Pathological changes in the central nervous system of diabetic animals
and human are usually subtle in the brain (Polanco et al., 2005)
selective vulnerability was reported especially in the cerebral cortex
and in the hypothalamus (the preoptic-suprachiasmatic nuclei) in diabetic
rats (Luo et al., 2002).
The hippocampus is a important structure for memory processing. It is
a particularly vulnerable and sensitive region of the brain that is also
very important for declarative and spatial learning and memory. Hippocampal
neurons are vulnerable to seizures, strokes and head trauma, as well as
responding to stressful experiences. At the same time they show remarkable
plasticity, involving long-term synaptic potentiation and depression,
dendrite remodeling (synaptic turnover and neurogenesis in the case of
the dentate gyrus (McEwen, 1999). The hippocampus has been implicated
in certain short-term memory. Indeed hippocampal lesions often produce
short-term memory deficits (Mumby et al., 2002). The hippocampus
is preferentially susceptible to a wide variety of toxic insults and disease
processes, including hypoxia-ischemia and hypoglycemia (Piotrowski et
al., 2001). Metabolic diseases such as diabetes and obesity have been
associated with increased vulnerability to stress (Damasceno et al.,
2002) and cognitive dysfunction (Nazer and Ramírez, 2000). Diabetes
mellitus can lead to functional and structural deficits in both the peripheral
and central nervous system. The pathogenesis of these deficits is multifactor
and may involve microvascular dysfunction and oxidative stress (Patil
et al., 2006). Cognitive deficits are also reported to occur in
animal models of diabetes (Stroptozotocin induced)which can be prevented,
but not fully reversed by insulin treatment (Kuhad et al., 2008).
Diabetes also induced morphological changes in the presynaptic mossy fiber
terminals (MFT) that form excitatory synaptic contacts with the proximal
CA3 apical dendrites (Magariños and McEwen, 2000).
Oxidative stress induced by chronic hyperglycemia contributes to cerebrovascular
complication in diabetes (Damasceno et al., 2002). Also diabetes
mellitus is associated with an increased risk for cerebrovascular disease
(Aragno et al., 2002). Accumulating data support the conclusion
that oxidative stress induced by chronic hyperglycemia plays a key role
in both microvascular and macrovascular complications of diabetes, including
stroke (Artola, 2008). Many deleterious events contribute to oxidative
damage to neurons in diabetes: because of high levels of polyunsaturated
lipids in the brain, direct lipperoxidation frequently occurs causing
lipid membrane disruption and consequent neurodegeneration (Ristow, 2004).
Moreover, oxidative stress increase tissue levels of highly reactive
and toxic substances and effects signal transduction pathways involved
in neuronal and endothelial cell function. Primary diabetic encephalopathy
is recognized as a late complication of both type 1 and type 2 diabetes
(Kinney et al., 2003). Impairments in learning, memory, problem
salving and mental and motor speed are more common in type 1 diabetic
patients than in the general population (Deregnier et al., 2000).
A diabetic duration dependent decline in cognitive function occurs independently
of hypoglycemic episodes (DeBoer et al., 2005) and impaired intellectual
and cognitive developments in type 1 diabetic children correlate with
diagnosis at young age, male sex and metabolic status. Cognitive deficits
(Ryan and Geckle, 2000) and poor performances in abstract reasoning and
complex psychomotor functioning occur in type 2 diabetes. Learning and
memory dysfunctions are more prominent in elderly type 2 diabetic patients
(Sinclair et al., 2000). It has not been determined whether this
is because of potentiation of the normal aging process, a function of
diabetes duration, or both. Notably Alzheimer disease is twice as prevalent
in the diabetic population as in nondiabetic subjects (Arvanitakis et
al., 2005). Several recent studies have implicated abnormal function
of the insulin/IGF axis in the early pathogenesis of Alzheimer`s
disease. Insulin and IGF-1 are believed to regulate β-amyloid levels
and tau phosphorylation (Gasparini and Xu, 2003).
Impaired spatial learning and memory occur in animal models of both type
1 and type 2 diabetes. In the hippocampus of STZ-induced rats, long-term
potentiation is impaired, whereas long-term depression is enhanced indicating
altered hippocampal synaptic plasticity, which are corrected by insulin
treatment (Gispen and Biessels, 2000).
