Neurotoxiclty Research. 2007, VOL. 12(3). pp. 163-169

F.P. Graham Publishing Co.

The Relationship Between Bcl-2 Gene Expressionand Learning & Memory Impairmentin Chronic Aluminum-exposed RatsQIAO NIU"-*, VANXU YANG', QINLI ZHANG'. PIYE NIU', SHUCHANG HE', MARIO DI GIOACCHINO^PIO CONTI^ atid PAOLO BOSCOLO''

"Public Health School of Shanxi Medical University, Taiyuan 030001. P.R. China; School of Medicine, UniversityofChieti, 66100 Chieti, Italy, pconti@unich.it

(Submitted 24 May 2007; Revised 30 July 2007; Jn final form 03 August 2007)

Aluminum (Al), a known neurotoxin, has beenimplicated in Alzheimer's Disease (AD),Amyotrophic Lateral Sclerosis (ALS),Parkinsooism Dementia Complex, etc., and itcauses extensive damage to the nervous system,including the impairment of learning and mem-ory. However, to date, the mechanism of Al neu-rotoxicity has not been fully elucidated. Neuronalapoptosis has become a focus of interest, as it hasbeen reported to play a key role in the impair-ment of learning and memory processes(Thompson, Science 267:1456, 1995). The Bcl-2gene acts as an important effector for inhibitingapoptosis. In the present study we observe neu-ronal apoptosis in association with learning andmemory impairment, as well as regional brainalterations in BcI-2 expression in rats chronicallyexposed to Al. The chronic Al-intoxicated modelwas established by i.p. injection of AICI3 in adultSprague Dawley rats for 3 successive days, withone-day intervals, for 60 days. After exposure,the step-down test was performed to examinethe behavioral reaction of the rats. Neuronalapoptosis and Bcl-2 protein expression in differ-ent regions of rat brain were then assessed by animmunohistochemical method. In the step-downtest, the latency of Al-exposed rats was signifi-cantly lower than that of controls. Also, the num-

ber of performance errors in 5 minutes of expo-sure was significantly higher than that of con-trols. Neuronal apoptosis was extensive in thebrain of Al-exposed groups, and the expressionsof Bcl-2 protein in frontal cortex, cerebellumand hippocampus of Al-exposed rats was stron-ger. In conclusion, chronic Al-exposure in rats isassociated with neuronal apoptosis in brain, andimpaired learning and memory. AugmentedBcl-2 protein expression may be a stimulatedcompensatory mechanism.

Keywords: Aluminum; Neuron apoptosis; Bcl-2;Learning and memory

INTRODUCTION

Aluminum (Al) is the third most common, and oneof the most widely distributed metallic elements inthe earth's crust (Balkissoon, 2006; Alvarez-Ayusoet ai, 2007). Although most Al exists as insolublealuminosilicates and Al oxides, it presents anopportunity for human exposure and provides ubiq-uitous contamination (He et al, 2003). At present itis a known neurotoxin. Many reports implicate Alwith Alzheimer's Disease (AD), AmyotrophicLateral Sclerosis (ALS), Parkinsonism Dementia

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ISSN 1029 8428 print; ISSN 1476-3524 online. © 2007 FP Graham Publishing Co., www.NeurotoxicityResearch.com

164 Q. NIU el al.

Complex, etc. (Choucair et al., 2006; Jiang et ai,2006; Staibano et ai, 2006). Al is capable of pro-ducing much damage to the nervous system, includ-ing the impairment of learning and memory (Niu etai, 2004). However, the exact mechanism has notyet been elucidated. Neuronal apoptosis has nowbecome a focus of interest because it has beenreported as performing a key role in the impairmentof learning and the memory processes (Thompsonand Sawtell, 2000), and is a key factor in neurode-generative disorders (Ekshyyan and Aw, 2004;Cantalupi et ai, 2006; Lanzilli et ai, 2006; LoMuzio et ai, 2006; Pugliesi et ai, 2006).Since Siem first reported the neurotoxicity of AI in

