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Innervation patterns and locally produced signal substances in the human patellar tendon: of importance when understanding the processes of tendinosis
Umeå University, Faculty of Medicine, Integrative Medical Biology.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Tendinosis is a condition of chronic pain that afflicts several human tendons, not least the patellar tendon, in which case it is often clinically referred to as ‘jumper’s knee’. The exact mechanisms behind tendinosis are yet not fully understood. One draw-back in the case of patellar tendinosis has been the lack of knowledge of the innervation patterns of the human patellar tendon. It cannot be excluded that the processes of tendinosis are influenced by nerve mediators, released from nerve endings or from stimulated cells inside the tendon.

Thus, the studies of the present thesis aimed to 1) map the general, sensory, cholinergic and sympathetic innervation patterns of the human patellar tendon, in both the tendon tissue proper and the loose paratendinous connective tissue surrounding the tendon, and 2) investigate the possible existence of a production of signal substances, traditionally associated with neurons, in non-neuronal tendon cells, and to see if there are signs of local cholinergic and catecholaminergic signaling pathways. Biopsies of both normal pain-free patellar tendons and patellar tendons from patients with chronic painful tendinosis were collected and investigated. The main method utilized was immunohistochemistry, using antibodies directed against synthesizing enzymes for acetylcholine and catecholamines, against muscarinic and adrenergic receptors, and against markers of general and sensory innervation. In situ hybridization (ISH) to detect mRNA for the cholinergic/catecholaminergic synthesizing enzymes was also used.

It was found that the loose paratendinous connective tissue of the patellar tendon was rather richly innervated by nerve structures. These consisted of large nerve fascicles, as well as perivascular innervation in the walls of some of the larger arteries and smaller blood vessels. It was found that part of the nerve structures corresponded to sensory afferents, and that some conformed to cholinergic and, especially, sympathetic nerve fibers. The tendon tissue proper was strikingly less innervated than the paratendinous tissue. The sparse innervation that was found in the tendon tissue proper was seen in narrow zones of loose connective tissue and blood vessels, interspersed between the collagen bundles. The overall impression was that the patterns of distribution of the general, sensory, and autonomic innervations of tendinosis tendon tissue were similar to those of normal tendon tissue proper.

The most pioneering findings were the immunohistochemical observations of an expression of enzymes related to production of both acetylcholine and catecholamines within the tendon cells (tenocytes) themselves, as well as of a presence of the receptors for these substances on the same cells; features that were predominantly seen in tendinosis tendons. The observations of the synthesizing enzymes for acetylcholine and catecholamines in tenocytes were confirmed by ISH findings of mRNA for these enzymes in the tenocytes. Immunoreactions for muscarinic and adrenergic receptors were also found in blood vessel walls and in some of the nerve fascicles.

In summary, this thesis presents novel information on the innervation patterns of the human patellar tendon, in healthy individuals with pain-free tendons as well as in patients with chronic painful tendinosis. Furthermore, it gives the first evidence of the presence of a local, non-neuronal production in the tendon tissue of signal substances normally seen in neurons, and a basis for these substances to affect the tenocytes as these cells also display muscarinic and adrenergic receptors. Thus, the results indicate an existence of autocrine and/or paracrine cholinergic/catecholaminergic systems in the tendon tissue; systems that seem to be up-regulated in tendinosis. This is of great interest as it is known that stimulation of receptors for both catecholamines and acetylcholine can lead to cell proliferation, interfere with pain sensation, influence collagen production, and take part in vasoregulation, as well as, in the case of adrenergic receptors, promote cell degeneration and apotosis. All these processes represent biological functions/events that are reported to be affected in tendinosis.

In conclusion, despite the fact that there is very limited innervation within the patellar tendon tissue proper, it is here shown that effects of signal substances traditionally associated with neurons seem to occur in the tissue, via a local production of these substances in tenocytes.

Place, publisher, year, edition, pages
Umeå: Integrativ medicinsk biologi , 2007. , 66 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1103
Keyword [en]
patellar tendon, innervation, tendinosis, tendinopathy, acetylcholine, catecholamines, muscarinic receptors, adrenergic receptors
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1156ISBN: 978-91-7264-319-2 (print)OAI: oai:DiVA.org:umu-1156DiVA: diva2:140418
Public defence
2007-06-08, sal BiA201 (Stora föreläsningssalen), Biologihuset, Umeå universitet, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2007-05-23 Created: 2007-05-23 Last updated: 2010-01-18Bibliographically approved
List of papers
1. Distribution of general (PGP 9.5) and sensory (substance P/CGRP) innervations in the human patellar tendon.
Open this publication in new window or tab >>Distribution of general (PGP 9.5) and sensory (substance P/CGRP) innervations in the human patellar tendon.
2006 (English)In: Knee Surgery, Sports Traumatology, Arthroscopy, ISSN 0942-2056, E-ISSN 1433-7347, Vol. 14, no 2, 125-132 p.Article in journal (Refereed) Published
Abstract [en]

