Shockwave effect on pain fibres by Kirk Andrew

Neuroscience 155 (2008) 138 –144

SELECTIVE LOSS OF UNMYELINATED NERVE FIBERS AFTER EXTRACORPOREAL SHOCKWAVE APPLICATION TO THE MUSCULOSKELETAL SYSTEM J. HAUSDORF,a M. A. M. LEMMENS,b,c K. D. W. HECK,b N. GROLMS,d H. KORR,b,d S. KERTSCHANSKA,d H. W. M. STEINBUSCH,b,c C. SCHMITZb,c* AND M. MAIERa

ical activity (Roxas, 2005; Thomas and Beaudreuil, 2006; Hume et al., 2006). Extracorporeal shockwave (ESW) application has become an established treatment for lithotripsy of kidney and urinary tract stones (Chaussy et al., 1980). Over the last decades ESW application was also introduced into the treatment of diseases of the musculoskeletal system (for review see Haupt, 1997; Thiel, 2001; Rompe et al., 2002; see also Maier and Schmitz, 2008). The efficacy of ESW application as treatment for these diseases is not uniform and depends on several circumstances, such as the anatomical site and the severity and chronicity of the disorder (for review see Seil et al., 2006; Sems et al., 2006). A number of prospective, randomized, controlled clinical trials were performed in the last years, of which some showed beneficial effect of ESW application to the musculoskeletal system (Gerdesmeyer et al., 2003; Rompe et al., 2003) whereas others did not (Haake et al., 2002, 2003; Speed et al., 2002a,b, 2003; Melikyan et al., 2003). Furthermore, long-term analgesia between several months and years after ESW application to the musculoskeletal system was reported in several clinical pilot studies (Rompe et al., 1996, 2001; Loew et al., 1999; Maier et al., 2000, 2001). Accordingly, ESW application may be a powerful tool in the treatment of chronic tendinopathies of the shoulder, elbow and heel. However, one of the major obstacles in further developing this treatment strategy is the fact that the molecular and cellular mechanisms of ESW on the musculoskeletal system are as yet largely unknown (Maier et al., 2002). Hence, the many variables that are present in this field, such as the energy flux density (EFD) of the applied ESW (for details see Ogden et al., 2001), the use of local anesthesia at the site of ESW application and the inclusion/exclusion criteria of patients, are being selected by (limited) clinical experience rather than by biological evidence. Consequently, these parameters vary substantially among studies, making comparisons a complicated matter. A number of recent studies indicated that the peripheral nervous system might play a central role in the longterm analgesia induced by ESW application (Ohtori et al., 2001; Maier et al.,2003; Takahashi et al., 2003, 2006; Murata et al., 2006; Ochiai et al., 2007; Hausdorf et al., 2008) and ESW-induced destruction of nerve fibers could contribute to this effect (Haist and Steeger, 1994; Loew et al., 1994; Schelling et al., 1994). Obviously, this could only be assessed as beneficial to the patient if the destruction of nerve fibers is selective and does not comprise nerve

Department of Orthopaedic Surgery, University of Munich, Klinikum Grosshadern, Marchioninistr. 15, 81377 Munich, Germany

School for Mental Health and Neurosciences, Division of Cellular Neuroscience, Maastricht University, P.O. Box 616, 6200 MD Maastricht, Netherlands

Europan Graduate School of Neuroscience (EURON), Maastricht, Netherlands

Department of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, 52057 Aachen, Germany

Abstract—Application of extracorporeal shockwaves (ESW) to the musculoskeletal system may induce long-term analgesia in the treatment of chronic tendinopathies of the shoulder, heel and elbow. However, the molecular and cellular mechanisms behind this phenomenon are largely unknown. Here we tested the hypothesis that long-term analgesia caused by ESW is due to selective loss of nerve fibers in peripheral nerves. To test this hypothesis in vivo, high-energy ESW were applied to the ventral side of the right distal femur of rabbits. After 6 weeks, the femoral and sciatic nerves were investigated at the light and electron microscopic level. Application of ESW resulted in a selective, substantial loss of unmyelinated nerve fibers within the femoral nerve of the treated hind limb, whereas the sciatic nerve of the treated hind limb remained unaffected. These data might indicate that alleviation of chronic pain by selective partial denervation may play an important role in the effects of clinical ESW application to the musculoskeletal system. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: analgesia, calcifying tendonitis, extracorporeal shockwaves, pain, plantar fasciitis, tennis elbow.

