Scholarly article on topic 'Aluminium-induced acute neurotoxicity in rats: Treatment with aqueous extract of Arthrophytum (Hammada scoparia)'

Aluminium-induced acute neurotoxicity in rats: Treatment with aqueous extract of Arthrophytum (Hammada scoparia) Academic research paper on "Biological sciences"

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Journal of Acute Disease
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{" Hammada scoparia " / Aluminium / Behaviour / Neurotoxicity / "Malic acid" / Neurodegeneration}

Abstract of research paper on Biological sciences, author of scientific article — Taïr Kaddour, Kharoubi Omar, Taïr Oussama Anouar, Hellal Nouria, Benyettou Iméne, et al.

Abstract Objective To study the antioxidative and protective properties of aqueous extract of Hammada scoparia (H. scoparia) against the effects of sub-chronic aluminium (Al) intoxication on mnemonic process and some neurochemical markers. Methods Al was administered intraperitoneally (50 mg/kg body weight, three times a week), and H. scoparia and malic acid were given orally by gavage at a daily dose (100 mg/kg body weight) to rats for 90 days. Results Al caused significant short-term and long-term memory disturbances, a decrease in locomotor activity, a significant inhibition of acetylcholinesterase activity in brain and a significant depletion of antioxidant enzymes (catalase, glutathione reductase and glutathione peroxidase) and glutathione. It significantly increased lipid peroxidation levels in cerebrum and cerebellum. However, treatment with H. scoparia extract protected efficiently the neurological functions of intoxicated rats by considerably increasing antioxidants levels and decreasing production of thiobarbituric acid reactive substances by 4.26% compared to untreated group. We noted some controversial results with malic acid. It showed some positive results but it was not as efficient as H. scoparia extract. Current results were consistent with histopathological observations including neurodegeneration and vacuolated cytoplasm (spongiosis) in Al treated sections when H. scoparia and malic acid treated sections showed marked neuroprotection signs. Conclusions This study strongly suggested that H. scoparia extract could possibly restore the altered neurological capacities and antioxidant power in rats, and it could even be a good alternative to chelating agents or other chemical medicines against Al-induced neurotoxicity.

Academic research paper on topic "Aluminium-induced acute neurotoxicity in rats: Treatment with aqueous extract of Arthrophytum (Hammada scoparia)"

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Original article

Aluminium-induced acute neurotoxicity in rats: Treatment with aqueous extract of Arthrophytum (Hammada scoparia)

Q2 Tai'r Kaddour1*, Kharoubi Omar1, Tair Oussama Anouar2, Hellal Nouria1, Benyettou Imene1, Aoues Abdelkader1

1 Laboratory of Experimental Biotoxicology, University of Ahmed Ben Bella "1", Oran, Algeria 2Faculty of Medicine, University of Ahmed Ben Bella "1", Oran, Algeria



Article history:

Received 6 Jun 2016

Received in revised form 28 Jul, 2nd

revised form 1 Aug 2016

Accepted 26 Aug 2016

Available online xxx


Hammada scoparia




Malic acid


1. Introduction

Aluminium (Al) is the most abundant metal present on the earth's crust. It is extensively used in daily life and was found in drinking water probably due to water purification procedures111. The new 20th century industrial products containing Al salts

*Corresponding author: Tai'r Kaddour, toxicology, University of Ahmed Ben Bella "1" Tel: +213 0696780294


All experimental procedures involving animals were conducted in accordance to the Guide for the Care and Use of Laboratory Animals (8th edition, 2011) and approved by the scientific committee of the university.

Peer review under responsibility of Hainan Medical College. The journal implements double-blind peer review practiced by specially invited international editorial board members.

Objective: To study the antioxidative and protective properties of aqueous extract of Hammada scoparia (H. scoparia) against the effects of sub-chronic aluminium (Al) intoxication on mnemonic process and some neurochemical markers. Methods: Al was administered intraperitoneally (50 mg/kg body weight, three times a week), and H. scoparia and malic acid were given orally by gavage at a daily dose (100 mg/kg body weight) to rats for 90 days.

Results: Al caused significant short-term and long-term memory disturbances, a decrease in locomotor activity, a significant inhibition of acetylcholinesterase activity in brain and a significant depletion of antioxidant enzymes (catalase, glutathione reductase and glutathione peroxidase) and glutathione. It significantly increased lipid peroxidation levels in cerebrum and cerebellum. However, treatment with H. scoparia extract protected efficiently the neurological functions of intoxicated rats by considerably increasing antioxidants levels and decreasing production of thiobarbituric acid reactive substances by 4.26% compared to untreated group. We noted some controversial results with malic acid. It showed some positive results but it was not as efficient as H. scoparia extract. Current results were consistent with histopathological observations including neurodegeneration and vacuolated cytoplasm (spongiosis) in Al treated sections when H. scoparia and malic acid treated sections showed marked neuroprotection signs.

Conclusions: This study strongly suggested that H. scoparia extract could possibly restore the altered neurological capacities and antioxidant power in rats, and it could even be a good alternative to chelating agents or other chemical medicines against Al-induced neurotoxicity.

like antiperspirants are another source of exposure; vaccines adjuvants, phosphate binders, dialysis, total parenteral nutrition solutions and foods provide easy exposure of Al to human


The toxicity of Al is directly linked to its bioavailability. In biological systems, this element has been shown to accumulate in many mammalian tissues such as brain, bone, liver and kid-ney18-101, and its elimination half-life from human brain is calculated to be seven years121.

Al is the most common neurotoxicant111-161, and the evidences about its implication in developing Alzheimer's disease are getting increased117-201. It was also found that this trivalent cation can participate as a factor in the development of neural tube defects in human1211. Many studies showed that there were neuropathological, neurobehavioral, neurophysical

Laboratory of Experimental , Oran 31000, Algeria.

