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Introduction: Sensory nerve endings transmit mechanical stimuli into afferent neural signals and form the basis of proprioception, giving rise to the self-perception of dynamic stability of joints. We aimed to analyze the three-dimensional structure of periarticular corpuscular sensory nerve endings in a carpal ligament to enhance our understanding of their microstructure. Methods: Two dorsal parts of the scapholunate ligament were excised from two human cadaveric wrist specimens. Consecutive cryosections were stained with immunofluorescence markers protein S100B, neurotrophin receptor p75, protein gene product 9.5 (PGP 9.5), and 4′,6-diamidino-2-phenylindole. Three-dimensional images of sensory nerve endings were obtained using confocal laser scanning microscopy, and subsequent analysis was performed using Imaris software. Results: Ruffini endings were characterized by a PGP 9.5-positive central axon, with a median diameter of 4.63 μm and a median of 25 cells. The p75-positive capsule had a range in thickness of 0.94 μm and 15.5 μm, consisting of single to three layers of lamellar cells. Ruffini endings were significantly smaller in volume than Pacini corpuscles or Golgi-like endings. The latter contained a median of three intracorpuscular structures. Ruffini endings and Golgi-like endings presented a similar structural composition of their capsule and subscapular space. The central axon of Pacini corpuscles was surrounded by S100-positive cells forming the inner core which was significantly smaller than the outer core, which was immunoreactive for p75 and PGP 9.5. Conclusion: This study reports new data regarding the intricate outer and intracorpuscular three-dimensional morphology of periarticular sensory nerve endings, including the volume, number of cells, and structural composition. These results may form a basis to differ between normal and pathological morphological changes in periarticular sensory nerve endings in future studies.

Sensory nerve endings are an integral part of the sensorimotor system, giving rise to the self-perception of dynamic stability of joints [1]. These specialized nerve endings detect mechanical stimuli and transmit the afferent neural signal to the central nervous system via pseudounipolar neurons, where it is processed as a spinal monosynaptic or oligosynaptic reflex to supraspinal proprioceptive centers [2, 3]. Specific types of sensory nerve endings have been analyzed in different types of human tissue including the skin [4], muscle [5], the periodontal ligament [6], vessels [7], and periarticular joint tissue [8]. Each type of sensory nerve ending possesses individual neurophysiological characteristics resulting in conscious proprioception, such as kinesthesia, joint position sense, and tensile strength, as well as unconscious proprioceptive qualities, including postural stability, joint stability, and feed-forward control [9].

Periarticular proprioception involves corpuscular sensory nerve endings, namely, Ruffini endings, Pacini corpuscles, and Golgi-like endings, according to the classification of Freeman and Wyke [10]. The morphological study of sensory nerve endings is significant, as it allows to enhance our knowledge on the proprioceptive qualities of tissues. Equally important are the clinical implications, given that specific effects of pathologies on sensory nerve endings have been described. A two-dimensional (2D) immunofluorescent study showed that in distal diabetic sensorimotor polyneuropathy the neural compartments of cutaneous Pacini corpuscles are disarranged and that axons lack expression of mechanoproteins, including PIEZO2, ASIC2, and TRPV4 [11]. A three-dimensional (3D) immunofluorescent study revealed a reduced density of Meissner corpuscles in Charcot-Marie-Tooth disease, and afferent myelinated fibers displayed signs of degeneration including fragmentation, swellings, and a variability in fiber caliber [12]. Furthermore, osteoarthritis induces severe structural and immunohistochemical alterations of sensory nerve endings in ligaments of the basal thumb. In healthy joints, Ruffini endings constitute the predominant type of corpuscle [13], whereas unclassifiable corpuscles are most prevalent in symptomatic osteoarthritis [14]. An intact proprioceptive function of the periarticular joint tissue is important for joint-protective ligamentous reflexes [15], with their degeneration leading to an impairment of neuromuscular control of the joint [16]. Therefore, analysis of the sensory nerve endings’ inner and outer microanatomy is essential to advance our understanding of the histopathological manifestations of diseases affecting the peripheral nervous system.