There is no information on the pattern of blood vessel innervation, and in principle no information on innervation in general, in the human patellar tendon. In the present study, biopsies from the proximal part of normal and pain-free patellar tendons (11 men, mean age 33 years) were examined. The specimens were evaluated by using antibodies against the general nerve marker protein gene-product 9.5 (PGP 9.5) and the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and immunohistochemistry. It was observed that the arteries, and to some extent the small vessels, in the loose paratendinous connective tissue were supplied with PGP 9.5- as well as SP- and CGRP-innervations. There was a marked PGP 9.5-like immunoreaction (LI), and to some extent also SP- and CGRP-LI, in the large nerve fascicles in this tissue. In the tendon tissue proper, PGP 9.5-LI was detected in nerve fibers located in the vicinity of some of the blood vessels and in thin nerve fascicles. There was a low degree of SP- and CGRP-innervation in the tendon tissue proper. The observations give a morphologic correlate for the occurrence of nerve-mediated effects in the patellar tendon. Particularly it seems as if there is a marked nerve-mediated regulation of the blood vessels supplying the tendon, at the level where they course in the loose paratendinous connective tissue.

Keyword
Adult, Arteries/innervation/metabolism, Biopsy, Calcitonin Gene-Related Peptide/*metabolism, Connective Tissue/innervation/metabolism, Humans, Immunohistochemistry, Male, Patella/innervation/metabolism, Substance P/*metabolism, Tendons/*innervation/*metabolism/pathology, Ubiquitin Thiolesterase/*metabolism
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-12697 (URN)doi:10.1007/s00167-005-0636-7 (DOI)15983834 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2017-12-14Bibliographically approved
2. Vascular NK-1 receptor occurrence in normal and chronic painful Achilles and patellar tendons: studies on chemically unfixed as well as fixed specimens.
Open this publication in new window or tab >>Vascular NK-1 receptor occurrence in normal and chronic painful Achilles and patellar tendons: studies on chemically unfixed as well as fixed specimens.
2005 (English)In: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 126, no 3, 173-181 p.Article in journal (Refereed) Published
Abstract [en]

It is not known as to whether the Achilles and patellar tendons contain neurokinin-1 (NK-1) receptors. This is a drawback when considering the fact that pain symptoms are frequent in these and as recent studies show that the pain symptoms might be cured via interference with blood vessel function. In the present study, the human Achilles and patellar tendons were examined concerning immunohistochemical expression of the NK-1 receptor. Chemically unfixed and fixed specimens, TRITC and PAP stainings and a battery of NK-1 receptor antibodies, including antibodies against the C-terminus and the N-terminal region, were utilized. NK-1 receptor immunoreaction could be detected in inner parts of the walls of large blood vessels and in the walls of small blood vessels. To some extent, NK-1 immunoreaction was also detectable in small nerve fascicles and in tenocytes. It was found to be of utmost importance to apply both chemically unfixed and fixed specimens. The use of chemically unfixed tissue was found advantageous in order to depict the immunoreactions in the blood vessel walls. The observations represent new findings and are of relevance as substance P (SP) is known to be of importance where neurogenic angiogenesis contributes to diseases and as SP on the whole has profound effects concerning blood vessel regulation.

Keyword
Achilles Tendon/blood supply/innervation/*metabolism, Adult, Biopsy, Female, Fixatives, Fluorescent Antibody Technique, Freezing, Humans, Male, Middle Aged, Patella, Receptors; Neurokinin-1/*biosynthesis, Rhodamines, Staining and Labeling/methods, Tendinopathy/*metabolism, Tendons/*blood supply/innervation/*metabolism
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-12703 (URN)doi:10.1016/j.regpep.2004.09.008 (DOI)15664664 (PubMedID)
Available from: 2007-10-19 Created: 2007-10-19 Last updated: 2017-12-14Bibliographically approved
3. Immunohistochemical and histochemical findings favoring the occurrence of autocrine/paracrine as well as nerve-related cholinergic effects in chronic painful patellar tendon tendinosis.
Open this publication in new window or tab >>Immunohistochemical and histochemical findings favoring the occurrence of autocrine/paracrine as well as nerve-related cholinergic effects in chronic painful patellar tendon tendinosis.
2006 (English)In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 69, no 10, 808-819 p.Article in journal (Refereed) Published
Abstract [en]

