27 Tendon Innervation and Neuronal Response After Injury

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Introduction

Tendon pain and injuries are frequent complications of repetitive motion and overuse syndromes and pose a huge burden on the health care system mainly due to the lack of specific treatment and long recovery time until return to work. The knowledge of the pathomechanisms under- lying tendon disorders is still limited. Although some mechanical risk factors have been identified, the relation- ship between pain and inflammation is debatable [54], and still requires a plausible biological explanation. Accumu- lating data support the hypothesis that the peripheral nervous system plays a major role in the regulation of pain and inflammation, maybe also in tissue repair.

In inflammatory conditions of the musculoskeletal system, pain is almost a constant feature. However, in degenerative conditions pain is highly variable. Whether inflammation is a prerequisite to pain in degenerative disease is unclear. Notably, spontaneous ruptures of tendons, presumed to be caused by degeneration, are often not preceded by pain. On the whole, the relation- ship between pain, inflammation and degeneration of the musculoskeletal system, and specifically tendons, remains obscure, a consequence of our limited knowledge of neu- roanatomy and pathophysiology.

Over the last decade, however, knowledge about specific neuronal signal substances involved in the regulation of pain, inflammation and tissue repair has evolved. This chapter discusses tendon innervation focus- ing on neuronal mediators, so-called neuropeptides, including opioids, in tendon proposing an endogenous peripheral system of pain inhibition. Moreover, recent findings on neuronal response to injury are presented, suggesting new pathways for tendon repair.

The novel data presented in this chapter considering tendon innervation and neuronal response to injury may suggest new targets for pain treatment and speciﬁc phar- macotherapy in tendon disorders that could shorten recovery time.

Tendon Innervation

Tendon innervation originates from 3 neighboring sources, cutaneous, muscular, and peritendinous nerve trunks. From the myotendinous junction the nerve fibers cross and enter the endotenon septa. In the paratenon, nerve fibers form rich plexuses and send branches that penetrate the epitenon. Most nerve fibers do not actually enter the tendon proper, but terminate as nerve endings on its surface.

The nerves of tendons are composed of myelinated, fast-transmitting Aa- and Ab-fibers and unmyelinated, slow-transmitting Ag-, Ad-, B- and C-fibers. The nerve endings of myelinated fibers (Aa, Ab) are type I-III spe- cialized mechanoreceptors, whose main function is to mediate physical energy (pressure, tension) into afferent nerve signals. The unmyelinated nerve endings of Ag-, Ad-, and C-fibers are type IVa fibers, so called nocicep- tors. These nerve endings mediate deep tissue pain and hyperalgesia that are typical features of tendon pain. The nerve endings of B-fibers, which are autonomic, consist of type IVb fibers mainly located in the walls of small arter- ies, arterioles, capillaries, and postcapillary veins and exert vasomotor actions.

Over the last decade, the peripheral nervous system in addition to classical functions such as nociception and vasoactivity, has been found to also participate in the regulation of a wide variety of efferent actions on cell proliferation, cytokine expression, inﬂammation, immune responses and hormone release [12,27,33,57,61,64,67].

The paradoxical “efferent” role of afferent nociceptive ﬁbers as suggested by Bayliss [11] more than one cen- tury ago is now widely recognized. So far, however, the innervation and neuronal regulation of periarticular tissues, and especially tendons, have received little atten- tion. In particular, this applies to the speciﬁc neuronal mediators, i.e. the signal substances.

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Tendon Innervation and Neuronal Response After Injury

Paul W. Ackermann, Daniel K-I. Bring, and Per Renström

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The mediators of the nervous system act through 2 principally different ways:

1) With fast transmitters, i.e. classical neurotransmitters (monoamines, acetylcholine, amino acids), which directly effectuate muscle contractions or afferently relay information on painful stimuli.

2) With a family of slow transmitters, neuropeptides, which slowly regulate physiological functions in the central as well as peripheral nervous system.

Neuropeptides

Neuropeptides act as chemical messengers and regulators in the central and peripheral nervous systems. They dif- fer from classical neurotransmitters in several respects.

