Characterization, Number, and Spatial Organization of Nerve Fibers in the Human Cervical Vagus Nerve and its Superior Cardiac Branch

Brain Stimulation 17 (2024) 510–524 Available online 25 April 2024 1935-861X/© 2024 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Characterization, number, and spatial organization of nerve fibers in the human cervical vagus nerve and its superior cardiac branch Bettina Kronsteiner a , b , c , 1 , Genova Carrero-Rojas c , 1 , Lukas F. Reissig c , Atieh Seyedian Moghaddam c , Karoline M. Schwendt c , Sylvia Gerges c , Udo Maierhofer d , Oskar C. Aszmann d , e , Angel M. Pastor f , Attila Kiss b , g , Bruno K. Podesser b , g , Wolfgang Birkfellner a , Francesco Moscato a , b , h , Roland Blumer c , * , Wolfgang J. Weninger c a Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Vienna, Austria b Ludwig Boltzmann Institute for Cardiovascular Research, Vienna, Austria c Division of Anatomy, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria d Clinical Laboratory for Bionic Extremity Reconstruction, Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Vienna, Vienna, Austria e Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Vienna, Vienna, Austria f Departamento de Fisiología, Facultad de Biología, Universidad de Sevilla, 41012, Sevilla, Spain g Center for Biomedical Research and Translational Surgery, Medical University of Vienna, Austria h Austrian Cluster for Tissue Regeneration, Vienna, Austria ARTICLE INFO Keywords: Human vagus nerve Superior cardiac branch of the human vagus nerve Vagal motor fibers Vagal sensory fibers Vagal sympathetic fibers Immunofluorescence Vagus nerve stimulation ABSTRACT Background: Electrical stimulation of the vagus nerve (VN) is a therapy for epilepsy, obesity, depression, and heart diseases. However, whole nerve stimulation leads to side effects. We examined the neuroanatomy of the mid-cervical segment of the human VN and its superior cardiac branch to gain insight into the side effects of VN stimulation and aid in developing targeted stimulation strategies. Methods: Nerve specimens were harvested from eight human body donors, then subjected to immunofluorescence and semiautomated quantification to determine the signature, quantity, and spatial distribution of different axonal categories. Results: The right and left cervical VN (cVN) contained a total of 25,489 ±2781 and 23,286 ±3164 fibers, respectively. Two-thirds of the fibers were unmyelinated and one-third were myelinated. About three-quarters of the fibers in the right and left cVN were sensory (73.9 ±7.5 % versus 72.4 ±5.6 %), while 13.2 ±1.8 % versus 13.3 ±3.0 % were special visceromotor and parasympathetic, and 13 ±5.9 % versus 14.3 ±4.0 % were sympathetic. Special visceromotor and parasympathetic fibers formed clusters. The superior cardiac branches comprised parasympathetic, vagal sensory, and sympathetic fibers with the left cardiac branch containing more sympathetic fibers than the right (62.7 ±5.4 % versus 19.8 ±13.3 %), and 50 % of the left branch contained sensory and sympathetic fibers only. Conclusion: The study indicates that selective stimulation of vagal sensory and motor fibers is possible. However, it also highlights the potential risk of activating sympathetic fibers in the superior cardiac branch, especially on the left side. 1. Introduction The vagus nerve (VN), the tenth cranial nerve, extends from the head to the abdomen [1]. It contains both sensory (afferent) and motor (efferent) fibers [2]. Different subtypes of motor and sensory fibers can be distinguished. According to traditional descriptions, these are special visceroefferent fibers for innervating the larynx muscles, parasympathetic (general visceromotor) fibers for innervating the organs, visceral sensory fibers that transmit sensations from the organs, and somatic sensory fibers that transmit sensations from the ear [2]. In * Corresponding author. E-mail address: [email protected] (R. Blumer). 