Diabetes mellitus (DM) is a systemic, chronic, and life-threatening disease that, according to WHO, affects 135 million individuals worldwide and may affect as many as 300 million by the year 2030 1. DM is a common metabolic disorder with well-known serious secondary complications. Diabetic peripheral neuropathy was long considered the only complication involving the nervous system, whereas the central nervous system (CNS) was believed to be relatively spared from the direct effects of DM 2,3. In the last decade, it has become clear that DM may be both primarily and secondarily responsible for CNS complications, with adverse functional and cognitive effects 4.
DM is a heterogeneous disease characterized by chronic hyperglycemia and requires long-term management. Chronic changes in the antecedent level of glycemia induce alterations in brain glucose metabolism and can lead to various complications, affecting the CNS 5. This complication is referred to as ‘diabetic encephalopathy’ and is characterized by impairments in cognitive functions and electrophysiological changes 6. These functional changes are accompanied by structural and neurochemical abnormalities as well as degenerative changes in the brain 7,8.
The streptozotocin (STZ)-induced diabetic rat serves as an excellent model to study the molecular, cellular, and morphological changes in the brain induced by stress in DM 5,9. Also, it provides a relevant example of endogenous chronic oxidative stress as a result of hyperglycemia 10. STZ is often used to induce DM in experimental animals because of its toxic effects on pancreatic β-cells 11. It is a potent alkylating agent that can methylate DNA and its cytotoxicity depends on DNA alkylation 12.
Several brain regions have been studied by biochemical analysis in DM. The cerebellum plays an important role in movement and posture. Malformation and lesions of the cerebellum disrupt motor coordination and impair balance. The cerebellum is also involved in a variety of nonmotor cognitive functions, including sensory discrimination, attention, learning, and memory 13. Also, few studies have been carried out to determine the ultrastructural features of DM in cerebellum. Therefore, this study aimed to characterize the ultrastructural changes in the cerebellar cortex in STZ-induced diabetic rats.
Materials and methods
Twenty adult male albino rats weighing 210–250 g were used for this experiment. These rats were obtained from animal house, Faculty of Medicine, Zagazig University. The rats were allowed to acclimatize for 2 weeks before the experiment. They were housed individually in separate cages under daily normal light/dark periods. Rats had free access to standard food and water ad libitum.
Experimental design and drug administration
The rats were divided randomly into three different groups:
- Group I: Five rats were not subjected to any procedure and served as a control.
- Group II: Five rats were administered a single intraperitoneal injection of 0.1 ml saline.
- Group III: Ten rats received STZ as the diabetogenic agent (Sigma Chemical Company, St Louis, Missouri, USA), and the vehicle for administration was the saline. Each rat was administered a single intraperitoneal injection of STZ at a dose of 60 mg/kg body weight freshly dissolved in 0.1 ml saline under anesthesia 14. Fasting blood samples were taken from the dorsal vein of rats’ tails. The blood glucose level of all rats was estimated by the glucose-oxidase method using (Accu-chek Active; Roche Diagnostics, Mannheim, Germany) before and 3 days after the STZ injection to confirm DM induction. All rats that presented with a fasting blood glucose level higher than 250 mg/dl were considered as diabetic 15. These rats were anesthetized with ether inhalation and scarified at 8 weeks 14 after the onset of DM. The skull was opened; the cerebellum was dissected out carefully and processed for the following procedures.
Light microscopic study
The specimens were fixed in 10% formol saline, processed into 5 μm-thick paraffin sections, and then stained with H&E 16.
Semithin and transmission electron microscopic studies
The specimens were immediately fixed in 2.5% glutaraldehyde buffered with 0.1 mol/l phosphate buffer at pH 7.4 for 2 h and postfixed in 1% osmium tetroxide in the same buffer for 1 h at 4°C. The specimens were processed and embedded in embed-812 resin in BEEM capsules (Polyscience, Warrington, Pennsylvania, USA) at 60°C for 24 h. Semithin sections were cut into 1 μm thickness using an ultramicrotome, stained with 1% toluidine blue, and examined by a light microscope. For electron microscopy, ultrathin sections (50–80 nm thick) were cut using the same ultramicrotome and stained with uranyl acetate and lead citrate 17. The sections were examined using a JEOL 1010 electron microscope (Japan) at Mycology and Regional Biotechnology Center, Al Azhar University, Cairo, Egypt.
