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Influences of paratendinous innervation and non-neuronal substance P in tendinopathy: studies on human tendon tissue and an experimental model of Achilles tendinopathy
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Pain of the musculoskeletal system is one of the most common reasons for people seeking medical attention, and is also one of the major factors that prevent patients from working. Chronic tendon pain, tendinopathy, affects millions of workers world-wide, and the Achilles tendon is an important structure often afflicted by this condition. The pathogenesis of tendinopathy is poorly understood, but it is thought to be of multifactoral aetiology. It is known that tendon pain is often accompanied not only by impaired function but also by structural tissue changes, like vascular proliferation, irregular collagen organisation, and hypercellularity, whereby the condition is called tendinosis. In light of the poor knowledge of tendinosis pathophysiology and recent findings of a non-neuronal signalling system in tendon tissue, the contributory role of neuropeptides such as substance P (SP) has gained increased interest. SP, known for afferent pain signalling in the nervous system, also has multiple efferent functions and has been described to be expressed by non-neuronal cells. As pain is the most prominent symptom of tendinopathy, the focus of the studies in this thesis was the innervation patterns of the tissue ventral to the Achilles tendon (i.e. the tissue targeted in many contemporary treatment methods) as well as the distribution of SP and its preferred receptor, the neurokinin-1 receptor (NK-1R), in the tendon tissue itself. It was hereby hypothesised that the source of SP affecting the Achilles tendon might be the main cells of the tendon tissue (the tenocytes) as well as paratendinous nerves, and that SP might be involved in tendinosis- development. The studies were conducted, via morphological staining methods including immunohistochemistry and in situ hybridisation, on tendon biopsies from patients suffering from Achilles tendinosis and on those from healthy volunteers. The hypothesis of the thesis was furthermore tested using an experimental animal model (rabbit) of Achilles tendinopathy, which was first validated. The model was based on a previously established overuse protocol of repetitive exercise. In the human biopsies of the tissue ventral to the Achilles tendon, there was a marked occurrence of sympathetic innervation, but also sensory, SP-containing, nerve fibres. NK-1R was expressed on blood vessels and nerve fascicles of the paratendinous tissue, but also on the tenocytes of the tendon tissue proper itself, and notably more so in patients suffering from tendinosis. Furthermore, the human tenocytes displayed not only NK-1R mRNA but also mRNA for SP. The animal model was shown to produce objectively verified tendinosis-like changes, such as hypercellularity and increased vascularity, in the rabbit Achilles tendons, after a minimum of three weeks of the exercise protocol. The contralateral leg of the animals in the model was found to be an unreliable control, as bilateral changes occured. The model furthermore demonstrated that exogenously administered SP triggers an inflammatory response in the paratendinous tissue and accelerates the intratendinous tendinosis-like changes such that they now occur after only one week of the protocol. Injections of saline as a control showed similar results as SP concerning hypercellularity, but did not lead to vascular changes or pronounced paratendinous inflammation. In summary, this thesis concludes that interactions between the peripheral sympathetic and sensory nervous systems may occur in Achilles tendinosis at the level of the ventral paratendinous tissue, a region thought to be of great importance in chronic tendon pain since many successful treatments are directed toward it. Furthermore, the distribution of NK-1R:s in the Achilles tendon described in these studies gives a basis for SP, whether produced by nerves mainly outside the tendon or by tenocytes within the tendon, to affect blood vessels, nerve structures, and/or tendon cells, especially in tendinosis patients. In light of this and of previously known SP-effects, such as stimulation of angiogenesis, pain signalling, and cell proliferation, the proposed involvement of SP in tendinosis development seems likely. Indeed, the animal model of Achilles tendon overuse confirms that SP does induce vascular proliferation and hypercellularity in tendon tissue, thus strengthening theories of SP playing a role in tendinosis pathology.

Place, publisher, year, edition, pages
Umeå: Umeå university , 2010. , 104 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1370
Keyword [en]
Achilles tendon, tendinopathy, tendinosis, paratenon, innervation, substance P, neuropeptides, neurokinin-1 receptor
National Category
Cell and Molecular Biology
Research subject
Human Anatomy
Identifiers
URN: urn:nbn:se:umu:diva-35917ISBN: 978-91-7459-067-8 (print)OAI: oai:DiVA.org:umu-35917DiVA: diva2:349953
Public defence
2010-10-01, Betula, Byggnad 6M, NUS, Umeå University, SE-901 87 Umeå, Sweden, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2010-09-13 Created: 2010-09-09 Last updated: 2010-09-13Bibliographically approved
List of papers
1. Nerve-related characteristics of ventral paratendinous tissue in chronic Achilles tendinosis
Open this publication in new window or tab >>Nerve-related characteristics of ventral paratendinous tissue in chronic Achilles tendinosis
2007 (English)In: Knee Surgery, Sports Traumatology, Arthroscopy, ISSN 0942-2056, E-ISSN 1433-7347, Vol. 15, no 10, 1272-1279 p.Article in journal (Refereed) Published
Abstract [en]

