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Authors: Nicole A. Pelot, J. Ashley Ezzell, Gabriel B. Goldhagen, Kara A. Clissold, Warren M. Grill
Group: SPARC
Summary: The protocol describes immunohistochemistry with anti-claudin-1, imaging, image segmentation, and image analysis methods to quantify human vagus nerve morphology.
Proper citation: Nicole A. Pelot, J. Ashley Ezzell, Gabriel B. Goldhagen, Kara A. Clissold, Warren M. Grill 2020. SPARC_Duke_PelotGrill_OT2-OD025340_PigVagusNerve_FibronectinIF_Morphology. protocols.io dx.doi.org/10.17504/protocols.io.bfwtjpen Copy
Authors: Charles Horn, Derek M. Miller, Stephanie Fulton, Bill J. Yates, Lee E. Fisher, Ameya C. Nanivadekar
Group: SPARC
Summary: This protocol is used for acute surgery and electrophysiological experimentation on the anesthetized ferret, specifically for manipulating the vagus nerve and gastrointestinal tract nerve stimulation and myoelectric recording.Funding: This protocol was developed with funding from the NIH Common Fund’s Stimulating Peripheral Activity to Relieve Conditions (SPARC) program (Award U18TR002205). To learn more about the SPARC program, visit https://sparc.science.
Proper citation: Charles Horn, Derek M. Miller, Stephanie Fulton, Bill J. Yates, Lee E. Fisher, Ameya C. Nanivadekar 2019. SPARC - Acute surgery and experimentation of the gastrointestinal tract and vagus nerve in the ferret. protocols.io dx.doi.org/10.17504/protocols.io.6a7hahn Copy
Authors: Shaina Robbins, Sean Nieves
Group: SPARC
Proper citation: Shaina Robbins, Sean Nieves 2020. 0.1% Cresyl Violet Stain (pH 4.3). protocols.io dx.doi.org/10.17504/protocols.io.wcjfaun Copy
Authors: Elisa Gonzalez-Rothi, Latoya Allen, Alec Simon, Yasin Seven, Marissa Ciesla, Gordon Mitchell
Group: SPARC
Summary: This protocol describes the procedures for conducting long-term exposure to various intermittent hypoxia (or normoxia) protocols in rats.
Proper citation: Elisa Gonzalez-Rothi, Latoya Allen, Alec Simon, Yasin Seven, Marissa Ciesla, Gordon Mitchell 2019. SPARC Long-term exposure to intermittent hypoxia (or normoxia) using a custom in-cage computer controlled system. protocols.io dx.doi.org/10.17504/protocols.io.2j3gcqn Copy
Authors: Lixin Wang, Collin Challis, Honghui Liang, Songlin Li, Charless Fowlkes, Aidan Sullivan, Kumar SR, Yvette Taché
Group: SPARC
Summary: Using a multicolor adeno-associate virus system to label the colonic enteric nervous system for digital tracing of individual neurons and nerve fibers in microcircuits in three-dimensions (3D). The methods include viral vectors retro-orbital injection in mice, preparation of colon tissues, microscopy and 3D digital tracing.
