Secondary Logo

Journal Logo

Original articles

Effect of streptozotocin-induced diabetes mellitus on the cerebellar cortex of adult male albino rats: histological and immunohistochemical study

Selim, Sally A.; Selim, Assmaa O.

Author Information
The Egyptian Journal of Histology: March 2013 - Volume 36 - Issue 1 - p 103-113
doi: 10.1097/01.EHX.0000424090.98199.b8
  • Free



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.

Immunohistochemical study

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.

Statistical study

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).


General observation

In the diabetic group, three rats died.

Morphometrical analysis

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).

Table 1
Table 1:
Mean and SD of the linear density of Purkinje cell/mm length of the folia in the groups studied
Histogram 1
Histogram 1:
Histogram 1. Purkinje cell number in the different groups studied.

Astrocyte number

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).

Table 2
Table 2:
Mean and SD of astrocyte number/20 000 µm2 in the groups studied
Histogram 2
Histogram 2:
Histogram 2. Astrocyte number in the different groups studied.

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).

Table 3
Table 3:
Mean and SD of apoptotic cell number/20 000 µm2 in the groups studied
Histogram 3
Histogram 3:
Histogram 3. Apoptotic cell number in the different groups studied.

Histological results

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).

Figure 1
Figure 1:
A photomicrograph of a section in the cerebellar cortex of a control rat (group I) showing that it is made up of clearly defined three layers. The molecular layer (ML) is formed of small stellate (sc) and basket (bc) cells. The Purkinje cell layer (PL) consisted of large pyriform cells (P) that arrange in a single row. The granular layer (GL) is composed of tightly packed small rounded cell nuclei. Note the presence of clear spaces (cerebellar islands) or glomerulus (ci).Figure 1. H&E, ×400.
Figure 2
Figure 2:
A photomicrograph of a semithin section in the cerebellar cortex of a control rat (group I) showing the Purkinje cells (P) arranged in a single row along the outer margin of the granular layer (GL). These neurons have pale nuclei (n) and a basophilic cytoplasm (*).Figure 2. Toluidine blue, ×1000.

Immunohistochemical staining for GFAP showed GFAP-positive astrocytes; they appeared small, with thin few processes, in the granular and molecular layers (Fig. 3).

Figure 3
Figure 3:
Immunohistochemical staining for the demonstration of glial fibrillary acidic protein (GFAP) showing GFAP-positive staining in the cytoplasm and processes of astrocytes; they appear small, with few processes (arrows).Figure 3. Immunoperoxidase technique for GFAP, ×400.

Immunohistochemical staining for apoptotic marker (Bax-protein) indicated a negative reaction in Purkinje neurons and granule cells (Fig. 4).

Figure 4
Figure 4:
A photomicrograph of a section in the cerebellum of a control rat showing a negative immunoreaction for Bax-protein in Purkinje neurons (black arrow) and granule cells (red arrow).Figure 4. Immunoperoxidase technique for Bax-protein, ×400.

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).

Figure 5
Figure 5:
An electron micrograph of a section in the cerebellar cortex of a control rat (group I) showing (a) Purkinje neuron (P) with a euchromatic nucleus (N) and a profile cytoplasm (P1) of other neurons. Bergman’s cell (B) with a demarcated nucleus (n) and an electron-lucent cytoplasm with glial filaments (*) can be observed. Purkinje neuron (P) with large somata and a euchromatic nucleus (N) can be seen. The nuclear envelope shows long, narrow dimples (arrow). Its cytoplasm is rich in organelles such as rough endoplasmic reticulum (r) and small mitochondria (m). (c) Mitochondria with intact cristae (m), cisternae of rough endoplasmic reticulum (r), and also free ribosomes (rs) observed in the cytoplasm of Purkinje neuron.Figure 5. (a) ×2000; (b) ×6000; (c) ×20 000.
Figure 6
Figure 6:
An electron micrograph of a section in the cerebellar cortex of a control rat (group I) showing (a) granule cells (G) with euchromatic nuclei (N). Several axons with intact myelin sheaths (arrows) can also be seen. (b) Granule cells (G) have nuclei with few scattered clumps of chromatins (N). These cells are surrounded by a thin shell of cytoplasm showing free ribosomes (*). Axons with intact mitochondria (m) and also myelin sheaths (arrows) can be seen.Figure 6. (a) ×6000; (b) ×12 000.
Figure 7
Figure 7:
An electron micrograph of a section in the cerebellar cortex of a control rat (group I) showing (a) axons of large Mossy fibers (a) with mitochondria (m) with regular intact cristae. Typical synapse (arrow) with presynaptic and postsynaptic membranes and also dendritic processes of granule cells (d) can be observed. (b) Many synapses (arrow) can be observed. Presynaptic and postsynaptic membranes with synaptic clefts can be seen clearly. Numerous, small easily identified vesicles (v) can also be observed.Figure 7. (a, b) ×40 000.

