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Physical Exercise Induces Excess Hsp72 Expression and Delays the Development of Hyperalgesia and Allodynia in Painful Diabetic Neuropathy Rats

Chen, Yu-Wen PhD*; Hsieh, Pei-Ling MS; Chen, Yu-Chung MS; Hung, Ching-Hsia PhD; Cheng, Juei-Tang PhD§║

doi: 10.1213/ANE.0b013e318274e4a0
Pain and Analgesic Mechanisms

BACKGROUND: The underlying mechanism of exercise on the development of diabetes-associated neuropathic pain is not well understood. We investigated in rats whether exercise regulates the functional recovery and heat shock protein 72 (Hsp72), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 expression in streptozotocin (STZ)-induced diabetes.

METHODS: Male Wistar rats were divided into 4 groups: normal sedentary rats, normal rats with exercise, sedentary STZ-diabetic (SS) rats, and STZ-diabetic rats with exercise. Diabetes was induced with STZ (65 mg/kg IV). The trained rats ran daily on a treadmill 30 to 60 min/d with an intensity of 20 to 25 m/min. We monitored thermal withdrawal latency and mechanical withdrawal threshold as well as Hsp72, TNF-α, and IL-6 expression in the 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. In contrast, diabetic rats undergoing exercise demonstrated delayed progress of tactile and thermal hypersensitivity. Exercise significantly suppressed diabetes-induced blood glucose levels and body weight loss, although they were not restored to control levels. Compared with normal sedentary rats, SS rats displayed significantly higher TNF-α and IL-6 levels in the spinal cord and peripheral nerves. The STZ-diabetic rats with exercise group showed greater Hsp72 expression and similar TNF-α or IL-6 level compared with the SS group in the spinal cord and peripheral nerves on day 14 after STZ treatment.

CONCLUSIONS: These results suggest that progressive exercise training markedly decreases diabetes-associated neuropathic pain, including thermal hyperalgesia and mechanical allodynia. In rats, this protective effect is related to the increase of Hsp72, but not TNF-α and IL-6, expression in the spinal cord and peripheral nerves of STZ-induced diabetes.

From the *Department of Physical Therapy, China Medical University, Taichung; Institute & Department of Physical Therapy, National Cheng Kung University, Tainan; Division of Physical Therapy, Department of Physical Medicine and Rehabilitation, Cheng Hsin General Hospital, Taipei; §Department of Medical Research, Chi-Mei Medical Center, Tainan; and Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

Accepted for publication September 5, 2012.

Supported by the National Science Council of Taiwan (NSC 98-2314-B-006-017-MY3 and NSC 100-2314-B-039-017-MY3).

The authors declare no conflicts of interest.

Drs. Yu-Wen Chen and Ching-Hsia Hung contributed equally to this work.

Reprints will not be available from the authors.

Address correspondence to Yu-Wen Chen, PhD, Department of Physical Therapy, China Medical University, No. 91 Hsueh-Shih Rd., Taichung 40402, Taiwan. Address e-mail to cywhwok@mail.cmu.edu.tw.

Diabetic peripheral neuropathic pain (DPNP) is typically a manifestation of distal symmetric sensorimotor polyneuropathy, described as an “aching, burning, stabbing, or tingling” sensation1 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 physiologic effects.1–4 The effective management of diabetes in patients with type 1 diabetes mellitus requires a daily balance 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 causes patient distress, and patients may dislike relying on medication. In contrast to medication, exercise is prescribed for different stages of diabetes mellitus to prevent the progress of diabetic syndromes and to increase or sustain physiologic and health-related fitness.6,7 We questioned whether exercise alone might have 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 had therapeutic and protective effects on diabetic peripheral neuropathy in rats with streptozotocin (STZ)-induced diabetes.11 Belotto et al.10 recommended aerobic exercise for the treatment of diabetes to improve vascular health and insulin sensitivity. Forced exercise markedly delays the progression of tactile hypersensitivity, but not thermal hyperalgesia, in experimentally induced diabetes in rats.8 Interestingly, low to moderate swimming reduces heat hyperalgesia caused by acute hyperglycemia in STZ-induced diabetes in female rats.14 However, the mechanisms underlying DPNP are not fully understood.

