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Vitamins C and E reverse melamine-induced deficits in spatial cognition and hippocampal synaptic plasticity in rats
NeuroToxicology 44 (2014) 132–139
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NeuroToxicology
Vitamins C and E reverse melamine-induced deficits in spatial cognition and hippocampal synaptic plasticity in rats Lei An a,b, Tao Zhang a,* a b
College of Life Sciences and Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, 300071 Tianjin, PR China Max-Planck Institute for Neurological Research, 50931 Cologne, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 April 2014 Accepted 13 June 2014 Available online 21 June 2014
Albeit the pathogenesis of cognitive impairment after exposure to melamine has not been fully elucidated, factors such as oxidative stress is thought to play potential roles. In the present study, we investigated the effect of treatment with vitamin C (150 mg/kg) and vitamin E (200 mg/kg) on the impairment induced by melamine. Three-week-old male Wistar rats were submitted to oral gavage with 300 mg/kg melamine in 1% carboxymethylcellulose (CMC) for 28 days (MEL-SAL group). After treatment with melamine, animals received administration of a combination of vitamin C and vitamin E once a day for 7 days (MEL-VIT group). Both control (CT-SAL) group and pair-fed (CT-VIT) group received the same dosage of CMC and vitamin complex, respectively. Melamine-treated rats presented a marked decrease in learning and memory in the Morris water maze (MWM) as well as a reduced efficiency to find the platform in the reversal learning task. The rats treated with vitamins E and C had part of the above effects rescued in MWM tests, with mitigating the melamine-induced deficit in the learning and memory but slightly improving the reversal learning ability. The vitamins C plus E regimen mitigated melamineinduced impairment of hippocampal synaptic plasticity. It showed that the modulation of oxidative stress with vitamins E and C reduced melamine-induced damage. The data suggested that there was a novel therapeutic strategy to the cognitive dysfunction observed in melamine-induced neuropathy. ß 2014 Published by Elsevier Inc.
Keywords: Melamine Oxidative damage Vitamin Spatial cognition Hippocampal synaptic plasticity
1. Introduction Melamine (2,4,6-triamino-s-triazine), a chemical intermediate product, is a resin widely used in thermosetting domestic plastics (Cremonezzi et al., 2004). It is a nitrogen-rich organic compound that has been deliberately added to food in order to increase the apparent protein content. Outbreaks of nephrolithiasis and acute kidney injury among children in China were closely linked to the ingestion of milk contaminated with melamine (Yoon et al., 2011). More than 50,000 infants were hospitalized as a result and 6 deaths have been confirmed. Besides in China, reports also showed that children were affected in Singapore, Vietnam and America (Brown et al., 2007; Ingelfinger, 2008). Thus, the toxic effects of melamine-tainted food stuffs are considered an international public health crisis. Several studies have investigated the toxic mechanism of melamine. Recently, oxidative stress in cellular and rodent experiments has been reported frequently (Guo et al., 2012a; Han et al., 2011; Wang et al., 2011). In several organ systems,
* Corresponding author. Tel.: +86 22 23500237. E-mail address: [email protected] (T. Zhang). http://dx.doi.org/10.1016/j.neuro.2014.06.009 0161-813X/ß 2014 Published by Elsevier Inc.
melamine may induce oxidative stress leading to generation of free radicals and alterations in antioxidants or reactive oxygen species (ROS) scavenging enzymes. For example, it was reported that melamine could induce cell death in PC12 cells, which were used as the model of dopamine neurons in vitro, and the damage of cell could be related to oxidative damage due to reducing superoxide dismutase (SOD) activity (Han et al., 2011). Another study also indicated that the toxicity of melamine was involved in breaking down redox balance and then the oxidation–antioxidation homeostasis (Ma et al., 2011). Notably, melamine caused apoptosis of the NRK-52e cells via excessive intracellular ROS (Guo et al., 2012b), decreased the levels of SOD and glutathione peroxidase (GSH-Px) and enhanced malonaldehyde (MDA) content in a dosedependent manner (Guo et al., 2012a). The aforementioned studies showed that oxidative stress could be an important component included in the mechanism of melamine-induced damage. The results of our previous studies proposed that pyramidal neurons of hippocampus were vulnerable to melamine (An et al., 2012a; Yang et al., 2010). Additionally, laboratory animal studies demonstrated that the hippocampus was one of the target sites of melamine (An et al., 2012a), in which the spatial learning and memory (An et al., 2011), and the synaptic plasticity (Yang et al., 2011) were
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impaired. Several mechanisms were proposed for neurobehavioral toxicity induced by melamine (An et al., 2011, 2013b; Yang et al., 2011), including oxidative stress (An et al., 2012a). For example, a study from our lab showed that residual melamine in hippocampus could result in the production increase of ROS, including superoxide radical anion and hydroxyl radical, as well as suppression of antioxidant defense mechanisms (An et al., 2012a). Of all the organs, the brain is thought to be vulnerable to oxidative damage due to its high content of polyunsaturated fatty acids in the membranes and low levels of enzymatic and non-enzymatic antioxidants (Halliwell, 2006). Consequently, it was critical to mitigate the oxidative damage in the central nervous system (CNS) induced by melamine. The cell has several ways of alleviating the effects of oxidative stress, either by repairing the damage of nucleotides and lipid peroxides (LPO) or by directly diminishing the occurrence of oxidative damage by means of enzymatic and non-enzymatic antioxidants (Guney et al., 2007). Whereas antioxidants were shown to decrease some of the biochemical measures of oxidative stress, relatively few studies evaluated the efficacy of antioxidants to rescue melamine neurobehavioral toxicity. Several studies assessed the ability of antioxidants to mitigate oxidative damage. Vitamin E is the most important lipophilic antioxidant and resides mainly in the cell membranes, thus helping to maintain membrane stability (Guney et al., 2007). In addition to its antioxidant effects, it is a scavenger of peroxyl radicals and quenches other free radicals, such as superoxide and hydroxyl radicals (Shirpoor et al., 2009). Vitamin E supplementation decreased alcohol-induced oxidative stress and apoptosis in the developing hippocampus (Marino et al., 2004). A high dose of vitamin E dietary supplementation was proposed to serve as a successful therapeutic strategy for the prevention or treatment of oxidative stress in neuropathic disease (Fariss and Zhang, 2003). Similarly, vitamin C is the most important hydrophilic free radical scavenger in extracellular fluids, trapping radicals in the aqueous phase and protecting biomembranes from peroxidative damage (Guney et al., 2007). Daily administration of vitamin C could reverse cognitive dysfunction observed in rats with seizures and decrease neuronal injury in rat hippocampus (Tome Ada et al., 2010). Particularly, it was involved in the regeneration of tocopherol from tocopheroxyl radicals in the membrane (Guney et al., 2007). Moreover, it was found that a combination of vitamins E and C reduced LPO caused by oxidative stress (Gultekin et al., 2001) and decreased oxidative damage both at biochemical and histological levels (Guney et al., 2007; Ornoy et al., 2009). It was likely that vitamins C and E acted in a synergistic manner, by vitamin E primarily being oxidized to the tocopheroxyl radical and then reduced back to tocopherol by vitamin C (Aydemir et al., 2004). Against the above background, the lipophilic antioxidant vitamin E and the hydrophilic antioxidant vitamin C together were used as antioxidative protection in the rat model for melamine-induced free radical production and lipid peroxidation in the hippocampus. It was reported that vitamins C and E administration provoked a selective action on the hippocampus with mitigating the impairment of oxidative damage (Green et al., 2005, 2006) and ameliorating hippocampal cellular apoptosis (McGoey et al., 2003). The present investigations were designed to examine the neurotoxic effects of melamine and potential rescue by vitamins C and E using a developing rat model. The animal model has been established during early adolescent period (Laviola et al., 2003) since the validity of adolescent rodent was endorsed for the purpose of comparison or extrapolation to the human case (Spear, 2000), which were more involved in the increasing risk of developing neuropathy-related problems (Laviola et al., 1999; Spear, 2000; Spear and Brake, 1983). The dose, duration and route of melamine treatment, which could induce cognitive defects (An et al., 2011, 2013b) and oxidative stress in the hippocampus (An et al., 2012a), were selected according to our
