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Chemosphere 151 (2016) 289e295

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Aluminum chloride induces neuroinflammation, loss of neuronaldendritic spine and cognition impairment in developing rat

Zheng Cao a, 1, Xu Yang a, 1, Haiyang Zhang a, Haoran Wang a, Wanyue Huang a, Feibo Xu a,Cuicui Zhuang a, Xiaoguang Wang b, Yanfei Li a, *

a College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, Chinab Suihua Food and Drug Administration, Suihua, 152000, China

h i g h l i g h t s

� AlCl3 caused neuroinflammation in hippocampus.� AlCl3 caused loss of dendritic spine in hippocampus.� Loss of spine may result in cognition impairment of AlCl3-treated rats.

a r t i c l e i n f o

Article history:Received 16 December 2015Received in revised form18 February 2016Accepted 21 February 2016Available online 15 March 2016

Handling Editor: Prof. A. Gies

Keywords:Aluminum chlorideNeuroinflammationDendritic spineCognition impairmentHippocampusRat

* Corresponding author. College of Veterinary MedUniversity, NO. 59 Mucai Street, Xiangfang District, H

E-mail address: liyanfei@neau.edu.cn (Y. Li).1 Both authors are contributed equally to this stud

http://dx.doi.org/10.1016/j.chemosphere.2016.02.0920045-6535/© 2016 Elsevier Ltd. All rights reserved.

a b s t r a c t

Aluminum (Al) is present in the daily life of humans, and the incidence of Al contamination increased inrecent years. Long-term excessive Al intake induces neuroinflammation and cognition impairment.Neuroinflammation alter density of dendritic spine, which, in turn, influence cognition function. How-ever, it is unknown whether increased neuroinflammation is associated with altered density of dendriticspine in Al-treated rats. In the present study, AlCl3 was orally administrated to rat at 50, 150 and 450 mg/kg for 90d. We examined the effects of AlCl3 on the cognition function, density of dendritic spine inhippocampus of CA1 and DG region and the mRNA levels of IL-1b, IL-6, TNF-a, MHC II, CX3CL1 and BNDFin developing rat. These results showed exposure to AlCl3 lead to increased mRNA levels of IL-1b, IL-6,TNF-a and MCH II, decreased mRNA levels of CX3CL1 and BDNF, decreased density of dendritic spineand impaired learning and memory in developing rat. Our results suggest AlCl3 can induce neuro-inflammation that may result in loss of spine, and thereby leads to learning and memory deficits.

© 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Worldwide, over 46 million people live with dementia (Martin,2015). It is estimated that about 75% of these people are affected byAlzheimer's disease (AD) (Morris, 1994). Although the etiologicalfactors of AD are not well understood, many studies suggestAluminum (Al) is a potential contributing factor (Bhattacharjeeet al., 2014; Exley, 2014). Aluminum is an accumulative toxicmetal that causes toxic effects on the brain, bone, liver, spleen(Willhite et al., 2014). Brain is the main target organ for Al

icine, Northeast Agriculturalarbin, 150030, China.

y.

accumulation (Kaneko et al., 2004). Al can penetrate the bloodebrain barrier (Zatta et al., 2002), and accumulate in all brain regions,most being in the hippocampus (Kaur et al., 2006). Epidemiologicalsurveys and animal studies showed that accumulation of Al in thehippocampus causes neurons apoptose, abnormal deposition of b-amyloid and neuroinflammation, resulting in hippocampus-dependent learning and memory ability impairments (Flaten,2001; Kiesswetter et al., 2009; Wang et al., 2014b; Oshima et al.,2013; Zaky et al., 2013). Al was widely used in water purifiers,food additives and pharmaceuticals (Tony et al., 2008; Fung et al.,2009; Anderson and Berkowitz, 2010; Bondy, 2014; Zhu et al.,2014; Gupta, 2014); it is also present in ambient and occupationalairborne particulates (Weinbruch et al., 2010; Boullemant, 2011). Inaddition, the growing prevalence of acid rain and bauxite minesexploitation can result in the discharge of larger amounts of Al salts

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from insoluble minerals, raising the risk of human contact with Al(Smith, 1996; Borgmann, 2007). Therefore, it is necessary todevelop further understanding of the mechanism of Al-inducedneurotoxicity.

Neuroinflammation is associated with pathogenesis of learningand memory deficits (Clark et al., 2010; Ze et al., 2014). The hip-pocampus with abundantly expressed receptors for pro-inflammatory cytokines such as Interleukin-1b (IL-1b),Interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) is espe-cially vulnerable to injury and inflammation (Teeling and Perry,2009). Pro-inflammatory cytokines can impair long-term potenti-ation (LTP), and inhibit neurotrophins which is important forneuronal survival/function, synaptic plasticity and memory for-mation (Poo, 2001; Lynch, 2002; Tyler et al., 2002; Pickering andO'Connor, 2007; Tong et al., 2008; Minichiello, 2009). In addition,exaggerated pro-inflammatory cytokine response in the hippo-campus is associated with altered density of dendritic spine (Bitzer-Quintero and Gonz�alez-Burgos, 2012; Hu et al., 2014; Le et al., 2014;Zou et al., 2015). Changes in dendritic spines govern alterations insynaptic plasticity, which, in turn, influence learning and memory(Segal, 2005). However, it is unknown whether increased pro-inflammatory cytokines is associated with loss of dendritic spinein Al toxicity exposed rats.

In this study we want to determine whether loss of dendriticspine is associated with the neuroinflammation and the cognitionimpairment in AlCl3-treated rats.