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).

Figure 8
Figure 8:
A photomicrograph of a section in the cerebellar cortex of a diabetic rat (group III) showing (a) a thin granular layer (GL), loss of most Purkinje neurons in the Purkinje layer (PL), leaving empty spaces (s), and a few of them (P) appeared to have dark stained nuclei. Prominent perineural spaces (arrows) can be seen around both basket (bc) and stellate (sc) cells in the molecular layer (ML). Congested blood vessel (bv) surrounded by a wide perivascular space (*) can also be seen. (b) Hemorrhage (Hg) in the granular layer (GL) and white matter (W).Figure 8. H&E, ×400.
Figure 9
Figure 9:
A photomicrograph of a semithin section in the cerebellar cortex of a diabetic rat (group III) showing (a) Purkinje neurons (P) with darkly hardly identified nuclei (n) and a darkly stained cytoplasm (*) in the Purkinje layer (pL). Many Bergmann astrocytes (A) with pale demarcated nuclei surrounded by a pale cytoplasm can be seen. The granular layer (GL) shows some granule cells (g) with darkly stained nuclei and also blood capillaries with wide perivascular spaces (bc). (b) Congested blood vessel (bv) in the granular layer (GL) can be observed.Figure 9. Toluidine blue, ×1000.

Immunohistochemical staining for GFAP showed that GFAP-positive astrocytes were more abundant and appeared larger in the three cerebellar cortical layers (Fig. 10).

Figure 10
Figure 10:
Immunohistochemical staining for the demonstration of glial fibrillary acidic protein (GFAP) in the cerebellar cortex of a diabetic rat (group III) showing GFAP-positive astrocytes; they appear greater in number, with multiple thick processes (arrows), and abundant distribution in the three layers: granular (GL), Purkinje (PL), and molecular (ML).Figure 10. Immunoperoxidase technique for GFAP, ×400.

Immunohistochemical staining for apoptotic marker (Bax-protein) showed a positive cytoplasmic reaction in Purkinje neurons and granule cells (Fig. 11).

Figure 11
Figure 11:
A photomicrograph of a section in the cerebellum of a diabetic rat (group III) showing a strong positive immunoreaction for Bax-protein in the cytoplasm of some Purkinje neurons (black arrows) and granule cells (red arrows).Figure 11. Immunoperoxidase technique for Bax-protein, ×400.

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).