2221-6189/Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Hainan Medical College. This is an open access article under the CC BY-NC-ND license (

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and neurochemical changes after Al exposure122-271. The brain is considered to be the most vulnerable to the toxic manifestation of Al, and it is particularly sensitive to oxidative stress due to increased levels of free radicals and decreased levels of antioxidants following toxicity128-321.

Oxidative events have frequently been linked to neurodegenerative disorders such as Alzheimer's disease133-361. Public, academic, and government interest in traditional medicines or their isolated bioactive constituents seems to become amplified because of the adverse drug reactions and economic burden of the modern system of medicine1371.

Hammada scoparia (Pomel) Iljin (H. scoparia) belongs to Chenopodiaceae family, and it is a glabrous, grey brown, woody, dwarf shrub usually turning darker or blackish when dried. It grows wild in dry habitats of the Mediterranean region and the Near East[38]. In Algeria, it is commonly known as "rimth". Tunisian colleagues reported that H. scoparia possessed a large spectrum of pharmacological and therapeutic activities and it has been shown recently that flavonoid-enriched fraction of the plant has a protective effect on hepatic ischemia/reperfu-sion injury. A hepatic damage usually happens during liver surgery and transplantation1391. Another study showed molluscicidal activity of the plant leaves extract against Galba truncatulam. It also possesses a potent antitumoral activity1411. On the other hand, malic acid (MA), a naturally occurring, nontoxic and organic dicarboxylic acid, and magnesium are both known to be involved in the processes of generating adenosine triphosphate through Krebs cycle, and they play a pivotal role in mitochondrial adenosine triphosphate synthesis1421. The MA-magnesium combination presented a big efficacy in treatment of patients having fibromyalgia when served as a dietary supplement1431. Additionally, the chelation abilities of MA against Al toxicity were assessed at the University of Barcelona when toxicologists administered MA to mice exposed to Al at about one-fourth of the LD50 level. LD50 is the concentration of compound that will kill 50% of the experimental animals. Compared to other chelators (oxalic acid, malonic acid and succinic acid), MA showed the best therapeutic effectiveness at the same level compared with synthetic defer-oxamine mesylate1441.

The lack of data information about the protective properties of H. scoparia against Al-induced neurotoxicity pushed us to conduct the present study as an answer to the wonders that if a nutritional strategy like chronic administration of aqueous extract of H. scoparia could efficiently prevent Al-induced neurotoxicity in terms of oxidative stress in rat brain as it could be with chelation therapy.

2. Materials and methods

2.1. Preparation of Arthrophytum plant extracts

Whole plants of H. scoparia [Arthrophytum scoparium (Pomel) Iljin, Haloxylon articulatum (Cav.) Bunge, Haloxylon scoparium (Pomel)1[45,461 were collected from the region of Ai'n Sefra, Algeria in June, 2013. The plant was subjected to the identification and authentication at the Herbarium of Botany Directorate in Ahmed Ben-Bella (Oran) University (voucher specimen No. LB0748). Twenty five grams of aerial parts of the plant were extracted with 250 mL of distilled water by the

method of continuous hot extraction at 60 °C. Once the filtrate recovered, it was lyophilized and the residue collected (yield 11%) was stored at -20 °C.

2.2. Animals and experimental design

A total of 24 male Wistar rats with weight of (150 ± 10) g were used for the study. The processes of protocols using the experimental animals were in accordance to the Guide for the Care and Use of Laboratory Animals (8th edition, 2011) and approved by the scientific committee of the university. The animals were housed in the cages with six per cage and fed ad libitum, and they were exposed to a 10 h light:14 h dark cycle and the room temperature was maintained at (23 ± 2) °C. Animals were divided into four groups of six animals each. Group 1 (control) served as untreated control and received a intraperitoneal injection of 0.9% saline solution (NaCl); Group 2 consisted of Al intoxicated rats which were given a dose of 50 mg/kg body weight (BW) of Al chloride (AlCl3-6H2O) three times a week; Group 3 was Arthrophytum (H. scoparia) treated group which received intragastrically aqueous extract at a dose of 100 mg/kg BW of H. scoparia simultaneously with an intraperitoneal injection of 50 mg/kg BW of Al chloride (AlCl3-6H2O); Group 4 was MA treated group which was given 100 mg/kg BW of MA by gavage in parallel with intraperitoneal injection of 50 mg/kg BW of Al chloride (AlCl3-6H2O).

All the groups were treated under the same housing conditions for a period of 90 days. The injection solution was prepared in sterilized saline solution. The animals were weighed and behavioural observations were recorded at the end of the experiment, then the animals were sacrificed under pentobarbital anaesthesia. The organs were removed, cleaned, washed with saline (0.9% of sodium chloride) then weighed and the organ weight ratio was estimated, and the relative weight of organs was calculated as g/100 g BW.

2.3. Tissue simple preparation

After anesthetization by intraperitoneal injection of pento-barbital, animals were sacrificed and the brains were immediately removed, placed in ice-cold isotonic saline and dissected into cerebrum and cerebellum which were stored at -80 °C. Later the brain regions were taken and minced into small pieces then homogenized with ten volumes of phosphate buffer (0.1 mol/L, pH 7.4) containing 0.3 mol/L sucrose and 0.08 mol/ L potassium chloride using WiseTis® (HG-15A) homogeniser, and the homogenates were then centrifuged at 7 600 r/min for 10 min at 4 °C and the resultant supernatant was further centrifuged at 12000 r/min for 10 min at 4 °C° to yield the supernatant which was later used for the estimation of anti-oxidants parameters [malondialdehyde (MDA), catalase (CAT), glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR)1 and acetylcholinesterase (AChE) activity.

2.4. Behavioural parameters

Behavioural tests were conducted in order to evaluate how Al intoxication can affect locomotor, learning and memory

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capacities in the different experimental groups and to assess the ability of either plant antioxidant molecules or chelators to restore a physiological homoeostasis. The following behavioural tests were used.

