Proprioceptors of the human pericardium

Abstract

In the human organism, all functions are regulated and, therefore, require a feedback mechanism. This control involves a perception of the spatial tensile state of cardiac tissues. The presence and distribution of respective proprioceptive corpuscles have not been considered so far. Therefore, a comprehensive study of the entire human fibrous pericardium was conducted to describe the presence of proprioceptors, their density, and distribution patterns. Eight human pericardial specimens gained from our body donation program were used to create a three-dimensional map of proprioceptors in the pericardium based on their histological and immunohistochemical identification. The 3D map was generated as a volume-rendered 3D model based on magnetic resonance imaging of the pericardium, to which all identified receptors were mapped. To discover a systematic pattern in receptor distribution, statistical cluster analysis was conducted using the Scikit-learn library in Python. Ruffini-like corpuscles (RLCs) were found in all pericardia and assigned to three histological receptor localizations depending on the fibrous pericardium's layering, with no other corpuscular proprioceptors identified. Cluster analysis revealed that RLCs exhibit a specific topographical arrangement. The highest receptor concentrations occur at the ventricular bulges, where their size reaches its maximum in terms of diameter, and at the perivascular pericardial turn-up. The findings suggest that the pericardium is subject to proprioceptive control. RLCs record lateral shearing between the pericardial sublayers, and their distribution pattern enables the detection of distinct dilatation of the heart. Therefore, the pericardium might have an undiscovered function as a sensor with the RLCs as its anatomical correlate.

Keywords: Distribution pattern; Pericardium; Proprioception; Ruffini-like corpuscles.

Fig. 1

Histological criteria of Ruffini-like corpuscles (RLCs) in the human pericardium. Histological criteria of these receptors are the presence of meandered vessels and scattered nerve fibers, the occurrence of pale epitheloid cells, a minor density of collagenous fibers compared to the surrounding area, and demarcation from the environment by straightened elastic fibers. Since not all criteria of an RLC are always in one histological plane, we show a variety of receptors to demonstrate all previously mentioned criteria. All fusiform structures within the pericardium that fulfill at least three of the histological criteria were counted as RLC. A Histological image (section thickness: 7 μm; stain: Elastica van Gieson; scale bar: 50 μm) of an RLC in the pericardium of an 80-year-old male donor. The arrowheads indicate the less dense collagen fibers within the receptor than the surrounding area. B Schematic drawing of the RLC shown in A) with all histological criteria depicted. The arrows point to the corresponding criteria in the histological images. C Histological image (section thickness: 7 μm; stain: Elastica van Gieson; scale bar: 50 μm) of an RLC in the pericardium of an 86-year-old female donor. The arrowheads direct to the epitheloid cells, and the arrows to the meandering vessels. D Histological image (section thickness: 7 μm; stain: Elastica van Gieson; scale bar: 50 μm) of an RLCs in the pericardium of an 80-year-old deceased man. The arrowheads indicate elastin fibers, which demarcate the receptor from the surrounding tissue. E Histological image (section thickness: 7 μm; scale bar: 50 μm) with immunochemical staining (pan-Neurofilament) of a RLC of an 83-year-old female donor. The arrowheads point to the receptors, specifically stained scattered nerve filaments. * = pericardium fibrosum, ** = lamina parietalis pericardii serosi

Fig. 2

Immunohistochemical characterisation of the pericardial Ruffini-like corpuscles (RLCs). To further confirm the diagnosis of RLCs and to characterise specific neuronal components within the receptors, immunohistochemical stainings were performed using antibodies against S-100 (a specific marker for sensory fibers), Calcitonine Gene-Related Peptide (CGRP, a peptide released by sensory fibers), Transient Receptor Potential Channel 4 (TRPC4, a non-selective calcium channel of sensory fibers), and tyrosine hydroxylase (a specific marker for efferent sympathetic fibers). A positive result is indicated by the red dye, labeled on the secondary antibody. The immunohistochemistries against S-100 (top), TRPC4 (middle), and tyrosine hydroxylase (bottom) yielded positive results. Staining against TRPC4 (middle) also marked the calcium channels of the smooth muscle of all vessels. The intensity of the surrounding environment was reduced to highlight the shape and extent of the RLCs

Fig. 3

Localization of Ruffini-like corpuscles (RLCs) within the fibrous pericardium. Classification of the localization of RLCs is based on the histological organization of the human fibrous pericardium, which can be divided into three sublayers of collagenous fibers interwoven with elastic fibers. By our definition, the pericardial sublayer facing the thoracic cavity is the lamina externa (III), the middle sublayer is the lamina intermedia (II), and the sublayer touching the pericardial cavity is the lamina interna (I). The histological structure of RLCs in all localizations was identical. Receptors outside the fibrous portion of the pericardium were not considered. A Histological image (section thickness: 7 μm; stain: Elastica van Gieson; scale bar: 50 μm) of the pericardium of an 87-year-old female body donor. The 1st receptor localization is between the lamina interna (I) and the lamina intermedia (II) of the pericardium. Arrowheads point to the RLC positioned between these layers. Receptors of this localization are rare (12.5%). B Histological image (section thickness: 7 μm; stain: Elastica van Gieson; scale bar: 50 μm) of the pericardium of an 80-year-old male body donor. The 2nd receptor localization is between the lamina intermedia (II) and the lamina externa (III) of the pericardium. Arrowheads direct to the RLC at this location. This receptor localization is most abundant (45.7%). C Histological image (section thickness: 7 μm; stain: Elastica van Gieson; scale bar: 50 μm) of the pericardium of a 70-year-old male body donor. The 3rd localization is summarized by RLCs mainly as part of the outer border of the lamina externa (III), touching the thoracic cavity. Arrowheads indicate the RLC inside the outermost pericardial sublayer. RLCs at this spot were detected second most (41.8%)

