Diabetes mellitus is a common, potentially serious metabolic disorder. Over the long term, diabetes exerts major effects on a number of tissues, especially those that are insulin insensitive, such as the retina, neurons, and kidneys. It also causes a variety of functional and structural disorders in the peripheral nervous system and increases the risk of central nervous system (CNS) disorders such as stroke, seizures, dementia, and cognitive impairment 1,2. These functional changes are accompanied by structural and neurochemical abnormalities as well as by degenerative changes in the brain 3,4.
Several brain regions have been studied by biochemical and structural analysis in diabetes; however, few studies have been carried out to determine the ultrastructural characteristics of diabetes in the cerebellar cortex.
The cellular mechanisms responsible for the increased risk of diabetes-induced brain disorders are incompletely understood. Astrocytes have been proven to be critical for normal CNS function. Glial interactions with neurons play vital roles during the ontogeny of the nervous system and in the adult brain. Physical and metabolic insults cause rapid changes in the glial cells. One of the important events during astrocyte differentiation is altered expression of the glial marker, glial fibrillary acidic protein (GFAP) 5–7.
Vitamin E is found naturally in some foods, added to others, and is available as a dietary supplement. ‘Vitamin E’ is the collective name for a group of fat-soluble compounds with distinctive antioxidant activities. Naturally occurring vitamin E exists in eight chemical forms (α-, β-, γ-, and δ-tocopherol and α-, β-, γ-, and δ-tocotrienol) that have varying levels of biological activity. α-Tocopherol is the only form that is known to meet human requirements. Serum concentrations of vitamin E (α-tocopherol) depend on the liver. Numerous foods such as nuts, seeds, and vegetable oils are the best sources of α-tocopherol 8,9.
The body forms reactive oxygen species (ROS) endogenously when it converts food into energy, and it may be exposed to free radicals from environmental exposures. Antioxidants might protect cells from the damaging effects of ROS. Vitamin E is a fat-soluble antioxidant that stops the production of ROS that is formed when fat undergoes oxidation. Scientists are investigating the role of vitamin E in preventing or delaying the complications of chronic or metabolic diseases associated with free radicals 10.
Researchers hypothesize that if cumulative free-radical damage to neurons over time contributes toward cognitive decline and neurodegenerative diseases, then ingestion of sufficient or supplemental antioxidants (such as vitamin E) might provide some protection 11. Learning and memory deficits occur in diabetes mellitus. Although the pathogenesis of cognitive impairment in diabetes has not been fully elucidated, factors such as metabolic impairments, vascular complications, and oxidative stress are believed to play possible roles 12.
This study aimed to characterize the structural, ultrastructural, and immunohistochemical changes in alloxan-induced diabetic rat cerebellar cortex and to determine the possible protective effect of vitamin E.
Materials and methods
Experimental animals and design
Twenty-four adult male albino rat weighing 150–250▒g were selected from the animal house of the Mansoura faculty of pharmacy for use in this study. Blood specimens were aspirated from the tail veins of all rats to measure the fasting blood glucose levels before alloxan injection to ensure that the rats were normoglycemic 13.
The animals were housed in single cages at 20°C on a 12▒h light/dark cycle and had free access to food and water. The animals were divided into three equal groups at random: group A (control), group B (alloxan-induced diabetic rats), and group C (alloxan-induced diabetic rats that received vitamin E). In rats of groups B and C, diabetes was induced by a single intraperitoneal injection of a freshly prepared aqueous solution of alloxan monohydrate (Sigma-Aldrich, Dorset, UK) at a dose of 150▒mg/kg body weight 13. Three days after induction, blood glucose levels from the tail veins were measured using blood glucose test strips (Finetest-autocoding Premium; Infopia Co. Ltd, Korea) and only rats with blood glucose levels above 200▒mg/dl were considered for further experiments 13–15. The control animals, group A, received equal amounts of isotonic saline intraperitoneally 13.
Vitamin E (Pharco, Alexandria, Egypt) was administered by an intragastric tube to the diabetic animals of group C, at a dose of 100▒mg/kg, once daily for 8 weeks 16.
