Experimental Neurology 267 (2015) 107–114
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Regular Article
Moderate exercise training attenuates inflammatory mediators in DRG of Type 1 diabetic rats HaeJee Yoon b, Vikram Thakur a, Danielle Isham b, Mona Fayad b, Munmun Chattopadhyay a,⁎ a b
Center of Excellence in Diabetes and Obesity, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA Department of Neurology, University of Michigan, Ann Arbor, MI, USA
a r t i c l e
i n f o
Article history: Received 10 October 2014 Revised 22 January 2015 Accepted 8 March 2015 Available online 14 March 2015 Keywords: Exercise Diabetes Neuropathy Pain Inflammation
a b s t r a c t Painful neuropathy is a long-term and difficult to treat complication of diabetes that affects 25% of diabetic patients and interferes with their quality of life. Unfortunately, available medical treatments are relatively ineffective due to dependency and addiction. Emerging research indicates that moderate-to-vigorous physical activity provides health-related benefits. However, adequate data is not available to determine whether regular exercise would prevent or delay the development of painful neuropathy in subjects with Type 1 diabetes. This study demonstrates the significance of moderate exercise in the amelioration of pain in animals with Type 1 diabetes after 6 weeks of exercise paradigm. After initial acclimatization, streptozotocin-diabetic animals were placed in motorized running wheels for 60 min per day, for five days a week for 6 weeks starting at one week after diabetes. A growing body of evidence suggests that the release of proinflammatory cytokines plays an important role in the development and persistence of pain. This study demonstrates that moderate exercise increases the expression of inhibitory neurotransmitter enkephalin and also reduces the presence of a number of proinflammatory cytokines in the dorsal root ganglia (DRG), subsequently impeding the development of neuropathy along with a decrease in the voltage gated ion channels in the DRG. Overall, the study suggests that exercise may provide an alternate route of treatment of painful neuropathy in Type 1 diabetic subjects by decreasing the use of pain medications, thereby providing a more useful and efficient way for pain management. © 2015 Published by Elsevier Inc.
Introduction Pain is a significant consequence of diabetic neuropathy. Diabetes mellitus is the most common cause of neuropathy in the United States, and 25% of diabetic patients with neuropathy suffer from neuropathic pain, resulting in a significant adverse effect on quality of life measures (Van Acker et al., 2009). Unfortunately, available medical treatment is relatively ineffective with limited efficacy and is complicated by side effects and dependency (Barbano et al., 2003). Accumulating evidence suggests that the activation of inflammatory cascades in the peripheral and central nervous systems plays a key role in the development and persistence of neuropathic pain states induced by physical or toxic injury to peripheral nerve (Gonzalez-Clemente et al., 2005; Herder et al., 2009; King, 2008). In diabetes, there is evidence of systemic immune activation. Patients with painful neuropathy show increased levels of IL-2 in blood and increased levels of TNFα mRNA and protein in blood (Uceyler et al., 2007). The elevated level of serum TNFα in Type 1 diabetes ⁎ Corresponding author at: Department of Biomedical Sciences, Center of Excellence in Diabetes and Obesity, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA. E-mail address: [email protected] (M. Chattopadhyay).
http://dx.doi.org/10.1016/j.expneurol.2015.03.006 0014-4886/© 2015 Published by Elsevier Inc.
patients suggests that TNFα may play a pathogenic role in the development of diabetic neuropathy (Gonzalez-Clemente et al., 2005; Kaul et al., 2010). Studies on patients with Type 2 with or without polyneuropathy exhibit a different immune profile and specific neuropathic deficits, suggesting that inflammation is associated with diabetic neuropathic impairments involving a number of immune mediators such as C-peptide, IL-2 receptor, IL1β and TNFα (Empl et al., 2001; Uceyler et al., 2007). Previously, we have shown that the inflammatory mediators in the dorsal root ganglia are altered with the development of pain in Type 2 model of diabetes (Galloway and Chattopadhyay, 2013). Although earlier studies have demonstrated the effects of moderate to intense physical exercise on pain perception (Rossi et al., 2011), not many have shown the effects of moderate exercise that may change the endogenous opioid content as well as the levels of inflammation in DRG of Type 1 diabetic animals with painful neuropathy. Hyperglycemia causes p38 mitogen-activated protein (p38) kinase activation (Igarashi et al., 1999), which can be induced by changes in the release of proinflammatory cytokines. Previously we have shown that viral vector mediated release of enkephalin modified the activation of p38 MAPK in Type 1 diabetic DRG (Chattopadhyay et al., 2008). This study also explores the possibility whether exercise can alter the endogenous opioid, enkephalin and stress associated markers, thus reducing pain related behaviors. It is well known that the heat shock protein (HSP) molecular
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chaperones protect cells from stressful insults. Diabetes is generally associated with lower HSPs (Padmalayam, 2014). A low HSP state promotes increased activation of inflammatory cytokines (Hooper and Hooper, 2009). Moreover, exercise training is a non-pharmacological and noninvasive treatment method. To avoid any intense exercise (up to 33 m/min for 20 min) induced increases in inflammatory markers and adverse effect on hyperglycemia in Type 1 diabetic animals (Bortolon et al., 2012), this study chose a moderate exercise regimen (10 m/min for 60 min). The current study explored the effect of moderate exercise on pain perception and also on the changes in enkephalin as well as the inflammatory mediators in the peripheral nervous system of Type 1 diabetic animals after 6 weeks of exercise regimen. Hence, moderate exercise may provide an alternative therapeutic approach for this disabling and difficult-to-treat complication of diabetes avoiding systemic side effects of the treatment.
pressure was applied through a cone-shaped plastic tip (diameter = 1 mm) onto the dorsal surface of the hindpaw. The tip was positioned between the third and fourth metatarsi, and force applied until the rat attempted to withdraw its paw (paw withdrawal threshold to pressure). For each animal, pain thresholds (mean of 3 consecutive responses, expressed in grams) were determined by a blinded observer.
Cold allodynia Animals were placed on a mesh floor 18 in. above the table, and after 20 min of acclimatization, 0.1 mL of acetone was sprayed onto the plantar surface of the hind paw using a 1 cc syringe. The latency of the response was measured as the delay to a withdrawal response of either flinching or licking with a cut off limit at 40 s. A total of 3 responses from each animal were assessed at 5 min intervals by a blinded observer.
Material and methods Experimental design Experiments were performed on male Sprague Dawley rats weighing approximately 250–280 g (Charles River, USA) in compliance with approved institutional animal care and use protocols. The rats were divided into four groups: naïve controls (n = 8), control exercise (n = 8), diabetic sedentary (n = 8) and diabetic exercise (n = 8) and were trained to run in the motorized wheels for six weeks. For the nociceptive analysis, animals from each group were tested after six weeks of exercise training by a blinded observer. After the completion of the study, all the animals were humanely euthanized; 5 animals per group were used for fresh tissue collection and 3 animals per group were used immunohistochemical studies. Diabetes induction Sprague Dawley rats were injected with 50 mg/kg intraperitoneal injection of streptozotocin (STZ) to induce diabetes. One week after the onset of diabetes, rats were tested for diabetes by measuring blood glucose levels, and animals with blood glucose levels ≥250 mg/dL were used as diabetic. Exercise training Before the start of the exercise regimen, animals were acclimatized in the motorized wheel without running for one day. The exercised groups were pre-trained the next 2 days for 20 min at a lower speed of 5 m/min. During the exercise regimen, animals were placed in motorized running wheels for 60 min per day at a speed of 10 m/min, five days a week, for 6 weeks starting at 1 week of diabetes under the supervision of designated researcher. They were given break for water after 20 min of running and were trained between 8 am to 12 pm every day. The naïve controls and diabetic sedentary groups were placed in the apparatus without exercise. Behavioral studies Thermal hyperalgesia Rats were placed in individual enclosures on a glass plate maintained at 30 °C. After a 30-min habituation period the plantar surface of the paw was exposed to a beam of radiant heat applied through the glass floor. The radiant bulb and timer simultaneously turned off by paw withdrawal or at the 20 s cut-off time. Testing was performed at 5 min intervals by a blinded observer in triplicates. Mechanical hyperalgesia Mechanical nociceptive thresholds were assessed by an analgesimeter (Ugo Basile, Comerio, VA, Italy). A linearly increasing
Blood collection The animals were deeply anesthetized before the terminal bleeding was performed. The chest cavity was opened to visualize the heart, before the perfusion or fresh tissue collection; a 21 gauge needle was introduced into the right ventricle to collect blood for the measurement of lipid profile.
