Physical exercise induces excess Hsp72 expression and delays
the development of hyperalgesia and allodynia in painful
diabetic neuropathy rats
Yu-Wen Chen1,*, Ph.D., Pei-Ling Hsieh2, M.S., Yu-Chung Chen3, M.S., Ching-Hsia
Hung2, Ph.D., Juei-Tang Cheng4,5, Ph.D.
1
Department of Physical Therapy, China Medical University, Taichung, Taiwan
Institute & Department of Physical Therapy, National Cheng Kung University,
Tainan, Taiwan
3
Division of Physical Therapy, Department of Physical Medicine and Rehabilitation,
Cheng Hsin General Hospital, Taipei, Taiwan
4
Department of Medical Research, Chi-Mei Medical Center, Tainan, Taiwan
5
Department of Pharmacology, College of Medicine, National Cheng Kung
2
University, Tainan, Taiwan
*Corresponding author:
Yu-Wen Chen, Ph.D.
Department of Physical Therapy, China Medical University, No.91 Hsueh-Shih
Road, Taichung 40402, Taiwan
Phone: 886-4-22053366 ext 7327
FAX: 886-4-22065051
Email: cywhwok@mail.cmu.edu.tw
Information for LWW regarding depositing manuscript into PubMed Central:
This paper does not need to be deposited in PubMed Central.
Submitted as a Research Report
1
Abstract
BACKGROUND: The underlying mechanism of exercise on the development of
diabetes-associated neuropathic pain is not well understood. We investigated whether
exercise regulates the functional recovery and heat shock protein 72 (Hsp72), tumor
necrosis factor-α (TNF-α) and interlukin-6 (IL-6) expression in streptozotocin
(STZ)-induced diabetic rats.
METHODS: Male Wistar rats were divided into 4 groups: normal sedentary rats (NS),
normal rats with exercise (NE), sedentary STZ-diabetic rats (SS), STZ-diabetic rats
with exercise (SE). Diabetes was induced with STZ (65 mg/kg, iv). The trained rats
ran daily on a treadmill 30-60 min/day with an intensity of 20-25m/min. We
monitored thermal withdrawal latency and mechanical withdrawal threshold as well
as Hsp72, TNF-α and IL-6 expression in spinal cord and peripheral nerves.
RESULTS: Two weeks after STZ injection, sedentary rats exhibited a marked and
sustained hypersensitivity to von Frey tactile and heat stimuli. By contrast, diabetic
rats undergoing exercise demonstrated a delayed progress of tactile and thermal
hypersensitivity that was independent of glucose control. TNF-α and IL-6 release was
significantly increased in spinal cord and peripheral nerves in SS compared with in
NS rats. The SE group showed greater Hsp72 expression and similar TNF-α or IL-6
level compared with the SS group in spinal cord and peripheral nerves on day 14 after
2
STZ treatment.
CONCLUSIONS: These results suggest that progressive exercise training markedly
decreases diabetes-associated neuropathic pain, including thermal hyperalgesia and
mechanical allodynia. This protective effect is related to the increase of Hsp72, but
not TNF-α and IL-6, expression in spinal cord and peripheral nerves of STZ-induced
diabetic rats.
KEY WORDS: Treadmill exercise; Streptozotocin; Diabetic neuropathic pain;
Tumor necrosis factor-α; Interlukin-6; Heat shock protein 72
3
INTRODUCTION
Diabetic peripheral neuropathic pain (DPNP) is typically a manifestation of
distal symmetrical sensorimotor polyneuropathy, and has been described as an
“aching, burning, stabbing, or tingling” sensation 1 characterized by hyperalgesia,
paresthesia and allodynia.2-4 Moreover, chronic pain serves no useful purpose, usually
negatively affects a person’s quality of life, and is associated with many deleterious
physiological effects.1-4 The effective management of diabetes in patients with type 1
diabetes mellitus requires a daily balancing of insulin administration, diet, and
exercise.5,6 Although many available pharmacotherapies (e.g., tricyclic
antidepressants and opioid analgesics) are effective for DPNP, these drugs produce
side effects.5 Furthermore, neuropathic pain from diabetes is making patient miserable,
and patients may hate to rely on medication. In contrast to medication, exercise is
prescribed in different stages of persons with diabetes mellitus to prevent the progress
of diabetic syndromes to increase or sustain physiological and health-related fitness.6,7
We wondered whether exercise alone might be an impact on the development of
diabetic complications (i.e., DPNP).