2. Methods

2.1. Animals and treatment

One hundred and twenty male Sprague Dawley (SD) rats (SPF,three-week old) were purchased from the Yisi Experimental Ani-mal Technology (Jilin, China). The license number was SCXK-2011-00004. The rats were kept in SPF animal laboratory of NortheastAgricultural University under controlled temperatures at 23 ± 1 �C,relative humidity at 55 ± 5% and in a 12 h light/dark cycle (lights onbetween 08:00 a.m and 20:00 p.m). The rats were kept in indi-vidual ventilated cages (Suhang Technology Equipment, China)with wood shavings (Xietong Organism, China). The size of thecages is 470 mm � 315 mm � 260 mm, and large enough for thegrowth of five rats. The cage was polypropylene PP material andstainless steel wire without aluminum. Throughout the experi-ment, wood shavings were renewed every three days and all ratwere allowed ad libitum access to food (Xietong Organism, China)andwater. Thewater, food andwood shavings were sterilizedwhenthese were used.

After 72 h of acclimatization, the rats (62±5 g) were randomlydivided into the control group and three AlCl3 treatment groups(n ¼ 30 per group). In AlCl3 treatment groups, the rats were treateddaily with AlCl3 (Aladdin, China) at doses of 50 mg/kg, 150 mg/kgand 450 mg/kg for 90d (8:00 a.me10:00 a.m), respectively. Incontrol group, the rats were treated daily with deionized waterinstead of AlCl3. The rats were treated daily with AlCl3 or deionizedwater at a volume of 5 ml/kg by gavage in SPF animal laboratory.Gavage was performed using a syringe with a ball-tipped gastric-feeding needle. The AlCl3-exposure is based on our previous study(Zhu et al., 2013). However, we modified the amount of AlCl3administered according to European Food Safety Authority rec-ommended Al doses for children (26.9e286.8 mg/kg-week) (EFSA,2013) and previous reported values of oral uptake that promotedneurotoxicity (Zhang et al., 2014). To maintain a constant AlCl3intake, we measured the body weight every five days and thenadjusted the dose accordingly. The experiment was carried outaccording to the Guiding Principles in the Use of Animals in

Toxicology, adopted by the Chinese Society of Toxicology. The ani-mal procedures were approved by the Animal Ethics Committee ofthe Northeast Agricultural University (Harbin, China).

2.2. Morris Water Maze

We used Morris Water Maze (MWM) to evaluate learning andmemory of control and AlCl3-exposed rats as described previously(Abdel-Aal et al., 2011). Briefly, the water maze was a black circularpool (160 cm in diameter, 50 cm high) filled with water (30 cm indepth) at 22 ± 1 �C. The pool was set in a moderately lit, circularenclosure made with black curtains. The pool was surrounded bytwo sets of cues. One set consisted of four cues were placed withinthe pool, a blue circle at the west wall, a red square at the southwall, a green triangle at the east wall, and a golden cross at thenorth wall; the other set consisted of two cues were placed exter-nally on the curtains, a white five-pointed star at the south, and awhite crescent at the north. These cues remained unchangedthroughout the testing period. To assess spatial learning the ratswent through an acquisition trial for 1e5 days, followed by 6thd probe trial to assess spatial memory. In the acquisition trial, weplaced a transparent round platform below the water surface ofnortheast quadrant in a circular pool. We then placed the rats(n ¼ 10 per group and saved other 20 per group for other studies)for 30s on this platform. We then placed the rats at a starting pointin the middle of the rim of a quadrant with their face to the wall.Rats swam freely until they reach platform where they stayed for30s. If the rats failed to reach the platform during the 90s, rats weguided the rats to the platform and allowed to remain for 30s. Wetrained the rats for 5 days with three trials per day (8:00 a.me12:00p.m). We kept the trials 10 min apart. We chose the start point ofquadrant (except northeast quadrant) in a quasi-random manner.We used video camera in conjunction with a computerized animaltracking system (Xinruan Information Technology, China) to recordlatency to the platform. We removed the escape platform in theprobe trial and placed the rats gently at the start point of southwestquadrant and allowed to swim freely for 90s. We recorded thenumber of times crossing original platform location and the timethat the rats spent swimming in each quadrant.

2.3. Tissue sample preparation

Because spatial learning increases the number of dendriticspines (Tronel et al., 2010), we chose the rats that were not exposedto the MWM to subsequent study. The rats were deeply anes-thetized by sodium pentobarbital (50 mg/kg, i.p.) (Lockman et al.,2005). The brain (n ¼ 10 per group) was quickly removed forGolgi-Cox staining. The hippocampus (n ¼ 10 per group) wasremoved and stored at �80 �C for qRT-PCR. The body and brainweight (n ¼ 20 per group) were measured. The brain index wascalculated bymultiplying the brainweight (g)/bodyweight (g) with100%.

2.4. Golgi-Cox staining

We processed the fresh brains from each group for Golgi-Coxstaining (FD NeuroTechnologies, USA) according to the manufac-turer's instructions. Briefly, we submerged the fresh brains inmixed solution A and B for 14 days and solution C for 3 days. Afterthat, we cut the brains into 150 mm thick sections (3 sections perbrain) and mounted them on the gelatin-coated slides. We thenstained the sections with solution D and E, dehydrated in gradedethanol, cleared in xylene, and covered with a coverslip. Finally, weused a Nikon E800 bright-field microscope (Nikon, USA) to visu-alize the Golgi-Cox stained sections at a magnification of 1000� for

Z. Cao et al. / Chemosphere 151 (2016) 289e295 291

spine density. We selected five representative neurons per region,per sections for tracing. The average value for each region, in eachsample (n ¼ 10 per group) was obtained and analyzed the neuronsfollowing previously described criteria (Tong et al., 2012; Le et al.,2014).

