(Investigative Ophthalmology and Visual Science. 2000;41:4055-4058.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Polyol Metabolism of Retrograde Axonal Transport in Diabetic Rat Large Optic Nerve Fiber
Masanori Ino-ue1,
Lixin Zhang1,
Hiroaki Naka2,
Hiroshi Kuriyama2 and
Misao Yamamoto1,3
1 From the Department of Ophthalmology, Kobe University, School of Medicine;
2 Research Laboratories, Senju Pharmaceutical Co. Ltd., Kobe; and the
3 Ophthalmic Division, Kobe Children’s Hospital, Japan.
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Abstract
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PURPOSE. The role of the polyol pathway metabolism in progressive impairment of
retrograde axonal transport was evaluated in the optic nerve of rats
with streptozotocin-induced diabetes.
METHODS. Rats with streptozotocin-induced diabetes received a low (3 mg/kg body
weight) or high dose (10 mg/kg body weight) of oral aldose reductase
inhibitor (ARI). At 1 and 3 months after induction of diabetes,
Fluoro-Gold (FG, Chemicon, Temecula, CA) was injected into the dorsal
lateral geniculate nucleus. Percentages of FG–labeled large, medium,
and small retinal ganglion cells (RGCs) per total population were
calculated in the retinas of ARI-treated diabetic, untreated diabetic,
and normal control rats.
RESULTS. Mean percentages of FG–labeled large RGCs per total population were
significantly decreased in nontreated diabetic rats compared with
control animals at 1 month of induced diabetes. This decrease in FG
labeling was not observed in both the low- and high-dose ARI-treated
diabetic rats. At 3 months of induced diabetes, FG labeling of both
large and medium RGCs was significantly decreased. This decrease was
completely ameliorated by high-dose ARI treatment.
CONCLUSIONS. These results indicate that diabetes affects retrograde axonal
transport progressively through selective impairment of RGCs and that
the polyol pathway metabolism is involved in such
impairment.
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Introduction
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Structural analyses of peripheral nerves in humans and
experimental animals with diabetes have shown that the main lesions
involve axonal atrophy and axoglial dysjunction.1
In the
diabetic optic nerve, a significant reduction in mean myelinated fiber
size has also been reported.2
3
Furthermore, retinal
ganglion cells (RGCs) and their proximal axons show development of
neuroaxonal dystrophy characterized by tubulovesicular changes,
neurofilament accumulation, and lamellar membrane profile
formation.2
These dystrophic changes were accompanied by
abnormalities in electrophysiological test results, suggesting
subclinical involvement of the optic nerve in diabetes.2
3
In the nervous system, retrograde axonal transport of nerve growth
factor (NGF) from target organs to neuronal cell bodies is required for
normal functioning.4
Reduction in retrograde transport of
NGF in the sciatic nerve of diabetic rats precedes the development of
distal axonopathy.1
5
Most studies regarding nerve
dysfunction in diabetes focus on the peripheral and autonomic nervous
system. Previous studies have also demonstrated retrograde axonal
transport impairment and reduction in the cross-sectional size of large
optic nerve fibers in diabetic rats.3
6
In the present
study, we evaluated the impairment of retrograde axonal transport in
RGCs of diabetic rats in relation to the duration of diabetes. The
effect of treatment with aldose reductase inhibitor (ARI;
2-[4-{4,5,7,-trifluorobenzothiazol-2-yl}
methyl-3-oxo-3,4-dihydro-2H-1.4-benzothiazin-2-yl] acetic acid) on the
retrograde axonal transport of the optic nerve in diabetic rats was
also evaluated.
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Materials and Methods
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Induction of Diabetes and ARI Treatment
Six-week-old male Wistar albino rats (Japan Clea, Osaka,
Japan), each weighing approximately 200 g, were maintained
in a room with an ambient temperature of 23°C. Rats were treated in
accordance with the Guidelines for Animal Experimentation at Kobe
University School of Medicine and in accordance with the ARVO Statement
for the Use of Animals in Ophthalmic and Vision Research.
