Effect of Eight Weeks of Exercise Training on the Hippocampal Tissue in Streptozotocin-Induced Diabetic Rats: A Histological Study

AUTHORS

Mohammad Rami ORCID 1 , * , Abdolhamid Habibi 1

1 Department of Sport Physiology, Faculty of Sport Sciences, Shahid Chamran University of Ahvaz, Ahvaz, Iran

How to Cite: Rami M, Habibi A. Effect of Eight Weeks of Exercise Training on the Hippocampal Tissue in Streptozotocin-Induced Diabetic Rats: A Histological Study, Zahedan J Res Med Sci. Online ahead of Print ; 22(2):e96841. doi: 10.5812/zjrms.96841.

ARTICLE INFORMATION

Zahedan Journal of Research in Medical Sciences: 22 (2); e96841
Published Online: May 19, 2020
Article Type: Research Article
Received: August 10, 2019
Revised: September 16, 2019
Accepted: September 22, 2019
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Abstract

Background: Glial cells perform several critical functions, and exercise can improve many of these functions. Therefore, careful study is needed to elucidate the mechanisms through which exercise affects brain function following diabetes.

Objectives: In this study, we evaluated the quantitative histological changes of glial cells in the hippocampus region of streptozotocin (STZ)-induced diabetic rats following exercise training.

Methods: Twenty-four adult male Wistar rats aged 10 weeks with an average weight of 256 ± 11.8 g were assigned into the four groups of diabetic, trained diabetic, control, and healthy trained groups. Diabetes was induced by a single dose intraperitoneal injection of STZ (45 mg/kg). Forty-eight hours after STZ injection and diabetes confirmation, moderate exercise activity was performed during an eight-week period, five sessions in a week. Forty-eight hours after the last training session, the rats were anesthetized, sacrificed and then the hippocampus tissue was removed. Sections with a thickness of 5 - 6 µ were prepared and stained with the hematoxylin and eosin staining method. Then, astrocyte, oligodendrocyte and microglia cells were counted in different regions of the hippocampus.

Results: Histological evaluations showed that after the training sessions the number of astrocyte and oligodendrocyte cells in different regions of the hippocampus in diabetic rats significantly decreased compared to healthy rats. However, the number of microglia cells in diabetic rats was significantly higher than healthy rats (P < 0.05). In addition, the numbers of astrocytes and oligodendrocytes significantly increased in the dentate gyrus, Cornu Ammonis and subiculum of the hippocampus tissue after endurance training compared to the control group, while the number of microglial cells significantly decreased (P < 0.05).

Conclusions: Moderating the mechanisms responsible for changes caused by diabetes in the hippocampal cells helps develop a treatment for impaired cognitive and memory functions in diabetic individuals. Our findings confirmed the potential effects of endurance exercise on diabetes. Thus, it seems that physical activity plays an important role in the improvement of the nervous complications in diabetic patients.

Copyright © 2020, Zahedan Journal of Research in Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.

1. Background

Diabetes is known as a chronic metabolic disorder with the two main forms of type 1 and 2 and a high index of hyperglycemia (1, 2). Another organ affected by diabetes is the central nervous system (3). Hyperglycemia-induced oxidative stress has been implicated in the development of diabetic neuropathy, one of the complications of diabetes (1). Cellular mechanisms that describe how diabetes mellitus negatively affects the structure and function of the central nervous system remain nebulous.

In recent years, numerous studies have shown that diabetes has a negative impact on the central nervous system in animal models (4). Research has shown that under advanced inflammatory conditions due to hyperglycemia the activity of microglia cells increases and leads to astrocytes hemichannels activity due to the release of pro-inflammatory cytokines. The activation of such hemichannels is the first step in the onset of neurotoxic intracellular cascades and the progression of cell death in astrocytes, neurons, and oligodendrocytes (5). Currently, there is still no effective therapy for neurodegenerative disorders despite extensive research and ongoing laboratory efforts (6). The therapeutic methods used to treat the process of neurodegeneration caused by diabetes have little or no effects or cause adverse effects such as acidosis and indigestion (7). Therefore, researchers are looking for alternative ways to prevent or treat this condition with fewer complications.