Peripheral neuropeptides are synthesized in the cell bodies, i.e. the dorsal root ganglia (sensory) and the sympathetic chain (autonomic), and transported distally. In contrast, classical neurotransmitters are synthesized in the axon terminals, thus exerting their effects more locally. Synthesis and turn-over of classical transmitters are more rapid than those of neuropeptides leading to more long-lasting regulatory effects of the neuropeptides.

Moreover, several neuropeptides are coreleased, also together with classical transmitters [34], which offers the opportunity of a variety of functional interactions.

The effects of neuropeptides and classical transmitters are elicited by different mechanisms, resulting in direct effects of classical transmitters, while neuropeptides act in a regulatory, “supervising” fashion.

Several neuropeptides have been identiﬁed in both the central and peripheral nervous system. So far, however, research on the occurrence and functions of neuropeptides in tendons has been very limited. Those hypothetically are important for nociception and tissue homeostasis in tendon can be classiﬁed in 3 groups;

sensory, opioid and autonomic, according to their function and original nerve ﬁber type ﬁnding.

Sensory Neuropeptides

Sensory primary afferents, C- and Ad-ﬁbers contain substance P (SP) claimed to transmit nociceptive signals [35,40,65]. Calcitonin gene-related peptide (CGRP) co- exists with and potentiates the effect of SP [56,74,77].

Both SP and CGRP have also been shown to exert pro- inﬂammatory effects such as vasodilation and protein extravasation [13,47,53]. In the periphery, release of SP leads to sensitization of surrounding primary afferents by enhancing cellular release of prostaglandins, histamines and cytokines [63,72]. Recently, however, SP also directly stimulates nociceptor endings [71]. CGRP, often colocal- ized with SP in unmyelinated C ﬁbers, facilitates the

release of SP and delays SP degradation in the spinal cord, thereby potentiating the nociceptive effect [38,62,77].

Sensory nerve fibers, interestingly, also seem to contain peptides with anti-nociceptive and anti- inflammatory effects counteracting the effects of SP and CGRP. Thus, galanin (GAL), somatostatin (SOM) as well as opioids, all of which occurring in primary afferents, inhibit inflammation and nociception [17,20,28,68,78].

These anti-nociceptive peptides probably exert their modulatory effect through inhibition of SP release [15,25,78,79].

Opioid Neuropeptides

The opioid neuropeptides and their receptors are, in sim- ilarity with sensory neuropeptides, produced by primary sensory neurons (dorsal root ganglia) and transported centrally and peripherally.

The endogenous opioid peptides consist of 3 families (enkephalins, dynorphins, and endorphins), which contain several biologically active products, e.g.

Met-enkephalin-Arg-Pro (MEAP) and dynorphin B (DYN B), that exert physiological actions by interacting with opioid receptors. Enkephalins are thought to be the ligands for the d-opioid receptor, and dynorphins for the k-receptor. Recent studies identiﬁed endomorphins as endogenous ligands for m-opioid receptors in addition to endorphins and enkephalins. A fourth opioid receptor was also demonstrated. This is genetically closely related to the others receptors and responds to the endogenous agonist nociceptin.

The endogenous opioids seem to provide a peripheral anti-nociceptive system. Thus, it was recently reported that intra-articular morphine after arthroscopic proce- dures had a signiﬁcant analgesic effect in a dose-response manner. Notably, morphine inhibits SP release from peripheral sensory nerve endings [15,79]. Opioid receptors are therefore not only conﬁned to the CNS, but also occur in the periphery, implication that pain can be inhibited at a peripheral level [29]. Indeed, opioid receptors have been demonstrated in peripheral sensory nerve terminals of the skin [19,66,73]. As for tendons, the neuronal occurrence of endogenous opioids and receptors, had, until recently, not been explored.

Autonomic Neuropeptides

Autonomic B-ﬁbers in the periphery contain several mediators with sympathetic and parasympathetic effects.

Neuropeptide Y (NPY) found in sympathetic ﬁbers is a potent vasoconstrictor. It often co-exists with the classical transmitter noradrenaline (NA) potentiating the vasoconstrictive action of the latter [45]. In parasympathetic nerve ﬁbers, vasoactive intestinal polypeptide

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(VIP) has been shown to be a potent vasodilator [44]. In addition to the well established vasoactive role of VIP, other important effects have been reported over the last years. Thus, VIP has been implicated in the regulation of immune cells and the expression of cytokines and growth factors.

