Neuroprotective Potential of Allium sativum against Monosodium Glutamate-Induced Excitotoxicity: Impact on Short-Term Memory, Gliosis, and Oxidative Stress.

This study evaluated the neuroprotective potential of Allium sativum against monosodium glutamate (MSG)-induced neurotoxicity with respect to its impact on short-term memory in rats. Forty male Wistar albino rats were assigned into four groups. The control group received distilled water. The second group was administered Allium sativum powder (200 mg/kg of body weight) orally for 7 successive days, then was left without treatment until the 30th day. The third group was injected intraperitoneally with MSG (4 g/kg of body weight) for 7 successive days, then left without treatment until the 30th day. The fourth group was injected with MSG in the same manner as the third group and was treated with Allium sativum powder in the same manner as the second group, simultaneously. Phytochemical analysis of Allium sativum powder identified the presence of diallyl disulphide, carvone, diallyl trisulfide, and allyl tetrasulfide. MSG-induced excitotoxicity and cognitive deficit were represented by decreased distance moved and taking a long time to start moving from the center in the open field, as well as lack of curiosity in investigating the novel object and novel arm. Moreover, MSG altered hippocampus structure and increased MDA concentration and protein expression of glial fibrillary acidic protein (GFAP), calretinin, and caspase-3, whereas it decreased superoxide dismutase (SOD) activity and protein expression of Ki-67 in brain tissue. However, Allium sativum powder prevented MSG-induced neurotoxicity and improved short-term memory through enhancing antioxidant activity and reducing lipid peroxidation. In addition, it decreased protein expression of GFAP, calretinin, and caspase-3 and increased protein expression of Ki-67 in brain tissues and retained brain tissue architecture. This study indicated that Allium sativum powder ameliorated MSG-induced neurotoxicity through preventing oxidative stress-induced gliosis and apoptosis of brain tissue in rats.


Introduction
Food additives are considered a big problem in the food industry and should be subjected to periodical evaluation. Monosodium glutamate (MSG) is a glutamic acid salt that act as a

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Allium sativum Powder
The phytochemical analysis of Allium sativum powder was performed by using a Trace GC1300-TSQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (30 m × 0.25 mm × 0.25 µm film thickness). The components were identified by comparing their retention times and mass spectra with those of the WILEY 09 and NIST 11 mass spectral databases.

Experimental Design
Forty male Wistar albino rats, 1 month of age (40 ± 5 g), were used in this experiment. Rats were fed with a standard laboratory diet and water ad libitum. The animals were housed in the animal house at Faculty of Medicine, Menoufia University, under a normal light/dark cycle and room temperature. The animals were assigned into 4 groups of 10 rats each.
First group: Rat received distilled water (DW) orally and injected intraperitoneally with 1ml of physiological saline/day for 7 successive days and kept as a normal control group.
Second group (Allium sativum group): Allium sativum powder was dissolved in DW at a dose of 200 mg/kg and administered orally for 7 successive days, then left without treatment until the 30th day of the experiment [28].
Third group (MSG group): Rats were injected intraperitoneally with MSG (Sigma-Aldrich Co, St. Louis, MO, USA.) Sigma-Aldrich,) dissolved in physiological saline 4 g/kg of body weight for 7 successive days, then left without treatment until the 30th day of the experiment [33].
Fourth group (Allium sativum-treated MSG group): Rats were injected with MSG, as was done for the third group, and were treated with Allium sativum powder, as was done for the second group, simultaneously. At the end of the experiment, memory and behavioral tests on modified T-maze and open field were performed. Then, rats were decapitated and brains were excised and dissected into two halves. One half was used for estimation of malondialdehyde (MDA) and superoxide dismutase (SOD). The other half was used for histopathological and immunohistochemical investigations.

Behavioral Tests
All tests were performed between 9:00 a.m. to 2:00 p.m. in a soundless observation room with normal daylight. Rats were habituated in the observation room for 1 h before the beginning of the tests. All tests were observed through a video camera (Samsung ST93 Digital Camera, Suwon, South Korea). The equipment were cleaned by 70% ethanol to avoid odor cues for animals [34].

