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The regulatory mechanisms involved in tendon homeostasis and repair are not fully understood. Accumulating data, however, demonstrate that the nervous system, in addition to afferent (sensory) functions, through efferent pathways plays an active role in regulating pain, inflammation, and tissue repair. In normal-, healing- and tendinopathic tendons three neurosignalling pathways consisting of autonomic, sensory and glutamatergic neuromediators have been established. In healthy tendons, neuromediators are found in the paratenon, whereas the proper tendon is practically devoid of nerves, reflecting that normal tendon homeostasis is regulated by pro- and anti-inflammatory mediators from the tendon surroundings. During tendon repair, however, there is extensive nerve ingrowth into the tendon proper and subsequent time-dependent appearance of sensory, autonomic and glutamatergic mediators, which amplify and fine-tune inflammation and tendon regeneration. In tendinopathy, excessive and protracted sensory and glutamatergic signalling may be involved in inflammatory, painful and hypertrophic tissue reactions. As our understanding of these processes improves, neuronal mediators may prove to be useful in the development of targeted pharmacotherapy and tissue engineering approaches to painful, degenerative and traumatic tendon disorders.
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[Frontiers in Bioscience 19, 1251-1278, June 1, 2014]
1251
Neuronal pathways in tendon healing and tendinopathy - update
Paul W Ackermann1, Sarah L Franklin2, Benjamin J F Dean2, Andrew J Carr2, Paul T Salo3, David A Hart3
1Integrative Orthopaedic Laboratory, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden,
2Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, Institute of
Musculoskeletal Sciences, Nuffield Orthopaedic Centre, Oxford UK, 3McCaig Institute for Bone and Joint Health, Department of
Surgery, University of Calgary, Calgary, Alberta, Canada
TABLE OF CONTENTS
1. Abstract
2. Introduction
3. Central neuronal signalling pathways
3.1. Autonomic regulation
3.1.1. Sympathetic innervation
3.1.2. Parasympathetic innervation
3.2. Sensory regulation
3.2.1. Sensory innervation
3.2.2. Opioid and opioid like signalling
3.3. Glutamatergic regulation
4. Peripheral neuronal signalling pathways
4.1. Autonomic regulation
4.1.1. Sympathetic innervation
4.1.2. Parasympathetic innervation
4.2. Sensory regulation
4.2.1. Sensory innervation
4.2.2. Opioid and opioid like signalling
4.3. Glutamatergic regulation
5. Neuronal response to tendon injury
5.1. Inflammatory phase
5.1.1. Autonomic regulation
5.1.2. Sensory regulation
5.1.3. Glutamatergic regulation
5.2. Proliferative phase
5.2.1. Autonomic regulation
5.2.2. Sensory regulation
5.3. Remodelling phase
5.3.1. Autonomic regulation
5.3.2. Sensory regulation
6. Neuronal contributions to tendinopathy
6.1. Autonomic regulation
6.2.1. Sympathetic innervation
6.2.2. Parasympathetic innervation
6.2. Sensory regulation
6.2.1. Sensory innervation
6.2.2. Opioid and opioid like signalling
6.3. Glutamatergic regulation
7. Molecular responses to neuropeptides and neuronal influences
7.1. Denervation effects on gene expression in healing ligament in vivo
7.1.1. Denervation increases mRNA levels for collagen I and III and TGF- ß1
7.1.2. Denervation increases mRNA levels for MMP-3, MMP-13 and uPA
7.1.3. Denervation increases mRNA levels for TIMP-1, TIMP-3 and TSP-1
7.2. Neuropeptide effects in tissue culture
7.2.1. Neuropeptides downregulate expression of some growth factors
7.2.2. Neuropeptides increase expression of inflammatory mediators
7.2.3. Neuropeptides decrease expression of matrix molecules
8. Healing response to altered neuronal pathways
8.1. Inadequate neuronal signalling
8.2. Stimulated neuronal signalling
9. Perspective
10. Acknowledgement
11. References
Neurosignalling in tendons
1252
1. ABSTRACT
The regulatory mechanisms involved in tendon
homeostasis and repair are not fully understood.
Accumulating data, however, demonstrate that the nervous
system, in addition to afferent (sensory) functions, through
efferent pathways plays an active role in regulating pain,
inflammation, and tissue repair. In normal-, healing- and
tendinopathic tendons three neurosignalling pathways
consisting of autonomic, sensory and glutamatergic
neuromediators have been established. In healthy tendons,
neuromediators are found in the paratenon, whereas the
proper tendon is practically devoid of nerves, reflecting that
normal tendon homeostasis is regulated by pro- and anti-
inflammatory mediators from the tendon surroundings.
During tendon repair, however, there is extensive nerve
ingrowth into the tendon proper and subsequent time-
dependent appearance of sensory, autonomic and
glutamatergic mediators, which amplify and fine-tune
inflammation and tendon regeneration. In tendinopathy,
excessive and protracted sensory and glutamatergic
signalling may be involved in inflammatory, painful and
hypertrophic tissue reactions. As our understanding of these
processes improves, neuronal mediators may prove to be
useful in the development of targeted pharmacotherapy and
tissue engineering approaches to painful, degenerative and
traumatic tendon disorders.
2. INTRODUCTION
Tissue repair after injury is a complex process,
and usually depicted as a series of three to four overlapping
sequences of molecular and cellular events (1, 2). These
events are influenced by the site of the injury, age, sex,
genetics, nutrition, co-morbidities, and a variety of other
factors (1-4). The outcome of this coordinated series of
events we label wound healing is usually tissue repair, not
tissue regeneration, a limitation which can compromise the
functional outcome of the healing process depending on the
site of the injury. For instance, insults to internal organs
such as the heart, lungs, liver, and kidneys can lead to
wound healing-like fibrotic processes that can lead to
deposition of scar-like material with little functional
capability (5). While most soft tissues (e.g. skin, ligaments,
tendons) heal via a repair process, some tissues (such as
bone) can regenerate after a fracture, in part due to the
nature of the bone remodeling process (3, 4).
The goal of most tissue repair in post-natal life is
the restoration of the integrity of the tissue even if it is
compromised. Thus, injury to the skin leads to development
of granulation tissue which remodels over time, but in the
meantime the scar tissue prevents loss of nutrients from the
organism, and limits the potential for infection.
Interestingly, tissue repair following certain types of injury
to specific tissues in utero can lead to outcomes more
resembling regeneration than the fibrotic repair discussed
above. The molecular and cellular basis for these
differences between post-natal healing and early in utero
healing has been the subject of intense investigation (6, 7),
and it would appear that at least some of the distinction is at
the level of differences in the inflammatory response of the
organism in the two environments (6, 8), with the
inflammatory response in utero being somewhat muted and
the involvement of specific cytokines/growth factors
altered compared to the post-natal situation (9, 10).
Whether this early in utero response is overt or is a
consequence of the altered immune status induced by the
mammalian fetal-maternal relationship during pregnancy is
not yet clear. It is clear that later in gestation (e.g.
equivalent to the third trimester of a human pregnancy),
tissue repair following injury becomes more and more
similar to post-natal responses (11).
Thus, a muted inflammatory response appears to
work in favour of better healing in some circumstances.
However, a compromised inflammatory response
associated with diseases such as diabetes (a prevalent co-
morbidity particularly in the older population with type 2
diabetes; discussed in Purkayastha and Cai, 2013 (12);
Hellman et al., 2012 (13); Mirza et al., 2013 (14)), can
exert a negative effect on healing, leading to chronic
wounds and life-threatening complications (15).
Oppositely, excessive or unregulated inflammation may
result in abnormal healing, with fibrosis or disruption of the
integrity of the tissue function (16). Thus, in a subset of
elbow injuries involving the joint capsule (for reasons
currently unknown), an abnormal, and apparently
exuberant inflammation results in compromised function
and joint contractures (17). Finally, “inflammation” can
be induced via endogenous or exogenous mechanisms,
and sometimes can be confused with tissue catabolism
since some of the same mediators may be involved (18).
Therefore, the key elements relate to regulation and the
“Yin and Yang” of balanced responses to yield effective
outcomes without loss of tissue integrity after an injurious
insult (19).
As indicated in the above discussion,
inflammation plays an important role in initiating and
regulating tissue repair following an injury. With the
exogenous inflammatory response into an injury site is an
influx of circulation-based inflammatory cells
(polymorphonuclear cells, monocytes/macrophages, mast
cells) with concomitant expression of a cascade of soluble
inflammatory mediators, cytokines and growth factors (20).
In response to this environment, the local fibroblasts and
other cells in the site together with recruited exogenous
mesenchymal stem cells or progenitor cells start to
proliferate, migrate and differentiate to synthesize a
provisional matrix. This is accompanied by
neovascularization to provide blood vessels to support the
metabolic activity. Subsequently, over time the matrix
remodels, many of the cells in the wound site disappear or
undergo apoptosis, and the neovasculature regresses.
Ultimately this leaves a somewhat modified matrix
compared to the original, and cells populating the matrix
that are different from those in the site originally. In tissues
highly adapted to perform specific mechanical functions,
such as a ligament or tendon, this modified matrix (or scar)
rarely functions as well as the original tissue, a situation
that could lead to impaired motor performance, or joint
dysfunction, including osteoarthritis and/or decreased
survival in an animal in the wild.
Neurosignalling in tendons
1253
Much of what we know about normal tissue
repair/wound healing has come from studies that have
focused on skin wound healing (1, 2, 21, 22), as well as the
healing of other soft tissues such as tendons and ligaments.
The emphasis has been on what many investigators believe
to be the major contributors to outcomes, namely the most
prominent cell types involved in the inflammatory
responses, the quantity and quality of the matrix formed,
and the regulation of the neovascularization events.
Conversely, how these components are altered in
compromised healing/repair due to co-morbidities such as
diabetes, associated with chronic wounds in patients who
are either aged and/or have compromised cardiovascular
systems, or in patients taking certain drugs for other
conditions, has also been the subject of intense
investigation due to the impact of loss of tissue repair
capabilities on survival, quality of life, and cost to the
health care system.
Recently, more attention has focused on a system
that has not normally been the topic of much research
attention as it is not prominent in many of these tissues
(e.g. skin, ligaments and tendons), and therefore the
contribution of neural elements to normal tissue function,
the response to injury in a variety of circumstances, and
compromised tissue repair is only now starting to become
the subject of more investigation. The view of the
peripheral nervous system as a passive “messenger”,
conveying information about tissue injury to higher centres,
has been superseded by the view that neural regulation is
actively involved in repair processes, even if the relative
contribution based on “cell” number in these tissues is
small, since a regulatory system by its nature should likely
be in such a situation so as not to interfere with function.
Normally, connective tissues such as ligaments, tendons,
menisci, and intervertebral discs are considered hyponeural,
and tissues such as articular cartilage are aneural, and do
not heal or repair well. However, while present at low
density, neural elements can contribute to tissue regulation
and response to injuries (both subclinical and overt) via
release of potent neuropeptides that can modify the activity
of normal fibroblast-like cells in such connective tissues
(23, 24), and interact with endogenous inflammatory cells
such as resident mast cells and macrophages to amplify
their influence (25-27) and regulate the functioning of the
microvasculature present in these tissues or occurring in
response to neovascularization stimuli.
An example of such “amplification” of neural
impact on normal tissue homeostasis, as well as during
normal and abnormal healing is what has been termed the
“Neural-Mast Cell-Myofibroblast/Fibroblast Axis” (17,
26). This postulated axis of influence has been partially
characterized during skin wound healing in domestic pigs
(28), flexor tendon healing in a rabbit model (29), in a
rabbit model of joint injury with joint contracture formation
(17) (30), and possibly in human tendinosis tissue (31).