Neuropeptides in Tendons

As information on neuropeptide prevalence in connective tissue was largely unexplored, we studied the neuropeptide occurrence in tendons as compared to ligaments [1]. With a combined approach based on radioimmunoassay (RIA), immunohistochemistry (IHC) and high performance liquid chromatography (HPLC), 3 main classes of neuropeptides, autonomic, sensory and opioid in normal rat Achilles tendon and medial collateral ligament have been detected. The combined quantitative and morphologic analyses have demonstrated 12 neuropeptides; sensory (SP, NKA, CGRP, GAL, SOM);

and opioid (LE, ME, MEAP, MEAGL, nociceptin), autonomic (NPY, VIP) in tendons [1].

Occurrence and Levels of Neuropeptides Sensory Peptides

Overall, several studies have disclosed the occurrence of sensory neuropeptides in both animal and human tendons [4,24,43]. In tendons, sensory neuropeptides with nociceptive and pro-inﬂammatory effects (SP and CGRP) as well as others (GAL, SOM), which are known to modulate these effects, are present. Thus, the relative concentrations of sensory mediators and modulators could suggest physiological nociceptive thresholds.

The concentrations of SP, CGRP vs. GAL were about 4 times higher in tendons compared to ligaments [4], which might reﬂect a tissue speciﬁc vulnerability to pain (Figure 27-1A). These levels can change in response to outer or inner stress [33], possibly explaining why pain syndromes in tendons are more common than in ligaments (Figure 27-1A).

In fact, increased levels of sensory neuropeptides are involved in painful and inﬂammatory conditions. Several studies have demonstrated increased levels of sensory peptides in patients with rheumatoid arthritis [50] and in tendons of rats with experimentally induced arthritis [14].

One report also showed increased levels of SP to corre- late with the pain caused by rotator cuff disease [24].

Other studies demonstrated increased levels of the neu- rotransmitter glutamate in painful patellar and Achilles tendons of patients with tendinopathy [8,9], indicating increased neuronal activity. Glutamate is known to exist

0 0,5

1 1,5

2 2,5

Tendon Ligament

Neuropeptide conc. (pmol/g tissue)

SP CGRP Galanin

0 0,2 0,4 0,6 0,8

Opioid conc. (pmol/g tissue)

MEAP NOC DYN B

0 1 2 3 4 5 6

Neuropeptide conc. (pmol/g tissue)

NPY VIP

Figure 27-1. Radioimmunoassay tissue concentrations (pmol/

g) of sensory (SP, CGRP and GAL) (A), opioid (MEAP, N/OFQ and DYN B) (B), and autonomic (NPY and VIP) (C) neuropeptides in the Achilles tendon and collateral ligaments of rat knee (mean ± s.e.m.). (Adapted from Ackermann WP, Finn A, Ahmed M. Sensory neuropetidergic pattern in tendon, ligament and joint capsule. A study in the rat. Neuroreport.

1999; 10(10): 2055–60. Reproduced from Ackermann et al.

Autonomic innervation of tendons, ligaments and joint cap- sules. A morphologic and quantitative study in the rat. J Orthop Res 2001; 19, 372–8, with permission from the Orthopaedic Research Society. Reproduced with permission from Acker- mann et al. An Opioid System in Connective Tissue: A Study of Achilles Tendon in the Rat. J Histochem Cytochem 2001;49:1387–96.)

A

B

C

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and injury. Notably, ligaments are more prone to rupture than tendons [76].

Altogether, quantitative analysis of tendons has demonstrated autonomic, sensory and opioid peptides presumed to be involved in nociception, inﬂammation and vasoactivity. The relative levels of counteracting neuropeptides, may represent the tissue homeostasis of different functions and, possibly the susceptibility to external stress. In fact, increased levels of pro-nociceptive sensory mediators have been shown in painful tendon conditions.

Morphological Distribution of Neuropeptides

Morphological studies by immunohistochemistry have veriﬁed the neuronal occurrence of 11 neuropeptides.

Sensory Fibers

Nerve fibers immunoreactive to SP, CGRP, NKA, GAL and SOM were consistently identified in the Achilles tendon of the rat [4]. The highest fiber density was observed in the surrounding tissues, i.e. the paratenon and loose connective tissue (Figure 27-2). The ratio of vascular vs non-vascular fibers was higher in the surrounding loose connective tissue than in the paratenon, which exhibited a large number of non-vascular free nerve endings (Figure 27-2C).