Pregnant women who suffer from diabetes are more likely to have a child
with memory problems; according to a new study. Fetal brain iron deficiency
occurs in human pregnancies complicated by diabetes mellitus or more common
in the offspring of these pregnancies. The aim of present research was
to induce maternal diabetes mellitus and to assess the effects of that
on Hippocampus structure (CA1, CA2, CA3) and measuring the neuronal density.
MATERIALS AND METHODS
Female Wistar rats, weighting 200-250 g, were hosed under standard laboratory
conditions and kept under natural 12 h light: 12 h dark cycle. The animals
procured from Razi Animal House, were housed 2 per cage with free access
to standard food and water. Rats were acclimatized to laboratory conditions
before testing. Animal were divided into 2 different groups for estiminat
the effect of maternal hyperglycemia in neonates.
||Normal-rats were not subjected to any procedures
||Diabetes-under STZ injection
All the experimental protocols were conducted in Faculty of Science,
Islamic Azad University of Mashhad, Iran (2008). All chemical used in
this study were purchased from Sigma (UK).
Induction of diabetes: Diabetes was induced in rats by a single
injection of STZ (60 mg kg-1) freshly dissolved in citrate
buffer (pH 4.5). Age-matched control animals were injected with citrate
buffer. Diabetes was confirmed after 8 weeks; only the animals with blood
glucose level above 400 mg dL-1 were included in the study.
The body weight was measured at the beginning and the end of the experiment.
All animals were checked for glucose blood concentration at the beginning
of the experiment. After 2 weeks from STZ-injection, as well as on the
day before the of experiment.
Tissue collection: Animals were anesthetized with sodium pentobarbital
(64 mg kg-1) and decapitated. The whole brain was removed and
fixed in 10% paraformaldehyde. NaCl was added to the fixative to make
the tissue float in order to overcome deformities during the fixation
period. Paraffin embedded tissue blocks were sectioned at 7 mμ thickness
coronally and stained with haematoxylin-eosin.
Measurement of neuronal density in hippocampus: Hemotoxylin-eosin-stained
serial paraffin sections were prepared from 8 hippocampi from individual
animals in each group. Regions of hippocampus (CA1 CA3) were identified
according to paxinos and watson. Tissue blocks containing samples (brains)
were serially cut throughout. Form several hundred sections per block,
of each 20 section 3 serial sections were obtained. For example for the
first series: 24, 25, 26th section and for the second series: 46, 47,
48th section and so on. Therefore we mounted every 3 section on a slide.
At a practical level, stereological methods are precise tools for obtaining
quantitative information about three-dimensional structures based mainly
on observations made on sections (Gunderson et al., 1988). All
experiments were performed a minimum of two times. Student`s t-test was
used for comparison when only 2 groups were analyzed. Statistical significance
was chosen as p<0.05. All results are reported as Mean ± SEM.
RESULTS AND DISCUSSION
Two weeks after STZ-injection the rat`s blood glucose level
was higher than 400 mg dL-1. At the end of the 10th week, the
diabetic rats showed an increase in blood glucose level (470 ±
18 mg dL-1).
||Neuronal density CA3 in neonate from diabetic
mothers compare to control The values are presented as Means ±
SEM n = 8 *p<0.05. Student`s t-test compare pups from Diabetic
dams with pups from controls. CA3-L: CA3 in left hemisphere. CA3-R:
CA3 in right hemisphere
||Neuronal density CA1 in neonate from diabetic mothers
compare to control. The values are presented as means ± SEM.
n = 8. *p<0.05 Student`s t-test compare pups from Diabetic dams
with pups from controls. CA1-L: CA1 in left hemisphere. CA1-R: CA1
in Right hemisphere
The body weight of diabetic animals increased to (230-260 g) during the
experimental time, whole in the normal to (270-340 g). In the urine, all
diabetic rats demonstrated abnormal results: glucosuria (+++), ketone
bodies (trace) and protein (trace). In the plasma all diabetic rats had
remarkable increase in Creatinine, Uric acid, Urea, Triglycerides, Cholesterol.
