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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 29  |  Issue : 4  |  Page : 944-953

Effect of exercise and/or melatonin on spatial learning and memory of d-galactose-treated rats


1 Physiology Department, Faculty of Medicine, Al Azhar University, Cairo, Egypt
2 Physiology Department, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission28-Mar-2015
Date of Acceptance07-Jun-2015
Date of Web Publication21-Mar-2017

Correspondence Address:
Marwa M Adel
MD of Clinical Physiology, Department of Clinical Physiology, Faculty of Medicine, Menoufia University, Shebin El-kom, Menoufia, 32511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.202490

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  Abstract 

Background
The antioxidant effect of swimming exercise and melatonin may combat against brain oxidative stress, hippocampal damage, and spatial learning and memory impairment induced by chronic exposure of rats to d-galactose.
Objective
This work aimed to study the effect of swimming exercise and/or melatonin on spatial learning and memory impairment induced by d-galactose in rats.
Materials and methods
Thirty adult male albino rats (each weighing 250–370 g) were divided into five equal groups: the control group (C), the d-galactose-treated group (G), the exercised d-galactose-treated group (EG), the melatonin and d-galactose-treated group (MG), and the combined exercised melatonin and d-galactose-treated group (CEMG). The duration of concomitant administration of treatments was 10 weeks. At the end of the 10th week of the study, spatial learning and memory of all groups were assessed using the Barnes maze test for 5 days; malondialdehyde and superoxide dismutase enzyme activity were estimated in brain tissue homogenate and histopathological changes in rat hippocampus were assessed.
Results
In the EG, MG, and CEMG groups, the mean errors and mean escape latency per day on day 1 of the acquisition phase and the probe phase of the Barnes maze test were significantly lower than that in the G group, denoting a significant improvement in spatial learning and memory. In addition, these three groups showed a significant decline in hippocampal damage and malondialdehyde level compared with the G group. In the EG and CEMG groups, superoxide dismutase activity in brain tissue homogenate was significantly higher than that in the G group, but it was nonsignificantly changed in the MG group.
Conclusion
Swimming exercise and/or melatonin reduced brain oxidative stress, hippocampal damage, and consequently improved spatial learning and memory impairment induced by d-galactose. Combined treatment showed better synergistic antioxidant and neuroprotective effect compared with either treatment alone.

Keywords: D-galactose, exercise, melatonin, memory


How to cite this article:
Ashour FA, Abdel-Razek H, Youssef GS, Ewida SF, Adel MM. Effect of exercise and/or melatonin on spatial learning and memory of d-galactose-treated rats. Menoufia Med J 2016;29:944-53

How to cite this URL:
Ashour FA, Abdel-Razek H, Youssef GS, Ewida SF, Adel MM. Effect of exercise and/or melatonin on spatial learning and memory of d-galactose-treated rats. Menoufia Med J [serial online] 2016 [cited 2020 Apr 4];29:944-53. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/944/202490


  Introduction Top


Oxidative stress is a major contributor to the aging process and neurodegenerative diseases [1]. Chronic exposure of rodents to d-galactose decreased the antioxidant enzyme activity, increased the production of free radicals in the brain, accelerated aging, caused hippocampal damage [2], and eventually led to progressive decline in learning and memory, specifically the spatial type [3–6]. In addition, d-galactose has been used for the induction of aging and memory impairment [2]. Exercise and melatonin may combat against oxidative stress-induced memory impairment due to their antioxidant effects [7]. Exercise is an effective way of strengthening antioxidative enzymes [8]. It may improve the performance on cognitive tasks and reduce the risk for neurodegenerative diseases as it may counteract the effects of reactive oxygen species [9]. Melatonin hormone is a neuroprotective molecule [10], which has an antiaging activity because of its powerful effects as an antioxidant, anti-inflammatory, and enhancer of mitochondrial activity [11]. Thus, this work aimed to study the effect of swimming exercise and/or melatonin on d-galactose-induced spatial learning and memory impairment in rats.