Throughout the past century, various methods have been employed to conduct research on the morphology of sensory nerve endings, yet many “methods begin to get slowly or quickly into cul-de-sac” [17]. With the aid of transmission electron microscopy, the ultrastructure of sensory nerve endings has been analyzed. However, this method has drawbacks as it is limited to 2D images and requires meticulous sample preparation [18], making it challenging to analyze entire anatomical structures such as ligaments. Within optical microscopy, immunohistochemical [19] and immunofluorescent protocols [8] have been developed, with the latter enabling simultaneous multiple immunostaining to visualize different structural components of sensory nerve endings. However, what major challenge does this vast field of research currently face? The main challenge of accurately analyzing the morphology of sensory nerve endings is the limitations set by performing 2D histological analysis on the intricate 3D structure, thereby causing insufficient visualization leading to the issue of unclassifiable sensory nerve endings [20]. By extending the visualization in the z-plane with 3D microscopy, the fine morphology of the neuronal structures is displayed more accurately [21].

A recently established method with 3D triple immunofluorescence has enabled the visualization of the microstructure of sensory nerve endings in human ligaments [22]. Therefore, the aim of the study was to investigate the precise anatomical microstructure of periarticular corpuscular sensory nerve endings, attempting to provide a basis for understanding pathological alterations.

Cadaver Specimens

The study was conducted on two dorsal parts of the scapholunate ligament (dSL), which were dissected from unfixed human cadaveric wrists of an 80-year-old female and a 90-year-old male with a postmortem delay of 2 days between death and tissue harvesting. The cadaveric tissue originated from the Institute of Anatomy of a university hospital. Causes of death were pneumonia due to lung cancer and acute cystitis due to prostate cancer, respectively. This ligament was chosen as past studies have confirmed the presence of corpuscular sensory nerve endings [23]. Furthermore, the dSL is the most frequently injured ligament of the human carpus, and as the primary stabilizer of the human wrist, it plays a fundamental role in the proprioceptive control of this joint [24]. Specimens were cooled at 3–4°C until dissection. Both wrists were assessed macroscopically prior to the dissection of ligaments to exclude ligament injuries, bony lesions, and structural abnormalities.

Dissection and Fixation of Ligaments

Dissection of the dSL was carried out with 2.5 loupe magnification. The skin was incised at the dorsal carpus, the extensor retinaculum, and extensor tendons excised, followed by a radial capsulotomy to expose the dSL [25]. The dSL was excised at its bone insertions at the lunate and scaphoid. The dorso-distal-scaphoidal region of the dSL was marked with a fine surgical suture for further anatomical orientation. Harvested dSL was fixed in 4% buffered paraformaldehyde (pH = 7.4), followed by cryoprotection in 30% sucrose buffer. Ligaments were mounted with optimal cutting temperature compound (Sakura Finetek Europe B.V., Zoeterwoude, Netherlands), frozen in −80°C isopentane, and stored at the same temperature.

Immunofluorescence Analysis

Seventy µm-thick cryosections were performed using a cryostat (Leica CM3050 S, Leica Microsystems, Mannheim, Germany). Immunofluorescent staining and 3D imaging were conducted as described in detail in a previous protocol [22]. Free-floating triple immunofluorescent staining was performed. Monoclonal rabbit antisera against S100B (1:250, RRID: AB_2819079) was used. The cytoplasmatic, calcium-binding protein S100B is a marker for Schwann-like cells [26]. Furthermore, the monoclonal mouse anti-nerve growth factor receptor p75 (1:250, RRID: AB_10663781) was used as a marker for the perineural cells in the capsule of sensory nerve endings [27]. The polyclonal guinea pig antisera against protein gene product 9.5 (PGP 9.5) (1:1,000, RRID: AB_10002332) was utilized as an axonal marker [28]. The secondary antibodies included AlexaFluor® 488 (1:800, donkey anti-mouse IgG, RRID: AB_2340846), Cy3 (1:800, donkey anti-rabbit IgG, RRID: AB_2307443), Cy5 (1:800, donkey anti-guinea pig IgG, RRID: AB_2340462), and 4′,6-diamidino-2-phenylindole (DAPI) (1:1,000). Positive and negative control stainings on human nerve and ligamentous tissue were performed in parallel as described by Meklef et al. [22].

Coverslips were prepared by pipetting fluorescent beads (TetraSpeck™ microspheres 0.1 μm, Thermo Fisher Scientific, Waltham, MA, USA) in a marked area prior to the mounting of the coverslip for later measurement of experimental point spread functions (PSFs). Stained sections were transferred onto glass slides, which contained a 70-μm-thick spacer to guarantee a consistent distance between the coverslip and the glass slide and were mounted with distilled water.