There is a growing body of evidence that exercise improves diabetic dysfunction
and neuropathic pain.8-14 For instance, swimming exercise training had therapeutic
and protective effects on diabetic peripheral neuropathy in rats with streptozotocin
4
(STZ)-induced diabetes.11 Belotto et al. reported that aerobic exercise has been
recommended for the treatment of diabetes in order to improve vascular health and
insulin sensitivity.10 Forced-exercise markedly delays the progression of tactile
hypersensitivity, but not thermal hyperalgesia, in experimentally-induced diabetic
rats.8 Interestingly, low to moderate swimming reduces heat hyperalgesia caused by
acute hyperglycemia in STZ-induced diabetic female rats.14 However, the
mechanisms underlying DPNP are not fully understood.
The application of exercise has since then been established as an effective and
safe integral approach to the management of diabetes.15,16 Hung et al. 12 clearly
showed that heat shock protein 72 (Hsp72) expression in the heart and nucleus tractus
solitarii of the brain was significantly increased in diabetic rats following exercise
training. Additionally, Hsp72 plays a neuroprotective role in attenuating
heatstroke-induced cerebral ischemia,17 circulatory shock in diabetic rats 12 and
neuropathic pain in rats after peripheral nerve injury.13 In addition, neuropathic pain
induces varying degrees of local inflammatory responses and overexpression
activated inflammatory cytokine release in activated macrophages and glial and
Schwann cells.18,19 It has been presumed that pro-inflammatory cytokines (i.e., tumor
necrosis factor-α, interlukin-6) could cause pain.20-23 By comparison, treatments with
inhibitors of pro-inflammatory cytokines or anti-inflammatory cytokines reduce
5
pain.24-27 However, only few experiments have evaluated the effects of treadmill
exercise on tactile allodynia, thermal hyperalgesia, pro-inflammatory cytokines and
Hsp72.
The purpose of the study was to evaluate whether treadmill exercise training, a
non-pharmacotherapy, reduces tactile allodynia, thermal hyperalgesia and
pro-inflammatory cytokines and increases Hsp72 expression after STZ treatment of
the spinal cord and peripheral nerves in a rat model of type 1 diabetes. Here, heat
hyperalgesia and tactile allodynia as well as tumor necrosis factor-α (TNF-α),
interleukin-6 (IL-6) and Hsp72 in nerves were examined in diabetic rats with/without
treadmill exercise.
6
METHODS
Animals
Male Wistar rats weighing 285-335 g were purchased from the Animal Center of
National Cheng Kung University, and then housed them, three rats per cage with free
access to food and water, in a climate-controlled room maintained at 21C and 50%
relative humidity, and on a 12-h light/dark cycle (light on at 6:00 a.m.) in the Animal
Center of China Medical University. The experimental protocols were approved by
the Institutional Animal Care and Use Committee of China Medical University,
Taiwan. Efforts were made to minimize discomfort of the animals and decrease the
number of experimental animals. All studies were conducted according to IASP
ethical guidelines.28
Inducing type 1 diabetes
After animals had fasted for 3 days, the rats were anesthetized with pentobarbital
sodium salt (35 mg/ kg). They were then given a single intravenous injection of STZ
(65 mg/kg; Sigma-Aldrich Co., St. Louis, MO), which destroys pancreatic cells and
causes an insulin deficiency. Three days after the STZ injection, we used a blood
glucose meter (Accu-Check Active; Roche Boehringer Mannheim Diagnostics,
Mannheim, Germany) to determine whether blood samples (obtained by tail prick)
had a glucose concentration  300 mg/dL, which was considered a confirmation of
7
STZ-induced diabetes.12
Groups and design
Rats were randomly divided into four groups: (1) normal sedentary rats (NS), (2)
normal rats with exercise (NE), (3) sedentary STZ-diabetic rats (SS) and (4)
STZ-diabetic rats with exercise (SE). Some rats were considered for the overall
behavioral analysis and body weight (n = 10, 10, 10, 10 for NS, NE, SS and SE,
respectively), while some part of rats were sacrificed for tissue analysis (TNF-alpha
and IL-6) on day 14 after STZ treatment (n = 5, 5, 5, 5 for NS, NE, SS and SE,
respectively), and other rats were sacrificed for Hsp72 analysis on day 14, 28, 56 after
STZ treatment (n = 12 [4, 4, 4], 12 [4, 4, 4], 12 [4, 4, 4], 12 [4, 4, 4] for NS, NE, SS
and SE, respectively).