Recent in vivo (32) and in vitro studies have supported the
concept using tendon cells or cells from human joint
contractures (Hildebrand et al., submitted) in fibroblast-
mast cell-neuropeptide collagen gel contraction studies.
Thus, the neural influence could be amplified via such
mechanisms and impact both normal and abnormal healing
if injured tendons and other connective tissues.
While the neural elements in normal and
repairing connective tissues are not present at high density,
due to the complexity of different types of neural elements
and their associated neuropeptides and functions, it is not
yet clear how the specific contributions of unique
components of the neural system are themselves regulated,
and how the integrity of these components regulate
outcomes of the repair process. Therefore, we present the
current state-of-the-art with regard to neural influences on
connective tissue healing, with a focus on tendons.
Obviously, there may be site- or tissue-specific
involvement of the neural system in maintenance and repair
of tendons, but there may also be some generalizations that
will provide clues to how the neural system contributes to
normal function and repair in a tissue-independent manner.
One key point that should be emphasized is that
neural influences are dynamic across the lifespan. It is
known that innervation of some tissues declines with age
(and may be influenced by genetics) (33, 34). It is also
known that sex/gender can influence inflammatory
pathways and neural impact (35). Thus, this dynamic nature
may complicate interpretation of findings, but it also offers
insights into regulatory points that may lead to new
approaches to overcome deficits or excessive involvement
in healing processes.
3. CENTRAL NEURONAL PATHWAYS
There are three major neuronal pathways by
which the central nervous system regulates the
inflammatory healing reflex that may be the most critical
step in the repair process. The autonomic and sensory
neuronal regulatory systems in combination with the
glutamatergic excitatory system modulate inflammatory
and trophic cellular response mechanisms through a
combined release of classic nerve transmitters and so called
neuropeptides (Table 1). The presence of contralateral
changes in animal models of tendinopathy is highly
suggestive of a key role of the central nervous system in
this process (36, 37). Furthermore, recent studies have also
indicated a contralateral effect during calcium deposition
following injury in a murine Achilles tendon injury model
(5). A mis-tuned inflammatory healing reflex by, for
example, inadequate or excessive neuronal signalling can
lead to deficient repair or even chronic tendon disorders
(38, 39). Evidence of central pain sensitization exists in
clinical studies of patients undergoing shoulder
acromioplasty surgery for tendinopathy of the rotator cuff
(40).
3.1. Autonomic regulation
3.1.1. Sympathetic innervation
The sympathetic nervous system regulates
inflammation at local and systemic levels through the
release of norepinephrine (noradrenaline) together with
neuropeptide Y (NPY). Activation of the sympathetic
outflow by flight-or-fight responses or pain can increase
local concentrations of adrenaline and noradrenaline,
Neurosignalling in tendons
1254
Table 1. Neuronal pathways in tendons
Pathway Signalling Mediator Receptor Actions
Noradrenaline alpha-,beta- adrenoceptors
Sympathetic Neuropeptide Y Y1, Y21,Y32 Pro-(anti)-inflammatory
Acetylcholine Nicotinic, muscarinic
Autonomic
Parasympathetic VIP VPAC1-22, PAC12 Anti-inflammatory
Substance P Neurokinin 1
CGRP CRLR, RAMP-1
Neurokinin A Neurokinin 22
Sensory
NKB*, NPK*, NPG* Neurokinin 32
Pro-inflammatory
Enkephalins: LE, ME, MEAP delta-opiod receptor
Dynorphins: DYN B1 kappa-opiod receptor1
Endomorphins1 mu-opiod receptor1
Nociceptin1 N/OFQ receptor2
Opioid like: Galanin Galanin receptor 1-32
Sensory
Opioid
Opioid like: Somatostatin Somatostatin receptor 1-52
Anti-inflammatory
NMDA
mGluR11, mGluR5-7
AMPA2
Excitatory Glutamatergic Glutamate
Kainate2
Cell-proliferative
1Not detected in tendon 2 Not yet assessed in tendon, VIP = vasoactive intestinal polypeptide, VPAC = Vasoactive intestinal
peptide receptor, PAC1 = Pituitary adenylate cyclase-activating polypeptide type I receptor, CGRP = calcitonin gene-related
peptide, CRLR = calcitonin receptor-like receptor,RAMP-1 = receptor activity-modifying protein 1, NKB = neurokinin B, NPK
= neuropeptide K, NPG =neuropeptide-g, LE = leucine enkephalin, ME = Methionine-enkephalin, MEAP = methionine-
enkephalin-arginine-phenylalanine N/OFQ = Nociceptin/orphanin FQ peptide, NMDA = N-methyl-D-aspartate receptor, mGluR
= metabotropic glutamate receptors, AMPA = alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor.
capable of suppressing inflammation (38). Adrenergic and
Y (NPY) receptors on immune cells allow the immune
system to respond to neuromediators released from
sympathetic nerves and thereby establish a neuro-immune
coupling.
3.1.2. Parasympathetic innervation
The parasympathetic nervous system likewise
regulates the inflammatory reflex at local and systemic
levels through the combined release of acetylcholine (Ach)
and the neuropeptide vasoactive intestinal polypeptide
(VIP). Parasympathetic activation of the vagus nerve is
called the ‘cholinergic anti-inflammatory pathway’ because
it leads to mediator release in multiple organs of the body
(38). Ach and VIP then interact respectively with nicotinic
(ACh receptor) and VIP-receptors on tissue macrophages,
which inhibit the release of pro-inflammatory cytokines
(eg. TNF, IL-1, HMGB1).
3.2. Sensory regulation
Interestingly the sensory nervous system seems
to act principally through release of slowly acting
mediators, i.e. neuropeptides. Recent research has
additionally disclosed the classical transmitter glutamate in
sensory nerve fibers demonstrating an interaction between
neuropeptides and classical transmitters in sensory fibers as
well.
3.2.1. Sensory innervation
The paradoxical “efferent” role of afferent
sensory fibers is now widely recognized (41). Sensitization
of the primary afferent nociceptive nerves gives rise to
altered stimulus–response coupling, leading to release of
sensory neuropeptides eliciting the so-called neurogenic
inflammation. The sensory neuropeptides consist of five
tachykinin peptides: substance P (SP), neurokinin A
(NKA), neuropeptide K, neuropeptide-g, and neurokinin B
and calcitonin gene-related peptide (CGRP), which coexists
with and potentiates the effect of SP (42-44). Immune cells
that appear during the different phases of healing express
sensory neuropeptide receptors, allowing them to be
regulated (45). SP and CGRP exert pro-inflammatory
effects such as vasodilation and protein extravasation (46,
47). An interesting example of the “efferent” role of
afferent sensory fiber stimulation is depicted in a unilateral
overuse model for Achilles tendinopathy. An increase in SP
also in the unexercised control legs of an animal model
points towards the involvement of descending neuronal
pathways in tendinopathy development (37).
3.2.2. Opioid and opioid like signalling
The sensory nerve fibers also contain peptides
with anti-inflammatory effects counteracting the effects of
SP and CGRP. Thus, galanin (GAL), somatostatin (SOM)
as well as opioid peptides (enkephalins, dynorphins,
endomorphins), all of them occurring in primary afferents,
inhibit inflammation and nociception (48-51).
3.3. Glutamatergic regulation
Accumulating data support the notion that
modulation of glutamate receptors, ionotropic (NMDA,
AMPA, Kainate) and metabotropic (mGlu), may have
potential for targeted therapy in several persistent pain
conditions, including neuropathic pain resulting from injury
and/or disease of central or peripheral nerves and
inflammatory or joint-related pain (e.g., rheumatoid
arthritis, osteoarthritis, tendinopathy). Persistent pain is
postulated to depend, at least in part, on long-term
increases in synaptic efficacy of glutamatergic signalling in
central nociceptive pathways, often called central
sensitization (52).
AMPA receptors are present in both myelinated
and unmyelinated sensory nerves of rat (GLUA1) and
human skin (GLUA2–3). Increased levels of glutamate and
the AMPA receptor have been shown in experimental
Neurosignalling in tendons
1255
Figure 1. A-C. Immunofluorescence micrographs of
longitudinal sections through the Achilles tendon after
incubation with antisera to Noradrenaline (NA) (A), NPY
(B) and VIP (C). NA-positive fibers are mainly found as
nerve terminals in outer layers of the blood vessel walls.
The NPY-positive fibers are arranged as nerve terminals in the
vessel walls. VIP-positive nerves are arranged as a “fence”,
surrounding the proper tendon, of small varicosities in the
paratenon. t = tendon tissue; Pt = paratenon; Bar = 50 µm.
Reproduced with permission from (58).
arthritis, suggesting that enhanced glutamate release acting
on peripheral AMPA receptors may contribute to the
initiation of nociceptive signalling. Activation of both
peripheral and central NMDA receptors as well has been
implicated in pain processing (53, 54). There also appears
to be an interaction between neuropeptides and glutamate.
Thus, SP has in the central nervous system been
demonstrated to remove a magnesium ion from the NMDA
receptor, which enables glutamate to bind, thereby
initiating nociceptive transmission.
Nociceptive afferents are mostly divided into two
groups: peptidergic (SP-positive) and non-peptidergic,
which typically express ATP-activated P2X3 receptors.
Kainate receptors immunoreactive to GLUK5 mostly display
P2X3- compared to SP-immunoreactivity, suggesting a
greater role for the kainate receptor (GLUK5) in neuropathic
than inflammatory pain conditions (52).
Also, the metabotropic receptors (mGlu1 and
mGlu5), which are known to potentiate the NMDA
receptor excitation, are expressed in dorsal root ganglia cell
bodies and peripheral terminal endings, suggesting that
there are multiple loci for modulation of primary afferent
sensory transmission.
4. PERIPHERAL NEURONAL PATHWAYS
The same pathways with nerve transmitters and
neuropeptides occurring centrally are also found
peripherally (Table 1). The presence of nerve fibers,
including various neuromediators in tendon, has been
demonstrated by combined quantitative and morphological
assessments with an almost identical neuroanatomy of rat
and human tendons (31, 55-57).
4.1. Autonomic pathways
4.1.1. Sympathetic innervation
The occurrence of sympathetic mediators has
been demonstrated in tendons of both animals and humans
(58-61). Noradrenaline (NA) and neuropeptide Y (NPY)
were mostly observed as networks around larger blood
vessels located in the loose connective tissue around the
main body of the tendon (Figure 1A-B). These observations
would reflect that the sympathetic tendon vasoregulation
predominantly occurs in the tendon envelope, i.e. the
adjacent loose connective tissue.
Adrenergic receptors have been identified on
tendon cells, blood vessel walls and on nerve fibers (59, 60,
62). Moreover receptors for NPY, the Y1-receptor has been
identified on tendon cells and blood vessel walls, whereas
the Y2-receptor was not identified in the tendon biopsies
(63). Adrenergic stimulation of tendons may be involved in
cell proliferation of fibroblasts, tenocytes, endothelial cells
and possibly nerve sprouting, which all are characteristics
seen in tendinosis.
Elevated sympathetic activity is also known to
act in an anti-inflammatory fashion by inhibiting
macrophage activation and suppressing synthesis of tumor
necrosis factor (TNF) and other cytokines (38).
Interestingly NPY has been demonstrated as a potent
immune mediator with both pro-inflammatory and anti-
inflammatory actions (64, 65).
Neurosignalling in tendons
1256
4.1.2. Parasympathetic innervation
The parasympathetic mediators acetylcholine and
VIP were both identified in tendon (58, 66, 67). Nerve
fibers displaying immunoreactivity to VIP have been
observed as long thin varicose nerve terminals forming a
“fence” in the paratenon (Figure 1C). The nerve fibers were
evenly distributed between vascular structures and free
nerve endings.