2.5. Hippocampal Al level

We determined the hippocampal Al level with graphite furnaceatomic absorption spectrophotometry as described previously (Zhuet al., 2013). 0.1 g hippocampus was dried in a dryer (80 �C) for 12 h.We added the dried tissue to a triangle flask, added 25ml nitric acidand perchloric acid mixture (volume ratio is 4:1), mixed everythingand let it sit overnight. Next day we heated the mixture slowly onan electric stove till it became colorless and transparent. Aftercooling it well, we transferred the samples to a 25 ml volumetricflask and added equal volume of 0.5% nitric acid. The Al standardsolution was made by mixing 1 ml Al standard reserve liquid and99 ml deionized water. The flame type was air-acetylene anddetermination line was 309.3 nm. We examined each sample(n ¼ 10 per group) in triplicate and calculated a mean value.

2.6. Quantitative real-time PCR

We detected the mRNA expressions of IL-1b, IL-6, TNF-a, Frac-talkine (CX3CL1), major histocompatibility complex class II (MHCII) and Brain Derived Neurotrophic Factor (BNDF) by quantitativereal-time reverse transcription-polymerase chain reaction asdescribed previously (Cao et al., 2015). Using Trizol reagent (Invi-trogen, USA) according to the manufacturer's instructions. Weextracted total RNA from AlCl3 administered and control rats andthen reversely transcribed each sample into cDNA using a reversetranscription kit (Trans Script First-Strand cDNA Synthesis SuperMix, Trans Gen Blotech, China). We used SYBR Green/Fluoresce inqPCR Master Mix and the 7000 real-time PCR detection system(ABI, USA) to examine gene expression. We used b-actin mRNA asinternal control to adjust the amount of mRNA in each sample. Weexamined each sample (n ¼ 10 per group) in triplicate and calcu-lated a mean value. We presented the data as the relative mRNAlevels. The primer sequences that we used for this study are asSupplementary Table 1.

2.7. Statistical analysis

We present the data as mean ± standard error (SE). We usedSPSS 22.0 software (SPSS Incorporated, USA) to analyze the data.Acquisition trials data were analyzed using Two-way analysis ofvariance (ANOVA) repeated measure to compare the differencesbetween the treatment group and control group. The other datawere analyzed using one-way ANOVA repeated measure withDunnett as the post test to compare the differences between thetreatment group and the control group. We considered p values ofless than 0.05 significant and less than 0.01 as markedly significant.

3. Results

3.1. Effects of AlCl3 on body weight, brain coefficients andhippocampal Al level

To verify the effects of AlCl3 on rats, we measured body weights,brain index and hippocampal Al level. For the body weights andbrain index, therewere no significant differences among the groups(F3,76 ¼ 0.721, p ¼ 0.542; F3,76 ¼ 0.091, p ¼ 0.965, respectively). Forthe hippocampal Al levels, there were significant differencesamong the groups (F3,36 ¼ 106.742, p < 0.001). The hippocampal Al

level of 50 mg/kg group was higher (p < 0.05) than that of controlgroup, and the hippocampal Al level of 150 mg/kg group and450 mg/kg group was significantly higher (p < 0.01) than that ofcontrol group (Table 1).

3.2. AlCl3 induces learning and memory deficits in rats

To assess the effects of AlCl3 toxicity on learning and memory,we subjected the AlCl3 administered rats to the MWM test. Inacquisition trial, Two-way ANOVA repeated measure revealed asignificant effect of AlCl3 (F3,36 ¼ 48.898, p < 0.001), training day(F4,144 ¼ 52.959; p < 0.001) and the interaction between AlCl3 andtraining day (F12,144¼ 2.339; p < 0.01) on the time to reach platform(Fig. 1A). Regarding AlCl3 effect, the time to reach platform of150 mg/kg group and 450 mg/kg group was longer than that ofcontrol group (p < 0.01), but the time to reach platform of 50mg/kggroup was similar to that of control group (p > 0.05). In addition,the time to reach platformwas no differences among the groups onfirst day (F¼ 1.403, p¼ 0.266). From the second day to fifth day, thetime to reach platform were differences among the groups(F ¼ 4.394, p < 0.05; F ¼ 12.614, p < 0.001; F ¼ 28.004, p < 0.001;F ¼ 41.942, p < 0.001, respectively). The multiple comparisonsrevealed the time to reach platform of 150 mg/kg group and450mg/kg groupwas longer than that of control group (for 150mg/kg group, p < 0.01, on the third, fourth and fifth day; for 450 mg/kggroup, p < 0.01, on second, third, fourth and fifth day), but the timeto reach platform of 50 mg/kg group was similar to that of control(p > 0.05, in all five days). These results indicate AlCl3 impair spatiallearning ability.

In probe trial, as shown as Fig. 1B, the time spent in targetquadrant was significant differences among the groups(F3,36 ¼ 6.741, p < 0.001). The time spent in target quadrant of50 mg/kg group was similar to that of control group (p > 0.05), butthe time spent in target quadrant of 150mg/kg group (p < 0.05) and450 mg/kg group (p < 0.01) was longer than that of control group.The time spent in target quadrant was longer (F3,36 ¼ 18.598,p < 0.001; F3,36 ¼ 13.331, p < 0.001; F3,36 ¼ 6.033, p < 0.001,respectively) than other quadrants in control group, 50 mg/kggroup and 150 mg/kg group, but no significant difference in450 mg/kg group (F3,36 ¼ 0.759, p ¼ 0.524). Additionally, thenumber of crossing the platform position was significant differ-ences among the groups (F3,36 ¼ 9.633, p < 0.001). The number ofcrossing the platform position in 50 mg/kg group was similar tothat in control group (p > 0.05), but the number of crossing theplatform position in 150 mg/kg group (p < 0.05) and 450 mg/kggroup (p < 0.01) was less than that in control group (Fig. 1C). Theseresults indicate AlCl3 impair spatial memory ability.

