = 4 per group) and mPFC (B, n = 4 per group) in the indicated groups. ***P < 0.001 vs vehicle (Veh) (one-way ANOVA followed by Tukey’s post hoc test).

Discussion

In the present work, we showed that OXA injections induced memory and emotional deficits (Fig. 1) were accompanied by reduced Mg2+ in serum and CSF (Fig. 3A, B). Importantly, we found that oral L-TAMS prevented both the Mg2+ deficiency and the behavioral disorders induced by OXA (Figs. 1 and 3). OXA impaired frequency facilitation at CA3–CA1 synapses and downregulated the NR2B subunit of NMDARs in the hippocampus, and the effects were again prevented by oral L-TAMS. Mg2+ deficiency activated TNF-α/NF-κB signaling and downregulated NR2B in cultured hippocampal slices (Fig. 3C, D). Oral L-TAMS prevented the activation of TNF-α/NF-κB signaling and microglial activation in the hippocampus and mPFC induced by OXA (Figs 4, 5, 6). Finally, we showed that inhibition of NF-κB by icv injection of PDTC substantially prevented the behavioral and molecular changes induced by OXA (Figs. 7, 8, and S1). The data demonstrate that activation of TNF-α/NF-κB signaling resulting from Mg2+ deficiency may underlie the memory and emotional deficits induced by OXA, and these adverse effects are prevented by chronic oral L-TAMS. This study may provide a novel approach to treat chemotherapy-induced memory and emotional deficits.

Here, we used L-TAMS to supplement Mg2+ because a previous report [18] showed that only L-TAMS but not MgCl2, Mg citrate, or Mg gluconate elevates Mg2+ in the CSF of rats. In patients, increasing the plasma Mg2+ by three-fold via intravenous infusion of MgSO4 does not elevate Mg2+ in the CSF [40].

The Activation of TNF-α/NF-κB Signaling in Neurons Resulting from Mg2+ Deficiency may Underlie the OXA-induced Memory and Emotional Deficits

At present, the mechanisms underlying chemotherapy-induced memory/emotional deficits are unclear. As noted in the Introduction, many anti-cancer agents cause hypomagnesemia in patients. Consistent with this, our previous work in rats showed that vincristine (another anticancer agent) reduces the Mg2+ level in serum and CSF [33]. The present work showed that OXA also reduced Mg2+ in serum and CSF (Fig. 3A) and that supplementation of Mg2+ by oral L-TAMS prevented both the Mg2+ deficiency (Fig. 3A) and the memory/emotional deficits induced by OXA (Fig. 1). Therefore, we conclude that Mg2+ deficiency may be the root cause of OXA-induced cognitive dysfunction. Furthermore, we found that the memory and emotional deficits induced by OXA were accompanied by long-lasting activation of TNF-α/NF-κB signaling in neurons of the hippocampus and mPFC. Reducing Mg2+ in the medium (from 0.8 to 0.6 mmol/L in the CSF of control and OXA-treated rats) activated TNF-α/NF-κB signaling in cultured hippocampal slices, and the molecular and the behavioral changes induced by OXA were prevented by icv injection of an NF-κB inhibitor. Therefore, the activation of TNF-α/NF-κB in neurons resulting from Mg2+ deficiency is responsible for the OXA-induced memory and emotional deficits.

Previous reports [32, 41] have shown that dysfunction of the NR2B subunit of NMDARs is involved in memory deficits and depression. The present work showed that activation of TNF-α/NF-κB signaling was accompanied by the downregulation of NR2B in vivo, and that in cultured hippocampal slices with lower Mg2+ the upregulation of TNF-α and NF-κB p-p65 was followed by downregulation of NR2B. These data indicate that the activation of TNF-α/NF-κB signaling resulting from Mg2+ deficiency may induce memory and emotional deficits via downregulation of NR2B.

