Exercise therapy normalizes BDNF upregulation and glial hyperactivity in a mouse model of neuropathic pain
Almeida, Cayoa; DeMaman, Alinea; Kusuda, Ricardoa; Cadetti, Flavianea; Ravanelli, Maria Idaa; Queiroz, André L.b; Sousa, Thais A.c; Zanon, Soniaa; Silveira, Leonardo R.d; Lucas, Guilhermea,*
Abstract
Abstract: Treatment of neuropathic pain is a clinical challenge likely because of the time-dependent changes in many neurotransmitter systems, growth factors, ionic channels, membrane receptors, transcription factors, and recruitment of different cell types. Conversely, an increasing number of reports have shown the ability of extended and regularphysical exercise in alleviating neuropathic pain throughout a wide range of mechanisms. In this study, we investigate the effect of swim exercise on molecules associated with initiation and maintenance of nerve injury–induced neuropathic pain. BALB/c mice were submitted to partial ligation of the sciatic nerve followed by a 5-week aerobic exercise program. Physicaltraining reversed mechanical hypersensitivity, which lasted for an additional 4 weeks after exercise interruption. Swim exercise normalized nerve injury–induced nerve growth factor, and brain-derived neurotrophic factor (BDNF) enhanced expression in the dorsal root ganglion, but had no effect on the glial-derived neurotrophic factor. However, only BDNF remained at low levels after exercise interruption. In addition, exercise training significantly reduced the phosphorylation status of PLCγ-1, but not CREB, in the spinal cord dorsal horn in response to nerve injury. Finally, prolonged swim exercise reversed astrocyte and microglia hyperactivity in the dorsal horn after nerve lesion, which remained normalized after training cessation. Together, these results demonstrate that exercise therapy induces long-lasting analgesia through various mechanisms associated with the onset and advanced stages of neuropathy. Moreover, the data support further studies to clarify whether appropriate exercise intensity, volume, and duration can also cause long-lasting pain relief in patients with neuropathic pain.
1. Introduction
Neuropathic pain is among the most difficult types of chronic pain to treat because of its complex etiology, and the involvement of several neurotransmitter systems, receptors, ionic channels, and cell types.30,76,78 Pharmacological management of neuropathic pain has been challenging to clinicians,35 whereas nonpharmacological approaches have been proven to significantly alleviate chronic pain.9,26,55 Transcutaneous electrical nerve stimulation, transcranial magnetic stimulation, acupuncture, ultrasound, and regular exercise, among others, are increasingly used as adjuvant therapies to treat pathological pain.54,74 However, an important advantage of exercisetherapy in many diseases is its effect on multiple organs and systems.15 Clinical studies have demonstrated that regular exercise alleviates chronic pain syndromes such as cancer pain, musculoskeletal pain, fibromyalgia, myofascial pain, diabetic neuropathy, low back pain, or pain after spinal cord injury.9,26,32,55,63Experimental studies have shown that a wide range of mechanisms underlie the beneficial effect of long-term exercise on acute and chronic pain. Thus, enhanced inhibitory activity mediated by opioids,3,47,66,73 adenosine,45 or serotonin47 are thought to underlie exercise-induced analgesia in neuropathic pain. Likewise, reduced excitatory synaptic transmission through glutamatergic NR1,71 and interleukin-6 receptors, interleukin-1β, tumor necrosis factor alpha,5,10or voltage-activated Ca2+ channel66 are also possible mechanisms contributing to the antinociceptive effect caused by long-term exercise training.
An increasing number of studies have suggested that exercise-induced normalization of neurotrophic growth factors plays an important role in promoting neurological recovery in many conditions such as brain and spinal cord injury, major depression, anxiety, dementia, schizophrenia, epilepsy, and stroke.1,27,79 Moreover, growth factors are among the first molecules in the nociceptive system to respond to nerve injury, and they trigger multiple mechanisms leading to neuropathic pain.70 In particular, increased activity of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) participate in pathophysiology of neuropathic pain by upregulating several pain-related genes, potentiating glutamatergic neurotransmission, and activating glial cells in the dorsal root ganglion and spinal cord dorsal horn.48,60,81,82 Moreover, BDNF and NGF mediate hypernociception by recruiting a number of intracellular signaling molecules including PLCγ-1, ERK1,2, and Akt,34,60,69 as well as the transcription factor CREB.19,42,51 Conversely, glial-derived neurotrophic factor (GDNF) activity in the dorsal root ganglion neurons induces antinociception through various mechanisms.7,83 However, the consequence and relevance of extended exercise training on neurotrophic factors and its signaling pathways after nerve injury are unknown. Therefore, we investigated the effect of regular swimming exercise on some of the early events associated with activity-dependent neuronal/glial plasticity in the DRG and spinal dorsal horn after nerve injury. We hypothesized that prolonged aerobic exercise can reverse neuropathic pain-induced, increase neurotrophin activity and signaling activation, normalizing gene transcription and glial hyperactivity in the spinal dorsal horn.