Western blot Pooled samples of L4–L6 DRG were homogenized with lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2% SDS, 10% glycerol, and 1:100 dilution of protease inhibitor mixture and phosphatase inhibitor mixture (Sigma)), the homogenized cells and tissues were centrifuged at 10,000 ×g for 10 min at 4 °C. Total protein from DRG (20 μg of protein per lane) was separated by PAGE, transferred to an Immobilon-P membrane (0.45 μm; Millipore), blocked with 5% nonfat milk, and then incubated with the primary antibody. Primary antibodies included an antibody against p-p38, GFAP, TNFα, IL1β (1:1000; EMDMillipore, USA), enkephalin (1:500; Abcam, USA), anti-VR1 and TRPM8 (1:1000; Santa Cruz Biotechnology, USA) followed by horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (1:5000; GE Healthcare) and visualized with ECL (Pierce) using a PC-based image analysis system (ChemiDoc XRS System; Bio-Rad Laboratories). The membranes were stripped and re-probed with mouse anti-β-actin (1: 2000; Sigma Aldrich) as a loading control. The intensity of each band was determined by quantitative chemiluminescence using a PC-based image analysis system (ChemiDoc XRS System, Bio-Rad Laboratories).
Immunocytochemistry One day after the behavioral analysis, rats were anesthetized with Ketamine–xylazine (i.p.; 100–10 mg/kg). Animals were then perfused transcardially with 0.9% NaCl followed by Zamboni's fixative. The DRG were removed, post-fixed with Zamboni's fixative for 2 h, and then cryo-protected with 30% sucrose in phosphate buffered saline (PBS) overnight. All tissues were cryostat sectioned at 10 μm, collected on gelatin-coated slides, fixed with 2% paraformaldehyde for 15 min, washed with PBS, and incubated with blocking solution (PBS with 1% normal goat serum and 0.3% Triton X-100) for 1 h, then washed once. The sections of DRG were incubated with the primary antibody for 2 h at room temperature and washed three times. After incubation in the secondary fluorescent antibody, Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Molecular Probes, Eugene, OR), for 1 h at room temperature, the specimens were washed 3 times and mounted in water-based Fluoromount G (Electron Microscopy Sciences, Fort Washington, PA).
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Statistical analysis
Exercise attenuated thermal hyperalgesia in the diabetic exercise group
The statistical significance of the difference between groups was determined by ANOVA (Systat 9) using Bonferroni's correction for the multiple post hoc analyses. All results are expressed as mean ± SEM.
Exercise also didn't alter the thermal sensitivity between the control sedentary and control exercised groups. On the other hand, there was a significant change in the thermal sensitivity between the control sedentary (11.17 ± 0.75 s) and diabetic sedentary (8.073 ± 0.52 s; *P b 0.05; Fig. 2b) groups. Significant differences were identified between the diabetic sedentary (8.073 ± 0.52 s) and diabetic exercise groups (12.21 ± 0.59 s; **P b 0.005).
Results Exercise did not alter body weight and blood glucose levels in STZ-diabetic animals
Exercise recessed the development of cold allodynia in diabetic exercise group
The Type 1 model of STZ-diabetic rats presented lower body weight compared to control rats. The change in body weight in the STZ-diabetic sedentary group was not significantly different than that of the STZdiabetic exercise group (Fig. 1a). Physical exercise did not decrease the blood glucose levels in diabetic exercised animals after 6 weeks of exercise regimen. Furthermore, the blood glucose level was also unaffected by exercise in control exercised rats (Fig. 1b). We measured blood glucose levels before and after 6 weeks of exercise which showed a minimal change in the diabetic exercise and sedentary groups.
The latency to withdraw from a cold stimulus was significantly decreased in the diabetic sedentary group compared to the control sedentary animals (dia-sed 1.55 ± 0.56 s; con-sed 12.51 ± 4.00 s; ##P b 0.001). The diabetic exercise group (5.52 ± 1.23 s) showed an improved withdrawal latency to the stimulus compared to the diabetic sedentary group (dia-sed 1.55 ± 0.56 s; **P b 0.005; Fig. 2c). Serum triglyceride levels decreased in exercised diabetic rats compared to sedentary rats
Exercise delayed the development of mechanical hyperalgesia in diabetic rats
The 6-week physical training program did not modify total cholesterol and HDL levels (data not shown); although LDL cholesterol was slightly decreased in the diabetic exercise group as compared to the sedentary group, but was not significantly different. Serum triglyceride level was increased in diabetic sedentary rats. Exercise decreased the serum triglyceride levels in the diabetic exercise group (80 ± 25 mg/dL) compared to that of the diabetic sedentary rats (191 ± 28 mg/dL; **P b 0.005, Fig. 3). The triglyceride levels of the control exercise group (con-ex 80 ± 20 mg/dL) were not different than that of the control sedentary group (con-sed 85 ± 14 mg/dL).
The sedentary STZ-diabetic group displayed significant alterations in the mechanical pain threshold as compared to the control sedentary and control-exercised groups. In a separate weekly study, diabetic animals tested for mechanical and thermal hyperalgesia every 2 weeks after the onset of diabetes, exhibited only mechanical hyperalgesia at 2 weeks after hyperglycemia (data not shown). In the current study, a significant reduction in paw withdrawal threshold by Randall Selitto test of mechanical hyperalgesia was detected in the diabetic sedentary group compared to the control sedentary group (con-sed 118 ± 4.36 g; dia-sed 62.67 ± 3.2 g; ##P b 0.001) at the end of 6 weeks post exercise regimen. The paw withdrawal threshold of the control exercise group (117.5 ± 2.78 g) rats was not different than that of the control sedentary group. The mechanical pain threshold was improved in the diabetic exercise group (87 ± 5.45 g; **P b 0.005; Fig. 2a).
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Exercise improved the levels of endogenous opioid enkephalin in the DRG Western blot analysis of DRG shows that diabetic sedentary animals had reduction in inhibitory neurotransmitter and endogenous opioid, enkephalin expression compared to the control exercised (***P b 0.005) and diabetic exercised groups (Figs. 4 a, b). Six weeks of
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Fig. 1. Changes in metabolic parameters before and after exercise. Control and diabetic animals gained weight significantly (*P b 0.001) 6 weeks after the onset of diabetes compared to their pre-diabetic states. STZ-diabetic rats presented lower body weight compared to control rats. The change in body weight in the STZ-diabetic sedentary group was not significantly different than that of the STZ-diabetic exercise group (a). Physical exercise did not decrease the blood glucose levels in diabetic exercised animals. Furthermore, the blood glucose level was also unaffected by exercise in control exercised rats (b). Animals treated with STZ have increased blood glucose level compared to the control animals (**P b 0.005) as measured 6 weeks post-diabetes.
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Fig. 2. Moderate exercise prevents the pain-related behaviors in diabetic animals. (a) Diabetic sedentary animals exhibited reduction in paw withdrawal threshold from mechanical pain stimuli after 6 weeks of exercise regimen compared to control-sedentary animals (con-sed 118 ± 4.36 g; dia-sed 62.67 ± 3.2 g; ##P b 0.001). The paw withdrawal threshold of the control exercise group (117.5 ± 2.78 g) rats was not different than that of the control sedentary group. The paw withdrawal threshold was improved in the diabetic exercise group (87 ± 5.45 g; **P b 0.005). (b) Diabetic-sedentary animals demonstrate thermal hyperalgesia as manifested by a decrease in withdrawal latency to noxious thermal stimuli compared to the controlsedentary (*P b 0.05) and control-exercised animals (**P b 0.005) six weeks after the exercise. There was a significant change between the diabetic sedentary (8.073 ± 0.52 s) and diabetic exercise groups (12.21 ± 0.59 s;**P b 0.005). (c) Diabetic-sedentary animals were more sensitive to cold stimuli as manifested by a decrease in withdrawal latency (dia-sed 1.55 ± 0.56 s; con-sed 12.51 ± 4.00 s; ##P b 0.001) compared to control-sedentary animals six weeks after the exercise regimen. The diabetic exercise group (5.52 ± 1.23 s) showed an improved latency to withdraw from the cold stimulus compared to the diabetic sedentary group (**P b 0.005; panel c). All data are presented as mean ± SEM, n = 8 per group.
moderate exercise demonstrated an increase in expression of enkephalin in the diabetic exercise group (***P b 0.001 compared to the sedentary group). There is a moderate increase in enkephalin also in the control exercise group (*P b 0.05 compared to the control sedentary group).
cytokine TNFα in the diabetic exercise group, which suggests that TNFα and IL1β may play a role in changing the pain threshold in these animals.
Exercise decreased levels of inflammatory mediators in the diabetic exercise group
Diabetic sedentary animals showed an increase in TRPM8 and TRPV1 (Transient Receptor Potential channel; activated by temperatures and chemical agents) in the DRG, whereas the diabetic exercise group showed a decrease in the TRPV1 (*P b 0.05 compared to the sedentary group) and TRPM8 ion channel expression after 6 weeks of exercise (Figs. 6 a, b). This correlates with our behavioral data where 6 weeks
Figs. 5 (a–e) show the levels of TNFα and IL1β in the DRG of diabetic and control rats with or without exercise at 7 weeks after STZ treatment. Western blot analysis of the diabetic sedentary animals showed an increase in TNFα and IL1β in the DRG compared to the control sedentary group, whereas the diabetic exercise group showed a decrease in TNFα as well as IL1β after 6 weeks of exercise. Immunohistochemical studies of DRG also showed a significant decrease in the proinflammatory
Exercise changed the levels of TRP ion channels in the DRG of the diabetic exercise group
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Fig. 3. Exercise prevents the increases in triglycerides in diabetic animals. The diabetic sedentary animals demonstrated increased serum triglycerides compared to control sedentary animals. Exercise decreased the serum triglyceride levels in the diabetic exercise group (80 ± 25 mg/dL) compared to those of diabetic sedentary rats (191 ± 28 mg/dL; **P b 0.005).
