advanced glycation end - products ( ages ) have been shown to accumulate in various tissues under diabetic conditions , and they participate in the development of vascular complications such as diabetic retinopathy [ 1 , 2 ] . our recent results showed that even a low concentration of ages , for example , 10  g / ml , can induce neuronal apoptosis in retinal neurons and decrease the number of regenerating neurites in cultures .
ages accomplish their effects by binding to specific cellular receptors [ 4 , 5 ] , such as receptors for ages ( rages ) .
these receptors have been found on neurons , mesangial cells , smooth muscle cells , and endothelial cells [ 68 ] .
the binding of rage to ages precursors generates intracellular oxidative stress which then induces receptor - mediated production of reactive oxygen species .
this then results in the activation of the free radical - sensitive transcription factor nuclear factor-b ( nf-b ) [ 9 , 10 ] .
the activated nf-b translocates into the nucleus and causes pathological changes in gene expression [ 9 , 1113 ] .
specificity protein 1 ( sp1 ) is a transcription factor that either activates or represses transcription in response to physiological and pathological stimuli .
it regulates the expression of a large number of genes involved in a variety of processes such as cell growth , apoptosis , differentiation , and immune responses .
several genes can be regulated by a combination of nf-b and sp1 , and in specific cases by direct interaction between the nf-b protein and sp1 protein .
tanaka et al .   found that ages can activate the rage gene through nf-b and sp1 , causing enhanced age - rage interactions in human vascular endothelial cells .
however , there are no reports demonstrating the relationship between nf-b / sp1 expression and regeneration in age exposed retinal neurons .
growing evidence indicates that neuronal abnormalities such as neuronal cell death and vascular abnormalities are associated with the development of early diabetic retinopathy .
because neuronal cell death is an irreversible change and can affect visual function of diabetic eyes , neuroprotective and regenerative therapies need to be determined .
however , no reports have been simultaneously compared the neuroprotective and regenerative effects of several neurotrophic factors including neurotrophin-4 ( nt-4 ) , hepatocyte growth factor ( hgf ) , glial cell line - derived neurotrophic factor ( gdnf ) , and tauroursodeoxycholic acid ( tudca ) in age exposed retinas cultured in the same system .
the purpose of this study was to examine the effect of high doses of ages on neuronal cell death and neurite regeneration in isolated rat retinas .
we also determined the neuroprotective and regenerative effects of four neurotrophic factors , namely , nt-4 , hgf , gdnf , and tudac in age exposed retinas .
in addition , we also examined whether the expressions of nf-b and sp1 were correlated with the neuroprotective and regenerative effects of different neurotrophic factors in ages exposed rat retinas .
seven - week - old male sprague - dawley ( sd ) rats ( japan slc co. , hamamatsu , japan ) were used .
all of the procedures were performed in accordance with the arvo statement for the use of animals in ophthalmic and vision research .
the retinas were isolated under sterile conditions and cut into square pieces of 0.16  mm with sharp razor blades . then the retinal explants were cultured on three - dimensional collagen gels as described in detail [ 1823 ] .
the retinal explants were incubated in 6 different types of media ; ( 1 ) serum - free control culture media , ( 2 ) 100  g / ml glucose - age - bsa ( cyclex co. , nagano , japan ) or glycolaldehyde - age - bsa ( cyclex co ) or glyceraldehyde - age - bsa ( cyclex co ) media , ( 3 ) glucose - age or glycolaldehyde - age or glyceraldehyde - age + 100  ng / ml nt-4 ( r&d systems , minneapolis , mn ) media , ( 4 ) glucose - age , glycolaldehyde - age , or glyceraldehyde - age + 100  ng / ml hgf ( r&d systems ) media , ( 5 ) glucose - age , or glycolaldehyde - age , or glyceraldehyde - age + 100  ng / ml gdnf ( r&d systems ) media , and ( 6 ) glucose - age , or glycolaldehyde - age , or glyceraldehyde - age + 100 m tudca ( wako , osaka , japan ) media .
