Neuroscience Letters 315 (2001) 57–60

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Shock wave application to rat skin induces degeneration and

reinnervation of sensory nerve fibres
Seiji Ohtori*, Gen Inoue, Chikato Mannoji, Takashi Saisu,
Kenji Takahashi, Shigeru Mitsuhashi, Yuichi Wada, Kazuhisa Takahashi,
Masatsune Yamagata, Hideshige Moriya
Department of Orthopaedic Surgery, School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8677, Japan
Received 25 August 2001; received in revised form 19 September 2001; accepted 22 September 2001

Abstract
There have been several reports on the use of extracorporeal shock waves in the treatment of pseudarthrosis, calcify-
ing tendinitis, and tendinopathies of the elbow. However, the pathomechanism of pain relief has not been clarified. To
investigate the analgesic properties of shock wave application, we analyzed whether it produces morphologic changes
in cutaneous nerve fibres. In normal rat skin, the epidermis is heavily innervated by nerve fibres immunoreactive for
protein gene product (PGP) 9.5 and by some fibres immunoreactive for calcitonin gene-related peptide (CGRP). There
was nearly complete degeneration of epidermal nerve fibres in the shock wave-treated skin, as indicated by the loss of
immunoreactivity for PGP 9.5 or CGRP. Reinnervation of the epidermis occurred 2 weeks after treatment. These data
show that relief of pain after shock wave application to the skin results from rapid degeneration of the intracutaneous
nerve fibres. q 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Shock wave; Protein gene product 9.5; Calcitonin gene-related peptide; Sensory nerve fibre; Degeneration; Reinnervation

With the use of shock waves for the treatment of and treated aseptically throughout the experiments. One
urolithiasis, a new treatment was introduced into medicine thousand shock wave impulses of an energy flux density
that allows the induction of effects inside the body and of 0.08 mJ/mm 2 were applied to the plantar skin of the
permits surgery without the use of a scalpel [1]. rats (Dornir Med. Tech.; Epos). The experimental protocol
Tendinopathies, so-called tennis elbow, golfer’s elbow, was conducted in accordance with the guidelines of the
or calcaneus spur are well-known painful orthopaedic Ethics Review Committee for Animal Experimentation.
diseases. Immobilization, physical therapy, radiotherapy Two days (n ¼ 3), 4 days (n ¼ 3), 7 days (n ¼ 3), 14 days
and steroid injections are symptomatic treatment for these (n ¼ 3), and 21 days (n ¼ 3) after application of shock
and various other orthopaedic conditions, while arthro- waves, the rats and control rats (n ¼ 3) (that did not receive
scopic or open surgery are reserved for patients with insuffi- shock waves) were anaesthetised with sodium pentobarbital
cient response to less invasive procedures. Since 1993, it has (40 mg/kg, i.p.) and perfused transcardially with 0.9% saline,
been reported that shock waves are effective therapy for followed by 500 ml of 4% paraformaldehyde in a phosphate
patients suffering from soft tissue pain of bone, enthesopa- buffer (0.1 M. pH 7.4). The plantar skins of the rat hind paws
thy, or epicondylitis [2,3,5–7]. However, the pathomechan- were resected. After storing in 0.01 M phosphate buffer saline
ism of pain relief from this therapy has not been clarified. (PBS) containing 20% sucrose for 20 h at 48C the specimens
We aimed to evaluate the morphologic changes of sensory were sectioned at 40 mm thickness on a cryostat.
nerve fibres induced by applied shock waves. Sections were collected in PBS. The specimens were then
Eighteen male Sprague–Dawley (SD) rats, weighing treated for 90 min in blocking solution, 0.01 M PBS contain-
250–300 g were anaesthetised with sodium pentobarbital ing 0.3% Triton X-100 and 1% normal goat serum, at room
temperature. They were processed for protein gene product
(PGP) 9.5 or calcitonin gene-related peptide (CGRP) immu-
* Corresponding author. Tel.: 181-43-226-2117; fax: 181-43- nohistochemistry by use of the free floating ABC technique,
226-2116. using rabbit antibody for PGP 9.5 (1:1000; Ultraclone, Isle
E-mail address: [email protected] (S. Ohtori).

0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S03 04 - 394 0( 0 1) 02 32 0- 5