Ultrasound and Doppler examination has shown high blood flow-neovascularisation inside and outside the ventral Achilles tendon in chronic painful tendinosis, but not in pain-free normal Achilles tendons. In patients with Achilles tendinosis, injections with the sclerosing substance polidocanol, targeting the areas with increased blood flow, have been demonstrated to give pain relief. A drawback when interpreting these findings is the fact that the pattern of nerve supply in the target area, i.e. the ventral area of the tendon, is so far unknown. In this study, therefore, tissue specimens from this area, obtained during surgical treatment of patients with chronic painful midportion Achilles tendinosis, were examined. In the examined area, containing loose connective tissue, the general finding was a presence of large and small arteries and nerve fascicles. The nerve fascicles were distinguished in sections processed for the pan-neural marker protein gene-product 9.5. The nerve fascicles contain sensory nerve fibers, as shown via staining for the sensory markers substance P (SP) and calcitonin gene-related peptide, and sympathetic nerve fibers as seen via processing for tyrosine hydroxylase. In addition, there were immunoreactions for the SP-preferred receptor, the neurokinin-1 receptor, in blood vessel walls and nerve fascicles. Some of the blood vessels were supplied by an extensive peri-vascular innervation, sympathetic nerve fibers being a distinct component of this innervation. There was also a marked occurrence of immunoreactions for the alpha1-adrenoreceptor in arterial walls as well as in the nerve fascicles. Altogether, these findings suggest that the area investigated is under marked influence by the nervous system, including sympathetic and sensory components. Thus, sympathetic/sensory influences may be involved in the pain mechanisms from this area. In conclusion, the nerve-related characteristics of the area targeted by the polidicanol injection treatment for Achilles tendinosis, are shown here for the first time.

Keyword
Achilles tendon, Tendinosis, Sympathetic innervation, Sensory innervation
National Category
Cell and Molecular Biology
Research subject
Human Anatomy
Identifiers
urn:nbn:se:umu:diva-8147 (URN)10.1007/s00167-007-0364-2 (DOI)17604979 (PubMedID)
Available from: 2008-01-15 Created: 2008-01-15 Last updated: 2017-12-14Bibliographically approved
2. Presence of substance P and the neurokinin-1 receptor in tenocytes of the human Achilles tendon
Open this publication in new window or tab >>Presence of substance P and the neurokinin-1 receptor in tenocytes of the human Achilles tendon
2008 (English)In: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 150, no 1-3, 81-87 p.Article in journal (Refereed) Published
Abstract [en]

Nerve signal substances, such as the tachykinin substance P (SP), may be involved in the changes that occur in response to tendinopathy (tendinosis). It is previously known that the level of SP innervation within tendon tissue is limited, but results of experimental studies have suggested that SP may have stimulatory, angiogenetic and healing effects in injured tendons. Therefore, it would be of interest to know if there is a local SP-supply in tendon tissue. In the present study, the patterns of expression of SP and its preferred receptor, the neurokinin-1 receptor (NK-1 R), in normal and tendinosis human Achilles tendons were analyzed by use of both immunohistochemistry and in situ hybridization. We found that there was expression of SP mRNA in tenocytes, and that tenocytes showed expression of NK-1 R at protein as well as mRNA levels. The observations concerning both SP and NK-1 R were most evident for tenocytes in tendinosis tendons. Our findings suggest that SP is produced in tendinosis tendons, and furthermore that SP has marked effects on the tenocytes via the NK-1 R. It cannot be excluded that the SP effects are of importance concerning the processes of reorganization and healing that occur for tendon tissue in tendinosis. In conclusion, it appears as if SPergic autocrine/paracrine effects occur in tendon tissue during the processes of tendinosis, hitherto unknown effects for human tendons.

National Category
Cell and Molecular Biology
Research subject
Human Anatomy
Identifiers
urn:nbn:se:umu:diva-19261 (URN)10.1016/j.regpep.2008.02.005 (DOI)18394729 (PubMedID)
Available from: 2009-03-05 Created: 2009-03-05 Last updated: 2017-12-13Bibliographically approved
3. Tenocyte hypercellularity and vascular proliferation in a rabbit model of tendinopathy: contralateral effects suggest the involvement of central neuronal mechanisms
Open this publication in new window or tab >>Tenocyte hypercellularity and vascular proliferation in a rabbit model of tendinopathy: contralateral effects suggest the involvement of central neuronal mechanisms
Show others...
2011 (English)In: British Journal of Sports Medicine, ISSN 0306-3674, E-ISSN 1473-0480, Vol. 45, no 5, 399-406 p.Article in journal (Refereed) Published
Abstract [en]

