Networks of Nerve Fibers, and Blood and Lymphatic Vessels in the Mouse Auricle: The Structural Basis of Ear Acupuncture.

Objective: Ear acupuncture, as a system for treating and preventing diseases through stimulation of points on the auricle, has been systematically introduced during the last 60 years. Although the auricular cartography was described somatotopically as an inverted fetus by Paul Nogier, MD, the underlying mechanism of auricular stimulation remains unclear. The aim of this research was to gain an understanding of the structural basis of auricular stimulation, as well as showing the distribution of the nerve fibers, and the blood and lymphatic vessels. Materials and Methods: The distribution of nerve fibers, and blood and lymphatic vessels was examined in whole-mount auricular skins of mice by combining the biomarkers protein gene product 9.5, cluster of differentiation 31, and lymphatic-vessel endothelial hyaluronan receptor-1 following tissue-clearing treatment with multiple immunofluorescent staining. Results: The labeled nerve fibers, and the blood and lymphatic vessels were distributed extensively in the inner and outer parts of the auricular skin. Auricular nerves aligning with blood vessels ran from the basal region to the peripheral region and crossed over lymphatic vessels, thus forming the neural, vascular, and lymphatic networks. Conclusions: As these are important tissue components of auricular skin, this result implies that the auricular nerve fibers, and blood and lymphatic vessels may coordinate with each other to respond directly to auricular stimulation.

Region-specific response of central microglial cells to sciatic nerve demyelination through sensory and motor pathways.

Peripheral nerve injury can cause changes in microglial cells on the spinal dorsal and ventral horns. This region-specific response implies that central microglial cells could be activated through both sensory and motor pathways. In order to further determine how peripheral nerve injury activates central microglial cells through neural pathways, the sciatic nerve was selected as the target for neural tract tracing and demyelination. Firstly, we used cholera toxin subunit B (CTB) to map the central sensory and motor territories of the sciatic nerve. Secondly, we applied lysophosphatidylcholine to establish the model of sciatic nerve demyelination and examined the distribution of activated microglial cells via immunofluorescence with ionized calcium-binding adapter molecule 1. It was shown that CTB labeling included the transganglionically labeled sensory afferents and retrogradely labeled somata of motor neurons along the sensory and motor pathways of the sciatic nerve ipsilateral to the injection, in which sensory afferents terminated on the gracile nucleus, Clarke's nucleus, and spinal dorsal horn, while motor neurons located on the spinal ventral horn. Consistently, after sciatic axon demyelination, the activated microglial cells were observed in the same territories as CTB-labeling, showing shortened processes and enlarged cell bodies. These results support the idea that central microglia might be activated by signals from the demyelinated sciatic nerve through both sensory and motor pathways.

Three-dimensional visualization of the lymphatic, vascular and neural network in rat lung by confocal microscopy.

In order to demonstrate the intricate interconnection of pulmonary lymphatic vessels, blood vessels, and nerve fibers, the rat lung was selected as the target and sliced at the thickness of 100 µm for multiply immunofluorescence staining with lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), alpha smooth muscle actin (α-SMA), phalloidin, cluster of differentiation 31 (CD31), and protein gene product 9.5 (PGP9.5) antibodies. Taking the advantages of the thicker tissue section and confocal microscopy, the labeled pulmonary lymphatic vessels, blood vessels, and nerve fibers were demonstrated in rather longer distance, which was more convenient to reconstruct a three-dimensional (3D) view for analyzing their spatial correlation in detail. It was clear that LYVE-1+ lymphatic vessels were widely distributed in pulmonary lobules and closely to the lobar bronchus. Through 3D reconstruction, it was also demonstrated that LYVE-1+ lymphatic vessels ran parallel to or around the α-SMA+ venules, phalloidin+ arterioles and CD31+ capillaries, with PGP9.5+ nerve fibers traversing alongside or wrapping around them, forming a lymphatic, vascular and neural network in the lung. By this study, we provide a detailed histological view to highlight the spatial correlation of pulmonary lymphatic, vascular and neural network, which may help us for insight into the functional role of this network under the physiological and pathological conditions.

[Application prospect of conditional gene editing animals and viral vectors in experimental research of acupuncturology].