Recently, strong evidence has shown that exercise contributes to the promotion of learning and memory, delay in the cognitive decline associated with age, and reduction in inflammatory factors and neurodegenerative disorders (8). Also, the previous evidence has shown that physical activity, in addition to promoting behavioral performance, improves synaptic plasticity in the hippocampus (9). It has been confirmed that physical activity in diabetic rats decreases the activity of microglia cells and subsequently reduces the levels of inflammatory cytokines in the hippocampus (10). In addition, the proliferation of astrocytes after exercise has been found to be associated with angiogenesis in the cortex of healthy mice. This event will protect the blood-brain barrier following brain damage (11). Studies have shown that neural adaptation following physical activity is crucial for the prevention and treatment of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and diabetic neuropathy (12). However, the possible mechanisms for the effect of physical activity on neurodegenerative disorders caused by diabetes are not well understood (13).

2. Objectives

Since the hippocampus region, which includes the Cornu Ammonis (CA), dentate gyrus (DG) and the subiculum (S) regions, is one of the critical areas of the central nervous system with various function such as learning and memory and is very sensitive to changes in glucose homeostasis (14), we aimed to investigate the effect of endurance exercise training on the histology of the hippocampus region in experimental diabetic rats.

3. Methods

3.1. Animals

Twenty-four adult male Wistar rats (aged 10 weeks, with a mean weight of 256 ± 11.8 g) were kept in Plexiglas cages under standard conditions. Five animals were housed in each cage in a controlled room (light-dark cycle 12:12 h) with free access to food and water. The rats were adapted with laboratory conditions and treadmill for 2 weeks (15). During the acquaintance stage, to get acquainted with the laboratory conditions, treadmill and manipulation, the animals travelled on the treadmill at the speed of 15 m/min for 15 minutes 5 days a week for 2 weeks.

3.2. Induction of Diabetes

After 12 hours of food deprivation, diabetes was induced by the intraperitoneal injection of 45 mg/kg of STZ solution (Sigma, St. Louis MO, USA) dissolved in fresh citrate buffer (0.5 M with pH 4.5). Non-diabetic rats were injected with the same volume of citrate buffer. After 48 hours, a drop of blood was placed on a glucometer tape by causing a small lesion on the venous tail vein, and blood glucose was read by a glucometer (Roche diagnostic, Japan). In this study, the rats with a blood glucose level of above 300 mg/dl were considered as diabetic rats (16).

3.3. Grouping and Exercise Program

After the familiarization stage, the rats were randomly divided into four groups as follows: (1) diabetic training group (DT, N = 6), in this group diabetes was induced by intraperitoneal injection of streptozotocin (STZ) (Sigma, USA) and completed 5 sessions of endurance training for 8 weeks. (2) Diabetic control group (DC, N = 6), this group did not exercise at any time. (3) Healthy training group (HT, N = 6), this group, similar to the DT Group, participated in a treadmill exercise program. (4) Control group (Co, N = 6), this group did not engaged in any activity.

3.4. Endurance Training Protocol

In the present study, moderate-intensity exercise was used for endurance training (15). The speed and duration of the treadmill gradually increased from 27 m/min for 20 - 30 minutes in the first week, to 27 m/min for 30 - 40 minutes in the second week, 27 m/min for 40 - 50 minutes in the third week, 27 m/min for 50 - 60 minutes in the fourth week and 27 m/min for 60 minutes in the fifth to eighth weeks. The treadmill slope was zero at all the stages. All the training variables were kept constant during the final week (eighth week) in order to obtain the adaptations resulting from the steady-state exercise.

3.5. Behavioral Measurements

3.5.1. Object Recognition Test

In the object recognition test, each rat was kept for 15 minutes in a white wooden box with dimensions of 40 x 40 cm and a height of 16 cm. After one week, two identical objects were placed in the box and each rat was housed in the box for 10 minutes. After 30 minutes, one of the objects was changed by another color-differentiated object, and each rat was kept in the box for 5 minutes, and the whole time was captured with the camera. When reviewing the films, the rat was considered as favorable and indifferent as the first move toward the new object, or the old object was considered as undesirable (17).

3.5.2. Tissue Extraction and Histological Staining

After eight weeks of training, the animals were anesthetized with ketamine (75 mg/kg) and xylazine (5 mg/kg), and after separating the head by guillotine, the hippocampus tissue was removed under sterile conditions and placed in 10% formalin solution. After 24 hours, the tissue of the hippocampus was removed and placed in fresh 10% formalin solution. Then, the hippocampal tissue was dehydrated with ethanol (70% for 24 hours, 90% for 1 hour and 100% for 1 hour) and then cleared with xylenol and placed in paraffin. In the next step, the samples were cut by a microtome (Leica RM2025, Germany) at a thickness of 5 microns, and then fixed onto a glass slide after the usual stages of tissue consolidation. In the end, the glass slides were stained with the usual Hematoxylin-Eosin method (H & E) (18).