2. Materials and methods
2.1. Animals
The experimental procedures were approved by the Ethical Committee for Animal Experimentation of Ribeirão Preto School of Medicine, University of São Paulo (protocol 201/209), and were conducted according to the ethical guidelines of the International Association for the Study of Pain.89 Male BALB/c mice weighing 23.5 ± 0.24 g (mean ± SEM) at the beginning of the experiments were used. They were housed in groups of 4 to 5 per cage and kept under 12-hour light–dark cycles (lights on at 6 AM). The animals had free access to food and water.
2.2. Neuropathic pain model
Surgical procedures were performed under anesthesia with a mixture of ketamine and xylazine (60 and 8 mg/kg, respectively). Partial nerve injury was produced by tight ligature with 8-0 silk suture around approximately 1/3 to 1/2 of the diameter of the left sciatic nerve.44,65Sham-operated mice had the nerve exposed, but not ligated. Development of mechanical allodynia and thermal hyperalgesia were investigated once a week from postsurgery day 7 to 70.
2.3. Research design
To investigate the effects of extended exercise on nerve injury–induced neuropathic pain, the exercise protocol was initiated 7 days after surgery. Trained animals swam for 5 weeks, whereas detrained mice returned to the sedentary condition for additional 4 weeks (Fig. 1). Thus, mice were divided into 7 groups and randomly assigned for: (1) partial sciatic nerve ligation (PNL group), (2) nerve ligation with exercise training (trained group), (3) nerve ligation followed by exercise training and 4-week detraining (detrained group), (4) sham-operated and sedentary animals (sham-group), (5) sham-operated animals with exercise training (sham-trained group), (6) sham-operation followed by exercise training and 4-week detraining (sham-detrained group), and (7) naive mice (naive group).
2.4. Exercise training
Mice were trained in a glass tank measuring 60 cm in diameter, 100 cm in width, and 50 cm in height. The tank was divided into 14 lanes with a surface area of 15 × 15 cm per lane and a depth of 35 cm. A heating system maintained the water temperature at 28°C ± 1°C, and a water filter with a flow capacity of 420 L/h was used to clean the swimming apparatus throughout each training session. Swimming exercise was continuously monitored to prevent floating behavior. The exercise training protocol is illustrated in Figure 1. Animals swam once a day for 5 consecutive days followed by 2 resting days over 5 weeks. Training sessions began with one 10-minute bout per day for 3 days. After that, the exercise period gradually increased for an additional 10 minutes every 3 sessions until the animals swam for 50 minutes per session. After each session, animals were gently dried. Five weeks of training was set up because of the observation that mechanical allodynia was completely reversed at that time point. Detrained mice returned to the sedentary condition for 2 or 4 weeks after training cessation.
2.5. Analysis of exercise-induced skeletal muscle adaptation
The transcriptional coactivator peroxisome proliferator-activated receptor-alpha coactivator (PGC-1α) mRNA expression. Total RNA was isolated from the left soleus muscle from naive, 5-week trained and 2- and 4-week detrained mice using the TRIzol reagent (Invitrogen, Carlsbad, CA). The PGC1α mRNA expression was determined by real-time PCR analysis where RNA was reverse transcribed using ImProm-II Reverse Transcription System (Promega, Madison, WI) and oligodT (Promega), according to standard protocols using 1.2 μg RNA treated with RQ1 DNAse (Promega). The cDNA obtained was used in quantitative PCR reactions containing SYBR-green florescent dye (SsoFast EvaGreen Supermix performed at CFX96 Real-Time PCR Detection System; Bio-Rad, Hercules, CA). The expression of β-actin mRNA was measured and remained stable along the tested period. Therefore, β-actin was used for normalization as the internal control gene, whereas the calibrator was the mean threshold cycle (CT) value for each experimental group. All reactions were run in triplicate. Relative expression of mRNAs was determined after normalization with β-actin using the 2−ΔΔCT method.39Quantitative PCR was performed using CFX96 Real-Time PCR Detection System (Bio-Rad). The PGC1α primers were designed as follows: PGC1α-sense (5′CAAGCCAAACCAACAACTTTATCTCT3′), PGC1α-antisense (5′CACACTTAAGGTTCGCTCAATAGTC3′), β-actin-sense (5′ACCTTCTACAATGAGCTGCG3′), and β-actin-antisense (5′CTGGATGGCTACGTACATGG3′).
