global incidence of diabetes mellitus ( dm ) estimates more than 171 million for 2000 and 366 million for 2030 .
the mortality and morbidity of dm are determined by various complications , such as diabetic vasculopathy , retinopathy , nephropathy , and peripheral neuropathy .
recently , many studies have indicated that dm also implicated the central nervous system ( cns ) and induced the brain pathological changes , named the diabetic encephalopathy , which is a complication of dm in the cns characterized by mild cognitive deficits and neuropathology [ 35 ] .
diabetic encephalopathy presents many symptoms , which can be described as the features of brain aging including brain atrophy , reactive oxygen species ( ros ) accumulation , cerebral vasculopathy , and impairment of cognition [ 6 , 7 ] .
clinical observation has shown that brain atrophy is more remarkable in diabetic patients than in age - matched controls .
animal experimental data have suggested that learning deficiency is associated with the distinct changes in synaptic plasticity in hippocampal slices in streptozotocin- ( stz- ) induced diabetic rats .
the affinity of glutamate for ampa but not for nmda receptors decreases in sprague - dawley ( sd ) rats at 6 to 8 weeks after stz injection .
the levels of malondialdehyde , xanthine oxidase , and nitric oxide in the hippocampus , cortex , cerebellum , brain stem , and spinal cord significantly increase in stz - induced diabetic - untreated rats , suggesting remarkable generation of ros in the brain .
hyperglycemia resulting from defective insulin secretion , resistance to insulin action , or both is a critical pathogenesis of dm .
willem hendrik gispen and geert - jan biessels have recently suggested that acute hyperglycemia is associated with mild cognitive dysfunction in population with type 1 or type 2 dm . another recent study has suggested that insulin is implicated in the pathogenesis of age - related memory decline and diabetic encephalopathy [ 13 , 14 ] .
insulin may act as a neuromodulator that regulates the release and reuptake of neurotransmitters and probably affects learning and memory .
impairments in the insulin signaling pathway in the periphery and brain have been implicated in alzheimer 's disease , diabetes , and aging [ 16 , 17 ] .
recent studies have revealed that impairments in cerebral glucose utilization and energy metabolism represent early abnormalities that precede or accompany the initial stages of cognitive impairment .
cerebral glucose utilization deficiency and insulin signaling decline are common features between dm and alzheimer 's disease ( ad ) .
ad is a progressive neurodegenerative disease characterized by the loss of memory and other cognitive functions , resulting in dementia .
the hallmarks of pathology of ad are a deposition and microtubule - associated protein tau overphosphorylation and formed the senile plaques in the extracellular matrix . additionally , some studies have indicated that the insulin signals are involved in regulation of a accumulation and tau phosphorylation [ 17 , 21 ] .
furthermore , the factors associated with high risk of ad are also involved in the development of dm , especially t2 dm .
clinical and experimental data clearly showed that diabetes affected the brain and these effects are similar to the acceleration of brain ageing .
however , whether or not diabetic encephalopathy at early onset shows ad - like pathology as aging dependent neurodegenerative disease remains unclear . in the present study ,
changes in the brain were indicated by hippocampal atrophy accompanied with beta - amyloid deposition , synapse loss , and learning behavioral deficiency at 4 months after stz injection .
fiftymale sprague - dawley ( sd ) rats ( 810 weeks old ) were obtained from the experimental animal center of lanzhou university .
the rats were kept in an animal house at 22  2c temperature , 65  10% relative humidity , and 12  h light / dark cycle .
fluoro - jade c ( fjc ) and mouse monoclonal anti - gapdh and rabbit polyclonal anti - a42 antibodies were purchased from millipore ( bellerica , ma , usa ) .
glucose , total cholesterol , triglyceride , and creatine enzymatic diagnostic kits were purchased from randox ( crumlin , county antrim , uk ) .
rabbit polyclonal anti - synaptophysin ( syn ) antibody was purchased from santa cruz biotechnology incorporation ( santa cruz , ca , usa ) .
thirty rats were fasted overnight and then injected with stz ( 65  mg / kg body wt . ) through the femoral vein .
one week after stz injection , blood samples were collected through the tail vein , and plasma glucose level and insulin level were measured by plasma glucose enzymatic diagnostic kits and insulin elisa kit , respectively .
rats with the plasma glucose level 300  mg / dl and symptoms of polyuria , polyphagia , and polydipsia were considered to be diabetic and used in the present study .
