Segmental branches emanating from saphenous nerve morphing into sympathetic trunks for innervation of saphenous artery and its clinical implication for arterial sympathectomy

Abstract

Sympathectomy of arteries has been adopted for the treatment of peripheral arterial disease and Raynaud's disease. However, the exact route for sympathetic axons to reach peripheral arteries awaits further investigation that could pave the way for development of new surgical strategies. In this study, saphenous neurovascular bundles from 10 neonatal Sprague-Dawley rats first were harvested for whole-mount immunostaining to show sympathetic innervation pattern of the artery. Secondly, 40 Sprague-Dawley male rats weighing 350 to 400 g were assigned to five groups, receiving either sham, perivascular sympathectomy, nerve-artery separation, nerve transection in the saphenous neurovascular bundle, or lumbar sympathectomy surgery that removes the lumbar sympathetic trunks. Immediately after surgery, the arterial perfusion and diameter were measured using laser speckling contrast imaging, and 1 week later the saphenous neurovascular bundles were harvested for immunostaining using antibodies against TH, neuron-specific β-tubulin (Tuj 1), and α-SMA to show the presence or absence of the TH-immuopositive staining in the adventitia. The differences among the five groups were determined using one-way analysis of variance (ANOVA). We found that an average of 2.8 ± 0.8 branches with a diameter of 4.8 ± 1.2 μm derived from the saphenous nerve that morphed into a primary and a secondary sympathetic trunk for innervation of the saphenous artery. Nerve-artery separation, nerve transection, and lumbar sympathectomy could eradicate TH-immunopositive staining of the artery, resulting, respectively, in a 12%, 36%, and 59% increase in diameter (P < .05), and a 52%, 63%, and 201% increase in perfusion compared with sham surgery (P < .01). In contrast, perivascular sympathectomy did not have a significant impact on the TH-immunopositive staining, the diameter, and perfusion of the distal part of the artery (P > .05). We conclude that the sympathetic innervation of an artery derives from segmental branches given off from its accompanying nerve. Nerve-artery disconnection is a theoretic option in sympathectomy of an artery.

Keywords: Raynaud's disease; arterial innervation; peripheral arterial disease; sympathectomy; sympathetic nerve.

FIGURE 1

Experimental design. The study was divided into two parts: morphologic observation and functional verification. In the part of panoramic morphologic observation, saphenous neurovascular bundles from 10 neonatal rats were harvested for whole‐mount immunostaining to observe the source of sympathetic innervation to the saphenous artery. Without the need for sectioning before staining, a panoramic picture of sympathetic innervation of the artery could be observed. In the part of surgical manipulations for verification, 40 rats were evenly assigned into five groups. A, Control group: only exposure of the saphenous neurovascular bundle was performed; B, perivascular sympathectomy: the small rectangle represents stripping of the adventitia for about 3 mm at the root of the saphenous artery; C, nerve‐artery disconnection: the green dotted line indicates severance of connections between the saphenous nerve and artery; D, nerve transection: the cross indicates transection of the saphenous nerve at the root; E, the two crosses indicate removal of the bilateral lumbar sympathetic trunks. Immediately after surgery, laser speckle contrast imaging (LSCI) was adopted for measurement of the inner diameter and perfusion intensity of the saphenous artery to determine whether haemodynamic changes were initiated by the surgical manipulations. One week later, the neurovascular bundles about 1.5 cm away from the root of the nerve indicated by the large rectangles were harvested, and conventional cryosection and immunostaining were performed to observe the presence or absence of immunopositive TH staining on the adventitia of the saphenous artery to evaluate the efficacy of sympathectomy

FIGURE 2

Presence of lumbar sympathetic trunks in the groove between the bilateral psoas majors. The arrowheads denote the usual four pairs of sympathetic ganglia that are removed during surgery. The first pair can be found underneath the crura of diaphragm, and the last pair can be found underneath the forking site of the abdominal aorta. In this study, all four pairs of the ganglia were removed. The arrow denotes the abdominal aorta and the inferior vena cava that are retracted aside to expose the lumbar sympathetic trunk. The two stars denote the psoas majors