Exercise has since 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 after exercise training. Additionally, Hsp72 has 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 Furthermore, neuropathic pain induces varying degrees of local inflammatory responses and overexpression of activated inflammatory cytokine release in activated macrophages and glial and Schwann cells.18,19 It has been presumed that proinflammatory cytokines (i.e., tumor necrosis factor [TNF]-α, interleukin [IL]-6) could cause pain.20–23 By comparison, treatments with inhibitors of proinflammatory cytokines or antiinflammatory cytokines reduce pain.24–27 However, only a few experiments have evaluated the effects of treadmill exercise on tactile allodynia, thermal hyperalgesia, proinflammatory cytokines, and Hsp72.

The purpose of this study was to evaluate whether treadmill exercise training, a nonpharmacotherapy, reduces tactile allodynia, thermal hyperalgesia, and proinflammatory cytokines and increases Hsp72 expression in the spinal cord and peripheral nerves in a rat model of type 1 diabetes. Heat hyperalgesia and tactile allodynia as well as TNF-α, IL-6, and Hsp72 in nerves were examined in diabetic rats with and without treadmill exercise.

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METHODS

Animals

Male Wistar rats weighing 285 to 335 g were purchased from the Animal Center of National Cheng Kung University, and housed, 3 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-hour light/dark cycle (lights on at 6:00 AM) 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 International Association for the Study of Pain ethical guidelines.28

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Inducing Type 1 Diabetes

After the rats had fasted for 3 days, they were anesthetized with pentobarbital sodium salt (35 mg/kg). They were then given a single IV injection of STZ (65 mg/kg; Sigma-Aldrich Co., St. Louis, MO), which destroys pancreatic cells and causes 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 STZ-induced diabetes.12

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Groups and Design

Rats were randomly divided into 4 groups: (1) normal sedentary (NS) rats, (2) normal rats with exercise (NE), (3) sedentary STZ-diabetic (SS) rats, and (4) STZ-diabetic rats with exercise (SE). Some rats were considered for overall behavioral analysis and body weight analysis (n = 10, 10, 10, and 10 for NS, NE, SS, and SE, respectively), some rats were killed for tissue analysis (TNF-α and IL-6) on day 14 after STZ treatment (n = 5, 5, 5, and 5 for NS, NE, SS, and SE, respectively), and other rats were killed for Hsp72 analysis on days 14, 28, and 56 after STZ treatment (n = 12 [4, 4, 4], 12 [4, 4, 4], 12 [4, 4, 4], and 12 [4, 4, 4] for NS, NE, SS, and SE, respectively).

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Treadmill Training Protocol

The exercise training protocol was performed according to previously described methods.12,17 In brief, animals were trained to run daily on a treadmill (CS-5515; Chanson, Taipei, Taiwan) for 8 weeks. The training program began with a treadmill speed of 20 m/min for 30 minutes. The speed and duration were then gradually increased to 20 m/min for 60 minutes during the initial 2 weeks. The rats ran for a warm-up period of 5 minutes at 15 m/min on each exercise day. If the rats’ feet were injured during the training protocol, they were withdrawn from this experiment.

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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 interpreted the decreases in thermal withdrawal latency and mechanical withdrawal threshold as hyperalgesia and allodynia, respectively. Unless otherwise specified, behavioral tests were conducted 3 days before STZ treatment, the day of STZ treatment, and on days 3, 7, 14, 21, 28, 35, 42, 49, and 56 after STZ treatment. All measurements were performed between 9:00 AM and 11:00 AM. 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 plexiglas chamber (23 cm [length] × 17 cm [width] × 14 cm [height]) and supported via a wire mesh floor (40 cm [width] × 50 cm [length]). A series of von Frey filaments (Linton Instruments, UK) was applied at the plantar surface of the rat’s right hindpaw, and the paw withdrawal threshold (grams) was recorded.13 The withdrawal response evoked by mechanical stimulation was determined by foot lifting, shaking, licking, and squeaking. Paw movements associated with weight shifting or locomotion were not counted. Mechanical stimulation was repeated 3 times at intervals of 5 minutes for each test and the median of the 3 tests was used for statistical analysis.