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Fig. 4. Exercise improved the expression of endogenous opioid in DRG of diabetic animals. Western blot analysis illustrated the reduction of enkephalin expression in the DRG of the diabetic sedentary group compared to the diabetic exercise group (*P b 0.001). Control exercise animals show a moderate alteration in expression of inhibitory neurotransmitter enkephalin in DRG compared to sedentary animals.
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Fig. 5. Exercise decreases TNFα and IL1β expression in DRG of diabetic animals after 6 weeks of exercise. (a) TNFα (red) immunostaining of DRG reveals increased expression of TNFα in DRG of diabetic sedentary animals. Six weeks of moderate exercise reduces TNFα expression in DRG of diabetic animals as shown by immunohistochemistry (bar = 50 μm) (b–e). Representative Western blots and bar graphs demonstrate the changes in TNFα (**P b 0.005) as well as the changes in IL1β in the DRG of exercise animals (*P b 0.05; n = 5 animals per group). Representative Western blots are showing two independent samples randomly chosen from the blot from each group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
after exercise, animals show an improvement in cold allodynia and thermal hyperalgesia. Exercise altered a number of stress related markers in the DRG of the diabetic exercise group
proinflammatory mediators TNFα and IL-1β in sedentary diabetic rats. Diabetic exercised animals exhibited increases in the expression of HSP70 in DRG (**P b 0.005; n = 5; Fig. 7 a). These changes in exercised animals may play a role in changing the pain threshold of these animals. Discussion
Diabetic animals show increased levels of stress related kinase p38 MAPK in the DRG. Six weeks of moderate exercise presented a decrease in the activation of the stress-related MAP kinase p38 in the diabetic exercise group (*P b 0.05 compared to the sedentary group; Fig. 7 b). The DRG exhibited low levels of HSP70 and increased
Our data clearly demonstrates that exercise delays the progression of thermal and mechanical hyperalgesia as well as cold allodynia in STZ-induced diabetes. This study focuses on the benefit of moderate exercise to reduce the risk of exercise induced hypoglycemia and
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Fig. 6. Exercise altered the expression of Transient Receptor Potential channels (TRP) in DRG of diabetic animals. (a) Western blot analysis illustrated the reduction of TRPV1 ion channel expression in the DRG of the diabetic exercise group compared to the diabetic sedentary group (*P b 0.05). (b) In diabetic sedentary animals thermal sensitivity has been altered and Western blot analysis of cold sensing channel TRPM8 showed an increase in expression in diabetic sedentary animals, whereas it was decreased in the diabetic exercised group (not statistically significant). Representative Western blots of DRG are showing random selection of two independent samples from each group.
inflammation in Type 1 diabetic animals. Studies have shown that there is a modest increase in IL-6, IL1β and TNF-α in Type 1 diabetic patients following intense exercise (Campbell et al., 2014; Nemet et al., 2002). The present study shows that moderate exercise may positively impact serum triglycerides; however our study was not designed to address this benefit. This study doesn't show any difference in HDL or LDL cholesterol. A number of previous studies also were unsuccessful to establish a significant improvement in the levels of HDL in diabetes patients, perhaps due to modest exercise intensities (Technical reviews, 2006). This study demonstrates that moderate exercise attenuates the increased levels of proinflammatory cytokines IL1β and TNF-α in the DRG of the STZ-diabetic animals. Elevated levels of inflammatory mediators such as triglycerides, proinflammatory cytokines and chemokines have been reported in association with diabetes (Brownlee, 2005). These mediators have been considered a link between inflammation and insulin resistance (Shoelson et al., 2006). Our results suggest that moderate exercise offers antinociceptive effects by increasing the release of the inhibitory neurotransmitter, enkephalin in the peripheral nervous system. This study also confirms previous findings that exercise may elevate the release of endogenous opioids, suggesting that these endogenous analgesics may be responsible for the antinociceptive effects observed in short-term exercise models (Koltyn, 2000; Shankarappa et al., 2011; Stagg et al., 2011). We have previously shown that HSV vector mediated transduction to release enkephalin in vitro or in vivo resulted in a reduction of p-PKC and concomitant reduction in p-p38, with a resultant reduction of pain in Type 1 diabetic animals (Chattopadhyay et al., 2008). Studies have also shown that overexpression of met-ENK in the spinal cord and pancreas significantly improves pancreatic inflammatory markers, and pain in a chronic pancreatitis model (Yang et al., 2008). Therefore it is possible that exercise induced release of enkephalin can prevent
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Con-Sed Con-Ex Dia-Sed Dia-Ex Fig. 7. Exercise reduces activation of p38 MAPK protein and improved the expression HSP70 in DRG of diabetic animals. (a) Western blot analysis illustrated the reduction in the expression of HSP70 in the DRG of the diabetic sedentary group and increased level of HSP70 in the diabetic exercise group (**P b 0.005; n = 5). (b) Western blot analysis of phospho-p38 of DRG reveals that diabetic sedentary animals have increased phosphorylation of p38 compared to control sedentary animals (*P b 0.05; n = 5). Exercise also decreased the activation of p38 MAPK in the DRG of the diabetic exercise group.
activation of p38 MAPK as well as decrease the inflammatory mediators TNFα and IL1β thereby reducing the pain in diabetic animals. Abnormal levels of pro-inflammatory cytokines are responsible for the development of vascular complications and increasing susceptibility to invasive microorganisms. Recently, Belotto et al. showed that moderate exercise improves leukocyte function and decreases inflammation in diabetic rats (Belotto et al., 2010). They also found that a moderate three-week exercise regimen on a treadmill decreases serum levels of TNF-α, CINC-2α/β, IL-1β, IL-6, CRP, and FFA in diabetic rats when compared to sedentary diabetic animals. Exercise also attenuated the increased responsiveness of leukocytes in diabetic patients when compared to those of controls, thereby diminishing ROS release by neutrophils and macrophages (Hatanaka et al., 2006). It has been shown that swimming for a long duration reduces thermal hyperalgesia in STZinduced diabetic female rats (Rossi et al., 2011). Therefore, exercise or lifestyle intervention strategies that include an exercise component may even delay or protect against the development of diabetic peripheral nerve complications. This study is focused on benefits of moderate exercise (10 m/min) in Type 1 diabetic animals. The vulnerability of hypoglycemia may be affected as a consequence of intense exercise (Chassin et al., 2007). Studies have also shown that diabetic rats 24 h after exercise exhibited an increase in the production of TNF-α and IL-1β resulting from intense exercise (up to 33 m/min in 20 min), which suggests that elevated inflammatory response were greater and lasted longer in diabetic animals than in non-diabetic control rats (Bortolon et al., 2012). This indicates that intense exercise in Type 1 diabetes can, not only elevate inflammatory mediators, but also cause hyperglycemia (Rosa et al., 2008a,b). Therefore it is not clear from different studies whether intense exercise
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can cause hypoglycemia or hyperglycemia. From the current study, it is evident that moderate exercise doesn't change blood glucose levels in Type 1 diabetic animals after 6 weeks of exercise regimen. Several studies have indicated that inflammation plays a role in the progression of diabetic microvascular complications such as retinopathy, nephropathy, neuropathy, macrovascular complications and atherosclerosis (Nguyen et al., 2012). It is well known that TNF-α, IL-1β, and CINC-2α/β have important effects on immune cells. At lower levels, these cytokines are important for the activation of endothelial cells and the expression of ligands for adhesion molecules for leukocyte integrins and endothelial selectins. Chronic inflammation results in fibrosis and loss of organ function, negatively affecting the health of diabetic patients (King, 2008). A 3-week moderate exercise regimen on a treadmill (30 min/day, 6 days a week) decreased serum levels of TNFα, CINC2α/β, IL1β, IL-6, CRP and FFA in Type 1 diabetic rats when compared with sedentary diabetic animals (Belotto et al., 2010). Therefore, it is important to select an exercise regimen for Type 1 diabetic subjects which can reduce inflammation. Our data from this study indicates that moderate exercise can reduce thermal hyperalgesia in STZ-hyperglycemic rats. Others have shown that low-intensity exercise decreases the nociception induced by thermal and chemical stimuli after exercise (Quintero et al., 2000); however, their methodology was different with respect to the intensity, the duration of exercise and the duration of the painful stimuli (Quintero et al., 2000). Moreover, Kuphal et al. have