Assessment of Motor Function
A wooden arena (100 × 100 × 60 height, brown wall, and floor) was divided into 25 squares (20 cm per square). Rat was put in the center of the arena for 15 min and freely allowed to explore it [34]. Latency to move from the center was observed, and the total distance moved by meter (m) was assessed by calculating the numbers of crossed squares. This consisted of three phases: habituation, training, and test phases. Habituation was carried out in the open field without any object for 15 min before testing short-term memory by 24 h to reduce anxiety and stress. Training and test phases were carried out for 5 min and 30 min intervals between the two phases in order to assess short-term memory. In the training phase, rats were allowed to recognize two identical cylindrical objects located 15 cm from the wall and 60 cm apart from each other. In the test phase, a novel cylindrical object replaced one of the familiar objects, as shown in Figure 1. The time spent per second by each rat to explore the objects was calculated [35].
Nutrients 2020, 11, x FOR PEER REVIEW 4 of 18 Latency to move from the center was observed, and the total distance moved by meter (m) was assessed by calculating the numbers of crossed squares. This consisted of three phases: habituation, training, and test phases. Habituation was carried out in the open field without any object for 15 min before testing short-term memory by 24 h to reduce anxiety and stress. Training and test phases were carried out for 5 min and 30 min intervals between the two phases in order to assess short-term memory. In the training phase, rats were allowed to recognize two identical cylindrical objects located 15 cm from the wall and 60 cm apart from each other. In the test phase, a novel cylindrical object replaced one of the familiar objects, as shown in Figure 1. The time spent per second by each rat to explore the objects was calculated [35].

Modified T-Maze
Modified T-maze was used to evaluate short-term spatial memory on the basis of rats' innate preferences to discover new areas that had not been previously discovered. The apparatus consisted of three wooden arms (50 cm long × 10 cm wide × 20 cm high) located 50 cm above the floor. The task consisted of two training trials and test trials of 5 min duration and 2h inter-trial interval. In the training trial, the novel arm was closed by removable door. Rats were located in the start arm facing the center, and they were allowed to move freely between the start and other arms. In the test trial, the novel arm was opened, and rats were free to move in all arms. Percentage of entry, as detected by the presence of four paws of rats in training and test trials in all arms as well as percentage of time (in seconds) spent in each arm, was calculated [36].

Modified T-Maze
Modified T-maze was used to evaluate short-term spatial memory on the basis of rats' innate preferences to discover new areas that had not been previously discovered. The apparatus consisted of three wooden arms (50 cm long × 10 cm wide × 20 cm high) located 50 cm above the floor. The task consisted of two training trials and test trials of 5 min duration and 2h inter-trial interval. In the training trial, the novel arm was closed by removable door. Rats were located in the start arm facing the center, and they were allowed to move freely between the start and other arms. In the test trial, the novel arm was opened, and rats were free to move in all arms. Percentage of entry, as detected by the presence of four paws of rats in training and test trials in all arms as well as percentage of time (in seconds) spent in each arm, was calculated [36].

Measurement of Brain Tissues Malondialdehyde Concentration and Superoxide Dismutase Activity
Brain tissues were taken and homogenized in normal saline solution (1:9 w/v). Then, the homogenate was centrifuged at 1800 × g/min for 10 min. The supernatant was used for detection of lipid peroxidation and antioxidant enzyme activity via biomarkers MDA and SOD, respectively. Malondialdehyde concentration and SOD activity were measured calorimetrically by using commercial kits (Biodiagnostic Company, Cairo, Egypt). Malondialdehyde was measured by using the thiobarbituric acid reaction [37]. Superoxide dismutase was measured as described by [38].

Histological Examination
Brain tissue samples of all groups were rapidly excised, cut into small pieces, and fixed in 10% neutral formalin. Then, tissue sections were dehydrated in ascending concentrations of alcohol, cleared in xylene, and finally embedded in paraffin. Serial sections of about 5µm in thickness were prepared and stained by hematoxylin and eosin (H&E) staining for demonstration of the histopathological changes in brain tissues [39].

