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Authors: James Fallon, Sophie Payne, Janet Keast, Peregrine Osborne
Group: SPARC
Summary: A pelvic nerve array is custom designed for implantation onto the pelvic nerve of male rats. This device is used for long-term (recovery) experiments. The device can electrically stimulate the pelvic nerve as well as record electrically evoked or spontaneous neural activity. Previously, we have successfully implanted rats for 2 months with no reports of biological adverse complications or damage sustained to the array. The surgical procedure 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: James Fallon, Sophie Payne, Janet Keast, Peregrine Osborne 2020. Implantation of a pelvic nerve array in rats. protocols.io dx.doi.org/10.17504/protocols.io.bjvxkn7n Copy   

•   Source: Protocols.io

Authors: Shaina Robbins, Sean Nieves
Group: SPARC

Proper citation: Shaina Robbins, Sean Nieves 2020. Staining/Dehydration 10 micron Rat Heart . protocols.io dx.doi.org/10.17504/protocols.io.webfban Copy   

•   Source: Protocols.io

Authors: Shaina Robbins, Alison Moss, Sean Nieves
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 2020. Molecular Phenotype Distribution of Single Rat ICN Neurons - Heart B. protocols.io dx.doi.org/10.17504/protocols.io.w56fg9e Copy   

•   Source: Protocols.io

Authors: Janet Keast, Peregrine Osborne, Nicole Wiedmann
Group: SPARC
Summary: This protocol is used for analysing expression pattens of immediate early gene products (e.g., c-Fos) in immunostained transverse sections of spinal cord.

Proper citation: Janet Keast, Peregrine Osborne, Nicole Wiedmann 2020. Image analysis of immediate early gene expression in spinal cord sections. protocols.io dx.doi.org/10.17504/protocols.io.bakmicu6 Copy   

