Intrahippocampal effects of Aluminum injection on affective and cognitive response in male Wistar rat: Potential role of oxidative stress

doi:10.21203/rs.3.rs-2602113/v1

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Intrahippocampal effects of Aluminum injection on affective and cognitive response in male Wistar rat: Potential role of oxidative stress

https://doi.org/10.21203/rs.3.rs-2602113/v1

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Aluminum (Al) is one of the more widespread metals in the environment used in various fields and the most abundant known for its neurotoxicity in both humans and animals and could be a potential factor inducing behavioral changes, oxidative stress (OS) and loss of synapses and neurons in the hippocampal and cerebral cortical areas. The main objective of this study is to determine the short-term impact of a single 2 μl intracerebral injection of AlCl₃ at different doses on the right hippocampus on affective and cognitive behaviour, on levels of oxidative stress and morphological changes in male Wistar rats. Rats were treated with a single intrahippocampal injection of 2 μL of NaCl (0.9%) (Control) or successively with 2 μL of AlCl₃ at 0.5 mg/Kg (Al-0.5), 1 mg/Kg (Al-1) and 2 mg/Kg (Al-2). Five days following surgical procedures, neurobehavioral tests were performed for all groups (OFT, EPM, FST, Y-maze and MWM) and the brain were taken to isolate the hippocampus from adjacent tissues to prepare homogenates for the determination of oxidative stress markers and to examine the morphological change in CA3 hippocampal area. The results clearly demonstrate that Al induced anxiety and depressive-like behaviours, cognitive deficit, increased lipid peroxidation (LPO), nitric oxide (NO) levels, decreased superoxide dismutase (SOD) activity in the hippocampus and mediates progressive alterations characterized by disorganization in the pyramidal cellular arrangement and a decrease in neuronal density in the CA3 hippocampal area. In conclusion, a single intrahippocampal injection of Al induced anxiety-like, depression-like, memory impairment, OS and morphological alterations in the hippocampus.

Aluminum

Neurobehavioral alterations

Oxidative stress

Hippocampus

Aluminium (Al) is one of the more widespread metals in the environment and widely present in soil, air, water, and plants. Al compounds are extensively used for various purposes in food, beverages, cosmetics, and medicines (medicaments, vaccination and dialysis). It has no physiological role of Al in metabolic processes but it can be toxic to humans and animals when the metal's body burden is elevated after natural or unnatural exposure. Currently, the provisional tolerable weekly intake (PTWI) is set at 2 mg/kg/wk [1, 2]

Al induce numerous toxic effects such as immunological alterations, genotoxicity, pro-inflammatory effect, peptide denaturation or transformation, enzymatic dysfunction, metabolic derangement, amyloidogenesis, membrane perturbation, iron dyshomeostasis, apoptosis, necrosis and dysplasia but the major toxic action of Al is to generate oxidative stress by producing reactive free radicals that may overwhelm the cell's antioxidant defenses to perpetrate cell damage [3–6].

Moreover, various tissues have different susceptibilities to oxidative stress. In fact, the relationship between the oxidative stress and the development of affective and cognitive disorders could arise from the vulnerability of central nervous system (CNS) to oxidative damage [7].

Many studies have reported that excessive exposure to Al induce behavioral changes such as spatial memory deficits, reduced locomotors activity and impaired active and passive avoidance behaviors in rodents. Additionally, Al is both a neurotoxic agent and a potential risk factor for neurodegenerative diseases [8].

The main purpose of this experimental study is to determine the short-term effect of a single intracerebral injection of 2 µl AlCl₃ at different doses on the right hippocampus on affective and cognitive behaviour, on levels of oxidative stress and on morphological assessment on the CA3 hippocampal area in male Wistar rats.

2.1. Animals and experimental conditions

This study was conducted on 28 male Wistar rats initially weighing 250 ± 50g from the breeding of the Faculty of life sciences, University Ibn Tofaïl. All rats were maintained under LD 12/12 (12 h Light/12 h Darkness) and at a standard temperature (21 ± 1°C) during which water and food were provided ad libitum. Animals were divided into four groups of seven animals in each and were housed in transparent cages (36 cm long, 20 cm wide and 15 cm high). 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 received an intrahippocampal injection on the right ventral hippocampus of 2 μL of NaCl (0.9%) or 2 µl of AlCl₃ (SEGMA-ALDRICH) at different doses (0.5, 1 and 2 mg/kg) as following:

Groups	Drugs and doses
Group I (Control)	NaCl 0,9 %
Group II (Al-0.5)	AlCl₃ (0.5 mg/kg)
Group III (Al-1)	AlCl₃(1 mg/kg)
Group IV (Al-2)	AlCl₃(2 mg/kg)

After the intrahippocampal surgery period and before the neurobehavioral tests, the rats were housed in individual cages for 5 days to promote their physiological recovery. At the end of the tests, the rats were killed by decapitation. Then, the brains were removed to isolate the hippocampus from adjacent tissues and prepare homogenates for the assay of oxidative stress markers.