the serum - free media contained 7.5  mm glucose , 5  g / ml insulin , 16.1  g / ml putrescine , 10% bovine serum albumin , 3.7  mg / ml nahco3 , 5.2  mg / l na2seo3 , and 3.6  mg / ml hepes in minimum essential medium as described [ 21 , 23 , 24 ] . one hundred
g / ml bsa was added to the control medium ( n + a ) as a control for the concentration of age - bsa . to determine whether apoptosis had occurred , the retinal explants were fixed in 4% paraformaldehyde after 7 days in culture and sectioned on a cryostat .
then , tdt - dutp terminal nick - end labeling ( tunel ) staining was carried out with an apoptosis detection kit ( chemicon international , temecula , ca ) according to the manufacturer 's instructions .
sections were costained with 4,6-diamidino-2-phenyl indole ( dapi , polyscience inc . , warrington , pa ) . for quantitative analyses ,
the ratio of the number of tunel - positive cells to the total number of dapi - staining nuclei in the ganglion cell layer ( gcl ) was determined .
a total of 18 sections from the 6 explants / group were studied , and the results were used for the statistical analyses .
the total number of nuclei counted was 405 ( n ) , 215 ( n + a ) , 816 ( ages ) , 472 ( nt-4 ) , 396 ( hgf ) , 512 ( gdnf ) , and 494 ( tudca ) . the retinal explants were fixed as described and cryosections were cut .
after blocking the sections in 5% goat serum and 3% bovine serum in 0.1  m phosphate buffer saline , they were incubated with antibodies against rabbit anti - phosphorylated nf-b ( p - nf-b ) and sp1 transcription factor ( santa cruz biotechnology , santa cruz , ca ) at 4c overnight .
then , the sections were incubated with fluorescein isothiocyanate - conjugated anti - rabbit igg for one hour .
the number of p - nf-b- and sp1-positive cells in the gcl was counted . for quantitative analyses ,
the number of immunopositive cells in the gcl was expressed relative to the total number of dapi - stained nuclei .
the total number of nuclei counted was 301 in n , 234 in ages , 198 in nt-4 , 254 in hgf , 186 in gdnf , and 276 in tudca .
the number of neurites regenerated from the explants was counted under a phase - contrast microscope after 7 days in culture when the number of regenerating neurites was very high [ 3 , 18 , 21 , 23 , 24 ] . branched neurites were counted as one .
the number of explants examined was 101 in the control group including that in serum - free media ( n , n + a , n + nt-4 , n + hgf , n + gdnf , n + tudca ) , 11 in the glucose - age - bsa group , 19 in the glycolaldehyde - age - bsa group , 18 in the glyceraldehyde - age - bsa group , 14 in the glucose - age - bsa + nt-4 , 12 in the glycolaldehyde - age - bsa + nt-4 , and 11 in the glyceraldehyde - age - bsa + nt-4 groups , 12 in the glucose - age - bsa + hgf , 14 in the glycolaldehyde - age - bsa + hgf , 13 in the glyceraldehyde - age - bsa + hgf groups , 12 in the glucose - age - bsa + gdnf , 13 in the glycolaldehyde - age - bsa + gdnf , 14 in the glyceraldehyde - age - bsa + gdnf groups , 10 in the glucose - age - bsa + tudca , 11 in the glycolaldehyde - age - bsa + tudca , and 10 in the glyceraldehyde - age - bsa + tudca groups .
to determine whether ages were toxic to the retinas in culture , the number of tunel - positive cells in the gcl was counted .
