Asiatic acid attenuates aluminium chloride-induced behavioral changes, neuronal loss and astrocyte activation in rats

Aluminium (Al) is a potent neurotoxic metal known to cause neurodegeneration. Al exposure causes oxidative stress by accumulation of reactive oxygen species, followed by the activation of neuronal cell death in the brain. Asiatic acid (AA), the major bioactive compound of Centella asiatica (a medicinal plant), act as multifunctional drug as well as an antioxidant. Thus, the present study aimed to investigate the potential neuroprotective effect of AA against Al neurotoxicity. Rats were orally administered aluminium chloride (AlCl3; 100 mg/kg b. wt.) dissolved in distilled water for 8 weeks or AA (75 mg/kg b. wt.) in combination with AlCl3. The results showed that AlCl3-intoxication causes significant impairment of memory, enhances anxiety-like behavior, acetyl cholinesterase (AChE) activity, malondialdehydes (MDA) level, and concomitant decrease in the activities of superoxide dismutase (SOD) and catalase (CAT) in the cortex and hippocampus regions of rat brain. In addition, AlCl3-intoxication enhanced neuronal loss and reactive astrogliosis in both regions. However, co-administration of AA with AlCl3 significantly attenuated the behavioral alterations, restored SOD and CAT activities, while reduced AChE activity and MDA content. Further, the study demonstrated that AA attenuates neuronal loss and reactive astrogliosis in rat brain. In conclusion, the study suggests that AA protects rat brain from Al neurotoxicity by inhibiting oxidative stress, neuronal loss and reactive astrogliosis.


