Gustav Andersson

Nerve-related characteristics of ventral paratendinous tissue in chronic Achilles tendinosis

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 α1-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.

Presence of substance P and the neurokinin-1 receptor in tenocytes of the human Achilles tendon

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.

Tenocyte hypercellularity and vascular proliferation in a rabbit model of tendinopathy: contralateral effects suggest the involvement of central neuronal mechanisms

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.

Substance P Is a Mechanoresponsive, Autocrine Regulator of Human Tenocyte Proliferation

It has been hypothesised that substance P (SP) may be produced by primary fibroblastic tendon cells (tenocytes), and that this production, together with the widespread distribution of the neurokinin-1 receptor (NK-1 R) in tendon tissue, could play an important role in the development of tendinopathy, a condition of chronic tendon pain and thickening. The aim of this study was to examine the possibility of endogenous SP production and the expression of NK-1 R by human tenocytes. Because tendinopathy is related to overload, and because the predominant tissue pathology (tendinosis) underlying early tendinopathy is characterized by tenocyte hypercellularity, the production of SP in response to loading/strain and the effects of exogenously administered SP on tenocyte proliferation were also studied. A cell culture model of primary human tendon cells was used. The vast majority of tendon cells were immunopositive for the tenocyte/fibroblast markers tenomodulin and vimentin, and immunocytochemical counterstaining revealed that positive immunoreactions for SP and NK-1 R were seen in a majority of these cells. Gene expression analyses showed that mechanical loading (strain) of tendon cell cultures using the FlexCell© technique significantly increased the mRNA levels of SP, whereas the expression of NK-1 R mRNA decreased in loaded as compared to unloaded tendon cells. Reduced NK-1 R protein was also observed, using Western blot, after exogenously administered SP at a concentration of 10−7 M. SP exposure furthermore resulted in increased cell metabolism, increased cell viability, and increased cell proliferation, all of which were found to be specifically mediated via the NK-1 R; this in turn involving a common mitogenic cell signalling pathway, namely phosphorylation of ERK1/2. This study indicates that SP, produced by tenocytes in response to mechanical loading, may regulate proliferation through an autocrine loop involving the NK-1 R.

Substance P accelerates hypercellularity and angiogenesis in tendon tissue and enhances paratendinitis in response to Achilles tendon overuse in a tendinopathy model

Background Tenocytes produce substance P (SP), and its receptor (neurokinin-1 receptor (NK-1R)) is expressed throughout the tendon tissue, especially in patients with tendinopathy and tissue changes (tendinosis) including hypercellularity and vascular proliferation. Considering the known effects of SP, one might ask whether SP contributes to these changes. Objectives To test whether development of tendinosis-like changes (hypercellularity and angiogenesis) is accelerated during a 1-week course of exercise with local administration of SP in an established Achilles tendinopathy model. Methods Rabbits were subjected to a protocol of Achilles tendon overuse for 1 week, in conjunction with SP injections in the paratenon. Exercised control animals received NaCl injections or no injections, and unexercised, uninjected controls were also used. Tenocyte number and vascular density, as well as paratendinous inflammation, were evaluated. Immunohistochemistry and in situ hybridisation to detect NK-1R were conducted. Results There was a significant increase in tenocyte number in the SP-injected and NaCl-injected groups compared with both unexercised and exercised, uninjected controls. Tendon blood vessels increased in number in the SP-injected group compared with unexercised controls, a finding not seen in NaCl-injected controls or in uninjected, exercised animals. Paratendinous inflammation was more pronounced in the SP-injected group than in the NaCl controls. NK-1R was detected in blood vessel walls, nerves, inflammatory cells and tenocytes. Conclusions SP accelerated the development of tendinosis-like changes in the rabbit Achilles tendon, which supports theories of a potential role of SP in tendinosis development; a fact of clinical interest since SP effects can be effectively blocked. The angiogenic response to SP injections seems related to paratendinitis.