2.5.1. Citrate synthase expression
Skeletal muscle oxidative capacity was monitored by Western blot analysis, as described previously.72 Briefly, the left soleus muscle was taken from naive, 5-week trained and 2- and 4-week detrained mice. Samples from trained mice were taken 24 hours after the last exercise training session. Total proteins (50 μg) were separated by SDS-PAGE in a 10% Tris-HCl gel and immunoblotted with an anticitrate synthase antibody (1:2000; Abcam ab96600). β-Actin loading control antibody (Santa Cruz Inc, Dallas, TX, 1:5000) was used to normalize citrate synthase expression between different groups.
2.5.2. Cardiac structure analysis
Twenty-four hours after the last exercise training session, exercise-detrained and naive mice were killed, the hearts were dissected out and washed with saline to removed excess blood. The weight of the heart and the left ventricular wall thickness were measured by a digital caliper to identify myocardial hypertrophy.
2.6. Evaluation of mechanical allodynia and thermal hyperalgesia
Mice were placed on an elevated meshed grid, which allowed full access to the ventral aspect of the hind paws. Then, the animals were habituated to the experimental environment (room and apparatus) for a period of at least 30 minutes. A logarithmic series of 9 filaments was applied to the left hind paw to determine the threshold stiffness required for 50% paw withdrawal according to Dixon up-and-down method.16 A single experimenter performed the test to reduce variability of the outcome.
Thermal hypersensitivity was measured using the plantar test (Analgesia Meter; IITC Inc/Life Science Instruments, Woodland Hills, CA). Before testing the behavioral responses to thermal stimuli, mice were also habituated to the test environment for at least 30 minutes. Then, a radiant heat source beneath a glass floor was aimed at the plantar surface of the left hind paw. Paw withdrawal latency was determined as the average of 3 measurements per paw over a 30-minute period. Stimulus intensity was adjusted to give approximately 10-second withdrawal latency in naive mouse. To avoid tissue damage, the cutoff time in the absence of a response was defined to be 20 seconds. Paw movements associated with weight shifting or locomotion were not considered as nocifensive behavior. Behavioral testing occurred on the day before surgery and once a week from postsurgery day 7 to 70 (Fig. 1). Animals submitted to exercisetherapy were always tested 20 to 24 hours after the last exercise session to avoid swim stress-induced analgesia.
2.7. Neurotrophic factor quantification
Soon after euthanasia, the left dorsal root ganglions (L4 and L5) were dissected out and immediately homogenized in lysis buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 1% NP40, 1 mM PMSF, 10% glycerol, 10 μg/mL aprotinin, 1 μg/mL leupeptin, 0.5 mM sodium vanadate, and 4% Triton X-100). Homogenates were centrifuged, and supernatants were collected. Total protein concentration of each sample was measured using a modified Lowry assay (DC Protein Assay, Bio-Rad). BDNF, NGF, and GDNF were quantified using an ELISA kit (Emax ImmunoAssay Systems, Promega) following the manufacturer's instructions. After normalization to total protein content, each protein was loaded in duplicate samples to a plate reader. Data are presented as the mean percentage of protein found in sham-operated sedentary animals, which was denoted as 100%.