Treadmill training protocol
The exercise training protocol was carried out according to previously described
methods.12,17 In brief, animals were trained to run daily on a treadmill (Chanson,
CS-5515, Taipei, Taiwan) for 8 weeks. The training program began with a treadmill
speed of 20 m/min for 30 min. The speed and duration were then gradually increased
to 20 m/min for 60 min during the initial 2 weeks. The rats ran for a warm-up period
of 5 min at 15 m/min on each exercise day. If the feet of rats were hurt during the
training protocol they were withdrawn from this experiment.
8
Tactile and heat responsiveness
Animals were examined for thermal hyperalgesia and mechanical allodynia after
a period of at least 3 days of habituation to the testing environment. We interpret the
decreases in thermal withdrawal latency and mechanical withdrawal threshold as
hyperalgesia and allodynia, respectively. Unless otherwise specified, behavioral tests
were conducted on 3 day before STZ-treated, the day of STZ-treated and on days 3, 7,
14, 21, 28, 35, 42, 49 and 56 after STZ-treated. All measurements were performed
between 9:00 a.m. and 11:00 a.m. For consistency, one experienced investigator who
was blinded to the groups was responsible for handling of all animals and behavioral
evaluations.
For tactile allodynia, rats were placed individually in a clear plexiglass chamber
(23 cm [length] x 17 cm [width] x 14 cm [height]) and supported via a wire mesh
floor (40 cm [width] x 50 cm [length]). A series of von Frey filaments (Linton
Instruments, UK) was applied at the plantar surface of the left hind paw of the rat, and
the paw withdrawal threshold (gram) was recorded.13 The withdrawal response
evoked by mechanical stimulation was determined including foot lifting, shaking,
licking and squeaking. Paw movements associated with weight shifting or locomotion
were not counted. The mechanical stimulation was repeated 3 times at intervals of 5
minutes for each test and the median of the three tests was used for statistical analysis.
9
Thermal withdrawal latency was examined according to the Hargreaves’ Method
with the plantar test.29 In brief, rats were placed individually in a clear plexiglass
chamber (23 cm [length] x 17 cm [width] x 14 cm [height]), and the animals stood on
a glass sheet with the temperature maintained at 30 ±1ºC to decrease the influence of
the temperature in different seasons. The plantar surface of the right hind paw of the
rat was exposed to a constant intensity radiant heat source (focused beam of light,
beam diameter: 0.5 cm, intensity: 20 I.R.) through a transparent perspex surface and a
Plantar Analgesia Meter (Ugo Basile, Milan, Italy), and the paw withdrawal latency
(second) was recorded. The withdrawal response evoked by thermal stimulation was
determined including foot lifting, shaking, licking and squeaking. The right hind paw
of each rat was tested three times at 5-min intervals, and the median of the three tests
was used for statistical analysis. A maximal automatic cut-off latency of 20 seconds
was used to avoid causing the rat excessive pain and to prevent tissue damage.29
Tissue Preparation
The animals were anesthetized with urethane (1.67 g/kg, i.p.) and sacrificed on
days 14, 28, 56 after STZ injection. Under aseptic conditions, skin was cut to expose
the L4–L6 region of rat spinal cord and the peripheral nerves (consisted of sural,
posterior tibial, and superficial peroneal nerves), proximal to the trifurcation, were
removed. The nerve specimen was immediately stored at −80℃ for the protein assay.
10
Ice cold (4ºC) homogenization buffer was added (300 μl/each sciatic nerve). The
homogenization buffer was freshly prepared by adding protease inhibitor (P 8340
cocktail, Sigma-Aldrich, St. Louis, MO) to T-PER™ Tissue Protein Extraction
Reagent (Pierce Chemical Co., Rockford, IL) prior to tissue lysis. After adding the
buffer, a homogenization probe (Tissue Tearor, Polytron; Biospec Products, Inc.,
Bartlesville, OK, USA) was applied for 20 seconds on ice at 21,000 rpm. Then the
homogenized samples were centrifuged for 40 minutes at a speed of 13,000 rpm at
4°C, stored at −80°C and used subsequently for protein quantification. The protein
concentration in the supernatant was quantified using the Lowry protein assay.