  Materials and Methods Top


Thirty adult male albino rats (each weighing 250–370 g) were divided into five equal groups (n = 6). In the control group (C), the rats were injected with sterile 0.9% saline once daily intraperitoneally for 10 weeks. In the d-galactose-treated group (G) the rats were injected intraperitoneally with d-galactose powder (S.D. Fine-Chem Ltd., Olympus, India) dissolved in sterile 0.9% saline at a dose of 100 mg/kg once daily for 10 weeks [2]. In the exercised d-galactose-treated group (EG) the rats underwent swimming exercise concomitantly with d-galactose injection of the same dose as previous group for 10 weeks. Exercise was started with 5 min/day followed by 5 days/week and was gradually increased over 10 weeks, until the rats swam continuously for 30 min/day at the last week of the training program [12]. In the melatonin and d-galactose-treated group (MG), melatonin powder (BioBasic Inc., Canada) was dissolved in 0.01% ethanol and then given to d-galactose-treated rats orally in drinking water (concentration 0.1 mg/ml) at a dose of 10 mg/kg daily for 10 weeks [13]. In the combined exercised melatonin and d-galactose-treated group (CEMG), d-galactose-treated rats underwent swimming exercise for 10 weeks concomitantly with melatonin. At the end of the 10th week of the study, spatial learning and memory of all groups were assessed using the Barnes maze test for 5 days. Thereafter, the rats were anesthetized and killed by means of cervical decapitation [14]. Skull vaults were removed by means of dissection and then the brains were extracted and each brain was divided into two equal halves: one half was weighed and prepared for tissue homogenization for the estimation of malondialdehyde (MDA) and superoxide dismutase (SOD), and the second half was fixed in 10% formalin saline and prepared for hematoxylin and eosin staining (H and E) with the routine technique [15] for histological and morphometric analyses.

Barnes maze test [16]

The paradigm consists of a circular platform that was especially designed at the Physiology Department (92 cm of diameter) with 20 equally spaced holes (5 cm diameter) along the perimeter and is elevated 105 cm above the floor. Bright light was used to reinforce rats to escape from the open platform surface to a target escape box located under the platform called the escape box (28 × 22 × 21 cm). The maze was located in a quiet test room surrounded by visual cues, which could be used by the rats for spatial orientation, and their locations were unchanged throughout the period of testing. All steps of the test were recorded using a Sony video camera for analysis and calculation of the number of errors (IBM, Chicago, USA) (total number of head deflections into incorrect holes) and escape latency (time interval to reach the target hole in seconds) that can be averaged in trials per day for the assessment of spatial learning in the acquisition phase and short-term spatial memory retention in the probe phase [16].

Spatial acquisition phase

Each trial took 3 min per rat, with an intertrial interval of 15 min and four trials per day. The trial was ended when the rat entered the target box or after 3 min had elapsed. Immediately after the rat entered the box, it was allowed to stay in it for 1 min.

Probe phase

On day 5, 24 h after the last training day, the probe trial was conducted. Each rat was allowed to explore the maze for 90 s. The probe trial was conducted to determine whether the animal remembered where the target hole was located.

Brain homogenate preparation [17]

Before dissection, the first half of the brain was perfused with PBS solution (Biodiagnostic Company, Egypt), pH 7.4 containing 0.16 mg/ml heparin to remove any red blood cells and clots and was homogenized in 5 ml cold buffer per gram tissue. Tissue homogenate was centrifuged at 4000 rpm for 15 min at 4°C and the supernatant was removed and stored at −80°C until the determination of MDA levels and SOD enzyme activity [17].

Biochemical estimation

Colorimetric estimation of MDA was carried out using thiobarbituric acid reactive substance for measuring the peroxidation of fatty acids [17]. Colorimetric estimation of SOD activity relies on the ability of the enzyme to inhibit the phenazine methosulphate-mediated reduction of nitroblue tetrazolium dye [18]. Kits for the estimation of both were obtained from Biodiagnostic Company.

Histological study

The other half of the brain from each rat was placed in 10% formalin and prepared for H and E staining with the routine technique [15] for histological and morphometric analysis.

Morphometric study

For quantitative assessment, two nonoverlapping fields of H and E-stained slides at a magnification of × 400 per se ction were randomly captured using a digital camera (Olympus, Japan) from regions (CA1, CA3, and dentate gyrus) of the hippocampus; the number of pyramidal cells and apoptotic cells were counted in the CA1 and CA3 regions and the number of granular cells and apoptotic cells were counted in the dentate gyrus. Fields taken from at least three anatomically comparable sections/animals were assessed using image J analyzer software (NIH Image, Maryland, USA) and the numbers for each cell type were averaged per field for each animal. The numbers calculated for at least five animals/experimental group were considered for comparison and statistical analyses. Data were statistically described in terms of mean ± SEM for area %.