3.3. AlCl3 increases inflammatory cytokines and decreasesimmunomodulatory factors and BNDF

To investigate the effect of AlCl3 toxicity on neuroinflammationin the hippocampus, we examined the mRNA level of pro-inflammatory cytokines (IL-1b, IL-6, TNF-a), marker of microgliaactivation (MHC II), immunomodulatory factors (CX3CL1) andBNDF. For the mRNA levels of IL-1b, IL-6, TNF-a and MHC II, therewere significant differences among the groups (F3,36 ¼ 55.567,p < 0.001; F3,36 ¼ 41.026, p < 0.001; F3,36 ¼ 117.396, p < 0.001;F3,36¼ 49.247, p < 0.001, respectively). The mRNA levels of IL-1b, IL-6, TNF-a and MHC II in 50 mg/kg group was similar to that ofcontrol group, and the mRNA levels of IL-1b, IL-6, TNF-a and MHC IIin 150 mg/kg group and 450 mg/kg group was significantly higherthan that of control group (p < 0.01, p < 0.05). Additionally, for themRNA levels of CX3CL1 and BNDF, there were significant differ-ences among the groups (F3,36 ¼ 17.726, p < 0.001; F3,36 ¼ 25.113,

Table 1Effects of AlCl3 on body weight, brain coefficients and hippocampal Al level.

Control group 50 mg/kg group 150 mg/kg group 450 mg/kg group

Body weight (g) 315.20 ± 3.39 312.35 ± 2.76 309.40 ± 2.91 310.75 ± 2.62Brain coefficients (10�4) 78.02 ± 1.62 77.61 ± 1.43 76.95 ± 1.74 77.18 ± 1.46Hippocampal Al level (mg/g) 3.80 ± 0.15 4.67 ± 0.15* 7.50 ± 0.31** 8.95 ± 0.28**

Values represent mean ± SE. *p < 0.05, **p < 0.01 vs. control value.

Fig. 1. Effects of AlCl3 on learning and memory by MWM test. Acquisition trials: meantime to reach platform (panel A, **p < 0.01 vs. control value in each day. ##p < 0.01 vs.control value.). Probe trials: mean time spent in each quadrant (panel B, *p < 0.05,**p < 0.01 vs. control value. ##p < 0.01 vs. target value in each group.) and the numberof crossed original platform location (panel C, *p < 0.05, **p < 0.01 vs. control value.).Values represent mean ± SE.

Fig. 2. Effects of AlCl3 on hippocampal gene expression by qRT-PCR. Values representmean ± SE. *p < 0.05, **p < 0.01 vs. control value.

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p < 0.001, respectively). The mRNA levels of I CX3CL1 and BNDF in50 mg/kg group was similar to that of control group (p > 0.05), andthe mRNA levels of CX3CL1 and BNDF in 150 mg/kg group and450 mg/kg group was significantly higher than that of control

group (p < 0.01, p < 0.05). These results suggest that AlCl3 inducehippocampal inflammation (Fig. 2).

3.4. AlCl3 induces loss of dendritic spine in the hippocampus CA1and DG region

To investigate the effects of AlCl3 toxicity on dendritic spinedensities in the hippocampus, we processed fresh brains for Golgi-Cox staining. For the dendritic spine densities of CA1 apical and CA1basal, there were significant differences among the groups(F3,36 ¼ 17.242, p < 0.001; F3,36 ¼ 20.875, p < 0.001, respectively).The dendritic spine densities of CA1 apical and CA1 basal in 50 mg/kg group were similar to those in control group (p > 0.05), and thedendritic spine densities of CA1 apical and CA1 basal in 150 mg/kggroup and 450 mg/kg group were less than those in control group(p < 0.01, p < 0.05). For the dendritic spine densities of DG, therewere significant differences among the groups (F3,36 ¼ 6.883,p < 0.001). The dendritic spine densities of DG in 50 mg/kg groupand 150 mg/kg group were similar to those in control group(p > 0.05), and the dendritic spine densities of DG in 450 mg/kggroup was significant less than those in control group (p < 0.01).These results suggest that AlCl3 induce loss of dendritic spine in thehippocampus (Fig. 3).

4. Discussion

Here we report that exposure to AlCl3 for 90 days leads toalteredmRNA levels of IL-1b, IL-6, TNF-a, MCH II, CX3CL1 and BDNF,decreased spine density in CA1 and DG region and impairedlearning and memory in rats. These data are the first to show thatAlCl3 induce s loss of dendritic spine in the CA1 and DG. Loss ofdendritic spine may underlie the deleterious effects of AlCl3 oncognitive function.

During this study, we observed that at the same time point inwhich learning and memory deficits are evident, the mRNA level ofMHC II, TNF-a, IL-1b and IL-6 were elevated in the hippocampus.MHC II expression is a marker for activated microglia (Perry et al.,2007; Lynch, 2009). Microglial cells are the resident pro-inflammatory cells of the central nervous system (CNS). They arenormally quiescent but when activated produce pro-inflammatorycytokines (Bitzer-Quintero and Gonz�alez-Burgos, 2012). Over-production of pro-inflammatory cytokines can negatively affect

Fig. 3. Effects of AlCl3 on spine density by Golgi-Cox staining. A. The neurons of CA1 and DG. B. Showing the dendritic spines (red arrow). C. AlCl3 significantly decreased the spinedensities of CA1 and DG in 150 mg/kg group and 450 mg/kg group. Values represent mean ± SE. *p < 0.05, **p < 0.01 vs. control value. (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of this article.)