Introduction
Aluminium (Al) is a highly abundant neurotoxic metal that affects the human population around the world. Besides the presence in nature, antacids, foils, cosmetics, deodorants, vaccines etc. are also sources of Al exposure. Hence, high abundance of Al in the environment makes human at high risk of its exposure via air, water and food (Krewski et al. 2007). Normal Al concentration in human body is in range of 0.1-0.4 µg Al/g tissue in dry wt. (Exley and House 2011). Beyond this limit, Al concentration causes multiple neurological defects like Alzheimer's disease, Autism, Multiple sclerosis (Exley and Clarkson 2020), Parkinson's disease and Dementia (Maya et al. 2016) including neurotoxicity (Exley 2014). The human population exposed to Al results in its accumulation in different tissues including the brain (Julka and Gill 1996). Moreover, animal studies demonstrated accumulation of Al in different brain regions following its longterm exposure (Nehru and Bhalla 2006;Sethi et al. 2008).
Accumulating body of evidence demonstrates that neurotoxic action of Al is associated with the impairment of memory and coordination, anxiety, tremor, and jerking movement in exposed workers (Kiesswetter et al. 2007). Multiple animal studies reported that long-term exposure of Al causes neurobehavioral, pathological and neurochemical changes in the brain and alters spatial memory and learning (Sethi et al. 2009;Junior et al. 2013;Zhao et al. 2020). Acetylcholine esterase (AChE), a prominent marker of neurotoxicity as well as a key modulator of memory and hippocampal plasticity has also shown to be affected following Al exposure (Kaizer et al. 2005). The brain is considered as a highly vulnerable organ to Al toxicity, owing to the presence of non-dividing neuronal cells. Moreover, the brain tissue has high polysaturated fatty acids content that makes it extremely sensitive to free radicals, following toxic insults (Joffre 2019). The emerging evidence for literature suggests that Al exposure generates reactive oxygen species (ROS) which in turn imbalance pro/ antioxidant system. Excessive ROS disrupt cellular antioxidant defense system in multiple neurodegenerative disorders (Lee et al. 2021). Malondialdehydes (MDA) are important marker of lipid peroxidation (LPO) and their elevated level depicts oxidative stress status of the cells. Enhanced oxidative stress causes damage to cellular biomolecules such as DNA, lipids and proteins, hence, cells commence under more oxidative stress (Singh et al. 2019). Earlier studies have shown that Al exposure causes LPO and protein oxidation along with decline in the activities of antioxidant enzymes viz. glutathione peroxidase, superoxide dismutase (SOD), catalase (CAT) etc. (Prakash and Kumar 2013;Nehru and Bhalla 2006).
Astrocytes play a crucial role in nourishing of neurons by supplying the glucose and neurotrophic factors as well as protect them from oxidative stress. In case of toxic insults or disease state, neural health gets affected, where astrocytes play vital role in survival and functioning of neurons (Sofroniew and Vinters 2010;Wang and Xu 2020). Evidence from animal studies indicates that Al toxicity causes astrogliosis and neuronal cell death (Junior et al. 2013;Laabbar et al. 2021). Hence, maintenance and protection of astrocytes can be of therapeutic interest that modulate functioning and degeneration of neurons (Jeong et al. 2014) in Al toxicity.
Centella asiatica (C. asiatica), an important medicinal herb from Apiaceae family, has been used from decades in Ayurvedic and Chinese medicines, and known as a memory enhancement tonic (Puttarak et al. 2017;Sun et al. 2020). This herb has been reported to have antioxidant, antiapoptotic, mitoprotective and neuroprotective properties (Prakash and Kumar 2013;Tabassum et al. 2013;Sun et al. 2020). Asiatic acid (AA) is a major bioactive compound responsible for antioxidative and therapeutic properties of C. asiatica (Nagoor Meeran et al. 2018;Krishnamurthy et al. 2009). AA attenuates cognitive deficits in animal model of monosodium glutamate-induced Dementia and protects SH-SY5Y cells against glutamate-induced apoptosis (Xu et al. 2012). In addition, AA exerts protective effect against spinal cord injury by suppressing inflammation and oxidative stress (Jiang et al. 2016). Recent studies investigated that AA mitigates Al-induced Alzheimer disease-associated pathologies such as behavioral impairments, Aβ burden and activation of inflammatory, and apoptotic pathways (Rather et al. 2018(Rather et al. , 2019. Therefore, in this study we examined the effect of AA in the spatial memory and learning, and anxiety in aluminium chloride (AlCl 3 )-exposed adult male Wistar rats. Furthermore, AChE activity, oxidative stress indices (LPO, and activities of SOD and CAT) and neuronal morphology were evaluated. The neuronal density and reactive astrogliosis were also assessed using immunohistochemical staining against neuronal nuclear protein (NeuN) and glial fibrillary acid protein (GFAP).

Animals and their care
Male Wistar rats weighing 180-200 g were procured from Central Laboratory of Animal Research, Jawaharlal Nehru University (JNU), New Delhi, and provided ad libitum supply of food and water. The animals were housed at standard hygienic conditions with a light/dark cycle of 12 h and 25-28 ℃ temperature. The use of rats in present study was approved (IAEC code: 27/2018) by the Institutional Animal Ethics Committee of JNU, New Delhi and all the experiments were conducted as per the Committee for the Purpose of Control and Supervision of Experiments on Animals guidelines.
Doses of AlCl 3 (Prakash and Kumar 2013) and AA (Krishnamurthy et al. 2009;Rather et al. 2018) were taken from literature. All administrations were given 5 days per week via oral gavage for 8 weeks. Literature suggests that 100 mg/kg b. wt. of AlCl 3 exposure to rats is reflecting adverse effects similar to the representative human dietary exposure of Al. Moreover, to effectively promote the adverse effects sufficient exposure time is required, hence, 8 weeks of AlCl 3 exposure period was chosen (Martinez et al. 2016(Martinez et al. , 2018. DW and CMC were employed as vehicle for AlCl 3 and AA respectively. Detailed schematic outline of the experimental design including studied parameters is given in Fig. 1.