1 BK and GCR contributed equally to the study. Contents lists available at ScienceDirect Brain Stimulation journal homepage: www.journals.elsevier.com/brain-stimulation https://doi.org/10.1016/j.brs.2024.04.016 Received 23 January 2024; Received in revised form 11 April 2024; Accepted 23 April 2024 Brain Stimulation 17 (2024) 510–524 511 addition, it has been reported, that the human VN contains sympathetic fibers [3–6]. Sympathetic fibers form clusters [3], but it is unclear whether other categories of vagal fibers are randomly distributed or anatomically organized. This lack of information limits our understanding of both, the function of the parasympathetic nervous system and clinical applications involving VN activation. Electrical stimulation of the cervical VN (cVN) has emerged as a promising non-pharmacological treatment method for various medical conditions, such as epilepsy, obesity, depression, and narcolepsy [7–9]. VN stimulation involves the placing of electrodes around the nerve [10], and today it is common practice to stimulate the entire nerve thus including all its constituents [7]. This non-selective mode of stimulation results in a heterogeneous spectrum of side effects, which limits the therapeutic efficacy of the treatment [11]. Currently, there is a lot of research being conducted on the activation of specific clusters of nerve fibers to achieve selective VN stimulation [7,12]. However, this requires precise knowledge of VN neuroanatomy, degree of myelination, and fiber counts, which is currently lacking. Because the VN is connected to the heart, VN stimulation has been identified as a potential therapy for cardiovascular disorders [13–15]. In fact, animal studies [16] and clinical studies in humans [13,17] have shown that VN stimulation decreases heart rate and improves ventricular function. While these studies are promising, it is important to note that the causality of several side effects and therapy resistance is not well understood [11,18]. One possible explanation for this is the dearth of knowledge regarding the fiber composition of the vagal cardiac branches. From these considerations, it is clear that comprehensive neuroanatomical information on the VN and its branches is crucial for understanding, optimizing, and specifying VN stimulation and for avoiding potentially life-threatening side effects [18]. We, therefore, aimed to generate highly detailed information on the quality, distribution, and numbers of the nerve fibers composing the mid-cervical segment of the VN and the superior cardiac branch. The results shall promote the establishment of new innovative VN stimulation methods, including targeted VN stimulation strategies, and help to further investigate the genesis of side effects and the definition of contraindications. In addition, they shall expand basic anatomic knowledge and challenge the anatomic principles of the human autonomic nervous system and the current concept of heart innervation. 2. Material and methods The cVN and its superior cardiac branches were harvested from eight body donors, comprising five females and three males, aged between 69 and 82 years (mean and standard deviation 79.5 ±5.8 years), who had no history of neurological disorders. The Division of Anatomy, Medical University of Vienna, had obtained written consent from all donors for the use of their bodies in teaching and research purposes. The study’s experiments were approved by the institutional review board of the Medical University of Vienna (approval protocol No. 1655/2022). 2.1. Dissection and tissue collection The cVN was bilaterally exposed by neck dissection. Across all body donors, only a single branch that could be traced towards the pericardium exited the VN in the mid-neck region. This branch was considered to be the superior cardiac branch. Other cardiac branches (2–4 per side) exited the VN caudal to the clavicle, at the thoracic level, consistent with a previous study [1]. A macroscopic image taken during the dissection shows the cVN and its superior cardiac branch (Fig. 1A and B). Three cm long tissue samples were taken from the cVN at the midcervical level and from the superior cardiac branch near its bifurcation (Fig. 1B). 