Next, 3D imaging was performed with confocal laser scanning microscopy (Leica TCS SP8, Leica Microsystems, Mannheim, Germany) and a ×40/1.10 water HC PL APO CS2 objective. A tile scan of the whole specimen was conducted to identify regions of interest. Hereafter, sensory nerve endings were imaged with a voxel size of 55 × 55 × 210 nm and an excitation wavelength 405 nm (DAPI), 488 (AlexaFluor®), 561 nm (Cy3), 633 nm (Cy5), a detection range 408–476 nm (DAPI), 491–547 nm (Alexa Fluor®), 564–652 nm (Cy3), and 639–749 nm (Cy5). To achieve optimal image sampling density, the image resolution was defined by the Nyquist criteria for a wavelength of 488 nm, the excitation wavelength of AlexaFluor®. In addition, imaging of fluorescent 100 nm beads was conducted with the same beforementioned settings, and the acquired data were analyzed by the PSF Distiller from Huygens Professional 20.10 (Scientific Volume Imaging, 2022/SVI, Hilversum, Netherlands, RRID: SCR_007370) to create a PSF for each recorded channel.

In 3D imaging with confocal laser scanning microscopy, light scattering is caused by optical aberrations. To enhance final image quality, deconvolution was performed with Huygens Professional 20.10, which involves restoring the location of point sources in a digital image using the measured experimental PSFs.

Analysis of the deconvolved images was conducted with Imaris version 9.7.2 (Oxford Instruments, Abingdon, UK, RRID: SCR_007370). Surface objects were generated to create 3D visualizations of sensory nerve endings for qualitative and quantitative analysis.

Analysis of Sensory Nerve Endings

Corpuscular sensory nerve endings were localized in each section plane and classified according to Freeman and Wyke [10], modified by Hagert [20], which includes Ruffini endings, Pacini corpuscles, Golgi-like endings, and free nerve endings. The purpose of this study was to analyze the microstructure of corpuscular sensory nerve endings, to which free nerve endings do not pertain. The first step of analysis was performed on the outer structure of the corpuscular sensory nerve endings and included measuring the length and width, the 3D volume, the total number of nuclei, and classifying the 3D shape. Next, the microstructure of the corpuscular sensory nerve endings was analyzed. For Ruffini endings, this included measuring the diameter of the central axon and its antibody distribution using histograms, the number of branching axons, the capsule thickness, and the subcapsular space. Afterward, the diameter of the central axon and its antibody distribution, the number of branching axons, the thickness, and antibody distribution of the inner and outer capsules were analyzed in Pacini corpuscles. Finally, the number of inner corpuscles and their antibody distribution, the capsule thickness, and the subcapsular space were recorded in Golgi-like endings.

Statistical Analysis

Median with minimum and maximum was given for descriptive statistics. The Kolmogorov-Smirnov test was performed to investigate data distribution. As statistical analysis of the diameter of the central axon of Ruffini endings and Pacini corpuscles as well as the statistical analysis of the inner and outer core of Pacini corpuscles showed a normal distribution, a two-tailed t test was performed with significance set at p ≤ 0.05. As the data of the capsule thickness and subscapular space in Ruffini endings and Golgi-like endings were found to not have a normal distribution, a Mann-Whitney test was performed, with the final level of significance of p ≤ 0.05. The corpuscular volume of all three sensory nerve endings showed a non-normal distribution. The subsequent statistical analysis was performed using the Kruskal-Wallis test followed by the Mann-Whitney test with the significance set at p ≤ 0.05.

In this study, a total of 18 Ruffini endings, 14 Golgi-like endings, and 11 Pacini corpuscles were analyzed in both dSL combined.