Morris water maze (MWM) test
Spatial learning and memory of rats (n = 6) was examined by MWM test (Morris 1984) with modifications. The water maze comprised of a circular black-painted tank (Diameter: 168 cm and Depth: 50 cm) with cues of various shapes. A circular (hidden) black-painted platform (Diameter: 15 cm) was placed in the middle of a quadrant, 2.0 cm beneath the water surface, so that rats can flee from swimming. The tank perimeter was labeled as east, west, north and south. Prior to start of experimentation rats were acclimatized to the setup and tested for their swimming capability. Rats were kept once in each quadrant of the tank, facing towards the wall for all four entry points. In case, rats were not able to find the hidden platform in 120 s, were guided to find the same and let them stay for 20 s. MWM test was started four days prior to the last doses, conducted for 5 consecutive days and recorded using a camera placed on the top of the tank. The test was performed amid 11:00 AM and 3:00 PM, to eliminate the variations.

Light and dark (L/D) test
At the end of treatment period, the L/D test was carried out in rats (n = 6) to access the anxiety-like behavior as described by Ramos et al. (2003). The light/dark setup of plexiglass (48 cm × 24 cm × 27 cm) was separated into two chambers through a barrier consisting of a doorway (10 cm × 10 cm). One chamber was made dark with a lid while other was illuminated by a bulb (60-W) placed above the apparatus. On the experiment day, rats were placed inside the dark room and remained undisturbed for 2 h. Afterwards, rats were centrally positioned in the lit chamber, facing opposite to the dark one. Then, rats were allowed to freely explore between both the chambers for 5 min and recorded. The defecation index, number of transitions between light/ dark chambers, and time spent in the light chamber were calculated.

Open field test (OFT)
Further, the OFT test was also performed to validate the anxiety-like behavior of rats (n = 6) in an open field arena. Rats were examined for their exploratory behavior as previously described by Sethi et al. (2009). Rats were positioned in the middle of arena and locomotor activity was analysed as the number of central and peripheral square entries. The number of squares entries done by rats within a span of 5 min were recorded and the number of faecal boli was counted to calculate defecation index. Prior to every trial, the floor was thoroughly cleaned with 70% ethanol.

Tissue homogenate preparation
Four rats from each group were euthanized by passing high flow rate of CO 2 (16 L/min) and decapitated. The intact brain was isolated followed by dissecting out cortex and hippocampus. The cortex and hippocampi of both sides (left and right) were combined to prepare one sample. All the samples were homogenized (10% w/v) using a tissue homogenizer in the homogenizing medium (0.1 mM Fig. 1 Schematic representation of the experimental design of the study EDTA, 0.32 M sucrose, 10 mM Tris-HCl, pH 7.4). The resulting mixture was then spun at 5000 x g for 10 min at 4 °C, supernatant was transferred to a fresh tube and stored for the biochemical analysis. The protein concentration of each sample was estimated by Bradford's (1976) method using BSA as standard.

AChE activity assay
AChE activity was estimated as per the method of Ellman et al. (1961). A reaction mixture comprising of 2.35 ml sodium phosphate buffer (0.1 M, pH 7.4), 0.2 ml DTNB (10 mM), 0.2 ml Triton X-100 (0.013%), 0.05 ml tissue homogenate and 0.2 ml ATch (10 mM) was prepared. Finally, the change in absorbance was measured spectrometrically at 412 nm and results were expressed as nmol ACh hydrolyzed/min/mg protein.

LPO assay
LPO was measured as described by Kaizer et al. (2005) with few modifications. Briefly, 50 µl tissue homogenate was mixed with 16.66 µl SDS (2.5%), 62.5 µl acetic acid (20%) and 37.5 µl TBA (1.33%). The reaction mixture was incubated for 1 h at 95℃ in water bath. Then, samples were cooled and centrifuged for 10 min (4000 x g, 4℃). Subsequently, the absorbance of thiobarbituric acid-reactive substances was recorded at 532 nm from the upper organic layer (supernatant). The results were expressed as nmol MDA/mg protein.