3.5.3. Histopathological Assessment of the Hippocampus

In the microscopic study, the number of astrocytes, oligodendrocytes, and microglia cells at x 40 magnification (5 microscopic slides for each sample) in the CA1, CA2, CA3, CA4, dentate gyrus, and subiculum areas were measured and counted. At this stage, the Dinocapture software and the Dino-Eye Microscope AM-423X (ANMO, Taiwan) were used. In H & E staining, astrocyte cells can be detected with respect to their oval nucleus (19). These cells have the largest nucleus among glia cells. Also, in the tissue section, the oligodendrocytes have small, dark, spherical nuclei (heterochromatin). In H & E staining, microglia can be detected by small nuclei and their heterochromatin (19). Using the Atlas of the central nervous system, different regions of the hippocampus were identified in the cross-section and the measurement and counting operations were performed (20).

3.6. Statistical Analysis

One-way ANOVA was used to compare changes in the number of cells and blood glucose between different groups. The Tukey’s post hoc test was used to analyze the variance for the pair-wise comparisons. For statistical analysis, SPSS software version 18 was used, and P value less than 0.05 was considered significant.

4. Results

4.1. Blood Glucose Level

At the beginning of the exercise program, 48 h after induction of diabetes, blood glucose levels increased significantly in the diabetic rats (P < 0.001), and after 8 weeks of endurance training, blood glucose level was significantly different compared to the healthy group (P < 0.001). At the end of the last session of training, blood glucose concentration in the training diabetic group was significantly lower than in the control group of diabetes (P < 0.001; Figure 1).

Changes in blood glucose levels in different groups. Dissimilar letters in each stage indicate a significant difference (P &lt; 0.05).
Figure 1. Changes in blood glucose levels in different groups. Dissimilar letters in each stage indicate a significant difference (P < 0.05).

4.2. Object Recognition Test

In the current study, cognitive function was assessed by the object recognition test in diabetic rats. Figures 2 showed the results of the object recognition test. According to this figure, at the end of the eighth week of training, the results showed that cognitive function was significantly improved in the diabetic group compared to the control group (P < 0.05). Exercise improved cognitive function in the healthy group as well.

Recognition of new object (sec). Dissimilar letters in each stage indicate a significant difference (P &lt; 0.05).
Figure 2. Recognition of new object (sec). Dissimilar letters in each stage indicate a significant difference (P < 0.05).

4.3. Histopathological Results of the Hippocampus

In Figure 3, histological examination of hematoxylin and eosin-stained sections of the hippocampus is shown. In this figure, different regions of the hippocampus are shown.

Sections from different regions of the hippocampus including the Cornu Ammonis (CA), as CA1, CA2, CA3 &amp; CA4 regions, and subiculum (S). Dentate gyrus (DG) is seen encircling CA4 by its upper &amp; lower limbs. (H &amp; E x4).
Figure 3. Sections from different regions of the hippocampus including the Cornu Ammonis (CA), as CA1, CA2, CA3 & CA4 regions, and subiculum (S). Dentate gyrus (DG) is seen encircling CA4 by its upper & lower limbs. (H & E x4).

As shown in Table 1, the results of ANOVA among different groups in the pyramidal and molecular layers of the hippocampus showed significant differences in the number of astrocytes, oligodendrocytes and microglia in different regions of the hippocampus (P < 0.05).

Table 1. Analysis of Variance Between Different Groups in the Two Layers of Hippocampus Including Pyramidal and Molecular (P < 0.05).
Cell TypeRegions
Pyramidal LayerMolecular Layer
DGSCA1CA2CA3CA4DGSCA1CA2CA3CA4
Astrocyte0.0010.0040.0340.0010.0130.0430.0010.0230.0020.0220.0410.017
Oligodendrocyte0.0020.0130.0010.0220.0020.0010.0010.0170.0460.0340.0250.043
Microglia0.0110.0010.0010.0010.0290.0010.0010.0410.0220.0010.0070.09

