Introduction

Aluminum (Al) is a very common agent in nature and the third component of the earth’s crust (8% of its weight) after oxygen (47%) and silicon (28%). It is the most used metal after iron in various fields and the most abundant known for its neurotoxicity [1]. It easily accesses the central nervous system and accumulates in different regions and could be a potential factor influencing affective and cognitive disorders in both humans and animals, it induces oxidative stress (OS) and loss of synapses and neurons in the hippocampal and cerebral cortical regions [2, 3].

In the context of prevention and/or control of the neurotoxicity effects of heavy metals in general and Al in particular, several researches in the recent years have been focusing on a pineal hormone, melatonin (MEL), as a molecule that attenuates and prevent effects on affective and cognitive disorders. In addition to its anxiolytic, antidepressant, anti-inflammatory, regulatory and immunomodulatory properties, which have already been shown in humans and rodents, this hormone and its metabolites have properties that can combat oxidative stress and stimulate the activation of molecules that help synaptic plasticity in the nervous system [4,5,6].

The objective of our study is to demonstrate in Wistar rats the possible neuroprotective effect of melatonin against the neurotoxicity of Al administered chronically by intraperitoneal injection and its negative impact on behavioral and biochemical capacities.

Materials and methods

Animals and experimental conditions

This study was performed on twenty male Wistar rats initially weighing 120 ± 20g. Animals, from the breeding of the Ibn Tofaïl University, were maintained under LD 12/12 (12 h Light/12 h Darkness) and at a standard temperature (21 ± 1°C). Water and food were provided ad libitum. All animal procedures were carried out in accordance with the Animal Scientific procedure and approved by the University Ethics Committee for Animal Experiments.

The rats are divided into four groups of five animals in each who received intraperitoneally and chronically one injection per day, between 16:00 and 17:00 during eight weeks, of physiological saline, AlCl3 (SEGMA-ALDRICH) or MEL (SIGMA France) as following:

Groups

Drugs and dose

Group I (control)

NaCl 0,9%

Group II (MEL)

MEL 4 mg/kg

Group III (Al)

AlCl3 1mg/kg

Group IV (Al + MEL)

AlCl3 1mg/kg + MEL 4 mg/kg

  1. MEL Melatonin, AlCl3 Aluminum chloride

Neurobehavioral tests

Anxiety-Like measurement

Open Field Test

The OFT is used to measure the anxiety-like behavior in rodents [7, 8]. The animal is placed in the arena and allowed to move freely for 10 minutes while being recorded by an overhead camera for subsequent analysis. The parameters measured are: The time spent in the center area (TCA), the number of returns to the center (NRC) and number of total squares (NTS). The entries in the central area and the time spent in this area by the rats are inversely proportional to the anxiety level. The time spent in the central area will be higher than the anxiety level of the animal will be lower. While the number of total squares is a reliable index of general locomotors activity. The apparatus was cleaned between each examination using 7% ethanol solution.

Elevated Plus Maze (EPM)

The EPM is used to assess anxiety-related behavior in rodent models of CNS disorders [9]. The rat ware placed on the central area of the maze facing an open arm and left to explore the EPM for 5 min and its movements while being recorded by a camera for later analysis. The parameters measured are: The time spent on the open arms (TOA), the entries into open arms (EOA) and the number of full entries into the arms (TAE). The TOA and EOA parameters are inversely correlated with the level of anxiety, whereas the TAE parameter is a reliable index of general locomotors activity. Reduced anxiety behavior is shown by a statistically significant increase in open-arm parameters (time and/or entries). The apparatus was cleaned between each examination using 7% ethanol solution.

Depression-like measurement

Forced Swimming Test (FST)

The FST is designed to evaluate the depressive-like behavior in rodent [10]. The rat are forced to swim for 5 min in glass cylinders (height = 50 cm; diameter = 30 cm) containing 30 cm of water at (23°C ± 2°C) while the camera is recording it for later analysis to determine the immobility times (TIM). Depressive-like behavior is characterized by an increase in immobility time (TIM) [10, 11].

Cognitive Measurement

Y-maze test

Is a behavioral test for measuring the willingness of rodents to explore new environments [12]. Rat ware placed on the central area of the Y-maze and left to explore the arena for 8 min. Rats typically prefer to investigate a new arm of the maze rather than returning to one that was previously visited. A rat would be making a triad when it visited all 3 arms consecutively. As a measure for working memory, the percentage of alternations that the rat made was calculated, being the number of triads divided by the maximum possible alternations (i.e. the total number of entries minus 2)*100. If a rat scored significantly above 50% alternations, this was indicative of functional working memory. The apparatus was cleaned between each examination using 7% ethanol solution.

