Pain due to tissue acidosis: a mechanism for inflammatory and ischemic myalgia?

ELSEVIER

Neuroscience Letters 208 (1996)191-194

NEUROSCIHCE [HT[llS

Pain due to tissue acidosis: a mechanism for inflammatory and ischemic myalgia? Ulrich Issberner a, Peter W. Reeh b, Kay H. Steen a,* aUniversitgits-Hautklinik und Poliklinik der UniversitgitBonn, Dermatophysiologie, Sigmund-Freud-Strafle 25, D-53105 Bonn, Germany blnstitut fiir Physiologie und Experimentelle Pathophysiologie der UniversitgitErlangen-Niirnberg, Universitiitsstrafle 17, D-91054 Erlangen, Germany Received 8 March 1996; revised version received 22 March 1996; accepted 22 March 1996

Abstract

To study the role of protons in ischemic muscle pain we employed the 'submaximal effort tourniquet technique' and, in a second attempt, an intramuscular pressure infusion of acid phosphate buffer. The pH measured in the forearm skin covering the muscles at work during the tourniquet test continuously dropped to a mean value of pH 7.00 + 0.26, starting 1 min after the contractions, while the pain increased in direct correlation with the hydrogen ion concentration (r = 0.96). After restoring the blood supply, the intradermal proton concentration decreased more slowly than the muscular pain. The same subjective quality of deep muscular pain was achieved with pressure infusion of acid phosphate buffer (pH 5.2) into the forearm muscles. Constant flow rates evoked constant, apparently nonadapting magnitudes of pain with a log-linear stimulus-response relationship (r = 0.93). Changes in flow rate were followed by changes in pain ratings with a certain phase lag. We conclude that muscular pain induced by infusion of acidic phosphate buffer and pain from ischemic contractions are generated through the same mechanisms based on the algogenic action of protons.

Keywords: Protons; pH; Muscle; Experimental algesimetry; Ischemia; Inflammation; Sensory; Human; Skin; Hydrogen ion concentration

In 1931, Lewis et al. [8] published that the characteristic claudication pain of patients with occlusive arterial disease in one leg can be mimicked by experimental ischemia and exercise in the healthy leg. The authors postulated a 'factor P' responsible for ischemic pain, accumulating in the ischemic muscles and rapidly disappearing upon restoration of the blood supply [8]. It has been shown that, as in the skin, the thin myelinated (group III or A6) and unmyelinated (group IV or C) fibres are responsible for transmitting muscular nociceptive information and that their endings are sensitive to inflammatory mediators such as bradykinin, histamine and serotonin [5, 9,10,27]. It is discussed that these substances, among others such as acetyicholine, potassium ions or adenosine, are responsible for the generation of ischemic pain if appearing solely or in combination [10,27]. In addition, protons are considered candidates for ischemic muscle pain, since they are able to excite unmyelinated nocicep* Corresponding author. Tel.: +49 228 2876969/2875518; fax: +49 228 2874333.

tors in skeletal and heart muscles [13,24,25]. However, it has been demonstrated in many tissues that the hormonal mediators show tachyphylaxis of their excitatory action [2,3,6,7], whereas protons produced non-adapting excitation of nociceptors [16,19]. Could protons be the hitherto unknown 'factor P' ? Nuclear magnetic resonance (NMR) examinations of patients with occlusive arterial disease and of healthy subjects under ischemic conditions showed only partial correlation in the muscle between intracellular pH and pain, though hydrogen ion concentrations as low as pH 6.0 were measured [1,4,23,26]. In contrast, determination of muscle surface, i.e. extracellular, pH by microprobes in individual patients revealed a good correlation between the extent of arterial vascular disease and hydrogen ion concentration. Patients with painful ischemic gangrene showed values in the lower leg muscle fascia as low as pH 6.8. On the other hand, patients without pain showed values between pH 7.3 and 7.5 [11,12]. To evaluate these inconsistencies about protons in the pathogenesis of ischemic pain we examined the intracutaneous pH of

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effort Fig. I. Averaged pain ratings on visual analog scale (VAS) of six subjects undergoing 'submaximal effort tourniquet ischemia' combined with intradermal pH recordings. (A) The bars represent means, grey shadows show SEM. 'Effort', muscle exercise as described in the text. Dotted line represents control data with the same six subjects undergoing tourniquet ischemia without 'effort'. (B) Correlation between the pH changes and the pain ratings shown in (A). The symbols represent mean values of pH changes related to each 10% segment of the rating scale, divided into the rising and falling phase of the ischemic pain. See text for methods and interpretation.

