Orthopedic Laboratory, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
paul.ackermann@ki.se
Dr. P.W. Ackermann is the Head of the Integrative Orthopedic Research Group, focusing his research on Neuronal Regulation of Pain and Musculo-skeletal Tissue Repair in collaboration with a global network of researchers. His mission in research is to understand and exploit neuronal pathways in the musculo-skeletal system for restoring joint function and alleviating pain.
Dr. P.W. Ackermann is a specialist in orthopedic surgery with focus on trauma. His mission is to develop and translate basic research findings into clinical practice. Dr. Ackermann has developed adjuvant treatment protocols with intermittent pneumatic compression to optimize the blood supply in patients at risk. By this means Dr. Ackermann is combining experience from basic and clinical research into new improved treatment protocols for patients.
Abstract: The aim of the study was to assess healing after capsaicin-induced substance P (SP) depletion during rat Achilles tendon repair by biomechanical testing. Capsaicin treatment reduced the concentrations of SP by ∼60% and calcitonin gene-related peptide by ∼40% as compared with the control group, as assessed by radioimmunoassay in the dorsal root ganglia, at 1 and 4 weeks post-tendon rupture. Also, the peripheral neuronal presence of SP and calcitonin gene-related peptide, as assessed by immunohistochemistry, was lower at both weeks 1 and 4. The decreased peripheral neuronal presence of SP at week 1 correlated with the corresponding levels in the dorsal root ganglia (r = 0.54, p = 0.018). The reduced presence of SP/calcitonin gene-related peptide after capsaicin treatment was verified by a decreased sensitivity to painful mechanical and thermal stimuli (p < 0.05). Correlation analyses between individual residual SP levels and biomechanical tissue properties were performed because of differences in failure mode between the groups and high individual variations in the SP levels after capsaicin treatment. Thus, the residual SP levels in the dorsal root ganglia correlated with transverse area, ultimate tensile strength, and stress at failure (r = 0.39, p = 0.036; r = 0.53, p = 0.005; and r = 0.43, p = 0.023, respectively). Furthermore, individual pain sensitivity at week 2 correlated with peripheral occurrence of SP and was correlated with tensile strength and stress at failure (r = 0.89, p = 0.006 and r = 0.78, p = 0.015) at week 4. In conclusion, rats with higher residual SP levels after capsaicin-induced neuropathy develop improved tensile strength and stress at failure in the healing of Achilles tendon.
Abstract: Tendon healing is characterized mostly by slow rehabilitation and, as in tendinopathy, aberrant, protracted sensory nerve ingrowth. This study investigated whether administration of the sensory neuropeptide substance P (SP) could enhance healing and modulate sensory nerve plasticity after Achilles tendon rupture. Fifty-four male Sprague-Dawley rats were allocated to three groups, all receiving six daily injections post-rupture of; (1) SP (10(-6) mol/kg body weight)+endopeptidase inhibitors captopril and thiorphan, (2) captopril/thiorphan only and (3) saline control. At 1, 3 and 6 weeks post-rupture tendon healing was evaluated by assessments of fibroblast proliferation, collagen III-LI (like) occurrence, diameter of newly organized collagen and sensory nerve fiber ingrowth. At 1 week, the SP-treated group exhibited increased occurrence of collagen III-LI (P=0.03) and of organized collagen (P=0.04) compared with control. At 3 weeks, the SP group notably displayed reduced SP-nerve fiber ingrowth (P=0.02), and higher fibroblast density (P=0.004). Both the SP and captopril/thiorphan groups demonstrated increase in collagen fiber organization compared with control (P=0.02 and 0.004, respectively). At 6 weeks, no significant differences were observed between the groups. SP supply in tendon repair promotes early tissue proliferation and regulation of endogenous sensory nerve ingrowth, suggesting implications for novel treatment in tendinopathy.