2.2. Tissue processing Nerve samples were fixed by immersion in 4 % paraformaldehyde in 0.1 M sodium phosphate buffer (PBS), pH 7.4 for 24 h at 4 ◦C. The time between death and tissue fixation (postmortem interval) was between 11 and 15 h to obtain optimal immunohistochemical results [19]. Following fixation, the nerve samples were washed in PBS for two days, cryoprotected in graded sucrose solutions (10 %, 25 %, and 40 %) in PBS, frozen, and stored at −80 ◦C according to our guidelines for cryo-embedding procedures [20]. Nerve cross sections at 10 μ m thickness were cut using a Cryotom (Leica CM3050 Cryostat, Leica Biosystems, Germany). Sections were mounted on Superfrost Ultra Plus®microscope slides (Thermo Scientific/Menzel) and subjected to conventional histology and multicolor immunofluorescence. 2.3. Conventional histology Cross sections of the cVN were labeled with hematoxylin-eosin using a standard protocol (hematoxylin 3 min and eosin 1 min). Hematoxylin- Fig. 1. Dissection of the cVN and its superior cardiac branch from a non-embalmed body donor. (A) The dissected left and right cVN and the left and right superior cardiac branches (sCB) are displayed in the frontal view. (B) Close-up of the right cVN and its superior cardiac branch. The upper green arrow indicates the site from which the cVN was harvested and the lower green arrow indicates the site from which the superior cardiac branch was harvested. The bars indicate the length of the tissue samples that were removed and analyzed. Common carotid artery (cCA), internal jugular vein (iJV), omohyoid muscle (OHM); cranial (cr), caudal (cd), medial (m), lateral (l). B. Kronsteiner et al. Brain Stimulation 17 (2024) 510–524 512 eosin-stained sections were analyzed and photodocumented with a slide scanner microscope (Olympus VS120, Olympus Europa SE & Co. KG, Hamburg, Germany). 2.4. Multicolor immunofluorescence 2.4. 1. Antibody characterization For immunolabeling, the following antibodies were used: anti-neu- rofilament (NF), anti-myelin basic protein (MBP), anti-tyrosine hydroxylase (TH), and anti-choline acetyltransferase (ChAT). The polyclonal NF antibody (AB5539, Merck Millipore) binds to neurofilament heavy chain. By immunofluorescence, it has been demonstrated that this antibody visualizes all types of nerve fibers in human tissue [21,22] and non-human tissue [23,24]. The monoclonal antibody against MBP (MAB386, Merck Millipore) is raised against the bovine myelin basic protein and was tested by Western blot in mouse brain (manufacturer’s datasheet). By immunofluorescence, it has been shown that the antibody visualizes myelinated fibers in the human spinal cord and non-human tissue [19,21,25]. Tyroxine hydroxylase is the rate-limiting enzyme for the synthesis of (nor) adrenaline and is present in postganglionic sympathetic fibers [26]. The polyclonal antibody against TH (AB152, Sigma Aldrich) has proven to label postganglonic sympathetic fibers in human tissue [20, 27,28]. The polyclonal goat antibody against ChAT (AB144P, Merck- Millipore) is raised against human placental ChAT. The antibody was validated in the human brain by pre-adsorption [29] and is the most commonly used marker for cholinergic (motor) neurons in human and non-human tissue [22,23,30]. 2.4. 