Download The effect of spinal cord injury on the neurochemical properties of

Articles in PresS. Am J Physiol Regul Integr Comp Physiol (April 8, 2015). doi:10.1152/ajpregu.00445.2014
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The effect of spinal cord injury on the neurochemical properties of
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vagal sensory neurons
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April N. Herritya,b, Jeffrey C. Petruskaa,b,c, David P. Stirlingb,c,d, Kristofer K. Raua,b,e and
Charles H. Hubschera,b.
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Department of Anatomical Sciences & Neurobiology, University of Louisville School of
Medicine, Louisville, KY 40202
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Kentucky Spinal Cord Injury Research Center, University of Louisville, Louisville, KY
40202
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Department of Neurological Surgery, University of Louisville, Louisville, KY 40202
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Department of Microbiology & Immunology, University of Louisville School of Medicine,
Louisville, KY 40202
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Department of Anesthesiology, University of Louisville, Louisville, KY 40202
Running head: SCI-induced vagal sensory plasticity
Number of pages: 39
Number of figures: 8
Number of Tables: 1
PROOFS AND CORRESPONDENCE TO:
Dr. Charles H. Hubscher
Dept. of Anatomical Sciences & Neurobiology
University of Louisville
511 S. Floyd Street
Louisville, KY 40202
Telephone: (502)852-3058
FAX: (502)852-6228
E-Mail: [email protected]
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Copyright © 2015 by the American Physiological Society.
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Abstract
The vagus nerve is comprised primarily of non-myelinated sensory neurons
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whose cell bodies are located in the nodose ganglion (NG). The vagus has widespread
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projections that supply most visceral organs, including the bladder. Due to its non-spinal
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route, the vagus nerve itself is not directly damaged from spinal cord injury (SCI).
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Because most viscera, including bladder, are dually innervated by spinal and vagal
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sensory neurons, an impact of SCI on the sensory component of vagal circuitry may
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contribute to post-SCI visceral pathologies. To determine if SCI, in male Wistar rats,
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might impact neurochemical characteristics of NG neurons, immunohistochemical
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assessments were performed for P2X3 receptor expression, IB4 binding, and
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Substance-P expression, three known injury-responsive markers in sensory neuronal
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subpopulations. In addition to examining the overall population of NG neurons, those
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innervating the urinary bladder also were assessed separately. All three of the
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molecular markers were represented in the NG from non-injured animals, with the
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majority of the neurons binding IB4. In the chronically injured rats, there was a
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significant increase in the number of NG neurons expressing P2X3 and a significant
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decrease in the number binding IB4 compared to non-injured animals, a finding that
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held true also for the bladder-innervating population. Overall, these results indicate that
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vagal afferents, including those innervating the bladder, display neurochemical plasticity
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post-SCI that may have implications for visceral homeostatic mechanisms and
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nociceptive signaling.
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Keywords: Vagus nerve; Nodose ganglion; Spinal Cord Injury; Bladder;
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Immunohistochemical phenotype
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Introduction
Spinal cord injury (SCI) results in deficits to sensorimotor systems and profoundly
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affects the functionality of the autonomic nervous system. Basic research focusing on
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improving pelvic-visceral outcomes following SCI is of great clinical importance, since
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complications such as bladder, bowel and sexual dysfunction affect health and quality of
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life for this population (4, 47, 67). Despite the direct immediate impact of injury to the
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spinal-derived autonomic supply of the pelvic viscera, most of the body’s visceral
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organs also are supplied by a non-spinal source through the vagus nerve. Since the
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vagus nerve does not travel directly through the spinal cord, its neurocircuitry is often
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considered intact following SCI. Nevertheless, there is some degree of indirect
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involvement of both vagal afferents and efferents. For example, following SCI,
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subsequent neuroplastic-responsive changes have been extensively described within
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the dorsal vagal complex controlling gastric function (79). Gastrointestinal (GI)
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alterations after human upper-thoracic SCI include conditions such as dysphagia (160),
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esophagitis (133), peptic ulcerations (62, 138), gastroparesis and overall dysmotility (87,
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123, 125, 159). Although the mechanisms of GI dysfunction in humans after SCI are not
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thoroughly understood, experimental studies in rats suggest that many of the delays in
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gastric emptying and transit may in part be attributed to vagally-mediated pathways (60,
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61, 79, 140). In fact, subdiaphragmatic vagotomy has been shown to prevent much of
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the SCI-induced GI sequelae (61).
