Analysis of Nerve Endings in the Superior Labrum-Biceps Complex by Fluorescence Immunohistochemistry and Confocal Laser Microscopy

Abstract

Objectives  The capsuloligamentous structures of the shoulder work as static stabilizers, together with the biceps and rotator cuff muscles, increasing the contact surface of the glenoid cavity. Free nerve endings and mechanoreceptors have been identified in the shoulder; however, there are a few studies that describe the presence of these nerves in the biceps' insertion. The present study aimed to describe the morphology and distribution of nerve endings using immunofluorescence with protein gene product 9.5 (PGP 9.5) and confocal microscopy.

Methods  Six labrum-biceps complexes from six fresh-frozen cadavers were studied. The specimens were coronally cut and prepared using the immunofluorescence technique. In both hematoxylin and eosin (H&E) and immunofluorescence, the organization of the connective tissue with parallel collagen fibers was described.

Results  In the H&E study, vascular structures and some nerve structures were visualized, which were identified by the elongated presence of the nerve cell. All specimens analyzed with immunofluorescence and confocal microscopy demonstrated poor occurrence of morphotypes of sensory corpuscles and free nerve endings. We identified free nerve endings located in the labrum and in the bicipital insertion, and sparse nerve endings along the tendon. Corpuscular endings with fusiform, cuneiform, and oval aspect were identified in the tendon.

Conclusion  These findings support the hypothesis that the generation of pain in the superior labral tear from Anterior to posterior (SLAP) lesions derives from the more proximal part of the long biceps cord and even more from the upper labrum. Future quantitative studies with a larger number of specimens may provide more information on these sensory systems.

Introduction

The superior glenoid labrum serves as the attachment site for the insertion of the biceps' long head tendon, thus being susceptible to the injury produced by its detachment from the glenoid, from anterior to posterior, known as superior labral tear from anterior to posterior (SLAP). ¹ Andrews et al. ² was the first to describe injuries in the superior labrum. These injuries can be frequently observed in young athletes of “throwing sports”, being secondary to repetitive micro lesions. ³ It may also occur in association with other traumatic situations, such as primary or recurrent glenohumeral dislocation. ⁴ Clinically, we observed that, subsequent to anatomical repairs of SLAP lesions, patients presented a prolonged period of pain when compared to those submitted to biceps release procedures. ^{5 6 7} The pathophysiological mechanism of pain, present in both lesion and surgical repair as well as in the eventual failure of treatment, has involved the presence of nociceptors. Histological studies of the labral complex have demonstrated the presence of free nerve endings and mechanoreceptors. ^{8 9} Symptoms such as pain, instability, and a sensation of joint “locking can be attributed to weakening of the proprioceptive reflex. ¹⁰ Although reports of SLAP injury are frequent in the literature, recent studies have reported a considerable increase in the number of surgical repairs of these lesions in the last decade. ¹¹ Currently, the advances in antibody markers specific to nerve endings, associated with confocal laser microscopy, allow the visualization and detailing of nerve structures with three-dimensional images. ^{12 13 14} The purpose of this investigation was to histologically evaluate the mid-portion of the superior labrum-biceps complex. We hypothesized the transition zone between the labrum and the long head of the biceps tendon would contain nerve endings and vessels. We look forward to establish a parallel between our findings and the pathophysiology of the SLAP lesion and tendinopathy of the long head of the biceps.

Methods

We used 6 superior labrum-biceps complex (SLBC) from frozen human cadavers (3 males, 3 females) aged from 20 to 70 years old. Approval for this project was granted and monitored through our institution by the ethics committee (No. 443.172). We used a selective pan-neuronal marker, the pan-axonal protein gene product 9.5 (PGP 9.5) (Thermo Fisher Scientific Inc., Rockford, IL), to highlight sensory innervation. The sections were washed with cold 0.1 M phosphate buffer solution (PBS, Laborclin, Pinhais, PR, Brazil) containing 3% Triton X-100 (TX-100, Inlab, Diadema, SP, Brazil). The tissues were washed and incubated in primary antibody for 2 hours; then, the secondary antibody was conjugated to a fluorescent tag (Alexa Fluor 488 goat anti-rabbit IgG, Thermo Fisher Scientific Inc., Rockford, IL, US). The sections were washed and sealed, and the slides were stored at -70 °C. All 36 sections of the specimens were examined with a confocal laser scanning microscope system (LSM710, Carl Zeiss Microscopy, Jena, Germany). We used hematoxylin & eosin (H&E) and Masson trichrome stain in the mid-portion of the SLBC specimens, and the median nerve was a quality control for immunofluorescence. The results are expressed as means and standard deviation (SD). The data were analyzed using software GraphPad Prism (version 6.0 for Windows, GraphPad Software, San Diego, California, USA, 2015).

Results

Light microscopy analysis of the sections showed easily distinguished the biceps tendon with scarce blood vessels, spaced apart, thin, and slightly wavy ( Fig. 1, A and B ). In the transition portion between the biceps and the labrum, we could observe complex structures, with elongated cells suggesting nerve cells ( Fig. 2 A, B and C ).

Fig. 1.

(A e B) Longitudinal and histological section (10 µm) stained with hematoxylin & eosin. Note the blood vessels (circles) on the biceps tendon.

Fig. 2.

Fusiform and conical nerves ending of conjunctive tissue in the transition zone between the labrum and biceps. Increase of 50x (A),100x (B) and 200x (C).Longitudinal histological section (10 µm) stained with hematoxylin & eosin.