Ruffini Endings

Ruffini endings were characterized by an ovoid 3D shape (shown in Fig. 1, 2), with a median size of 62.5 × 31.7 μm and a total median cell count of 25 (range: 15–103). They appeared both individually and in small clusters of 2–3 nerve endings. The central axon was visualized accurately with the immunofluorescent marker for PGP 9.5, but also showed immunoreactivity for S100 and p75 and had a median diameter of 4.63 μm (range: 1.21–10.4 μm) (shown in Fig. 3). The size of the central axon in Ruffini endings and Pacini corpuscles was statistically nonsignificant. In 5 out of 18 corpuscles (28%), the central nerve was traced after entering the capsule and branched out after a median distance of 14 μm (range: 3.73–25.10) into multiple PGP 9.5-positive dendritic endings. The dendritic nerve endings were surrounded by S100-positive cells, with the flat nuclei ordered in a globular fashion. The outer sheath of Ruffini endings displayed a strong, characteristic immunofluorescence for p75 (shown in Fig. 4). The dense outer sheath encapsulated the whole Ruffini ending with a variable thickness of 0.94–15.5 μm and contained single to three layers of lamellar cells. The microstructure of the capsule showed bud-like projections, and beneath the capsule, a subscapular space with a range of 0–8.66 μm was measured (shown in Table 1).

Fig. 1.

Ruffini ending. A 3D visualization of a Ruffini ending displays the distribution of S100 (a), PGP 9.5 (b), p75 (c), DAPI (d), and all immunofluorescent markers (e) simultaneously. The central axon is surrounded by S100-positive Schwann cells (arrow in a). The central axon is precisely visualized with PGP 9.5 (arrow in b) and branches out into dendritic terminal nerve endings. The capsule shows strong immunoreactivity for p75 (c); scale bar 50 μm.

Fig. 1.

Ruffini ending. A 3D visualization of a Ruffini ending displays the distribution of S100 (a), PGP 9.5 (b), p75 (c), DAPI (d), and all immunofluorescent markers (e) simultaneously. The central axon is surrounded by S100-positive Schwann cells (arrow in a). The central axon is precisely visualized with PGP 9.5 (arrow in b) and branches out into dendritic terminal nerve endings. The capsule shows strong immunoreactivity for p75 (c); scale bar 50 μm.

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Fig. 2.

Histogram Ruffini ending. A section of a Ruffini ending is displayed as the deconvolved CLSM image (a) and as a reconstructed 3D visualization (b). The histogram (c) graphically represents the antibody intensity along the white lines in a and b. The distribution indicates that p75 is mainly found in the capsule, S100 is distributed in the inner part of the corpuscle and in the capsule, whereas PGP 9.5 is located in the inner part exclusively; scale bar 15 μm.

Fig. 2.

Histogram Ruffini ending. A section of a Ruffini ending is displayed as the deconvolved CLSM image (a) and as a reconstructed 3D visualization (b). The histogram (c) graphically represents the antibody intensity along the white lines in a and b. The distribution indicates that p75 is mainly found in the capsule, S100 is distributed in the inner part of the corpuscle and in the capsule, whereas PGP 9.5 is located in the inner part exclusively; scale bar 15 μm.

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Fig. 3.

Microstructure Ruffini ending. a The p75-positive capsule is rendered transparent to visualize the underlying PGP 9.5-positive dendritic nerve endings. b The central axon divides into two main branches, with a diameter of 5.14 μm and 3.77 μm, respectively. The capsule is cropped in the z-axis to visualize its circular arrangement around the dendritic nerve endings (c) with a subscapular space of about 4 μm (d). Scale bar 50 μm (a), 20 μm (b), 40 μm (c), 10 μm (d).

Fig. 3.

Microstructure Ruffini ending. a The p75-positive capsule is rendered transparent to visualize the underlying PGP 9.5-positive dendritic nerve endings. b The central axon divides into two main branches, with a diameter of 5.14 μm and 3.77 μm, respectively. The capsule is cropped in the z-axis to visualize its circular arrangement around the dendritic nerve endings (c) with a subscapular space of about 4 μm (d). Scale bar 50 μm (a), 20 μm (b), 40 μm (c), 10 μm (d).

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Fig. 4.

Capsule Ruffini ending. a The capsule of a Ruffini ending is cropped in the z-axis to display the inner core of the corpuscle with a globular arrangement of nuclei. The capsule is multi-layered (white arrows in b) with flattened cells and has bud-like projections and a subscapular space (star in b). The capsule may extend with projections into the corpuscle (red arrow in b). Scale bar 35 μm (a), 15 μm (b).

Fig. 4.

Capsule Ruffini ending. a The capsule of a Ruffini ending is cropped in the z-axis to display the inner core of the corpuscle with a globular arrangement of nuclei. The capsule is multi-layered (white arrows in b) with flattened cells and has bud-like projections and a subscapular space (star in b). The capsule may extend with projections into the corpuscle (red arrow in b). Scale bar 35 μm (a), 15 μm (b).

