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 cellular and molecular basis of liver regeneration after acute pericentral injury.

Liver injuries often occur in a zonated manner. However, detailed regenerative responses to such zonal injuries at cellular and molecular levels remain largely elusive. By using a fate-mapping strain, Cyp2e1-DreER, to elucidate liver regeneration after acute pericentral injury, we found that pericentral regeneration is primarily compensated by the expansion of remaining pericentral hepatocytes, and secondarily by expansion of periportal hepatocytes. Employing single-cell RNA sequencing, spatial transcriptomics, immunostaining, and in vivo functional assays, we demonstrated that the upregulated expression of the mTOR/4E-BP1 axis and lactate dehydrogenase A in hepatocytes contributes to pericentral regeneration, while activation of transforming growth factor ß (TGF-ß1) signaling in the damaged area mediates fibrotic responses and inhibits hepatocyte proliferation. Inhibiting the pericentral accumulation of monocytes and monocyte-derived macrophages through an Arg-Gly-Asp (RGD) peptide-based strategy attenuates these cell-derived TGF-ß1 signalings, thus improving pericentral regeneration. Our study provides integrated and high-resolution spatiotemporal insights into the cellular and molecular basis of pericentral regeneration.

Fixed-time terminal sliding mode control for uncertain robot manipulators.

This paper proposes a fixed-time tracking control for robot manipulators in the presence of parametric uncertainties and disturbances. An auxiliary function is first proposed for constructing a fixed-time sliding manifold. Benefited from this fixed-time sliding manifold, a singularity-free robust control is proposed to evade the effects of algebraic loop problem of the commonly-used sliding mode controls (SMC). The key advantages of the proposed approach are: (i) exact fixed-time stability featuring the convergence time does not relate to the initial conditions and is acquired in advance; (ii) the singularity and algebraic loop problems are eliminated completely; (iii) a simple and intuitive control structure is used for easy implementation of trajectory tracking control for uncertain robot manipulators with faster transient and higher steady-state precision. Simulations and experimental comparisons validate the improved tracking performance of the proposed approach.

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.

The Intergenerational Transmission of Risk Preferences: Evidence from Field Experiments in China and Korea.

In this study, we conduct field experiments with 196 worker-parent pairs from two companies in China and South Korea and explore factors that affect the similarity of risk preferences between parents and their offspring. In the Chinese data, we show more similar risk preferences between parents and their offspring when there are higher levels of parental involvement and financial parenting. In contrast, in the Korean data, a more demanding parenting style contributes to intergenerational transmission. These effects are mainly captured by the intergenerational transmission from Chinese mothers to their offspring and from Korean fathers to their offspring. In addition, we find that in our study, same-gender transmission contributes highly to intergenerational transmission, and the risk preferences of Chinese workers and their parents are more similar than those of Korean workers and their parents. We also discuss potential differences in the intergenerational transmission of risk preferences between China and Korea and Western countries. Our study provides a better understanding of the formation of individuals' risk preferences.

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.

Microbial diversity of milk ghee in southern Gansu and its effect on the formation of ghee flavor compounds.

Ghee is a traditional Tibetan dairy product with high-fat content, low yield, plasticity, caseation, and rich nutrition. In this study, we analyzed the diversity of microbial communities in yak milk and ghee samples at high and low altitudes, especially the Lactobacillus genus, and further used metabolomic techniques to compare the differences in metabolites in yak ghee at different altitudes. The results showed that the increase in altitude had a significant and generally inhibitory effect on the microbial community diversity in milk ghee, and yak milk at high altitude was abundant in nutrients, which could antagonize the negative impact of increased altitude. Using non-targeted metabolomics, we infer the composition of flavor compounds in ghee: nine kinds of carboxylic acids, 11 kinds of esters, six kinds of ketones, two kinds of alcohols, and four kinds of alkene compounds, among which the key flavor compounds are dl-2-(acetylamino)-3-phenylephrine acid, 1-(4-methoxyphenyl)-2-propanone, sebacic acid, Lysope 18:1, and uracil 1-beta-d-arabinofuranoside. These flavor substances are found in Lactococcus, Lactobacillus, and Streptococcus. With the participation of Lactobacillus, it is synthesized through biosynthesis of alkaloids derived from ornithine, lysine, and nicotine acid and glyoxylate and decarboxylate metabolism, among which Lactococcus plays a key role. In this study, a variety of lactic acid bacteria related to ghee fermentation were screened out, revealing the composition of volatile flavor compounds in Gannan yak milk ghee in the Qinghai-Tibet Plateau and providing a reference for further key volatile flavor compounds and the formation mechanism of flavor compounds.