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previous studies (An et al., 2011, 2012a, 2013b) and also the investigations from other labs (Burns, 2007; Jingbin et al., 2010; Kobayashi et al., 2010; Rumbeiha et al., 2010; Stine et al., 2011; Xie et al., 2010). The treatment method of vitamins was referenced from the previous studies (Gultekin et al., 2001; Karaoz et al., 2002), which reported that the dose of vitamins could effectively reduce lipid peroxidation and rescue antioxidant enzymes activities caused by chlorpyrifos-ethyl. It is well known that the hippocampus plays crucial roles in encoding and consolidating memory (Morris et al., 1982; Squire, 1992). Activity-dependent plasticity of hippocampal glutamatergic synapses, particularly long-term potentiation (LTP) and longterm depression (LTD), was proposed as the primary cellular substrate to fulfill these cognitive functions (Bliss and Collingridge, 1993; Martin et al., 2000). Accordingly, our investigation was performed to evaluate deleterious effects of melamine on hippocampus via oxidative damage, manifesting as physical changes, cognitive deficits and synaptic plasticity impairment, after that antioxidants could prevent such damage, using both MWM test and electrophysiological recordings. 2. Materials and methods 2.1. Reagents Melamine (purity > 99.5%) was purchased from Yingda Sparseness & Nobel Reagent Chemical Factory, Tianjin, PR China. Melamine assay kit was purchased from Huaan Magnech BioTech Co., Ltd., Beijing, PR China. Other reagents were of A.R. grade. 2.2. Animals and treatment Healthy male Wistar rats, three-week-old (prenatal day (PND) 21), were obtained from the Laboratory Animal Center, Academy of Military Medical Science of People’s Liberation Army, and reared in the animal house of Medical School in Nankai University. After the adaptation period, animals were randomly assigned into one of four experimental groups, eight rats each as follows. In melamine (MEL-SAL) group, the rats were gavaged with melamine solution (30 mg/mL, dissolved in 1% carboxymethylcellulose (CMC)) at a dose of 300 mg/kg/day for 28 consecutive days (from PND 22 to PND 49) and intraperitoneal injected saline for the next 7 consecutive days (from PND 50 to PND 56). In control (CT-SAL) group, the rats were gavaged with the same dose of 1% CMC for 28 consecutive days and intraperitoneal injected saline for the next 7 consecutive days. In melamine-vitamins (MEL-VIT) group, after 28 consecutive treatment days, each rat received vitamins C and E, dissolved in sterile endotoxin-free isotonic saline, intraperitoneally once a day for 7 consecutive days at 150 and 200 mg/kg, respectively. In pair-fed (CT-VIT) group, the rats were gavaged with the same dose of 1% CMC on 28 consecutive days and then each rat received vitamins C and E saline solution intraperitoneally once a day for 7 consecutive days at 150 and 200 mg/kg, respectively. The rats were individually housed in transparent plastic cages in a temperature controlled (21 8C) colony room under a 12/12 h light/ dark cycle (lights on at 07:00 h) with ad libitum access to food and water. All experiments were conducted during the light phase and performed according to the protocols approved by the Committee for Animal Care at Nankai University and in accordance with the practices outlined in the NIH Guide for the Care and Use of Laboratory Animals. 2.3. Morris water maze experiment On PND 57, rats were examined for spatial cognition performance in the MWM test, using procedures described previously
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with modifications (An et al., 2013a; Han et al., 2014). The water maze consisted of a 1.5-m-diameter circular tank divided into four equal quadrants (I, II, III, and IV), and there was a 10-cm-diameter platform submerged 2 cm below the water surface in the center of quadrant III. The water was made opaque using nontoxic black ink and maintained at 25 1 8C. Movement of rats in the maze was monitored by a computerized video tracking system (Ethovision 2.0, Noldus, Wagenigen, Netherlands) connected to a personal computer, through which data were collected for off-line analyzing. Briefly, the task consisted of three consecutive stages, which were initial training (IT), space exploring test (SET) and reversal training (RT). In IT stage, animals were subjected to four swimming trial per day for 4 consecutive days covering PND 57 to PND 60. In each day, the order of starting points was used pseudorandom and each rat was located in the same position on each trial at one of four starting quadrant points. Animals were allowed to swim freely and trained to find a hidden platform in a circular water tank. The times required to find the platform (escape latency), path length and the swimming speed were recorded. If an animal failed to locate the platform within 60 s, it was placed on it for 10 s, and escape latency was recorded as 60 s. Interval between two trials was approximately 10 min. In SET stage, rats were given the probe trial test at 9 am on PND 61. The platform was removed from the tank. Rats were released individually into water from the starting point of quadrant I and allowed to swim for 60 s as probe test. Quadrant dwell time (the percentage of time spent in the target quadrant) and platform crossings (numbers passing platform area) were recorded. In RT stage, a rat was given four trials on each day during two training days, which was from PND 62 to PND 63. The platform was placed in the center of quadrant I, which was opposite to quadrant III. Rats were trained to find the hidden platform in an opposite position and the reversal learning ability was examined. In RT stage, each rat was given four trials of each training day and tested for 2 days, which was from PND 62 to PND 63. The method used and parameters recorded were the same as those in IT stage. 2.4. In vivo electrophysiological test Electrophysiological tests were performed after MWM assessment. The animals were anesthetized with 30% urethane with a dosage of 4 mL/kg by intraperitoneal injection. And then they were placed in a stereotaxic frame (SN-3, Narishige, Japan) for surgery and the recordings were made as described previously (An et al., 2012b; An and Zhang, 2013). Briefly, at the left side of rat’s head, two small holes were drilled in the skull for the recording and stimulating electrodes inputting respectively. According to The Rat Brain in Stereotaxic Coordinates, the bipolar stimulating electrode was implanted into the hippocampal Schaffer collaterals region (4.2 mm posterior to the bregma, 3.5 mm lateral to midline, 2.5 mm ventral below the dura), and the recording electrode was implanted into hippocampal CA1 region (3.5 mm posterior to the bregma, 2.5 mm lateral to midline, 2.0 mm ventral below the dura). Test stimuli were delivered to the Schaffer collaterals every 30 s at an intensity that evoked a response of 70% of its maximum (range 0.3–0.5 mA). Once the response stabilized, sampling was made under low-frequency stimulations (0.05 Hz) for 20 min as the baseline. After recording baseline, high frequency stimulation (HFS) (8 pulses at 100 Hz for 6 s repeated 30 times) was delivered and then the field excitatory postsynaptic potentials (fEPSPs) were amplified (100), filtered at 5–5 kHz, digitized and collected at 20 kHz sample frequency (Scope software, PowerLab; AD Instruments, Australia) every 60 s for 60 min. Initial data measurement was performed in Clampfit 9.0 (Molecular Devices, Sunnyvale, CA, USA). The fEPSP slope (20–80% level of the falling phase) was used to measure synaptic efficacy. After recording baseline low frequency stimulation (LFS) (900 pulses of 1 Hz for 15 min) was delivered and then the fEPSPs were recorded on the other side of
the hemisphere. The classic hematoxylin–eosin (HE) staining was used to evaluate electrode placements. Only data obtained from rats with correct locations were included in the statistical analysis. 2.5. Biochemical assay in hippocampus After electrophysiological test, the brain of each rat was removed and weighted, and then the hippocampus in the right side of brain was dissected out and rinsed in 0.1 M phosphate buffer (pH 7.4). After being weighed, the hippocampus was homogenized with ice-cold saline to be 10% (w/v) homogenates. The mixtures were homogenized using a glass homogenizer for 5 min and centrifuged at 300 g at 4 8C for 15 min. The supernatant was collected and stored at 70 8C for biochemical assay. The content of melamine in hippocampus was determined following the instructions provided by the manufacturer of the kit. 2.6. Statistical analysis All data were presented as mean S.E.M. A repeated measures ANOVA was applied for analysis of differences between four groups during IT and RT stages followed by Bonferroni post hoc test. One-way measures ANOVA was performed on the data from SET stage, electrophysiological recording, biochemical tests and the weight data followed by Bonferroni post hoc test. Statistical differences were taken when P < 0.05. The analyses were performed using SPSS 16.0 software.
3. Results 3.1. Results of physical changes The effects of melamine and the combination of vitamins C and E treatment on body weight were shown in Fig. 1A. It was found that rats gained weight in all four groups during the 4 weeks of oral gavage treatment with melamine. A one-way ANOVA showed that there were statistical differences of day (F(6,196) = 135.538, P < 0.001), day group interaction (F(18,196) = 23.969, P < 0.001) and group (F(3,196) = 30.066, P < 0.001). Except from PND 22 to PND 26, there was a significant decrease of body weight in MEL-SAL group (PND 43, 50 and 53, P < 0.05; PND 57, P < 0.01) compared to that in MEL-VIT group (PND 43, 50 and 53, P < 0.05; PND 57, P < 0.01) and CT-SAL group, respectively. However, there was no difference of body weight changes either between CT-SAL group and CT-VIT group or between MEL-SAL group and MEL-VIT group. The ratios of brain and hippocampal weights to body weight in each group were presented in Fig. 1B and C, respectively. A oneway ANOVA showed that there was a significant difference of group in hippocampus (F(3,28) = 13.925, P < 0.01). Bonferroni post hoc test confirmed that there was a statistical decrease of hippocampal weight in MEL-SAL group compared to that in CTSAL group (Fig. 1C, P < 0.05). However, there was no difference of hippocampal weight either between CT-SAL group and MEL-VIT group or between MEL-SAL group and MEL-VIT group. In addition, no statistical difference of hippocampal weight was found between CT-SAL and CT-SAL groups. Additionally, the concentration of melamine in the hippocampus of each group was presented in Table 1. A one-way ANOVA showed a statistical difference of group (F(3,12) = 167.690, P < 0.001). Bonferroni post hoc test confirmed that there were abnormal higher levels of melamine in both MEL-SAL (P < 0.001) and MEL-VIT (P < 0.001) groups compared to that in CT-SAL group, while no statistical difference was found either between CT-SAL group and CT-VIT group or between MEL-SAL group and MEL-VIT group.
