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Elevated concentrations of circulating cytokines and correlations with nerve conduction velocity in human peripheral nerves
Journal of Neuroimmunology 277 (2014) 134–139
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Elevated concentrations of circulating cytokines and correlations with nerve conduction velocity in human peripheral nerves David J. Allison ⁎, Lara A. Green, David A. Gabriel, Brian D. Roy, J. Greig Inglis, David S. Ditor Department of Kinesiology, Brock University, St. Catharines, Ontario L2S 3A1, Canada
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Article history: Received 19 May 2014 Received in revised form 2 July 2014 Accepted 13 September 2014 Keywords: Channelopathy Nerve conduction velocity Interleukin-6 Interleukin-1ra Interleukin-10 Tumor necrosis factor-alpha
a b s t r a c t The purpose of the current study was to quantify the potential relationship between various cytokines and peripheral nerve function in humans, in-vivo. Measures of nerve conduction velocity (NCV) were examined prior to and following the induction of a cytokine spike. A significant negative correlation was found between the change in IL-1ra and the change in NCV at 24 h post-exercise (r = −0.65, p = 0.02) while a significant positive correlation was found between the change in IL-6 and the change in NCV at 2 h post-exercise (r = 0.61, p = 0.048). It may be possible that different cytokines induce a unique neural influence at elevated concentrations. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cytokines have traditionally been characterized as signaling proteins of the immune system which act to govern the inflammatory response following tissue damage (Davies et al., 2007). A growing number of additional roles have since been established including an important role in energy regulation. Cytokines (termed myokines) have been shown to be secreted by skeletal muscle following the depletion of muscle glycogen stores for the purpose of providing alternative fuel sources via increased hepatic glucose release and lipolysis (Ostrowski et al., 1998; Febbraio et al., 2004; Pedersen and Hojman, 2012). Despite these critical roles in inflammation, tissue repair, and energy regulation, research has shown that certain cytokines may possess neuromodulatory properties which could pose detriments to neurological function when at elevated concentrations (Jüttler et al., 2002). It has been proposed that cytokines may interfere with neuronal membrane channels causing a cytokineinduced channelopathy (Gutmann and Gutmann, 1996; Davies et al., 2006). An interference with sodium (Na+) and potassium (K+) ion channels may block the exchange of ions across the membrane thereby having the potential to disrupt normal membrane depolarization and/or
⁎ Corresponding author at: Department of Kinesiology, Faculty of Applied Health Science, Brock University, 500 Glenridge Ave., St. Catharines, ON L2S 3A1, Canada. Tel.: +1 289 668 0656; fax: +1 905 688 8364. E-mail addresses: [email protected] (D.J. Allison), [email protected] (L.A. Green), [email protected] (D.A. Gabriel), [email protected] (B.D. Roy), [email protected] (J.G. Inglis), [email protected] (D.S. Ditor).
http://dx.doi.org/10.1016/j.jneuroim.2014.09.010 0165-5728/© 2014 Elsevier B.V. All rights reserved.
repolarization and reduce nerve excitability (Gutmann and Gutmann, 1996). The potential neuromodulatory properties of inflammatory mediators have been demonstrated within nociceptive fibers in studies examining neuropathic pain. Alterations in ion channel trafficking and expression have been demonstrated in both injured as well as structurally healthy sensory fibers (Hudson, 2001). Direct insult to primary sensory neurons may cause cell death and compromise signal transmission leading to ectopic firing (von Hehn et al., 2012). The fact that structurally healthy nociceptors experience a similar neuropathological state suggests a molecularly induced effect. Specifically, the cytokines interleukin-1β (IL-1 β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) have each been suggested to be sufficient to reduce ion channel activation thresholds and induce peripheral sensitization (Sommer and Kress, 2004). The rapid onset of pain following the injection of IL-1β in animal models has also contributed to the concept that inflammatory mediators may influence nerves via direct mechanisms (Fukuoka et al., 1994). Evidence of a cytokine-induced channelopathy within spinal cord axons has also been demonstrated. A study by Davies et al. (2006), examined the effects of tumor necrosis factor-α (TNF-α) on the electrophysiological properties of an excised guinea pig spinal cord. Upon introducing a solution containing TNF-α to the ex-vivo tissue, a dosedependent, reversible reduction in the compound action potential amplitude as well as a depolarization of the resting membrane potential were observed. The fact that these effects were immediately reversible upon washout provided further evidence of a channelopathic influence rather than structurally damaging effects. In addition to these findings,
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when the study was reproduced using heat denatured TNF-α, no such response was induced. Such a finding provides evidence that it was in fact the cytokine TNF-α, and not the presence of any associated proteins which caused the observed effects. These potential cytokine induced channelopathic effects had however, yet to be examined in humans. In the present study it was possible to safely and effectively examine the relationship between elevated cytokine levels and nerve function in humans, in-vivo, based on the known cytokine kinetics during and following exercise. Long duration, aerobic exercise has been shown to lead to a release of cytokines (termed myokines) by skeletal muscle (Pedersen and Febbraio, 2008). Through the use of healthy able-bodied individuals it was therefore possible to achieve baseline measures of peripheral nerve function under basal cytokine levels, prior to exercise, as well as post-measures under elevated cytokine levels following an exercise bout. The ability to acutely elevate cytokine levels in such a manner allowed the present study to examine the potential influence of cytokines on nerve function in the peripheral nerves of humans. It was hypothesized that each of the cytokines of interest would be associated with similar detrimental neural effects as reflected by a reduction in nerve conduction velocity (NCV).