2.2. Stereotaxic Surgery and Injection Procedures

Rats were placed in a stereotaxic frame and the skull was oriented according to the protocol adapted by Ferry et al. [9]. Stereotaxic coordinates chosen for target the right ventral hippocampus are as follows: anteroposterior (AP): − 2.4 mm; mediolateral (ML): + 1.6 mm; dorsoventral (DV): − 3.4 mm; using bregma as a reference [10].

A Hamilton syringe with a cannula (0.3 mm diameter) was used to inject 2 μL of AlCl₃ or saline solution depending on the group studied. The injections were carried out for 2 min at a rate of 1 μL/min.

2.3. Neurobehavioral tests

2.3.1. Anxiety-Like Measurement

Open Field Test. The OFT is used to assess the anxiety-like behavior in rats following the administration of Al [11,12]. The rats were gently placed in the central part of the apparatus (100-L × 100-W × 40-H cm) and the following parameters were monitored for 10 min by video-tracking software animal: (a) The time spent in the central area (TCA), (b) the number of returns to the central area (NRC) and (c) 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 device was cleaned between each rat with 7% alcohol solution to remove odor clues.

Elevated Plus Maze (EPM). The EPM is used for assessing anxiety behavior in rodents [13]. The test was conducted in the second day after the OFT with an EPM apparatus (2 open arms (50 × 10 cm), 2 closed arms (50 × 10 × 40 cm), and a central area (10 × 10 cm). The rats were allowed to explore the maze for 5 min. A connected camera monitored the number of entries in open arms (EOA), the time spent in these arms (TOA) and the number of full entries into the arms (TAE). The TOA and EOA parameters were reported as the criteria of open space-induced anxiety-like behavior. 7 % alcohol was used to clean the apparatus prior to the introduction of each rat.

2.3.2. Depression-Like Measurement

Forced Swimming Test (FST). The FST, also known as behavioral despair test, is used to test the depressive-like behavior in rodents [14]. The rats were individually forced to swim in a cylindrical transparent glass (50 cm high and 30 cm in diameter) filled with purified water (35 cm, 23 ± 2°C), and the immobility time (TIM) is recorded by a video camera for 5 min. Depressive-like behavior is characterized by an increase in the TIM.

2.3.3. Cognitive Measurement

Y-maze test Spatial working memory was tested by the Y-maze spontaneous alternation test [15]. Each rat was placed in the central zone of the Y-maze and allowed to explore freely through the three arms (A, B and C; 61 × 35 × 12 cm³) for 8 min. The sequences of arms entries were recorded (i.e., ACBCABACABCABAC, etc.). Spontaneous alternation behavior that reflect spatial working memory was defined as an entry into the three different arms on consecutive choices. The % of spontaneous alternation was calculated by the following formula: % of Spontaneous alternation = [(Number of alternations)/(Total arms entries-2)] × 100.

Morris Water Maze Test (MWM). The water maze [16] consisted of a big round water tank (110 cm in diameter and 20 cm high) that was filled with opaque water and maintained a temperature at 22°C. The apparatus was divided into four quadrants of equal areas with a circular platform (13 cm high, 9 cm in diameter), submerged 0.5 cm beneath the water surface and placed at the center of the northeast zone. Rats were tested in two phases: acquisition and probe test [17]. During acquisition (4 trials/day for 4 days), each rat was placed individually into the water maze facing the wall of the pool and allowed to locate the hidden platform within a maximum swimming time of 60 seconds. If the rat did not locate the platform, it was guided and allowed to stay on it for 10 s. The time to find the platform was recorded using a video camera. On the fifth day, rats were tested for spatial memory in a 60 s probe trial [18].

2.4. 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 a 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 [19]. The TBARS levels in hippocampus is presented as nmol/g of tissue and were determined by the absorbance at 535 nm [20].