2. Immunolabeling Cross sections of the cVN and the superior cardiac branch were immunolabeled in multiple colors. A total of four doubleimmunofluorescence staining experiments and one tripleimmunofluorescence staining experiment were conducted. In three double-labeling experiments anti-NF was combined with [1] anti-MPB [2], anti-TH, or [3] anti-ChAT. The fourth double-labeling experiment combined anti-ChAT and anti-MBP. The triple-labeling experiment used anti-NF along with anti-MBP and anti-TH. In all staining combinations, except for anti-ChAT/anti-MBP, anti-NF was used as a panneuronal marker. Using this method, it was possible to visualize all nerve fibers and specific ones simultaneously. Table 2 demonstrates how different types of nerve fibers can be identified through immunofluorescence. Before immunolabeling, nerve cross-sections were air-dried for 30 min. Sections were blocked for 1 h with 10 % normal goat serum or 10 % normal rabbit serum (depending on the staining protocol) followed by incubation with the primary antibodies diluted in PBS containing 0.1 % Triton X (PBS-T) for 24 h at 4 ◦C. After washing in PBS, sections were incubated with Alexa Fluor (AF) 488, AF 568, and AF 647 conjugated secondary antibodies for 2 h at 37 ◦C. Secondary antibody concentration was between 1:200 and 1:500. Sections were rinsed again in PBS and coverslipped with fluorescence mounting medium (DakoCytomation, Santa Clara, CA, USA). The primary antibodies including working dilutions, RRID numbers, and suppliers are listed in Table 1. For negative controls, the primary antibody was omitted, and the secondary antibody was used alone. The omission of the primary antibody resulted in no immunolabeling. 2.5. Confocal laser scanning microscopy Fluorescently labeled sections were analyzed with a confocal laser scanning microscope (CLSM Olympus FV3000). Images were captured using dry lenses with 20×magnification or oil lenses with 40×and 60× magnification for panoramic views (20×) and close ups (40x and 60x). A series of virtual CLSM sections of 1–2 μ m thickness were cut through the structures of interest. Each section was photo-documented with a 1024 ×1024 pixel resolution and 3D projections were rendered using Image J software (NIH, Bethesda, MA, USA). Double-colored images were generated using lasers with excitation wavelengths of 488 and 568 nm and triple-colored images using an additional laser with an excitation wavelength of 640 nm. 2.6. Quantification of nerve fibers in the cVN and superior cardiac branch To quantify the different axonal components within the cVN and the superior cardiac branch, we utilized a quantitative immunofluorescence technique that encompasses multicolor immunolabeling and semiautomated fiber segmentation. Using this technique, we determined the number of NF-, MBP-, TH-, and ChAT-positive nerve fibers in the right and left cVN (n =4 individuals) and the right and left cardiac branches (n =4 individuals). All imaging datasets were preprocessed and analyzed in tiff format using the free Fiji software [31]. 2.6. 1. Image preprocessing and nerve fiber segmentation The individual color channels of the multi-colored images were separated and displayed at their maximum intensity. The images were converted to RGB format (Image >Color >RGB to Luminance) and underwent contrast enhancement (Process >Enhance contrast >saturated pixels: 0.35 %). Subsequently, the nerve fibers were automatically thresholded using the Otsu filter of Fiji software (Image >Adjust > Threshold >Otsu >Apply), resulting in a binary image (black and white). 2.6. 2. Manual counts of nerve fibers Fiber counts were manually performed in three cVNs and three cardiac branches using Fiji’s Cell Counter plugin (Plugins >Analyze > Cell Counter). These manual counts served as reference data for developing and evaluating the semi-automated counting method. Comparisons between the manual and semi-automated counts on identical images demonstrated consistent results. After this, only semi-automated counts were performed on all of the remaining images of the nerve specimens. Table 1 List of markers used in the present study including working dilution, RRID number, and suppliers. Markers Working dilution RRID number Supplier General Neuronal Markers chicken anti-neurofilament (NF) 1:2000 AB_177520 Merck/ Millipore Specific Neuronal Markers rat anti-myelin basic protein (MBP) 1:100 AB_94975 Merck/ Millipore rabbit anti-tyrosine hydroxylase (TH) 1:250 AB_390204 Sigma Aldrich goat anti-choline acetyltransferase (ChAT) 1:100 AB_2079751 Merck/ Millipore Table 2 Types of axons identified by antibody expression. Antineurofilament (NF) Antimyelin basic protein (MBP) Anti-tyrosine Hydroxylase (TH) Anti-choline acetyl transferase (ChAT) Myelinated axons þ þ ¡ ¡ Unmyelinated axons þ ¡ ¡ ¡ Sympathetic axons þ ¡ þ ¡ Motor axons þ+/¡ ¡ þ Sensory axons þ þ /¡ ¡ ¡ B. Kronsteiner et al. Brain Stimulation 17 (2024) 510–524 513 2.6. 