Thus, the abundance of immunopositive vascular fibers in the surrounding loose connective tissues may reflect an important role in the regulation of blood flow to the proper structures. Both SP and CGRP, in particular the latter, are potent vasodilators [13]. In addition, they have pro-inflammatory effects, e.g. by enhancing protein extravasation and leukocyte chemotaxis. The occurrence of sensory free nerve endings unrelated to vessels predominantly seen in the paratenon suggests a nociceptive role.

Opioid Peptides and Receptors

Morphological studies demonstrated the neuronal presence of several enkephalins, i.e. LE, ME, MEAP, and MEAGL, in peripheral nerve ﬁbers of the rat Achilles tendon (Figure 27-3D) [2]. All 4 enkephalins predominantly occurred in the loose connective tissue, the paratenon and musculotendinous junction, and no enkephalins were found in the tendon proper. Hypothet- ically, this difference in anatomical distribution might suggest that regulation of painful disorders of the Achilles tendon mainly occurs in the surrounding tissues, which during normal condition also harbor the sensory and autonomic neuropeptides.

The existence of an opioid system in connective tissues, as demonstrated by neuronal immunoreactivity to enkephalins, was supported by opioid receptor studies peripherally and to interact with SP in nociception [18].

SP potentiates the effect of glutamate [18,42], which was also demonstrated in Achilles tendons of patients with tendinopathy [7].

The occurrence of sensory neuropeptides in tendons complies with nociceptive effects, possibly in combina- tion with traditional neurotransmitters, and the neuropeptide levels indicate a vulnerability to develop painful disorders.

Opioid Peptides

A peripheral neuronal source of anti-nociception to modulate the sensory system could be the endogenous opioid neuropeptides. Notably, data on opioids in the peripheral nervous system and speciﬁcally tendons are quite scarce.

However, an opioid system in the Achilles tendon of rats was recently detected (Figure 27-1B) [2].This was the ﬁrst observation on the occurrence of opioids including nociceptin in periarticular tissues. The results clearly demonstrated the existence of opioid peptides (enkephalin and nociceptin) in the tendon.

The physiological role of enkephalins like MEAP can be assumed to be anti-nociceptive [17,46], anti-inﬂam- matory [31,39], vasodilatory [23,52], immunosuppressive [16] and trophic [75,80]. Notably, the release of SP from afferents in the cat knee joint is inhibited by intra- articular enkephalin-analogue injections [79]. Also, nociceptin can reduce mechanosensitivity in the rat knee joint through inhibition of SP release [48,49].

The lower concentrations of MEAP and nociceptin in tendons compared to ligaments (Figure 27-1B) could reﬂect a greater susceptibility to pain and inﬂammation, in particular when considering also the high levels of SP found in tendons. The combined quantitative data obtained suggest a balance between nociceptive and anti-nociceptive peptides under normal conditions. This balance may be altered in pathological conditions.

Autonomic Peptides

The presence of autonomic peptides has been demonstrated in tendons of both animals [5] and humans [43].

The autonomic peptides in sympathetic (NPY) and parasympathetic (VIP) nerve ﬁbers exert opposite effects in the regulation of vasoactivity.

One study showed that the concentration of NPY compared to VIP was 15 times higher in ligaments and twice as high in tendons (Figure 27-1C). The observation would seem to reﬂect that the sympathetic tone is higher than the parasympathetic in tendons and ligaments. Such a difference between tissues may be assumed to be of im- portance for blood-ﬂow, and hypothetically for healing capacity, possibly also for susceptibility to degeneration

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based on binding assays and immunohistochemistry [2].

Of the 3 opioid receptors (d-,k-,m-) studied, however, only the d-opioid receptor (DOR) could be detected by immunohistochemistry (Figure 27-3D). Double staining disclosed coexistence of each of the enkephalins

with DOR in the nerve ﬁbers (Figure 27-3D), which is in accordance with other studies suggesting that enkephalins are the main ligands for DOR [21]. DOR exerts a potent inhibitory effect on SP release [30,79].

