Background: Although the tendon of the long head of the biceps is a well-known source of shoulder pain, the pathophysiological basis of this pain has yet to be explained. The aim of this study was to detect and characterize any nervous element of the tendon and to determine a possible explanation for pain originating from this structure.
Methods: The nature of the neuronal innervation of the tendon of the long head of the biceps was studied immunohistochemically, in four tendons from different human cadavers, with use of neurofilament antibody 2H3, neurofilament-like antibody 3A10, calcitonin gene-related peptide, substance P, and tyrosine hydroxylase.
Results: A large neuronal network, asymmetrically distributed along the length of the tendon with a higher degree of innervation at the tendon origin, was identified by the neurofilament and neurofilament-like antibodies 2H3 and 3A10. This innervation was found to be positive for calcitonin gene-related peptide and substance P, suggesting the presence of thinly myelinated or unmyelinated sensory neurons. It was also positive for tyrosine hydroxylase, suggesting a post-ganglionic sympathetic origin.
Conclusions and Clinical Relevance: These findings demonstrate that the tendon of the long head of the biceps is innervated by a network of sensory sympathetic fibers, which may play a role in the pathogenesis of shoulder pain.
1 Department of Orthopaedics-Traumatology (K.A. and A.H.) and Neurosciences Laboratory (D.McL. and D.K.), University of Crete, 711 10, Heraklion, Crete, Greece
2 1 Pindarou Street, 713 05 Heraklion, Crete, Greece. E-mail address: email@example.com
Currently, the pathophysiology of chronic tendon pain is not fully understood1-3. Overuse tendon injuries are infrequently characterized by the presence of inflammatory cells. Instead, degenerative changes resulting from a failure at self-repair are commonly found2-4. Several authors have presented data suggesting that the nervous system plays a major role not only in nociception but also in communication with the immune system with regard to degeneration and inflammation3,4. A large number of studies have demonstrated that the sympathetic innervation of the musculoskeletal system is involved in the regulation of blood flow and may participate in the inflammatory reaction5-7. In addition, the role of neuropeptides in the process of healing has recently been recognized8,9. Besides transmitting pain, neuropeptides such as substance P and calcitonin gene-related peptide, which are present in sensory nerves, are responsible for vasodilation and plasma extravasation. This process leads to so-called neurogenic inflammation5-7,10.
Immunohistochemical studies of the nerve distribution in the shoulder joint capsule8,11, in the subacromial bursa9, and in the coracoacromial ligament12 have been published, but we are not aware of any investigations of the neural structures innervating the tendon of the long head of the biceps. The aim of the present study was to detect and characterize the nervous elements of the long head of the biceps and to determine a possible explanation for pain originating from this structure.
Materials and Methods
The tendon of the long head of the biceps was harvested from the shoulders of four fresh male cadavers (age range at the time of death, fifty-five to eighty-one years). We did not include tissue from female cadavers only because they were unavailable. The tendons showed no signs of degeneration on hematoxylin and eosin staining and viewing under normal light microscopy. Each tendon was divided into three equal parts (Fig. 1), which were marked, fixed overnight in a solution of 4% paraformaldehyde, and embedded in paraffin. Longitudinal 10-μm tissue sections were cut with a microtome, mounted on super-frost glass slides, and immunostained with the avidinbiotin system according to the manufacturer's instructions (Vector Laboratories, Burlingame, California). The sections were washed twice, for ten minutes each time, with phosphate-buffered saline solution plus 0.1% Triton X. Subsequently, the sections were incubated with 10% fetal calf serum in phosphate-buffered saline solution for sixty minutes to block nonspecific binding before incubation with primary antibodies for substance P (1:5000 rabbit polyclonal; Chemicon International, Temecula, California), calcitonin gene-related peptide (1:100 rabbit polyclonal; Abcam, Cambridge, United Kingdom), tyrosine hydroxylase (1:100 mouse monoclonal; Biotrend Chemikalien, Cologne, Germany), neurofilament-like antibody 3A10 (1:100 mouse monoclonal; Developmental Studies Hybridoma Bank, Iowa City, Iowa), and neurofilament antibody 2H3 (1:100 mouse monoclonal; Developmental Studies Hybridoma Bank) overnight at 4°C.
After incubation with primary antibody, the sections were washed three times, for ten minutes each time, with phosphate-buffered saline solution plus 0.1% Triton X and then incubated with the secondary antibody (1:100 anti-mouse IgM-biotinylated [Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania] for the neurofilament antibody and tyrosine hydroxylase and 1:250 anti-rabbit IgG-biotinylated [Vector Laboratories] for substance P and calcitonin gene-related peptide) for two hours at room temperature.