•   Source: Protocols.io

Authors: Leah Reznikov
Group: SPARC
Summary: RNA Sequencing of Porine Ganglia and brainstem Animals. A total of 44 piglets (Yorkshire Landrace breed, 2–3 days of age) were fed commercial milk replacer (Soweena Litter Life) and allowed a 36–48-h acclimation period before interventions began. The University of Florida Animal Care and Use Committee approved all procedures. Procedures were completed in accordance with federal policies and guidelines. A total of 12 piglets were reserved for RNA sequencing experiments (6 male and 6 female). Airway instillation. After acclimation, piglets were anesthetized with 8% SevoThesia (Henry Schein). The piglets’ airways were accessed with a laryngoscope; a laryngotracheal atomizer (MADgic) was passed directly beyond the vocal folds, as previously described to aerosolize either a 500 μl 0.9% saline control or 1% acetic acid in 0.9% saline solution to the airway. This procedure results in widespread distribution of aerosolized solutions throughout the piglet airway, including the lung. Consistent with acid as a cough-evoking stimulus acetic acid induced cough in 90% or greater of the piglets. Nodose ganglia and brain stem isolation. Forty-eight hours after instillations, piglets were euthanized with a 90 mg/kg intravenous Euthasol solution (Henry Schein). Nodose ganglia were carefully dissected using methods that we previously developed. Ganglia were removed, placed in TRIzol, and stored at −80°C until RNA extraction. Any connective tissues, inflammatory cells, satellite cells, or blood cells covering the ganglia were not removed. A region of the brain stem encompassing the nucleus tractus solitarius, nucleus ambiguous, and dorsal motor nucleus was removed. Briefly, a blunt scissor was used to cut through the sagittal suture of the skull post mortem, and the brain was delicately excised. The cerebellum was removed, and the obex was identified. A section of the brain stem encompassing 2 mm rostral and 2 mm caudal to the obex was then removed and placed in RNAlater (Thermo Fisher Scientific). The block was further trimmed to extend 2 mm in depth and 2 mm lateral from the obex, while still submerged in RNAlater. RNA isolation. RNA from thenodose ganglia, and brain stem were isolated using RNeasy Lipid Tissue kit (Qiagen) with optional DNase digestion (Qiagen). RNA concentrations were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). RNA sequencing. RNA concentration was determined on Qubit 2.0 Fluorometer (ThermoFisher/Invitrogen), RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA with 28S/18S > 1 and RNA integrity number (RIN) ≥ 7 were used for RNAseq library construction. RNAseq library were constructed using NEBNext Ultra Directional RNA library prep kit for Illumina (New England Biolabs) following the manufacturer’s recommendations. Briefly, 1,000 ng of total RNA was used for mRNA isolation using NEBNext Ploy(A) mRNA magnetic isolation module (New England Biolabs, cat. no. E7490). RNA library construction was achieved using NEBNext Ultra Directional RNA library prep kit for Illumina (New England Biolabs; cat. no. E7420). Thirty-six barcoded libraries were sized on the bioanalyzer and quantitated by QUBIT. Libraries were pooled in an equimolar ratio and sequenced by Illumina HiSeq 3000 2X100 cycles run for total of six runs (Illumina). RNA library construction was performed at the Interdisciplinary Center for Biotechnology Research (ICBR) Gene Expression & Genotyping Core, University of Florida (UF). HiSeq 3000 sequencing run was performed at the ICBR NextGen DNA Sequencing core, UF. The nodose ganglia and brain stem of three individual piglets for each condition were prepared separately and used. Mapping and differential gene expression. The quality of the RNA-Seq sequence data was first evaluated using FastQC before further downstream analysis. Low-quality sequences were removed, and the poor-quality part of the reads were trimmed using Trimmomatic. Star Aligner was used to map high-quality paired-end reads to Sus scrofa genome, Sscrofa11.1. Expression was obtained using RSEM. The expected read counts and fragments per kilobase of transcript per million-mapped reads (FPKM) were extracted for further analysis. The estimated read counts were taken as input for edgeR to perform differential expression analysis using generalized linear models. The threshold for calling significantly differentially expressed genes was set at FDR 0.05 with the average FPKM for at least one of each comparison group being higher than 0. Additional criteria using P This protocol accompanies the following publication 3. Reznikov LR, Liao YSJ, Gu T, Davis K, Kuan SP, Atanasova KR, Dadural JS, Collins EN, Guevara MV, Vogt KM (2019) Sex-specific airway hyperreactivity and sex-specific transcriptome remodeling in neonatal piglets challenged with intra-airway acid. AJP-Lung. Jan 1;316(1):L131-L143. doi: 10.1152/ajplung.00417.2018. Epub 2018 Nov 8. https://www.physiology.org/doi/full/10.1152/ajplung.00417.2018#

Proper citation: Leah Reznikov 2019. RNA Sequencing of Porine Ganglia and brainstem. protocols.io dx.doi.org/10.17504/protocols.io.w5sfg6e Copy   

•   Source: Protocols.io

Authors: Deborah Jaffey, Terry Powley, Jennifer Mcadams, Robert Phillips
Group: SPARC
Summary: This protocol describes the methods used to trace and enable morphometric quantification of preganglionic efferent neurites in the rat stomach. A mixture of dextran conjugates was injected into the dorsal motor nucleus of the vagal nerve (dmnX) of young adult Sprague-Dawley rats and after a survival period of 19 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 efferent 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: Deborah Jaffey, Terry Powley, Jennifer Mcadams, Robert Phillips 2019. High resolution labeling of vagal efferent fibers using Dextran-Biotin with counterstaining. protocols.io dx.doi.org/10.17504/protocols.io.2iqgcdw Copy   

•   Source: Protocols.io

Authors: James Fallon, Sophie Payne, Janet Keast, Peregrine Osborne
Group: SPARC
Summary: A pelvic nerve array is custom designed for implantation onto the pelvic nerve of male rats. This device is used for long-term (recovery) experiments. The device can electrically stimulate the pelvic nerve as well as record electrically evoked or spontaneous neural activity. Previously, we have successfully implanted rats for 2 months with no reports of biological adverse complications or damage sustained to the array. The surgical procedure 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: James Fallon, Sophie Payne, Janet Keast, Peregrine Osborne 2020. Implantation of a pelvic nerve array in rats_original from SP. protocols.io dx.doi.org/10.17504/protocols.io.bfwxjpfn Copy   

•   Source: Protocols.io

Authors: Janet Keast, Peregrine Osborne
Group: SPARC
Summary: This collection describes the procedures required to label, visualise, characterise and quantify neurons that innervate the lower urinary tract tissues 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: Fluorescence immunohistochemistry of ganglion cryosections.