SOD activity assay
The activity of SOD was estimated as per the method of Marklund and Marklund (1974). Briefly, 1 ml of pyrogallol solution (20 mM) was mixed with equal volume of tris-buffer (50 mM), followed by addition of 50 µl of tissue homogenate. The absorbance of reaction mixture was measured at 420 nm. Enzyme needed for 50% inhibition of pyrogallol autoxidation was considered as 1 U and the results were expressed as U/mg protein.

CAT activity assay
The activity of CAT was estimated as per the method of Koroliuk et al. (1988), with slight modifications. The assay mixture was prepared with 0.1 ml tissue homogenate and 0.2 ml potassium phosphate buffer (50 mM, pH 7.0) consisting of 0.1 ml H 2 O 2 (30%). Then, 0.2 ml ammonium molybdate (32.4 mM) was added to stop the reaction. The absorbance was measured at 405 nm and results were expressed as µmol H 2 O 2 oxidized/min/mg protein.

Sample preparation for immunofluorescence and histopathology
Three rats from each group were perfused transcardially with normal saline (0.9% NaCl) and 2% paraformaldehyde (PFA). Afterwards, brain was post-fixed with 2% PFA overnight, passed through 10, 20, and 30% of sucrose solutions and stored in 30% sucrose solution. Coronal Sect. (15 μm; IF and 30 μm; CV staining) of brain tissue were cut with the help of a Cryostat (Leica CM 1860, Germany), affixed on gelatin-coated slides and stored at -20 ℃ for histopathological and immunofluorescence analysis.

Histopathological study
Histopathological examination of brain tissue was carried out by CV staining. Briefly, the tissue sections were stained with CV after air-drying for few min. Afterwards, a graded series of alcohol was used to dehydrate the sections and cleared with xylene by dipping twice (5 min each). Lastly, slides were mounted with DPX, coverslipped and observed under a light microscope (Motic Instruments Co. Ltd., Chengdu, China).

Immunofluorescence examination
For immunoreactivity study, sections were air-dried at room temperature for few min followed by 3 washes of phosphate buffer saline (PBS) for 5 min each. Next, sections were incubated for 10 min with 0.5% Triton-X100, followed by three washes of PBS. Nonspecific antigens were blocked by 3% normal goat serum. Then, sections were covered with mouse anti-GFAP (3% BSA, 1:100 dilutions, Invitrogen, Carlsbad, USA) and mouse anti-NeuN (3% BSA, 1:100 dilutions, GenTex, Inc. CA, USA) monoclonal antibodies and incubated overnight at 4 °C. After 3 washes, sections were covered with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (3% BSA, 1:200 dilutions, Invitrogen, Carlsbad, USA) and incubated in dark for 2 h at room temperature, then washed with PBS thrice. Next, sections were stained with DAPI for 10 min to visualize nucleus, followed by PBS washes (3 times). Finally, sections were mounted with Fluoromount™ Aqueous Mounting Medium and cover slipped. Sections were observed under a fluorescence microscope (Nikon Eclipse 90i, Tokyo, Japan), images were captured and analysed.

Statistical analysis
The statistical analyses of data were carried out using Sig-maStat 3.5 (Systat Software Inc., San Jose, CA, USA) and were represented as mean ± SEM. Statistical differences among the groups were analysed using one-way analysis of variance (ANOVA) followed by Tukey test for MWM and Holm-Sidak test for rest of the assays. The P value of ≤ 0.05 was considered statistically significant for all the assays.

MWM test
The MWM test was performed to examine memory consolidation by monitoring average escape latency to locate a camouflaged platform in experimental rats. Results demonstrated that AlCl 3 -intoxicated rats display significantly extended escape latency on 2nd, 3rd, 4th, and 5th days than respective control rats. However, co-administration of AA along with AlCl 3 significantly reduced the escape latency as compared to AlCl 3 -intoxicated rats on respective days. The rats treated with AA alone exhibited no significant difference in escape latency with respect to control rats. These results suggest that supplementation of AA rescued spatial learning and memory deficits in AlCl 3 -intoxicated rats (Fig. 2). Table 1 AlCl 3 -intoxicated rats exhibited a significant increase in defecation index, number of transitions between light/dark chambers and reduced time spent in light chamber as compare to controls. These results suggest anxiety-like behavior of AlCl 3 -intoxicated rats. Co-administration of AA with AlCl 3 significantly decreased defecation index and increased number of transitions, and time spent in the light chamber with respect to AlCl 3 -intoxicated rats. While, the rats treated with AA alone showed no significant differences in these parameters in comparison to controls. Overall, these results suggest that AA supplementation improved anxiety-like behavior in AlCl 3 -intoxicated rats.

