Fresh cadavers of male (n = 3) and female (n = 4) pigs (domestic pigs, n = 7, nerve harvest 1–2 h post mortem (p.m.)., 36–64 kg, age of 6 months) as well as female rabbits (New Zealand White, n = 3, nerve harvest 1–2 h p.m., 2.5–3.3 kg body weight, age of 3–4 months) were donated immediately after euthanasia for dissection and sampling of fresh autonomic nerve specimen including superior cardiac vagal branches (3R principle). All experimental procedures were ethically reviewed and approved by the Ethics Committee for Laboratory Animal Research of the Medical University of Vienna (pigs: GZ 2021-0.615.897//rabbits: 0030-V/3b/2018) and of the Austrian Federal Ministry of Science, Research, and Economy. Animals were treated in accordance with the “Guide for the Care and use of Laboratory Animals” (NIH publication 85–23, revised 1985). Out of all cadavers (n = 7 pigs, n = 3 rabbits) given to our study, 11 pig nerves (n = 5 left, n = 6 right nerves) and 5 rabbit nerves (n = 2 left nerves, n = 3 right nerves) could be retrieved for use in this study. A schematic diagram of the surgical field was created using the vector graphics tool Inkscape (Inkscape, 2016) as shown in Figure 1. In pigs, the nerves were dissected and harvested as is depicted in Figure 1, demonstrating that the superior cardiac branch was only dissected. A similar surgical window was used in rabbits as well.

For dissection, cadavers were placed in the dorsal recumbence position. A ventral skin incision (15–20 cm in pigs, 7–9 cm in rabbits) was cut on both sides next to the trachea starting at the level of the mandibles and extended further to the sternal notch. Tissue was dissected and the carotid sheath was opened to expose the common carotid artery, internal jugular vein, and the VN as well as the ST. Nerve dissection was started at the level of the nodose ganglion (NG) (Figure 1, “position.1”) and extended caudally to the superior cardiac branch (Figure 1 “position 4”), which originates approximately 2–5 cm cranial to the subclavian artery on both sides. The inferior cardiac branches were not dissected and included into this study since their anatomical branching point within the epicardial fat pad is not practically suitable for selective cardiac VNS. Branches emerging from and terminating in the VN were tracked to their origin structures. Both recurrent laryngeal nerves were identified as running close to the trachea and looping back around the subclavian arteries. Subsequently, the chest was opened by median sternotomy followed by gentle exposure of the heart. Then, the surgical preparation of the VN was continued towards the heart, and the superior CBs (one CB per nerve) were dissected starting from the cardiac branching points at the VN towards the insertion points at the heart. Care was taken not to cut any of the cross-connections between the VN and the ST, also referred to in this study, as “communicating branches”. The macroscopic branching and merging positions between the VN, ST, and superior cardiac branching were highlighted in-situ (Figures 2A,B). Throughout dissection, isotonic sodium chloride solution (NaCl, 0.9%) was used to prevent the nerves from drying out. Finally, the dimensions of the VN, from the nodose ganglion down to the insertion points of the cardiac branches into the heart were measured using a ruler and captured in a photo Figures 2A,B. In the following, the superior CB will be briefly named “CB”.

To maintain the anatomical in-situ orientations of the nerves after dissection and harvesting, surgical sutures (5–0 silk suture, Silkam^® B-Braun Aesculap, Tutlingen, Germany), were fixed onto the epineurium of the VN labeling the ventral side starting just below the nodose ganglion.

After complete dissection, nerves were placed and fixed onto a polystyrene plate with V-shaped incisions labeling the medial site, cut edges labeling the caudal site, and the sutures labeling the ventral sites of the VN, thus maintaining the anatomical in-situ orientation of the nerve specimen. Nerve specimens were briefly washed in PBS followed by fixation in 4% phosphate-buffered 4% PFA (Roti-Histofix, Roth, Karlsruhe, Germany) at 4°C for 16–18 h in case of pig nerves, and 14 h for rabbit nerves, respectively. After fixation, nerve samples were briefly washed again and stored in freshly made PBS at 4°C until staining for µCT imaging.