demonstrated that extended swimming exercise reduces hyperalgesia induced by intraplantar injection of formalin, or by partial injury in rodent peripheral nerves (Kuphal et al., 2007). In conclusion, the present study demonstrates that 6 weeks of exercise training for 60 min/day, 5 days/week reduces neuroinflammation in the DRG of the STZ-diabetic rats, an experimental model of Type 1 diabetes. This study also suggests that exercise training prevents increases in TRP associated channels and reduces the production of proinflammatory cytokines TNFα and IL1β. The changes in expression of TRPV1 and TRPM8 may have contributed to the altered thermal sensitivity. Other studies have previously shown that DRG neurons dissociated from diabetic hyperalgesic mice exhibited a change in TRPM8-mediated currents as compared to non-diabetic mice (Pabbidi and Pharmacology, S.I.U.a.C., 2007). It is also known that TNF-α can directly activate TNF-α receptors on peripheral nerve terminals, amplify hyperalgesic responses (Cunha et al., 1992; Junger and Sorkin, 2000), and release enzymes to enhance inflammation. It has been shown that TNF-α mediated increase in TRPV1 promotes painful inflammatory processes locally, impacting subsequent responses in the immune system (Spicarova and Palecek, 2010). Kochukov et al. also demonstrated up-regulation of TRP channel in human synovial cells after preincubation with TNFα (Kochukov et al., 2009). Our studies suggest that the decrease in inflammatory mediators in the DRG with exercise may reduce the expression of TRP channels, thereby decreasing pain sensitivity. The pathogenesis of diabetic neuropathy is multifactorial. Chronic hyperglycemia plays an important role in the development of neuropathy causing hyperalgesia, followed by hypoalgesia due to degeneration of the peripheral nerves. Recent studies have shown that treadmill training for 10 weeks improves hind limb motor function and prevents morphometric alterations in diabetic rats subjected to sciatic nerve crush (Malysz et al., 2010). Selagzi and colleagues investigated the effects of swimming training on development of peripheral neuropathy in STZinduced diabetic male rats (Selagzi et al., 2008). Similar to our findings, they observed that swimming exercise protocol did not influence blood glucose levels; however, exercise restored body weight, compound muscle action potential amplitude and potential latency which are the parameters that define motor dysfunction in diabetic animals. In contrast to previous studies, we evaluated male rats for heat, cold and mechanical sensitivity rather than motor dysfunction. Our findings reveal new aspects of how exercise influences Type 1 diabetic animals. HSP levels are inversely correlated with insulin resistance (Hooper and
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Hooper, 2009), inflammatory cytokines, GLUT4 levels, and mitochondrial function (Bruce et al., 2003). A low HSP state consequently promotes increased activation of inflammatory cytokines. Recent studies on treadmill exercise showed increases in expression of Hsp72 in the spinal cord and peripheral nerves (Chen et al., 2013). p38 mitogenactivated protein kinase (MAPK) is known to play a significant role in inflammatory processes in diabetes. Other studies suggest that p38 MAPK inhibition in early stages of diabetes can prevent sensory nerve function (Cheng et al., 2010). This study shows that exercise increases HSP70 levels in DRG and inhibits the activation of p38 MAPK. In addition, moderate exercise training for 6 weeks reduces TRP ion channels, thereby attenuating pain behaviors in STZ-induced diabetic rats. Therefore, we conclude that moderate physical exercise can induce the release of enkephalin which may reduce inflammatory mechanisms in DRG of diabetic rats offering an efficient strategy to protect diabetics against vascular and peripheral nerve complications. Furthermore, we also found that moderate exercise training reduced the levels of inflammatory mediators while improving hyperalgesia without influencing blood glucose levels. This study suggests that exercise may provide a better way to improve diabetic neuropathy, eventually leading to a novel therapeutic approach for this debilitating condition. Authors' contributions HY carried out the behavior tests and biochemical assays. DI and MF carried out the behavior tests and exercise regimen. VT carried out biochemical assays and reviewed the manuscript. MC contributed to the design and analysis of the study, behavior test and wrote the manuscript. Conflict of interests The authors declare that they have no conflict of interests. Acknowledgments This work was funded by American Diabetes Association (Grant #712-BS-021) to MC. We acknowledge Jessica Meyers, Ryan Pattnaik and Valerie Zeer for helping with the animal exercise. References Barbano, R., Hart-Gouleau, S., Pennella-Vaughan, J., Dworkin, R.H., 2003. Pharmacotherapy of painful diabetic neuropathy. Curr. Pain Headache Rep. 7, 169–177. Belotto, M.F., et al., 2010. Moderate exercise improves leucocyte function and decreases inflammation in diabetes. Clin. Exp. Immunol. 162, 237–243. Bortolon, J.R., et al., 2012. Persistence of inflammatory response to intense exercise in diabetic rats. Exp. Diabetes Res. 213986. Brownlee, M., 2005. The pathobiology of diabetic complications: a unifying mechanism. Diabetes 54, 1615–1625. Bruce, C.R., Carey, A.L., Hawley, J.A., Febbraio, M.A., 2003. Intramuscular heat shock protein 72 and heme oxygenase-1 mRNA are reduced in patients with type 2 diabetes: evidence that insulin resistance is associated with a disturbed antioxidant defense mechanism. Diabetes 52, 2338–2345. Campbell, M.D., et al., 2014. Metabolic implications when employing heavy pre- and postexercise rapid-acting insulin reductions to prevent hypoglycaemia in type 1 diabetes patients: a randomised clinical trial. PLoS One 9, e97143. Chassin, L.J., Wilinska, M.E., Hovorka, R., 2007. Intense exercise in type 1 diabetes: exploring the role of continuous glucose monitoring. J. Diabetes Sci. Technol. 1, 570–573. Chattopadhyay, M., Mata, M., Fink, D.J., 2008. Continuous delta-opioid receptor activation reduces neuronal voltage-gated sodium channel (NaV1.7) levels through activation of protein kinase C in painful diabetic neuropathy. J. Neurosci. 28, 6652–6658. Chen, Y.W., Hsieh, P.L., Chen, Y.C., Hung, C.H., Cheng, J.T., 2013. Physical exercise induces excess hsp72 expression and delays the development of hyperalgesia and allodynia in painful diabetic neuropathy rats. Anesth. Analg. 116, 482–490. Cheng, H.T., et al., 2010. p38 mediates mechanical allodynia in a mouse model of type 2 diabetes. Mol. Pain 6, 28. Cunha, F.Q., Poole, S., Lorenzetti, B.B., Ferreira, S.H., 1992. The pivotal role of tumour necrosis factor alpha in the development of inflammatory hyperalgesia. Br. J. Pharmacol. 107, 660–664. Empl, M., et al., 2001. TNF-alpha expression in painful and nonpainful neuropathies. Neurology 56, 1371–1377.
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Galloway, C., Chattopadhyay, M., 2013. Increases in inflammatory mediators in DRG implicate in the pathogenesis of painful neuropathy in type 2 diabetes. Cytokine 63, 1–5. Gonzalez-Clemente, J.M., et al., 2005. Diabetic neuropathy is associated with activation of the TNF-alpha system in subjects with type 1 diabetes mellitus. Clin. Endocrinol. 63, 525–529. Hatanaka, E., Monteagudo, P.T., Marrocos, M.S., Campa, A., 2006. Neutrophils and monocytes as potentially important sources of proinflammatory cytokines in diabetes. Clin. Exp. Immunol. 146, 443–447. Herder, C., et al., 2009. Subclinical inflammation and diabetic polyneuropathy: MONICA/ KORA Survey F3 (Augsburg, Germany). Diabetes Care 32, 680–682. Hooper, P.L., Hooper, P.L., 2009. Inflammation, heat shock proteins, and type 2 diabetes. Cell Stress Chaperones 14, 113–115. Igarashi, M., et al., 1999. Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways. J. Clin. Invest. 103, 185–195. Junger, H., Sorkin, L.S., 2000. Nociceptive and inflammatory effects of subcutaneous TNFalpha. Pain 85, 145–151. Kaul, K., Hodgkinson, A., Tarr, J.M., Kohner, E.M., Chibber, R., 2010. Is inflammation a common retinal–renal–nerve pathogenic link in diabetes? Curr. Diabetes Rev. 6, 294–303. King, G.L., 2008. The role of inflammatory cytokines in diabetes and its complications. J. Periodontol. 79, 1527–1534. Kochukov, M.Y., et al., 2009. Tumor necrosis factor-alpha (TNF-alpha) enhances functional thermal and chemical responses of TRP cation channels in human synoviocytes. Mol. Pain 5, 49. Koltyn, K.F., 2000. Analgesia following exercise: a review. Sports Med. 29, 85–98. Kuphal, K.E., Fibuch, E.E., Taylor, B.K., 2007. Extended swimming exercise reduces inflammatory and peripheral neuropathic pain in rodents. J. Pain 8, 989–997. Malysz, T., et al., 2010. Beneficial effects of treadmill training in experimental diabetic nerve regeneration. Clinics (Sao Paulo) 65, 1329–1337. Nemet, D., Oh, Y., Kim, H.S., Hill, M., Cooper, D.M., 2002. Effect of intense exercise on inflammatory cytokines and growth mediators in adolescent boys. Pediatrics 110, 681–689. Nguyen, D.V., Shaw, L.C., Grant, M.B., 2012. Inflammation in the pathogenesis of microvascular complications in diabetes. Front. Endocrinol. 3, 170. Pabbidi, R.M., Pharmacology, S.I.U.a.C., 2007. Role of Transient Receptor Potential Channels in Diabetic Peripheral Neuropathy. Southern Illinois University at Carbondale.