Abstract: Achilles tendon ruptures are treated with an initial period of immobilization, which obstructs the healing process partly by a reduction of blood circulation. Intermittent pneumatic compression (IPC) has been proposed to enhance tendon repair by stimulation of blood flow. We hypothesized that daily IPC treatment can counteract the deficits caused by 2 weeks of immobilization post tendon rupture. Forty-eight Sprague-Dawley SD) rats, all subjected to blunt Achilles tendon transection, were divided in three equal groups. Group A was allowed free cage activity, whereas groups B-C were immobilized at the operated hindleg. Group C received daily IPC treatment. Two weeks postrupture the rats were euthanatized and the tendons analyzed with tensile testing and histological assessments of collagen organization and collagen III-LI occurrence. Immobilization significantly reduced maximum force, energy uptake, stiffness, tendon length, transverse area, stress, organized collagen diameter and collagen III-LI occurrence by respectively 80, 75, 77, 22, 47, 65, 49, and 83% compared to free mobilization. IPC treatment improved maximum force 65%, energy 168%, organized collagen diameter 50%, tendon length 25%, and collagen III-LI occurrence 150% compared to immobilization only. The results confirm that immobilization impairs healing after tendon rupture and furthermore demonstrate that IPC-treatment can enhance proliferative tendon repair by counteracting biomechanical and morphological deficits caused by immobilization.
Abstract: It was hypothesized that mobilization vs immobilization after injury would promote tissue healing by regulating gene expression for molecules associated with repair. Cast immobilization vs free mobilization was studied after rat Achilles tendon rupture. Reverse transcriptase-polymerase chain reaction was performed at 8 and 17 days post-rupture to assess different growth factors [brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1)] and inflammatory mediators [cyclooxygenase 1 and 2 (COX 1 and COX 2), inducible nitric oxide synthase and hypoxia-inducible factor-1alpha (HIF-1alpha)] in the healing region. At 8 days post-injury, tendon mRNA levels were comparable in both groups. However, by day 17, the mRNA levels for BDNF, bFGF, COX 1 and HIF-1alpha in the mobilized group had increased significantly. Corresponding mRNA levels in the immobilized group decreased during the same period. There were no significant differences in the expression of NGF, IGF-1 or COX 2 between the different groups, indicating that injury-associated expression of these molecules is not overtly influenced by loading. This study supports the notion that prolonged immobilization post-rupture hampers the healing process by compromising the up-regulation of repair gene expression in the healing tendon. It might be speculated that a shorter period of immobilization, i.e. 1 week, would not impair the healing process significantly. The findings support the current development of earlier and more active rehabilitation programs after tendon injuries.
Abstract: Elevated levels of the neurotransmitter glutamate and the presence of its receptor, N-methyl-d-aspartate receptor type 1 (NMDAR1), have been established in patients with tendinopathy, i.e. chronic tendon pain and degeneration. However, whether NMDAR1 is up- or down-regulated in tendinopathy and co-localized with glutamate is still unexplored. We hypothesize that an alteration in tissue expression and in the coexistence of NMDAR1 and glutamate occurs in tendinopathy and might play a role in nociception and possibly also progression of tendon degeneration (tendinosis). We therefore examined the tissue distribution and levels of NMDAR1 and glutamate in biopsies from patients with patellar tendinopathy (n=10) and from controls (n=8). The biopsies were single- and double-stained immunohistochemically for glutamate and NMDAR1 and assessed subjectively and semi-quantitatively. The chronic painful tendons exhibited a significant elevation of NMDAR1 (ninefold), which was independent of the observed increase in glutamate (10-fold). This up-regulation of NMDAR1 and glutamate was found to be co-localized on nerve fibers as well as on morphologically altered tenocytes and blood vessels. None of the controls exhibited neuronal coexistence of glutamate and NMDAR1. The neuronal coexistence of glutamate and NMDAR1, observed in painful tendinosis but not in controls, suggests a regulatory role in intensified pain signalling.
