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Authors: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill
Group: SPARC
Summary: Protocol for collection, histological processing, and imaging of rat vagus nerves.
Proper citation: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill 2020. SPARC_Duke_PelotGrill_OT2-OD025340_RatVagusNerve_Collection_Histology_Microscopy. protocols.io dx.doi.org/10.17504/protocols.io.bh4bj8sn Copy
Authors: Janet Keast, Peregrine Osborne, Nicole Wiedmann
Group: SPARC
Summary: This collection describes the procedures required to visualize and characterize lumbosacral spinal neurons that are activated by cystometry of awake adult male and female Sprague-Dawley rats. This collection includes protocols for:STAGE 1: Surgery to cannulate the bladder, followed by recovery then cystometrySTAGE 2: Intracardiac perfusion with fixative to preserve the spinal cord tissueSTAGE 3: Immunohistochemical labelling of spinal cord sections to visualise immediate early gene expression in specific spinal regions and neuronal populationsSTAGE 4: Microscopy and image analysis to assess patterns of immediate early gene expression in different spinal cord regions
Proper citation: Janet Keast, Peregrine Osborne, Nicole Wiedmann 2020. Immediate Early Gene (IEG) mapping of spinal cord neurons activated by cystometry-induced micturition in rats [keast-002]. protocols.io dx.doi.org/10.17504/protocols.io.bakxicxn Copy
Authors: Jeffrey Ardell
Group: SPARC
Summary: Neural analysis using linear micrarray electrodes.
Proper citation: Jeffrey Ardell 2019. Pig-Neural recording and analysis-workflow. protocols.io dx.doi.org/10.17504/protocols.io.2i4gcgw Copy
Authors: Janet Keast, Peregrine Osborne, Nicole Wiedmann
Group: SPARC
Summary: This protocol is used for bladder cannulation and cystometry in an experimental adult male or female rat. The surgery is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation, including methods of anesthesia, surgical environment, and post-operative monitoring and care.
Proper citation: Janet Keast, Peregrine Osborne, Nicole Wiedmann 2020. Cystometry in awake rats. protocols.io dx.doi.org/10.17504/protocols.io.bakjicun 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 to surgically implant gastrointestinal planar electrodes and vagus nerve cuff electrodes for chronic electrophysiological recording and stimulaiton in the ferret. Electrode lead wiring is tunneled subcutaneously to a connector embedded in a headcap affixed to the cranium.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 - Chronic implantation of gastrointestinal and vagus nerve electrodes in the ferret. protocols.io dx.doi.org/10.17504/protocols.io.6crhav6 Copy
Authors: Janet Keast, Peregrine Osborne
Group: SPARC
Summary: This protocol describes confocal microscopy and image analysis procedures for characterizing neuronal cell bodies and their associated synaptic boutons in thick (50 µm) cryosections. The protocol has been applied to rat pelvic ganglia, where neuronal cell bodies have been identified using immunohistochemical markers of specific neuron populations and/or fluorescent retrograde tracer.
Proper citation: Janet Keast, Peregrine Osborne 2020. Confocal microscopy and characterization of synaptic boutons associated with ganglion neurons. protocols.io dx.doi.org/10.17504/protocols.io.bakdics6 Copy
Authors: Jeffrey Ardell
Group: SPARC
Summary: Yorshire pigs are anthesized for ICN/ neuromodulation recodings. Montior hemodynics A1mid-sternotomy
Proper citation: Jeffrey Ardell 2019. Pig ICN recording. protocols.io dx.doi.org/10.17504/protocols.io.2jugcnw Copy
Authors: Lauren Segers, Kendall Morris, Donald Bolser
Group: SPARC
Proper citation: Lauren Segers, Kendall Morris, Donald Bolser 2019. Morris USF Lab protocol. protocols.io dx.doi.org/10.17504/protocols.io.58vg9w6 Copy
Authors: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill
Group: SPARC
Summary: The protocol describes immunohistochemistry with anti-choline acetyltransferase, as it has been applied to cervical and abdominal vagus nerve samples from rats, pigs, and humans.
Proper citation: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill 2020. SPARC_Duke_Grill_OT2-OD025340_VagusNerve_IHC_ChAT. protocols.io dx.doi.org/10.17504/protocols.io.bi9tkh6n Copy
Authors: Pradeep Rajendran, John Tompkins, Kalyanam Shivkumar
Group: SPARC
Proper citation: Pradeep Rajendran, John Tompkins, Kalyanam Shivkumar 2019. Pig- Heart Neuonal and Fiber Immunocytochemistry. protocols.io dx.doi.org/10.17504/protocols.io.2n9gdh6 Copy
Authors: Elizabeth Smith
Group: SPARC
Summary: Immunohistochemistry protocol used for staining with fluorescent secondary antibodies to highlight specific tissue structures and amplify specific antibody signals.
Proper citation: Elizabeth Smith 2019. IHC-Amplified Fluorescent Frozen Sections. protocols.io dx.doi.org/10.17504/protocols.io.8rmhv46 Copy
Authors: Maci Heal, Susan Tappan
Group: SPARC
Summary: Convert binary mask tracings from Nikon's NIS Elements software into MBF XML format.
Proper citation: Maci Heal, Susan Tappan 2020. SPARC Segmentation Conversion: Binary mask tracings to MBF XML. protocols.io dx.doi.org/10.17504/protocols.io.bchjit4n Copy
Authors: Clara Leung, Maci Heal, Shaina Robbins, Alison Moss, Corey Monteith, Susan Tappan
Group: SPARC
Summary: This protocol describes the process of using the Tissue Mapper software to map single neurons of the intrinsic cardiac nervous system, annotate or "trace" key cardiac anatomy on select histological sections of an image volume, and to visualize the mapped neurons and traced anatomy in a 3D reconstructed heart.