Padmalayam, I., 2014. The heat shock response: its role in pathogenesis of type 2 diabetes and its complications, and implications for therapeutic intervention. Discov. Med. 18, 29–39. Quintero, L., et al., 2000. Long-lasting delayed hyperalgesia after subchronic swim stress. Pharmacol. Biochem. Behav. 67, 449–458. Rosa, J.S., et al., 2008a. Altered kinetics of interleukin-6 and other inflammatory mediators during exercise in children with type 1 diabetes. J. Investig. Med. Off. Publ. Am. Fed. Clin. Res. 56, 701–713. Rosa, J.S., et al., 2008b. Sustained IL-1alpha, IL-4, and IL-6 elevations following correction of hyperglycemia in children with type 1 diabetes mellitus. Pediatr. Diabetes 9, 9–16. Rossi, D.M., Valenti, V.E., Navega, M.T., 2011. Exercise training attenuates acute hyperalgesia in streptozotocin-induced diabetic female rats. Clinics (Sao Paulo) 66, 1615–1619. Selagzi, H., Buyukakilli, B., Cimen, B., Yilmaz, N., Erdogan, S., 2008. Protective and therapeutic effects of swimming exercise training on diabetic peripheral neuropathy of streptozotocin-induced diabetic rats. J. Endocrinol. Investig. 31, 971–978. Shankarappa, S.A., Piedras-Renteria, E.S., Stubbs Jr., E.B., 2011. Forced-exercise delays neuropathic pain in experimental diabetes: effects on voltage-activated calcium channels. J. Neurochem. 118, 224–236. Shoelson, S.E., Lee, J., Goldfine, A.B., 2006. Inflammation and insulin resistance. J. Clin. Invest. 116, 1793–1801. Spicarova, D., Palecek, J., 2010. Tumor necrosis factor alpha sensitizes spinal cord TRPV1 receptors to the endogenous agonist N-oleoyldopamine. J. Neuroinflammation 7, 49. Stagg, N.J., et al., 2011. Regular exercise reverses sensory hypersensitivity in a rat neuropathic pain model: role of endogenous opioids. Anesthesiology 114, 940–948. Technical reviews. Diabetes Care 29 (Suppl. 1), S70–S72. Uceyler, N., Rogausch, J.P., Toyka, K.V., Sommer, C., 2007. Differential expression of cytokines in painful and painless neuropathies. Neurology 69, 42–49. Van Acker, K., et al., 2009. Prevalence and impact on quality of life of peripheral neuropathy with or without neuropathic pain in type 1 and type 2 diabetic patients attending hospital outpatients clinics. Diabetes Metab. 35, 206–213. Yang, H., et al., 2008. Enkephalin-encoding herpes simplex virus-1 decreases inflammation and hotplate sensitivity in a chronic pancreatitis model. Mol. Pain 4, 8.
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Regular Article
Moderate exercise training attenuates inflammatory mediators in DRG of Type 1 diabetic rats HaeJee Yoon b, Vikram Thakur a, Danielle Isham b, Mona Fayad b, Munmun Chattopadhyay a,⁎ a b
Center of Excellence in Diabetes and Obesity, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA Department of Neurology, University of Michigan, Ann Arbor, MI, USA
a r t i c l e
i n f o
Article history: Received 10 October 2014 Revised 22 January 2015 Accepted 8 March 2015 Available online 14 March 2015 Keywords: Exercise Diabetes Neuropathy Pain Inflammation
a b s t r a c t Painful neuropathy is a long-term and difficult to treat complication of diabetes that affects 25% of diabetic patients and interferes with their quality of life. Unfortunately, available medical treatments are relatively ineffective due to dependency and addiction. Emerging research indicates that moderate-to-vigorous physical activity provides health-related benefits. However, adequate data is not available to determine whether regular exercise would prevent or delay the development of painful neuropathy in subjects with Type 1 diabetes. This study demonstrates the significance of moderate exercise in the amelioration of pain in animals with Type 1 diabetes after 6 weeks of exercise paradigm. After initial acclimatization, streptozotocin-diabetic animals were placed in motorized running wheels for 60 min per day, for five days a week for 6 weeks starting at one week after diabetes. A growing body of evidence suggests that the release of proinflammatory cytokines plays an important role in the development and persistence of pain. This study demonstrates that moderate exercise increases the expression of inhibitory neurotransmitter enkephalin and also reduces the presence of a number of proinflammatory cytokines in the dorsal root ganglia (DRG), subsequently impeding the development of neuropathy along with a decrease in the voltage gated ion channels in the DRG. Overall, the study suggests that exercise may provide an alternate route of treatment of painful neuropathy in Type 1 diabetic subjects by decreasing the use of pain medications, thereby providing a more useful and efficient way for pain management. © 2015 Published by Elsevier Inc.
Introduction Pain is a significant consequence of diabetic neuropathy. Diabetes mellitus is the most common cause of neuropathy in the United States, and 25% of diabetic patients with neuropathy suffer from neuropathic pain, resulting in a significant adverse effect on quality of life measures (Van Acker et al., 2009). Unfortunately, available medical treatment is relatively ineffective with limited efficacy and is complicated by side effects and dependency (Barbano et al., 2003). Accumulating evidence suggests that the activation of inflammatory cascades in the peripheral and central nervous systems plays a key role in the development and persistence of neuropathic pain states induced by physical or toxic injury to peripheral nerve (Gonzalez-Clemente et al., 2005; Herder et al., 2009; King, 2008). In diabetes, there is evidence of systemic immune activation. Patients with painful neuropathy show increased levels of IL-2 in blood and increased levels of TNFα mRNA and protein in blood (Uceyler et al., 2007). The elevated level of serum TNFα in Type 1 diabetes ⁎ Corresponding author at: Department of Biomedical Sciences, Center of Excellence in Diabetes and Obesity, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA. E-mail address: [email protected] (M. Chattopadhyay).
http://dx.doi.org/10.1016/j.expneurol.2015.03.006 0014-4886/© 2015 Published by Elsevier Inc.
patients suggests that TNFα may play a pathogenic role in the development of diabetic neuropathy (Gonzalez-Clemente et al., 2005; Kaul et al., 2010). Studies on patients with Type 2 with or without polyneuropathy exhibit a different immune profile and specific neuropathic deficits, suggesting that inflammation is associated with diabetic neuropathic impairments involving a number of immune mediators such as C-peptide, IL-2 receptor, IL1β and TNFα (Empl et al., 2001; Uceyler et al., 2007). Previously, we have shown that the inflammatory mediators in the dorsal root ganglia are altered with the development of pain in Type 2 model of diabetes (Galloway and Chattopadhyay, 2013). Although earlier studies have demonstrated the effects of moderate to intense physical exercise on pain perception (Rossi et al., 2011), not many have shown the effects of moderate exercise that may change the endogenous opioid content as well as the levels of inflammation in DRG of Type 1 diabetic animals with painful neuropathy. Hyperglycemia causes p38 mitogen-activated protein (p38) kinase activation (Igarashi et al., 1999), which can be induced by changes in the release of proinflammatory cytokines. Previously we have shown that viral vector mediated release of enkephalin modified the activation of p38 MAPK in Type 1 diabetic DRG (Chattopadhyay et al., 2008). This study also explores the possibility whether exercise can alter the endogenous opioid, enkephalin and stress associated markers, thus reducing pain related behaviors. It is well known that the heat shock protein (HSP) molecular
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chaperones protect cells from stressful insults. Diabetes is generally associated with lower HSPs (Padmalayam, 2014). A low HSP state promotes increased activation of inflammatory cytokines (Hooper and Hooper, 2009). Moreover, exercise training is a non-pharmacological and noninvasive treatment method. To avoid any intense exercise (up to 33 m/min for 20 min) induced increases in inflammatory markers and adverse effect on hyperglycemia in Type 1 diabetic animals (Bortolon et al., 2012), this study chose a moderate exercise regimen (10 m/min for 60 min). The current study explored the effect of moderate exercise on pain perception and also on the changes in enkephalin as well as the inflammatory mediators in the peripheral nervous system of Type 1 diabetic animals after 6 weeks of exercise regimen. Hence, moderate exercise may provide an alternative therapeutic approach for this disabling and difficult-to-treat complication of diabetes avoiding systemic side effects of the treatment.
pressure was applied through a cone-shaped plastic tip (diameter = 1 mm) onto the dorsal surface of the hindpaw. The tip was positioned between the third and fourth metatarsi, and force applied until the rat attempted to withdraw its paw (paw withdrawal threshold to pressure). For each animal, pain thresholds (mean of 3 consecutive responses, expressed in grams) were determined by a blinded observer.
Cold allodynia Animals were placed on a mesh floor 18 in. above the table, and after 20 min of acclimatization, 0.1 mL of acetone was sprayed onto the plantar surface of the hind paw using a 1 cc syringe. The latency of the response was measured as the delay to a withdrawal response of either flinching or licking with a cut off limit at 40 s. A total of 3 responses from each animal were assessed at 5 min intervals by a blinded observer.