Immunohistochemical Investigations
Glial fibrillary acidic protein, caspase-3, and Ki-67 proteins were localized immunohistochemically by using avidin-biotin complex (ABC) immunoperoxidase technique. After blocking the endogenous peroxidase, brain sections were incubated with primary antibodies for 20 min at room temperature (the primary anti-GFAP antibody at 1:100 dilution; caspase-3 antibodies at 3 µg/mL with 1:200 dilution, and Ki67 antibody at 1:100 dilution). The primary GFAP and Ki-67 antibodies were mouse monoclonal antibodies, (GFAP) Ab-1 (Clone GA-5), specific to the astrocytes obtained from Lab Vision Corporation, Medico Co., Egypt (catalog. #MS-280-B0 and for Ki-67). Caspase-3 antibody reacts broadly with all known caspase-3 variants of human, rat, and mouse origin by immunohistochemistry (Lab Vision Corporation, Fremont, CA, USA). Then, the slides were washed with diluted phosphate-buffered saline (PBS) and incubated with the secondary anti-mouse antibody universal kits for 30 min in a humid chamber at room temperature.
To demonstrate the immunoreactivity of calretinin (CR) in neurons, indirect immunohistochemical peroxidase-antiperoxidase reaction (PAP) was performed. Brain sections were treated with 0.4% H 2 O 2 at room temperature for 30 min to inhibit the endogenous peroxidase. After rinsing in 0.5 M trisaminomethane (TRIS) buffer (TBS, pH = 7.6), the sections were incubated in normal goat serum at room temperature for 20 min in order to eliminate background staining. A set of antibodies and reagents (Sigma-Aldrich, St. Louis, MO, USA) were diluted with 0.5 M TBS according to the producer's recommendations and were used to conduct immunohistochemical PAP reaction. The primary antibody was specific monoclonal rabbit anti-CR antibody (incubation for 48 h at 4 • C), whereas the secondary antibody was monoclonal goat anti-immunoglobulin G (IgG) antibody (Sigma-Aldrich). Finally, the monoclonal peroxidase-antiperoxidase complex was applied.
All sections were stained by incubation with 3,3'-diaminobenzidine (DAB), a substrate chromogen, for 5-10 min. One DAB tablet was dissolved in 10 mL PBS. Chromogen resulted in brown-colored precipitate at the antigen sites, and Mayer's haematoxylin was used as a counter stain. Positive control was Cellosaurus cell line (IMR5) cells in the brain. For negative controls, incubation was carried out with the removal of the primary antiserum. The positive reactivity of GFAP, caspase-3, Ki-67, and calretinin were exhibited as different grades of reactivity (i.e., weak, moderate, and strong), according to the intensity of staining. Their positive reactivity was indicated by a brown-colored reaction [40].
For image capturing, a colored video camera (Panasonic Color CCTV camera, Matsushita Communication (Industrial Co. Ltd., Tokyo, Japan) fixed on a light microscope (Olympus BX-40, Olympus Optical Co. Ltd., Tokyo, Japan) was used. Images were taken at X400 magnification and 2.6 zoom. Photomicrographs were analyzed by using Software Image J program, a public domain image processing and analysis program (http://rsb.info.nih.gov/ij/) [41].

Statistical Analysis
The data are expressed as mean ± standard error of the mean (SEM). The statistical analysis was conducted by using SPSS version 22. The behavioral tests on the open field and modified T-maze were performed by using Kruskal-Wallis test and Mann-Whitney test. Other results were analyzed by using one-way ANOVA (analysis of variance), followed by post hoc (Tukey's) test. The difference of variance at p < 0.05 was considered significant.
The data are expressed as mean ± standard error of the mean (SEM). The statistical analysis was conducted by using SPSS version 22. The behavioral tests on the open field and modified T-maze were performed by using Kruskal-Wallis test and Mann-Whitney test. Other results were analyzed by using one-way ANOVA (analysis of variance), followed by post hoc (Tukey's) test. The difference of variance at p < 0.05 was considered significant.

