Nerve Regeneration After Radiofrequency Application

The American Journal of Sports Medicine http://ajs.sagepub.com/

Nerve Regeneration After Radiofrequency Application Nobuyasu Ochiai, James P. Tasto, Seiji Ohtori, Norimasa Takahashi, Hideshige Moriya and David Amiel Am J Sports Med 2007 35: 1940 originally published online July 16, 2007 DOI: 10.1177/0363546507304175 The online version of this article can be found at: http://ajs.sagepub.com/content/35/11/1940

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Nerve Regeneration After Radiofrequency Application Nobuyasu Ochiai,* MD, PhD, James P. Tasto,† MD, Seiji Ohtori,‡ MD, PhD, ‡ ‡ § Norimasa Takahashi, MD, PhD, Hideshige Moriya, MD, PhD, and David Amiel,* PhD From the *Department of Orthopedic Surgery, University of California San Diego, † La Jolla, California, San Diego Sports Medicine & Orthopedic Center, San Diego, California, ‡ and the Department of Orthopedic Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan

Background: Many patients with chronic tendinosis have experienced early pain relief after application of bipolar radiofrequency treatment. It is hypothesized that the mechanism of action may be the acute degeneration and/or ablation of sensory nerve fibers. Hypothesis: After ablation or degeneration by bipolar radiofrequency, nerve fibers will have the ability to regenerate with time. Study Design: Controlled laboratory study. Methods: Eighteen Sprague-Dawley rats were used in this study. These rats were divided into 3 groups (30, 60, and 90 days after bipolar radiofrequency). These rats were treated with 2 points of bipolar radiofrequency applications to the left hindpaws with the Topaz microdebrider device. Right hindpaws were used as the contralateral control. Tissues were processed for neural class III β-tubulin or calcitonin gene-related peptide immunohistochemistry by using the free-floating avidin biotin complex technique. The numbers of neural class III β-tubulin–immunoreactive and calcitonin gene-related peptide-immunoreactive nerve fibers in the epidermis were counted and compared with those in the contralateral control. Results: Although the numbers of nerve fibers demonstrated by both the antibodies of neural class III β-tubulin and calcitonin gene-related peptide were significantly decreased (P < .0001) until 60 days after bipolar radiofrequency treatment, regeneration of the epidermal nerve fibers occurred 90 days after treatment. Conclusion: Bipolar radiofrequency treatment induced degeneration of sensory nerve fibers immediately after treatment, but by 90 days posttreatment, there was evidence of complete regeneration. Clinical Relevance: Early degeneration followed by later regeneration of nerve fibers after bipolar radiofrequency treatment may explain long-term postoperative pain relief after microtenotomy for tendinosis. Keywords: bipolar radiofrequency; chronic tendinosis; microtenotomy; pain relief

Chronic tendinopathy, like medial and lateral epicondylitis and patellar and Achilles tendinopathy, is a well-known painful orthopaedic disease. The characteristic pathologic characteristics of all these tendinosis conditions are linked to microrupture of the tendon,4,5 granulation tissue,9 and degenerative changes.13,15 Nonsteroidal anti-inflammatory medication, physical therapy, steroid injections, and

orthoses are symptomatic treatment for these and various other orthopaedic conditions, while arthroscopic or open surgery is reserved for patients with insufficient response to less invasive procedures. Neovessels and accompanying free nerve endings have been observed in tendinosis tissue using Protein Gene Product 9.5 immunohistochemistry.2 Furthermore, the tendon insertion, which is supplied with substance P (SP) and calcitonin gene-related peptide (CGRP) innervation, has been implicated in the origins of tennis elbow.11 The rat model of Achilles tendinosis showed an increased number of nerve filaments and increased immunoreactivity for SP and CGRP.12 Neuropeptides SP and CGRP are not only involved in transmitting nociceptive information to the spinal cord, but also in peripheral effects, including microvascular leakage and local edema formation.21 Pathological nerve ingrowth may have been involved in the origin of tendinosis.11 On the

§ Address correspondence to David Amiel, PhD, Department of Orthopedic Surgery, Connective Tissue Biochemistry, 9500 Gilman Drive, Department 0630, La Jolla, CA 92093-0630 (e-mail: [email protected]). One or more of the authors has declared a potential conflict of interest: Drs Tasto and Amiel are members of the Medical and Scientific Advisory Board for ArthroCare Corp.

