Sture Forsgren

In vivo microdialysis and immunohistochemical analyses of tendon tissue demonstrated high amounts of free glutamate and glutamate NMDAR1 receptors, but no signs of inflammation, in Jumper's knee.

This investigation describes, to our knowledge, the first experiment where the microdialysis technique was used to study certain metabolic events in human patellar tendons in combination with immunohistochemical analyses of tendon biopsies. In five patients (four men and one woman) with a long duration (range 12‐36 months) of pain symptoms from Jumpers knee (localized tenderness in the patellar tendon verified as tendon changes with ultrasonography or MRI), and in five controls (four men and one woman) with normal patellar tendons, a standard microdialysis catheter was inserted into the patellar tendon under local anestesia. The local concentrations of glutamate (excitatory neurotransmitter) and prostaglandin E2 (PGE2) were registered under resting conditions. Samplings were done every 15 min during a 2 h period. In all individuals (patients and controls) biopsies were taken for immunohistochemical analyses.

The innervation pattern of the human Achilles tendon: studies of the normal and tendinosis tendon with markers for general and sensory innervation

Pain-free normal Achilles tendons and chronic painful Achilles tendons were examined by the use of antibodies against a general nerve marker (protein gene-product 9.5, PGP9.5), sensory markers (substance P, SP; calcitonin gene-related peptide, CGRP), and immunohistochemistry. In the normal tendons, immunoreactions against PGP9.5 and against SP/CGRP were encountered in the paratendinous loose connective tissue, being confined to nerve fascicles and to nerve fibers located in the vicinity of blood vessels. To some extent, these immunoreactions also occurred in the tendon tissue proper. Immunoreaction against PGP9.5 and against SP/CGRP was also observed in the tendinosis samples and included immunoreactive nerve fibers that were intimately associated with small blood vessels. In conclusion, Mechanoreceptors (sensory corpuscles) were occasionally observed, nerve-related components are present in association with blood vessels in both the normal and the tendinosis tendon.

Neurokinin 1-receptors and sensory neuropeptides in tendon insertions at the medial and lateral epicondyles of the humerus. Studies on tennis elbow and medial epicondylalgia

There is no information on the sensory innervation at the flexor muscle origin at the medial epicondyle of the humerus and it is not known if substance P receptors (Neurokinin 1‐receptors, NK1‐R) are present in tendon insertions in general. In the present investigation, we have studied the muscle origin in patients suffering from medial epicondylalgia and tennis elbow. Immunohistochemistry and antibodies to substance P (SP) and CGRP as well as the general nerve marker PGP 9.5 were used. Specific immunoreactions were observed in nerve bundles and as free nerve fibers. The immunoreactive structures were partly seen in association with some of the blood vessels. The observations constitute a morphological correlate for the occurrence of nerve mediated effects in this region. By using immunohistochemistry and antibodies to NK1‐R, the distribution of this receptor was studied at the insertion of the proximal tendon of the extensor carpi radialis brevis muscle at the lateral epicondyle. Specific immunoreactions were seen as varicose fibers occurring as single fibers or grouped into bundles, indicating that SP has effects in the nerves in this region. The results give further evidence for a possible neurogenic involvement in the pathophysiology of tennis elbow and in medial epicondylalgia.

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.

Glutamate NMDAR1 receptors localised to nerves in human Achilles tendons. Implications for treatment

In this investigation, we show the presence of both free glutamate (microdialysis) and glutamate NMDAR1 receptors (immunohistochemical analyses of tendon biopsies), in tendons from patients with chronic Achilles tendon pain (Achilles tendinosis) and in controls (pain-free tendons). The NMDAR1 immunoreaction was usually confined to acetylcholinesterase-positive structures, implying that the reaction is present in nerves. Glutamate is a potent pain mediator in the human central nervous system, and in animals it has been shown that peripherally administered glutamate NMDA receptor antagonists diminish the response to formalin-induced nociception. Our present finding of glutamate NMDA receptors in human Achilles tendons might have implications for pain treatment.

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.

Immunoreactive atrial and brain natriuretic peptides are co-localized in Purkinje fibres but not in the innervation of the bovine heart conduction system

SummaryRecently, we observed that atrial natriuretic peptide (ANP) immunoreactivity was present in Purkinje fibres and nerve fibre varicosities in the conduction system of the bovine heart. In order to elucidate further the morphological correlation between natriuretic peptides and the conduction system, the distribution of brain natriuretic peptide (BNP) was examined. The different parts of the conduction system in the bovine heart were dissected out and processed for immunohistochemistry with antisera against BNP and ANP. BNP immunoreactivity was frequently observed in Purkinje fibres of the atrioventricular bundle, whereas only a few Purkinje fibres in the ventricular part of the conduction system showed immunoreaction. BNP immunoreactivity was detected in regions of the Purkinje fibres which also showed ANP immunoreactivity. BNP immunoreactivity was not observed in nerve fibre varicosities. Methodologically, a larger number of small BNP immunofluorescent granular structures was observed by using an elution-restaining technique instead of conventional immunohistochemistry. The present study shows that BNP and ANP immunoreactivities frequently occur in the atrioventricular bundle and that they are co-localized in Purkinje fibres, but not in nerve fibre varicosities, in the conduction system. As previously has been proposed for ANP, the present observations suggest that also BNP may act in an autocrine and/or paracrine way in the conduction cells.

Vascular NK-1 receptor occurrence in normal and chronic painful Achilles and patellar tendons : studies on chemically unfixed as well as fixed specimens.

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.

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.

Levels of gastrin-releasing peptide and substance P in synovial fluid and serum correlate with levels of cytokines in rheumatoid arthritis

It is well known that cytokines are highly involved in the disease process of rheumatoid arthritis (RA). Recently, targeting of neuropeptides has been suggested to have potential therapeutic effects in RA. The aim of this study was to investigate possible interrelations between five neuropeptides (bombesin/gastrin-releasing peptide (BN/GRP), substance P (SP), vasoactive intestinal peptide, calcitonin-gene-related peptide, and neuropeptide Y) and the three cytokines tumour necrosis factor (TNF)-α, IL-6, and monocyte chemoattractant protein-1 in synovial fluid of patients with RA. We also investigated possible interrelations between these neuropeptides and soluble TNF receptor 1 in serum from RA patients. Synovial fluid and sera were collected and assayed with ELISA or RIA. The most interesting findings were correlations between BN/GRP and SP and the cytokines. Thus, in synovial fluid, the concentrations of BN/GRP and SP grouped together with IL-6, and SP also grouped together with TNF-α and monocyte chemoattractant protein-1. BN/GRP and SP concentrations in synovial fluid also grouped together with the erythrocyte sedimentation rate. In the sera, BN/GRP concentrations and soluble TNF receptor 1 concentrations were correlated. These results are of interest because blocking of SP effects has long been discussed in relation to RA treatment and because BN/GRP is known to have trophic and growth-promoting effects and to play a role in inflammation and wound healing. Furthermore, the observations strengthen a suggestion that combination treatment with agents interfering with neuropeptides and cytokines would be efficacious in the treatment of RA. In conclusion, BN/GRP and SP are involved together with cytokines in the neuroimmunomodulation that occurs in the arthritic joint.