The Phytochemical Components of Allium sativum Powder
Phytochemical analysis of Allium sativum powder indicated the presence of diallyl disulphide, carvone, diallyl trisulfide, allyl tetrasulfide, and 1-allyl-3-(2-(allylthio) propyl) trisulfane ( Figure 2, Table 1 and File S1).   Figure 3 showed the effect of MSG and/or Allium sativum powder on rats' motor functions. MSG significantly (p < 0.001) reduced the locomotor activity of rats by increasing the latency time to move from the center ( Figure 3A) and decreased the total distance moved in open field test compared to control group ( Figure 3B). However, simultaneous administration of MSG and Allium sativum powder to rats of the fourth group significantly (p < 0.001) decreased the latency time to move from the center and increased the total distance moved in open field test compared with the MSGadministered group (third group) ( Figure 3A,B).   Figure 3 showed the effect of MSG and/or Allium sativum powder on rats' motor functions. MSG significantly (p < 0.001) reduced the locomotor activity of rats by increasing the latency time to move from the center ( Figure 3A) and decreased the total distance moved in open field test compared to control group ( Figure 3B). However, simultaneous administration of MSG and Allium sativum powder to rats of the fourth group significantly (p < 0.001) decreased the latency time to move from the center and increased the total distance moved in open field test compared with the MSG-administered group (third group) ( Figure 3A,B).  Figure 3 shows the effect of MSG and/or Allium sativum on short-term spatial memory of rats. In the novel object discrimination task in open field, MSG significantly (p < 0.05 and p < 0.001, respectively) decreased the time for investigation of objects 1 and 2 during the training phase compared with the control group ( Figure 3C). In the test phase, the time for investigation significantly (p < 0.001 and p < 0.01, respectively) decreased for both the familiar and novel objects compared with the control group ( Figure 3D). In contrast, administration of MSG and Allium sativum powder simultaneously to rats (fourth group) significantly (p < 0.01) increased the time for investigation of objects 1 and 2 in the training phase and the time for investigation of both the familiar and novel objects in the test phase compared with rats administered with MSG (third group) ( Figure C,D).  Figure 3 shows the effect of MSG and/or Allium sativum on short-term spatial memory of rats. In the novel object discrimination task in open field, MSG significantly (p < 0.05 and p < 0.001, respectively) decreased the time for investigation of objects 1 and 2 during the training phase compared with the control group ( Figure 3C). In the test phase, the time for investigation significantly (p < 0.001 and p < 0.01, respectively) decreased for both the familiar and novel objects compared with the control group ( Figure 3D). In contrast, administration of MSG and Allium sativum powder simultaneously to rats (fourth group) significantly (p < 0.01) increased the time for investigation of objects 1 and 2 in the training phase and the time for investigation of both the familiar and novel objects in the test phase compared with rats administered with MSG (third group) ( Figure 3C,D).

Allium sativum Modulated the Effect of MSG on Short-Term Spatial Memory of Rats
In the T-maze test, MSG significantly decreased (p < 0.001) the number of entries into the start and other arms in the training and test phases as well as the novel arm in the test phase compared with the control rats ( Figure 4A,B). In addition, it significantly increased (p < 0.001) the time spent and percentage of time spent in the start arm, whereas it significantly decreased (p < 0.001) the time spent in the other arm during the training phase compared with the control group. In the test phase, the MSG-treated group showed insignificant increase in the time spent and percentage of time spent in the start and other arms, but showed a significant (p < 0.01) decrease in the time spent and percentage of time spent in the novel arm compared to both control and powder groups ( Figure 4C-F). However, treatment of MSG-administered rats with Allium sativum powder (fourth group) significantly (p < 0.01) increased the total number of entries into different arms compared with the MSG-treated group (third group) during training and test phases ( Figure 4A,B). Additionally, Allium sativum powder (fourth group) significantly (p < 0.01) increased the time spent and percentage of time spent exploring the novel arm compared to the MSG-treated group (third group) during test phases ( Figure 4D,F).  Monosodium glutamate significantly increased (p < 0.05) the brain tissue lipid peroxidation biomarker MDA content, whereas it significantly decreased the activity of SOD in brain tissue compared with the control group. However, administration of MSG and Allium sativum powder simultaneously to rats significantly (p < 0.05) decreased concentration of MDA while increasing the activity of SOD in brain tissue compared with the MSG-administered group (third group) ( Table 2).

Allium sativum Modified MSG-Induced Alterations in Brain Tissue Architecture of Rats
Microscopic examination of brain tissues of both control and powder-treated rats stained with H&E revealed normal histological structure of the hippocampus ( Figure 5). However, administration of rats with MSG significantly decreased the thickness of the pyramidal cell layer and cell count in Cornu Ammonis (CA1 and CA3) areas compared with those of the control group. In addition, pyramidal cells showed disarrangement, degeneration, and pyknosis of their nuclei with loss of pyramidal cells, leaving empty spaces filled with vacuolations in the surrounding neutrophils. The dentate gyrus of this group showed marked reduction in number of cells of all three layers with disarrangement of granular cells, appearance of many apoptotic cells, and darkly stained nuclei with vacuolations of their cytoplasm (V) ( Figure 5).
Treatment of MSG rats with Allium sativum powder significantly increased the thickness of pyramidal cell layer and cell count in CA1 area compared with the MSG group. The pyramidal layer of CA1 and CA3 areas revealed improvement of the histological picture, and most of the pyramidal cells appeared normal with euchromatic vesicular nuclei. However, some pyramidal cells were seen in the polymorphic layer, and a few other cells showed pyknosis of their nuclei with dilated blood vessels. Dentate gyrus of the same group revealed improvement of the histological picture of all layers ( Figure 5).