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Fig. 1. Measurement of physical changes. (A) The changes of rat’s body weight. (B) The ratio of brain weight to body weight on PND 64. (C) The ratio of hippocampal weight to body weight on PND 64. Data are presented as mean S.E.M. *P < 0.05, **P < 0.01: comparison between MEL-SAL group vs. CT-SAL group. %P < 0.05, %%P < 0.01: comparison between MEL-VIT group vs. CT-SAL group. n = 8 per group.
3.2. MWM experiment results Fig. 2 showed the data obtained from the Morris water maze test in four groups on each test day. During IT stage, the average escape latencies were decreased remarkably with training (Fig. 2A). A repeated measures ANOVA showed that there were statistical differences of day (F(3,112) = 71.106, P < 0.001), day group interaction (F(9,112) = 3.352, P < 0.01) and group (F(3,112) = 23.782, P < 0.001), as all four groups did improve over the 4 days of training. Bonferroni post hoc test showed that escape latency was significantly longer in MEL-SAL group than that in CTSAL group (day 2, P < 0.05; day 3, P < 0.01; day 4, P < 0.01), while the escape latency in MEL-VIT group was markedly reduced compared with those in MEL-SAL group (day 3, P < 0.05; day 4, P < 0.01). In addition, a repeated measures ANOVA showed that there were statistical differences of day (F(3,112) = 65.521, P < 0.001), day group interaction (F(9,112) = 3.214, P < 0.01)
Table 1 The concentration of melamine in the hippocampus of each group.. Group CT-SAL Concentration (mg/g)
0.19 0.06
CT-VIT 0.22 0.03
MEL-SAL 23.36 0.55
MEL-VIT aaa
25.71 0.82aaa
The data are represented as the average levels of melamine in rats’ hippocampal homogenate on PND 64. All data are presented as mean S.E.M. aaaP < 0.001 versus CT-SAL group. n = 4 per group.
and group (F(3,112) = 22.005, P < 0.001) in the path length. Bonferroni post hoc test showed that the path length was significantly longer in MEL-SAL group compared to that in CTSAL group (day 2, P < 0.05; day 3, P < 0.01; day 4, P < 0.01), while it was significantly shorter in MEL-VIT group than that in MEL-SAL group (day 3, P < 0.05; day 4, P < 0.01). Similar results were shown on swimming speeds and there was no marked difference of day, day group or group (Fig. 2C). In SET stage, one-way ANOVA revealed statistical differences of group in both platform crossing (F(3,28) = 24.517, P < 0.001) and quadrant dwell time (F(3,28) = 31.628, P < 0.001). Statistical results confirmed that both the platform crossings (Fig. 2D, P < 0.01) and the quadrant dwell time (Fig. 2E, P < 0.01) were reduced in MEL-SAL group compared to that in CT-SAL group. It could be found that both the platform crossings (Fig. 2D, P < 0.05) and the quadrant dwell time (Fig. 2E, P < 0.01) were obviously increased in MEL-VIT group compared to that in MEL-SAL group. In addition, there was no significant difference of them between MEL-VIT group and CT-SAL group. With respect to RT stage, mean escape latency was calculated for each rat on each of two training days. A repeated measures ANOVA confirmed statistical difference of day (F(1,56) = 60.924, P < 0.001), day group (F(3,56) = 3.415, P < 0.05), and group (F(3,56) = 14.423, P < 0.001), as all three groups did improve over the 2 days of reversal training (Fig. 2A). Bonferroni post hoc test showed that there was statistically greater escape latency in MEL-SAL group than that in CTSAL group (day 6, P < 0.01; day 7, P < 0.01). In addition, escape latency was dramatically prolonged in MEL-VIT group compared to
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Fig. 2. Rats’ performance in MWM test. (A) Mean escape latency calculated for each day in IT and RT stages. (B) Mean path length in IT and RT stages. (C) Mean swimming speed in IT and RT stages. (D) Mean number of platform area crossings in SET stage. (E) Mean percentage of time in target quadrant in SET stage. Data are presented as mean S.E.M. *P < 0.05, **P < 0.01: comparison between MEL-SAL group vs. CT-SAL group. #P < 0.05, ##P < 0.01: comparison between MEL-VIT group vs. MEL-SAL group. % P < 0.05, %%P < 0.01: comparison between MEL-VIT group vs. CT-SAL group. n = 8 per group.
that in CT-SAL group (day 6, P < 0.05; day 7, P < 0.01). A repeated measures ANOVA showed that there were statistical differences of day (F(1,56) = 53.739, P < 0.001), day group (F(3,56) = 2.977, P < 0.05), and group (F(3,56) = 13.762, P < 0.001) in the path length. Bonferroni post hoc test showed that the path length was longer in MEL-SAL group than that in CT-SAL group (day 6, P < 0.01; day 7, P < 0.01), so did MEL-VIT group (day 6, P < 0.05; day 7, P < 0.01). There was no significant difference of escape latency between
MEL-SAL group and MEL-VIT group throughout the whole RT stage. Again, a repeated measures ANOVA showed that there was no significant difference of swimming speed in day or group. 3.3. LTP and LTD from Schaffer collaterals to CA1 Both LTP and LTD results in the hippocampal CA1 region were presented in Fig. 3. In the LTP test, stimulation of Schaffer
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Fig. 3. Electrophysiological results from hippocampal Schaffer collateral to CA1 area. (A) Changes in fEPSP slopes after HFS. The fEPSP slope is plotted as a percentage change against the baseline before HFS. Arrow represents application of HFS. (B) Magnitude of LTP is determined as responses between 40 and 60 min after HFS. (C) Changes in fEPSP slopes after LFS. The fEPSP slope is plotted as a percentage change against the baseline before LFS. Two-way arrow represents application of LFS. (D) Magnitude of LTD is determined as responses between 40 and 60 min after LFS. Data are presented as mean S.E.M. **P < 0.01: comparison between MEL-SAL group vs. CT-SAL group. #P < 0.05, ## P < 0.01: comparison between MEL-VIT group vs. MEL-SAL group. %P < 0.05: comparison between MEL-VIT group vs. CT-SAL group. n = 8 per group.
collaterals evoked a basal fEPSP in hippocampal CA1, while HFS induced LTP of the stimulated synapses for at least 1 h. Results representing the time courses of fEPSP slopes normalized to the 20 min baseline period were shown in Fig. 3A. The fEPSP slopes were increased immediately after HFS and stabilized to a level above the baseline period. The magnitude of LTP was dramatically smaller in MEL-SAL group compared to that in CT-SAL group during whole the recordings after HFS. A one-way ANOVA showed that there was a statistical difference of group (F(3,28) = 43.725, P < 0.001). Bonferroni post hoc test showed that the fEPSP slopes were dramatically decayed in MEL-SAL group compared to that in CT-SAL group (Fig. 3B, P < 0.01). Furthermore, there was a statistical difference of the fEPSP slopes between MEL-VIT group and MEL-SAL group, indicating that vitamins treatment did efficiently reversed the melamine-induced LTP impairment (Fig. 3B, P < 0.01). The LTD of stimulated synapses was induced by LFS for at least 1 h and the time course of fEPSP slopes was shown in Fig. 3C. A one-way ANOVA showed that there was statistical difference of group (F(3,28) = 47.965, P < 0.001). The fEPSP slope values were significantly higher in MEL-SAL group than that in CT-SAL group (Fig. 3D, P < 0.01). Administration of vitamin complex was due to the fact that the magnitude of LTD was greater in MEL-VIT group compared to that in MEL-SAL group (Fig. 3D, P < 0.01), while there was a dramatically depressed effect on LTD in MEL-VIT group compared to that in CT-SAL group (Fig. 3D, P < 0.05). In addition, the data of CT-VIT group was indistinguishable to that of CT-SAL group throughout the whole electrophysiological experiments.