the majority of the tunel - positive cells were detected in the gcl because all of the retinal ganglion cells ( rgcs ) were axotomized to isolate the retina [ 3 , 18 , 21 , 2426 ] . in retinas added 100  g / ml bsa more to the control medium ( n + a ) , the number of tunel - positive cells was not significantly different from the control medium ( n ) ( 9.3  2.3% versus 9.7  6.8% , p = 0.244 ) . in retinas cultured in glucose - age - bsa , glycolaldehyde - age - bsa , and glyceraldehyde - age - bsa ,
the number of tunel - positive cells in the gcl was significantly higher than that in the serum - free control medium ( 38.1  6.2% versus 9.7  6.8% , p <
0.0001 ; 36.1  4.9% versus 9.7  6.8% , p < 0.0001 ; and 39.9  9.4% versus 9.7  6.8% , p < 0.0001 , resp . ; figure 1 ) .
the addition of nt-4 decreased the number of tunel - positives cells more than in glucose - age - bsa without nt-4 ( 17.8  7.2% versus 38.1  6.2% ; p = 0.0002 ) , in glycolaldehyde - age - bsa without nt-4 ( 12.8  4.2% versus 36.1  4.0% ; p = 0.0046 ) , and in glyceraldehyde - age - bsa without nt-4 ( 13.4  3.6% versus 39.9  9.4% ; p = 0.0078 ; figure 1 ) .
addition of hgf in age - bsa did not decrease the number of tunel - positives cells compared to the number in glucose - age - bsa without hgf ( 34.0  5.5% versus 38.1  6.2% ; p = 0.7861 ) and glyceraldehyde - age - bsa without hgf ( 29.1  11.0% versus 39.9  9.4% ; p = 0.1722 ) but
it significantly decreased in glycolaldehyde - age - bsa without hgf ( 25.4%  7.3% versus 36.1  4.0% ; p = 0.0039 ; figure 1 ) .
addition of gdnf did not decrease the number of tunel - positives cells compared to the number in glucose - age - bsa without gdnf ( 32.4  3.0% versus 38.1  6.2% ; p = 0.0935 ) and in glycolaldehyde - age - bsa without gdnf ( 35.2%  5.1% versus 36.1  4.0% ; p = 0.7373 ) and in glyceraldehyde - age - bsa without gdnf ( 31.2  9.78% versus 39.9  9.4% ; p = 0.1293 ; figure 1 ) .
the addition of tudca decreased the number of tunel - positives cells more than that in glucose - age - bsa without tudca ( 28.2  6.5% versus 38.1  6.2% ; p = 0.0184 ) and in glyceraldehyde - age - bsa without tudca ( 26.6  8.3% versus 39.9  9.4% ; p = 0.0294 ) but it did not decrease in glycolaldehyde - age - bsa without tudca ( 31.6  3.3% versus 36.1  4.0% ; p = 0.45 ; figure 1 ) .
the sections were immunostained for p - nf-b to determine whether nf-b was activated in retinas exposed to ages .
the effects of the neurotrophic factors on the activation of nf-b were also examined . in retinas
cultured with glucose - age - bsa , the number of p - nf-b immunopositive cells was higher than in serum - free control medium ( 53.2  7.2% versus 31.6  16.1% ; p = 0.0146 ; figures 2 and 3 ) .
addition of nt-4 decreased the number of immunopositive cells more than in the serum - free media ( 11.3  6.9% versus 31.6  16.1% ; p = 0.0315 ) and more than in glucose - age - bsa without nt-4 ( 31.5  5.3% versus 53.2  7.2% ; p = 0.0133 ; figures 2 and 3 ) .
addition of hgf decreased the number of immunopositive cells more than in serum - free media ( 26.8  5.3% versus 31.6  16.1% ; p = 0.049 ) and in glucose - age - bsa without hgf ( 42.6  2.7% versus 53.2  7.2% ; p = 0.0114 ; figures 2 and 3 ) .
addition of gdnf decreased the number of immunopositive cells more than in serum - free media ( 13.3  5.0% versus 31.6  16.1% ; p = 0.0495 ) and in glucose - age - bsa without gdnf ( 25.8  5.3% versus 53.2  7.2% ; p = 0.0011 ; figures 2 and 3 ) .