Allium sativum Reduced MSG-Induced GFAP and Calretinin Protein Expression While Increasing Ki-67 Protein Expression in Brain Tissue of Rats
Administration of MSG to rats significantly increased GFAP immune reaction in the hippocampus, indicating an increase in astrocyte number and cell processes. This was shown by the intensity of brown color that meant active gliosis compared with the control group. However, treatment of MSG-administered rats (fourth group) with Allium sativum powder significantly decreased GFAP immunoreactivity in astrocytes and their processes compared to MSG-administered rats (third group) ( Figure 6A-D).
In addition, MSG induced strong calretinin immune reactivity in neurons, which was indicated by the presence of cytoplasmic and nuclear reaction. However, brain section of rats of the normal control, second, and fourth groups showed mild reaction ( Figure 6E-H).
Investigating the protein expression of Ki-67 in brain sections of different groups showed positive nuclei in the sub-granular zone of the dentate gyrus of the control and Allium sativum-treated groups (first and second groups). The number of Ki-67 immunoreactive nuclei was significantly decreased in the MSG-administered group (third group) compared to the control group. However, treatment of the MSG-administered group with Allium sativum powder (fourth group) normalized the expression of Ki-67 protein in brain tissue ( Figure 6I-L).
The immunohistochemical detection of caspase-3 in brain tissue of different groups showed that the expression of caspase-3 protein in CA1, CA3, and dentate gyrus of brain tissue of the control and Allium sativum groups was mild. Administration of rats of the third group with MSG increased caspase-3 protein expression in CA1, CA3, and dentate gyrus. However, treatment of MSG-administered rats (fourth group) with Allium sativum powder reduced caspase-3 protein expression in CA1, CA3, and dentate gyrus compared to that of the third group (Figure 7).  Apoptotic and pyknotic nuclei of pyramidal cells surrounded with pericellular haloes. Dilated blood vessels and massive vacuolation of the cytoplasm (V) can be also seen. Dentate gyrus of the same group showed many apoptotic cells (arrows), darkly stained nuclei, vacuolated cytoplasm (V), and disorganized blood vessels (BV). Allium sativum-treated MSG (AS-treated MSG) group was more or Administration of MSG to rats significantly increased GFAP immune reaction in the hippocampus, indicating an increase in astrocyte number and cell processes. This was shown by the intensity of brown color that meant active gliosis compared with the control group. However, treatment of MSG-administered rats (fourth group) with Allium sativum powder significantly decreased GFAP immunoreactivity in astrocytes and their processes compared to MSG-administered rats (third group) ( Figure 6A-D). In addition, MSG induced strong calretinin immune reactivity in neurons, which was indicated by the presence of cytoplasmic and nuclear reaction. However, brain section of rats of the normal control, second, and fourth groups showed mild reaction ( Figure 6E-H).
Investigating the protein expression of Ki-67 in brain sections of different groups showed positive nuclei in the sub-granular zone of the dentate gyrus of the control and Allium sativum-treated groups (first and second groups). The number of Ki-67 immunoreactive nuclei was significantly

Discussion
Monosodium glutamate is one of the food additives that has been used over the centuries, especially in Chinese and Asian dishes [42]. The results of the current study revealed that injection of rats with MSG induced excitotoxicity and cognitive deficit, which was indicated by hypoactivity in the form of decreased distance moved in open field, taking a long time to start moving from the center, and lack of curiosity in investigating novel object and novel arm during the test for short-term spatial memory by open field and T-maze. These results were in line with that of [43], who demonstrated that MSG has an excitotoxic effect on the brain with significant long-term damage of many brain areas, especially if administered in early life. In addition, Hassaan et al. [44] reported that MSG intake in early life induced neurotoxicity that impairs the short-term memory and affects the exploratory behavior in mice. These exitotoxic effects of MSG may be due to the dissolving of MSG in water, dissociating it into sodium and glutamate ions, which increases the plasma glutamate levels up to 17-fold higher than the normal basal level [43]. Further, injection of animals with MSG increases the extracellular brain glutamate concentration and induces motor and behavioral alterations [15] due to glutamate reacting with its receptors, as well as initiating apoptosis and necrosis of neuronal cells through over-activation of glutamate receptors. Activation of glutamate receptors releases Ca +2

