D-galactose induced dysfunction in mice hippocampus and the possible antioxidant and neuromodulatory effects of selenium

Aging is an ultimate reality that everyone has to face. D-galactose (D-gal) has been used extensively to develop aging model. Trace elements such as selenium (Se) have been used as a potential antioxidant for neuro-protection. The present work aims to develop therapeutic agents such as Se for the treatment of aging-induced neurological ailments such as anxiety, depression, and memory impairment. For this purpose, mice were treated with D-gal at a dose of 300 mg/ml/kg and various doses of Se (0.175 and 0.35mg/ml/kg) for 28 days. Behavioral tests were monitored after treatment days. After the behavioral assessment, mice were decapitated and their brains were collected. Hippocampi were removed from the brain for biochemical, neurochemical, and histopathological analysis. The present findings of behavioral analysis showed that D-gal-induced anxiety- and depression-like symptoms were inhibited by both doses of Se. D-gal-induced memory alteration was also prevented by repeated doses of Se (0.175 and 0.35mg/ml/kg). Biochemical analysis showed that D-gal-induced increase of oxidative stress and inflammatory markers and decrease of antioxidant enzymes and total protein contents in the hippocampus were prevented by Se administration. An increase in the activity of acetylcholinesterase was also diminished by Se. The neurochemical assessment showed that D-gal-induced increased serotonin metabolism and decreased acetylcholine levels in the hippocampus were restored by repeated treatment of Se. Histopathological estimations also exhibited; normalization of D-gal induced neurodegenerative changes. It is concluded that D-gal-induced dysfunction in mice hippocampus caused anxiety, depression, memory impairment, oxidative stress, neuro-inflammation, and histological alterations that were mitigated by Se via its antioxidant potential, anti-inflammatory property, and modulating capability of serotonergic and cholinergic functions.


Introduction
Aging is a gradual functional impairment at the organismal and cellular levels (Wagner et al. 2016). These physiological dysfunctions are attributed to increased genomic instability, altered metabolism, and the loss of regenerative potential (Benn and Woolf 2004;Baker et al. 2016). The process of aging is associated with a variety of disorders that cause tissue homeostasis imbalance, memory and learning impairments (Bonfili et al. 2020), and depression-and anxiety-like behaviors (Samad et al. 2019a). Extensive evidence shows that aging is progressed by oxidative stress which is induced by a discrepancy between reactive oxygen species formation and antioxidant system (Cabello-Verrugio et al. 2016). Moreover, a decrease in oxidative stress and its associated homeostatic imbalance is a valuable measure to increase life expectancy (Pérez et al. 2009). The process of aging is linked with pathophysiology by declining redox status and is a challenge for therapeutic gerontology (Franco and Vargas 2018).
D-galactose (D-gal) is a reducing sugar (Timal et al. 2012) which is mostly present in dairy foods, some vegetables, certain fruit pectin, chestnuts, and in few herbs (Fernandes et al. 2006). Extensive studies show that the accumulation of D-gal alters the antioxidant system and speeds up the process of aging (Du et al. 2019). Excessive D-gal may also cause rapid reactive oxygen species formation which hinders the cellular antioxidant defense system (Samad et al. 2020). Furthermore, it is revealed that intoxication of D-gal is responsible for neurochemical and behavioral alterations (Samad et al. 2019b). Repeated D-gal treatment mimics the consequence of natural aging in an animal model (Ho et al. 2003). It is reported that the intake of D-gal induces anxiety (Kaviani et al. 2017), depression (Liaquat et al. 2017), and memory impairment (Guo et al. 2018). Oxidative stress induced by chronic administration of D-gal was mitigated by blueberry (Çoban et al. 2015), Ocimum Basilicum (Garabadu and Singh 2020), ascorbic acid (Nam et al. 2019), quercetin (Dong et al. 2017), lithium chloride (Samad et al. 2019a), n-3 polyunsaturated fatty acid (Guo et al. 2018), and caffeine (Ullah et al. 2015) to ameliorate the D-gal-induced behavioral and cognitive deficits.
Trace elements and minerals are important micronutrients (Qu et al. 2016) that can regulate the aging-associated alternations in physiological, homeostatic, and metabolic pathways (Méplan 2011). Selenium (Se) is a well-known mineral due to its antioxidant potential (Oliveira et al. 2012). It is a novel and probable remedy for aging and aging-associated diseases such as tumors, cardiovascular syndromes, and skin aging (Cai et al. 2019). It carried out its most biological roles via seleno-proteins (Kryukov et al. 2003). Se exerts neuroprotective effects by homeostasis regulations through several mechanisms such as alleviation of oxidative stress (Cosín-Tomàs et al. 2019), inflammatory pathway regulation (Ataizi et al. 2019), decrease in DNA destruction (Kryukov et al. 2003), increasing telomere length and activity (Suñol et al. 2019;Cao et al. 2017), and modulation of numerous neurotransmitter (Yan et al. 2019). Moreover, Se intricates in the physiopathology of various psychiatric illnesses (Nogueira and Rocha 2011) by modulating serotonergic systems (Besckow et al. 2020).
Several shreds of evidence suggest that besides its antioxidant effects, Se plays a vital role in the treatment of various psychiatric ailments Bampi et al. 2019). Considering its therapeutic role, it is assumed that Se may mitigate D-gal-induced dysfunction in mice hippocampus resulting in anti-anxiety-and/or depression-like behavior and improvement in memory functions. For the said purpose, low (0.175mg/ml/kg) and high (0.35mg/ml/kg) doses of sodium selenite were selected and used for the possible mitigation of D-gal-induced psychiatric illnesses in mice.