Sections were incubated with a monoclonal antibody against Bax protein (Dako, Carpinteria, California, USA) 18 and glial fibrillary acidic protein (GFAP; Sigma, St Louis, Missouri, USA) 19. Detection of the antibody was carried out using a biotin–streptavidin detection system with 3-amino 9-ethyl carbazole as a chromogen (Dako) for Bax and with 0.05% diaminobenzidine as a chromogen (Amersham, Little Chalfont, UK) for GFAP.
Quantitative morphometric study
Images were analyzed using computer-based image analysis software (Leica Qwin 500; Imaging Systems, Cambridge, UK). All Purkinje cells were counted in each of the 10 cerebellar lobules of each section using light microscopy at ×200 magnification. Then the average value for the 10 lobules was calculated for each section. Also, astrocytes and apoptotic cells were counted in an area of 20 000 μm2 and selected randomly in the GFAP-stained and Bax-protein stained sections using light microscopy at ×400 magnification.
The number of Purkinje cells, GFAP-positive astrocytes, and also apoptotic cells were presented as mean±SD. Statistical analysis was carried out using unpaired student’s t-test, where the level of significance (P) value was set at 0.05 (one-way analysis of variance).
In the diabetic group, three rats died.
Purkinje cell number
In the diabetic group III, the mean number of Purkinje cells decreased significantly (P<0.005) compared with the control groups I and II (Table 1 and Histogram 1).
In the diabetic group III, the mean number of astrocytes increased highly significantly (P<0.0001) compared with the control groups I and II (Table 2 and Histogram 2).
Apoptotic cell number
In the diabetic group III, the mean number of apoptotic cells increased insignificantly (P>0.005) compared with the control groups I and II (Table 3 and Histogram 3).
Control group I and II
Light microscopic examination of both control groups I and II showed no observable differences.
The H&E-stained and toluidine-stained sections showed the usual architecture of the cerebellum. The cerebellar cortex was made up of molecular, Purkinje cells, and granular layers. The molecular layer was formed of few small stellate cells located superficially and basket cells were found in the deeper parts near Purkinje cell bodies (Fig. 1). The Purkinje cell layer was observed to be arranged in a single row along the outer margin of the granular layer. It consisted of large pyriform somata of Purkinje neurons with clear vesicular nuclei, prominent nucleoli, and a basophilic cytoplasm (Figs 1 and 2). The granular layer was composed of tightly packed small rounded cells with deeply stained nuclei and also interspersed among these cells were clear spaces (glomerulus or cerebellar islands), where synapses occur between axons entering the cerebellum from outside and dendrites of granule cells (Fig. 1).
Immunohistochemical staining for GFAP showed GFAP-positive astrocytes; they appeared small, with thin few processes, in the granular and molecular layers (Fig. 3).
Immunohistochemical staining for apoptotic marker (Bax-protein) indicated a negative reaction in Purkinje neurons and granule cells (Fig. 4).
Ultrathin sections of the cerebellar cortex showed that Purkinje cells were distinguished by their position, large size, and well-defined euchromatic nuclei with long dimples. The cytoplasm was rich in mitochondria with intact membranes and regular cristae, free ribosomes, and cisternae of rough endoplasmic reticulum (Fig. 5a–c). Bergmann astrocytes were observed between the Purkinje cells with their demarcated euchromatic nuclei and electron-lucent cytoplasm, with few organelles as glial filaments (Fig. 5a). Granule cells were observed with their euchromatic nuclei and a shell of cytoplasm that showed free ribosomes. Several axons with intact mitochondria and also myelin sheaths were observed (Fig. 6a and b). Axons of large Mossy fibers with mitochondria with regular intact cristae were observed. Typical synapses with a synaptic cleft, and presynaptic and postsynaptic membranes were observed. Axonal terminals with easily identifiable small synaptic vesicles were observed (Fig. 7a and b).
Diabetic group III
The H&E-stained and toluidine blue-stained sections showed a marked reduction in the number of Purkinje cells. The Purkinje neurons showed an irregular outline, a darkly stained cytoplasm, and hardly identified nuclei (Figs 8a and 9a). Prominent perineuronal spaces were observed in the molecular layer around both basket and stellate cells (Fig. 8a). Vascular congestion with wide perivascular spaces or swelling and also hemorrhage in the granular layer and white matter were observed (Figs 8a, b, and 9b). Many Bergmann astrocytes with demarcated pale nuclei surrounded by a pale cytoplasm were observed (Fig. 9a).