3. Semi-automated counts of nerve fibers Counts were conducted semi-automatically on binary images of each immunolabeling. To avoid false-positive counts of background particles, a line was drawn around each fascicle (using the polygon tool) and saved as a single Region of Interest (ROI). The Analyze Particles plugin of Fiji software was subsequently used to count the nerve fibers in each fascicle by encircling objects of interest (nerve fibers) and assigning them a number. The nerve fibers detected by the analysis software were then visually verified against the original image. Supplementary Figs. 1 and 2 display the software’s nerve fiber detection in the cVN and the superior cardiac branch, respectively. For the quantification of NF-positive (all nerve fibers) and TH- positive (sympathetic) fibers, merging fibers were separated by applying the watershed function (Process >Binary >Watershed) of Fiji software. For MBP-positive and ChAT-positive fibers, we made manual adjustments to the particle sizes, which were included in the Particle Fig. 2. Structural organization and number of nerve fascicles in the human cVN. (A) Hematoxilin-eosin staining of a human right cVN cross-section. The cVN consists of serval nerve fascicles each surrounded by epineurium, and the whole nerve is surrounded by a layer of perineurium. (B) The bar chart displays the mean number and standard deviation of nerve fascicles (n =8) on the right and left sides. Although the mean number was higher on the right side, this difference was not statistically significant (paired t-test, t [14] =0.727, p =0.479). Scale bar: 200 μ m in A. Table 3 Total numbers and percentage of different types of axons in the human right and left cervical vagus nerve. NF: neurofilament, MBP: myelin basic protein, TH: tyrosine hydroxylase, ChAT: choline acetyltransferase. Right cervical vagus nerve Cases NF + (n)/(%) MBP + (n)/ (%) MBP − (n)/(%) TH + (n)/ (%) ChAT + thick fibers (n)/ (%) ChAT + thin fibers (n)/ (%) ChAT + total (n)/ (%) Sensory fibers n/ % Case 1 25,581/ 100.0 7461/29.2 18,120/70.8 2201/8.6 452/1.8 2719/10,6 3171/12.4 20,209/79.0 Case 2 22,860/ 100.0 8247/36.1 14,613/63.9 1829/8.0 523/2.3 2424/10,6 2947/12.9 18,084/79.1 Case 3 24,201/ 100.0 8443/34.9 15,758/65.1 3586/14.8 435/1.8 2317/9,6 2752/11.4 17,890/73.8 Case 4 29,314/ 100.0 9472/32.3 19,842/67.7 6012/20.5 590/2.0 4013/13,7 4603/15.7 18,699/63.8 Mean 25,489/ 100.0 8405.8/33.1 17,083,3/ 66.9 3407.0/ 13.0 500/2.0 2,868,3/11,1 3368.3/13.1 18,721/74.0 SD 2781.5/0.0 827.9/3.1 2348,3 1894.1/5.9 71.1/0.2 781.9/1.8 840.8/1.8 1050.5/8.9 Left cervical vagus nerve Cases NF + (n)/(%) MBP + (n)/ (%) MBP − (n)/(%) TH + (n)/ (%) ChAT + thick fibers (n)/ (%) ChAT + thin fibers (n)/ (%) ChAT + total (n)/ (%) Sensory fibers n/ % Case 1 27,526/ 100.0 7601/27.6 19,925/72,4 4642/16,9 475/1.7 2.843/10.3 3318/12.1 19,556/71.0 Case 2 19,863/ 100.0 5309/26.7 14,554/73,3 2286/11.5 572/2.9 2198/11.1 2770/14.0 14,807/74.5 Case 3 22,815/ 100.0 7513/32.9 15,302/65.1 4231/18.5 555/2.4 3365/14.7 3924/17.2 14,660/64.3 Case 4 22,940/ 100.0 8591/37.4 19,842/67,1 2356/10.3 477/2.1 1824/8.0 2301/10.0 18,283/79.7 Mean 23,286/ 100.0 7253.5/31.2 16,032.5/ 68.9 3378.8/ 14.3 519.8/2.3 2557/11.0 3,078,3/13,3 16,825.5/72.4 SD 3164.2/0.0 827.9/4.3 2275.2/3.9 1233.2/4.0 51/0.5 683.3/2.8 700.5/3.0 2141.5/5.6 B. Kronsteiner et al. Brain Stimulation 17 (2024) 510–524 514 (caption on next page) B. Kronsteiner et al. Brain Stimulation 17 (2024) 510–524 515 Analyzer. Thus, the automatic counts used the size of the smallest myelinated and ChAT-positive fibers as a reference for the range of fiber size. This ensures objectivity and precision in the measurements. Particles labeled as positive for fibers were examined by the investigators, with any missing or false positive annotations corrected at the end of the quantification process. 