OFT test
Anxiety-like behavior in experimental rats was further validated by OFT. The results showed that Al-intoxicated rats exhibit significantly increased defecation index and decreased locomotor activity when compared with controls, hence, validate anxiety-like behavior in Al-intoxicated rats. AA co-administration significantly reduced the defecation index and increased locomotor activity in comparison to AlCl 3 -intoxicated rats. While, the alone treatment of AA caused no significant changes in these parameters in comparison to control rats (Table 1). Thus, these results suggest that AA may have reversed the adverse effects of AlCl 3 exposure.

Effect of AA on AChE activity
The results exhibited significantly enhanced AChE activity in Al-intoxicated rats as compare to controls in the cortex and hippocampus of the brain. Co-administration of AA effectively declined the AChE activity in both regions as compare to Al-intoxicated rats. The alone treatment of AA showed no significant difference with respect to control rats (Fig. 3a). Thus, the results suggest that AA significantly decreased the AChE activity in AlCl 3 -intoxicated rats.

Effect of AA on LPO
The effect of AA on LPO in AlCl 3 -intoxicated rats is presented in Fig. 3b. Results showed significantly increased level of LPO in the cortex and hippocampus of AlCl 3 -intoxicated rats as compare to controls. Contrary to this, AA co-administered with AlCl 3 displayed a significant decline of LPO in the cortex and hippocampus regions as compare to AlCl 3 -intoxicated rats. The treatment of AA alone caused no significant difference as compare to controls. Thus, it can be observed from these results that AA reduces AlCl 3 -induced LPO.

Effect of AA on antioxidant enzymes
A significant decline of SOD activity in the cortex and hippocampus was observed in AlCl 3 -intoxicated rats with respect to controls; whereas rats treated with AA along with AlCl 3 exhibited a significant increase of SOD activity when compared to AlCl 3 -intoxicated rats (Fig. 4a). The Fig. 2 Latency plot for platform search in AlCl 3 and AA-treated rat. The latency to locate the platform is more in AlCl 3 -intoxicated rats as compare to controls, while decreased in AA co-treated rats with respect to AlCl 3 -intoxicated rats. Each data point represents mean ± SEM of 6 rats. *** P ≤ 0.05 significantly different from control group and ### P ≤ 0.05 significantly different from AlCl 3 -intoxicated group Similarly, the activity of CAT was also significantly declined in the cortex and hippocampus regions of AlCl 3 -intoxicated rats; whereas rats treated with AA along with AlCl 3 exhibited a significant increase of CAT activity as compared to AlCl 3 -intoxicated rats (Fig. 4b). The alone treatment of AA exhibited no significant difference of CAT activity with respect to control rats. Overall, these results suggest that AA significantly restored the activity of antioxidant enzymes in AlCl 3 -intoxicated rats.

Effects of AA on histopathology
As presented in Fig. 5, CV-stained microscopic images demonstrate severe degeneration of neurons, as evident Data are mean ± SEM of four rats. *** P ≤ 0.001 significantly different from control group and ## P ≤ 0.01; ### P ≤ 0.001 significantly different from AlCl 3 -intoxicated group from pyknotic appearance and less intact cells in the cortex and hippocampus of AlCl 3 -intoxicated rats with respect to controls. Whereas, co-administration of AA with AlCl 3 attenuated these neuronal changes in AlCl 3 -intoxicated rats. However, alone treatment of AA has shown no significant changes in both brain regions. Hence, CV staining results suggest that AA treatment remarkably prevented neuronal damage in the cortex and hippocampus of the rat brain.