Thermal withdrawal latency was examined according to the plantar test (Hargreaves method).29 In brief, rats were placed individually in a clear plexiglas chamber (23 cm [length] × 17 cm [width] × 14 cm [height]), and the animals stood on a glass sheet with the temperature maintained at 30°C ± 1°C to decrease the influence of the temperature in different seasons. The plantar surface of the right hindpaw was exposed to a constant-intensity radiant heat source (focused beam of light, beam diameter: 0.5 cm, intensity: 20 IR) through a transparent Perspex surface, and paw withdrawal latency (seconds) was recorded by a Plantar Analgesia Meter (Ugo Basile, Milan, Italy). The withdrawal response evoked by thermal stimulation was determined including foot lifting, shaking, licking, and squeaking. The right hindpaw of each rat was tested 3 times at 5-minute intervals, and the median of the 3 tests was used for statistical analysis. A maximal automatic cut-off latency of 20 seconds was used to avoid causing rats excessive pain and to prevent tissue damage.29

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Tissue Preparation

The animals were anesthetized with urethane (1.67 g/kg, intraperitoneally) and killed on day 14, 28, or 56 after STZ injection. Under aseptic conditions, skin was cut to expose the L4-6 region of the spinal cord, and peripheral nerves (consisting of sural, posterior tibial, and superficial peroneal nerves), proximal to the trifurcation, were removed. The nerve specimen was immediately stored at −80°C for protein assay.

Ice cold (4°C) homogenization buffer was freshly prepared by adding protease inhibitor (P 8340 cocktail; Sigma-Aldrich) to T-PER™ Tissue Protein Extraction Reagent (Pierce Chemical Co., Rockford, IL) before tissue lysis. After adding the buffer (300 μL/each nerve), a homogenization probe (Tissue Tearor, Polytron; Biospec Products, Inc., Bartlesville, OK) was applied for 20 seconds on ice at 21,000 rpm. The homogenized samples were then centrifuged for 40 minutes at 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) 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.

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Hsp72 Evaluation

The protein samples (30 μg/lane) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis at a constant voltage of 75 V.13 These electrophoresed proteins were transferred to a polyvinylidene difluoride membrane with a 0.45-μm pore size (Millipore, Bedford, MA) by a transfer apparatus (Bio-Rad, Hercules, CA). The polyvinylidene difluoride membrane was incubated in 5% milk in tris-buffered saline (TBS) buffer. The membrane was then 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, BC, Canada) was diluted to 1:500 in antibody binding buffer overnight at 4°C. The membrane was then washed 3 times with TBS (10 minutes per wash) and incubated for 1 hour with horseradish peroxidase–conjugated goat antimouse secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and diluted 500-fold in TBS buffer at 4°C. The membrane was washed 3 times in TBS buffer for 10 minutes. Immunodetection for Hsp72 was performed by the enhanced chemiluminescence Western blotting luminal reagent (Santa Cruz Biotechnology), and the membrane was quantified by a Fujifilm LAS-3000 chemiluminescence detection system (Tokyo, Japan).

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Cytokine Assay

The concentrations of TNF-α and IL-6 in the supernatants were determined13 using 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 4-parameter logistics curve-fit. Data were expressed in pg/mg protein of duplicate samples.

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Statistical Analysis

Data are presented as the mean ± SEM of n observations unless noted otherwise. Statistical significance among multiple experimental groups was determined via 1-way or 2-way analysis of variance (ANOVA) with a Bonferroni multiple-comparison post hoc analysis. In each case, statistical significance was set at P < 0.05.

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RESULTS

Physical Exercise Reduces 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 decrease in weight gain, agreeing with Hung et al.12 who showed that body mass in type 1–like diabetic rats was markedly less than that of normal rats. A similar but less exaggerated decrease was observed with STZ-induced diabetic rats undergoing physical exercise (Fig. 1A). In other words, body weight loss in SE rats was not as serious as that in SS rats.

