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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 https://dx.doi.org/10.17504/protocols.io.bakmicu6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.2iqgcdw Copy   

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Authors: Gemma Mazzuoli-Weber, Kristin Elfers, Anna Katharina Filzmayer
Group: SPARC
Summary: This protocol is to investigate the sensitivity of enteric neurons to mechanical stimulation in the inner submucosal plexus of the porcine colon using ultrafast neuroimaging and immunhistochemical techniques. Ultrafast neuroimaging technique combined with a voltage sensitive dye was used to characterize neuronal responses to mechanical stimuli compression and tension. Immunohistochemistry was subsequently used to determine the neurochemical coding of mechanosensitive submucosal neurons.

Proper citation: Gemma Mazzuoli-Weber, Kristin Elfers, Anna Katharina Filzmayer 2021. Mechanosensitive enteric neurons: incidence and abundance in the porcine submucosal plexus with ultrafast neuroimaging and immunhistochemical techniques. protocols.io https://dx.doi.org/10.17504/protocols.io.btv3nn8n Copy   

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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 https://dx.doi.org/10.17504/protocols.io.w3gfgjw Copy   

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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 serotonin 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 Serotonin (5-HT) Immunohistochemistry Protocol in Rat Tissues Labeled with Cholera Toxin B-fragment. protocols.io https://dx.doi.org/10.17504/protocols.io.2kggctw Copy   

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Authors: Nicole A. Pelot, J. Ashley Ezzell, Kara A. Clissold, Warren M. Grill
Group: SPARC
Summary: Protocol for collection, histological processing, and imaging of human vagus nerves.

Proper citation: Nicole A. Pelot, J. Ashley Ezzell, Kara A. Clissold, Warren M. Grill 2020. SPARC_Duke_PelotGrill_OT2-OD025340_HumanVagusNerve_Collection_Histology_Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.bh4cj8sw Copy   

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Authors: Shaina Robbins, Sean Nieves
Group: SPARC

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

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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 https://dx.doi.org/10.17504/protocols.io.7ayhifw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bgrmjv46 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.6a8hahw Copy   

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Authors: Shaina Robbins, Sean Nieves
Group: SPARC

Proper citation: Shaina Robbins, Sean Nieves 2020. 0.1% Cresyl Violet Stain (pH 4.3). protocols.io https://dx.doi.org/10.17504/protocols.io.wcjfaun Copy   

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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 https://dx.doi.org/10.17504/protocols.io.6a7hahn Copy   

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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 serotonin 7 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 Serotonin 7 Receptor (5-HT7) Immunohistochemistry Protocol in Rat Tissues Labeled with Cholera Toxin B-fragment. protocols.io https://dx.doi.org/10.17504/protocols.io.2kjgcun Copy   

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Authors: Gemma Mazzuoli-Weber, Kristin Elfers, Anna Katharina Filzmayer
Group: SPARC
Summary: This protocol is to investigate the sensitivity of enteric neurons to mechanical stimulation in the inner submucosal plexus of the porcine colon using ultrafast neuroimaging and immunhistochemical techniques. Ultrafast neuroimaging technique combined with a voltage sensitive dye was used to characterize neuronal responses to mechanical stimuli compression and tension. Immunohistochemistry was subsequently used to determine the neurochemical coding of mechanosensitive submucosal neurons.

Proper citation: Gemma Mazzuoli-Weber, Kristin Elfers, Anna Katharina Filzmayer 2020. Mechanosensitive enteric neurons: incidence and abundance in the porcine submucosal plexus with ultrafast neuroimaging and immunhistochemical techniques. protocols.io https://dx.doi.org/10.17504/protocols.io.bpcamise Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bgm5ju86 Copy   

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Authors: Deborah Jaffey, Terry Powley, Logan Chesney
Group: SPARC
Summary: This protocol describes how to prepare a rat stomach for micro-CT imaging to enable a 3D description of the organ. Iodine is used to stain the tissue post-perfusion. By varying the meal size and the delay time between feeding and perfusion, it is possible to determine how stomach volume changes, both overall and for stomach compartments. The DICOM file output can be used to generate a 3D scaffold of the stomach for modeling purposes.