2.8. Western blot
Spinal cord dorsal horn extracts were collected 24 hours after the last exercise training to avoid an acute effect of the training. Samples were taken from sham-operated mice submitted to sedentary conditions (control group), sham-operated mice submitted to exercise training, sham-operated mice submitted to detraining, nerve injury mice submitted to sedentary conditions, nerve injury mice submitted to exercise training, and nerve injury animals submitted to detraining. Thus, the dorsal quadrant of the lumbar (L4-L6) spinal cord ipsilateral to the lesion was dissected and homogenized in lysis buffer containing 137 mM NaCl, 20 mM Tris, 1% Igepal CA-630 (Sigma-Aldrich, St. Louis, MO), 10% glycerol, 2 mM sodium orthovanadate, 1% sodium dodecyl sulfate, 50 mM sodium fluoride, 2 mM EDTA, and protease inhibitor cocktail (Sigma-Aldrich) at pH 7.4. Tissue homogenates were centrifuged at 40.000 rpm for 10 minutes at 4°C, and supernatant was collected for analysis. Protein concentration in tissues homogenates was determined by a modified Lowry assay (DC Protein Assay, Bio-Rad) and spectrophotometry (μ-Quant; BioTek Instruments Inc, Winooski, VT). Aliquots containing 30 μg of protein were dissolved in loading buffer and boiled at 70°C for 10 minutes. Proteins were separated by 10% or 12% Tris-glycine SDS-PAGE (GE Healthcare-Bioscience Corp, Piscataway, NJ) and transferred to PVDF membranes (Amersham Biosciences, Piscataway, NJ). Immunoblots were blocked with bovine serum albumin for 1 hour at room temperature followed by incubation with the primary antibody overnight at 4°C. Primary antibodies included monoclonal rabbit anti-mouse phospho-CREB (both 1:1000; Cell Signaling Technology, Beverly, MA), and rabbit anti-mouse phospho-PLCγ (1:500), anti-GFAP (1:40,000), and anti-Iba1 (1:1000). After incubation, the membrane was washed and incubated with secondary antibody (1:2000; ECL anti-Rabbit IgG, GE Healthcare Ltd, Buckinghamshire, United Kingdom) for 1 hour at room temperature. Labeled proteins were detected by chemiluminescence. For stripping and reprobing, stripping buffer (100 mM 20-mercaptoethanol, 2% sodium dodecyl sulfate, 62.5 mM Tris-HCl, pH 6.8) was applied to the membrane for 10 minutes at 50°C, washed for 90 minutes under tap water, rehydrated with methanol and washed with TBS-T before blocking. Thereafter, membranes were reprobed with primary antibody against rabbit anti-mouse anti-CREB (both 1:1000; Cell Signaling Technology, Beverly, MA), and rabbit anti-mouse anti-PLCγ (1:2000). Finally, blots were reprobed against anti-β actin (1:15,000) antibody for loading control. Western blots were quantitated with image analysis system (Molecular Imaging Systems, Eastman Kodak Company, Rochester, NY). After normalization with β-actin, data are presented as mean percentages of the ratio of phosphorylated or total protein to their respective signal intensity levels found in sham-operate/sedentary animals, which were indicated as 100%.
2.9. Statistical analysis
All data are presented as mean ± SEM. Data were analyzed by 2-way repeated-measures analysis of variance (multiple groups × time) followed by Bonferroni post hoc test for multiple comparisons. The Student t test was applied to compare 2 groups. In each case, significance was defined as P < 0.05. Statistical analysis was performed with GraphPad Prism software, version 5 (La Jolla, CA).
3. Results
3.1. Exercise-induced aerobic adaptation
The expression of PGC-1α and citrate synthase was measured in the soleus muscle, as markers of muscle oxidative activity, which regulate metabolic pathways in skeletal muscle toward an oxidative muscle type.2,59 Thus, under the exercise protocol used herein, PGC-1α mRNA was significantly increased in trained animals, which persisted for 2 weeks after exercise interruption (Fig. 2A). Similarly, citrate synthase expression was also increased after exercise training and gradually returned to naive values after training interruption (Fig. 2B). In addition, our exercise protocol did not induced undesirable cardiovascular effects such as myocardial hypertrophy and hyperplasia (Fig. 2C and D) commonly associated high-intensity exercise training.17 Together, both markers indicate that the training protocol promotes muscle oxidative adaptation without a major cardiac overwork.
3.2. Assessment of pain-like behavior
Partial ligation of the sciatic nerve induces long-lasting mechanical allodynia and thermal hyperalgesia as compared with sham-operated mice (Fig. 3A and C). Nerve injury animals maintained under sedentary conditions showed mechanical hypersensitivity from postsurgery day 7 to 70. However, mice submitted to swimming exercise showed partial recovery from mechanical allodynia 4 weeks after exercise initiation and were completely recovered at postinjury day 42 (Fig. 3B). Thermal hypernociception occurred from postinjury day 7 to 28 (Fig. 3C). From day 35 to 70 after nerve damage, latency response to thermal noxious stimulation was similar to control animals. In trained animals, thermal hyperalgesia was abolished faster than in nontrained animals (Fig. 3D). From day 28 after nerve damage, animals showed no sign of thermal hypersensitivity. Moreover, exercise training induced no change in paw withdrawal mechanical threshold or thermal latency in sham-operated mice as compared with sedentary animals (Fig. 3A–D).
