diabetic retinopathy ( dr ) is a leading cause of blindness among working group in developed countries .
it is estimated that the number of diabetes patients will achieve 366 million by 2030 , the majority of which will be type 2 diabetes mellitus ( t2 dm ) . after initial diagnosis , over 80% of t2 dm patients
dr is a prototypical microvascular disorder characterized by microaneurysms , capillary nonperfusion , intraretinal hemorrhages , intraretinal microvascular abnormalities , and neovascularization [ 46 ] .
however , recent studies have identified neuroretinal abnormalities in diabetics , even before the evidence of visible microvascular changes [ 710 ] .
dr patients usually exhibit reduced responses in full - field and multifocal electroretinography , decreased blue - yellow color sensitivity , and contrast sensitivity before the microvascular lesions occurrence .
large amount of neuronal cells is detected to be damaged in the very early stage of the disease , while microvascular lesions are usually difficult to be identified by fundus photography or ffa , which would cause easily clinical missed diagnosis followed by worsen progression .
retinal neurodegeneration has also been identified in diabetic rodent models , which reproduces most aspects of the early stages of dr .
lewis rats with streptozotocin - induced type 1 diabetes have shown the most accelerated loss of retinal ganglion cells ( rgcs ) at 8 months after the onset of diabetes .
the spontaneous development of diabetes in ins2akita mice for 5 - 6 months also reveals the changes in neurosensory retina , including rgcs apoptosis as well as marked alterations to the morphology of surviving cells , reduction of cholinergic and dopaminergic amacrine cells , and a distinct thinning of the inner plexiform layer and the inner nuclear layer [ 1416 ] .
furthermore , morphologic alterations of astrocytes and microglial cells in the inner retina and impaired glutamate metabolism by mller cells are found in other rodents [ 1719 ] .
type 1 diabetic animal models were used in the majority of these researches , and to date retinal neural structural and functional studies of type 2 diabetic animal model are scarce .
the db / db mouse ( bks / db/ ) is a typical model of type 2 diabetes that exhibits hyperglycemia associated with obesity a few 48 weeks after birth .
physiological studies have revealed early inner retinal neuronal dysfunction including prolonged latencies of the oscillatory potentials and impaired b - wave at 24 weeks of age .
an increased apoptosis of rgcs and other retinal neurons was observed in 60 weeks old db / db mice .
however , the structural and functional features of inner retina on db / db mice during the early stage of diabetes remain unclear .
this study was designed to follow the development of neurosensory retina damage from both structural and functional aspects in the early stages of diabetes in db / db mice .
thirty - seven 8-week - old male db / db mice ( bks / db/ ) and 37 age - matched nondiabetic littermates ( bks / db+/+ ) were provided by the animal laboratory center of nanjing university and were kept in our spf - certified vivarium and exposed to a 12-hour light - dark cycle .
all experiments conformed to standard environmental conditions ( at room temperature of 23c and humidity of 60% ) . to assess the chronological sequence of the retinal abnormalities associated with diabetes , 6 diabetes db / db mice were compared with 6 age - matched nondiabetic mice at difference ages ( 12 , 16 , 20 , 24 , and 28 weeks , resp . ) .
visual functions were measured using pattern electroretinography ( perg ; roland consult , germany ) and in vivo retinal structures were visualized with spectral domain oct ( cirrus hd - oct , zeiss meditec , germany ) . at last ,
mice were anesthetized with intraperitoneal injection of a mixture containing 5% urethane and 10% chloralic hydras ( 1  :  1 ) ( 1020  mg  kg body weight ) .
all animal procedures were performed according to guide for the care and use of laboratory animals published by the institute for laboratory animal research .
, mice ensured to natural light - adapted state were weighted and anesthetized as above mentioned .
self - modified electrodes were made of acupuncture needles , welded to the signal - recorded wire . a recording electrode ( 0.25  mm diameter silver wire configured to a semicircular loop of 2  mm radius )
reference and ground electrodes were inserted under the skin of ipsilateral pars buccalis and tail , respectively .
the pupils were not dilated , and eyes were not refracted for the viewing distance since the mouse eye has a large depth of focus .
visual stimuli consisted of horizontal bars with a contrast of 99% at 0.05 cycles / degree spatial frequency , 1  hz temporal frequency .
perg waveforms consisted of two main waves : a positive wave with a peak latency of 90 to 100  ms ( p1 ) followed by a broad negative with peak latency in the range of 200 to 300  ms ( n1 ) .