Conditional gene editing animals and viral vectors have been widely applied in the research fields of biology and medicine. Recently, they are also used as the effective approaches to reveal the underlying mechanism of acupuncture from the nervous system to the specific molecules. In order to further understand the application of conditional gene editing animals and viral vectors, in this article, we analyze their characteristics, advantages and recent development in the field of acupuncture research and discuss their potential roles and prospect in the future.

The immunolocalization of cluster of differentiation 31, phalloidin and alpha smooth muscle actin on vascular network of normal and ischemic rat brain.

Cluster of differentiation 31 (CD31), phalloidin and alpha smooth muscle actin (α-SMA) have been widely applied to label the cerebral blood vessels in the past years. Although CD31 is mainly used as endothelial marker in determining the cerebral capillaries, it seems likely that its labeling efficiency is closely correlated with the antibodies from the polyclonal or monoclonal one, as well as the conditions of blood vessels. In order to test this phenomenon, we compared the labeling characteristics of goat polyclonal anti-CD31 (gP-CD31) and mouse monoclonal anti-CD31 (mM-CD31) with those of phalloidin and α-SMA on the rat brain in health and ischemia/reperfusion (I/R) with the middle cerebral artery occlusion. By multiple immunofluorescence staining, it was found that gP-CD31 labeling expressed extensively on the cerebral capillaries forming the vascular networks on the normal and ischemic regions, but mM-CD31 labeling mainly presented on the capillaries in the ischemic region. In contrast to the vascular labeling with gP-CD31, phalloidin and α-SMA were mainly expressed on the wall of cortical penetrating arteries, and less on that of capillaries. By three-dimensional reconstruction analysis, it was clearly shown that gP-CD31 labeling was mainly located on the lumen side of vascular wall and was surrounded by phalloidin labeling and α-SMA labeling. These results indicate that gP-CD31 is more sensitive than mM-CD31 for labeling the cerebral vasculature, and is highly compatible with phalloidin and α-SMA for evaluating the cerebral vascular networks under the physiological and pathological conditions.

Temporal alteration of microglia to microinfarcts in rat brain induced by the vascular occlusion with fluorescent microspheres.

Microglia, the resident immune cells in the central nervous system, can monitor the microenvironment and actively respond to ischemic stroke and other brain injuries. In this procedure, microglia and neurons can cross-talk via transmembrane chemokine, Fractalkine (CX3CL1), to impact one another. We used a rat model of multifocal microinfarcts induced by the injection of fluorescent microspheres into the right common carotid artery and examined the morphological alteration of blood vessels, microglia, astrocytes, and neurons at 6 h, 1, 7, and 14 days after modeling, along with neurobehavioral tests and the staining of CX3CL1 in this study. Our results demonstrated that in the infarcted regions, astrocytes and microglia activated in response to neuronal degeneration and upregulation of cleaved caspase-3, which occurred concurrently with vascular alteration and higher expression of CX3CL1. We provided sequential histological data to shed light on the morphological changes after modeling, which would help in the identification of new targets and the choice of the ideal time window for therapeutic intervention in ischemic stroke.

Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches.

Skin innervation is an important part of the peripheral nervous system. Although the study of the cutaneous nerve fibers has progressed rapidly, most of the understanding of their distributional and chemical characteristics comes from conventional histochemical and immunohistochemical staining on thin tissue sections. With the development of the tissue clearing technique, it has become possible to view the cutaneous nerve fibers on thicker tissue sections. The present protocol describes multiple fluorescent staining on tissue sections at a thickness of 300 µm from the plantar and dorsal skin of rat hindfoot, the two typical hairy and glabrous skin sites. Here, the calcitonin gene-related peptide labels the sensory nerve fibers, while phalloidin and lymphatic vessel endothelial hyaluronan receptor 1 label the blood and lymphatic vessels, respectively. Under a confocal microscope, the labeled sensory nerve fibers were followed completely at a longer distance, running in bundles in the deep cutaneous layer and freestyle in the superficial layer. These nerve fibers ran in parallel to or surrounded the blood vessels, and lymphatic vessels formed a three-dimensional (3D) network in the hairy and glabrous skin. The current protocol provides a more effective approach to studying skin innervation than the existing conventional methods from the methodology perspective.

Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle.

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of three essential histological components: the pre-synaptic motor axon terminals, post-synaptic nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs). In order to demonstrate the morphological characteristics of NMJ, the rat medial gastrocnemius muscle was selected as the target-tissue and examined by using multiple fluorescent staining with various kinds of biomarkers, including neurofilament 200 (NF200) and vesicular acetylcholine transporter (VAChT) for the motor nerve fibers and their pre-synaptic terminals, alpha-bungarotoxin (α-BTX) for the post-synaptic nicotinic AchRs, and S100 for the PSCs. In this study, staining was performed in two groups: in the first group, samples were stained with NF200, VAChT, and α-BTX, and in the second group, samples were stained with NF200, α-BTX, and S100. It was shown that both protocols can effectively demonstrate the detailed structure of NMJ. Using the confocal microscope, morphological characteristics of the pre-synaptic terminals, post-synaptic receptors, and PSC were seen, and their Z-stacks images were reconstructed in a three-dimensional pattern to further analyze the spatial correlation among the different labeling. From the perspective of methodology, these protocols provide a valuable reference for investigating the morphological characteristics of NMJ under physiological conditions, which may also be suitable to evaluate the pathological alteration of NMJ, such as peripheral nerve injury and regeneration.

Sensory and autonomic innervation of the local tissues at traditional acupuncture point locations GB14, ST2 and ST6.

OBJECTIVE: To visualize and compare the sensory and autonomic innervation of the local tissues at the sites of different traditional acupuncture points in the rat forehead and face by histochemical examination. METHODS: GB14 (Yangbai), ST2 (Sibai) and ST6 (Jiache) were selected as the representative traditional acupuncture points in this study, and the local tissues at these sites were dissected in rats after perfusion followed by double or triple fluorescent histochemical staining. Here, calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT) were used to label the sensory, sympathetic and parasympathetic nerve fibers, respectively. RESULTS: The CGRP+ sensory, TH+ sympathetic and VAChT+ parasympathetic nerve fibers were simultaneously demonstrated in the local tissues at GB14, ST2 and ST6. Although the three kinds of nerve fibers ran in parallel or intermingled with each other, by the analysis from the view of three-dimensional reconstruction, it was clear that each of them distributed in an independent pattern to their corresponding target tissues including the blood vessels, hair follicles, arrector pili and subcutaneous muscles, as well as sebaceous glands. CONCLUSION: Our study demonstrated the sensory and autonomic innervation of the local tissues at GB14, ST2 and ST6, providing neurochemical evidence indicating that the CGRP+ sensory, TH+ sympathetic and VAChT+ parasympathetic nerve fibers form a neural network at these point locations that may respond to acupuncture stimulation.

Temporal alterations in pericytes at the acute phase of ischemia/reperfusion in the mouse brain.

Pericytes, as the mural cells surrounding the microvasculature, play a critical role in the regulation of microcirculation; however, how these cells respond to ischemic stroke remains unclear. To determine the temporal alterations in pericytes after ischemia/reperfusion, we used the 1-hour middle cerebral artery occlusion model, which was examined at 2, 12, and 24 hours after reperfusion. Our results showed that in the reperfused regions, the cerebral blood flow decreased and the infarct volume increased with time. Furthermore, the pericytes in the infarct regions contracted and acted on the vascular endothelial cells within 24 hours after reperfusion. These effects may result in incomplete microcirculation reperfusion and a gradual worsening trend with time in the acute phase. These findings provide strong evidence for explaining the "no-reflow" phenomenon that occurs after recanalization in clinical practice.

Alexa Fluor 488-conjugated cholera toxin subunit B optimally labels neurons 3-7 days after injection into the rat gastrocnemius muscle.

Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries. However, it is not always clear which tracer should be used to yield optimal results. In this study, we examined the use of Alexa Fluor 488-conjugated cholera toxin subunit B (AF488-CTB). This was injected into the gastrocnemius muscle of rats, and it was found that motor, sensory, and sympathetic neurons were labeled in the spinal ventral horn, dorsal root ganglia, and sympathetic chain, respectively. Similar results were obtained when we injected AF594-CTB into the tibialis anterior muscle. The morphology and number of neurons were evaluated at different time points following the AF488-CTB injection. It was found that labeled motor and sensory neurons could be observed 12 hours post-injection. The intensity was found to increase over time, and the morphology appeared clear and complete 3-7 days post-injection, with clearly distinguishable motor neuron axons and dendrites. However, 14 days after the injection, the quality of the images decreased and the neurons appeared blurred and incomplete. Nissl and immunohistochemical staining showed that the AF488-CTB-labeled neurons retained normal neurochemical and morphological features, and the surrounding microglia were also found to be unaltered. Overall, these results imply that the cholera toxin subunit B, whether unconjugated or conjugated with Alexa Fluor, is effective for retrograde tracing in muscular tissues and that it would also be suitable for evaluating the regeneration or degeneration of injured nerves.

Histochemistry of microinfarcts in the mouse brain after injection of fluorescent microspheres into the common carotid artery.

The mouse model of multiple cerebral infarctions, established by injecting fluorescent microspheres into the common carotid artery, is a recent development in animal models of cerebral ischemia. To investigate its effectiveness, mouse models of cerebral infarction were created by injecting fluorescent microspheres, 45-53 µm in diameter, into the common carotid artery. Six hours after modeling, fluorescent microspheres were observed directly through a fluorescence stereomicroscope, both on the brain surface and in brain sections. Changes in blood vessels, neurons and glial cells associated with microinfarcts were examined using fluorescence histochemistry and immunohistochemistry. The microspheres were distributed mainly in the cerebral cortex, striatum and hippocampus ipsilateral to the side of injection. Microinfarcts were found in the brain regions where the fluorescent microspheres were present. Here the lodged microspheres induced vascular and neuronal injury and the activation of astroglia and microglia. These histopathological changes indicate that this animal model of multiple cerebral infarctions effectively simulates the changes of various cell types observed in multifocal microinfarcts. This model is an effective, additional tool to study the pathogenesis of ischemic stroke and could be used to evaluate therapeutic interventions. This study was approved by the Animal Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (approval No. D2021-03-16-1) on March 16, 2021.

Kininogen-Nitric Oxide Signaling at Nearby Nonexcited Acupoints after Long-Term Stimulation.

Acupuncture treatment is based on acupoint stimulation; however, the biological basis is not understood. We stimulated one acupoint with catgut embedding for 8 weeks and then used isobaric tags for relative and absolute quantitation to screen proteins with altered expression in adjacent acupoints of Sprague Dawley rats. We found that kininogen expression was significantly upregulated in the stimulated and the nonstimulated adjacent acupoints along the same meridian. The enhanced kininogen expression was meridian dependent and was most apparent among small vessels in the subcutaneous layer. Enhanced signals of nitric oxide synthases, cGMP-dependent protein kinase, and myosin light chain were also observed at the nonstimulated adjacent acupoints along the same meridian. These findings uncover biological changes at acupoints and suggest the critical role of the kininogen-nitric oxide signaling pathway in acupoint activation.

Comparison of Sensory and Motor Innervation Between the Acupoints LR3 and LR8 in the Rat With Regional Anatomy and Neural Tract Tracing.

OBJECTIVE: This study aimed to investigate the sensory and motor innervation of "Taichong" (LR3) and "Ququan" (LR8) in the rat and provide an insight into the neural relationship between the different acupoints in the same meridian. METHODS: The LR3 and LR8 were selected as the representative acupoints from the Liver Meridian and examined by using the techniques of regional anatomy and neural tract tracing in this study. For both acupoints, their local nerves were observed with regional anatomy, and their sensory and motor pathways were traced using neural tract tracing with single cholera toxin subunit B (CTB) and dual Alexa Fluor 594/488 conjugates with CTB (AF594/488-CTB). RESULTS: Using the regional anatomy, the branches of the deep peroneal nerve and saphenous nerve were separately found under the LR3 and LR8. Using single CTB, the sensory neurons, transganglionic axon terminals, and motor neurons associated with both LR3 and LR8 were demonstrated on the dorsal root ganglia (DRG), spinal dorsal horn, Clarke's nucleus, gracile nucleus, and spinal ventral horn corresponding to their own spinal segments and target regions, respectively. Using dual AF594/488-CTB tracing, it was shown that the sensory and motor neurons associated with LR3 were separated from that of LR8. CONCLUSION: This study demonstrates that LR3 and LR8 are innervated by different peripheral nerves, which originated from or terminated in their corresponding spinal segments and target regions independently through the sensory and motor pathways. These results provide an example for understanding the differential innervation between the different acupoints in the same meridian.