2.4.1. Open field

The open field test provides simultaneous measures of locomotion and anxiety1471. The open field used was a square wooden arena (90 cm 90 cm 25 cm). The floor was divided by white lines into 36 smaller squares (15 cm 15 cm). The open field maze was cleaned between each rat to avoid odour cues. The rats were carried to the test room in their home cages and tested once at a time period of 30 min each. Other parameters of exploratory activity such as rearing, grooming and sniffing were carefully observed and time spent in performing each behaviour was recorded. These parameters were defined as follows: rearing was defined as standing on hind legs with paws pressing against the wall of the arena; sniffing was defined as continuously placing nose against the floor for at least 2 s; grooming was defined as using paws or tongue to clean/

scratch body1481.

2.4.2. Elevated T-maze

The test was realized according to the procedure described by Viana et al.1491. This behavioural task assessed an effective memory of the experimental model related to the environment due to the open arms of the elevated T-maze apparatus149-531 which was elevated by 50 cm from the ground and composed of two open arms of equal dimensions (50 cm 10 cm) and two enclosed arms surrounded by 15 cm high walls. Rodents were found aversive to the characteristics and keeping a vivid memory of the aversive situation through measuring the time spent in the open arms during

the test150,531.

Right after the open field test, rats were placed at the end of the enclosed arms and the time (latency) taken to withdraw from the arm was recorded over 300 s (a cut off time if no changes have been noted). This time was called baseline. Afterwards this step was repeated two successive times at about 30 s of intervals between all attempts and the times to get out from the closed arms were recorded, and these were called inhibitory avoidances (inhibitory avoidance 1, inhibitory avoidance 2). The escape test was performed following the inhibitory avoidance 2, and it was represented by the time used for animal to withdraw from the open arms. To assess long-term memory, inhibitory avoidance and escape were

measured again 72 h later150,531.

2.5. Lipid peroxidation (LPO) levels, reduced GSH and antioxidant enzyme activities

2.5.1. LPO levels [thiobarbituric acid reactive substances (TBARS)]

The LPO levels in cerebrum and cerebellum homogenates were measured colourimetrically as described by Okhawa et al. 1541 by measuring MDA formation. This is a method based on the reaction of thiobarbituric acid with some products of lipid peroxidation in acidic environment at increased temperature.

The formed product was coloured in pink which enabled its spectrophotometric determination.

In brief, 0.2 mL of supernatant prepared from homogenized tissues using 9 mL potassium chloride (1.15%) was added with 0.2 mL of sodium dodecyl sulphate, 1.5 mL of acetic acid and 1.5 mL of thiobarbituric acid. After completing volume with

4 mL of distilled water, the samples were heated in boiling water bath for 60 min, and the samples were then cooled and centri-fuged at 4000 r/min for 10 min. Absorbance was measured at 535 nm. The amount of MDA was calculated using a molar extinction coefficient of 1.56 x ^ 105 mol/L/cm.

2.5.2. Determination of CAT (EC 1.11.16) levels

CAT was assayed by the method of Aebi[55] with slight modification. The rate of H2O2 decomposition was followed by monitoring absorption at 420 nm. In brief, 250 mL of phosphate buffer (0.066 mol/L), 250 mL of cerebrum and cerebellum homogenates and 250 mL of 0.03 mol/L H2O2 (prepared in phosphate buffer, 0.066 mol/ L, pH 7.0) were added in a cuvette. After incubation for

5 min, TiOSO4 was added to the mixture and absorbance was directly measured against phosphate buffer as a blank, and one unit of CAT is equal to 1 mmol H2O2 degraded/ mg of protein.

2.5.3. Reduced GSH levels

Reduced GSH was determined using a colourimetric technique as described by Sedlak and Lindsay[56]. The principle was based on reaction of compounds containing sulfydryl groups with 5,5' dithiobis(2-nitrobenzoic acid) (DTNB) which produced yellow coloured product that absorbed at 412 nm. In brief, 1 mL of cerebrum and cerebellum supernatant (homogenates) was prepared after treatment with 1 mL of 50% trichloroacetic acid-distilled water (1:4), and the supernatant obtained after centrifugation at 2 400 r/min for 15 min was mixed with 0.02 mL of 0.01 mmol/L DTNB and an amount of Tris buffer (0.4 mol/L, pH 8.5). Total GSH content was expressed as nanomoles of GSH per milligram of protein.

2.5.4. GR

GR catalyses the conversion of oxidized glutathione employing nicotinamide adenine dinucleotide phosphate (NADPH) as a substrate, and it was assayed by the procedure adopted by David and Richard[57].

The amount of NADPH utilized was a direct measure of enzyme activity in our tissue homogenates. In brief, the assay system contained 1 mL of phosphate buffer (0.12 mmol/L, pH 7.2), 0.1 mL of 15 mmol/L ethylene diamine-tetra-acetic acid, 0.1 mL of sodium azide (10 mmol/L), 0.1 mL of oxidized glutathione and 0.1 mL of supernatant (cerebrum and cerebellum homogenates) and the volume was made up to 2 mL with distilled water. The reaction was started by the addition of NADPH solution, and the absorbance was read at 340 nm and the enzyme activity was expressed as mmol NADPH oxidized/ mg of protein.

2.5.5. Activity of GPx

GPx (EC activity in brain tissues was assessed by the method of Rotruck et al.[58]. Briefly the reaction mixture

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contained 0.2 mL of Tris-HCl buffer (0.4 mol/L, pH 7.0), 0.2 mL of reduced GSH (1 mmol/L), 0.1 mL of sodium azide (10 mmol/L), 0.1 mL of H2O2 (1 mmol/L) and 0.2 mL of tissue sample.

After incubation at 37 °C° for 10 min, reaction was stopped by the addition of 0.4 mL of 10% trichloroacetic acid, and tubes were subjected to centrifugation at 2400 r/min for 10 min. The supernatant (0.2 mL) was then added with 0.1 mL Ellman's reagent (0.019 8 g of DTNB prepared in 0.1% sodium citrate). Absorbance was recorded at 340 nm.