[P3+], [Ca2+] (p<0.05).
||Neuronal density CA2 in neonate from diabetic
mothers compare to control. The values are presented as means ±
SEM. n = 8. *p<0.05 Student`s t-test compare pups from Diabetic
dams with pups from controls. CA2-L: CA2 in left hemisphere. CA2-R:
CA2 in Right hemisphere.
Maternal hyperglycemia produced evoked significant neuronal loss in CA3
of neonate brains (Fig. 1). This decrease was 0.25 ±
0.01 in control to 0.23 ± 0.00 in neonates from diabetic mothers
We estiminat left and right regions of hippocampus and the results were
the same. Although there was remarkable decreased in CA1 and CA2 but they
were not meaningful (Fig. 2, 3).
In CA1 the mean of neuronal density was 0.24 ± 0.01
in control to 0.22 ± 0.02 in neonates from diabetic mothers (p
ND in CA2 was 0.24 ± 0.00 in control to 0.23 ±
0.00 in experimental groups (p<0.08).
In morphometric study, there was more changes in neuronal shape in hippocampus
regions of neonates from diabetic mothers camper to controls (Fig.
In our morphometric studies on pyramidal cells in CA1, CA2 and CA3, it
has been shown that the experimental maternal hyperglycemia in rats results
neuronal loss and damage expressed maximally in CA3. Although in experimental
groups there is a remarkable neuronal density decrease in all hippocampus
sectors (Fig. 1-3), but it is meaningful
in CA3 (Fig. 1).
||Photomicrograph of the brain section of neonate at the
region of the midhippocampus. (a) hippocampus of neonate from diabetic
mother and (b) Control. (X40)
Diabetes causes morphometric neuronal changes (Sinclair et al.,
2000). This observation has allowed us to postulate that the neuronal
death in the infant from diabetic mothers proceeds on an apoptotic pathway
(Sima and Li, 2005). Gestational conditions increase fetal iron demand
for erythropoiesis beyond placental iron transport capacity. Diabetes
mellitus can result in a 30-40% reduction in neonatal brain iron (Damasceno
et al., 2002). Iron in the form of cytochoromes is a required component
of cellular oxidative metabolism in the brain and is thus essential for
normal neuronal function (Ornoy, 2007).
A higher prevalence of cognitive impairment has been documented in infants
born to mothers with hyperglycemia gestational conditions (Gao and Gao,
2007). Iron deficiency could have a direct effect on brain development
or could potentiate the effects of other adverse prenatal events. The
cognitive nature of the long- term impairments of infants of diabetic
mothers (Nold and Georgieff, 2004) and growth-retarded infants suggests
that factors targeting the developing hippocampus may be particularly
important. The hippocampus in necessary for normal cognitive function,
especially for processing recognition memory and transferring short-term
memory items into long-term storage (McEwen, 1999).
In humans and animal models, the hippocampus appears to be particularly
vulnerable to prenatal hypoxia-ischemia an event that occurs more commonly
in gestations complicated by maternal diabetes mellitus or fetal growth
retardation. We hypothesized that prenatal brain iron deficiency that
occur in diabetic pregnancy increases the vulnerability of the hippocampus
to hypoxic-ischemic injury (Barnes-Powell, 2007).
Iron deficiency of the heart, liver and skeletal muscle has been shown
to affect cellular energy production and organ performance. Arguably,
sever iron deficiency may lead to similar deficits in cellular energy
metabolism and organ performance in the brain, resulting in a reduced
ability to respond to restriction of oxygen and perfusion and in greater
hippocampus damage. The hippocampus, especially the DG, is one of the
iron rich areas of the rat brain, finding suggest that the integrity of
the prenatal hippocampus and its cholinergic input are important for normal
development of memory and learning (Ryan and Geckle, 2000). In other hand,
this area of the brain (hippocampus) particularly the dentate region is
also vulnerable to damage when glucocorticoids are elevated as occurs,
for example, when an organism is stressed (Gispen and Biessels, 2000).