4. Discussion A previous study provided an evidence that exposure to melamine induced oxidative damage in the developing brain (An et al., 2012a). The present investigation tested the hypothesis that administration with vitamins C plus E antioxidant compounds could mitigate the deficits of spatial cognition and the impairments in hippocampal synaptic plasticity induced by melamine. In the study, the effects of the combination of vitamins C with E on body weight, brain weight and hippocampal weight, performance in the learning and reversal learning of the MWM tests, and hippocampal synaptic plasticity were determined in melaminetreated and vitamin-treated adolescence rats. Our previous studies reported that there was a trend decreasing in the average weight of brain and a statistical difference in the hippocampal weight after exposure to melamine (An et al., 2011), which was in line with the selective neurotoxicity of melamine in hippocampus (An et al., 2012a). Our data indicated that the two antioxidant vitamins selectively and effectively prevented melamine-induced growth restriction in the adolescent hippocampus. The combination of vitamins C and E might make such a selective action on the hippocampus by potentially mitigating oxidative damage (Green et al., 2006) and affecting the regulation of hippocampal pyramidal cell apoptosis in the neonate (Green et al., 2005; McGoey et al., 2003). Additionally, the abnormal melamine levels that found in melamine-treated rats’ hippocampus indicated that melamine was able to accumulate in brain, which was consistent with our previous findings (An et al., 2011, 2012a).
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Due to impaired spatial cognition as a consequence of hippocampal injury or degeneration, MWM test, which was widely used as an effective behavioral approach for examining cognition in rodents (D’Hooge and De Deyn, 2001), was performed. Melamine-treated rats showed that there were considerably deficits in IT, SET and RT stages of MWM performance compared with that in control group. These data were consistent with our previous studies that melamine caused learning and memory deficits and reversal learning deficit in the MWM task (An et al., 2011, 2013b). Administration of vitamins C plus E attenuated melamine-induced deficit in spatial learning and memory performance in IT and SET stages, but did not totally ameliorate the performance of reversal learning in RT stage. The involvement of the hippocampus in learning and memory was well established. Several studies suggested that the alterations of synaptic plasticity were associated with cognitive behavior (Collingridge et al., 2010; Malenka and Nicoll, 1999). Two major forms of long-lasting synaptic plasticity, LTP and LTD, play a crucial role in the function of synaptic transmission (Bliss and Collingridge, 1993; Collingridge et al., 2010; Morris, 2003). To further investigate the underlying mechanism for the results obtained from the MWM tests, the electrophysiological changes in hippocampus were recorded. It was found that the administration of vitamins C plus E effectively reduced the impairments of both LTP and LTD induced by melamine, which expressed the recuperative effect of vitamins C plus E on melamine-induced cognitive deficit in MWM tasks. The dramatic impairments of synaptic plasticity in melamine-treated rats may be due to the persistent loss of hippocampal CA1 neurons (An et al., 2012a). Administration of vitamins C plus E may provide the structural revival of the hippocampus from melamine-induced injury, as demonstrated by oxidative damage (An et al., 2014). Although the impairments of synaptic plasticity in hippocampus were notably retrieved by a combination of vitamins C and E, rat’s performance in RT stage was not significantly improved. Meanwhile, the impairment of LTD was not entirely diminished, which suggested that LTD played a crucial role in the spatial learning and memory capability, especially reversal learning ability. Recent study showed that lipid peroxidation affected oxidative phosphorylation, maintenance of mitochondrial membrane potential and mitochondrial Ca2+ buffering capacity (Ott et al., 2007). Since hippocampus is one of the critical regions for spatial learning and memory (Eichenbaum, 2004; Geinisman et al., 2004), the occurrence of hippocampal neural apoptosis via affecting oxidation–antioxidation homeostasis provided supportive evidence for the impaired performance of rats’ in behavioral tests. One of our previous studies revealed that the combination of vitamins C plus E effectively reduced the oxidative damage in PC12 cell, with decreasing the levels of free radicals and ameliorating the cellular apoptosis (An et al., 2014). The restoration of hippocampal neuron apoptosis could revive the decreased connection between neurons and improve neuronal transmission (An et al., 2014). As a result, the impairments of synaptic plasticity and cognitive functions were markedly reduced in the present study. In addition, other studies reported that superoxide could serve as a cellular messenger molecule in normal LTP, and the ROS were necessary for LTP expression (Hu et al., 2007). However, with abnormal increased ROS during aging, the activated cellular signaling pathways during normal LTP could be altered by the oxidation of certain critical LTP effector molecules (Kamsler and Segal, 2003; Serrano and Klann, 2004). Nevertheless, a further investigation is still required. Furthermore, there were no statistical differences of the indexes, obtained from either MWM test or synaptic plasticity recordings, between CT-VIT group and CT-SAL group, suggesting that administration of vitamins C plus E did not induce cognitive deficits and hippocampal synaptic transmission impairment. The
result showed the relative safety pharmaceutical effect on attenuating melamine-induced CNS dysfunction. To sum up, the treatment with the complex of vitamins C and E produces positive effects in melamine-induced rats but the mechanism remains to be explored. In conclusion, the administration of vitamins C plus E rescued melamine-induced decrement in the performances of learning and memory and hippocampal long-term synaptic plasticity, and partly attenuated melamine-induced deficits in the reversal learning ability. Meanwhile, the regimen of vitamins C plus E did not produce cognitive deficits. In view of this safety issue, the administration of vitamins C plus E against cognitive deficits induced by melamine is reasonable. Conflict of interest The authors declare there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (31171053, 11232005) and Tianjin Research Program of Application Foundation and Advanced Technology (12JCZDJC22300) and 111 Project (B08011). References An L, Li Z, Yang Z, Zhang T. Cognitive deficits induced by melamine in rats. Toxicol Lett 2011;206:276–80. An L, Li Z, Yang Z, Zhang T. Melamine induced cognitive impairment associated with oxidative damage in rat’s hippocampus. Pharmacol Biochem Behav 2012a;102:196–202. An L, Li Z, Zhang T. Reversible effects of vitamins C and E combination on oxidative stress-induced apoptosis in melamine-treated PC12 cells. Free Radic Res 2014;48:239–50. An L, Liu S, Yang Z, Zhang T. Cognitive impairment in rats induced by nano-CuO and its possible mechanisms. Toxicol Lett 2012b;213:220–7. An L, Yang Z, Zhang T. Imbalanced synaptic plasticity induced spatial cognition impairment in male offspring rats treated with chronic prenatal ethanol exposure. Alcohol Clin Exp Res 2013a;37:763–70. An L, Yang Z, Zhang T. Melamine induced spatial cognitive deficits associated with impairments of hippocampal long-term depression and cholinergic system in Wistar rats. Neurobiol Learn Mem 2013b;100:18–24. An L, Zhang T. Spatial cognition and sexually dimorphic synaptic plasticity balance impairment in rats with chronic prenatal ethanol exposure. Behav Brain Res 2013;256:564–74. Aydemir O, Naziroglu M, Celebi S, Yilmaz T, Kukner AS. Antioxidant effects of alpha-, gamma- and succinate-tocopherols in guinea pig retina during ischemia–reperfusion injury. Pathophysiology 2004;11:167–71. Bliss TV, Collingridge GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 1993;361:31–9. Brown CA, Jeong KS, Poppenga RH, Puschner B, Miller DM, Ellis AE, et al. Outbreaks of renal failure associated with melamine and cyanuric acid in dogs and cats in 2004 and 2007. J Vet Diagn Invest 2007;19:525–31. Burns K. Researchers examine contaminants in food, deaths of pets. J Am Vet Med Assoc 2007;231:1636–8. Collingridge GL, Peineau S, Howland JG, Wang YT. Long-term depression in the CNS. Nat Rev Neurosci 2010;11:459–73. Cremonezzi DC, Diaz MP, Valentich MA, Eynard AR. Neoplastic and preneoplastic lesions induced by melamine in rat urothelium are modulated by dietary polyunsaturated fatty acids. Food Chem Toxicol 2004;42:1999–2007. D’Hooge R, De Deyn PP. Applications of the Morris water maze in the study of learning and memory. Brain Res Rev 2001;36:60–90. Eichenbaum H. Hippocampus: cognitive processes and neural representations that underlie declarative memory. Neuron 2004;44:109–20. Fariss MW, Zhang JG. Vitamin E therapy in Parkinson’s disease. Toxicology 2003;189:129–46. Geinisman Y, Ganeshina O, Yoshida R, Berry RW, Disterhoft JF, Gallagher M. Aging, spatial learning, and total synapse number in the rat CA1 stratum radiatum. Neurobiol Aging 2004;25:407–16.