For contrast enhancement of the different types of tissue, nerves were stained in 14 ml 0.5% (rabbit nerves) or- 1% (pig nerves) Lugol’s solution for 24 h. Lugol’s solution is one part iodide and two parts potassium iodide in an aqueous solution. To produce a concentration of 1%, potassium iodide is dissolved in double-distilled water as described in a previous study [(Heimel et al., 2019)]. μCT scans were performed using a SCANCO μCT 50 (SCANCO Medical AG, Brutistellen, Switzerland) specimen μCT scanner. To avoid movement artifacts nerve specimens were placed in 15 ml conical centrifuge tubes in the residual staining solution with the polystyrene plate used for fixation. For overview scans low-resolution scanning was performed with 70 kVp (200μA, 0.5 mm Al filter, 550 projections, 100 ms integration time) at an isotropic resolution of 20 μm. The VNs from the pigs were divided into two nerve specimens of 7 ± 2 cm long half-sections, followed by digital alignment between scans over the entire length of the nerves (= cranial and caudal nerve specimen). Anatomical orientation for proper alignment of the two parts was given by a ventral suture that was placed to the ventral site of the nerve and a polystyrene plate on which the nerve was placed onto with its dorsal side. The medial site was labeled by the site where the cardiac branch left the VN trunk. The shorter rabbit nerves were scanned over the total length with 7 ± 1.3 cm of length and also the polystyrene plate labelling the dorsal site and the CB on the medial site. Therefore, it did not need any alignment of the scans afterwards.

High-resolution scans of both species were performed 2 days after the low-resolution scans at the site of the cardiac branching point with 70 kVp (85 μA, 0.5 mm Al filter, 850 projections, 350 ms integration time) at an isotropic resolution of 8.9 μm over 1 cm of nerve length. To maintain full saturation of fascicles with Lugol’s staining solution, nerves were re-stained 24 h prior to performing high-resolution scans.

High-resolution data were analyzed at the cardiac branching point and investigated whether a greater number of cardiac fascicles could be detected when higher resolution was applied to the nerves

Raw images from µCT scans were provided as DICOM and tiff formats while only the DICOM format was exported for contrast-based segmentation of VN using Materialize Mimics Research 23.0 software (Materialize, Leuven, Belgium). The fascicles of the VN, the ST, and the CB were manually segmented and isolated, owing to partly low contrast between nerve and connective tissue, from the complete VN using split function to trace the course of the CB fascicles within the cervical VN from the CB point to the isolated cardiac branch. Particular focus was placed on the identification of the position where the fascicles of the CB traveled topographically in the cervical VN and whether they crossed or joined other fascicles. The STL file generated was then imported to Materialize 3-Matic 15.0 software (Materialize, Leuven, Belgium) for performing a remesh, necessary for reducing the size of the file by keeping the quality of the data basically unchanged. Here the connective tissue was further separated from the VN and CB. The segmented 3D models will provide data for subsequent elaboration e.g., simulation of electrical-field spatial distributions during stimulation.

All µCT data sets were available as tiff format and as dicom format. Firstly, µCT imaging stacks and single cross-sections were analyzed as TIFF files in XY, YZ, and XZ direction using Fiji software [(Schindelin et al., 2012), (Bergmeister et al., 2017)] for delineation, tracing and quantification (e.g., areas, fascicle counts). Firstly, each stack was rotated and aligned with the dorsal side of the nerves being on the upper part of the images. The dorsal and the medial sites were then labeled in each stack. For digital alignment of the whole pig nerves, both stacks (stack of the cranial and caudal part of the nerve) were opened in Fiji and the positions of nerve the fascicles on the last slides of the cranial part and those on the first slides of the caudal part were compared. The positions of the cardiac branch were labeled on the last slides of the cranial part and the first slides of the caudal part to maintain anatomical orientation. The contrast of the nerve cross-sections was manually enhanced for proper quantification of nerve fascicles using the brightness/contrast function based on tissue-dependent differences in gray values of nerve fascicles and background. Tracing of the fascicles of the cardiac branch was performed using the lasso tool in XY and XZ views. This was done in both species, pigs and rabbits. Here, the course of the cardiac branch fascicles was tracked from the cross-slice position of the CB point (position 4, Figure 1) up to the cervical VN, where the cardiac fascicles started either merging, splitting, or both with the cervical VN fascicles so that it was not possible to further distinguish them as single cardiac branch. This cross-slice position was labeled and the distance Figure 5A from the CB point up to this position was measured for each nerve (Figure 5A). The same procedure was then performed for mapping the distance of the first fascicle exchange position between the ST and the VN cranial to the cardiac branching point.