addition of tudca decreased the number of immunopositive cells more than in serum - free media ( 18.6  3.97% versus 31.6  16.1% ; p = 0.0024 ) and in glucose - age - bsa without tudca ( 36.2  7.2% versus 53.2  7.2% ; p = 0.0034 ; figures 2 and 3 ) .
the sections were immunostained for sp1 transcription factor to determine whether it was expressed in retinas exposed to ages .
we also examined the effect of neurotrophic factors on the expression of sp1 transcription factor . in retinas
cultured with glucose - age - bsa , the number of sp1 immunopositive cells was higher than in serum - free control medium ( 32.2  6.8% versus 6.3  1.2% ; p < 0.0001 ; figures 4 and 5 ) .
addition of nt-4 increased the number of immunopositive cells more than in serum - free media ( 13.0  4.3% versus 6.3  1.2% ; p = 0.0315 ) but did not decrease the number of immunopositive cells in glucose - age - bsa without nt-4 ( 30.2  8.4% versus 32.2  6.8% ; p = 0.6753 ; figures 4 and 5 ) .
addition of hgf increased the number of immunopositive cells more than in serum - free media ( 19.7  5.16% versus 6.3  1.2% ; p = 0.0001 ) but did not decrease the number of immunopositive cells in glucose - age - bsa without hgf ( 24.0  6.7% versus 32.2  6.8% ; p = 0.0926 ; figures 4 and 5 ) .
addition of gdnf increased the number of immunopositive cells more than in the serum - free media ( 12.3  1.4% versus 6.3  1.2% ; p < 0.0001 ) but it decreased compared to in glucose - age - bsa without gdnf ( 19.7  6.4% versus 32.2  6.8% ; p = 0.0007 ; figures 4 and 5 ) .
addition of tudca increased the number of immunopositive cells more than in serum - free media ( 10.2  3.9% versus 6.3  1.2% ; p = 0.0454 ) but it decreased compared to in glucose - age - bsa without tudca ( 19.5  5.5% versus 32.2  6.8% ; p = 0.0075 ; figures 4 and 5 ) . in retinas added 100  g / ml bsa more to the control medium ( n + a ) , the number of neurites was not significantly different from the control medium ( n ) ( 94.6  24.0/mm versus 97.5  34.9/mm , p = 0.948 ) . in retinas incubated with ages ( glucose - age , glycolaldehyde - age , and glyceraldehyde - age ) , the number of regenerating neurites was less than in retinas without age ( 45.0  27.5/mm versus 97.5  34.9/mm , p = 0.0046 ; 29.4  29.4/mm versus 97.5  34.9/mm ,
p = 0.0003 ; 25.0  15.6/mm versus 97.5  34.9/mm , p < 0.0001 ; figures 6 and 7 ) .
all of the retinas incubated in the neurotrophic factors ( nt-4 , hgf , gdnf , and tudca ) had an increase in the number of regenerating neurites in serum - free media ( 541.9  77.5/mm versus 97.5  34.9/mm , p < 0.0001 ; 207.5  49.1/mm versus 97.5  34.9/mm , p < 0.0001 ; 211.3  70.6/mm versus 97.5  34.9/mm , p < 0.0001 ; 229.4  33.8/mm versus 97.5  34.9/mm , p < 0.0001 ; figures 6 and 7 ) . in addition , all of the neurotrophic factors increased the number of regenerated neurites in ages exposed retinas , but the most significant regenerative effect was found in the nt-4 group : 379.4  178.0/mm versus 45.0  27.5/mm , p < 0.0001 , in nt-4 group ; 281.3  100.6/mm versus 45.0  27.5/mm , p < 0.0001 , in hgf group ; 148.8  35.0/mm versus 45.0  27.5/mm , p < 0.0001 , in the gdnf group ; 247.5  56.9/mm versus 45.0  27.5/mm , p < 0.0001 , in tudca group supplemented with glucose - age incubated retinas ; 440.0  165.0/mm versus 29.4  29.4/mm , p
< 0.0001 , in nt-4 group ; 196.3  89.4/mm versus 29.4  29.4/mm , p < 0.0001 , in hgf group ; 161.1  53.9/mm versus 29.4  29.4/mm , p <
0.0001 , in gdnf group ; 238.1  33.1/mm versus 29.4  29.4/mm , p < 0.0001 , in tudca group supplemented to glycolaldehyde - age incubated retinas , 230.6  116.9/mm versus 25.0  15.6/mm , p < 0.0001 , in nt-4 group ; 123.8  29.4/mm versus 25.0  15.6/mm , p < 0.0001 , in hgf group ; 137.5  48.8/mm versus 25.0
 15.6/mm , p < 0.0001 , in gdnf group ; 178.8  39.4/mm versus 25.0
 15.6/mm , p < 0.0001 , in tudca group supplemented to glyceraldehyde - age incubated retinas ( figures 6 and 7 ) .