of subjects during tourniquet ischemia. Placement of the pH probe inside the contracting muscle was not practicable because of unavoidable bleeding and clotting on the probe surface. Working ischemic forearm muscles cause constantly rising pain as long as ischemia is maintained. The 'submaximal effort tourniquet technique', as introduced by Smith et al. [14], was induced on the non-dominant arm of 12 volunteers (age 23-34 years, mean 28 years; nine male, three female). With the arm raised, an Esmarch bandage was wrapped from the fingers to the elbow and a blood pressure cuff around the upper arm was inflated to 250 mmHg. At that time, PC recording was started and the Esmarch bandage removed. To measure skin pH, a 17G indwelling cannula was placed about 2 mm deep intradermally in the palmar forearm directly over the flexor muscles. The mandrin was extracted and the plastic tube was left in position in order to guide a sterilized pH needle electrode of 20G (IC 401, S. Agulian, Hamden, CT, USA). The electrode was connected to a portable pH meter (portamess 654; Knick, Berlin, Germany) and data was transmitted every 10s to the computer [16]. The subjects were then prompted every 4 s by an acoustic signal to squeeze a hand exerciser (14 N) for 2 s. After repeating the exercise 20 times (80 s duration), the subjects laid their arm on an armrest. For the test duration subjects were requested to rate their actual pain every 10 s

on a visual analog scale (VAS) ranging from 'pain threshold' (>0%) to 'unbearable pain' (100%). When a subject evaluated pain as 100% (or after a 20 min cut-offtime), the cuff was deflated. Only six trials were suitable for analysis: one subject stopped after 3 min because of indisposition and three others showed artefactual pH measurements. Two further subjects ended after 20 rain without reaching 100% VAS; intracutaneous pH was only 7.4 and 7.3, respectively, probably because of insufficient exercise. Six subjects ended with ischemia durations of between 7 and 10 min (mean _+SEM, 8 min _+30 s) until reaching 100% pain on the VAS. At first, the intracutaneous pH remained stable; however, about 1 min after the start of the subject's muscle exercise ('effort') the intradermal pH began to drop below normal values (Fig. 1A). About 40 s later, on average, pain ratings started to occur and rise; this was after the end of exercise in all cases. The temporal difference probably reflects the time needed to reach the pH threshold for muscle nociceptors to be excited. As has been reported for rat skin nociceptors [19], this threshold is likely to be below pH 6.9. As shown in Fig. 1A, the mean intradermal pH value dropped continuously to a minimal value of pH 7.06, on average (6.67-7.30, _+0.26 SEM), until about 3 min after the beginning of the exercise, but then stabilized until the cuffs were deflated. Roughly 1 min later than the pH values, the mean pain-

U. Issberner et al. / Neuroscience Letters 208 (1996) 191-194

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Fig. 2. Individualpain ratings of a subject (upper trace) and flow rate of intramuscular infusion of isotonic phosphate buffer (pH 5.2; lower trace). The pain ratings follow upon changes in the flow rate with certain phase-lags that are exphfinedin the text. ratings, too, approached a level that did not rise much further until deflation of the cuffs. The similar time courses of pain and pH resulted in a perfectly linear correlation (Fig. 1B; r = 0.96, P < 0.001) during the phase of increasing pain and proton concentrations. After deflation of the cuffs, the pain of each individual volunteer vanished totally within 120 s (mean _+SEM, 60 _ 18 s). On average, the ratings dropped with a time constant (r) of about 2 min, whereas the pH recovered to normal values much more slowly with T - 9 min. Accordingly, the previously good correlation between pain and hydrogen ion concentration is lost (Fig. 1B). The discrepancy between pain and pH recovery has previously been noticed in experiments with cutaneous acidosis and was explained by a particular buffering capacity of the nociceptive nerve endings themselves that possess an amiloride-sensitive sodium-dependent mechanism of proton extrusion [16, 22]. In addition, in the present study the different compartments (pain in muscle versus pH measurements in skin) may serve to explain the difference in recovery: The post-ischemic increase in muscular blood flow should help the acidotic muscle to reach normal pH values faster than the overlaying skin. The finding that the skin became acidic in the first place is most likely explained by the muscle contractions, which pumped muscular venous blood of low pH into the empty superficial veins of the forearm, a common clinical manoeuvre to make venous puncture easier. In control experiments (n = 6) with cuff inflation at rest (dotted line in Fig. 1A, lower inset), only a small initial drop of the intradermal pH was recorded which recovered during continued ischemia and did not lead to any pain sensations. To eliminate the problem with the two different compartments we applied the previously introduced technique