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hippocampal function by impairing synaptic plasticity, reducingneurogenesis and apoptosis, result in learning and memory deficits(Lynch and Lynch, 2002; Sheng et al., 2005; Shaftel et al., 2008;Weiet al., 2011; Donzis and Tronson, 2014). IL-1b, IL-6 and TNF-a areamong the most commonly studied pro-inflammation cytokines inthe brain (Capuron and Miller, 2011) and cause the pathogenesisassociated with learning and memory deficits in aging, dementia,epilepsy, surgery and exposure to neurotoxic substance(Annamaria and Tiziana, 2005; Richwine et al., 2008; Fonken et al.,2011; Le et al., 2014). Thus, these results indicate that neuro-inflammation is involved in learning and memory deficits of AlCl3-treated rat. In addition, immunomodulatory factor CX3CL1 isexpressed in the brain and is crucial in down-regulating inflam-mation and microglial activation during aging, stress and neuro-degenerative diseases (Cardona et al., 2006; Wynne et al., 2010;Jurgens and Johnson, 2010; Pabon et al., 2011). BNDF is well-

known for their role in neuron survival and dendritic spine for-mation (Fritzsch et al., 2004; Verpelli et al., 2010; Hiester et al.,2013). It also help maintain microglia in a resting state (Neumannet al., 1998). Down-regulation of CX3CL1 and BDNF further indi-cate neuroinflammation is related to learning and memory deficitsof AlCl3-treated rat.

Al toxicity induces hippocampal dysfunctions, which leads toirreversible learning and memory deficits. However, most of the Alneurotoxicity studies focus on changes in the function of hippo-campal neurons. To date, few studies have investigated Al toxicityaffected structures of the hippocampus, particularly dendriticspines. In this study, our data for the first time show that AlCl3toxicity causes loss of spine density in hippocampal CA1 and DGregion. Dendritic spines of neurons were easily affected by theenvironment (Petralia et al., 2014). Loss of dendritic spines is alsoassociatedwith neuroinflammation disorders and the pathogenesis

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of learning andmemory deficits associated with Alzheimer disease,postoperative cognitive dysfunction, and exposure to neurotoxicsubstance such as lead, alcohol, cocaine (Shen et al., 2009; Spigaet al., 2014; Hu et al., 2014; Le et al., 2014; Zou et al., 2015). Thus,the neuroinflammation that we observed may result in loss ofspine, which in turn leads to learning and memory deficits in AlCl3-treated rat.

Dendritic spines are the locus of the vast majority of excitatoryglutamergic synapses containing N-methyl-D-aspartic acid (NMDA)and a-amino-3-hydroxy-5-methyl-4-isoxazole- propionic acid(AMPA) receptors (Bellot et al., 2014). NMDA and AMPA receptorsaffect synaptic plasticity and conduction between synapses in theCNS (Castellani et al., 2001). Many studies have indicated thatexposure to Al hampered the expression of glutamate receptors andhippocampal function, although the underlying mechanismsremain elusive (Yuan et al., 2011; Song et al., 2014). Our presentstudy provides histological evidence to support the notion that Alexposure reduces the expression of glutamate receptors, inducesabnormal conduction between synapse and then impairs learningand memory. Moreover, LTP is acknowledged to be a well-knownsynaptic model of learning and memory (Malenka, 1994; Adamset al., 2000). LTP induces a significant increase of spine densitymediated by clustering and remodeling of spine F-actin (Colicoset al., 2001; Harvey and Svoboda, 2007). Many studies Al expo-sure attenuating the population spike amplitude of LTP from thehippocampal CA1 region in the rats (Wang et al., 2014a; Zhanget al., 2014). Our current study provides further structural evi-dence for the reduction of LTP.

In conclusion, our results suggest AlCl3 can induce neuro-inflammation that may result in loss of spine, and thereby leads tolearning and memory deficits. In addition, that may underlie thedeleterious effects of AlCl3 on cognitive function and have impli-cations for the novel clinical treatment strategies.

Acknowledgments

This workwas supported by the following grant: Ph.D. ProgramsFoundation of Ministry of Education of China (20132325110001)and Natural Science Foundation of Heilongjiang Province of China(C201136).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.chemosphere.2016.02.092.

References

Abdel-Aal, R.A., Assi, A.A.A., Kostandy, B.B., 2011. Memantine prevents aluminum-induced cognitive deficit in rats. Behav. Brain Res. 225, 31e38.

Adams, J.P., Roberson, E.D., English, J.D., Selcher, J.C., Sweatt, J.D., 2000. MPAKregulation of gene expression in the central nervous system. Acta. Neurobiol.Exp. 60, 377e394.

Anderson, M.A., Berkowitz, J., 2010. Aluminum polymers formed following alumtreatment of lake water. Chemosphere 81, 832e836.

Annamaria, V., Tiziana, G., 2005. Brain inflammation in epilepsy: experimental andclinical evidence. Epilepsia 11, 1724e1743.

Bellot, A., Guivernau, B., Tajes, M., Bosch-Morat�o, M., Valls-Comamala, V.,Mu~noz, F.J., 2014. The structure and function of actin cytoskeleton in matureglutamatergic dendritic spines. Brain Res. 1573, 1e16.

Bhattacharjee, S., Zhao, Y., Hill, J.M., Percy, M.E., Lukiw, W.J., 2014. Aluminum and itspotential contribution to Alzheimer's disease (AD). Front. Aging Neurosci. 6, 62.

Bitzer-Quintero, O.K., Gonz�alez-Burgos, I., 2012. Immune system in the brain: amodulatory role on dendritic spine morphophysiology? Neural. Plast. 26,92e98.