Statistical analysis

The Statistical Package for the Social Sciences (SPSS) version 16 statistical tool was used for analysis of data. The results were expressed as mean ± SEM. The significance of differences between groups was determined using the Kruskal–Wallis one-way analysis of variance test for nonparametric not normally distributed data [19] and one-way analysis of variance for parametric normally distributed data, and the post-hoc Tukey's test was carried out for multiple comparisons between groups. The significance of differences was determined at P value less than 0.05 [20].


  Results Top


Barnes maze test results

Acquisition phase results

The mean number of errors in the G group on day 1 was significantly higher (P < 0.001) than that in the C group. In the EG group, it was significantly (P < 0.001) lower than that in the G group, but nonsignificantly changed (P > 0.05) when compared with the C group. It was significantly (P < 0.001) lower in the MG group than in the G group, but nonsignificantly changed (P > 0.05) when compared with the C and EG groups. In the CEMG group, it was significantly (P < 0.001) lower than that in the G group, but nonsignificantly changed (P > 0.05) when compared with the C, EG, and MG groups [Figure 1]a.
Figure 1: (a) The mean number of errors and (b) mean escape latency in seconds (s) per day in 4 days (D1–D4) of the acquisition phase of the Barnes maze test for all groups. Data are expressed as mean ± SEM (n = 6). The Kruskal–Wallis one-way analysis of variance (ANOVA) test: *P < 0.05 versus the C group; #P < 0.05 versus the G group; ◻P < 0.05 versus the EG group; ♦P < 0.05 versus the MG group.

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The mean escape latency (s) on day 1 in the G group was significantly longer (P < 0.05) than that in the C group. It was significantly (P < 0.001) shorter in the EG group than in the G group, but nonsignificantly changed (P > 0.05) when compared with the C group. In the MG group, it was significantly (P < 0.001) shorter than that in the G group, but nonsignificantly changed (P > 0.05) when compared with the C and EG groups. In the CEMG group, it was significantly (P < 0.001) shorter than that in the G group, but nonsignificantly changed (P > 0.05) when compared with the C, EG, and MG groups [Figure 1]b. There were no significant changes between groups (P > 0.05) on days 2, 3, and 4 of the acquisition phase as regards the mean number of errors and mean escape latency per day [Figure 1]a and [Figure 1]b.

Probe phase results

The mean number of errors in the probe phase of the G group was significantly higher (P < 0.05) than that in the C group. In the EG group, it was significantly (P < 0.05) lower than that in the G group. However, it was nonsignificantly changed (P > 0.05) when compared with the C group. It was nonsignificantly (P > 0.05) changed in the MG group when compared with the G, C, and EG groups. In the CEMG group, it was significantly (P < 0.05) lower than that in the G group and was nonsignificantly (P > 0.05) changed when compared with the C, EG, and the MG groups [Figure 2]a.
Figure 2: (a) The mean number of errors and (b) the mean escape latency in seconds (s) in the probe phase of the Barnes maze test for all groups. Data are expressed as mean ± SEM (n = 6). The Kruskal–Wallis one-way analysis of variance (ANOVA) test: *P < 0.05 versus the C group; #P < 0.05 versus the G group; ◻P < 0.05 versus the EG group; ♦P < 0.05 versus the MG group.

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The mean escape latency (s) in the probe phase of the G group was significantly longer (P < 0.001) than that in the C group. It was significantly (P < 0.001) shorter in the EG group than in the G group. It was nonsignificantly changed (P > 0.05) when compared with the C group. It was significantly (P < 0.05) shorter in the MG group than in the G group and was nonsignificantly (P > 0.05) changed when compared with the C and EG groups. In the CEMG group, it was significantly (P < 0.001) shorter compared with the G group and was nonsignificantly (P > 0.05) changed when compared with the C, EG, and MG groups [Figure 2]b.