Immunohistochemical staining for GFAP showed that GFAP-positive astrocytes were more abundant and appeared larger in the three cerebellar cortical layers (Fig. 10).
Immunohistochemical staining for apoptotic marker (Bax-protein) showed a positive cytoplasmic reaction in Purkinje neurons and granule cells (Fig. 11).
Some Purkinje neurons with heterochromatic and others with apoptotic nuclei were observed. Numerous vacuoles, dilated cisternae of rough endoplasmic reticulum, and also swollen organelles, mainly mitochondria, were observed (Fig. 12a–c). Bergman’s astrocytes had demarcated nuclei and an electron-lucent cytoplasm with numerous glial filaments (Fig. 13a and b). Granule cells had nuclei with increased condensation of heterochromatin (Fig. 14b) and apparently increased area of myelinated axons (Fig. 14a) compared with the control groups (Fig. 6a). Arbitrarily myelin alterations were observed in axons of STZ-induced rats. These alterations were defined on the basis of myelin disarrangement either with a local disarrangement (type I), a diffuse local disarrangement of myelin sheath resembling a ‘collar’ but preserving the structural arrangement (type II), and also a diffuse disarrangement (type III) (Fig. 14a). Axonal abnormalities in the form of axonal swelling with dispersed, ill-defined presynaptic vesicles were observed. Mitochondrial abnormalities in the form of swollen, rupture, loss of cristae, and also myelin-like formation from degenerated mitochondria were clearly observed (Fig. 15a and b).
Many neurodegenerative disorders, such as diabetic encephalopathy and Alzheimer’s disease, are associated with the types I and II DM. Manifestations of these disorders include alterations in neurotransmission, electrophysiological abnormalities, structural changes, and cognitive deficit 20. Many approaches and tools have been used to study the etiology and pathogenesis of DM and its associated neural disorders, and their diagnosis and treatment. The most perspective approaches are based on a combined use of the methods of biochemistry, molecular biology, and physiology; they include clinical investigations of diabetic patients and experimental models and their complications, such as the model of DM I induced by STZ 21.
The present study described the alterations in the histological structure of rat cerebellar cortex resulting from chronic uncontrolled DM. There was a statistical loss of Purkinje neurons; a few of them had apoptotic nuclei. Chronic hyperglycemia causes oxidative stress in tissues prone to complications 22. Moreover, other investigations have also reported that severe hyperglycemia in DM I, mild hyperglycemia typical of DM II, and recurrent hypoglycemia induced by inadequate insulin therapy are the major factors responsible for the development of CNS complications. The brain is mainly a glucose-dependent organ, which can be damaged by hyperglycemia as well as hypoglycemia 23.
Although the mechanisms leading to cerebellar dysfunction associated with DM are not completely understood, brain cells are particularly vulnerable to oxidative stress. Oxidative stress leads to an increased production of reactive oxygen species and lipid peroxidation. Hyperglycemia causes autoxidation of glucose, glycation of proteins, and activation of polyol metabolism. These changes accelerate the generation of ROS to increase oxidative modifications of lipids, DNA, and proteins 24,25. Oxidative stress occurs in a cellular system when the production of free radicals exceeds antioxidant capacity. If cellular antioxidants do not remove free radicals, radicals attack and damage proteins, lipids, and nucleic acids. The oxidized or nitrosylated products of free radical attack have decreased biological activity, leading to loss of energy metabolism, cell signaling, transport, and other functions. These products are also targeted for proteosome degradation, further decreasing cellular functions. These radicals cause a cell to die through necrotic or apoptotic mechanisms 22. In addition, hyperglycemia effectively makes more substrate available for aerobic glycolysis in the brain, leading to acidosis 26 and enhanced free radical formation by a reduction in the levels of protective endogenous antioxidants 27.
Also, hyperglycemia increases the accumulation of advanced glycation end products (AGE), products formed by the nonenzymatic reaction between sugars and amino groups, which may lead to cellular and molecular damage. In this respect, both AGE and oxidative stress have been identified as potential contributors toward DM-induced brain aging. Among the actions of AGE are effects on extracellular matrix proteins and basement membrane components that can cause or facilitate vascular complications. In addition, the generation of AGE increases proinflammatory mechanisms in the vessels that enhance oxidative stress 28. In the current work, congested blood vessels with perivascular swelling in diabetic rats could induce altered blood–brain barrier functions. This swelling could represent swollen pericytes, astrocytes, or adipocytes 29. This result was in agreement with those of other investigators who reported that the disturbances of neuronal glucose transport and metabolism in both hyperglycemia and hypoglycemia can induce vascular damages 23.