2.5.6. Estimation of tissue AChE

AChE belongs to cholinesterase family. The enzyme activity was assessed according to the procedure of Ellman et al.[591. Acetylthiocholine was hydrolysed by AChE to acetic acid and thiocholine. The catalytic activity was measured by following the increase of yellow anion, 5-thio-2-nitrobenzoate, produced from thiocholine when it reacted with DTNB[591 at 410 nm.

In brief, an aliquot of cerebrum and cerebellum homogenate (0.02 mL) was added to tubes containing 3 mL of phosphate buffer (100 mmol/L, pH 8.0), 0.02 mL of acetylthiocholine solution (75 nmol/L) and 0.1 mL of DTNB.

2.5.7. Protein estimation

Protein was measured by the method of Lowry et al.[601 using bovine serum albumin as a standard, and necessary dilutions were realized to get the correct concentrations of the proteins present in tissues.

2.6. Histopathological studies [haematoxylin and eosin (H&E) staining]

Samples (entire brains: cerebrum and cerebellum) from each group were selected, transversely cut and fixed in 10% buffered formaldehyde solution, then conserved in paraffin. Four-micrometre tissue sections were realized and dried at adequate temperature to get paraffin removed from the glass slides. The next step was to rehydrate sections then stain them with hae-matoxylin and eosin as nuclear and cytoplasmic stains. The sections were analysed using Leica®DM5000B microscope and photographed with Leica EC3 digital camera.

2.7. Statistical analysis

Data were expressed as mean ± SEM with six rats in each group. Data comparisons were carried out by using One-way

ANOVA followed by least significant difference (LSD) test to compare means between the different treatment groups, and results were considered statistically significant when P < 0.05.

3. Results

3.1. Effect of treatment on body, cerebrum and cerebellum weights

As shown in Table 1, there was a significant difference in BWs of all experimental animals compared to controls. However, there was no significant change between Groups 2 and 3. The relative whole brain and cerebrum weights were also significantly lower in Group 2 than in the control group, and administration of H. scoparia in parallel with Al produced a recovery in relative whole brain compared to Group 2. MA had the same effect on relative cerebrum weight.

3.2. Effect of treatment on behavioural parameters

The Al chloride treatment induced significantly decreased (P < 0.05) locomotor activity as shown in Figure 1. This was concluded through the significant decrease in numbers of crossed squares and the highly significant decrease in rearing and sniffing performed by the animals in intoxicated group compared to the control one (Figure 1C,D).

Treatment with H. scoparia aqueous extract during Al exposure showed a very protective effect by significantly improving some of the previously altered scores in intoxicated rats, and this result was available for MA treatment group.

The mean inhibitory avoidance latency (IAL) and escape latency (ESL) of the elevated plus maze task presented some adverse variations. The IAL was significantly higher in intoxicated animals compared to control (Figures 2-5) (P < 0.05), and the rats permanence time (IAL1) was three times much longer in enclosed arms compared to baseline. In the next attempt (avoidance 2 test), the results showed a highly significant decrease in term of latencies in the protected arms (169 s was spent by intoxicated rats when controls stayed there over 300 s).

Treatment with Al and H. scoparia simultaneously presented a recovery in term of learning-short term memory capacities, and the animals had been stationary for about 30.8 s more than those in Al treated group.

Table 1

The effects of H. scoparia and MA on BW, absolute whole brain, cerebrum and cerebellum weights of control and rats treated with AlCl3 after 90 days of treatment.

Groups Initial BW Final BW Absolute Relative whole Absolute

whole brain weight cerebrum

brain weight (g/100 g BW) weight

Relative cerebrum weight (g/100 g BW)

Absolute cerebellum weight

Relative cerebellum weight (g/100 g BW)

Control AlCl3 AlCl3+ H.


AlCl3 + MA 150.00 ± 8.06 237.50

150.12 ± 4.92 270.52 : 150.85 ± 7.45 230.85 : 151.10 ± 12.61 237.60 :


2.015 ± 0.086 0.842 ± 0.166 1.498 ± 0.061 0.640: 1.879 ± 0.096 0.757 ±0.166* 1.415 ± 0.093 0.553: 1.936 ± 0.057 0.841 ± 0.160* 1.453 ± 0.020 0.588:

0.021 0.517 0.002* 0.456 0.019 0.482

0.031 0.198 ± 0.014 0.022 0.198 ± 0.016 0.034 0.196 ± 0.018

17.14* 1.999 ± 0.060 0.798 ± 0.166 1.547 ± 0.043 0.632 ± 0.016# 0.462 ± 0.039 0.208 ± 0.008

Values are given as mean ± SEM each group. : P < 0.05 compared with control group; : P < 0.05 compared with AlCl3 group.

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60 50 40 30 20 10 0

Control AICIj Aid, + H. sc AICIj+MA

Aid, Ala, + H sc Aid, + MA


Figure 1. Open field results following AlCl3 treatment during 90 days. The open field parameters crossing (A), rearing (B), grooming (C) and sniffing (D) were evaluated in the test. Values are given as mean ± SEM. : P < 0.05, : P < 0.01, : P < 0.001 compared with control group. #: P < 0.05, ###: P < 0.001 compared with AlCl3 group. $: P < 0.05 compared with AlCl3 + H. scoparia group. H. sc: H. scoparia.

For the first escape trial, rats joined the closed arms of the apparatus within 17 s, 36.61 s, 16.65 s, and 87.88 s for Groups 1 -4 respectively.

The long-term memory tests (avoidance 3: IAL3 and escape 2: ESL2) was performed at 3 days after avoidance 2 and escape 1 tests. Control and H. scoparia treated rats performed good scores, which were very close to each other even there was no statistical differences between values of both IAL3 and ESL2 for the two groups, when those of Al intoxicated group clearly indicated an impairment in long-term memory process through a highly significant decrease in IAL3 compared to controls (Figure 2).

Finally, MA treatment procedure presented some contradiction since there was no statistical difference between avoidances

fr 160

of Al+ H. scoparia treated group and Al intoxicated group, but their ESL2 values were favourable for amelioration of the memory process.