the fasting plasma insulin level in stz - induced dm rats was 1.32  0.6 
pmol / l and was markedly lower than that of the normal rats ( 166.2  4.3 
the metabolic parameters were tested according to our previous methods . in brief , the rats were placed in metabolic cages and raised for 24  h after 8  h of fasting .
urine/24  h , water/24  h , and food consumption/24  h were measured , and fasting plasma glucose , triglyceride , total cholesterol , and creatinine were quantified using respective enzymatic diagnostic kits .
creatinine clear ratio ( ccr ) was calculated according to urine volume/24  h and body weight ; the ccr was calculated as follows : 
 ( 1)urine  creatinineurine  volume  ( ml/24  h)(plasma 
finally , the total metabolic data of 18 rats in each group were recorded after four months .
six rats in each group were sacrificed with an overdose of 10% chloral hydrate and then transcardially perfused with 0.9% saline solution followed by 4% ice - cold phosphate - buffered paraformaldehyde ( pfa ) .
the brains were removed , postfixed in 4% pfa for 12  h , and then immersed sequentially in 20% and 30% sucrose solutions in 0.1  m phosphate buffer ( ph 7.4 ) until they sank .
coronal sections with a thickness of 40  m were cut at 2.2  mm to 4.80  mm from the bregma using a freezing microtome ( leica , germany ) and then stored at 20c in a cryoprotectant solution . to evaluate brain
atrophy , the forebrain and hippocampal volumes were measured according to our previous methods . in brief , for forebrain volume , six sections at 2.28  mm to 0.12  mm from the bregma ( interval at 400  m ) were selected and then stained with cresyl violet .
the forebrain volume was calculated as 
 ( 2)[s1a+s2a2+s2a+s3a2+s3a+s4a2  + s4a+s5a2+s5a+s6a2]400 , 
 where s1a to s6a represent the forebrain areas in sections 1 to 6 , respectively . for hippocampal volume , six sections at 2.2  mm to 4.6  mm from the bregma ( interval at 400  m ) were selected and then stained with cresyl violet .
the hippocampal volume was calculated as 
 ( 3)[s1a+s2a2+s2a+s3a2+s3a+s4a2  + s4a+s5a2+s5a+s6a2]400 , 
 where s1a to s6a represent the hippocampal areas in sections 1 to 6 , respectively .
to evaluate the neurodegeneration , the fjc staining was used according to our previous methods . in brief , six sections at 2.28  mm to 0.12  mm and six sections at 2.2  mm to 4.6  mm from the bregma ( at 400  m intervals ) were selected , respectively .
dried sections were dipped in 80% ethanol solution that contains 1% sodium hydroxide , 70% ethanol , and 0.06% potassium permanganate for 5 , 2 , and 10  min , respectively .
the sections were rinsed with distilled water and then incubated with 0.0004% fjc in 0.1% acetic acid for 20  min .
fjc staining was detected under a fluorescent microscope at 480  nm excitation and 525  nm emission .
images were acquired through a 20 objective , and the number of fjc - positive cells in the frontal cortex , hippocampus , and hypothalamus was counted .
the total number of positive cells in the frontal cortex was calculated as 
 ( 4)[s1+s22  + s2+s32+s3+s42+s4+s52+s5+s62]10 , 
 where s1 to s6 represent the fjc - positive cell numbers in sections 1 to 6 , respectively .
the total number of positive cells in the hippocampus and hypothalamus was calculated as 
 ( 5)[s7+s82+s8+s92+s9+s102  + s10+s112+s11+s122]10 , 
 where s7 to s12 represent the fjc - positive cell numbers in hippocampus or hypothalamus in sections 7 to 12 , respectively . for a42 immunostaining ,
three sections at 2.28  mm , 0.12  mm , and 2.2  mm from the bregma were selected and then incubated with 0.3% h2o2 for 30  min .
the sections were placed in blocking buffer that contains 10% normal goat serum and 0.3% triton x-100 in 0.01  m phosphate - buffered saline ( ph 7.2 ) for 30  min at 37c and then incubated overnight with antibodies against rabbit polyclonal anti - a42 ( 1  :  500 ) at 4c .
the sections were then incubated with corresponding biotinylated secondary antibodies ( 1  :  200 ) at 37c for 1  h and then with avidin - biotin - peroxidase ( 1  :  200 ) at 37c for 1  h. immunoreactivity was visualized with 0.05% 3 , 3-diaminobenzidine as chromogen .
negative controls received the same treatment without the primary antibodies and showed no specific staining .
six rats were sacrificed by decapitation , and their brains were quickly removed and placed on ice - cold glass plates .