Bondy, S.C., 2014. Prolonged exposure to low levels of aluminum leads to changesassociated with brain aging and neurodegeneration. Toxicology 315, 1e7.

Borgmann, U., Couillard, Y., Grapentine, L.C., 2007. Relative contribution of food andwater to 27 metals and metalloids accumulated by caged hyalella azteca in tworivers affected by metal mining. Environ. Pollut. 145, 753e765.

Boullemant, A., 2011. PM2.5 emissions from aluminum smelters: coefficients andenvironmental impact. J. Air. Waste Manag. Assoc. 61, 311e318.

Cao, Z., Fu, Y., Sun, X., Zhang, Q., Xu, F., Li, Y., 2015. Aluminum trichloride inhibitsosteoblastic differentiation through inactivation of Wnt/b-catenin signalingpathway in rat osteoblasts. Environ. Toxicol. Pharmacol. http://dx.doi.org/10.1016/j.etap.2015.11.023.

Capuron, L., Miller, A.H., 2011. Immune system to brain signaling: neuro-psychopharmacological implications. Pharmacol. Ther. 130, 226e238.

Cardona, A.E., Pioro, E.P., Sasse, M.E., Kostenko, V., Cardona, S.M., Dijkstra, I.M.,Huang, D., Kidd, G., Dombrowski, S., Dutta, R., Lee, J.C., Cook, D.N., Jung, S.,Lira, S.A., Littman, D.R., Ransohoff, R.M., 2006. Control of microglial neurotox-icity by the fractalkine receptor. Nat. Neurosci. 9, 917e924.

Castellani, G.C., Quinlan, E.M., Cooper, L.N., Shouval, H.Z., 2001. A biophysical modelof bidirectional synaptic plasticity: dependence on ampa and nmda receptors.Proc. Natl. Acad. Sci. U. S. A. 98, 12772e12777.

Clark, I.A., Alleva, L.M., Vissel, B., 2010. The roles of TNF in brain dysfunction anddisease. Pharmacol. Ther. 128, 519e548.

Colicos, M.A., Collins, B.E., Sailor, M.J., Goda, Y., 2001. Remodeling of synaptic actininduced by photoconductive stimulation. Cell 107, 605e616.

Donzis, E.J., Tronson, N.C., 2014. Modulation of learning and memory by cytokines:signaling mechanisms and long term consequences. Neurobiol. Learn. Mem.115, 68e77.

European Food Safety Authority (EFSA), 2013. Dietary Exposure to Aluminium-containing Food Additives. EFSA viewed 15 Dec 2015. www.efsa.europa.eu/publications.

Exley, C., 2014. What is the risk of aluminium as a neurotoxin? Expert. Rev. Neu-rother. 14, 589e591.

Flaten, T.P., 2001. Aluminium as a risk factor in Alzheimer's disease, with emphasison drinking water. Brain Res. Bull. 55, 187e196.

Fonken, L.K., Xu, X., Weil, Z.M., Chen, G., Sun, Q., Rajagopalan, S., Nelson, R.J., 2011.Air pollution impairs cognition, provokes depressive-like behaviors and altershippocampal cytokine expression and morphology. Mol. Psychiatr. 16,987e995.

Fritzsch, B., Tessarollo, L., Coppola, E., Reichardt, L.F., 2004. Neurotrophins in the ear:their roles in sensory neuron survival and fiber guidance. Prog. Brain Res. 146,265e278.

Fung, K.F., Carr, H.P., Poon, B.H.T., Wong, M.H., 2009. A comparison of aluminumlevels in tea products from Hong Kong markets and in varieties of tea plantsfrom Hong Kong and India. Chemosphere 75, 955e962.

Gupta, A., 2014. Ferric citrate hydrate as a phosphate binder and risk of aluminumtoxicity. Pharmaceuticals 7, 990e998.

Harvey, C.D., Svoboda, K., 2007. Locally dynamic synaptic learning rules in pyra-midal neuron dendrites. Nature 450, 1195e1200.

Hiester, B.G., Galati, D.F., Salinas, P.C., Jones, K.R., 2013. Neurotrophin and wntsignaling cooperatively regulate dendritic spine formation. Mol. Cell. Neurosci.56, 115e127.

Hu, F., Xu, L., Liu, Z.H., Ge, M.M., Ruan, D.Y., Wang, H.L., 2014. Developmental leadexposure alters synaptogenesis through inhibiting canonical wnt pathwayin vivo and in vitro. PLoS One 9, e101894.

Jurgens, H.A., Johnson, R.W., 2010. Dysregulated neuronal-microglial cross-talkduring aging, stress and inflammation. Exp. Neurol. 233, 40e48.

Kaneko, N., Yasui, H., Takada, J., Suzuki, K., Sakurai, H., 2004. Orally administratedaluminum-maltolate complex enhances oxidative stress in the organs of mice.J. Inorg. Biochem. 98, 2022e2031.

Kaur, A., Joshi, K., Minz, R.W., Gill, K.D., 2006. Neurofilament phosphorylation anddisruption: a possible mechanism of chronic aluminium toxicity in Wistar rats.Toxicology 219, 1e10.

Kiesswetter, E., Sch€aper, M., Buchta, M., Schaller, K.H., Rossbach, B., Kraus, T.,Letzel, S., 2009. Longitudinal study on potential neurotoxic effects of aluminum:II. Assessment of exposure and neurobehavioral performance of Al welders inthe automobile industry over, 4 years. Int. Arch. Occup. Environ. Health 82,1191e1210.