The pathogenesis of the pain in patellar tendon tendinosis ("jumper's knee") is unclear. We have recently presented new information about the sensory nervous system in the human patellar tendon, but there is very little information regarding the possible occurrence of a cholinergic system in this tendon. In the present study, specimens of pain-free normal tendons and chronically painful tendinosis tendons were examined by different immunohistochemical and histochemical methods. Antibodies against the M(2) receptor, choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT) were applied, and staining for demonstration of activity of acetylcholinesterase (AChE) was also utilized. It was found that immunoreactions for the M(2) receptor could be detected intracellularly in both blood vessel cells and tenocytes, especially in tendinosis specimens. Furthermore, in the tendinosis specimens, some tenocytes were seen to exhibit immunoreaction for ChAT and VAChT. AChE reactions were seen in fine nerve fibers associated with small blood vessels in both the normal control tendons and the tendinosis tendons. The observations suggest that there is both a nerve related and a local cholinergic system in the human patellar tendon. As ChAT and VAChT immunoreactions were detected in tenocytes of tendinosis tendons, these cells might be a source of local acetylcholine (Ach) production. As both tenocytes and blood vessel cells were found to exhibit immunoreactions for the M(2) receptor, it is likely that both of these tissue cells may be influenced by ACh. Thus, in conclusion, there appears to be an upregulation of the cholinergic system, and an occurrence of autocrine/paracrine effects in this system, in the tendinosis patellar tendon.

Keyword
Acetylcholine/biosynthesis, Acetylcholinesterase/analysis, Adult, Autocrine Communication, Blood Vessels/metabolism/pathology, Choline O-Acetyltransferase/analysis, Cholinergic Fibers/*chemistry/pathology, Chronic Disease, Female, Humans, Immunohistochemistry, Male, Middle Aged, Pain/pathology, Paracrine Communication, Patella/pathology, Receptor; Muscarinic M2/analysis, Tendinopathy/metabolism/*pathology, Vesicular Acetylcholine Transport Proteins/analysis
Identifiers
urn:nbn:se:umu:diva-12694 (URN)doi:10.1002/jemt.20351 (DOI)16830327 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2017-12-14Bibliographically approved
4. Extensive expression of markers for acetylcholine synthesis and of M2 receptors in tenocytes in therapy-resistant chronic painful patellar tendon tendinosis - a pilot study.
Open this publication in new window or tab >>Extensive expression of markers for acetylcholine synthesis and of M2 receptors in tenocytes in therapy-resistant chronic painful patellar tendon tendinosis - a pilot study.
2007 (English)In: Life Sciences, ISSN 0024-3205, E-ISSN 1879-0631, Vol. 80, no 24-25, 2235-2238 p.Article in journal (Refereed) Published
Abstract [en]

We have recently obtained evidence favoring the occurrence of an up-regulation of a non-neuronal cholinergic system in chronic painful patellar tendon tendinosis. It seems possible that this up-regulation to a certain degree may be involved in the manifestations of the disease. Today, there is a new, very successful, line of treatment of patellar tendinosis in the form of Doppler guided sclerosing injections. However, a few patients seem resistant to this therapy. Therefore, we have in this pilot study investigated biopsies from the patellar tendon of three such therapy-resistant patients, using immunohistochemistry. In situ hybridization was also applied. Comparisons were made with a material of specimens from both normal (n=16) and tendinosis (n=7) tendons, also previously examined. The study showed that there were extensive immunoreactions for choline acetyltransferase (ChAT) and vesicular acetylcholine transporter, as well as for the M(2) muscarinic acetylcholine receptor, in the overwhelming majority of the tenocytes. The immunoreactions were more pronounced than those generally obtained in the tendinosis tissue of the previously studied patients and clearly more pronounced than those of patellar tendon tissue of controls. Also, for the first time, we here present findings of mRNA for ChAT within tenocytes. In conclusion, it appears as if there is an excessive local acetylcholine (ACh) production and an occurrence of marked ACh effects in cases of severe tendinosis. An excessive production of local ACh might be related to pain sensation and the processes that occur in tendinosis development, such as cell proliferation. Thus, the results of this pilot study suggest that non-neuronal ACh is highly involved in the pathology of therapy-resistant patellar tendinosis.