Generation of multicellular tumor spheroids with micro-well array for anticancer drug combination screening based on a valuable biomarker of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis. More than 30% of patients with diagnosed HCC have abnormally high expression of fibroblast growth factor receptor 4 (FGFR4). Currently, clinical trials for a variety of FGFR4-specific inhibitors have started. However, the effect of these inhibitors is not ideal, and it is necessary to find a drug combination to synergistically exert anti-tumor effects. We found strong correlations between FGFR4 and HCC clinicopathological characteristics in the present study. After grouping patients according to FGFR4 expression, the key gene signatures were inputted the drug-gene related databases, which predicted several potential drug candidates. More importantly, to achieve the reliable and high throughput drug cytotoxicity assessment, we developed an efficient and reproducible agarose hydrogel microwells to generate uniform-sized multicellular tumor spheroids, which provide better mimicry of conventional solid tumors that can precisely represent anticancer drug candidates' effects. Using high content screening, we quickly evaluated the enhanced anti-tumor effects of these combinations. Finally, we demonstrated that Parthenolide is a potential drug that can significantly enhance the clinical efficacy of FGFR4 receptor inhibitors. In general, we offered a new therapeutic way for FGFR4 positive HCC patients.

Integration of single-cell RNA sequencing and bulk RNA sequencing to reveal an immunogenic cell death-related 5-gene panel as a prognostic model for osteosarcoma.

Background: Despite the comparatively low prevalence of osteosarcoma (OS) compared to other cancer types, metastatic OS has a poor overall survival rate of fewer than 30%. Accumulating data has shown the crucial functions of immunogenic cell death (ICD) in various cancers; nevertheless, the relationship between ICD and OS was not previously well understood. This research aims to determine the function of ICD in OS and construct an ICD-based prognostic panel. Methods: Single cell RNA sequencing data from GSE162454 dataset distinguished malignant cells from normal cells in OS. The discrepancy in ICD scores and corresponding gene expression was intensively explored between malignant cells and normal cells. Using the RNA sequencing data of the TARGET-OS, GSE16091, GSE21257, and GSE39058 datasets, the molecular subtype of OS was determined by clustering seventeen ICD-related genes obtained from the literature. Differentially expressed genes (DEGs) between different molecular subtypes were identified to develop a novel ICD-associated prognostic panel. Results: The malignant cells had a remarkable decrease in the ICD scores and corresponding gene expression compared with normal cells. A total of 212 OS patients were successfully stratified into two subtypes: C1 and C2. C1-like OS patients were characterized by better prognostic outcomes, overexpression of ICD genes, activation of the ICD pathway, high inflitration abundance of immunocytes, and low expression levels of immune checkpoint genes (ICGs); however, the reverse is true in C2-like OS patients. Utilizing the limma programme in R, the DEGs between two subtypes were determined, and a 5-gene risk panel consisting of BAMBI, TMCC2, NOX4, DKK1, and CBS was developed through LASSO-Cox regression analysis. The internal- and external-verification cohorts were employed to verify the efficacy and precision of the risk panel. The AUC values of ROC curves indicated excellent prognostic prediction values of our risk panel. Conclusions: Overall, ICD represented a protective factor against OS, and our 5-gene risk panel serving as a biomarker could effectively evaluate the prognostic risk in patients with OS.

The role and mechanism of NDST1/NULP1 regulating right ventricular hypertrophy in hypoxic pulmonary hypertension.