Samples were pipetted as duplicates (1 μl/50 μl/well) in a 96-well microtiter plate
(Costar). Each plate was inserted into a plate reader (Molecular Device Spec 383,
Sunnyvale, CA, USA) to read the optical density of each well at an absorbance of 750
nm. Data were analyzed using Ascent Software (London, UK) for iEMS Reader.
Hsp72 evaluation
The protein samples (30 μg/lane) were separated by 12% SDS polyacrylamide
gel electrophoresis (SDS-PAGE) at a constant voltage of 75 V.13 These
electrophoresed proteins were transferred to a polyvinylidene difluoride (PVDF)
membrane with a 0.45 μm pore size (Millipore, Bedford, MA) by a transfer apparatus
(Bio-Rad, Hercules, CA, USA). The PVDF membrane was incubated in 5% milk in
11
TBS buffer. Then the membrane was blocked in TBS (20 mM Tris, 500 mM NaCl,
and 0.1% Tween 20, pH 7.5) containing 5% skim milk (Difco, Detroit, MI) for 1 hour.
Mouse monoclonal anti-Hsp72 primary antibody (SPA 810; StressGen
Biotechnologies, Victoria, British Columbia, Canada) was diluted to 1:500 in
antibody binding buffer overnight at 4ºC. The membrane was then washed 3 times
with TBS (10 minutes per wash) and incubated for 1 hour with horseradish
peroxidase-conjugated goat anti-mouse secondary antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) and diluted 500-fold in TBS buffer at 4°C. The
membrane was washed in TBS buffer for 10 minutes 3 times. Immunodetection for
Hsp72 was performed by the enhanced chemiluminescence ECL Western blotting
luminal reagent (Santa Cruz Biotechnology) and then the membrane was quantified
by a Fujifilm LAS-3000 chemiluminescence detection system (Tokyo, Japan).
Cytokine assay
The concentrations of TNF-α and IL-6 in the supernatants were determined 13 by
the DuoSet® ELISA Development Kit (R&D Systems, Minneapolis, MN). All
experimental procedures were performed in accordance with the manufacturer’s
instructions. Plates were individually inserted into the plate reader for reading optical
density by a 450-nm filter. Data were then analyzed using Ascent Software for iEMS
Reader and a four-parameter logistics curve-fit. Data were expressed in pg/mg protein
12
of duplicate samples.
Statistical analysis
Data are presented as the mean ± SEM of N observations unless noted otherwise.
Statistical significance between multiple experimental groups was determined via
one-way or two-way ANOVA with a Bonferroni multiple comparison post-hoc
analysis. In each case, statistical significance was set at P < 0.05.
13
RESULTS
Physical exercise reduces the body weight loss in STZ-induced experimental
diabetes
The body weight of the NS and NE rats constantly increased over the course of
the experiment (Fig. 1A). By comparison, the STZ-induced diabetic rats showed a
regular decline in body weight gain, agreeing with the study showed that the body
mass in type I-like diabetic rats was markedly lower than that of normal rats.12 A
similar, but less exaggerated decline was observed with STZ-induced diabetic rats
undergoing physical exercise (Fig. 1A). In other words, the body weight loss in SE
rats was not as serious as that in SS rats.
Physical exercise does not reverse the high blood glucose in STZ-induced
experimental diabetes
After a single intravenous injection of STZ, male rats revealed a marked
elevation of blood glucose (> 300 mg/dL) that was sustained throughout this 8-week
study (Fig. 1B). By comparison, NS rats maintained blood glucose levels between 90
and 100 mg/dL (Fig. 1B). Rats ran continuously on a treadmill displayed nearly
identical sustained increases (> 300 mg/dL) in blood glucose (Fig. 1B), although the
level of blood glucose of SE rats showed a significant decrease on days 3, 7, 14, 21,
42, 49 and 56 after STZ treatment when compared with that of SS rats. Therefore, the
14
blood glucose level was similarly unaffected by continuous exercise in NE rats (Fig.
1B).