We found that OXA also induced microglial activation, which is critical for chemotherapy-induced cognitive impairment [39]. Our previous work showed that vincristine activates TNF-α and NF-kB only in neurons but not in microglia in the spinal dorsal horn [33]. The present work showed that OXA also did the same in the hippocampus and mPFC. As TNF-α/NF-kB are not activated in microglia in the case of chemotherapy, the relationship between microglial activation and TNF-α/NF-κB might be indirect. We speculated that the release of inflammatory cytokines such as TNF-α from neurons may stimulate microglia. Conversely, the microglia may activate neuronal NF-κB via the release of gliotransmitters. The complicated neuron-glial interaction may contribute to the lasting TNF-α/NF-kB activation after the termination of OXA injection. Further studies are needed to clarify the mechanisms underlying the positive feedback.

Our finding that Mg2+ deficiency is the root cause of the memory and emotional deficits induced by OXA is clinically important, as it indicates that the mechanism underlying the side-effects is different from its anticancer effect. In fact, accumulating evidence shows that NF-κB signaling plays critical roles in many aspects of cancers, such as cancer cell proliferation and survival, the epithelial-to-mesenchymal transition, and resistance to anticancer agents (see [17] for a review). Epidemiological studies have shown that high Mg2+ in drinking water reduces the risk of esophageal cancer [42] and liver cancer [43]. Therefore, Mg2+ supplementation may not only prevent the side-effects of chemotherapy but also be beneficial to the prevention and treatment of cancer. At present, how Mg2+ deficiency activates NF-κB signaling is unknown. As the second most abundant intracellular cation, Mg2+ is essential for more than 600 enzymatic reactions [44]. The dysfunction of some enzymes may be responsible for the activation of NF-κB. Further studies are needed to elucidate this issue.

We showed that an NF-κB inhibitor was also able to prevent the OXA-induced cognitive dysfunction (Fig. 7) and TNF-α upregulation in the hippocampus and mPFC (Fig. 8). However, TNF-α/NF-κB signaling has many physiological functions, such as memory storage [45] and immunity [17], so insufficient activation of the pathway may also impair these functions. It is difficult to regulate the pathway at a proper level via an NF-κB inhibitor. As noted above, anti-cancer agents lead to Mg2+ deficiency in both patients and in rats, and lowering extracellular Mg2+ leads to over-activation of TNF-α/NF-κB signaling. Accordingly, supplementary Mg2+ can normalize the pathway. Oral L-TAMS that can enhance brain Mg2+ may be a better way to prevent the chemotherapy-induced side-effects.

Impairment of Synaptic Transmission Contributes to the Chemotherapy-Induced Memory and Emotional Deficits

We found that the memory and emotional deficits induced by OXA were paralleled by impairment of frequency facilitation in synaptic transmission and downregulation of NR2B protein in the hippocampus. Both were prevented by oral L-TAMS. This is consistent with a previous report showing that L-TAMS improves memory and upregulates NR2B in the hippocampus of naïve rats [18]. It is well established that NR2B subunit-containing NMDARs at the postsynaptic density are important for the strengthening of synaptic connections [46] and memory formation [32]. Furthermore, our previous work showed that the impairment of frequency facilitation at CA3–CA1 synapses is associated with a reduction of the density of presynaptic boutons [31]. Therefore, both presynaptic and postsynaptic impairment may cause a failure to follow repetitive afferent inputs in CA3–CA1 synapses, leading to the memory deficits induced by anticancer agents. Prevention of the synaptic dysfunction may contribute to the therapeutic effects of L-TAMS on the memory and emotional deficits induced by OXA.

In conclusion, the excessive activation of TNF-α/NF-κB signaling resulting from Mg2+ deficiency contributes to OXA-induced memory and emotional deficits. Supplementary Mg2+ by chronic oral L-TAMS is effective for preventing the TNF-α/NF-κB activation, and the memory and emotional deficits induced by OXA.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 245 kb) (245.7KB, pdf)

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31771166).

Conflict of interest

The authors claim that there are no conflicts of interest.

Footnotes

Xin Zhou, Zhuo Huang and Jun Zhang have contributed equally to this work.

Contributor Information

Hui Zhang, Email: zhanghui@gd2h.org.cn.

Xian-Guo Liu, Email: liuxg@mail.sysu.edu.cn.

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