Hypoxia leads to hypoxic pulmonary hypertension (HPH), causing right ventricular hypertrophy (RVH). RVH becomes a significant and nonnegligible public health issue in the world. In our study, we successfully established the HPH rat model and found that RVH happened in HPH, and then we observed an increased inflammation response in the heart tissue of HPH-induced RVH rats. Moreover, increased N-deacetylase-N-sulfotransferase-1 (NDST1) and decreased nuclear localized protein 1 (NULP1) were found in the heart tissue of HPH-induced RVH rats. An in vitro cell experiment showed that inhibition of NDST1 expression enhanced cell viability, reduced cell apoptosis, alleviated cardiomyocyte hypertrophy, decreased inflammation and increased phosphorylated AKT level, however, over-expression of NDST1 had opposite effects on these aspects. NULP1 reversed the effects of NDST1 on these regulations. Finally, we found that up-regulated NDST1 reduced NULP1 expression and down-regulated NDST1 increased NULP1 expression. Our study confirmed that inhibition of the NDST1/NULP1 pathway might contribute to the attenuation of HPH-induced RVH, and the mechanism may be related to the reduction of inflammation, cardiomyocyte apoptosis, and AKT phosphorylation.

Comprehensive characterization of extracellular matrix-related genes in PAAD identified a novel prognostic panel related to clinical outcomes and immune microenvironment: A silico analysis with in vivo and vitro validation.

The extracellular matrix (ECM) is a vital component of the tumor microenvironment, which interplays with stromal and tumor cells to stimulate the capacity of cancer cells to proliferate, migrate, invade, and undergo angiogenesis. Nevertheless, the crucial functions of ECM-related genes (ECMGs) in pancreatic adenocarcinoma (PAAD) have not been systematically evaluated. Hence, a comprehensive evaluation of the ECMGs is required in pan-cancer, especially in PAAD. First, a pan-cancer overview of ECMGs was explored through the integration of expression profiles, prognostic values, mutation information, methylation levels, and pathway-regulation relationships. Seven ECMGs (i.e. LAMB3, LAMA3, ITGB6, ITGB4, ITGA2, LAMC2, and COL11A1) were identified to be hub genes of PAAD, which were obviously up-regulated in PAAD and considerably linked to tumor stage as well as prognosis. Subsequently, patients with PAAD were divided into 3 clusters premised on ECMG expression and ECM scores. Cluster 2 was the subtype with the best prognosis accompanied by the lowest ECM scores, further verifying ECM's significant contribution to the pathophysiological processes of PAAD. Significant differences were observed for oncogene and tumor suppressor gene expression, immune microenvironment, and chemotherapy sensitivity across three ECM subtypes. After applying a variety of bioinformatics methods, a novel and robust ECM-associated mRNA-lncRNA-based prognostic panel (ECM-APP) was developed and validated for accurately predicting clinical outcomes of patients with PAAD. Patients with PAAD were randomly categorized into the train, internal validation, and external validation cohorts; meanwhile, each patient was allocated into high-risk (unfavorable prognosis) and low-risk (favorable prognosis) populations premised on the expression traits of ECM-related mRNAs and lncRNAs. The discrepancy in the tumor mutation burden and immune microenvironment might be responsible for the difference in prognoses across the high-risk and low-risk populations. Overall, our findings identified and validated seven ECMGs remarkably linked to the onset and progression of PAAD. ECM-based molecular classification and prognostic panel aid in the prognostic assessment and personalized intervention of patients with PAAD.

Synthesis and Corrosion Inhibition Performance of Mannich Bases on Mild Steel in Lactic Acid Media.

Heterocyclic Mannich bases, N-(3-oxo-3-phenylpro-pyl)thiazol-2-aminium chloride (DTZA) and N-(3-oxo-3-phenylpropyl)-1H-pyrazol-3-aminium chloride (DPZA), were developed for the corrosion inhibition of N80 steel in a 15 wt % lactic acid solution. Weight loss measurements, electrochemical techniques, surface characterization, and theoretical calculations were combined to investigate their anticorrosion performance and mechanism. The results showed that DTZA exhibited a satisfactory inhibitor efficiency of 97.56% with a dosage of 0.15% at 363 K, while DPZA achieved only 58.3% under the same conditions. Adsorptions of both inhibitors on the metal surface followed the Langmuir model with physical and chemical adsorptions. Based on X-ray photoelectronic spectroscopy (XPS) analysis, DFT calculations, and molecular dynamics (MD) simulations, stronger interactions between DTZA and iron than those in the case of DPZA were revealed, leading to the formation of a compact protective film on the metal surface, which is attributed to the presence of a thiazole ring in the DTZA chemical structure.

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.

Kallikrein-related peptidase-8 (KLK8) aggravated hypoxia-induced right ventricular hypertrophy by targeting P38 MAPK/P53 signaling pathway.

