diabetic retinopathy ( dr ) is a chronic complication of diabetes mellitus ( dm ) , the leading cause of blindness in adults , affecting 51 million people all over the world [ 1 , 2 ] . all types of cells within the retina are involved in an array of pathophysiological processes even at the early stages of diabetes . a growing number of studies suggested that dr is also the lesion of the retinal neurons [ 8 , 9 ] . the newly onset clinical features of dr , breakdown of the blood - retinal barrier ( brb ) , and various visual deficits all support this concept in human studies [ 1013 ] . because of gradual and accelerating deterioration , intervention should be implemented before irreversible dr occurs [ 14 , 15 ] . although the mechanism remains unclear , it is generally believed that excessive reactive oxygen species ( ros ) from mitochondria induce apoptosis . with the consequent oxidative damages the importance of mitochondria in regulating apoptosis has been well documented . under hyperglycemic conditions , inflammation   and excessive ros damage mitochondria and thus promote the release of apoptosis - inducing factors such as cytochrome c , which further activates the neuronal apoptosis process [ 2123 ] . therefore , alleviation of mitochondrial oxidative damage may reduce apoptosis of retinal neurons and thus provide a new strategy for treatment of newly onset dr . one effective method to reduce mitochondrial oxidative damage is lowering ros . sod , an important antioxidant enzyme , combines and disproportionates superoxide anion and thus reduces their effects on lowering sod activity . therefore , it is very important to understand and restore sod activity to protect mitochondria . it remains unknown which one plays a key role in preventing retinal neurons apoptosis . in this study , by examining the activity and mrna expressions of mn sod and cu - zn sod in retinal neuron of rats with diabetes , we hope to demonstrate the relationship of retinal neurons apoptosis and expression / activity of mn sod in rats at the early phase of diabetes . a total of 72 male healthy sprague - dawley ( sd ) rats ( 8 weeks old , weighted at 230250  g ) were provided by pla academy of military medical experimental animal center , grouping into normal controls and diabetics . stz 60  mg / kg body weight was injected intraperitoneally to establish diabetic models . a blood glucose > 16.7  mmol / l after 48 hours was regarded as diabetic . the rats were kept at 12  hr day - night cycle room , with free access to chaw diet and water . the left or right eyeballs from 6 rats in each group were used for histopathology he staining , tdt - mediated dna nick end dutp label ( tdt - mediated dutp nick end labeling , tunel ) detection , sod activity detection , and tissue rna extraction and detection respectively . xanthine oxidase enzymatic detection kit for sod detection was purchased from nanjing jiancheng bioengineering institute . primers used were as follows : for cu - zn sod , forward : 5-gtt ccg agg ccg cgc gt-3 and reverse : 5-gtc ccc ata ttg atg gac-3 ; for mn sod , forward : 5-ggc caa ggg aga tgt tac aa-3 and reverse : 5-gct tga tag cct cca gca ac-3 ; and for rat -actin , forward : 5-cct gct tgc tga tcc aca-3 and reverse : 5-ctg acc gag cgt ggc tac-3. rna extraction kit was from tiandz , inc . , reverse - transcription kit was from promega , usa , and real master mix ( sybr green ) was from tiangen biotech ( beijing ) co. , ltd . left eyes were quickly removed and fixed in 4% paraformaldehyde for 1520  min . the vitreous were then carefully removed and the remaining  eye cup  was fixed for 2 more hours and then followed by graded ethanol dehydration , xylene , and embedded wax . starting at 2  mm from the optic nerve , the sections were made ( 4  m thick ) and stained with he . as described in our previous study , the paraffin was dewaxed , and then the tissues were treated with 3% h2o2 at room temperature for 10  min , followed by proteinase k treatment at 37c for 10  min and then with tdt and dig - d - utp . the biotinylated antidigoxin antibody was added and incubated at 37c for 30  min and then 1  :  100 diluted streptavidin - biotin complex reagents were added ; the sample was further processed with 3 , 3-diaminobenzidine coloration , slightly redyed with mayer hematoxylin , then washing , dehydration , transparent and mounting . three slices from each sample were used to count cell number with 5 independent fields for each . the apoptosis index of neurons in retinal ganglion cell layer was calculated as follows : 