1886, studies have been focused on its mechanism.Learning and memory deficits have become a hall-mark of serious damage to central nervous systemfunction (Ballerini et ai, 2006; Di Lorenzo andPlacido, 2006; Di Pietro et ai, 2006; Marcucci etai, 2006; Zhu et ai, 2006). We performed neurobe-havioral tests on AJ-exposed workers to discovertheir learning and memory impairments (He et ai,2003), and we also observed the behavioral deficitin Al-exposed rats (Niu et ai, 2004). However, theexact mechanism is still not yet clear. Many studiesshow that AD is closely related to neuronal apopto-sis (Li and Gu, 1998). Currently the Bcl-2 gene isconsidered as the most important suppressor geneof apoptosis (Li et ai, 2005), and the Bcl-2 gene isreportedly connected with learning and memoryability in the CNS (Phelps, 1990; Linnik et ai,1995; Wei and Zhang, 1999; Wei et ai, 2000),therefore study of the relationship between learningand memory deficits, neuron apoptosis, and varia-tions of the expression of the Bcl-2 protein in dif-ferent regions of the rat brain exposed to aluminummay be important for the exploration of the cellularand molecular mechanisms of Al-induced learningand memory deficits.

MATERIALS AND METHODS

SubjectsAduh male Spraque-Dawley rats (n=40) wereobtained from the experimental animal center inShanxi Medical University. All rats were housed ina clean, quiet room maintained at 18*'-23''C withrelative humidity in a range of 40%-60%.

Aluminum AdministrationThe 40 rats in this study were divided into fourgroups matched by body weight after one week'sfeeding. Rats in three of the groups were adminis-tered 0.2 ml aluminum chloride (AICI3; AP,Shanghai, China) in distilled water, at a dose of2.0, 4.0 and 8.0 mg Al/kg-bw, respectively, for 60days by i.p. injection. For better absorption, theinjections were made on 3 successive days withone-day intervals. The fourth group of animals wasdesignated as control, and received 0.2 ml saline inthe same way and at the same time as theAl-administered rats.

Assessment of Learning and MemoryA passive avoidance test (step-down test) was per-formed to assess the behavioral reaction of rats. Theapparatus consisted of five boxes equipped withcopper bars at the bottom and one plastic platform.Before the formal test, rats were trained.

1. Training Process:Each rat was individually placed on the copper barsin every box and 3-min later the copper bars wereelectrified. Electricity-shocks rats jumped upon theplastic platfonn to avoid the electric current. Therats then descended again to the copper bar aftersome minutes, and quickly jumped onto the plasticplatfonn again after being shocked again. All therats were trained by this process for 5-min.

2. Formal Testing:After 24 h, rats were individually placed on theplastic platforms of the boxes while the copper barswere again electrified. The interval between place-ment on the platform and step-down onto the gridwas recorded as "Latency". The number of step-downs onto the grid in a 5-min session was record-ed as "number of errors". "Latency" and "No. oferrors in 5 min" were utilized as indicators of learn-ing and memory.

Tissue Sampling and ImmunohistochemicalExamination for Neuronal Cell Apoptosis andBcl-2 Protein ExpressionFollowing completion of the learning and memorytasks, controls and Al-exposed rats were anesthe-tized with sodium pentobarbital (40 mg/kg, i.p.)and perfused intra-aortically with 200 ml saline,

APOPTOSIS & Bft'2 IN AL-INDUCED LEARNING DAMAGE 165

followed by 500 ml of an ice-cold 4% paraformal-dehyde in 0.1 M phosphate buffer (PB). After per-fusion, the brains were quickly extracted and theright cerebral hemisphere of each rat was immobi-lized in 10% formaldehyde solution for 72 h. Next,tissues were processed and embedded in paraffmblocks by routine procedures. Each paraffm speci-men was cut into 9 transverse sections of 4 jimthickness. One section was stained by the hematox-ylin-eosin method. Remaining sections were pro-cessed for inununohistochemistry, with 4 sectionsfor observation of neuronal cell apoptosis, and theother 4 sections for observation of Bcl-2 proteinexpression. The hippocampus CA3 area wasobserved, using visually recognizable boundaries,according to Li et ai (2007) (FIG. 1).

