Neural degeneration in the retina of the streptozotocin-induced type 1 diabetes model.
ABSTRACT Diabetic retinopathy, a vision-threatening disease, has been regarded as a vascular disorder. However, impaired oscillatory potentials (OPs) in the electroretinogram (ERG) and visual dysfunction are recorded before severe vascular lesions appear. Here, we review the molecular mechanisms underlying the retinal neural degeneration observed in the streptozotocin-(STZ-) induced type 1 diabetes model. The renin-angiotensin system (RAS) and reactive oxygen species (ROS) both cause OP impairment and reduced levels of synaptophysin, a synaptic vesicle protein for neurotransmitter release, most likely through excessive protein degradation by the ubiquitin-proteasome system. ROS also decrease brain-derived neurotrophic factor (BDNF) and inner retinal neuronal cells. The influence of both RAS and ROS on synaptophysin suggests that RAS-ROS crosstalk occurs in the diabetic retina. Therefore, suppressors of RAS or ROS, such as angiotensin II type 1 receptor blockers or the antioxidant lutein, respectively, are potential candidates for neuroprotective and preventive therapies to improve the visual prognosis.
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ABSTRACT: To test the hypothesis that increased expression of proinflammatory cytokine high-mobility group box-1 (HMGB1) in epiretinal membranes and vitreous fluid from patients with proliferative diabetic retinopathy and in retinas of diabetic rats plays a pathogenetic role in mediating diabetes-induced retinal neuropathy. Retinas of 1-month diabetic rats and HMGB1 intravitreally injected normal rats were studied using Western blot analysis, RT-PCR and glutamate assay. In addition, we studied the effect of the HMGB1 inhibitor glycyrrhizin on diabetes-induced biochemical changes in the retina. Diabetes and intravitreal injection of HMGB1 in normal rats induced significant upregulation of HMGB1 protein and mRNA, activated extracellular signal-regulated kinase 1 and 2 (ERK1/2), cleaved caspase-3 and glutamate; and significant downregulation of synaptophysin, tyrosine hydroxylase, glutamine synthetase, and glyoxalase 1. Constant glycyrrhizin intake from the onset of diabetes did not affect the metabolic status of the diabetic rats, but it significantly attenuated diabetes-induced upregulation of HMGB1 protein and mRNA, activated ERK1/2, cleaved caspase-3, and glutamate. In the glycyrrhizin-fed diabetic rats, the decrease in synaptophysin, tyrosine hydroxylase, and glyoxalase 1 caused by diabetes was significantly attenuated. These findings suggest that early retinal neuropathy of diabetes involves upregulated expression of HMGB1 and can be ameliorated by inhibition of HMGB1.Mediators of Inflammation 01/2014; 2014:746415. · 3.88 Impact Factor
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ABSTRACT: Retinal neurodegeneration is a key component of diabetic retinopathy (DR), although the detailed neuronal damage remains ill-defined. Recent evidence suggests that in addition to amacrine and ganglion cell, diabetes may also impact on other retinal neurons. In this study, we examined retinal degenerative changes in Ins2Akita diabetic mice. In scotopic electroretinograms (ERG), b-wave and oscillatory potentials were severely impaired in 9-month old Ins2Akita mice. Despite no obvious pathology in fundoscopic examination, optical coherence tomography (OCT) revealed a progressive thinning of the retina from 3 months onwards. Cone but not rod photoreceptor loss was observed in 3-month-old diabetic mice. Severe impairment of synaptic connectivity at the outer plexiform layer (OPL) was detected in 9-month old Ins2Akita mice. Specifically, photoreceptor presynaptic ribbons were reduced by 25% and postsynaptic boutons by 70%, although the density of horizontal, rod- and cone-bipolar cells remained similar to non-diabetic controls. Significant reductions in GABAergic and glycinergic amacrine cells and Brn3a+ retinal ganglion cells were also observed in 9-month old Ins2Akita mice. In conclusion, the Ins2Akita mouse develops cone photoreceptor degeneration and the impairment of synaptic connectivity at the OPL, predominately resulting from the loss of postsynaptic terminal boutons. Our findings suggest that the Ins2Akita mouse is a good model to study diabetic retinal neuropathy.PLoS ONE 01/2014; 9(5):e97970. · 3.53 Impact Factor