3.3. Effect of treatment on AChE activity

Acute/sub-chronic Al chloride administration in rats produced a significant (P < 0.05) decrease in cerebrum AChE activity by 19.13% as compared to control rats. However, the Arthrophytum (100 mg/kg) treatment improved modestly the alteration in AChE activity in the region mentioned above when compared to Al treated rats (Figure 6).

300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0

Baseline Avoidance 1 Avoidance 2 Avoidance 3 i- Control »AICI .AICI3 + H. sc*AICI3 + MA

Figure 2. Elevated T-maze results following AICI3 treatment during 90 days. Values are given as mean ± SEM. *: P < 0.05, ***: P < 0.001 compared with control group. ###: P < 0.001 compared with AlCl3 group. $: P < 0.05, $$$: P < 0.001 compared with AlCl3 + H. scoparia group. H. sc: H. scoparia.

Control AICIj AICI, + H. sc AICI3 + MA

□ Baseline □ Avoidance 1 □ Avoidance 2 ■ Avoidance 3

Figure 3. Comparative analysis of avoidance tasks inside each experimental group (elevated T-maze). Values are given as mean ± SEM. : P < 0.001 compared with baseline. ###: P < 0.001 compared with Avoidance 1.$$$: P < 0.001 compared with Avoidance 2. H. sc: H. scoparia.

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*** ###

Escape 1 a Control üAICI,

Escape 2

Figure 4. Effects of AlCl3 treatment during 90 days on escape from the open arm of the elevated T-maze. Escape 1 was measured in all experimental groups immediately after inhibitory avoidance training. Escape 2 was measured 72 h later. Values are given as mean ± SEM. : P < 0.05, : P < 0.001 compared with control group. ###: P < 0.001 compared with AlCl3 group. $$$: P < 0.001 compared with AlCl3 + H. scoparia group. H. sc: H. scoparia.

Control AICI3 AICI3 + H. sc AICI3 + MA ■ Escape 1 . Escape 2

Figure 5. Comparative analysis of escape tasks inside each experimental group (elevated T-maze). Values are given as mean ± SEM. No significant differences between Escape 1 and Escape 2 were recorded. H. sc: H. scoparia.

10 9 8 7 6 5 4 3 2 1 0

3.4. Effect of treatment on lipid peroxidation and GSH contents in cerebrum and cerebellum

Changes in TBARS and GSH levels were illustrated in Figures 7 and 8, and a significant increase in TBARS levels by 53.43% in cerebrum of intoxicated rats was noted when compared to controls. A highly significant (P < 0.001) increase was also noted in cerebellum of exposed rats, and these results were accompanied by a reduction in GSH levels in cerebrum and cerebellum of Al treated rats (59.72% and 5.04% respectively) in comparison with those of controls.

The co-administration of H. scoparia and Al decreased the TBARS production by a rate of 4.26% in cerebrum and 72.36% in cerebellum. This treatment alleviated significantly GSH levels in brain regions when compared to intoxicated rats. The plant extract showed more efficient results in term of restoring normal values of some altered parameters than the chelation strategy did.

3.5. Effect of treatment on antioxidant enzymes activities in cerebrum and cerebellum

Exposure to Al produced significant changes in the cerebrum and cerebellum redox status. A very significant decrease (P < 0.01) in CAT levels, GR and GPx activities was recorded in intoxicated group compared to controls (Figures 9-11).

Oral administration of aqueous H. scoparia extract during Al exposure showed an amelioration in CAT, GR and GPx by significantly increasing their values (61.79%, 43.74% and 48.47% respectively) in cerebrum when compared to those in Al treated group.

Treatment with MA failed again in establishing normal balance between oxygen reactive species (ROS) generating and antioxidants.

3.6. Effect of treatment on brain histopathological changes

Pathological changes in the cerebrum and cerebellum of Al intoxicated rats were examined under light microscopy. We noted that there were a neuronal loss, a spongy degeneration and vacuolated cytoplasm when sections were found to be intact in control group. These changes were minimized in Al+ H. scoparia and Al + MA treated groups (Figure 12).

4.5 4.0 3.5 3.0 2.5 2.0

1.5 1.0 0.5 0.0

Control AICI3 AICI3 + H. sc AICI3 + MA

AICI3 AICI3 + H. sc AICI3 + MA

Figure 6. Effects of treatment with H. scoparia and MA on AChE (mmol/min/mg protein) activity in cerebrum (A) and cerebellum (B) of control and Al intoxicated rats after 90 days of exposure. Values are given as mean ± SEM. Significant differences: : P < 0.05, : P < 0.01 compared with control group.

H. sc: H. scoparia.

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AICI3 + H. sc AICI3 + MA


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## $$$

Figure 7. Effects of H. scoparia and MA on TBARS (nmol/mg of protein) in cerebrum (A) and cerebellum (B) of control and rats treated with Al after 90

days of treatment. Values are given as mean ± SEM. Significant differences: : P < 0.01, : P < 0.001 compared with control group. P < 0.001 compared with AlCl3 group. $$$: P < 0.001 compared with AlCl3 + H. scoparia group. H. sc: H. scoparia.

: P< 0.01,

AICI3 + H. sc AICI3 + MA

AICI3 + H. sc AICI3 + MA

Figure 8. Effects of treatment with H. scoparia and MA on GSH levels in cerebrum (A) and cerebellum (B) of control and rats treated with Aluminium after 90 days of exposure. Values are given as mean ± SEM. ***: P < 0.001 compared with control group. #: P < 0.05 compared with AlCl3 group. $$: P < 0.01 compared with AlCl3 + H. scoparia group. H. sc: H. scoparia.

AICI3 AICI3 + H. sc AICI3 + MA

AICI3 AICI3 + H. sc AICI3 + MA

Figure 9. Effects of H. scoparia and MA on CAT level (mmol H2O2/mg of protein) in cerebrum (A) and cerebellum (B) of control and rats treated with Al

after 90 days of exposure. Values are given as mean ± SEM. with AlCl3 group. H. sc: H. scoparia.