the hippocampus was rapidly dissected , frozen , and then stored in a deep freezer at 80c until assayed .
the frozen tissues were homogenized in 4 volumes of buffer a that contains 50  mm tris - hcl ( ph 7.6 ) , 150  mm nacl , and a protease inhibitor cocktail ( complete ; roche diagnostics , mannheim , germany ) with 10 strokes of a teflon glass homogenizer .
the supernatant was used as the soluble fraction , and the pellet was solubilized by sonication in buffer a that contains 6  m guanidine - hcl .
the solubilized pellet was centrifuged at 20,000  g for 20  min at 4c .
the supernatant was diluted 12-fold to reduce the concentration of guanidine - hcl and used as the insoluble fraction .
golgi staining was performed on 4-month - old diabetic rats and age - matched normal rats according to the previous methods .
briefly , freshly dissected brains were immersed in a golgi - cox solution for 2 weeks at room temperature .
the golgi - cox solution was replaced , and immersion was continued for 2 weeks .
the pyramidal neurons in cortical layers ii / iii and in the ca1 region of the hippocampus were analyzed .
a minimum of two segments were randomly selected per neuron from the apical oblique and basal shaft dendrites .
fourrats in each group were sacrificed by decapitation , and their brains were quickly removed and placed on ice - cold glass plates . the cerebral cortex was dissected and frozen in liquid nitrogen .
total rna in the left cerebral cortex was extracted by trizol reagent , and rna concentration was measured using a spectrofluorometer .
total protein in the right cerebral cortex was extracted by ripa buffer , and protein concentration was measured using the bradford assay .
the frontal cortex and hippocampus of the rats were placed on ice - cold glass plates .
proteins ( 50 g ) were fractionated on 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis and then transferred into polyvinylidene fluoride membranes .
the membranes were blotted with anti - synaptophysin ( 1  :  1000 ) and anti - gapdh ( 1  :  5000 ) antibodies , as well as with horseradish peroxidase - conjugated second antibody ( 1  :  5000 ) .
morris water maze was performed as previously described . briefly , at 4 months after stz injection , twelve rats were selected according to the open field test and then allowed to perform a learning task in the morris water maze with the blind method .
the maze consisted of a black pool ( 148  cm diameter ) filled with water ( 26  2c ) .
a circular black platform was submerged 2  cm below the water surface in the middle of the target quadrant .
the behavior of the rats in the pool was traced with a camera connected to a wmt-100 analysis system ( taimeng , chinese instruments ) .
the swimming speed , distance , and latency of the rats to find the platform were measured .
the pool was divided into four quadrants ( i.e. , quadrants 1 to 4 ) .
the quadrants had different shapes , different colors , and three rings ( inner , middle , and outer ) .
the platform was placed in a constant location in the middle ring of quadrant 3 .
each day involved training the rats in the four quadrants with 30  min intervals .
each trial was started by placing a rat with its back facing toward the platform at the starting points .
when the rat did not find the platform within 60  s , it was guided on the platform for 15  s. during acquisition ( days 1 to 4 ) of the spatial navigation test , all groups were given one session of four trials each day . on each day after training
, the rat was removed from the pool , dried , and then returned to its cage .
briefly , the ia box consisted of a lighted ( safe ) compartment and a dark ( shock ) compartment separated by a door . in the dark compartment ,
the rats received a footshock of 0.6  ma for 2  s through a constant current scrambler circuit delivered through the grid floor .
rats were placed in the lighted compartment , as specified in each experiment , facing away from the door .
the door leading to the dark chamber was opened after 10  s. once the animals fully entered the dark chamber ( all four limbs contacting the grid in the dark chamber ) , the door was closed , and a footshock was delivered for 2  s after a 2  s delay .
the rats were returned to their home cage 10  s later and were tested 48  h after training .
mann - whitney u test was used for each evaluation , and unpaired student 's t - test was used for other data .
the results of consecutive metabolic test showed that the body weight ( figure 1(a ) ) of the diabetic rats was reduced compared with that of the age - matched rats .
this test also demonstrated that the water consumption ( figure 1(b ) ) , food consumption ( figure 1(c ) ) , and urine production ( figure 1(d ) ) of the diabetic rats increased compared with those of the age - matched rats .
consecutive plasma examination showed that the levels of glucose ( figure 1(e ) ) , triglyceride ( figure 1(f ) ) , and cholesterol ( figure 1(g ) ) were higher in the diabetic rats than in the age - matched rats .