Identifiers
urn:nbn:se:umu:diva-12688 (URN)doi:10.1016/j.lfs.2007.01.005 (DOI)17289083 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2017-12-14Bibliographically approved
5. Studies on the importance of sympathetic innervation, adrenergic receptors, and a possible local catecholamine production in the development of patellar tendinopathy (tendinosis) in man.
Open this publication in new window or tab >>Studies on the importance of sympathetic innervation, adrenergic receptors, and a possible local catecholamine production in the development of patellar tendinopathy (tendinosis) in man.
2007 (English)In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 70, no 4, 310-324 p.Article in journal (Refereed) Published
Abstract [en]

Changes in the patterns of production and in the effects of signal substances may be involved in the development of tendinosis, a chronic condition of pain in human tendons. There is no previous information concerning the patterns of sympathetic innervation in the human patellar tendon. In this study, biopsies of normal and tendinosis patellar tendons were investigated with immunohistochemical methods, including the use of antibodies against tyrosine hydroxylase (TH) and neuropeptide Y, and against alpha(1)-, alpha(2A)-, and beta(1)-adrenoreceptors. It was noticed that most of the sympathetic innervation was detected in the walls of the blood vessels entering the tendon through the paratendinous tissue, and that the tendon tissue proper of the normal and tendinosis tendons was very scarcely innervated. Immunoreactions for adrenergic receptors were noticed in nerve fascicles containing both sensory and sympathetic nerve fibers. High levels of these receptors were also detected in the blood vessel walls; alpha(1)-adrenoreceptor immunoreactions being clearly more pronounced in the tendinosis tendons than in the tendons of controls. Interestingly, immunoreactions for adrenergic receptors and TH were noted for the tendon cells (tenocytes), especially in tendinosis tendons. The findings give a morphological correlate for the occurrence of sympathetically mediated effects in the patellar tendon and autocrine/paracrine catecholamine mechanisms for the tenocytes, particularly, in tendinosis. The observation of adrenergic receptors on tenocytes is interesting, as stimulation of these receptors can lead to cell proliferation, degeneration, and apoptosis, events which are all known to occur in tendinosis. Furthermore, the results imply that a possible source of catecholamine production might be the tenocytes themselves. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc.

Identifiers
urn:nbn:se:umu:diva-12692 (URN)doi:10.1002/jemt.20413 (DOI)17206652 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2017-12-14Bibliographically approved
6. In situ hybridization studies confirming recent findings of the existence of a local nonneuronal catecholamine production in human patellar tendinosis.
Open this publication in new window or tab >>In situ hybridization studies confirming recent findings of the existence of a local nonneuronal catecholamine production in human patellar tendinosis.
2007 (English)In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 70, no 10, 908-911 p.Article in journal (Other academic) Published
Abstract [en]

We have in recent studies presented unexpected immunohistochemical evidence favoring the existence of a local production of catecholamines, and an occurrence of adrenergic receptors on the tendon cells (tenocytes), in the human patellar tendon. This was particularly noticed for tendons from patients suffering from tendinosis (chronic tendon pain), which has led us to propose an involvement of this autocrine/paracrine system in the development of tendinosis, especially since catecholamines have been reported to be modulators of tissue remodeling and pain processes. However, the findings concerning catecholamine production have so far only been noted at the level of protein detection, and for this reason, the aim of the present study was to confirm the previous immunohistochemical results by using in situ hybridization (ISH) technique. A ssDNA probe detecting human mRNA for the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) was applied. The ISH results revealed that there were clear reactions indicating the existence of mRNA for TH in tenocytes of tendinosis specimens. It was generally noted that disfigured tenocytes were the ones with the most distinct reactions, while normally looking tenocytes hardly displayed any reactions at all. In conclusion, this study presents the first evidence at the mRNA level of the existence of a local nonneuronal production of catecholamines in human patellar tendon tissue. The findings add to recent observations of the occurrence of a local production in tendons of signal substances traditionally related to neurons.

(c) 2007 Wiley-Liss, Inc.

Keyword
Adult, Catecholamines/*biosynthesis, Female, Humans, In Situ Hybridization, Male, Patellar Ligament/cytology/enzymology/*metabolism, RNA; Messenger/genetics/metabolism, Tendinopathy/enzymology/*metabolism, Tyrosine 3-Monooxygenase/*biosynthesis/genetics
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-8146 (URN)10.1002/jemt.20495 (DOI)17661370 (PubMedID)
Available from: 2008-01-15 Created: 2008-01-15 Last updated: 2017-12-14Bibliographically approved
7. Marked sympathetic component in the perivascular innervation of the dorsal paratendinous tissue targeted in sclerosing Polidocanol injection therapy of patellar tendinosis
Open this publication in new window or tab >>Marked sympathetic component in the perivascular innervation of the dorsal paratendinous tissue targeted in sclerosing Polidocanol injection therapy of patellar tendinosis
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-2432 (URN)
Available from: 2007-05-23 Created: 2007-05-23 Last updated: 2010-06-23Bibliographically approved

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