Modulation of the axon-reflex response to local heat by reactive oxygen species in subjects with chronic fatigue syndrome

Abstract

Local cutaneous heating causes vasodilation as an initial first peak, a nadir, and increase to plateau. Reactive oxygen species (ROS) modulate the heat plateau in healthy controls. The initial peak, due to C-fiber nociceptor-mediated axon reflexes, is blunted with local anesthetics and may serve as a surrogate for the cutaneous response to peripheral heat. Chronic fatigue syndrome (CFS) subjects report increased perception of pain. To determine the role of ROS in this neurally mediated response, we evaluated changes in cutaneous blood flow from local heat in nine CFS subjects (16–22 yr) compared with eight healthy controls (18–26 yr). We heated skin to 42°C and measured local blood flow as a percentage of maximum cutaneous vascular conductance (%CVC_max). Although CFS subjects had significantly lower baseline flow [8.75 ± 0.56 vs. 12.27 ± 1.07 (%CVC_max, CFS vs. control)], there were no differences between groups to local heat. We then remeasured this with apocynin to inhibit NADPH oxidase, allopurinol to inhibit xanthine oxidase, tempol to inhibit superoxide, and ebselen to reduce H₂O₂. Apocynin significantly increased baseline blood flow (before heat, 14.91 ± 2.21 vs. 8.75 ± 1.66) and the first heat peak (69.33 ± 3.36 vs. 59.75 ± 2.75). Allopurinol and ebselen only enhanced the first heat peaks (71.55 ± 2.48 vs. 61.72 ± 2.01 and 76.55 ± 5.21 vs. 58.56 ± 3.66, respectively). Tempol had no effect on local heating. None of these agents changed the response to local heat in control subjects. Thus the response to heat may be altered by local levels of ROS, particularly H₂O₂ in CFS subjects, and may be related to their hyperesthesia/hyperalgesia.

chronic fatigue syndrome (CFS) is characterized by incapacitating fatigue, joint pain, myalgias, unrefreshing sleep, tender lymph nodes, sore throat, and headache (9). Importantly, many subjects with CFS and fibromyalgia describe excessive pain as a cardinal symptom with allodynia, a heightened sensitivity of their skin to thermal or mechanical stimulation (28, 29, 50). These sensations are transduced by cutaneous nociceptors that sense temperature and painful stimuli (50). Because these stimuli elicit an increased response in those with allodynia, we chose to investigate the response of the skin of subjects with CFS to the application of local heat and measured the resulting changes in cutaneous blood flow with particular regard to the first heat peak that is neurally mediated.

The response of skin to local heating results in three distinct phases of cutaneous blood flow: an initial rapid phase (first peak), a nadir, and an increase to a plateau. The first heat peak is predominantly mediated by neurogenic reflexes through local sensory nerves, can be attenuated by local anesthetics (2, 30, 31), and informs on the acute responsiveness of skin to local thermal stimulation. In contrast, the plateau, which occurs after 20–30 min of heating, is NO dependent (19, 31, 43, 45) and represents a more temporally chronic response to locally applied heat.

Increased oxidative stress levels have been reported in CFS subjects (21), and ROS, including superoxide anions (SO), hydrogen peroxide (H₂O₂), and hydroxyl radicals, may play a role in cutaneous inflammatory hyperalgesia (18). ROS can modulate transient receptor potential vanilloid type 1 (TRPV1) channel-mediated increases in skin blood flow (18, 41). TRPV1 channels, thought to be both heat and pain sensing, are localized within sensory afferents and contribute to the first heat peak in response to local cutaneous heating (53).

We therefore measured the cutaneous vascular response to local heat in CFS subjects to test the hypothesis that ROS alter the response to local heating. Particular attention was paid to the first heat peak that is due to neurogenic reflexes mediated through the activity of local sensory nerves (53). We used allopurinol and apocynin to inhibit xanthine oxidase and NADPH oxidase, respectively, and also reduced local cutaneous levels of superoxide (SO) with tempol and H₂O₂ with ebselen because of the reported vasoactive and sympathetic effects of these compounds (49, 52, 56).

METHODS

Subjects.