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The vagus nerve, with sensory cell bodies primarily located in the nodose ganglia
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(NG), provides innervation to the thoraco-abdominal structures. Despite the view that
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the vagus nerve does not innervate viscera caudal to the transverse colon, numerous
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experimental studies have demonstrated that it also provides sensory innervation to the
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majority of the pelvic viscera (2, 28, 38, 57, 77, 81, 84, 111, 149). Even though the
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vagus nerve has such widespread projections, SCI does not disconnect the anatomical
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relationship the nerve has with the tissue it innervates. However, SCI does lead to
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pathological changes and dysfunction of below-level target organs, such as the bladder
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(45, 46, 94), and can thereby influence neuronal phenotype (122, 145, 158, 167, 176).
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Furthermore, various classes of primary sensory neurons, including vagal afferents,
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have been shown to alter their phenotype and the expression of different receptors in
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response to nerve injury and tissue inflammation (11, 78, 103, 107, 155, 173).
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In this experiment, P2X3 receptor and SP immunoreactivity (ir) as well as IB4
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binding was examined in NG neurons. These particular markers were selected based
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on their presence in the NG, involvement in the spinal and vagal circuitry,
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responsiveness in other sensory neurons to injury and/or inflammation, and the
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potential physiological role these markers may play in nociceptive signaling (11, 25, 30,
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40, 42, 43, 83, 97, 103, 109, 146, 154, 163, 169, 172). In addition, anatomical evidence
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that the vagus nerve provides sensory innervation to the bladder in male rats (77) and
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the presence of these cellular markers in bladder tissue (6, 19, 100) adds to the
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importance of understanding the relationship between target-organ tissue and its
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innervating neurons. It is therefore hypothesized that following a spinal transection
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injury which removes any potential sources of spinal input rostrally and isolates vagal
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afferent fibers, the expression profile of known injury-responsive factors P2X3, IB4 and
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SP would be altered in the NG in general and also for the subset of NG neurons
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innervating the bladder.
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Materials and Methods
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Animals
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All experimental procedures were carried out according to NIH guidelines and
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protocols reviewed and approved by the Institutional Animal Care and Use Committee
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at the University Of Louisville School Of Medicine. All adult male Wistar rats (n=16,
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Harlan Sprague Dawley, Inc, Indianapolis, IN), approximately 250 grams in weight, were
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individually housed in an animal room with a 12-hour light and dark cycle. They had ad
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libitum access to water and food (Laboratory Rodent Diet). Groups were either naïve
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(n=8) or spinal cord injured (n=8). Each group had a subset (n=4 each) which received
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retrograde neural tracer injected into the bladder to enable identification of single NG
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neurons which innervated the bladder.