Fig. 4

Visualization of Ruffini-like corpuscles (RLCs) in the human pericardium. To visualize the distribution of RLCs, all receptors of the eight pericardia examined were matched to a “standard-pericardium”. Using the histologically detected corpuscles with the adjacent environment and magnetic resonance imaging of the “standard-pericardium” in all three spatial planes, the receptors were transferred to the “standard-pericardium” by matching prominent anatomical landmarks. A Anatomical preparation of the heart within pericardium dissected from a 91-year-old formalin-fixed female body donor, which was utilized as the “standard-pericardium”. All tissue around the pericardium was carefully removed to maintain the heart’s natural in situ position on top of the diaphragm. An epoxy resin frame ensured the natural form of the diaphragm, whereas hard fixation and filling stabilized the heart within its pericardium to allow radiological 3D reconstruction by MRI and photogrammetry. Scale bar: 5 cm. B Exemplary histological image (section thickness: 7 μm; stain: Elastica van Gieson; scale bar: 800 μm) of the pericardium of an 80-year-old male body donor. All discovered RLCs within the pericardium (center) were matched to the corresponding position on the “standard-pericardium,” considering the myocardial topography (left) and the surrounding tissue (right). C Radiological volume rendering model of the “standard-pericardium” (top), to which all RLCs were assigned using the 3D Slicer program. The three forms of the yellow dots represent the different localizations of the RLCs within the fibrous pericardium (triangle = 1st localization, circle = 2nd localization, quadrate = 3rd localization). The three spatial planes included in the radiological sequence (bottom) were used for orientation in the “standard-pericardium” to achieve the most anatomically correct transfer of the receptors. Scale bar: 5 cm

Fig. 5

Distribution of Ruffini-like corpuscles (RLCs) in the human pericardium. Mapping and visualization of the proprioceptors were performed by digitally transferring all RLCs of the eight pericardia studied to a three-dimensional volume-rendering model used as a "standard- pericardium”. This three-dimensional model was acquired by magnetic resonance imaging from a formalin-fixated heart, including the pericardium and diaphragm of a 91-year-old female body donor. Transfer of all receptors was implemented using 3D-Slicer software. Each receptor location was mapped according to anatomical landmarks and relative to size, considering the adjacent environment and cardiac topography. Symbols represent the three pericardial localizations of corpuscles (triangle = 1st localization, circle = 2nd localization, quadrate = 3rd localization). A total number of 1476 RLCs was detected. Scale bars: 5 cm. A Distribution of RLCs as seen from the front of the pericardium. B Distribution of RLCs as seen from the posterior side of the pericardium. C Distribution of RLCs as seen from the left and lower sides of the pericardium. D Distribution of RLCs seen from above the pericardium

Fig. 6

Dendrogram of hierarchical agglomerative clustering. A dendrogram partitioning the data in a logical distribution of Ruffini-like corpuscles using hierarchical agglomerative clustering with WARD linkage. We chose a cluster number of four based on the dendrogram, as indicated by the black dashed line. The x-axis displays the number of points in a given node, while the y-axis shows the cluster distance

Fig. 7

Cluster analysis of possible partitions and hotspots within the pericardial proprioceptors. Based on their relative coordinates, proprioceptors were divided into prospective partitions and areas of higher density using cluster analysis. The Python Scikit-learn package was utilized for this analysis, while the Plotly module was used for the 3D scatter plots. We used mean shift clustering in addition to the hierarchical method to find the local maxima of neighborhood density. Cluster analysis found four potential partitions and regions of increased density in the pericardial proprioceptors. The arrangement of pericardial receptor partitions correlates to the compartmentalization of the heart into four different chambers (blue: right atrium, red: right ventricle, green: left ventricle, yellow: left atrium). Hotspots of high and low receptor density within the pericardium were discovered by Mean shift function analysis of the created heatmap. Four hotspots of pericardial proprioceptors are exposed according to the four selected clusters. A “Standard pericardium” shown from a ventral perspective, displaying four partitions of pericardial proprioceptors determined through cluster analysis (blue = right atrium, red = right ventricle, green = left ventricle). The interventricular septum separates the two ventricles (red and green clusters), while the valvular plane separates the atria from the corresponding ventricles (blue and red clusters and green and yellow clusters (not visible from this side). Scale bar: 5 cm. B “Standard pericardium” viewed from a dorsal perspective with four partitions of pericardial proprioceptors identified by cluster analysis ((blue = right atrium, green = left ventricle, yellow = left atrium). The valvular plane separates the left atria (yellow) from the corresponding left ventricles (green). Scale bar: 5 cm. C) Heat map of the distribution of pericardial proprioceptors from a ventral perspective. Two hotspots of high receptor density are evident: at the exit of the aorta and pulmonary trunk and the right ventricular bulge, where the cardiac radius is widest. A low receptor density is found along the interventricular septum and at the cardiac apex. D Legend of the heat map shown in C and E. The legend displays the distance to the density center. E Heat map of the distribution of pericardial proprioceptors from a dorsal perspective. Two more hotspots of high receptor density are distinct: at the left ventricular bulge, where the cardiac radius is widest, and in the area of the left atrium touching the esophagus