after anesthetizing , mice were dilated with tropicamide 1% and underwent spectral domain oct ( cirrus hd - oct , zeiss meditec , germany ) as reported elsewhere [ 26 , 27 ] . in brief , mice were placed on the three - dimensional table .
the mouse eyeball was coated with viscoelastic material and covered with a coverslip to form a plano - concave lens .
then the 90  dpt noncontact slit lamp lens ( volk , superfield nc , volk optical inc . ,
macular cube scanning in the cirrus hd - oct was performed with the 512  128 scan pattern where a 6  6  mm area on the retina is scanned with 128 horizontal lines , each consisting of 512 a - scans per line with the scanning speed of 27000 a - scans per second . during the analysis procedure
then retinal thicknesses are averaged in nine retinal subfields which are an area of a 6  mm diameter circle centered on the mouse optic disk .
the eyes were dipped in ffa ( mixture of 95% ethyl alcohol , 10% neutral formalin , and glacial acetic acid , 17  :  2  :  1 ) for 1  min and fixed in 0.1  m buffer phosphate ( ph 7.4 ) containing 4% paraformaldehyde for 2 hours at 4c .
then , the anterior segment of the eye was dissected away , the vitreous cleared , and the eyecup was left for further fixation overnight .
serial sections 7  m thick were prepared for the assessment of retinal morphology , immunofluorescence staining , and tunel immunoreactivity .
after deparaffinization and rehydration , the sections were stained with hematoxylin and eosin ( h&e ) .
the measurements were taken at the regions of the retina ( at 480  m form ora serrata ) and were examined to ensure similar locations of measurements for all eyes .
image analyses of ten sections of each eye were used to quantify total retinal thickness , the thickness of the inner nuclear layer ( inl ) and outer nuclear layer ( onl ) .
images of h&e sections were captured with a microscope ( olympus ) using the program image j for quantification .
the terminal deoxynucleotidyl transferase - mediated biotinylated utp nick - end labeling ( tunel ) assay was performed with dapi ( 4,6-diamidino-2-phenylindole ) staining .
the prepared tissue sections were deparaffinized in xylene for 10  min and hydrated through a graded ethanol series .
the tunel assay was then carried out following the protocol described by the manufacturers ( in situ cell death detection ; roche molecular biochemicals ) . after staining retinal sections were mounted with fluorescence mounting medium and images
quantitative observations of retinal cells count from semithin section were defined as five hp fields ( 40  10 ) since 200  um up from the optic papilla .
after deparaffinization and rehydration , the sections were incubated with ionized calcium binding adaptor molecule 1 ( iba-1 ; dilution 1  :  150 , wako , neuss , germany ) and f4/80 ( m-300 ; dilution 1  :  200 , santa cruz ) at 48c for 2 days and then rinsed in pbs ( 3  1  hr ) .
then , the retina was incubated at 48c in sheep anti - mouse and anti - rabbit antibodies conjugated to fluorescein - isothiocyanate ( fitc ) ( boehringer mannheim gmbh , germany ) dilution 1  :  30 in 0.1% triton x-100 in 0.01  m pbs at ph 7.4 , for 3 days , and rinsed again .
the retina was mounted on a slide in glycerol diluted with pbs at a ratio of 1  :  9 .
whole retina thicknesses obtained from all oct devices were compared one to each other using unpaired student 's t - test .
measurements of blood glucose ( vein blood of tail using contour ts ) and body weight were conducted in diabetic and nondiabetic mice before visual function testing or they were killed for other experiments at each time point .
when the result of blood glucose value was beyond the upper limit of detection ( 33.3  mmol / l ) , we set 33.3  mmol / l as the statistical default .
there were significant statistic differences of body weight and blood glucose level between the two groups , and hyperglycemia in diabetic mice runs in parallel with a significant increase of weight .
p1 amplitudes , measured from the peak of the p1 wave to the trough of the n1 wave , were slightly reduced in diabetes mice at the age of 12 and 16 weeks , but they were significantly reduced in diabetes mice when compared with controls at the age of 20 , 24 , and 28 weeks ( figure 2(a ) ) . besides , a significant delay of p1 latency recorded in the diabetic mice at the ages of 24 and 28 weeks was observed when compared with the age - matched control mice ( figure 2(b ) ) .
these data showed abnormal perg responses in diabetic db / db mice for 20 weeks , which also suggested that rgcs dysfunction might have occurred in early period of db / db mice .
measurements of retinal thickness in diabetic and in nondiabetic mice at 20 , 24 , and 28 weeks are shown in figure 3 .
total retinal thickness ( measured from inner limiting membrane to bruch 's membrane ) was significantly decreased in diabetic mice in comparison with nondiabetic mice at 28 weeks .