Chronic tendinopathies of the shoulder, elbow and heel are common painful musculoskeletal disorders (Uhthoff, 1996; Crawford, 2002; Assendelft et al., 2003). Although current treatment strategies have improved, most of the conservative and surgical approaches are of limited benefit and many patients keep suffering from pain and impaired phys*Correspondence to: C. Schmitz, School for Mental Health and Neurosciences, Division of Cellular Neuroscience, Maastricht University, P.O. Box 616, 6200 MD Maastricht, Netherlands. Tel: ⫹31-43-3884108; fax: ⫹31-43-367-1096. E-mail address: c.schmitz@np.unimaas.nl (C. Schmitz). Abbreviations: BW, body weight; CGRP, calcitonin gene-related peptide; EFD, energy flux density; ESW, extracorporeal shock waves; FN, femoral nerves; LMNF, large myelinated nerve fibers; RM, repeated measures; SMNF, small myelinated nerve fibers; SN, sciatic nerves; UMNF, unmyelinated nerve fibers.

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fibers responsible for motor function (i.e. particularly large myelinated nerve fibers (LMNFs)). We therefore tested in the present study the hypothesis that high-energy ESW application to the ventral side of the distal femur of rabbits resulted in a selective loss of unmyelinated and small myelinated nerve fibers (SMNFs) within the femoral nerve (FN), without effects on the sciatic nerve (SN).

EXPERIMENTAL PROCEDURES Animals Seven female 1-year-old chinchilla bastard rabbits (Charles River; Kissleg, Germany) with a body weight (BW) between 4.3 kg and 4.9 kg and closed femoral growth plates were used in the present study. During the experimental period, the animals were maintained at the central animal house of the Institute for Surgical Research of the University of Munich (Germany) on a 12-h light/ dark cycle with lights on at 06:00 am, at 21 °C and with ad libitum access to standard food pellets and water. For each animal the right hind limb was selected for ESW application with the left hind limb serving as control. All research and animal care procedures were approved by the district government of Munich (Germany) according to the German animal protection law and considering international standards. Efforts were undertaken to minimize the number of animals used and their suffering.

Anesthesia The animals were anesthetized by i.v. injection of a combination of xylazine hydrochloride (1.5 mg/kg BW) and ketamine (6 mg/kg BW). Anesthesia was maintained by the i.v. administration of xylazine hydrochloride (2.4 mg/kg BW/h) and ketamine (10 mg/kg BW/h) using a perfusion pump. During anesthesia, the animals were supplied with oxygen by means of an oxygen mask. The administration of i.v. drugs was stopped immediately after ESW application.

Application of ESWs After shaving both hind limbs, the animals were positioned for ESW application as described in detail elsewhere (Delius et al., 1995). ESW application to the distal femoral region of the selected hind limb was carried out using an electrohydraulic shockwave source (XL1; Dornier MedTech, Wessling, Germany). The shockwave device was coupled to the distal femur by means of a waterbath. Focusing of the shockwaves to the distal femur (with the ventral side of the femur facing the shockwave source) was controlled by two laser pointers adjusted in two planes (Delius et al., 1995). ESW were applied as 1500 pulses with EFD⫽0.9 mJ/mm2 at a frequency of 1 Hz over 25 min. The EFD was measured with a laser hydrophone before ESW application.