Diabetes was induced by an intraperitoneal injection of streptozotocin
(Sigma, St. Louis, MO) dissolved in 0.01 M citrate (pH 4.5) at a dosage
of 70 mg/kg body weight. Age-matched normal Wistar rats served as
control subjects. Rats with blood glucose levels of more than 400 mg/dl
were used in the study. The diabetic rats were randomly divided into
ARI-treated and ARI-untreated groups. An oral daily low (3 mg/kg body
weight [BW]; n = 16) or high (10 mg/kg BW; n =
16) dose of ARI (SG-210) suspended in 0.5% carboxymethyl cellulose
(CMC) solution was administered to the diabetic rats. The ARI-untreated
(n = 18) and normal control rats (n = 16)
received CMC solution without ARI for the same duration. Body weights
and blood glucose levels were monitored at 2-week intervals throughout
the experiment.
Retrograde Fluorescent Labeling of RGCs
One and 3 months after induction of diabetes, rats treated with
ARI (n = 8 per dosage group per interval) were subjected to
retrograde fluorescence labeling. The ARI-untreated (n = 9
per interval) and normal control (n = 8 per interval) groups
were also subjected to retrograde fluorescence labeling at the same
time intervals. Retrograde fluorescence labeling of RGCs was performed
as described previously.6
Briefly, rats were anesthestized
with an intraperitoneal injection of 5% pentobarbital sodium (0.5
ml/kg BW). Glass micropipettes (20 µm diameter) were loaded with 4%
Fluoro-Gold (FG; Chemicon, Temecula, CA) dissolved in distilled water,
and stereotaxically lowered into the dorsal lateral geniculate nuclei
(dLGN). An arbitrary volume of dye was injected iontophoretically with
a high-voltage current source and a 3-µA charge applied
intermittently (7 seconds on, 7 seconds off) for 10 minutes. For
adequate observation of FG-labeled RGCs, rats were killed 72 hours
after FG injection. Eyes in rats showing adequate labeling of the
contralateral dLGN without involvement of neighboring nuclei were
enucleated. Retinas were separated from the pigmented epithelium and
mounted vitreal side up on a nonfluorescent gelatin-coated glass slide.
To visualize the distribution of FG-labeled RGCs in the entire retina,
specimens were viewed by fluorescence microscopy with a UV filter
system (main excitation wavelength: 360 nm), and photographed
(Optiphotograph-EF; Nikon, Tokyo, Japan).
Estimation of FG-Labeled RGCs
Photographic images of the entire retina at 1 and 3 months after
induction of diabetes were saved and converted into Macintosh tagged
information format (TIF) files and then exported to the public domain
NIH Image 1.44 program for further analysis on a Macintosh computer
(Apple Computer, Cupertino, CA). With the imaging program, soma sizes
of each RGC were traced, measured, and classified into three types:
large, more than 20 µm; medium, 16 to approximately 20 µm; and
small, 15 µm or less.6
The FG-labeled RGCs were counted
and classified by two reviewers (LX, HN) in a masked fashion. Because
of the variations in the actual numbers of labeled RGCs, mean
percentage values of labeled cells per total population for each RGC
type were calculated and used for statistical analysis. Representative
photomicrographs of control, untreated diabetic, low-dose ARI-treated
diabetic, and high-dose ARI-treated diabetic retinas at the 3-month
interval are shown in Figure 1
.
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Figure 1. Retinal photomicrographs demonstrating FG labeling of RGCs 3 months
after induction of diabetes. (A) Control, (B)
untreated diabetic, (C) low-dose ARI-treated diabetic, and
(D) high-dose ARI-treated diabetic retinas. FG-labeled RGCs
demonstrated intense gold fluorescence within the cell bodies. The
FG-labeled large and medium RGCs are indicated by large and
small arrows, respectively. The
arrowheads indicate small RGCs. The number of FG-labeled
large and medium RGCs was decreased in the untreated diabetic rat
retina (B) compared with the control (A). There
was an observable increase in the number of FG-labeled large and medium
RGCs in the ARI-treated diabetic retinas (C, D)
compared with the untreated diabetic retina (B). Scale bar,
20 µm.
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Statistical Analysis
The percentage of each type of FG-labeled RGC per total population
in the control, ARI-treated, and ARI-untreated diabetic groups was
analyzed with Student’s t-test. P < 0.05
was considered significant.
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Results
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Diabetic State
Body weights of the ARI-treated and ARI-untreated diabetic rats
were significantly reduced throughout the experimental period, whereas
body weights of the control rats increased progressively (Table 1)
. Blood glucose concentration levels were significantly
elevated in the ARI-treated and -untreated diabetic rats compared with
control animals (Table 1)
.