Confocal microscopic revealed that free nerve endings through the long head of the biceps tendon, from one to six μm in length, parallel to the collagen bundles, also dissociated from the presence of blood vessels ( Fig. 3 ). In the mid-portion of the SLBC, we identified nerve fibers measuring between 60 and 70 μm in diameter, branching in smaller nerve bundles ( Fig. 4 ).

Fig. 3.

Section of the biceps tendon (50 µm thick). Observe the free nerve endings (arrows) in the conjunctive tissue, analyzed by confocal laser microscope (immunofluorescence, scale 50 µm).

Fig. 4.

Transition zone between the labrum and biceps. Note the nerve fiber in the deep layer measuring between 60 and 70 µm in diameter, bifurcating into smaller nerve bundles (immunofluorescence, scale 50 µm).

In addition, the mid-portion of the SLBC, we also observed nerve fibers of smaller caliber, from seven to ten micrometers μm in diameter, close to the vessel, exhibiting peripheral and intraluminal immunoreactivity to PGP 9.5. In the slides submitted to the antigenic recovery technique, the presence of complex nerve endings with variable dimensions, ranging from 150 to 350 μm in length and 80 to 100 μm in width. In the articular face of the samples, next to the labral region and the labrum-biceps transition, we observed axons with between 10 and 20 μm of thickness, and different spatial formats, with predominance of spindle, conical and oval shapes ( Fig. 5 A, B and C ).

Fig. 5.

Nerves endings with different shapes, ranging from 150 to 350 µm in length and 80 to 100 µm in width (Antigenic recovery technique, scale 50 µm).

Discussion

The knowledge of the neuroanatomy of the passive stabilizing structures of the shoulder helps to understand the proprioceptive mechanisms of joint protection and stabilization. The tendon of the long head of the biceps has been studied as a cause of pain in the glenohumeral joint, either in tendinopathies. ¹⁵ Alpantaki et al. ¹⁶ was the first to study these neural elements in the long biceps' tendon, which he described as containing a large network of sympathetic nerve fibers and sensors, not associated with blood vessels, and with neural distribution predominantly close to its insertion. Our findings were partially compatible with those reported by Alpantaki et al. ¹⁶ We have found a few fine nerve fibers, following their own pathways, isolated from the vessels and dispersed along the collagen fiber structure. We also observed, proximally, larger fibers at the labrum-biceps transition around vascular structures, as reported by Boesmueller et al., ¹⁷ who also demonstrated a density of nerves in the proximal segment of the long biceps tendon, similar to the anterior portion of the superior labrum. In agreement with these authors, we observed the presence of neural structures occurring predominantly in the more proximal portion of the biceps tendon. In the samples submitted to the antigenic recovery technique, we observed complex nerve endings near the labral portion and labrum-bicipital transition, which are predominantly distributed in the layers closest to the glenoid joint, making it possibly the first region to be stimulated by contact with the humeral head during shoulder movement. Information exists on the location of neural structures in the upper labrum and bicipital anchor. Among the descriptions, Hashimoto et al. ¹⁸ showed the isolated presence of free nerve endings in the labrum and capsular transition; according to Vangsness et al., ⁸ we have only free nerve endings; Witherspoon et al. ⁹ describe only nervous fascicles in the periphery of the anteroinferior and posteroinferior labrum. In relation to the pathophysiological aspect of the SLAP lesion in the shoulder, it seems evident that the bicipital tendon acts as a potential pain generator, having a greater density of neural structures (neurofilaments) in the proximal parts, as observed in others studies. ^{17 19 20} We identified nerve endings in the three regions (biceps tendon, labrum transition and biceps, upper labrum) of the complex formed by the superior labrum and bicipital insertion, with well differentiated aspects. In the proximal segment of the biceps' tendon, we found fine nerve fibers, without association with vascular structures, and no complex nerve endings were identified. In the transition zone between the labrum and the biceps, we found thick nerve bundles, accompanying arterial blood vessels present in this area. In the labral region and in the labrum-bicipital transition, we found complex nerve endings with oval, conical and fusiform structures. ²¹ There is a need for new studies to confirm our findings, as well as to accurately identify the mechanoreceptors, including testing other antibodies and cellular markers, thus increasing the number of individuals in the sample, seeking to comprehend all factors related to the interaction of biomechanics and proprioceptive system of the shoulder. The present preliminary study shows the morphology of nerve endings and accurately identifies mechanoreceptors by immunofluorescence. However, the number of specimens and including other antibodies and cellular markers, it would be interesting to compare the anatomical and pathological conditions.

Conclusion

We identified nerve endings in the three regions (biceps tendon, labrum transition and biceps, upper labrum) of the complex formed by the superior labrum and the bicipital insertion, , with well differentiated aspects. In the proximal segment of the biceps' tendon, we found fine nerve fibers, without association with vascular structures, and no complex nerve endings were identified. In the transition zone between the labrum and the biceps, we found thick nerve bundles, accompanying arterial blood vessels present in this area. In the labral region and in the labrum-bicipital transition, we found complex nerve endings, and it was possible to identify them in relation to the spatial format, which consisted of oval, conical, and fusiform structures.

Funding Statement

Suporte Financeiro Este trabalho teve suporte do Instituto de Pesquisa e Ensino Home, Brasília, DF, Brasil.