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ABSTRACT: Insulin receptor substrate-2 (Irs2) mediates peripheral insulin action and is essential for retinal health. Previous investigations have reported severe photoreceptor degeneration and abnormal visual function in Irs2-deficient mice. However, molecular changes in the Irs2-/- mouse retina have not been described. In this study, we examined retinal degenerative changes in neuronal and glial cells of adult (9- and 12-week old) Irs2-/- mice by immunohistochemistry. 9-week old Irs2-/- mice showed significant thinning of outer retinal layers, concomitant to Müller and microglial cell activation. Photoreceptor cells displayed different signs of degeneration, such as outer/inner segment atrophy, redistribution of rod- and cone-opsins and spatial disorganization of cone cells. This was accompanied by synaptic changes at the outer plexiform layer, including the retraction of rod-spherules, reduction of photoreceptor synaptic ribbons and synaptic remodeling in second order neurons (i.e. loss and sprouting of dendritic processes in rod bipolar and horizontal cells). By 12 weeks of age, the thickness of inner retinal layers was severely affected. Although inner plexiform layer stratification remained unchanged at this stage, rod bipolar cell axon terminals were significantly depleted. Significant loss of Brn3a+ retinal ganglion cells occurred in 12-week old Irs2-/- mice, in contrast to younger ages. Adult Irs2-/- mice showed clear hallmarks of neurodegeneration and disruption of the inner retina with increasing age. Pharmacological stimulation of Irs2 signaling pathway may provide additional neuroprotection in certain degenerative retinopathies.Experimental Eye Research 01/2014; · 3.03 Impact Factor
Hindawi Publishing Corporation
Experimental Diabetes Research
Volume 2011, Article ID 108328, 7 pages
theStreptozotocin-InducedType1 Diabetes Model
Yoko Ozawa,1,2Toshihide Kurihara,1,2Mariko Sasaki,1,2NorimitsuBan,1,2KenyaYuki,1,2
ShunsukeKubota,1,2and Kazuo Tsubota2
1Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-Ku, Tokyo 160-8582, Japan
2Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-Ku, Tokyo 160-8582, Japan
Correspondence should be addressed to Yoko Ozawa, firstname.lastname@example.org
Received 12 July 2011; Accepted 26 August 2011
Academic Editor: N. Cameron
Copyright © 2011 Yoko Ozawa et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Diabetic retinopathy, a vision-threatening disease, has been regarded as a vascular disorder. However, impaired oscillatory
potentials (OPs) in the electroretinogram (ERG) and visual dysfunction are recorded before severe vascular lesions appear. Here,
we review the molecular mechanisms underlying the retinal neural degeneration observed in the streptozotocin-(STZ-) induced
type 1 diabetes model. The renin-angiotensin system (RAS) and reactive oxygen species (ROS) both cause OP impairment and
reduced levels of synaptophysin, a synaptic vesicle protein for neurotransmitter release, most likely through excessive protein
degradation by the ubiquitin-proteasome system. ROS also decrease brain-derived neurotrophic factor (BDNF) and inner retinal
Therefore, suppressors of RAS or ROS, such as angiotensin II type 1 receptor blockers or the antioxidant lutein, respectively, are
potential candidates for neuroprotective and preventive therapies to improve the visual prognosis.