In the first series of experiments, we measured the cutaneous heat response alone in eight healthy volunteer subjects aged 18–26 yr, mean age 21.9 ± 1.8 yr (3 men and 5 women). In a second series of experiments, we determined the effects of NADPH-oxidase inhibition, xanthine-oxidase inhibition, superoxide reduction, and H₂O₂ reduction. These studies were then performed in subjects with CFS, aged 16–22 yr, with a mean age of 17.8 ± 1.9 yr (2 men and 7 women), who fulfilled the Centers for Disease Control criteria for CFS (9) and reported symptoms of heightened skin sensitivity to mechanical stimulation and/or heat. All CFS subjects were diagnosed with postural tachycardia syndrome (POTS), which is a common CFS comorbidity (42).

All subjects had normal physical examinations, normal EKG, and echocardiographic evaluations and were free from heart disease and systemic illness. Subjects with CFS included those with persistent or relapsing chronic fatigue of new or definite onset and four or more of the following concurrent symptoms that have persisted or recurred during six consecutive months and have not predated the fatigue: 1) impairment in memory or concentration severe enough to cause substantial reduction of previous levels of educational, social, or personal activities; 2) muscle pain or multiple joint pain; 3) headaches of a new type, pattern, or severity; 4) unrefreshing sleep; and 5) postexertional malaise lasting more than 24 h.

Subjects were not taking any medications and refrained from alcohol and caffeinated beverages for at least 24 h before the study. There were no smokers or trained competitive athletes. Written informed consent was obtained, and the Committee for the Protection of Human Subjects (Institutional Review Board) of New York Medical College approved this protocol. Female subjects were enrolled without regard to the phase of their menstrual cycle except that none were menstruating during testing procedures.

Instrumentation.

All testing was conducted in a temperature-controlled room (25°C) at least 2 h after a light breakfast. All measurements were made in the left calf, and because all experiments were performed with the subject supine, the leg was at the level of the heart throughout all procedures. Subjects had microdialysis catheters placed in the dermal space of the lateral aspect of the left calf after each site was cooled with ice to reduce discomfort of catheter insertion. Each microdialysis catheter (MD-2000 Linear Microdialysis Probes; Bioanalytical Systems, West Lafayette, IN) has a 10-mm microdialysis membrane section that is placed in the intradermal space using a 25-gauge needle as an introducer. The molecular mass cutoff is nominally 30,000 Da. Following placement, all catheters were initially perfused with Ringer solution at 2 μl/min. An integrating laser Doppler flow probe (Probe 413; Perimed) containing seven individual probe tips (each contains a separate transmitting and receiving fiber) was then placed directly over each microdialysis catheter to measure skin blood flow, designated as laser Doppler flow (LDF). LDF was thereafter recorded until values were similar to those measured over the same area before catheter insertion. The return of LDF to approximately preinsertion values indicated recovery from the trauma of the catheter emplacement and usually occurred by 60–90 min (1, 27). When necessary, longer times were allowed until preinsertion LDF was reached. Baseline untreated LDF was then recorded during local heating and 30 min post-heat recovery.

Local heating.

Once baseline LDF values were obtained, the areas under each laser were gradually heated at 1°C/10 s to 42°C, which was maintained for at least 30 min until a plateau was reached. The area underneath the heating unit is 3 cm². Heat was turned off to allow for recovery to baseline LDF.

Blood pressure measurements.

Blood pressure was measured by finger plethysmography (Finometer), intermittently recalibrated against oscillometry. Mean arterial pressure was obtained by averaging the signal over 5 min and compared with oscillometry [using the formula mean arterial pressure = (systolic arterial pressure + 2× diastolic arterial pressure)/3], as previously described (46). Finometer and oscillometric blood pressure were in agreement.

General protocol.

During experiments, four microdialysis catheters were placed to infuse drugs locally into the intradermal space of the calf; the drugs that were perfused were randomly assigned to each catheter. Before the microdialysis catheter insertion, LDF was measured over each of the four insertion sites to estimate baseline flows for later use in determining when the area had recovered from the trauma of catheter insertion. Laser probes were removed and the four microdialysis catheters were inserted. After recovery, LDF was measured while perfusing the catheters with lactated Ringer's solution, and values were recorded for 10 min. Following this, LDF was recorded during local heating at each site with continued perfusion of lactated Ringer's solution. A recovery from heat period followed, requiring 30–60 min. After recovery, catheters were perfused with drugs dissolved in lactated Ringer's solution, and local heating was repeated using the heat-reheat protocol described below.