Physical exercise decreases diabetes-induced blood glucose levels, although it does not decrease to control levels.

After a single IV injection of STZ, male rats had a marked increase 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). The data shown in Figure 1B clearly demonstrate a significant separation between the SS and SE groups in glucose level (400–500 mg/dL vs 300–400 mg/dL) in most days after STZ treatment. Furthermore, the blood glucose level was similarly unaffected by continuous exercise in NE rats (Fig. 1B).

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Physical Exercise Produces Short-Term but Not Long-Term Effects on Diabetes-Associated Thermal Hyperalgesia

Patients with diabetes frequently have abnormal and bothersome perceptions of thermal stimuli, called thermal hyperalgesia, which may be an indicator of early DPNP.30 In Figure 2A, thermal withdrawal latencies (15.7 ± 0.6 seconds, 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 thermal withdrawal latencies were evaluated on day 14 after STZ injection, SS rats uniformly experienced thermal hyperalgesia, indicated through a significant decrease in paw withdrawal latencies (P < 0.05), compared with NS rats. Thermal hyperalgesia coincided with severe hyperglycemia in STZ-diabetic rats (Fig. 1B). These results are in agreement with the study by Calcutt et al.,31 which indicated that 1 month after the onset of STZ-induced diabetes, rats 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 compared with NS rats, suggesting an inhibition of thermal hyperalgesia (Fig. 2A). Long-term physical activity did not seem to increase thermal withdrawal latencies (12.8 ± 0.7 seconds, n = 10) in SE rats compared with SS rats (12.4 ± 0.8 seconds, n = 10) on day 56 after STZ injection.

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Physical Exercise Delays 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). NE rats had similar sensitivities (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; 2-way repeated-measures ANOVA). By comparison, 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 was 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 STZ32,33 and lasted for up to 2 months.34 Furthermore, in the first week, STZ-treated rats that exercised on the treadmill displayed paw withdrawal thresholds of 8.4 ± 1.3 g (n = 10), less than NS rats and higher than SS rats (Fig. 2B). Moreover, this physical exercise regimen significantly (P < 0.05; 2-way repeated-measures ANOVA) delayed neuropathic tactile allodynia between 7 and 21 days after STZ injection (Fig. 2B) by decreasing blood glucose levels (>300 mg/dL; Fig. 1B). From week 4 until week 8 after STZ treatment, there was no significant difference in mechanical withdrawal threshold between the SS and SE groups.

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Physical Exercise Enhances Hsp72 Expression in the Spinal Cord and Peripheral Nerves

Figure 3 depicts the expression of Hsp72 in the spinal cord and peripheral nerves on days 14, 28, and 56 after STZ treatment in the 4 different groups. Hsp72 levels in the spinal cord and peripheral nerve were significantly increased in the NE group (P < 0.05) and SE group (P < 0.05) on days 14, 28, and 56 after STZ treatment compared with the NS and SS groups, respectively (Fig. 3).

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Physical Exercise Suppresses Cytokine Levels in the Spinal Cord and Peripheral Nerves

Figure 4 (A–D) shows the levels of TNF-α and IL-6 in the spinal cord and peripheral nerves of NS, NE, SS, and SE rats on day 14 after STZ treatment. NS and NE rats had similar cytokine levels in the spinal cord and peripheral nerves, suggesting that the exercise regimen in our 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 the spinal cord and peripheral nerves was increased in the SS group (P < 0.05) and SE group (P < 0.05) compared with the NS and NE groups, respectively, as shown in Figure 4 (A–D). The levels of TNF-α and IL-6 in the spinal cord and peripheral nerve were not significantly different between the SS and SE groups (Fig. 4, A–D).

