diabetes mellitus type 1 ( dm1 ) is an endocrine disorder which is characterized by lack of insulin and hyperglycemia . for a long period , it was believed that the central nervous system ( cns ) as an insulin independent organ is spared from diabetic complications ; however , in recent decades , studies have provided evidence that indicates the deleterious effects of dm1 on structure and functions of the brain [ 24 ] .
although the mechanisms through which hyperglycemia might mediate these effects are not completely understood , it seems that hyperglycemia increases oxidative stress and free radicals generation . increased free radicals damage cellular membrane ( lipid per oxidation ) and initiates neuronal death signaling pathways [ 5 , 6 ] .
it is the common hallmark of diabetes mellitus type 1 ( dm1 ) and neurodegenerative disorders like alzheimer [ 57 ] .
it is a complex part of the limbic system which plays a pivotal role in cognitive functions like navigation , memory , and learning in animals and human as well .
it is divided into dentate gyrus ( dg ) and ca13 . among the hippocampus subfields , dg and ca3
experimental studies showed that uncontrolled hyperglycemia increases oxidative stress markers in hippocampus and subsequently accelerates neuronal death in dentate gyrus ( dg ) and ca3 [ 8 , 1113 ] .
additionally , suppressed cell proliferation in dg and morphological changes like simplification / retraction of dendrites of ca3 pyramidal neurons have been reported in experimental model of dm1 [ 9 , 12 ] . in vivo
neuroimaging study ( voxel - based morphometry , vbm ) could also demonstrate significant volume reduction of grey matter density of hippocampus in diabetic cases . although neuronal apoptosis as the main cause of diabetic central neuropathy has been reported in the hippocampus , a great challenge in evaluation of diabetic cases that has remained to be answered is the contribution of neuronal loss in volume reduction of brain particularly in the hippocampus . in other words , most of the experimental studies have dealt with each area of the hippocampus separately , and functional / anatomical connectivity of adjacent regions has been ignored .
dg and ca3 are connected directly together and can be considered an anatomical module . based on the anatomical module concept , two strongly connected regions should respond to an insult contemporarily .
so calculated response index of dg / ca3 can be used instead of considering each area separately .
the aims of this study were ( 1 ) to assess the role of neuronal apoptosis in volumetric change and ( 2 ) the efficiency of response index ( ri dg / ca3 ) in interpretation and predicting of dm1-related pathologies of the hippocampus .
male wistar rats ( 8 weeks old ) were randomly divided into 4 groups ( n = 10 ) .
all rats were maintained in animal house and allowed free access to drinking water and standard rodent diet . in 3 groups ,
diabetes mellitus was induced by single intraperitoneal ( ip ) injection of streptozotocin ( sigma ) at a dose of 60  mg / kg .
animals were considered diabetic with fasting blood glucose level above 250  mg / dl four days after diabetes induction [ 12 , 13 ] .
six weeks after diabetes approval , diabetic animals were treated as follows : group ( 1 ) insulin treated ( 35  u ) ( nph , exir , iran ) , group ( 2 ) antioxidant treated ( vitamin c ) ( darou pahksh , iran ) ( 80  mg / kg , ip ) , and group ( 3 ) diabetic . the treatments were conducted for 2 weeks . at the end of 8 weeks
, animals were anesthetized by chloroform and subsequently 5  ml blood was collected from the heart of each animal in order to measure the plasma cu level as an oxidative stress marker , and then animals were perfused transcardially with 100  ml of saline followed by 200  ml of fixative containing 2% glutaraldehyde and 2% paraformaldehyde in 0.1 phosphate buffer ( ph : 7.4 ) .
serial coronal sections ( thickness = 5 and 10 microns ) were obtained through the entire rostrocaudal extent of paraffin embedded hippocampi in the left and right hemispheres by a microtome ( 1212 ernst leitz gmbh wetzlar , germany ) . serial coronal sections ( a thickness of 10 microns )
the volumes of the hippocampi in 4 groups were estimated using cavalieri 's principle [ 15 , 16 ] .
the volumes of dentate gyrus and ca3 were estimated by cavalieri 's method as follows : 
 ( 1)v = tapp , 
 where t is the thickness , a / p is the area of each point , and p is the total number of hit points .
tunel assay was done by in situ cell death detection kit , pod ( rouch ) , according to the recommended manual ( rouch ) .
paraffin sections of hippocampus ( 5  m ) were treated for apoptosis detection as briefly as follows : deparaffinization , absolute ethanol , graded series of 7095% 3 minutes , washing in pbs 5 minutes , proteinase k 15 minutes , washing in distilled water 4 ( each 2 minutes ) , hydrogen peroxide 3% , pbs 2 , reaction mixture at room temperature ( 60  min ) , pbs 3 , convertor - pod 30 minutes in 37c , pbs 3 , adding dab 5  min , pbs 3 , counterstaining with toluidine blue , washing in running water 3 , graded series alcohol ( 70100 ) , xylene , and mounting . the density of apoptotic neurons in the granular layer and pyramidal layers was estimated by physical dissector principle .
we used an olympus microscope ( bx 51 , japan ) , equipped with a software program , digital 5 analysis .
after registrations of paired samples with 5  m intervals , counting frame was superimposed on the captured pictures , and counting was performed according to the dissector principle .
apoptotic neurons density in unit volume was estimated by the following formula : 
 ( 2)nv=1ahqp , 
 where n is the apoptotic neuronal density , a is the area of applied frame , is linear magnification , q is the number of apoptotic neurons , and p is the number of counted frames .
the collected blood samples were transferred to the laboratory and centrifuged at 3000  rpm  10 minute .
based on the anatomical connectivity between the dentate gyrus and ca3 region , we tried to develop a response index between the parameters of volume and neuronal death of the two regions of interest .
the ratios of mean volumes of dg to ca3 ( vdg / vca3 ) and numerical density of apoptotic neurons ( nddg / ndca3 ) were calculated ( table 1 ) .
all data are expressed as mean  sd . comparisons of data between groups were made using one - way anova and tukey 's post hoc test .
at the end of 8 weeks , the bgs of vitamin c ( 487.5  83.35  mg / dl ) and diabetic ( 568  45.2  mg
/ l ) groups showed significant level of difference to the control ( p < 0.001 ) .
the bg of insulin treated group ( 114.5  15.3  mg / dl ) showed no difference in comparison to the control group ( 101  6.3  mg / dl ) ( p > 0.05 ) ( figure 1 ) . the free plasma cu level of control ( 7.29  0.08  g / dl ) , insulin ( 7.29  0.1  g / dl ) , and vitamin c ( 7.22  0.11  g / dl ) groups showed no significant level of difference ( p > 0.05 ) ( figure 2 ) .
the volume of dg in diabetic ( 1.58  0.04  mm ) and vitamin c ( 1.64  0.04  mm ) groups showed significant level of difference compared with control ( 2.00  0.09  mm ) and insulin ( 1.99  0.07  mm ) groups ( p < 0.001 ) .
the volume of ca3 in vitamin c ( 2.06  0.12  mm ) and diabetic groups ( 1.98  0.08  mm ) showed significant level of difference compared with control ( 3.15  0.2  mm ) and insulin ( 3.08  0.2  mm ) groups ( p < 0.001 ) . the volumes of dg and ca3 showed strong connection with bg level ( r = 0.9 ) . the connections between volumes of dg and ca3 with cu were r = 0.6 and r = 0.7 , respectively .
comparison between the total volume of dg and ca3 showed significant level of difference in control group ( p < 0.001 ) . the number of apoptotic neurons in dg of diabetic group ( 9615  264  mm ) showed significant level of difference ( p < 0.001 ) in comparison with those of control ( 945    106  mm ) , insulin ( 1074  154  mm ) , and vitamin c ( 1119  220  mm ) groups .
the numbers of apoptotic neurons in ca3 of diabetic group ( 18055  3842  mm ) showed significant level of difference ( p < 0.001 ) in comparison to the control ( 613  230  mm ) , insulin ( 616  159  mm ) , and vitamin c ( 798  137  mm ) groups .
the comparison between the number of apoptotic neuron in dg and ca3 was significant in control group ( p < 0.05 ) .
the connection between the rate of apoptosis in dg and bg was r = 0.72 and for ca3 was r = 0.75 .
a strong connection was found between the rate of apoptosis in dg with plasma cu level ( r = 0.99 ) and in ca3 region ( r = 0.99 ) .
ri of volumes ( vdg / vca3 ) of vitamin c and diabetic groups showed a clear difference in comparison with control ( p < 0.05 ) , while in contrast ri of apoptosis ( nddg / ndca3 ) in diabetic group showed significant level of difference ( p < 0.05 ) ( table 1 ) .
based on the equations , 0.08 is a critical point in which two curves met together .
the results of this study showed that ( 1 ) dg and ca3 volume reduction begins and progresses independently of neuronal loss .
( 2 ) volumetric index is more sensitive to diabetes mellitus than neuronal loss index . ( 3 ) shifting of both indices toward 0.8 of ri is a critical reference point in which volumetric changes and neuronal loss become coincident .