moreover , ccr was higher in the diabetic rats than in the age - matched control rats ; this result was more pronounced at 2 months after stz injection , suggesting that the kidney function was normal and that muscle wasting increased in the diabetic rats ( figure 1(h ) ) . to evaluate the brain
results showedthe forebrain volume is not significantly different in the diabetic rats compared to the age - matched control rats at 2 and 4 months after stz injection ( figures 2(a ) and 2(b ) ) . the hippocampal volume in the diabetic rats decreased at 4 months after stz injection compared with that in the age - matched control rats ( figures 2(c ) and 2(d ) ) .
the concentrations of rna and protein are shown in figures 2(e ) and 2(f ) .
the total rna concentrationin the cerebral cortex of the diabetic rats decreased at 4 months after stz injection ( 3.032  1.05  g/l ) compared with that in the cerebral cortex of the age - matched control rats ( 4.56  1.21  g/l )
. the total protein concentration in the cerebral cortex of the diabetic rats also decreased at 4 months after stz injection ( 16.535  2.76  g/l ) compared with that in the cerebral cortex of the age - matched control rats ( 19.805  2.49  g/l ) .
the effect of the persistent metabolic disorder on neuronal survival was determined by conducting fjc staining to evaluate the neurodegeneration in discrete brain regions .
the number of fjc - positive cells was higher in the frontal cortex ( figures 3(a ) and 3(b ) , 1570  350 ) , hypothalamus ( figures 3(c ) and 3(d ) , 2800  385 ) , and hippocampus ( figures 3(e ) and 3(f ) , 3235  502 ) of the diabetic rats at 4 months after stz injection than in those of the age - matched control rats .
a deposition is the hallmark characteristic in ad and ad - like brain aging ; in the present study , a deposition was analysis .
immunohistochemistry revealed the presence of a42 immunoreactivity in the frontal cortex ( figure 4(a ) ) and hippocampus ( figure 4(b ) ) of the diabetic rats at 4 months after stz injection .
quantitative analysis of a42 in the frontal cortex and hippocampal tissues by elisa showed no significant difference in the soluble fraction of hippocampal tissues between the diabetic rats and age - matched control rats .
however , the amount of a42 in the insoluble fraction increased in the diabetic rats at 4 months after stz injection compared with that in the age - matched control rats ( figures 4(c ) and 4(d ) ) . to evaluate the synaptic plasticity , dendritic spine density and syn expression were measured .
results showed that the dendritic spine density of the frontal cortex decreased in the diabetic rats at 4 months after stz injection compared with that in the age - matched control rats ( figures 5(a ) and 5(c ) ) .
consistently , the synaptophysin expression of the frontal cortex was lower in the diabetic rats than in the control rats ( figures 5(d ) and 5(f ) ) . similarly , the dendritic spine density of the hippocampus decreased in the diabetic rats at 4 months after stz injection compared with that in the age - matched rats ( figures 5(b ) and 5(c ) ) .
the synaptophysin level in the hippocampal tissues decreased in the diabetic rats compared with that in the age - matched control rats ( figures 5(e ) and 5(f ) ) . at 1 
d after training , no significant difference in the time to find the platform ( escape latency ) was found between the diabetic rats and age - matched control rats at 4 months after stz injection . at 2 , 3 , and 4  d after training
, the escape latency exhibited by the diabetic rats was significantly longer than that exhibited by the control rats ( figures 6(a ) and 6(b ) ) .
the velocity of swimming has no significant difference in both groups ( figure 6(c ) ) .
no significant difference in training of ia was observed between the diabetic rats and age - matched control rats at 4 months after stz injection .
however , at 24 , 48 , and 72  h after training , the freezing duration exhibited by the diabetic rats was notably shorter than that exhibited by the control rats ( figure 6(d ) ) .
in this study , cognitive impairment accompanying hippocampal atrophy was observed at 4 months after stz injection .
brain atrophy , as a general feature during brain aging , is involved in complicated mechanisms .
stz - induced animal models are characterized by insulin deficiency accompanied with polydipsia , polyphagia , polyuria , and weight loss .
insulin receptors are distributed in several brain regions , including the hippocampus , hypothalamus , olfactory bulb , and cortex . and
plasma insulin can pass the blood - brain barrier , binding with its receptor to play important roles . in the cns
, insulin is involved not only in regulating the glucose metabolism , but also in maintaining the neuronal survival and development . in the hippocampus
the administration of insulin enhances performance in a passive - avoidance memory task , and spatial memory training alters the expression levels of insulin receptors in the hippocampus .