Marked sympathetic component in the perivascular innervation of the dorsal paratendinous tissue of the patellar tendon in arthroscopically treated tendinosis patients

During the recent years, a few studies have shed new light on the innervation patterns of the human patellar tendon, but the area of the loose paratendinous connective tissue dorsal to the proximal tendon proper has yet not been investigated. That is a drawback, since this is the area targeted in promising treatment regimens of chronic painful patellar tendinosis, namely sclerosing Polidocanol injection therapy, and a new surgical method conforming to ultrasound and color Doppler guided arthroscopic shaving, directed at neovessels found in the region. The present study thus aimed at investigating the paratendinous area dorsal to the proximal patellar tendon proper in seven patients being operated for tendinosis. Biopsies were collected through the new arthroscopic technique, approaching the tendon from the dorsal side. Samples were investigated using immunohistochemistry with antibodies delineating general (PGP 9.5), sensory (SP/CGRP), and sympathetic (TH/NPY) nerve patterns, and also antibodies against α1- and α2A-adrenoreceptors. Both small and large blood vessels had a marked perivascular innervation (PGP 9.5). Surprisingly, this perivascular innervation was found only to a very limited extent to correspond to sensory nerves, while there were marked immunoreactions for sympathetic markers. Adrenoreceptor immunoreactions frequently occurred in blood vessel walls. In conclusion, this study demonstrates, for the first time, the innervation patterns of the area dorsal to the patellar tendon in man. It shows that the area investigated is under marked influence by the sympathetic nervous system. Thus, sympathetic effects are likely to occur for blood vessels of the area, which is interesting since color Doppler has revealed that vessels of this area (“neovessels”) display a pathologically high blood flow in tendinosis. The findings are discussed in relation to aspects of vascular regulation, and to pain symptoms of tendinosis.

Short-chain aliphatic acids in the interdigital gland secretion of reindeer (Rangifer tarandus L.), and their discrimination by reindeer

Interdigital gland secretion from reindeer (Rangifer tarandus L.) was analyzed by thin-layer chromatography and gas chromato-graphy. The short-chain acid fraction consisted of acetic, propionic, isobutyric,n-butyric, isovaleric, 2-methylbutyric,n-valeric, isocaproic, andn-caproic acids. The short-chain acids were produced by sterol esters when hydrolyzed in the gland-probably by microorganisms. Triglycerides present did not contain any short-chain acids. By testing isovaleric acid and isobutyric acid applied on small filter papers placed in a pen and measuring the number of sniffings on and towards the samples, we elicited good response at 1 ng application compared with the blanks, while pivalic acid gave no response under the same conditions.

Volatile compounds from the tarsal scent gland of reindeer (Rangifer tarandus)

Volatile compounds from the tarsal scent gland of reindeer (Rangifer tarandus) were isolated by a precolumn technique and analyzed on a combined gas chromatograph-mass spectrometer. Saturated aldehydes and alcohols were found in both male and female tarsal scent gland.

Volatile compounds from the interdigital gland of reindeer (Rangifer t. tarandus L.)

The isolation, identification, and synthesis of volatile compounds from the interdigital gland of reindeer (Rangifer t. tarandus L.) are reported. Two of the major constituents have been identified as 1-hydroxy-7-methyl-3-octanone and 7-methyl-1-octen-3-one.

HUMAN TENOCYTES ARE STIMULATED TO PROLIFERATE BY ACETYLCHOLINE THROUGH AN EGFR SIGNALLING PATHWAY

Studies of human patellar and Achilles tendons have shown that primary tendon fibroblasts (tenocytes) not only have the capacity to produce acetylcholine (ACh) but also express muscarinic ACh receptors (mAChRs) through which ACh can exert its effects. In patients with tendinopathy (chronic tendon pain) with tendinosis, the tendon tissue is characterised by hypercellularity and angiogenesis, both of which might be influenced by ACh. In this study, we have tested the hypothesis that ACh increases the proliferation rate of tenocytes through mAChR stimulation and have examined whether this mechanism operates via the extracellular activation of the epidermal growth factor receptor (EGFR), as shown in other fibroblastic cells. By use of primary human tendon cell cultures, we identified cells expressing vimentin, tenomodulin and scleraxis and found that these cells also contained enzymes related to ACh synthesis and release (choline acetyltransferase and vesicular acetylcholine transporter). The cells furthermore expressed mAChRs of several subtypes. Exogenously administered ACh stimulated proliferation and increased the viability of tenocytes in vitro. When the cells were exposed to atropine (an mAChR antagonist) or the EGFR inhibitor AG1478, the proliferative effect of ACh decreased. Western blot revealed increased phosphorylation, after ACh stimulation, for both EGFR and the extracellular-signal-regulated kinases 1 and 2. Given that tenocytes have been shown to produce ACh and express mAChRs, this study provides evidence of a possible autocrine loop that might contribute to the hypercellularity seen in tendinosis tendon tissue.