Abstract: Substance P (SP) has been shown in vitro to stimulate both formation and resorption of bone. This seemingly contradictory observation could be explained by in vivo variations in skeletal loading and rate of bone turnover, features which may be explored during different phases of fracture healing. In 50 SD rats, the right tibia was fractured and fixed with an intramedullary pin in straight alignment and in anterior angulation resulting in a convex and concave side under different load. Fracture repair was assessed by radiography, histology, and semi-quantitative immunohistochemistry of SP nerve fiber occurrence at days 7, 21, 35, 56, and 84 post-fracture. During regeneration, days 7-35, abundant SP-nerve ingrowth was observed in the fracture callus reaching a side-symmetrical peak at day 21 in straight fractures. In angulated fractures, the SP peak was also observed at day 21 on the concave loaded side, but not until day 35 on the convex unloaded side. Each SP-peak coincided with cortical bridging. During remodeling, days 35-84, a side-symmetrical disappearance of SP-positive fibers was seen in straight fractures. The same pattern was seen on the concave loaded side of angulated fractures. However, on the convex unloaded side, where resorption now took place, SP-fibers remained until the end of the experiment. Our study suggests that neuronal SP during bone regeneration has a stimulatory role on bone formation, while during remodeling increased SP fiber density in unloaded areas may be related to bone resorption.
Abstract: Autonomic neuropeptide Y (NPY) is involved in local bone remodeling via the central nervous system. However, the role of peripheral neuronal NPY in fracture healing is not known. We investigated the relationship between bone healing and side-specific occurrence of NPY in angular and straight fractures.
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 neuronal pathways plays an active role in regulating pain, inflammation, and tissue repair processes. Thus, in normal-, healing- and tendinopathic tendons three major neuronal signalling pathways consisting of autonomic, sensory and glutamatergic neuromediators have been established. In healthy tendons, these neural elements are found in the paratenon, whereas the proper tendon is practically devoid of nerves, reflecting that normally 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. In a future perspective, neuronal mediators may prove to be useful in targeted pharmacotherapy and tissue engineering in painful, degenerative and traumatic tendon disorders.
Abstract: A few studies have shown that eccentric exercise is effective for prevention and treatment of muscle injuries. Most earlier studies on eccentric exercises have used training with advanced equipment. Forward lunges are considered eccentric exercises, and they may be performed without any equipment. These exercises are commonly used by sprint runners. We performed a prospective, randomized, 6-week training study comparing the effects of walking or jumping forward lunges on hamstring and quadriceps strength and function. Thirty-two soccer players were included in the study. The forward lunge training was done as an addition to ordinary soccer training twice a week for 6 weeks. The outcome was measured by the maximal hamstring and quadriceps strength tests and by functional tests with 1-leg hop tests and 30-m sprint runs. Overall muscle pain was evaluated using a visual analogue scale score, and local pain was estimated with an algometer. Whereas the walking lunge improved hamstring strength, the jumping lunge resulted in sprint running improvements. Algometer testing showed a general increase in the pain detection thresholds of all subjects, including the controls. Thus, precautions should be taken when algometers are used for temporal studies of pain. Walking and jumping forward lunges can be used for improving hamstring strength and running speed in young soccer player. The findings may have relevance when designing protocols for prevention and rehabilitation of muscle injuries.
Abstract: Healing after mobilization versus immobilization was assessed in a model of rat Achilles tendon rupture, by RT-PCR at 8 and 17 days and by histological analyses at 14 and 28 days postrupture. The expression of mRNA for extracellular matrix (ECM) molecules (collagen type I and type III, versican, decorin, and biglycan), and the subjective histological maturation of the healing area were analyzed. Effects of immobilization on healing were related to changes in the peripheral expression of substance P (NK(1))- and calcitonin gene-related peptide (CRLR and RAMP-1)- receptors. At 8 days postinjury, mRNA levels for ECM molecules were equal in both groups. However, by day 17, the ECM mRNA expression in the mobilized group had increased up to approximately 14x that of the immobilized group, which were comparable to intact tendon values. Histological analysis confirmed a higher regenerating activity in the mobilized group, with an increased amount of blood vessels, fibroblasts, and new collagen. The expression of sensory neuropeptide receptors in the mobilized group exhibited a significant increase from 8 to 17 days postinjury similar to the increased ECM mRNA expression, whereas the immobilized group at 17 days exhibited levels comparable to the intact tendon values. Therefore, immobilization postrupture appears to hamper tendon healing, a process which may prove to be directly linked to a downregulated peripheral sensitivity to sensory neuropeptide stimulation.