Nitrite/nitrate assay: The nitric oxide (NO) activity was assayed by the method of [21]. 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 [22]. SOD activity is reported as U/g of hippocampal tissue.

2.5. Histological analysis

For histological studies, brains were collected in 4% paraformaldehyde for proper fixation. Sections of the hippocampus (slices of 30 mm) were made with a vibratome and stained with cresyl violet. Following, the slices were mounted in gelatinized slides, dehydrated and mounted on a coverslip. We selected the CA3 area for light microscopic analyses at magnifications of ×20. After, the photomicrographs were taken and analyzed using ImageJ software [23,24].

The experimental design of all methodological steps of the study are summarized in Fig. 1.

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, biochemical and histological 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 repeated 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.

3.1. Effect of Al on the levels of anxiety-like measured in OFT (Fig. 2):

The results obtained show that the TCA and NRC parameters was significantly affected by Al treatment compared to control group [(Control/Al-1 and Control/Al-2: p < 0.001 for TCA parameter); (Control/Al-1: p < 0.01 and Control/Al-2: p < 0.001 for NRC parameter)]. Moreover, there is a statistically significant difference between the Al-treated groups [(Al-0.5/Al-1: p < 0.05; Al-0.5/Al-2: p < 0.001 and Al-1/Al-2: p < 0.01 for TCA parameter); (Al-0.5/Al-2: p < 0.001 and Al-1/Al-2: p < 0.01 for NRC parameter)]. In contrast, the NTS parameter which represents locomotors activity was unaffected.

3.2. Effect of Al on the levels of anxiety-like measured in EPM (Fig. 3):

Statistical analysis showed that the TOA and EOA parameters was significantly affected by Al treatment compared to their controls [(Control/Al-1 and Control/Al-2: p < 0.001 for TOA parameter); (Control/Al-2: p < 0.001 for EOA parameter)]. Additionally, there is a statistically significant difference between the Al-treated groups [(Al-0.5/Al-1: p < 0.01; Al-0.5/Al-2: p < 0.001 and Al-1/Al-2: p < 0.01 for TOA parameter); (Al-0.5/Al-2: p < 0.01 for EOA parameter)]. No statistically significant difference was found between the control and al-treated groups for TEA parameter.

3.3. Effect of Al on depressive-like performances measured by FST (Fig. 4):

Statistical analysis showed that TIM was significantly affected by the Al treatment. Al increases the TIM compared with the control group (Control/Al-1: p < 0.001 and Control/Al-2: p < 0.001). In addition, there is a statistically significant difference between Al-0.5/Al-1, Al-0.5/Al-2 and Al-1/Al-2 groups (p < 0.001).

3.4. Al Effect on memory:

3.4.1. Y-Maze Test (Fig. 5):

Spontaneous alternation percentage was affected by Al treatment. This latter significantly affects this parameter in dose-dependent manner, since at doses of 0.5, 1 and 2 mg/kg it decreases the spontaneous alternation percentage compared with the control group (p < 0.01, p < 0.001 and p < 0.001 respectively). In addition, there is a statistically significant difference between the Al-treated groups Al-0.5/Al-1, Al-1/Al-2 (p < 0.01) and Al-0.5/Al-2 (p < 0.001).

3.4.2 Morris Water Maze (Fig. 6):

Spatial learning (Fig. 6(A))

The latency to reach the hidden platform on each of the 4 days of learning phase in the Morris water maze was affected by Al treatment. We observed that the groups Al-1 and Al-2 spent significantly more time in finding the hidden platform compared to control (p < 0.01 and p < 0.001 respectively). In addition, there is a statistically significant difference between the Al-treated groups Al-0.5/Al-2: p < 0.001 and Al-1/Al-2: p < 0.05. In contrast, no difference was noted between Al-0.5/Al-1 groups (p < 0.05).

Percentage time spent in the correct quadrant during the probe test (Fig. 6(B)).

The statistical analysis showed that the percentage of time spent in the correct quadrant is significantly affected by Al treatment in dose-dependent manner, since at doses of 0.5, 1 and 2 mg/kg it decreases this parameter compared with the control group (p < 0.001, p < 0.001 and p < 0.001 respectively). In addition, there is a statistically significant difference between the Al-treated groups Al-0.5/Al-1 and Al-0.5/Al-2 (p < 0.001). In contrast, no difference was noted between Al-1/Al-2 groups (p < 0.05).