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Twelve healthy, college-aged males were recruited for participation in this study. Participants were all between the ages of 20 and 24 years (23.3 ± 1.1 yrs) and weights varied from 67.9 to 96.5 kg (80.9 ± 9.5 kg). All participants were recreationally active, but did not participate in competitive sports at an elite or varsity level, and all were healthy, with no signs of neuromuscular disease or contraindications to vigorous physical activity, as ensured through the completion of a PAR-Q form prior to participation. Informed consent was obtained from all participants. The study received ethical approval from the Brock University Bioscience Research Ethics Board.
wattage and RPM as opposed to heart rate during the exercise session allowed for a more consistent means of monitoring intensity as cardiac drift was not a confounding factor. Cardiac drift involves a gradual increase in HR, despite the maintenance of a consistent work intensity, due to factors such as changes in temperature, hydration levels, and muscle tissue activation. As hydration levels may affect measures of nerve function, fluid loss over the initial bout of exercise was also determined. Each participant was weighed prior to and following the exercise session and fluid loss was estimated based on weight loss. The estimated fluid loss was then used as a means of determining necessary fluid intake to be consumed during the exercise session on Day 2. Participants were allowed to consume only water during testing. The exercise session on Day 2 was performed for a minimum of 72 h after the familiarization session to provide an adequate time period for any elevations in cytokine concentrations to return to basal values (Ostrowski et al., 1998). During the exercise session participants consumed water at 6 time points (every 10 min) over the span of 1 h in order to ensure that hydration levels were maintained (as determined by water loss during the familiarization session). Temperature has also been shown to affect nerve conduction velocity, and accordingly both exercise sessions were conducted in a temperature controlled room that was 20 °C for both sessions. Further, skin temperature over the thenar muscles was measured prior to each nerve conduction measurement to ensure that adequate time had been provided for temperature to return to resting levels. Finally, in order to help limit exercise induced changes in muscle temperature as well as local metabolite production as confounding factors, exercise was performed by the legs while all measures of nerve conduction were performed on the nondominant arm with a minimum of 1 h of rest before the first round of post-measures. Each exercise session began with a 5 minute warm-up period and ended in a 5 minute cool-down period in which the participant was instructed to cycle at a light intensity of their choosing. All participants were required to maintain the set intensity and complete the 1 hour bout of cycling in its entirety, or their results were excluded from the study.
2.2. Exercise protocol
2.3. Measurement of serum cytokine concentrations
The exercise protocol consisted of two bouts of cycling at approximately 65% VO2max for a duration of 1 h per session. This particular form and volume of exercise has been previously shown to be an appropriate method of inducing an increase in circulating cytokines as shown by the achievement of IL-6 elevations ranging in magnitude of approximately 5 to 9-fold (Pedersen and Febbraio, 2008). The first bout of exercise was performed on Day 1 as a familiarization session that served two purposes. First, the familiarization period was used to determine an appropriate relative intensity for each participant's actual exercise session (on Day 2). Second, the familiarization period allowed for the measurement of fluid loss and determination of necessary fluid intake during the actual exercise session as dehydration may affect measures of nerve function. The relative intensity for each participant was determined through the use of heart rate reserve (HRR), which was deemed an appropriate method of assessing intensity as it has been shown to be highly correlated (r = .990) with %VO2max (Swain et al., 1998). Age predicted maximum heart rate was first calculated through the use of the Miller equation (Miller et al., 1993). The HRR of each participant was then calculated using the age predicted maximum heart rate in conjunction with measured resting heart rate. It was then possible to calculate target heart rate using the Karvonen formula and a set intensity corresponding to 65% VO2max (Strath et al., 2000). At the midpoint (30 min) of the initial exercise session, the wattage and revolutions per minute (RPM) that each participant was working at in order to maintain their target heart rate were recorded. These wattage and RPM were then used as a measure of intensity for the exercise session on Day 2. The use of
Blood draws (10 ml) were taken from the antecubital vein of each participant before and 30 min following the cessation of the exercise session performed on Day 2. The cytokines of interest included interleukin-1 receptor antagonist (IL-1ra), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α). These cytokines were chosen due to their unique pro- and antiinflammatory properties. As different cytokines reach peak levels at unique times following exercise, it was necessary to perform postexercise blood draws at an appropriate time to ensure that each cytokine of interest was adequately elevated. The cytokines IL-6, IL-10, and TNF-α have all been shown to peak immediately following the cessation of exercise, whereas the receptor antagonist IL-1ra has been shown to peak 1 h after the cessation of exercise (Ostrowski et al., 1998). As both IL-6 and IL-10 levels begin to decline rapidly immediately after peaking, it was necessary to obtain the blood draws no later than 30 min following the cessation of exercise. Thus obtaining blood draws 30 min after the cessation of exercise allowed time for IL-1ra levels to become putatively elevated while IL-6 and IL-10 levels had yet to substantially decline. Cytokine levels were assessed at only one time point following the cessation of exercise whereas measures of NCV were assessed at multiple time points. This is due to the fact that there is no literature to suggest a time course for cytokines to travel from the blood to the nerve to induce any potential effects. Blood samples were collected in anticoagulant-free tubes (for serum preparation) and allowed to clot at room temperature for 20 min. Samples were centrifuged for 15 min at 1000 ×g and the serum was extracted and stored at
2. Materials and methods 2.1. Participants
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− 80 °C until the cytokine analysis was performed via enzymelinked immunosorbent assay (Quantikine kit R&D systems, Minneapolis, U.S.A.). All assays were performed by a single examiner at Brock University. 2.4. Measurement of nerve conduction velocity Nerve conduction velocity was assessed using M-wave recordings by means of stimulation of the median nerve and the recording of the corresponding motor response of the thenar muscles. Subjects lay in a supine position with the elbow in full extension. Prior to performing the test, the electrode locations were prepared by shaving the skin, removing any dead skin cells with an abrasive gel, and disinfecting the areas with rubbing alcohol. Surface electrodes were placed in a monopolar configuration with the recording electrode directly over the motor point of the thenar muscles, the reference electrode over the tendon of the interphalangeal joint, and the ground on the palm of the hand. Stimulation of the median nerve was performed distally at the wrist between the flexor tendons of the hand, as well as proximally on the medial side of the biceps brachii. The evoked responses were amplified using a bandwidth of 10 Hz–1 kHz passband and displayed via oscilloscope. A total of 10 trials were taken and averaged for each stimulation site and M-wave onset was used to determine latency (Fig. A.1). Measures of NCV were assessed at 1 h, 2 h, and 24 h post-exercise. Multiple post-measures were necessary as there is no literature to suggest how delayed the potential neural influence of cytokines may be. As serum cytokine concentrations were measured there may be some delay before they travel to, and influence the nerve. Once collected all NCV data was analyzed via Matlab (The Mathworks Inc., Natick, MA). NCV has been shown to have a high degree of intra-examiner reliability by a number of studies (Chaudhry et al., 1991, 1994; Sleivert et al., 1994).