2.7. Statistical analyses The paired t-test was used at a significance level of p <0.05 to compare the number of nerve fascicles in the right and left cVN, as well as the percentage of MBP-, TH-, ChAT-positive, and sensory fibers between the right and left cVN and right and left cardiac branch. To compare the diameter of small-sized, medium-sized, and large-sized myelinated fibers as well as unmyelinated fibers, the one-way ANOVA was used at the same level of significance followed by the post hoc Holm- Sidak method. The statistic was performed using SigmaPlot 11 software (Systat Software, Inc., Chicago, IL, USA). Data are presented as the mean ±standard deviation. 3. Results 3.1. Structural organization of the cVN The cVN had a diameter of 1994 ±556 μ m (n =8) and consisted of 1–13 fascicles of various sizes, which were surrounded by connective tissue (Fig. 2A). The fascicle number differed across individuals as well as between the right and left sides of the same individual. The mean number of nerve fascicles was slightly higher on the right than on the left side, although the differences did not reach statistical significance (8.6 ±2.0 versus 7.4 ±4.4, n =8, Fig. 2B). 3.2. Characterization, number, and distribution of nerve fibers within the cVN The total number of nerve fibers in the right and left cVN was similar (25,489 ±2781 versus 23,286 ±3164, Table 3). The staining combination of anti-NF/anti-MBP demonstrated that the human cVN contained myelinated (MBP-positive) and unmyelinated (MBP-negative) fibers with unmyelinated fibers forming groups between the myelinated ones (Fig. 3A – A ″ , 3B–3B ″ ). Both cVNs had a ratio of 2:1 for unmyelinated to myelinated fibers. The right cVN had 66.9 ±3.1 % unmyelinated and 33.1 ±3.1 % myelinated fibers, while the left cVN had 68.9 ±3.9 % unmyelinated and 31.2 ±4.3 % myelinated fibers. Whilst unmyelinated fibers were small, with a diameter of 0.8 μ m (±0.2 μ m), three groups of myelinated fibers - small-sized, medium-sized, and large-sized - were identified (Fig. 3B–B ″ ). The measurements included the axon and the myelin sheath. Significant differences in size were found among the three groups. In particular, the mean diameter was 11.3 ±1.2 μ m for large-sized, 5.8 ±0.7 μ m for medium-sized, and 2.2 ±0.4 μ m for smallsized myelinated fibers (one-way ANOVA test, F (3,213) =2181.345, P <0.001; bar chart in Fig. 3E. Large-sized myelinated fibers formed clusters at the periphery of individual nerve fascicles, while small and medium-sized myelinated fibers as well as unmyelinated fibers were dispersed throughout the nerve (Fig. 3B–B ″ , 3C–3C”, clusters are within the stippled boundaries in Fig. 3B and C). The antibody against TH was used to visualize postganglionic sympathetic fibers. TH-expressing fibers were found in all nerve fascicles (Fig. 4A – 4A”; 4B–4B ″ , 5A–5A ″ ). The distribution pattern was nonuniform and concentrations of TH-positive fibers (arrow in Fig. 4A) and occasionally small groupings were observed (Fig. 4C–C ″ ). The proportion of TH-positive fibers exhibited inter-individual variations, ranging from 8 % to 20.5 % in the right and 10.3 %–18.5 % in left the cVN (Table 3, Supplementary Fig. 3). There was no significant difference between the right and left cVN in the average proportion of TH-positive fibers (13 ±5.9 % versus 14.3 ±4.0 %; paired t-test, t [3] =0.328, p = 0.764; bar chart Fig. 4D). Labeling with anti-NF together with anti-MBP and anti-TH revealed that all TH-positive fibers were unmyelinated, as indicated by the absence of MBP expression. All myelinated nerve fibers and a population of unmyelinated nerve fibers were TH negative (Fig. 5B – 5B ″ , 5C–5C”). To visualize motor fibers, we utilized double labeling with anti-NF/ anti-ChAT. The enzyme ChAT is responsible for the synthesis of