Figure 12
Figure 12:
An electron micrograph of a section in the cerebellar cortex of a diabetic rat (group III) showing (a) a Purkinje neuron (P) with a heterochromatic nucleus (N) and dilated some cisternae of endoplasmic reticulum (r). Other apoptotic neuron (P1) with large vacuolated cytoplasm (v) can be seen. Some granule cells (G) with apoptotic (n) and others with a euchromatic (n1) nuclei can be seen. (b) Apoptotic Purkinje neuron (P) with an ill-distinct nucleus can be seen. Bergman’s astrocytes have demarcated nuclei (N) and an electron-lucent cytoplasm (*) can be observed. Also, a granule cell (G) with a euchromatic nucleus (n) can be observed. (c) Mitochondria with destructed cristae (m) and relatively dilated cisternae of endoplasmic reticulum (r) can be observed in the cytoplasm of Purkinje neuron.Figure 12. (a, b) ×8100; (c) ×20 000.
Figure 13
Figure 13:
An electron micrograph of a section in the cerebellar cortex of a diabetic rat (group III) showing (a) Bergman’s astrocytes (A) with demarcated nuclei (N) and an electron-lucent cytoplasm (*). (b) Bergman’s astrocytes (A) with demarcated euchromatic nuclei (N) and an electron-lucent cytoplasm with numerous Glial filaments (gf) can be observed.Figure 13. (a) ×6000; (b) ×12 000.
Figure 14
Figure 14:
An electron micrograph of a section in the cerebellar cortex of a diabetic rat (group III) showing (a) disorganizing periodic myelin patterns in the form of local disarrangement (thin arrow), diffused local disarrangement (thick arrows), and diffused disarrangement (double arrows). (b) Granule cells (G) with nuclei (N) studded with coarse clumps of heterochromatin can be observed. Mitochondria (m) without cristae in the surrounding neuropil can be seen.Figure 14. (a) ×8000; (b) ×12 000.
Figure 15
Figure 15:
An electron micrograph of a section in the cerebellar cortex of a diabetic rat (group III) showing (a) swollen axons of large Mossy fibers (a) with dispersed hardly identified synaptic vesicles (v). Some mitochondria (m) with normal paralleled cristae and other myelin-like formations (M) can be seen. (b) Mitochondria alterations in the form of destroyed cristae (m) and swollen rupture (m1) can be observed in swollen axons.Figure 15. (a, b) ×40 000.


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.

No title available.


Conflicts of interest

There is no conflict of interest to declare.