of 210.9 ± 157.5 pg/ml (p = 0.05). Interleukin-6 was significantly elevated from an average resting value of 6.7 ± 7.8 pg/ml to an average post-exercise value of 14.3 ± 7.1 pg/ml (p = 0.02). Interleukin10 levels underwent a significant elevation from an average resting value of 10.9 ± 10.3 pg/ml to an average post-exercise value of 27.5 ± 13.6 pg/ml (p = 0.001). Tumor necrosis factor alpha reached significantly elevated levels with an average resting value of 5.7 ± 5.3 pg/ml and an average post-exercise value of 16.0 ± 15.9 pg/ml (p = 0.03).
3.3. Correlations between the changes in cytokine concentrations and changes in NCV Pearson's r correlations were performed to determine if any correlations existed between the exercise-induced changes in cytokine concentrations and the changes in nerve conduction velocity prior to and 1 h, 2 h, and 24 h post-exercise (Table 1).
3.3.1. Interleukin-1 receptor antagonist and nerve conduction velocity A significant negative correlation was found between the exercisedinduced change in IL-1ra concentrations and the change in NCV from baseline to 24 h post-exercise (such that, the greater the increase in IL-1ra, the greater the decrease in NCV) (r = − 0.65, p = 0.02; Fig. B.1). The corresponding r2 value of 0.43 suggests that 43% of the variability in NCV was accounted for by the variability in the exerciseinduced changes in IL-1ra. There were also trends between the exercise-induced change in IL-1ra concentrations and the change in NCV at 1 h post-exercise (r = − 0.55, r2 = 0.306, p = 0.06) and 2 h post-exercise (r = − 0.48, r2 = 0.24, p = 0.11). Respective r2 values of 0.31 and 0.24 suggest that 31% and 24% of the variability in NCV were accounted for by the variability in the exercise-induced change in IL-1ra.
2.5. Statistical analysis The change in each cytokine concentration prior to and following the single bout of cycling exercise was examined using a Student's t-test. The change in electrophysiological measures was determined via 1-way repeated measures ANOVA (with 4 levels for time; preexercise as well as 60 min, 120 min, 24 h post-exercise). Pearson's r correlation analysis was used to determine if a correlation existed between the exercise-induced changes in cytokine concentrations and changes in NCV. Statistical significance was set at p ≤ 0.05 for all tests which were conducted using Statistica software (Statsoft, Inc., MA, version 10). 3. Results
3.3.2. Interleukin-6 and nerve conduction velocity A significant positive correlation was found between the exerciseinduced change in IL-6 concentrations and the change in NCV from baseline to 2 h post-exercise (r = 0.61, p = 0.048; Fig. B.2). The corresponding r2 value of 0.37 suggests that 37% of the variability in NCV was accounted for by the variability in the exercise-induced changes in IL-6. There was also a trend for a positive correlation found between the exercise-induced change in IL-6 and the change in NCV from baseline to 1 h post-exercise (r = 0.59, p = 0.058), such that the greater the increase in IL-6, the greater the increase in NCV. The corresponding r2 value of 0.34 suggests that 34% of the variability in NCV was accounted for by the variability in the exercise-induced changes in IL-6.
3.1. Exercise performance All participants completed the 1 hour cycle in its entirety with no adverse events. Fluid levels were maintained based on the previously calculated estimated fluid loss from the familiarization session. Following exercise, adequate time was provided for body temperature to return to similar levels as seen during initial baseline testing. As pre- and post-exercise measures of skin temperature over the thenar muscles were shown to be similar (pre: 31.7 ± 1.3 °C, post: 31.7 ± 1.9 °C; p N 0.05), it is likely that muscle temperature was not a confounding factor for any observed change in NCV. 3.2. Cytokine concentrations There was a significant increase in interleukin-1 receptor antagonist following the exercise session as values increased from an average resting value of 121.8 ± 37.0 pg/ml to an average post-exercise value
Table 1 Nerve conduction velocity (m/s). Subject
Baseline
1 h post
2 h post
24 h post
1 2 3 4 5 6 7 8 9 10 11 12 Mean SD
52.96 49.46 52.69 53.88 58.41 61.19 64.09 58.64 59.57 54 50 55.65 55.88 4.53
49.14 53.46 53.4 58.4 55.43 66 56.3 59.09 62.73 56.25 56.6 58.18 57.08 4.39
49.14 55 56.2 59.2 60.95 64.5 60.91 59.32 62.95 56.25 54.6 58.18 58.10 4.18
50.89 53.4 52.96 55.6 55.63 55.65 55.65 61.14 60.68 46.07 52.88 56.82 54.78 4.07
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3.3.3. Interleukin-10 and nerve conduction velocity No significant correlations were found between the exerciseinduced change in IL-10 concentrations and the change in NCV before and 1 h, 2 h, or 24 h post-exercise. 3.3.4. Tumor necrosis factor alpha and nerve conduction velocity No significant correlations were found between the exerciseinduced change in TNF-α concentrations and the change in NCV before and 1 h, 2 h, or 24 h post-exercise. 4. Discussion 4.1. Major findings The current study was the first to examine the effects of acutely elevated cytokine concentrations on peripheral nerve function in humans. Two major findings existed in the current study. First, the changes in IL-1ra levels were shown to be negatively correlated with the change in NCV, 24 h after the cessation of exercise. Second, the changes in IL-6 levels were shown to be positively correlated with the change in NCV, 2 h after the cessation of exercise. The negative correlation found between the change in IL-1ra and the change in NCV may suggest that IL-1ra is involved in slowing nerve conduction via potential channelopathic effects. Although the mechanisms behind such channelopathies are not fully understood, evidence for several potential mechanisms has been demonstrated. The proinflammatory cytokine TNF-α has been shown to increase membrane permeability to Na+, allowing it to accumulate intracellularly (van der Goot et al., 1999). This may be sufficient to induce membrane depolarization and conduction failure as demonstrated ex-vivo by Davies et al. (2006). Additionally, as intracellular Na+ accumulation is tied to calcium (Ca+) import (Li et al., 2000), the corresponding accumulation of Ca+ may result in the production of reactive oxygen species which may further act to block conduction (Kapoor et al., 1999; Paschen, 2003). Although no correlations were demonstrated between TNF-α and NCV in the current study it is possible to speculate that other cytokines, such as IL-1ra, may have the potential to act in a similar fashion. The positive correlation found between the change in IL-6 and the change in NCV may suggest IL-6 to be involved in a process which acts to enhance nerve function as opposed to producing the negative channelopathic