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Table 1.

Analysis of sensory nerve endings

CriteriaSensory nerve endings
RuffiniPaciniGolgi-like
Dimensions, µm 62.5 × 31.7 107 × 54.4 156 × 45.2 
3D volume, ×104 μm³ 2.85 7.61 9.61 
Cells, N 25 55 69 
Diameter central axon, µm 4.63 3.67 
Thickness capsule, µm 0.94–15.5 1.13–9.72 
Thickness subscapular space, µm 0–8.66 1.12–9.33 
Thickness inner core, µm 4.43–86 
Thickness outer core, µm 17.1–168 
Inner corpuscles, n 
CriteriaSensory nerve endings
RuffiniPaciniGolgi-like
Dimensions, µm 62.5 × 31.7 107 × 54.4 156 × 45.2 
3D volume, ×104 μm³ 2.85 7.61 9.61 
Cells, N 25 55 69 
Diameter central axon, µm 4.63 3.67 
Thickness capsule, µm 0.94–15.5 1.13–9.72 
Thickness subscapular space, µm 0–8.66 1.12–9.33 
Thickness inner core, µm 4.43–86 
Thickness outer core, µm 17.1–168 
Inner corpuscles, n 

Pacini Corpuscles

Pacini corpuscles had a range of different shapes, mostly globular, ovoid, branching, and lamellar in shape (shown in Fig. 5). The median dimension was 107 × 54.4 μm, and the median number of total cells was 55 (range: 26–70). Very defining for these endings was a central axon, with a median diameter of 3.67 μm (range: 1.91–6.24 μm), which displayed immunoreactivity mainly for PGP 9.5 and to a lesser extent also p75 and S100 (shown in Fig. 6). The central axon was surrounded by an inner core of S100-positive cells, which constituted the inner core of the corpuscle, which had a range in thickness of 4.43–86 μm. These cells were surrounded by concentric, lamellar layers of the outer core, with a range in thickness of 17.1–168 μm. The outer core was significantly thicker than the inner core (p < 0.001). Flat cell bodies of the lamellar cells were embedded into the capsule in a circular pattern. The lamellated capsule displayed immunofluorescence for p75 and PGP 9.5. Analysis of the microstructure showed that p75-positive structures ensheathed PGP 9.5-positive structures. Leading to the Pacini corpuscle was the S100- and PGP 9.5-positive parent axon, which contained a p75-positive perineurium.

Fig. 5.

Pacini corpuscle. The 3D visualization of a Pacini corpuscle shows immunostaining with S100 (a), PGP 9.5 (b), p75 (c), DAPI (d), and all immunofluorescent markers (e) simultaneously. The parent axon shows immunoreactivity for PGP 9.5 and S100 (arrows in a, b) and is covered by p75-positive perineurium (arrow in c). The inner core shows immunoreactivity mainly for S100 (arrowhead in a). The lamellae are immunoreactive for p75 and PGP 9.5 (arrowhead b, c). Scale bar 50 μm.

Fig. 5.

Pacini corpuscle. The 3D visualization of a Pacini corpuscle shows immunostaining with S100 (a), PGP 9.5 (b), p75 (c), DAPI (d), and all immunofluorescent markers (e) simultaneously. The parent axon shows immunoreactivity for PGP 9.5 and S100 (arrows in a, b) and is covered by p75-positive perineurium (arrow in c). The inner core shows immunoreactivity mainly for S100 (arrowhead in a). The lamellae are immunoreactive for p75 and PGP 9.5 (arrowhead b, c). Scale bar 50 μm.

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Fig. 6.

Central axon Pacini corpuscle. The 3D microscopic image (a) is used to render a 3D visualization (b) of a Pacini corpuscle. Two markers are set across the central axon of the Pacini corpuscle (arrow in b), and a histogram (c) displays the intensity values of the antibodies across the marked positions in b. The central axon mainly shows immunofluorescence for PGP 9.5 and to a lesser extent S100 and p75. Scale bar 15 μm (a), 20 μm (b).

Fig. 6.

Central axon Pacini corpuscle. The 3D microscopic image (a) is used to render a 3D visualization (b) of a Pacini corpuscle. Two markers are set across the central axon of the Pacini corpuscle (arrow in b), and a histogram (c) displays the intensity values of the antibodies across the marked positions in b. The central axon mainly shows immunofluorescence for PGP 9.5 and to a lesser extent S100 and p75. Scale bar 15 μm (a), 20 μm (b).