Physical exercise shows short- but not long-term effects on diabetes-associated
thermal hyperalgesia
Diabetic patients frequently have abnormal and bothersome perceptions of
thermal stimuli, called thermal hyperalgesia, were suggested as an indicator of early
DPNP.30 In Fig. 2A, the thermal withdrawal latencies (15.7 ± 0.6 s, n = 10) obtained
from NS rats were not significantly different from those of NE rats (15.7 ± 0.3 s, n =
10) after a 2 week exercise training program. When the thermal withdrawal latencies
were evaluated on day 14 after STZ injection, SS rats uniformly experienced thermal
hyperalgesia as indicated through a significant decrease in paw withdrawal latencies
(P < 0.05), compared with NS rats. The thermal hyperalgesia coincided with severe
hyperglycemia in STZ-diabetic rats (Fig. 1B). These results are in agreement with that
Calcutt et al. study 31 on rats indicated that one month after the onset of STZ-induced
diabetes they developed thermal hyperalgesia, which lasted for at least 2 months. In
contrast, SE rats displayed minimal changes in thermal withdrawal latencies on day
14 after STZ treatment as compared with NS rats, suggesting an inhibition of thermal
hyperalgesia (Fig. 2A). Long-term physical activity did not exhibit to increase thermal
withdrawal latencies (12.8 ± 0.7 s, n = 10) in SE rats compared with those (12.4 ± 0.8
15
s, n = 10) in SS rats on day 56 after STZ injection.
Physical exercise retards the development of diabetes-associated tactile allodynia
On day 7, the NS rats displayed paw withdrawal threshold sensitivities of 14.0 ±
0.7 g (n = 10) that were maintained over the 8-week course of the experiment (Fig.
2B). The NE rats showed similarly (14.1 ± 0.7 g, n = 10) to tactile stimulation on day
7, suggesting that this exercise program performed in this experiment does not alter
tactile sensitivity (P > 0.05; two-way repeated measures ANOVA). By comparison,
those SS rats on day 7 after STZ treatment exhibited an aggrandized sensitivity to
innocuous von Frey stimuli (4.6 ± 0.7 g, n = 10) that were sustained over the 8-week
course of the experiment (Fig. 2B). Consistent with previous studies, significant
tactile allodynia in rats began 7 days after they had been injected with STZ 32,33 and
lasted for up to 2 months.34 Furthermore, STZ-treated rats at first week underwent
treadmill exercise displayed paw withdrawal thresholds of 8.4 ± 1.3 g (n = 10), lower
than those of NS rats and higher than these of SS rats (Fig. 2B). This physical exercise
regimen significantly (P < 0.05; two-way repeated measures ANOVA) retarded
neuropathic tactile allodynia in rats between 7-21 days after STZ injection (Fig. 2B)
without altering high blood glucose levels (> 300mg/dL; Fig. 1B). From week 4 until
week 8 after STZ treatment, there was no significant difference in mechanicl
withdrawal threshold between the SS and SE groups.
16
Physical exercise enhances Hsp72 expression in spinal cord and peripheral nerves
Figure 3 depicts the expression of Hsp72 in spinal cord and peripheral nerves on
days 14, 28 and 56 after STZ treatment in four different groups. It can be seen that the
Hsp72 levels in spinal cord and peripheral nerve were significantly increased in the
NE (P < 0.05) and SE groups (P < 0.05) on days 14, 28 and 56 after STZ treatment
compared with the NS and SS groups, respectively (Fig. 3).
Physical exercise suppresses cytokine levels in spinal cord and peripheral nerves
Figure 4 A-D reveal the levels of TNF-α and IL-6 in spinal cord and peripheral
nerves of NS, NE, SS and SE rats on day 14 after STZ treatment. The NS and NE rats
demonstrated similarly cytokine levels in spinal cord and peripheral nerves,
suggesting that this exercise regimen in this experiment does not alter TNF-α and IL-6
levels. On day 14 after STZ treatment, the expression of TNF-α and IL-6 in spinal
cord and peripheral nerves was increased in the SS group (P < 0.05) or SE (P < 0.05)
group compared with the NS or NE group, respectively, as shown in Fig. 4A-D. The
levels of TNF-α and IL-6 in spinal cord and peripheral nerve were not significantly
different between the SS and SE groups (Fig. 4 A-D).