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Spinal cord injury
Half of the animals (n=8) were anesthetized with a mixture of ketamine (80mg/kg)
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and xylazine (10mg/kg), injected intraperitoneally, for spinal transection. All surgeries
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were performed under aseptic conditions and the body temperature was maintained
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within the range of 36-37ºC via a warm water recirculator (Gaymar T/Pump, Gaymar
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Industries Inc, Orchard Park, NY) throughout the surgery and recovery period. Following
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our previously published protocol (86), a dorsal longitudinal incision was made to
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expose the T7 vertebra and a laminectomy was performed in order to expose the
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underlying T8 spinal cord. The overlying dura was reflected laterally and the spinal cord
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cut using a pair of surgical microdissecting scissors. Gentle suction with an air vacuum
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was used to carefully elevate the cut stump in order to verify the completion of the
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lesion. Gelfoam (Pharmacia & Upjohn Company, Kalamazoo, MI) soaked in topical
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hemostat solution (Henry Schein Inc., Melville, NY) was placed in the lesion cavity. The
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incision was closed using 4-0 nylon suture for the muscle layers and fascia and surgical
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clips for the skin. Animals were given subcutaneous injections of ketoprofen (Ketofen,
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2.5mg/kg, Fort Dodge Animal Health, Fort Dodge, IA) for analgesia twice a day for 2
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days, 0.5ml of dual penicillin (Penicillin G coupled with Procaine, PenJect ®, The Butler
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Company, Columbus, OH) in a single dose peri-operatively as a general prophylactic
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and 5mg/kg gentamicin (GentaFuse®, Butler Schein, Dublin, OH) once per day for 5
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days to prevent bladder infections. After surgery each animal was housed individually.
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The urinary bladder was emptied by manual crede every 8 hours until the micturition
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reflex occurred automatically, typically 6–12 days after surgery (82). Animals survived
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for six weeks followed by euthanasia and tissue removal.
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Retrograde tracer injection
At five weeks post injury, four spinally-transected rats and four age-matched
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naïve control rats were anesthetized with a mixture of ketamine (80mg/kg) and xylazine
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(10mg/kg). They received a ventral/caudal midline peritoneal incision to expose the
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urinary bladder which was subsequently manually voided by pressure. Using an
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established protocol (77, 124), the fluorescent tracer FAST DiI™ oil (1,1’-dilinoleyl-
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3,3,3’,3’,-tetramethylindo-carbocyanine perchlorate, 5mg dye dissolved in 0.1ml
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methanol, Molecular Probes, Inc., Eugene, OR) was injected into the bladder wall with a
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dye-dedicated 33-gauge needle coupled to a Hamilton microsyringe (Fisher Scientific,
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Pittsburgh, PA). Note that the abbreviation DiI is used throughout the
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text/figures/legends to refer to FAST DiI™ oil. Dye injections were made to the
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circumference of the trigone, body and dome areas (10μl volume per animal divided into
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10 injections of 1ul each; (77)).
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Animal body temperature was maintained within the range of 36-37°C during
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surgery via a warm water recirculator (Gaymar T/Pump, Gaymar Industries Inc, Orchard
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Park, NY). After each injection, the needle was removed slowly and any dye-leakage
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was removed by cotton-tipped applicators. After injections were completed, the exposed
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viscera were hydrated as necessary with 5% Dextrose Lactated Ringers, the abdominal
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musculature was sutured closed (Ethicon 4-0 non-absorbable surgical suture), the skin
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closed with Michel clips (Fine Science Tools, Foster City, CA), and a topical antibiotic
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(Bacitracin, Actavis Mid Atlantic LLC, Lincolnton, NC) applied. Following surgery,
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animals were placed on a heating pad and core temperature monitored. Post-operative
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medication was provided as per spinal transection surgery. All animals were monitored
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daily to inspect the surgical incision and identify any changes in an animal’s general
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condition.