Proper citation: Janet Keast, Peregrine Osborne 2019. Immunohistochemical classification of sensory and autonomic neurons projecting to the lower urinary tract in rats [keast-001]. protocols.io dx.doi.org/10.17504/protocols.io.w3gfgjw Copy   

•   Source: Protocols.io

Authors: Elizabeth Butterworth, Wesley Dickerson, Vindhya Vijay, Kristina Weitzel, Julia Cooper, Eric W. Atkinson, Jason E. Coleman, Kevin Otto, Martha Campbell Thompson
Group: Human Cell Atlas Method Development Community, Optical Clearing of Tissue, SPARC
Summary: Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to 500 μm using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth >150 μm with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Proper citation: Elizabeth Butterworth, Wesley Dickerson, Vindhya Vijay, Kristina Weitzel, Julia Cooper, Eric W. Atkinson, Jason E. Coleman, Kevin Otto, Martha Campbell Thompson 2020. Human Pancreas PACT Optical Clearing and High Resolution 3D Microscopy. protocols.io dx.doi.org/10.17504/protocols.io.9gbh3sn Copy   

•   Source: Protocols.io

Authors: Muriel Larauche, Yushan Wang, Po-Min Wang, James Dunn, Wentai Liu, Mulugeta Million
Group: SPARC
Summary: This protocol describes a process for the measurement of acute electrical stimulation-induced effects on colonic motility in anesthetized young adult Yucatan minipigs. Signals recorded from manometry probes inserted into the proximal, transverse and distal colonic regions were used to measure the effect of stimulation by serosally placed electrodes opposed to the colonic tissue at multiple sites in an acute anesthetized preparation. The effect of stimulation was quantified as motility index assessments before, during and after stimulation, and the data was used to create a functional map of colonic motor response to localized colon stimulation.

Proper citation: Muriel Larauche, Yushan Wang, Po-Min Wang, James Dunn, Wentai Liu, Mulugeta Million 2020. Tache_Mulugeta_OT2OD024899_Colon tissue electrical stimulation and colonic motility measurements. protocols.io dx.doi.org/10.17504/protocols.io.3rmgm46 Copy   

•   Source: Protocols.io

Authors: Janet Keast, Peregrine Osborne
Group: SPARC
Summary: This protocol is used to visualise sensory and autonomic neurons innervating the bladder body (dome), bladder trigone or proximal urethra in an experimental adult male or female rat. The protocol 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 2019. Use of tracer dyes to label neural projections to lower urinary tract organs [keast-001-stage01]. protocols.io dx.doi.org/10.17504/protocols.io.w3dfgi6 Copy   

•   Source: Protocols.io

Authors: Nathan Lee
Group: SPARC

Proper citation: Nathan Lee 2020. Abdominal Emitter Implantation. protocols.io dx.doi.org/10.17504/protocols.io.bpzbmp2n Copy   

•   Source: Protocols.io

Authors: Pradeep Rajendran, Guy Salama, Ching Zhu, Peter Hanna
Group: SPARC

Proper citation: Pradeep Rajendran, Guy Salama, Ching Zhu, Peter Hanna 2020. Dual Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart during Optogenetic Stimulation of the ICNS. protocols.io dx.doi.org/10.17504/protocols.io.bcdtis6n Copy   

•   Source: Protocols.io

Authors: James Fallon, Sophie Payne
Group: SPARC
Summary: Recording electrically-evoked compound action potentials (ECAPs) is performed to confirm that the electrode array is appropriately placed along the nerve. Recording ECAPS also allows confirmation that electrical stimulation is above neural threshold i.e. that electrical stimulation activates neural activity. The procedure is performed under anesthesia (immediately post-surgery) or in awake animals, and the procedure should incorporate all local requirements for standards of animal experimentation.