: P < 0.01, : P < 0.001 compared with control group. #: P < 0.05, ###: P < 0.001 compared


AICI3 AICI3 + H. sc AICI3 + MA

AICI3 AICI3 + H. sc AICI3 + MA

Figure 10. Effects of H. scoparia and MA on GR (mmol NADPH oxidized/min/mg of protein) activity in cerebrum (A) and cerebellum (B) of control and rats treated with Al after 90 days. Values are given as mean ± SEM. Significant differences: : P < 0.05, : P < 0.001 compared with control group. ###: P < 0.001 compared with AlCl3 group. $$$: P < 0.001 compared with AlCl3 + H. scoparia group. H. sc: H. scoparia.

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Tair Kaddour et al/Journal of Acute Disease 2016; u(u): 1-13

10 11 12

20 21 22

60 61 62

Ü 1.2

IM a 0.5

*c 0.4

<i 0.3

PL C 0.2

AICI3 AICI3 + H. sc AICI3 + MA

Control AICI3 AICI3 + H. sc AICI3 + MA Figure 11. Effects of H. scoparia and MA on GPx (nmol/min/mg of protein) activity in cerebrum (A) and cerebellum (B) of control and rats treated with Al

after 90 days of exposure. Values are given as mean ± SEM. : P < 0.05, : P < 0.01, with AlCl3 group. H. sc: H. scoparia.

: P < 0.001 compared with control group. : P < 0.001 compared

Figure 12. Effects of H. scoparia on Al-induced histological changes in cerebral cortex and cerebellum of control and experimental rats. A and B (control): Sections of cerebral cortex (A) and cerebellum (B) showing normal histo-architecture (H&E, 20x); C and D (Al: 50 mg/kg BW): Sections of cerebral cortex (C) and cerebrum (D) showing neuronal spongiosis, gliosis with apparent vacuoles in both regions in addition to disorganization in cerebellum layers (H&E, higher magnification, C: 40x; B: 10x); E and F (Al + H. scoparia 100 mg/kg BW): Sections of cerebrum (E) and cerebellum (F) showing very reduced vacuolar spaces around the pyramidal cells in cerebral cortex, a modest loss in Purkinje's cells and a minimized spongiosis in cerebellum (H&E, 20x); G and H (Al + MA 100 mg/kg BW): Cerebrum and cerebellum section showing less alterations in the histoarchitecture compared to Al group especially in cerebellum, some vacuolated neuronal cells still exist in cerebrum (H&E, 20x). GL: Granular layer; ML: Molecular layer; GC: Glial cell; PM: Pia mater; PC: Purkinje's cell; Py: Pyramidal cells; VPy: Vacuolated pyramidal cell; PCL: Purkinje's cell loss; WM: White matter; S: Spongiosis; OD: Oligo dendrocyte.

4. Discussion

The results from the present research indicate that Al exposure has changed the BW and relative weights of the whole brain and cerebrum, which reveal a possible detrimental effect of Al on the body and brain weight as compared to the control (Table 1). These results concur with many previous researches. Julka et al.[611 reported that sub-acute Al exposure of rats generated a loss of about 27.8 g in animals BW. Tripathi et al.[621 also noticed a reduction in terminal body weights of animals administered Al during 90 days while Sharma et al.[351 noticed

a significant reduction of about 50.44% gain in weight in Al treated rats. The loss of brain weight after sub-acute Al treatment could be a result of the spongiosis of the neuropil resulting in retarded development of the animals[611. This agrees with our results about histology of studied organs.

In this study, we have investigated the behavioural and the potential neuropathological effect of acute/sub-chronic experimental exposition of rats to Al chloride, and tested animals presented neurological disorders including learning impairments and memory deficits as well as neuronal loss when compared to controls.

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Tair Kaddour et al/Journal of Acute Disease 2016; u(u): 1-13

1 In the present study, Al chloride was found to decrease the

2 crossing scores according to the results obtained after perform-

3 ing in the open field apparatus. Even secondary parameters

4 evaluated in this test were altered including rearing, grooming

5 and sniffing. Similar observations have also been reported by Lal

6 et al.1631 who noticed significant reduction in the spontaneous

7 locomotor activity after daily Al treatment of rats (500 mg A!/

8 L in drinking water) for 180 days. Sharma et al.1351 made the

9 same observations after an intragastrically administration of Al

10 lactate to rats for 12 weeks. Yellamma et al.1641 also reported

11 an hypokinesia (reduced locomotor activity) in rats

12 administered with sub lethal dose of Al once a day for 25

13 days continuously. Elsewhere, Abd-Elhady et al.1131 did not

14 find any distinct effects of Al on crossing or rearing done by

15 the animals.

16 The elevated T-maze apparatus which reflects learning and

17 memory abilities by measuring inhibitory avoidance revealed

18 that intoxicated animals presented differences in IAL compared

19 to normal control animals. This refers to possible learning pro-

20 cess failures and memory deficits caused by the neurotoxicant.

21 These results are supported by those described in previous

22 works when peripheral and oral administrations of Al to rodents

23 lead to learning and memory deficits165-671, and the same result

24 was found in studies using other forms of Al which showed

25 that administration of Al citrate to rats decreased IAL

26 values1531. Similar results that indicate impairments of rats

27 receiving Al in drinking water in the passive avoidance task

28 have been reported1681. Abd-Elhady et al.1131 also demonstrated

29 that Al caused a deterioration in learning and memory

30 functions in passive avoidance task after Al treatment. The

31 latencies of animals in the closed arms of the previously

32 described apparatus seem to be higher when compared the

33 values of inhibitory avoidance of the intoxicated group to their

34 baseline latency and the IAL 1 values of control group. This

35 matches with anterior works reporting that Al citrate

36 administration impaired inhibitory avoidance performance1531.

37 While others have indicated that the stay of animals is

38 normally longer in the closed arms in attempts following the

39 first trial because they prefer to explore more open space than

40 confine and protect ones150,531.