Biochemical results

MDA level (nm/g tissue) in the G group was significantly higher (P < 0.001) than that in the C group. It was significantly (P < 0.001) lower in the EG and MG groups compared with the G group, whereas it was nonsignificantly changed (P > 0.05) when compared with the C group. In the CEMG group, it was significantly (P < 0.001) lower than that in the G group, whereas it was nonsignificantly changed (P > 0.05) when compared with the C, EG, and MG groups ([Table 1]).
Table 1 Malondialdehyde (nm/g tissue) level and superoxide dismutase enzyme activity (U/g tissue) in brain tissue homogenate of all groups

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However, SOD level (U/g tissue) of the G group was significantly lower (P < 0.001) than that in the C group. It was significantly (P < 0.001) higher in the EG group than in the G group, whereas it was significantly lower (P < 0.05) than that in the C group. In the MG group, it was nonsignificantly changed (P > 0.05) when compared with the G and EG groups, whereas it was significantly lower (P < 0.05) than that in the C group. It was significantly (P < 0.001) higher in the CEMG group than in the G, EG, and MG groups, whereas it was nonsignificantly changed (P > 0.05) when compared with the C group ([Table 1]).

Histological results

The hippocampus was identified as a C-shaped structure, which is formed of three major areas (CA1, CA2, and CA3) and the dentate gyrus (DG) in the C group [Figure 3]a,[Figure 3]b,[Figure 3]c,[Figure 3]d,[Figure 3]e.
Figure 3: (a) A section of the hippocampus of a control rat (group C) showing the three zones of the cornu ammonis (CA1, CA2, and CA3) and parts of the dentate gyrus [two blades ↑, apex (a) and hilum (h)] (H and E × 40). (b) The three layers of the CA1 area: the outer polymorphic layer (o), the middle pyramidal layer (p), and the inner molecular layer (m). The pyramidal cells are closely packed together. (c) The three layers of CA3: the outer polymorphic layer (o), the middle pyramidal layer (p), and the inner molecular layer (m). The pyramidal cells are not densely packed as CA1 cells. (d) A section of rat dentate gyrus showing the apex and parts of the two blades. It consists of three layers (from outward to inward): the molecular layer (m) formed of small neurons, the granular layer (g) formed of outer mature granular cells (↑), and the inner immature granular cells (thick arrow) and the polymorphic layer called hilum (h) formed of neurons of different shapes. (e) A section of the dentate gyrus showing the hilum with its well-organized hilar cells showing vesicular rounded nuclei and long processes (thick arrow). Notice also microglia cells (↑) and some apoptotic cells with pyknotic nuclei (curved arrow) (H and E × 400).

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Sections of the hippocampus of the G group showed infiltration of edema fluid, sloughing of capillary endothelium, and hypocellularity with disarrangement in pyramidal cell layer, and some pyramidal cells showed apoptosis with pyknotic nuclei [Figure 4]a and [Figure 4]b. The DG of this group showed disarrangement of the granular layer cells with many apoptotic cells with pyknotic nuclei. Numbers of astrocytes and microglia increased denoting gliosis [Figure 4]c. Many apoptotic cells were seen among hilar cells [Figure 4]d and [Figure 7]d. In the EG, MG, and CEMG groups, most of the pyramidal cells of the hippocampus appeared normal and well arranged. Few cells showed apoptosis with pyknotic nuclei [Figure 5], [Figure 6] and [Figure 7]a and [Figure 7]b. The DG showed densely packed mature granular cells with few immature granular cells. The edema fluid and capillary congestion were still evident, but diminished [Figure 5], [Figure 6] and [Figure 7]c.
Figure 4: A section of the hippocampus of a d-galactose-treated rat. (group. G). (a) The CA1 area with hypocellularity of the pyramidal cell layer with many apoptotic cells with darkly stained nuclei. (↑). The outer polymorphic layer showing many astrocytes. (curved arrow) (gliosis), edema fluid. (*), and sloughing of capillary endothelium. (c). (b) The CA3 area with disarrangement of the pyramidal cell layer with many pyramidal cells in the polymorphic cell layer. (↑). Other pyramidal cells show apoptosis with pyknotic nuclei and short axons. (curved arrow). Notice also sloughing of capillary endothelium. (c) and presence of edema fluid separating the axons of neurons. (thick arrow). (c) A section of the dentate gyrus of a d-galactose-treated rat. (group. G) showing marked disarrangement of the granular cell layer with many apoptotic cells with darkly stained nuclei. (↑), apparently increased number of astrocytes and microglia. (gliosis) (curved arrow), filtration of edema fluid, and sloughing of capillary endothelium. (c). (d) A section of the dentate gyrus of a d-galactose-treated rat. (group. G) showing the hilum containing many apoptotic cells with darkly stained nuclei and short axons. (↑). Numerous astrocytes (curved arrow) and microglia (arrow head) are also seen (H and E ×400).

