Maternal Hyperglycemia Proliferate Choroids Plexus and Enlarge the Lateral Ventricle in Brain of Newborn Rats
It is well known that structural and functional alterations
of microvascular systems are responsible for the most devastating complications
of diabetic patients. Alterations of electrolyte composition of CSF in
hyperglycemic condition may reflect the microvascular deleterious of choroid
plexus. In the present study it is examined the effects of maternal hyperglycemia
on the microvascular structure of choroid plexus and the volume of lateral
ventricle in newborn Wistar rats. At 7th day of pregnancy hyperglycemia
was induced by a single injection (i.p.) of streptozotocin (55 mg kg-1).
Control animals were given an equal volume of citrate buffer. After parturition
2 pups were randomly selected from each litter, their brain dissected,
fixed in 10% formalin, sectioned in 7 μm thickness and stained by
H.E. By applying stereological techniques and systematic random sampling
scheme the volume of choroid plexus, length density of capillaries and
the volume of the lateral ventricles were estimated. In comparison with
controls, statistical analysis showed significant increases (p<0.01)
in the length density of capillaries as well as volumes of choroid plexus
and lateral ventricle. In conclusion it seems that, similar to the microvascular
systems of retina and kidney, the choroid plexus probably is a vulnerable
microvascular system in hyperglycemia.
Diabetes mellitus is a metabolic disorder that produces various dysfunction
in the body. Oxidative stress induced by chronic hyperglycemia may play
a key role in both microvascular and macrovascular complications of diabetic
patients (Aragno et al., 2005). Underlying diabetic complications
such as nephropathy, neuropathy, retinopathy, cardiovascular disease and
peripheral vascular disease progress gradually before an actual diagnosis
is made (Spijkeman and Dekker, 2003). Cardiovascular disorders may result
from an inadequate blood supply which cause complications such as slurred
speech, aphasia, paralysis of a limb or blindness (Boulton, 2005) or structural
changes in blood vessels such as infarction (hemorrhages) of retinal vessels,
weakened blood vessel walls (microaneurysms) leakage of fluid (edema or
exudate) and loss of circulation (Sharma et al., 1985). Diabetes
exerts a heavy toll on the vascular system. The hallmark of diabetic macrovascular
disease is accelerated by atherosclerosis involving the aorta and large
and medium-sized arteries.
Diabetes is also considered to be a risk factor for Alzheimer,s
disease and other neurodegenerative diseases (Arvanitakis et al.,
2004). Although, much has been learned about peripheral changes related
to diabetes, changes induced in the CNS are not well characterized. Epidemiological
studies and experiments in rodent embryos show that there is an increased
risk of fetal malformations and spontaneous abortion in diabetic pregnancies
Infants of hyperglycemic mothers remain at risk for fetal hyperinsulinemia,
consequent increase fetal adiposity and often excess fetal size (macrosomia)
which increases the likelihood of birth trauma and operative delivery
and pancreatic dysfunction and neurologic complications. Extensive experimental
and clinical evidence indicates that metabolic disturbances in the mother
contribute to virtual all the adverse effects of DM on the offspring (Jarvis
et al., 2005). In addition to a number of developmental and functional
abnormalities in CNS, it has been reported that diabetes increases the
CSF pressure (Egelton et al., 2003) which is responsible for the
excessive retention or production of CSF from epithelial cells of choroid
plexus (Segal, 2000).
In the present study it is investigated the effect of streptozotocin
(STZ) induced gestational hyperglycemia on the length density of capillaries
of choroid plexus, CSF electrolytes composition and the volume of lateral
ventricle in newborn rat.
MATERIALS AND METHODS
Experimental design: All experiment was conducted in Faculty of
Science, Islamic Azad University of Mashhad, Iran (2008). All chemical
used in this study were purchased from Sigma (UK).
Animals were cared for and handled in accordance with the Iranian Society
of Animal Care (member of International Animal Care Society) and also
local Islamic Azad University of Mashhad animal ethics directions.
Young female rats (approximately 250 g), purchased from Razi Institute
(Mashhad, Iran), were maintained in a temperature controlled room (20°C)
and a 12 h light:dark cycle. They were allowed access to food and water
ad libitum. They were mated with normal males and the morning of appearance
of vaginal plug was considered as day zero of pregnancy. At 7 days of
gestation (dg), diabetes was induced by a single injection (i.p.) of streptozotocin
(55 mg kg-1) dissolved in sterile phosphate buffered saline
(Rosa et al., 1997). Control group received only buffer. Induction
of diabetes was confirmed by blood glucose level (glycemia>400).