3.5. Al Effect on oxidative stress (Fig. 7):

LPO in hippocampus (Fig. 7(A)).

Statistical analysis showed that LPO reflected by TBARS levels was significantly affected by the Al treatment. As shown in Fig. 7A, there was a significant increase in lipid peroxidation (TBARS level) in the hippocampus of Al-treated groups as compared to the control (Control/Al-1: p < 0.01 and Control/Al-2: p < 0.001), whereas at 0.5 mg/kg Al was not effective (p > 0.05). Additionally, there is a statistically significant difference between the Al-treated groups Al-0.5/Al-1: p < 0.01, Al-0.5/Al-2: p < 0.001 and Al-1/Al-2: p < 0.01.

NO concentrations in hippocampus (Fig. 7(B)).

NO level was affected by Al treatment. At doses of 1 and 2 mg/kg, was associated with a significant increase of NO levels compared to control group (Control/Al-1: p < 0.01 and Control/Al-2: p < 0.001), while at dose of 0.5 mg/kg, Al did not provoke any significant change (p > 0.05). As well, there is a statistically significant difference between the Al-treated groups Al-0.5/Al-1: p < 0.01, Al-0.5/Al-2 and Al-1/Al-2: p < 0.001.

Superoxide Dismutase (SOD) activity in hippocampus (Fig. 7(C)).

SOD activity was affected by Al treatment. Al at dose of 1 and 2 mg/kg, was associated with a significant decrease of SOD activity compared to control group (p < 0.001), while at doses of 0.5 mg/kg this metal did not induce any significant change (p > 0.05). Moreover, there is a statistically significant difference between the Al-treated groups Al-0.5/Al-1: p < 0.01, Al-0.5/Al-2 and Al-1/Al-2: p < 0.001.

3.6. Histological analysis

Figure 8A showed that the number of intact pyramidal neurons in CA3 region of the hippocampus was affected by Al treatment. The single intrahippocampal injection of Al at the dose of 2 mg/kg induced a significant decrease in number of intact pyramidal neurons in CA3 region of the hippocampus compared to control group (p < 0.01), while at doses of 0.5 and 1 mg/kg this metal did not induce any significant change (p > 0.05). on the other hand, as illustrated by Fig. 8B, the control and Al-0.5 groups showed normal pyramidal neurons in CA3 sub-region, whereas, a neuronal loss and cellular disorganization were observed in Al-1 and Al-2 treated groups compared to their controls.

The main purpose of this experimental study is to determine the short-term effect of a single intrahippocampal injection of 2 µl AlCl₃ at different doses on affective and cognitive behavior, on levels of oxidative stress and morphological assessment on the CA3 hippocampal area in male Wistar rats.

Based on the neurobehavioral tests results, we found that compared to the control group, a single intrahippocampal injection of Al to rats induced anxiety-like, depression-like, as well as memory and learning disorders. The maximum effect is obtained at 2 mg/kg. Anxiogenic properties is based on the fact that Al decreasing the TCA and NRC parameters in the OFT and TOA and EOA parameters in EPM without modifying the locomotors activity. As well, Al exerts depressive effects in the FST that result in an increase in TIM parameter. In addition, data obtained through the MWM and Y-maze tests show that Al at the doses used causes cognitive impairment characterized by deficiency of working memory and spatial learning performance. These findings were parallel to our previous published paper, in which Al administrated chronically by intraperitoneal injection modulates anxiety-like, depression-like and memory impairment in male and female Wistar rats [25]. Consistent with the observations of other investigators stated that administration of aluminum chloride (AlCl₃) to rats has negative effects on behavioral endpoints (anxiety-like and depression-like) and can impair learning and memory abilities [6, 26–30].

On the other hand, biochemical parameters of OS in hippocampal tissue clearly demonstrate that Al-induced brain OS, this can be explained by the fact that exposure to Al significantly increase the levels of TBARS and NO although a decrease in SOD activity. Similar effects were also observed in other studies. Benyettou et al., reported that AlCl₃ is able to cause marked alterations in some behavioral and biochemical parameters by inducing an oxidative damage (increase TBARS and NO) and inhibiting the antioxidant enzymes activities (SOD, CAT, GPx and GR) [29]. Also, Auti and Kulkarni showed that AlCl₃ treatment increase OS in the brain which was indicated by decrease in level of CAT, SOD, GR, and increase in malondialdehyde (MDA) level leading to neuronal damage [31].

