Intrahippocampal dose-dependent effects of aluminum injection on affective and cognitive response in male Wistar rat: potential role of oxidative stress

ABSTRACT Aluminum (Al) is one of the more widespread metals in the environment 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 areas. The main objective of this study is to determine the impact of a single intracerebral injection of AlCl3 at different doses on the right hippocampus on affective and cognitive behaviors, on levels of OS 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 (Al-0.5), 1 (Al-1) and 2 mg/kg (Al-2). Five days following surgical procedures, neurobehavioral tests were performed for all groups and the brain were taken to evaluate OS markers and morphological changes in the hippocampus. The results clearly demonstrate that Al induced anxiety and depressive-like behaviors, cognitive deficit, increased lipid peroxidation, nitric oxide levels, decreased superoxide dismutase activity 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


Introduction
Aluminum (Al) is one of the most abundant metals in the environment.It is found in soil, air, water and plants.Al compounds are widely used for various purposes in food, beverages, cosmetics and pharmaceuticals (drugs, vaccines and dialysis).It has no physiological role in metabolic processes; however, it can be toxic to humans and animals if the body burden of the metal is increased after natural or unnatural exposure through oral, inhalation, and dermal routes.Currently, the provisional tolerable weekly intake (PTWI) is set at 2 mg/kg/wk [1,2].
nasal cavity, this heavy metal is distributed to all regions of the brain, mainly to the hippocampus [4,5].
Recent studies have shown that Al is associated with epilepsy [6], Alzheimer's disease [7] and autism [8] in humans.In animals, our team has demonstrated that daily administration of Al induces anxiety, depression and cognitive impairment in rats after two months of treatment [9].These effects are similar to those induced by other heavy metals such as Ni, Pb and Cu [10][11][12].
Generally, these Al-provoked behavioral alterations might involve a range of mechanisms including, neuronal death [13], disruption of neurotransmission implicated in emotional processes [14], and amyloid plaques deposition [15].However, the particular mechanisms by which Al induces behavioral disorders are still debatable.Oxidative stress (OS) has been proposed as a mechanism for the detrimental effects of this metal.
A growing body of evidence indicates that OS is connected to the etiology of the affective and cognitive dysfunctions [10][11][12].In fact, the brain is very vulnerable to OS, especially the hippocampus which plays a very important role in mood and memory functions [16].In this sense, previous studies have shown that Al favors the generation of free radicals and the dysfunction of antioxidant defense systems, resulting in the oxidation of lipids and proteins [17], which may contribute to the development of neurological disorders.
However, there is still a gap in the field to fully understand the mechanisms underlying the action of Al on behavior and OS markers.For this reason, this experiment aims to determine the short-term effect of a single intracerebral injection of 2 μl AlCl 3 at different doses into the right hippocampus on affective and cognitive behavior, on OS levels and on morphological assessment on the CA3 hippocampal area in male Wistar rats.

Animals and experimental conditions
This study was conducted on 28 male Wistar rats initially weighing 250 ± 50 g from the breeding of the Faculty of sciences, University Ibn Tofaïl.All rats were maintained under LD 12/12 (12 h Light/12 h Dark) 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  The rats received an intrahippocampal injection on the right ventral hippocampus of 2 μL of NaCl (0.9%) or 2 µl of AlCl 3 (SEGMA-ALDRICH) at different doses (0.5, 1 and 2 mg/kg) as following:

Stereotaxic surgery and injection procedures
The rats were placed in a stereotaxic frame and the skull was oriented according to the protocol adapted from Ferry et al. [18].The stereotaxic coordinates chosen to target the right ventral hippocampus are the following: anteroposterior (AP): −2.4 mm; mediolateral (ML): +1.6 mm; dorsoventral (DV): −3.4 mm; using bregma as a reference [19].Depending on the group studied, 2 μL of AlCl 3 or saline was injected using a Hamilton syringe with a 0.3 mm diameter cannula.Injections were performed for 2 minutes at a rate of 1 μL/min.
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.

Open Field Test
The OFT has been used to assess the anxietylike behavior in rats following the Al administration [20,21].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 videotracking 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 anxiety level is inversely proportional to the number of entries and time spent in the central area.To remove odor cues, the apparatus was cleaned with 7% alcohol solution between each rat.

Elevated Plus Maze (EPM)
The EPM has been used for assess anxiety behavior in rodents [22].The test was conducted on the second day after OFT.The EPM apparatus consisted of 2 open arms (50 × 10 cm), 2 closed arms (50 × 10 × 40 cm), and a central area (10 × 10 cm).Rats were allowed to explore the maze for 5 min.A connected camera monitored the number of entries into the 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 for open space-induced anxiety-like behavior.Seven percent alcohol was used to clean the apparatus prior to the introduction of each rat.