Animals
Thirty-six male adult albino BALB/c mice were taken to conduct the present study. Animals with free access to water and food were housed individually at 27±1°C on 12-h day and night cycle. The experiment was carried out thoroughly in an optimal setting to overcome the effect of time and order. All experimental protocols were carried out after approval by the Departmental Bioethical Committee (Ref No# Biochem/ D/162/2020; Dated: January 2, 2020).

Experimental schedule
Thirty-six male adult albino mice (9 weeks old; weighing 25-30g) were randomly separated in six groups (six mice in each group): (i) water + water, (ii) water + Se (0.175mg/ml/ kg), (iii) water + Se (0.35mg/ml/kg), (iv) water + D-gal (300mg/ml/kg) (Samad et al. 2019a;Haider et al. 2020), (v) D-gal + Se (0.175mg/ml/kg), and (vi) D-gal + Se (0.35mg/ml/kg). The unit expression for doses is same as reported previously (Samad et al. 2019b;Liaquat et al. 2017Liaquat et al. , 2019. The control animals were given distilled water (1ml/kg) as vehicle, while D-gal and Se were dissolved in distilled water and injected intraperitoneally daily for 28 days. Behavioral activity was performed after 28 days including light dark box activity and elevated plus maze activity (for anxiety), Morris water maze (for memory), and forced swim test (for depressive symptoms). After behavioral tests, mice were decapitated without using rodent anesthesia (Samad and Haleem 2009), and their brains were taken out and immediately dipped in ice-cold saline and then placed in brain slicer with ventral side up to dissect out the hippocampus as mentioned previously (Haider et al. 2016) by inserting the blade into the slots of the brain slicer just above and below the hypothalamus, to cut the brain into three slices which were then shifted to a petri dish placed on ice, moistened with chilled saline (0.9% NaCl). The middle slice of the dissected brain was used to obtain hippocampus bilaterally with the help of sharp scalpel blade. All the hippocampus samples were stored at −40°C immediately for bio-and neurochemical and histopathological estimations.

Evaluation of various behaviors
Elevated plus maze Elevated plus maze was firstly used by Handley and Mithani (1984). Elevated plus maze consists of a plus sign maze having four arms with a central stage (a) in which two are opened (b) and two are closed (c) with the dimensions 5×9×5cm (a), 16×9×5cm (b), and 16×9×5×9×15cm (c), respectively, while the height of maze is 26 cm from the floor. Open arm and height are used to induce anxiety in rodents by elevated plus maze. One hour after the last treatment, every mouse separately holds at the central stage of elevated plus maze and percent time spent in open arm recorded for 5 min (Samad et al. 2019c;Samad and Haleem 2009).

Light dark activity
Light dark transition test was originally described by Crawley and Goodwin (1980). Light dark activity box consists of a quadrilateral case separated by an impervious layer into two compartments: (a) dark compartment and (b) light compartment. The magnitude of two chambers was 26×26×26cm with a transportation connectivity of 12×12cm in-between partition. Light compartment is made up of transparent plastic, while dark box is made up of translucent plastic. Mice were placed to light box 1 h after the treatment of drugs and percent time spent in light box recorded for 5 min (Haider et al. 2015;Samad et al. 2005).

