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Authors: Brett Hanzlicek, Ben Abelson, Margot Damaser, Dennis Bourbeau
Group: SPARC
Summary: This protocol for cat surgery to expose the badder for inactive device insertion.
Proper citation: Brett Hanzlicek, Ben Abelson, Margot Damaser, Dennis Bourbeau 2021. SPARC Cat acute UroMOCA implantation surgery. protocols.io dx.doi.org/10.17504/protocols.io.bf2pjqdn Copy
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 dx.doi.org/10.17504/protocols.io.bax7ifrn Copy
Authors: Martha Campbell Thompson
Group: Human Cell Atlas Method Development Community, Optical Clearing of Tissue, SPARC
Summary: DRAFTPurpose:The purpose of this protocol is to outline proper procedures for fixed specimen processing using an automatic paraffin processor and sectioning and staining for histopathological review of normalcy. Scope:This protocol applies to human pancreas specimens that will be processed to paraffin blocks for HuBMAP Tissue Mapping Centers.Expected Outcome:Pancreas samples are processed properly and efficiently to whole slide images for histopathology review. Nuclei should stain blue. Cytoplasm of acinar cells should stain pink with blue zymogen granules. The large blood vessels should demonstrate clear differentiation of RBCs when present, surrounding collagen, and endothelial nuclei with scant cytoplasm.
Proper citation: Martha Campbell Thompson 2021. Human Pancreas Histopathology Assessment. protocols.io dx.doi.org/10.17504/protocols.io.br7tm9nn Copy
Authors: Martha Campbell Thompson, Malavika Nair
Group: Optical Clearing of Tissue, SPARC
Summary: Optical clearing of tissues to improve deep tissue microscopy was described over a century ago and methods have improved considerably following publication of the CLARITY method from Karl Deisseroth in 2013. Improvements in organic and inorganic methods are constantly reported for multiple species and organs. Large volume imaging was needed for human pancreas studies due to known heterogeneity in normal islet sizes and proportions of endocrine cells. Lobularity in islet beta-cell loss and islet inflammation is also seen in patients with type 1 diabetes. The iDISCO protocol allows for rapid clearing, easy staining, and high resolution confocal imaging. Human pancreas samples were cleared using iDISCO as originally published. This protocol describes steps to clear 4mm x 6mm x 400um deep sections of 4% paraformaldehyde fixed human pancreas using iDISCO-like reagents purchased from Visikol.
Proper citation: Martha Campbell Thompson, Malavika Nair 2020. Human Pancreas Optical Clearing by iDISCO and VISIKOL . protocols.io dx.doi.org/10.17504/protocols.io.ba4qigvw Copy
Authors: Seoeun Lee, Lori Zeltser
Group: Optical Clearing of Tissue, SPARC
Summary: This protocol describes how to stain Brown Adipose Tissue using iDisco protocol and visualize the result via confocal microscope.
Proper citation: Seoeun Lee, Lori Zeltser 2019. iDisco immunolabeling in brown adipose tissue (BAT). protocols.io dx.doi.org/10.17504/protocols.io.wqmfdu6 Copy
Authors: Janet Keast, Peregrine Osborne
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 2019. Intracardiac perfusion with fixative for anatomical studies [keast-001-stage02]. protocols.io dx.doi.org/10.17504/protocols.io.w3ffgjn Copy
Authors: Lixin Wang, Pu-Qing Yuan, Honghui Liang, Yvette Tache
Group: SPARC
Summary: A table listed the information of antibodies used in the mouse colon. The protocol described the methods of colon tissues sampling, whole mount preparations, passive clarity technique, and respective fluorescent Immunohistochemistry for the antibody tests in different tissue preparations.