Forced Swimming Test (FST)
The FST, also known as the behavioral despair test, is used to test the depressive-like behavior in rodents [23].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.

Y-maze test
Spatial working memory was tested using the Y-maze spontaneous alternation test [24].Each rat was placed in the central zone of the Y-maze and allowed to freely explore the three arms (A, B and C; 61 × 35 × 12 cm 3 ) for 8 min.The sequences of arms entries were recorded (i.e.ACBCABACABCABAC, etc.).Spontaneous alternation behavior, reflecting spatial working memory, was defined as an entry into the three different arms on consecutive choices.The % of spontaneous alternation was calculated using the following formula: % of Spontaneous alternation = [(Number of alternations)/(Total arms entries-2)] × 100.

Morris Water Maze Test (MWM)
The water maze [25] 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 in the center of the northeast zone.Rats were tested in two phases: acquisition and probe test [26].During acquisition (4 trials/day for 4 days), each rat was individually placed in the water maze facing the pool wall and allowed to locate the hidden platform within a maximum swimming time of 60 seconds.If the rat failed to locate the platform, it was guided and allowed to remain on the platform for 10 s.The time to find the platform was recorded with a video camera.On the fifth day, the rats were tested for spatial memory in a 60 s probe trial [27].
At the end of the tests, the rats were killed by decapitation.Brains were then removed to isolate the hippocampus from adjacent tissues and to prepare homogenates for oxidative stress marker assay and histologic analysis.

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 kept on ice at a low temperature.Hippocampus was quickly and gently removed and separated from the 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 for the biochemical assays.

Lipid peroxidation assay
The formation of lipid peroxides during the lipid peroxidation process was analyzed by measuring the thiobarbituric-acid-reacting substances (TBARS) in cells, as previously described by Draper and Hardley [28].Briefly, the samples were mixed with 10% trichloroacetic acid and 0.67% thiobarbituric acid and heated in a boiling water bath for 15 minutes.After adding butanol (2:1 v/v) to the solution and centrifuging at 800 g for 5 minutes, TBARS levels in hippocampus are expressed as nmol/ g of tissue and were determined by the absorbance at 535 nm [29].

Nitrite/nitrate assay
The nitric oxide (NO) activity was assayed by the method of [30].Briefly, samples (500 µL) of the hippocampus supernatant was pipetted into tubes.An equal volume of Griess reagent was added to each tube, which contained 1% sulphanylamide (1 mL) and 0.1% N-1-naphtylethylenediamine dihydrochloride (1 mL) in 2.5% orthophosphoric acid.The tubes were incubated for 30 minutes at room temperature to allow the Griess reagent to react with nitrite and form a colored product.After incubation, the absorbance of the colored product was measured at 540 nm using a spectrophotometer.Nitrite concentrations were calculated using linear regression analysis.Tissue nitrite levels are 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 [31].The method for measuring SOD activity was based on the inhibition of nitroblue tetrazolium (NBT) reduction.The process involves illuminating riboflavin in the presence of O 2 and an electron donor such as methionine, which generates superoxide anions.The reaction mixture, containing 0.94 mL of 50 mM phosphate buffer (pH 7.4), 12 mM methionine, 75 µM NBT, 0.1 mM EDTA, 0.025% Triton X-100, 2 µM riboflavin, and 0.06 mL of supernatant, was placed in yellow light for 10 min.The reduction of NBT to formazan by superoxide radicals was monitored at 560 nm, and the SOD activity was expressed as the amount of enzyme required to inhibit the reduction of NBT by 50% under the given conditions, which is defined as one unit of SOD activity.It is important to note that a control without the enzyme source was included in the assay.SOD activity is reported as U/g of hippocampal tissue.
Hippocampal sections (slices of 30 mm) were prepared using a vibratome (VT 1000 S, Leica Microsystems).The slices were then mounted in gelatinized slides, stained with cresyl violet, dehydrated and mounted on a coverslip.The CA3 area selected for light microscopic analysis at a magnification of × 20 (Optika microscope, Italy).Photomicrographs were then taken and analyzed using ImageJ software [33,34].
The experimental design of all methodological steps of the study are summarized in Figure 1.

Statistical analysis
All data are expressed as the mean ± standard error of the mean (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 was 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.

Effect of Al on depressive-like performances measured by FST (Figure 4)
Statistical analysis showed that TIM was significantly affected by the Al treatment.Al increases the TIM compared to 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).