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flow rate (mllh) Fig. 3. Individual pain ratings in relation to flow rate of isotonic phosphate buffer connected to display stimulus-response curves. Average values from the last minute before changing the flow were taken as measures. Large dots represent median values used to calculate a regression line (r = 0.93). of continuous infusion of acid phosphate buffer [15-18, 21] to the forearm flexor muscles under investigation. Nine volunteers gave their written consent to this examination (n = 9, age 24-29 years, mean 26 years; eight male, one female). A 27G needle was stuck vertically into the belly of the flexor carpi radialis muscle, placing the cannula tip just below the muscle fascia. In addition, the pH was measured in the overlaying skin as described above, at about 10 mm vertical distance to the tip of the infusion needle. An isotonic phosphate buffer solution (pH 5.2), containing Na2HPO 4 (3.43 mM) and NaH2PO 4 (137.3 mM), was prepared under sterile conditions and infused with a syringe pump at various flow rates into the muscle (Fig. 2). Infusion rates of 5-40 mi/h (mean +_ SEM, 16 _+3.7 ml/h) were needed to achieve pain ratings of around 20% of the VAS extension. All subjects developed dull-aching or stinging muscular pain, the magnitude of which showed log-linear correlation with the flow rate (Fig. 3). The pain quality was described as being the same as in the ischemia sessions. With an infusion rate of 5 ml/h, the pain started after a delay of 70 s on average (20-160 s) and reached a stable plateau after 4 min, on average (40 s-6 min), without any signs of adaptation. Increasing the flow rate led to increasing individual pain ratings again after 90 s on average (40-180 s). Decreasing the flow rate led to a drop in pain after a delay of 170 s on average (40 s-7 min). The phase lags are partly due to the elastic compliance of the polyethylene infusion tubing and to the buffering capacity of the tissue [16]. At all infusion rates, the intradermal pH measurement did not reveal any significant pH changes, certainly because no congestion of muscular blood in cutaneous veins could occur. The present results provide evidence for a linear corre-

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lation between the intramuscular infusion rate of acidic buffer and muscle pain. This finding is analogous to the pH-induced sustained graded pain in human skin, as previously published [15]. Raising the infusion rate leads to increasing pain by lowering the local pH more effectively and by increasing the tissue volume in which the proton concentration exceeds the threshold to excite nociceptors [16]. Both effects converge in increasing temporal and spatial summation of nociceptor discharge [19]. Thus, tissue pH and magnitude of pain are closely correlated in this model. If, in addition, one accepts the intradermal pH measurements during ischemic contractions to reflect the muscular acidosis, one can safely conclude that it is the muscle pH that causes the acute ischemia pain. In quantitative respect, this does not exclude a contribution from hormonal mediators that can actually modulate the pH sensitivity of nociceptors [20,21]. The previous NMR investigations, measuring pH inside the muscle cells, could not reveal the correlation between pH and pain, because it is probably the pH inside the nociceptive nerve ending that is responsible for the pain and this is dependent on the interstitial, extracellular hydrogen ion concentration [16,19]. The NMR findings that the intracellular muscle pH even decreases during pain relief after ischemia [1] is therefore not in disagreement with our data, but is a reminder of a similar discrepancy between pain and pH in the recovery phase of our ischemia experiments. The neurobiological mechanism described above (and [22]) to explain this divergence is probably supported by a human psychological tendency to estimate decreasing pain less intensely than increasing pain. Supported by the Deutsche Forschungsgemeinschaft (Ste 593/1-2) to the third author. [1] Hands, L.J., Bore, P.J., Galloway, G., Morris, P.J. and Radda, G.K., Muscle metabolism in patients with peripheral vascular disease investigated by 31p nuclear magnetic resonance spectroscopy, Clin. Sci., 71 (1986) 283-290. [2] Handwerker, H.O., Reeh, P.W. and Steen, K.H., Effects of 5HT on nociceptors. In J.M. Besson (Ed.), Serotonin and Pain, Elsevier, Amsterdam, 1990, pp. 1-15. [3] Kanaka, R., Schaible, H.-G. and Schmidt, R.F., Activation of fine articular afferent units by bradykinin, Brain. Res., 327 (1985) 8190. [4] Keller, U., Oberhtinsli, R., Huber, P., Widmer, L.K., Aue, W.P., Hassink, R.I., M~iller, S. and Seelig, J., Phosphocreatine content and intracellular pH of calf muscle measured by phosphorus NMR spectroscopy in occlusive arterial disease of the legs, Eur. J. Clin. Invest., 15 (1985) 382-388. [5] Kumazawa, T. and Mizumura, K., Thin-fibre receptors responding to mechanical, chemical and thermal stimulation in the skeletal muscle of the dog, J. Physiol. (London), 273 (1977) 179-194. [6] Kumazawa, T., Mizumura, K. and Sato, J., Response properties of polymodal receptors studied using in vitro testis superior spermatic nerve preparations of dogs, J. Neurophysiol., 57 (1987) 702-711. [7] Lang, E., Novak, A., Reeh, P.W. and Handwerker, H.O., Chemosensitivity of fine afferents from rat skin in vitro, J. Neuro-