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Contents lists available at ScienceDirect
NeuroToxicology
Vitamins C and E reverse melamine-induced deficits in spatial cognition and hippocampal synaptic plasticity in rats Lei An a,b, Tao Zhang a,* a b
College of Life Sciences and Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, 300071 Tianjin, PR China Max-Planck Institute for Neurological Research, 50931 Cologne, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 April 2014 Accepted 13 June 2014 Available online 21 June 2014
Albeit the pathogenesis of cognitive impairment after exposure to melamine has not been fully elucidated, factors such as oxidative stress is thought to play potential roles. In the present study, we investigated the effect of treatment with vitamin C (150 mg/kg) and vitamin E (200 mg/kg) on the impairment induced by melamine. Three-week-old male Wistar rats were submitted to oral gavage with 300 mg/kg melamine in 1% carboxymethylcellulose (CMC) for 28 days (MEL-SAL group). After treatment with melamine, animals received administration of a combination of vitamin C and vitamin E once a day for 7 days (MEL-VIT group). Both control (CT-SAL) group and pair-fed (CT-VIT) group received the same dosage of CMC and vitamin complex, respectively. Melamine-treated rats presented a marked decrease in learning and memory in the Morris water maze (MWM) as well as a reduced efficiency to find the platform in the reversal learning task. The rats treated with vitamins E and C had part of the above effects rescued in MWM tests, with mitigating the melamine-induced deficit in the learning and memory but slightly improving the reversal learning ability. The vitamins C plus E regimen mitigated melamineinduced impairment of hippocampal synaptic plasticity. It showed that the modulation of oxidative stress with vitamins E and C reduced melamine-induced damage. The data suggested that there was a novel therapeutic strategy to the cognitive dysfunction observed in melamine-induced neuropathy. ß 2014 Published by Elsevier Inc.
Keywords: Melamine Oxidative damage Vitamin Spatial cognition Hippocampal synaptic plasticity
1. Introduction Melamine (2,4,6-triamino-s-triazine), a chemical intermediate product, is a resin widely used in thermosetting domestic plastics (Cremonezzi et al., 2004). It is a nitrogen-rich organic compound that has been deliberately added to food in order to increase the apparent protein content. Outbreaks of nephrolithiasis and acute kidney injury among children in China were closely linked to the ingestion of milk contaminated with melamine (Yoon et al., 2011). More than 50,000 infants were hospitalized as a result and 6 deaths have been confirmed. Besides in China, reports also showed that children were affected in Singapore, Vietnam and America (Brown et al., 2007; Ingelfinger, 2008). Thus, the toxic effects of melamine-tainted food stuffs are considered an international public health crisis. Several studies have investigated the toxic mechanism of melamine. Recently, oxidative stress in cellular and rodent experiments has been reported frequently (Guo et al., 2012a; Han et al., 2011; Wang et al., 2011). In several organ systems,
* Corresponding author. Tel.: +86 22 23500237. E-mail address: [email protected] (T. Zhang). http://dx.doi.org/10.1016/j.neuro.2014.06.009 0161-813X/ß 2014 Published by Elsevier Inc.
melamine may induce oxidative stress leading to generation of free radicals and alterations in antioxidants or reactive oxygen species (ROS) scavenging enzymes. For example, it was reported that melamine could induce cell death in PC12 cells, which were used as the model of dopamine neurons in vitro, and the damage of cell could be related to oxidative damage due to reducing superoxide dismutase (SOD) activity (Han et al., 2011). Another study also indicated that the toxicity of melamine was involved in breaking down redox balance and then the oxidation–antioxidation homeostasis (Ma et al., 2011). Notably, melamine caused apoptosis of the NRK-52e cells via excessive intracellular ROS (Guo et al., 2012b), decreased the levels of SOD and glutathione peroxidase (GSH-Px) and enhanced malonaldehyde (MDA) content in a dosedependent manner (Guo et al., 2012a). The aforementioned studies showed that oxidative stress could be an important component included in the mechanism of melamine-induced damage. The results of our previous studies proposed that pyramidal neurons of hippocampus were vulnerable to melamine (An et al., 2012a; Yang et al., 2010). Additionally, laboratory animal studies demonstrated that the hippocampus was one of the target sites of melamine (An et al., 2012a), in which the spatial learning and memory (An et al., 2011), and the synaptic plasticity (Yang et al., 2011) were
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impaired. Several mechanisms were proposed for neurobehavioral toxicity induced by melamine (An et al., 2011, 2013b; Yang et al., 2011), including oxidative stress (An et al., 2012a). For example, a study from our lab showed that residual melamine in hippocampus could result in the production increase of ROS, including superoxide radical anion and hydroxyl radical, as well as suppression of antioxidant defense mechanisms (An et al., 2012a). Of all the organs, the brain is thought to be vulnerable to oxidative damage due to its high content of polyunsaturated fatty acids in the membranes and low levels of enzymatic and non-enzymatic antioxidants (Halliwell, 2006). Consequently, it was critical to mitigate the oxidative damage in the central nervous system (CNS) induced by melamine. The cell has several ways of alleviating the effects of oxidative stress, either by repairing the damage of nucleotides and lipid peroxides (LPO) or by directly diminishing the occurrence of oxidative damage by means of enzymatic and non-enzymatic antioxidants (Guney et al., 2007). Whereas antioxidants were shown to decrease some of the biochemical measures of oxidative stress, relatively few studies evaluated the efficacy of antioxidants to rescue melamine neurobehavioral toxicity. Several studies assessed the ability of antioxidants to mitigate oxidative damage. Vitamin E is the most important lipophilic antioxidant and resides mainly in the cell membranes, thus helping to maintain membrane stability (Guney et al., 2007). In addition to its antioxidant effects, it is a scavenger of peroxyl radicals and quenches other free radicals, such as superoxide and hydroxyl radicals (Shirpoor et al., 2009). Vitamin E supplementation decreased alcohol-induced oxidative stress and apoptosis in the developing hippocampus (Marino et al., 2004). A high dose of vitamin E dietary supplementation was proposed to serve as a successful therapeutic strategy for the prevention or treatment of oxidative stress in neuropathic disease (Fariss and Zhang, 2003). Similarly, vitamin C is the most important hydrophilic free radical scavenger in extracellular fluids, trapping radicals in the aqueous phase and protecting biomembranes from peroxidative damage (Guney et al., 2007). Daily administration of vitamin C could reverse cognitive dysfunction observed in rats with seizures and decrease neuronal injury in rat hippocampus (Tome Ada et al., 2010). Particularly, it was involved in the regeneration of tocopherol from tocopheroxyl radicals in the membrane (Guney et al., 2007). Moreover, it was found that a combination of vitamins E and C reduced LPO caused by oxidative stress (Gultekin et al., 2001) and decreased oxidative damage both at biochemical and histological levels (Guney et al., 2007; Ornoy et al., 2009). It was likely that vitamins C and E acted in a synergistic manner, by vitamin E primarily being oxidized to the tocopheroxyl radical and then reduced back to tocopherol by vitamin C (Aydemir et al., 2004). Against the above background, the lipophilic antioxidant vitamin E and the hydrophilic antioxidant vitamin C together were used as antioxidative protection in the rat model for melamine-induced free radical production and lipid peroxidation in the hippocampus. It was reported that vitamins C and E administration provoked a selective action on the hippocampus with mitigating the impairment of oxidative damage (Green et al., 2005, 2006) and ameliorating hippocampal cellular apoptosis (McGoey et al., 2003). The present investigations were designed to examine the neurotoxic effects of melamine and potential rescue by vitamins C and E using a developing rat model. The animal model has been established during early adolescent period (Laviola et al., 2003) since the validity of adolescent rodent was endorsed for the purpose of comparison or extrapolation to the human case (Spear, 2000), which were more involved in the increasing risk of developing neuropathy-related problems (Laviola et al., 1999; Spear, 2000; Spear and Brake, 1983). The dose, duration and route of melamine treatment, which could induce cognitive defects (An et al., 2011, 2013b) and oxidative stress in the hippocampus (An et al., 2012a), were selected according to our