The American Journal of Sports Medicine, Vol. 35, No. 11 DOI: 10.1177/0363546507304175 © 2007 American Orthopaedic Society for Sports Medicine


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other hand, CGRP is important for formation of new vessels during wound healing.10 Furthermore, CGRP is vasoactive in normal rabbit medial collateral ligaments, suggesting that it may participate in the maintenance of ligament homeostasis and the promotion of soft tissue healing.7 Recently extracorporeal shock wave therapy (ESWT)17 and bipolar radiofrequency (bRF) microtenotomy20 have been used for the treatment of chronic tendinosis. Clinically, the majority of patients with chronic lateral epicondylitis received at least some pain relief within the first 7 to 10 days, and it lasted at least 2 years after bRF microtenotomy. The pathomechanism of pain relief after ESWT14 and bRF microtenotomy18 is thought to be acute degeneration of epidermal nerve fibers in the early period. A previous study showed that bRF treatment induced acute degeneration or ablation of the sensory nerve fibers for at least 2 weeks.18 Although regeneration of the epidermal nerve fibers was observed 2 weeks after ESWT,14 regeneration after bRF is still unknown. We hypothesized that microtenotomy with bRF-induced degeneration of the nerve fibers would be followed by regeneration. Regeneration of nerve fibers would be necessary to resume normal tendon condition that leads to long-term effect by bRF, similar to the process of wound healing.10 The purpose of this study was to evaluate the regeneration potential of peripheral nerve fibers after bRF application using a rat model.

MATERIALS AND METHODS Following Institutional Animal Care and Use Committee and our institution’s Animal Subjects Committee approval, we used 18 male Sprague-Dawley rats (weight, 250-300 g) divided into 3 groups (30, 60, and 90 days after bRF). They were anesthetized with isofluorane (VEDCO Inc, St Joseph, Mo) and sodium pentobarbital (Abbott Labs, Chicago, Ill) and treated aseptically throughout bRF applications. Two separate points of bRF were equally applied (at a distance of 1-3 mm from each other for 500 ms) to the middle 2 footpads of the left hindpaw of the 18 rats. The TOPAZ Microdebrider device (ArthroCare, Sunnyvale, Calif) connected to a System 2000 generator at a setting of 4 V-RMS (voltage-root metered squared) was used to perform the bRF application. In this process, bRF energy is used to excite the electrolytes in normal saline, and the energized particles have sufficient energy to break molecular bonds, ablating soft tissue at low temperatures (40°-70°C). After treatment, the rats were allowed activity as tolerated. Rats treated by bRF after 30 (n = 6), 60 (n = 6), and 90 days (n = 6) were anesthetized with ketamine (80 mg/kg intraperitoneal) and isofluorane and perfused transcardially with 0.9% saline, followed by 500 mL 4% paraformaldehyde in a phosphate buffer (0.1 1M, pH 7.4). The footpads of both rat hindpaws were resected (left for study group, right for the contralateral control group). After being stored in 0.01 M phosphate buffered saline (PBS) containing 20% sucrose for 20 hours at 4°C, the specimens were sectioned at a 30µm thickness on a cryostat. Twenty sections were collected in PBS. Half of them were processed for neural class III βtubulin (TUJ-1) for immunochemistry, and the other half were processed for CGRP immunohistochemistry1 by using

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the free-floating avidin biotin complex (ABC) technique. They were incubated for 20 hours at 4°C with mouse antibody for TUJ-1 (1:500; Convance, Berkeley, Calif) or rabbit antibody for CGRP (1:500; ImmunoStar Inc, Hudson, Wis) diluted with a blocking solution in 0.01 M PBS containing 0.3% Triton X-100, 5% skim milk (Becton, Dickinson and Company, Sparks, Md), and 0.05% bovine serum albumin (Sigma-Aldrich, St Louis, Mo). After thorough washing, sections were incubated for 90 minutes in an Alexa Fluor 488 labeled goat anti-mouse IgG (1:100; Molecular Probes, Eugene, Ore) or Alexa Fluor 488 labeled anti-rabbit IgG (1:100; Molecular Probes). The sections were then viewed with fluorescent light microscopy by 1 observer in our laboratory (N.O.), who was blinded to the experimental group and time period of each sample, and nerve fibers that passed through the basement membrane of the epidermis were counted. Branching occurred within the epidermis, but these additional branches were not counted. The numbers of TUJ1-immunoreactive (IR) and CGRP-IR fibers were counted for each section per 16.3 × 10-1 mm2 of epidermis in a footpad. After counting all sections in all time periods, the numbers of TUJ1-IR and CGRP-IR fibers in the 10 sections of each rat were summed and then averaged in each group. Statistical analysis of results was performed using StatView 5.0 (Statsoft, Tokorozawa, Japan). Results were presented as mean ± standard deviation (SD) in each group. The mean numbers of nerve fibers between the bRF study groups were matched with their respective contralateral control group and were analyzed using a paired t test. The mean numbers of nerve fibers between the bRF study group on day 30, day 60, and day 90 were compared using a nonpaired Student t test. Significance was defined as P < .05.