Forced swim test
Forced swim test is one of the most widely employed methods by Porsolt (1981), for the evaluation of depressant/antidepressant behavior of the rodents in a restraining environment. In our experimental setting, we used a glass tank with a dimension of 45cm height and 30cm radius for the assessment of the antidepressant activity of the test substances. The apparatus was filled with water (25°C) up to a certain level where the feet of the animal should not get the support from the basement and simultaneously inescapable from the tank. In a trial session of 5 min each, all animals were allowed to forced swim. In the experimental session, the treated mice were challenged to the environment of inescapability, and the difference between the states of mobility vs immobility was observed by lateral recording which subsequently was analyzed by the blinded observer to the experiments. The mice will be considered in a phase of depression where it spent most of the time in a phase of immobility and makes virtually no efforts to escape and merely tries to keep its head above the water .

Morris water maze test
The Morris water maze (MWM) is a behavioral test that is developed by Morris (1981) and used to evaluate several aspects of cognitive functions in rodents (Haider et al. 2012). This test requires an animal to use spatial learning and memory to locate a hidden platform just below the surface of a circular pool of water and also to remember its location as in the previous trial. It is a circular pool of a diameter of 122 cm and a depth of 76 cm. The pool is a metal cylinder painted white on the inner surface, and the escape platform is also made of metal cylinder with flat metallic. In the pool, mice were trained to swim to find a platform concealed (1.5 cm) under water surface. The pool is filled with water (23 ± 2°C) and made opaque with milk in order to obscure the platform and to allow proficient tracking of the swim paths of the mice. Animals placed in the circular pool have to swim until they find a hidden platform submerged in the pool (Haider et al. 2011). Learning and memory are measured by escape latency and time spent in the target quadrant in which the platform is hidden. It is reported that the animal used cue in order to locate the hidden platform. The performance of animals improved across trials (indicated by a reduction in latencies to locate the platform) despite starting from multiple locations. Such a procedure mitigates the use of egocentric navigation and promotes the use of allocentric navigation. Each trial started by placing the mouse gently in the water, facing the edge of the circular pool, at the designated position. After learning training, mouse was given a probe trial in which the platform was removed and memory for platform location was assessed by quantifying the time spent in the maze quadrant that previously contained the platform ("target quadrant"). The Morris water maze task comprised three components: In order to familiarize the mice with the maze and the escape procedure, the training session was performed initially during which each mouse was placed into the water in such a way that their face was towards the wall of the tank. After placing, 120 s was given to each animal to find and mount onto the hidden platform; if the mouse located the platform, it was allowed to stay on it for 10 s. If it failed to locate the platform during the allocated time, then it is usually picked up and was guided gently onto the platform (Haider et al. 2011) and placed on it for~15 s. Place or spatial learning is the most basic MWM procedure. The concept behind it is that the animal must learn to use distal cues to navigate a direct path to the hidden platform when started from different, random locations around the perimeter of the tank. If there are no proximal cues available, the use of distal cues provides the most effective strategy to accomplish this. Each mouse was given four consecutive trials in the maze to locate the hidden platform with the hidden escape platform being located in the NW quadrant. The interval between trials was 15 min. Starting positions from arbitrarily assigned compass locations were randomized for each mouse on each trial. We used four start locations: NE, SE, NW, and SW (target quadrant). These positions are designed so that the animal is not able to learn a specific order of right or left turns to locate the platform while using each of the four start positions. The platform was kept in a constant position for all mice throughout all trials. The time taken to reach the platform (escape latency) was noted.
Probe trial: To assess reference memory at the end of learning, a probe (transfer) trial was performed. In the present study we have performed two probe trials, one was performed 1 h after the last acquisition trial, and the other was performed after 24 h of last hidden platform acquisition trial. During the probe trial, the platform was removed, and the mice were allowed to swim for 2 min. The parameters that were measured during this trial included the time spent in the target quadrant (NW) and the number of entries over the target quadrant (NW) as an index of reference memory (Haider et al. 2016).

Analysis of biochemicals in the hippocampus
Hippocampus was washed with 0.9% saline solution and weighted (averagely 35 mg). A 10% w/v tissue homogenate was prepared with 0.1M phosphate buffer (pH 7.4) and centrifuged at 10,000×g for 10 min at 4°C. The supernatant was used for following biochemical analysis.