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previous studies (An et al., 2011, 2012a, 2013b) and also the investigations from other labs (Burns, 2007; Jingbin et al., 2010; Kobayashi et al., 2010; Rumbeiha et al., 2010; Stine et al., 2011; Xie et al., 2010). The treatment method of vitamins was referenced from the previous studies (Gultekin et al., 2001; Karaoz et al., 2002), which reported that the dose of vitamins could effectively reduce lipid peroxidation and rescue antioxidant enzymes activities caused by chlorpyrifos-ethyl. It is well known that the hippocampus plays crucial roles in encoding and consolidating memory (Morris et al., 1982; Squire, 1992). Activity-dependent plasticity of hippocampal glutamatergic synapses, particularly long-term potentiation (LTP) and longterm depression (LTD), was proposed as the primary cellular substrate to fulfill these cognitive functions (Bliss and Collingridge, 1993; Martin et al., 2000). Accordingly, our investigation was performed to evaluate deleterious effects of melamine on hippocampus via oxidative damage, manifesting as physical changes, cognitive deficits and synaptic plasticity impairment, after that antioxidants could prevent such damage, using both MWM test and electrophysiological recordings. 2. Materials and methods 2.1. Reagents Melamine (purity > 99.5%) was purchased from Yingda Sparseness & Nobel Reagent Chemical Factory, Tianjin, PR China. Melamine assay kit was purchased from Huaan Magnech BioTech Co., Ltd., Beijing, PR China. Other reagents were of A.R. grade. 2.2. Animals and treatment Healthy male Wistar rats, three-week-old (prenatal day (PND) 21), were obtained from the Laboratory Animal Center, Academy of Military Medical Science of People’s Liberation Army, and reared in the animal house of Medical School in Nankai University. After the adaptation period, animals were randomly assigned into one of four experimental groups, eight rats each as follows. In melamine (MEL-SAL) group, the rats were gavaged with melamine solution (30 mg/mL, dissolved in 1% carboxymethylcellulose (CMC)) at a dose of 300 mg/kg/day for 28 consecutive days (from PND 22 to PND 49) and intraperitoneal injected saline for the next 7 consecutive days (from PND 50 to PND 56). In control (CT-SAL) group, the rats were gavaged with the same dose of 1% CMC for 28 consecutive days and intraperitoneal injected saline for the next 7 consecutive days. In melamine-vitamins (MEL-VIT) group, after 28 consecutive treatment days, each rat received vitamins C and E, dissolved in sterile endotoxin-free isotonic saline, intraperitoneally once a day for 7 consecutive days at 150 and 200 mg/kg, respectively. In pair-fed (CT-VIT) group, the rats were gavaged with the same dose of 1% CMC on 28 consecutive days and then each rat received vitamins C and E saline solution intraperitoneally once a day for 7 consecutive days at 150 and 200 mg/kg, respectively. The rats were individually housed in transparent plastic cages in a temperature controlled (21 8C) colony room under a 12/12 h light/ dark cycle (lights on at 07:00 h) with ad libitum access to food and water. All experiments were conducted during the light phase and performed according to the protocols approved by the Committee for Animal Care at Nankai University and in accordance with the practices outlined in the NIH Guide for the Care and Use of Laboratory Animals. 2.3. Morris water maze experiment On PND 57, rats were examined for spatial cognition performance in the MWM test, using procedures described previously
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with modifications (An et al., 2013a; Han et al., 2014). The water maze consisted of a 1.5-m-diameter circular tank divided into four equal quadrants (I, II, III, and IV), and there was a 10-cm-diameter platform submerged 2 cm below the water surface in the center of quadrant III. The water was made opaque using nontoxic black ink and maintained at 25 1 8C. Movement of rats in the maze was monitored by a computerized video tracking system (Ethovision 2.0, Noldus, Wagenigen, Netherlands) connected to a personal computer, through which data were collected for off-line analyzing. Briefly, the task consisted of three consecutive stages, which were initial training (IT), space exploring test (SET) and reversal training (RT). In IT stage, animals were subjected to four swimming trial per day for 4 consecutive days covering PND 57 to PND 60. In each day, the order of starting points was used pseudorandom and each rat was located in the same position on each trial at one of four starting quadrant points. Animals were allowed to swim freely and trained to find a hidden platform in a circular water tank. The times required to find the platform (escape latency), path length and the swimming speed were recorded. If an animal failed to locate the platform within 60 s, it was placed on it for 10 s, and escape latency was recorded as 60 s. Interval between two trials was approximately 10 min. In SET stage, rats were given the probe trial test at 9 am on PND 61. The platform was removed from the tank. Rats were released individually into water from the starting point of quadrant I and allowed to swim for 60 s as probe test. Quadrant dwell time (the percentage of time spent in the target quadrant) and platform crossings (numbers passing platform area) were recorded. In RT stage, a rat was given four trials on each day during two training days, which was from PND 62 to PND 63. The platform was placed in the center of quadrant I, which was opposite to quadrant III. Rats were trained to find the hidden platform in an opposite position and the reversal learning ability was examined. In RT stage, each rat was given four trials of each training day and tested for 2 days, which was from PND 62 to PND 63. The method used and parameters recorded were the same as those in IT stage. 2.4. In vivo electrophysiological test Electrophysiological tests were performed after MWM assessment. The animals were anesthetized with 30% urethane with a dosage of 4 mL/kg by intraperitoneal injection. And then they were placed in a stereotaxic frame (SN-3, Narishige, Japan) for surgery and the recordings were made as described previously (An et al., 2012b; An and Zhang, 2013). Briefly, at the left side of rat’s head, two small holes were drilled in the skull for the recording and stimulating electrodes inputting respectively. According to The Rat Brain in Stereotaxic Coordinates, the bipolar stimulating electrode was implanted into the hippocampal Schaffer collaterals region (4.2 mm posterior to the bregma, 3.5 mm lateral to midline, 2.5 mm ventral below the dura), and the recording electrode was implanted into hippocampal CA1 region (3.5 mm posterior to the bregma, 2.5 mm lateral to midline, 2.0 mm ventral below the dura). Test stimuli were delivered to the Schaffer collaterals every 30 s at an intensity that evoked a response of 70% of its maximum (range 0.3–0.5 mA). Once the response stabilized, sampling was made under low-frequency stimulations (0.05 Hz) for 20 min as the baseline. After recording baseline, high frequency stimulation (HFS) (8 pulses at 100 Hz for 6 s repeated 30 times) was delivered and then the field excitatory postsynaptic potentials (fEPSPs) were amplified (100), filtered at 5–5 kHz, digitized and collected at 20 kHz sample frequency (Scope software, PowerLab; AD Instruments, Australia) every 60 s for 60 min. Initial data measurement was performed in Clampfit 9.0 (Molecular Devices, Sunnyvale, CA, USA). The fEPSP slope (20–80% level of the falling phase) was used to measure synaptic efficacy. After recording baseline low frequency stimulation (LFS) (900 pulses of 1 Hz for 15 min) was delivered and then the fEPSPs were recorded on the other side of
the hemisphere. The classic hematoxylin–eosin (HE) staining was used to evaluate electrode placements. Only data obtained from rats with correct locations were included in the statistical analysis. 2.5. Biochemical assay in hippocampus After electrophysiological test, the brain of each rat was removed and weighted, and then the hippocampus in the right side of brain was dissected out and rinsed in 0.1 M phosphate buffer (pH 7.4). After being weighed, the hippocampus was homogenized with ice-cold saline to be 10% (w/v) homogenates. The mixtures were homogenized using a glass homogenizer for 5 min and centrifuged at 300 g at 4 8C for 15 min. The supernatant was collected and stored at 70 8C for biochemical assay. The content of melamine in hippocampus was determined following the instructions provided by the manufacturer of the kit. 2.6. Statistical analysis All data were presented as mean S.E.M. A repeated measures ANOVA was applied for analysis of differences between four groups during IT and RT stages followed by Bonferroni post hoc test. One-way measures ANOVA was performed on the data from SET stage, electrophysiological recording, biochemical tests and the weight data followed by Bonferroni post hoc test. Statistical differences were taken when P < 0.05. The analyses were performed using SPSS 16.0 software.
3. Results 3.1. Results of physical changes The effects of melamine and the combination of vitamins C and E treatment on body weight were shown in Fig. 1A. It was found that rats gained weight in all four groups during the 4 weeks of oral gavage treatment with melamine. A one-way ANOVA showed that there were statistical differences of day (F(6,196) = 135.538, P < 0.001), day group interaction (F(18,196) = 23.969, P < 0.001) and group (F(3,196) = 30.066, P < 0.001). Except from PND 22 to PND 26, there was a significant decrease of body weight in MEL-SAL group (PND 43, 50 and 53, P < 0.05; PND 57, P < 0.01) compared to that in MEL-VIT group (PND 43, 50 and 53, P < 0.05; PND 57, P < 0.01) and CT-SAL group, respectively. However, there was no difference of body weight changes either between CT-SAL group and CT-VIT group or between MEL-SAL group and MEL-VIT group. The ratios of brain and hippocampal weights to body weight in each group were presented in Fig. 1B and C, respectively. A oneway ANOVA showed that there was a significant difference of group in hippocampus (F(3,28) = 13.925, P < 0.01). Bonferroni post hoc test confirmed that there was a statistical decrease of hippocampal weight in MEL-SAL group compared to that in CTSAL group (Fig. 1C, P < 0.05). However, there was no difference of hippocampal weight either between CT-SAL group and MEL-VIT group or between MEL-SAL group and MEL-VIT group. In addition, no statistical difference of hippocampal weight was found between CT-SAL and CT-SAL groups. Additionally, the concentration of melamine in the hippocampus of each group was presented in Table 1. A one-way ANOVA showed a statistical difference of group (F(3,12) = 167.690, P < 0.001). Bonferroni post hoc test confirmed that there were abnormal higher levels of melamine in both MEL-SAL (P < 0.001) and MEL-VIT (P < 0.001) groups compared to that in CT-SAL group, while no statistical difference was found either between CT-SAL group and CT-VIT group or between MEL-SAL group and MEL-VIT group.
