Objectives This study was performed to investigate the innervations related to acupuncture point PC8 in rats using a neural tracing technique.
Methods After 6 μL of 1% cholera toxin subunit B (CTB) was injected into the site between the second and third metacarpal bone in rats, a corresponding site to acupuncture point PC8 in the human body, CTB labelling was examined with immunofluorescence and immunohistochemistry in the dorsal root ganglia (DRG), spinal cord and brainstem.
Results All CTB labelling appeared on the ipsilateral side of the injection. The labelled sensory neurons distributed from cervical (C)6 to thoracic (T)1 DRG, while the labelled motor neurons were located on the dorsolateral part of the spinal ventral horn ranging from the C6 to T1 segments. In addition, the transganglionically-labelled axonal terminals were found to be dense in the medial part of laminae 3–4 from C6 to the T1 spinal dorsal horn, as far as in the cuneate nucleus.
Conclusions These results indicate that sensory and motor neurons associated with PC8 distribute in a distinct segmental pattern. The sensory information from PC8 could be transganglionically transported to the spinal dorsal horn and cuneate nucleus.
Statistics from Altmetric.com
PC8 (Laogong) is a well recognised point in acupuncture treatment. Although its definition and characterisation have been studied in traditional Chinese medical theory, understanding of the anatomical structures for PC8 still remain at the gross anatomical level.1 ,2 Over the past several decades, a great number of researchers have attempted to demonstrate the morphology of acupuncture points. Although no specific structures have been clearly linked to acupuncture points, increasing evidence suggests that the nervous system closely corresponds with acupuncture points.3–8 For the morphological study of acupuncture, the neural tracing technique has been used as an effective approach to reveal the neuroanatomical characteristics of acupuncture points.9–12 In this study, cholera toxin subunit B (CTB) was chosen as a sensitive tracer for investigating the neuroanatomical basis for PC8.13–15 Through systematically revealing the distribution of the neural labelling associated with PC8, regular connections between the individual acupuncture point and the nervous system can be determined, which should benefit the understanding of the distal neural mechanism of acupuncture point stimulation.
The experiments were performed on five young adult male Sprague–Dawley rats (8–10 weeks, weight 225±25 g). Experimental animals were provided by the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (license number SCKX (JUN) 2007-004). All animals were housed in a 12 h light/dark cycle with controlled temperature and humidity, and allowed free access to food and water. This study was approved by the ethics committee at the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences. The handling and care of experimental animals conformed to the regulations imposed by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, USA, 1996).
Microinjection of CTB
Microinjection was carried out at PC8 in the rat. On the human body, PC8 is located between the second and third metacarpal bones of the hand palm. According to the principle of comparative anatomy, a corresponding site on the rat is situated between the second and third metacarpal bone of the forefoot planta pedis (figure 1). Under anaesthesia with ether (Beijing Chemical Plant, Beijing, China), a total of 6 μL 1% CTB (List Biological Labs, Campbell, California, USA) solution was injected into PC8 using a fine-tipped Hamilton microsyringe. The depth of injection was about 2–3 mm. In this study, the injection of tracer was not limited to the muscle layer or dermal layer. According to the depth of injection and the path of the needle, CTB could be diffused around the injection site and absorbed by local axonal terminals in the adjacent structures, such as the interosseous muscles and the subcutaneous tissues. In order to prevent leakage of solution, the needle was kept in place for 1 min after injection and then pulled out slowly. When the rats awoke from anaesthesia, they were put back into their raising box.
After surviving 3 days post injection, rats were deeply anaesthetised with ether and transcardially perfused with 100 mL of 0.9% saline immediately followed by 300 mL of 4% paraformaldehyde in 0.1 M phosphate-buffered solution (PB, pH 7.4). The brain stem, cervical (C) and thoracic (T) spinal cord and associated dorsal root ganglia (DRG) were dissected out and stored in 25% sucrose PB at 4°C and allowed to sink.
Serial sagittal sections of DRG were cut at a thickness of 20 μm on a cryostat (Thermo, Microm International FSE, Walldorf, Germany) and mounted on silane-coated glass slides. In addition, serial transverse sections of brain stem and spinal cord were cut at a thickness of 40 μm on a freezing microtome (Thermo, Microm International GmbH) and collected in order in a six-well Petri dish with 0.1 M PB (pH 7.4).
Immunofluorescence and immunohistochemistry staining for CTB
Cryostat-sectioned DRG were conducted with immunofluorescence staining. First, the mounted sections were incubated in a blocking solution containing 3% normal rabbit serum and 0.3% Triton X-100 in 0.1 M PB for 1 h, then transferred to goat anti-CTB (List Biological Labs) at a dilution of 1 : 1000 in 0.1 M PB containing 1% rabbit serum and 0.3% Triton X-100 for overnight at 4°C. On the following day, after washing three times with 0.1 M PB, sections were exposed to rabbit anti-goat Alexa 488 secondary antibody (1 : 500, Molecular Probes, Eugene, Oregon, USA) for 2 h and then washed with 0.1 M PB. Slides were coverslipped using Immu-mount (Thermo Shandon, Pittsburgh, PA, USA) to improve visualisation of labelling.