17
DISCUSSION
This study reveals that physical exercise markedly prevents abnormal weight loss
and retards the progression of thermal hyperalgesia and tactile allodynia, the
behavioral measures of painful neuropathy, though exercise-facilitated analgesia
occurs independent of blood glucose control in STZ-induced diabetic rats. The
exercise apparently induces excess Hsp72 expression in spinal cord and peripheral
nerves of trained rats, in a time of day-dependent manner, compared with sedentary
controls. This is in resemblance to our previous study that exercise increases the
expression of Hsp72 in the heart and nucleus tractus solitarii to protect against
cardiovascular dysfunction induced by lipopolysaccharide administration in diabetic
rats.12 In spinal cord and peripheral nerves, diabetes induced inflammatory cytokine
(TNF-α and IL-6) overexpression that was not attenuate by exercise training. Overall,
these results suggest that treadmill exercise training reduces the symptoms of
diabetes-associated neuropathic pain, in part, possibly relating to induce excess Hsp72,
but not cytokines, expression in spinal cord and peripheral nerves.
Physical exercise alters STZ-induced diabetic thermal and tactile hypersensitivity
Painful diabetic polyneuropathy is difficult to treat in humans. Current
management of affected patients primarily involves alleviation of discomfort and
glycemic control.35 In this study, STZ-treated rats developed within 2 weeks
18
aggrandized plantar responsiveness to mechanical and heat stimuli as shown in Fig. 2.
In fact, the application of exercise has since then been established as a safe and
effective integral approach to the management of diabetes.15,16 Early clinical studies
show a significant independent association between the occurrence of diabetic
complications, including neuropathy, and declining exercise capacity among diabetic
patients.36 Our present data demonstrated that treadmill exercise attenuates diabetic
peripheral neuropathic pain, including heat hyperalgesia and tactile allodynia, caused
by acute hyperglycemia in STZ-induced diabetic rats. These findings are in agreement
with the report by Shankarappa et al.,8 who reported that forced-exercise delays the
development of tactile allodynia in experimentally-induced diabetic rats, in part,
through increasing opioidergic tone thereby inhibiting diabetes-associated modulation
of Ca2+ channels in dorsal root ganglion sensory neurons. Additionally, it has been
shown that swimming training for a long duration in STZ-induced diabetic female rats
reduces thermal hyperalgesia in a hot plate test.14 Therefore, exercise,37 or lifestyle
intervention strategies that include an exercise component,38 may even delay or
protect against the development of diabetic peripheral nerve complications.
It is interesting to observe that there is temporal window in this study, namely
from day 0 to day 14, where the tactile allodynia was reduced or, better, the
withdrawal threshold was increased, while after day 14 the beneficial effect of
19
treadmill running is progressively abolished by continued exercise. In the meanwhile
treadmill training completely prevented thermal hyperalgesia at 4 weeks, while after
day 56 (8 weeks) the beneficial effect of treadmill running is progressively decreased.
This seems to suggest that a treadmill exercise protocols of short duration are
favourable to sensory recovery while, at difference, long-duration exercise protocols
are detrimental. It could be the result of exercise or uneven stratification of animals so
that the exercised group was less severely diabetic.
It has been proposed that long-term rhythmic exercise activates central opioid
systems by triggering discharges from mechanosensitive afferent nerve fibers in
contracting muscles.39 Endorphin secretion requires strenuous physical activity.40 By
comparison, swimming exercise in cold water is analgesic in animals with a blocked
endogenous opioid analgesic system, which indicates that there are multiple analgesic
systems.41 We did not verify whether the endogenous opioid system is involved in the
analgesia in this study. However, it has been proved that forced-exercise significantly
delays the onset of diabetes-induced neuropathic pain, in part, possibly via altering
opioidergic tone.8
Diabetic rats with exercise exhibits a delayed onset of neuropathic pain that is
independent of glucose control
It is well established that the peripheral nerve damage develops in diabetes in
20
close association with poorly controlled chronic hyperglycemia.5 Furthermore, acute
periods of hyperglycemia may precipitate neuropathic pain. We showed that an acute
period (2-week) of sustained hyperglycemia caused a sensory hypersensitivity to the
thermal or tactile stimuli. Our results are in agreement with that hyperglycemia may
lead to persistent alterations in the pain threshold in diabetic rats.42 Additionally, pain
thresholds are markedly decreased following glucose administration to experimental
animals 43 and to healthy volunteers.44 Furthermore, we demonstrated that the
STZ-induced diabetic rats exhibited a regular body weight loss. In contrast, exercise
retarded the body weight loss in STZ-induced experimental diabetes. In agreement
with Selagzi et al., swimming restored body weight, compound muscle action
potential (CMAP) amplitude, CMAP latency, and motor dysfunction but did not affect
glycemic control in diabetic rats.11
To optimize outcome and prevent the microcirculatory and neuropathic effects of
hyperglycemia, the blood glucose level should be maintained within an acceptable
range, which can be accomplished through frequent evaluation and adjustment of the
overall treatment regimen.5-7 In our experiment, the trained diabetic rats still displayed
hyperglycemia (300mg/dL), suggesting that exercise does not alter the distribution of
pancreatic hormones, including insulin, in STZ-induced diabetic rats.45 Whereas
exercise training did not significantly reduce blood glucose levels (Fig. 1), changes in
21
trophic insulin or C peptide content are not anticipated to play a significant role in
exercise-facilitated analgesia.45
We revealed that diabetic rats undergoing exercise running exhibited normal
sensitivity to heat stimuli (Fig. 2A) despite elevated levels of blood glucose (Fig. 1B).