The results of the post hoc test showed that the number of astrocytes and oligodendrocytes in different regions of the hippocampus in diabetic rats, both in the pyramidal and molecular layers, significantly decreased compared to healthy rats (P < 0.05). However, the number of microglia cells in the pyramidal and molecular layers in diabetic rats was significantly higher than in healthy rats (P < 0.05; Table 2). Also, after the end of eight weeks of training, the results of microscopic observation showed that the mean number of astrocytes and oligodendrocyte cells in the pyramidal and molecular layers of different regions of the hippocampus in the diabetic training group did not show a significant difference compared to the control group (P > 0.05). However, the results proved that the number of microglia cells in the pyramidal and molecular layers in diabetic trained rats was significantly lower than that of the diabetic control rats (P < 0.05). In Figures 4-6, changes in the cells of different regions of the cross-section of the hippocampus tissue in the four groups are schematically shown.

Table 2. Mean ± SEM of One-Way ANOVA with Tukey’s Post Hoc Test of the Number of Astrocytes, Oligodendrocytes and Microglia in Different Layersa
RegionLayerCellDiabetes ControlDiabetes TrainingControlHealth Training
Dentate gyrusPyramidalAstrocyte15.6 ± 1.5 A27.4 ± 4 B22.3 ± 1 B25.7 ± 1.2 B
Oligodendrocyte17.8 ± 0.7 A19.9 ± 3.5 A10.5 ± 1.6 B17.6 ± 2.8 A
Microglia2.8 ± 0.6 A2.3 ± .5 A3.2 ± 2.7 A4.2 ± .5 A
MolecularAstrocyte24.5 ± 1.5 A28.9 ± 1.1 A19.1 ± 2.3 B25.9 ± 1.7 A
Oligodendrocyte11.8 ± 1.8 A25.2 ± 2.7 B16.3 ± .8 C18.2 ± 1.5 C
Microglia5.5 ± .6 A2.6 ± .6 B2.3 ± 1.6 B2.6 ± .3 B
Cornu Ammonis (CA1-CA4)PyramidalAstrocyte10.2 ± 2.9 A14.9 ± 3.5 B16.3 ± .4 B20.7 ± 4.1 C
Oligodendrocyte7.8 ± 2.2 A16.9 ± 2.4 B12.5 ± 1.9 C16.6 ± 1.3 B
Microglia8.8 ± 1.6 A2.8 ± .8 B4.3 ± 2.7 B3.2 ± 1.1 B
MolecularAstrocyte11.5 ± 3.5 A13.9 ± 2.1 A15.5 ± 2.8 A21.4 ± 5.3 B
Oligodendrocyte9.8 ± 2.4 A14.2 ± 3.7 B16.3 ± .8 B20.2 ± 4.5 C
Microglia9.5 ± 3.7 A3.6 ± 1 B4.3 ± 1.8 B2.9 ± 1.2 B
SubiculumAstrocyte13.1 ± 2.5 A16.1 ± 1.22 B17.2 ± 1.21 B19.6 ± 5.3 B
Oligodendrocyte11.8 ± .7 A15.9 ± 3.5 B18.5 ± 1.3 C20.6 ± 2.1 C
Microglia6.4 ± 1.9 A2.9 ± 1.5 B3.2 ± 2.1 B4.4 ± .5 B

aDissimilar letters in each row indicate a significant difference (P < 0.05).

Selective histological sections of the pyramidal and molecular layers of the dentate gyrus (DG) region of the hippocampus tissue. A) Diabetes Control; B) Diabetes training; C) Health control; D) Health training. (Black arrows show astrocyte cells, red arrows show oligodendrocyte cells, and blue arrows show microglia cells). (H &amp; E x 40).
Figure 4. Selective histological sections of the pyramidal and molecular layers of the dentate gyrus (DG) region of the hippocampus tissue. A) Diabetes Control; B) Diabetes training; C) Health control; D) Health training. (Black arrows show astrocyte cells, red arrows show oligodendrocyte cells, and blue arrows show microglia cells). (H & E x 40).
Selective histological sections of the pyramidal and molecular layers of the Cornu Ammonis (CA) regions of the hippocampus tissue. A) Diabetes Control; B) Diabetes training; C) Health control; D) Health training. (Black arrows show astrocyte cells, red arrows show oligodendrocyte cells, and blue arrows show microglia cells). (H &amp; E x 40).
Figure 5. Selective histological sections of the pyramidal and molecular layers of the Cornu Ammonis (CA) regions of the hippocampus tissue. A) Diabetes Control; B) Diabetes training; C) Health control; D) Health training. (Black arrows show astrocyte cells, red arrows show oligodendrocyte cells, and blue arrows show microglia cells). (H & E x 40).
Selective histological sections of the pyramidal and molecular layers of the subiculum (S). A) Diabetes Control; B) Diabetes training; C) Health control; D) Health training. (Black arrows show astrocyte cells, red arrows show oligodendrocyte cells, and blue arrows show microglia cells). (H &amp; E x 40).
Figure 6. Selective histological sections of the pyramidal and molecular layers of the subiculum (S). A) Diabetes Control; B) Diabetes training; C) Health control; D) Health training. (Black arrows show astrocyte cells, red arrows show oligodendrocyte cells, and blue arrows show microglia cells). (H & E x 40).