As opposed to sympathetic (NA/NPY) nerve
terminals occurring mostly in larger vessels, VIP has
predominantly been found around smaller vessels (58,
61, 68). This observation may reflect that NA and NPY
(vasoconstrictive) predominantly regulate the main
blood flow to the tendon proper, whereas VIP
(vasodilatory) is responsible for the fine-tuning of blood
flow at a microlevel (58).
The non-vascular distribution of VIP would
seem to comply with an anti-inflammatory effect in the
periphery (Figure 1C) (69-71). The strong anti-
inflammatory role of VIP has been suggested to act
through inhibition of T cell proliferation and migration
(64, 71-73).
Acetylcholine (Ach) receptors have been
identified on tendon cells and blood vessels (66, 67),
while VIP receptors (VPAC1, VPAC2 and PAC1) have
yet to be explored in tendons. Ach receptors located on
tissue macrophages may be involved in the so called
cholinergic anti-inflammatory pathway, inhibiting the
release of pro-inflammatory cytokines (38).
Interestingly, the expression of the nicotinic
receptor-alpha7, which is an essential regulator of
inflammation by inhibiting tumor necrosis factor release
from macrophages, has in tendon been found during
early development and hence could be important during
embryogenesis (74). Also, nicotinic receptors have been
detected at the myotendinous junction and are suggested
to promote repair by regulating cell fusion (75). The
occurrence of nicotinic receptors in tendons and their
function, however, need further exploration.
Overall, mapping of para-/sympathetic
mediators in tendon has opened a link to increased
understanding of autonomic neuro-immune modulation in
tendon homeostasis.
4.2. Sensory pathways
4.2.1. Sensory innervation
The occurrence of sensory neuropeptides SP,
CGRP and NKA in tendons of both animals and humans
has been disclosed (Figure 2A) (Table 1) (61, 76-79).
Sensory nerve fibers have been found in the tendon
envelope, i.e. the paratenon, endotenon and surrounding
loose connective tissue, whereas the tendon proper,
notably, during normal condition is devoid of nerve fibers
(Figure 3) (80). This would reflect that the neuronal
regulation of tendons highly depends on the innervation of
the tendon envelope.
Thus, the abundance of vascular sensory nerve
fibers detected in the surrounding loose connective tissues
may reflect an important role in the regulation of blood
flow to the tendon structures. Both SP and CGRP, in
particular the latter, have been reported to be potent
vasodilators (81). In addition, they have also been
demonstrated to exert pro-inflammatory effects, for
example by enhancing protein extravasation, leukocyte
chemotaxis and cytokine production (46, 47). The
occurrence of sensory free nerve endings unrelated to
vessels predominantly seen in the paratenon (Figure 2A)
suggests nociceptive, trophic and immune regulatory
roles.
4.2.2. Opioid and opioid like signalling
The peripheral sensory nervous system exhibits
an opioid and opioid-like source of anti-inflammatory
and anti-nociceptive neuropeptides to modulate the
sensory system (82-85). However, data on opioids in the
peripheral nervous system and specifically tendons are
quite scarce (Table 1). Notably, the existence of an
opioid system in tendons has been established in the rat
(86). The results clearly demonstrated the occurrence of
opioid peptides (enkephalins and nociceptin) and
opioid-like peptides (GAL, SOM) in the tendon (Figure
2B-C).
Thus, all four enkephalins detected (LE, ME,
MEAP, MEAGL), as well as GAL and SOM,
predominantly occurred in sensory C-fibers localized in
the tendon envelope, i.e. the paratenon, loose connective
tissue, and musculo-tendinous junction, whereas no
opioids were found in the proper tendon tissue (Figure
2C). This difference in anatomical distribution might
suggest that regulation of painful disorders of the tendon
mainly occurs in the surrounding tissues, which, during
normal conditions, also harbour the sensory and
autonomic neuropeptides.
In the loose connective tissue surrounding the
tendon, the opioid and opioid-like neuropeptides appear
as free nerve endings around the walls of both large and
small blood vessels, which may reflect involvement in
both vasodilatory actions (87) and anti-inflammatory
responses (Figure 2D) (88, 89). In the paratenon and the
musculo-tendinous junction, the opioid and opioid-like
peptides mostly occurred in free nerve terminals without
any relationship to blood vessels suggesting a paracrine
or an autocrine function in the regulation of nociception
(Figure 2C) (48, 90, 91). Such a regulation is probably
executed in close interaction with the sensory nervous
system. Thus, the release of the sensory neuropeptide SP
from afferents in the cat knee joint is inhibited by intra-
articular enkephalin-analogue injections (92). Whether
there is an inherent balance between opioid and sensory
neuropeptides under normal conditions is unknown.
The existence of an opioid system in tendons
consisting of neuronal enkephalins was supported by the
identification of opioid receptor analyses based on binding
assays and immunohistochemistry (56). Of the three opioid
Neurosignalling in tendons
1257
Figure 2. A-D. Immunofluorescence micrographs of
longitudinal sections through the Achilles tendon after
double staining (co-localization) with antisera to SP and
CGRP (A), SP and GAL (B) LE and DOR (C) and
incubation with antisera to ME (D). A co-existence of SP
and CGRP is seen in nerve fibers localised in the paratenon
(A), indicating possible pro-inflammatory actions.
Moreover, SP is also co-localised with GAL (B), which
may reflect anti-inflammatory actions. The
immunoreactivity displaying co-existence of LE and DOR
is seen as free nerve endings in the paratenon (C), which
indicates a potential peripheral anti-nociceptive system.
ME immunoreaction is localised in a vessel wall (D). t =
tendon tissue; Pt = paratenon; Bar = 50 µm. Reproduced
with permission from (76, 86).
receptors (DOR, KOR, MOR) studied, however, only DOR
could be detected by immunohistochemistry (Figure 2C).
Double staining disclosed co-existence of each of the
enkephalins with DOR in the nerve fibers, in accordance
with other studies suggesting that enkephalins are the main
ligands for DOR (93).
The presence of peripheral opioid receptors was
corroborated by receptor binding analysis, showing that
tendon tissue could bind the competitive opioid receptor
agonist, naloxone, in a specific and saturable way. DOR
activity has been demonstrated to exert a potent inhibitory
effect on SP-release (92, 94). Treatment with delta opioid
agonists in the periphery elicits both anti-inflammatory and
anti-nociceptive effects in models of inflammation (95).
Thus, peripheral acting opioid agonists may prove effective
in preventing the symtoms associated with tendinopathy
(96).
Depiction of sensory and opioid pathways in the
tendon envelope suggests an intricate homeostatic balance
in nociception, trophic actions and immune regulation
occurring in the tendon surrounding structures.
4.3. Glutamatergic regulation
Glutamate transmission, which occurs in the
central nervous system, has recently also been shown to
take place in the peripheral nervous system through
interaction with its ionotropic (AMPA, NMDA, kainate)
and metabotropic receptors (mGlu) (97).
In tendon, both the ligand glutamate and the
NMDA- as well as several mGlu receptors have been
identified (Table 1). Both glutamate and its receptors have
been identified in nerve fibers, blood vessels and tendon
cells. Likely sources of glutamate production may be nerve
fibers and, in tendinopathy, the tenocytes themselves (98-
100). More information is available in the section on
neuronal responses to tendinopathy. Peripheral
glutamatergic regulation may be involved in the
maintenance of tendon strength, cell proliferation,
differention and in pathological cell transformation (97,
99). Moreover, the peripheral glutamate signalling has been
implicated in tendon tissue repair (101, 102).
Overall, in tendons there seems to exist similar
neuronal pathways consisting of different autonomic,
sensory, opioid and glutamate neuromediators as observed
in other organs of the body. One important conclusion on
tendon neuroanatomy is that the tendon proper during
normal conditions is devoid of nerve fibers, while
innervation is found in the tendon envelope, i.e. the
paratenon, endotenon and surrounding loose connective
tissue (Figure 3A-C).
Another vital feature of the neuronal
pathways which appears is the balance between
different mediators, i.e. pro- and anti-inflammatory
peptides. These observations would suggest that
homeostatic regulation of healthy tendon tissue is
highly dependent on balanced neuro-immune-mediator
modulation occurring in the tendon envelope
Neurosignalling in tendons
1258
Figure 3. A-C. Overview micrographs of longitudinal sections through the Achilles tendon built up 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 localization of NPY is depicted in (B), whereas the free nerve endings are typical localization of
SP (C). The immunoreactivity is seen in the paratenon and surrounding loose connective tissue, whereas the proper tendinous
tissue, notably, is almost devoid of nerve fibers pt = paratenon. Reproduced with permission from (80).
5. NEURONAL RESPONSE TO TENDON INJURY
There is anatomic evidence of dynamic
peripheral neuronal responses to tendon injury in a rat
model of Achilles tendon rupture. Axonal sprouting and
growth and a time dependent expression of neuropeptides
have been found to occur during healing of Achilles
tendon rupture in the rat (Figure 4-6) (80, 103).
Similar reactions can be observed in other tendons
and in human tendon repair as well (29, 102, 104).
The study presented was conducted using
immunohistochemistry including a semi-quantitative
assessment focusing on the rupture site of the proper
tendinous tissue. Neuronal markers for regenerating
and mature fibers, ie. growth associated protein 43
(GAP) and protein gene product 9.5. (PGP),
respectively, were analyzed at different time points (1
to 16 weeks) post-rupture. The temporal expressions
of sensory and autonomic neuromediators were
assessed at the same time points.
5.1. Inflammatory phase
In the first week post rupture, the increased
occurrence of neuronal markers indicates nerve
regeneration both in original nerve fibers of the tendon
envelope, i.e. paratenon and surrounding loose
connective tissue, and, notably, in new nerve fibers in
the proper tendon tissue of the rupture site (Figure 4).
In the proper tendinous tissue, normally devoid of
nerves, there was a clear GAP-immunoreactivity
suggesting new nerve fiber ingrowth, which is consistent
with reports on GAP levels in dorsal root ganglia after
peripheral nerve injury (Figure 5) (105, 106). GAP may be
involved in nerve regeneration by regulating growth cone
motility and axon guidance signals (107). These
observations of early nerve regeneration are in line with
observations on bone, ligament and skin healing indicating
that nerve ingrowth is a fundamental aspect of tissue
healing (22, 108-111).
5.1.1. Autonomic regulation
At 1 week post injury, there was only weak scanty
expression of sympathetic (NPY) and parasympathetic
(VIP) mediators. These findings suggest suppressed anti-
inflammatory effects of the autonomic nervous system
during the inflammatory phase.
5.1.2. Sensory regulation
At 1 week, SP and CGRP nerve fibers were
predominantly located in blood vessel walls surrounded by
inflammatory cells in the loose connective tissue (Figure
8A). The findings comply with the nociceptive role of
sensory neuropeptides, but also with a pro-inflammatory
role. Thus, SP release enhances vasopermeability, probably
to stimulate recruitment of leukocytes and cytokine
production (112-115). The opioid like mediator GAL only
Neurosignalling in tendons
1259
Figure 4. A-B. Overview micrographs of longitudinal sections through the Achilles tendon two weeks post rupture built up 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 abundantly observed in the
healing proper tendon tissue. Reproduced with permission from (80).
Figure 5. Area occupied by nerve fibers (%) immunoreactive to GAP and PGP 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 (80).
showed weak expression, reflecting low anti-inflammatory
actions (Figure 6).
5.1.3. Glutamatergic regulation
During the first week post tendon injury,
microarray- followed by real-time PCR analyses
demonstrated an up-regulation of glutamatergic signalling
molecules involved in activation of the metabotropic
glutamate receptor type 1 and the NMDA receptor (116).