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Figure 5: A section of the hippocampus of a exercised d-galactose-treated rat (group. EG): (a) the CA1 area with many pyramidal cells appear on the same line; however, few cells appear in the polymorphic layer (thick arrow). They appear with vesicular rounded nuclei and long axons. However, there are excess numbers of astrocytes and neuroglia (gliosis) (curved arrow), some edema, and sloughing of the capillary endothelium (star shape) (b) A section of the CA3 area of the hippocampus of a EG group rat showing many pyramidal cells with nearly normal appearance. However, some cells show apoptosis with darkly stained nuclei (↑). Some edema and capillary endothelial sloughing are also seen (c) A section of one blade of the dentate gyrus of EG group rat showing densely packed mature granular cells (thick arrow) with few immature granular (curved arrow); notice also the capillary congestion (↑) and the edema fluid cells (*) (d) A section of the hilar region of the EG group showing nearly normal well-organized hilar cells with vesicular nuclei (↑) (H and E × 400).

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Figure 6: A section of the hippocampus of a melatonin and d-galactose-treated rat (group. MG) (a) The CA1 area with most pyramidal cells appears on the same line with vesicular nuclei (thick arrow); however, other pyramidal cells show pyknotic nuclei (↑). Excessive number of microglia and astrocytes (curved arrow) are also seen. Filtration of edema fluid and sloughing of capillary endothelium are still obvious (b) A section of CA3 of rat hippocampus of the MG group showing many pyramidal cells appearing normal with vesicular nuclei (thick arrow); however, other cells show apoptosis with darkly stained nuclei (↑). Excess number of astrocytes and microglia (gliosis) is still evident (curved arrow). Capillary congestion and edema are also seen (c) A section of the dentate gyrus of a MG rat showing one of its blade with few number of immature granular cells (↑) and edema fluid (thick arrow) (d) A section of the dentate gyrus of a MG rat showing the hilum with well-organized hilar cells (↑) having euchromatic vesicular nuclei. Some astrocytes appear among hilar cells (curved arrow) (H and E × 400).

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Figure 7: A section of the hippocampus of a combined exercised melatonin and d-galactose-treated (group. CEMG). (a) The CA1 area with well-organized pyramidal cells (thick arrow). Most pyramidal cells appear normal; however, there are few pyramidal cells that appear apoptotic with darkly stained nuclei (curved arrow) (b) A section of the CA3 area of the hippocampus of a CEMG group rat showing pyramidal cells with normal arrangement (↑). Gliosis (curved arrow) and some edema are also seen (star) (c) A section of the dentate gyrus of a CEMG group rat showing well-organized mature granular cells (thin arrow), few immature granular cells (curved arrow), some edema (star), and capillary sloughing (thick arrow) (d) A section of the hilum of the dentate gyrus of a CEMG group rat showing the hilar cells with vesicular nuclei (thick arrow) (H and E × 400).

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Morphometric results

Pyramidal cell count for area % in the CA1 region of the G group was significantly lower (P < 0.001) than that in the C group. It was significantly (P < 0.001) higher in the EG, MG, and CEMG groups compared with the G group, whereas it was significantly lower (P < 0.05) compared with the C group. However, apoptotic cell count for area % in the CA1 region of the G group was significantly higher (P < 0.001) than that in the C group. It was significantly (P < 0.001) lower in the EG, MG, and CEMG groups compared with the G group, whereas it was nonsignificantly changed (P > 0.05) when compared with the C group ([Table 2]).
Table 2 Pyramidal and apoptotic cell count for area % in the CA1 region of the hippocampus in all groups

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Pyramidal cell count for area % in the CA3 region of the G group was significantly lower (P < 0.001) compared with the C group. It was significantly (P < 0.05 and P < 0.001) higher in the EG and MG groups, respectively, compared with the G group, whereas it was nonsignificantly changed (P > 0.05) when compared with the C group. In the MG group, it was significantly (P < 0.05) higher than that in the EG group. In the CEMG group, it was significantly (P < 0.001 and P < 0.05) higher than that in the G and EG groups, respectively. It was nonsignificantly (P > 0.05) changed when compared with the C and MG groups. However, apoptotic cell count for area % in the CA3 region of the G group was significantly higher (P < 0.001) compared with the C group. It was significantly (P < 0.05, P < 0.001, and P < 0.05) lower in the EG, MG, and CEMG groups, respectively, compared with the G group, whereas it was nonsignificantly changed (P > 0.05) when compared with the C group ([Table 3]).
Table 3 Pyramidal and apoptotic cell count for area % in the CA3 region of the hippocampus in all groups