After completion of the staining steps for both primary and secondary antibody, the sections were washed three times, for ten minutes each time, with phosphate-buffered saline solution plus 0.1% Triton X and incubated with avidin-biotinylated enzyme complex (ABC) solution (Vector Laboratories) for thirty minutes. After the sections were washed again three times, for ten minutes each time, with phosphate-buffered saline solution plus 0.1% Triton X, they were stained in diaminobenzidine-H2O2 solution (DAB peroxidase substrate; Sigma Chemical, St. Louis, Missouri) for fifteen minutes. Immunohistochemical controls were prepared by incubating sections with 10% fetal calf serum in phosphate-buffered saline solution instead of the primary antibody. The following steps were identical to those previously described for the experimental sections. Each section was observed with light microscopy, and all structures were photographed with use of a reversal camera.
Both the neurofilament (2H3) and the neurofilament-like (3A10) antibodies revealed a rich innervation, which was more common at the proximal insertion to the bone than at the distal musculotendinous junction. The staining patterns with the two antibodies appeared to be identical, showing a thin interconnected network of nerve fibers (described as a “net-like pattern”) with neuronal buttons, suggesting terminal/secretory endings. These nerve-fiber bundles, which made up the main trunk of the network, gave rise to smaller bundles and fine individual nerves (Figs. 2-A and 2-B).
When a number of different antibodies were used on consecutive sections, positive labeling was found, in overlapping positions of each section, for calcitonin gene-related peptide, substance P, and tyrosine hydroxylase. In each case, a net-like pattern that was identical to that seen with the neurofilament antibody was observed. Staining of consecutive sections with hematoxylin and eosin revealed no defects in tissue integrity. The morphological structure of this network of nerve fibers was similar in the four tendons that we studied. A more prominent neural network was found in the proximal third than in the middle third, and the network was even less prominent in the distal third of each specimen.
The neurofilament antibody 2H3 and neurofilament-like antibody 3A10 that were used as general neuronal markers in this study revealed a prominent neuronal structure. The two antibodies displayed an identical net-like pattern, which was not observed in any of our control specimens (in which the primary antibody was omitted).
To explain the nature of this nerve network, we examined the expression of calcitonin gene-related peptide, which is colocalized with substance P in sensory nerve fibers5-7, in consecutive sections. The autonomic innervation was visualized by the use of tyrosine hydroxylase antibodies. An identical net-like pattern of innervation, in which thick and coarse-appearing nerve fibers gave rise to thinner ones, was displayed.
Trophic effects of autonomic and sensory innervation on tendon origin have previously been described4-7. A growing number of studies have raised the question of whether the nervous system plays a functional role in the pathogenesis of pain and degeneration in this area6-8,13-17. These studies have shown that tendons are supplied by nerve fibers containing sensory and sympathetic neurotransmitters, which, apart from nociception, participate in vasoregulation and immune responses. However, these nerve fibers were detected in association with blood vessels and within nerve bundles5-7.
Substance P and calcitonin gene-related peptide are considered to be the most important mediators of neurogenic inflammation6,7,13,18. Substance P is involved in vascular permeability, vasodilation, and the release of cytokines and growth factors from immune cells. Calcitonin gene-related peptide plays an important vasoactive role, leading to vasodilation and plasma extravasation. It also enhances the vasodilatory effect of substance P4-7,14-18. Substance P coexists with calcitonin generelated peptide in tendons and ligaments5-7, and both have been implicated in the healing process of ruptured tendons in association with new nerve ingrowth and a specific temporal pattern of neuropeptide occurrence18. On the other hand, there is evidence that sympathetic innervation participates in the pathogenesis of both skeletal and tendon pain by interacting with sensory afferent nerve fibers and immune cells19. Imbalances between vasoconstrictor mediators (tyrosine hydroxylase) and vasodilator mediators (substance P and calcitonin gene-related peptide) may lead to abnormal perfusion, nutritional deficiency, ischemia, and hypoxic degeneration5-7.
The current study demonstrated that the tendon of the long head of the biceps in humans contains a network of sensory and sympathetic nerve fibers. Additionally, it appears that these structures are not distributed homogeneously throughout the tendon body, but rather are primarily located in its proximal part. The reason for this heterogeneity is not clear.
In conclusion, the major new findings of this study were (1) the tendon of the long head of the biceps contains a large network of sensory and sympathetic nerve fibers, (2) this type of innervation is not associated with blood vessels (as it was described to be in previous studies3,6,7), and (3) the innervation is not distributed evenly throughout the tendon but rather is found predominantly near its insertion. ▪
Investigation performed at the Department of Orthopaedics-Traumatology and Neurosciences Laboratory, University of Crete, Heraklion, Crete, Greece
The authors did not receive grants or outside funding in support of their research or preparation of this manuscript. They did not receive payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.
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