Y-Maze Test (Figure 5)
Spontaneous alternation percentage was affected by Al treatment.This latter significantly  affects this parameter in a dose-dependent manner, since at doses of 0.5, 1 and 2 mg/kg it decreases the spontaneous alternation percentage compared to 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).

Morris Water Maze (Figure 6)
• Spatial learning (Figure 6(a)) The latency to reach the hidden platform on each of the 4 days of the learning phase in the Morris water maze was affected by Al treatment.We observed that the groups Al-1 and Al-2 groups spent significantly more time in finding the hidden platform compared to the 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 found between the Al-0.5/Al-1groups (p < 0.05).
• Percentage time spent in the correct quadrant during the probe test (Figure 6(b)).
Statistical analysis showed that the percentage of time spent in the correct quadrant is significantly affected by Al treatment in a dose-dependent manner, since at doses of 0.5, 1 and 2 mg/kg it decreases this parameter compared to 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 found between the Al-1/Al-2 groups (p < 0.05).
Statistical analysis showed that LPO, as reflected by TBARS levels, was significantly affected by the Al treatment.As shown in Figure 7a, there was a significant increase in lipid peroxidation (TBARS levels) 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 levels were affected by Al treatment.At doses of 1 and 2 mg/kg, was associated with a significant increase in NO levels compared to SOD activity was affected by Al treatment.Al at doses of 1 and 2 mg/kg was associated with a significant decrease in SOD activity compared to the 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.

Discussion
The main purpose of this experimental study is to determine the short-term effect of a single intrahippocampal injection of 2 μl AlCl 3 at different doses on affective and cognitive behavior, on levels of OS and on morphological assessment of the CA3 hippocampal area in male Wistar rats.Based on the neurobehavioral test results, we found that compared to the control group, a single intrahippocampal injection of Al in rats induced anxiety-like, depression-like, as well as memory and learning disorders.The maximum effect is obtained at 2 mg/kg.The anxiogenic properties are based on the fact that Al reduces the TCA and NRC parameters in the OFT and the TOA and EOA parameters in the EPM without modifying locomotor activity.Al also exerts depressive effects in the FST, resulting in an increase in the TIM parameter.In addition, data from the MWM and Y-maze tests show that Al, at the doses used, causes cognitive impairment characterized by deficits in working memory and spatial learning performance.These findings were in parallel with our previously published work in which chronic administration of Al by intraperitoneal injection modulated anxiety-like, depression-like and memory impairment in male and female Wistar rats [9].Consistent with the observations of other investigators, it was found that administration of AlCl 3 to rats has negative effects on behavioral endpoints (anxiety-like and depression-like) and may impair learning and memory.A study by Benyettou et al. found that AlCl 3 exposure induced anxiety-like behavior in rats [35], while another study by Rebai et al. reported that AlCl 3 exposure caused depression-like behavior in rats [36].Similarly, a study by Tair et al. found that AlCl 3 exposure impaired spatial memory in rats [37], while a study by Cao et al. reported that AlCl 3 exposure impaired both spatial and recognition memory in rats [38].Moreover, the effects of AlCl 3 on behavior in rats may be influenced by various factors, such as age, gender, route and duration of exposure.Overall, the evidence suggests that AlCl 3 exposure may have negative effects on behavior in rats.The difference between this study and the previous ones is that a single intrahippocampal injection of AlCl 3 was the cause of the range of disorders, including anxiety, depression, and memory impairment.These findings have important implications for human health, as Al is a widely used metal and exposure to it is common in daily life.The mechanisms underlying these effects are complex and involve multiple pathways, including OS, inflammation and alterations in neurotransmitter signaling.
On the other hand, biochemical parameters of OS in hippocampal tissue clearly demonstrate that Al-induced brain OS, which can be explained by the fact that exposure to Al significantly increases the levels of TBARS and NO despite a decrease in SOD activity.Similar effects have been observed in other studies.Benyettou et al., reported that AlCl 3 is able to cause marked alterations in some behavioral and biochemical parameters by inducing an oxidative damage (increase in TBARS and NO) and inhibiting the antioxidant enzymes activities (SOD, CAT, GPx and GR) [35].Auti and Kulkarni also showed that AlCl 3 treatment increased OS in the brain, which was indicated by a decrease in the levels of CAT, SOD, GR, and an increase in level of malondialdehyde (MDA), leading to neuronal damage [39].Several investigators have suggested a link between OS and the etiology of affective and cognitive impairments.
Al can infiltrate in the brain through the BBB and accumulate in all brain regions of the rat, with maximum accumulation in the hippocampus, which is implicated in behavioral comportments, information storage and memory consolidation processes of the brain [40].Moreover, the brain is particularly vulnerable to oxidative damage due to its high lipid content, increased levels of free radicals and low antioxidant enzymes concentration.It is well known that Al produces free radicals, enhances mitochondrial activity and increased electron chain activity in the brain, causing a loss of cellular homeostasis and leading to neurotoxicity mainly by triggering OS [41], 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 accumulates in both neurons and astrocytes, causing mitochondrial dysfunction and antioxidant defense system impairment that may lead to generation of OS [42].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 [43].In fact, increased LPO in brain cells leads to membrane damage and neuronal death preferentially in pyramidal neurons in the cortex and hippocampus area [44].At the level of astroglial cells, Al induced profound morphological changes with cell body shrinkage, nuclear condensation and DNA fragmentation leading to cell death [45].
In addition, Al is likely capable of inducing NO production in the rat brain [46], which may be due to the ability of this metal to increase the expression of NO synthase (NOS) [47].At high levels, NO can react with the superoxide ion (O 2-) to form the ion peroxynitrite (ONOO − ), a reactive toxic molecule that can elicit cellular damage by initiating the LPO formation, leading to neuronal cell damage [48], and consequently, an altered behavior.
Additionally, Al exposure affects axonal transport, induces inflammatory responses and leads to changes in the expression or activation of several intracellular signaling molecules (PKA, PKC, CaMKII, ERK, etc.) related to the LTP in the CA1 area of the hippocampus, which may cause synaptic structural abnormalities, brain metabolic disorder and disturbance of ion metabolism especially calcium homeostasis, which resulting in profound memory loss [49][50][51].Furthermore, AlCl 3 exposure in rats increased ROS levels, activated astrocytes and microglia, increased proinflammatory cytokines and mRNA expressions, and decreased anti-inflammatory cytokine levels in the hippocampus [42].
Histological analyses reveal that AlCl 3 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 correlated with those reported by Bittencourt et al.,;Fernandes et al.,and Maya et al. [34,40,52].
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.

