Sympathetic components in left and right human cervical vagus nerve: implications for vagus nerve stimulation

Figures

• 

  FIGURE 1. Fascicle composition of vagus nerve at mid-cervical level. (A1,A2) Staining with myeline basic protein (MBP) showing ten individual fascicles (A1), inset is shown magnified in panel (A2). Note that within this fascicle, several subgroups can be recognized. (B) Branching and joining of fascicles in a stretch of 2.8 mm of the cervical vagus. Only two fascicles (arrowheads) did not show any change in fiber composition. (C1,C2) Reconstruction of fascicle composition of 20 mm of the cervical vagus seen from medial (C1) and lateral (C2). Cranial is to the left. White arrows depict branching of fascicles, blue arrows indicate joining of fascicles. A video showing a 3D-rotation of this segment of cervical vagus is found as Supplementary Video 1.

• 

  FIGURE 2. Examples of redistribution of small parts of a fascicle to another fascicle at mid-cervical level. (A1,A2) Here, several tens of myelinated fibers travel more than 500 μm in a transverse direction to join another large fascicle. (A2) Shows a magnification of the inset in panel (A1). Arrows indicate strands of fibers that jump from one fascicle to another. (B,C) Two other examples where only a small part of a fascicle (arrows) detaches itself to join another fascicle at virtually the same cranio-caudal level of the vagus nerve.

• 

  FIGURE 3. Vagus nerve fascicles contain TH-positive structures. (A) Vagal fascicles at mid-cervical level demonstrate evenly distributed MBP-labeling indicating a generally uniform density of myelinated fibers throughout all vagal fascicles. (A1) MBP-labeling of mouse forebrain embedded within the same gelatin block. Note the MBP-staining of the corpus callosum (black arrow), anterior commissure (white arrow) and internal capsule fascicles in the caudate-putamen. (B) Consecutive section stained for TH. Note distributed punctate labeling within most fascicles. (B1) TH-labeling of mouse forebrain. Note absence of labeling in the corpus callosum (black arrow) and anterior commissure (white arrow). Caudate-putamen now shows dense TH-labeling in its neuropil as dopaminergic terminals also are positive for TH. (C,D) Magnification of inset shown in panels (A,B), respectively. (E) Detail of punctate TH-labeling. Larger punctae can be seen to be made up of several smaller ones with diameters of 1–2 μm.

• 

  FIGURE 4. TH-positive structures are unmyelinated. (A) Overview of mid-cervical vagus nerve section double stained for MBP (green) and TH (red). (B) Magnification of framed area shown in panel (A). (C) Magnification of framed area shown in panel (B). Note that TH-positive structures are not surrounded by myeline (i.e., green) but are scattered between the myelinated fibers. Blue staining indicates DAPI, labeling cell nuclei.

• 

  FIGURE 5. TH-positive structures represent unmyelinated nerve fibers. (A) Double fluorescent labeling of neurofilament (NF, green) and TH-positive structures (red) in a joint preparation of the superior cervical ganglion (SCG; left) and vagus nerve (nX; right). Note that all somata in the SCG are TH-positive, but only a few are also labeled for NF (yellow, arrows). (A1,A2) Magnification of framed areas in panel (A). Virtually all fibers within the SCG are TH- and NF-positive (yellow), whereas in the vagus nerve many large profiles are NF positive, in selected areas small profiles are TH- and NF-positive. (B1-B3) Triple labeling of myeline (MBP, blue), neurofilament (NF, green) and tyrosine hydroxylase (TH, red) of part of a vagal fascicle. Note that the red TH profiles in panel (B3) match the location of the green NF profiles in panel (B2) (although they do not have the exact same shape) and represent unmyelinated fibers as shown in panel (B1). All myelinated fibers are TH-negative (cf. B1,B2).