Abstract: Intermittent pneumatic compression (IPC) is a treatment method to decrease venous stasis and stimulate blood flow. Recently, it was hypothesized that IPC may exert positive effects on tissue healing, a process highly dependent upon adequate circulation. In this study, we investigated the effects of daily 1-h IPC treatment during 2 and 4 weeks post-rat Achilles tendon rupture. The tendons were subjectively and semiquantitatively analyzed for collagen organization, fibroblast density, angiogenesis, and the occurrence of sensory neuropeptides, substance P (SP) and calcitonine gene related peptide (CGRP), as well as for a nerve regeneration marker, growth associated protein 43 (GAP-43). After 2 weeks of treatment, fibroblast density increased by 53% (p = 0.0004), vessel density by 64% (p = 0.022), and the occurrence of SP by 110% (p = 0.047) and CGRP by 47% (p = 0.0163) compared to untreated controls. Following 4 weeks of treatment, both the occurrence of sensory neuropeptides and the vessel density remained significantly higher (p < 0.05), whereas fibroblast density returned to normal. However, at 4 weeks the treated tendons displayed a higher degree of organized parallel collagen fibers, a sign of increased maturation. Daily IPC treatment improves neurovascular ingrowth and fibroblast proliferation in the healing tendon and may accelerate the repair process.
Abstract: In a rat model of tendon rupture using semiquantitative methodology, healing was assessed according to the diameter of newly organized collagen and the occurrence of the sensory neuropeptides (SP, CGRP) in relation to different levels of physical activity. Normally, innervation of the Achilles tendon is confined to the paratenon. After rupture new nerve fibers grow into the tendon proper, but disappear after healing. In a first experiment to establish peak tissue and nerve regeneration after rupture, tendon tissues from freely moving rats were collected consecutively over 16 weeks. A peak increase in organized collagen and nerve ingrowth was observed between week 2 to 4 post rupture. Therefore, in a second experiment week 4 was chosen to assess the effect of physical activity on tendon healing in three groups of rats, that is, wheel running, plaster treated, and freely moving (controls). In the wheel-running group, the diameter of newly organized collagen was 94% ( p = 0.001) greater than that in the plaster-treated group and 48% ( p = 0.02) greater than that in the controls. Inversely, the neuronal occurrence of CGRP in the tendon proper was 57% ( p = 0.02) lower in the wheel-running group than that in the plaster-treated group and 53% ( p = 0.02) lower than that in the controls, suggesting an earlier neuronal in-growth and disappearance in the more active group. Physical activity speeds up tendon healing, which may prove to be linked to accelerated neuronal plasticity.
Abstract: BACKGROUND: The occurrence of nerve ingrowth and its relation to chronic tendon pain (tendinopathy) are still largely unknown. In healthy tendons, the innervation is confined to the paratenon, whereas the tendon proper is devoid of nerve fibers. In this study on the pathogenesis of tendinopathy, the authors examined sensory and sympathetic nerve fiber occurrence in the patellar tendon. HYPOTHESIS: Nerve ingrowth and altered expression of sensory and sympathetic neuromediators play a major role in the pathophysiology of pain in patellar tendinopathy. STUDY DESIGN: Case control study; Level of evidence, 3. METHODS: Biopsies from the patellar tendon in patients with patellar tendinopathy (n = 10) were compared with biopsies from a control group (n = 10) without any previous or current knee symptoms compatible with patellar tendinopathy. The biopsies were stained immunohistochemically for sensory and autonomic nerve markers. The biopsies from the 2 groups were compared using subjective and semiquantitative methods. RESULTS: Chronic painful patellar tendons exhibited increased occurrence of sprouting nonvascular sensory, substance P-positive nerve fibers and a decreased occurrence of vascular sympathetic nerve fibers, positive to tyroxin hydroxylase, a marker for noradrenaline. CONCLUSION: The altered sensory-sympathetic innervation suggests a role in the pathophysiology of tendinopathy. Ingrowth of sprouting substance P fibers presumably reflects a nociceptive and maybe a proliferative role, possibly as reactions to repeated microtraumata, whereas the decreased occurrence of tyroxin hydroxylase may represent a reduced antinociceptive role. These findings could be used to develop targeted pharmacotherapy for the specific treatment of tendinopathy.