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Perfusion and Tissue Collection
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All 16 animals were deeply anesthetized with a ketamine (80mg/kg body
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weight)/xylazine (10mg/kg) mixture and transcardially exsanguinated with heparinized
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saline, followed by 4% paraformaldehyde perfusion. Each vagus nerve was identified
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adjacent to the carotid artery and gently separated from surrounding tissues and traced
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rostrally to the NG, which was excised. The bladder was dissected free from the
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prostate and extracted at the most distal aspect of the neck. Before weighing the
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bladder, any residual intravesicular urine was allowed to flow out. Superior cervical
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ganglia were identified on both sides at the bifurcation of the common carotid artery and
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removed to be used as control tissue. For the transected group of rats, following a
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dorsal spinal incision, removal of the spinal cord tissue 1cm above and below the
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transection site was performed. All tissues were placed in individually-labeled tubes of
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4% paraformaldehyde for at least 48h, followed by immersion in a cryoprotectant
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solution of 30% sucrose/phosphate buffer solution with 1% sodium azide for at least
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24h. Following removal from the cryoprotectant solution, NG’s were embedded in OCT®
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compound (Baxter Scientific) and 12-14μm sections were cut on a cryostat (Leica CM
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1850). During retrieval of the bladder, the abdominal cavity and surrounding viscera
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were inspected for tracer leakage.
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Histology of the lesion epicenter
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The lesion cavity, coated in embedding media, was cut into 18µm sagittal
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sections using a Leica CM 1850 cryostat and mounted onto gelatin-coated histological
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slides (Azer Scientific, Morgantown, PA). The slides were then stained with both Luxol
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fast blue and cresyl violet (Kluver-Barrera method) to observe myelin and Nissl
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substance, respectively. Spot Advanced software (Diagnostic Instruments, Sterling
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Heights, MI) and the Nikon E400 microscope were used to image the lesion cavity and
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verify the completeness of the spinal transection (86). The percentage of spared white
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matter from the transection lesion was calculated using Nikon Elements software. The
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boundary between spared tissue at the ventral portion of the cord and the lesion cavity
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was identified. The first anatomical region of interest (ROI) outlined was the portion of
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spared tissue at the ventral aspect of the spinal cord and the second ROI outlined was
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the entire lesion cavity, encompassing both spared and non-spared tissue and
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extending from both the rostral and caudal cord stumps. Area was determined for each
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ROI and the percentage of spared tissue was calculated by dividing these areas (95).
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Hindlimb assessment for lesion completeness
The Basso-Beattie-Bresnahan (BBB) scale (14), an open-field locomotor
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assessment, was used to evaluate hindlimb function as an assessment of post-injury
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spinal cord function. Each animal was placed in an open-field and tested for 4 minutes
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by the same two scorers, who were presented with injured and non-injured animals in
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random order. The 21-point BBB scale was used to assess hindlimb coordination and
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rated parameters such as individual joint movements (0-7), weight support (8-13) and
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paw placement (14-21). Intact animals should demonstrate a locomotor score of 21.
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Animals that receive a completeT8 transection have been shown to exhibit BBB scores
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of 3 (extensive movement of 2 joints) on average (13, 130). To prevent any functional
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connections across the lesion site from potential spared tissue, gelfoam was placed
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between the two cut stumps. It has been demonstrated that as little as 4-5% sparing
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(primarily in the ventrolateral funiculi) was sufficient for attaining a BBB score of 7
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following “complete” spinal transection (no gelfoam used across lesion) (53).
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Immunofluorescence histochemistry
Sections were thaw-mounted onto slides and allowed to air-dry. They were then
encircled with hydrophobic resin (PAP Pen, Research Products International Corp).
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Slide-mounted sections were incubated at room temperature for 2h in a solution of 2%
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Triton X-100® in phosphate buffered saline (PBS). This pre-treatment step improves the
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quality of P2X3-ir (116). The slides were rinsed in distilled water and then incubated for
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30min in a solution of 10% normal donkey serum (Jackson Immuno Research, West
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Grove, PA) in PBS with 0.3% Triton X-100 (MP Biomedicals, LLC, Solon, OH) to block
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non-specific antibody binding. The immunohistochemical reagents and the labeling
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procedures are summarized in Table 1. Incubations in primary antisera were performed
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overnight (14–18 hours) at 4ºC. All steps were followed by multiple rinses with PBS. All
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fluorescent secondary antisera were diluted 1:100 and incubations were 2 hours at
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room temperature. The tyramide signal-amplification (TSA™) reagent kit (Sigma-
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Aldrich, St. Louis, MO) (20-22) was used at 1:100 and incubations were for 4-5min
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(124). Once all steps were completed, the slides were coverslipped with a glycerol-
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based photobleach-protective medium (Fluoromount-G, Southern Biotech).