The localisation of the NMDA receptor on tendon cells was
further evidenced by immunohistochemical staining.
It is plausible that the glutamatergic system is
used to coordinate some aspects of tendon healing as has
been shown in development and maintenance of bone tissue
(97). An interesting observation was the fact that the
Neurosignalling in tendons
1260
Figure 6. 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 with permission from (103).
expression of glutamatergic signalling molecules
during tendon repair demonstrated a temporal relationship
to genes involved in embryonic development (116).
5.2. Proliferative phase
From 1 to 6 weeks post rupture, there was a
striking shift in neuronal occurrence from the surrounding
loose connective tissue into the proper tendinous tissue.
This would seem to reflect the transition of a
predominantly inflammatory into a proliferative phase. The
peak expression of GAP-immunoreactivity at the rupture
site occurred between week 2 and 6, while that of PGP
occurred somewhat later, i.e. between weeks 4 and 6
(Figure 5). The extensive ingrowth of new nerve fibers into
the rupture site probably represents a neuronal involvement
in tendon repair. The observed free nerve endings among
fibroblasts in the tendinous tissue may reflect a stimulatory
role in cell proliferation (Figure 4B) (117). The occurrence
of free nerve endings around newly formed blood vessels at
the rupture site suggests a role in vasoregulation, and
possibly in angiogenesis (Figure 7) (118).
5.2.1. Autonomic regulation
During the proliferative phase, the occurrence of
the autonomic mediators NPY and VIP was sparse until
week 4. Subsequently, the expression increased to reach a
peak at the end of the regenerative phase, ie. at about week
6, followed by a successive decrease.
The observations of low sympathetic (NPY)
innervation would reflect that vasoconstriction is
downregulated during tissue repair. The balance between
the vasoconstrictive actions of NPY and the vasodilatory
actions of CGRP is of decisive importance for the supply of
oxygen and nutrients to the healing area. The high ratio of
CGRP to NPY in the proliferative phase of healing
probably represents highly perfused vessels, a necessity for
tissue repair. Similar observations on the ratio CGRP to
NPY have been made in studies on reinnervation of skin
flaps, where CGRP immunoreactivity emerged at 2 weeks
postoperatively and that of NPY 2 weeks later (119, 120).
The low levels of NPY observed may also promote
angiogenesis (121).
The reduced expression of parasympathetic VIP
appears surprising considering the need of vasodilation
during tissue repair. However, the low occurrence of VIP
possibly pertains to its inhibitory action on the pro-
inflammatory effects of SP and CGRP (69). Thus, the
observation suggests less inhibition of SP and CGRP,
which are presumed to be important regulators of early
tissue repair.
5.2.2. Sensory regulation
During weeks 1 to 6, the expression of SP and
CGRP peaked (Figure 6). Notably, this peak occurred at the
rupture site of the proper tendon, while the sensory
neuropeptide expression in the surrounding loose
connective tissue declined. The latter would seem to
comply with decreased pain 3-4 weeks after injury, which
is substantiated by a recent microdialysis study
demonstrating resolving inflammation after two weeks post
tendon rupture (122). The most conspicuous finding,
however, was the occurrence of SP and CGRP in free
sprouting nerve endings among fibroblasts in the healing
tendinous tissue (Figure 8B). The observation might reflect
a stimulatory role of sensory neuropeptides on cell
proliferation, as demonstrated in cultured fibroblasts (117,
123). SP and CGRP are also known to stimulate
proliferation of endothelial cells (124-126). The
Neurosignalling in tendons
1261
Figure 7. A-H. Photomontage of typical high power images of immunohistochemically stained nerve fibers (arrows) at different
time points after medial collateral ligament injury in the rabbit. Scale bar = 20 microns At two weeks CGRP fibers appear very
fine in the early scar. At 6 weeks there is the appearance of CGRP-immunoreactive growth cone like structures (large arrow
head) and sprouting as well as a predominantly perivascular distribution of fibers. Fourteen weeks post-injury there are fewer
CGRP-immunoreactive fibres in the scar with most found again in the epiligament. All SP positive profiles were found to be
perivascular at 2 weeks after injury. Similar to the appearance at 2 weeks, SP positive profiles were only found associated with
proliferating vessels within the scar at later time points. The letter v indicates a vessel lumen. Scale bar at bottom right = 20
microns and applies to the entire montage. Typical image of NPY containing fibres associated with small arterial vessels in a 6
week post-injury scar. The letter v indicates a vessel lumen. Six weeks post-injury, staining for the marker PGP 9.5., which
stains all nerve fibres, revealed a pattern that combined aspects of the other markers, with most fibres in a perivascular location,
and some free in the scar matrix. Reproduced with permission from (78).
observation of free sprouting SP- and CGRP fibers around
newly formed blood vessels in the rupture site would
comply with a role in angiogenesis (Figure 7). Recently, SP
has also been demonstrated to exert an important role in
stem cell mobilization during tissue repair (127).
The reduced occurrence of the opioid-like GAL
presumably pertains to its inhibitory action on the pro-
inflammatory effects of SP and CGRP (128-130).
5.2.3. Glutamatergic regulation
At two weeks post tendon injury, microdialysis
followed by quantification demonstrated a 3-times up-
regulation of glutamate levels in the paratenon of the
healing tendon (102). Of all metabolites assessed during
healing glutamate exhibited the highest elevation (102).
This observation further strengthens the conception that the
glutamatergic system is involved in regulating some
aspects of proliferative tendon healing. Hypothetically,
glutamate signalling molecules may be involved in
regulating cell proliferation and differentiation as has been
shown in other tissues (97).
5.3. Remodelling phase
During weeks 6 to 16 post-rupture the nerve
fibers appeared to regress from the proper tendon tissue
(Figure 5). While GAP-immunoreactivity almost
completely disappeared during this phase, that of PGP
successively returned to normal in the paratenon and
surrounding loose connective tissue. The process appeared
to end simultaneously with the completion of paratenon
repair.
5.3.1. Autonomic regulation
Between week 4 and 6, corresponding to the
transition of the proliferative into the remodeling phase,
there was a dramatic increase in the expression of the
autonomic neuropeptides VIP and NPY. The increase in the
immunoreactivity for VIP and NPY was observed both
around vessels and in free nerve endings in a “border zone”
enveloping the healing tendon.
The upregulation of parasympathetic VIP may be
explained by its inhibitory effect on immune cells
expressing pro-inflammatoy cytokines (131). The
increased occurrence of sympathetic NPY during this
phase, mostly seen around vessels, should probably be
attributed to its vasoconstrictive actions. Notably,
increased vasoconstriction leads to a relative hypoxia,
which enhances the tensile strength of the tendon by
switching production of collagen from type III to type I
(132).
Neurosignalling in tendons
1262
Table 2. Neuronal alterations in tendinopathy
Pathway Signalling Mediator Receptor Actions
Noradrenaline alpha-,beta- adrenoceptors Cell proliferation / differentiation
Sympathetic Neuropeptide Y2 Y1 Cell proliferation / differentiation
Acetylcholine Nicotinic2,muscarinic Cell proliferation / differentiation
Autonomic
Parasympathetic VIP2 VPAC1-22, PAC12 Cell proliferation / differentiation
Substance P Neurokinin 1
CGRP2 CRLR2, RAMP-12
Sensory
Neurokinin A2 Neurokinin 22
Tenocyte / endothelial cell proliferation, MMP-3
Enkephalins2 delta-opiod receptor2
Cannabinoids Cannabinoid receptor 1
Galanin2 Galanin receptor 1-32
Sensory
Opioid
Somatostatin2 Somatostatin receptor 1-52
NMDA
Phosfo-NMDA1
mGluR5
Excitatory Glutamatergic Glutamate
mGluR6-7
Cell proliferation / differentiation
2 Not yet assessed in tendinopathy, VIP = vasoactive intestinal polypeptide, VPAC = Vasoactive intestinal peptide receptor,
PAC1 = Pituitary adenylate cyclase-activating polypeptide type I receptor, CGRP = calcitonin gene-related peptide, CRLR =
calcitonin receptor-like receptor,RAMP-1 = receptor activity-modifying protein 1, NKB = neurokinin B, NPK = neuropeptide K,
NPG =neuropeptide-g, LE = leucine enkephalin, ME = Methionine-enkephalin, MEAP = methionine-enkephalin-arginine-
phenylalanine N/OFQ = Nociceptin/orphanin FQ peptide, NMDA = N-methyl-D-aspartate receptor, mGluR = metabotropic
glutamate receptors, AMPA = alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor.
5.3.2. Sensory regulation
Between week 4 and 6, matching the
upregulation of autonomic neuropeptides, an increased
occurrence of the opiod like GAL was observed both
around vessels and in free nerve endings enveloping the
healing tendon (Figure 6). The emergence of GAL, known
to modulate the effect of sensory neuropeptides, would
seem to comply with inhibition of the early inflammatory
and nociceptive response to injury. Thus, GAL has been
demonstrated to mitigate the proinflammatory and
nociceptive effects of SP (49-51, 128).
Subsequent to the elevated expression of GAL
and the autonomic neuropeptides, a significantly decreased
expression of SP and CGRP followed (Figure 6). Thus, the
early remodelling phase after tendon injury seems to be
characterized by an increased expression of GAL, VIP and
NPY, all of which are known to modulate the effects of SP
and CGRP. It may well be that this modulation is required
to end the nociceptive, inflammatory and reparative
processes, thereby permitting entry to and maintenance of
the remodelling phase.
5.3.3. Glutamatergic regulation
At six weeks post tendon injury, microdialysis
followed by quantification demonstrated resolving
glutamate levels as compared to the proliferative phase in
the paratenon of the healing tendon (Ackermann et al. -
unpublished data). The temporal expression of glutamate
signalling during tendon healing seems to correspond to
that of the sensory mediators. This observation may suggest
that the glutamatergic system could interact with sensory
mediators in regulating cell proliferation and differentiation
during proliferative tendon healing.
On the whole, the observations during tendon healing
clearly demonstrate the capability of the peripheral nervous
system to adapt and respond to an injury. This plasticity is
characterized by nerve fiber ingrowth into the rupture site,
and a peak nerve fiber expression during the proliferative
phase followed by nerve fiber withdrawal. Interestingly,
new nerve ingrowth provides a delivery route for neuronal
mediators that are required for tissue repair. Subsequently,
the temporal expression of the different neuropeptides
studied implies specific actions to regulate the
inflammatory, proliferative and remodelling healing
phases. Thus, the end effect on tissue healing will depend
on the nerve fiber localization, temporal neuropeptide
expression and cellular receptor expression.
6. NEURONAL CONTRIBUTIONS TO
TENDINOPATHY
Interestingly, in tendinopathies with chronic pain
and a failed healing response, altered neural elements have
been noted in a variety of tendons from various locations
(Table 2). However, the cause-and-effect relationship is
currently an area under investigation in most instances for
the nerve endings and neuropeptides in the pathology.
With that limitation, immunohistochemistry
analysis of tissue samples does demonstrate similar patterns
of innervation in tendinopathy tissue as is seen during the
proliferative phase of healing after tendon injury. Thus,
chronic painful tendons exhibit new ingrowth of sensory
nerve fibers (Figure 9) (78, 79, 133), which is also
observed during tissue proliferation in healing tendons (80).