the importance of ages in the pathogenesis of diabetic complications has been shown in animal models .
two unrelated age inhibitors were found to partially block some functional and structural changes in retinas , neuronal tissues , and kidneys of diabetic animal models [ 2729 ] .
our previous study showed that even a low concentration of ages , for example , 10  g / ml , induced neuronal apoptosis in the gcl and decreased the number of regenerated neurites in culture . higher doses ( 100  g / ml ) of ages had similar effects as low concentrations and decreased the number of tunel - positive cells and significantly blocked neurite regeneration .
the results of this study confirmed that the apoptosis in the gcl were caused by ages and also that neurite regeneration was significantly suppressed .
all examined neurotrophic factors were able to increase the number of regenerative neurites ; however the most significant regenerative effect was observed with nt-4 .
in addition , all examined neurotrophic factors suppressed the expression of nf-b expression in ages exposed retinas .
nf-b is a primary transcription factor that plays an important role in regulating cellular responses , that is , a transcription factor that is present in cells in an inactive state and does not require new protein synthesis to become activated .
other members of this family include transcription factors such as c - jun , stats , and nuclear hormone receptors .
some aspects of the mechanism by which nf-b protects cells against toxins have been identified .
for example , tumor necrosis factor- has been shown to protect hippocampal neurons against excitatory amino acid toxicity through nf-b activation by inducing bcl-2 and bcl - x expression .
sp1 transcription factor belongs to a group of factors that are associated with gc - rich promoters that are involved in basal promoter activity .
sp1 regulates the expression of different genes , including the vascular endothelial growth factor , fibrogenic cytokine , and many matrix genes .
several studies reported that the interactions between age and rage cause phenotypic changes in the microvascular endothelial cells and pericytes [ 3238 ] .
our results showed that ages increase the expression of the transcription factor nf-b and sp1 in retinal neurons .
this suggests that ages enhanced the expression of the rages gene in retinal neurons through the increased expression of nf-b and sp1 .
an upregulation of nf-b and sp1 proteins in retinal neurons suggests that different protective mechanisms are important for the protection of these cells against ages - mediated cell death .
tudca is a member of a group of compounds that modulate the endoplasmic reticulum ( er ) function , protecting the cells against er stress - induced apoptosis .
earlier studies showed that tudca had protective effects on damaged retinal neurons under diabetic stress as an anti - er reagent .
the neuroprotective effect of tudca was correlated with the suppression of phosphorylated jnk and phosphorylated c - jun expression [ 18 , 26 ] .
it is also a potent mitogen and differentiation factor for endothelial cells , and it had neurotrophic and neuroprotective activity for central nervous system neurons [ 41 , 42 ] .
.   used an optic nerve axotomy model and demonstrated that hgf prevented rgc apoptosis in vivo in a concentration - dependent manner .
found that hgf promoted long - term survival and axonal regeneration of rgc after optic nerve injury .
recently , gdnf was found to be a growth and survival factor for neuronal cells , and it promoted the survival of peripheral sensory and sympathetic neurons and also motor neurons .
koeberle and ball   studied the effects of gdnf on rgc survival and apoptosis after optic nerve transection .
nt-4 is a member of the nerve growth factor family which acts on different types of nerve cells , for example , sensory , cortical , and hippocampal neurons .