There are several reports demonstrating that treatment

A

tendon muscle

B

tendon muscle

C

Figure 27-2. Overview micrographs of longitudinal sections through the Achilles tendon obtained by putting together computerized images of smaller micrographs. Incubation with antisera to general nerve marker (PGP). Micrographs depict the proximal half of the Achilles tendon at increasing magnification in figures (A–C). Arrows denote varicosities and nerve terminals. The typical vascular location of NPY is depicted in (B), whereas the free nerve endings are typical location of SP (C). The immunoreactivity is seen in the paratenon and surrounding loose connective tissue. The main body of the tendon is notably almost devoid of nerve fibres pt = paratenon. (Reproduced with permission from Ackermann WP, Ahmed M, Kreicbergs A, Early nerve regeneration after Achilles tendon rupture—a prerequisite for healing? J Orthop Res 2002; 20(4): 849–56.)

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with delta opioid agonists in the periphery elicit both anti-inflammatory and anti-nociceptive effects in models of inflammation [55,69,81]. Presumably, the coexistence of enkephalins and DOR in vascular nerve fibers reflects inhibition of neurogenic pro-inflammatory actions, whereas the co-existence in free nerve endings reflects inhibition of nociception.

The present ﬁndings suggesting a neuronal source of opioids in the periphery probably reﬂect an antinocicep- tive system in tendons, which may be exploited in the therapeutic setting by drugs acting selectively in the periphery.

Autonomic Fibers

Both sympathetic (NPY, NA) and parasympathetic (VIP) fibers were identified in the tendon [5]. The autonomic fibers were mostly observed as networks around blood vessels located in the loose connective tissue around the tendon proper (Figure 27-2). Overall, these observations reflect that the regulation of blood flow to tendons predominantly occurs in the adjacent loose connective tissue.

Altogether, there seems to exist a complex neuropep- tidergic network in tendons with a specific ratio of potentiating and inhibitory mediators. The observations may well reflect unique tissue requirements under normal conditions including the susceptibility to stress. The most conspicuous findings pertained to the abundance of sensory fibers in the surrounding tissues of the tendon, whereas the tendon proper, notably, was almost devoid of nerve fibers (Figure 27-2). This would seem to reflect that the neuronal regulation of tendons highly depends on the innervation of the surrounding tissues.

t

D

[Leu]-enkephalin and d-opioid receptor

t

pt

SP and CGRP

C B A

Figure 27-3. Immunofluorescence micrographs of longitudinal sections through the Achilles tendon after incubation with antisera to NPY (A), VIP (B) and doubble staining (co- localization) of SP and CGRP (C), LE and DOR (D). The NPY- positive fibres are arranged as nerve terminals in the vessel walls. VIP-positive nerves are arranged as a “fence”, surrounding the main body of the tendon, of small varicosities in the paratenon. Co-existence of SP and CGRP is evident in the paratenon, indicating possible pro-inflammatory actions.

The immunoreactivity displaying co-existence of LE and DOR is seen as free nerve endings in the paratenon, which indicates a potential peripheral anti-nociceptive system. t = tendon tissue;

Pt = paratenon; Bar = 50 mm. (Adapted from Ackermann WP, Finn A, Ahmed M. Sensory neuropetidergic pattern in tendon, ligament and joint capsule. A study in the rat. Neuroreport 1999;

10(10): 2055–60. Reproduced from Ackermann et al.Autonomic innervation of tendons, ligaments and joint capsules. A mor- phologic and quantitative study in the rat. J Orthop Res 2001;19, 372–8, with permission from the Orthopaedic Research Society.

Reproduced with permission from Ackermann et al. An Opioid System in Connective Tissue: A Study oof Achilles Tendon in the Rat. J Histochem Cytochem 2001;49:1387–96.)

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Neuronal Response After Tendon Injury

Having established the normal occurrence of nerve ﬁbers and their expression of different neuropeptides mainly localized in the surrounding structures of the tendon, i.e. the paratenon, this section aims to explore the distribution of nerve ﬁbers and their expression of neuropeptides after injury and during healing of tendinous tissue.

In addition to cytokines and growth factors, neuropeptides in the periphery may participate in tissue repair [12,27,57,61].