In normal tendon repair, sensory nerve ingrowth is
correlated with increased nociception (103). This
inflammatory phase is followed by peripherally acting
autonomic and opioid-like signalling, coinciding with
decreased nociception (103). Hence, the neuronal
dysregulation in tendinopathy, characterized by aberrant
increase of sensory nerve sprouting and a deficient
autonomic and opioid-like modulation, presumably triggers
pain signalling and possibly also the
hyperproliferative/degenerative changes associated with
tendinopathy (Figure 10). The ongoing morphologic
alterations may reflect protracted or failed healing. An
important point related to this concept is the role of nerves
Neurosignalling in tendons
1263
Figure 8. A-B. Immunofluorescence micrograph of
longitudinal sections through healing Achilles tendon 1-
(A) and 2- (B) weeks post rupture after incubation with
antisera to CGRP. Nerve fibers immunoreactive to CGRP
at week 1 are seen as vascular and free nerve endings in the
loose connective tissue (A). At week 2, CGRP-
immunoreactivity occurs mainly in the healing tendinous
tissue as sprouting free nerve fibers (B). v = blood vessel;
lct = loose connective tissue; t = proper tendon tissue; Bar
= 50 µm. Reproduced with permission from (103).
and neuropeptides during an initial response to an insult
leading to the first symptoms of tendinopathy versus their role
during the chronic phases of the condition, phases that can lead
to overt degeneration, and, in some instances, rupture of the
tendon. Some neuropeptides may serve different functions
during these different “phases”, or different neuropeptides
could be involved. It has been postulated that a
nerve/neuropeptide-mast cell-tenocyte axis may play a role in
tendinopathies and over-use syndromes (25-27), and recently it
has been confirmed that higher mast cell numbers are present
in human patellar tendinopathy tissue samples of patients with
disease of long standing as compared to samples from more
recent onset (134), and there may be a destruction of a fine
equilibrium with the alterations to the neural components.
Thus, some aspects of tendinopathy may be independent of
such an axis, while others may involve this axis to amplify the
impact on the tendon tissue.
6.1. Autonomic regulation
6.1.1. Sympathetic innervation
Chronic painful tendons exhibit a decreased
occurrence of sympathetic nerve fibers, immunopositive to
noradrenaline. Microscopic analysis demonstrated that
sympathetic nerves related to blood vessels were
distinctively decreased in patients with tendinopathy
(Figure 11) (58). Computerized image analysis confirmed a
50% drop in vascular nerve fibers immunoreactive to
noradrenaline in the painful tendons (58). The reduction in
vasoregulatory noradrenalin suggests a reduced blood flow
and a suppressed anti-nociceptive function. A recent study
demonstrated that noradrenaline release leads to secretion
of opioids from leukocytes (90). Similarly, patients with
painful rheumatoid arthritis exhibit a decrease in vascular
innervation expressing noradrenaline (135). To counteract
the decreased sympathetic innervation, immune cells in the
synovia from rheumatoid arthritis patients respond by
upregulating noradrenaline. Likewise, in tendinopathy
patients an upregulated noradrenaline production has been
suggested in morphologically altered tenocytes (60, 136).
Adrenoreceptors for noradrenaline have also been
identified in tendinopathy. Immunoreaction for the alpha1-
adrenoreceptor has been detected in blood vessel walls,
nerve fascicles and tenocytes (60, 136). Adrenegic
activation of alpha1/2-adrenoreceptors has been
demonstrated to stimulate cell proliferation and
differentiation (137, 138). Thus, upregulated noradrenaline
in tendon cells may stimulate the alpha1/2-adrenoreceptors
and contribute to tenocyte excessive cell proliferation and
differentiation. In contrast, decreased vascular innervation
expressing noradrenaline may be compensated by the
observed increased alpha1-adrenoreceptor
immunoreactions in blood vessel walls of tendinopathic
patients (60, 136). More recently, alpha1-adrenoreceptor
expression in tendon cells has been found to be associated
with NPY expression (139), concluding a strong role of the
sympathetic nervous system in peripheral tissues.
6.1.2. Parasympathetic innervation
The cholinergic innervation in tendinopathy
appears to be relatively scarce. Neuronal immunoreaction
to choline acetyltransferase, vesicular acetylcholine
transporter and acetylcholinesterase in tendons is reported
to be limited compared to other tissues investigated.
However, whether cholinergic innervation in tendinopathy
is significantly lower than that of healthy tendons is still
unclear (66, 67, 140).
Immunohistochemical studies have identified
activated markers of cellular acetylcholine production in
human tendinopathy, most prominently seen in
morphologically altered tenocytes (66, 67, 140).
Immunoreaction to choline acetyltransferase and vesicular
acetylcholine transporter has recently been detected in
normal Achilles tenocytes (141). This suggests that resident
tenocytes have the capability to produce acetylcholine,
which is increased during tendinosis. This process implies
that acetylcholine either is involved in regulating the
tenocyte transformation seen in tendinopathy (142), or that
acetylcholine synthesis is initiated in tenocytes in response
to development of tendinopathy.
Parasympathetic muscarinic acetylcholine
receptors (M2) have been identified in human
tendinopathy. Thus, M2-receptors have been identified in
Neurosignalling in tendons
1264
Figure 9. A-B. Immunofluorescence micrographs of
longitudinal sections of healthy Achilles tendon (A) and
tendinosis tissue (B) after immunostaining for SP. Arrows
denote varicosities and nerve terminals. The micrograph
illustrates SP-positive nerve fibres in close vicinity to a
proliferated vessel (B). v = blood vessel. Bar = 50 µm.
Reproduced with permission from (79).
tendon cells, blood vessel walls and nerve fibers (66, 67).
The difference between normal and tendinopathic tendons
consisted in an intense M2-immunostaining in
morphologically altered tenocytes, which was not seen in
normal tenocytes. The observations demonstrate an
endogenous autocrine and/or paracrine acetylcholine and
M2-receptor signalling in transformed tenocytes. M2-
receptors have also been found to contribute to an increase
in cell proliferation and hypercellularity, through an
autocrine loop, which is replicative of the early stages of
tendon healing (141).
6.2. Sensory regulation
6.2.1. Sensory innervation
Immunohistochemical and semi-quantitative
assessments have clearly demonstrated that tendinopathic
tendons exhibit an increased number of sensory SP-positive
nerve fibers (Figure 9) (78, 79, 133). Closer analysis
revealed that the nerve fibers occurred mainly as thin,
varicose, sprouting nerve terminals within the tendon
proper. The observation of increased ingrowth of sensory
nerves into the painful tendon proper, seen as sprouting free
nerve endings, possibly represents nociceptors responding
to mechanical stimuli by initiating pain signalling.
The increase of the sensory neuropeptide SP in
tendinopathy may, in addition to its role in nociception,
reflect pro-inflammatory and trophic actions (143). Thus,
SP has been found to participate in inflammatory actions
such as vasodilation, plasma extravasation, and release of
cytokines. SP has also been reported to stimulate
proliferation of fibroblasts (143) and endothelial cells, as
well as the production of transforming growth factor beta in
fibroblasts. Hence, SP may well contribute to the
morphologic changes observed in early tendinopathic
patients, that is, tenocyte transformation, hypercellularity,
and presumably neovascularization, in conjunction with
mechanical loading (37, 144), and may therefore precede
tendinosis.
The implications of the above mentioned effects
of SP are all plausible with respect to tendon pathology
since its receptor, NK-1, has been detected in tenocytes,
blood vessel walls and in nerve fibers in tendinopathy
(145). SP has further been shown to directly stimulate
nociceptor endings in an autocrine/ paracrine manner.
Similar actions could presumably occur in tendinopathy
since the NK-1 receptor is present.
6.2.2. Opioid and opioid like signalling
To date there are very few publications on opioid
and opioid-like signalling in tendinopathy. In line with a
depressed opioid and opioid-like signalling in
tendinopathy, immunohistochemical analysis detected a
low occurrence of galanin (unpublished data, Ackermann et
al.). However, a recent publication detected increased
cellular expression of cannabinoid receptors in
tendinopathy (146).
6.3. Glutamate pathways
Elevated interstitial levels of glutamate have also
been found in tendinopathy by microdialysis (147).
Furthermore, the specific localization for the increased
glutamate levels has just recently been established in
tendinopathic patients (100, 148). Thus, up-regulated
glutamate occurrence is observed in morphologically
altered tenocytes, in the endothelial and adventitial layers
of blood vessel walls and in nerve fibers. Injection of
glutamate has also been shown to provoke and maintain
local prolonged tendon pain (149), potentially through both
the ionotropic and metabotropic receptors.
One receptor for glutamate, NMDA receptor 1
(NMDAR1) has been identified in tendinopathy. Recently,
both subjective and quantitative assessments demonstrated
a 9-fold increased NMDAR1 occurrence in tendinopathic
patients (98, 99). This finding was corroborated by a study
on rat supraspinatus tendon overuse likewise demonstrating
NMDAR1 upregulation (116).
Maybe the most intriguing finding regarding
glutamatergic signalling is the recent report demonstrating
that elevated glutamate co-existed with its up-regulated
receptor NMDAR1 in nerve fibers, morphologically altered
Neurosignalling in tendons
1265
Figure 10. A-B. Hematoxylin and eosin micrographs of
longitudinal sections through the patellar tendon of healthy
control (A) and painful tendinopathy (B). Arrows denote
tenocytes. The healthy tendon is homogeneous, with
organized parallel collagen structure and thin, elongated
tenocytes (A). The tendinopathy, on the other hand, is
marked by collagen disorganization, increased cell count,
activated tenocytes, and vascular ingrowth in the tendon
proper (B). V = blood vessel. Bar = 50 µm. Reproduced
with permission from (79).
tenocytes and blood vessels (98), which may reflect cell-
hyperexcitation involved in cell
proliferation/differentiation. However, none of the controls
exhibited neuronal co-existence of glutamate and
NMDAR1 in contrast to prominent neuronal occurrence in
all the painful tendons, which strongly suggests a role in
pain regulation (98).
Overall, the neuronal mediator contributions to
tendinopathy seem to involve an intricate regulation of cell
proliferation/survival and differentiation. However, at
present it is unclear to what extent this is regulated by a
cellular origin of neuromediators versus a neuronal origin
of neuromediators. Current and emerging data may prove
that a balance in neuromediator levels is critical for tendon
tissue integrity and that dysregulated neurosignalling
contributes to the morphological features, i.e.
cellproliferation and celltransformation, associated with
tendinopathy.
7. MOLECULAR RESPONSES TO
NEUROPEPTIDES AND NEURONAL INFLUENCES
Denervation impairs the healing of skin, bone,
ligament and tendon in a variety of animal models (150-
155). In the medial collateral ligament, a tissue with many
structural and functional similarities to tendon, denervation
significantly attenuates the response to injury. Scars from
denervated ligaments exhibit lower blood flow, diminished
angiogenesis and decreased mechanical strength when
compared to their normally innervated counterparts
(155).
7.1. Denervation effects on gene expression in healing
ligament in vivo
In rabbits, denervation induced significant
differences in the mRNA levels for many genes of interest
in the injured MCL at two weeks post-injury (156).
7.1.1. Denervation increases mRNA levels for collagen I
and III and TGF- ß1
In the denervated injury group, mRNA levels for the matrix
components collagen I and III were both increased at two-
weeks post-injury, in comparison to the non-denervated
animals (p0.0.1). Significant differences were not detected
at any of the other time points assessed.
Similarly, mRNA levels for the growth factor
TGF-ß were increased at two-weeks post-injury, in
comparison to non-denervated (p0.0.1) (Figure 2). VEGF
and NGF mRNA levels were not significantly altered at
any time points post-injury (p>0.0.1).
7.1.2. Denervation increases mRNA levels for
angiogenesis-associated matrix metalloproteinases
(MMP-3, MMP-13 and uPA)
By two weeks post-injury, the mRNA level for
angiogenesis-associated collagenase MMP-13 was
increased almost 3-fold in the denervated group, in
comparison to the non-denervated group (p0.0.1), and was
20 times the level found in normal ligaments. MMP-3 and
uPA mRNA levels appeared increased in the denervated
injured ligaments at 2 weeks, but these apparent differences
between the experimental groups did not achieve statistical
significance at any time point.