Proper citation: Clara Leung, Maci Heal, Shaina Robbins, Alison Moss, Corey Monteith, Susan Tappan 2020. Single-Cell ICN Neuron Mapping and 3D Heart Reconstruction with Tissue Mapper. protocols.io dx.doi.org/10.17504/protocols.io.bdz5i786 Copy
Authors: Max Novelli
Group: SPARC
Summary: Protocol for dataset “RNEL Bladder January 2019”Lower urinary track nerve responses to high-density epidural spinal cord stimulation ( RNEL January 2019 )
Proper citation: Max Novelli 2021. SPARC RNEL Bladder January 2019 protocol. protocols.io dx.doi.org/10.17504/protocols.io.xszfnf6 Copy
Authors: Christina M Wright, Robert O Heuckeroth
Group: SPARC
Summary: INTRODUCTION: 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 (OBJECTIVES: To obtain a suspension of single nuclei from human colon myenteric plexus that could be used for RNA-seq.METHODS: 1) Human colon "myenteric plexus" is dissected from the bowel wall after labeling with a live cell dye and then frozen in OCT. 2) Frozen "myenteric plexus" is sectioned on a cryostat to generate small fragments and disrupt muscle and connective tissue. 3) RNA quality is assessed from a few sections using an Agilent Bioanalyzer. 4) Nuclei are obtained from frozen sections using Dounce homogenization and labeled with Hoechst 33342 dye. 5). Nuclei are separated from other cell debris by FACS. 6) RNA-seq data were obtained using the 10X Genomic single cell sequencing platform.RESULTS: Live dye labeling works well on living tissue to visualize the ENS in human colon. Careful dissection is required to micro-dissect "myenteric plexus" from from within bowel wall muscle layers. A suspension of single nuclei is efficiently isolated by freezing isolated tissue in OCT, frozen sectioning, Dounce homogenizing, and FACS sorting Hoechst 33342 stained nuclei. The isolated nuclei are suitable for sequencing on the 10X Genomics single cell sequencing platform. This procedure dramatically enriched for cells in the region of the myenteric plexus, but unfortunately only a small percentage of the cells isolated are myenteric neurons. In addition to myenteric neurons, we obtained data from enteric glia, smooth muscle, muscularis macrophage, interstitial cells of Cajal, and PDGFRalpha+ cells that are known to be closely associated with myenteric plexus.CONCLUSION: We tried many approaches to optimize this procedure including additional steps to enrich for myenteric neuron nuclei. These additional procedures invariably reduced total cell yield without enriching significantly for neuronal nuclei. We do not know yet why neuronal nuclei remain a small component of our total nuclei population, but suspect that neuronal nuclei are more fragile than other nuclei isolated. This approach generated valuable data at a single nucleus RNA-seq level for all cell types within the muscle wall known to control bowel motility.
Proper citation: Christina M Wright, Robert O Heuckeroth 2021. Processing Human Colon "Myenteric Plexus" for Single Nuclei RNA-seq. protocols.io dx.doi.org/10.17504/protocols.io.w26fghe Copy
Authors: Billie Hunne, Martin Stebbing, Rachel M. McQuade, John B. Furness
Group: SPARC
Summary: Enteroendocrine cells are important regulators of gastrointestinal, digestive and metabolic function. Here we describe protocols for identifying, mapping and characterising these cells in the rat gastric mucosa using immunohistochemistry and high resolution microscopy techniques
Proper citation: Billie Hunne, Martin Stebbing, Rachel M. McQuade, John B. Furness 2019. Immunohistochemistry and high resolution microscopy of rat gastric enteroendocrine cells. protocols.io dx.doi.org/10.17504/protocols.io.xeyfjfw Copy
Authors: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill
Group: SPARC
Summary: Protocol for collection, histological processing (paraffin embedding, Masson's trichrome staining), and imaging of pig vagus nerves.
Proper citation: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill 2020. SPARC_Duke_PelotGrill_OT2-OD025340_PigVagusNerve_Collection_Histology_Microscopy. protocols.io dx.doi.org/10.17504/protocols.io.6bqhamw Copy
Authors: Elizabeth Smith
Group: SPARC
Summary: Immunohistochemistry protocol used for staining with fluorescent secondary antibodies to highlight specific tissue structures.
Proper citation: Elizabeth Smith 2019. IHC Fluorescent Frozen Sections. protocols.io dx.doi.org/10.17504/protocols.io.7ayhifw Copy
Authors: Janet Keast, Peregrine Osborne, Nicole Wiedmann
Group: SPARC
Summary: This protocol is suitable for preserving tissues for anatomical studies of organs, ganglia, spinal cord or brain in adult rats. The protocol is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation.
Proper citation: Janet Keast, Peregrine Osborne, Nicole Wiedmann 2020. Intracardiac perfusion with fixative for anatomical studies. protocols.io dx.doi.org/10.17504/protocols.io.bahzib76 Copy
Authors: James Fallon, Sophie Payne, Peregrine Osborne, Janet Keast
Group: SPARC
Summary: This collection describes the procedures required to implant a pelvic nerve array and bladder catheter into male Sprague-Dawley rats as well as cystometry and electrophysiological testing in awake animals. This collection includes protocols for:STAGE 1: Implantation of a pelvic nerve array in ratsSTAGE 2: Cystometry in awake ratsSTAGE 3: Electrophysiological recording of electrically-evoked compound action potentials
Proper citation: James Fallon, Sophie Payne, Peregrine Osborne, Janet Keast 2020. Pelvic nerve implantation, testing and processing in awake rats. protocols.io dx.doi.org/10.17504/protocols.io.bgrmjv46 Copy
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