physiol., 63 (1990) 887-901. [8] Lewis, T., Pickering, G.W. and Rothschild, P., Observations upon muscular pain in intermittent claudication, Heart, 15 (1931) 359383. [9] Mense, S. and Schmidt, R.F., Activation of group IV afferent units from muscle by algesic agents, Brain. Res., 72 (1974) 305310. [10] Newham, D.J., Edwards, R.H.T. and Mills, K.R., Skeletal muscle pain. In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, Churchill Livingstone, Edinburgh, 1994, pp. 423. [11] O'Donnell, T.F., Clowes, G.H.A., Browse, N.L., Ryan, N.T. and Blackburn, G.L., A metabolic approach to the evaluation of peripheral vascular disease, Surg. Gynecoi. Obstet., 144 (1977) 5157. [12] O'Donnell, T.F., Raines, J.K. and Darling, R.C., Relationship of muscle surface pH to noninvasive hemodynamic studies, Arch. Surg., 114 (1979) 600-604. [13] Rotto, D.M. and Kaufman, M.P., Effect of metabolic products of muscular contraction on discharge of group II1 and IV afferents, J. Appl. Physiol., 64 (1988) 2306-2313. [14] Smith, G.M., Egbert, L.D., Markowitz, R.A., Mosteller, F. and Beecher, H.K., An experimental pain method sensitive to morphine in man: the submaximum effort tourniquet technique, J. Pharmacol. Exp. Ther,, 154 (1966) 324-332. [15] Steen, K.H. and Reeh, P.W., Sustained graded pain and hyperalgesia from harmless experimental tissue acidosis in human skin, Neurosci. Lett., 154 (1993) 113-116. [16] Steen, K.H., Issbemer, U. and Reeh, P.W., Pain due to experimental acidosis in human skin: evidence for non-adapting nociceptor excitation, Neurosci. Lett., 199 (1995) 29-32. [17] Steen, K.H., Reeh, P.W. and Kreysel, H.W., Dose-dependent competitive block by topical acetylsalicylic and salicylic acid of low pH-induced cutaneous pain, Pain, 64 (1996) 71-82. [18] Steen, K.H., Reeh, P,W. and Kreysel, H.W., Topical acetylsalicylic, salicylic acid and indomethacin suppress pain from experimental tissue acidosis in human skin, Pain, 62 (1995) 339-347. [19] Steen, K.H., Reeh, P.W., Anton, F. and Handwerker, H.O., Protons selectively induce lasting excitation and sensitization to mechanical stimulation of nociceptors in rat skin, in vitro, J. Neurosci., 12 (1992) 86-95. [20] Steen, K.H., Steen, A.E. and Reeh, P.W., A dominant role of acid pH in inflammatory excitation and sensitization of nociceptors in rat skin, in vitro, J. Neurosci., 15 (1995) 3982-3989. [21] Steen, K.H., Steen, A.E., Kreysel, H.W. and Reeh, P.W., Inflammatory mediators potentiate pain induced by experimental tissue acidosis, Pain, (1996) in press. [22] Steen, K.H., Wegner, H., Kreysel, H.W. and Reeh, P.W., The pHresponse of rat cutaneous nociceptors correlates with extracellular [Na+] and is increased under amiloride, in vitro, Soc. Neurosci. Abstr., 21 (1995) 648. [23] Taylor, D.J., Bore, P.J., Styles, P., Gadian, D.G. and Radda, G.K., Bioenergetics of intact human muscle: a 31p nuclear magnetic resonance study, Mol. Biol. Med., 1 (1983) 77-94. [24] Uchida, Y. and Murao, S., Acid-induced excitation of afferent cardiac sympathetic nerve fibers, Am. J. Physiol., 228 (1975) 2733. [25] Victor, R.G., Bertocci, L.A., Pryor, S.L. and Nunnally R.L., Sympathetic nerve discharge is coupled to muscle cell pH during exercise in humans, J. Clin. Invest., 82 (1988) 1301-1305. [26] Zatina, M.A., Berkowitz, H.D., Gross, G.M., Maris, J.M. and Chance, B., 31p nuclear magnetic resonance spectroscopy: noninvasive biochemical analysis of the ischemic extremity, J. Vasc. Surg., 3 (1986)411-420. [27] Zimmermann, M., Physiological mechanisms of pain in the musculo-skeletal system. In M. Emre and H. Mathies (Eds.), Muscle Spasms and Pain, Parthenon, Carnforth, 1988, pp 7-17.