7.1.3. Denervation increases mRNA levels for MMP
inhibitors (TIMP-1 and TIMP-3) and the angiogenesis
inhibitor thrombospondin-1 (TSP-1)
At two weeks post-injury, mRNA levels for the
angiogenic inhibitors TIMP-3 and TSP-1 in the
denervated injury group were significantly increased
compared to those in the non-denervated injury group
(p0.0.1). Notably, mRNA levels for TSP-1 were
significantly elevated in the denervated injured group at
sixteen weeks post-injury (p0.0.1). Levels of mRNA
for TIMP-1 were not significantly different between
innervated and denervated groups at any time points
Neurosignalling in tendons
1266
Figure 11. A-B. Immunofluorescence micrographs of
longitudinal sections through the patellar tendon of healthy
control (A) and painful tendinopathy (B) stained for TH (a
marker for noradrenaline). Arrows denotes nerve fibers. In
the healthy tendon, a strong relation is seen between blood
vessels and TH positive nerves (A). In painful
tendinopathy, a decreased number of TH positive nerves,
which are blood vessel related is seen. V = blood vessel.
Bar = 50 µm. Reproduced with permission from (79).
The study demonstrated that, in a denervated
MCL, mRNA levels for many angiogenic and repair-
associated genes are increased during the early stages of
healing. The majority of the significant differences
between denervated and innervated ligaments were
detected at 2 weeks following injury, where levels of six of
eleven genes tested were significantly altered. Two weeks
post-injury corresponds to the proliferative stage of wound
healing in this ligament injury model. Levels of mRNAs for
repair-associated molecules are at their highest 2-3 weeks
following injury in the rabbit MCL (157).
The denervation-induced increases in mRNA
levels for numerous matrix-molecule and angiogenesis-
associated genes are difficult to reconcile with the previous
work in the same model showing that denervated ligaments
heal poorly. Based on the current findings, several possible
explanations exist. Firstly, a loss of neuropeptide
stimulation may substantially diminish cellular proliferation in
the denervated scar, negating the effect of increased mRNA
levels. Secondly, the increased levels of mRNA for TIMP-1
and TSP-1 in denervated injured ligament could have an
inhibitory affect on cell function and more specifically,
angiogenesis (158). Finally, the timing and regulation of
expression of various interacting genes are likely very
important in the production of an organized, mechanically
adequate scar, and the data suggested that denervation
disrupted the timing and regulation of mRNA changes for
many molecules important in healing.
7.2. Neuropeptide effects in tissue culture
Because of the complexity of the wound healing
environment, investigators have sought to gains insights from
the observation of cells or tissue specimens in culture where
variables inherent to the in vivo environment (blood flow,
mechanical loading, etc.) are controlled or eliminated. Cellular
responses in the form of mRNA or protein production can be
assayed in the presence or absence of specific mediators.
Neuropeptide effects on normal and injured ligament in
culture
Recently it was reported that tissue cultured
specimens of injured ligament respond to the addition of
specific neuropeptides to the culture medium (159). In this
study, mRNA levels for numerous healing-associated
molecules were significantly altered by individual
neuropeptides.
7.2.1. Neuropeptides downregulate expression of some
growth factors
TGF-ß1 mRNA levels were significantly
depressed in 2 week post-injury ligament specimens
cultured with 10-7 M CGRP and 10-7 M NPY compared to
untreated specimens. SP had no detectable effect on TGF-ß
mRNA levels in injured ligament. TGF-ß1 mRNA levels
in normal uninjured ligament and in 3 days post-injury
ligament explants were not significantly affected by any of
the neuropeptides employed.
The mRNA levels for bFGF were also responsive
to neuropeptide exposure, with significant depression
detected at 3 days post-injury with NPY, and at 2 weeks
post-injury with SP and CGRP.
Similarly, the mRNA levels for VEGF were
significantly depressed by CGRP and NPY only at two
weeks post-injury.
7.2.2. Neuropeptides increase expression of
inflammatory mediators
IL-1 mRNA levels were significantly increased
by NPY and SP in the 2 week post-injury specimens.
Similarly, NPY increased the mRNA levels for both TNF-α
and COX-2 in 2 week post-injury specimens.
7.2.3. Neuropeptides decrease expression of matrix
molecules
Type I Collagen mRNA levels were significantly
depressed by SP in normal ligament specimens, by CGRP
Neurosignalling in tendons
1267
at 3 days post-injury, and by CGRP and NPY at two weeks
post-injury.
Type III Collagen mRNA levels were
significantly depressed by all three neuropeptides in the 2
week post-injury specimens.
Biglycan mRNA levels were significantly
lowered by CGRP in both the 3 day and 2 week post-injury
specimens, and by NPY in the 2 week post-injury
specimens. Lumican mRNA levels were significantly
lowered by NPY in uninjured specimens but were not
affected by any neuropeptide in any of the injured
specimens.
Thus, SP and NPY can induce increased mRNA
levels for inflammatory mediators in specimens of injured
ligament placed in culture at 2 weeks after injury. In
contrast, all three neuropeptides tested induced
significantly lower mRNA levels for several molecules
associated with healing in MCL scar, including growth
factors, matrix molecules, excluding lumican, and some
angiogenesis-associated proteins. These data are consistent
with the results of the denervation study, and support the
idea that the effects of denervation are largely the result of
the loss of neuropeptide stimulation. However, the results
of both studies seem at odds with the previous in vivo data,
showing that denervation impaired ligament healing in the
rabbit model (155).
A number of possible explanations could account
for this apparent paradox. The three neuropeptides tested
promote increased blood flow and/or angiogenesis (160-
164). As angiogenesis is widely accepted to be the key
determinant of the outcome of wound healing, neuropeptide
induced increases in blood flow and accelerated
angiogenesis might be more important to the outcome of
healing than the observed changes in mRNA levels for
matrix molecules and growth factors (165-170).
Neuropeptides are also known to increase cellular
proliferation (118, 121, 171, 172). Cells stimulated to
proliferate would potentially downregulate or stop
producing matrix molecules or growth factors until their
proliferative phase was completed. Increased cellularity
would likely subsequently lead to the formation of a larger,
stronger scar in vivo, and could account for the superior
healing of innervated ligaments.
Importantly, not all potentially important
neuropeptides known to be present in articular tissues were
tested. Vasoactive intestinal polypeptide (VIP),
somatostatin (SOM) and met-enkephalin, all present in
articular tissues (76, 86, 173, 174), are also likely important
modulators of cellular metabolism in healing ligament.
Future studies should address the influence of these
mediators on healing and scar cell behavior, as the
neuropeptide milieu in the healing ligament is likely very
complex. Furthermore, most of the significant effects on
mRNA levels were observed at two weeks post-injury.
This corresponds to the late inflammatory and early
proliferative phase of healing in the rabbit MCL. The way
cells in the scar respond to neuropeptides appears to be
time dependent, with different effects seen at different
times after injury (175). Thus, testing specimens retrieved
at longer intervals after ligament injury, with a broader
variety of mediators, might reveal additional differences in
response to neuropeptide stimulation.
Only one concentration (10-7 M) of each
neuropeptide was tested. Physiologic levels of
neuropeptides in healing ligaments or tendons are
unknown. The effects of these agents may be dose-
dependent and different effects might be seen at lower
concentrations.
In vivo, neuropeptides are produced in a
temporally and spatially regulated manner by nerve fibers
in close proximity to existing or newly forming vessels (76,
176). During development, angiogenesis can be highly
regulated by neuronal factors (177). It is not known
whether a similar relationship is found in healing ligament,
tendon, or other wounds. However, a highly localized
down-regulation of matrix production would facilitate
angiogenesis, which depends on MMP-mediated matrix
digestion to produce a channel for proliferating endothelial
cells to migrate into. Since all the neuropeptides tested to
date are associated with increased blood flow and
angiogenesis, the results would indicate that blood vessels
and endothelium are likely the primary targets of these
neuropeptides in early ligament healing (161-164, 170).
7.3. Diversity of fibroblast phenotypes
In recent years a number of investigations have
revealed that fibroblast behavior and activity is strongly
determined by genetic and developmental factors, which
are retained to a considerable extent in the tissue culture
environment. For example, one early study showed that
when grown in three dimensional collagen gels, fibroblasts
derived from cornea, tendon and dermis oriented very
differently after 7 days, with corneal fibroblasts orienting
into orthogonal sheets, tendon fibroblasts forming parallel
bundles, and dermal fibroblasts remaining randomly
oriented (178).
Much of our information about fibroblast
behavior in tissue culture, particularly in the context of
wound healing, comes from studies of dermal fibroblasts.
Even within dermis, fibroblasts in derive from different
embryonic precursors. Evidence continues to accumulate
that there is considerable phenotypic diversity between
fibroblasts found in different locations within a given tissue
or organ (179). Thus neuropeptide effects on fibroblast
behavior likely are specific to the tissue of origin, and this
mandates a cautious approach to the interpretation of
results derived from tissue culture based experiments.
8. HEALING RESPONSE TO ALTERED
NEURONAL PATHWAYS
8.1. Inadequate neuronal signalling
Several investigations have indicated that
denervation impairs the mechanical properties of both
normal and injured ligament. A chemical sympathectomy
Neurosignalling in tendons
1268
with continuous systemic administration of guanethidine
(40 mg/kg/day) leads to degradation of the mechanical
properties of the intact medial collateral ligament (MCL) of
the knee joint in rats after only ten days of treatment (180).
Ligaments from treated animals had a larger cross-sectional
area, a higher wet weight, a decreased modulus of elasticity
and a decreased stress at failure. Some of these structural
changes might be explained by the significantly increased
mRNA levels for the matrix degrading enzymes MMP-13
and cathepsin K, and increased ligament blood flow
induced by chemical sympathectomy.
As noted in the previous section, femoral nerve
transection impairs healing of the medial collateral
ligament in rabbits (155). In that study, blood flow,
angiogenesis and mechanical strength of the ligament scar
were all significantly decreased in denervated limbs
compared to normally innervated limbs, 6 weeks after
injury. Similar results have since been reported in rats,
where surgical sympathectomy or femoral nerve transection
each reduced failure loads of healing MCLs by 50%
compared to normally innervated healing MCLs, at 2
weeks after injury (181).
One study assessed Achilles tendon healing after
performing a specific sensory denervation using Spanish
pepper (capsaicin), which reduced the concentrations of SP
by ~60% (182). The study demonstrated that the residual
SP levels after denervation correlated with the
biomechanical tissue properties, i.e. transverse area,
ultimate tensile strength, and stress at failure (182). Thus,
higher residual SP levels after sensory neuropathy are
associated with improved tensile strength and stress at
failure in the healing of Achilles tendon.
Taken together these studies strongly support the
idea that neuronal derived factors have a powerful
influence on the structure, function and healing capacity of
dense connective tissues such as ligament and tendon.
8.2. Stimulated neuronal signalling
Recent work has explored the possibility that the
exogenous administration of neuropeptides or neurotrophic
factors lead to improved tendon and ligament healing.
Local injections of SP combined with the neutral
endopeptidases thiorphan and captopril were used as a
treatment for Achilles tendon rupture in rats (183, 184).
This resulted in dose dependent increases in fibroblast
number at the injury site between 1 and 6 weeks after
injury. Histological parameters of collagen type III
occurrence and collagen organization were enhanced in the
SP group as compared to the control group (184).