After 8 weeks, the animals of all groups were perfused through the heart apex with 100▒ml isotonic saline, followed by 250▒ml of 2.5% glutaraldehyde in 0.1▒mol/l cacodylate buffer (pH 7.3). The rats were then sacrificed and the cerebellum was removed immediately.
Light microscopic study
The cerebellum was cut and fixed in 10% neutral-buffered formalin and processed for light microscopic study to obtain paraffin sections of 5▒µm thickness. Sections were stained with:
- Immunohistochemical staining (Purchased from Lab Vision, Michigan, USA) for GFAP of astrocytes.
Serial paraffin sections of 5▒µm thickness were deparaffinized and dehydrated, including positive control sections from the rat cerebral white matter. The endogenous peroxidase activity was blocked with 0.05% hydrogen peroxide in absolute alcohol for 30▒min. The slides were washed for 5▒min in PBS at pH 7.4. To unmask the antigenic sites, sections were placed in 0.01▒mol/l citrate buffer (pH 6) in a microwave for 5▒min. The slides were incubated in 1% BSA dissolved in PBS for 30▒min at 37°C in order to prevent nonspecific background staining. Two drops of ready-to-use primary antibodies were applied to the sections, except for the negative control; then they were incubated for 1½▒h at room temperature. GFAP was applied to the sections. The slides were rinsed with PBS, and then incubated for 1▒h with anti-mouse immunoglobulins (secondary antibody) conjugated to a peroxidase-labeled dextran polymer (Dako, Denmark). In order to detect the reaction, the slides were incubated in 3,3-diaminobenzidine for 15▒min. The slides were counterstained by Mayer’s hematoxylin, and then dehydrated, cleared, and mounted by DPX 17.
Transmission electron microscopy
Immediately after sacrifice, thin elongated strips from the cerebellar cortices from the rats of all groups were dissected and immersed in 2.5% gluteraldehyde in 0.1▒mol/l cacodylate buffer (pH 7.3) for 4▒h and then postfixed in 1% osmium tetroxide in 0.1▒mol/l cacodylate buffer (pH 7.3) for 2▒h. The specimens were dehydrated in ascending grades of alcohol, and then passed through two changes of propylene oxide to be finally embedded in epon in an orientation that allowed better examination of all layers. Semithin sections (1▒µm thick) were stained with toluidine blue to select the proper sites for ultrathin sections (60–80▒nm thick), which were cut, double stained with 2% uranyl acetate and 2% lead citrate, and examined using a transmission electron microscope 18.
Morphometric and statistical study
The numbers of GFAP-positive astrocytes in the granular layers of the cerebellar cortices of the rats of all groups were counted. Twenty-five fields from five sections of each rat from each group were examined by a high-power lens (×40). Data entry and analysis was carried out using the software statistical package of social science ‘version 16’ SPSS (Chicago, Illinois, USA). All data were expressed as mean±SD. One-way analysis of variance test with Tukey’s post-hoc test were used. Values of P less than 0.05 were considered significant.
Light microscopic results
H&E-stained paraffin sections of the control group showed that the cerebellar cortex was formed of three layers: an outer molecular layer, a middle Purkinje layer, and inner granular layers. The molecular layer was formed mainly of fibers with few glial cells. Purkinje cells of the middle layer were arranged in one row, and appeared pyriform in shape with thick and long apical dendrites. The granular layer showed a large number of small cells with deeply stained nuclei and non cellular areas in between the cells, representing the cerebellar islands (Fig. 1).
In the cerebellar cortex of alloxan-induced diabetic rats, the Purkinje cells were shrunken, with an eosinophilic cytoplasm and fragmented nuclei surrounded by halos of empty spaces and numerous neuroglia. Many apoptotic granular cells with an eosinophilic cytoplasm and eccentric nuclei were observed (Fig. 2).
In vitamin E-treated diabetic rats, some of the Purkinje cells regained their flask shape with long apical dendrites surrounded by neuroglia cells. No apoptotic cells were detected in the granular layer (Fig. 3).
Immunohistochemical staining for glial fibrillary acidic protein
Examination of positive control sections of the rat cerebral white matter immunohistochemically stained for the detection of GFAP showed the presence of GFAP-positive fibrous astrocytes with long and thin processes. Smaller oligodendrocytes with short and few processes and spindle-shaped microglia were also observed (Fig. 4).