Objective To determine whether there are objective findings of tendinosis in a rabbit tendinopathy model on exercised and contralateral (non-exercised) Achilles tendons. Design Four groups of six New Zealand white rabbits per group were used. The animals of one (control) group were not subjected to exercise/stimulation. Interventions Animals were subjected to a protocol of electrical stimulation and passive flexion-extension of the right triceps surae muscle every second day for 1, 3 or 6 weeks. Main Outcome Measures Tenocyte number and vascular density were calculated. Morphological evaluations were also performed as well as in-situ hybridisation for vascular endothelial growth factor (VEGF) messenger RNA. Results There was a significant increase in the tenocyte number after 3 and 6 weeks of exercise, but not after 1 week, in comparison with the control group. This was seen in the Achilles tendons of both legs in experimental animals, including the unexercised limb. The pattern of vascularity showed an increase in the number of tendon blood vessels in rabbits that had exercised for 3 weeks or more, compared with those who had exercised for 1 week or not at all. VEGF-mRNA was detected in the investigated tissue, with the reactions being more clearly detected in the tendon tissue with tendinosis-like changes (6-week rabbits) than in the normal tendon tissue (control rabbits). Conclusions There were bilateral tendinosis-like changes in the Achilles tendons of rabbits in the current model after 3 weeks of training, suggesting that central neuronal mechanisms may be involved and that the contralateral side is not appropriate as a control.

National Category
Cell and Molecular Biology
Research subject
Human Anatomy
Identifiers
urn:nbn:se:umu:diva-35363 (URN)10.1136/bjsm.2009.068122 (DOI)20605910 (PubMedID)
Note

E-pub ahead of print.

Available from: 2010-08-13 Created: 2010-08-13 Last updated: 2017-12-12Bibliographically approved
4. Substance P induces tendinosis-like changes in a rabbit model of Achilles tendon overuse
Open this publication in new window or tab >>Substance P induces tendinosis-like changes in a rabbit model of Achilles tendon overuse
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

BACKGROUND: In previous studies we found evidence favouring that human Achilles tendon cells (tenocytes) are capable of producing the neuropeptide substance P (SP). Furthermore, the preferred receptor for SP (the neurokinin-1 receptor, NK-1 R) was widely expressed throughout the tendon, especially in patients suffering from chronic tendon pain (tendinopathy) with tissue changes (tendinosis) including hypercellularity and vascular proliferation. Considering known effects of SP, one might ask whether SP contributes to tendon cell proliferation and neovascularisation in tendinosis. We have an established animal (rabbit) model of Achilles tendinopathy based on overuse in the form of repetitive exercise. Recent studies with this model have shown that tendinosis-like changes are present after 3 weeks of exercise, but not after only 1 week. The current study aimed to test whether the development of tendinosis-like changes would be accelerated during a 1 week course of exercise with repetitive local administration of SP.

MATERIAL AND METHODS: Four groups of animals (5-6 New Zealand white rabbits per group) were used. Three groups were subjected to the previously established protocol of Achilles tendon overuse for 1 week. One of these groups was given repetitive SP injections in the paratendinous tissue of the Achilles tendon, whereas one group (‘NaCl controls’) was given an equivalent schedule of saline injections. Two additional control groups existed: One in which the animals were neither subjected to the overuse protocol nor to any injections (‘untrained controls’), and one in which the animals trained for 1 week but were not given any injections (‘1 week controls’). Tenocyte number, vascular density, and the possible occurrence of paratendinous inflammation were evaluated. Immunohistochemistry and in situ hybridisation to detect NK-1 R were also conducted.

RESULTS: There was a significant increase in tenocyte number in the SP-injected group compared to both untrained controls and 1 week controls. However, the same phenomenon was noticed for NaCl controls, i.e. tenocyte number was significantly increased in response to NaCl injections compared to untrained controls. There was an increase in the number of tendon blood vessels in the SP-injected group as compared to untrained controls, and this increase in vascularity was not seen for the NaCl controls or the 1 week controls. Paratendinous inflammation, as evidenced by invasion of inflammatory cells in the paratenon, was clearly more pronounced in the SP-injected group than in the NaCl controls. NK-1 R was detected in blood vessel walls, on nerves, on inflammatory cells, and on tenocytes.

DISCUSSION AND CONCLUSIONS: The observations suggest that SP induces tenocyte proliferation and angiogenesis in the rabbit Achilles tendon, thus supporting a potential role of this neuropeptide in the processes that occur in tendinosis. The study corroborates findings on the human Achilles tendon in that NK-1 R was expressed on tenocytes and tendon blood vessel walls, thereby providing a potential anatomic basis for the observed effects of SP on the development of tendinosis. The hypercellularity observed in response to NaCl injections might be due increased tissue pressure or to stimulation of endogenous SPproduction, a phenomenon not unheard of. The angiogenic effect of SP injections, on the other hand, appeared to be more specifically related to an induction of inflammation in the paratendon.

Keyword
Tendinopathy; Neuropeptides; Neurokinin-1 Receptor; Animal model; Overuse injuries
National Category
Cell and Molecular Biology
Research subject
Human Anatomy
Identifiers
urn:nbn:se:umu:diva-35909 (URN)
Available from: 2010-09-09 Created: 2010-09-09 Last updated: 2010-09-13Bibliographically approved

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