Abstract: OBJECTIVES: To detect neuropeptides in human skeletal muscle at rest and after eccentric exercise. METHOD: Eight healthy subjects participated in the study. Microdialysis of the distal part of the vastus lateralis of the quadriceps muscle and pain evaluation were performed immediately after eccentric exercise, after two days, and at rest. Calcitonin gene related peptide (CGRP) and neuropeptide Y (NPY), representatives of the sensory and autonomic nervous system, were analysed by radioimmunoassay. RESULTS: Overall, the measured concentrations were low, some even below the limit of detection. At rest, CGRP was detected in two of seven samples, but after eccentric exercise it was detected in 27 of 30 samples. At rest, all NPY concentrations were below the limit of detection, but after exercise it was found in six of 30 samples. CONCLUSION: The significant increase in detectability of CGRP after eccentric exercise may be related to the increased experience of pain. Therefore the occurrence of CGRP after heavy eccentric exercise may be associated with the regulation of delayed onset muscle soreness and possibly also the stimulation of tissue regeneration.
Abstract: The Achilles tendon in rats with adjuvant arthritis was analyzed by radioimmunoassay (RIA) and semi-quantitative immunohistochemistry for the occurrence of two sensory neuropeptides, substance P (SP) and calcitonin gene related peptide (CGRP), and a sensory modulating peptide, galanin (GAL). The tissue concentration of SP and CGRP in the Achilles tendon and its envelope, i.e. the paratenon and bony insertion, as assessed by RIA was increased by 22% and 71%, respectively, compared to normal controls, whereas the level of GAL was unchanged. Semi-quantitative immunohistochemistry applied to different regions of the tendon in arthritic rats disclosed an increased occurrence of SP and CGRP positive nerve fibers in the paratenon and bone tendinous junction, whereas GAL fibers were only increased at the bone tendinous junction. Notably, neither neuropeptides nor inflammatory cells were seen in the tendon proper. The increased occurrence of SP and CGRP in the tendon envelope presumably reflects inflammatory actions, whereas that of GAL implies an endogenous anti-inflammatory response. The observed SP and CGRP upregulation in the paratenon and bony insertion suggests a pathophysiological role in paratenonitis and enthesitis often seen in patients with rheumatoid arthritis. Presumably Achillodynia originates in the tendon envelope rather than the tendon proper. The observations could be used to define new pharmacological targets for mitigating symptoms from tendons in rheumatoid arthritis and possibly also in other disorders. Whether a neuronal pathogenic mechanism underlies tendon overuse disorders in non-arthritic tendinopathies and the development of degeneration, i.e. tendinosis, remains to be studied.
Abstract: Skeletal ligaments are well characterized as mechanical
stabilizers of diarthrodial joints. New evidence now suggests that the
normal regulation of ligament and joint function may occur through
a neural and microvascular ‘‘axis,’’ where the physiology of normal
ligaments is influenced by heterogeneous cellular, neural, and microvasculature
elements. Within ligament tissue, complex networks of
cellular processes linked by gap junctions allow the direct cell-to-cell
transfer of signaling molecules, whereas sensory innervation and
neurovascular reflexes contribute to motor control and affect ligament
mechanical properties. The application of new imaging technologies
may assist in determining the functional implications of an integrated
neural and microvascular axis. Aging and gender related differences
in ligament function are also discussed.
Abstract: BACKGROUND: The use of sports massage is very common in the athletic community. However, only a few studies have shown any therapeutic effect of massage. HYPOTHESIS: Sports massage can improve the recovery after eccentric exercise. STUDY DESIGN: Prospective randomized clinical trial. METHODS: Sixteen subjects performed 300 maximal eccentric contractions of the quadriceps muscle bilaterally. Massage was given to 1 leg, whereas the other leg served as a control. Subjects were treated once daily for 3 days. Maximal strength was tested on a Kin-Com dynamometer, and functional tests were based on 1-leg long jumps. Pain was evaluated using a visual analog scale. RESULTS: There was a marked loss of strength and function of the quadriceps directly after exercise and on the third day after exercise. The massage treatment did not affect the level or duration of pain or the loss of strength or function following exercise. CONCLUSION: Sports massage could not improve the recovery after eccentric exercise.