41 In Arthrophytum treated group and MA treated group, we

42 found that IAL 1 is particularly closed to IAL of control group,

43 which reflects an effective action of plant molecules and

44 chelating elements on restoring normal state.

45 In the present work, short-term memory was seen to be

46 significantly affected by Al exposure referring to values of

47 Avoidance 2. The amnestic effect of Al was also consolidated by

48 performances done 72 h after avoidance 2 when tested IAL3 in

49 AlCl3 group was reduced in comparison with control group

50 suggesting a long-term memory alterations.

51 Obtained results showed that one-way escape from the open

52 arms of the T-maze was not affected by Al intoxication as it was

53 with IAL trials when compared intoxicated group to control

54 group. Additionally it was shown that memory in this task was

55 not impaired. Thus, different types of memory seem to exist,

56 each having specific underlying brain mechanisms. This can be

57 explained by the fact that some brain structures (like amygdala

58 complex) lesions attenuate expression of emotion, behaviour and

59 memory whereas their integrity is not required for other types of

60 memory150,691. Based on our results, Al-induced neuro-

61 degeneration seems to be verified, and this is in accordance with

62 the findings that memory impairments along with compromised

learning behaviour are the major neurodegeneration disorders1701 63

such as Alzheimer's disease affecting particularly some brain 64

structures like hippocampus1711 and amygdala1721. 65

Al exposure is known to produce neurotransmission 66

disruption and cholinotoxicity173-751, and acetylcholine is 67

usually related to short-term memory. Our finding demon- 68

strated that Al causes disturbances in cholinergic neurotrans- 69

mission, and H. scoparia extract co-administrated with Al 70

revealed a better effect on learning in animals since passive 71

avoidance in this group were improved in comparison with 72

intoxicated animals. These results concur with previously re- 73

ported data indicating that a co-administration of some plant 74

preparation like Vitis vinifera extract with Al showed a re- 75

covery from amnestic troubles1761. 76

AChE is usually located in membranes (erythrocytes) of 77

vertebrates and non-vertebrates. The enzyme controls ionic 78

current in excitable membranes and plays an essential role in 79

nerve conduction process at the neuromuscular junction1771 and 80

motor function1781. That's why some previous studies reported 81

that Al altered the muscular-locomotion activities by 82

decreasing them, which can explain our result about behaviour 83

(crossing task values) since high levels of Al not only interfered 84

with the memory but also attenuated the motor functions and led 85

to decreased motor activities and grip strength in mice1791. 86

However, giving H. scoparia antioxidant extract could restore 87

altered motor function and acquisition-memory process (closed 88

to normal) by modulating AChE activity. 89

Although AChE enzyme always receives a big attention in 90

the study of Al neurotoxicity, the elevated acetylcholine levels 91

are known to improve learning and memory1801 and AChE plays 92

an essential role in cognitive functions1811 by two mechanisms 93

including elevating acetylcholine levels and promoting the 94

cholinergic neurogenesis1821. Results of previous studies 95

showed that Al has biphasic effect on AChE activity 96

(increased at 4 and 14 days and decreased at 60 days of 97

intoxication)1831. Lakshmi et al.1761 have also reported an 98

decrease in AChE activity in brain as a response to Al 99

intoxication. 100

AChE activity measured in the present study was observed to 101

be decreased after Al exposure. This could be explained by an 102

accumulation of the metal in rat brain. Authors mentioned that 103

exposure of rats to Al chloride by intubation for 60 days pre- 104

sented accumulation of the neurotoxicant at different levels in 105

the brain1831 affecting even serotoninergic neurotransmission. 106

Similar results suggested that the decrease of 5-HT level in 107

hippocampus while causing cholinergic hypofunction by 108

administration of neurotoxin AF64A is a direct result of losing 109

cholinergic input1731. Also, it has been observed that Al 110

influences the metabolism of acetyl-CoA which leads to a 111

possible reduction in the formation of acetylcholine and hence 112

the substrate for AChE enzyme1841. These results were also 113

described by Ravi et al.1851. 114

Other works highlighted the relation between Al exposure 115

and glucose since production of acetyl-CoA is widely linked to 116

pyruvate, a key molecule in glycolysis process1861. Our results 117

about AChE activity disagree with some others indicating that 118

Al exposure led to an increase in AChE activity in brain of 119

rats187-891. This could be explained by the hypothesis of the 120

biphasic effect of Al related to the metal exposure duration 121

established by Kumar1831. 122

H. scoparia extract administered in parallel with Al improved 123

modestly the decreased enzyme activity in brain regions. Similar 124

Tair Kaddour et ai/Journal of Acute Disease 2016; 1-13

1 reports have been noted by Kakad et al.[901 and Lakshmi et al.[761

2 when administration of Vitis vinfera, a black grapes fruit from

3 India, to Al exposed rats alleviated AChE activity. Also

4 Kumar et al.[251 have found that chronic oral treatment with

5 curcumin (30 mg/kg and 60 mg/kg) significantly ameliorated

6 the reduction in AChE activity compared to Al chloride

7 treated group.

8 The positive response of cholinergic neurons in term of

9 system reactivation has been explained by two hypothesis:

10 antioxidant plant extract/flavonoids administration changed the

11 configuration of AChE or corrected the impaired metabolism of

12 glucose'76,911.

13 Since it was reported that the manifestation of oxidative

14 stress generation in brain was a response to sub-chronic expo-

15 sure to Al[32,74,891, we undertook the present study based on

16 measuring LPO levels and quantitating endogenous

17 antioxidants (CAT, GR, GSH, GPx).

18 Now, it is well documented that Al-induced oxidative stress

19 in neurons involves an imbalance between generation of ROS

20 and antioxidants[92,931.

21 Lipid oxidation products are one of the main consequences

22 associated with oxidative stress and brain is considered to be the

23 most sensitive target to be damaged due to the high level of lipid

24 content and tissue oxygen consumption[321.