It has generally been suggested that hyperglycemia enhances neuronal damage, in addition to neurons; astrocytes may also be the target 30. The current work showed that the number of GFAP-positive astrocytes increased significantly in STZ-induced rats. Our result is similar to that of previous studies 31,32. The alterations in astrocyte number are possibly because of oxidative stress 33 and free radical formation 34. Also, these findings were in agreement with those of a study that deduced that mechanical and chemical insults to the brain stimulate the proliferation and hypertrophy of astrocytes with increased synthesis of intermediate glial filaments. This phenomenon is called reactive gliosis, which is a universal reaction of astrocytes with specific structural and functional changes 35. During reactive gliosis, astrocytes secrete neurotoxic substances such as inflammatory cytokines and free radicals, which actively attack protein molecules within neurons, resulting in neuronal damage, and contribute toward the pathogenesis of neurodegenerative diseases 36. These evidences indicate that altered astrocyte activity contributes toward the CNS pathophysiology in DM 32.
In the current work, similar mitochondrial abnormalities were observed in both cellular bodies and neuropil. An arbitrary classification of disarray of mitochondria cristae, swelling, rupture, and also myelin-like formation from degenerated mitochondria in order to suggest different degrees of mitochondrial lesions were observed. These findings were consistent with previous ultrastructural published studies in rats 30. These alterations have been related to oxidative stress. This mechanism may be related to diabetic rat brain; however, it cannot be ruled out that the oxidative stress could be a result of a previous mitochondrial damage by increased intracellular glucose or by the effects of other damage inducers during DM. As interesting observation, unaltered mitochondria, was often found, besides the damage mitochondria, suggesting a selective mitochondrial resistance to the diabetic injury or a mitochondrial compensatory effect with an increase in their number 37. The damage to the mitochondria may activate mitochondria-initiated cell death pathways, resulting in DNA fragmentation and ischemic cell death 30.
The current work found an increased area of myelinated axons, with diverse degrees of neuronal and vascular swellings. The cellular swelling was represented by a local cytoplasm swelling, dilated cisternae of endoplasmic reticulum, and also swollen mitochondria. In this respect, brain edema is the most common serious complication of diabetic ketoacidosis in children, where mechanisms of rapid changes in serum osmolality during therapy and others such as brain ischemia have been suggested 38. In diabetic rats, hyperglycemia could induce brain damage. Thus, hyperglycemia may cause brain acidosis and dehydration, both involved in reduced cerebral blood flow and ischemia 39. Ischemia-related edema involves stimulation of the brain Na–K–Cl-cotransporter system, facilitating edema formation and swelling of endothelial cells 40.
In the current work, several grades of myelin alterations were observed. These myelin abnormalities may be involved in a decreased propagation of nerve impulse and contribute toward the brain disorders observed in DM. Several mechanisms have been suggested in myelin alterations in the brain of STZ-diabetic rats, such as decreased myelin-associated glycoprotein, autoantibodies to myelin basic protein, and myelin damage induced by nitric oxide 8,41. Atherosclerosis in the brain is one of the prominent changes in DM. Studies have shown that obstruction of the feeding vessels of nerves causes the death of nerve bundles and myelin destruction 42. Also, a decrease in nerve Na–K–ATPase activity is effective in the pathogenesis of diabetic neuropathy 43. Also, in this study, swollen axonal terminals with a high dispersion or ill-defined synaptic vesicles were observed. These ultrastructural findings suggest an altered synaptic transmission and could contribute toward abnormal synaptic plasticity and cognitive impairments 44,45. Dispersion of synaptic vesicles could be an early alteration, as similar findings have been reported in 9-day diabetic STZ rats in the presynaptic hippocampal mossy fiber terminals 46.
Our results support the previous findings and provide new information about the cerebellar ultrastructural alterations in the diabetic rat brain. Also, the cerebellar cortex was particularly susceptible to hyperglycemia-induced oxidative stress and could have contributed toward the neuronal damage.
Conflicts of interest
There is no conflict of interest to declare.
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