RESULTS Although the RF-treated footpad had a dimple on the surface just after bRF application, no macroscopic difference was detected between the bRF foot and the contralateral control foot at 30, 60, and 90 days after bRF. The rats were observed ambulating with a normal gait immediately after the bRF applications. Figure 1A shows the nerve fibers in the epidermis of the footpad (the area in which the bRF was applied), with immunoreactivity of the TUJ-1 in the contralateral control sample. The contralateral control epidermis is richly innervated by TUJ1-IR nerve fibers. After bRF application, nerve fibers were rarely seen in the epidermis at day 30 (Figure 1B) and day 60 (Figure 1C). There was a significant difference between the contralateral control and bRF group in the numbers of TUJ1IR–stained nerve fibers (mean number of nerve fibers ± SD) at day 30 (172.9 ± 20.4 vs 91.6 ± 20.5, P < .0001) and at day 60 (169.9 ± 15.2 vs 119.1 ± 20.5, P < .0001). There was no significant difference between the contralateral control and bRF group in the numbers of TUJ1-IR–stained nerve fibers at day 90 (169.2 ± 20.5 vs 164.7 ± 24.0, P = .6557) (Figure 1D; Figure 2).


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Figure 1. Immunofluorescent light micrographs showing nerve fibers immunoreactive against TUJ-1 in the epidermis. (A) Micrograph showing nerve fibers innervating the epidermis of the contralateral control footpad (white arrows) of the rat. White bar (—) indicates 100 µm. (B) Left middle footpads at 30 days after bRF treatment. A micrograph shows few nerve fibers in the epidermis. (C) Left middle footpads at 60 days after bRF treatment. A micrograph shows slightly more nerve fibers in the epidermis than at 30 days. (D) Left middle footpads at 90 days after bRF treatment. No differences between Figures 1A and 1D are seen. TUJ1-IR nerve fibers

Contralateral control bRF treated

Number of nerve fibers/ per unit area of bRF-treated tissue

250 200

*

*

*

7

14

30

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150 100 50 0 60

90

(post days after bRF treatment)

Figure 2. Line graphs showing the mean value ± standard deviation of TUJ1-IR nerve fibers at 7, 14, 30, 60, and 90 days after bRF treatment. Although there were significant differences in the number of nerve fibers between the contralateral group and bRF-treated group at 30 and 60 days (P < .0001), no significant differences were observed between the contralateral group and 90 days post-bRF (P = .6557). There was also significant difference in the number of nerve fibers among day 30, day 60, and day 90 time periods (day 30 vs day 60, P < .0001; day 30 vs day 90, P < .0001; day 60 vs day 90, P < .0001). *P < .05. The values of 7 and 14 days were previously described.18 The reaction of CGRP-IR nerve fibers after RF application was similar to that of TUJ1-IR nerve fibers. Figure 3A demonstrates the immunofluorescent micrographs showing

Figure 3. Immunofluorescent light micrographs showing CGRP-IR nerve fibers. (A) Micrograph showing nerve fibers innervating the epidermis of the contralateral control footpad (white arrows) shows the richly innervated left middle footpad of the rat. (B) Left middle footpads at 30 days after bRF treatment. A micrograph shows few nerve fibers in the epidermis. (C) Left middle footpads at 60 days after bRF treatment. A micrograph shows slightly more nerve fibers in the epidermis than at 30 days. (D) Left middle footpads at 90 days after bRF treatment. No differences between Figures 3A and 3D are seen. CGRP-IR nerve fibers (white arrows) innervating the middle footpad of the contralateral control. Figure 3B shows few fibers seen in the epidermis at 30 days after bRF treatment. Figure 3C shows slightly more fibers seen in the epidermis at 60 days after bRF treatment compared with 30 days post-bRF. There were significantly more CGRP-IR nerve fibers present in the contralateral control epidermis when compared on day 30 to bRF-treated nerve fibers (78.6 ± 12.3 vs 46.6 ± 7.9, P < .0001) and at day 60 (80.6 ± 8.7 vs 55.0 ± 10.0, P < .0001). There was no significant difference between contralateral control and bRF group on the numbers of CGRP-IR–stained nerve fibers at day 90 (84.7 ± 11.5 vs 81.6 ± 12.5, P = .9331) (Figure 3D; Figure 4). There was a significant difference in the number of nerve fibers among day 30, day 60, and day 90 time periods for both the TUJ1-IR group (day 30 vs day 60, P < .0001; day 30 vs day 90, P < .0001; day 60 vs day 90, P < .0001) (Figure 2) and the CRGP-IR group (day 30 vs day 60, P = .0013; day 30 vs day 90, P < .0001; day 60 vs day 90, P < .0001) (Figure 4). This result demonstrates nerve regeneration after the bRF treatments occurred gradually from 30 days to 90 days.