Neuropeptides and Repair

Neuropeptides elicit and convey responses to stress and injury [33], responding very speciﬁcally both in the central and peripheral nervous system to peripheral inﬂammation and nerve injury. A number of experimental studies on arthritis have demonstrated a local increase in SP and CGRP one week after induction with adjuvans.

After peripheral nerve lesion, a number of studies show that the expression of different neuropeptides follows a speciﬁc temporal pattern in accordance with the healing phases [36,59]. This would seem to reﬂect a regulatory role of neuropeptides in response to injury, which possibly also could play a role in tendon repair.

New Nerve Fiber Ingrowth After Injury

Both human [24,60,70] and animal studies [10,22,51,58]

indicate specific nerve fiber response to tendon injury, where nerve fiber ingrowth often is associated with increased experience of pain. Our studies of tendon injury specifically addressed the question of nerve regeneration during healing of experimental Achilles tendon rupture. Nerve regeneration at different time points (1–16 weeks) was investigated by immunohistochemistry, including semi-quantification of neuronal markers for new and mature fibers [3].

In the first week after rupture, corresponding to the inflammatory phase, there was increased nerve fiber activity in the surrounding loose connective tissue. From 1 to 6 weeks after rupture (regenerative phase), there was a striking shift in neuronal occurrence from the surrounding loose connective tissue into the rupture site of the tendon proper (Figures 27-4 and 27-5), in normal conditions notably devoid of nerve fibers. The peak expression of new nerve fibers at the rupture site occurred between weeks 2 and 6, while mature nerve fibers were seen somewhat later, between weeks 4 and 6. During weeks 8 to 16 after rupture (remodeling phase), the nerve fibers decreased significantly at the rupture site (Figure 27-4). They successively returned to normal in the paratenon and surrounding loose connective tissue, reflecting the plasticity of the peripheral nervous system.

In summary, tendon injury is characterized by nerve fiber ingrowth at the rupture site, a peak nerve fiber expression during the regenerative phase, followed by nerve fiber withdrawal during the remodeling phase. The ingrowth and later withdrawal of nerve fibers in the tendon proper after rupture point towards a fundamental role of neuronal supply for tendon tissue healing. Pre- sumably, new nerve ingrowth is a prerequisite for delivery of neuronal mediators required for tissue repair.

Temporally Orchestrated Neuropeptide Occurrence During Repair

So far there are very limited data on the peripheral expression of neuropeptides after injury of musculoskeletal tissues. Increased expression of SP and CGRP 2 weeks after injury has been observed in fracture and skin healing [32,37,41]. Grönblad et al. studied SP and CGRP expression 4 and 14 weeks after ligament rupture, and found an increase at week 4 and a decrease at week 14 [26]. Altogether, the temporal expression of neuropeptides indicates their involvement in not only the acute response to injury, but also in tissue regeneration.

Bearing this in mind, we studied the temporal expression of speciﬁc neuropeptides at different time points (1 to 16 weeks) after rupture using the same experimental Achilles tendon rupture mentioned above. The study entailed morphological and semi-quantitative analyses by immunohistochemistry of sensory (SP, CGRP), sensory modulating (GAL), and autonomic (NPY, VIP) neuropeptides [1,6].

0 0,5 1 1,5 2 2,5 3

0 2 4 6 8 10 12 14 16

Week after rupture

Area fraction of PGP (%)

0 0,25 0,5 0,75 1 1,25 1,5

Area fraction of GAP (%)

PGP GAP

Figure 27-4. Area occupied by nerve fibers (%) immunoreactive to GAP (regenerating nerve fibers) and PGP (mature nerve fibers) in relation to total area, in the mid third of the tendon, over 16 weeks post rupture (mean ± s.e.m.). (Reproduced with permission from Ackermann WP, Ahmed M, Kreicbergs A, Early nerve regeneration after Achilles tendon rupture—a pre- requisite for healing? J Orthop Res 2002; 20(4): 849–56.)

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One week post rupture (inflammatory phase), SP and CGRP fibers were predominantly located in blood vessel walls surrounded by inflammatory cells in the loose connective tissue (Figure 27-6A). This complies with a nociceptive and pro-inflammatory role. During week 2 to 6 (regenerative phase), the expression of SP and CGRP peaked (Figure 27-7). Notably, this peak occurred at the muscle

tendon rupture

connective tissue Week 2 – post rupture

A

tendon

rupture B

Figure 27-5. Overview micrographs of longitudinal sections through the Achilles tendon 2 weeks after rupture obtained by putting together computerized images of smaller micrographs.