In marked contrast, the degree of reduction (Fig. 2B) in decreased mechanical von
Frey thresholds (< 50%) by exercise was quite small and shows the relevance of the
findings in relation to neuropathic pain that is still present. Interestingly, the
protective effect of physical exercise was found to be short, with diabetic rats
ultimately developing tactile allodynia and thermal hyperalgesia by the eighth week
of sustained hyperglycemia. These data propose that treadmill exercise facilitated
analgesia occurs through a mechanism that is largely independent of blood glucose
control.
Treadmill exercise increases the expression of Hsp72 in spinal cord and peripheral
nerves
Previous studies testified that exercise-induced modulation of heat shock factor-1
(HSF-1, a HSPs transcription factor) aggregation, subsequently expression of Hsp72
in multiple organs or neurons of rats.17,46-48 Hsp72 had been shown to have the
neuroprotective effect to repair the damaged nerves.17 A recent experiment reported
that treadmill and swimming exercises increase Hsp72 expression in sciatic nerve of
22
CCI rats and ameliorate neuropathic pain.13 Our findings showed that treadmill
training alleviated DPNP and increased neuronal expression of Hsp72 with the
observation that remedial gymnastics relieves pain only temporarily. Although we did
not provide direct evidence of the mechanism of Hsp72 which reduced diabetic
neuropathic pain in this study, accumulated evidence shows that HSPs corrects
folding of many proteins in a cell requires protein-folding machinery and the
molecular chaperones 49 as well as chaperones repair denatured proteins or promote
their degradation.50
Exercise running dose not alter diabetic-induced increased levels of
pro-inflammatory cytokines in nerves
Dysregulation of systemic inflammatory status is now believed to play an
important role 51-53 while the molecular mechanisms that link chronic hyperglycemia
to diabetic complications are still incompletely defined. Evidences have been
presented that STZ, the ability to cause β-cell damage and hyperglycemia, can induce
activation of microglia, and release pro-inflammatory cytokines in spinal dorsal
horn.54 TNF-α plays a central role in diabetic neuropathy and its inhibition by
infliximab, a TNF-α antagonist, leads to the amelioration of this major complication
of diabetes.55 In vivo studies have shown that elevated systemic IL-6 concentrations
have been documented in animal diabetic models and for a variety of human
23
conditions;56-58 in vitro, cultured cells exposed to hyperglycemia increases their IL-6
secretion.53,59 Interestingly, we showed that the expression of TNF-α and IL-6 in
spinal cord and peripheral nerves was markedly increased in STZ-diabetic rats
compared with normal rats on day 14 after STZ treatment (Fig. 4). In some sense, this
appears similar to the “inflammatory component” caused by hyperglycemia: the
inflammatory component is susceptible to correction through strict glycemic
control.60,61 A logical extension of our results is the question of whether mean
hyperglycemia over a more prolonged period of time may also similarly regulate
inflammatory status.