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Granular cell count for area % in the DG of the G group was significantly lower (P < 0.001) than that in the C group. In the EG group it was significantly (P < 0.001) higher than that in the G group, whereas it was significantly lower (P < 0.001) than that in the C group. In the MG group, it was significantly (P < 0.001) higher than that in the G and EG groups. However, it was nonsignificantly (P > 0.05) changed when compared with the C group. In the CEMG group, it was significantly (P < 0.001 and P < 0.05) higher than that in the G and EG groups, respectively. It was nonsignificantly (P > 0.05) changed when compared with the C and MG groups. However, apoptotic cell count for area % in the DG of the G group was significantly higher (P < 0.001) than that in the C group. It was significantly (P < 0.001) lower in the EG, MG, and CEMG groups than in the G group, whereas it was nonsignificantly changed (P > 0.05) when compared with the C group ([Table 4]).
Table 4 Granular and apoptotic cell count for area % in the dentate gyrus of the hippocampus in all groups

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  Discussion Top


In the present work, the G group showed spatial learning and memory impairment. This is in accordance with other studies [3–6] and was expressed in different ways according to the evaluation method. For example, Hua et al. [5] indicated that d-galactose-treated rats significantly required more training trials and made significantly more errors compared with controls in passive avoidance and Y maze tests.

The memory impairment induced by d-galactose can be attributed to its reaction with the free amine groups of neuronal proteins and neuropeptides to form advanced glycation end-products [21], which undergo chemical oxidation and degradation through advanced glycation end-product-receptor binding and activation of signaling pathways to form free radicals and cause oxidative stress. The free radicals generated from the oxidation of d-galactose overrun the capacity of cells to clean them and in turn result in the chain reaction of lipid peroxidation and production of a large quantity of MDA, which combines with protein as well as phospholipids, causing cellular damage and impairment of the central nervous system [22]. In the present work, d-galactose caused a significant increase in MDA level and a significant decrease in SOD level in brain tissue that caused neuronal damage, especially in the hippocampus [23]. These findings are in agreement with those of Wang et al. [6], who demonstrated that the hippocampus, which plays a major role in memory and learning [24], might have a higher sensitivity to chronic d-galactose-induced impairment of the antioxidant system, as it might be more sensitive to oxidative stress during various pathological states due to the multiplicity of apoptosis-related pathways in it. Hence, an increased production of reactive oxygen species has a high possibility of degrading neurons in the hippocampus [25] and deteriorating cognitive and memory functions [26], and these facts confirm the present histopathological results that are in agreement with those of Cui et al. [2], who reported that four sections of the hippocampal formation of d-galactose-treated rats had increased in pyknotic nuclei, nuclei of cells undergoing apoptosis, and TUNEL-positive cells, or cells with DNA degradation.

The EG group showed a significant improvement in spatial learning and memory performance compared with the G group. This is in agreement with the findings of Alaei et al. [27], who reported that exercised rats had shorter escape latencies and swim paths in Morris water maze (MWM) compared with controls. Another study by Shih et al. [28] reported that exercise improved spatial memory performance in rats after brain ischemia. This improvement may be due to the antioxidant effect of exercise, as it led to a significant decrease in MDA level to the control level, and SOD level was significantly higher than that in the G group. These results are in agreement with the findings of Chae and Park [29], who reported that regular exercise following d-galactose injection resulted in the positive effect of reducing MDA concentration in the hippocampus and could lead to more defensive antioxidant enzymatic activities such as increased SOD, glutathione [30], catalase [31], and glutathione peroxidase [32]. Therefore, it is proposed that regular exercise could strengthen the antioxidant protection of the hippocampus, and protect it from oxidative stress-induced damage [28]. It was shown in H and E-stained cross sections of the hippocampus of this group. This is in agreement with the findings of Chae and Kim [33], who reported a significant decrease in the level of TUNEL-positive cells in the exercised group compared with the nonexercised group.

The MG group showed a significant improvement in spatial learning and memory performance compared with the G group. This is in agreement with the findings of Shen et al. [13], who reported that melatonin shortened the latencies and reduced the number of errors in MWM of d-galactose-treated mice.