Diabetic retinopathy, a vision-threatening disease, has long
according to the proliferative status of the retinal vasculature
. The disorder involves hemorrhage, vascular obliteration,
and the resulting neovascularization; these events subse-
quently cause fibrovascular proliferation and then retinal
detachment, all of which can secondarily cause retinal neu-
ral degeneration. However, impaired visual function is re-
over, the atrophic appearance and dysfunction of the neural
retina continue to worsen clinically after the vascular lesions
become quiescent. Therefore, the diabetes-induced retinal
neural degeneration may progress independently of the vas-
On the other hand, current treatments for diabetic ret-
inopathy are mostly intended to regulate vascular changes
mediated by the action of vascular endothelial growth factor
(VEGF), by laser treatment to attenuate the hypoxic retinal
cells that produce VEGF, and by anti-VEGF drugs, as well
as blood glucose level. The future generation of therapies
is expected to target neural tissue and to elicit a better
visual prognosis. In the retina, a light stimulus activates
the phototransduction pathway in the photoreceptor cells
which span the outer layer of the retina, and is converted
into electric signals which are then processed through the
synaptic network system in the inner layer of the retina
to finally transmit the signals to the brain to form visual
function. This series of electric reactions in the retina can
be recorded as an electroretinogram (ERG), which is used
to evaluate retinal function objectively, both clinically in
humans and experimentally in animals [2, 3]. Diabetes-
related changes appear in the oscillatory potentials (OPs) in
the ERG of human patients; these changes represent inner
reported in the 1960s [2–4]. However, the molecular mech-
anisms underlying the diabetes-induced neural degeneration
and dysfunction of the retina are still not well understood,
and elucidating them is a current hot topic in the field of
2 Experimental Diabetes Research
diabetes research. In this paper, we review the molecular
mechanisms of neural degeneration revealed in the retina of
the streptozotocin-(STZ-) induced type 1 diabetes models.
Streptozotocin is a glucosamine-nitrosourea compound that
was originally identified as an antibiotic, but is also known
to have an anticancer effect. It is cytotoxic to pancreatic beta
cells after being transported through glucose transporter 2
model animals. Experimental diabetes can be induced in
both rats and mice by the intraperitoneal injection of STZ
[6–9]. In the mouse model, the blood glucose level reaches
over 500mg/dL after 1 month of diabetes, compared with
about 100–120mg/dL in control mice [6–9].
Although the above method is often used to make a
diabetes model, there do not appear to be pathological neo-
vascularization caused by vascular obliteration in these an-
imals, which is typical finding in severe diabetic patients.
However, this model shows apoptosis of the inner retinal
neurons, such as ganglion cells and amacrine cells, and the
can all contribute to the inner retinal dysfunction detected as
OP changes in the ERG. Interestingly, VEGF, which is found
at a high level in the diabetic retina [7, 12–15], is generally
protective for neurons. Thus, the current treatments, which
target the vascular impairments and VEGF, may not be
effective for the neural pathogenesis. To develop new treat-
ment approaches, it is therefore important to elucidate the
molecular mechanisms underlying the neurodegenerative
changes in diabetic retinopathy. For these studies, the STZ-
induced model is useful, because it mimics the diabetes-
induced OP change in the ERG observed in humans [6, 8,
In the following sections, we describe the molecular
mechanisms of neural degeneration in the retina of the STZ-
induced diabetes model, focusing on the renin-angiotensin
system (RAS) [6, 7, 18, 19] and oxidative stress [8, 20, 21].
3.The RAS inthe Neural Retinaof Diabetes
The RAS was originally recognized as a regulator of the sys-
temic blood pressure. One of its principal effectors, angi-
otensin II, was originally reported to be converted from a
precursor peptide, angiotensinogen, which is produced in
the liver and then sequentially converted into angiotensin II
by enzymes, renin in the kidney and angiotensin-converting
enzyme (ACE) in the lung, after it enters the circulation.
Angiotensin II promotes vessel contraction to elevate the
blood pressure, and this is known as systemic RAS. On
the other hand, recent studies have revealed that all the
RAS components can be generated within a given tissue
or organ. This is called tissue RAS and is found in the
heart, blood vessels, kidney, adrenal gland, pancreas, central
nervous system, reproductive system, and lymphatic and
adipose tissue , as well as the retina [23, 24]. Tissue RAS
can be regulated independently of the systemic RAS .