effects originally hypothesized. Such an influence has not been previously demonstrated within somatic nerves, however, there is strong evidence concerning the ability for cytokines to enhance nociceptor excitability (Sommer and Kress, 2004). Cytokines such as IL-1β have been shown to enhance nociceptor excitability and induce ectopic firing by means of complex signaling cascades and the production of secondary mediators such as nitric oxide, bradykinin, and prostaglandins (Poole et al., 1999). However, IL-1β has also been demonstrated to excite nociceptive fibers within 1 min of administration, in-vivo, suggesting a more direct mechanism (Fukuoka et al., 1994). IL-1β has the ability to increase neuronal excitability within nociceptors by enhancing both Ca + and nonselective cation currents, in addition to inhibiting the extracellular flow of K+ (Desson and Ferguson, 2003; Shu et al., 2007). The ability of IL-6 to induce similar effects is less clear and conflicting results have been reported whereby enhanced excitability, reduced excitability, and a lack of effects have each been demonstrated following injection in animal models (Cunha et al., 1992; Członkowski et al., 1993; Flatters et al., 2004). This may be attributed to the ability of IL-6 to exert opposing inflammatory responses in different conditions. The high degree of variability regarding the influence of IL-6 on nociceptors and lack of studies examining its influence on somatic nerve function in-vivo, make it difficult to speculate a particular mechanism of action. However, it may be possible that under different circumstances, certain cytokines could impose unique influences
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on somatic nerves similar to that demonstrated in nociceptors. The current study utilized a model of muscle-derived cytokines whereby IL-6 is released, in-part, in response to an energy crisis following muscle glycogen depletion. In this situation IL-6 is responsible for inducing an elevation in hepatic glucose output, providing working skeletal muscle with an alternative fuel source (Febbraio et al., 2004). It may therefore be fitting that in addition to aiding working skeletal muscle by supplying an additional fuel source, IL-6 could also act to enhance neural drive to allow for continued force output during prolonged activity. If cytokines are to influence somatic nerve function, the effects may be highly variable depending on the cytokine, source, and environment. The fact that the elevations in both IL-10 and TNF-α, in the current study, were shown to have no correlation with any change in NCV may further add to the argument that different cytokines may induce unique influences or lack thereof. It is important to note that as exercise was utilized to induce a spike in circulating cytokines it is possible that the changes in nerve conduction can be attributed, in part, to other exercise related factors. Although factors such as hydration levels, body temperature, and local metabolite production were controlled for, their influences cannot be completely discounted. However, the authors have provided the r2 values that correspond to the correlations found. As such, we are not suggesting cytokines to be the sole cause of any alterations in NCV, but rather have attempted to quantify the contribution of cytokines to the exerciseinduced changes in NCV. 4.2. Physiological/clinical significance The current study achieved elevations in cytokines similar to those reported in chronic inflammatory conditions such as spinal cord injury (SCI) (Davies et al., 2007). However, it must be taken into consideration that the current study observed acute elevations in cytokines, whereas a number of clinical conditions such as, SCI, multiple sclerosis, obesity, diabetes and depression, are associated with chronic elevations (Hayes et al., 2002; Schiepers et al., 2005; Navarro and Mora, 2006; Davies et al., 2007; Miles et al., 2012). In addition, such conditions commonly present as comorbidities placing these populations under even more exaggerated levels of inflammation. Such factors may make these populations more susceptible to alterations in nerve conduction than that of healthy individuals. It will be necessary for future studies to be performed on such populations in order to examine the potential neuromodulatory influence of chronically elevated cytokines. It is possible to speculate that more severe effects may occur under chronic conditions. Additionally, any small alterations in nerve conduction may be particularly magnified within populations with preexisting neural deficits such as spinal cord injury and multiple sclerosis. It will also be important for future studies to examine a wider variety of cytokines and other inflammatory mediators to assess the potential unique influence of various molecules. 5. Conclusion The current study was the first to examine the association between acutely elevated systemic cytokines and peripheral nerve function in humans. The results demonstrated a significant negative correlation between the change in IL-1ra and the change in NCV as well as a significant positive correlation between the change in IL-6 and the change in NCV. Additionally, IL-10 and TNF-α were found to have no correlation with NCV. These results may indicate the potential for different cytokines to participate in unique neuromodulatory roles. The findings of this study warrant further research, potentially examining special populations with chronically elevated cytokine concentrations such as those with spinal cord injury or multiple sclerosis. The fact that such populations have
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persistent elevations in systemic cytokines on top of pre-existing neurological deficits may make these populations particularly susceptible to the potential effects of channelopathy.
Acknowledgments This study was funded by the Ontario Neurotrauma Foundation (2011-ONF-RHI-MT-894).
Appendix A
Fig. B.2. Representation of the significant positive correlation between the change in IL-6 and the change in NCV at 2 h post exercise (r = 0.61, p = 0.048).
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Fig. A.1. Representation of typical M-wave used in the calculation of NCV. The M-wave is represented by the largest of the three waveforms on each graph (additional 2 smaller waveforms represent muscle fiber conduction (not assessed in this study)). The upper graph represents the waveforms produced upon stimulation of the median nerve from the proximal location while the lower graph represents the waveforms produced upon stimulation from the distal location.
Fig. B.1. Representation of the significant negative correlation between the change in IL-1ra and the change in NCV at 24 h post exercise (r = −0.65, p = 0.02).