Morris Water Maze Test (MWM)

The MWM is the most commonly test used to evaluate cognitive functions related with learning and memory in rodents [13]. Tempera paint is added into the water (22◦C) until it becomes opaque. A hidden platform, from a Plexiglas cylinder (13.75 cm high, 9 cm in diameter), is placed about 0.5 cm below the water surface making it invisible. The MWM The water maze was in a room with posters and furniture around the walls, which served as additional visual cues. Rats were tested in two phases: acquisition and probe trial test [14].

During acquisition (4 trials/day for 4 days), rats were trained to swim to a hidden platform located in the northeast quadrant. Each rat was released from a randomly assigned starting point (East, North, South or West). If the rat did not find the platform within 60 s, it was guided to the platform and allowed to remain there for approximately 10 s. On the fifth day, the rats spatial memory was tested in a 60 s probe trial with no platform present [15].

Biochemical Examination

One day after the end of the behavioral tests, all animals were firstly anesthetized and then sacrificed by decapitation. Brains were quickly removed and maintained at low temperature on ice. The hippocampus was rapidly and gently removed and separated from surrounding tissues and homogenized in phosphate buffer at PH: 7.4 (w/v), centrifuged at 1500 rpm for 10 min, and the resulting supernatant was used in the biochemical assays.

Lipid peroxidation assay

The formation of lipid peroxides during lipid peroxidation process was analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in cells, as previously described by Draper and Hardley [16]. The TBARS levels in hippocampus is presented as nmol/g of tissue and were determined by the absorbance at 535 nm [17].

Nitrite/nitrate assay

The nitric oxide (NO) activity was assayed by the method of [18]. Tissue nitrite levels is expressed as µmol/g of tissue.

Determination of Superoxide Dismutase (SOD) activity

The superoxide dismutase (SOD) activity was determined according to the method described by Beauchamp and Fridovich [19]. SOD activity is reported as U/g of hippocampal tissue.

Statistical analysis

All data are expressed as the means ± standard error of the means (S.E.M.). To determine the differences between experimental groups in behavioral data and biochemical parameters, statistical analysis was performed by two-way ANOVA using SPSS (version 22 SPSS). Post hoc comparisons were made using the Tukey’s test. ANOVA repeat measures were used for the Morris water maze test. Differences were considered significant when p<0.05, very significant when p<0.01 and highly significant when p<0.001.

Results

Effect of Al and MEL on the levels of anxiety-like measured in the OFT (Fig. 1)

Fig. 1
figure 1

Effect of Aluminum, Melatonin and their combination on (A): Total amount time spent in the center (TCA), (B): Number of return into center area (NRC) and (C): Number of total squares (NTS) measured in OFT in male rats treated with NaCl 0.9% (Control), 4 mg/kg of melatonin (MEL), 1 mg/Kg of AlCl3 (Al) and simultaneously with 4mg/kg of MEL and 1mg/kg of AlCl3 (Al+ MEL). Results are represented as mean ± SEM. The significance level is 0.05. *p<0.05, **p<0.01, ***p<0.001. *P, significant change with respect to control; $P, significant change with respect to Al

Full size image

Statistical analysis showed that the TCA and NRC parameters was significantly affected by Al treatment compared to their controls (p<0.001). Moreover, MEL administration did not induce any significant change in this parameter compared with control group (p>0.05). Additionally, rats treated with the combination of Al and MEL (Al+MEL) show a significantly elevated in those parameters compared to Al-treated groups (p<0.01 and p<0.05 respectively). Furthermore, locomotors activity represented by the NTS was unaffected by any treatment (p>0.05).

Effect of Al an MEL on anxiety levels measured in EPM (Fig. 2)

Fig. 2
figure 2

Effect of Aluminum, Melatonin and their combination on (A): Total amount of time spent in exposed arms (TOA); (B): Number of entries in exposed arms (EOA) and (C): Total number of arms entries (TEA) measured in EPM test in male rats treated with NaCl 0.9% (Control), 4 mg/kg of melatonin (MEL), 1 mg/Kg of AlCl3 (Al) and simultaneously with 4mg/kg of MEL and 1mg/kg of AlCl3 (Al+MEL). Results are represented as mean ± SEM. The significance level is 0.05. *p<0.05, **p<0.01, ***p<0.001. *P, significant change with respect to control; $P, significant change with respect to Al

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The results obtained showed that TOA parameter was significantly affected by Al treatment compared to control group (p<0.001) and no difference was noted between Al+MEL-treated groups and Al-treated groups (p>0.05). In addition, the EOA and TEA parameters was unaffected by any treatment. No statistically significant difference was found between the control and treated groups (p>0.05).

Effect of Al and MEL on depressive-like performances measured by FST (Fig. 3)

Fig. 3
figure 3

Effect of Aluminum, Melatonin and their combination on depression-like behaviors parameters measured in FST in male rats treated with NaCl 0.9% (Control), 4 mg/kg of melatonin (MEL), 1 mg/Kg of AlCl3 (Al) and simultaneously with 4mg/kg of MEL and 1mg/kg of AlCl3 (Al+ MEL). Results are represented as mean ± SEM. The significance level is 0.05. *p<0.05, **p<0.01, ***p<0.001. *P, significant change with respect to control; $P, significant change with respect to Al

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For the TIM parameter, statistical analysis showed that chronic injection of Al significantly increased the immobility time compared to control group (p<0.001). For the Al+MEL-treated groups, a significant decrease in TIM was observed compared to Al-treated groups (p<0.01).

Al and MEL effect on memory

Y-maze test (Fig. 4)

Fig. 4
figure 4

Spontaneous alternation percentage evaluated in Y-maze test, in male rats treated with NaCl 0.9% (Control), 4 mg/kg of melatonin (MEL), 1 mg/Kg of AlCl3 (Al) and simultaneously with 4mg/kg of MEL and 1mg/kg of AlCl3 (Al+ MEL). Results are represented as mean ± SEM. The significance level is 0.05. *p<0.05, **p<0.01, ***p<0.001. *P, significant change with respect to control; $P, significant change with respect to Al

Full size image

The spontaneous alternation percentage was significantly affected by Al treatment compared to control groups (p<0.01). In the same, the statistical analysis shows that treatment with MEL alone did not significantly increase this parameter compared to control groups (p>0.05). Similarly, a significant increase in this parameter is observed in Al+MEL-treated groups compared to Al-treated groups (p<0.01).

Morris water maze

Acquisition phase (Fig. 5A)

Fig. 5(A) shows that during acquisition phase, the latency to reach the hidden platform on each of the 4 days is decreased in all treated groups. Statistical analysis indicates a highly significant difference between Al-treated groups compared to control rats (p<0.001). Paradoxically to Al-treated groups, the statistical analysis for MEL groups indicates a non-significant decrease in rats compared to their controls (p>0.05). With respect to Al+MEL groups, statistical analysis shows a significant decrease in this parameter compared to Al-treated groups (p<0.05).

Fig. 5
figure 5

A): Latency to reach the hidden platform on each of the 4 days of learning phase, (B): Percentage of time spent in the correct quadrant measured in MWM tests in male rats after 8 weeks of treatment with NaCl 0.9% (Control), 4 mg/kg of melatonin (MEL), 1 mg/Kg of AlCl3 (Al) and simultaneously with 4mg/kg of MEL and 1mg/kg of AlCl3 (Al+ MEL). Results are represented as mean ± SEM. The significance level is 0.05. *p<0.05, **p<0.01, ***p<0.001. *P, significant change with respect to control; $P, significant change with respect to Al

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Percentage time spent in the correct quadrant (Fig. 5B)

The percentage of time spent in the correct quadrant was significantly affected by Al treatment. It is observed that Al group rats spend significantly less time in the correct quadrant (NO) than control rats (p<0.01), with an estimated decrease of 50%. A significant increase in the percentage of time spent in the correct quadrant is noted in MEL-treated groups compared to Al-treated groups (p<0.05), as well as in Al+MEL-treated groups compared to Al-treated groups (p<0.01).

Al and MEL effect on oxidative stress (Fig. 6)

Fig. 6
figure 6

A) the lipid peroxidation levels in hippocampus, TBARS levels expressed in nmol/g of tissue; (B) The nitric oxide level in hippocampus, expressed in µmol/g of tissue; (C) Changes in Superoxide Dismutase (SOD) activity in hippocampus, expressed in U/g of tissue in male rats after 8 weeks of treatment with NaCl 0.9% (Control), 4 mg/kg of melatonin (MEL), 1 mg/Kg of AlCl3 (Al) and simultaneously with 4mg/kg of MEL and 1mg/kg of AlCl3 (Al+ MEL). Results are represented as mean ± SEM. The significance level is 0.05. *p<0.05, **p<0.01, ***p<0.001. *P, significant change with respect to control; $P, significant change with respect to Al

Full size image

Lipid Peroxidation (LPO) in Hippocampus reflected by TBARS levels was significantly affected by Al treatment compared with control group (p<0.01). Even though it is statistically not significant, the treatment with MEL reduced this parameter compared to control (p>0.05). For the Al+MEL combination, we note a decrease in TBARS levels in hippocampus compared to Al-treated groups, but no significant difference was observed.

NO concentrations in hippocampus was significantly affected by treatment. The chronic injection of Al significantly increased the NO levels compared to control group (p<0.001). As well, chronic injection of MEL reduced this parameter compared to control, but no statistically significant difference was observed (p>0.05). For the Al+MEL combination, a significant decrease in NO concentrations in hippocampus was observed compared to Al-treated groups (p<0.001).

Superoxide Dismutase (SOD) Activity in Hippocampus was affected by treatment. The results summarized in Fig. 6(C) showed the following: Al affect SOD activity in rat hippocampus compared with the control group (p<0.01). In addition, the treatment with Al+MEL combination significantly increased the SOD activity in rat hippocampus compared with Al-treated groups (p<0.05).

Discussion

The aim of this study was to demonstrate, in addition to the neurotoxic effect of chronic Al administration, the neuroprotective impact of MEL via chronic injections on neurobehavioral functions in male rats.

Analysis of the different parameters showed that chronic exposure of male rats to Al causes:

  • Anxiety-like disorders. This anxiogenic effect is based on the fact that Al decreases the TCA and NRC parameters in the OFT, and EBO and TOA parameters in EPM.

  • Depressive-like disorders evaluated by the FST, whose results clearly show a very significant increase in TIM parameter.

  • Cognitive disorders characterized by the impairment of working memory due to the decrease in the percentage of alternations (Y-maze test) and the affection of spatial learning performance causing a deficit in retention of spatial memory (MWM).

The behavioral outcomes presented here are consistent with current literature. These observations corroborate with findings from our previous paper that investigated the effect of chronic Al injection at different doses for 8 weeks on affective and cognitive disorders in male Wistar rat [20]. Another study by Tair et al., [21] evaluated the protective effect of an aqueous extract of Hamda scoparia against the neurotoxic effects of chronic intraperitoneal Al exposure for 90 days in rats, reported that Al intake causes anxiety in the OFT. Also, in adult Wistar rats, decreased TOA in the EPM was obtained after oral exposition to AlC3 for 8 weeks [22]. Regarding the depressive effect of Al assessed by the FST, similar effects were observed in mice after chronic exposure to Al [23]. The same results were observed in male Prague Dawley rats who received for 5 weeks an intraperitoneal injection of AlCl3 at 70 mg/kg [24].

Several studies on memory disorders showing that Al causes cognitive dysfunction and negatively affects spatial learning and memory capacities of rats [25]. Our data was consistent with results obtained in Wistar rats treated with Al lactate intragastrically for 12 weeks [26]. As well as in albino mice treated with AlCl3 in distilled water for 3 months at a dose of 50 mg/kg/d [23]. In addition, Ribes et al., [27] study showed that low oral exposure of Al (Al lactate at 0 and 1 mg/g feed for 120 days) altered spatial learning and memory in transgenic mice of Alzheimer's disease.

The results of our study, supported with the works mentioned above, confirmed that Al induces depression-like and anxiety-like behaviors, as well as memory and learning disorders. Since no single mechanism of Al neurotoxicity has been identified, some interconnected hypotheses have been suggested. These hypotheses include the alteration of intracellular signal transduction pathways and exacerbation of OS.

Several investigators have suggested a link between OS and the etiology of cognitive and affective impairments [28,29,30,31]. OS can alter brain activity including neurotransmission, provokes neuronal cell death, leading to an altered behavior [32]. In this context, several studies reported that behavioral abnormality in rats treated with Al is associated to enhanced OS in the brain, especially in the hippocampus [33,34,35] which plays a crucial role in emotion regulation and in learning and memory processes [36, 37].

In our work, chronic exposure to Al showed a marked increase in OS in the hippocampus, which was indicated by a decrease in SOD activity, and an increase in TBARS (LPO) levels. These findings are consistent with recent reports showing similar results [33, 34, 38]. Under OS, the LPO provoked is followed by alterations in membrane permeability and consequently deleterious effects on neuronal function [39]. The increased LPO level is, at least in part, due to an inhibition of SOD and CAT enzymes in the hippocampus following Al exposure. These two antioxidant enzymes can control the harmful effects of free radicals [40]. Al was found to induce decline in the mRNA expression of endogenous antioxidants [41].

On the other hand, the reduction in SOD activity may indicate inability of this enzyme to cope with the influx of free radicals such NO generated due to administration of Al as shown in this study. The reports of Al-Amin et al., are in line with our results; Al treatment enhanced the level of LPO and NO in the hippocampus as well as a decrease in antioxidant status [35, 42]. Since Al is implicated in free radicals generation, it is possible that it does execute its effects by impairing mitochondrial functions [43]. As known, the changes in mitochondrial functions are responsible for generation of OS. Mitochondria are considered to be the major sites producing free radicals. High levels of free radicals such as NO will induce oxidative damage to unsaturated fatty acids, proteins, and DNA in the mitochondria [44, 45], leading to cell damage and death [46]. It has been demonstrated that chronic Al exposure (10 mg/kg) for 3 months increases free radicals generation and is implicated in the impairment of mitochondrial functions in the various regions of rat brain [43]. These findings may explain the increased level of NO and LPO observed in this experiment following Al administration. In addition, Al is likely capable of inducing NO production in the rat brain [47], which may be due to the ability of this metal to enhance the expression of NO synthase (NOS) [48]. At high levels, NO can reacts with the superoxide ion (O2-) to form the ion peroxynitrite (ONOO-), a reactive toxic molecule which can elicit cellular damages by initiating LPO formation, leading to neuronal cell damage [49], and consequently, an altered behavior.

To reduce the neurotoxic effects of Al, our research relied on the chronic administration of MEL in combination with Al. In our study, in rats treated with MEL in combination with Al, levels of anxiety and depression were significantly reduced compared to rats treated with Al alone. Similar results have been observed in other studies [50, 51]. In addition, the anxiolytic and antidepressant effects of MEL have also been reported in recent years [52,53,54]. Thus, learning and memory performances are high in the Y-Maze and the MWM tests. Some recent data indicate that MEL may reduce hippocampal-dependent memory deficit in mice [53, 55, 56]. Additionally, MEL improves spatial memory in ovariectomized adult rats with cognitive impairment [57]. The results thus obtained confirmed that MEL as a neuroprotective substance has both anxiolytic and antidepressant effects, as well as a positive effect on memory and learning in rat.

Because OS and neurobehavioral changes are strongly correlated [58,59,60,61], the anti-OS property of MEL provides one possible mechanism by which this molecule abolishes Al-induced impairment of emotional process and learning and memory [62]. In the present study, chronic administration of MEL was found to improve not only the affective and cognitive functions but also reduced Al induced-oxidative damage in the hippocampus by attenuating the rise in LPO and NO levels and also by restoring the SOD activity. These findings supported earlier observation about attenuation of brain OS by MEL in Al treated animals [62]. Al-Olayan et al. also found that treatment with MEL (10 mg/kg) for 7 days, decreased LPO and NO levels and increased antioxidant enzymes activities in brains of Al treated rats [48]. MEL might protect against the Al induced neurotoxicity through reducing OS and improving mitochondrial function. This indolamine and its metabolites provide on-site protection to mitochondria from OS, thus, preserving optimal functions of mitochondria and preventing the cell death [63]. Generally, the on-site production of Mel could provide local protection of the mitochondria [64]. Interestingly, exogenously administered MEL can quickly penetrate the blood-brain-barrier and accumulate at high levels in each neuron compartment [65]. Compared to conventional antioxidants such as vitamin C and E, MEL is more effective in reducing OS in every neuronal compartment [64, 65]. MEL can act at two levels; firstly, as a direct antioxidant by its scavenging actions, MEL detoxify free radicals and their derivatives such ONOO– [66, 67], and secondly as an indirect antioxidant, this hormone up-regulates gene expression of antioxidant enzymes, such as SOD [68, 69]. Collectively, these reports confirmed the protective effect of MEL against oxidative damage.

Conclusions

In summary, our results confirmed some previous findings of the neurotoxic effect of Al and here we revealed that chronic administration of Al induced anxiety-like, depression-like, memory impairment and hippocampal OS in male rats. Moreover, MEL attenuates affective and cognitive disorders induced via Al intoxication possibly by inhibiting OS in the hippocampus. This investigation reinforces the neuroprotective potential of MEL.