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DISCUSSION

This study reveals that in rats, physical exercise markedly prevents abnormal weight loss, decreases diabetes-induced blood glucose levels, and delays the progression of thermal hyperalgesia and tactile allodynia, the behavioral measures of painful neuropathy, in STZ-induced diabetes. The exercise apparently induces excess Hsp72 expression in the spinal cord and peripheral nerves of trained rats, in a manner dependent on the time of day, compared with sedentary controls. These results are similar to our previous study that found that exercise increases the expression of Hsp72 in the heart and nucleus tractus solitarii, which protects against cardiovascular dysfunction induced by lipopolysaccharide administration in diabetic rats.12 In the spinal cord and peripheral nerves, diabetes-induced inflammatory cytokine (TNF-α and IL-6) overexpression was not attenuated by exercise. Overall, these results suggest that treadmill exercise reduces the symptoms of diabetes-associated neuropathic pain due to the suppression of diabetes-induced blood sugar levels and increase of Hsp72, but not cytokine, expression in the spinal cord and peripheral nerves.

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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, within 2 weeks, STZ-treated rats developed aggrandized plantar responsiveness to mechanical and heat stimuli as shown in Figure 2. In fact, the use 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 decreasing exercise capacity among diabetic patients.36 Our present data demonstrated that treadmill exercise attenuates DPNP, including heat hyperalgesia and tactile allodynia, caused by acute hyperglycemia in STZ-induced diabetes. These findings are in agreement with the report by Shankarappa et al.,8 who demonstrated that forced exercise delays the development of tactile allodynia in experimentally induced diabetes in 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 for a long duration in STZ-induced diabetes in female rats reduces thermal hyperalgesia in a hotplate 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 was a temporal window in this study, namely from day 0 to day 14, when tactile allodynia was reduced or the withdrawal threshold was increased, whereas after day 14 the beneficial effect of treadmill running was progressively abolished by continued exercise. In addition, treadmill exercise completely prevented thermal hyperalgesia at 4 weeks, whereas after day 56 (8 weeks) the beneficial effect of treadmill running was progressively decreased. These results demonstrate the beneficial effects of exercise by delaying the onset of thermal hyperalgesia by several weeks and delaying the onset of mechanical allodynia by approximately 2 weeks (Fig. 2B); however, the exercised group could have been less severely diabetic because of exercise or an uneven stratification of animals in our study.

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 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 was involved in the analgesia in this study. However, it has been proven that forced exercise significantly delays the onset of diabetes-induced neuropathic pain, possibly via altering opioidergic tone.8

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Diabetic Rats That Exercise Have Lower Blood Glucose Levels

Peripheral nerve damage develops in diabetes in close association with poorly controlled chronic hyperglycemia.5 Furthermore, acute periods of hyperglycemia may precipitate neuropathic pain. In this study, we showed that an acute period (2 weeks) of sustained hyperglycemia caused a sensory hypersensitivity to thermal or tactile stimuli. Our results show agreement with Lee and McCarty42 that hyperglycemia may lead to persistent alterations in the pain threshold in diabetic rats. Additionally, pain thresholds are markedly decreased after glucose administration to experimental animals43 and to healthy volunteers.44 Furthermore, we demonstrated in rats that STZ-induced diabetes causes regular body weight loss. In contrast, exercise delayed body weight loss in STZ-induced experimental diabetes. In agreement with the report by Selagzi et al.,11 swimming restored body weight, compound muscle action potential amplitude and latency, and motor dysfunction in diabetic rats.

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 experienced hyperglycemia (300 mg/dL) although exercise significantly suppressed diabetes-induced blood sugar levels, suggesting that exercise is somewhat effective in altering the distribution of pancreatic hormones, including insulin, in STZ-induced diabetes.45 However, Howarth et al.45 disclosed that a heavy exercise program did not significantly reduce blood glucose levels, changes in trophic insulin, or C peptide content and may not have a significant role in exercise-facilitated analgesia.

We found that exercising diabetic rats exhibited normal sensitivity to heat stimuli (Fig. 2A) despite increased 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 showed the relevance of the findings in relation to existing neuropathic pain. 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.