Le, Y., Liu, S., Peng, M., Tan, C., Liao, Q., Duan, K., Ouyang, W., Tong, J., 2014. Agingdifferentially affects the loss of neuronal dendritic spine, neuroinflammationand memory impairment at rats after surgery. PLoS One 9, e106837.

Lockman, P.R., Van, D.S.C.J., Abbruscato, T.J., Allen, D.D., 2005. Chronic nicotineexposure alters blood-brain barrier permeability and diminishes brain uptakeof methyllycaconitine. J. Neurochem. 94, 37e44.

Lynch, M.A., 2002. Interleukin-1 beta exerts a myriad of effects in the brain and inparticular in the hippocampus: analysis of some of these actions. Vitam. Horm.64, 185e219.

Lynch, M.A., 2009. The multifaceted profile of activated microglia. Mol. Neurobiol.40, 139e156.

Lynch, A.M., Lynch, M.A., 2002. The age-related increase in IL-1 type I receptor in rathippocampus is coupled with an increase in caspase-3 activation. Eur. J. Neu-rosci. 15, 1779e1788.

Malenka, R.C., 1994. Synaptic plasticity in the hippocampus: LTP and LTD. Cell 78,535e538.

Martin, P., 2015. World Alzheimer Report 2015: The Global Impact of Dementia.Alzheimer's Disease International viewed 14 Dec 2015. http://apo.org.au/node/56882.

Minichiello, L., 2009. TrkB signalling pathways in LTP and learning. Nat. Rev. Neu-rosci. 2009, 850e860.

Morris, J.C., 1994. Differential diagnosis of Alzheimer's disease. Clin. Geriatr. Med.10, 257e276.

Z. Cao et al. / Chemosphere 151 (2016) 289e295 295

Neumann, H., Misgeld, T., Matsumuro, K., Wekerle, H., 1998. Neurotrophins inhibitmajor histocompatibility class II inducibility of microglia: involvement of thep75 neurotrophin receptor. Proc. Natl. Acad. Sci. U. S. A. 95, 5779e5784.

Oshima, E., Ishihara, T., Yokota, O., Nakashima-Yasuda, H., Nagao, S., Ikeda, C.,Naohara, J., Terada, S., Uchitomi, Y., 2013. Accelerated tau aggregation, apoptosisand neurological dysfunction caused by chronic oral administration ofaluminum in a mouse model of tauopathies. Brain Pathol. 23, 633e644.

Pabon, M.M., Bachstetter, A.D., Hudson, C.E., Gemma, C., Bickford, P.C., 2011. Cx3cl1reduces neurotoxicity and microglial activation in a rat model of parkinson'sdisease. J. Neuroinflamm. 8, 9.

Perry, V.H., Cunningham, C., Holmes, C., 2007. Systemic infections and inflammationaffect chronic neurodegeneration. Nat. Rev. Immunol. 7, 161e167.

Petralia, R.S., Mattson, M.P., Yao, P.J., 2014. Communication breakdown: the impactof ageing on synapse structure. Ageing Res. Rev. 14, 31e42.

Pickering, M., O'Connor, J.J., 2007. Pro-inflammatory cytokines and their effects inthe dentate gyrus. Prog. Brain Res. 163, 339e354.

Poo, M.M., 2001. Neurotrophins as synaptic modulators. Nat. Rev. Neurosci. 2,24e32.

Richwine, A.F., Parkin, A.O., Buchanan, J.B., Chen, J., Markham, J.A., Juraska, J.M.,Johnson, R.W., 2008. Architectural changes to CA1 pyramidal neurons in adultand aged mice after peripheral immune stimulation. Psychoneuroendocrinol-ogy 33, 1369e1377.

Segal, M., 2005. Dendritic spines and long-term plasticity. Nat. Rev. Neurosci. 6,277e284.

Shaftel, S.S., Griffin, W.S., O'Banion, M.K., 2008. The role of interleukin-1 in neu-roinflammation and Alzheimer disease: an evolving perspective.J. Neuroinflamm. 5, 7.

Shen, H.W., Toda, S., Moussawi, K., Bouknight, A., Zahm, D.S., Kalivas, P.W., 2009.Altered dendritic spine plasticity in cocaine-withdrawn rats. J. Neurosci. 29,2876e2884.

Sheng, W.S., Hu, S., Ni, H.T., Rowen, T.N., Lokensgard, J.R., Peterson, P.K., 2005. TNF-alpha-induced chemokine production and apoptosis in human neural precursorcells. J. Leukoc. Biol. 78, 1233e1241.

Smith, R.W., 1996. Kinetic aspects of aqueous aluminum chemistry: environmentalimplications. Coord. Chem. Rev. 149, 81e93.

Song, J., Liu, Y., Zhang, H.F., Zhang, Q.L., Niu, Q., 2014. Effects of exposure toaluminum on long-term potentiation and ampa receptor subunits in ratsin vivo. Biomed. Environ. Sci. 27, 77e84.

Spiga, S., Talani, G., Mulas, G., Licheri, V., Fois, G.R., Muggironi, G., Masala, N.,Cannizzaro, C., Biggio, G., Sanna, E., Diana, M., 2014. Hampered long-termdepression and thin spine loss in the nucleus accumbens of ethanol-dependent rats. Proc. Natl. Acad. Sci. U. S. A. 111, 3745e3754.

Teeling, J.L., Perry, V.H., 2009. Systemic infection and inflammation in acute CNSinjury and chronic neurodegeneration: underlying mechanisms. Neuroscience158, 1062e1073.

Tong, L., Balazs, R., Soiampornkul, R., Thangnipon, W., Cotman, C.W., 2008. Inter-leukin-1 beta impairs brain derived neurotrophic factor-induced signal trans-duction. Neurobiol. Aging 29, 1380e1393.