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Fig. 1. Measurement of physical changes. (A) The changes of rat’s body weight. (B) The ratio of brain weight to body weight on PND 64. (C) The ratio of hippocampal weight to body weight on PND 64. Data are presented as mean S.E.M. *P < 0.05, **P < 0.01: comparison between MEL-SAL group vs. CT-SAL group. %P < 0.05, %%P < 0.01: comparison between MEL-VIT group vs. CT-SAL group. n = 8 per group.
3.2. MWM experiment results Fig. 2 showed the data obtained from the Morris water maze test in four groups on each test day. During IT stage, the average escape latencies were decreased remarkably with training (Fig. 2A). A repeated measures ANOVA showed that there were statistical differences of day (F(3,112) = 71.106, P < 0.001), day group interaction (F(9,112) = 3.352, P < 0.01) and group (F(3,112) = 23.782, P < 0.001), as all four groups did improve over the 4 days of training. Bonferroni post hoc test showed that escape latency was significantly longer in MEL-SAL group than that in CTSAL group (day 2, P < 0.05; day 3, P < 0.01; day 4, P < 0.01), while the escape latency in MEL-VIT group was markedly reduced compared with those in MEL-SAL group (day 3, P < 0.05; day 4, P < 0.01). In addition, a repeated measures ANOVA showed that there were statistical differences of day (F(3,112) = 65.521, P < 0.001), day group interaction (F(9,112) = 3.214, P < 0.01)
Table 1 The concentration of melamine in the hippocampus of each group.. Group CT-SAL Concentration (mg/g)
0.19 0.06
CT-VIT 0.22 0.03
MEL-SAL 23.36 0.55
MEL-VIT aaa
25.71 0.82aaa
The data are represented as the average levels of melamine in rats’ hippocampal homogenate on PND 64. All data are presented as mean S.E.M. aaaP < 0.001 versus CT-SAL group. n = 4 per group.
and group (F(3,112) = 22.005, P < 0.001) in the path length. Bonferroni post hoc test showed that the path length was significantly longer in MEL-SAL group compared to that in CTSAL group (day 2, P < 0.05; day 3, P < 0.01; day 4, P < 0.01), while it was significantly shorter in MEL-VIT group than that in MEL-SAL group (day 3, P < 0.05; day 4, P < 0.01). Similar results were shown on swimming speeds and there was no marked difference of day, day group or group (Fig. 2C). In SET stage, one-way ANOVA revealed statistical differences of group in both platform crossing (F(3,28) = 24.517, P < 0.001) and quadrant dwell time (F(3,28) = 31.628, P < 0.001). Statistical results confirmed that both the platform crossings (Fig. 2D, P < 0.01) and the quadrant dwell time (Fig. 2E, P < 0.01) were reduced in MEL-SAL group compared to that in CT-SAL group. It could be found that both the platform crossings (Fig. 2D, P < 0.05) and the quadrant dwell time (Fig. 2E, P < 0.01) were obviously increased in MEL-VIT group compared to that in MEL-SAL group. In addition, there was no significant difference of them between MEL-VIT group and CT-SAL group. With respect to RT stage, mean escape latency was calculated for each rat on each of two training days. A repeated measures ANOVA confirmed statistical difference of day (F(1,56) = 60.924, P < 0.001), day group (F(3,56) = 3.415, P < 0.05), and group (F(3,56) = 14.423, P < 0.001), as all three groups did improve over the 2 days of reversal training (Fig. 2A). Bonferroni post hoc test showed that there was statistically greater escape latency in MEL-SAL group than that in CTSAL group (day 6, P < 0.01; day 7, P < 0.01). In addition, escape latency was dramatically prolonged in MEL-VIT group compared to
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Fig. 2. Rats’ performance in MWM test. (A) Mean escape latency calculated for each day in IT and RT stages. (B) Mean path length in IT and RT stages. (C) Mean swimming speed in IT and RT stages. (D) Mean number of platform area crossings in SET stage. (E) Mean percentage of time in target quadrant in SET stage. Data are presented as mean S.E.M. *P < 0.05, **P < 0.01: comparison between MEL-SAL group vs. CT-SAL group. #P < 0.05, ##P < 0.01: comparison between MEL-VIT group vs. MEL-SAL group. % P < 0.05, %%P < 0.01: comparison between MEL-VIT group vs. CT-SAL group. n = 8 per group.
that in CT-SAL group (day 6, P < 0.05; day 7, P < 0.01). A repeated measures ANOVA showed that there were statistical differences of day (F(1,56) = 53.739, P < 0.001), day group (F(3,56) = 2.977, P < 0.05), and group (F(3,56) = 13.762, P < 0.001) in the path length. Bonferroni post hoc test showed that the path length was longer in MEL-SAL group than that in CT-SAL group (day 6, P < 0.01; day 7, P < 0.01), so did MEL-VIT group (day 6, P < 0.05; day 7, P < 0.01). There was no significant difference of escape latency between
MEL-SAL group and MEL-VIT group throughout the whole RT stage. Again, a repeated measures ANOVA showed that there was no significant difference of swimming speed in day or group. 3.3. LTP and LTD from Schaffer collaterals to CA1 Both LTP and LTD results in the hippocampal CA1 region were presented in Fig. 3. In the LTP test, stimulation of Schaffer
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Fig. 3. Electrophysiological results from hippocampal Schaffer collateral to CA1 area. (A) Changes in fEPSP slopes after HFS. The fEPSP slope is plotted as a percentage change against the baseline before HFS. Arrow represents application of HFS. (B) Magnitude of LTP is determined as responses between 40 and 60 min after HFS. (C) Changes in fEPSP slopes after LFS. The fEPSP slope is plotted as a percentage change against the baseline before LFS. Two-way arrow represents application of LFS. (D) Magnitude of LTD is determined as responses between 40 and 60 min after LFS. Data are presented as mean S.E.M. **P < 0.01: comparison between MEL-SAL group vs. CT-SAL group. #P < 0.05, ## P < 0.01: comparison between MEL-VIT group vs. MEL-SAL group. %P < 0.05: comparison between MEL-VIT group vs. CT-SAL group. n = 8 per group.
collaterals evoked a basal fEPSP in hippocampal CA1, while HFS induced LTP of the stimulated synapses for at least 1 h. Results representing the time courses of fEPSP slopes normalized to the 20 min baseline period were shown in Fig. 3A. The fEPSP slopes were increased immediately after HFS and stabilized to a level above the baseline period. The magnitude of LTP was dramatically smaller in MEL-SAL group compared to that in CT-SAL group during whole the recordings after HFS. A one-way ANOVA showed that there was a statistical difference of group (F(3,28) = 43.725, P < 0.001). Bonferroni post hoc test showed that the fEPSP slopes were dramatically decayed in MEL-SAL group compared to that in CT-SAL group (Fig. 3B, P < 0.01). Furthermore, there was a statistical difference of the fEPSP slopes between MEL-VIT group and MEL-SAL group, indicating that vitamins treatment did efficiently reversed the melamine-induced LTP impairment (Fig. 3B, P < 0.01). The LTD of stimulated synapses was induced by LFS for at least 1 h and the time course of fEPSP slopes was shown in Fig. 3C. A one-way ANOVA showed that there was statistical difference of group (F(3,28) = 47.965, P < 0.001). The fEPSP slope values were significantly higher in MEL-SAL group than that in CT-SAL group (Fig. 3D, P < 0.01). Administration of vitamin complex was due to the fact that the magnitude of LTD was greater in MEL-VIT group compared to that in MEL-SAL group (Fig. 3D, P < 0.01), while there was a dramatically depressed effect on LTD in MEL-VIT group compared to that in CT-SAL group (Fig. 3D, P < 0.05). In addition, the data of CT-VIT group was indistinguishable to that of CT-SAL group throughout the whole electrophysiological experiments.