Proper citation: Lixin Wang, Collin Challis, Honghui Liang, Songlin Li, Charless Fowlkes, Aidan Sullivan, Kumar SR, Yvette Taché 2020. Multicolor adeno-associate virus labeling and 3D digital tracing of enteric plexus in mouse proximal colon. protocols.io dx.doi.org/10.17504/protocols.io.bqavmse6 Copy
Authors: Latoya Allen, Marissa Ciesla, Yasin Seven, Elisa Gonzalez-Rothi, Gordon Pool
Group: SPARC
Summary: This protocol describes the procedure for retrogradely labeling bilateral phrenic motor neuron pools in the cervical spinal cord using intrapleural injections of cholera toxin B fragment
Proper citation: Latoya Allen, Marissa Ciesla, Yasin Seven, Elisa Gonzalez-Rothi, Gordon Pool 2019. SPARC Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons Using Intrapleural Injections of Cholera Toxin B Fragment. protocols.io dx.doi.org/10.17504/protocols.io.2kpgcvn Copy
Authors: Pu-Qing Yuan, Yvette Taché
Group: SPARC
Proper citation: Pu-Qing Yuan, Yvette Taché 2019. Tache_Yuan_OT2OD024899_CLARITYAnd3DImagingOfColonicENSintheMouseAndPig_1_2019-Pig_Protocol. protocols.io dx.doi.org/10.17504/protocols.io.4r9gv96 Copy
Authors: Pu-Qing Yuan, Lixin Wang, Yvette Taché
Group: SPARC
Proper citation: Pu-Qing Yuan, Lixin Wang, Yvette Taché 2019. Tache_Yuan_OT2OD024899_CLARITYAnd3DImagingOfColonicENSintheMouseAndPig_1_2019-Mouse_Protocol (Annotation Copy). protocols.io dx.doi.org/10.17504/protocols.io.4sagwae Copy
Authors: Christina M Wright, Robert O Heuckeroth
Group: SPARC
Summary: ABSTRACTINTRODUCTION: The human enteric nervous system (ENS) is a complex network of neurons and glia that extends throughout the length of the bowel. There are many neuron and glia types, but very little is known about the human ENS compared to other species. There are two main ENS layers. The myenteric plexus neurons are clustered into ganglia with thick nerve fiber bundles running between ganglia. Submucosal neuron ganglia are scattered throughout the region between circular muscle and the epithelial lining of the bowel. One key problem with studying human ENS is that these cells are a very minor component of the bowel wall (< 1: 10,000 cells) and they are surrounded by other tissue. The bowel wall is not easily dissociated making it challenging to isolate cells of the human ENS for any type of analsysis.
OBJECTIVES: To enrich for neurons from human colon myenteric plexus using PHOX2B antibody and FACS
METHODS: 1) Human colon muscle was dissected from the bowel wall and then frozen in OCT. 2) Frozen muscle was sectioned on a cryostat to generate small fragments and disrupt muscle and connective tissue. 3) RNA quality was assessed from a few sections using an Agilent Bioanalyzer. 4) Nuclei were obtained from frozen sections using Dounce homogenization and stained with antibody to PHOX2B. 5). Nuclei were separated from other cell debris by FACS. 6) RNA-seq data were obtained using the 10X Genomic single cell sequencing platform.
RESULTS: This procedure worked well to isolate nuclei from human colon muscle. PHOX2B antibody labeling led to a small population of nuclei that appeared to be antibody stained under the microscope and have attached antibody by FACS. Unfortunately, single nucleus RNA-seq demonstrated that we had not enriched for myenteric neurons with this approach. RNA-seq data were obtained from many other cell types within the muscle layers of the bowel wall.
CONCLUSION: This approach generated valuable data at a single nucleus RNA-seq level for many cell types within the muscle wall known to control bowel motility, but did not enrich for neuronal nuclei, even though PHOX2B antibody selectively stains myenteric plexus nuclei within the human bowel myenteric plexus
Proper citation: Christina M Wright, Robert O Heuckeroth 2021. Processing Human Colon muscle layers for Single Nuclei RNA-seq using PHOX2B antibody immunoselection. protocols.io dx.doi.org/10.17504/protocols.io.w3afgie Copy
Authors: Thomas Taylor-Clark, Seol-Hee Kim
Group: SPARC
Summary: Mice are euthanized, perfused with fixative and the vagal ganglia extracted. Vagal ganglia are then cyosectioned. Slices are stained for protein expression using immunohistochemistry. Expression of specific proteins and reporter proteins isarevisualized using microscopy.
Proper citation: Thomas Taylor-Clark, Seol-Hee Kim 2019. Dissection and immunohistochemistry of mouse vagal ganglia. protocols.io dx.doi.org/10.17504/protocols.io.baumieu6 Copy
Authors: Nicole A. Pelot, Gabriel B. Goldhagen, Jake E. Cariello, Warren M. Grill
Group: SPARC
Summary: The protocol describes image segmentation and image analysis methods to quantify rat vagus nerve morphology from Masson's trichrome-stained nerve cross sections.