Abstract: Nerve regeneration and the occurrence of three neuropeptides; i.e. substance P (SP), calcitonin gene related peptide (CGRP) and galanin (GAL), were studied during healing of tendon rupture in the rat by semi-quantitative immunohistochemistry. The neuronal findings were related to nociception as assessed by hindpaw withdrawal latencies at thermal and mechanical tests.Experimental rupture of rat Achilles tendon--normally devoid of nerves--elicited extensive nerve ingrowth into the rupture site in the early phase of healing followed by almost complete fiber disappearance (weeks 12-16). The ingrowth of SP and CGRP positive fibers, seen already at weeks 1-2, was associated with increased nociception. Subsequently, the occurrence of GAL positive fibers at weeks 4-6 was associated with decreased nociception. An even stronger relationship to nociception during healing was observed when the rate of change in neuropeptide expression instead of the expression in absolute terms was considered, according to the "cascade" formula of SP(')+CGRP(')-GAL(').It may prove that the observed temporal occurrence of different neuropeptides reflects a role of the peripheral nervous system in regulating synchronously nociception and healing.
Abstract: Nerve regeneration during healing of Achilles tendon rupture in the rat was studied by immunohistochemistry including semi-quantitative assessment. Neuronal markers for regenerating and mature fibers, ie., growth associated protein 43 (GAP-43) and protein gene product 9.5 (PGP 9.5), respectively, were analyzed at different time points (1-16 weeks) post-rupture. In the paratenon, both the ruptured and intact contralateral tendon (control) consistently exhibited immunoreactivity to the two neuronal markers. However, in the proper tendinous tissue only the ruptured tendon showed immunoreactivity to GAP-43 and PGP 9.5. This expression was seen already at week 1 post-rupture to reach a peak at week 6 followed by a successive drop till week 16. Also the occurrence of sensory and autonomic fibers according to immunoreactivity for calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY), respectively, was analyzed. CGRP-positivity was abundantly seen from weeks 2-6 in both perivascular and sprouting free nerve endings in the proper tendon tissue undergoing healing. NPY appeared later, at weeks 6-8 post-rupture around blood vessels mainly located in the surrounding loose connective tissue. Apart from a role in vasoaction (CGRP, vasodilatory; NPY, vasoconstrictory). both neuropeptides have been implicated in fibroblast and endothelial cell proliferation required for angiogenesis. The present study shows that early healing of ruptured tendons is characterized by an orchestrated, temporal appearance of nerve fibers expressing peptides with different actions. The observed pattern of neuronal regeneration and neuropeptide expression may prove to be important for normal connective tissue healing.
Abstract: We analyzed the neuronal occurrence of autonomic transmitters; noradrenaline (NA), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), in the Achilles tendon, medial and lateral collateral ligaments and knee joint capsule in the rat--by immunohistochemistry (IHC). In addition, the tissue concentrations of the sympathetic neuropeptide, NPY, and the parasympathetic peptide, VIP, were determined by radioimmunoassay (RIA). IHC demonstrated nerve fibers containing sympathetic vasoconstrictors--NA and NPY--and the parasympathetic vasodilator, VIP, in all tissues. NPY- and NA-positive nerve fibers were predominantly observed in larger blood vessels, whereas, nerve fibers immunoreactive to VIP were found in smaller vessels. In many nerve fibers a co-localization of the transmitters was seen. RIA showed that the concentration of NPY compared to VIP was 15-times higher in ligaments and twice as high in tendons and capsules. The differences noted may reflect a difference in vulnerability to degenerative conditions. In pathological conditions, dysregulation of autonomic transmitters in hypovascularized tissues subjected to repetitive mechanical load may contribute to tissue hypoxia leading to degeneration and rupture of tendons and ligaments.