Moreover, collagen fiber orientation and angiogenesis were
also improved by SP treatment (183). A second report,
originating from the same laboratory utilizing the same
model, showed that treatment with SP also increased stress
at maximal load and work to maximal load in healing
Achilles tendon, although stiffness was not improved
compared to controls, and actual failure loads were not
reported (185). An interesting observation from one of
these studies was that the SP treated group exhibited an
accelerated withdrawal of sensory nerve fibers from the
injury site during healing (184). This observation may be
interesting in the context of human tendinopathy with
protracted, pathological sensory nerve fiber ingrowth in the
tendon. It may prove that SP injections could promote
sensory nerve fiber withdrawal.
Another recent study assessed the effect of
exogenous neuropeptide administration on the healing of
denervated ligaments in rats (181). The aim was to restore
or improve the healing responses of the medial collateral
ligament in joints partially denervated by femoral nerve
transection or surgical sympathectomy. In this study SP,
VIP and NPY all improved the healing of denervated
MCLs. In the case of SP and VIP treatment, several
mechanical properties, including failure load and failure
stress were reported to be higher than those of intact,
uninjured MCL. Treatment with SP, VIP and NPY also
significantly improved the histological appearance of
healing MCLs. Treated ligaments showed more organized
scars and the appearance of increased matrix production.
Interestingly, CGRP did not improve any of the measured
histological or mechanical parameters of MCL healing in
this model.
Another approach to the augmentation of
neuronal factors in wound healing has been the use of nerve
growth factor (NGF). NGF was initially described and
characterized as a trophic factor for specific neuronal
populations in the peripheral nervous system (186).
Subsequent studies have shown that NGF displays an
extended spectrum of biological functions (138), and
promotes skin wound healing in both rodents and humans
(187-189). NGF can promote ligament healing in rats
(190). In this study, NGF was continuously administered
to the injury site for 7 days via an implanted mini-osmotic
pump. At 7 days after injury, the fractional area occupied
by blood vessels was increased, indicating that
angiogenesis was promoted by NGF. By 14 days, in
addition to increased vascular density, an increase in nerve
fiber density was noted in the NGF treated specimens,
although no differences in the mechanical properties of the
ligament were detected. By 42 days after injury, nerve fiber
and blood vessel densities were progressively increased,
and scar mechanical properties were also significantly
improved in the NGF group. Thus, early exposure of
injured tissues to exogenous NGF can lead to improved
mechanical outcomes, a critical factor for structurally
important tissues such as tendons and ligaments. Once
optimized, this approach could have broad clinical
implications. In this context it is interesting to notice that
blockage of NGF with humanised monoclonal antibodies,
eg. Tanezumab, in stage 3 clinical trials provides much
more effective pain relief than traditional therapy with
NSAIDs (191). However, balancing the inhibition of NGF-
mediated pain and NGF-mediated regulation of tissue
metabolism and repair will be a turning point whether
NGF-targeted therapies will ever reach the patients in need
(192).
Tendon repair is reported to be promoted also by
physical activity, which recently was shown to be linked to
accelerated neuronal plasticity (193, 194). Morover, it has
been demonstrated that physical activity and training leads
Neurosignalling in tendons
1269
to increased levels of various neuromediators, including SP
and CGRP, which may be involved in regulating the
healing response (193, 195). Interestingly, not only the
neuromediator ligands are influenced by physical activity,
but maybe more importantly, so are the neuromediator
receptors. Thus, mRNA-levels for the SP- and CGRP-
receptors are in mobilized tendons significantly increased
at 17 days post tendon injury compared to immobilized
controls (194). It may prove that enhanced tendon repair
after physical activity is related to an increased peripheral
sensitivity to sensory neuropeptide stimulation, implying an
up-regulation of sensory neuropeptide receptors. Whether
the sensory neuropeptide receptors elicit different responses
or affect different cell types depending on loading
conditions after injury has yet to be investigated.
Other physical means of stimulating tendon repair
include intermittent pneumatic compression, which applied
1 hour daily for two weeks post tendon injury induced a
substantially elevated occurrence of SP by 110% and
CGRP by 47% and simultaneously increased the fibroblast
density by 53% and vessel density by 64% (196).
Moreover, it was recently demonstrated that intermittent
pneumatic compression could promote tendon repair in
immobilized condition by increasing the biomechanical
tissue properties; maximum force increased 65%, energy
increased 168% and tendon length increased 25% (197).
These results demonstrate that the compression treatment
may have a substantial clinical impact, considering that
immobilization is the basic principle in the treatment of
ruptured tendons. In fact, clinical translation of the
preclinical data has demonstrated that adjuvant intermittent
pneumatic compression applied at least 6 hours daily for
two weeks post human Achilles tendon rupture upregulated
the concentration of local essential metabolites in the
paratenon of the healing tendon (102). The metabolite
exhibiting the largest elevation, 1.5.-times, after adjuvant
compression treatment was glutamate (102).
On the whole, our data indicate several interesting
pharmacological and physical strategies of promoting
neuronal pathways that could be further developed and
employed to enhance tendon repair.
9. PERSPECTIVE
From the above discussions, it should be apparent
that the neural contributions to normal tissue function,
repair, and in some instances, pathology, support the
contention that these neural regulatory components play
essential, but as yet incompletely defined roles in
mechanically active tissues such as tendons. While not
present at high density, the neural elements are part of a
highly complex integrative system that likely serves
multiple roles in such tissues, contributing to homeostasis,
proprioception and proper functioning of the various tissues
in a joint to ensure that it works as a smooth machine (e.g.
the joint as a mechanical organ concept). It is also likely
that there are some aspects in common between the repair
functions that such neural elements play in different tissues.
However, considerable more investigation is needed to
provide clarity with regard to the dynamic aspects of their
role(s), and how neural elements are modulated by
genetics, aging, and sex and co-morbidities. Some of this
complexity likely serves fundamental roles in repair and
restoration of function, but it is clear also from the work of
Salo et al (33, 34, 198) that innervation of normal joint
tissues can decline with age in both rats and mice, and thus
elevate risk for degenerative joint disease. Whether similar
changes occur in tendons and ligaments throughout the
body during aging and following events such as
menopause, remains to be delineated. Similarly, how such
changes impact the repair process in tendons and ligaments,
as well as other connective tissues, also remain to be
clarified.
In particular, how impaired neural contributions
resulting from aging, co-morbidities such as diabetes or
genetic variables impact tissue repair will be critical to
elucidate. Conversely, excessive or aberrant neural
involvement in healing could also be detrimental to
functional outcomes. Clearly, the key missing piece to the
puzzle is “what regulates the regulators?” and how does our
understanding of the neural responses interface with the
other elements of the host response to injury. Hopefully a
raised awareness of neural contributions to effective tissue
repair will stimulate increased research in this area.
From the above discussion, nerves certainly play
an essential role in response to injury and healing following
an injury. This conclusion is reinforced from preclinical
animal studies and from patient populations. The
hierarchy of influences on healing is most certainly
complex, with redundant systems operative to ensure a
reasonable outcome even if the system as a whole is
compromised by disease or aging. Interestingly, neural
influences likely rank high in such a hierarchy based on
what is known regarding connective tissue homeostasis,
breakdown and healing in spinal cord injured people or
preclinical models (199). Thus, spinal cord injured
individuals exhibit alterations in these processes below the
level of the injury, but retain regulatory control above the
level of the injury. While likely multifactorial, this is
evidence for the importance of the neural system in
regulation.
While nerves are likely central to regulation, one
new area of research could also provide additional insights
into chronic conditions such as tendinosis or other chronic
abnormal “healing” situations where the responder cells
(e.g. fibroblasts, myofibroblasts, and others) become
altered. Thus, understanding the impact of epigenetics on
responsiveness to regulatory systems such as nerves is
currently emerging. Unlike genetics, which is the genome
everyone inherits, epigenetic processes occur during life
and result in modifications to the genome which in turn
affect regulation of gene expression and function ( ). Thus,
in conditions of chronic stress of various sorts (200),
conditions of chronic exposure to environmental factors
(201-203), and due to chronic stimulation, such as in
rheumatoid pannus (204, 205) as examples, DNA can be
modified by methylation or derivatization affecting
outcomes. Whether such changes can occur with some
regularity in tendinosis, or abnormal healing situations in
Neurosignalling in tendons
1270
tendons, ligaments and other connective tissues, or even be
facilitated by neural influences, remains to be determined.
However, it is likely that such alterations may relate to the
neural regulation of tissue responses, and thus be relevant
to better understanding both normal and “deviant” healing
processes.
In summary, considerable progress has been made
on several of the “fronts” discussed above, to document the
involvement and potential involvement of innervation to
regulate and modulate repair of tendons and related connective
tissues. It is also clear that further progress in this area will
require a multi- or trans-disciplinary approach, bringing
together molecular and cell biologists with neuroscientists and
neurophysiologists, biomedical engineers, and clinician-
scientists to advance the field. The field is certainly poised to
make significant advances, and such advances may well be
translated into new interventions to treat specific patient
populations via the basic information generated, as well as
identify those at risk (via genomic and epigenomic screening
and other approaches) for loss of neural contributions to
maintaining connective tissue function and integrity.
10. ACKNOWLEDGEMENT
Extensive parts of this work were supported by
the regional agreement on medical training and clinical
research (ALF) between Stockholm County Council and
Karolinska Institutet (project nr. SLL20100168), the
Swedish National Centre for Sports Research, a COREF-
Sweden grant (Salo/Bray-Ackermann), and an Alberta
Innovates Health Solutions OA Team Grant (DAH and CH)
and the Swedish Research Council (project nr. 2012-3510).
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Key Words: Neurotransmitters, Neuropeptides, Glutamate,
Opioids, Tendon, Healing, Pain, Tendinopathy, Review
Send correspondence to: Paul Ackermann, Orthopedic
Department, Karolinska University Hospital, 171 76
Stockholm, Sweden, Tel: 468-517-717-34, Fax: 468-517-
761-03, E-mail: paul.ackermann@karolinska.se
... Increase in collagen type III / decrease in type I Biomechanical stability decreases 52,52,72 Fibre arrangement: tendon thickening, disorganization of collagen fibrils Change in biomechanical properties (> stiffness, < elasticity) 70,71,73 Increased angiogenesis, irregular arrangement Impaired biomechanical properties, VEGF↑, MMP-3↑ 69,72 Increased nerve ingrowth Pain 74 Misrouted SC differentiation Ossification, adipogenesis 75,76,77 Transdifferentiation, e.g. myofibroblast transition Contraction, scarring 78,79 AT, Achilles tendon; MMP, matrix metalloproteinase; SC, stem cell; VEGF, vascular endothelial growth factor. ...
... 125 Nerve ingrowth into the tendon fascicles, along with time-dependently emerging sensory, autonomic, and glutamatergic mediators, amplifies and modulates tendon inflammation and healing. 74 Nerve branches follow newly formed blood vessels into the healing area of the tendon, but relocate later during the remodelling and reorganization process to the surrounding AT, particularly the paratenon. 74,125 However, painful degenerated tendons remaining after unsuccessful healing might reflect an uncoupled reinnervation process. ...
... 74 Nerve branches follow newly formed blood vessels into the healing area of the tendon, but relocate later during the remodelling and reorganization process to the surrounding AT, particularly the paratenon. 74,125 However, painful degenerated tendons remaining after unsuccessful healing might reflect an uncoupled reinnervation process. 74 ECM remodelling. ...