Proper citation: Nicole A. Pelot, Gabriel B. Goldhagen, Jake E. Cariello, Warren M. Grill 2020. SPARC_Duke_PelotGrill_OT2-OD025340_RatVagusNerve_Morphology. protocols.io dx.doi.org/10.17504/protocols.io.y6hfzb6 Copy
Authors: Terry Powley, Jennifer Mcadams, Robert Phillips
Group: SPARC
Summary: This protocol describes the methods used to trace and enable morphometric quantification of vagal afferent neurites in the rat stomach. A mixture of dextran conjugates was injected into the nodose ganglia of young adult Sprague-Dawley rats and after a survival period of 14 days for optimal tracer transport, stomachs were removed and processed as whole mounts. ABC-DAB was used to create a permanent gold-brown stain of all labeled afferent neurites. Subgroups of samples were also counterstained with either the panneuronal chromogen cuprolinic blue or with nNOS antibodies and steel gray chromogen to label nitrergic cells.
Proper citation: Terry Powley, Jennifer Mcadams, Robert Phillips 2019. High resolution labeling of vagal afferent fibers using Dextran-Biotin with counterstaining. protocols.io dx.doi.org/10.17504/protocols.io.2ipgcdn Copy
Authors: Shaina Robbins, Sean Nieves
Group: SPARC
Summary: This is the straightforward procedure we follow when sectioning rat heart.
Proper citation: Shaina Robbins, Sean Nieves 2020. Sectioning Rat Heart. protocols.io dx.doi.org/10.17504/protocols.io.w53fg8n Copy
Authors: Janet Keast, Peregrine Osborne
Group: SPARC
Summary: This collection describes the procedures required to visualise and characterize synaptic boutons associated with functionally classified pelvic ganglion autonomic neurons of adult male and female Sprague-Dawley rats. This collection includes protocols for:STAGE 1: Surgery to micro-inject fluorescent retrograde tracer dyes into one or more sites within the lower urinary tractSTAGE 2: Intracardiac perfusion with fixative to preserve neural tissues of interestSTAGE 3: Immunohistochemical labeling of thick cryosections of pelvic gangliaSTAGE 4: Confocal microscopy and image analysis of synaptic boutons associated with ganglion neurons.
Proper citation: Janet Keast, Peregrine Osborne 2020. Light microscopic analysis of synaptic input to neurons in the rat major pelvic ganglion [keast-003]. protocols.io dx.doi.org/10.17504/protocols.io.bakiicue Copy
Authors: Colleen clancy, Pei-Chi, Parya Aghasafari
Group: SPARC
Summary: Multi-scale computational modeling is a major branch of computational biology as evidenced by the US federal interagency Multi-Scale Modeling Consortium and major international projects. It invariably involves specific and detailed sequences of data analysis and simulation, often with multiple tools and datasets, and the community recognizes improved modularity, reuse, reproducibility, portability and scalability as critical unmet needs in this area. Scientific workflows are a well-recognized strategy for addressing these needs in scientific computing. While there are good examples if the use of scientific workflows in bioinformatics, medical informatics, biomedical imaging and data analysis, there are fewer examples in multi-scale computational modeling in general and cardiac electrophysiology in particular. Cardiac electrophysiology simulation is a mature area of multi-scale computational biology that serves as an excellent use case for developing and testing new scientific workflows. In this dataset, we develop, describe and test a computational workflow that serves as a proof of concept of a platform for the robust integration and implementation of a reusable and reproducible multi-scale cardiac cell and tissue model that is expandable, modular and portable. The workflow described leverages Python and Kepler-Python actor for plotting and pre/post-processing. During all stages of the workflow design, we rely on freely available open-source tools, to make our workflow freely usable by scientists.
Proper citation: Colleen clancy, Pei-Chi, Parya Aghasafari 2019. A multi-scale model of cardiac electrophysiology. protocols.io dx.doi.org/10.17504/protocols.io.5nkg5cw Copy
Authors: Nicole A. Pelot, Gabriel B. Goldhagen, Jake E. Cariello, Warren M. Grill
Group: SPARC
Summary: The protocol describes image segmentation and image analysis methods to quantify pig vagus nerve morphology from Masson's trichrome-stained nerve cross sections.