• 

  FIGURE 6. TH-positive fibers from the sympathetic trunk enter the vagus nerve and take a caudal course. “Direction” of fibers should be seen in relation to the normal flow of information (i.e., direction of actionpotentials) through the axons. (A1–A4) Series of sequential sections (distance between sections 600 μm) from cranial (A1) to caudal (A4). SCG is shown on the left, vagus nerve on the right. In panels (A2,A3) the SCG is attached to the vagus nerve and small bundles of TH-fibers can be observed to enter the nerve. At (A1) (cranial) and (A4) (caudal-most level) the SCG is not connected to the vagus. Insets show details, red arrowheads indicate TH-fibers. Note that in panel (A1) no TH-fibers can be observed, which indicates that the TH-fibers upon entering the vagus nerve take a caudal route. (B1–B8) Series of sequential sections from cranial (B1) to caudal (B8). The vagus nerve is demarcated by a striped line at the left side, whereas the SCG is positioned at the right side. In panel (B8) two fascicles detach from the sympathetic trunk and course in cranial direction as they can be followed from the section shown at (B8) to the level shown at (B3) (single arrowhead; inset in panel…

• 

  FIGURE 7. Density distribution of TH-positive fibers through sequential levels of the cervical vagus. (A) The density distribution is shown at nine levels, representing 45 mm shown from cranial (top) to caudal (bottom) of the right mid-cervical vagus (386R). Note that small fascicles present at the top distribute themselves throughout multiple fascicles. (B) Shows the density distribution at twelve levels, representing 90 mm of mid-cervical vagus 000R. Here too, the TH-positive fibers do not take up a fixed position, but may drift throughout the vagus, yet they tend to stay clustered.

• 

  FIGURE 8. Determining surface area of the TH-labeled fibers using Fiji software. (A) Starting image captured with Nanozoomer (Hamamatsu). (B) Conversion to gray values using automatic adjustment for contast and brightness and manual delineation of the fascicles. (C,D) Thresholding and determination of the positive surface area within the individual fascicles and final calculation of summed fascicle area and percentage coverage by TH-labeling. Data for this particular section were: number of fascicles: 8, total fascicle surface area: 1.294 mm2. and average TH-coverage: 15.66%.

• 

  FIGURE 9. Fascicle number and surface area of left and right cervical vagus nerves. (A) Number of fascicles observed in the 20 pairs of examined vagus nerves at the four levels. On the x-axis the blue dots represent the left vagus nerve and the matching orange dots represent the right vagus nerve of all individual pairs. Note that in levels 3 and 4, generally more fascicles were found in right nerves. Also note that data were not always available for every level. (B) Average number of fascicles for all left and right nerves for every level. Bars represent standard deviation. (C) The number of fascicles per nerve was not correlated to the age of the specimens. (D) Box-whisker plot of the ratio left/right nerve fascicle number at the four levels. Note that at cranial-most levels the average ratio is close to 1.0 whereas at the caudal levels most ratio’s fall below 1.0, indicating there are more fascicles present in the right vagus nerve as compared to the left one. (E) Average surface area of the summed fascicle areas at the four examined levels. Note high variability at the cranial-most level. No significant statistical differences were noted between left and right vagus nerve, although…

• 

  FIGURE 10. Coverage and distribution of TH-positive fibers in left and right vagus nerves. (A) Average surface percentage of total fascicle area ( = %TH-coverage) of the left (blue) and right (orange) vagus nerve related to the age of the specimens. Note large differences between individuals and between individual left and right nerves. (B) % TH-coverage related to post-embalming period. Note that the longest post-embalming periods (51–27 months) demonstrated the least coverage. (C) Relation between the averaged and summed surface areas of the vagus nerve and of the averaged and summed surface areas of the TH-compartments. (D) Average TH-surface area at the four examined cervical levels. Note that the surface generally diminished for left vagus nerves but remained at the same overall value for the right vagus nerve. (E) TH-surface area as related to the total effective nerve surface area of the summed fascicle surfaces. From panels (C,D) it can be surmised that the TH-coverage of the left nerve kept pace with the diminishing surface of the nerve whereas this increased for the right nerve. (F,G) Box-whisker plots of the standard deviations of the covering percentage of the individual…