Proper citation: James Fallon, Sophie Payne 2020. Electrophysiological recording of electrically-evoked compound action potentials. protocols.io dx.doi.org/10.17504/protocols.io.bfwyjpfw Copy   

•   Source: Protocols.io

Authors: Zachariah Sperry, Kyounghwan Na, Mihaly Vöröslakos, Saman Parizi, James Jun, Tim M. Bruns, Euisik Yoon, John P. Seymour
Group: SPARC
Summary: Dorsal root ganglia (DRG) are components of spinal roots containing sensory cell bodies, and hold significant promise as neural recording sites for sensory neuroprostheses.This protocol details the fabrication and use of a high-density flexible polyimide electrode array into the sacral DRG of an anesthetized feline for neural recordings.

Proper citation: Zachariah Sperry, Kyounghwan Na, Mihaly Vöröslakos, Saman Parizi, James Jun, Tim M. Bruns, Euisik Yoon, John P. Seymour 2021. High-Density Penetrating Microelectrode Recordings from Anesthetized Feline Dorsal Root Ganglia. protocols.io dx.doi.org/10.17504/protocols.io.w5nfg5e Copy   

•   Source: Protocols.io

Authors: Nicole A. Pelot, J. Ashley Ezzell, Gabriel B. Goldhagen, Jake E. Cariello, 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, Jake E. Cariello, Kara A. Clissold, Warren M. Grill 2020. SPARC_Duke_PelotGrill_OT2-OD025340_HumanVagusNerve_Claudin1IHC_Morphology. protocols.io dx.doi.org/10.17504/protocols.io.bh4dj8s6 Copy   

•   Source: Protocols.io

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 2019. SPARC_Duke_Grill_OT2-OD025340_RatVagusNerveCollectionHistologyMicroscopy. protocols.io dx.doi.org/10.17504/protocols.io.ww3ffgn Copy   

•   Source: Protocols.io

Authors: Shaina Robbins, Alison Moss, Sean Nieves
Group: SPARC
Summary: This protocol utilizes Fluidigm's Biomark system which performs high-throughput real-time PCR that can assay 48 or 96 genes for 48 or 96 samples respectfully. This protocol is used for gene expression targeting samples at the single-cell scale and can be used with the 48.48 Dynamic Array integrated fluidic circuit (IFC) or the 96.96 Dynamic Array IFC. Note that this protocol assumes that single-cell samples are captured using the Arcturus Laser Capture Microdissection system.

Proper citation: Shaina Robbins, Alison Moss, Sean Nieves 2020. BioMark Single Cell Protocol (Two-Step RTSTA). protocols.io dx.doi.org/10.17504/protocols.io.wdsfa6e Copy   

•   Source: Protocols.io

Authors: Janet Keast, Peregrine Osborne
Group: SPARC
Summary: This protocol is used to visualise sensory and autonomic neurons innervating the bladder body (dome), bladder trigone or proximal urethra in an experimental adult male or female rat. The protocol 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 2020. Use of tracer dyes to label neural projections to lower urinary tract organs. protocols.io dx.doi.org/10.17504/protocols.io.w2xfgfn Copy   

•   Source: Protocols.io

Authors: Charles Horn, Derek M. Miller, Stephanie Fulton, Bill J. Yates, Lee E. Fisher, Ameya C. Nanivadekar
Group: SPARC
Summary: This protocol provides the steps for recording the gastrointestinal myoelectric responses and stimulation of the abdominal vagus nerve from the behaving ferret. These tests occur after an animal is surgically implanted with gastrointestinal and vagus nerve electrodes (see the protocol "SPARC - Chronic implantation of gastrointestinal and vagus nerve electrodes in the ferret" for implantation methods).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 - Gastrointestinal myoelectric recordings from the behaving ferret. protocols.io dx.doi.org/10.17504/protocols.io.6a8hahw Copy   

•   Source: Protocols.io