Conclusion
In conclusion, our results confirmed some previous findings on the neurotoxic effect of Al, and here we showed that a single intrahippocampal injection of Al-induced anxiety-like, depression-like, memory impairment, OS and morphological changes in the hippocampus.
Further research is needed to fully understand the mechanisms underlying aluminum chloride-induced behavioral changes in rats and to develop effective strategies for prevention and treatment.

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

3 )Figure 1 .
Figure 1.Diagram of the experimental design shown in a timeline from intrahippocampal injection to sacrifice.After intrahippocampal injection, the rats were subjected to behavioural tests for evaluation by open field test (OFT), elevated plus maze (EPM), forced swimming test (FST), Y-maze and Morris water maze (MWM) test.Animals were euthanized and the hippocampus was harvested.Markers of oxidative stress (lipid peroxidation (LPO), nitric oxide (NO) levels and superoxide dismutase (SOD) activities) and histopathological of the CA3 area of the hippocampus were then evaluated.

Figure 6 .
Figure 6.(a): Latency to reach the hidden platform on each of the 4 days of the learning phase in the morris water maze; (b): Percentage of time spent in the correct quadrant in the probe trial of the morris water maze expressed as % in male rats after an intrahippocampal injection of 2 μL of NaCl (0.9%) (Control) or 2 μL of 0.5 mg/kg (Al-0.5), 1 mg/kg (Al-1) and 2 mg/kg (Al-2) of AlCl 3 .Results are expressed as mean ± SEM.The significance level is 0.05.*p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8 (
Figure 8(a)  shows that the number of intact pyramidal neurons in CA3 region of the

Figure 8 .
Figure 8. a.Effect of treatment with different doses of Al on the number of neurons in the 0.18 mm 2 area of the CA3 region of the hippocampus in male rats.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.b.Light micrographs of cresyl violet-stained hippocampal CA3 cell layer in control (a), Al-0.5 (b), Al-1 (c), and Al-2 (d) male rat groups, magnified ×20.Bar represents 100μm.
Al: Aluminum; AlCl3: Aluminum chloride; BBB: blood-brain barrier; CA: Cornu Ammonis; CNS: Central nervous system; EOA: Entries in open arms; EPM: Elevated Plus Maze; FST: Forced Swimming Test; LPO: Lipid peroxidation; MEL: Melatonin; MWM: Morris Water Maze; NO: Nitric oxide; NRC: Number of returns to the center; NTS: Number of total squares; OFT: Open Field Test; OS: Oxidative stress; ROS: Reactive oxygen species; SOD: Superoxide dismutase; TAE: Number of full entries into the arms; TCA: Time spent in the center area; TIM: Immobility times; TOA: Time spent in these arm.
long, 20 cm wide and 15 cm high).All experimental procedures were carried out strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the