FIGURE 3

Whole‐mount staining of a saphenous neurovascular bundle. 1, 2, and 3 represent the saphenous nerve, artery, and vein, respectively; 4 and 5 indicate the primary and secondary sympathetic trunks formed by the segmental sympathetic branches. Note the dense sympathetic network on the adventitia of the saphenous artery in comparison to the scarce one on the adventitia of the saphenous vein; the arrowheads denote the segmental sympathetic branches given off from the nerve to the artery; the arrows denote the transverse branches given off from the primary sympathetic trunk that cross the artery to form the secondary sympathetic trunk. The scale bar represents 500 μm

FIGURE 4

Speckling images of the saphenous vessels after surgical manipulations and the statistical bar charts. The green arrowhead denotes the saphenous arteries; the red arrowheads denote the saphenous veins. A, B, C, D, and E represent the perfusion images of the saphenous vessels collected using LSCI from the control, perivascular sympathectomy, nerve‐artery disconnection, nerve‐transection, and lumbar sympathectomy groups, respectively. In the nerve‐artery disconnection group, because of the reason that the nerve and artery should be slightly separated by pinching using microsurgical forceps before spring scissors could be inserted to disrupt connections between the nerve and artery, a slightly undulated surface could be observed on the saphenous artery. The diameter of the saphenous artery in this group was calculated by averaging the inner calibre measured at the dented site and that measured not at the dented site. The arrow indicates the saphenous nerve that was retracted aside after manipulation. The scare bar represents 0.5 cm. F statistical comparisons of the inner diameter among the five groups. There were significant differences among the five groups regarding both the inner calibre and perfusion (P < .001). * indicates that significant differences of the inner diameter and perfusion of the saphenous artery could be found compared with those of the saphenous artery in the control group (P < .05). ∆ indicates that no significant difference of the inner diameter and perfusion of the saphenous artery could be found between the control and perivascular sympathectomy groups (P > .05). # indicates the inner diameter and perfusion of the saphenous artery in the lumbar sympathectomy were significantly larger than that those of the saphenous artery in the nerve‐artery disconnection and nerve‐transection groups (P < .05)

FIGURE 5

TH, Tuj1, and α‐SMA‐immunostaining for sympathetic axons (green), pan‐axons (blue), and smooth muscle cells (red) of cross sections of the saphenous neurovascular bundles from the five groups. The green, red, and blue arrowheads denote the saphenous nerve, artery, and vein, respectively. A, a representative image from the control group. Note the evenly scattered distribution of sympathetic fibres among other non‐adrenergic axons in the saphenous nerve; dense and scarce sympathetic network could also be observed, respectively, over the adventitia of the saphenous artery and vein; B, after perivascular sympathectomy, the sympathetic fibres in the adventitia about 1.5 cm distal to the surgical site were not obviously affected. C, after disruption of the connections between the nerve and artery, the nerve was not affected, whereas the sympathetic fibres disappeared in the adventitia; D, after transection of the saphenous nerve at the root, all axons in the nerve underwent degeneration, and the sympathetic fibres in the adventitia disappeared; E, after lumbar sympathectomy, sympathetic fibres disappeared in both the nerve and the adventitia, whereas other non‐adrenergic axons in the nerve still existed; F. the strong green staining of the tunica intima is a consistent artefact that manifests even when no primary or secondary antibodies are added when a 488 nm laser line is used. The scale bar represents 250 μm

FIGURE 6

Schematic drawing of the pathway for sympathetic fibres to reach and innervate a peripheral artery. As illustrated, the lower centre of the sympathetic nervous system is located in the intermediolateral nucleus of the grey matter of the spinal cord. The axons leaving the intermediolateral nucleus (pre‐ganglionic neurons) are named pre‐ganglionic fibres, which travel in the anterior root of the spinal nerve and course to the paravertebral sympathetic trunks through the white rami communicante. In the paravertebral trunks, the pre‐ganglionic fibres can synapse with post‐ganglionic neurons, whose axons, named post‐ganglionic fibres, return to spinal nerves through the grey rami communicante, and then travel with the spinal nerves to the peripheral nerves, such as the femoral nerve and radial nerve, exiting the peripheral nerves as segmental sympathetic branches, which then morph into a primary and secondary sympathetic trunk that send off filaments to form sympathetic networks that are dense over the artery and scarce over the vein

FIGURE 7

Disconnection between the digital artery and nerve is a theoretic way to achieve sympathectomy of the digital artery. In clinic practice, the vascular sheath shrouding the proper digital vessels and nerves can be opened first, and then scissors could be used conveniently to transect all connections between the proper digital artery and nerve. The same operation can be applied to the upper‐order artery and nerve