Spatial topological analysis of sympathetic neurovascular characteristic of acupoints in Ren meridian using advanced tissue-clearing and near infrared II imaging

Abstract

Acupuncture has been used for treating various medical conditions in traditional Chinese medicine. Both manual and electro-acupuncture stimulate specific acupoints to obtain local and systemic biological effects, but the underlying mechanisms remain unclear. Here, we used three-dimensional tissue-clearing technology to study acupoints on the Ren meridian of mice to reveal the distribution, density, branching, and relationships between blood vessels and nerves. Using topological Mapper methods, we found that sympathetic neurovascular networks were denser in the CV 4 acupoint compared with surrounding non-acupoints. Furthermore, high resolution in vivo real-time vascular imaging using the near infrared-II probe LZ-1105 demonstrated increased blood flow in the CV 4 acupoint compared with neighboring non-acupoints after manual or electro-acupuncture. Consistent with earlier findings, our research indicated that acupuncture could enhance local blood flow, and our high-resolution 3D images show for the first time the important role of sympathetic neurovascular networks in the CV 4 acupoint.

Keywords: 3D visualization; Acupoint; Acupuncture; In-vivo NIR-II imaging; Tissue-clearing; Topology.

Graphical abstract

Fig. 1

Experimental methodology flow. EA, electroacupuncture; MA, manual electroacupuncture; OVX, ovariectomy; CUBIC, clear, unobstructed brain/body imaging cocktails and computational analysis; H&E staining, hematoxylin and eosin staining.

Fig. 2

The location and stratification of the neurovascular 3D structure of acupoints and surrounding non-acupoints from a randomly chosen mouse a). Location, sampling, and CUBIC treatment of Ren meridian tissues consisting of CV 3, 4, 5, and 7. b). 3D structure of the vasculature in the Ren meridian highlighting acupoints and non-acupoints. The anatomic landmarks of the linea alba and the two IAVs (inferior abdominal vessels) are shown as the white dashed line and arrows, respectively. Insets show enlarged acupoints with vessels and capillaries. c). The stratified neurovascular 3D structure of CV 4. Left panel: Acupoints could be divided into the upper skin layer and lower muscle layer. Middle panel: Blood vessels (Red) and sympathetic nerves (Green) were identified. The upper skin layer consisted of non-parallel and non-intermingled nerves and vessels. The lower muscle layer could be divided into the upper region with parallel vessels and nerves, whereas the lower region had perpendicular vessels and nerves. Right panel: Horizontal view of the neurovascular relationships at different depths. Thin arrows: vessels and nerves in the skin layer. Thick arrows: parallel capillaries and nerves in the upper region of muscle. Arrowhead: perpendicular vessels and nerves in the lower muscle layer. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Denser sympathetic neurovascular networks in the CV 4 acupoint compared with the neighboring non-acupoints 3D structure of CV 4 and surrounding non-acupoints showing a) vascular networks (red, lectin; blue, DAPI), b) neural networks (green, TH; blue, DAPI), and c) merged staining. d). Denser capillary branching in CV 4 compared with surrounding non-acupoints. e). Representative graphs showing vascular staining in CV 4 and CV 4-R. Capillary branching density in muscle = number of vessels with branching/total number of vessels f). Ratio of overlapping vessels and nerves. g). representative graphs showing denser neurovascular networks in CV4 compared to CV 4-L. n = 4/group, *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Topological analysis of the neurovascular 3D structure of acupoints and non-acupoints from a representative mouse from the control group. a). 3D view of the vascular structure of CV 4. b). Tracing of the topological skeleton centered in CV 4. (Red, CV 4; white, CV 4-R; gray, CV 4-U; black, CV 4-L) c). Blood vessel length in relation to branching level. At the acupoint, fewer branching points and longer vessel lengths were found. d). Representative levels of branching points found in acupoints and non-acupoints. e). Representative images for different Mapper analyses comparing non-acupoints with CV 4. The number on the node shows the amount of contained data. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

NIR-II real-time imaging of blood flow in CV 4 and surrounding non-acupoints. a). Nude mice underwent control, MA, and EA treatment at CV 4. b). Blood flow monitoring during the 30 min MA or EA treatment and for 30 min after removal of the needle. The dotted circle indicates the CV 4 region. c). Restricted cubic spline curves of the real-time blood flow changes in CV 4 and non-acupoints at different times after MA or EA treatment. n = 6/group.