Discussion
Monosodium glutamate is one of the food additives that has been used over the centuries, especially in Chinese and Asian dishes [42]. The results of the current study revealed that injection of rats with MSG induced excitotoxicity and cognitive deficit, which was indicated by hypoactivity in the form of decreased distance moved in open field, taking a long time to start moving from the center, and lack of curiosity in investigating novel object and novel arm during the test for short-term spatial memory by open field and T-maze. These results were in line with that of [43], who demonstrated that MSG has an excitotoxic effect on the brain with significant long-term damage of many brain areas, especially if administered in early life. In addition, Hassaan et al. [44] reported that MSG intake in early life induced neurotoxicity that impairs the short-term memory and affects the exploratory behavior in mice. These exitotoxic effects of MSG may be due to the dissolving of MSG in water, dissociating it into sodium and glutamate ions, which increases the plasma glutamate levels up to 17-fold higher than the normal basal level [43]. Further, injection of animals with MSG increases the extracellular brain glutamate concentration and induces motor and behavioral alterations [15] due to glutamate reacting with its receptors, as well as initiating apoptosis and necrosis of neuronal cells through over-activation of glutamate receptors. Activation of glutamate receptors releases Ca +2 from its stores, which causes mitochondrial over-activation and stimulates a number of intracellular enzymes such as endonucleases, phospholipases, and proteases. These enzymes damage cell structures including the cytoskeleton, cell membrane, and DNA [6]. Another study demonstrated that treatment of rats with MSG leads to cognitive deficit with hypoactivity and irritability, as it induced hippocampal degeneration, impaired synaptic plasticity, and caused a deficit in short-term and long-term potentiation [45]. Another possible cause of the excitotoxic effect of MSG is the oxidative stress that induced in the hippocampus, evidenced by elevation of MDA concentration and concomitant decrease in SOD activity in brain tissues ( Table 2) because oxidative stress is a characteristic feature of neurodegenerative disease. It easily affects the brain because the brain has a high metabolic activity with low antioxidant capacity. Released free radicals cause peroxidation of cell membranes and DNA, causing cell damage and apoptosis [46]. The presence of these free radicals activates signaling pathways of inflammation and cell damage, and may cause disruption of the blood-brain barrier by affecting the endothelial basement membrane [43]. Moreover, the released free radicals contribute to excitotoxicity. Free radicals are generated from mitochondria following calcium influx due to glutamate receptor stimulation [47]. On contrast, the results of the current study indicated that Allium sativum modulated MSG-induced behavioral abnormality, as treatment of MSG-administered rats with Allium sativum powder improved motor activity and behavior and spatial memory, indicating a protective effect of Allium sativum against MSG-induced excitotoxicity. Behavioral and motor improvement induced by Allium sativum may be attributed to its contents of diallyl disulphide (DAD), diallyl trisulfide (DAT), and allyl tetrasulfide (AT) (Figure 2 and Table 1), as Tang et al. [48] found that DAT, an Allium sativum active constituent, inhibits colonic smooth muscle contraction in male rats that act as a channel blocker that inhibits the L-type calcium channel. The other possible cause of the improvement effects of Allium sativumonon on the behavior of MSG administrated rats is the antioxidant activities of its components, as [49] reported that garlic has strong antioxidant, neuroprotective, and antiapoptotic effects against many neurotoxins, and also has a neurotrophic effect on hippocampal cells due to it containing allicin [46]. Thus, we postulated that DAD and DAT decreased Ca +2 influxes to hippocampal neurons by blocking the L-type calcium channel, which may reduce excitotoxicity and neuronal death.