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 58 S. Ohtori et al. / Neuroscience Letters 315 (2001) 57–60

of Wight, UK) or for CGRP (1:2000; Chemicon, Temecula, terminating in the epidermis. Shock waves produced degen-
CA, US) diluted with a blocking solution for 20 h at 48C, eration of these nerve fibres. All rats exhibited denervation
biotinylated goat anti rabbit IgG (1:100; Vector Labs, in shock wave-treated skin during the first week after appli-
Burlingame, CA, US), and fluorescein iso-thiocyanate cation, and nerve fibres were rarely observed. Reinnervation
avidin D (1:100; Vector Labs). The sections were then of the epidermis by PGP 9.5-ir nerve fibres began during the
observed with a fluorescent microscope, and nerve fibres 2- and 3-week periods after the application of shock waves,
were counted as they passed through the basement and by day 14 and 21, the number of PGP 9.5-ir nerve fibres
membrane of the epidermis. Branching occurred within in shock wave-treated skin was not significantly different
the epidermis, but the number of increased branches was from that of the non-treated skin (Fig. 3).
not counted. The numbers of PGP 9.5-immunoreactive (ir) Some CGRP-ir nerve fibres were found in the control rat
and CGRP-ir fibres were counted for each section per skin (Fig. 2). The number of CGRP-ir nerve fibres on day 2,
25 £ 10 23 mm 2 of epidermis. We counted, and then aver- 4, and 7 was significantly less than that of the non-treated
aged, the numbers of PGP 9.5- and CGRP-ir fibres in the 20 skin. However, reinnervation of the epidermis by CGRP-ir
serial sections. nerve fibres began during the second and third week after
Thermal hyperalgesia or hypoalgesia of the shock wave- shock wave and the number of CGRP-ir nerve fibres on day
applied paw and that of the control rat were evaluated by a 14 and 21 was not significantly different from that of the
hot plate test daily, three times at 3-min intervals, for 3 non-treated skin (Fig. 3).
weeks. A control group (n ¼ 3) received no shock waves. Hyperalgesia or hypoalgesia did not occur after applica-
The data were compared by non-paired Welch’s t-test. A P- tion of shock waves, with significant differences in latency
value of less than 0.05 was considered statistically signifi- between shock wave-applied hind paws and that of control
cant. rat on all days (P . 0:05, Fig. 4).
In non-treated skin, PGP 9.5-ir nerve fibres are abundant In the present study, we demonstrated that there was nearly
in the subepidermal neural plexus, which lies just below the complete degeneration of epidermal nerve fibres in shock
basement membrane (Fig. 1). The epidermis here is richly wave-treated skin, as indicated by the loss of immunoreac-
innervated by fibres that originate from the subepidermal tivity for PGP 9.5 and CGRP. We also showed that reinner-
plexus and project up through the basement membrane, vation of the epidermis occurred 2 weeks after treatment.

Fig. 1. Denervation and reinnervation of epidermis by PGP 9.5-ir nerve fibres in the rat hind paw. Samples were taken from shock wave-
treated skin at 4, 7, and 21 days after application and from normal untreated skin. Bar scale: 100 mm. Lamina I indicates the epidermis.
Lamina II indicates the dermis.

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 S. Ohtori et al. / Neuroscience Letters 315 (2001) 57–60 59

Fig. 2. Denervation and reinnervation of epidermis by CGRP-ir nerve fibres in the rat hind paw. Samples were taken from shock wave-
treated skin at 4, 7, and 21 days after application and from normal untreated skin. Bar scale: 100 mm. Lamina I indicates the epidermis.
Lamina II indicates the dermis.

It has been reported that capsaicin decreased sensitivity to Reinnervation of the epidermis began during the 2- and 3-
pain produced by sharp mechanical stimuli and nearly week period after the application of shock waves. The shock
eliminated heat-evoked pain within the injected area.
There was nearly complete degeneration of epidermal
nerve fibres and the subepidermal neural plexus in capsai-
cin-treated skin, as indicated by the loss of immunoreactiv-
ity for PGP 9.5 and CGRP. Limited reinnervation of the
epidermis and partial return of sensation occurred 3 weeks
after treatment [8]. These data show that sensory dysfunc-
tion after capsaicin application to the skin results from rapid
degeneration of intracutaneous nerve fibres. The mechanism
of depression of pain sensitivity applied by shock waves
may be similar to that induced by capsaicin application.
There was a discrepancy in the current study, however,
compared with capsaicin results. We could not show signif-
icant hypoalgesia after shock wave application. Possible
reasons for the discrepancy are that: (1) the intensity of
shock wave-stimulation was weaker than that of capsaicin
application, therefore, apparent hyposensitivity to pain
could not be induced; (2) it has been reported that after
denervation in rats, the amounts of CGRP increased in the
remaining small-sized sensory nerve fibres and emerged in
large-sized sensory nerve fibres, resulting in hyperalgesia
Fig. 3. The number of PGP 9.5-ir or CGRP-ir sensory nerve fibres
contrary [4]. In the present study, although sensory nerve
over time. The number of PGP 9.5-ir or CGRP-ir nerve fibres on
fibres decreased in the rat skin, hypoalgesia might not have day 2, 4, and 7 was significantly less than that of the non-treated
been observed because the remaining nerve fibres changed skin. However, on day 14 and 21 no significant differences in
their phenotype. these values were observed.

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 60 S. Ohtori et al. / Neuroscience Letters 315 (2001) 57–60

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We wish to thank Miss Keiko Kitajo for technical assis-

tance.

Temístocles dias de Oliveira Neto - [email protected] - CPF: 876.218.601-97