Angiotensin II has long been known to have a role in dia-
betic complications, such as nephropathy and retinopathy,
with evidence indicating that it affects the vascular system
of each organ or tissue [7, 18, 25] via tissue RAS, in a
paracrine fashion [25, 26]. Thus, the common mechanism
of these complications has been believed to be the effects of
angiotensin II on the vascular system [7, 18, 25]. However,
recent studies using the STZ-induced diabetes model have
begun to reveal the influences of angiotensin II on neural
cells [6, 27].
3.1. Influence of the RAS on Synapses. In STZ-induced dia-
betes model mice, angiotensin II and its type 1 receptor
(AT1R) are upregulated in the retina [6, 7]. Interestingly,
AT1R is abundantly coexpressed with synaptophysin, a syn-
aptic vesicle protein critical for neurotransmitter release, in
the inner layer of the normal mouse retina (Figure 1) ,
consistent with AT1R signaling role in modulating synaptic
activity in the central nervous system .
Kurihara et al. analyzed the role of AT1R in the diabetic
retina by administering the AT1R blockers telmisartan and
valsartan to STZ-induced diabetes model mice . AT1R
blockers suppressed the OP change in this model that
reflects impaired inner retinal function. Therefore, the visual
functional impairment in this STZ-induced diabetes model
reduced synaptophysin expression in a posttranscriptional
fashion in the retina of the diabetes model mice. This ob-
servation is consistent with the fact that the visual dysfunc-
tion in this model originates in the inner layer of the retina,
where the synaptic network system is located.
3.2. Direct Influence of the RAS on Neurons. The molecular
mechanism underlying the decrease in synaptophysin pro-
tein was analyzed using a neuronal cell line originated from a
pheochromocytoma, PC12D. In this cell line, AT1R is coex-
pressed with synaptophysin which is posttranscriptionally
decreased by adding angiotensin II to the culture medium
. This decrease is inhibited by both AT1R blockers and
AT1R knockdown using shRNA. Downstream of AT1R, ERK
is activated, which was also confirmed using inhibitors and
shRNA. This posttranscriptional reduction of synaptophysin
protein results from its excessive degradation through the
experiment showed that angiotensin II has a direct effect
on neuronal cells. ERK is activated in the diabetic retina
in vivo, suggesting that the same pathway is involved in
the neuronal pathogenesis in the retina of the STZ-induced
diabetes model. In another report on the effect of an ACE
inhibitor in these model animals, it was concluded that the
drug inhibited retinal degeneration by reducing the blood
pressure and blood glucose level . However, the in vivo
results of Kurihara et al. were obtained under conditions in
which the blood pressure and blood glucose levels did not
change, further supporting the idea that RAS has a direct
effect on neurons in vivo.
The role of angiotensin II in diabetic retinopathy has
tan Trials (DIRECTs) [31, 32], in which the effect of an
Experimental Diabetes Research3
Figure 1: Expression of angiotensin II type 1 receptor and syn-
aptophysin in the mouse retina. Expression of AT1R (green) in the
normal retina (A–C). The boxed area in A is magnified in B and C
(merged in C), and the boxed areas in C are magnified in the insets
of C. AT1R is coexpressed with synaptophysin (pink), a presynaptic
vesicle protein, in the IPL (arrow in the upper inset of C) and OPL
(arrows in B and the lower inset of C). AT1R, angiotensin II type 1
receptor; GCL, ganglion cell layer; IPL, inner plexiform layer; INL,
inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear
AT1R blocker on the incidence and progression of diabetic
Retinopathy Study (ETDRS) scale. Although this scale has
been established to evaluate vascular lesions of diabetic
retinopathy , the effect of the drug may also involve
improving the retinal neural condition taking into account
the data from the animal model .