 ( 1)apoptotic  index  = ( apoptotic  cell  number)(total  cell  number  within  the  same  field)100% . retina tissue was homogenized in lysis buffer . after being centrifuged at 2,000  rpm for 15  min , the protein concentration was measured by use of bradford method ; t - sod and cu - zn sod activities were detected by use of xanthine oxidase enzymatic detection according to the instruction manual . mn sod activity was calculated by subtracting cu - zn sod activity from t - sod activity . reverse transcription was performed according to the instruction manual ( sybr green master mix , abi ) . a 7500 quantitative pcr machine was used and the relative quantitative ( rq ) values were used for analysis . pearson correlation was used to detect the relationship of mrna levels of cu - zn and mn sod with apoptosis of the retina neurons . the results were summarized in table 1 . in the controls , body weights of the rats were about 230250  g after week 8 . the coat is smooth and supple . fasting blood glucose was between 4.2 and 6.3  mmol / l and urine sugar test was negative . there was an upward trend for body weight with time , not statistically significant . in the diabetics , the lens was cloudy , which was worsening with time . in the controls , at week 4 ( figure 1(a ) ) , week 8 ( figure 1(b ) ) , and week 12 ( figure 1(c ) ) , all the retinal layers were clear and arranged in order ; the retinal ganglion cells were arranged in a layer ; the nucleus of rgcs was big , circle- or oval - shaped with light staining ; the inner plexiform layer was net - shaped with loose structure ; the inner nuclear cell layer consisted of 3 to 5 layers of cells ; the nucleus was big with dark staining ; the outer plexiform layer was thinner than the inner plexiform layer ; the outer nuclear layer was thicker and arranged tightly with 8 to 10 layers of cells ; the nucleus was small with dark staining ; and the rods and cones layers were thicker . there is no difference in the diabetics at week 4 ( figure 1(d ) ) or week 8 ( figure 1(e ) ) . at week 12 ( figure 1(f ) ) , the intercellular space increased and the number of the cells decreased in retinal ganglion cells layer , and the inner nuclear layer was thinner . in the controls , rgcs ( figure 2(a ) ) showed a big but low electronic density nucleus , with abundant organelles including mitochondria ( with normal structure and clear mitochondrial ridge , without swollen ) , endoplasmic reticulum , and ribosome . the cells of the inl were circle- or oval - shaped with big nucleus and more organelles ( figure 2(b ) ) and were arranged tightly ( figure 2(c ) ) ; the outer nuclear layer of retina composed of the nucleus of the photoreceptor and arranged tightly and trimly , with uniform nuclear chromatin distribution ( figure 2(d ) ) . in the diabetics at week 4 , there was mitochondrial swollen in some rgcs ( figure 2(e ) ) , with undefined ridge , shorter than normal or diminished ; some chromatin of inl cells was distributed asymmetrically ( figure 2(f ) ) or condensed as crescent ( figure 2(g ) ) with undefined cell membrane ( figure 2(h ) ) . there was no significant difference in the outer nuclear layer of retina compared with the controls . in the diabetics at week 8 , more mitochondrial rgcs were swollen ( figure 2(i ) ) , and some rgcs were shrunk with condensed cytoplasm and decreased organelles ( figure 2(j ) ) . some inner nuclear cells became smaller with undistinguishable nuclear membrane but with more vacuolization in cytoplasm ( figure 2(k ) ) . there was no significant difference at the outer nuclear layer compared with controls . in the diabetic at week 12 , some retinal ganglion cells became smaller ( figure 2(l ) ) with barely defined nuclear membrane ( figure 2(m ) ) , even with nucleus collaps ( figure 2(m ) ) ; there were more vacuolization in some fragments of the nucleus in the inner nuclear cells ( figure 2(n ) ) ; the electrical density of the cytomembrane was asymmetrical and condensed with asymmetrical chromatin in onl ( figure 2(o ) ) . for the controls , at week 4 , 8 , and 12 , there was no apoptosis of neurons in retinal ganglion cell layer ( figures 3(a)3(c ) ) . at week 4 of the diabetics , no apoptotic cells appeared in ganglion cell layer or outer nuclear layer cells ( figure 3(d ) ) with an apoptosis index of ( 0.48  0.53)% . after week 8 , there were more apoptotic cells within the inner nuclear layer of retina ganglion cells . there were apoptotic cells within the neurons of retinal ganglion cell layer ( figure 3(e ) ) , with an apoptotic index of ( 5.66  2.1)% . at week 12 , there was dramatic increase of apoptotic cells within neurons of retinal ganglion cell layer and inner nuclear layer ( figure 3(f ) ) , with an apoptotic index of ( 11.83  1.58)% . the difference of apoptosis index between the controls and diabetics was not