Malondialdehyde (MDA)
Estimation of MDA levels was essentially the same as described by Chow and Tappel (1972) with slight modifications. In a reaction mixture, 300 μl homogenate was taken, and 2 ml of TCA (15%)-TBA (0.375%) mixture was added. The mixture was boiled for 20 min in water bath, cooled with ice cold water at 4°C, and then centrifuged at 3500 rpm for 10 min. Supernatant of light pink color was then collected, and absorbance was taken at 532 nm. Lipid peroxidation was expressed as mM of MDA/mg protein.

Superoxide dismutase (SOD)
The SOD was estimated by the method of Naskar et al. (2010). An aliquot of brain tissue homogenate (10%) was treated with 0.75 ml of ethanol and 0.15 ml of ice chilled chloroform and then centrifuged. Then 0.5 ml of EDTA (0.6 mM) and 1.0 ml of carbonate-bicarbonate (0.1M; pH 10.2) buffer were added in 0.5 ml of supernatant. The reaction was started by adding 0.5 ml of epinephrine (1.8 mM), and the absorbance was measured for 3 min at 480 nm. Blank contained all reagents except supernatant. Activity of SOD was expressed as U/mg protein.

Catalase (CAT)
The activity of the CAT enzyme was calculated through the previously reported procedure of Pari and Latha (2001). The tissue homogenate was prepared through a 0.1 M phosphate buffer (pH 7.0). The reaction mixture for the CAT activity analysis was mixed in test tubes separately having tissue homogenate (0.1 ml) with 1 ml of phosphate buffer (pH 7.4). Then H 2 O 2 (0.4ml) was added. Incubation was then performed at 37°C for 90 s. To prevent the reaction, potassium dichromate/acetic acid (2 ml) was added in all test tubes, and after adding the reagent, the reaction mixture turned blue. Now at 100°C, incubate again for 15 min; then the color of the mixture turned solid blue to green. This change of color was due to the chromic acetate synthesis. The control test tube contained no tissue homogeneous, while the test tube having distilled water in place of tissue homogenate is called as blank.
With the help of a spectrophotometer, absorbance was noted at 570nm. The activity of CAT in tissue sample was expressed as μmoles of H 2 O 2 consumed/min/mg protein.

Glutathione peroxidase (GPx)
GPx activity was assessed via method given by Flohe and Gunzler (1984). A reaction mixture was formed in each test tube by combining 0.1ml of sodium azide, 0.2ml of brain tissue supernatant, 0.2ml of glutathione, and 0.3 ml of phosphate buffer. The reaction mixture was incubated for 15 min at 37°C. To stop the reaction, TCA was inserted in all test tubes having each tissue sample reaction mixture. Again, the centrifugation was done at 15000 rpm for 5 min. The further reaction was performed by collecting the supernatant after centrifugation. Disodium hydrogen phosphate buffer (0.2ml), DTNB (0.7ml), and supernatant of each tissue sample (0.1ml) were assorted in each tube. Absorbance was noted at 420nm. The activity of GPx was expressed as μmol of residual GSH /mg protein.

Acetylcholinesterase (AChE)
AChE activity was assessed via methods given by Ellman et al. (1961). The reaction mixture was formulated by adding acetylcholine iodide as enzyme substrate. Tissue homogenate of tissue (0.4 ml) was added in 2.6 ml phosphate buffer having 8.0 pH, and DTNB (100μl) was mixed separately and now mixed well with aerated air. Absorbance was noted at 420nm when the reaction mixture was stabilized with the use of a spectrophotometer. The acetylcholine iodide (5.2μl) was then added in every test tube, and then the absorption was noted after every 2min from 0 to10 min for each sample. The activity AChE in a sample of the hippocampus was described as μmol/min/mg protein.

Total protein contents
The protein content was estimated using a previously reported method (Lowry et al. 1951). The tissue homogenate was mixed with an equal amount of 15 % perchloric acid (HClO4; PCA) and stored at 4°C overnight. PCA-treated samples were then centrifuged (semi cold centrifuge) at 2000 g for 10 min at 4°C. The precipitate (pallet) was used for determination of protein content. The precipitate was dissolved in 1ml of 0.1N NaOH and set aside for 10 min at room temperature; then 0.5 ml of Folin reagent was added, and again set aside for 10 min at room temperature for complete color development. The absorbance was measured at 610 nm. Protein levels were calculated using standard bovine serum solution, 200 mg in 100 ml of distilled water.