DISCUSSION In the present study, we demonstrated that regeneration of the epidermal nerve fibers occurred gradually from 30 days to 90 days. Finally it became obvious that there was no statistical difference in the number of nerve fibers with


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CGRP-IR nerve fibers

Contralateral control bRF treated

Number of nerve fibers/ per unit area of bRF-treated tissue

140 120

* * *

100

*

80 60 40 20 0 7

14

30

60

90

(post days after bRF treatment)

Figure 4. Line graph showing the mean value ± standard deviation of CGRP-IR nerve fibers at 7, 14, 30, 60, and 90 days after bRF treatment. Although there were significant differences in the number of nerve fibers between the contralateral group and bRF-treated group at 30 and 60 days (P < .0001), no significant differences were observed between the contralateral group and 90 days post-bRF (P = .9331). There was significant difference in the number of nerve fibers among day 30, day 60, and day 90 time periods (day 30 vs day 60, P = .0013; day 30 vs day 90, P < .0001; day 60 vs day 90, P < .0001). *P < .05. The values of 7 and 14 days were previously described.18

the contralateral control groups. Regeneration of TUJ1-IR and CGRP-IR nerve fibers seemed to be similar to each other. Neovascularity has been proposed to be one of the factors in the origin of tendinosis. Hypovascularity induces not only necrosis and fibrillation in the tendon1 but also induces the rupture of the tendon. Lack of vascularity compromises the nutrition required by tendon cells, making it more difficult for those cells to synthesize extracellular matrix necessary for repair and remodeling for fatiguedamaged tendon.20 Regarding the effect of bRF, histologic evidence of neovessel formation and an increase in angiogenic markers demonstrated that bRF microtenotomy can increase tissue vascularity when properly applied to tendons, leading to healing of the tendon.20 Although the results revealed macroscopic and microscopic changes in the early term, bRF-treated tendons resumed a normal appearance by 90 days.20 Neural class III β-tubulin antibody, which recognizes class III β-tubulin exclusively, reveals the axons and their terminations, as well as significant injury-related alterations.8 In this study, we demonstrated that bRF applications significantly decrease the number of TUJ1-IR nerve fibers by 60 days, which gradually increase by 90 days. This increase after decrease in numbers of nerve fibers was also found after ESWT to the rat skin but resumed rapidly at 2 weeks.14 Calcitonin gene-related peptide has previously been reported also to promote angiogenesis and tissue regeneration,3,6,10 partly by stimulating proliferation of endothelial cells and fibroblasts.3,10 The effect of CGRP on endothelial cells has a potential effect in angiogenesis, including formation of new vessels in wound healing.10 The increase in

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immunoreactivity for CGRP-IR nerve fibers may result in changing the hypovascularity condition such as occurs in tendinosis to be healed with the increase responses of the vascularity. Although this study did not clarify the confusion in the literature about the vascularity,2,16 we demonstrated that the CGRP-IR nerve fibers regenerate by 90 days and resumed normal appearance. We postulated that the regeneration of the CGRP-IR nerve fibers accompanied by the increase of vascularity is important for the healing process in tendinosis that may in turn be partially caused by hypovascularity. Clinically, the majority of patients with chronic lateral epicondylitis received at least some pain relief within the first 7 to 10 days that lasted at least 2 years after bRF microtenotomy.19 Although degeneration of sensory nerve fibers would lead to early pain relief,18 our study explained that the nerve fibers regenerated, along with the healing process, and with potential of no return of the pathological condition. One of the limitations of this study is using rat skin instead of pathological tendon. There are no suitable animal models for assessing effectiveness of bRF treatment of tendinosis. We analyzed the abundant nerve fibers in the rat sole for a response to bRF. Extracorporeal shock wave therapy study has already showed the validity of using this model.14 We assume that the effects of bRF would be the same for normal epidermal nerves and the pathologic nerves found in tendinosis. The long-lasting effectiveness of bRF in clinical cases19 would correlate with the regeneration of the nerve fibers the same as in the contralateral control. Further study is necessary to determine the effects of bRF on pathological conditions and to confirm the expected same mechanism. We concluded that bRF treatment induced degeneration and regeneration of the nerve fibers in the rat sole. These results may explain the rapid pain relief as well as one of the healing responses after bRF treatment in tendinosis.

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