Effects of AA on neuronal loss and astrocytes activation
The number of NeuN positive cells was significantly decreased in the AlCl 3 -intoxicated rats, whereas AA coadministration with AlCl 3 resulted in higher NeuN positive cells in the cortex and hippocampus of rats. The alone treatment of AA resulted in no remarkable change in the number of NeuN positive cells as compare to control rats (Fig. 6). These results suggest that AA treatment rescued the neuronal loss caused by AlCl 3 .
Al exposure also increased the number of activated astrocytes as evident from significantly increased GFAP immunoreactivity in the cortex and hippocampus of AlCl 3 -intoxicated rats. While, AA co-administered with AlCl 3 significantly decreased GFAP immunoreactivity in the cortex and hippocampus of rats. Treatment of AA alone caused no significant change in the immunoreactivity of GFAP as compare to controls (Fig. 7).

Discussion
Al excessively occurs in the environment and human are easily exposed to it via gastrointestinal tract, lung, skin etc. Thus, Al readily enters into the blood circulatory system from where it can travel to the brain by crossing the bloodbrain-barrier (Kawahara et al. 2007). The half-life of Al is very high, hence, on exposure to human it accumulates in different body organs including the brain and causes neurodegeneration (Yokel 2006;Wu et al. 2012).
Several human and animal studies indicate that Al causes severe behavioral alterations such as the memory and learning impairment, and anxiety-like symptoms (Kumar and Gill 2009;Sethi et al. 2009;Krewski et al. 2007;Exley and House 2011). The MWM test analyses latency period to find a hidden platform, which is a standard test for determining the consolidation of memory (D'Hooge and De Deyn 2001). Our results observed that AA ameliorates memory impairment caused by Al in rats. These results are in agreement with the study of Rather et al. (2018), suggesting that AA consolidates memory impairment in Al neurotoxicity. Moreover, OFT and light/dark tests are designed to evaluate the anxiety-like behavior in rodents. We observed that AA treatment reduces anxiety-like behavior in Al-intoxicated rats. Overall, these findings suggest anxiolytic and memory enhancing potential of AA in Al-induced neurotoxicity.
Acetylcholine (ACh) metabolism plays pivotal role in cognitive functions such as the spatial learning and memory. The change in ACh concentration is associated with pathophysiology of multiple brain disorders (Luchicchi et al. 2014), where altered AChE activity is known to regulate the cholinergic transmission. Al exposure has been reported to alter the AChE activity in peripheral and central nervous system (Maya et al. 2016). In the present study, we observed elevated AChE activity in Al-exposed rats whereas co-treatment of AA caused a significant decline of AChE activity in the cortex and hippocampus. Some previous studies have also demonstrated that AA actively competes with ACh at the esteratic site of AChE and inhibits the enzymatic activity (Prakash and Kumar 2013;Rather et al. 2018). Hence, it can be speculated that suppression of AChE activity may attenuate Al-induced neurobehavioral changes especially the learning and memory deficits.
Several evidence indicates the role of oxidative stress on Al neurotoxicity (Kumar and Gill 2009). Moreover, Al-induced oxidative stress can be effectively ameliorated by restoring the impairment of pro/antioxidant ratio (Kumar and Gill 2014). Hence, to evaluate the pro/antioxidant status of experimental animals, we estimated LPO and the activities of SOD, and CAT. Our results demonstrated that administration of AA results in alteration of oxidative stress and cellular antioxidant indices. We found significant increase of MDA content in the cortex and hippocampus of AlCl 3 -intoxicated rats. Further, decline in SOD and CAT activities indicate disruption of pro/ antioxidant balance. Treatment of AA significantly reduced MDA content and enhanced SOD and CAT activities. These results are in accordance with earlier findings (Rather et al. 2019;Loganathan and Thayumanavan 2018;Chen et al. 2019) and demonstrate the attenuation of oxidative stress and cellular antioxidants by AA.