Article
Full-text available
Tendon is a bradytrophic and hypovascular tissue, hence, healing remains a major challenge. The molecular key events involved in successful repair have to be unravelled to develop novel strategies that reduce the risk of unfavourable outcomes such as non-healing, adhesion formation, and scarring. This review will consider the diverse pathophysiological features of tendon-derived cells that lead to failed healing, including misrouted differentiation (e.g. de- or transdifferentiation) and premature cell senescence, as well as the loss of functional progenitors. Many of these features can be attributed to disturbed cell-extracellular matrix (ECM) or unbalanced soluble mediators involving not only resident tendon cells, but also the cross-talk with immigrating immune cell populations. Unrestrained post-traumatic inflammation could hinder successful healing. Pro-angiogenic mediators trigger hypervascularization and lead to persistence of an immature repair tissue, which does not provide sufficient mechano-competence. Tendon repair tissue needs to achieve an ECM composition, structure, strength, and stiffness that resembles the undamaged highly hierarchically ordered tendon ECM. Adequate mechano-sensation and -transduction by tendon cells orchestrate ECM synthesis, stabilization by cross-linking, and remodelling as a prerequisite for the adaptation to the increased mechanical challenges during healing. Lastly, this review will discuss, from the cell biological point of view, possible optimization strategies for augmenting Achilles tendon (AT) healing outcomes, including adapted mechanostimulation and novel approaches by restraining neoangiogenesis, modifying stem cell niche parameters, tissue engineering, the modulation of the inflammatory cells, and the application of stimulatory factors. Cite this article: Bone Joint Res 2022;11(8):561–574.
... 2 Peripheral nerve endings at the site of injury can release potent neuropeptides (e.g., Substance P) with the ability to modify the function of fibroblast-like cells in injured tendon tissue. [3][4][5] The released neuropeptides may affect the function of resident mast cells (MCs) and macrophages. 2,6 Conversely, MCs contain substances that, when released from the granules, may alter the function of both the peripheral nervous system and tissue cells. ...
... 12 However, the composition of these subunits can vary depending on different cell types and affects their functional properties. 3 In patients with tendon pain, a 10-fold upregulation of NMDAR1 expression has been observed in transformed tenocytes, in the endothelial and adventitial layers of neovessel walls and in nerve fibers presumed to be sprouting. 13 Nerve ingrowth, in combination with the expression of different pain signaling molecules, may be important for pain regulation in tendinopathy, tendon healing, and inflammation. ...
... All 3D images were captured with z-stacks and adjusted with IMARIS software (Bitplane, Zürich, Switzerland) for making 3D movies. 3 H-glutamate binding assay Assays for the binding of 3 H-labeled glutamate (PerkinElmer, Stockholm, Sweden) to the MCs (1 × 10 6 cells) were performed in 1.5-ml microcentrifuge tubes using 100 nM 3 H-labeled glutamate (specific activity = 48.6 Ci/mmol) for different incubation periods (5,20,40,60, and 180 min) at 4°C. ...
... However, emerging evidence suggests that alarmins released from necrotic cells can constitute important triggers for the ensuing inflammatory response [25]. As part of ongoing inflammation and delayed healing, the histologic appearance will contain irregularities from healthy tendon tissue such as calcifications and sprouting of nerves and vessels along with the upregulation of signaling peptides and receptors such as substance P, calcitonin gene-related peptide (CGRP), and N-methyl-Daspartate (NMDA) receptors [5,10,[26][27][28][29][30][31][32]. ...
... In peripheral tissues, the peripheral nerve system has a key role in regulating inflammation and pain signaling of the damaged tissue via afferent to efferent pathways [42]. In the healthy tendon, nerve fibers are localized in the tendon sheath, so called interfibrillar matrix, whereas the tendon proper, also denoted the intrafibrillar matrix, is devoid of nerves [26]. During the early healing phase, extensive ingrowth of nerve fibers has been demonstrated into the tendon proper [43]. ...
... The close location of mast cells and peripheral nerve endings raises the possibility that mast cells can be activated by different neurotransmitters that may be released from peripheral nerves in response to tendon injury. Such neurotransmitters include substance P, glutamate, CGRP, and neurokinin A (NKA) [26,87,88]. In line with a potential role for neurological mechanisms in mast cell activation, mast cells are known to express several receptors for neurotransmitters, e.g., neurokinin 1 receptor (NK1; receptor for substance P) and calcitonin receptor-like receptor (receptor for CGRP) [89][90][91]. ...
Article
Full-text available
Understanding the links between the tendon healing process, inflammatory mechanisms, and tendon homeostasis/pain after tissue damage is crucial in developing novel therapeutics for human tendon disorders. The inflammatory mechanisms that are operative in response to tendon injury are not fully understood, but it has been suggested that inflammation occurring in response to nerve signaling, i.e., neurogenic inflammation, has a pathogenic role. The mechanisms driving such neurogenic inflammation are presently not clear. However, it has recently been demonstrated that mast cells present within the injured tendon can express glutamate receptors, raising the possibility that mast cells may be sensitive to glutamate signaling and thereby modulate neurogenic inflammation following tissue injury. In this review, we discuss the role of mast cells in the communication with peripheral nerves, and their emerging role in tendon healing and inflammation after injury.
... 2 Peripheral nerve endings at the site of injury can release potent neuropeptides (e.g., Substance P) with the ability to modify the function of fibroblast-like cells in injured tendon tissue. [3][4][5] The released neuropeptides may affect the function of resident mast cells (MCs) and macrophages. 2,6 Conversely, MCs contain substances that, when released from the granules, may alter the function of both the peripheral nervous system and tissue cells. ...
... 12 However, the composition of these subunits can vary depending on different cell types and affects their functional properties. 3 In patients with tendon pain, a 10-fold upregulation of NMDAR1 expression has been observed in transformed tenocytes, in the endothelial and adventitial layers of neovessel walls and in nerve fibers presumed to be sprouting. 13 Nerve ingrowth, in combination with the expression of different pain signaling molecules, may be important for pain regulation in tendinopathy, tendon healing, and inflammation. ...
... All 3D images were captured with z-stacks and adjusted with IMARIS software (Bitplane, Zürich, Switzerland) for making 3D movies. 3 H-glutamate binding assay Assays for the binding of 3 H-labeled glutamate (PerkinElmer, Stockholm, Sweden) to the MCs (1 × 10 6 cells) were performed in 1.5-ml microcentrifuge tubes using 100 nM 3 H-labeled glutamate (specific activity = 48.6 Ci/mmol) for different incubation periods (5,20,40,60, and 180 min) at 4°C. ...
Article
Full-text available
Mast cells are emerging as players in the communication between peripheral nerve endings and cells of the immune system. However, it is not clear the mechanism by which mast cells communicate with peripheral nerves. We previously found that mast cells located within healing tendons can express glutamate receptors, raising the possibility that mast cells may be sensitive to glutamate signaling. To evaluate this hypothesis, we stimulated primary mast cells with glutamate and showed that glutamate induced the profound upregulation of a panel of glutamate receptors of both the ionotropic type (NMDAR1, NMDAR2A, and NMDAR2B) and the metabotropic type (mGluR2 and mGluR7) at both the mRNA and protein levels. The binding of glutamate to glutamate receptors on the mast cell surface was confirmed. Further, glutamate had extensive effects on gene expression in the mast cells, including the upregulation of pro-inflammatory components such as IL-6 and CCL2. Glutamate also induced the upregulation of transcription factors, including Egr2, Egr3 and, in particular, FosB. The extensive induction of FosB was confirmed by immunofluorescence assessment. Glutamate receptor antagonists abrogated the responses of the mast cells to glutamate, supporting the supposition of a functional glutamate–glutamate receptor axis in mast cells. Finally, we provide in vivo evidence supporting a functional glutamate–glutamate receptor axis in the mast cells of injured tendons. Together, these findings establish glutamate as an effector of mast cell function, thereby introducing a novel principle for how cells in the immune system can communicate with nerve cells.
... play an important role in the repair of bone and tendon injuries. 1,23 The sensory nerves are distributed widely in different bone and tendon tissues, including the rotator cuff 34 and patellar tendon, 5 and seem to act principally in the healing process via the release of neuropeptides. 7 Calcitonin gene-related peptide (CGRP) is considered one of the most important osteoanabolic neuropeptides 32,36 and is produced by both central and peripheral neurons. ...
Article
Full-text available
Background Calcitonin gene-related peptide (CGRP), which has been shown to play an important role in osteogenesis during fracture repair, is also widely distributed throughout the tendon and ligament. Few studies have focused on the role of CGRP in repair of the bone-tendon interface (BTI). Purpose To explore the effect of CGRP expression on BTI healing in a rabbit partial patellectomy model. Study Design Controlled laboratory study. Methods A total of 60 mature rabbits were subjected to a partial patellectomy and then randomly assigned to CGRP, CGRP-antagonist, and control groups. In the CGRP-antagonist group, the CGRP receptor antagonist BIBN4096BS was administered to block CGRP receptors. The patella–patellar tendon complex was harvested at 8 and 16 weeks postoperatively and subjected to radiographic, microlaser Raman spectroscopy, histologic, and biomechanical evaluation. Results Radiographic data showed that local CGRP expression improved the growth parameters of newly formed bone, including area and volumetric bone mineral density ( P < .05 for both). Raman spectroscopy revealed that the relative bone mineral composition increased in the CGRP group compared with in the control group and the CGRP-antagonist group ( P < .05 for both). Histologic testing revealed that the CGRP group demonstrated better integration, characterized by well-developed trabecular bone expansion from the residual patella and marrow cavity formation, at the 8- and 16-week time points. Mechanical testing demonstrated that the failure load, ultimate strength, and stiffness in the CGRP group were significantly higher than those in the control group ( P < .05 for all), whereas these parameters in the CGRP-antagonist group were significantly lower compared with those in the control group at 16 weeks after surgery ( P < .05 for all). Conclusion Increasing the local concentration of CGRP in the early stages of BTI healing enhanced osteogenesis in a rabbit partial patellectomy model and promoted healing of the BTI injury, whereas treatment using a CGRP antagonist had the opposite effect. However, exogenous CGRP expression did not induce novel bone remolding. Clinical Relevance CGRP may have potential as a new therapy for BTI injuries or may be added to postoperative regimens to facilitate healing.
... Neuronal cells which process information from the Achilles tendon are localized in the lumbar intumescence at the L5 segment 18,19 . Few studies have evaluated how the spinal cord responds to injury in the Achilles tendon 20,21 . Furthermore, it remains unclear if Achilles tendon injuries are able to trigger in ammatory response on the spinal cord of injured patients. ...
Preprint
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Rupture of Achilles tendon is a common accident affecting professional and recreational athletes. Acute and chronic pains are symptoms commonly observed in ruptured patients. Despite that, no studies have described whether Achilles tendon rupture is able to promote disorders in CNS. Based in these finds, the current study aimed to evaluate nociceptive alterations and inflammatory response in L5 lumbar segment of Balb/c mice spinal cord after Achilles tendon rupture. We demonstrated increased algesic response in the paw of ruptured group on the 7th and 14th days post tenotomy when compared with control group. This phenomenon was accompanied by over expression of COX-2 and NOS-2 as well as hyperactivation of astrocytes and microglia in nociceptive areas of L5 spinal cord as evidenced by intense GFAP and IBA-1 immunostaining, respectively. Biochemical studies also demonstrated increased levels of nitrite in the L5 spinal cord of tenotomized animals when compared with control group. Thus, we have demonstrate for the first time that total rupture of the Achilles tendon induces inflammatory response, nitrergic and glial activation in the CNS at L5 spinal cord region.
... Furthermore, using ibuprofen, a first-line and commonly used NSAID for the treatment of inflammatory TMD [24][25][26][27], we validate the MMTL-produced myogenic TMD model. Given that the surrounding structures, such as paratenon and endotenon, are innervated by nerve fibers and the innervation could be damaged by tendon ligation and that there is potential nerve ingrowth into the tendon during tendon repair [42], neuropathic factors may be also involved in the MMTL-produced myogenic TMD. ...
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