Analysis of neurochemicals in the hippocampus
Analysis of 5-HT and its metabolite 5-Hydroxy indole acetic aci d (5-HIAA) and 5hydroxytryptamine (5-HT) levels in the hippocampus were estimated using the method described by Samad et al. (2019aSamad et al. ( , 2019b. Reversed phase high performance liquid chromatography (HPLC) with an electrochemical detector (Shimadzu LEC 6A detector) was performed to detect levels of biogenic amines in brain samples. The electrochemical (EC) detector was operated at a potential of +0.8 V. The stationary phase used for separation is a 5-μ Shim-pack octadecyl silane (ODS) column having an internal diameter of 4.0 mm and a length of 150 mm. The mobile phase that passes through a column with a pump pressure of 2000-3000 psi contains octyl sodium sulfate (0.023%) in 0.1 M phosphate buffer at pH 2.9.

Analysis of acetylcholine (ACh)
The level of ACh concentration was determined as described by Liaquat et al. (2019) and was presented as μmol/g of the hippocampus. The tissue sample was boiled to release the bound ACh and inactivate the enzyme. The reaction mixture was then mixed with ferric chloride 1% (1000 μl) to form a brown color complex and read at 540 nm against the reagent blank.

Histopathological studies
Hisopathological studies were performed on hippocampus that was first static in 10% formalin solution after that fixed in paraffin wax. 5 μm thickened section stained with eosin and hematoxylin as reported previously Thenmozhi et al. (2015). Alteration in tissue sections was examined at 200× under the light microscope.

Statistical analysis
The data are presented as mean ± SD (n=6). All the behavioral and biochemical data were analyzed by two-way ANOVA. Escape latencies of training trial in MWM were analyzed by three-way ANOVA (repeated measure). Multiple comparisons of all the data were evaluated by Tukey's test using SPSS V. 21. p< 0.05 was taken as significant.  35mg/ml/kg)-administered mice [mean ± SD (n = 6)]. Data was analyzed by Tukey's test. **p<0.01 when compared with their respective control group and ++p<0.0 when compared with water + water-and water + Se (0.35 mg/ml/kg)-administered animals. Fig. 2 Percent immobility time monitored in forced swim test apparatus in water-, D-gal-. and Se (0.175 and 0.35mg/ml/kg)administered mice [mean ± SD (n = 6)]. Data was analyzed by Tukey's test. **p<0.01when compared with respective control group and ++p<0.01 when compared with water + water-and water + Se (0.175 and 0.35 mg/ml/kg)-administered animals.

Effect of Se on D-gal induced depression-like symptoms
p< 0.01] were found by two-way ANOVA. According to Tukey's test, percent immobility time during FST was substantially elevated by D-gal treatment. Percent immobility period was decreased at both doses of Se as compared to waterand D-gal-treated mice. Percent immobility time was greater in D-gal + Se (0.35mg/ml/kg) than water + Se (0.35mg/ml/ kg)-treated groups.
Effect of Se on D-gal induced cognitive impairment Figure 3 shows the effect of Se on cognitive function in D-gal and water-treated mice that evaluated in MWM. Mice from all treatment groups were experienced to four acquisition trials after a preliminary training in MWM. To evaluate the change in escape latencies of mice during acquisition trials, three-way ANOVA (repeated measures) was done. Final data showed substantial effects of groups [(F1,30) Figure 3A). Escape latencies of all groups and treatment crosswise trials revealed substantial (p<0.001) increase in escape latencies in D-gal-treated animals. Results suggest that all mice largely improved at the same rate over the course of training, with the exception of the D-gal mice given 0.175mg/kg Se.
Probe trials were performed to evaluate the reference memory. Two probe trials were performed; one was conducted 1 h after the last trial, while other was performed 24 h after the last training trial via monitoring the time spent in the target quadrant (NW) and the number of entries in the target quadrant. Tukey's test showed that entries in target quadrant decreased following D-gal administration. On the other hand, entries were increased in all Se treated than water-and D-gal-treated mice ( Figure 3C). Effect of Se on D-gal induced alteration in total protein contents Tukey's test exhibited that D-gal treatment reduced total protein contents in the brain tissue of mice. Administration of Se at both doses enhanced protein contents in water-as well as D-galtreated mice.     water-, D-gal-. and Se (0.175 and 0.35 mg/ml/kg)-administered mice [mean ± SD (n = 6)]. A Escape latencies of mice in MWM to evaluate memory function during acquisition training trials (120 s for each trial). Two-way ANOVA (repeated measure) was performed followed by post hoc Tukey's test for multiple comparisons, and significant differences were expressed as **p<0.01 from respective control and ++p<0.01 when compared with water + water and water + Se (0.175 mg/ml/kg)-administered animals. B Time spent in target quadrant after 1 h and 24 h of acquisition trials was evaluated by two-way ANOVA followed by Tukey's test for multiple comparisons. Significant differences were expressed as **p<0.01 and *p<0.05 from respective control and ++p<0.01 and +p<0.05 when compared with water + water and water + Se (0.175 mg/ml/kg)-administered animals. C Number of entries in target quadrant after 1 h and 24 h of acquisition trials was evaluated by two-way ANOVA followed by Tukey's test for multiple comparisons. Significant differences were expressed as **p<0.01 from respective control and ++p<0.01 when compared with water + water and water + Se (0.175 mg/ml/kg)-administered animals. Fig. 4 Oxidative stress marker in mice hippocampus in water-, Dgal-, and Se (0.175 and 0.35mg/ml/kg)-administered mice [mean ± SD (n = 6)]. Data was analyzed by Tukey's test. **p<0.01 and *p<0.05 when compared with respective control group and ++p<0.01 when compared with water + wateradministered animals.