1. Tobin KW, Chaum E, Priya Govindasamy V, Karnowski TP. Detection of anatomic structures in human retinal imagery. IEEE Trans Med Imaging. 2007;26:1729–1739
2. Sima AAF, Kamiya H, Li ZG. Insulin, C-peptide, hyperglycemia, and central nervous system complications in diabetes. Eur J Pharmacol. 2004;490:187–197
3. Kodl CT, Seaquist ER. Cognitive dysfunction and diabetes mellitus. Endocr Rev. 2008;29:494–511
4. Biessels GJ, Deary IJ, Ryan CM. Cognition and diabetes: a lifespan perspective. Lancet Neurol. 2008;7:184–190
5. Kumar TP, Antony S, Gireesh G, George N, Paulose CS. Curcumin modulates dopaminergic receptor, CREB and phospholipase C gene expression in the cerebral cortex and cerebellum of streptozotocin induced diabetic rats. J Biomed Sci. 2010;17:43
6. Allen KV, Frier BM, Strachan MWJ. The relationship between type 2 diabetes and cognitive dysfunction: longitudinal studies and their methodological limitations. Eur J Pharmacol. 2004;490:169–175
7. McCall AL. Cerebral glucose metabolism in diabetes mellitus. Eur J Pharmacol. 2004;490:147–158
8. Mastrocola R, Restivo F, Vercellinatto I, Danni O, Brignardello E, Aragno M, Boccuzzi G. Oxidative and nitrosative stress in brain mitochondria of diabetic rats. J Endocrinol. 2005;187:37–44
9. Aragno M, Parola S, Brignardello E, Mauro A, Tamagno E, Manti R, et al. Dehydroepiandrosterone prevents oxidative injury induced by transient ischemia/reperfusion in the brain of diabetic rats. Diabetes. 2000;49:1924–1931
10. Low PA, Nickander KK, Tritschler HJ. The roles of oxidative stress and antioxidant. Treatment in experimental diabetic neuropathy. Diabetes. 1997;46(Suppl 2):S38–S42
11. Junod A, Lambert AE, Stauffacher W, Renold AE. Diabetogenic action of streptozotocin: relationship of dose to metabolic response. J Clin Invest. 1969;48:2129–2139
12. Tjalve H. Streptozotocin: distribution, metabolism and mechanisms of action. Uppsala J Med Sci. 1983;88(Suppl 39):145–157
13. Qiao X, Chen L, Gao H, Bao S, Hefti F, Thompson RF, Knusel B. Cerebellar brain-derived neurotrophic factor-trkB defect associated with impairment of eyeblink conditioning in stargazer mutant mice. J Neurosci. 1998;18:6990–6999
14. Tehranipour M, Khakzad MR. Effect of maternal diabetes on hippocampus neuronal density in neonatal rats. J Biol Sci. 2008;8:1027–1032
15. Suthagar E, Soudamani S, Yuvaraj S, Ismail Khan A, Aruldhas MM, Balasubramanian K. Effects of streptozotocin (STZ)-induced diabetes and insulin replacement on rat ventral prostate. Biomed Pharmacother. 2009;63:43–50
16. Bancroft JD, Gamble M Theory and practice of histological techniques. 20076th ed London Churchill Livingstone:179
17. Hayat MA Principles and techniques of electron microscopy: biological applications. 20004th ed Edinburgh, UK Cambridge University Press:37–59
18. Zhang T-J, Hang J, Wen D-X, Hang Y-N, Sieber FE. Hippocampus bcl-2 and bax expression and neuronal apoptosis after moderate hypothermic cardiopulmonary bypass in rats. Anesth Analg. 2006;102:1018–1025
19. Saad EL-Dien HM, EL Gamal DA, Mubarak HA, Saleh SM. Effect of fluoride on rat cerebellar cortex: light and electron microscopic studies. Egypt J Histol. 2010;33:245–256
20. Biessels GJ, Smale S, Duis SEJ, Kamal A, Gispen WH. The effect of gamma-linolenic acid-alpha-lipoic acid on functional deficits in the peripheral and central nervous system of streptozotocin-diabetic rats. J Neurol Sci. 2001;182:99–106
21. Shafrir E. Contribution of animal models to the research of the causes of diabetes. World J Diabetes. 2010;1:137–40
22. Rösen P, Nawroth PP, King G, Möller W, Tritschler H-J, Packer L. The role of oxidative stress in the onset and progression of diabetes and its complications: a summary of a congress series sponsored by UNESCO-MCBN, the American diabetes association and the German diabetes society. Diabetes Metab Res Rev. 2001;17:189–212
23. Scheen AJ. Central nervous system: a conductor orchestrating metabolic regulations harmed by both hyperglycaemia and hypoglycaemia. Diabetes Metab. 2010;36(Suppl 3):S31–S38
24. Ouyang L, Wang J, Zhu X. Diagnostic efficacy of glutamic acid decarboxylase antibody and islet cell antibody in type I diabetes mellitus. Zhonghua Nei Ke Za Zhi. 2000;39:674–676
25. Osawa T, Kato Y. Protective role of antioxidative food factors in oxidative stress caused by hyperglycemia. Ann N Y Acad Sci. 2005;1043:440–451