Uncontrolled experimental diabetes induced by (STZ) in rats is an endogenous
chronic stressor that produces retraction and simplification of apical
dendrites of hippocampal CA3 pyramidal neurons (Damasceno et
One effect, synaptic vesicle depletion and dendrite atrophy occurs in
diabetes as well as after repeated stress and cort treatment. These changes
occurred in concert with adrenal hypertrophy and elevated basal cort release
as well as hypersensitivity and defective shut off of cort secretion after
stress. Thus as an endogenous stressor STZ diabetes not only accelerates
the effects of exogenous stress to alter hippocampal morphology: it also
produce structural changes that overlap only partially with those produced
by stress and cort in the nondiabetic state (Magariños and McEwen,
The hippocampal morphological changes induced by stress are mediated
by interactions between Gc secretion, excitatory amino acid and are also
correlated with deficits in hippocampal dependent memory (DeBoer et
These results and present results confirm that an oxidative imbalance
occurs in the hippocampus of neonates born from diabetic rats as we have
earlier shown in diabetic rats. Several studies have pointed out that
NF-kB (nuclar factor) activation is inhibited by a variety of antioxidants,
such as N-acetyl-cystein, butylanted hydroxyl anisole, vitamin E and lipoic
acid (Aragno et al., 2000). These data suggest that antioxidants
effect some steps of signaling events leading to phosphorylation, ubiquination
and degradation (Patil et al., 2006). The role of oxidative stress
and Nk-kB activation on diabetic complications is well documented, moreover
antioxidant treatment exerts a beneficial effect in experimental models
of chronic injury in diabetes and treatment with antioxidants can significantly
reduce diabetic complications (Magariños and McEwen, 2000). Reactive
oxygen species activates a variety of target genes linked to the development
of diabetic complications (Rakeshwar et al., 2006).
In addition, the loss of arachidonic acid content of the synaptosomal
membrane, induced by diabetes and by transient cerebral ischemia, making
the membrane more resistant to oxidative stress. Oxidative stress induced
by chronic hyperglycemia directly can damage ionic homeostasis and membrane
transport systems in the brain (Aragno et al., 2000) and may be
this is one of the reasons for hippocampal neurons death. Apoptosis in
diabetes has been ascribed to hyperglycemia and oxidative stress (Mumby
et al., 2002).
However, other experimental studies on streptozotocin induced rat diabetic,
showed pathological changes, such as so-called dark neurons and neuronal
loss, in different cerebral regions, especially in the hippocampus. A
dominant opinion is that hyperglycemia aggravates ischaemic brain damage
in experimental STZ-diabetes with transient cerebral ischemia in rats
(Piotrowski et al., 2001). It has been suggested that diabetes
and ischaemia evoke the oxidative stress following an impairment of the
respiratory chain in mitochondria and an overproduction of the Reactive
Oxygen Species (ROS). ROS are considered as a main factor in the pathogenesis
on neuronal death (Piotrowski et al., 2001).
The other reason for neuronal death in diabetic pregnancy is Iscemia.
It has been postulated that neurons with higher oxygen consumption in
normal conditions are more susceptible to ischemia insult (Ristow, 2004).
Some studies shown that there is differences in the neural death between
diabetic and Ischaemia (Piotrowski et al., 2001) that differences
in the neuronal death pathomechanism among hippocampal sectors in diabetes
and ischaemia may be related to the different duration and intensity of
the oxidative stress in both applied models (acute in ischaemia and chronics
in STZ-induced diabetes). It has been postulated that in cerebral ischaemia
neuronal apoptosis may be followed by necrotic phase (Patil et al.,
2006). Pathomechanism of degenerative changes and neuronal loss through
apoptosis or necrosis is not clear until now. It has been suggested that
changes in intracellular calcium concentrations in oxidative stress may
indicate the pathway of cell death. It is suggested that more sever injury
with high intracellular calcium concentration (Ca+2) promotes
necrotic cell death, where low (Ca+2) and milder injury promotes
cell death through apoptosis (Misumi et al., 2008). Studies on
antioxidative treatment would deliver further data important in the exploration
of neuronal death in diabetes and ischemia.
In total, it is concluded that maternal diabetes induces some changes
in hippocampus neuronal structure and density. Statistical analysis show
significant decrease in neuronal density (ND) of CA3 in neonates
from diabetic mothers compare to control. Although ND in CA1, CA2 have
showed decrease but it is not meaningful. Maybe neurogenic process in
hippocampus is responsible for these changes. Therefore, it is better
in diabetic pregnant mother the level of glucose was maintained under