Oxidative stress can activate apoptotic cell death pathway and causes neurodegeneration (Kim et al. 2015). Hence, we studied the effect of Al and AA treatments on the brain morphology of rats. Control rats depicted normal cellular architecture in the cortex and hippocampus with well-differentiated and healthy neurons. CV-stained sections from AlCl 3 -intoxicated rats showed histological disorganization of neurons in both regions of the brain. Further, the tissue sections from AlCl 3 -intoxicated rats represented decreased cell density, along with darkly stained nuclei and shrunken cytoplasm. These pathological changes are indicative of neuronal injury and are in line with results of biochemical indices. Sections obtained from rats co-treated with Al and AA exhibited remarkable improvement of cell density in the cortex as well as hippocampus, and attenuated other architectural changes. These results suggest that neurons have recovered to their characteristic shapes. Evidence from previous studies have also demonstrated remarkable restoration of histological alterations of the brain by AA treatment in different pathological conditions (Tabassum et al. 2013;Nagoor Meeran et al. 2018). The effect of AA on neuronal loss was further validated by immunohistochemical examination of NeuN, a marker of postmitotic mature neurons (Mullen et al. 1992). Al exposure has significantly decreased the protein level of NeuN and these results are in accordance with some previous reports (Junior et al. 2013;Laabbar et al. 2021). Further, the study showed that AA supplementation increases the protein level of NeuN in AlCl 3 -intoxicated rats that suggests the protection of neurons in both regions (cortex and hippocampus). Previously, AA has also been reported to protect neurons by alleviation of oxidative stress (Lee et al. 2014;Jiang et al. 2016;Rather et al. 2019). Moreover, AA has been reported to prevent C2-ceramide-induced cell death in primary cortical neurons, by stimulating antioxidant defense system (Zhang et al. 2012), and improve the learning and memory by modulating cholinergic and GABAergic neurotransmission (Nasir et al. 2011). The hippocampus is an important neurogenic region in adult brain (Aimone et al. 2011;Sahay et al. 2011). It has been suggested that hippocampal adult neurogenesis enhanced by AA may have also contributed to the learning and memory consolidation.
Astrocytes play critical role in normal functioning of neurons by serving as "second line of defense" of the brain (Verhoog et al. 2020). Reactive astrogliosis is a process that occurs in various pathological conditions and toxic insults, where elevated expression of GFAP is the hallmark of activated astrocytes (Pekny and Pekna 2014). Compelling evidence suggests that Al intoxication activates astrocytes (Ekong et al. 2017;Laabbar et al. 2021). In this study, we observed enhanced expression of GFAP positive cells in the cortex and hippocampus of AlCl 3 -intoxicated rats, that is indicative of reactive astrogliosis. While, AA treatment reduced Fig. 7 Effect of AA treatment on astrocytes activation in the cortex and hippocampus of AlCl 3 -intoxicated rats. (a) GFAP immunostaining in the cortex and hippocampus. (b) Quantitative analy-sis of GFAP immuno-stained cells. Data are mean ± SEM of three rats. *** P ≤ 0.001 significantly different from control group and ### P ≤ 0.001 significantly different from AlCl 3 -intoxicated group the expression of GFAP in both brain regions, indicating that AA attenuates the process of astroglial activation that may have also contributed to the protection of neurons. Numerous studies demonstrated that the impairment of astroglial functions can cause neuronal degeneration (Aremu and Meshitsuka 2005;Meshitsuka and Aremu 2008). Hence, attenuation of reactive astrogliosis by AA may have contributed in the repairing processes of neurodegeneration caused by Al.

Conclusions
In summary, our observations suggest that AA treatment mitigates AlCl 3 -induced neurotoxicity by altering oxidative stress, neuronal loss and astroglial activation. Hence, AA might be beneficial against Al-induced neurotoxicity, however, thorough research is needed to know its exact mechanism of action. Hence, further studies are warranted to explore the link between AlCl 3 -mediated oxidative stress and associated neurodegeneration to establish the neuroprotective role of AA.