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Scientific Reports Dec 2017The blood-nerve barrier (BNB), formed by tight junction-forming microvessels within peripheral nerve endoneurium, exists to regulate its internal microenvironment...
The blood-nerve barrier (BNB), formed by tight junction-forming microvessels within peripheral nerve endoneurium, exists to regulate its internal microenvironment essential for effective axonal signal transduction. Relatively little is known about the unique human BNB molecular composition. Such knowledge is crucial to comprehend the relationships between the systemic circulation and peripheral nerves in health, adaptations to intrinsic or extrinsic perturbations and alterations that may result in disease. We performed RNA-sequencing on cultured early- and late-passage adult primary human endoneurial endothelial cells and laser-capture microdissected endoneurial microvessels from four cryopreserved normal adult human sural nerves referenced to the Genome Reference Consortium Human Reference 37 genome browser, using predefined criteria guided by known transcript or protein expression in vitro and in situ. We identified 12881 common transcripts associated by 125 independent biological networks, defined as the normal adult BNB transcriptome, including a comprehensive array of transporters and specialized intercellular junctional complex components. These identified transcripts and their interacting networks provide insights into peripheral nerve microvascular morphogenesis, restrictive barrier formation, influx and efflux transporters with relevance to understanding peripheral nerve homeostasis and pharmacology, including targeted drug delivery and the mediators of leukocyte trafficking in peripheral nerves during normal immunosurveillance.
Topics: Adult; Blood-Nerve Barrier; Cells, Cultured; Endothelial Cells; Female; Gene Expression Profiling; Humans; Laser Capture Microdissection; Male; Middle Aged; Primary Cell Culture; Sciatic Nerve; Sequence Analysis, RNA; Sural Nerve; Transcriptome
PubMed: 29234067
DOI: 10.1038/s41598-017-17475-y -
The Journal of Craniofacial SurgeryTo obtain further understanding of the eyelid lymphatic anatomy.
OBJECTIVE
To obtain further understanding of the eyelid lymphatic anatomy.
METHOD
Thirty-two halves of eyelids from 16 fresh fetus cadavers were studied by microdissection using a mixture of 3% Prussian blue and chloroform to visualize the lymphatic vessels.
RESULTS
Three layers of lymphatic plexuses were demonstrated in the eyelids: a superficial or preorbicularis muscle plexus; a pretarsal or postorbicular muscle plexus; and a deep or posttarsal plexus. Furthermore, communicating branches among these plexuses were also spotted.
CONCLUSIONS
The study results demonstrated the topographic distribution of the eyelid lymphatic vessels and confirmed the existence of communicating branches. This discovery will be conducive to understanding the route and mechanism by which inflammation of the eyelid spreads and cancer disseminates. It also provides anatomical insights to apply during eyelid surgery with regard to the prevention of possible eyelid lymphatic injury.
Topics: Cadaver; Eyelids; Humans; Lymphatic System; Lymphatic Vessels; Microdissection
PubMed: 34224459
DOI: 10.1097/SCS.0000000000007759 -
Current Protocols in Immunology Nov 2018Ex vivo cell culture models are of particular interest for neurobiologists, as these allow the study of brain cells in an isolated environment. Organotypic brain slice...
Ex vivo cell culture models are of particular interest for neurobiologists, as these allow the study of brain cells in an isolated environment. Organotypic brain slice cultures allow growth of three-dimensional brain tissue that conserves the cellular architecture. This unit describes the preparation and culturing of organotypic brain slices from mice. In three basic protocols, the general procedure, the specific preparation of chopper slices, and slicing of whole-brain vibratome sections are described. Support protocols explain the use of postnatal or adult mice, the preparation of coronal or sagittal slices, the preparation of co-cultures, post-processing of slices, the use of extra filter membranes, and the re-use of membrane inserts. This unit provides an easy-to-understand methodological introduction to brain slice cultures. © 2018 by John Wiley & Sons, Inc.
Topics: Animals; Animals, Newborn; Brain; Mice; Microdissection; Organ Culture Techniques
PubMed: 30311739
DOI: 10.1002/cpim.59 -
Lab on a Chip Jul 2022Spatial proteomics holds great promise for revealing tissue heterogeneity in both physiological and pathological conditions. However, one significant limitation of most...
Spatial proteomics holds great promise for revealing tissue heterogeneity in both physiological and pathological conditions. However, one significant limitation of most spatial proteomics workflows is the requirement of large sample amounts that blurs cell-type-specific or microstructure-specific information. In this study, we developed an improved sample preparation approach for spatial proteomics and integrated it with our previously-established laser capture microdissection (LCM) and microfluidics sample processing platform. Specifically, we developed a hanging drop (HD) method to improve the sample recovery by positioning a nanowell chip upside-down during protein extraction and tryptic digestion steps. Compared with the commonly-used sitting-drop method, the HD method keeps the tissue pixel away from the container surface, and thus improves the accessibility of the extraction/digestion buffer to the tissue sample. The HD method can increase the MS signal by 7 fold, leading to a 66% increase in the number of identified proteins. An average of 721, 1489, and 2521 proteins can be quantitatively profiled from laser-dissected 10 μm-thick mouse liver tissue pixels with areas of 0.0025, 0.01, and 0.04 mm, respectively. The improved system was further validated in the study of cell-type-specific proteomes of mouse uterine tissues.