The effects on the heating response of ROS inhibition using tempol, a superoxide dismutase mimetic (10 μM), xanthine oxidase inhibition using allopurinol (10 μM), NADPH-oxidase inhibition using apocynin (100 μM), and H₂O₂ inhibition using ebselen (100 μM) was evaluated by perfusing each of these drugs through individual microdialysis catheters dissolved in lactated Ringer's solution. After a 30-min run-in period, local heating and heating recovery were recorded at all sites. Following this, maximum blood flow and conductance were elicited by perfusing 28 mM sodium nitroprusside through each of the four microdialysis catheters. The sequence of events for these determinations is shown in Fig. 1.

Fig. 1.

Sequence of events for experimental maneuvers.

We used concentrations of tempol, allopurinol, apocynin, and ebselen as previously described in healthy volunteers (26). None of the inhibitors used influenced the response of skin blood flow to SNP; thus they did not alter maximal CVC.

Use of heat-reheat assessment.

In these experiments, we employed a heat-reheat protocol in which we measured the cutaneous response to locally applied heat up to three times while allowing for the skin blood flow to return to “baseline” in between, as we (26, 47, 48) and others (17) have employed. The variability between the plateau phase of the local heating response during sequential heat-reheat in each catheter is significantly less than that between local heating plateaus of different catheters placed in the same subject. This is in contrast to another study that showed repeated heating of the skin can affect its responses (7).

Data and statistical analysis.

Laser-Doppler skin blood flows were measured in arbitrary perfusion units (pfu). Continuous LDF data were collected at a sampling rate of 200 Hz during experiments. Data from the lasers were multiplexed and interfaced to a personal computer through a analog-to-digital converter (DI-720; Dataq, Milwaukee, WI) using custom data acquisition software. LDF data were converted to units of CVC by dividing by the mean arterial blood pressure.

CVC measurements were then converted to a maximum cutaneous vascular conductance (%CVC_max) by dividing CVC by the CVC_max achieved after the administration of 28 mM sodium nitroprusside at the end of experiments. This fraction was converted to a percentile by multiplication by 100. Conductance data are therefore displayed as %CVC_max. Changes in baseline LDF before and after drugs were compared by two-way ANOVA. Results are shown and reported as mean ± SE. Other comparisons were made by repeated-measures ANOVA, with a Bonferroni post hoc test to look at differences in the local heating response between pre- and postdrug infusion using the particular microdialysis catheter as the within factor. Results were calculated using Statistical Package for the Social Sciences software version 11.0. The value for significance was P < 0.05.

RESULTS

Effects of local heating on cutaneous vascular conductance.

The application of local heat, increased at a rate of 1°C/10 s to 42°C, resulted in the characteristic three distinct phases of cutaneous blood flow: an initial rapid phase (first peak), a nadir, and an increase to a plateau. These results are shown in Fig. 2, comparing the magnitude of the heat response in control subjects to CFS subjects expressed as %CVC_max for baseline, first thermal peak, nadir, and plateau. CFS subjects had significantly lower baseline (preheat) cutaneous blood flow compared with that measured in control subjects. There were, however, no differences between %CVC_max measured in control and CSF subjects for the first thermal peak, nadir, and plateau.

Fig. 2.

Response of skin in control subjects and chronic fatigue syndrome (CFS) subjects to local heating. Data are expressed as percentage of maximum cutaneous vascular conductance (%CVC_max) and are averaged over all subjects. In this figure, averaged data ± SE are shown at baseline, first thermal peak, nadir, and heat plateau.

Effects of antioxidants on LDF.

To evaluate whether ROS are involved in the cutaneous response to local heating in CFS, we first measured local heating, and then local heating during the infusion of apocynin, allopurinol, ebselen, or tempol. The characteristic multiphasic response of skin measured during the 30-min application of local heat is shown in Fig. 3. The effects of NADPH-oxidase inhibition using apocynin on the heat response are also shown. In CFS subjects, in the presence of NADPH-oxidase inhibition due to apocynin, %CVC_max for baseline (14.91 ± 2.21 vs. 8.75 ± 1.66) and the first heat peak (69.33 ± 3.36 vs. 59.75 ± 2.75) were increased significantly (P < 0.05) compared with measurements made in CFS subjects with and without drug, respectively. In control subjects apocynin had no effect on the response to local heat (data not shown) and, in contrast to CFS subjects, had no effect on the first heat peak (Table 1).

Fig. 3.

Response of skin in CFS subjects to local heating alone and after administration of apocynin through microdialysis catheters. Data are expressed as %CVC_max and are averaged over all subjects. In this figure, averaged data ± SE are shown at baseline, first thermal peak, nadir, and heat plateau. *Significantly different from heat alone (P < 0.05).

Table 1.

Magnitudes of heat response (%CVC_max)

	CFS (n = 9)	Control (N = 8)	Control (Apo)	Control (Allo)	Control (Ebsel)	Control (Temp)
Baseline	8.75 ± 0.56*	12.27 ± 1.07	10.36 ± 2.16	11.91 ± 3.52	10.07 ± 2.20	9.29 ± 1.84
First heat peak	60.84 ± 1.75	57.01 ± 1.77	59.29 ± 4.79	60.77 ± 3.63	63.78 ± 3.83	59.92 ± 4.31
Nadir	43.35 ± 1.74	41.69 ± 1.81	47.46 ± 3.83	44.74 ± 1.86	42.89 ± 2.93	39.77 ± 4.49
Plateau	79.87 ± 2.18	83.14 ± 2.49	76.44 ± 4.88	79.54 ± 3.03	80.89 ± 4.60	76.67 ± 4.79

Values shown are mean ± SE. Apo, apocynin; Allo, allopurinol; Ebsel, Ebselen; Temp, Tempol.

^*P < 0.01 significantly different firm control, comparing percentage of maximum cutaneous vascular conductance (%CVC_max).

The effects of xanthine oxidase inhibition using allopurinol on the heat response are shown in Fig. 4. The response to local heating in CFS subjects and xanthine oxidase inhibition due to allopurinol was similar to that seen with apocynin in that the first heat peak was significantly increased (71.55 ± 2.48 vs. 61.72 ± 2.01) (P < 0.05) comparing CFS subjects with and without drug, respectively. Again, as shown in Table 1, allopurinol had no effect on the magnitude of the first heat peak in control subjects.

Fig. 4.

Response of skin in CFS subjects to local heating alone and after administration of allopurinol through microdialysis catheters. Data are expressed as %CVC_max and are averaged over all subjects. In this figure, averaged data ± SE are shown at baseline, first thermal peak, nadir, and heat plateau. *Significantly different from heat alone (P < 0.05).

Figure 5 shows similar data comparing the cutaneous heat response in CFS subjects without drug with that measured with the glutathione peroxidase mimetic ebselen. Reducing H₂O₂ also resulted in a significantly increased first heat peak (76.55 ± 5.20 vs. 58.56 ± 3.66) (P < 0.05) comparing CFS subjects with and without drug, respectively; whereas the baseline, nadir, and the heat plateau remained the same. There was no effect of ebselen on the first heat peak in control subjects (Table 1).

Fig. 5.

Response of skin in CFS subjects to local heating alone and after administration of ebselen through microdialysis catheters. Data are expressed as %CVC_max and are averaged over all subjects. In this figure, averaged data ± SE are shown at baseline, first thermal peak, nadir, and heat plateau. *Significantly different from heat alone (P < 0.05).

The effects of reduced superoxide were tested using tempol, a superoxide dismutase mimetic. Figure 6 shows that there were no differences in the heat response comparing CFS with and without drug for %CVC_max measured at baseline, first heat peak, nadir, or plateau.

Fig. 6.

Response of skin in CFS subjects to local heating alone and after administration of tempol through microdialysis catheters. Data are expressed as %CVC_max and are averaged over all subjects. In this figure, averaged data ± SE are shown at baseline, first thermal peak, nadir and heat plateau.

DISCUSSION

Many CFS subjects report a heightened sensitivity to heat or touch, or allodynia, thus an investigation of their response to locally applied heat was performed. In addition, because we have reported that ROS modulate aspects of this response in subjects with POTS, a common comorbidity of CFS (42), an understanding of factors that influence this response may prove beneficial for these individuals. The results of our study show direct ROS modulation of the cutaneous sensory nerve-dependent neurogenic response (11), the so-called first heat peak, caused by local heating. The first heat peak being due to C-fiber nociceptor-mediated axon reflex (25, 35, 37) that results in vasodilatation is presumed to occur through the local release of calcitonin gene-related peptide (CGRP) (35, 51), substance P (5, 51), and neuropeptide Y (13). This phase of the heat response can be blunted with the application of local anesthetics (31) but not by proximal neural blockade or muscarinic receptors blockade (30, 31). All phases of the local heating response, especially the plateau phase, are thought to be nitric oxide dependent (19, 31).

A study of the effect of age in healthy subjects showed that the initial heat peak was significantly diminished in older subjects (32), suggesting that healthy aging influences the nerves that mediate the axon reflex or alters vascular responsiveness to the neurotransmitters released from these nerves. A recent study showed that the first heat peak is attenuated in subjects with chronic kidney disease (CKD), compared with healthy controls, and the antioxidant, ascorbic acid, returned this response to control values suggesting that oxidative stress may influence neurovascular and microvascular function in this population (8). Studies have also shown that exogenous antioxidants can reverse the effects of aging on the response to both local heating (15) and cooling (54).

We recently showed in healthy control subjects that ROS, produced via NADPH and xanthine oxidase pathways, modulates the response of skin to the application of heat and thus contributes to the control of local cutaneous blood flow (26). However, little information exists regarding the role of ROS in subjects with CFS. The present study, by demonstrating augmentation of the first heat peak with decreased ROS in CFS subjects suggests that oxidant-stress affords cutaneous vascular control in these subjects.

Our results, with the exception of tempol showing that reducing ROS resulted in an increased first heat peak, implies that ROS afford a suppressive influence on this response. A study in rats showed that ROS are involved in mediating TRPV1-mediated vasodilatation (41); however, in contrast to our findings, they demonstrated that reducing ROS diminished the magnitude of the first heat peak. Furthermore, following inhibition of the first heat peak with catalase and SOD, the addition of apocynin showed that ROS derived from NADPH oxidase is produced in response to neuropeptides (41). Similar to our present study, these investigators also showed that tempol had no effect on neurogenic vasodilation, in this case induced by substance P, suggesting that superoxide has little effect on this response; the reason for these findings is unclear but may be related to increased levels of H₂O₂.

The results of our study suggest that the ROS responsible for suppression of the heat response is likely to be H₂O₂ and not superoxide. This is based on the lack of effect of tempol in reducing superoxide production and the finding that ebselen, the glutathione peroxidase mimetic, significantly increased the magnitude of the first heat peak. This is also consistent with our finding that apocynin and allopurinol increased the height of the first heat peak, because endothelial cell NOX4 (NADPH Oxidase, type 4) (3, 4) and xanthine oxidase (12) can each produce H₂O₂.

The ability of ROS to modulate the heat response in this cohort of subjects may be related to allodynia reported by many CFS subjects. Increased ROS levels have been implicated in the generation and maintenance of pain-associated symptoms (21) and myalgias (10) in subjects with CFS. Increased ROS has been shown to induce persistent neuropathic spinal cord pain (23, 55) and that reducing spinal cord microglial ROS level attenuated mechanical allodynia and thermal hyperalgesia in mice (22). It is possible therefore that altered local ROS, particularly H₂O₂, can influence local sensory nerve activity and may explain allodynia, experienced by many subjects with CFS and fibromyalgia (28, 29, 50). Although the data suggest that the use of antioxidants in CFS subjects may result in increased skin blood flow, until the relationship between local skin blood flow and nociception is defined in this population, the therapeutic utility of antioxidants remains unknown.

The first heat peak may be due to TRPV-1 channel activation acting as a local heat sensor through cutaneous sensory nerve depolarization during heating. Numerous studies have demonstrated the effects of ROS on ion channel activity (6, 16, 39) and its effect on transmembrane potential (36, 39). In addition ROS can influence the excitability of amygdala neurons and thus alter central pain mechanisms (24). In contrast to these studies, our findings are somewhat contradictory because reducing local levels of ROS resulted in increased local blood flow due to the application of local heat. Although the relationship between increased local blood flow and the perception of pain remains to be determined in this unique population, peripheral nociception in CFS subjects may in part be controlled by ROS-related modulation of receptors.

In the present study we did not evaluate the perception of heat in our study subjects, but rather we measured heat-induced changes in skin blood flow. It is possible that CFS subjects perceived this stimulus differently than controls; however, because there were no differences in the %CVC_max for either the first heat peak and plateau, comparing CFS to controls, this was likely not the case. Therefore, we cannot comment on the central perception of pain, but peripheral nociceptor activity is likely altered in this group of CFS subjects. We used local heating that gradually increases the temperature of the skin at a rate of 1°C/10 s to a maximum of 42°C, which based on previous studies in healthy controls and those with CFS/POTS, is a temperature that is perceived as being warm, but nonpainful, and results in increased local blood flow due to heat in the absence of systemic stimulation. Additionally, we did not evaluate the degree of allodynia in any of the current subjects with dolorimetry (pressure algometry) because of inherent biases introduced by this method due to anticipation of a painful stimulus or as a result of generalized psychological hypervigilance (33, 34).

In previous studies of POTS patients, most of whom also had CFS, subjects were partitioned into three groups; low, normal, and high flow on the basis of calf blood flow (44). We showed that compared with controls, low flow POTS subjects have reduced skin blood flow for both baseline and local heat-induced plateau values (43, 46). In contrast, normal flow POTS subjects had baseline skin blood flow that was similar to controls but the plateau skin blood flow was significantly higher (45). Although the emphasis of the present study is on the first heat peak, we did measure a significant reduction of baseline skin blood flow in CFS subjects compared with control. It is possible that this reduced baseline skin blood flow is due to increased vasoconstrictor tone mediated through the actions of AT1R activation of NADPH oxidase. The finding that apocynin, by inhibiting NADPH oxidase, increases baseline blood flow in CFS subjects compared with that measured in the absence of drug supports this and suggests that ROS may play a role in the control of cutaneous blood flow at room temperature. However, because enrollment in this study was based on the diagnosis of CFS and not measurements of calf blood flow, it is difficult to know if these differences are related to the 3 POTS subgroups.

The lack of effect of tempol is in contrast to what we have shown previously in healthy controls that inhibition of superoxide can partially mitigate the vasoconstrictive effects of Ang II through its effect in the heat plateau (26). Interestingly, none of these agents had any effect on the plateau phase of the local heat response measured in CFS subjects. Several investigations have demonstrated a role for ROS and in the regulation of nitric oxide manifested by alterations in the plateau phase of the response to local heat in both healthy subjects (26) and those with hypercholesterolemia (14). It is possible therefore that the role of local ROS in the regulation of the NO-dependent heat plateau phase in CFS is minimal.

Limitations

In the present study, we enrolled women without regard to menstrual cycle. This was likely a limitation because the phase of the menstrual cycle can affect NO-dependent mechanisms and may also exert influence on ROS-dependent functions (38). We also combined data from women and men, and this may have obscured sex-related differences in the control of skin blood flow. This is suggested by a recent study showing sex-specific differences in the response of skin blood flow to adrenergic stimulation (40).

We did not evaluate the degree of either mechanical or thermal allodynia in this cohort of CFS subjects. Although these measures would have been interesting, the nonpainful nature of the applied thermal stimulus may provide a relatively unbiased measure of altered cutaneous responsiveness in this patient population.

Microdialysis is invasive and alters the interstitial milieu. Studies suggests that flow responses return to baseline levels within ∼1 h (1). In pilot experiments, we measured baseline flows, removed the LDF probes, instrumented the same site with microdialysis catheters, replaced the probes, waited ∼1 h, and repeated the LDF measurements with similar results (on average). Also, the present studies utilized our heat-reheat protocol, which yields reproducible results (26, 47, 48), as confirmed by others (17). Other investigators, however, have reported that repeated heating of the skin can affect its responses (7). This discrepancy is likely due to methodological differences between their study design and ours, which include use of a different anatomical site and a dissimilar source of heat and laser measuring device.

Other differences between the present results and those reported by others may be due in part to variability among subjects and may also be related to our use of the lower leg as the site for investigation, compared with the forearm that is more commonly used (15, 20, 32). One additional limitation of these studies is that they only inform on the vasoregulatory response of skin to the application of local heating. Therefore any speculation regarding the role of ROS in modulation or influencing skin blood flow should be considered within this context and may not be applicable to whole body response to heat.

In summary, although using skin as surrogate for the investigation of vascular control, we studied subjects with CFS who experience allodynia. In these subjects, we found that a response to localized skin heating, the magnitude of the first peak, may be altered by local levels of ROS and may be related to hyperesthesia/hyperalgesia reported by many individuals with CFS.

GRANTS

This work was supported by the National Heart, Lung, and Blood Institute Grants 1-RO1-HL-074873, 1-RO1-HL-087803, and 1-F30-HL-097380 and a grant from the Chronic Fatigue and Immune Dysfunction Syndrome Association.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: M.S.M., A.A., and J.M.S. conception and design of research; M.S.M., A.A., and Z.R.M. performed experiments; M.S.M., A.A., Z.R.M., and J.M.S. analyzed data; M.S.M., A.A., and J.M.S. interpreted results of experiments; M.S.M. prepared figures; M.S.M. drafted manuscript; M.S.M. edited and revised manuscript; M.S.M., A.A., Z.R.M., and J.M.S. approved final version of manuscript.