3.3. Exercise induced trophic factors plasticity
Peripheral nerve injury marked increases the expression and activity of trophic factor such as NGF, BDNF, and GDNF in the DRG.60,77Likewise, exercise training also influences the expression of those molecules in a number of brain areas, under different stimuli.29,79Therefore, we speculate that the benefits of prolonged exercise for chronic pain conditions could be mediated by those molecules. Indeed, partial ligation of the sciatic nerve markedly increased the expression of NGF, BDNF, and GDNF in the DRG. Nerve growth factor and BDNF upregulation was observed from postinjury day 7 to 70, whereas increased GDNF expression occurred only in the initial phase of the sensory disorder (Fig. 4). Extended swimming exercise reduced, but not normalized, NGF increased expression after nerve lesion, whereas BDNF returned to normal after 5 weeks of training (Fig. 4A and B). However, after exercise cessation the levels of BDNF remained similar to sham-operated mice, whereas NGF expression was still significantly high. Conversely, nerve injury–induced GDNF upregulation was observed only at postsurgery days 7 and 14 and was normalized by postinjury day 42 (Fig. 4C).
3.4. Exercise therapy influences nerve injury–increased PLCγ-1 phosphorylation
A pronociceptive role for PLCγ-1 in sensory neurons and increased phosphorylation of PLCγ-1 have been demonstrated in the spinal dorsal horn after peripheral nerve injury.6,21,34 Seven days after partial ligation of the sciatic nerve, PLCγ-1 phosphorylation was markedly increased in the dorsal horn ipsilateral to the lesion (Fig. 5). This increase was observed not only 7 days after nerve lesion but also at days 42 and 70 after surgery. In trained mice, nerve injury–increased PLCγ-1 phosphorylation was fully abolished as compared with sham-operated animals. However, increased phosphorylation was observed again when the exercise training was interrupted (Fig. 5).
3.5. Exercise effect on CREB phosphorylation
Increased CREB phosphorylation in the spinal dorsal horn represents a consistent phenomenon in different pain models and facilitates pain transmission.43,51 Indeed, 7 days after nerve injury, CREB phosphorylation was significantly increased in the spinal dorsal horn ipsilateral to the lesion (Fig. 6). At days 42 and 70 after injury, its phosphorylation status was normalized, suggesting a time-dependent regulation of CREB activity after nerve injury. Trained mice showed a similar pattern, however, at postlesion day 42, there was reduced CREB phosphorylation as compared with nontrained animals. Thus, exercise training had an effect on the nerve-injured induced CREB phosphorylation by shortening its normalization time. In addition, these data demonstrate an important dissociation between mechanical allodynia and increased CREB phosphorylation in long-term maintenance of neuropathic pain.
3.6. Exercise effect on spinal astrocyte and microglia hyperactivity
Astrocyte and microglia activation in the spinal dorsal horn play a fundamental role in the development of mechanical allodynia, hypernociception, and spontaneous pain after nerve injury.11,22,87GFAP and Iba-1 are protein markers for astrocytes and microglia, respectively, and their expression was marked increased at postinjury day 7 (Fig. 7A and B). This increased glial hyperactivity persisted at days 42 and 70 after nerve lesion. However, extended swimming exercise normalized GFAP and Iba-1 expression not only during exercise training but also 4 weeks after exercise cessation.
4. Discussion
The current data show that swimming exercise fully reversed mechanical allodynia, and alleviated thermal hyperalgesia in a mice model of neuropathic pain. Another critical finding in this study was that the benefits of exercise therapy persisted for at least 4 weeks after training interruption. We further show that exercise training reverses BDNF-increased expression and reduced NGF upregulation in the DRG. Moreover, extended exercise normalized PLCγ-1 phosphorylation in the spinal dorsal horn and reversed astrocyte and microglia hyperactivity after nerve injury. Together, these data provide evidence that prolonged swimming treatment reverses or attenuates multiple mechanisms underlying neuropathic pain not only during exercise training but also after exercise interruption.