Effect of Se on D-gal induced alteration in 5-HT metabolism
HIAA were enhanced in Se + water-and Se + D-gal-treated animals.
Histopathological estimation in control-, D-gal-, and Se-treated mice Figure 11 shows the analysis of histopathology of the hippocampus in water-, D-gal-, and Se-treated animals. In the water-treated mice, hippocampus showed usual basophilic staining and normal appearance. Se (0.175 and 0.35mg/ml/ kg)-administered mice showed vacuolation at few places and normal nuclear material of pyramidal cells. D-galadministered mice showed a clear decrease in basophilic properties of pyramidal neuron, maximum cellular degeneration, and degeneration of neurons in the hippocampus because of pyknotic nuclei. D-gal + Se (0.175 and 0.35 mg/ml/kg)-administered mice did not show pyramidal neuron degeneration in the hippocampus which indicate the normalization through the onset of pyknosis.

Discussion
D-gal is known for the induction of aging in animals (Wang et al. 2018). The present research work is aimed to evaluate the effects of Se on D-gal-induced aging-associated neurological ailments, i.e., anxiety, depression, and cognitive abnormality. It was observed that repeated treatment with D-gal at a dose of 300 mg/ml/kg produced behavioral deficits that were similar to anxiety-and depression-like behavior, and memory impairment as well. Furthermore, imbalancing in oxidant and antioxidant enzyme status indicates oxidative stress and reduced cholinergic/serotonergic metabolism. Nevertheless, Se independently produced anxiolytic, antidepressant, and memory enhancing effects as well as inhibited above mentioned negative effects of D-gal at both (0.175 and 0.35 mg/ml/kg) doses.
D-gal is widely used to prompt neurotoxicity in agingrelated therapeutic research (Kumar et al. 2011). In experimental studies, an exogenous dose of D-gal raised the oxidative stress and influenced the typical physiological process which ultimately elevated the process of aging in the brain (Ullah et al. 2015) which may lead various neurological disorders. Results of the present study showed that D-gal increased percent immobility time in forced swim test suggesting depression-like behavior (Figure 2) (Zhu et al. 2020) and stimulation of 5-HT-2C receptor at the dentate gyrus of the hippocampus (Sant'Ana et al. 2019). It might be possible that increased serotonin in the brain cause upregulation of 5-HT-2C and downregulation of 5-HT-1A receptor and produced anxiety condition. It is also notable that imbalancing of oxidant and antioxidant status increased the oxidative stress which could be a reason for anxiety. Besides that, it is examined that Se at both low (0.175 mg/ml/kg) and high (0.35 mg/ml/kg) doses inhibits anxiety-and depression-like behaviors and produces anxiolytic and antidepressant effects.
Se is a trace element and potential antioxidant having neuroprotective effects which were observed in various clinical (Vinceti et al. 2018) and pre-clinical ) studies. It was evaluated from current research work that treatment of Se increased 5-HT and its metabolite 5-HIAA ( Figure 8) and decreased percent immobility time in forced swim test in water- Fig. 5 Total protein contents in the hippocampus of mice treated with water, D-gal, and Se (0.175 and 0.35mg/ml/kg) [mean ± SD (n = 6)]. Data was analyzed by Tukey's test. **p<0.01 when compared with respective control group and ++p<0.01 when compared with water + wateradministered animals. and D-gal-treated animals, suggesting the antidepressant effect of Se. It has been earlier reported that low levels of serum selenium levels produce depressive behaviors (Samad et al. 2019a), while administration of Se increased serotonin metabolism and produced antidepressant effects (Nogueira and Rocha 2011) as observed in control-and D-gal-treated groups. It is reported that Se modulated serotonergic neurotransmission as an inhibitor of monoamine oxidase A, a degradative enzyme of serotonin, subsequently increased the synaptic availability of serotonin (Brüning et al. 2019). It is indicated that Se as a powerful antioxidant can inhibit D-gal-instigated oxidative stress and modulates serotonergic mechanism.