26. Biessels GJ, Kappelle AC, Bravenboer B, Erkelens DW, Gispen WH. Cerebral function in diabetes mellitus. Diabetologia. 1994;37:643–650
27. Baydas G, Canatan H, Turkoglu A. Comparative analysis of the protective effects of melatonin and vitamin E on streptozocin-induced diabetes mellitus. J Pineal Res. 2002;32:225–230
28. Wang S-H, Sun S-L, Guo Y-J, Yuan Y, Yang B-Q. Diabetes impairs hippocampal function via advanced glycation end product mediated new neuron generation in animals with diabetes-related depression. Toxicol Sci. 2009;111:72–79
29. Guzik TJ, Marvar PJ, Czesnikiewicz-Guzik M, Korbut R. Perivascular adipose tissue as a messenger of the brain-vessel axis: role in vascular inflammation and dysfunction. J Physiol Pharmacol. 2007;58:591–610
30. Ding C, He QP, ALi P. Diabetes mellitus causes early ultrastructural changes to the nuclei and mitochondria of neurons and astrocytes in rats subjected to a brief period of cerebral ischemia. Microsc Microanal. 2005;11:1010–1011
31. Jahanshahi M, Golalipour MJ, Afshar M. The effect of Urtica dioica extract on the number of astrocytes in the dentate gyrus of diabetic rats. Folia Morphol (Warsz). 2009;68:93–97
32. Golalipour MJ, Ghafari S, Latifimoghadam MH, Kaboli S. Alteration of dentate gyrus astrocytes in diabetic rats: protective role of Urtica dioica. Int J Morphol. 2011;29:1307–1312
33. Baydas G, Reiter RJ, Yasar A, Tuzcu M, Akdemir I, Nedzvetskii VS. Melatonin reduces glial reactivity in the hippocampus, cortex, and cerebellum of streptozotocin-induced diabetic rats. Free Radic Biol Med. 2003;35:797–804
34. Baydas G, Nedzvetskii VS, Tuzcu M, Yasar A, Kirichenko SV. Increase of glial fibrillary acidic protein and S-100B in hippocampus and cortex of diabetic rats: effects of vitamin E. Eur J Pharmacol. 2003;462:67–71
35. Baydas G, Ozer M, Yasar A, Koz ST, Tuzcu M. Melatonin prevents oxidative stress and inhibits reactive gliosis induced by hyperhomocysteinemia in rats. Biochemistry (Mosc). 2006;71(Suppl 1):S91–S95
36. Bates KA, Fonte J, Robertson TA, Martins RN, Harvey AR. Chronic gliosis triggers Alzheimer’s disease-like processing of amyloid precursor protein. Neuroscience. 2002;113:785–796
37. Zhou J, Wang L, Ling S, Zhang X. Expression changes of growth-associated protein-43 (GAP-43) and mitogen-activated protein kinase phosphatase-1 (MKP-1) and in hippocampus of streptozotocin-induced diabetic cognitive impairment rats. Exp Neurol. 2007;206:201–208
38. Edge JA, Ford-Adams ME, Dunger DB. Causes of death in children with insulin dependent diabetes 1990-96. Arch Dis Child. 1999;81:318–323
39. Ding D, Moskowitz SI, Li R, Lee SB, Esteban M, Tomaselli K, et al. Acidosis induces necrosis and apoptosis of cultured hippocampal neurons. Exp Neurol. 2000;162:1–12
40. O’Donnell ME, Tran L, Lam TI, Liu XB, Anderson SE. Bumetanide inhibition of the blood–brain barrier Na-K-Cl cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke. J Cereb Blood Flow Metab. 2004;24:1046–1056
41. Kawashima R, Kojima H, Nakamura K, Arahata A, Fujita Y, Tokuyama Y, et al. Alterations in mRNA expression of myelin proteins in the sciatic nerves and brains of streptozotocin-induced diabetic rats. Neurochem Res. 2007;32:1002–1010
42. Khaksar Z, Jelodar GA, Hematian H. Morphometric study of cerebrum in fetuses of diabetic mothers. Iranian J Vet Res. 2011;12:199–204
43. Scarping E, Bianchi R, Moggio M, Sciacco M, Fiori MG, Scarlato G. Decrease of nerve Na+,K+-ATPase activity in the pathogenesis of human diabetic neuropathy. J Neurol Sci. 1993;120:159–167
44. Grillo CA, Piroli GG, Wood GE, Reznikov LR, McEwen BS, Reagan LP. Immunocytochemical analysis of synaptic proteins provides new insights into diabetes-mediated plasticity in the rat hippocampus. Neuroscience. 2005;136:477–486
45. Artola A. Diabetes-, stress- and ageing-related changes in synaptic plasticity in hippocampus and neocortex. The same metaplastic process? Eur J Pharmacol. 2008;585:153–162
46. Magariños AM, McEwen BS. Experimental diabetes in rats causes hippocampal dendritic and synaptic reorganization and increased glucocorticoid reactivity to stress. Proc Natl Acad Sci USA. 2000;97:11056–11061

cerebellum; immunohistochemical; streptozotocin; structurel

© 2013 The Egyptian Journal of Histology