Free-floating sections from every third transverse section of the brain stem and spinal cord were used for visualising the CTB labelling with immunohistochemistry. The procedure before the reaction with secondary antibody was similar to the immunofluorescence staining as mentioned above. Afterwards, the sections were exposed to biotinylated rabbit anti-goat IgG (1 : 500, Vector Laboratories, Burlingame, California, USA) for 2 h at room temperature and processed with avidin-biotin-peroxidase complex (1 : 100, Vectastain ABC Kit, Vector Laboratories) for 1 h. After washing in 50 mM Tris buffer (pH 7.4), sections were treated with 0.02% 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma, St Louis, Missouri, USA) and 0.01% H2O2 in 50 mM Tris buffer (pH 7.4) for about 2–5 min in order to visualise immunolabelling. After the reaction was completed, sections were washed in Tris buffer (50 mM, pH 7.4), mounted on silane-coated glass slides, air dried overnight, dehydrated in a series of alcohols, cleared in xylene and coverslipped with Entellan (Merck, Whitehouse Station, New Jersey, USA).
CTB labelling detected by immunofluorescence and immunohistochemistry methods were examined, respectively, using a laser scanning confocal microscope (FV1000, Olympus, Tokyo, Japan) and a light microscope (Eclipse, Nikon Co., Tokyo, Japan) equipped with a digital camera (DP 70, Olympus, Tokyo, Japan). Digital images were then processed with Adobe Photoshop CS2 (Adobe Systems, San Jose, California, USA). The anatomical structure of tissue sections from brain stem and spinal segments was determined cytoarchitecturally based on Paxinos and Watson,16 and segments of DRG were counted as the location of vertebra.
Data was expressed as mean±SD, and processed with the statistical software Stata V.7.0 (Stata Corp., College Station, Texas, USA).
Survival 3 days post injection was sufficient for producing intense CTB reaction products in the corresponding DRG, spinal cord and brain stem. The CTB-labelled sensory neurons in DRG were demonstrated in fluorescent green by immunofluorescence staining (figure 2), while the CTB-labelled motor neurons and axonal terminals in the spinal dorsal horn and brain stem were shown in dark brown with immunohistochemical reaction (figures 3 and 4).
As the primary sensory neurons, CTB-labelled ganglionic cells distributed from C6 to T1 DRG with high concentration in C8 DRG, in which CTB labelling presented in the cell body of different-sized ganglion neurons (figure 2A,B). The number of CTB-labelled ganglionic cells was counted in three sections from each DRG in all experimental rats, and were arranged in the order of C8: 114±21 > C7: 87±16 > C6: 59±11 > T1: 31±8 (mean±SD, n=5). According to the size of soma diameter, the labelled sensory neurons were assigned to the large, medium or small group with soma diameters of >50 μm, between 30 and 50 μm and <30 μm, respectively, and each represented 18%, 55% and 27% of the neurons.
In the spinal cord, CTB-labelled motor neurons located at the dorsolateral part of the ventral horn and congregated to form a longitudinal column from C6 to T1 with a high concentration at the C8 segment (figure 3A,B). Since it was difficult to clearly distinguish the spinal segments from the transverse section, comparisons on the number of labelled motor neurons at each spinal segment were not carried out. According to the size of soma diameter, the labelled motor neurons were divided into either the large (soma diameter >25 μm) or small (soma diameter ≤25 μm) group, belonging to α and γ motor neurons, respectively. Here, a total of 419 labelled motor neurons were counted from 30 representative sections, of which large-sized and small-sized motor neurons represented 81.4% and 18.6%, respectively.
As well as CTB-labelled sensory and motor neurons, CTB-labelled small dotted terminals were also observed in the spinal dorsal horn and the brain stem (figures 3A,C and 4A,B). In the spinal cord, labelled terminals distributed densely in the medial part of laminae 3–4 and scatted in lamina 5, which was ranged from C6 to T1 with a high concentration at the C8 segment (figure 3A,C). In the brain stem, CTB-labelled terminals were found to be concentrated on the ventromedial part of the cuneate nucleus (figure 4A,B), and scatted on external parts of the cuneate nucleus (not shown).
In addition, it should be noted that CTB-labelled sensory, motor neurons and transganglionic axonal terminals in all experimental rats were located ipsilaterally on the injection side, and were distributed with the same segmental and regional pattern in the DRG, spinal cord and cuneate nucleus as shown in figure 5.