the neurotransmitter acetylcholine which is typically for motoneurons. The antibody against ChAT identifies motor neurons including preganglionic sympathetic neurons in both the human central and peripheral nervous system [22,23,32]. In the VN, ChAT-positive fibers are considered as “vagal motor fibers” - a term, which summarizes special visceromotor and parasympathetic fibers. A similar proportion of nerve fibers within the right and left cVN expressed ChAT (13.2 ±1.8 % versus 13.3 ±3.0 %; paired t-test, t [3] = 0.0698, p =0.949; bar chart in Fig. 6E). ChAT-positive fibers formed clusters that either filled a segment at the nerve fascicle periphery or almost the entire nerve fascicle (Fig. 6A and B). Other nerve fascicles displayed only a few scattered vagal motor fibers or lacked them at all. The clusters of vagal motor fibers contained thick and thin fibers that were spatially separated, with thick fibers located peripherally, and thin fibers located centrally (Fig. 6C–C”). In the right cVN, ChAT-positive thick fibers accounted for 2.0 ±0.2 % and, in the left cVN, they accounted for 2.3 ±0.5 %. ChAT-positive thin fibers accounted for 11.1 ±1.8 % and 11.0 ±2.8 %, respectively (bar chart in Fig. 6E–Table 3). No significant differences were found in the percentage of ChAT-positive thick and thin fibers in the right and left cVN (paired t-test, t [3] =1.702, p =0.150; paired t-test, t [3] =0.0472, p =0.965 respectively). All thick vagal motor fibers and many of the thin vagal motor fibers expressed MBP (Fig. 6D–D ″ ). However, a considerable number of thin vagal motor fibers did not express MBP (Fig. 6D–D”). Thus, based on the staining combinations anti-NF/anti-ChAT and anti-MBP/anti-ChAT, three groups of vagal motor fibers ere distinguishable: myelinated vagal motor fibers of small and large caliber, as well as unmyelinated vagal motor fibers. Most nerve fibers within the cVN lacked ChAT (Fig. 6A and B). However, not all ChAT-negative fibers could be classified as vagal sensory fibers because the distribution area of ChAT-negative fibers overlapped with TH-positive sympathetic fibers, which represent postganglionic sympathetic fibers. This overlap was seen by comparing consecutive sections of the same nerve in NF/TH/MBP staining (Fig. 5A–A”) and NF/ChAT staining (Fig. 6B). To determine the Fig. 3. Visualization, percentage, and size specification of myelinated and unmyelinated fibers in the human cVN. (A – A”, B – B ″ , C – C ″ ) CLSM images of cVN crosssections immunolabeled with anti-NF and anti-MBP. In these and other figures, the color channels are separated. The combination of the color channels is displayed in the overlay. (A – A ″ ) Panoramic images of a right cVN. The images demonstrate that all nerve fascicles contain myelinated (MBP-positive) and unmyelinated (MBP- negative) fibers. (B – B ″ ) Magnified images of the insert in A”. The nerve fascicle contains myelinated fibers of variable size and unmyelinated fibers. Large-sized myelinated fibers form a cluster (the cluster is within the stippled boundary shown in B) at the periphery of the nerve fascicle. The zoom-in (lower right) of the insert shows myelinated fibers of different sizes. A single large-sized myelinated fiber (lower left), a single medium-sized myelinated fiber (upper left), and several small-sized myelinated fibers can be distinguished. Additionally, unmyelinated fibers without MBP expression are seen. (C – C ″ ) Images of a single nerve fascicle. Several clusters of large-sized myelinated fibers (the clusters are within stippled boundaries shown in C) are at the fascicle periphery. (D, E) Bar charts showing the percentage of MBP-positive fibers in the right and left cVN (n =4) and (D) the fiber diameters of unmyelinated fibers, small-sized, medium-sized, and large-sized myelinated fibers (E). Data are presented as mean value and standard deviation. The different symbols (*, ◦, ●) show significant differences between the diameter of all fiber groups (one-way ANOVA test, F (3,213) =2181.345, P <0.001). Scale bars: 200 μ m in A ″ , 50 μ m in B ″ and C ″ , and 5 μ m in the lower right insert of B”. B. Kronsteiner et al. Brain Stimulation 17 (2024) 510–524 516 Fig. 4. Visualization and percentage of sympathetic fibers in the human cVN. (A – A ″ , B – B ″ , C – C ″ ) CLSM images of cVN cross sections immunolabeled with anti-NF and anti-TH. (A – A ″ ) Panoramic images of a right cVN illustrate that TH-positive sympathetic fibers are present in all nerve fascicles. The TH-positive fibers are irregularly distributed with areas of higher concentration (arrow). (B – B ″ ) Magnified images of the insert in A”. Scattered, TH-positive fibers of small diameter are present in the nerve fascicle. The zoom-in (upper left) shows that exclusively thin fibers are TH-positive. Other thin fibers and all thick fibers are TH-negative. (C – C ″ ) Showing a cluster of TH-positive axons at the periphery of a nerve fascicle. (D) Bar charts showing the percentage of TH-positive fibers in the right and left cVN (n = 4) along with mean values and standard deviation. Scale bars: 200 μ m in A ″ , 50 μ m in B ″ , 5 μ m in the insert of B ″ and 50 μ m in D”. B. Kronsteiner et al. Brain Stimulation 17 (2024) 510–524 517 proportion of vagal sensory fibers, we subtracted the values counted for sympathetic fibers from the values of the ChAT-negative fibers. Based on this calculation, we concluded that the proportion of vagal sensory fibers was 74.0 ±7.5 % in the right cVN and 72.4 ±5.6 % in the left cVN (paired t-test, t [3] =0.257, p =0.814; bar chart in Fig. 6F–Table 3). By applying additional criteria such as MBP expression, morphologically different types of sensory fibers were distinguishable within the human cVN. This includes myelinated fibers of small and medium size as well as unmyelinated fibers. For a detailed discussion of criteria used to classify morphological types of vagal sensory fibers see Supplementary Fig. 4. 3.3. Characterization, Number, and Distribution of Nerve Fibers within the Vagal Superior Cardiac Branch. Fig. 5. Simultaneous visualization of myelinated and sympathetic fibers in the human cVN. (A – A ″ , B – B ″ , C – C ″ ) CLSM images of cVN cross sections immunolabeled with anti-NF along with anti-MBP and anti-TH. In the images (A, B, C and A′, B′, C′) two color channels are merged while in the overlay (A ″ , B ″ , C ″ ) three color channels are merged. (A – A ″ ) The panoramic images of a left cVN show that a variable number of TH-positive fibers are present in all nerve fascicles. (B – B ″ ) Magnified images of the insert in A”. TH-positive fibers are found in between MBP-positive fibers (B′, B ″ ). (C – C ″ ) High-resolution images. Only a portion of MBP- negative fibers express TH (C′, C ″ ). Another portion of MBP-negative fibers (arrows in C ″ ) and all MBP-positive fibers lacks TH-immunoreactivity. Scale bars: 200 μ m in A ″ , 100 μ m in B ″ , and 25 μ m in C”. B. Kronsteiner et al. Brain Stimulation 17 (2024) 510–524 518 Fig. 6. Visualization and percentage of motor fibers in the human cVN. (A, B, C – C ″ ) CLSM images of cVN cross sections immunolabeled with anti-NF and anti-ChAT. (A, B) Panoramic images of a right (A) and a left cVN (B). ChAT-positive motor fibers form clusters in some nerve fascicles. (C – C ″ ) Magnified images of a single nerve fascicle. The nerve fascicle contains thick and thin fibers expressing ChAT, which run in separate compartments. The compartment of the ChAT-positive thick fibers is indicated by the white stippled line. (D – D ″ ) High-resolution images of the cVN labeled with anti-ChAT and anti-MBP. A single thick fiber (arrow in D ″ ) and thin fibers expressing ChAT and MBP are seen. Additionally, ChAT-positive fibers without MBP signals are visible. (E, F) The bar charts display the percentage of thin and thick fibers expressing ChAT (E) and the percentage of sensory fibers (F) in the right and left cVN (n =4). Scale bars: 200 μ m in A and B, 50 μ m in C ″ , and 10 μ m in D”. B. Kronsteiner et al.