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.

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.

[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.

[Expression, purification and identification of the domain III of DENV II envelop protein in Escherichia coli].

OBJECTIVE: To express the domain III of DENV II envelop protein in Escherichia coli, obtain the purified recombinant protein and identify its immunoreactivity. METHODS: Suckling mice were inoculated with live DENV II in the brain. The total RNA was extracted from the brain of the infected mice, and the envelope protein DNA fragment was amplified by RT-PCR and ligated into pMD 18-T to construct pMD 18-T-DV2-E. The domain III DNA fragment of the envelope protein was amplified by PCR with pMD 18-T-DV2-E as the template and cloned into pET-32a(+) to construct the expression plasmid pET-32a(+)-DV2-E-DIII. The recombinant plasmid was transformed into E.coli BL21(DE3) and induced by IPTG, and the expressed products were analyzed by SDS-PAGE and Western blotting. RESULTS: After RT-PCR amplification, a specific DNA fragment of about 1.5 kb was obtained and ligated into pMD 18-T to construct pMD 18-T-DV2-E. With pMD 18-T-DV2-E as the template, the domain III DNA fragment about 320 bp in length was amplified and the expression plasmid pET-32a(+)-DV2-E-DIII was successfully constructed. After induction with IPTG, a specific soluble protein with a relative molecular mass of 29000 was obtained and the expression product accounted for 52.50 percent; of the total protein of the cell lysate. Western blotting demonstrated reactivity of the recombinant protein with His-Tag McAb and DENV (Type I-IV) McAb. CONCLUSION: The recombinant plasmid can be highly expressed in E.coli BL21(DE3) in a soluble form and the recombinant protein can react with DENV (Type I-IV) McAb.

[Isolation and identification of Bacillus anthracis in an accidental case].

During June to July 2005, a few farmers in Chengde county of Hebei province were got ill after eating beef of sick cattle. The cattle could be infected with Bacillus anthracis. One beef sample and one soil sample contaminated with cattle blood were collected and used for pathogen isolation and identification in laboratory. Two bacteria strains were isolated from beef and soil sample, respectively, and showed typical morphology of Bacillus anthracis on blood agar and under microscope with Gram stain. The two bacteria strains were also positive to standard positive serum of Bacillus anthracis by slide agglutination test. Biochemical characteristics of the two bacteria were tested using API CHB/E strip and analyzed by API software (version 3.3), result showed that the two isolated bacteria were Bacillus anthracis. Polymerase chain reaction (PCR) was used to further characterize the two isolated bacteria strains. Three pairs of primer were designed and used for PCR, and these primers exactly matched the protective antigen gene, edema factor gene and capsule gene, respectively. By analyzed on agarose gel, PCR products were 423bp, 494bp and 397bp, respectively, and this result showed that the two isolated bacteria contained two plasmids, pX01 and pX02, which encoded anthrax toxin and capsule, respectively. Anthrax toxin and capsule were very important virulent factors for Bacillus anthracis. PCR products were purified and then cloned to T vector, positive clone was chose and sequenced. By BLAST with GenBank, sequence of the three genes of the two bacteria strains had a similarity of 99% with Bacillus anthracis A2012 strain, Ames Ancestor strain and A16R strain. Based on results of colonial morphology, serum test and biochemistry characterization, the two bacteria strains are Bacillus anthracis. They can encode anthrax toxin and capsule, and are virulent to animal and human.