5. Discussion

Diabetic hyperglycemia causes complications such as DNA damage, chronic oxidative stress, and neuronal death (21). Increased glial response is a common symptom of diabetes mellitus (22). Therefore, in the present study, the protective effects of 8 weeks of moderate activity against hyperglycemia and oxidative stress and glial reactions were studied. The results of this study showed that 8 weeks of moderate-intensity training significantly reduced blood glucose concentration in the diabetic training group. Also, increased cognition of the new object indicates the effect of physical activity on preventing object recognition disorders in STZ-induced diabetic rats. Recent research has shown that hyperglycemia and oxidative damage in the hippocampus were reduced due to activity with sub-maximal severity and regular exercise can promote memory performance through anti-oxidative effects (23).

Astrocyte cells are considered as energy storage sources by storing glycogen and releasing glucose (19). Oligodendrocytes in the white matter, with the formation of myelin sheath around axons, accelerate the transfer of active potentials in the central nervous system (19). The present study confirmed that the number of astrocyte and oligodendrocyte cells of the hippocampus in diabetic rats decreased compared to the control group. While, eight weeks of endurance training on treadmill prevented the decrease in the number of astrocyte and oligodendrocyte cells and improved the number of these cells. Our results are consistent with those of De Senna et al. despite the difference in the type and volume of exercise (19). Endurance training limits indices such as neurogenesis (24), long-term potentiation and synaptic plasticity (25), and increased expression of neurotrophic factor (26), and prevents oxidative stress (27). In contrast, hyperglycemia associated with diabetes leads to a decrease in neuronal function (28) and increased oxidative stress and neuronal death (29).

In response to central nervous system damages, microglia cells proliferate and show defensive and even cytotoxic properties. These cells appear to play a role in important diseases such as viral infections, autoimmune and neurodegeneration disorders, such as diabetes (10). Orellana et al. reported that during inflammatory processes in neurodegenerative diseases, opening the hemi channels reduces neuronal immunity (30). Increased activity of astrocytes and neuronal hemi channels leads to the release of neurotoxic molecules such as glutamate and ATP, which can release more cytokines in microglia leading to cell death and degradation of the CNS function, as seen in diabetes mellitus and Alzheimer’s disease (30, 31). The results of this study showed that endurance activity reduces microglia cells in the hippocampal tissue, in both layers of pyramidal and molecular. Our results are in agreement with those of Yoo et al., which revealed that exercise activity on treadmill significantly reduced microglial cells and moderated the increase in inflammatory factors such as TNF-α, IL-6 and IL-1β induced by diabetes (10).

Also, De Senna et al. showed that the levels of glial cells in the hippocampal CA region were increased in diabetic animals following physical activity on the treadmill (19). In their study, they stated that the proliferation of astrocytes after exercise was coupled with the angiogenesis process and may cause the development of a neuro-vascular unit, a structure that includes capillary endothelial cells, astrocytes, neurons, and extracellular matrix (19). Therefore, exercise activities may protect the blood-brain barrier from the damages caused by diabetes or other types of brain damage (19, 32).

5.1. Conclusion

In conclusion, our results demonstrated that considerable changes occur in the histology of the hippocampus of diabetic rats following exercise programs, which can be indicative of the beneficial effect of basic exercise activities on the central nervous system homeostasis. The results of this study showed that endurance activity reduces microglia cells and thus moderates the increase in inflammatory factors. Therefore, these results can be used to prevent and treat disorders caused by nerve degradation in diabetic patients.

Acknowledgements

Footnotes

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