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Treadmill Exercise Increases the Expression of Hsp72 in the Spinal Cord and Peripheral Nerves

Previous studies stated that exercise-induced modulation of heat shock factor-1 (a heat shock protein transcription factor) aggregation, subsequently expressed Hsp72 in multiple organs or neurons of rats.17,46–48 Hsp72 has been shown to have neuroprotective effects that repair damaged nerves.17 A recent experiment showed that treadmill and swimming exercises increase Hsp72 expression in the sciatic nerve of chronic constriction injury rats and ameliorates neuropathic pain.13 Our findings showed that treadmill exercise alleviated DPNP and increased neuronal expression of Hsp72 with the observation that remedial gymnastics relieves pain only temporarily. We did not provide direct evidence for the mechanism of Hsp72 that reduced diabetic neuropathic pain in this study. However, accumulative evidence has shown that heat shock proteins act as molecular chaperones49 to correct folding of many proteins, repair denatured proteins, or promote their degradation.50

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Running Does Not Alter Diabetic-Induced Increased Levels of Proinflammatory Cytokines in Nerves

Dysregulation of the systemic inflammatory status is now believed to have an important role51–53 in diabetes whereas the molecular mechanisms that link chronic hyperglycemia to diabetic complications are still incompletely defined. Evidence has been presented that STZ, and its ability to cause β-cell damage and hyperglycemia, can induce activation of microglia and release proinflammatory cytokines in the spinal dorsal horn.54 Furthermore, TNF-α has 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 increased systemic IL-6 concentrations have been documented in animal diabetic models and for a variety of human conditions;56–58 in vitro, cultured cells exposed to hyperglycemia increase their IL-6 secretion.53,59 Interestingly, we showed that the expression of TNF-α and IL-6 in the spinal cord and peripheral nerves was markedly increased in STZ-diabetic rats compared with normal rats on day 14 after STZ treatment (Fig. 4). This seems similar to the “inflammatory component” caused by hyperglycemia, which is susceptible to correction through strict glycemic control.60,61 Our results also cause us to question whether levels of mean hyperglycemia over a more prolonged period of time may also similarly regulate inflammatory status.

Exercise is believed to be mediated, at least in part, by an overall reduction in systemic inflammation in both healthy and type 1 diabetic individuals.62,63 A recent experiment showed that serum IL-6 secretion is reduced in the context of aerobic exercise in hyperglycemia compared with euglycemia in patients with well-controlled type 1 diabetes.64 Because TNF-α in the nervous system modulates neuronal signaling related to various kinds of degenerative diseases,65 it is reasonable to expect that diminishing TNF-α signaling may improve diabetic neuropathy. However, the relationships among cytokines, pain, and exercise have not been analyzed. We showed that treadmill exercise did not suppress TNF-α and IL-6 overexpression in the spinal cord and peripheral nerves on day 14 after STZ treatment. Contrary to the long-term effects of exercise, however, each exercise session actually exerts an acute proinflammatory effect, reflected by transient increases of inflammatory cytokines such as TNF-α, IL-1β, and IL-6.66–68 We did note that the observations in this study on thermal hyperalgesia, mechanical allodynia, TNF-α, IL-6, and Hsp72 are, at present, merely coincidental.

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Summary

We conclude that physical exercise delays the process of DPNP. Elucidating the methods by which exercise enhances Hsp72 expression, but not inhibition of inflammatory cytokine (TNF-α or IL-6) overexpression, in the spinal cord and peripheral nerves may present new opportunities for the development of nonpharmacologic adjunctive therapeutic strategies for the management of painful diabetic neuropathy.

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DISCLOSURES

Name: Yu-Wen Chen, PhD.

Contribution: This author helped design the study, conduct the study, and analyze the data.

Attestation: Yu-Wen Chen has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Pei-Ling Hsieh, MS.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Pei-Ling Hsieh has seen the study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Yu-Chung Chen, MS.

Contribution: This author helped design the study, conduct the study, and analyze the data.

Attestation: Yu-Chung Chen has seen the study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Ching-Hsia Hung, PhD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Ching-Hsia Hung has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Juei-Tang Cheng, PhD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Juei-Tang Cheng has seen the study data, reviewed the analysis of the data, and approved the final manuscript.

This manuscript was handled by: Jianren Mao, MD, PhD.

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ACKNOWLEDGMENTS

The authors gratefully acknowledge the financial support provided for this study by the National Science Council of Taiwan.

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