After birth 2 pups of each mother were selected randomly, one for CSF
collection and the other for stereological analysis of C.P.
Blood and CSF assays: After delivery, blood samples were collected,
via the retro orbital sinus, from mothers and the serum levels of glucose,
uric acid, urea, Ca and P were measured by autoanalyser.
CSF extraction: Pups CSF was collected from cisterna magna by
a glass micropipette. The blood contaminated samples were discarded. Samples
were centrifuged at 10,000 rpm for 10 min and then stored at -70°C
until electrolyte measurements. The CSF [Na], [K] and [Ca] concentrations
were determined by autoanalyzer (Nabiyouni et al., 2004).
Dissection of the brains and sections sampling: Under the pentobarbital
anesthesia the brain of the second pupsn was rapidly removed and fixed in 10% paraformaldehyde for histological
evaluation. By applying a systematic random sampling scheme, the paraffin
blocked brains were sectioned coronally. Then sections were stained with
hematoxylin and eosin.
Stereological methods: The volumes of lateral ventricles and choroid
plexus were measured with Cavalieri technique (Gunderson et al.,
1988) and the length density of capillaries was determined by counting
the number of capillaries profiles as described by Gundersen et al.
Statistical analysis: Student`s t test was used for comparison
when only two groups were analyzed and a one-way ANOVA followed by a Scheffe`f
test when more than two groups were analyzed. Statistical significance
was chosen as p<0.05. All results are reported as mean±SEM.
Blood biochemistry and CSF electrolyte measurements: The incidence
of diabetes was assessed by monitoring the blood glucose levels in both
PBS and STZ injected rats (Table 1). There was a significant
increase (p<0.001) in blood glucose levels from 100±5 mg dL-1
in control to 470±18 mg dL-1 in STZ injected rats. The
serum levels of cholesterol, urea, uric acid, [P3+] and [Ca2+]
were also significantly (p<0.05) higher in diabetic dams than controls.
The CSF levels of [Na+], [K+] and [Ca2+]
in one day old pups belong to hyperglycemic dams were significantly higher
(p<0.05) than pups born from controls (Table 2).
The volumes of lateral ventricles and choroids plexus and length density
of choroids plexus capillaries: In comparison with control pups, the
volume of lateral ventricle was significantly increased (p<0.01) in
newborns from hyperglycemic mothers (0.614±0.99 mm3
in control and 1.042±0.2 mm3 in newborns from hyperglycemic
mothers) (Fig. 1). Also the comparison of choroid plexus
volume measurements showed significant increase in pups from diabetic
mothers (from 0.314±0.027 mm3 in controls to 0.556±0.036
in experimental pups; p<0.01) (Fig. 2, 4).
The length density of choroid plexus capillaries in experimental group
was increased significantly as well (from 1974±228 in controls
to 5068±430 mm mm-3 in experimental group; p<0.01)
(Fig. 3, 4).
||Serum concentrations of different metabolites in normal
and hyperglycemic dams
|The values are presented as means±SEM. The numbers
of animals in each group were 6, *p<0.05 Student`s t-test compare
hyperglycemic dams with control
||The CSF levels of [Na+], [K+]
and [Ca2+] in neonates from hyperglycemic and control dams
|The values are presented as means±SEM (n = 6),
*p<0.05 Student`s t-test compare pups from hyperglycemic dams with
pups from controls
||The volume of lateral ventricles in the neonates from
diabetic and control pups
||The volume of choroids plexus in the neonates from diabetic
and control pups
Maternal hyperglycemia produced evoked significant lateral ventricle
volume increase in neonate that obviously could see. Also in Fig.
4, choroids plexus volume in neonates from hyperglycemic mothers was
||The length density of choroids plexus capillaries in
neonates from diabetic and control dams
||Cross section of lateral ventricle and choroids plexus
(x6) in control (up) and experimental (down) neonate
When lateral ventricle enlarged it compress that region surround them
and this compression induced degenerative disorders in this neonates (Fig.
