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 21C 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. 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Effect of intense exercise on inflammatory cytokines and growth mediators in adolescent boys. Pediatrics 2002;110:681-9. 68. Pedersen BK, Akerstrom TC, Nielsen AR, Fischer CP. Role of myokines in exercise and metabolism. J Appl Physiol 2007;103:1093-8. 36 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