Next, we compared the findings regarding branching and merging patterns in µCT scans vs. macroscopically in-situ, out of which the following types could be differentiated in the pigs. The first type noted was “contact”, describing the situation where two nerves were closely adjacent to each other albeit there was still epineurium present between the nerves, thus no exchange of fascicles occurred. In some nerves, an additional layer of epineurium was surrounding the two nerves together. The next definition used in this study was “merged” fascicles representing two fascicles from each nerve fusing together and forming one fascicle surrounded by one layer of perineurium that further moved cranially as one fascicle, which was found between fascicles of the VN and ST at the mid-cervical level and between the VN and CB at the lower cervical level, from where fascicles of the CB merged with the fascicles of the cervical VN, thus, were not further distinguishable and traceable as “isolated” CB fascicles in the µCT scans. The third pattern, “communicating” branches”, also referred to as cross-connections, was observed between VN and ST at the mid-cervical level, where single nerve branches crossed the surgical field from one nerve to the other.

The next steps of image analysis of the functional anatomy were conducted only in the multifascicular nerves of pigs using also Fiji software (Schindelin et al., 2012). The area of the nerves and fascicle number was measured for each nerve at the cross-slice positions of interest using the freehand selection tool followed by manual counts of the fascicle numbers using the multipoint tool and the freehand tool. Measurements on cross-sections were performed at five different positions as shown in Figure 1, starting from just below the nodose ganglion (position 1) down to just below the cardiac branch (position 5). Positions 2 and 3 were located at the mid-cervical level approximately 2 cm apart from each other. Position 4 was the position of the cardiac branching point (CB point).

Data were analyzed using Graphpad Prism software (GraphPad Software, San Diego, CA, United States) and MATLAB (version R2021a, Mathworks, Natick, Massachusetts, United States).

Topographical measurements of the cardiac branching patterns were analyzed using descriptive statistics and are presented as absolute and relative values.

Normal distribution of data was assessed by the Shapiro-Wilk test. Non-normally distributed data is presented as median and interquartile range (IQR). Side-related comparisons were performed using the Kruskal–Wallis test for the variables fascicle numbers and area at level of the CB point. Statistical significance was accepted if p < 0.05.

Dissections of the VN and the superior cardiac branch revealed that the branching position of the superior CB (“position 4”, Figure 1) varied among individuals in pigs and between left and right side. However, we could find the branching position of the superior cardiac branch to be at 2–5 cm cranial to the subclavian artery. In rabbits, the superior cardiac branch was found to branch off closer (approximately 1–3 cm) to the level of the subclavian artery in all animals.

To determine whether VN and ST use separate nerve fibers or, conversely, share them in order to send signals from the cervical level downwards to the heart and other organs, surgical dissection and macroscopic examinations were applied to fresh swine cadavers as well as those of rabbits.

The results of the surgical dissection in pigs revealed different merging and branching patterns among individuals (Table 1). Close contact between or even merging between VN and ST was macroscopically observed in 7/11 (64%) nerves, of which 3/7 (43%) nerves were on the left side, and 4/7 (57%) nerves were to the right. In contrast, when it came to the rabbit nerves, no prominent differences in branching patterns were observed and no difference was determined between the left and right sides. Hence, unlike the situation in pigs, in rabbits, the fascicles in the VN and ST were clearly separated from each other in the cardiac branches towards the heart, and it would be interesting to gain further insight into the situation in pigs using more advanced tomography.

To investigate whether fascicle number in the cardiac branches (CBs) differed between the two scanning protocols low-resolution vs. high-resolution scans, all nerve specimens were firstly scanned in low-resolution (pig: n = 11; rabbit: n = 5) over the entire length (pigs: 7 ± 2 cm, rabbits 7 ± 1.3 cm) followed by high-resolution scans over 16.906 ± 2.803 mm at level of the cardiac branching point.

In both scans fascicles were clearly separated from the rest of the nerve tissue, nevertheless, boundaries between single fascicles were better visible in high-resolution scans.

The direct comparison between the low-resolution and high-resolution scans revealed a better distinguishability of single fascicles in HR scans, but it did not change the detectable number of cardiac fascicles at the level of the CB point (Figure 3).

To understand further the anatomical structure of the cardiac autonomic innervation in both species, µCT scans were also 3D-rendered and an anatomical model of both nerves was developed. Data have shown that the 3D rendering of the multi-fascicular anatomy of the pig VN (Figure 4A) and the mono-fascicular anatomy of the rabbit VN (Figure 4B). In both species, the cardiac branch (indicated in red) enters VN (indicated in green) on the medial side, then travels in ventromedial position in the cervical VN towards cranial adjacent to other fascicles until it merges with other fascicles, as depicted in Figure 4.

Table 1 shows the three types of branching patterns we identified in this study. Therefore, the following patterns were defined: 1) contact between VN and ST, 2A) merged fascicles between VN and ST and 2B) merged fascicles between VN and CB fascicles, and 3) cross-connections between VN and ST, referred to as “communicating branches”. In addition, close contact or merging of VN fascicles with ST was macroscopically investigated during surgical dissections (Table 1 , column 4).