The granular layer of the control cerebellar cortex showed protoplasmic astrocytes with thick processes (Fig. 5).
An apparent increase in the number of GFAP-positive astrocytes with relatively longer processes was observed in alloxan-induced diabetic rats (Fig. 6), whereas vitamin E-treated diabetic rats showed relatively fewer number astrocytes with thin processes (Fig. 7).
Transmission electron microscopic study
Examination of the control cerebellar cortex by transmission electron microscopy showed large flask-shaped Purkinje cells with large rounded nuclei, abundant nuclear sap, and prominent nucleoli. Its cytoplasm showed short rough endoplasmic reticulum (rER) cisternae, mitochondria, and a well-developed Golgi apparatus. Thick apical dendrites were observed (Figs. 8 and 9). In addition, granule cells with darker and smaller nuclei and Golgi type II cells with larger and lighter nuclei were detected. The cytoplasm of Golgi II cells contained profiles of rER cisternae. Myelinated fibers with spherical mitochondria and mossy rosettes were observed (Figs. 8 and 10).
The cerebellar cortex of alloxan-diabetic rats showed irregular Purkinje cells. Irregular nuclei with enfolding of the nuclear membrane and small and eccentric nuclei were observed. The cytoplasm contained numerous lysosomes, variable in size, shape, and electron density, abnormal mitochondria with a heterogenous matrix, few short rER cisternae, and dilated Golgi saccules containing electron-dense granules. Empty halos and irregularly arranged myelin surrounded the Purkinje cells (Figs. 11–13). The granular layer showed Golgi cells with vesicular dilatation of rER cisternae. The myelinated fibers showed splitting of myelin sheaths, wide spaces in the axons, and peculiar-shaped mitochondria. The mossy rosettes showed abnormal mitochondria and disrupted vesicles (Figs. 14 and 15).
The cerebellar cortex of diabetic rats that received vitamin E showed normal flask-shaped Purkinje cells with a large nucleus, profiles of rER cisternae, mitochondria, and lysosomes. The granular layer showed normal granule cells with relatively darker nuclei. Normal Golgi type II cells with lighter nuclei, rER cisternae, and some cytoplasmic spaces were observed. Myelinated nerve fibers and a few fibers with axonal spaces were observed. Normal Mosssy rosettes with numerous spherical mitochondria were observed (Figs. 16–18).
Three days after induction of diabetes, a significant increase in the blood glucose level was recorded in rats of group B as compared with the control. However, there was no detectable significant change in group C versus diabetic untreated rats (Table 1).
A significant increase in the number of GFAP-positive astrocytes compared with the control was observed 8 weeks after induction of diabetes. Moreover, a significant decrease was found in animals that received vitamin E as compared with group B. Meanwhile, compared with the control animals, this was significantly increased (Table 2).
The CNS is highly susceptible to oxidative stress and ROS. The body forms ROS endogenously when it converts food into energy. ROS contribute toward a number of pathological processes including aging and apoptosis 19,20. The mitochondrial electron transport chain is the main source of ROS during normal metabolism and the rate of ROS production is increased in a variety of pathologic conditions 21 and may play an important role in the pathogenesis of degenerative diseases and cancer 22. ROS production was reported to increase in diabetic rats as hyperglycemia initiates nonenzymatic glycolysis in the cells, leading to oxidative stress, which results in the release of free radicals that alter lipid and protein structures 23,24. Moreover, prolonged hyperglycemia has been proposed to reduce the supply of antioxidants to the heart, retina, and brain 25.
Most of the ROS-dependent central nervous disorders in diabetes have been observed to be actually triggered by the presence of free radicals, which are molecules that contain unshared electrons. The body is also exposed to free radicals from environmental exposures, such as cigarette smoke, air pollution, and ultraviolet radiation 26. Antioxidants protect cells from the damaging effects of free radicals. Vitamin E is a fat-soluble antioxidant that stops the production of ROS formed when fat undergoes oxidation. Scientists are investigating whether, by limiting free-radical production and possibly through other mechanisms, vitamin E might help prevent or delay the chronic or metabolic diseases associated with free radicals 10.