Exercise training is believed to be mediated, at least in part, by an overall
reduction in systemic inflammation in both healthy and type 1 diabetes
individuals.62,63 A recent experiment showed that serum IL-6 secretion is reduced in
the context of aerobic exercise in hyperglycaemia compared with euglycaemia in
patients with well-controlled type 1 diabetes.64 Because TNF-α elaborates important
signals in nervous system in various kinds of degenerative diseases,65 it is reasonable
to expect that lack of TNF-α signals in the peripheral nervous system may prevent or
ameliorate diabetic neuropathy. However, the relationships among cytokines, pain and
exercises have not been analyzed. The practical relevance of our observations is
apparent that treadmill exercise training did not attenuate TNF-α and IL-6 expression
24
in spinal cord and peripheral nerves on day 14 after STZ treatment. Contrary to the
long-term effects of exercise training, however, each exercise bout actually exerts an
acute proinflammatory effect, reflected by transient elevations of inflammatory
cytokines such as TNF-α, IL1β and IL-6.66-68 In this study, we did note that the
observations in this study on thermal hyperalgesia, mechanical allodynia, TNF-α, IL-6
and Hsp72 are, at present, merely coincident.
Summary
We conclude that physical exercise delays the process of diabetic peripheral
painful neuropathy. Elucidating the methods by which exercise enhances Hsp72
expression, but not inhibition of inflammatory cytokine (TNF-α or IL-6)
overexpression, in spinal cord and peripheral nerves may present new opportunities
for the development of non-pharmacological adjunctive therapeutic strategies for the
management of painful diabetic neuropathy.
25
ACKNOWLEDGMENTS
We gratefully acknowledge the financial support provided by grants NSC
98-2314-B-006-017-MY3 and NSC 100-2314-B-039-017-MY3 from the National
Science Council, Taiwan.
26
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FIGURE LEGENDS
Fig. 1. Body weight (A) and blood glucose (B) change in NS, NE, SS and SE rats. NS
= normal sedentary rats; NE = normal rats with exercise; SS = sedentary
streptozotocin-diabetic rats; SE = streptozotocin-diabetic rats with exercise. Data are
presented as mean ± SEM for 10 rats per group. The asterisk (*) indicates P < 0.05
when the SE group was compared with the SS group; the pus symbol (+) indicates P
< 0.05 when the SE or SS group was compared with the NS group (2-way ANOVA of
repeated measures followed by post hoc Bonferroni’s test).
Fig. 2. Time courses of thermal withdrawal latency (A) and mechanical withdrawal
threshold (B) in NS, NE, SS and SE rats, where NS = normal sedentary rats; NE =
normal rats with exercise; SS = sedentary streptozotocin-diabetic rats; SE =
streptozotocin-diabetic rats with exercise. The thermal withdrawal latency (s) and
mechanical withdrawal threshold (g) to heat and mechanical stimulation were not
significantly different between the NS or NE group. Data are presented as mean ±
SEM for 10 rats per group. The asterisk (*) indicates P < 0.05 when the SE group was
compared with the SS group; the pus symbol (+) indicates P < 0.05 when the SE or
SS group was compared with the NS group (2-way ANOVA of repeated measures
followed by post hoc Bonferroni’s test).
Fig. 3. The expression of Hsp72 in spinal cord (A–C) and peripheral nerves (D–F) on
37
days 14 (A, D), 28 (B, E), 56 (C, F) after STZ treatment in different groups of rats:
NS, NE, SS and SE (NS = normal sedentary rats; NE = normal rats with exercise; SS
= sedentary streptozotocin-diabetic rats; SE = streptozotocin-diabetic rats with
exercise). The values are presented as mean ± SEM for 4 rats per group. Symbols
(*,**and***) indicate P < 0.05, P < 0.01 and P < 0.001when the NE or SE group was
compared with the NS or SS group, respectively (1-way ANOVA followed by post
hoc Bonferroni’s test).
Fig. 4. The levels of TNF-α and IL-6 on day 14 after STZ treatment in spinal cord (A,
C) and peripheral nerves (B, D) in NS, NE, SS and SE rats, where NS = normal
sedentary rats; NE = normal rats with exercise; SS = sedentary streptozotocin-diabetic
rats; SE = streptozotocin-diabetic rats with exercise. The values are presented as mean
± SEM for 5 rats per group. Symbols (*,**and***) indicate P < 0.05, P < 0.01 and P
< 0.001when the SS or SE group was compared with the NS group, respectively
(1-way ANOVA followed by post hoc Bonferroni’s test).
38