3.3. RAS and UPS. Angiotensin II induces pathological UPS
activity in various tissues, including vascular smooth mus-
cle . Angiotensin II promotes the expression of atrogin-
1, an E3 ubiquitin ligase selective for skeletal muscle wast-
ing . Notably, another animal model of retinal tissue
inflammation, endotoxin-induced uveitis and retinitis, in
ges , shows the involvement of UPS-mediated exces-
sivedegradation ofrhodopsin, avisualsubstance,intheneu-
ronal dysfunction of the retina [36, 37]. Given that the
RAS contributes to various kinds of diabetic complications,
the UPS may have important roles in the diabetes-induced
pathogenesis, a possibility meriting further study.
the Neural Retinaof Diabetes
Oxidative stress is another key modulator of diabetic com-
plications [8, 20, 38]. Reactive oxygen species (ROS) can be
produced in mitochondria through the electron transport
chain [39, 40]. Keeping the levels of mitochondrial ROS
at normal levels by pharmacological method prevents the
activation of protein kinase C, the formation of advanced
glycation end products, sorbitol accumulation, and NF-κB
activation, all of which are coupled with diabetes-induced
vascular endothelial cell damage . In this section, we in-
troduce the mechanism of oxidative stress-induced neural
degeneration observed in the retina of the STZ-induced dia-
betes model .
4.1. Influence of Oxidative Stress on Synapses. The retinal
neuronal cells are influenced by ROS: in the STZ-induced
diabetes model mice, both the decrease in synaptophysin
protein and the ERG impairment are suppressed under the
constant administration of an antioxidant, lutein, which
suppresses the local ROS (Figure 2) and ERK activation in
the diabetic retina . This influence, shown by Sasaki et
al., is observed as early as 1 month from diabetic onset, the
same time as the RAS influence reported by Kurihara et al.
Since lutein also did not change the blood glucose level in
this analysis, but reduced the ROS level in the retina with
diabetes, the effect of lutein in the retina was, at least in part,
through the reduction of the diabetes-induced local ROS.
Another influence of ROS related to neuronal activity
is exerted on the expression of brain-derived neurotrophic
factor (BDNF). This factor regulates axonal growth and
synaptic activity as well as neuronal survival . The level
of BDNF in the diabetic retina is decreased [8, 42], but lutein
treatment prevents this reduction . The BDNF level is
regulated by neuronal synaptic activity , suggesting that
the mechanism of its preservation in the retina of diabetic
mice by lutein administration may involve the preservation
of synaptophysin protein and the subsequent protection of
neuronal synaptic activity.
In addition to promoting BDNF expression , synap-
tic activity promotes neuronal survival through depolar-
ization of the cell membrane and increasing the level of
intracellular calcium in the neuronal cells . Therefore,
the synaptic change observed in the diabetic retina affects,
at least in part, retinal neuronal survival and visual function.
Sasaki et al. showed that the thickness of the inner layer of
the retina, including the retinal ganglion cells and amacrine
cells, in the diabetes model mice is reduced 4 months
after diabetes onset , consistent with the previous report
showing similar changes in the model rats 7.5 months after
diabetes onset . However, in lutein-administered mice,
in which the synaptophysin and BDNF levels are preserved
after 1 month of diabetes, the thickness is preserved, and
the neuronal cells survive by constant treatment . Thus,
the antioxidative treatment by lutein appears to protect the
neuronal cells from apoptosis.
The antioxidant, lutein, is presently being studied for
its preventive effects on the progression of age-related
macular degeneration (AMD), a vision-threatening disease,
as a micronutrient supplement. It is a yellow pigment and
can filter blue light, which has a high energy level that is
toxic to the retina. However, interestingly, the preventive
effect of lutein observed in the retina of the STZ-induced
diabetes model occurs by the ROS reduction rather than
the filtering of light energy. Moreover, the fact that lutein is
physiologically delivered to the retinal neurons  suggests
that it might act directly in the retina, and not only as an
antioxidant. Further studies aimed at elucidating whether
lutein’s effects involve pathways other than the antioxidative
pathway should be performed.
4 Experimental Diabetes Research
Figure 2: Oxidative stress in the retina of the STZ-induced diabetes model mouse. Dihydroethidium (DHE) indicates ROS in the retina (a).
The level of diabetes-induced ROS in the retina is decreased by constant lutein treatment. Fluorescence intensity in the INL relative to that
of nondiabetic mice was measured by the Image J program (b). DHE, dihydroethidium; ROS, reactive oxygen species; GCL, ganglion cell
layer; IPL, inner plexiform layer; INL, inner nuclear layer (original copyright; , Figure 1, reproduced with the kind permission of Springer
Science + Business Media.)