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Table 1. Body Weight and Blood Glucose Levels in ARI-Treated Diabetic,
ARI-Untreated Diabetic, and Normal Control Rats
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FG Labeling of RGCs at 1 Month
Labeling of RGCs was highest 72 hours after FG injection. One
month after induction of diabetes, the mean percentage of FG-labeled
RGCs per total population in control subjects was calculated as 0.67%
± 0.18% for large, 8.20% ± 2.40% for medium, and 91.34% ± 2.39%
for small RGCs (Fig. 2)
. In ARI-untreated diabetic rat retinas, the mean percentage of large
RGCs was 0.31% ± 0.15%, representing a significant decrease compared
with age-matched control retinas (P = 0.0126). No
significant decrease in the mean percentage of medium and small RGCs
was observed between diabetic and controls retinas. The mean percentage
values of FG-labeled large RGCs in both low- and high-dose ARI-treated
diabetic rats was significantly improved (P = 0.0275,
P = 0.0028).
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Figure 2. Proportion of FG-labeled RGCs in ARI-treated diabetic, ARI-untreated
diabetic and normal control groups after 1 month of induced diabetes.
White: control group; black:
ARI-untreated diabetic group; gray: low-dose (3 mg/kg
BW) ARI-treated diabetic group; hatching: high-dose (10
mg/kg BW) ARI-treated diabetic group. Data are means ± SD.
*P < 0.05 versus control; ** P < 0.05 versus untreated diabetic rats.
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FG-Labeling of RGCs at 3 Months
The mean percentages of FG-labeled large (P <
0.0001) and medium (P = 0.0174) RGCs calculated from
the retinal photographs were significantly decreased in diabetic rats
compared with control subjects (Fig. 3)
. High-dose ARI treatment improved the decrease in mean percentage
values of FG-labeled large and medium RGCs to almost normal
(P = 0.0368). However, treatment with low-dose ARI only
partially improved the percentage values of FG-labeled large RGCs
(P < 0.0001) and had no effect in mean percentage
values of medium RGCs.
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Figure 3. The proportion of FG-labeled RGCs in ARI-treated diabetic,
ARI-untreated diabetic, and normal control groups after 3 months of
induced diabetes. All representations are as in Figure 2
.
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After observation by fluorescence microscopy, the retinal sections were
Nissl stained and viewed again. Significant cell loss and degeneration
were not observed in either ARI-treated or -untreated diabetic rat
retinas (data not shown).
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Discussion
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The pathophysiology of diabetic neuropathies has been investigated
extensively.1
The polyol or sorbitol hypothesis is the
most widely cited pathometabolic mechanism for diabetic neuropathy. The
hypothesis relates to accumulation of sorbitol in the nerves, with a
compensatory reduction in myo-inositol content, which leads to
impairment in nerve function, and ultimately, to structural
neuropathy.1
7
The morphologic structure of the neuron
complements its capability of transmitting impulses over long
distances. The anterograde axonal transport system is responsible for
transporting proteins associated with axonal structure and synaptic
transmitter function to the axon and its terminals. In the opposite
direction is the retrograde axonal transport system, which carries
neurotrophic factors that influence steady state activities in the cell
body.4
Because neurotrophic factors are known to promote
survival, maintenance, and regeneration of neurons, their role in
diabetic neuropathy has been given consideration.1
Serum
NGF levels were observed to be decreased both in humans with diabetes
and experimental diabetic rats.1
8
Therefore, the
disruption in normal expression of NGF under hyperglycemic states may
lead to diabetic neuropathy.
The present study demonstrated a progressive deficit in the retrograde
axonal transport of selective RGCs to the optic nerve. This impairment
may cause reduction in expression of neurotrophic factors, which leads
to downregulation in the synthesis of factors such as neurofilaments
and substance P.1
A concomitant deficit in the anterograde
axonal transport system of large myelinated optic nerve fibers is
suggested to cause neuroaxonal dystrophic changes in the RGCs and the
optic nerve.1
2
3
ARI or myo-inositol supplementation prevents development of deficits in
the orthograde axonal transport system.9
The structural
and functional impairments in peripheral and optic nerves were also
reportedly improved by ARI treatment.1
3
10
These findings
suggest an etiopathogenetic role for the polyol pathway and its induced
alteration of myo-inositol metabolism in axonal transport deficits. In
this study, ARI treatment prevented impairment in retrograde axonal
transport in a dose-dependent manner. Low-dose ARI partially prevented
the early deficit in retrograde axonal transport of large RGCs. In
contrast, high-dose ARI treatment prevented deficits not only in large,
but also in medium, RGCs. The effect of high-dose ARI treatment on
axonal transport at the 3 month interval led to amelioration of the
neuroaxonal dystrophic changes previously observed in the optic nerve
fibers.3
The present study revealed a progressive deficit
in the retrograde axonal transport of selective RGCs from 1 to 3 months
of induced diabetes. Twelve-months of induced diabetes may affect
retrograde axonal transport in the smaller-sized RGCs and may cause a
more severe form of axonal atrophy resistant to ARI
treatment.10
In conclusion, we demonstrated that the impairment in retrograde axonal
transport of RGCs in diabetic rats is related to the duration of
diabetes, with the impairment initially occurring in large RGCs and
progressively affecting the medium RGCs. The impairment in retrograde
axonal transport and neuroaxonal changes occurring with diabetes was
prevented by ARI treatment in a dose-dependent manner. It is also
interesting to note that our findings of abnormalities of large RGCs in
the diabetic optic nerve are analogous to those seen not only in
peripheral diabetic neuropathies, but also in glaucoma.
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Acknowledgements
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The authors thank Naomi Kurumatani and Michael Francis Teraoka
Escaño for valued assistance.
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Footnotes
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Submitted for publication April 3, 2000; revised August 15, 1000; accepted August 25, 2000.
Commercial relationships policy: P (HN, HK); N (MI, LZ, MY).
Corresponding author: Masanori Ino-ue, Department of Ophthalmology, Kobe University School of Medicine, 7-5-2, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. myman956{at}mailgate.kobe-u.ac.jp
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References
|
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-
Greene, DA, Sima, AA, Stevens, MJ, Feldman, EL, Lattimer, SA (1992) Complications: neuropathy, pathogenetic considerations Diabetes Care 15,1902-1925[Abstract]
-
Sima, AA, Zhang, WX, Cherian, PV, Chakrabarti, S. (1992) Impaired visual evoked potential and primary axonopathy of the optic nerve in the diabetic BB/W-rat Diabetologia 35,602-607[Medline][Order article via Infotrieve]
-
Ino-ue, M, Yokogawa, H, Yamamoto, M, Naka, H, Kuriyama, H. (1998) Structural impairments in optic nerve of diabetic rats ameliorated with the aldose reductase inhibitor Exp Eye Res 66,397-401[Medline][Order article via Infotrieve]
-
Sidenius, P, Jakobsen, J. (1981) Retrograde axonal transport: a possible role in the development of neuropathy Diabetologia 20,110-112[Medline][Order article via Infotrieve]
-
Jakobsen, J, Brimijoin, S, Skau, K, Sidenius, P, Wells, D. (1981) Retrograde axonal transport of transmitter enzymes, fucose-labeled protein, and nerve growth factor in streptozotocin-diabetic rats Diabetes 30,797-803[Medline][Order article via Infotrieve]
-
Zhang, L, Ino-ue, M, Dong, K, Yamamoto, Y. (2000) Retrograde axonal transport of large- and medium-sized retinal ganglion cells in diabetic rat Curr Eye Res 20,131-136[Medline][Order article via Infotrieve]
-
Greene, DA, Lattimer, SA, Sima, AA (1987) Sorbitol, phosphoinositides, and sodium-potassium-ATPase in the pathogenesis of diabetic complications N Engl J Med 316,599-606[Abstract]
-
Faradji, V, Sotelo, J. (1990) Low serum levels of nerve growth factor in diabetic neuropathy Acta Neurol Scand 81,402-406[Medline][Order article via Infotrieve]
-
Mayer, JH, Tomlinson, DR (1983) Prevention of defects of axonal transport and nerve conduction velocity by oral administration of myo-inositol or an aldose reductase inhibitor in streptozotocin-diabetic rats Diabetologia 25,433-438[Medline][Order article via Infotrieve]
-
Kamijo, M, Cherian, PV, Sima, AA (1993) The preventive effect of aldose reduction inhibition on diabetic optic neuropathy in the BB/W-rat Diabetologia 36,893-898[Medline][Order article via Infotrieve]
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Abstract
Introduction