Material and methods Experimental design Experiments were performed on male Sprague Dawley rats weighing approximately 250–280 g (Charles River, USA) in compliance with approved institutional animal care and use protocols. The rats were divided into four groups: naïve controls (n = 8), control exercise (n = 8), diabetic sedentary (n = 8) and diabetic exercise (n = 8) and were trained to run in the motorized wheels for six weeks. For the nociceptive analysis, animals from each group were tested after six weeks of exercise training by a blinded observer. After the completion of the study, all the animals were humanely euthanized; 5 animals per group were used for fresh tissue collection and 3 animals per group were used immunohistochemical studies. Diabetes induction Sprague Dawley rats were injected with 50 mg/kg intraperitoneal injection of streptozotocin (STZ) to induce diabetes. One week after the onset of diabetes, rats were tested for diabetes by measuring blood glucose levels, and animals with blood glucose levels ≥250 mg/dL were used as diabetic. Exercise training Before the start of the exercise regimen, animals were acclimatized in the motorized wheel without running for one day. The exercised groups were pre-trained the next 2 days for 20 min at a lower speed of 5 m/min. During the exercise regimen, animals were placed in motorized running wheels for 60 min per day at a speed of 10 m/min, five days a week, for 6 weeks starting at 1 week of diabetes under the supervision of designated researcher. They were given break for water after 20 min of running and were trained between 8 am to 12 pm every day. The naïve controls and diabetic sedentary groups were placed in the apparatus without exercise. Behavioral studies Thermal hyperalgesia Rats were placed in individual enclosures on a glass plate maintained at 30 °C. After a 30-min habituation period the plantar surface of the paw was exposed to a beam of radiant heat applied through the glass floor. The radiant bulb and timer simultaneously turned off by paw withdrawal or at the 20 s cut-off time. Testing was performed at 5 min intervals by a blinded observer in triplicates. Mechanical hyperalgesia Mechanical nociceptive thresholds were assessed by an analgesimeter (Ugo Basile, Comerio, VA, Italy). A linearly increasing
Blood collection The animals were deeply anesthetized before the terminal bleeding was performed. The chest cavity was opened to visualize the heart, before the perfusion or fresh tissue collection; a 21 gauge needle was introduced into the right ventricle to collect blood for the measurement of lipid profile.
Western blot Pooled samples of L4–L6 DRG were homogenized with lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2% SDS, 10% glycerol, and 1:100 dilution of protease inhibitor mixture and phosphatase inhibitor mixture (Sigma)), the homogenized cells and tissues were centrifuged at 10,000 ×g for 10 min at 4 °C. Total protein from DRG (20 μg of protein per lane) was separated by PAGE, transferred to an Immobilon-P membrane (0.45 μm; Millipore), blocked with 5% nonfat milk, and then incubated with the primary antibody. Primary antibodies included an antibody against p-p38, GFAP, TNFα, IL1β (1:1000; EMDMillipore, USA), enkephalin (1:500; Abcam, USA), anti-VR1 and TRPM8 (1:1000; Santa Cruz Biotechnology, USA) followed by horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (1:5000; GE Healthcare) and visualized with ECL (Pierce) using a PC-based image analysis system (ChemiDoc XRS System; Bio-Rad Laboratories). The membranes were stripped and re-probed with mouse anti-β-actin (1: 2000; Sigma Aldrich) as a loading control. The intensity of each band was determined by quantitative chemiluminescence using a PC-based image analysis system (ChemiDoc XRS System, Bio-Rad Laboratories).
Immunocytochemistry One day after the behavioral analysis, rats were anesthetized with Ketamine–xylazine (i.p.; 100–10 mg/kg). Animals were then perfused transcardially with 0.9% NaCl followed by Zamboni's fixative. The DRG were removed, post-fixed with Zamboni's fixative for 2 h, and then cryo-protected with 30% sucrose in phosphate buffered saline (PBS) overnight. All tissues were cryostat sectioned at 10 μm, collected on gelatin-coated slides, fixed with 2% paraformaldehyde for 15 min, washed with PBS, and incubated with blocking solution (PBS with 1% normal goat serum and 0.3% Triton X-100) for 1 h, then washed once. The sections of DRG were incubated with the primary antibody for 2 h at room temperature and washed three times. After incubation in the secondary fluorescent antibody, Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Molecular Probes, Eugene, OR), for 1 h at room temperature, the specimens were washed 3 times and mounted in water-based Fluoromount G (Electron Microscopy Sciences, Fort Washington, PA).
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Statistical analysis
Exercise attenuated thermal hyperalgesia in the diabetic exercise group
The statistical significance of the difference between groups was determined by ANOVA (Systat 9) using Bonferroni's correction for the multiple post hoc analyses. All results are expressed as mean ± SEM.
Exercise also didn't alter the thermal sensitivity between the control sedentary and control exercised groups. On the other hand, there was a significant change in the thermal sensitivity between the control sedentary (11.17 ± 0.75 s) and diabetic sedentary (8.073 ± 0.52 s; *P b 0.05; Fig. 2b) groups. Significant differences were identified between the diabetic sedentary (8.073 ± 0.52 s) and diabetic exercise groups (12.21 ± 0.59 s; **P b 0.005).
Results Exercise did not alter body weight and blood glucose levels in STZ-diabetic animals
Exercise recessed the development of cold allodynia in diabetic exercise group
The Type 1 model of STZ-diabetic rats presented lower body weight compared to control rats. The change in body weight in the STZ-diabetic sedentary group was not significantly different than that of the STZdiabetic exercise group (Fig. 1a). Physical exercise did not decrease the blood glucose levels in diabetic exercised animals after 6 weeks of exercise regimen. Furthermore, the blood glucose level was also unaffected by exercise in control exercised rats (Fig. 1b). We measured blood glucose levels before and after 6 weeks of exercise which showed a minimal change in the diabetic exercise and sedentary groups.
The latency to withdraw from a cold stimulus was significantly decreased in the diabetic sedentary group compared to the control sedentary animals (dia-sed 1.55 ± 0.56 s; con-sed 12.51 ± 4.00 s; ##P b 0.001). The diabetic exercise group (5.52 ± 1.23 s) showed an improved withdrawal latency to the stimulus compared to the diabetic sedentary group (dia-sed 1.55 ± 0.56 s; **P b 0.005; Fig. 2c). Serum triglyceride levels decreased in exercised diabetic rats compared to sedentary rats
Exercise delayed the development of mechanical hyperalgesia in diabetic rats
The 6-week physical training program did not modify total cholesterol and HDL levels (data not shown); although LDL cholesterol was slightly decreased in the diabetic exercise group as compared to the sedentary group, but was not significantly different. Serum triglyceride level was increased in diabetic sedentary rats. Exercise decreased the serum triglyceride levels in the diabetic exercise group (80 ± 25 mg/dL) compared to that of the diabetic sedentary rats (191 ± 28 mg/dL; **P b 0.005, Fig. 3). The triglyceride levels of the control exercise group (con-ex 80 ± 20 mg/dL) were not different than that of the control sedentary group (con-sed 85 ± 14 mg/dL).
The sedentary STZ-diabetic group displayed significant alterations in the mechanical pain threshold as compared to the control sedentary and control-exercised groups. In a separate weekly study, diabetic animals tested for mechanical and thermal hyperalgesia every 2 weeks after the onset of diabetes, exhibited only mechanical hyperalgesia at 2 weeks after hyperglycemia (data not shown). In the current study, a significant reduction in paw withdrawal threshold by Randall Selitto test of mechanical hyperalgesia was detected in the diabetic sedentary group compared to the control sedentary group (con-sed 118 ± 4.36 g; dia-sed 62.67 ± 3.2 g; ##P b 0.001) at the end of 6 weeks post exercise regimen. The paw withdrawal threshold of the control exercise group (117.5 ± 2.78 g) rats was not different than that of the control sedentary group. The mechanical pain threshold was improved in the diabetic exercise group (87 ± 5.45 g; **P b 0.005; Fig. 2a).
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Exercise improved the levels of endogenous opioid enkephalin in the DRG Western blot analysis of DRG shows that diabetic sedentary animals had reduction in inhibitory neurotransmitter and endogenous opioid, enkephalin expression compared to the control exercised (***P b 0.005) and diabetic exercised groups (Figs. 4 a, b). Six weeks of
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Fig. 1. Changes in metabolic parameters before and after exercise. Control and diabetic animals gained weight significantly (*P b 0.001) 6 weeks after the onset of diabetes compared to their pre-diabetic states. STZ-diabetic rats presented lower body weight compared to control rats. The change in body weight in the STZ-diabetic sedentary group was not significantly different than that of the STZ-diabetic exercise group (a). Physical exercise did not decrease the blood glucose levels in diabetic exercised animals. Furthermore, the blood glucose level was also unaffected by exercise in control exercised rats (b). Animals treated with STZ have increased blood glucose level compared to the control animals (**P b 0.005) as measured 6 weeks post-diabetes.
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Fig. 2. Moderate exercise prevents the pain-related behaviors in diabetic animals. (a) Diabetic sedentary animals exhibited reduction in paw withdrawal threshold from mechanical pain stimuli after 6 weeks of exercise regimen compared to control-sedentary animals (con-sed 118 ± 4.36 g; dia-sed 62.67 ± 3.2 g; ##P b 0.001). The paw withdrawal threshold of the control exercise group (117.5 ± 2.78 g) rats was not different than that of the control sedentary group. The paw withdrawal threshold was improved in the diabetic exercise group (87 ± 5.45 g; **P b 0.005). (b) Diabetic-sedentary animals demonstrate thermal hyperalgesia as manifested by a decrease in withdrawal latency to noxious thermal stimuli compared to the controlsedentary (*P b 0.05) and control-exercised animals (**P b 0.005) six weeks after the exercise. There was a significant change between the diabetic sedentary (8.073 ± 0.52 s) and diabetic exercise groups (12.21 ± 0.59 s;**P b 0.005). (c) Diabetic-sedentary animals were more sensitive to cold stimuli as manifested by a decrease in withdrawal latency (dia-sed 1.55 ± 0.56 s; con-sed 12.51 ± 4.00 s; ##P b 0.001) compared to control-sedentary animals six weeks after the exercise regimen. The diabetic exercise group (5.52 ± 1.23 s) showed an improved latency to withdraw from the cold stimulus compared to the diabetic sedentary group (**P b 0.005; panel c). All data are presented as mean ± SEM, n = 8 per group.
moderate exercise demonstrated an increase in expression of enkephalin in the diabetic exercise group (***P b 0.001 compared to the sedentary group). There is a moderate increase in enkephalin also in the control exercise group (*P b 0.05 compared to the control sedentary group).
cytokine TNFα in the diabetic exercise group, which suggests that TNFα and IL1β may play a role in changing the pain threshold in these animals.
Exercise decreased levels of inflammatory mediators in the diabetic exercise group
Diabetic sedentary animals showed an increase in TRPM8 and TRPV1 (Transient Receptor Potential channel; activated by temperatures and chemical agents) in the DRG, whereas the diabetic exercise group showed a decrease in the TRPV1 (*P b 0.05 compared to the sedentary group) and TRPM8 ion channel expression after 6 weeks of exercise (Figs. 6 a, b). This correlates with our behavioral data where 6 weeks
Figs. 5 (a–e) show the levels of TNFα and IL1β in the DRG of diabetic and control rats with or without exercise at 7 weeks after STZ treatment. Western blot analysis of the diabetic sedentary animals showed an increase in TNFα and IL1β in the DRG compared to the control sedentary group, whereas the diabetic exercise group showed a decrease in TNFα as well as IL1β after 6 weeks of exercise. Immunohistochemical studies of DRG also showed a significant decrease in the proinflammatory
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Fig. 3. Exercise prevents the increases in triglycerides in diabetic animals. The diabetic sedentary animals demonstrated increased serum triglycerides compared to control sedentary animals. Exercise decreased the serum triglyceride levels in the diabetic exercise group (80 ± 25 mg/dL) compared to those of diabetic sedentary rats (191 ± 28 mg/dL; **P b 0.005).
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Fig. 4. Exercise improved the expression of endogenous opioid in DRG of diabetic animals. Western blot analysis illustrated the reduction of enkephalin expression in the DRG of the diabetic sedentary group compared to the diabetic exercise group (*P b 0.001). Control exercise animals show a moderate alteration in expression of inhibitory neurotransmitter enkephalin in DRG compared to sedentary animals.
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Fig. 5. Exercise decreases TNFα and IL1β expression in DRG of diabetic animals after 6 weeks of exercise. (a) TNFα (red) immunostaining of DRG reveals increased expression of TNFα in DRG of diabetic sedentary animals. Six weeks of moderate exercise reduces TNFα expression in DRG of diabetic animals as shown by immunohistochemistry (bar = 50 μm) (b–e). Representative Western blots and bar graphs demonstrate the changes in TNFα (**P b 0.005) as well as the changes in IL1β in the DRG of exercise animals (*P b 0.05; n = 5 animals per group). Representative Western blots are showing two independent samples randomly chosen from the blot from each group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
after exercise, animals show an improvement in cold allodynia and thermal hyperalgesia. Exercise altered a number of stress related markers in the DRG of the diabetic exercise group
proinflammatory mediators TNFα and IL-1β in sedentary diabetic rats. Diabetic exercised animals exhibited increases in the expression of HSP70 in DRG (**P b 0.005; n = 5; Fig. 7 a). These changes in exercised animals may play a role in changing the pain threshold of these animals. Discussion
Diabetic animals show increased levels of stress related kinase p38 MAPK in the DRG. Six weeks of moderate exercise presented a decrease in the activation of the stress-related MAP kinase p38 in the diabetic exercise group (*P b 0.05 compared to the sedentary group; Fig. 7 b). The DRG exhibited low levels of HSP70 and increased
Our data clearly demonstrates that exercise delays the progression of thermal and mechanical hyperalgesia as well as cold allodynia in STZ-induced diabetes. This study focuses on the benefit of moderate exercise to reduce the risk of exercise induced hypoglycemia and
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Fig. 6. Exercise altered the expression of Transient Receptor Potential channels (TRP) in DRG of diabetic animals. (a) Western blot analysis illustrated the reduction of TRPV1 ion channel expression in the DRG of the diabetic exercise group compared to the diabetic sedentary group (*P b 0.05). (b) In diabetic sedentary animals thermal sensitivity has been altered and Western blot analysis of cold sensing channel TRPM8 showed an increase in expression in diabetic sedentary animals, whereas it was decreased in the diabetic exercised group (not statistically significant). Representative Western blots of DRG are showing random selection of two independent samples from each group.
inflammation in Type 1 diabetic animals. Studies have shown that there is a modest increase in IL-6, IL1β and TNF-α in Type 1 diabetic patients following intense exercise (Campbell et al., 2014; Nemet et al., 2002). The present study shows that moderate exercise may positively impact serum triglycerides; however our study was not designed to address this benefit. This study doesn't show any difference in HDL or LDL cholesterol. A number of previous studies also were unsuccessful to establish a significant improvement in the levels of HDL in diabetes patients, perhaps due to modest exercise intensities (Technical reviews, 2006). This study demonstrates that moderate exercise attenuates the increased levels of proinflammatory cytokines IL1β and TNF-α in the DRG of the STZ-diabetic animals. Elevated levels of inflammatory mediators such as triglycerides, proinflammatory cytokines and chemokines have been reported in association with diabetes (Brownlee, 2005). These mediators have been considered a link between inflammation and insulin resistance (Shoelson et al., 2006). Our results suggest that moderate exercise offers antinociceptive effects by increasing the release of the inhibitory neurotransmitter, enkephalin in the peripheral nervous system. This study also confirms previous findings that exercise may elevate the release of endogenous opioids, suggesting that these endogenous analgesics may be responsible for the antinociceptive effects observed in short-term exercise models (Koltyn, 2000; Shankarappa et al., 2011; Stagg et al., 2011). We have previously shown that HSV vector mediated transduction to release enkephalin in vitro or in vivo resulted in a reduction of p-PKC and concomitant reduction in p-p38, with a resultant reduction of pain in Type 1 diabetic animals (Chattopadhyay et al., 2008). Studies have also shown that overexpression of met-ENK in the spinal cord and pancreas significantly improves pancreatic inflammatory markers, and pain in a chronic pancreatitis model (Yang et al., 2008). Therefore it is possible that exercise induced release of enkephalin can prevent
0
Con-Sed Con-Ex Dia-Sed Dia-Ex Fig. 7. Exercise reduces activation of p38 MAPK protein and improved the expression HSP70 in DRG of diabetic animals. (a) Western blot analysis illustrated the reduction in the expression of HSP70 in the DRG of the diabetic sedentary group and increased level of HSP70 in the diabetic exercise group (**P b 0.005; n = 5). (b) Western blot analysis of phospho-p38 of DRG reveals that diabetic sedentary animals have increased phosphorylation of p38 compared to control sedentary animals (*P b 0.05; n = 5). Exercise also decreased the activation of p38 MAPK in the DRG of the diabetic exercise group.