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Elevated concentrations of circulating cytokines and correlations with nerve conduction velocity in human peripheral nerves David J. Allison ⁎, Lara A. Green, David A. Gabriel, Brian D. Roy, J. Greig Inglis, David S. Ditor Department of Kinesiology, Brock University, St. Catharines, Ontario L2S 3A1, Canada
a r t i c l e
i n f o
Article history: Received 19 May 2014 Received in revised form 2 July 2014 Accepted 13 September 2014 Keywords: Channelopathy Nerve conduction velocity Interleukin-6 Interleukin-1ra Interleukin-10 Tumor necrosis factor-alpha
a b s t r a c t The purpose of the current study was to quantify the potential relationship between various cytokines and peripheral nerve function in humans, in-vivo. Measures of nerve conduction velocity (NCV) were examined prior to and following the induction of a cytokine spike. A significant negative correlation was found between the change in IL-1ra and the change in NCV at 24 h post-exercise (r = −0.65, p = 0.02) while a significant positive correlation was found between the change in IL-6 and the change in NCV at 2 h post-exercise (r = 0.61, p = 0.048). It may be possible that different cytokines induce a unique neural influence at elevated concentrations. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cytokines have traditionally been characterized as signaling proteins of the immune system which act to govern the inflammatory response following tissue damage (Davies et al., 2007). A growing number of additional roles have since been established including an important role in energy regulation. Cytokines (termed myokines) have been shown to be secreted by skeletal muscle following the depletion of muscle glycogen stores for the purpose of providing alternative fuel sources via increased hepatic glucose release and lipolysis (Ostrowski et al., 1998; Febbraio et al., 2004; Pedersen and Hojman, 2012). Despite these critical roles in inflammation, tissue repair, and energy regulation, research has shown that certain cytokines may possess neuromodulatory properties which could pose detriments to neurological function when at elevated concentrations (Jüttler et al., 2002). It has been proposed that cytokines may interfere with neuronal membrane channels causing a cytokineinduced channelopathy (Gutmann and Gutmann, 1996; Davies et al., 2006). An interference with sodium (Na+) and potassium (K+) ion channels may block the exchange of ions across the membrane thereby having the potential to disrupt normal membrane depolarization and/or
⁎ Corresponding author at: Department of Kinesiology, Faculty of Applied Health Science, Brock University, 500 Glenridge Ave., St. Catharines, ON L2S 3A1, Canada. Tel.: +1 289 668 0656; fax: +1 905 688 8364. E-mail addresses: [email protected] (D.J. Allison), [email protected] (L.A. Green), [email protected] (D.A. Gabriel), [email protected] (B.D. Roy), [email protected] (J.G. Inglis), [email protected] (D.S. Ditor).
http://dx.doi.org/10.1016/j.jneuroim.2014.09.010 0165-5728/© 2014 Elsevier B.V. All rights reserved.
repolarization and reduce nerve excitability (Gutmann and Gutmann, 1996). The potential neuromodulatory properties of inflammatory mediators have been demonstrated within nociceptive fibers in studies examining neuropathic pain. Alterations in ion channel trafficking and expression have been demonstrated in both injured as well as structurally healthy sensory fibers (Hudson, 2001). Direct insult to primary sensory neurons may cause cell death and compromise signal transmission leading to ectopic firing (von Hehn et al., 2012). The fact that structurally healthy nociceptors experience a similar neuropathological state suggests a molecularly induced effect. Specifically, the cytokines interleukin-1β (IL-1 β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) have each been suggested to be sufficient to reduce ion channel activation thresholds and induce peripheral sensitization (Sommer and Kress, 2004). The rapid onset of pain following the injection of IL-1β in animal models has also contributed to the concept that inflammatory mediators may influence nerves via direct mechanisms (Fukuoka et al., 1994). Evidence of a cytokine-induced channelopathy within spinal cord axons has also been demonstrated. A study by Davies et al. (2006), examined the effects of tumor necrosis factor-α (TNF-α) on the electrophysiological properties of an excised guinea pig spinal cord. Upon introducing a solution containing TNF-α to the ex-vivo tissue, a dosedependent, reversible reduction in the compound action potential amplitude as well as a depolarization of the resting membrane potential were observed. The fact that these effects were immediately reversible upon washout provided further evidence of a channelopathic influence rather than structurally damaging effects. In addition to these findings,
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when the study was reproduced using heat denatured TNF-α, no such response was induced. Such a finding provides evidence that it was in fact the cytokine TNF-α, and not the presence of any associated proteins which caused the observed effects. These potential cytokine induced channelopathic effects had however, yet to be examined in humans. In the present study it was possible to safely and effectively examine the relationship between elevated cytokine levels and nerve function in humans, in-vivo, based on the known cytokine kinetics during and following exercise. Long duration, aerobic exercise has been shown to lead to a release of cytokines (termed myokines) by skeletal muscle (Pedersen and Febbraio, 2008). Through the use of healthy able-bodied individuals it was therefore possible to achieve baseline measures of peripheral nerve function under basal cytokine levels, prior to exercise, as well as post-measures under elevated cytokine levels following an exercise bout. The ability to acutely elevate cytokine levels in such a manner allowed the present study to examine the potential influence of cytokines on nerve function in the peripheral nerves of humans. It was hypothesized that each of the cytokines of interest would be associated with similar detrimental neural effects as reflected by a reduction in nerve conduction velocity (NCV).