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Golgi-Like Nerve Endings

Golgi-like nerve endings were ovoid and fusiform in shape with median dimensions of 156 × 45.2 μm and a median number of cells of 69 (range: 16–158) (shown in Fig. 7). The corpuscle was surrounded by a thin, p75-positive capsule with a range in thickness of 1.13–9.72 μm and a subscapular space with a range of 1.12–9.33 μm. The difference of the thickness of the capsule as well as of the width of the subscapular space between Ruffini endings and Golgi-like endings was statistically not significant. The inner part of Golgi-like endings consisted of small, ramified, corpuscular structures, which were the characteristic feature of these sensory nerve endings (shown in Fig. 8). On average, we found a median of 3 (range: 1–7) inner corpuscles in Golgi-like endings. These smaller corpuscular structures contained cell bodies arranged in concentric patterns and displayed positive immunoreactivity for p75 on their surface, while S100- and PGP 9.5-positive structures were located beneath. These structures were groups of nerve endings, which branched out in the inner part of Golgi-like endings.

Fig. 7.

Golgi-like ending. A Golgi-like ending is displayed as a 3D visualization showing S100 (a), PGP 9.5 (b), p75 (c), DAPI (d), and all immunofluorescent markers (e) simultaneously. The Golgi-like ending contains multiple smaller corpuscles which are concentric in shape and show immunoreactivity for S100, p75, and PGP 9.5 (arrows in a-c). Scale bar 50 μm.

Fig. 7.

Golgi-like ending. A Golgi-like ending is displayed as a 3D visualization showing S100 (a), PGP 9.5 (b), p75 (c), DAPI (d), and all immunofluorescent markers (e) simultaneously. The Golgi-like ending contains multiple smaller corpuscles which are concentric in shape and show immunoreactivity for S100, p75, and PGP 9.5 (arrows in a-c). Scale bar 50 μm.

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Fig. 8.

Histogram Golgi-like ending. A section of an inner corpuscle of a Golgi-like ending is displayed as the deconvolved CLSM image (a) and as a reconstructed 3D visualization (b). The histogram (c) graphically represents the antibody intensity along the white lines in a, b and shows that p75 is mainly located on the surface of the inner corpuscle, S100 is found on the surface, and PGP 9.5 is also found in the inner part of the corpuscle. Scale bar 30 μm.

Fig. 8.

Histogram Golgi-like ending. A section of an inner corpuscle of a Golgi-like ending is displayed as the deconvolved CLSM image (a) and as a reconstructed 3D visualization (b). The histogram (c) graphically represents the antibody intensity along the white lines in a, b and shows that p75 is mainly located on the surface of the inner corpuscle, S100 is found on the surface, and PGP 9.5 is also found in the inner part of the corpuscle. Scale bar 30 μm.

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Volume

Both Pacini corpuscles (7.61 [2.3–54.3] × 104 μm³; p = 0.002) and Golgi-like endings (9.61 [1.69–37.4] × 104 μm³; p = 0.002) were significantly larger than Ruffini endings (2.85 [0.96–24.2] × 104 μm³) (shown in Fig. 9).

Fig. 9.

Volume measurements of corpuscular sensory nerve endings. The ovoid volume of a Ruffini (a), the globular volume of a Pacini corpuscle (b), and the fusiform volume of a Golgi-like ending (c) are shown. Scale bar 20 μm.

Fig. 9.

Volume measurements of corpuscular sensory nerve endings. The ovoid volume of a Ruffini (a), the globular volume of a Pacini corpuscle (b), and the fusiform volume of a Golgi-like ending (c) are shown. Scale bar 20 μm.

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With the use of a 3D immunofluorescent method, this study reports an exact analysis of the microstructure of corpuscular sensory nerve endings. Complete visualization of sensory nerve endings was achieved allowing to report new data such as the number of cells that constitute a sensory nerve ending, its volume, and the outer and intracorpuscular 3D morphology.