furthermore , a reduction in onl was observed in diabetic mice in comparison with nondiabetic mice at 28 weeks .
the retinal thickness was measured by oct noninvasively in 7 diabetic db / db mice and 7 nondiabetic mice at 28 weeks .
one eye was selected at random in each mouse and only that eye was included in the analysis . as shown in table 1 ,
oct revealed a significant thinner in total retinal thickness of db / db mice than the control mice ( p < 0.01 ) .
specifically , the differences were significant also for all quadrants . moreover , both the inner and outer sectors thicknesses of superior as well as nasal quadrants in db / db mice were significantly thinner than nondiabetic mice . additionally , significant thinning occurred in inner sector of the temporal quadrant in diabetic mice compared with control mice , despite the fact that no significant difference was detected in the temporal quadrant between the two groups .
after examinations of perg , ffa were conducted which monitor the flow of a fluorescent dye through the retinal vasculature .
all db / db as well as control mice did not show any alternations corresponding to relevant vascular abnormalities at each endpoint .  
figure 4 shows the images of ffa from db / db and db/+ mice at 28 weeks and there was no visible neovascularization and cellular capillaries in the retina .
retinal sections were stained for dying photoreceptors by tunel staining to further determine the histological retina changes . a significant increase in tunel - positive immunofluorescence
was observed in diabetic mice in comparison with retinas from nondiabetic mice at 20 , 24 , and 28 weeks ( figure 5 ) .
tunel - positive cells were mainly localized in the rgcs layer , as well as the inl , implying the rgcs apoptosis which was also suggested by preg results . in addition , the number of apoptosis cells was increased from 1 - 2/hp at 20 weeks to 8 - 9/hp at 28 weeks ( figure 5 ) . to confirm the possible neuroinflammation in retinal neurodegeneration , iba-1-labeled microglia / macrophages and f4/80-labeled mature macrophages staining were performed . as shown in figure 6 , no positive expression of the two markers was observed in either 20-week - old diabetics or nondiabetics mice .
however , db / db mice at 24 and 28 weeks showed obvious fluorescence of positive iba-1 and f4/80 expression in rgcs layer and inl , while no specific immunofluorescence exhibited in the retina of control mice ( figure 6(a ) ) . moreover , positive f4/80 was also expressed in the onl of db / db mice ( figure 6(b ) ) .
these results confirmed the presence of iba-1 and f4/80 positive cells in rgcs layer and inl by the time of 24 weeks .
when compared with normal heterozygous littermates , the db / db ( lepr ) mice can be affected with hyperglycemia , hyperlipidemia , and accelerated degeneration of the retinal capillaries and pericytes .
it has been reported that 26-week - old db / db mice showed early features of dr , manifesting pericyte and endothelial cell loss , bruch 's membrane thickening , and increased blood flow in the retina [ 2931 ] .
however , rodent models can reproduce most aspects of the early stages of dr but fail to develop retinal neovascularization or other advanced lesions ( retinal hemorrhages and microaneurysms ) of retinopathy .
although cheung et al .   had reported that 15-month - old db / db mice showed increased density of retinal capillaries in the inner nuclear layer , in current study , we did not found any obvious microvascular changes using ffa in db / db mice alive for 28 weeks .
moreover , the reduction of retinal thickness in the db / db mice by using oct also supports that there was no retinal edema and/or retinal blood barrier damage .
the relatively short lifespan of the mice and thus shorter duration of diabetes ( most studies are < 1 year ) were probable reason for failing to observe neovascularization in these models .
therefore , the results of our study were supporting previous researches which implied that db / db mice developed capillary changes under microscope even in the early period of life span , but the lesion was not severe enough to cause the breakdown of retinal blood barrier . the results of perg presented in our study indicated that diabetic condition acts to be injurious events that lead to exacerbation of rgcs death .
perg signal depends on the structural and functional integrity of rgcs and will greatly reduce when rgcs were selectively degenerated after optic nerve lesion .
perg , therefore , is extensively used to probe rgc function in clinical and experimental models of diabetes and optic neuropathies [ 3436 ] and may be more sensitive to assess early damage of dr .
previous studies showed that perg impairment could be detected in diabetic patients without or with minimal signs of retinopathy .
this suggests that retina functional loss may occur in the early stage of this disease .
therefore , we used perg to evaluate the changes in rgcs function and we found that the p1 amplitudes significantly reduced at the follow - up examinations for 20 weeks in db / db mice ; then a significantly increased latency was found later .
all these results indicated that the deficit function of rgcs in db / db mice had certainly occurred .