25 The significant increased cerebrum and cerebellum levels of

26 MDA found in our study reflect the efficiency of Al in acti-

27 vation of lipid production process. These results corroborated

28 the previous findings that Al exposure enhanced iron-

29 dependant LPO in rat brain[94,951. While administration of Al

30 through intraperitoneal injection increased LPO in cerebrum

31 and cerebellum.

32 GSH is an important intracellular non-enzymatic antioxidant,

33 and it is considered as the most important scavenger of free

34 radicals and cofactor of many detoxifying enzymes against

35 oxidative stress like GPx, GR and others. It is able to regenerate

36 the most important antioxidants, vitamins C and E, back to their

37 active forms[96,971.

38 In our case, we noted decreased GSH levels in cerebrum and

39 cerebellum of intoxicated rats compared to those found in

40 controls. Antioxidant enzymes are the first cellular molecules

41 required for defence against ROS generation. Thus, in the

42 present work, increased oxidative stress and brain injury were

43 evident by decreased GPx, GR activities and CAT level in

44 cerebrum and cerebellum. All of these records are in agreement

45 with the fact that Al reduced the total antioxidant parameter

46 levels enhancing imbalance between pro-oxidant and antioxi-

47 dant potentials. Results similar to ours made it clear that Al

48 chloride intraperitoneally administered at dosages of 0.7 and

49 35 mg/kg BW for 14 days resulted in higher Al concentration

50 in hippocampus and cerebellum in the Al treated group

51 compared to the control[261. Also, Azadeh and Abdollahi[981

52 reported that most studies on Al toxicity demonstrated a

53 decrease in both the activity of GPx and the concentration of

54 GSH. Nayak et al.[991 found the same results with GR and

55 GSH when co-exposure of rats to both ethanol and Al fav-

56 oured the development of Al-induced oxidative stress in

57 cerebrum.

58 H. scoparia administration to Al treated rats was found to

59 significantly re-equilibrate antioxidant parameters back to normal

60 values. This positive effect of Algerian Arthrophytum on oxidative

61 stress defence is probably due to its secondary metabolites

62 composition including alkaloids, polyphenols and flavonoids.

Benkrief et al.[1001 reported that H. scoparia from Algeria contained 63

the alkaloids carnegine, and N-methylisosalsoline as major 64

tetrahydroisoquinoline alkaloids in addition to isosalsoline, N- 65

methylcoryaldine, dehydrosalsolidine, isolsalsolidine and N- 66

methyltryptamine as minor alkaloids, while others reported that 67

H. scoparia from Algeria Nabors regions contained quercetin- 68

galactose-rhamnose commonly called rutin as the major and 69

most active flavonoid[421. 70

Similarly, recent studies highlighted the determinant role of 71

some specified isolated secondary metabolites such as resvera- 72

trol[1011 and quercetin[351 in protection and remission from 73

respectively Al-induced brain neuroinflammation and cognitive 74

impairments/neurotransmission dysfunction related to oxidative 75

damage. Similar studies using bioflavonoids from plant and fruit 76

extracts such as pomegranate peel showed decreased Al accu- 77

mulation and stimulated anti-apoptotic proteins against Al 78

exposure in rat brain[1021. Prakash et al.[1031 also found that fisetin, 79

a natural flavonol, can attenuate increased LPO and reduced 80

GSH levels in brains of Al chloride treated mices. Similar 81

findings were recorded when curcumin supplementation 82

helped to normalize the levels of some oxidative stress 83

parameters including reduced GSH following chronic 84

administration of Al to rats[1041. In the other hand, some 85

authors claim that the supplementation of rodents by some 86

trace element metals as antioxidants like selenium is suitable 87

for removing Al toxicity[1051. 88

The examination of H&E stained sections revealed that 89

Al can cause marked histopathological abnormalities in 90

brain tissues (cerebrum and cerebellum) including neuronal 91

vacuolization, spongiosis, gliosis and cellular rarefaction 92

in cerebral cortex. These results are correlated to those 93

claimed by many authors. Bhadauria[91, Prakash et al.[1031, 94

Bihaqi et al.[1061, Matyja[1071 and Sumathi et al.[1081 all 95

reported the same modifications induced by Al on cerebral 96

cortex histoarchitecture. Therefore, these alterations are 97

associated with learning-memory impairments11091. Others 98

reported that vacuolated cells are a striking feature in both 99

ageing and Al-treated brain parenchyma11101, and they may 100

be considered as the initial stages of dying cells producing 101

a swollen appearance with indistinct boundaries1110,1111. 102

In Al+ H. scoparia group, spongiosis and alterations were 103

really minimized and loss of cell degeneration appeared clearly. 104

Histological observations of control cerebellum H&E stained 105

sections showed typical cell characteristics and organizations 106

including pia mater, molecular and granular cell layer and Pur- 107

kinje's cell layer. In Al-treated group, spongiosis was also 108

noticed in addition to disorganization in layers and some Pur- 109

kinje's cells loss. It was documented that these cells are 110

responsible for motor co-ordination through their projections 111

until cerebral cortex and any damage in the cell layer may 112

change the motor co-ordination11121. This could explain the 113

decreased locomotor activity found in Al-teated rats in the pre- 114

sent study. 115

The co-administration of Al and H. scoparia showed better 116

improvement in cerebrum and cerebellum histology than that in 117

Al + MA group. 118

In conclusion, the results of the present study indicate that 119

H. scoparia extract is a potential formulation which can be 120

used for treatment of Al neurotoxicity. It shows more effi- 121

cient recovery from the toxicant-induced oxidative damage, 122

histopathological changes and AChE activity inhibition than 123

MA. 124

Täir Kaddour et al/Journal of Acute Disease 2016; ш(и): 1-13

10 11 12

20 21 22

60 61 62

Conflict of interest statement

The authors report no conflict of interest. Acknowledgments

The authors thank gratefully Professor Yammouni/Beldjilalli

Yamina and Professor Tou Abdenacer.


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