Moreover, our data showed that MSG administration decreased the number of pyramidal cell layers in CA1 and CA3 areas of the hippocampus due to neuronal cell death. Additionally, most of the nerve cells were distorted in shape ( Figure 5). These histopathological changes in brain tissues may be related to MSG-induced over-expression of caspase-3 protein and decreased Ki-67 protein expression in brain tissue of the MSG group (Figures 6 and 7). The increased expression of caspase-3 protein in the hippocampus of MSG-administered rats can explain the pyramidal cell loss and neuronal degeneration. Activation of caspases had been linked to neuronal apoptosis and considered a key stimulator of cell death. Neuronal injury under the effect of neurotoxins leads to activation of cell death signals [50]. MSG-induced apoptosis in the hippocampus may be attributed to oxidative stress that leads to mitochondrial damage with release of cytochrome C and activation of caspases. However, Ki-67 is a protein marker of active cell proliferation in the subgranular zone of dentate gyrus, the active site of neurogenesis in the brain [51], indicating downregulation of neurogenesis in the MSG-administrated group. These degenerative effects greatly affected memory because they disrupted the normal flow of information from the dentate gyrus to CA3 through mossy fibers, and also may have affected the flow of information from CA3 via the Schaffer's fibers to CA1 [26]. Our findings were in agreement with Rycerz et al. [25], who found that administration of MSG to rats in postnatal week revealed loss of 11.5% of pyramidal neurons in the hippocampus. On the other hand, MSG significantly increased astrogliosis, which was evidenced by increased GFAP expression (Figure 6), a hallmark of increased astrocyte activity as a result of injury to the central nervous system [52]. Previous data indicated that MSG administration induced hypertrophy of astrocytes and microglial cells in the hippocampus, also increasing in neuronal apoptosis in CA1 and CA3 areas, leading to cognitive impairment [43]. Therefore, the use of antioxidants is a vital process in inhibiting neuronal damage. Diallyl disulfide is an important natural antioxidant capable of recovering normal redox activity and enhances certain signaling pathways that favor protection of homeostatic response of the cell [50]. Diallyl disulfide also has been proven to be neuroprotective because it protects the brain against hypoxically and ischemically induced brain injury [53,54] in rats. This neuroprotective activity of Allium sativum is attributed to the ability of garlic-derived organosulfur compounds-DAD and DAT-to modulate redox activity and to reduce oxidative stress and their incorporation in many signaling pathways [47]. Diallyl disulfide was proven as a strong lipid peroxidation terminator [55], thus providing neuronal protection and preventing neuronal damage by excitotoxins. The protective effect of Allium sativum against MSG-induced hippocambal neurons agreed with the finding of Hwang et al. [55], who reported that garlic protected dopaminergic neurons from degeneration in a Parkinson rat model. The authors also found that it decreased astrocyte activation and neural inflammation. Another study by Rojas et al. [6] indicated that Allium sativum powder protected mice from neurotoxicity and oxidative damage by free radical scavenging effects and preservation of SOD activity. Diallyl trisulfide also protects motor neurons and mediates cell survival by enhancing autophagy and stimulating antioxidant enzymes [56]. Additionally, Mostafa [57] reported that DAT protected neurons and improved cognitive functions in streptozotocin-induced diabetic rats through H 2 S release. In addition, Allium sativum was found to significantly decrease the expression of caspase-3 pro-apoptotic protein marker and up-regulate the anti-apoptotic genes because it contains many sulfur-containing compounds and volatile oil [58]. Beside its antioxidant effect, Allium sativum reduces cell damage through an anti-inflammatory property and decreases mitochondrial disorders occurring in different diseases [59]. Furthermore, our results indicated that the expression of calretinin protein was increased in the brain tissue of the MSG-treated group, which is a neuroprotective protein that regulates calcium homeostasis by buffering excess calcium in cases of glutamate-induced excititoxicity [25]. Treatment with Allium sativum powder significantly reduced the expression of calretinin, as it reduced MSG-induced excitotoxicity.

Conclusions
Monosodium glutamate severely affected the short-term spatial memory of rats through oxidative stress-induced degenerative changes and apoptosis of brain tissue. However, Allium sativum ameliorated MSG-induced degenerative and apoptotic changes in brain tissue through reducing the oxidative stress and gliosis-induced structural damage of the hippocampus and by increasing the protein expression of Ki-67 in brain tissue. This study suggested that Allium sativum is a potent neuroprotective agent against pollutant-induced nervous tissue damage. Funding: This work was funded by Researchers Supporting Project number RSP 2019/121, King Saud University, Riyadh, Saudi Arabia. Data availability statement: All data will be available when required.