4.2. Other Influences of Oxidative Stress in the Retina. The
influence of diabetes on neural retinal cells has also been
analyzed from other viewpoints. Under diabetic conditions,
the increase in superoxide anions in the neural retina leads to
a decreased bioavailability of nitric oxide which is originally
induced in the retina for tissue protection; the decrease in
nitric oxide action increases the formation of peroxynitrite,
factor, nerve growth factor (NGF), and neuronal survival
M¨ uller glial cells, which contribute to the homeostasis
of the retina, are also damaged by oxidative stress [47, 48].
These cells originate from a common retinal progenitor cell
the damage of these cells can also influence visual function.
The influences of oxidative stress on M¨ uller glial cells involve
the downregulation of a potassium channel, Kir4.1, a water
channel, aquaporin 4 , and matrix metalloproteinase-7
(MMP-7) which converts a toxic factor for neurons, pro-
nerve growth factor (pro-NGF), to a neuroprotective fac-
tor, NGF . Therefore, there are multiple pathways down-
stream of oxidative stress that can cause retinal neural cell
damage and decrease visual function.
Synaptophysin protein is decreased in the retina of the
STZ-induced diabetes model through both the AT1R-ERK
axis and the ROS-ERK axis, suggesting the involvement
of cross talk between angiotensin II and ROS signals in
the diabetic retina (Figure 3). Angiotensin II can activate
nicotinamide adenine dinucleotide phosphate (NAD(P)H)
oxidase via AT1R stimulation and produces ROS, as shown
in the pathogenesis of atherosclerosis [49–51]. Moreover,
angiotensin 1-7, which are cleaved from angiotensin II and
act as its negative regulators, are reduced in the retina with
diabetes , and their overexpression reduces diabetes-
induced oxidative stress in the retina, suggesting that their
reduction might also be related to the cross talk.
Indeed, the effect of angiotensin II on the retinal leu-
kostasis in diabetes occurs at least partly through NAD(P)H
Experimental Diabetes Research5
(a synaptic vesicle protein)
synaptic network activity
Visual function impairment
Figure 3: Model of retinal neural degeneration and visual im-
pairment in the STZ-induced diabetes model mouse. Cross-talk
between AT1R signaling and ROS is involved in the neural degen-
eration of the diabetic retina. Downstream of both these effectors,
ERK activation reduces synaptophysin, while ROS also decrease
BDNF. These changes impair visual function most probably
through synaptic network abnormalities and neuronal apoptosis.
AT1R, angiotensin II type 1 receptor; ROS, reactive oxygen species;
BDNF, brain-derived neurotrophic factor.
oxidase-related ROS generation, as shown using apocynin,
an antioxidant . The ROS generated downstream of
AT1R further upregulate multiple inflammatory cytokines,
such as TNF-α, IL1-β, and IFN-γ, which further induce ROS
. Collectively, these findings support the idea that RAS-
ROS cross talk has a large role in the pathogenesis of the
diabetes-induced degeneration of neural tissue.
Another outcome of the cross talk in the diabetic retina
may be the expression of VEGF. M¨ uller glial cells are the
responsible cells for VEGF induction in the diabetic retina,
and this induction is regulated by hypoxia-inducible factor-
1α (HIF-1α) , which can be activated by oxidative stress
. VEGF induction in the diabetic retina is also regulated
Further studies are required to assess the contribution of
the RAS-ROS interaction in the diabetic retinal degeneration
that leads to impaired visual function and its possibility as a
therapeutic target for this impairment.
Retinal neural cell loss is involved as one of the main causes
for visual deficits in diabetes, and given that the retinal neu-
ral tissue eventually regulates diabetes-induced vascular dis-
orders , it is essential to understand the molecular
mechanisms underlying the pathology in the retinal neural
cells to establish the next generation of therapies. To the best
antioxidants are the most promising candidates for future
neuronal cell survival and normalizing the microenviron-
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