Abstract: The occurrence of endogenous opioids and their receptors in rat achilles tendon was analyzed by immunohistochemistry (IHC), radioimmunoassay (RIA), and in vitro binding assays. The investigation focused on four enkephalins, dynorphin B, and nociceptin/orphanin FQ. Nerve fibers immunoreactive to all enkephalins (Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg-Gly-Lys, Met-enkephalin-Arg-Phe) were consistently found in the loose connective tissue and the paratenon, whereas dynorphin B and nociceptin/orphanin FQ could not be detected. The majority of enkephalin-positive nerve fibers exhibited varicosities predominantly seen in blood vessel walls. Measurable levels of Met-enkephalin-Arg-Phe and nociceptin/orphanin FQ were found in tendon tissue using RIA, whereas dynorphin B could not be detected. In addition to the endogenous opioids identified, delta-opioid receptors on nerve fibers were also detected by IHC. Binding assays to characterize the opioid binding sites showed that they were specific and saturable for [3H]-naloxone (Kd 7.01 +/- 0.98 nM; Bmax 23.52 +/- 2.23 fmol/mg protein). Our study demonstrates the occurrence of an opioid system in rat achilles tendon, which may be assumed to be present also in other connective tissues of the locomotor apparatus. This system may prove to be a useful target for pharmacological therapy in painful and inflammatory conditions by new drugs acting selectively in the periphery.
Abstract: The normal occurrence of sensory neuropeptides in tendons, ligaments and joint capsules in the rat was analyzed by immunohistochemistry and radioimmunoassay (RIA). Nerve fibres immunoreactive to substance P (SP), calcitonin gene-related peptide (CGRP), neurokinin A, galanin and somatostatin were identified in the Achilles tendon as well as the collateral ligaments and joint capsule of the knee. The neuropeptidergic fibres were predominantly found in the epiligament and paratenon. However, SP- and CGRP-positive fibres were also seen in the proper ligament and tendon tissues. RIA showed higher concentrations of SP and CGRP in tendons than in ligaments and capsules. The morphological and quantitative data obtained on sensory neuropeptides in normal tendons, ligaments and joint capsules may be used as a reference for tissue analysis in painful and inflammatory conditions of the locomotor apparatus.
Abstract: Both occupational and sports-related tendon pain
and degeneration pose a huge problem, mainly
because of the lack of specific treatment subsequent
to limited knowledge of the pathomechanisms
involved. Recent studies show that a variety of
signal substances (e.g. neuropeptides) harbored by
the peripheral nervous system have an important
role in the regulation of pain, inflammation, vasoactivity,
and tissue repair. This chapter presents novel
findings considering tendon innervation, expression
of neuropeptides, and neuronal response to
injury as studied in the rat Achilles tendon.
Based on structural and quantitative analysis,
the neuronal presence of autonomic, sensory, and
opioid peptides in the tendon was disclosed. This is
the first detection of a peripheral musculoskeletal
anti-nociceptive system consisting of opioid ligands
and receptors. Abundant nerve fibers were observed
in the paratenon, whereas the proper tendon was
practically devoid of nerves, suggesting that tendon
pain and inflammation is regulated from the surrounding
structures.
Nerve regeneration and neuropeptide expression
at different time points (1–16 weeks) were analyzed
in the healing of experimental Achilles tendon
rupture. During weeks 1–2 (inflammatory phase),
an extensive nerve fiber ingrowth into the rupture
site (i.e. the tendon proper) was seen which peaked
during weeks 2–6 (regenerative phase). Nerve
ingrowth may represent a prerequisite for delivery
of neuronal mediators required for tissue repair.
During the regenerative phase, a peak expression
of the sensory neuropeptides substance P (SP)
and calcitonin gene-related peptide (CGRP) in the
proper tendon, seen as free sprouting nerve endings
among fibroblasts and new vessels, may have a role
in repair.
The present study demonstrates that tendons
are supplied with a complex peptidergic network,
which presumably takes part in maintaining tissue
homeostasis, but also by its plasticity (i.e. nerve
ingrowth and temporal alteration in neuropeptide
expression is capable of responding to injury). In a
future perspective, neuronal mediators may prove
to be useful in targeted pharmacotherapy and tissue
engineering in painful and degenerative tendon
disorders.