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Twelve healthy, college-aged males were recruited for participation in this study. Participants were all between the ages of 20 and 24 years (23.3 ± 1.1 yrs) and weights varied from 67.9 to 96.5 kg (80.9 ± 9.5 kg). All participants were recreationally active, but did not participate in competitive sports at an elite or varsity level, and all were healthy, with no signs of neuromuscular disease or contraindications to vigorous physical activity, as ensured through the completion of a PAR-Q form prior to participation. Informed consent was obtained from all participants. The study received ethical approval from the Brock University Bioscience Research Ethics Board.
wattage and RPM as opposed to heart rate during the exercise session allowed for a more consistent means of monitoring intensity as cardiac drift was not a confounding factor. Cardiac drift involves a gradual increase in HR, despite the maintenance of a consistent work intensity, due to factors such as changes in temperature, hydration levels, and muscle tissue activation. As hydration levels may affect measures of nerve function, fluid loss over the initial bout of exercise was also determined. Each participant was weighed prior to and following the exercise session and fluid loss was estimated based on weight loss. The estimated fluid loss was then used as a means of determining necessary fluid intake to be consumed during the exercise session on Day 2. Participants were allowed to consume only water during testing. The exercise session on Day 2 was performed for a minimum of 72 h after the familiarization session to provide an adequate time period for any elevations in cytokine concentrations to return to basal values (Ostrowski et al., 1998). During the exercise session participants consumed water at 6 time points (every 10 min) over the span of 1 h in order to ensure that hydration levels were maintained (as determined by water loss during the familiarization session). Temperature has also been shown to affect nerve conduction velocity, and accordingly both exercise sessions were conducted in a temperature controlled room that was 20 °C for both sessions. Further, skin temperature over the thenar muscles was measured prior to each nerve conduction measurement to ensure that adequate time had been provided for temperature to return to resting levels. Finally, in order to help limit exercise induced changes in muscle temperature as well as local metabolite production as confounding factors, exercise was performed by the legs while all measures of nerve conduction were performed on the nondominant arm with a minimum of 1 h of rest before the first round of post-measures. Each exercise session began with a 5 minute warm-up period and ended in a 5 minute cool-down period in which the participant was instructed to cycle at a light intensity of their choosing. All participants were required to maintain the set intensity and complete the 1 hour bout of cycling in its entirety, or their results were excluded from the study.
2.2. Exercise protocol
2.3. Measurement of serum cytokine concentrations
The exercise protocol consisted of two bouts of cycling at approximately 65% VO2max for a duration of 1 h per session. This particular form and volume of exercise has been previously shown to be an appropriate method of inducing an increase in circulating cytokines as shown by the achievement of IL-6 elevations ranging in magnitude of approximately 5 to 9-fold (Pedersen and Febbraio, 2008). The first bout of exercise was performed on Day 1 as a familiarization session that served two purposes. First, the familiarization period was used to determine an appropriate relative intensity for each participant's actual exercise session (on Day 2). Second, the familiarization period allowed for the measurement of fluid loss and determination of necessary fluid intake during the actual exercise session as dehydration may affect measures of nerve function. The relative intensity for each participant was determined through the use of heart rate reserve (HRR), which was deemed an appropriate method of assessing intensity as it has been shown to be highly correlated (r = .990) with %VO2max (Swain et al., 1998). Age predicted maximum heart rate was first calculated through the use of the Miller equation (Miller et al., 1993). The HRR of each participant was then calculated using the age predicted maximum heart rate in conjunction with measured resting heart rate. It was then possible to calculate target heart rate using the Karvonen formula and a set intensity corresponding to 65% VO2max (Strath et al., 2000). At the midpoint (30 min) of the initial exercise session, the wattage and revolutions per minute (RPM) that each participant was working at in order to maintain their target heart rate were recorded. These wattage and RPM were then used as a measure of intensity for the exercise session on Day 2. The use of
Blood draws (10 ml) were taken from the antecubital vein of each participant before and 30 min following the cessation of the exercise session performed on Day 2. The cytokines of interest included interleukin-1 receptor antagonist (IL-1ra), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α). These cytokines were chosen due to their unique pro- and antiinflammatory properties. As different cytokines reach peak levels at unique times following exercise, it was necessary to perform postexercise blood draws at an appropriate time to ensure that each cytokine of interest was adequately elevated. The cytokines IL-6, IL-10, and TNF-α have all been shown to peak immediately following the cessation of exercise, whereas the receptor antagonist IL-1ra has been shown to peak 1 h after the cessation of exercise (Ostrowski et al., 1998). As both IL-6 and IL-10 levels begin to decline rapidly immediately after peaking, it was necessary to obtain the blood draws no later than 30 min following the cessation of exercise. Thus obtaining blood draws 30 min after the cessation of exercise allowed time for IL-1ra levels to become putatively elevated while IL-6 and IL-10 levels had yet to substantially decline. Cytokine levels were assessed at only one time point following the cessation of exercise whereas measures of NCV were assessed at multiple time points. This is due to the fact that there is no literature to suggest a time course for cytokines to travel from the blood to the nerve to induce any potential effects. Blood samples were collected in anticoagulant-free tubes (for serum preparation) and allowed to clot at room temperature for 20 min. Samples were centrifuged for 15 min at 1000 ×g and the serum was extracted and stored at
2. Materials and methods 2.1. Participants
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− 80 °C until the cytokine analysis was performed via enzymelinked immunosorbent assay (Quantikine kit R&D systems, Minneapolis, U.S.A.). All assays were performed by a single examiner at Brock University. 2.4. Measurement of nerve conduction velocity Nerve conduction velocity was assessed using M-wave recordings by means of stimulation of the median nerve and the recording of the corresponding motor response of the thenar muscles. Subjects lay in a supine position with the elbow in full extension. Prior to performing the test, the electrode locations were prepared by shaving the skin, removing any dead skin cells with an abrasive gel, and disinfecting the areas with rubbing alcohol. Surface electrodes were placed in a monopolar configuration with the recording electrode directly over the motor point of the thenar muscles, the reference electrode over the tendon of the interphalangeal joint, and the ground on the palm of the hand. Stimulation of the median nerve was performed distally at the wrist between the flexor tendons of the hand, as well as proximally on the medial side of the biceps brachii. The evoked responses were amplified using a bandwidth of 10 Hz–1 kHz passband and displayed via oscilloscope. A total of 10 trials were taken and averaged for each stimulation site and M-wave onset was used to determine latency (Fig. A.1). Measures of NCV were assessed at 1 h, 2 h, and 24 h post-exercise. Multiple post-measures were necessary as there is no literature to suggest how delayed the potential neural influence of cytokines may be. As serum cytokine concentrations were measured there may be some delay before they travel to, and influence the nerve. Once collected all NCV data was analyzed via Matlab (The Mathworks Inc., Natick, MA). NCV has been shown to have a high degree of intra-examiner reliability by a number of studies (Chaudhry et al., 1991, 1994; Sleivert et al., 1994).
of 210.9 ± 157.5 pg/ml (p = 0.05). Interleukin-6 was significantly elevated from an average resting value of 6.7 ± 7.8 pg/ml to an average post-exercise value of 14.3 ± 7.1 pg/ml (p = 0.02). Interleukin10 levels underwent a significant elevation from an average resting value of 10.9 ± 10.3 pg/ml to an average post-exercise value of 27.5 ± 13.6 pg/ml (p = 0.001). Tumor necrosis factor alpha reached significantly elevated levels with an average resting value of 5.7 ± 5.3 pg/ml and an average post-exercise value of 16.0 ± 15.9 pg/ml (p = 0.03).
3.3. Correlations between the changes in cytokine concentrations and changes in NCV Pearson's r correlations were performed to determine if any correlations existed between the exercise-induced changes in cytokine concentrations and the changes in nerve conduction velocity prior to and 1 h, 2 h, and 24 h post-exercise (Table 1).
3.3.1. Interleukin-1 receptor antagonist and nerve conduction velocity A significant negative correlation was found between the exercisedinduced change in IL-1ra concentrations and the change in NCV from baseline to 24 h post-exercise (such that, the greater the increase in IL-1ra, the greater the decrease in NCV) (r = − 0.65, p = 0.02; Fig. B.1). The corresponding r2 value of 0.43 suggests that 43% of the variability in NCV was accounted for by the variability in the exerciseinduced changes in IL-1ra. There were also trends between the exercise-induced change in IL-1ra concentrations and the change in NCV at 1 h post-exercise (r = − 0.55, r2 = 0.306, p = 0.06) and 2 h post-exercise (r = − 0.48, r2 = 0.24, p = 0.11). Respective r2 values of 0.31 and 0.24 suggest that 31% and 24% of the variability in NCV were accounted for by the variability in the exercise-induced change in IL-1ra.
2.5. Statistical analysis The change in each cytokine concentration prior to and following the single bout of cycling exercise was examined using a Student's t-test. The change in electrophysiological measures was determined via 1-way repeated measures ANOVA (with 4 levels for time; preexercise as well as 60 min, 120 min, 24 h post-exercise). Pearson's r correlation analysis was used to determine if a correlation existed between the exercise-induced changes in cytokine concentrations and changes in NCV. Statistical significance was set at p ≤ 0.05 for all tests which were conducted using Statistica software (Statsoft, Inc., MA, version 10). 3. Results
3.3.2. Interleukin-6 and nerve conduction velocity A significant positive correlation was found between the exerciseinduced change in IL-6 concentrations and the change in NCV from baseline to 2 h post-exercise (r = 0.61, p = 0.048; Fig. B.2). The corresponding r2 value of 0.37 suggests that 37% of the variability in NCV was accounted for by the variability in the exercise-induced changes in IL-6. There was also a trend for a positive correlation found between the exercise-induced change in IL-6 and the change in NCV from baseline to 1 h post-exercise (r = 0.59, p = 0.058), such that the greater the increase in IL-6, the greater the increase in NCV. The corresponding r2 value of 0.34 suggests that 34% of the variability in NCV was accounted for by the variability in the exercise-induced changes in IL-6.
3.1. Exercise performance All participants completed the 1 hour cycle in its entirety with no adverse events. Fluid levels were maintained based on the previously calculated estimated fluid loss from the familiarization session. Following exercise, adequate time was provided for body temperature to return to similar levels as seen during initial baseline testing. As pre- and post-exercise measures of skin temperature over the thenar muscles were shown to be similar (pre: 31.7 ± 1.3 °C, post: 31.7 ± 1.9 °C; p N 0.05), it is likely that muscle temperature was not a confounding factor for any observed change in NCV. 3.2. Cytokine concentrations There was a significant increase in interleukin-1 receptor antagonist following the exercise session as values increased from an average resting value of 121.8 ± 37.0 pg/ml to an average post-exercise value
Table 1 Nerve conduction velocity (m/s). Subject
Baseline
1 h post
2 h post
24 h post
1 2 3 4 5 6 7 8 9 10 11 12 Mean SD
52.96 49.46 52.69 53.88 58.41 61.19 64.09 58.64 59.57 54 50 55.65 55.88 4.53
49.14 53.46 53.4 58.4 55.43 66 56.3 59.09 62.73 56.25 56.6 58.18 57.08 4.39
49.14 55 56.2 59.2 60.95 64.5 60.91 59.32 62.95 56.25 54.6 58.18 58.10 4.18
50.89 53.4 52.96 55.6 55.63 55.65 55.65 61.14 60.68 46.07 52.88 56.82 54.78 4.07
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3.3.3. Interleukin-10 and nerve conduction velocity No significant correlations were found between the exerciseinduced change in IL-10 concentrations and the change in NCV before and 1 h, 2 h, or 24 h post-exercise. 3.3.4. Tumor necrosis factor alpha and nerve conduction velocity No significant correlations were found between the exerciseinduced change in TNF-α concentrations and the change in NCV before and 1 h, 2 h, or 24 h post-exercise. 4. Discussion 4.1. Major findings The current study was the first to examine the effects of acutely elevated cytokine concentrations on peripheral nerve function in humans. Two major findings existed in the current study. First, the changes in IL-1ra levels were shown to be negatively correlated with the change in NCV, 24 h after the cessation of exercise. Second, the changes in IL-6 levels were shown to be positively correlated with the change in NCV, 2 h after the cessation of exercise. The negative correlation found between the change in IL-1ra and the change in NCV may suggest that IL-1ra is involved in slowing nerve conduction via potential channelopathic effects. Although the mechanisms behind such channelopathies are not fully understood, evidence for several potential mechanisms has been demonstrated. The proinflammatory cytokine TNF-α has been shown to increase membrane permeability to Na+, allowing it to accumulate intracellularly (van der Goot et al., 1999). This may be sufficient to induce membrane depolarization and conduction failure as demonstrated ex-vivo by Davies et al. (2006). Additionally, as intracellular Na+ accumulation is tied to calcium (Ca+) import (Li et al., 2000), the corresponding accumulation of Ca+ may result in the production of reactive oxygen species which may further act to block conduction (Kapoor et al., 1999; Paschen, 2003). Although no correlations were demonstrated between TNF-α and NCV in the current study it is possible to speculate that other cytokines, such as IL-1ra, may have the potential to act in a similar fashion. The positive correlation found between the change in IL-6 and the change in NCV may suggest IL-6 to be involved in a process which acts to enhance nerve function as opposed to producing the negative channelopathic effects originally hypothesized. Such an influence has not been previously demonstrated within somatic nerves, however, there is strong evidence concerning the ability for cytokines to enhance nociceptor excitability (Sommer and Kress, 2004). Cytokines such as IL-1β have been shown to enhance nociceptor excitability and induce ectopic firing by means of complex signaling cascades and the production of secondary mediators such as nitric oxide, bradykinin, and prostaglandins (Poole et al., 1999). However, IL-1β has also been demonstrated to excite nociceptive fibers within 1 min of administration, in-vivo, suggesting a more direct mechanism (Fukuoka et al., 1994). IL-1β has the ability to increase neuronal excitability within nociceptors by enhancing both Ca + and nonselective cation currents, in addition to inhibiting the extracellular flow of K+ (Desson and Ferguson, 2003; Shu et al., 2007). The ability of IL-6 to induce similar effects is less clear and conflicting results have been reported whereby enhanced excitability, reduced excitability, and a lack of effects have each been demonstrated following injection in animal models (Cunha et al., 1992; Członkowski et al., 1993; Flatters et al., 2004). This may be attributed to the ability of IL-6 to exert opposing inflammatory responses in different conditions. The high degree of variability regarding the influence of IL-6 on nociceptors and lack of studies examining its influence on somatic nerve function in-vivo, make it difficult to speculate a particular mechanism of action. However, it may be possible that under different circumstances, certain cytokines could impose unique influences
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on somatic nerves similar to that demonstrated in nociceptors. The current study utilized a model of muscle-derived cytokines whereby IL-6 is released, in-part, in response to an energy crisis following muscle glycogen depletion. In this situation IL-6 is responsible for inducing an elevation in hepatic glucose output, providing working skeletal muscle with an alternative fuel source (Febbraio et al., 2004). It may therefore be fitting that in addition to aiding working skeletal muscle by supplying an additional fuel source, IL-6 could also act to enhance neural drive to allow for continued force output during prolonged activity. If cytokines are to influence somatic nerve function, the effects may be highly variable depending on the cytokine, source, and environment. The fact that the elevations in both IL-10 and TNF-α, in the current study, were shown to have no correlation with any change in NCV may further add to the argument that different cytokines may induce unique influences or lack thereof. It is important to note that as exercise was utilized to induce a spike in circulating cytokines it is possible that the changes in nerve conduction can be attributed, in part, to other exercise related factors. Although factors such as hydration levels, body temperature, and local metabolite production were controlled for, their influences cannot be completely discounted. However, the authors have provided the r2 values that correspond to the correlations found. As such, we are not suggesting cytokines to be the sole cause of any alterations in NCV, but rather have attempted to quantify the contribution of cytokines to the exerciseinduced changes in NCV. 4.2. Physiological/clinical significance The current study achieved elevations in cytokines similar to those reported in chronic inflammatory conditions such as spinal cord injury (SCI) (Davies et al., 2007). However, it must be taken into consideration that the current study observed acute elevations in cytokines, whereas a number of clinical conditions such as, SCI, multiple sclerosis, obesity, diabetes and depression, are associated with chronic elevations (Hayes et al., 2002; Schiepers et al., 2005; Navarro and Mora, 2006; Davies et al., 2007; Miles et al., 2012). In addition, such conditions commonly present as comorbidities placing these populations under even more exaggerated levels of inflammation. Such factors may make these populations more susceptible to alterations in nerve conduction than that of healthy individuals. It will be necessary for future studies to be performed on such populations in order to examine the potential neuromodulatory influence of chronically elevated cytokines. It is possible to speculate that more severe effects may occur under chronic conditions. Additionally, any small alterations in nerve conduction may be particularly magnified within populations with preexisting neural deficits such as spinal cord injury and multiple sclerosis. It will also be important for future studies to examine a wider variety of cytokines and other inflammatory mediators to assess the potential unique influence of various molecules. 5. Conclusion The current study was the first to examine the association between acutely elevated systemic cytokines and peripheral nerve function in humans. The results demonstrated a significant negative correlation between the change in IL-1ra and the change in NCV as well as a significant positive correlation between the change in IL-6 and the change in NCV. Additionally, IL-10 and TNF-α were found to have no correlation with NCV. These results may indicate the potential for different cytokines to participate in unique neuromodulatory roles. The findings of this study warrant further research, potentially examining special populations with chronically elevated cytokine concentrations such as those with spinal cord injury or multiple sclerosis. The fact that such populations have
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persistent elevations in systemic cytokines on top of pre-existing neurological deficits may make these populations particularly susceptible to the potential effects of channelopathy.
Acknowledgments This study was funded by the Ontario Neurotrauma Foundation (2011-ONF-RHI-MT-894).
Appendix A
Fig. B.2. Representation of the significant positive correlation between the change in IL-6 and the change in NCV at 2 h post exercise (r = 0.61, p = 0.048).
References
Fig. A.1. Representation of typical M-wave used in the calculation of NCV. The M-wave is represented by the largest of the three waveforms on each graph (additional 2 smaller waveforms represent muscle fiber conduction (not assessed in this study)). The upper graph represents the waveforms produced upon stimulation of the median nerve from the proximal location while the lower graph represents the waveforms produced upon stimulation from the distal location.
Fig. B.1. Representation of the significant negative correlation between the change in IL-1ra and the change in NCV at 24 h post exercise (r = −0.65, p = 0.02).
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