, we investigated the neuroprotective and regenerative effects of nt-4 on retinal neurons under diabetic condition [ 3 , 18 , 21 , 26 ] .
our results showed that nt-4 promoted the survival and the regeneration of neuronal cells in the retinas incubated in high glucose media .
the neuroprotective and regenerative effects of nt-4 were correlated with the reduction in the activation of caspase-9 and -3 , expression of perk and chop , and c - jun and jnk expression .
the results of the present study showed that all examined neurotrophic factors decreased the number of nf-b immunopositive cells in glucose - age - bsa exposed retina , but nt-4 had the highest significant effect .
however , the number of sp1 immunopositive cells was increased by the addition of neurotrophic factors in serum - free media and more significantly in the hgf and nt-4 groups .
thus after the addition of neurotrophic factors to glucose - age - bsa media , the level of sp1 transcription factor remained high in the nt-4 and hgf group .
kanda et al . found that prostaglandin e2 promoted innervation in skin lesions by the induction of nt-4 , and the induction was mediated by sp1 .
their results showed that the ep3 , g - protein - coupled receptors , mediated by prostaglandin e2 transcription of nt-4 was dependent on the activity of sp1 .
thus , our findings suggest that sp1 may be related to neuronal survival and regeneration .
it is possible that the increased expression of sp1 may result from an enhanced phosphorylation of sp1 , because the sp1 promoter contains several sp1-binding elements and is positively regulated by its own gene product , sp1 protein .
further studies are needed to identify the connection between sp1 expression and nt-4 transcription . to find a possible decrease in the effect of the ages on the retina is important .
our findings that a suppression of nf-b expression in retinal neurons by several neurotrophic factors can result in neuroprotection , and reducing inflammation and oxidative stress suggests therapeutic potential of neuroprotective therapy in various ocular pathologies associated with ages accumulation . in conclusion ,
high - dose ages inhibit neurite regeneration which is correlated with increased expression of nf-b and sp1 .
nt-4 enhances neurite regeneration in ages exposed retinas more than other neurotrophic factors such as hgf , gdnf , and tudca .
our results indicate the therapeutic potentials of the neurotrophic factors as axoprotectants in ages exposed retinas .
 to determine the effect of advanced glycation end - products ( ages ) on neurite regeneration , and also to determine the regenerative effects of different neurotrophic factors ( ntfs ) on rat retinal explants , the retinas of sd rats were cultured in three - dimensional collagen gels and incubated in 6 types of media : ( 1 ) serum - free control culture media ; ( 2 ) 100  g / ml ages - bsa media ; ( 3 ) ages - bsa + 100  ng / ml neurotrophin-4 ( nt-4 ) media ; ( 4 ) ages - bsa + 100  ng / ml hepatocyte growth factor media ; ( 5 ) ages - bsa + 100  ng / ml glial cell line - derived neurotrophic factor media ; or ( 6 ) ages - bsa + 100  m tauroursodeoxycholic acid media . 
 after 7 days , the number of regenerating neurites was counted . 
 the explants were immunostained for nuclear factor-b ( nf-b ) and specificity protein 1 ( sp1 ) . 
 statistical analyses were performed by one - way anova . in retinas incubated with ages , the numbers of neurites were fewer than in control . 
 all of the ntfs increased the number of neurites , and the increase was more significant in the nt-4 group . 
 the number of nf-b and sp1 immunopositive cells was higher in retinas exposed to ages than in control . 
 all of the ntfs decreased the number of nf-b immunopositive cells but did not significantly affect sp1 expression . 
 these results demonstrate the potential of the ntfs as axoprotectants in ages exposed retinal neurons . 