[Application of Adobe Illustrator for drawing schematic diagrams of anatomical structure of acupoints -- taking Five Shu-points of Jueyin Meridian as an example].

The schematic diagram is an indispensable part of research article in life science，which can effectively and intuitively show the specific content of the article with simple composition. As an intuitive expression of the interdisciplinary subject at the early stage, the schematic diagram in the literature has gained an all-round improvement in the quality along with the deve-lopment of computer technology and modern drawing tools. In contrast，the level of the schematic diagram appears to be lagged behind in the field of acupuncture research papers. In order to improve the drawing level of this field, we took the Five Shu-points of Hand and Foot Jueyin Meridian as an example, and drew some vector diagrams of their anatomical structures including the body surface, skeleton, nerves, arteries and veins by using the Adobe Illustrator image software, through which we sum up a simple and easy-to-learn process including application skills and key points needing attention. We hope these methods can play a role in the acupuncture research in the future.

[Characteristics of "deqi" and myoelectricity in different tissue structures of acupoint].

OBJECTIVE: To investigate the effect of acupuncture in different tissue structures on deqi and the electromyography of acupoint area. METHODS: Twenty healthy subjects, respectively accepted 4 kinds of needling stimulation, i.e. stimulating skin at Zusanli (ST36), stimulating ST36 with and without skin anesthesia using compound lidocaine cream, and stimulating at Dubi (ST35) without skin anesthesia. Deqi sensation of the acupuncturist and subjects were measured according to MGH Acupuncture Sensation Scale (MASS) during needling, and the myoelectricity around the acupoints was recorded simultaneously. The occurrence rate and intensity of the different deqi sensations, the relationship between the acupuncturist's and subjects' deqi sensations, and the integrated electromyogram (iEMG) were analyzed. RESULTS: Sharp pain and tingling were the main sensations during skin needling at ST36. Fullness, dull pain, soreness and acupuncturist's tightness were the main sensations during needling with or without skin anesthesia at ST36. Fullness was the main sensation during needling at ST35, while the intensity was lower than that during needling at ST36. A positive correlation in the intensity was found between subjects' fullness and acupuncturist's tightness during needling with or without skin anesthesia at ST36. The subjects' fullness appeared earlier about 5 seconds than acupuncturist's tightness. The iEMGs during subjects' fullness and acupuncturist's tightness were 2-3 times of that before needling. CONCLUSION: Deqi sensations such as subjects' fullness, dull pain, soreness and acupuncturist's tightness are mainly related to the activity of the muscles under the acupoints. Subjects' fullness and acupuncturist's tightness always appear together. Acupuncturist's tightness may be mediated by the muscle stretch reflex induced by needling stimulation.

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing.

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the cranial dura mater using immunofluorescence, three-dimensional (3D) reconstruction and retrograde tracing technique. Here, the nerve fibers and blood vessels were stained using immunofluorescence and histochemistry techniques with CGRP and fluorescent phalloidin, respectively. The spatial correlation of dural CGRP-immuoreactive nerve fibers and blood vessels were demonstrated by 3D reconstruction. Meanwhile, the origin of the CGRP-immunoreactive nerve fibers were detected by neural tracing technique with fluorogold (FG) from the area around middle meningeal artery (MMA) in the cranial dura mater to the trigeminal ganglion (TG) and cervical (C) dorsal root ganglia (DRGs). In addition, the chemical characteristics of FG-labeled neurons in the TG and DRGs were also examined together with CGRP using double immunofluorescences. Taking advantage of the transparent whole-mount sample and 3D reconstruction, it was shown that CGRP-immunoreactive nerve fibers and phalloidin-labeled arterioles run together or separately forming a dural neurovascular network in a 3D view, while the FG-labeled neurons were found in the ophthalmic, maxillary, and mandibular branches of TG, as well as the C2-3 DRGs ipsilateral to the side of tracer application in which some of FG-labeled neurons presented with CGRP-immunoreactive expression. With these approaches, we demonstrated the distributional characteristics of CGRP-immunoreactive nerve fibers around the blood vessels in the cranial dura mater, as well as the origin of these nerve fibers from TG and DRGs. From the perspective of methodology, it may provide a valuable reference for understanding the complicated neurovascular structure of the cranial dura mater under the physiological or pathological condition.