Immunohistochemical examination was conductedusing the following procedure. Tissue sections werede-paraffmized with xylene and re-hydrated withdegraded ethanoU then incubated in hydrogen per-oxide block for 10 min at room temperature.Subsequently, sections were heated in a microwaveoven with target retrieval solution. After washingwith a solution of 0.01 mol/1 PBS, tissue sectionswere incubated in Ultra V Block for 10 min at roomtemperature, to block non-specific backgroundstaining. Sections were then incubated with apopto-sis monoclonal antibody or BcI-2 monoclonal anti-body, separately, at 4''C overnight. Two types ofmonoclonal antibodies were separately used as aprimary antibody (Santa Cruz Co.) 1:50 dilutionwith 0.01 mol/1 phosphate-buffered saline (PBS).

Sections were rinsed in a buffer solution and incu-bated with biotinylated goat anti-rabbit IgG (SantaCruz Co., USA) for 30 min at room temperature.After washing with buffer solution, tissue sectionswere incubated with avidin-biotin-peroxidase com-plex (ABC) (Santa Cruz Co.) for 30 min at roomtemperature. Finally, sections were washed withbuffer solution, and the peroxidase reaction was

developed with diaminobenzidine (DAB) (SantaCruz Co.). Nuclei were counter-stained with hema-toxylin. After the immunostaining procedure, tis-sue sections were dehydrated and coverslippedwith DPX.

Sections processed for immunohistochemistrywere observed by optical microscopy, and con-trasted with HE-stained sections. The number ofpositively-stained neurons in each region (cerebralcortex, hippocampus and cerebellum) of rat brainwas counted with higher clarity optical microscopy.In brief, the number of positively stained neurons in10 visual fields of each section under higher mag-nification optical microscopy were averaged. Then,the mean number of apoptotic cells or Bcl-2-postivecells was determined. Apoptotic neuronal cell num-ber was determined only in the hippocampus, whilenumbers of Bcl-2 positive cells were determined inthe three brain regions under study.

Data AnalysisLearning and memory test scores were analyzed bythe Kruskal-Watlis non-parametric analysis of vari-ance. One-way analysis of variance (ANOVA) wasperformed on immunohistochemical data. P <0.05was considered to be statistically significant.

RESULTS

Assessment of Learning and MemoryIn the step-down task, "Latency" of Al-exposed ratswas shorter {P <0.05) and "No. of errors in 5 min"was more frequent {P <0.01), particularly in mid-dose and high-dose groups (Table I). These findingsindicate that there was a reduction in learning andmemory in Al-treated rats.

Determination of Neuronal Apoptosisand Bcl-2 Protein ExpressionUnder microscopic observation, brown stained

Table I Effect of i.p. aluminum on the passive avoidance condition response in ratsGroup Dose N Latency No. of errors in 5 min

(mg Al/kg) [s)ABCD

0.02.04.08.0

10101010

162.3 ± 145.826-0 ± 35.5*18.0 ± 16.3'8.3 ± 4.0*

0.10 + 0.320.50 ± 0.711.43 ± 0.98'3.00 ± 0.82'

*P <0.05, **P <0.01, V.S control group (A).

166 Q. NILU/a/.

CA2 CAt

D CA3

CA4

FIGURE 1 Area location in hippocampus.

FIGURE 2 Cellular apoptosis in CA3 areaof hippocampus of control rats. 200X

FIGURE 4 Bcl-2 protein in pyramidal cellsin CA3 area of hippocampus of control rats. 200X

FIGURE 5 Bcl-2 protein in pyramidal cellsin CA3 area of hippocampus of rats treated with A13*

at the dosage of 8.0 mg AP^/kg bw. 200X

FIGURE 3 Cellular apoptosis in CA3 areaof hippocampus of A13+, 8 mg/kg group. 200X

APOPTOSIS & Bcl-2 IN AL-INDUCED LEARNING DAMAGE 167

Table II Number of apoptotic cells in hippocampus CA3 area of Al-treated rats

Group

ABCD

Dose(mg Al/kg)0.02.04.08.0

N

10101010

No. of apoptotic cells

14.2±3.1016.5±4.5128.5±3.32*39.8±7.76**

"P <0.05, **P O.Ol, vs control group (A).