Proper citation: Deborah Jaffey, Terry Powley, Logan Chesney 2020. Micro-CT imaging of iodine-stained rat stomach. protocols.io https://dx.doi.org/10.17504/protocols.io.95ih84e Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bfxvjpn6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.6bvhan6 Copy   

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Authors: Matthew Ward, Bartek Rajwa, John M Wo, Anita Gupta, John Furness, Terry Powley, Thomas V Nowak
Group: SPARC
Summary: Protocol Title: A simple, non-invasive approach to detect vagal nerve response patterns that predict a positive treatment response to gastric electrical stimulation therapy for gastroparesis [1]Background: Gastric electrical stimulation (GES) can be a life-changing, device-based treatment option for drug-resistant nausea and vomiting associated with diabetic or idiopathic gastroparesis (GP).Despite over two decades of clinical use, the mechanism of action remains unclear.We hypothesize a vagal mechanism, whereby GES activates vagal sensory afferents that project to the nucleus of the solitary tract and onward to influence brain structures that mediate the biological processes leading to nausea and vomiting (e.g., area postrema). Here, we describe a noninvasive method to investigate vagal nerve involvement in GES therapy in human subjects.We have developed transcutaneous recording of cervical vagal activity that is synchronized with GES in conscious subjects, along with methods of discriminating the activity of different nerve fiber groups that are activated.We are refining the system of recording and analysis so that it can be applied in the clinic.New Method: Sixty-six adults receiving GES therapy (Medtronic Enterra I/II) to treat refractory nausea and vomiting secondary to diabetic, idiopathic, or postoperative GP were enrolled in the study (IRB #:1206008988). Subjects completed a GCSI symptom survey upon enrollment. The left and right cutaneous vagal electroneurograms (vENG) were collected from the skin surface over the left and right cervical vagal nerves, respectively, without changing the prescribed GES parameters. The mean response to GES was computed for each subject and classified according to the Letter System for nerve fiber classification. Symptom scores were compared between groups of subjects with or without significant Ab, Ag, Ad, or B fiber responses, defined as volleys in the mean response to GES whose peak amplitude is significantly different from 0 V ata=0.05. Nerve responses were correlated with GP symptoms.Results: Of the 66 subjects, 28 had diabetic GP (type 1:9; type 2:19), 35 had idiopathic GP, and 3 had postsurgical GP. Stimulus pulse current and charge did not predict treatment efficacy, but did predict a significant increase in total symptom score in type 1 diabetics as GES stimulus charge per pulse increased (p p Conclusions: Cutaneous vagal CNAP analysis is a useful technique to unmask relationships among GES parameters, vagal recruitment, efficacy and side-effect management. Our results suggest that CNAP-guided GES optimization will provide the most benefit to patients with idiopathic and type 1 diabetic GP, especially when tuned for left vagal Ag and right vagal Ad/B fiber responses. The possible side effects associated with left vagal Abactivation in type 1 diabetics underscore the need to consider disease etiology and fiber recruitment profiles in the patient and parameter selection process.Funding AcknowledgmentThis work is supported, in part, by NIH SPARC OT2OD028183 and NIH SPARC OT2OD023847.Publication/Abstract Reference:[1] Ward M.P., Rajwa B., Wo J.M., Gupta A., Furness J.B., Powley T.L., and T.V. Nowak. An emerging method to noninvasively measure and identify vagal response markers to enable bioelectronic control of gastroparesis symptoms with gastric electrical stimulation. Journal of Neuroscience Methods 336, pp. 1-13, Feb 2020. PMID:32087238DOI:10.1016/j.jneumeth.2020.108631

Proper citation: Matthew Ward, Bartek Rajwa, John M Wo, Anita Gupta, John Furness, Terry Powley, Thomas V Nowak 2020. A simple, non-invasive approach to detect vagal nerve response patterns that predict a positive treatment response to gastric electrical stimulation therapy for gastroparesis. protocols.io https://dx.doi.org/10.17504/protocols.io.bax7ifrn Copy   

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Authors: Dennis Bourbeau, Brett Hanzlicek, Margot Damaser
Group: SPARC
Summary: In this study, we will be using Yorkshire Pigs, 50-70kg. The purpose of this study is to develop a tool to measure bowel fullness and activity. This system will eventually be able to provide continuous, wireless, battery−powered data and ultimately be translated for use in research and clinicalapplications of bowel function. We will measure porcine bowel compliance, pressures, and dimensions in acute live animal studies.

Proper citation: Dennis Bourbeau, Brett Hanzlicek, Margot Damaser 2021. SPARC Pig1 acute wired ColoMOCA implantation. protocols.io https://dx.doi.org/10.17504/protocols.io.bfxbjpin Copy   

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