Progress on the Experimental Research of Sciatic Nerve Injury with Acupuncture.

OBJECTIVE: To collect and summarize relevant literatures on the experimental researches of sciatic nerve injury (SNI) with acupuncture during the last decade providing a guideline for effectively treating SNI with acupuncture in the future. METHODS: The Chinese and English databases including China National Knowledge Infrastructure (CNKI), Wanfang Data Knowledge Service Platform (WanFang Data), VIP Information Chinese Journal Service Platform (VIP Date), and PubMed were searched from 2009 to 2020 with keywords of "acupuncture and moxibustion OR acupuncture OR electroacupuncture OR scalp acupuncture OR wrist-ankle acupuncture OR acupoint injection OR ear acupuncture" AND "sciatic nerve OR sciatic nerve injury OR sciatic injury OR SNI." The collected data were mainly evaluated in the items of animal model of SNI, type of interventions, selection of acupuncture points (acupoints), course of treatment and its frequency, and approaches of assessment. RESULTS: A total of 89 studies were included in this analysis. Among them, the most commonly used animal models of SNI were produced by the clamp or transverse injury in the rats; the most frequently used intervention was electroacupuncture with dilatational wave of 2/100 Hz; the frequency of acupuncture was mainly performed once per day lasting for more than 2 weeks; the mainly selected acupoints were Huantiao (GB30), Zusanli (ST36), and Yanglingquan (GB34); and the approaches of assessment were contained with behavioral, functional, morphological, histological, cellular, and molecular measurements. CONCLUSION: The results indicated that the experimental researches of SNI with acupuncture has made marked progress in recent years, which may provide important clues for further investigating the underlying mechanisms of acupuncture for the treatment of SNI in the future.

Effects of Three Needling Manipulations of Zusanli (ST 36) on Deqi Sensations and Surface Myoelectricity in Healthy Participants.

OBJECTIVE: To investigate the effects of different acupuncture manipulations on Deqi sensations and surface myoelectricity, and explore the correlation between Deqi sensations and needling manipulations. METHODS: Forty-five healthy participants accepted twirling, lifting-thrusting, and twirling plus lifting-thrusting manipulanions at right Zusanli (ST 36), respectively. The acupuncturist's and participants' Deqi sensations were collected by MGH Acupuncture Sensation Scale (MASS). The intensity and occurrence rate of soreness, dull pain, pressure, heaviness, fullness, numbness, sharp pain, warmth, coolness, and throbbing feelings of participants, and tightness, smooth, and tangle feelings of acupuncturist were measured. The correlation between the acupuncturist's and participant's Deqi sensations was analyzed. Surface electromyogram (EMG) were recorded before, during and after needling in 30 participants. The integrated EMG (iEMG), mean power frequency (MPF) and media frequency (MF) were analyzed. RESULT: Both fullness and soreness of participants and tightness of acupuncturist were the most frequently occurred ones. A positive correlation between participants' fullness and acupuncturist's tightness was observed during the three aforementioned needling manipulations (P<0.05, OR>1). Almost all the needling sensations measured in the present study could be induced by the three needling manipulations. However, strength of Deqi sensations was exhibited as lifting-thrusting > twirling plus lifting-thrusting > twirling according to MASS index. The iEMG values were increased and MPF, MF values were decreased during needling compaired to those before needling, especially during lifting-thrusting (P<0.01). CONCLUSIONS: The intensity and occurrence rate of the different Deqi sensations induced by different needling manipulations were basically similar. The fullness and soreness were both the most frequently induced Deqi sensations. The strongest Deqi sensation could be induced by lifting-thrusting manipulation. There is a positive correlation between participants' fullness and acupuncturist's tightness during the three needling manipulations. The myoelectricity around the acupoint is related to Deqi responses. (Registration No. AMCTR-IOR-20000314).