Prior to this research, only one other 3D immunofluorescence method existed, which solely utilized the axonal marker PGP 9.5 and a section thickness of 50 μm to analyze sensory nerve endings in the human dorsal radiocarpal ligament [21]. By performing triple immunofluorescence in this study, more structures of the sensory nerve endings were visualized and the subsequent digitalized models enabled a comprehensive analysis. Alternative microscopic techniques for immunofluorescent 3D imaging include two-photon microscopy, which offers higher tissue penetration depth and less phototoxicity [29], yet does not provide higher resolution than CLSM [30]. Advancements in light-sheet fluorescence microscopy have made it possible to increase scanning speeds of millimeter-thick cleared tissue samples at the cost of a low axial resolution, making it unsuitable to image individual sensory nerve endings precisely [31]. Recently, a new technique using focused ion beam scanning electron microscopy has been reported to visualize avian Meissner corpuscles in 3D [32]. The advantage over light microscopy-based techniques is the high resolution of several nanometers, while it is extremely time and cost intensive, involving imaging of a single sensory nerve ending over several days up to weeks.

Two-dimensional immunohistochemical and immunofluorescent studies of pathologic morphological changes in sensory nerve endings faced the problem of high amounts of unclassifiable corpuscles [8, 33]. These findings are related to morphological changes and alterations in immunoreactivity of sensory nerve endings due to age [34, 35] or pathologies [11, 14] as well as technical difficulties of visualization including the thin section thickness employed in 2D microscopy [22]. The precise analysis given with a 3D model of sensory nerve endings might address this problem, by providing a complete visualization of sensory nerve endings.

Ruffini endings were surrounded by a p75-positive capsule, with no uniform thickness and with single to multiple layers of glial cells. The varying thickness of the capsule may result in regional mechanosensitive differences within the corpuscles. In periarticular tissue, the slowly adapting sensory nerve endings mainly register changes in axial rather than compressive loading [36] and give rise to joint position sense [9]. The exact mechanism of mechanotransduction has not been encoded, yet past histochemical studies have demonstrated the presence of mechanosensitive epithelial sodium channels in periodontal Ruffini endings [37] as well as calcium-dependent processes [38].

Beneath the capsule of Ruffini endings and Golgi-like, we found a subcapsular space of varying thickness. Our study showed that there was no significant difference in the size of the subscapular space between both sensory nerve endings. Studies performed in cutaneous located Pacini and Meissner corpuscles showed that the fluid-filled space between the lamellar cells is filled with a rich and complex extracellular matrix, with the cytoskeletal proteins influencing mechanotransduction [39]. Further studies are required to analyze the structural composition of the extracellular matrix in periarticular Ruffini and Golgi endings. This might have clinical implications, as, for example, mutations in the transmembrane protein USH2A have been shown to reduce vibration sensitivity in human Meissner corpuscles [40].

Pacini corpuscles possess a characteristic 3D structure consisting of a p75- and PGP 9.5-positive, thick, lamellated capsule surrounding a central axon concentrically, which displays positive immunoreactivity mainly for PGP 9.5 and to a lesser extent for S100 and p75. Contrary to 2D immunofluorescence studies [13, 41], PGP 9.5 immunofluorescence was not only found in the central axon but also in the capsule. The diverging results may arise out of the more exact visualization of the microstructure of Pacini corpuscles with confocal laser scanning microscopy and 3D analysis as employed in the present study. It has been described that the cells of the outer core are of perineurial origin and are not S100-positive [42], which our study also confirms. Furthermore, S100 is a useful marker to study pathologies affecting Pacini corpuscles, as reduced levels of the Schwann-like cell marker have been reported in diabetic sensorimotor polyneuropathy indicating denervation [11], as well as after peripheral nerve injuries [43]. Cobo et al. [44] have provided a comprehensive review of proteins detected in Pacini corpuscles, demonstrating the capacity for selectively visualizing the different structural components of the corpuscle.

The data of our study indicate that Pacini corpuscles have a large size span and a variable size of their outer and inner core. The size of Pacini corpuscles is affected by certain diseases of the hand, including Pacini corpuscle hyperplasia in palmar fibromatosis [45]. However, the exact pathomechanism of hyperplasia still remains unclear, yet the most significant factor is local or distant repetitive trauma [46]. Furthermore, Halata et al. [47] performed transmission electron microscopy on Pacini corpuscles in the human knee joint and divided Pacini corpuscles into small and large according to their size, the number of layers of their capsule, and their localization in the joint. We have also seen a range in size of Pacini corpuscles, yet we performed an intraligamentous analysis and therefore cannot report on the size of Pacini corpuscles of the whole wrist joint.