4.1. Behavioral changes induced by exercise therapy
The effect of exercise on acute and chronic pain is still very discrepant likely because of different exercise types, training protocol, pain model, and species used. Short-term swim exercise reduces formalin-induced nociception,8,33 whereas extended training reduces nerve injury–induced cold allodynia and thermal hyperalgesia in rats and mice.33 However, these studies focused on the prophylactic effect of exercise rather than its therapeutic use for an established chronic pain syndrome. Conversely, our data are in agreement with a number of reports showing that prolonged exercise can reduce or fully reverse mechanical allodynia after spinal cord contusion,29 chronic constriction injury of the sciatic nerve,10,12,67 streptozotocin-induced diabetic neuropathic pain,66 complex regional pain syndrome type I,45a prediabetes painful neuropathy,24 and noninflammatory chronic muscle pain.3 An important finding in this study was that after exercise cessation, mechanical withdrawal threshold remains normalized for at least 4 weeks. Similar adaptation has been previously reported after interruption of treadmill or running wheel exercise in nerve-injured animals and in a chronic muscle pain model.5,71 The mechanisms underlying this phenomenon remain to be determined and are under current investigation. We propose that depending on the exercise type, frequency, and duration, nerve injury–induced mechanical allodynia and thermal hyperalgesia can be completely reversed even after exercise cessation.
4.2. Exercise effect on nerve injury–induced neurotrophic growth factor expression
Previous studies show that trophic factors such as NGF, BDNF, and GDNF play an important role in pain transmission under physiological and pathological conditions of neuropathic pain.60,70 Nerve growth factor is upregulated in the DRG, and spinal dorsal horn after axotomy, chronic constriction injury of the sciatic nerve, or in a painful neuropathy induced by high-fat diet.24,61,80 In addition, administration of anti-NGF antibodies reduced mechanical hyperalgesia after CCI, SNL, and spinal cord injury.20,25,28 Together, these data indicate that NGF facilitates pain transmission in the DRG and the spinal dorsal horn. In this study, NGF concentration was also significantly increased in the DRG after partial sciatic nerve ligation, which parallels mechanical hypersensitivity up to postinjury day 70. Neurons and activated satellite glial cells are important sources of NGF in the DRG85 where upregulation of several pain-related genes such as TRPV-1, substance P, calcitonin gene-related peptide, and Nav1.8 and Nav1.9 sodium channels, has been suggested to be mediated by NGF.18,38,53,84 Thus, we propose that exercise-induced normalization of NGF in the DRG contributes to behavioral recovery after nerve injury. It is noteworthy that after exercise cessation, NGF expression increased again, but the animals showed no sign of sensory abnormality. Together, these results indicate that the antinociceptive mechanisms induced by exercise become preponderant over those mechanisms facilitating pain transmission after never injury.
Brain-derived neurotrophic factor is well known for its role in pain modulation, facilitating nociceptive responses.40,41 In the periphery, BDNF is released from DRG neurons and satellite as well as Schwann glial cells, whereas in the spinal dorsal horn, BDNF is released from primary afferent terminals and from activated glial cells. After nerve injury, BDNF is upregulated both in the DRG and in the spinal cord.20,58 In the spinal cord, BDNF increases dorsal horn excitability and facilitates pain transmission after nerve injury by modulating both glutamatergic excitatory, GABAergic/glycinergic inhibitory neurotransmission,40,41,49 and increasing the transcription of pronociceptive genes such as c-fos, c-Jun, Krox-24, and CREB.50Then, we speculate that swimming exercise-induced BDNF normalization in DRG represents one of the mechanisms responsible for reversing mechanical allodynia after nerve injury. In agreement with this view, treadmill running reduced BDNF mRNA expression after nerve injury and promoted functional recovery.12
A number of reports demonstrated that GDNF can counteract neuroplastic changes that occur in peripheral nerves after injury, hence reducing neuropathic pain.60 Exogenous administration of GDNF causes analgesia in nerve-injured animals and a number of mechanisms have been proposed.7,64,83 For instance, GDNF normalizes the expression of Nav1.3 and 1.8 sodium channels in peripheral nerves and restores the expression of nociceptive-specific receptors and markers in DRG neurons including NPY, CGRP, somatostatin, and ATP-activated purinergic P2X3 receptor.83However, we found that GDNF increased expression only at days 7, and 14 after injury, whereas it was fully recovered by day 35. Thus, we propose that GDNF has a neuroprotective effect in an early phase, but not in the late phase of neuropathy.35,62,68 Likewise, treadmill running did not change GDNF mRNA in the DRG of rats submitted to peripheral nerve injury.12,13
4.3. Exercise effect on nerve injury–induced signaling alterations
Previous studies showed that PLCγ-1 phosphorylation at tyrosine 783 is necessary for BDNF-induced release of glutamate through the TrkB/Src/PLCγ-1 signaling pathway.56,57,86 In DRG neurons, PLCγ is required for the TRPV1-mediated sensitizing effect of heat responses21 and NGF-induced mechanical hyperalgesia.6 In addition, treatment with the nonselective PLC inhibitor U73122 significantly reduced mechanical allodynia induced by peripheral nerve injury.69 We extended these findings by showing that PLCγ-1 plays an important role in the spinal dorsal horn after nerve injury.34 Our results support the hypothesis that increased activation of PLCγ-1 plays a role in maintenance of chronic pain by inducing long-term potentiation of glutamatergic synaptic transmission.31,56,57,86 Thus, exercise-induced normalization of PLCγ-1 phosphorylation in the dorsal horn may contribute to the recovery of mechanical allodynia after nerve lesion.