activation of p38 MAPK as well as decrease the inflammatory mediators TNFα and IL1β thereby reducing the pain in diabetic animals. Abnormal levels of pro-inflammatory cytokines are responsible for the development of vascular complications and increasing susceptibility to invasive microorganisms. Recently, Belotto et al. showed that moderate exercise improves leukocyte function and decreases inflammation in diabetic rats (Belotto et al., 2010). They also found that a moderate three-week exercise regimen on a treadmill decreases serum levels of TNF-α, CINC-2α/β, IL-1β, IL-6, CRP, and FFA in diabetic rats when compared to sedentary diabetic animals. Exercise also attenuated the increased responsiveness of leukocytes in diabetic patients when compared to those of controls, thereby diminishing ROS release by neutrophils and macrophages (Hatanaka et al., 2006). It has been shown that swimming for a long duration reduces thermal hyperalgesia in STZinduced diabetic female rats (Rossi et al., 2011). Therefore, exercise or lifestyle intervention strategies that include an exercise component may even delay or protect against the development of diabetic peripheral nerve complications. This study is focused on benefits of moderate exercise (10 m/min) in Type 1 diabetic animals. The vulnerability of hypoglycemia may be affected as a consequence of intense exercise (Chassin et al., 2007). Studies have also shown that diabetic rats 24 h after exercise exhibited an increase in the production of TNF-α and IL-1β resulting from intense exercise (up to 33 m/min in 20 min), which suggests that elevated inflammatory response were greater and lasted longer in diabetic animals than in non-diabetic control rats (Bortolon et al., 2012). This indicates that intense exercise in Type 1 diabetes can, not only elevate inflammatory mediators, but also cause hyperglycemia (Rosa et al., 2008a,b). Therefore it is not clear from different studies whether intense exercise
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can cause hypoglycemia or hyperglycemia. From the current study, it is evident that moderate exercise doesn't change blood glucose levels in Type 1 diabetic animals after 6 weeks of exercise regimen. Several studies have indicated that inflammation plays a role in the progression of diabetic microvascular complications such as retinopathy, nephropathy, neuropathy, macrovascular complications and atherosclerosis (Nguyen et al., 2012). It is well known that TNF-α, IL-1β, and CINC-2α/β have important effects on immune cells. At lower levels, these cytokines are important for the activation of endothelial cells and the expression of ligands for adhesion molecules for leukocyte integrins and endothelial selectins. Chronic inflammation results in fibrosis and loss of organ function, negatively affecting the health of diabetic patients (King, 2008). A 3-week moderate exercise regimen on a treadmill (30 min/day, 6 days a week) decreased serum levels of TNFα, CINC2α/β, IL1β, IL-6, CRP and FFA in Type 1 diabetic rats when compared with sedentary diabetic animals (Belotto et al., 2010). Therefore, it is important to select an exercise regimen for Type 1 diabetic subjects which can reduce inflammation. Our data from this study indicates that moderate exercise can reduce thermal hyperalgesia in STZ-hyperglycemic rats. Others have shown that low-intensity exercise decreases the nociception induced by thermal and chemical stimuli after exercise (Quintero et al., 2000); however, their methodology was different with respect to the intensity, the duration of exercise and the duration of the painful stimuli (Quintero et al., 2000). Moreover, Kuphal et al. have demonstrated that extended swimming exercise reduces hyperalgesia induced by intraplantar injection of formalin, or by partial injury in rodent peripheral nerves (Kuphal et al., 2007). In conclusion, the present study demonstrates that 6 weeks of exercise training for 60 min/day, 5 days/week reduces neuroinflammation in the DRG of the STZ-diabetic rats, an experimental model of Type 1 diabetes. This study also suggests that exercise training prevents increases in TRP associated channels and reduces the production of proinflammatory cytokines TNFα and IL1β. The changes in expression of TRPV1 and TRPM8 may have contributed to the altered thermal sensitivity. Other studies have previously shown that DRG neurons dissociated from diabetic hyperalgesic mice exhibited a change in TRPM8-mediated currents as compared to non-diabetic mice (Pabbidi and Pharmacology, S.I.U.a.C., 2007). It is also known that TNF-α can directly activate TNF-α receptors on peripheral nerve terminals, amplify hyperalgesic responses (Cunha et al., 1992; Junger and Sorkin, 2000), and release enzymes to enhance inflammation. It has been shown that TNF-α mediated increase in TRPV1 promotes painful inflammatory processes locally, impacting subsequent responses in the immune system (Spicarova and Palecek, 2010). Kochukov et al. also demonstrated up-regulation of TRP channel in human synovial cells after preincubation with TNFα (Kochukov et al., 2009). Our studies suggest that the decrease in inflammatory mediators in the DRG with exercise may reduce the expression of TRP channels, thereby decreasing pain sensitivity. The pathogenesis of diabetic neuropathy is multifactorial. Chronic hyperglycemia plays an important role in the development of neuropathy causing hyperalgesia, followed by hypoalgesia due to degeneration of the peripheral nerves. Recent studies have shown that treadmill training for 10 weeks improves hind limb motor function and prevents morphometric alterations in diabetic rats subjected to sciatic nerve crush (Malysz et al., 2010). Selagzi and colleagues investigated the effects of swimming training on development of peripheral neuropathy in STZinduced diabetic male rats (Selagzi et al., 2008). Similar to our findings, they observed that swimming exercise protocol did not influence blood glucose levels; however, exercise restored body weight, compound muscle action potential amplitude and potential latency which are the parameters that define motor dysfunction in diabetic animals. In contrast to previous studies, we evaluated male rats for heat, cold and mechanical sensitivity rather than motor dysfunction. Our findings reveal new aspects of how exercise influences Type 1 diabetic animals. HSP levels are inversely correlated with insulin resistance (Hooper and
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Hooper, 2009), inflammatory cytokines, GLUT4 levels, and mitochondrial function (Bruce et al., 2003). A low HSP state consequently promotes increased activation of inflammatory cytokines. Recent studies on treadmill exercise showed increases in expression of Hsp72 in the spinal cord and peripheral nerves (Chen et al., 2013). p38 mitogenactivated protein kinase (MAPK) is known to play a significant role in inflammatory processes in diabetes. Other studies suggest that p38 MAPK inhibition in early stages of diabetes can prevent sensory nerve function (Cheng et al., 2010). This study shows that exercise increases HSP70 levels in DRG and inhibits the activation of p38 MAPK. In addition, moderate exercise training for 6 weeks reduces TRP ion channels, thereby attenuating pain behaviors in STZ-induced diabetic rats. Therefore, we conclude that moderate physical exercise can induce the release of enkephalin which may reduce inflammatory mechanisms in DRG of diabetic rats offering an efficient strategy to protect diabetics against vascular and peripheral nerve complications. Furthermore, we also found that moderate exercise training reduced the levels of inflammatory mediators while improving hyperalgesia without influencing blood glucose levels. This study suggests that exercise may provide a better way to improve diabetic neuropathy, eventually leading to a novel therapeutic approach for this debilitating condition. Authors' contributions HY carried out the behavior tests and biochemical assays. DI and MF carried out the behavior tests and exercise regimen. VT carried out biochemical assays and reviewed the manuscript. MC contributed to the design and analysis of the study, behavior test and wrote the manuscript. Conflict of interests The authors declare that they have no conflict of interests. Acknowledgments This work was funded by American Diabetes Association (Grant #712-BS-021) to MC. We acknowledge Jessica Meyers, Ryan Pattnaik and Valerie Zeer for helping with the animal exercise. References Barbano, R., Hart-Gouleau, S., Pennella-Vaughan, J., Dworkin, R.H., 2003. Pharmacotherapy of painful diabetic neuropathy. Curr. Pain Headache Rep. 7, 169–177. Belotto, M.F., et al., 2010. Moderate exercise improves leucocyte function and decreases inflammation in diabetes. Clin. Exp. Immunol. 162, 237–243. Bortolon, J.R., et al., 2012. Persistence of inflammatory response to intense exercise in diabetic rats. Exp. Diabetes Res. 213986. Brownlee, M., 2005. The pathobiology of diabetic complications: a unifying mechanism. Diabetes 54, 1615–1625. Bruce, C.R., Carey, A.L., Hawley, J.A., Febbraio, M.A., 2003. Intramuscular heat shock protein 72 and heme oxygenase-1 mRNA are reduced in patients with type 2 diabetes: evidence that insulin resistance is associated with a disturbed antioxidant defense mechanism. Diabetes 52, 2338–2345. Campbell, M.D., et al., 2014. Metabolic implications when employing heavy pre- and postexercise rapid-acting insulin reductions to prevent hypoglycaemia in type 1 diabetes patients: a randomised clinical trial. PLoS One 9, e97143. Chassin, L.J., Wilinska, M.E., Hovorka, R., 2007. Intense exercise in type 1 diabetes: exploring the role of continuous glucose monitoring. J. Diabetes Sci. Technol. 1, 570–573. Chattopadhyay, M., Mata, M., Fink, D.J., 2008. Continuous delta-opioid receptor activation reduces neuronal voltage-gated sodium channel (NaV1.7) levels through activation of protein kinase C in painful diabetic neuropathy. J. Neurosci. 28, 6652–6658. Chen, Y.W., Hsieh, P.L., Chen, Y.C., Hung, C.H., Cheng, J.T., 2013. Physical exercise induces excess hsp72 expression and delays the development of hyperalgesia and allodynia in painful diabetic neuropathy rats. Anesth. Analg. 116, 482–490. Cheng, H.T., et al., 2010. p38 mediates mechanical allodynia in a mouse model of type 2 diabetes. Mol. Pain 6, 28. Cunha, F.Q., Poole, S., Lorenzetti, B.B., Ferreira, S.H., 1992. The pivotal role of tumour necrosis factor alpha in the development of inflammatory hyperalgesia. Br. J. Pharmacol. 107, 660–664. Empl, M., et al., 2001. TNF-alpha expression in painful and nonpainful neuropathies. Neurology 56, 1371–1377.
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