Aging is characterized by alteration in brain anatomy and physiology which causes various neurological diseases (Frazzini et al. 2006). Aberrations in the antioxidant system initiate aging, and aging linked pathological conditions (Jadeja et al. 2020). Previously it is reported that administration of D-gal impaired the learning and memory behaviors conducted via water maze and increased acquisition trials and decreased time spent and entries in target quadrant during probe trial (Haider et al. 2020). In the present work, increased escape latencies following acquisition trials ( Figure 3A), decreased time spent (Figure 3B), and entries ( Figure 3C) in the target quadrant after 1 h and 24 h in D-gal-treated mice Fig. 6 Assessment of hippocampal antioxidant enzymes in water-, D-gal-, and Se (0.175 and 0.35mg/ml/kg)administered mice [mean ± SD (n = 6)]. Data was analyzed by Tukey's test. **p<0.01 and *p<0.05 when compared with respective control group and ++p<0.01 when compared with water + water-administered animals. Fig. 7 Assessment of hippocampal inflammatory markers in water-, D-gal-, and Se (0.175 and 0.35mg/ml/kg)administered mice [mean ± SD (n = 6)]. Data was analyzed by Tukey's test. **p<0.01 and *p<0.05 when compared with respective control group and ++p<0.01 when compared with water + water-and water + Se (0.175 mg/ml/kg)-administered animals. Fig. 8 Activity of hippocampal acetylcholinesterase enzyme in water-, D-gal-, and Se (0.175 and 0.35mg/ml/kg)-administered mice [mean ± SD (n = 6)]. Data was analyzed by Tukey's test. **p<0.01 when compared with respective control group and ++p<0.01 when compared with water + water-administered animals.
indicate that D-gal impaired learning and memory behaviors. Acetylcholine (ACh) is a prototype neurotransmitter associated with cognitive functions (Papandreou et al. 2011). Acetylcholinesterase (AChE), a degradative enzyme, hydrolyzes ACh and alters the transmission of Ach at cellular and molecular level. Aging induces an increase in the activity of AChE . Previously it is reported that D-gal impaired the process of normal signaling pathways such Fig. 9 Hippocampal acetylcholine levels in water-, Dgal-, and Se (0.175 and 0.35mg/ml/kg)-administered mice [mean ± SD (n = 6)]. Data was analyzed by Tukey's test. **p<0.01 when compared with respective control group and +p<0.05 when compared with water + water-administered animals. Fig. 10 Hippocampal serotonin metabolism in water-, D-gal-, and Se (0.175 and 0.35mg/ml/kg)administered mice [mean ± SD (n = 6)]. Data was analyzed by Tukey's test. **p<0.01 and *p<0.05 when compared with respective control group and ++p<0.01 when compared with water + water-and water + Se (0.175 mg/ml/kg)-administered animals.
as reduced levels of ACh by stimulating the activity of AChE (Kumar et al. 2011), which is also consistent with our results. The recent study elaborates that D-gal elevates the activity of AChE ( Figure 8) and decreases the level of Ach (Figure 9) which may be responsible for D-gal-induced memory impairment ( Figure 3A, B, C). Previous reports showed that other trace elements such as manganese (Biswas et al. 2019) and zinc (Mecocci et al. 2018) improved cognition with the reduction of oxidative stress and inflammatory markers. At the central levels, Se enhanced cholinergic function with the improvement of neuronal plasticity (Demirci et al. 2017) and by inhibition of inflammatory cytokines (Tang et al. 2019), which can increase the availability of ACh at the synaptic level and reduce AChE activity. In the present study, Se (0.35 mg/ml/kg) administration decreased the acquisition trials in both control-and D-gal-treated mice. The decreasing behavior of acquisition trial in water maze is less in Se (0.175 mg/ml/kg)+D-gal than water + water-treated mice which may be due to less adaptive behavior to the tasks of repeated training trials ( Figure 3A). In the probe test conducted after 1 h and 24 h, Se (0.175 and 0.35 mg/ml/kg) treatment exhibited improvement in time spent ( Figure 3B) and entries ( Figure 3C) in the target quadrant in control-and D-gal-treated animals with reduced activity of AChE (Figure 8), inflammatory markers, i.e., TNF-α and IL-6 (Figure 7), and enhanced ACh (Figure 9) in control-and D-gal-treated mice. It is suggested that as an antioxidant, anti-inflammatory agent, and modulator of cholinergic transmission, Se improves cognitive functions.