This improvement may be attributed to the antioxidant effect of melatonin as evidenced by its ability to cause a significant decrease in MDA level to that of controls; this is in agreement with the findings of Ismailoglu et al. [34], who found that melatonin treatment reduced MDA level in the brain tissue of rats after cerebral cortical injury. This indicated that melatonin may improve the oxidative injury induced by d-galactose as it is an elective free radical scavenger and antioxidant [35] that is protective in the brain against oxidative deterioration [36].

In this group, there was no statistically significant difference as regards SOD level in brain tissue when compared with the G and EG groups, whereas it was significantly lower than that in the C group. These results are in agreement with the findings of Gönenç et al. [37], who reported that melatonin did not affect the SOD level in the brain tissue of rats with spatial memory impairment induced by acute ethanol treatment. Moreover, others did not find changes in SOD activity after melatonin administration [38]. However, another study by Nogues et al. [39] reported that melatonin is an antioxidant molecule that promoted a decrease in SOD in senescence-accelerated mice (SAMP mice). Some authors reported that melatonin increases SOD activity in various tissues of different animal species [13]. The effect that melatonin has on SOD activity may depend on the redox status and the activity of the other antioxidant defense systems. Thus, if the oxidative stress is high and the amount of melatonin available is insufficient to counteract it, SOD activity will probably increase. However, if there is sufficient melatonin to scavenge free radicals, SOD activity has no need to increase and may even decrease [39], as in our results. In experimentally induced oxidative brain damage models [40], melatonin was effective in reducing the damage of the brain because of its inhibitory effects on lipid peroxidation [41]. The hippocampal histopathological picture of this group revealed a decrease in hippocampal damage. This is in agreement with in-vitro experiments, which showed that melatonin-attenuated Alzheimer's disease induced apoptosis [42].

The CEMG group showed a significant improvement in spatial learning and memory performance compared with the G group, whereas there was no statistically significant difference between the CEMG group and the C, EG, and MG groups. These nonsignificant results between the combination of treatments or either treatment alone was different from the results of García-Mesa et al. [43], who reported that exercised melatonin-treated transgenic model of Alzheimer disease mice showed a better acquisition compared with either treatment alone. Learning and memory appeared to be improved with either physical exercise or melatonin, and also the combined treatment; however, the combined treatment facilitated acquisition, and melatonin alone produced the best outcome in the retention of learning of the platform position of MWM.

Differential responses as regards memory and learning results are probably derived from difference in the method of induction and assessment of memory impairment, the degree of memory impairment of the different animal models and its interplay with the neuroprotective mechanisms triggered by physical exercise and melatonin, the longer duration of treatment, and the difference in exercise protocol and dosage of melatonin of García-Mesa and colleagues compared with that of this study. García-Mesa and colleagues used mice, whereas rats were used in this study.

There was a significant improvement in spatial learning and memory in the CEMG group compared with the G group. This may be attributed to the antioxidant effect of both swimming exercise and melatonin that decreased MDA in brain tissue to almost the control level. However, as regards SOD level in brain tissue, combination therapy seems to have a synergistic effect as it significantly increased its level compared with either exercise or melatonin treatment alone. Interestingly, melatonin alone had a nonsignificant effect on SOD level, but its combination with exercise increased the SOD level to the control level. A study by García-Mesa et al. [43] found that CuZnSOD activity was improved by either melatonin or exercise and MnSOD activity was increased by either melatonin or exercise or both. All treatments reduced the elevated lipid peroxidation levels of the transgenic mice.

In the present work, synergistic antioxidant effect of swimming exercise and melatonin caused improvement in hippocampal histological and morphometric picture of this group compared with the G group. Pyramidal cell count in CA3 and granular cell count in DG showed a significant improvement in the CEMG group compared with exercise treatment alone. Hence, it seems that the effect of combination gave more protection for hippocampal neurons from damage compared with exercise alone. This may be attributed to the synergistic antioxidant effect of exercise and melatonin and/or melatonin's antiapoptotic activities [42].


  Conclusion Top


The antioxidant and antiapoptotic effects of swimming exercise and melatonin make those treatments beneficial for the protection of oxidative stress-induced spatial learning and memory impairment and gives better outcome if used for prophylaxis and treatment of neurodegenerative diseases.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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