The large size and the intracorpuscular grouped nerve endings located in the corpuscle were characteristic for Golgi-like nerve endings. In electron microscopic studies conducted on Kowaris and sheep, Ruffini- and Golgi-like endings featured a similar ultrastructure, resulting in some authors to assign both to the same group of spray-like endings, suggesting that spray-like endings represent a spectrum of simple Ruffini endings to complex Golgi-like endings, with the complexity increasing especially in dense connective tissue [48, 49]. Our study shows that morphological similarities exist between Golgi-like endings and Ruffini endings, including the size of the capsule and the subscapular space, whereas Golgi-like endings were significantly larger than Ruffini endings. However, Ruffini endings and Golgi-like endings were found throughout the entire dSL. Therefore, our data do not support the hypothesis that the density of connective tissue influences the complexity of Ruffini endings or Golgi-like endings.

We have shown that sensory nerve endings possess an intricate outer and inner 3D morphology. Tomita et al. [21] attempted to classify sensory nerve endings of the dorsal radiocarpal ligament of the wrist by assigning nine shapes to describe the morphological appearance, with 6.9% possessing an unclassifiable shape. Another approach to further characterize ligamentous sensory nerve endings was conducted by studying the level gene expression of S100B, calcitonin gene-related peptide, and neurofilament medium chain in ligaments of the rabbit knee [35]. Possibly, further differentiation of sensory nerve endings of periarticular joints is achieved by studying the occurrence and distribution of mechanosensitive ion channels, as in Pacini corpuscles of the human palmar fascia [45].

Limitations of the present study include the high age of specimens. In ligaments of the talocrural joint, the number of Ruffini endings correlated negatively with age [33]. Furthermore, human Pacini corpuscles of the skin have displayed altered immunofluorescent patterns in specimens over 60 years old [34]. It is therefore desirable to conduct 3D analysis of sensory nerve endings in younger specimens. Despite using distinct excitation and detection wavelengths, sequential scanning of markers, and experimentally measured PSFs for deconvolution, overlapping of immunofluorescent signals was registered, for example, in the inner corpuscles of Golgi-like endings or the central axons of Ruffini endings and Pacini corpuscles. Previous studies have reported an overlap of S100 and p75 in the central axon of Pacini corpuscles [27] and of all three neuronal markers in Golgi-like endings [8] may be caused by the close proximity of the markers in the sensory nerve endings [13]. A prospective improvement for future studies employing multiplex imaging is the use of fluorescent markers with more distinct excitation and emission peaks to mitigate the overlap of signals and enhance the specificity of staining, as well as enhancing post-image processing of multichannel images by correcting for chromatic aberration [50]. Furthermore, we have reported on the intracorpuscular analysis of sensory nerve endings, yet future studies should aim to visualize the entire neuronal intraligamentous network. This may be achieved with histological clearing techniques to enable a higher penetration depth of microscopes [22].

In conclusion, we reported new data regarding the intricate outer and intracorpuscular 3D morphology of periarticular sensory nerve endings. With the use of digitalized models, we precisely analyzed sensory nerve endings, including their structural composition, number of cells, and their volume, thereby yielding novel insights into their microstructure. This 3D immunofluorescent method may be useful for studies attempting to visualize morphological changes of sensory nerve endings in pathologies.

We would like to thank Matthias Behr, PhD (Leipzig, Germany), and Anja Doering (Leipzig, Germany) for their generous assistance in the laboratory work. Furthermore, we thank Christian Retschke for logistic support.

All protocols in the present study were approved by the Ethics Committee of the medical chamber of Saxony-Anhalt (ethics approval number: 33/17). Written informed consent was obtained from donors to participate in studies.

The authors have no conflicts of interest to declare.

This work was supported by Deutsche Gesetzliche Unfallversicherung, Sankt Augustin, Germany (Grant No. FR-0272), and Bauerfeind AG, Zeulenroda-Triebes, Germany (Grant No. 8344). The funders had no role in the design, data collection, data analysis, and reporting of this study.

The development of the experimental design was conducted by S.R., R.A.M., and J.K. Experiments were performed by S.R. and R.A.M. All authors (S.R., R.A.M., J.K., and T.K.) made substantial contributions to this study, analyzed and discussed the results, and contributed to the final version of the manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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