Injection of CTB into PC8 in rats resulted in the effective retrograde and transganglionic labelling of the corresponding targets including DRG, spinal cord and brain stem. These results demonstrate that CTB can be used as a new choice for morphological study of acupuncture points, and provide further evidence to understand the neuroanatomical basis of the acupuncture point at the cellular level.
An important tool, the neural tracing technique is widely used for neuroanatomical studies;17–19 30 years have passed since it was first introduced into acupuncture research.9 Currently, the neural tracing technique has been considered as an effective approach to reveal the neuroanatomical basis of acupuncture points.9–12 Although numerous tracers have paved the way for this work, to the best of our knowledge, CTB has not been used alone for defining the neuroanatomical characteristics of an acupuncture point.
Similar to the peripheral application of horseradish peroxidase (HRP),9 ,10 ,20 CTB can retrogradely label sensory and motor neurons, as well as transganglionically label axonal terminals.13 ,14 However, CTB-labelled axonal terminals tend to distribute in the deep layers of the spinal dorsal horn,13 ,14 while HRP-labelled axonal terminals preferentially locate in the superficial layers of the spinal dorsal horn.20 ,21 However, unlike HRP application, CTB-labelled animals are usually fixed with routine fixative, which is more convenient for obtaining clear neural labelling by using immunofluorescence or immunoperoxidase methods. These features make CTB more suitable for double labelling with other antigens expressed on different tracers or neuronal phenotypes.14 ,22 Compared with the advantages and disadvantages of tracers previously used in this field, the CTB method could be another important choice for potential use in revealing the neuroanatomical basis of acupuncture points.
In addition, it is worth noting that, by using a CTB technique, we only focused on the relationship of PC8 with the nervous system. Future studies should address the correlation between PC8 and the pericardial meridian.
Correlation between PC8 and nervous system
It is clear that every acupuncture point, as a peripheral area on the body, should have its own innervation. In the present study, we set up a practical experimental model to map the neural connections at an individual acupuncture point. As an important approach, it is helpful to reveal regular connections between numerous acupuncture points and the nervous system at the cellular level. Based on our analysis of the present neuroanatomical evidence, it was demonstrated that the CTB labelling associated with PC8 distributed in segmental and regional patterns at the corresponding areas in the rat nervous system, and mainly correlated with spinal cervical enlargement and the cuneate nucleus. As a comparison, innervations on the acupuncture points from the hind leg, such as KI3, BL57, BL64 and KI4, mainly correlated with spinal lumbar enlargement and the gracile nucleus.10–12 These regular connections between different acupuncture points and the nervous system is consistent with somatotopic arrangement, as demonstrated in previous neural tracing studies and the classic reference text, Gray's Anatomy.23–25 Therefore, we speculated that there is ‘somatotopic arrangement’ between different acupuncture points and the nervous system at the levels of the spinal cord and the brain. It is worth emphasising that in this study we pay more attention to the peripheral and the central neural mechanisms of acupuncture stimulation. If this ‘somatotopic arrangement’ could be elucidated for different acupuncture points, it would play an instructive role in selecting proper acupuncture points to treat regional and distal ailments according to clinical demands.
The organised neural labelling with CTB observed in the present study contained sensory and motor elements. These results indicate that the sensory and motor systems serve as two important pathways to receive and relay the acupuncture stimulation information from acupuncture points to the central nervous system. Although the functional significance of the acupuncture point is still far from clear, the findings from PC8 provide a clue as to the neuroanatomical basis of the path from acupuncture point via neural pathway to cellular targets, which should be helpful for understanding the underlying neural mechanisms of the effects caused by acupuncture stimulation. From the perspective of neuroanatomy, it provides a valuable reference for understanding the distal effect induced by acupuncture point stimulation at the cellular level. In addition, according to the size of labelled cells, the sensory and motor neurons associated with PC8 can be further divided into subtype groups. Nevertheless, the roles of these subtype neurons in acupuncture stimulation are still important issues for future research.
In summary, we found that the CTB neural tracing technique can be effectively used for investigating the neuroanatomical characteristics of an individual acupuncture point. Via this technique, we revealed the sensory and motor innervations of PC8, which may provide insight into the peripheral and central neural mechanism of action of acupuncture point stimulation.
Cholera toxin B is a novel tracer for the neural connections of acupuncture points.
PC8 is mainly connected at C8 spinal segmental level.
Contributors The work presented here was carried out in collaboration between all authors. W-ZB designed the study. J-JC, L-JH, X-LZ, HS, F-CW and X-HJ carried out the laboratory experiments and analysed the data. W-ZB and J-JC wrote the paper. All authors read and approved the final manuscript.
Funding This study was funded by the National Natural Science Foundation of China (project code no. 81072759) and the National Basic Research Program of China (973 program, no. 2010CB530507; no. 2011CB505201).
Competing interests None.
Ethics approval This study was approved by the ethics committee at Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences.
Provenance and peer review Not commissioned; internally peer reviewed.
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.