Proper citation: Lixin Wang, Pu-Qing Yuan, Honghui Liang, Yvette Tache 2021. Immunofluorescent methods for antibody test in mouse colon. protocols.io dx.doi.org/10.17504/protocols.io.bqi2muge Copy
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_Grill_OT2-OD025340_HumanVagusNerve_Claudin1IHC_Morphology. protocols.io dx.doi.org/10.17504/protocols.io.6fzhbp6 Copy
Authors: Lauren Segers, Kendall Morris, Donald Bolser
Group: SPARC
Proper citation: Lauren Segers, Kendall Morris, Donald Bolser 2020. Morris USF Lab protocol. protocols.io dx.doi.org/10.17504/protocols.io.bci8iuhw Copy
Authors: Kun-Han Lu, Zhongming Liu, Jaiyue Cao
Group: SPARC
Summary: Briefly, a gadolinium‐based contrast agent was mixed with the animal’s meal in order for chyme to appear “bright” in MRI scans, thereby delineating the gastric and intestinal volume. A multi‐slice MRI sequence was used to scan the GI volume with high spatial resolution, and a similar sequence with a smaller spatial coverage was used to scan antral contractions with high temporal resolution. Measurements of gastric functions and physiology included the overall change in GI volume, gastric emptying, forestomach volume, corpus volume, antral volume, antral contraction frequency, antral peristaltic wave velocity, antral contraction amplitude, pyloric opening size, intestinal filling and, indirectly, absorption.
Proper citation: Kun-Han Lu, Zhongming Liu, Jaiyue Cao 2019. Contrast-enhanced magnetic resonance imaging of gastric emptying and motility in rats. protocols.io dx.doi.org/10.17504/protocols.io.wvxfe7n Copy
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 dx.doi.org/10.17504/protocols.io.bpcamise Copy
Authors: Janet Keast, Peregrine Osborne, Nicole Wiedmann
Group: SPARC
Summary: This protocol is used for immunohistochemical visualisation of immediate early gene expression (c-Fos or Egr-1) in cryosections of rat lumbosacral spinal cord. Free-floating sections are processed in a double labelling protocol to distinguish immediate early gene expression in different neurochemical classes of spinal cord neurons:ChAT [choline acetyltransferase]: preganglionic neuronsTH [tyrosine hydroxyls]: dopaminergic neuronsPax2: inhibitory interneurons
Proper citation: Janet Keast, Peregrine Osborne, Nicole Wiedmann 2020. Immunohistochemical labelling of spinal cord neurons involved in bladder activity. protocols.io dx.doi.org/10.17504/protocols.io.bakkicuw Copy
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 dx.doi.org/10.17504/protocols.io.95ih84e Copy
Authors: Anna Rietsch, Brett Hanzlicek, Margot Damaser, Dennis Bourbeau
Group: SPARC
Summary: This is a procedure for an acute wired UroMOCA implant cat experiment. A cystotomy is performed and an active, wired (for power) UroMOCA is placed in the bladder. The bladder is sutured closed, and urodynamics is performed to collect wireless data from the UroMOCA. This protocol includes basic surgery, UroMOCA implantation, urodynamics and imaging.
Proper citation: Anna Rietsch, Brett Hanzlicek, Margot Damaser, Dennis Bourbeau 2021. SPARC Cat acute UroMOCA implantation. protocols.io dx.doi.org/10.17504/protocols.io.bf2kjqcw 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 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 dx.doi.org/10.17504/protocols.io.2kjgcun 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 serotonin 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 Serotonin 2A Receptor (5-HT2AR) Immunohistochemistry Protocol in Rat Tissues Labeled with Cholera Toxin B-fragment. protocols.io dx.doi.org/10.17504/protocols.io.2khgct6 Copy
Authors: Shaina Robbins, Alison Moss
Group: SPARC
Summary: This is the standard procedure we use when sacrificing our Sprague-Dawley rats.
Proper citation: Shaina Robbins, Alison Moss 2020. Sacrificing Rat. protocols.io dx.doi.org/10.17504/protocols.io.ww6ffhe Copy
Authors: Shaina Robbins
Group: SPARC
Proper citation: Shaina Robbins 2020. Embedding Rat Heart. protocols.io dx.doi.org/10.17504/protocols.io.w52fg8e Copy
Authors: Janet Keast, Peregrine Osborne
Group: SPARC
Summary: This protocol describes immunohistochemical procedures applied to thick (50 µm) cryosections mounted directly on slides. It is used when the structures to be analysed are too large to remain intact within thin (10-20 µm) cryosections. Antibodies have been selected to distinguish different neurochemical classes of autonomic ganglion neurons and synaptic boutons associated with these neurons. The protocol can also be used to characterize neurons containing retrograde tracer.
Proper citation: Janet Keast, Peregrine Osborne 2020. Immunohistochemical labeling of thick cryosections from pelvic ganglia. protocols.io dx.doi.org/10.17504/protocols.io.bakcicsw 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 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 dx.doi.org/10.17504/protocols.io.2kggctw Copy
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