Topics: Animals; Laser Capture Microdissection; Mice; Proteome; Proteomics; Specimen Handling; Workflow
PubMed: 35838077
DOI: 10.1039/d2lc00384h -
Molecular & Cellular Proteomics : MCP Dec 2022Despite their diminutive size, islets of Langerhans play a large role in maintaining systemic energy balance in the body. New technologies have enabled us to go from...
Despite their diminutive size, islets of Langerhans play a large role in maintaining systemic energy balance in the body. New technologies have enabled us to go from studying the whole pancreas to isolated whole islets, to partial islet sections, and now to islet substructures isolated from within the islet. Using a microfluidic nanodroplet-based proteomics platform coupled with laser capture microdissection and field asymmetric waveform ion mobility spectrometry, we present an in-depth investigation of protein profiles specific to features within the islet. These features include the islet-acinar interface vascular tissue, inner islet vasculature, isolated endocrine cells, whole islet with vasculature, and acinar tissue from around the islet. Compared to interface vasculature, unique protein signatures observed in the inner vasculature indicate increased innervation and intra-islet neuron-like crosstalk. We also demonstrate the utility of these data for identifying localized structure-specific drug-target interactions using existing protein/drug binding databases.
Topics: Islets of Langerhans; Proteomics; Proteins; Laser Capture Microdissection
PubMed: 36244662
DOI: 10.1016/j.mcpro.2022.100426 -
The Journal of Contemporary Dental... May 2018Oral cancer is one of the most significant public health concerns in the world. The past decades have witnessed the trend of molecular profiling for diagnostic and...
Oral cancer is one of the most significant public health concerns in the world. The past decades have witnessed the trend of molecular profiling for diagnostic and prognostic implications.
Topics: Humans; Laser Capture Microdissection; Molecular Diagnostic Techniques; Mouth Neoplasms
PubMed: 29807954
DOI: No ID Found -
Expert Review of Proteomics Oct 2021Laser Capture Microdissection (LCM) uses a laser to isolate, or capture, specific cells of interest in a complex heterogeneous tissue section, under direct microscopic... (Review)
Review
INTRODUCTION
Laser Capture Microdissection (LCM) uses a laser to isolate, or capture, specific cells of interest in a complex heterogeneous tissue section, under direct microscopic visualization. Recently, there has been a surge of publications using LCM for tissue spatial molecular profiling relevant to a wide range of research topics.
AREAS COVERED
We summarize the many advances in tissue Laser Capture Proteomics (LCP) using mass spectrometry for discovery, and protein arrays for signal pathway network mapping. This review emphasizes: a) transition of LCM phosphoproteomics from the lab to the clinic for individualized cancer therapy, and b) the emerging frontier of LCM single cell molecular analysis combining proteomics with genomic, and transcriptomic analysis. The search strategy was based on the combination of MeSH terms with expert refinement.
EXPERT OPINION
LCM is complemented by a rich set of instruments, methodology protocols, and analytical A.I. (artificial intelligence) software for basic and translational research. Resolution is advancing to the tissue single cell level. A vision for the future evolution of LCM is presented. Emerging LCM technology is combining digital and AI guided remote imaging with automation, and telepathology, to a achieve multi-omic profiling that was not previously possible.
Topics: Artificial Intelligence; Laser Capture Microdissection; Lasers; Precision Medicine; Proteomics
PubMed: 34607525
DOI: 10.1080/14789450.2021.1984886 -
Reproductive Biology Jun 2018Non-obstructive azoospermia (NOA) is the most severe form of male infertility, defined by lack of spermatozoa in the ejaculate caused by impaired spermatogenesis. The... (Review)
Review
Non-obstructive azoospermia (NOA) is the most severe form of male infertility, defined by lack of spermatozoa in the ejaculate caused by impaired spermatogenesis. The chance of biological fatherhood of these men has been improved since the introduction of microdissection testicular sperm extraction (MD-TESE) combined with intracytoplasmic sperm injection. A thorough patient evaluation preoperatively is essential to recognize any underlying conditions, and to assist in patient counseling on the sperm recovery rate and pregnancy results. This review article summarizes the present data on MD-TESE to reach optimal results is treating men with NOA.
Topics: Azoospermia; Female; Humans; Male; Microdissection; Pregnancy; Pregnancy Rate; Sperm Injections, Intracytoplasmic; Sperm Retrieval; Testis
PubMed: 29602610
DOI: 10.1016/j.repbio.2018.03.003 -
Hypertension (Dallas, Tex. : 1979) Oct 2020
Topics: Denervation; Humans; Hypertension; Kidney; Microdissection; Nervous System
PubMed: 32903105
DOI: 10.1161/HYPERTENSIONAHA.120.15834 -
Journal of Visualized Experiments : JoVE Nov 2019Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such...
Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.
Topics: Animals; Cochlea; Epithelium; Hair Cells, Auditory; Hair Cells, Auditory, Inner; Mice; Microdissection; Organ of Corti
PubMed: 31762458
DOI: 10.3791/60299