Preparation and fixation of tissue Six weeks after ESW application the animals were killed by an overdose of pentobarbital. Both FNs were prepared under the inguinal ligament and removed from the cadaver. Then, a portion with a length of approximately 0.5 cm was cut from each nerve, covering the entire cross-sectional area of the FN. Then, both SNs were prepared above the sacrospinal ligament (after removal of the gluteus maximus muscle), and two portions with a length of approximately 0.5 cm each were cut from each SN, also covering the entire cross-sectional area of this nerve. One of these portions was longitudinally split into two or three fiber bundles according to the individual morphology of the SNs. These fiber bundles and the portions of the FNs were fixed in 3.9% glutaraldehyde in 0.1 M phosphate buffer at room temperature for 3 h. The remaining portions of the SNs were fixed with 4% paraformaldehyde in 0.1 M Tris–HCl buffer at 4 °C for 10 days.

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Preparation of sections After post-fixing the fiber bundles of the SNs and the portions of the FNs in buffered osmium tetroxide (2% OsO4 in 0.2 M phosphate buffer containing 13% sucrose), they were embedded in araldite. Semithin (0.5 ␮m) and ultrathin (90 nm) cross-sections were cut with a rotation microtome (Reichert Ultracut S, Leica, Wien, Austria). Semithin sections were mounted on slides (Süsse, Gudensberg, Germany) and stained with Toluidine Blue. Ultrathin sections were mounted on nickel grids (300 mesh, Plano GmbH, Wetzlar, Germany) and additionally stained with lead citrate for 8 min and 5% uranyl acetate for 15 min. The remaining portions of the SNs were embedded in paraffin, and 5 ␮m thick cross-sections were cut with a motor-driven rotation microtome (Leica RM 2065, Leica Instruments, Nu␤loch, Germany) using 25-mm Ralph type glass knives prepared by a LKB 2078 HistoKnife Maker (LKB Producter, Bromma, Sweden). Sections were mounted on slides (Süsse), deparaffinized with xylol, stained with hematoxylin, and covered with a coverslip using DePeX (Serva, Heidelberg, Germany).

Analysis of femoral and SNs The cross-sectional areas of the FNs (semithin araldite sections) and the SNs (paraffin sections) were delineated and measured with a stereology workstation, consisting of a modified light microscope (type BX50; Olympus, Tokyo, Japan) with 10⫻ Olympus UPlanApo objective (numerical aperture⫽0.40), motorized specimen stage for automatic sampling (Ludl Electronic Products; Hawthorne, NY, USA), electronic microcator (Ludl), CCD color video camera (Hitachi Kosukai Electric, Japan), PC with frame-grabber board and stereology software (StereoInvestigator, MBF Bioscience, Williston, VT, USA). Within the ultrathin sections of the FNs and the fiber bundles of the SNs, the densities of the myelinated nerve fibers and the unmyelinated nerve fibers (UMNFs) were evaluated by inspection of 63 consecutive electron microscopic images on average in case of each FN, and of 139 consecutive electron microscopic images on average in case of the SNs. In both cases the localization of the consecutive images on the cross-sectional areas of the nerves (or fiber bundles, respectively) was randomly selected. Electron microscopy was carried out with a Phillips EM 300 transmission electron microscope (Phillips 300, Eindhoven, Netherlands) at a total magnification of ⫻7700. Density estimates were performed by placing an unbiased counting frame (Gundersen, 1977) on the electron microscopic photomicrographs. Furthermore, the size of these myelinated nerve fibers which were found within the unbiased counting frame was assessed on the electron microscopic photomicrographs. Nerve fibers with a total cross-sectional area (including the myelin sheet) of more than 10 ␮m2 were classified as LMNFs, and those with less than 10 ␮m2 as SMNFs. The large myelinated fibers are considered to have mainly motor functions, whereas the SMNFs are sensory (Kandel et al., 2000). Finally, total numbers of LMNFs, SMNFs and UMNFs were calculated from the corresponding fiber densities and the cross-sectional areas of the nerves.