Table n i Number of Bcl-2 protein positive cells in different regions of rat brains.Group Dose N No. of Bcl-2 protein positive cells

(mg Al/Kg) Frontal cortex CA3 area of Cerebellumhippocampus

ABCD

ao2.04.08.0

10101010

2.15+ 1.5614.80± 6.09*19.80+ 7.06-22.30 + 10.48*

1.50+1.892.70+4.08*3.80 + 3.32"8. 55±6. 3 r

0. 75+0. 795.35+3.28*6.40±2. 59*9.02+4.61*

*PO.05, **P <0.01, compared with control group (A).

small bodies in nuclei represent cellular apoptosis.The cytoplasm of some neurons also appearedbrown and these are the expression of Bcl-2 proteinpositive neurons. Generally, in Al-exposed ratsthere was an increase in numbers of apoptotic cellsand numbers of Bcl-2-positive cells. As shown inFIG. 1, numbers of apoptotic cells in the hippocam-pus of Al-exposed rats was increased versus con-trols (Table H; FIGs. 2 & 3). Also, the number ofBcl-2-protein positive cerebellar Purkinje cells,hippocampal pyramidal cells and neiu'ons in cere-bral cortex was increased in Al-treated rats (TableIII, FIGs. 4 & 5).

DISCUSSION

It is well-known that Bcl-2 protein plays an impor-tant role in suppressing neuronal apoptosis(Hockenbery et ai, 1990; Boise et ai, 1993; Zhonget ai, 1993; Casteliani et ai, 2006a,b; Greco et ai,2006; Marino et ai, 2006; Midulla et ai, 2006).Bcl-2 over-expression inhibits neuronal apoptosisinduced by diverse causes, including ischemia,anoxia and hyperthermia (Capoluongo et ai, 2006;Passam et ai, 2006). Also, Bcl-2 preserves learningand memory by preventing neuronal injury. Undernormal conditions, Bcl-2 regelates cellular apopto-

sis, in opposition to the pro-apototic cellular proteinBax (Cadoni et ai, 2007; Deepak et ai, 2007),thereby preserving cellular function (Lanzilli et ai,2006; Lazarova et ai, 2006; Staibano et ai, 2006).Over-expression of Bcl-2 is associated with preser-vation of learning and memory, as well. However,when Bcl-2 is over-expressed, the regulation of cellapoptosis may change. Takao et ai (1995) foundthat the level of Bcl-2 protein is increased in thehippocampus and cortex of people with dementia ofAD (Bei et ai, 2006; Lanzilli et ai, 2006: Masottiet ai, 2006). These findings suggest that Bcl-2 pro-tein may have a role in compensation responses toAD pathology, perhaps affording, to the remainingneurons, a margin of protection from apoptosis.Our study shows, for the first time, that, with theincrement of Al-exposure, the expression of Bcl-2protein in cerebral cortex neurons and cerebellumPurkinje's cells is increased in association with theincrement of cellular apoptosis. This suggests thatAl may lead to extensive toxic effects which accel-erate neuronal apoptosis, known to associated withCa^^ overload and lipid peroxidation (Raff, 1992;D'Alimonte et ai, 2007; di Lorenzo et ai, 2007;Zhu et ai, 2007). Bcl-2 is rapidly expressed, andmay suppresses apoptosis by inhibiting Ca ^ releaseor by increasing clearance of oxygen-derived free

168

radicals from endoplasmic reticulum (ER). Ourresults coincide with the report by Liu et ai (2000)who showed an elevation of Bcl-2 protein contentin the hippocampus and frontal cortex neurons of arat model of AD. By virtue of an RT-PCR method,Wei et ai (2000) detected an over-expression ofBcl-2 in the brain of 11-month old SAMP8, a typeof senescence accelerated mouse with impairedlearning and memory.Our results also demonstrate a high level of Bcl-2expression in frontal cortex cells (Table IU), sug-gesting that this anti-apoptotic response of frontalcortex is stronger than in other brain regions.According to the general rule "more effect, moreresponse" in biological procedure, we speculatethat the apoptotic process of this area was greater,which means more apoptotic effect had been stimu-lated by Al. However, whether greater Bcl-2 expres-sion equates with more serious apoptosis is notknown, and this aspect needs further study. Thepathways of cellular apoptosis in brain of Al-exposedrats is still in need of further study. Nevertheless,our fmdings indicate that in Al-exposed rats, Bcl-2protein is involved in the course of learning andmemory impairment and plays a protective role asa suppressor gene of neuron apoptosis.

A cknowledgement

This work was supported by research funds fromthe National Natural Science Foundation of China(NSFC.No.30371203).

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