Investigators have reported that streptozotocin-induced diabetes led to decreased locomotor activity and cognitive impairment in rats compared with normoglycemic controls 16. Although the pathogenesis of cognitive impairment in diabetes has not been fully elucidated, factors such as metabolic impairment, vascular complications, and oxidative stress are believed to play possible roles 12. In the current study, considerable degenerative changes were observed in the diabetic rat cerebellar cortex. The Purkinje cells as well as the granular cells showed characteristics of apoptosis. Ultrastructurally, Purkinje cells appeared irregular with irregular and eccentric nuclei, numerous lysosomes, dilated Golgi saccules, and peculiar-shaped mitochondria. Degenerated Golgi cells and splitting of myelin sheaths were also observed. This was in agreement with a previous study that observed structural and functional changes in the cerebellar cortex of poorly controlled diabetic rats. Increased neuronal death was the prominent characteristic 27. In addition, similar structural and ultrastructural changes have been observed by many investigators 28,29. They found that uncontrolled diabetes is associated with degenerative changes in the cerebellar cortical neurons, neuroglia as well as disarrangement of the myelin sheath. They postulated that oxidative stress in diabetes coexists with a reduction in the antioxidant status, which can further increase the deleterious effects of free radicals 29. Moreover, chronic hyperglycemia involves a direct neuronal damage that leads to altered neurotransmitter functions and decreased motor activity 30.
In diabetic rats that received vitamin E, these morphological changes were ameliorated to a huge degree. Some authors have reported that neuronal ischemic damage in diabetic rats was less prominent after vitamin E supplementation and consumption of edible palm oil 16,31. Moreover, a combination of a group of antioxidants, vitamin C, vitamin E, l-carnitine, and melatonin, improved hepatic discomfort and reversed learning and memory deficits in diabetic rats 12,32. The potent effect of vitamin E in ameliorating diabetes-induced neuronal degeneration can be linked not only to the antioxidant action 33,34 but also to its superior effect in reducing chronic hyperglycemia 12,16,32.
With the expanding list of astrocyte functions, the role of astrocytes in diabetes-induced CNS disorders remains of interest. In the present study, a significant increase in the number of GFAP-positive astrocytes was found after alloxan-induced diabetes as compared with the control animals. Some investigators have reported an increased GFAP content in different areas of the brain tissue of diabetic rats, mostly in the hippocampus and the cerebellar cortex 6,27,35. This finding indicated that diabetes alters the degradation and production of GFAP, which is a marker of reactive astrocytosis. Thus, determination of GFAP expression may be a relevant marker for studying neurodegenerative changes in diabetes. In contrast, other investigators have reported decreased GFAP expression in the cerebral cortex, hippocampus, and cerebellar cortex of untreated diabetic rats 36. This controversy clearly warrants further experimental and clinical investigations.
Gliosis that occurs in diabetes might be mediated at least indirectly by the formation of free radicals 6,27,35 and antioxidants may prevent this reactive gliosis possibly by reducing the damaging effect of ROS in the CNS. On the basis of this postulation, the use of vitamin E in the present study reduced significantly GFAP expression in diabetic rat cerebellar cortex. A previous study has reported that vitamin E alters the GFAP content in different brain regions of diabetic rats as it influences glycemic parameters and has a direct neruroprotective effect in reducing neuroglial damage in the CNS 37. Thus, it might influence and ameliorate the functions of CNS astrocytes in prolonged diabetes.
From the results of the current work, it can be concluded that alloxan-induced diabetes resulted in a variety of structural and ultrastructural changes in the rat cerebellar cortex associated with increased GFAP expression, which is considered a marker of reactive astrocytosis. These ultrastructural and immunohistochemical changes are found to be corrected with the administration of vitamin E. Therefore, vitamin E can be used as an adjuvant therapy for the prevention and treatment of the CNS complications of diabetes.
Vitamin E might represent a new potential alternative for the prevention of impaired cognitive functions associated with diabetes and this may warrant further clinical investigations.
Conflicts of interest
There is no conflict of interest to declare.
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