Statistical analysis Mean and standard error of the mean (S.E.M.) were calculated for each investigated variable. Differences between the left and the right side in the mean cross-sectional areas of the nerves were tested with Student’s paired t-test. Differences between the left and the right side in all other investigated variables were tested with two-way repeated measures analysis of variance (two-way RM ANOVA) (with the results of the left and the right sides as related values) and Bonferroni post-tests to compare for replicate means by side (i.e. left vs. right). Tests were performed separately for the FNs and the SNs. Statistical significance was established as P⬍0.05. All

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(Fig. 1c to f). The same was found for the UMNFs within the SN (Fig. 1d, f). In contrast, both the mean densities and the mean total numbers of the UMNFs within the FN were substantially reduced on the treated side compared with the untreated side (reduction by 57% in the mean density and by 59% in the mean total number, respectively) (Figs. 1c, e and 2). Two-way RM ANOVA showed significant differences between the left and the right side for the FN but not the SN, between the type of the nerve fibers within both the FN and the SN, and for the interaction between the side and the type of the nerve fibers for the FN but not the SN (all P-values are shown in Table 1). Bonferroni post-tests showed significant differences between the treated and the untreated side only in the mean density and the mean total number of the UMNFs within the FN (DF: t⫽5.384; P⬍0.001; NF: t⫽4.949; P⬍0.001). Accordingly, ESW treatment (with EFD of 0.9 mJ/mm2 to the distal rabbit femur, with the ventral side of the femur facing the shockwave source) resulted in a significant reduction in both the mean density and the mean total number of the UMNFs within the FN of the treated side. ESW treatment had no influence on the size of the myelinated nerve fibers themselves and their myelin sheet, respectively (Fig. 1i, k and Table 1). In addition, we did not observe signs of Wallerian degeneration in the myelinated nerve fibers. Obviously this does not exclude the possibility that some of the SMNFs underwent Wallerian degeneration during the first 6 weeks after ESW application. However, this could not be tested in the present study.

DISCUSSION Fig. 1. Mean and S.E.M. of the nerve cross-sectional areas (AN) (a, b) the density of nerve fibers (DF) (c, d), the total number of nerve fibers (NF) (e, f), the total cross-sectional areas of the individual myelinated nerve fibers (i.e. including the myelin sheet) (AM) (g, h) and the cross-sectional areas of the individual myelinated nerve fibers without consideration of the myelin sheet (AF) (i, k) in the FNs (a, c, e, g, i) and SNs (b, d, f, h, k) 6 weeks after ESW application with EFD of 0.9 mJ/mm2 to the right distal rabbit femur, with the ventral side of the femur facing the shockwave source. Closed bars, data from the left (i.e. untreated) hind limbs. Open bars, data from the right (i.e. treated) hind limbs. LMNF, LMNFs, large myelinated nerve fibers (i.e. AM⬎10 ␮m2). SMNF, small myelinated nerve fibers (i.e. AM⬍10 ␮m2). UMNF, unmyelinated nerve fibers. *** P⬍0.001.

calculations were carried out using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA).

RESULTS The FNs had smaller mean cross-sectional areas than the SNs (Fig. 1a, b). However, there were no significant differences between the untreated (i.e. left) and the treated (i.e. right) side (FN: t⫽0.164; P⫽0.874; SN: t⫽0.692; P⫽0.515) (Fig. 1a, b). Both the mean densities and the mean total numbers of the LMNFs, the SMNFs and the UMNFs were similar between the FN and the SN on the left side (Fig. 1c to f). In addition, both the mean densities and the mean total numbers of the LMNFs and the SMNFs were similar within either the FN or the SN between the left and the right side