4. Discussion A previous study provided an evidence that exposure to melamine induced oxidative damage in the developing brain (An et al., 2012a). The present investigation tested the hypothesis that administration with vitamins C plus E antioxidant compounds could mitigate the deficits of spatial cognition and the impairments in hippocampal synaptic plasticity induced by melamine. In the study, the effects of the combination of vitamins C with E on body weight, brain weight and hippocampal weight, performance in the learning and reversal learning of the MWM tests, and hippocampal synaptic plasticity were determined in melaminetreated and vitamin-treated adolescence rats. Our previous studies reported that there was a trend decreasing in the average weight of brain and a statistical difference in the hippocampal weight after exposure to melamine (An et al., 2011), which was in line with the selective neurotoxicity of melamine in hippocampus (An et al., 2012a). Our data indicated that the two antioxidant vitamins selectively and effectively prevented melamine-induced growth restriction in the adolescent hippocampus. The combination of vitamins C and E might make such a selective action on the hippocampus by potentially mitigating oxidative damage (Green et al., 2006) and affecting the regulation of hippocampal pyramidal cell apoptosis in the neonate (Green et al., 2005; McGoey et al., 2003). Additionally, the abnormal melamine levels that found in melamine-treated rats’ hippocampus indicated that melamine was able to accumulate in brain, which was consistent with our previous findings (An et al., 2011, 2012a).
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Due to impaired spatial cognition as a consequence of hippocampal injury or degeneration, MWM test, which was widely used as an effective behavioral approach for examining cognition in rodents (D’Hooge and De Deyn, 2001), was performed. Melamine-treated rats showed that there were considerably deficits in IT, SET and RT stages of MWM performance compared with that in control group. These data were consistent with our previous studies that melamine caused learning and memory deficits and reversal learning deficit in the MWM task (An et al., 2011, 2013b). Administration of vitamins C plus E attenuated melamine-induced deficit in spatial learning and memory performance in IT and SET stages, but did not totally ameliorate the performance of reversal learning in RT stage. The involvement of the hippocampus in learning and memory was well established. Several studies suggested that the alterations of synaptic plasticity were associated with cognitive behavior (Collingridge et al., 2010; Malenka and Nicoll, 1999). Two major forms of long-lasting synaptic plasticity, LTP and LTD, play a crucial role in the function of synaptic transmission (Bliss and Collingridge, 1993; Collingridge et al., 2010; Morris, 2003). To further investigate the underlying mechanism for the results obtained from the MWM tests, the electrophysiological changes in hippocampus were recorded. It was found that the administration of vitamins C plus E effectively reduced the impairments of both LTP and LTD induced by melamine, which expressed the recuperative effect of vitamins C plus E on melamine-induced cognitive deficit in MWM tasks. The dramatic impairments of synaptic plasticity in melamine-treated rats may be due to the persistent loss of hippocampal CA1 neurons (An et al., 2012a). Administration of vitamins C plus E may provide the structural revival of the hippocampus from melamine-induced injury, as demonstrated by oxidative damage (An et al., 2014). Although the impairments of synaptic plasticity in hippocampus were notably retrieved by a combination of vitamins C and E, rat’s performance in RT stage was not significantly improved. Meanwhile, the impairment of LTD was not entirely diminished, which suggested that LTD played a crucial role in the spatial learning and memory capability, especially reversal learning ability. Recent study showed that lipid peroxidation affected oxidative phosphorylation, maintenance of mitochondrial membrane potential and mitochondrial Ca2+ buffering capacity (Ott et al., 2007). Since hippocampus is one of the critical regions for spatial learning and memory (Eichenbaum, 2004; Geinisman et al., 2004), the occurrence of hippocampal neural apoptosis via affecting oxidation–antioxidation homeostasis provided supportive evidence for the impaired performance of rats’ in behavioral tests. One of our previous studies revealed that the combination of vitamins C plus E effectively reduced the oxidative damage in PC12 cell, with decreasing the levels of free radicals and ameliorating the cellular apoptosis (An et al., 2014). The restoration of hippocampal neuron apoptosis could revive the decreased connection between neurons and improve neuronal transmission (An et al., 2014). As a result, the impairments of synaptic plasticity and cognitive functions were markedly reduced in the present study. In addition, other studies reported that superoxide could serve as a cellular messenger molecule in normal LTP, and the ROS were necessary for LTP expression (Hu et al., 2007). However, with abnormal increased ROS during aging, the activated cellular signaling pathways during normal LTP could be altered by the oxidation of certain critical LTP effector molecules (Kamsler and Segal, 2003; Serrano and Klann, 2004). Nevertheless, a further investigation is still required. Furthermore, there were no statistical differences of the indexes, obtained from either MWM test or synaptic plasticity recordings, between CT-VIT group and CT-SAL group, suggesting that administration of vitamins C plus E did not induce cognitive deficits and hippocampal synaptic transmission impairment. The
result showed the relative safety pharmaceutical effect on attenuating melamine-induced CNS dysfunction. To sum up, the treatment with the complex of vitamins C and E produces positive effects in melamine-induced rats but the mechanism remains to be explored. In conclusion, the administration of vitamins C plus E rescued melamine-induced decrement in the performances of learning and memory and hippocampal long-term synaptic plasticity, and partly attenuated melamine-induced deficits in the reversal learning ability. Meanwhile, the regimen of vitamins C plus E did not produce cognitive deficits. In view of this safety issue, the administration of vitamins C plus E against cognitive deficits induced by melamine is reasonable. Conflict of interest The authors declare there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (31171053, 11232005) and Tianjin Research Program of Application Foundation and Advanced Technology (12JCZDJC22300) and 111 Project (B08011). References An L, Li Z, Yang Z, Zhang T. Cognitive deficits induced by melamine in rats. Toxicol Lett 2011;206:276–80. An L, Li Z, Yang Z, Zhang T. Melamine induced cognitive impairment associated with oxidative damage in rat’s hippocampus. Pharmacol Biochem Behav 2012a;102:196–202. An L, Li Z, Zhang T. Reversible effects of vitamins C and E combination on oxidative stress-induced apoptosis in melamine-treated PC12 cells. Free Radic Res 2014;48:239–50. An L, Liu S, Yang Z, Zhang T. Cognitive impairment in rats induced by nano-CuO and its possible mechanisms. Toxicol Lett 2012b;213:220–7. An L, Yang Z, Zhang T. Imbalanced synaptic plasticity induced spatial cognition impairment in male offspring rats treated with chronic prenatal ethanol exposure. Alcohol Clin Exp Res 2013a;37:763–70. An L, Yang Z, Zhang T. Melamine induced spatial cognitive deficits associated with impairments of hippocampal long-term depression and cholinergic system in Wistar rats. Neurobiol Learn Mem 2013b;100:18–24. An L, Zhang T. Spatial cognition and sexually dimorphic synaptic plasticity balance impairment in rats with chronic prenatal ethanol exposure. Behav Brain Res 2013;256:564–74. Aydemir O, Naziroglu M, Celebi S, Yilmaz T, Kukner AS. Antioxidant effects of alpha-, gamma- and succinate-tocopherols in guinea pig retina during ischemia–reperfusion injury. Pathophysiology 2004;11:167–71. Bliss TV, Collingridge GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 1993;361:31–9. Brown CA, Jeong KS, Poppenga RH, Puschner B, Miller DM, Ellis AE, et al. Outbreaks of renal failure associated with melamine and cyanuric acid in dogs and cats in 2004 and 2007. J Vet Diagn Invest 2007;19:525–31. Burns K. Researchers examine contaminants in food, deaths of pets. J Am Vet Med Assoc 2007;231:1636–8. Collingridge GL, Peineau S, Howland JG, Wang YT. Long-term depression in the CNS. Nat Rev Neurosci 2010;11:459–73. Cremonezzi DC, Diaz MP, Valentich MA, Eynard AR. Neoplastic and preneoplastic lesions induced by melamine in rat urothelium are modulated by dietary polyunsaturated fatty acids. Food Chem Toxicol 2004;42:1999–2007. D’Hooge R, De Deyn PP. Applications of the Morris water maze in the study of learning and memory. Brain Res Rev 2001;36:60–90. Eichenbaum H. Hippocampus: cognitive processes and neural representations that underlie declarative memory. Neuron 2004;44:109–20. Fariss MW, Zhang JG. Vitamin E therapy in Parkinson’s disease. Toxicology 2003;189:129–46. Geinisman Y, Ganeshina O, Yoshida R, Berry RW, Disterhoft JF, Gallagher M. Aging, spatial learning, and total synapse number in the rat CA1 stratum radiatum. Neurobiol Aging 2004;25:407–16.
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