significant at week 4 ( p = 0.405 ) but was statistically significant at weeks 8 and 12 ( f = 112.896 , p < 0.001 ) . there were significant differences for the activities of t - sod , cu - zn sod , and mn sod , between the controls and the diabetics ( t - sod : f = 40.907 , p < 0.05 ; cu - zn sod : f = 15.735 , p < 0.05 ; mn sod : f = 17.636 , p < 0.05 ) . as shown in table 2 , there was no significant change of the activities of cu - zn sod or mn sod in the controls at all - time points . while the activities of t - sod , cu - zn sod , and mn sod decreased with time in the diabetics , which mainly occurred at weeks 4 and 8 for mn sod and weeks 8 and 12 for cu - zn sod . as shown in table 3 , there was no significant difference for mrna expressions ( rq values ) of cu - zn sod and mn sod in the controls at any time point . while in the diabetics , both mrna expressions of cu - zn sod and mn sod decreased gradually with time . there was significant difference between controls and diabetics ( cu - zn sod : f = 10.917 , p < 0.05 ; mn sod : f = 75.579 , p < 0.05 ) . the mrna expression of mn sod decreased at week 4 , 8 and 12 , while that of cu - zn sod decreased at week 8 and 12 . there was a negative correlation between mn sod mrna and apoptosis of the retina neurons but not with cu - zn sod mrna ( p < 0.001 , r = 0.89 ) . diabetic retinopathy includes retinal neuropathy and microvascular pathology , both causing retinal lesion and visual function defect . in our study , we investigated the onset , the morphological features of retinal neuron apoptosis , and the mitochondrial structure features along with the development of diabetes and provided an evidence of retinal neuron apoptosis in newly onset diabetic rats . our study showed that there were increased intercellular space , decreased cell number in retinal ganglion cell layer with time , and thinner inner nuclear layer at week 12 in the diabetic rats . apoptosis of retinal neurons is the initial presentation in diabetic retinopathy . in previous studies [ 31 , 32 ] , retinal ganglion cells were labeled by injection of 3% fluorogold into both sides of superior colliculus and the numbers and distribution of rgc were observed . our study confirmed that there was a series of ultrastructure changes of retinal neurons in newly onset diabetic rats , varying in mitochondrial swollen , cytoplasm condensation , chromatin margination , and apoptosis . although em provides direct vision of apoptosis , it can not be used to quantify apoptosis . terminal deoxynucleotidyl transferase - mediated dutp nick end labeling ( tunel ) is an in situ method for detecting the 3-oh ends of dna exposed during the internucleosomal cleavage during apoptosis . it can be used to quantify apoptotic degree sensitively and specifically , especially for newly onset apoptosis . our research demonstrated that apoptotic cells were mainly located at retinal ganglion cell layer and inner nuclear layer , getting more significant with the development of diabetes . showed there were 22% and 14% reductions in the thickness of the inner plexiform and inner nuclear layers in rats with 7.5 months of diabetes . if the mechanism of apoptosis can be identified , it may be very helpful for preventing or alleviating diabetic retinopathy . two classical cellular apoptotic pathways were identified : extracellular ( cell surface death receptor ) and intracellular ( mitochondrial - mediated ) pathways . the reactive oxidant intermediates can trigger the release of cytochrome c from mitochondria , a key event in activation of caspase-3 and a downstream pivotal step in the initiation of apoptosis [ 35 , 36 ] . the mitochondrial electron transport chain is a major source of superoxide , converting up to 5% of molecular o2 to superoxide normally but even more under pathologic conditions [ 37 , 38 ] . studies suggested that reactive oxygen species ( ros ) in nmol level improve cell proliferation , cause apoptosis in pmol level , and induce cell necrosis in mmol level . at low density , it is a normal physiological process . at high density , hyperglycemia increases oxidative stress and plays an important role in the onset and development of diabetic complications . mitochondrion is vulnerable to oxidative stress and thus releases more ros , which accelerates oxidative stress and leads to vicious circle . excessive ros causes mitochondrial swollen and excessive mitochondrial permeability thus induces release of cytochrome c and activates the mitochondrial - mediated apoptosis . under hyperglycemic conditions , oxidative stress is increased in retina and isolated retinal capillary cells ( both endothelial cells and pericytes ) . in another rat model of dr induced