Several reports in the literature suggested that ESW application to the musculoskeletal system might result in the destruction of nerve fibers (Haist and Steeger, 1994; Loew et al., 1994; Schelling et al., 1994). In line with this hypothesis a number of recent studies indicated that the peripheral nervous system might play a central role in long-term analgesia induced by ESW application (Ohtori et al., 2001; Maier et al.,2003; Takahashi et al., 2003, 2006; Murata et al., 2006; Ochiai et al., 2007; Hausdorf et al., 2008). Furthermore, it has been hypothesized that ESW-induced long-term analgesia might be due to the inability of sensory nociceptive neurons to generate an adequate receptor potential after ESW application (Schelling et al., 1994). However, studies testing this hypothesis have not been published. Recent studies on animal models focusing on the peripheral nervous system after ESW application to the musculoskeletal system in vivo pointed specifically to a reduction of two substances that are involved in pain perception, namely calcitonin gene-related peptide (CGRP) and substance P (Ohtori et al., 2001; Maier et al.,2003; Takahashi et al., 2003, 2006; Ochiai et al., 2007; Hausdorf et al., 2008). (CGRP is a neuropeptide that is known for its major vasodilatation potency and is found in sensory nerves [Ghatta and O’Rourke, 2006]. Substance P is present in both unmyelinated C-fibers and a subpopulation of lightly myelinated A-␦ nerve fibers and is released at central and

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Fig. 2. Representative electron microscopic photomicrographs of nerve fibers within the left (a, b) and right (c, d) femoral nerve of a rabbit 6 weeks after ESW application with EFD of 0.9 mJ/mm2 to the right distal femur, with the ventral side of the femur facing the shockwave source. Asterisks indicate the myelin sheet of large myelinated nerve fibers, arrows point to small myelinated nerve fibers and arrowheads to unmyelinated nerve fibers. Note the substantial reduction in the number of unmyelinated nerve fibers within the right femoral nerve. Scale bar⫽2 ␮m.

peripheral terminals of sensory nociceptive neurons after stimulation [Keen et al., 1982; Malcangio and Bowery, 1999; Snijdelaar et al., 2000]). Specifically, Ohtori et al. (2001) reported an almost complete loss of immunoreactivity for CGRP within the plantar skin of rats 4 days after

low-energy ESW application (EFD⫽0.08 mJ/mm2; 1000 pulses at unknown frequency). The authors interpreted this finding as indicating a nearly complete degeneration of epidermal nerve fibers in the shockwave-treated skin. In a follow-up study on the same animal model, Takahashi

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Table 1. Results of statistical analysis (P values) with two-way repeated measures ANOVA followed by Bonferroni post-tests to compare replicate means by side (i.e., left vs. right)

Femoral nerve S T I LMNF SMNF UMNF Sciatic nerve S T I LMNF SMNF UMNF

0.002 ⬍0.001 0.002 ⬎0.05 ⬎0.05 ⬍0.001

0.004 ⬍0.001 0.004 ⬎0.05 ⬎0.05 ⬍0.001

0.122 ⬍0.001 0.228 ⬎0.05 ⬎0.05 —

0.060 ⬍0.001 0.210 ⬎0.05 ⬎0.05 —

0.969 ⬍0.001 0.999 ⬎0.05 ⬎0.05 ⬎0.05

0.216 ⬍0.001 0.506 ⬎0.05 ⬎0.05 ⬎0.05

0.524 ⬍0.001 0.581 ⬎0.05 ⬎0.05 —

0.450 ⬍0.001 0.567 ⬎0.05 ⬎0.05 —

P values smaller than 0.05 are shown in boldface. DF, density of the nerve fibers; NF, total number of the nerve fibers; AM, total cross-sectional areas of the individual myelinated nerve fibers (i.e. including the myelin sheet); AF, cross-sectional areas of the individual myelinated nerve fibers without consideration of the myelin sheet; S, side (i.e. left or right); T, type of the nerve fibers (i.e. large myelinated nerve fibers (LMNF), small myelinated nerve fibers (SMNF) and unmyelinated nerve fibers (UMNF), respectively); I, interaction between the side and the type of nerve fiber; —, no data available.