Several features were noted, as follows (Table 1): 1) Tight contact between the VN and ST was observed in 7/11 (64%) nerves, with 3/11 (27%) nerves on the left and 4/11 (36%) nerves on the right side. 2A) Merging fascicles, VN and ST were observed in 3/11 (27%) nerves, with 1/11 (9%) observations on the left side and 2/11 (18%) observations on the right side, respectively. 2B) Merging fascicles between VN and CB were observed in 10/11 (90%) nerves, of which 4/10 (40%) nerves were located on the left and 6/10 (60%) nerves on the right side. 3) In 6/11 (55%) of nerves communicating branches were found, of which 2/11 (18%) nerves were located on the left and 4/11 (36%) on the right side. 4) Macroscopical investigations of the branching- and merging patterns in-situ revealed 7/11 (64%) observations, with 3/7 (43%) on the left and 4/7 (57%) on the right side. A comparison of the branching patterns between the left and the right side displayed slightly more branching patterns on the right side.

To measure the traceable length of the fascicles of the cardiac branch in the cervical VN trunk in both species, the cardiac branch was traced starting from the cardiac branching point in cranial direction (Figure 5B ) In total, the cardiac branch could be traced cranially in 10/11 (90.9%) of pig nerves and in all rabbit nerves (5/5). The median traceable length of the pig cardiac branch was 22.19 mm (IQR 14.02–41.30 mm) and 7.68 mm in rabbits (IQR 4.06–12.77 mm).

To explore differences in merging and branching patterns of fascicles between ST and VN in pig nerves, fascicle-merging events were counted manually in individual nerves between VN and ST using the µCT scans (Figure 6).

Merging of fascicles between VN and CB (VN_CB fascicles) were visible in 10/11 (90.0%) nerves. Out of these 10 nerves, 4/10 (40%) observations were located on the left side, and right 6/10 (60%) observations on the right side. In contrast, merging of single fascicles between VN and ST (VN_ST fascicles) on the upper cervical level was counted in 3/11 (27%) animals. The median distance of the fascicle merging positions between VN and ST was 68.98 mm (IQR 42.32–71.54 mm) above the cardiac branching point. The median distance for merging of fascicles between VN and CB was 24.82 mm (IQR 22.24–31.37 mm) on the left side and 9.66 mm (IQR 4.09–17.86 mm) on the right side. The calculated p-value for the group comparison left vs. right side “merging fascicles” was not significant (p = 0.361).

To examine whether there was a correlation between fascicle numbers and anatomical positions of the VN, the median values for the two features were measured for the VN, ST, and CB point at different positions (Figure 1) and are listed in Table 2.

The comparisons at different levels show that the widest median area of the cervical VN was found at position 1, and at position 3 in the ST. The smallest median area of the VN was at position 3 and that of the ST was at position 4. The largest median area of the cardiac branch was found just at the cardiac branching point (pos.4). The measurements for these comparisons taken at the level of the cardiac branching point (pos. 4) are shown in Figures 7A,B, presenting the side-related comparisons of the median values of the parameters “fascicle numbers” and “nerve area” at the level of the CB point. The median area of the left and right VN was 2.47 mm² (IQR 1,7 to 3.2 mm²) and 1.86 mm² (IQR 1.2–2.7 mm²), of the left ST 0.38 mm² (IQR 0.3–0.4 mm²), and right 0.38 mm² (IQR 0.3–1.2 mm²), and of the left vs. right CB 0.22 mm² (0.1–0.3 mm²) and 0.24 mm² (0.1–0.3 mm²). The calculated p-values for side comparisons in each of the three nerves in terms of the area were not statistically significant (VN p = 0.39; ST p = 0.46; CB p = 0.67). The median values of VN counted fascicles at the level of the CB point were n = 28 on the left side (IQR 20 to 32 fascicles) and n = 24 fascicles on the right side (IQR 16 to 28 fascicles), in the ST n = 3 (IQR 3 to 5 fascicles) on the left and n = 4 fascicles (IQR 3 to 7 fascicles) on the right side, respectively and in the CB n = 2 (IQR 1 to 5 fascicles) on the left and n = 4 (IQR 2 to 4 fascicles) on the right side. The calculated p-values for side comparisons in each of the three nerves in terms of the fascicle distributions were not statistically significant (VN p = 0.24; ST p = 0.43; CB p = 0.44).