The most important of all the results was histopathological photomicrographs (Figure 11). Se prevented D-gal-induced morphological alteration. Results were strengthened by observing histopathological results in which the cells are darkly stained in D-gal-treated aging model which clearly indicates oxidative stress (Miyata et al. 2010). Vacuolization around the neuron was present in the hippocampus of D-gal-treated mice. Coban et al. (2015) observed marked vacuolar alteration, edema, and inflammation in cortical areas of D-gal-treated rats indicating neurodegeneration. Furthermore, irregular nerve fiber arrangement within the neuron in the hippocampal area was also observed following repeated D-gal administration (Sumathi et al. 2015). Result of the present study also supported with the histopathological examination in which cells were observed in oxygen deprivation state due to oxidative stress in Fig. 11 Photomicrographs representing histopathological alterations using hematoxylin and eosin staining at 200× magnification in the hippocampus of water-, D-gal-, and Se (0.175 and 0.35mg/ml/kg)-administered mice. D-gal-treated mice, which also indicate the D-gal-induced neurotoxicity. Administration of Se appears clear and obvious image of hippocampal area at both doses with no degenerative signs, indicating its antioxidant effect.
Oxidative stress impairs normal physiological and neurological functions (Grimm et al. 2016) and induces aging (Barnham et al. 2004). An important antioxidant enzyme SOD interacts with superoxide radical and changes it into water and H 2 O 2 which detoxifies by CAT and GPx enzymes (Saso and Firuzi 2014). Along with these redox enzymes, certain endogenous antioxidants primarily GSH and proteins also provide defense against reactive oxygen species via maintaining redox homoeostasis (Li et al. 2008). Previous studies showed that chronic administration of D-gal elevated oxidative deterioration by diminishing the contents of antioxidant enzyme which alters the homeostasis and impairs many physiological processes (Benn et al. 2004). The reactive oxygen species generation by peroxidation of membrane lipids (Mladenov et al. 2006) can be disintegrated into different carbonyl compounds like MDA, so, the level of MDA is considered oxidative stress biomarker. Administration of D-gal enhanced inflammatory cytokines such as IL-1, IL-6, TNF-α, and β (Mohammadi et al. 2018). Like previous findings (Saso and Firuzi 2014;Benn and Woolf 2004), the recent study also revealed that repeated D-gal treatment reduces the activities of SOD, CAT, and GPx ( Figure 6) and enhances the brain content of MDA ( Figure 4) and inflammatory markers (Figure 7) in mice as compared to control ones. It is reported that Se supplementation in rats can decrease oxidative stress and inflammatory cytokines (Dominiak et al. 2017) by increasing/restoring the endogenous antioxidant (protein contents) (Wu et al. 2021;Gatineau et al. 2018) and activity of antioxidant enzymes (Aydoğan et al. 2013). Our study is also in agreement with above mentioned work that Se administration at both doses reduces oxidative stress and neuroinflammation by increasing endogenous antioxidant protein and antioxidant enzyme activities in mice treated with D-gal which could be due to antioxidant potential of Se (Huang et al. 2012a, b). It is suggested that antioxidant property of Se may be responsible for its antidepressant, memory improving, and anxiolytic effects.
In conclusion, the observed D-gal-induced anxiety, depression cognitive impairment, and neurodegeneration are associated with increased oxidative stress, neuroinflammation, and declined cholinergic/serotonergic neurotransmission in the brain (hippocampus). In addition to this, our results also contribute that Se as a powerful antioxidant and neurotransmitter modulator can inhibit D-gal-instigated anxiety, depression, and memory impairment. The present study confirms the neuroprotective role of Se and proves it as an anxiolytic, antidepressant, memory enhancer, antioxidant, anti-inflammatory, and neurotransmitter (serotonin and acetylcholine) modulator. The current work therefore suggests the use of Se as dietary source and/or supplement as an effective therapy for D-galinduced psychiatric illnesses and associated disorders.