et al. (2003) found that the number of CGRP immunoreactive neurons in the dorsal root ganglia L4 and L5 (innervating the most middle foot pad of the hind paw) was substantially reduced after ESW treatment compared with untreated animals. Likewise, application of high-energy ESW to the distal rabbit femur (EFD⫽0.9 mJ/mm2; 1500 pulses at a frequency of 1 Hz) led to a reduced concentration of substance P in the femoral periosteum 6 weeks after ESW application (Maier et al., 2003). Furthermore, for the same animal model Hausdorf et al. (2008) reported that the number of neurons immunoreactive for substance P in the dorsal root ganglion L5 (innervating the ventromedial side of the upper hind paw) was substantially reduced after ESW treatment compared with the untreated hind limbs of the same animals. Altogether these studies point to a possible mechanism underlying the long-term analgesia observed in patients after ESW application to the musculoskeletal system, namely degeneration of CGRP- and substance P-positive nerve fibers. The results of the present study are in line with these earlier observations and represent the first direct evidence of shockwave-induced destruction of nerve fibers at the electron-microscopic level (unfortunately we were not able to test whether the treated rabbits showed reduced pain perception in the innervation zone of the right FN). Of particular importance is the fact that the destruction of these nerve fibers was limited to UMNFs within the FN, whereas the myelinated nerve fibers within the FN and all nerve fibers within the SN remained unaffected by the shockwaves. The large amount of destruction of the UMNFs might be explained by the relatively small rabbit femur compared with the size of the shockwave source

used, and by the high EFD of the shockwaves. It should be mentioned that there is a possibility of (partial) reinnervation of UMNFs later than 6 weeks after application of ESW. This should be tested in future experimental studies. In clinical use, long-term analgesia after ESW application to the musculoskeletal system has usually been explained by the concept of hyperstimulation analgesia (Melzack and Wall, 1965; Rompe et al., 1996, 1998). However, studies testing this hypothesis have not been published. Considering the results of the present study, it is reasonable to hypothesize that hyperstimulation analgesia is (if at all involved) most probably not the only mechanism inducing long-term analgesia after ESW application to the musculoskeletal system. Rather selective destruction of UMNFs within the focal zone of the shockwaves might contribute to this analgesia. Importantly, these small unmyelinated fibers, the so-called C fibers, are known to be responsible for throbbing, chronic pain (Kandel et al., 2000). Patients suffering from chronic painful diseases like calcifying tendonitis of the shoulder, tennis elbow and chronic plantar fasciitis could generally benefit from selective destruction of UMNFs resulting in hypalgesia (note in particular that surgical denervation has been described as beneficial in tennis elbow after failed conservative treatment; Kaplan, 1959; Wittenberg et al., 1992; Wilhelm, 1996). Furthermore, there is evidence that chronic inflammation contributes to the etiology of pain at least in tennis elbow and chronic plantar fasciitis (Nirschl, 1977; Kamien, 1990; LeMelle et al., 1990; Schepsis et al., 1991; Roetert et al., 1995), although this is still a controversial issue (Boyer and Hastings, 1999; Lemont et al., 2003). It is important to note that Uchio et al. (2002) revealed the contribution of neuropeptides in the pathogenesis of tennis elbow, without apparent infiltration of inflammatory cells. These neuropeptides cause a so-called neurogenic inflammation, which is an inflammation that results from the release of substances (e.g. substance P, CGRP) from primary sensory nerve terminals (Holzer, 1988; Richardson and Vasko, 2002). Denervation can lead to a reduction of these substances and therefore decrease the observed inflammation and concomitant pain. In this regard, denervation or depletion of substance P has repeatedly been shown to reduce experimentally induced inflammation of paws and joints in laboratory animals (Lam and Ferrell, 1989, 1991; Rees et al., 1994; Cruwys et al., 1995; Shakhanbeh and Abo-Galyon, 1996; Garrett et al., 1997; Gentili et al., 1999).

CONCLUSION In summary, long-term analgesia and reduction of neurogenic inflammation by shockwave-induced selective denervation could be an important, novel target in the treatment of diseases such as calcifying tendonitis of the shoulder, tennis elbow and chronic plantar fasciitis. Acknowledgments—The authors wish to thank M. Nicolau (Aachen) for excellent technical assistance. This study was supported by funds from the Friedrich-Bauer-Stiftung (Munich, Germany) and the Deutsche Forschungsgemeinschaft (DFG) (to C.S. and M.M.).

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(Accepted 24 March 2008) (Available online 7 April 2008)