it was reported that rgcs die in a compartmentalized manner such that the mechanisms responsible for death of the rgcs body and dendrites may be different from those responsible for axonal injury ; both of them compromised the rgcs dysfunction .
oct was performed as an indicator of progressive neural retinal pathology in animal models of retinal degeneration [ 38 , 39 ] . in the present study
, we found a significant reduction in overall retinal thickness as well as all four quadrants , depending on the oct scanning , in db / db mice when compared with control mice at the age of 28 weeks .
moreover , the results of h&e showed that total retinal thickness as well as onl ( at 480  m form ora serrata ) was significantly decreased in diabetic mice in comparison with nondiabetic mice at only 28 weeks , but there was no difference between the 2 groups at 20 and 24 weeks .
these results are mainly due to the apoptosis of the ganglion cell layer and photoreceptors . in a recent study , bogdanov et al .   reported that total retinal thickness in both central and peripheral retina was significantly decreased in db / db diabetic mice at 16 and 24 weeks , and a thinning in both onl and inl as well as a reduction in the number of cells in the gcl
unfortunately , in our study only the db / db mice at 28 weeks were measured by oct , with the original intention to validate the reduced perg response . whether there existed oct alternations before 28 weeks
so further studies are needed to determine the early changes of oct in db / db mice and evaluate the correlation between oct measured and histologically measured changes in retinal thickness following the life periods .
recent studies have approached a common view that cell apoptosis is the base of diabetic neurodegeneration in cytopathogenesis .
reportedly , various retinal neurocytes in early dr have been in apoptosis state , in particular the rgcs [ 4244 ] . accompanying cell apoptosis ,
inflammation has been considered to be an important pathogenesis in early stages of experimental dr , which has shown both of microglial and macrophages reaction represented during the alternations of functional and structural in retina [ 4547 ] . here , to further confirm the rgcs dysfunction determined by perg
previous study reported that 15-month - old db / db mice had increased apoptosis of rgcs and other cells in the neural retina . in our study , apoptotic rgcs were observed in 20-week - old db / db mice , consistent with the reduced perg amplitudes .
moreover , the number of apoptotic cells was increasing with age , accompanied with evident induction of iba-1 and f4/80 expression , both of which were considered as established marker for microglia / macrophages in the retina .
additionally , iba-1 and f4/80 positive cells as well as apoptosis cells were also found in the inl , which was implied to be affected by db / db mice .
all these findings indicated that db / db mice undergoing diabetes in 20 weeks or longer had developed neurodegeneration and neuroinflammation . in conclusion
, we demonstrated that functional and structural retinal neurodegeneration exactly occurred in db / db mice with type 2 diabetes mellitus as early as 20 weeks , preceding any detectable alternations of blood vessel visibly .
the retina lesions that develop in type 2 diabetes are similar to those that develop in type 1 diabetes , although the severity and/or incidence may be different .
thus , it is prominently important to trace the degenerated phases and change rule of diabetic nerve cells in the early stage of dr and timely block and reverse these cells ' degeneration .
these actions would play a significant role in prevention of early diabetic retinal neuropathy and nerve protection treatment , which are promising to be a potently effective target for the early intervention of diabetic retinopathy in future .
 
 purpose . to describe both the functional and pathological alternations in neurosensory retina in a murine model of spontaneous type 2 diabetes ( db / db mouse ) 
 . methods . 
 db / db ( bks / db/ ) mice and heterozygous littermates ( as control group ) at various ages ( 12 , 16 , 20 , 24 , and 28 weeks ) were inspected with pattern electroretinogram ( perg ) , fundus fluorescein angiography ( ffa ) , and optical coherence tomography ( oct ) . 
 histological markers of neuroinflammation ( iba-1 and f4/80 ) were evaluated by immunohistochemistry . 
 in addition , levels of retinal ganglion cell death were measured by terminal dutp nick - end labeling ( tunel ) . results . 
 significant alternations of perg responses and increased retinal ganglion cells ( rgcs ) apoptosis were observed in diabetic db / db mice for 20-week period when compared with control group . 
 iba-1 and f4/80 expression in microglia / macrophages became evidently for 24-week period , thus supporting the perg findings . furthermore , obvious thinning of nasal and dorsal retina in 28-week - old db / db mice was also revealed by oct . 
 no visible retinal microvascular changes were detected by ffa throughout the experiments on db / db mice . 
 conclusions . 
 diabetic retina underwent neurodegenerative changes in db / db mice , which happened at retinal ganglion cell layer and inner nuclear layer . 
 but there was no obvious abnormality in retinal vasculature on db / db mice . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion