biochemicals associated with pain and inflammation are elevated in sites near to ando remote from...

7/27/2019 Biochemicals Associated With Pain and Inflammation Are Elevated in Sites Near to Ando Remote From Active Myof

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ORIGINAL ARTICLE

Biochemicals Associated With Pain and Inflammation areElevated in Sites Near to and Remote From Active MyofascialTrigger Points

Jay P. Shah, MD, Jerome V. Danoff, PhD, PT, Mehul J. Desai, MD, Sagar Parikh, BA,Lynn Y. Nakamura, MD, Terry M. Phillips, PhD, DSc, Lynn H. Gerber, MD

ABSTRACT. Shah JP, Danoff JV, Desai MJ, Parikh S,Nakamura LY, Phillips TM, Gerber LH. Biochemicals associ-ated with pain and inflammation are elevated in sites near toand remote from active myofascial trigger points. Arch PhysMed Rehabil 2008;89:16-23.

Objectives: To investigate the biochemical milieu of theupper trapezius muscle in subjects with active, latent, or absentmyofascial trigger points (MTPs) and to contrast this with thatof the noninvolved gastrocnemius muscle.

Design: We used a microanalytic technique, including nee-dle insertions at standardized locations in subjects identified as

active (having neck pain and MTP), latent (no neck pain butwith MTP), or normal (no neck pain, no MTP). We followed apredetermined sampling schedule; first in the trapezius muscleand then in normal gastrocnemius muscle, to measure pH,bradykinin, substance P, calcitonin gene-related peptide, tumornecrosis factor alpha, interleukin 1 (IL-1), IL-6, IL-8, sero-tonin, and norepinephrine, using immunocapillary electro-phoresis and capillary electrochromatography. Pressure algom-etry was obtained. We compared analyte concentrations amonggroups with 2-way repeated-measures analysis of variance.

Setting: A biomedical research facility.Participants: Nine healthy volunteer subjects.Interventions: Not applicable.Main Outcome Measures: Preselected analyte concentrations.Results: Within the trapezius muscle, concentrations for all ana-

lytes were higher in active subjects than in latent or normal subjects(P.002); pH was lower (P.03). At needle insertion, analyte con-centrations in the trapezius for the active group were always higher(pH not different) than concentrations in the gastrocnemius muscle.At all times within the gastrocnemius, the active group had higherconcentrations of all analytes than did subjects in the latent andnormal groups (P.05); pH was lower (P.01).

Conclusions: We have shown the feasibility of continuous,in vivo recovery of small molecules from soft tissue withoutharmful effects. Subjects with active MTPs in the trapeziusmuscle have a biochemical milieu of selected inflammatorymediators, neuropeptides, cytokines, and catecholamines dif-ferent from subjects with latent or absent MTPs in their trape-zius. These concentrations also differ quantitatively from aremote, uninvolved site in the gastrocnemius muscle. Themilieu of the gastrocnemius in subjects with active MTPs in thetrapezius differs from subjects without active MTPs.

Key Words: Inflammation; Microdialysis; Myofascial pain;

Rehabilitation; Trigger points, myofascial. 2008 by the American Congress of Rehabilitation Medi-

cine and the American Academy of Physical Medicine andRehabilitation

MYOFASCIAL PAIN SYNDROME (MPS), a commontype of nonarticular musculoskeletal pain, is a conditionassociated with regional pain and muscle tenderness character-ized by the presence of hypersensitive nodules, also calledmyofascial trigger points (MTPs).1 MPS affects up to 95% ofpeople with chronic pain disorders and is a common finding inpatients in specialty pain management centers.2

MTPs are easily identified through physical examination ashyperirritable nodules within taut bands of skeletal muscle, thepalpation of which can produce a muscle twitch and referredpain.1,2 Active MTPs are associated with pain, are acutelytender to palpation, and may contribute to general motor dys-function (stiffness and restricted range of motion). LatentMTPs, which have similar physical findings, are often associ-ated with motor dysfunction and muscle tenderness, but with-out spontaneous pain. Normal or uninvolved muscle does notcontain taut bands or MTPs.1

In acute muscle pain, local tenderness is often caused by theperipheral sensitization of local muscle nociceptors.3 Nociceptiveterminals in muscle have a multitude of different receptors in theirmembranes, including matched receptors for molecules that arereleased from damaged tissue such as bradykinin, serotonin, pro-

tons (H

), and prostaglandins.3

Furthermore, continuous activa-tion of nociceptors by these and other endogenous substances canlead to central sensitization of dorsal horn neurons.3 The contin-ued presence of these inflammatory mediators and other bio-chemicals may be necessary for persistent pain conditions such asMPS.3 There has been little documentation, however, of the bio-chemical differences between the local tissue milieu of normalmuscle and muscle with nonpainful or painful MTPs.

Although the physical findings of myofascial pain involve thelocal muscle tissue, we have observed that standard treatmentapproaches have been largely empirical and suboptimal. Medica-tions may provide moderate symptom improvement without com-plete resolution of the symptoms or the elimination of the MTP.4

See commentary, p 157.

From the Rehabilitation Medicine Department, National Institutes of Health, Clin-ical Center, Bethesda, MD (Shah, Danoff, Parikh); Departments of Exercise Science(Danoff) and Anesthesiology and Critical Care Medicine (Desai), George Washington

University, Washington, DC; National Rehabilitation Hospital, Washington, DC(Nakamura); National Institutes of Health, National Institute of Biomedical Imagingand Bioengineering, Ultramicro Analytical Immunochemistry Resource, Bethesda,MD (Phillips); and College of Health and Human Services, Center for the Study ofChronic Illness and Disability, George Mason University, Fairfax, VA (Gerber).

Supported by the Intramural Research Program, National Institutes of Health(NIH), the Clinical Center and Office of the Director, NIH.

A commercial party having a direct financial interest in the results of the researchsupporting this article has conferred or will confer a financial benefit upon the authoror 1 or more of the authors. Shah, Danoff, Phillips, and Gerber have filed a patentapplication for the device used in this study.

Correspondence to Jay P. Shah, MD, Rehabilitation Medicine Dept, ClinicalResearch Center, National Institutes of Health, 10 Center Dr, Rm 1-1469, MSC 1604,Bethesda, MD, 20892,e-mail: [email protected].

0003-9993/08/8901-00646$34.00/0doi:10.1016/j.apmr.2007.10.018

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Arch Phys Med Rehabil Vol 89, January 2008


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Eliciting a local twitch response (LTR)* through dry needling of

MTPs often has a therapeutic benefit.

5

Mechanisms for this arehypothetical and have not been established. Associations betweenendogenous substances and muscle pain have been shown, but thepathogenesis of myofascial pain remains unclear. Assaying thelocal milieu before, during, and after an LTR could potentiallydisclose changes in bioactive substances that may contribute topain. This could then lead to the development of treatments thattarget underlying mechanisms.

In a previous study,6 we explored the local milieu using anovel microanalytical system, including a minimally invasive30-gauge needle, that enabled the in vivo collection of smallvolumes (0.5L) and sub-nanogram sizes (75kDa) of sol-utes from muscle tissue (fig 1). This system gave us thecapability to safely explore and measure the local biochemicalmilieu of MTPs before, during, and after an LTR.

We found differences in the local biochemical milieu (eg,inflammatory mediators, neuropeptides, catecholamines, cyto-kines) of subjects with active MTPs, compared with subjectswith latent MTPs and those without MTPs, at a standardizedlocation in the upper trapezius muscle.6 Furthermore, the localmilieu does appear to change with the occurrence of an LTR,and these changes can be monitored almost continuously byusing short (1030s) collection intervals. An important, unan-swered question is whether the differences in the biochemicalmilieu between active, latent, or no MTPs pertain to the uppertrapezius exclusively, or whether these differences are alsopresent in remote, uninvolved sites.

Our purposes in this study were: (1) to determine the repro-ducibility of previous findings that showed the unique bio-

chemical milieu of substances associated with pain and inflam-mation in an active MTP in the upper trapezius muscle, and (2)to compare the similarity of analyte levels sampled from thebiochemical milieu in the upper trapezius of subjects withactive, latent, and no MTPs with those levels in a remoteuninvolved site in the upper medial gastrocnemius muscle.

METHODS

Participants

Subjects were recruited from members of the staff of theclinical center at the National Institutes of Health (NIH). Allsubjects underwent a thorough musculoskeletal evaluation soas to rule out potential causes of their symptoms other thanMTPs in the upper trapezius muscle. Exclusion criteria in-

cluded a history of the following conditions: fibromyalgia,cervical or lumbar radiculopathy, other pain syndromes, orcancer. Subjects with knee pain, pathology or infection, previ-ous injection treatments, bleeding disorders, and psychologicissues (eg, fear of needles), or who were taking medications orwere smokers, were excluded. Subjects who had calf pain orMTPs in the upper medial gastrocnemius were also excluded.None of the subjects had active or latent trigger points in theregion of the gastrocnemius in which the needle was inserted.Two clinicians examined all subjects (JPS, MJD). The seniorexaminer (JPS) is a practicing physician experienced in theevaluation and treatment of musculoskeletal disorders and is aninstructor in these techniques.

Based on their history and physical findings in the upper tra-pezius muscle, 9 subjects were recruited. The 9 were then assigned

to 1 of 3 groups: (1) active (MTP present, idiopathic cervical painof3mo duration; 3 subjects); (2) latent (MTP present, no cer-vical pain; 3 subjects); and (3) normal (MTP absent, no cervicalpain; 3 subjects). A fourth subject initially recruited for the activegroup was disqualified. Post hoc review of her medications andclinical history (ie, an unknown cervical radiculopathy) led to thedecision that she did not qualify for the active group.

An institutional review board at the NIH approved the studyprotocol and all subjects signed an informed consent documentapproved by that board.

Instrumentation

We have previously described our instrumentation.6 In brief,we used a microdialysis technique to sample the substances ofinterstitial fluid, which diffused through a semi-permeable mem-brane at the tip of a 30-gauge stainless steel hypodermic needle.6,7

Analyte levels in the samples were quantified using immunoaf-finity capillary electrophoresis and capillary electrochromatogra-phy analysis, 2 techniques known to provide reliable results.8-10

Procedures

Subjects completed a visual analog scale (VAS) to documentself-reported pain intensity, and underwent pressure algometrya

at GB-21 in the upper trapezius and LV-7 in the medialgastrocnemius.11 GB-21 is a standard acupuncture location inthe suprascapular region, midway between the tip of the acro-mion process and below the spinous process of the C7 vertebra.LV-7 is located on the medial side of the leg, inferior to themedial condyle of the tibia, in the upper portion of the medial

head of the gastrocnemius muscle. We selected these points tostandardize procedures for all subjects. We used pressure al-gometry to measure local pain (pressure pain threshold [PPT])bilaterally in all subjects. Algometry has produced valid andreliable data relative to local pain.11

Six microdialysis needles were fabricated for this study.They were gas-sterilized between uses. The microdialysis sys-tem was calibrated as in the previous study.6

For the experimental procedures, subjects were comfortablypositioned prone on a standard clinical exam table, with 2 pillowsfor support and stabilization. The microdialysis needle was theninserted into the upper trapezius muscle at point GB-21 withoutpenetrating the MTP (if present). Muscle penetration was con-

*Local twitch response refers to the brief local contraction that occurs when atrigger point is stimulated by snapping palpation or needle penetration. Dry needlingrefers to stimulation of a trigger point, usually with a narrow object such as a bluntneedle or empty hypodermic needle.

Fig 1. Microdialysis needle.

17BIOCHEMICALS IN ACTIVE MYOFASCIAL TRIGGER POINTS, Shah



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firmed by change in tissue resistance at needle contact with themuscle and by surface electromyography pickup. The needle waskept stationary in situ for 1 minute, after which collection of thesample commenced. Five minutes after insertion, the needle wasadvanced into the muscle until an LTR was obtained in the activeand latent subjects. Again, this was confirmed by surface electro-myography. Depth of penetration was estimated to be between 0.5and 1.0cm. Equivalent needle advancement was used in the nor-mal subjects although an LTR was not observed. The dialysate(physiologic saline) was pumped through the system at a flow rateof 5L a minute for the entire procedure. Dialysate was sampledaccording to the following schedule: every minute for the first 4minutes, then every 20 seconds for 4 minutes (minutes 48), thenevery minute for another 6 minutes (minutes 814). Total collec-tion time was 14 minutes. The collection schedule is summarizedin appendix 1.

After 14 minutes, the needle in the trapezius muscle was re-moved and subjects were repositioned in a comfortable supineposition with the knees supported by a pillow. A second sterilemicrodialysis needle was then inserted into the upper medial

gastrocnemius muscle at LV-7 and held in this position for 10minutes, with samples being collected every minute. Depth ofpenetration was approximately 0.5cm. The needle remained sta-tionary during this procedure because a local twitch response wasnot desired (and was not expected to occur in normal muscle). Aflow rate through the system of 5L a minute was maintainedduring the calf procedure.

All samples (22 from the trapezius, 10 from the gastrocne-mius for each subject) were analyzed by immunoaffinity cap-illary electrophoresis, capillary electrochromatography, andmicro-pH (a modified microcombination electrode in combi-nation with an Orion model 370b pH meter) for pH and 9different analytes including bradykinin, substance P (SP), cal-citonin gene-related peptide (CGRP), tumor necrosis factoralpha (TNF-), interleukin-1 (IL-1), IL-6, IL-8, serotonin

(5-HT), and norepinephrine.Statistical Design and Analysis

We used 2, 1-way analyses of variance (ANOVAs) to com-pare VAS scores and algometry values among the 3 groups.

Analyte concentrations for all subjects were averaged at eachtime point. This produced 3 concentration versus time curves foreach analyte in the trapezius and 3 curves in the gastrocnemiusmuscles. Eight of the analytes recovered in this study were alsorecovered from the trapezius in our previous work,6 in which weused subjects with inclusion and exclusion criteria similar to thoseof the current study. These were pH, SP, CGRP, bradykinin,TNF-, IL-1, 5-HT, and norepinephrine. Concentration curves

of these analytes from the trapezius muscles of subjects in thisstudy were graphically compared with data from our previousstudy.6 These data were also compared through 2-way repeated-measures ANOVA using time (repeated variable: early, mid, late)and study (current vs previous).

Data from the previous and current samples were thenpooled to evaluate analyte concentrations; we used 2-wayrepeated-measures ANOVA with time as the repeated variableand group (active, latent, normal) as a nonrepeated variable.

We did a third analysis with the current data to compareanalyte concentrations in the trapezius muscle with analyteconcentrations in the gastrocnemius muscle. The between-group variable again had 3 levels: active, latent, and normal.The within-group (repeated) variable included 3 collectionevents for the trapezius and 1 for the gastrocnemius (table 1).

Post hoc testing of significant ANOVA findings was donewith protected t tests subject to Bonferroni adjustments of levels. Data variability has been represented on the figures bythe standard error of the mean, which is the dispersion ofobservations around a sample mean.

RESULTS

Subjects in the active group reported greater pain levels

(P

.001) on the VAS than did subjects in the latent and normalgroups. The active group also had a lower PPT in both thetrapezius and gastrocnemius muscles, which would indicate agreater sensitivity to direct pressure. The PPT differences werenot significant, however.

In comparing data from subjects previously sampled6 withthe subjects in this study, we found close agreement of bio-chemical concentrations. Figure 2 shows, as examples, pH andSP. ANOVA indicated that with data from both studies com-bined, concentrations of all analytes for the active group werehigher than concentrations in the latent and normal groups (pHlevel is lower). There were no statistical differences betweenanalyte concentrations from the previous study and this study.

The averaged data for the remaining biochemical concentra-tions from subjects in this study are displayed in figures 3

0

3

6

9

pHunits

0.00

100.00

200.00

300.00

400.00

500.00

600.00

0.00 5.00 10.00 15.00

Time (min)

pg/L

Set 1 Active

Set 1 Latent

Set 1 Normal

Set 2 Active

Set 2 Latent

Set 2 Normal

A

B

Fig 2. Analyte concentrations in the trapezius combining previousand current data. Collection for (A) pH and (B) SP.

Table 1: Labels and Definitions of Events Used in Repeated-Measures ANOVA

Event

Designation Time (min) Location Description

Pre 1:00

2:00

Trapezius Needle

insertion

Peak 5:00

5:20

Trapezius Needle

advancement

and LTRPost 10:00

11:00

Trapezius Recovery

Calf* 2:00

3:00

Gastrocnemius Needle

insertion

*Calf event times were independent of trapezius event times.

18 BIOCHEMICALS IN ACTIVE MYOFASCIAL TRIGGER POINTS, Shah



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through 6 (trapezius muscle only). Examining the ANOVAmain factor group is equivalent to collapsing the time factor (ie,combining all time levels together). This enabled us to compare

the overall analyte concentrations in the trapezius muscleacross the 3 groups. Concentrations for all analytes in thetrapezius, except pH, were significantly higher in active than inlatent or normal subjects (P.002). In the active group, the

recovery (post) amounts of SP and CGRP were significantlylower than the baseline (pre) levels (P.02). For pH, the activegroup was significantly lower (P.03). There were no overalldifferences between the latent and normal subjects.

We also found differences in analyte concentrations in thegastrocnemius muscles among the 3 groups. In general, the activegroup had higher concentrations of all analytes than did those inthe latent and normal groups (P.05). In the active group, pH waslower (P.01). Figures 7 through 9 show comparisons of gastroc-nemius concentrations with trapezius concentrations. Statisticalrelationships are summarized in table 2.

We used repeated-measures ANOVA to compare analyte con-centrations within the trapezius at specified events (see table 1)with those in the gastrocnemius. We used post hoc testing toidentify individual level differences. In all cases, significance was

adjusted, using the Bonferroni method, to be at least less than .05.For the active group, analyte concentrations in the gastrocnemiuswere almost always lower than concentrations in the trapezius. Forthe latent group, gastrocnemius concentrations were consistentlylower than the peak trapezius concentrations, but not necessarilyfor other time levels. The normal group was similar to the latentgroup except there were no significant differences in TNF- andnorepinephrine. Table 3 summarizes the comparisons of trapeziusand gastrocnemius levels.

DISCUSSION

We have confirmed that biochemicals associated with painand inflammation are elevated in soft tissue in the vicinity of

0

225

450

675

0.00 5.00 10.00 15.00

pg/mL

Active

Latent

Normal

pM/L

300

0

100

200

0.00 5.00 10.00 15.00

Time (min)

A

B

Fig 3. Analyte concentrations in the trapezius for (A) CGRP and (B)bradykinin.

0

250

500

0.00 5.00 10.00 15.00

pg/mL

0

250

500

750

0.00 5.00 10.00 15.00

Time (min)

pg/mL

Active

LatentNormal

A

B

Fig 4. Analyte concentrations in the trapezius for (A) TNF- and (B)IL-1.

0

300

600

900

0.00 5.00 10.00 15.00

pg/mL

0

200

400

0.00 5.00 10.00 15.00

Time (min)

pg/mL

Active

LatentNormal

A

B

Fig 5. Analyte concentrations in the trapezius for (A) IL-6 and (B)IL-8.




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active MTPs. The concentrations of these biochemicals, includ-ing protons (equivalent to inverse pH), bradykinin, SP, CGRP,TNF-, IL-1, 5-HT, and norepinephrine differentiate the ac-tive group from the latent and normal groups. Two additional

analytes not previously sampled, IL-6 and IL-8, were alsosignificantly higher in the active group.We have also shown that the concentrations of these bio-

chemicals in the upper trapezius, adjacent to active MTPs,differ quantitatively from the remote, uninvolved site we chosein the gastrocnemius muscle. There are also consistent differ-ences in the biochemical milieu among active, latent, andnormal groups in the gastrocnemius. This suggests that sub-stances associated with pain and fatigue are not limited to localareas of MTPs or a single anatomic locus.

These findings suggest that subjects with active MTPs have agreater presence of inflammatory mediators, neuropeptides, cat-echolamines, and cytokines within the local milieu of the triggerpoint. The elevated levels of these sensitizing substances and ahigher proton concentration (lower pH) in the active MTP support

Simons integrated hypothesis1

of an area of relative localischemia and hypoxia compared to latent and normal muscle.According to the integrated hypothesis, . . . a central MTrP

[myofascial trigger point] has multiple muscle fibers with end-plates releasing excessive acetylcholine, and it shows histopatho-logical evidence of regional sarcomere shortening.12(p100) Sarco-mere shortening requires high oxygen to maintain continuousmuscle contractile activity. The combination of this increasedmetabolic demand and the ischemia from compromised circula-tion caused by increased tension of the involved sarcomeres couldaccount for the severe local hypoxia. Consequently, the relativeischemia and hypoxia would cause the elevated levels of sensi-tizing substances we found in active MTPs. Furthermore, rela-

tively higher levels of these substances in active MTPs helpexplain the local tenderness and referred pain of active MTPs.12

Issberner et al13 showed a positive correlation between painand local acidity. An acidic milieu alone (without muscledamage) is sufficient to cause profound changes in the prop-erties of the pain matrix such that alterations in pH would besufficient to modify the threshold sensitivity of the nociceptor.An acidic pH stimulates the production of bradykinin duringlocal ischemia and inflammation; therefore, a local acidic mi-lieu may explain the pain associated with an active MTP.

Mechanical hyperalgesia is a hallmark of an MTP. Ongoingnociceptive activity, however, is not necessary to cause me-chanical hyperalgesia. In a rat model,14 repeated injections ofacidic saline boluses into 1 gastrocnemius muscle producedbilateral, long-lasting mechanical hypersensitivity (ie, hyperal-gesia) of the paw. Furthermore, the study found that the per-sistent mechanical hyperalgesia was not caused by muscletissue damage and was not maintained by continued nocicep-tive input from the site of muscle injury.14 This model clearlydemonstrates that secondary mechanical hyperalgesia may bemaintained by neuroplastic changes in the central nervoussystem (eg, in spinal dorsal neurons and thalamic neurons).14

Hong et al15 suggest that an integrative mechanism at the spinal

cord level in response to sensitized nociceptors has a role indevelopment of active MTPs and should be considered in anypathogenetic hypotheses.

In an expansion of Simons integrated hypothesis1, Gerwin etal16 postulate that the acidic pH may also modulate the motorendplate by inhibiting acetylcholinesterase. This would result inan increased concentration of acetylcholine at the synaptic cleftthat would lead to sarcomere contraction and formation of a tautband.

0

13

25

0.00 5.00 10.00 15.00

nM/L

0

6

12

0.00 5.00 10.00 15.00

Time (min)

nM/L

Active

LatentNormal

A

B

Fig 6. Analyte concentrations in the trapezius for (A) 5-HT and (B)norepinephrine.

100

200

300

0.00 5.00 10.00 15.00 0.00 5.00 10.00

Time (min)

pM/L

0

4

8

pHUnits

Trapezius ---------------

----

Gastrocnemius--

Active

LatentNormal

A

B

Fig 7. Analyte concentrations in the trapezius compared with thegastrocnemius for (A) pH and (B) bradykinin.




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We found significantly elevated levels of SP and CGRP in thevicinity of the active MTPs. The orthodromic and antidromicrelease of these substances is greatly increased in response tonociceptor activation, (eg, by H and bradykinin binding to theirmatched receptors).17 This dynamic phenomenon may lead to

neuroplastic changes in the dorsal horn and profound changes inneuronal activity and the perception of pain.The goal of trigger point needling of the active MTP is to

elicit multiple LTRs.4,18 In our active group, SP and CGRPwere the only 2 analytes for which concentrations during therecovery period (post) after the LTR were significantly belowtheir concentrations at baseline (pre). This corresponds with thecommonly observed decrease (at least temporarily) in pain andlocal tenderness after the release of an MTP by needling.Physiologically, this may be caused by interference with noci-ceptor membrane channels or transport mechanisms associatedwith a briefly augmented inflammatory response. The levels ofthese analytes may also fall because of a local increase in bloodflow. Additional study of these analytes would be needed toexplain the nature of this phenomenon.

SP causes mast cell degranulation with the release of histamine,serotonin, and upregulation of both proinflammatory (eg, TNF-,IL-6) and anti-inflammatory cytokines (eg, IL-4, IL-10). TNF- isthe only cytokine prestored in the mast cell and is released im-mediately after mast cell degranulation. TNF- stimulates norepi-nephrine production. We found significantly elevated levels ofserotonin and norepinephrine in subjects with active MTPs. Theincreased levels of norepinephrine may be associated with in-creased sympathetic activity in the motor end plate region.

Levels of TNF- and IL-1 were significantly elevated in thetrapezius of subjects with active MTPs. In a rat model, TNF-produces a time- and dose-dependent muscle hyperalgesia withinseveral hours after injection into the gastrocnemius or biceps

brachii muscles. This hyperalgesia was completely reversed bysystemic treatment with the nonopioid analgesic metamizol.19

Furthermore, TNF- did not cause histopathologic tissue damage

or motor dysfunction. One day after injection of TNF-, therewere elevated levels of CGRP, nerve growth factor, and prosta-glandin E2 in the muscle. Therefore, TNF- and other proinflam-matory cytokines such as IL-1 may have a role in the develop-ment of muscle hyperalgesia, and the targeting ofproinflammatory cytokines might be beneficial for the treatmentof muscle pain syndromes.19 This may depend, however, on thetime course of the injury and inflammatory response. Hoheiselsdata20 suggest that TNF- has a dual action when released intra-muscularly. Specifically, . . . it suppresses neuronal hyperexcit-ability early after release but contributes to neuronal hyperexcit-ability in a later phase.20(p174)

We found significantly elevated levels of IL-6 and IL-8 inthe trapezius of the active group. IL-6 has both pro- andanti-inflammatory effects and like TNF-, IL-1, and IL-8, it

produces a dose- and time-dependent mechanical hypernoci-

0

6

12

0.00 5.00 10.00 15.00 0.00 5.00 10.00

Time (min)

nM/L

Trapezius ---------------

----

Gastrocnemius--

Active

LatentNormal

0

300

600

pg/L

A

B

Fig 8. Analyte concentrations in the trapezius compared with thegastrocnemius for (A) SP and (B) norepinephrine.

5.00

0

250

pg/mL

500

Trapezius ---------------

----

Gastrocnemius--

Active

LatentNormal

0.00 10.00 15.00 0.00 5.00 10.00

0

300

600

Time (min)

p

g/mL

A

B

Fig 9. Analyte concentrations in the trapezius compared with thegastrocnemius for (A) TNF- and (B) IL-6.

Table 2: Significant Analyte Differences in Gastrocnemius,Comparison of Groups (average at 2 and 3min)

Analyte Differences -Level (P)

pH Active latent, normal .01

SP, CGRP, TNF- IL-1, IL-6,

IL-8, norepinephrine

Active latent, normal .01

Bradykinin Active normal .01

5-HT Active latent normal .05




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ception in rats. Cytokines and chemokines play a crucial role inmediating inflammatory and neuropathic pain in rat models oftissue injury. Furthermore, the cascade of cytokines releasedfollows 2 distinct pathways with different final mediators (ie,prostanoids and sympathetic amines). For example, . . .TNF-, IL-6, and IL-1 sequentially precede the release ofprostanoids to induce hypernociception in rats.21(p123)

Conversely, the chemokine IL-8 participates in a separate(ie, cyclo-oxygenase [COX] independent) pathway and coor-dinates the sympathetic components of the inflammatory hy-pernociception after tissue injury. Moreover, IL-8 induces adose- and time-dependent mechanical hypernociception. Ele-vated levels of IL-8 in active MTPs may mediate inflammatoryhypernociception, muscle tenderness, and pain through thispathway, which is inhibited by adrenergic receptor antago-nists; COX inhibitors, however, do not inhibit this pathway.21

Analysis of samples taken from both muscles found that allanalyte concentrations in the trapezius of the actives weregenerally higher than concentrations in their gastrocnemiusmuscles. There were fewer differences in the latent group andalmost none in the normal group. The differences between theactive and the other groups in the trapezius were more dramatic

(ie, greater slope of the curve and higher peak and amplitude)than the differences in the gastrocnemius muscle. Concentra-tions in the gastrocnemius measurements remained relativelyconstant over the 10-minute sampling period.

The striking differences between analyte concentrations in thetrapezius versus gastrocnemius muscles might be explained byseveral inherent muscle properties and functions. The trapeziusand gastrocnemius have different muscle fiber types, mechanicalproperties, functions, and structures. Given the nature and densityof the gastrocnemius muscle, analyte concentration locally may beinsufficient to generate a noxious stimulus sufficient to produce asubstantial rise in inflammatory analytes. Additionally, the trape-zius is among the muscles most commonly affected by MPS

because of its unique antigravity function. This puts it at high riskfor overuse and susceptibility to irritation.

The trapezius and gastrocnemius muscles demonstrate dif-ferent responses to needle insertion. The reaction during thepre-time level also suggests sensitivity to the needle, with theactives having the largest response, the latents a smaller re-sponse, and the normals having the least response to needleinsertion. Additionally, the gastrocnemius muscles of allgroups showed the least response to needle insertion. This maybe associated with hyperirritability of the trapezius muscle inpeople with active MTPs, which is not evident in normals or inthe gastrocnemius muscle.

Concentrations at the post-time level represent a recoveryphase after a rapid drop in analyte levels associated with needleadvancement. We thought that gastrocnemius concentrationswould represent baseline levels for unaffected muscle and thatpost-trapezius data would trend toward these unaffected musclelevels. This was not the case in the trapezius of the actives,where all analytes except CGRP remained significantly higherthan in the gastrocnemius in the recovery phase (post-level,approximately 5 minutes after needle advancement) (see table1). This suggests that the muscle with an active MTP is morebiochemically reactive to needle perturbation than latent or

normal muscle tissue.Although there were no trigger points in the upper medialgastrocnemius, our results indicate that even in this muscle,analyte levels were always significantly higher in the activegroup than in the normal group and generally higher than in thelatent group. This suggests that analyte abnormalities may notbe limited to local areas of active (painful) MTPs in the uppertrapezius, but are present in unaffected muscles remote fromthe active MTP, although at a lower level than in the trapezius.The slightly elevated analyte levels in the gastrocnemius maybe a widespread phenomenon in the active group. This may berelated to a central sensitization in the active group, whichlowers the threshold to stimuli and leads to a higher sensitivityto mechanical stimuli at the gastrocnemius, or to a systemicsusceptibility to inflammation. There is a possibility that wide-

spread elevation of analytes is a precursor to development ofactive (painful) MTPs. Conversely, people who are predis-posed to develop active MTPs might have elevated baselinelevels in muscles throughout their bodies. An ongoing naturalhistory study could determine whether the relatively elevatedanalyte levels in the gastrocnemius in the active group followthe development of an active MTP, or if there is a baselinelow-level elevation of these analytes that precedes the devel-opment of an active MTP.

We believe that further research in this area should includestudying the natural history of subjects with normal muscle andthose with MTPs. This type of research will determine whetherMTPs resolve spontaneously or evolve into the active formsfrom latent or normal conditions. Microdialysis sampling of thelevels of inflammatory mediators, neuropeptides, catecholamines,

pro-inflammatory cytokines, and other substances (eg, anti-inflammatory cytokines, peripheral opioids) may lead to animproved biochemical characterization of the MTP and iden-tify people who are at risk for developing persistent symptoms.Furthermore, discovering whether and which measurable sub-stances are predictive of pain could lead to focused therapies.

Study Limitations

There are several limitations to this study. First, the sample sizewas small. Because of this, statistical power was low, whichwould result in an increased probability of a type II error. A typeII error, however, can only occur when findings do not achievesignificance, so we could not have made this error. Further, we

Table 3: Analyte Concentrations at the Trapezius Time Levels(pre, peak, post) Compared With Gastrocnemius Concentrations

Trapezius Versus

Gastrocnemius

Analytes by Group Pre* Peak* Post*

Active group

pH NS TG NS

SP, bradykinin, TNF-,IL-1, IL-6, IL-8, 5-HT,

norepinephrine

T

G T

G T

G

CGRP TG TG NS

Latent group

pH NS TG NS

Bradykinin, IL-1, IL-8, 5-HT,

norepinephrine

NS TG NS

SP, CGRP, TNF- TG TG TG

IL-6 NS TG TG

Normal group

pH NS NS NS

CGRP, IL-1, IL-6, IL-8 NS TG NS

SP, 5-HT NS TG TG

Bradykinin TG TG NSTNF-, norepinephrine NS NS NS

NOTE. See table 1 for definitions of time levels.Abbreviations: G, gastrocnemius; NS, not significant; T, trapezius.*All differences significant at P.05.




8/8

used conservative analysis (Bonferroni adjustment) to minimizetype I error (ie, inappropriate significant differences), and we aretherefore confident that our differences are real.

The depth of needle penetration was not standardized inter-subject or within subjects because a stylus was not used. Therewas, however, a consistent initial end point in all groups, asdetermined by a change in tissue resistance at needle contactwith the muscle. A consistent second end point for the activeand latent groups was determined by the LTR and was approx-imated in the normal group as closely as possible.

We studied only 2 muscles. There is a possibility that othermuscles might provide different findings. We believe, how-ever, that the trapezius and gastrocnemius muscles are reason-able models for more generalized conclusions.

The physical findings and symptoms still may have somedegree of clinical variance, as evidenced by the subject whowas disqualified post hoc. There may be many different, and asyet undetermined, physiologic or pharmacologic phenomenapresent, and future studies will need to be specifically designedto seek these answers.

CONCLUSIONS

Our microdialysis system, utilizing samples of less than

1L, is capable of continuous, near real-time, in vivo recoveryof molecules 75kDa and smaller directly from the soft tissueenvironment without harmful effects on subjects.

There is a unique biochemical milieu of substances asso-ciated with pain and inflammation in the vicinity of an activeMTP in the upper trapezius that includes elevated concen-trations of protons, SP, CGRP, bradykinin, TNF-, IL-1,IL-6, IL-8, 5-HT, and norepinephrine. Concentrations ofanalytes from the milieu of the upper trapezius differ quan-titatively from a remote uninvolved site in the medial gas-trocnemius muscle. Furthermore, compared with the othergroups, subjects with active MTPs in the trapezius hadelevated levels of inflammatory mediators, neuropeptides,catecholamines, and cytokines in the gastrocnemius muscle.This suggests that elevations of biochemicals associated

with pain and inflammation may not be limited to localizedareas of active MTPs.

APPENDIX 1: COLLECTION SCHEDULE FOR

DIALYSATE SAMPLES

Minute Collections

0 Needle inserted

1 Sample (1) at minute 1



4 Samples (46) at minute 4 , 4:20, 4:40

5 Samples (79) at minute 5, 5:20, 5:40

(needle advanced into muscle

at 5:05)

6 Samples (1012) at 6:00, 6:20, 6:40

7 Samples (1315) at 7:00, 7:20, 7:40








Needle removed

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Suppliersa. Pain Diagnostics & Therography, 233 E Shore Rd, Ste 108, Great

Neck, NY 11023-2433.b. Orion, 16117 Covello St, Van Nuys, CA 91406.



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