Proper citation: Nicole A. Pelot, Gabriel B. Goldhagen, Jake E. Cariello, Warren M. Grill 2020. SPARC_Duke_PelotGrill_OT2-OD025340_PigVagusNerve_Morphology. protocols.io dx.doi.org/10.17504/protocols.io.6bvhan6 Copy
Authors: Elisa Gonzalez-Rothi, Yasin Seven, Latoya Allen, Marissa Ciesla, Gordon Mitchell
Group: SPARC
Summary: This protocol describes the immunofluorescent labeling technique used to identify adenosine 2a receptor expression in CtB-labelled phrenic motor neurons and within a defined region of interest surrounding phrenic motor neurons.
Proper citation: Elisa Gonzalez-Rothi, Yasin Seven, Latoya Allen, Marissa Ciesla, Gordon Mitchell 2019. SPARC Adenosine 2A Receptor Immunohistochemistry Protocol in Rat Tissues Labeled with Cholera Toxin B-fragment. protocols.io dx.doi.org/10.17504/protocols.io.2kfgctn Copy
Authors: Terry Powley, Zhenjun Tan, Matthew Ward
Group: SPARC
Summary: This protocol describes a process for the measurement of electrical stimulation-induced effects on duodenal motility in young adult Sprague-Dawley rats. Signals recorded from strain gauges attached to the proximal duodenal surface were used to measure the effect of stimulation by patch electrodes implanted at multiple sites across the rat stomach in an acute anesthetized preparation. The effect of stimulation was quantified as the ratio of various motility assessments during and after stimulation vs. before stimulation, and the data was used to create a functional map of duodenal motor response to localized gastric stimulation.
Proper citation: Terry Powley, Zhenjun Tan, Matthew Ward 2020. Measurement of duodenal motility using implanted strain gauges. protocols.io dx.doi.org/10.17504/protocols.io.2irgcd6 Copy
Authors: Donald Bolser
Group: SPARC
Summary: Cervical vagotomy sections both lower airway and recurrent laryngeal afferent fibers and eliminates cough induced by stimulation of sensory afferents in the lower trachea and intrathoracic airways. In the guinea pig, recurrent laryngeal afferent fibers are primarily responsible for cough in response to mechanical or chemical stimulation of the upper trachea and larynx. Further, it has been proposed that lower airway slowly adapting receptors have a permissive effect on the cough reflex. The objective of this study was to determine the role of lower airway vagal afferent fibers on the cough reflex in the cat. We hypothesized that cough induced by mechanical or chemical stimuli applied to the lower airways or the larynx of this species would be depressed after vagotomy below the branch of the recurrent laryngeal nerve. Experiments were conducted in four anesthetized, bilaterally thoracotomized, and artificially ventilated cats. Artificial ventilation was accomplished via a tracheal cannula placed through an incision in the tenth trachea ring. Mechanical (via a flexible cannula through a small port in the trachea tube) or chemical (capsaicin, 0.5 cc, 10 µM) stimulation of the intrathoracic airway was employed during mechanical ventilation. Thoracic vagotomy virtually eliminated the cough response to mechanical stimulation of the lower airways. The cough response to mechanical stimulation of the larynx was also depressed after thoracic vagotomy. Capsaicin introduced into the intrathoracic trachea only elicited cough in two animals but this response was eliminated in both animals by thoracic vagotomy. The results support an important role of lower airway sensory feedback in the production of cough in the anesthetized cat. Further, in the presence of intact recurrent laryngeal nerves, the excitability of cough induced from the larynx is strongly influenced by sensory feedback from the lower airways.
Proper citation: Donald Bolser 2020. SPARC_Bolser_intrathoracic vagotomy and cough . protocols.io dx.doi.org/10.17504/protocols.io.bgm5ju86 Copy
Authors: Shaina Robbins, Alison Moss, Sean Nieves, Sirisha Achanta
Group: SPARC
Summary: This collection of protocols were used to obtain the current data for the Blackfynn Dataset Molecular Phenotype Distribution of Single Rate ICN Neurons, which we refer to as Rat Heart B.
Proper citation: Shaina Robbins, Alison Moss, Sean Nieves, Sirisha Achanta 2020. Molecular Phenotype Distribution of Single Rat ICN Neurons - Heart B. protocols.io dx.doi.org/10.17504/protocols.io.bfxvjpn6 Copy
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