Abstract: Painful musculo-skeletal disorders pose a tremendous burden on the healthcare system. One main reason is
the lack of specific treatment due to limited knowledge of the underlying pathomechanisms. Hypothetically,
the peripheral nervous system in the musculo-skeletal tissues plays an important role in the regulation of
pain, inflammation, vasoactivity and tissue repair. The present study in the rat was designed to explore the
expression of neuropeptides in periarticular tissues under normal conditions and after injury.
A combined approach based on radioimmunoassay (RIA), immunohistochemistry (IHC) and high
performance liquid chromatography (HPLC), disclosed the presence of autonomic (NPY, VIP, NA);
sensory (SP, NKA, CGRP, GAL, SOM); and opioid (LE, ME, MEAP, MEAGL, N/OFQC) mediators in
tendon, ligament and joint capsule. In tendon, δ-opioid receptors were also detected. The peptidergic nerve
fibres occurred in abundance in the paratenon, epiligament and beneath the lining layer of the synovium,
whereas the proper structures were almost devoid of nerve fibres. This suggests that the neuronal regulation
of periarticular tissues highly depends on the innervation of the surrounding structures. RIA showed that the
ratio of NPY (sympathetic)/ VIP (parasympathetic) was 8-times higher in ligaments than in tendons and in
capsules. The ratio of SP and CGRP (nociceptive)/ GAL, MEAP, N/OFQ (anti-nociceptive) was 10-times
higher in tendons and twice as high in capsules compared to ligaments. The distribution and levels in the
different tissues may represent the homeostatic state of vasoactivity and nociception, and also indicate the
susceptibility to physico-chemical stress.
In healing of experimental Achilles tendon rupture, nerve regeneration and neuropeptide expression at
different time points (1-16 weeks) were analysed by IHC including semi-quantification. Analysis of
markers for new (GAP) and mature fibres (PGP) disclosed extensive nerve fibre ingrowth into the rupture
site. A peak nerve fibre expression during weeks 2-6 (regenerative phase) was followed by nerve fibre
withdrawal during weeks 8-16 (remodeling phase). New nerve ingrowth is presumably a prerequisite for
delivery of neuronal mediators required for tissue repair. Analysis of specific neuropeptides showed an
increased number of SP and CGRP fibres one week post-rupture (inflammatory phase) around blood vessels
surrounded by inflammatory cells. This complies with a nociceptive and pro-inflammatory role. During the
regenerative phase, the expression of SP and CGRP peaked in the rupture site of proper tendon, seen as free
sprouting nerve endings among fibroblasts and newly formed vessels, which may prove to reflect a role in
cell proliferation. An initial sparse occurrence of GAL, NPY and VIP was followed by a peak expression at
the early remodeling phase, i.e., around week six, which may represent modulatory effects on SP and CGRP
required to end the nociceptive, inflammatory and regenerative processes.
Nociception according to hind paw withdrawal at thermal and mechanical stimuli was related to the
expression of sensory neuropeptides during healing as assessed by semi-quantitative IHC. Increased
expression of SP/CGRP during weeks 1-2 coincided with increased thermal sensitivity on the injured side.
Conversely, increased GAL expression during weeks 4-6 coincided with decreased thermal sensitivity, and
notably, also with increased mechanical sensitivity. A strong relationship between neuropeptide expression
and thermal sensitivity was found by considering the combined rates of change of the three neuropeptides
(SP + CGRP – GAL) during healing. The finding indicates that nociception is regulated by the speed by
which the expression of potentiating and inhibitory neuropeptides is altered, rather than the absolute tissue
levels.
From the present study, it seems obvious that periarticular tissues are supplied with a complex peptidergic
network, which presumably takes part in maintaining tissue homeostasis, but also by its plasticity, i.e. nerve
ingrowth and temporal alteration in neuropeptide expression, is capable of responding to injury.
Interestingly, ingrowth of new nerves seems to be a fundamental feature of tissue repair. In a future
perspective, neuronal mediators may prove to be useful in targeted pharmacotherapy and tissue engineering
of painful and degenerative musculo-skeletal disorders.