It seems that maternal hyperglycemia is a risk factor for normal-pressure
hydrocephalus (Casmiro et al., 1989). It is obvious that ion transport
in the choroid plexus plays a critical role in the production of CSF and
thus homeostasis of the brain (Egleton et al., 2003). Changes in
the number or dysfunction of ion pumps and ion permeases (symporters and
antiporters) are probably common events in diabetes (Egleton, 2003). In
this respect, although the results of some animal studies indicated that
the BBB structure remains intact in hyperglycemia, it has been shown that
the BBB permeability is increased in STZ diabetic animals (Sharma et
al., 1985). The BBB has both paracellular and transcellular transport
systems and the regulation of the rate of their transportation and/or
permeabilities are affected by hyperglycemia. While the paracellular pathway
is so limited to macromolecules, ions and most small water-soluble molecules
the transcellular movement of lipid soluble molecules and organic ions
can be highly regulated by the number of different transporters and efflux
proteins. The etiology of deleterious in CSF electrolytes composition
in different conditions may reflect the altered transcellular transport
system (Egleton et al., 2003).
The results obtained from the present study show a significant increase
in CSF [Na+], [K+] and [Ca2+] levels
and the volume of lateral ventricle in pups born from experimental diabetic
mother, when compared with control (Table 2, Fig.
1). These results may indicate to the up regulation of ions transporters
at the CSF- blood- barrier in hyperglycemic condition. It is obvious that
the elevated rate of ion transports may increases the CSF osmolality which
follows by excess water flow via aquaporin channels (Malcolm, 2000). It
has been shown that in hyperglycemic condition the number of aquaporine
channales in choroid plexus epithelial cells is increased (Malcolm et
al., 2000). Furthermore, the blockade of ion transporters decreases
CSF production (Pollay et al., 1985; Javaheri and Wagner, 1993).
Hyperglycemia may induce ion transporter expression through a number of
post-transnational modifications (Eglton et al., 2003).
Ischemia is a common indication in diabetes. It has been demonstrated
that retinal microvascular endothelium cell replication increase threefold
in diabetic rats (Sharma et al., 1985). In response to a prolonged
hyperglycemia, alteration in vasculature may provide specific ligands
for vascular targeting in type 1 diabetes (Stitt et al., 2005).
In contrast to the retina, the effects of diabetes on microvasculature
in neural tissues are not well described. It has been suggested that angiogenic
remodeling occurs in the central nervous system in response to stimuli
such as exercise traumatic or ischemic injury and metabolic disorders
such as diabetes may insert profound effects on brain`s peripheral and
central vasculature, as well as neurons (Sharma et al., 1985).
For instance, it has been proposed that the risk of stroke and other types
of cerebrovascular accidents may increase in diabetes and structural and/or
functional changes observed in the microvasculature in diabetic neuropathy
may be involved in the resulting nerve damage (Boulton, 2005).
The choroid plexus microvasculature can be a vulnerable target in diabetes
and in our knowledge the effects of hyperglycemia on choroid plexus has
not been fully explored so far. In consistent with the above findings,
the results obtained from the present research show that the choroid plexus
volume measurement and the length density of choroid plexus capillaries
estimation significantly increase in newborns of hyperglycemic dams (p<0.05)
(Fig. 2). The increase in capillary length density is
probably due to the high proliferation rate of capillary which may be
induced by elevated metabolism and hypoxia in hyperglycemic condition
(Freinkel, 1980). These factors act as angiogenic factors and induce the
production of endothelial growth factors such as VEGF, TGF (Nakamura et
al., 1993; Yamanaka et al., 1993) that correlate with finding
in this study (Fig. 3). It is obvious that a chronic
deficiency in brain new-vessel formation in the face of increasing ischemia
makes a major contribution toward progression to the sight threatening
proliferative stages of diabetic diseases. Angiogenesis may be the result
of an imbalance between stimulatory and inhibitory factors that presumably
accurse from the elevated expression of local angiogenic factor induced
by ischemia (Duenas et al., 1999). Therapeutic intervention may
therefore be directed to the abnormal vasculature as well as the nerve.
In total, it is concluded that maternal hyperglycemic condition could
induced remarkable alterations in lateral ventricle structures and the
region was surrounded them. Study shows meaningful increase in Electrolytes
concentration of CSF in neonates from hyperglycemic mothers that may be
related to changes in Blood Brian Barrier structure. Also maternal hyperglycemia
evoke an increase in choroids plexus volume and capillary length that
obviously are seen in figures. Possibility hyperglycemia active angiogenic
factors that proliferate capillary of choroids plexus. It is commended
that researchers investigate how angiogenesis mechanism act. Now we know
increase lateral ventricle and choroids plexus volume is dangerous for
normal physiological actions Therefore, it is better in diabetic pregnant
mothers the level of glucose was maintained under normal condition.
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