consistently , recent animal studies have suggested that delivery of insulin to the hippocampus modulates hippocampal memory processes .
therefore , insulin deficiency may play pilot roles in development of diabetic encephalopathy in stz - induced animal model .
our results indicated that the dendritic spine density in the frontal cortex and hippocampus decreased in the stz - induced diabetic rats compared with the age - matched control rats .
synaptophysin ( syn ) , a crucial presynaptic protein , also decreased in the diabetic rats compared with age - matched control rats . aside from hyperglycemia due to insulin deficiency
brain is the most cholesterol - rich organ in the body ; most of this cholesterol is produced in situ .
the notion that decreased cholesterol biosynthesis alters the brain function is supported by the results of in vitro studies that cholesterol is essential for synaptogenesis and synapse function . in our present experiment ,
the insulin deficiency and aberrant lipid metabolism may contribute to the decline of synaptic plasticity . in the present study , a42 deposition in the hippocampus
however , it did not form plaques in the extracellular matrix at 4 months after stz injection . at early onset , a aggregated in the cytoplasm .
furthermore , a formed plaques and deposited in the extracellular matrix with the decline in a clearance .
alternatively , the autopsy studies in humans do not find increased amyloid and tau pathology in relation to diabetes .
but many studies indicated that the a aggregation was regulated by insulin signals in vivo and in vitro .
once insulin has bound to its receptor , phosphoinositide 3-kinase is phosphorylated , and protein kinase b is activated , which in turn phosphorylates and thereby inhibits gsk-3 .
gsk-3 is a serine / threonine kinase encoded by two genes with high sequence homology ( gsk-3 and gsk-3 ) .
also , insulin improves a clearance through insulin - degrading enzyme , one of the main proteases involved in a degradation . in the present study ,
the mechanisms of a deposition induced by dm remain to be elucidated in future work .
in addition , our unpublished work indicated that the gsk3 activity increased in the hippocampus and frontal cortex of stz - induced diabetic rats .
diabetes is characterized by marked peripheral alterations in glucose homeostasis , which leads to hyperglycemia .
excess glucose is metabolized through alternative metabolic pathways , which may have direct adverse effects on the brain through the generation of potentially toxic metabolic by - products and through the depletion of important metabolic cofactors .
in addition , diabetes is associated with long - term complications in other organ systems , wherein vascular disease can cause secondary damages to the brain .
identifying the brain injury and cognitive impairment resulting only from central nervous systemic pathology is difficult because vascular pathology and metabolic toxicity may be important in diabetic encephalopathy development .
after all , more and more evidence indicates that population with the type 1 or type 2 dm are prevalence in suffering from ad . and the rodent animal model with type 1 or type 2 dm can be observed the ad pathological changes including a deposition and tau - phosphorylation [ 40 , 41 ] . in conclusion , aberrant metabolism including hyperglycemia and hyperlipidemia following insulin deficiency caused hippocampal atrophy , neurodegeneration , a deposition , and declined dendritic spine density in stz - induced diabetic rats .
 
 objective . 
 numerous epidemiological studies have linked diabetes mellitus ( dm ) with an increased risk of developing alzheimer 's disease ( ad ) . however 
 , whether or not diabetic encephalopathy shows ad - like pathology remains unclear . 
 research design and methods . 
 forebrain and hippocampal volumes were measured using stereology in serial coronal sections of the brain in streptozotocin- ( stz- ) induced rats . 
 neurodegeneration in the frontal cortex , hypothalamus , and hippocampus was evaluated using fluoro - jade c ( fjc ) . 
 a aggregation in the frontal cortex and hippocampus was tested using immunohistochemistry and elisa . 
 dendritic spine density in the frontal cortex and hippocampus was measured using golgi staining , and western blot was conducted to detect the levels of synaptophysin . 
 cognitive ability was evaluated through the morris water maze and inhibitory avoidant box . 
 results . 
 rats are characterized by insulin deficiency accompanied with polydipsia , polyphagia , polyuria , and weight loss after stz injection . 
 the number of fjc - positive cells significantly increased in discrete brain regions of the diabetic rats compared with the age - matched control rats . 
 hippocampal atrophy , a aggregation , and synapse loss were observed in the diabetic rats compared with the control rats . 
 the learning and memory of the diabetic rats decreased compared with those of the age - matched control rats . 
 conclusions . 
 our results suggested that aberrant metabolism induced brain aging as characterized by ad - like pathologies . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion