The Roles of Oxidative Stress and Antioxidant
Treatment in Experimental Diabetic Neuropathy
Phillip A. Low, Kim K. Nickander, and Hans J. Tritschler
Oxidative stress is present in the diabetic state. Our
work has focused on its presence in peripheral nerves.
Antioxidant enzymes are reduced in peripheral nerves
and are further reduced in diabetic nerves. That lipid
peroxidation will cause neuropathy is supported by evi-
dence of the development of neuropathy de novo when
normal nerves are rendered et-tocopherol deficient and
by the augmentation of the conduction deficit in dia-
betic nerves subjected to this insult. Oxidative stress
appears to be primarily due to the processes of nerve
ischemia and hyperglycemia auto-oxidation. The
indexes of oxidative stress include an increase in
nerve, dorsal root, and sympathetic ganglia lipid
hydroperoxides and conjugated dienes. The most reli-
able and sensitive index, however, is a reduction in
reduced glutathione. Experimental diabetic neuropathy
results in myelinopathy of dorsal roots and a vacuolar
neuropathy of dorsal root ganglion. The vacuoles are
mitochondrial; we posit that lipid peroxidation causes
mitochondrial DNA mutations that increase reduced
oxygen species, causing further damage to mitochon-
drial respiratory chain and function and resulting in a
sensory neuropathy, a-lipoic acid is a potent antioxidant
that prevents lipid peroxidation in vitro and in vivo. We
evaluated the efficacy of the drug in doses of 20, 50, and
100 mg/kg administered intraperitoneally in preventing
the biochemical, electrophysiological, and nerve blood
flow deficits in the peripheral nerves of experimental
diabetic neuropathy, a-lipoic acid dose- and time-
dependently prevented the deficits in nerve conduc-
tion and nerve blood flow and biochemical abnormali-
ties (reductions in reduced glutathione and lipid per-
oxidation). The nerve blood flow deficit was 50% (P <
0.001). Supplementation dose-dependently prevented
the deficit; at the highest concentration, nerve blood
flow was not different from that of control nerves. Dig-
ital nerve conduction underwent a dose-dependent
improvement at 1 month (P < 0.05). By 3 months, all
treated groups had lost their deficit. The antioxidant
drug is potentially efficacious for human diabetic sen-
sory neuropathy. Diabetes 46 (Suppl. 2):S38-S42, 1997
From the Department of Neurology (P.A.L., K.K.N.), Mayo Clinic and Mayo
Foundation, Rochester, Minnesota; and Asta Medica (H.J.T.), Frankfurt, Ger-
many.
Address correspondence and reprint requests to Dr. Phillip A. Low,
Department of Neurology, Mayo Clinic and Mayo Foundation, Rochester, MN
55905.
Accepted for publication 19 December 1996.
GSH, reduced glutathione; SOD, superoxide dismutase.
Nothing in this publication is meant to imply that the Mayo Foundation
endorses the products of ASTA Medica.
S38
T
he twin problems of chronic hyperglycemia and
endoneurial hypoxia result in excessive oxidative
stress (1), which may be important in the patho-
genesis of diabetic neuropathy. If this is so, antiox-
idants could be particularly important in the prevention and
treatment of diabetic neuropathy.
This discussion will be focused on answering the follow-
ing five questions: 1) What are the free radical defenses of
nerve? 2) Is there increased oxidative stress in experimental
diabetic neuropathy? 3) What are the best indexes of
increased lipid peroxidation in nerves? 4) Does lipid perox-
idation cause neuropathy? 5) Do antioxidants work?
FREE RADICAL DEFENSES
Peripheral nerves have a number of cytosolic and lipophilic
antioxidants, which are often subdivided into preventive and
chain-breaking antioxidants (2). Preventive antioxidants
block chain initiation. Chain-breaking antioxidants suppress
free radical chain oxidation by molecular oxygen and com-
prise water-soluble and lipid-soluble antioxidants. The key
enzymatic scavengers are superoxide dismutase (SOD), cata-
lase, glutathione peroxidase, and glutathione reductase (2).
Reduced glutathione (GSH) is particularly important in mem-
brane because it is the main scavenger in the blocking of chain
propagation (2). The cytosolic and membrane antioxidants
work in concert in a well-organized, interacting chain. Ample
activity of all components is needed to maintain these antiox-
idants in their reduced state (3). There is much literature on
free radical biology, but information on peripheral nerves, and
in particular diabetic peripheral nerves, is quite limited.
Of great interest is the marked and selective reduction in
antioxidant defenses in rat sciatic nerve. GSH and GSH-con-
taining enzymes scavengers (glutathione peroxidase and
reductase) in rodent sciatic nerve are only about 10% that of
the brain, whereas the activities of other enzymes, such as
SOD, are approximately the same in brain and nerve (4). The
diabetic state results in a reduction in both SOD and glu-
tathione peroxidase. Cuprozinc SOD is reduced, and this
reduction is improved by insulin treatment (5). The very low
activity of nerve glutathione peroxidase is further reduced in
experimental diabetic neuropathy (6). Plasma and leukocyte
ascorbic acid are reduced, and oxidation of this antioxidant
is increased (7,8).
OXIDATIVE STRESS IN EXPERIMENTAL DIABETIC
NEUROPATHY
There are a number of potential sources of free radical gen-
eration in diabetes: 1) ischemia, 2) hyperglycemia by autox-
DIABETES, VOL. 46, SUPPL. 2, SEPTEMBER 1997
 P.A. LOW, K.K. NICKANDER, AND H.J. TRITSCHLER

idative peroxidation, 3) increased mitochondrial leak, 4) cat- other than nerve (e.g., 1,5,21-23). Evidence of increased
echolamine oxidation, and 5) leukocytes. Most of the nerve lipid peroxidation in vivo is now available. Diabetic
research to date has been concentrated on the roles of peripheral nerves have increased conjugated dienes (5,25),
ischemia and hyperglycemia. The concept of increased mito- reduced GSH (25), and reduced glutathione peroxidase (6).
chondrial leak is discussed briefly in the context of our Among the indexes of increased oxidative stress in a chronic
recent findings involving the pathology of vacuolar degener- in vivo situation, malondialdehyde, conjugated dienes, and
ation of dorsal root ganglion neurons. lipid hydroperoxides are increased in the peripheral nerves
in experimental diabetic neuropathy (5,25,26), and the
ISCHEMIA increase is more consistent in lumbar dorsal root and supe-
The suggested pathogenetic scheme has been previously rior cervical ganglia (25,26). The most reliable index of
described (1,9,10). Diabetic neuropathy is a metabolic dis- increased oxidative stress is reduction in GSH (25,26).
order in which the brunt of the insult is borne by the We argued that to demonstrate that oxidative stress causes
microvessels; the endothelial cell is perceived as being a neuropathy, it is necessary to satisfy the following four cri-
dynamic tissue responding to perturbations in nitric oxide, teria: 2) that an increase in lipid peroxidation in previously
endoneurial eicosanoids, advanced glycation end products, normal nerves results in neuropathy; 2) that an increase in
oxidative stress, growth factors, and neurotransmitters/neu- lipid peroxidation in diabetic neuropathic nerves further
romodulators. Endoneurial hypoxia is secondary to a reduc- worsens function; 3) that antioxidant therapy improves or
tion in nerve blood flow and increased endoneurial vascular prevents neuropathy; and 4) that this improvement or pre-
resistance (9,11), which begins by the 1st week of diabetes vention is associated with a reduction or improvement in
(12) and affects the cell body as well as the axon (13). The sug- the indexes of lipid peroxidation of peripheral nerve.
gested scheme can be briefly summarized. The major meta- To approach the first question of whether increased oxida-
bolic effect of hyperglycemia in chronic progressive diabetic tive stress causes neuropathy in previously normal nerves, we
neuropathy is suggested to be on microvessels. Hyper- undertook the experimental depletion of tissue a-tocopherol
glycemia acts via rheological mechanisms, advanced glyca- in previously normal nerves (25). Weaned rats were fed an a-
tion end products (14,15), and a reduction in nitric oxide tocopherol-deficient diet. Plasma a-tocopherol became
(16), resulting in impaired microvascular tone and reduced immeasurable. Endoneurial lipid peroxidation resulted, as
nerve blood flow and endoneurial hypoxia. A vicious cycle indicated by a reduction in GSH and increased conjugated
supervenes in which a reduction in nitric oxide and certain dienes and lipid hydroperoxides. These findings were associ-
eicosanoids (especially a reduction in the prostaglandin: ated with the development of a sensory neuropathy in previ-
thromboxane Ag ratio) (17), leukotrienes, advanced glycation ously normal nerves (25). A second line of evidence derives
end products, and oxygen free radicals and continued rheo- from the worsening of neuropathy in experimental diabetic neu-
logical mechanisms are important. Insulin per se may also ropathy (25) associated with an increase in lipid peroxidation.
have an atherogenic role (1,18). The progressive microvas-
cular and macrovascular pathology is suggested to result in LEUKOCYTES AND CATECHOLAMINE OXIDATION
the nerve-fiber pathology of multifocal fiber loss. The roles of the leukocyte and catecholamine oxidation is
The effects of oxidative stress depend on balancing oxida- uncertain. Leukocytic infiltration is not a feature in most
tive stress, pro-oxidant status, and free radical defenses (19). cases of diabetic neuropathy. In human diabetic neuropathy,
Changes in all three areas occur in diabetic peripheral there are some indications of an immune-mediated process,
nerves. Diabetic pro-oxidant status is increased, since diabetic as suggested by the presence of iritis and inflammatory infil-
sciatic nerve has increased polyunsaturated fatty acids with trates in sympathetic ganglia (27-29). Some forms of dia-
excessive lipolysis (20). It has also been suggested that met- betic neuropathy, such as acute autonomic neuropathy and
als, especially copper, might be increased (21,22). the subacute proximal neuropathies, might be associated
with round cell infiltration (30).
HYPERGLYCEMIA GLYCATION/OXIDATION
Chronic hyperglycemia per se results in autoxidative glyca- ROLE OF MITOCHONDRIAL LEAK
tion/oxidation and lipid peroxidation (21-23). Hyper- We have completed a study on the neuropathology of chronic
glycemia results in increased arachidonic acid flux and mal- (12-18 months) experimental diabetic neuropathy (13).
ondialdehyde formation. We have recently demonstrated Changes are most prominent in the nerve roots and lumbar
that hyperglycemia per se will cause lipid peroxidation of dorsal root ganglion. The changes in ventral root seem to be
peripheral nerve in vitro (24). We undertook studies of in vitro an exaggeration of the aging process causing a vesicular
lipid peroxidation of brain and sciatic nerve using an in vitro myelinopathy. There are more pronounced changes, how-
lipid peroxidation model with an ascorbate-iron-EDTA sys- ever, in the dorsal root ganglion and dorsal root. The dorsal
tem. When we used an incubation medium containing 20 root shows myelinopathy, and the dorsal root ganglion
mmol/1 glucose, lipid peroxidation increased fourfold, indi- shows pronounced vacuolar degeneration of dorsal root gan-
cating that hyperglycemia causes rapid and marked lipid glion cells. The vacuoles are almost entirely degenerating
peroxidation (24). mitochondria (Fig. 1) associated with excessive lipofuscin
deposition.
LIPID PEROXIDATION AND NEUROPATHY The vacuoles observed in our study may be related to dys-
Oxidative stress is part of the price mammals pay for breath- functional mitochondria (31). Oxidative stress has been sug-
ing oxygen. There is good evidence that oxidative stress is gested to be a major pathogenetic mechanism of aging, and
increased in the diabetic state. Most studies in human and the mitochondrion is a selective focus of its action (32,33).
experimental diabetes have been done on plasma or tissue Mitochondrial DNA is unusually susceptible to oxidative
DIABETES, VOL. 46, SUPPL. 2, SEPTEMBER 1997 S39
 OXIDATIVE STRESS AND ANTIOXIDANTS IN NEUROPATHY

a-lipoic acid is a powerful lipophilic free radical scavenger

of peripheral nerve in vitro and in vivo (3,25,41). It has addi-
tional actions, including increasing glucose entry into periph-
eral nerve (unpublished observations), stimulating nerve
growth factor (42), and promoting fiber regeneration (43).
The roles of these additional effects on diabetic neuropathy
remain to be ascertained. To evaluate if a-lipoic acid will
reduce oxidative stress in diabetic peripheral nerve and
improve neuropathy, we used the model of streptozotocin
experimental diabetic neuropathy and evaluated the efficacy
of a-lipoic acid supplementation in improving nerve blood
flow, electrophysiology, and indexes of oxidative stress in
peripheral nerve of experimental diabetic neuropathy at 1
and 3 months after onset of diabetes and in age-matched con-
trol rats (26). a-lipoic acid, in doses of 20, 50, and 100 mg/kg,
was administered intraperitoneally,fivetimes per week, after
onset of diabetes. Nerve bloodflowin experimental diabetic
neuropathy was reduced by 50%; a-lipoic acid did not affect
FIG. 1. Electronmicrography of L5 dorsal root ganglion neurons of rat nerve blood flow of normal nerves but improved that of
after 12 months of diabetes, showing vacuolar degeneration and nerves in experimental diabetic neuropathy in a dose-depen-
prominent lipofuscin granules. dent manner. After 1 month of treatment, a-lipoic acid-sup-
plemented rats (100 mg/kg) had normal nerve blood flow
(Fig. 2). Mean nerve blood flow of untreated diabetic nerves
damage with aging (31), which leads to increased mutations; was 51% that of controls. The corresponding values for
these dysfunctional mitochondria have increased free radical treated nerves at 20, 50, and 100 mg/dl were 51, 72, and 92%,
leakage (31). Shigenaga et al. (31) have suggested that the respectively, being significantly improved with treatment (P <
vicious cycle of oxidative damage to inner membrane proteins 0.05). The most sensitive and reliable indicator of oxidative
(of mitochondria) leads to imbalances in the electron trans- stress was a reduction in GSH, which was significantly
port chain, which results in increased superoxide and hydro- reduced in streptozotocin-diabetic and a-tocopherol-defi-
gen peroxide production, which in turn further damages cient nerves; it was improved in a dose-dependent manner in
membrane proteins, and the cycle repeats. a-lipoic acid-supplemented rats (P < 0.001; Fig. 3). Mean GSH
value of untreated diabetic nerves was 63% of controls. The
ANTIOXIDANT THERAPY IN EXPERIMENTAL DIABETIC corresponding values for treated nerves at 20, 50, and 100
NEUROPATHY mg/dl were 73, 83, and 96% of normal, respectively. The con-
Several antioxidants have shown promise in the treatment of duction velocity of digital nerve was reduced in streptozotocin-
experimental diabetic neuropathy. diabetic neuropathy (P < 0.001) and was significantly
Probucol, a powerful free radical scavenger (34), normal- improved by a-lipoic acid (Fig. 4). At 1 month, treatment
izes both nerve blood flow and electrophysiology (35,36). resulted in a significant improvement (P < 0.05); by 3
These studies also reported an improvement in nerve perfu-
sion with 1% vitamin E diet.
Improvement in experimental diabetic neuropathy has
been reported following treatment with GSH (37). Bravenboer
et al. (37) administered intravenous glutathione 200 mg/kg for Nerve 12
the 10-week study and were able to prevent some of the con- blood
flow
duction slowing. Delayed administration resulted in only (mL/100 g/min) 8

minor improvement.
One percent butylated hydroxytoluene for 2 months com- Con Con50 STZ STZ20 STZ50 STZ100
pletely prevented the conduction deficit in experimental dia-
betic neuropathy (38). Carvidilol, an antioxidant and
P<0.05
vasodilator, is reported to be efficacious (39). Cameron and r25
Cotter (40) have reported improvement in both nerve blood
flow and nerve conduction in experimental diabetic neu- Nerve
ropathy following treatment with the iron chelator desfer- vascular
resistance
roximine. (mm Hg/mL/100 g/min)
While these important studies in experimental diabetic
neuropathy have shown improvement of the electrophysio- Con Con50 STZ STZ20 STZ50 STZ100 CA-H
logical deficit following treatment with antioxidants, in none
of these studies were measurements of lipid peroxidation FIG. 2. Nerve blood flow and nerve vascular resistance of control
done, so it is not known if these agents actually reduced (Con), streptozotocin-diabetic neuropathy (STZ), and animals sup-
neural lipid peroxidation. Some of these antioxidants have a plemented with lipoic acid at doses of 20 mg/kg (STZ20), 50 mg/kg
(Con50; STZ50), and 100 mg/kg (STZ100). Lipoic acid supplementation
number of non-antioxidant actions that could also affect results in normal nerve blood flow and nerve vascular resistance.
peripheral nerve function. From Nagamatsu et al. (26).

S40 DIABETES, VOL. 46, SUPPL. 2, SEPTEMBER 1997

 P.A. LOW, K.K. NICKANDER, AND H.J. TRITSCHLER

I 8H T T
2

Q.
a
T T T FIG. 3. Sciatic nerve GSH concentrations
"5 in controls (Con) and on restricted
caloric intake (Con[R])( streptozotocin-
I »H diabetic (STZ), a-tocopherol-depleted (-)
and supplemented with lipoic acid at 20
X
mg/kg, 50 mg/kg, and 100 mg/kg. Lipoic
U) acid supplementation resulted in a dose-
Con Con[R] ConlOO Con[-] Con[-]100 STZ STZ20 STZ50 STZ100 dependent prevention of GSH.

months, all treated nerves were significantly improved com- ACKNOWLEDGMENTS

pared with untreated nerves (P < 0.01), and treated nerves
were not significantly slowed compared with untreated
nerves. These studies suggest that a-lipoic acid improves
streptozotocin-diabetic neuropathy, in significant part, by
reducing the effects of oxidative stress. We have also found
that modified SOD will prevent the deficit in nerve blood flow
(unpublished observations).
In conclusion, mammalian nerve is particularly prone to
oxidative stress because of its high content of phospholipids
and its marked reduction in free-radical defenses. The dia-
betic state results in hyperglycemic autoxidation-glycation
and endoneurial hypoxia-associated lipid peroxidation. At
the dorsal root and ganglion, hypoxia also occurs; peroxi-
dation-induced DNA mutation of mitochondria! membrane
is suggested with vacuolar degeneration of mitochondria, and
increased mitochondrial leak is suggested as a mechanism
of the sensory neuropathy of diabetes. Antioxidants, espe-
cially lipophilic antioxidants, are efficacious in preventing
diabetic neuropathy.
1 Month
_ 45-]
¥ •P<0.05
^ 40-
8 35-
a>
30
3 Months
B P < 0.01
P<0.01
^ 45-
1 40-
§ 35-
30
Con Con50 STZ STZ20 STZ50 STZ100
FIG. 4. Digital nerve conduction velocity at 1 month (A) and 3 months
of supplementation intraperitoneally with lipoic acid at 0 mg/kg (Con,
STZ), 20 mg/kg (STZ20), 50 mg/kg (Con50; STZ50), and 100 mg/kg
(STZ100). Lipoic acid supplementation improved conduction velocity.
From Nagamatsu et al. (26).
This work was supported in part by grants from the National
Institute of Neurological Disorders and Stroke (PO1
NS32352, NS22352, NS30534), Mayo Funds, and a grant-in-aid
from Asta Medica.
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