Right ventricular (RV) hypertrophy and further heart failure are major co-morbidities, resulting in the premature death of patients with hypoxic pulmonary hypertension (HPH). The regulatory effects of kallikrein-related peptidase (KLK) family members on cardiac function have been extensively studied. However, to the best of the authors' knowledge, the regulatory effects of KLK8 on RV hypertrophy caused by HPH have yet to be reported. The aim of the present study was to assess KLK8 expression in the RV tissue of HPH-modeled rats, and to further explore the effects and underlying mechanism of KLK8 in regulating the hypertrophy of hypoxia-induced H9c2 cardiomyocytes. In HPH model rats, increases in the right ventricle hypertrophy index, the right ventricular systolic pressure, cardiac output, as well as pulmonary artery wall thickness were observed. Western blot analysis revealed that KLK8 expression and MAPK/p53 signaling activity were enhanced in the RVs of rats in an RV HPH rat model. In hypoxia-induced H9c2 cardiomyocytes, KLK8 overexpression promoted cardiomyocyte hypertrophy, whereas KLK8 silencing showed the opposite results. KLK8 overexpression increased the expression levels of ventricular hypertrophy markers, including atrial natriuretic peptide, brain natriuretic peptide and myosin heavy chain 7, which were blocked upon addition of the p38 MAPK inhibitor, SB202190. Conversely, KLK8 silencing caused a decrease in the expression levels of the ventricular hypertrophy markers, which were further reduced via inhibition of the p38 MAPK/p53 signaling pathway. Taken together, the results of the present study have shown that KLK8 may subtly regulate RV hypertrophy, and therefore KLK8 may be a promising therapeutic target for treating HPH-induced RV hypertrophy.

Yak Gut Microbiota: A Systematic Review and Meta-Analysis.

The yak (Bos grunniens) is closely related to common cows (Bos taurus), but is clearly a distinct species. Yaks are of substantial importance to food and leather production in certain high-altitude regions of Asia. The animal is increasing elsewhere as well, mainly because of the perceived health benefits of its milk. Like all ruminants, the animal harbors a complex community of microbial cells in its gut, crucial for its physiology. Despite yaks being important domestic animals, the composition of its gut microbiota and how the composition is guided by its specific high-altitude environment remains largely uncategorized. Hence, online databases (Embase, Medline ALL, Web of Science Core Collection, Cochrane Central Register of Controlled Trials, and Google Scholar) were searched for articles on yak intestinal microbiota. The pooled taxonomic abundance was compared between regions, sexes, different age groups, and feeding patterns. The gut microbiota distribution across different yak intestinal segments was established through pooled average taxonomic abundance. A total of 34 studies met the inclusion criteria and yielded information on 982 unique yak gut microbiota samples. An analysis of overall pooled microbiota revealed a segmented microbial community composition of the yak gut. Yak rumen microbiota was significantly influenced by difference in region, sex, and feeding patterns, the latter factor being dominant in this respect. Yak microbiome is shaped by the feeding strategy and provides an obvious avenue for improving health and productivity of the animal. More generally, the current segmental description of physiological gut microbiome provides insight into how the microbiology of this animal has adapted itself to help comping yaks with its high-altitude habitat.

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.

Investigating Ramadan Like Fasting Effects on the Gut Microbiome in BALB/c Mice.

Recently we reported that in healthy volunteer Ramadan-associated intermittent fasting (RAIF) remodels the gut microbiome and resulted in an increase in small chain fatty acid producing bacteria concomitant with improved metabolic parameters. As interpretation of these results is hampered by the possible psychological effects associated with the study, we now aim to investigate RAIF in experimental animals. To this end, 6-week male BALB/c mice were subjected to RAIF (30 days of a 16-h daily fasting; n = 8) or provided with feed ad libitum (n = 6). Fecal samples were collected before and the end of fasting and bacterial 16S rRNA sequencing was performed. We found that RAIF remodeled the composition of gut microbiota in BALB/c mice (p < 0.01) and especially provoked upregulation of butyrate acid-producing Lachnospireceae and Ruminococcaceae (p < 0.01), resembling the effects seen in human volunteers. Hence we conclude that the effects of RAIF on gut microbiome relate to the timing of food intake and are not likely related to psychological factors possibly at play during Ramadan.

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.