by galactose , it was reported that long term administration of antioxidants inhibits the development of dr [ 41 , 43 ] . this further supported the importance of oxidative stress in the development of dr . under normal condition , the excessive ros can be scavenged by antioxidative defense system . whereas , in diabetes , the activity and defense system of the antioxidative enzymes were impaired significantly , and thus the damage of ros was worsened . sod was considered as an important antioxidative enzyme , which combines superoxide anion and converts them into nonreactive products . consistent with our results , it is shown that the activity of sod is decreased in diabetic retina [ 44 , 45 ] . overexpression of sod reduces oxidative stress , decreases release of cytochrome c and apoptosis of neurons , and prevents diabetes - induced glomerular injury , suggesting its major role of regulating apoptosis [ 4649 ] . due to its location , mn sod is considered as the first line to prevent oxidative damage . thus , it is very important to investigate the change of mn sod to clarify the apoptosis pathway . however , in previous studies , the focus has been placed on total sod or cu - zn sod . our analysis demonstrated that both activities of cu - zn and mn sod of rat retina decreased with extended course of diabetes , suggesting that the longer the course of diabetes , the worse oxidative damage of the retina and the less the anti - oxidative capacity of sod . although the content of cu - zn sod in t - sod was higher than that of mn sod , its activity decreased at a quite late stage . in contrast , there was significant decrease for the activity of mn sod even at week 4 , further confirming its frontier activity against oxidative stress under diabetic conditions . more interestingly , this is consistent with the reduction of its mrna expression , suggesting the possibility cause of decreased activity . our study showed that mrna overexpression of mn sod could enhance its activity and antioxidative capacity . study has reported that mn sod activity in nontransfected retinal endothelial cells was 20% of the total sod activity and increased to 60% in mn sod - transfected cells . overexpression of mn sod prevented glucose - induced increase of oxidative stress and apoptosis of retinal endothelial cells [ 27 , 51 ] . it also plays an important role in preventing oxidative damage but may not influence the newly onset mitochondrial - mediated apoptosis because its changes only occurred at a rather late stage . thus , in newly onset dr , overexpression or increased activity of mn sod is more significant than that of cn - zn sod and t - sod . it may challenge the concept that cu - zn sod plays an irreplaceable role in scavenging ros because of its large proportion in t - sod . there are other studies on antioxidants , such as vitamin c and vitamin e. however , their antioxidative effects were not promising . this may be due to the fact that they only change cu - zn sod activity and are short of protection for mitochondria . in summary , our study suggested a protective role for mn sod in the apoptosis of retinal neurons and , ultimately , in the pathogenesis of diabetic retinopathy . it may elucidate a better understanding of mn sod in modifying the course of diabetic retinopathy and suggest an important molecular target for future pharmacological interventions .
this paper aims to explore the relationship of retinal neuron apoptosis and manganese superoxidase dismutase ( mnsod ) at early phase of diabetic retinopathy . 
 sprague - dawley rats were grouped into normal controls and diabetics . 
 data were collected after 4 , 8 , and 12 weeks ( n = 12 ) . 
 the pathological changes and ultrastructure of the retina , the apoptosis rate of retinal neurons by tdt - mediated dutp nick end label ( tunel ) , mrna expressions of mnsod and copper - zinc superoxide dismutase ( cu  zn sod ) , and the activities of total sod ( t - sod ) and subtypes of sod were tested . for the controls 
 , there was no abnormal structure or apoptosis of retinal neurons at any time . 
 there was no change of structure for rats with diabetes at 4 or 8 weeks , but there was a decrease of retinal ganglion cells ( rgcs ) number and thinner inner nuclear layer ( inl ) at 12 weeks . 
 the apoptosis ratio of rgcs was higher than that of the controls at 8 and 12 weeks ( p < 0.001 ) . 
 the activity and mrna levels of mnsod were lower in diabetics at 4 , 8 , and 12 weeks ( p < 0.05 ) . in summary , 
 the apoptosis of the retinal neurons occurred at 8 weeks after the onset of diabetes . 
 retinal neuron apoptosis in early diabetic rats may be associated with the decreased activity and mrna of mnsod .