( 4 ) neuronal loss is more related to increased oxidative stress ( plasma cu level ) , while in contrast volume changes are more related to glycemic level . ( 5 ) interestingly , 2 weeks of therapy with vitamin c maintained the cu level and neuronal loss rate at a normal range but in contrast had no effect on bg and volume changes of the two subfields of interest .
( 6 ) insulin therapy prevents degenerative changes of the dentate gyrus and ca3 regions and also maintains plasma cu at normal level .
( 7 ) dm1 induces a higher neuronal loss in ca3 region in contrast to normal condition . for the first time
the index is based on functional and anatomic connectivity of two regions , dg to ca3 .
in fact , the index represents the degree of impression of two regions from each other in the presence of a disorder like diabetes mellitus .
the index of the numerical density of neuronal loss indicates that ( 1 ) neuronal loss and volumetric changes at first are two separate scenarios .
( 2 ) the tone of neuronal loss in ca3 takes over that of dg , while the tone of volume changes in the dg and ca3 regions is more orchestrated in an insulin - dependent reductive manner . according to our results , the neuronal death could not be considered the main leading cause of volume change of the subfields of interest of hippocampus because by antioxidant therapy , vitamin c , in spite of the decreased rate of neuronal loss and normal plasma cu level , the volumes of the two regions showed a clear reduction in comparison with insulin treated group . at present , we can not explain the relation between the volume of the two regions of interest and exact mechanism(s ) of exogenous insulin , but one of the possible mechanisms could be the dysregulation of hypothalamo - pituitary - adrenal ( hpa ) axis and subsequently an increase in circulating cortisol levels . in part of our study , we weighed the adrenal glands of the animals and found that hyperglycemic animals had massive hypertrophy of the adrenal glands ( unpublished data ) .
so increased weight of adrenal gland ( hypertrophy ) could be considered as a mirror of hpa hyperactivity and lack of insulin . according to the results of this study , neuronal loss and atrophy are two different but relevant scenarios .
it seems that the lack of insulin imposed negative effects on granular cells of dentate gyrus and more vulnerability of pyramidal neurons of ca3 [ 18 , 19 ] .
our results , in line with other studies , are indicative of neurotropic / protective role(s ) of insulin on dentate gyrus and ca3 region and lack of insulin as a possible factor of neurodegeneration .
studies have reported that insulin replacement could reverse electrophysiological and morphological changes of hippocampus [ 2022 ] .
dg undergoes continuous proliferation through the life , and the rate of proliferation / death could be influenced by strenuous physical activity and hyperglycemia .
hyperglycemia accelerates free radical generation and nonenzymatic degradation of ceruloplasmin which resulted in increased free plasma cu level .
the underlying repercussion of the increased free radical generation will result in more neuronal death through the complex mechanisms which can be considered as a hallmark of neurodegeneration . at present ,
morphometric changes are detectable in clinical neuroimaging examinations , but neuronal loss even in large scale would not be discernible
. therefore , generalization of microscopic findings to neuroimaging findings can be worthwhile in evaluation of diabetic cases .
the results of our study can be considered as a window to a comprehensive insight into the hippocampus encephalopathy .
although the generalization of this little study to human should be taken cautiously , more studies are needed in the future .
 
 background . 
 neuronal apoptosis is the major cause of diabetes central neuropathy , but its role in volumetric changes of hippocampus has not been clarified . 
 the aims of this study were to assess the role of apoptosis in volumetric changes of dentate gyrus ( dg ) and ca3 region of hippocampus and to determine a reference point in which these neuropathological changes reach a meaningful level . 
 methods and materials . 
 diabetes was induced in male wistar rats ( n = 10 ) by streptozotocin ( 60  mg / kg ) . 
 six weeks after diabetes , verification animals were divided into four groups as follows : diabetic treated with insulin ( 35  u ) , diabetic treated with vitamin c ( 80  mg / kg ) , and diabetic and control groups . at the end of 8 weeks , numerical density of apoptotic neurons and volume of dentate gyrus and ca3 were calculated by stereological methods . 
 results . 
 the number of apoptotic neurons in dg and ca3 in diabetic group showed significant level of difference in comparison with the control ( p < 0.001 ) . 
 the volume of dg and ca3 in diabetic and vitamin c showed significant level of difference compared with control ( p < 0.001 ) . 
 conclusion . 
 our results suggest that dg and ca3 volume reduction begins and progresses independently of neuronal loss . 
1. Introduction
2. Method and Materials
3. Results
4. Discussion