Al can infiltrate in the brain through the blood-brain barrier and accumulate in all brain regions of rat, the maximum being in the hippocampus, which is implicated in behavioral comportments, information storage and memory consolidation process of brain [32]. Moreover, the brain is particularly vulnerable to oxidative damage due to high lipid content, increased level of free radicals and low antioxidant enzymes concentration. It is well known that Al produced free radicals, enhanced mitochondrial activity and increased electron chain activity in the brain that cause a loss of cellular homeostasis and leads to neurotoxicity mainly by triggering OS [33], which can be hypothesized that OS could be one of the contributing factors for neurobehavioral alterations such as anxiety-like, depression-like and memory impairment.

Al accumulated in both neurons and astrocytes caused mitochondrial disturbance functions and antioxidant defense system impairment that may lead to generation of OS [34]. An increase in OS is related to an increase in LPO, which is an important biomarker of OS and in a decrease in antioxidant enzymes activities, which play a crucial role in preventing free radical damage such as SOD, CAT, GR and GPx [35]. In fact, the increased LPO in brain cells, leading to membrane damage and neuron death preferentially in pyramidal neurons in cortex and hippocampus area [36]. At the level of astroglial cells, Al induced profound morphological changes with the shrinkage of cell bodies, nuclear condensation and the fragmentation of DNA, which then leads to cell death [37].

Additionally, exposure to Al affects the axonal transports, induces inflammatory responses and leads to changes in the expression or activation of several intracellular signal transduction molecules (PKA, PKC, CaMKII, ERK, etc.) related to the LTP in CA1 area of hippocampus that can causes synaptic structural abnormalities, brain metabolic disorder and disturbance of ion metabolism especially calcium homeostasis, which resulting in profound memory loss [38–40]. Moreover, AlCl₃ exposure to rat increased ROS level, activated astrocytes and microglia, increased proinflammatory cytokines and mRNA expressions, and diminished anti-inflammatory cytokines contents in the hippocampus [34].

Histological analyses reveal that AlCl₃ mediates progressive alterations characterized by disorganization in the pyramidal cellular arrangement and a decrease in neuronal density in the CA3 hippocampal area as compared with the control group, which correlated with the biochemical changes revealed in this work. These findings were correlated to those claimed by Bittencourt et al., ; Fernandes et al., and Maya et al., [24, 32, 41]

These neurotoxic effects of Al may be justified by the tropism that this metal has for CNS structures and cause oxidative damage to hippocampal cells.

In summary, our results confirmed some previous findings of the neurotoxic effect of Al and here we revealed that a single intrahippocampal injection of Al induced anxiety-like, depression-like, memory impairment, OS and morphological alterations in the hippocampus. Al administration has been reported to induce oxidative stress by inflicting damage to membrane lipid, proteins and antioxidant enzyme defense system.

Acknowledgements

The authors would like to express their heartfelt and deepest condolences to family and friends of Professor Ali Ouichou. The neuroscience family in Morocco will miss one of its significant members, and the University Ibn Tofail has lost a great academic professor, who has contributed to the growth of the neuroscience community in Morocco. May his soul rest in peace.

Funding

No fund was received or no funding organization was involved in this study.

Conflict of interest

There are no conflicts of interests with other organizations.

Ethics approval

The experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee (Local Institutional Research Committee).

Author Contributions

Oussama Zghari and Sofia Azirar performed the experiments, analyzed the data and wrote the paper. Mouloud Lamtai participated in behavioral analysis and statistical significance. Aboubaker El Hessni reviewed and provided comments on the content and interpretation of the manuscript. Ali Ouichou and Abdelhalem Mesfiouisupervised the work, revised and approved the manuscript.

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Intrahippocampal effects of Aluminum injection on affective and cognitive response in male Wistar rat: Potential role of oxidative stress

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Abstract

Figures

1. Introduction

2. Materials And Methods

3. Results

3.1. Effect of Al on the levels of anxiety-like measured in OFT (Fig. 2):

3.2. Effect of Al on the levels of anxiety-like measured in EPM (Fig. 3):

3.3. Effect of Al on depressive-like performances measured by FST (Fig. 4):

3.4. Al Effect on memory:

3.4.1. Y-Maze Test (Fig. 5):

3.4.2 Morris Water Maze (Fig. 6):

3.5. Al Effect on oxidative stress (Fig. 7):

3.6. Histological analysis

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

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