Incubation with antisera to a nerve growth marker, GAP-43.

Micrographs depict the proximal half of the Achilles tendon at increasing magnification in figures (A–B). Arrows denote varicosities and nerve terminals. The GAP-positive fibers, indicating new nerve fiber ingrowth, are abundant in the healing main body of the tendon. Some GAP-fibers are also located in the loose connective tissue, but most are located as free nerve endings within the healing main body of the tendon. (Repro- duced with permission from Ackermann WP, Ahmed M, Kre- icbergs A, Early nerve regeneration after Achilles tendon rupture—a prerequisite for healing? J Orthop Res 2002; 20(4):

849–56.)

A

B

Figure 27-6. Immunoﬂuorescence micrograph of longitudinal sections through healing Achilles tendon 1- (A) and 2- (B) weeks after rupture after incubation with antisera to CGRP.

Nerve ﬁbers immunoreactive to CGRP are seen as vascular and free nerve endings in the loose connective tissue (A). CGRP- immunoreactivity occurs mainly in the healing tendinous tissue as sprouting free nerve ﬁbers. v = blood vessel; lct = loose connective tissue; t = main body of the tendon; Bar = 50 mm. (Repro- duced from Ackermann et al. Early nerve regeneration after achilles tendon rupture—a prerequisite for healing? J Orthop Res 2002; 20: 849–56, with permission from the Orthopaedic Research Society.)

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rupture site of the tendon proper, seen as free sprouting nerve endings among fibroblasts and newly formed vessels (Figure 27-6B), which may reflect a role for SP/CGRP in cell proliferation. Up to week 4, the occurrence of GAL, NPY and VIP was sparse. Subsequently, the expression of GAL, NPY and VIP increased to reach a peak at the end of the regenerative phase, around week 6, followed by their successive decrease, SP and CGRP included, until the end of the experiment (week 16) (Figure 27-7). The early appearance of SP and CGRP in tissues surrounding the tendon would seem to comply with a nociceptive and pro-inflammatory role, whereas the later occurrence at the rupture site may prove to reflect a role in cell proliferation.The emergence of GAL, VIP and NPY in the early remodelling phase may, in addition to regulation of vasoactivity, represent modulatory effects on SP and CGRP. This modulation may be required to switch from the nociceptive, inflammatory and regenerative processes to the remodeling phase.

Summary

Tendinous tissues are supplied with a complex network of neuronal mediators, which may be involved in the regulation of nociception, vasoactivity, and inﬂammation. In response to injury, the expression of neuropeptides is sig- niﬁcantly altered in a temporally orchestrated manner, suggesting a well synchronized mechanism in nociception and tissue repair.

The relevance of the present ﬁndings pertains to the possibility of intervening in different efferent mecha-

nisms of the peripheral nervous system, which presumably are implicated in a wide variety of functions such as vasoactivity, nociception, inflammatory responses, and regeneration. Specific neuronal intervention, however, presupposes that the innervation of the musculoskeletal system according to specific mediators is clarified.

Tendons are supplied with a complex peptidergic network, which presumably takes part in maintaining tissue homeostasis. Ingrowth of new nerves seems to be a fundamental feature of tissue repair.

Whether neuronal mediators could be utilized in tissue engineering has so far not been explored. As for phar- macotherapy, it may prove that this can be targeted to speciﬁc neuronal mediators to enhance or inhibit their effects in painful conditions of the tendons.

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0 0,5

0,4

0,3

0,2

0,1

0 2 4 6 8 10 12 14 16

Week

Area fraction (%)

GAL SP CGRP

Figure 27-7. Area occupied by nerve ﬁbers (%) immunoreactive to SP, CGRP and GAL in relation to total area, in the mid third of the tendon, over 16 weeks post rupture (mean ± s.e.m.).

(Reproduced from Ackermann et al. Neuronal plasticity in rela- tion to nociception and healing of rat achilles tendon. J Orthop Res 2003; 21, 432–41, with permission from the Orthopaedic Research Society.)

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