Increased CREB phosphorylation at Ser133 has been reported in the spinal dorsal horn under different neuropathic pain models.43,51Moreover, inhibition of CREB by antisense oligonucleotides attenuated nerve injury–induced mechanical allodynia.42 Multiple extracellular signals may converge to CREB activation, including BDNF, nitric oxide, and CGRP.46 Intracellular kinase pathways, such as PKA, ERK1,2, and PLCγ-1, can result in CREB phosphorylation, which initiates transcription of many genes associated with maintenance of neuronal hypersensitivity such as BDNF, P2X3, CGRP, NK1. In cultured cortical neurons, BDNF-induced CREB phosphorylation is linked to activation of the trkB/PLCγ-1 pathway.52 Whether a similar pathway is recruited in the spinal dorsal horn neurons remains to be determined; however, we speculate that the beneficial effect of exercise observed in this study can be, at least partially, associated with normalization of PLCγ-1/CREB activity.
4.4. Exercise effect on nerve injury induced glial hyperactivity
Activation of spinal microglia and astrocyte sensitizes dorsal horn neurons by a number of mechanisms including releasing of pronociceptive molecules such as TNF-α, IL-1β, IL-6, prostaglandin, endocannabinoid, or BDNF.4,11,14,22,23,75 Moreover, previous reports have shown that exercise may have a marked effect on the glial cell activity. In a genetic model of neuroinflammation, chronic endurance training reduced astrocyte and microglia activation in an exercise intensity-dependent manner, and normalized TNF-α, IL-1β, IL-6, COX-2, and iNOS expression in the trained mice brain.36 In addition, treadmill exercise reduced hypoxia-induced astrocyte and microglia activation and restored BDNF levels in the rat hippocampus.37 Our results showed that aerobic exercise reverses both microglia and astrocyte hyperactivity in the spinal cord, which is consistent with behavior normalization of mechanical allodynia. We propose that extended exercise induces long-lasting recovery of glial activity in the spinal dorsal horn, restoring the sensory function even after exercise interruption.
4.5. Conclusions
This study demonstrates that regular and prolonged swimming exercise can fully reverse tactile stimulus–induced neuropathic hypersensitivity beyond the exercise period. The beneficial effect of extended exercise normalized multiple mechanisms in both the DRG and the spinal cord, which may provide new therapeutic directions for the treatment of nerve injury–induced chronic pain. However, the efficacy of exercise therapy depends very much on the exercise type, intensity, frequency, and duration. Therefore, further studies are required to determine appropriate exercise protocols to achieve the most effective pain relief for different types of chronic pain.
Conflict of interest statement
The authors have no conflicts of interest to declare.
This work was supported by grants from the São Paulo State Research Foundation (FAPESP grant 09/16926-4). A. DeMaman was supported by a postdoctoral fellowship from FAPESP (grant 11/08364-6), and C. Almeida was a recipient of a Brazilian Ministry of Education (CAPES Foundation) research scholarship. C. Almeida and A. DeMaman contributed equally.
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Keywords:
Neuropathic pain; Exercise; BDNF; NGF; PLCγ-1; CREB; Astrocyte; Microglia
© 2015 International Association for the Study of Pain
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