Tong, J., Huang, C., Bi, F., Wu, Q., Huang, B., Zhou, H., 2012. XBP1 depletion precedesubiquitin aggregation and Golgi fragmentation in TDP-43 transgenic rats.J. Neurochem. 123, 406e416.

Tony, M.A., Zhao, Y.Q., Fu, J.F., Tayeb, A.M., 2008. Conditioning of aluminium-basedwater treatment sludge with Fenton's reagent: effectiveness and optimisingstudy to improve dewaterability. Chemosphere 72, 673e677.

Tronel, S., Fabre, A., Charrier, V., Oliet, S.H., Gage, F.H., Abrous, D.N., 2010. Spatiallearning sculpts the dendritic arbor of adult-born hippocampal neurons. Proc.Natl. Acad. Sci. U. S. A. 107, 7963e7968.

Tyler, W.J., Alonso, M., Bramham, C.R., Pozzo-Miller, L.D., 2002. From acquisition toconsolidation: on the role of brain-derived neurotrophic factor signaling inhippocampal-dependent learning. Learn. Mem. 9, 224e237.

Verpelli, C., Piccoli, G., Zanchi, A., Gardoni, F., Huang, K., Brambilla, D., Di Luca, M.,Battaglioli, E., Sala, C., 2010. Synaptic activity controls dendritic spinemorphology by modulating eEF2-dependent BDNF synthesis. J. Neurosci. 30,5830e5842.

Wang, B., Zhao, J., Yu, M., Meng, X., Cui, X., Zhao, Y., Xing, W., Guan, Y., 2014a.Disturbance of intracellular calcium homeostasis and CaMKII/CREB signaling isassociated with learning and memory impairments induced by chronicaluminum exposure. Neurotox. Res. 26, 52e63.

Wang, L., Hu, J., Zhao, Y., Lu, X., Zhang, Q., Niu, Q., 2014b. Effects of aluminium on b-amyloid (1-42) and secretases (APP-cleaving enzymes) in rat brain. Neurochem.Res. 39, 1338e1345.

Wei, H., Zou, H., Sheikh, A.M., Malik, M., Dobkin, C., Brown, W.T., Li, X., 2011. IL-6 isincreased in the cerebellum of autistic brain and alters neural cell adhesion,migration and synaptic formation. J. Neuroinflamm. 8, 1e10.

Weinbruch, S., Benker, N., Koch, W., Ebert, M., Drabløs, P.A., Skaugset, N.P.,Ellingsen, D.G., Thomassen, Y., 2010. Hygroscopic properties of the workroomaerosol in aluminium smelter potrooms: a case for transport of HF and SO2 intothe lower airways. J. Environ. Monit. 12, 448e454.

Willhite, C.C., Karyakina, N.A., Yokel, R.A., Yenugadhati, N., Wisniewski, T.M.,Arnold, I.M., Momoli, F., Krewski, D., 2014. Systematic review of potential healthrisks posed by pharmaceutical, occupational and consumer exposures tometallic and nanoscale aluminum, aluminum oxides, aluminum hydroxide andits soluble salts. Crit. Rev. Toxicol. 44 (Suppl. l), 1e80.

Wynne, A.M., Henry, C.J., Yan, H., Cleland, A., Godbout, J.P., 2010. Protracteddownregulation of CX3CR1 on microglia of aged mice after lipopolysaccharidechallenge. Brain Behav. Immun. 24, 1190e1201.

Yuan, C.Y., Hsu, G.S., Lee, Y.J., 2011. Aluminum alters NMDA receptor 1A and 2A/Bexpression on neonatal hippocampal neurons in rats. J. Biomed. Sci. 18, 1e9.

Zaky, A., Mohammad, B., Moftah, M., Kandeel, K.M., Bassiouny, A.R., 2013. Apurinic/apyrimidinic endonuclease 1 is a key modulator of aluminum-induced neuro-inflammation. BMC Neurosci. 14, 1e12.

Zatta, P., Kilyen, M., Kiss, T., 2002. In vivo and in vitro effects of aluminum on theactivity of mouse brain acetylcholinesterase. Brain Res. Bull. 59, 41e45.

Ze, Y., Sheng, L., Zhao, X., Hong, J., Ze, X., Yu, X., Pan, X., Lin, A., Zhao, Y., Zhang, C.,Zhou, Q., Wang, L., Hong, F., 2014. TiO2 nanoparticles induced hippocampalneuroinflammation in mice. PLoS One 9, e92230.

Zhang, L., Jin, C., Lu, X., Yang, J., Wu, S., Liu, Q., Chen, R., Bai, C., Zhang, D., Zheng, L.,Du, Y., Cai, Y., 2014. Aluminium chloride impairs long-term memory anddownregulates cAMP-PKA-CREB signalling in rats. Toxicology 323, 95e108.

Zhu, Y., Han, Y., Zhao, H., Li, J., Hu, C., Li, Y., Zhang, Z., 2013. Suppressive effect ofaccumulated aluminum trichloride on the hepatic microsomal cytochromep450 enzyme system in rats. Food Chem. Toxicol. 51, 210e214.

Zhu, Y., Li, Y., Miao, L., Wang, Y., Liu, Y., Yan, X., Cui, X., Li, H., 2014. Immunotoxicity ofaluminum. Chemosphere 104, 1e6.

Zou, C., Montagna, E., Shi, Y., Peters, F., Blazquez-Llorca, L., Shi, S., Filser, S.,Dorostkar, M.M., Herms, J., 2015. Intraneuronal app and extracellular ab inde-pendently cause dendritic spine pathology in transgenic mouse models ofalzheimer's disease. Acta Neuropathol. 129, 1e12.