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Cell Quantification
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To view labeled sections, imaging was performed using the Nikon Eclipse TiE
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inverted microscope with NIS Elements software. Initially, images were captured using a
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10x lens (APO DIC N1 10x/0.45 NA, Nikon) with consistent exposure times and
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computationally stitched together to visualize whole ganglion sections. Imaging of
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individual fluorophores was achieved with a mercury-arc lightsource and the following
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filter sets: for Cy3 [543/22nm excitation, 593/40nm emission, 562nm dichroic, Semrock];
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for Alexa Fluor 488 [470/40nm excitation, 525/50nm emission, 495nm dichroic, Nikon];
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for Alexa Fluor 350 [350/50nm excitation, 460/50nm emission, 400nm dichroic, Nikon];
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for Cy5 [615/70nm excitation, 700/74nm emission, 660nm dichroic, Nikon].
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In order to obtain unbiased percentages of P2X3, SP and IB4 positive neurons in
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the NG, the physical dissector method was applied (37, 113). Across the entire
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ganglion, assembled by automated stitching, counts of all singly-, multi- as well as non-
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labeled/other (collectively comprising total neuronal counts) NG neurons were made by
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a scorer blinded to treatment groups. Starting with a random section, neurons with a
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clearly visible nucleus and definable soma were counted only if they were not present in
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an adjacent “look up” serial section. As an added measure to avoid double counting
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single neurons, the counting-sections were at least 60 microns apart (every 5th section).
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To differentiate background from foreground pixels, threshold values also were obtained
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based off the image histogram for each marker and held constant for each image
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quantified. Note that non-labeled/other cells (NeuN+ only) were quantified and
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represented the total neuron population (59, 75, 105). Images of tissue without
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application of the primary antibody were taken and utilized as baseline controls (images
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not shown). Using Nikon Elements software, cell diameter between groups also was
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determined by estimating the average of two cross-sectional diameters (longest and
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shortest axis).
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Positive neuron counts were expressed as a percentage of the total number of
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neurons (NeuN+ only) from within the entire stitched ganglion as well as a percentage
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of all labeled neurons. An Olympus 3 Laser scanning confocal microscope with
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Fluoview 500 software (Mellville, NY) and a Nikon A1R MP+ confocal microscope with
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Elements software were used to collect high resolution, serial optical sections of NG
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neurons.
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Statistics
Analyses were performed using SPSS v19-20 (IBM, North Castle, NY). Levene’s
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statistic was applied for homogeneity of variances and data are expressed as mean ±
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standard deviation (SD). A one-way analysis of variance (ANOVA) with Tukey HSD post
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hoc t-tests was performed for the assessment of all histochemical markers and NG
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bladder labeling within groups. For the analysis of the histochemical markers between
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groups, data were normalized as percentages of total NG cells and analyzed via a two-
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way ANOVA with Tukey HSD post hoc t-tests. For all other analyses, two-tailed
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Student's t-tests were performed assuming equal variance. Statistical significance was
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defined as p≤.05.
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Results
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Immunohistochemical signature of NG neurons
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IB4 binding in NG neurons was localized to the plasma and axonal membranes
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as well as the Golgi complex. Immunoreactivity (-ir) for P2X3 and SP was present in the
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cytoplasm and P2X3-ir also could be found in the plasma and axonal membranes. All of
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the patterns of staining for these markers were consistent with previous studies in both
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NG and DRG (8, 10, 83, 116, 151). When examining the total percentage of labeled NG
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neurons, all of the markers were well represented (Figure 1A), with the majority of NG
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neurons binding IB4 (IB4, 65.5 ± 6.2% versus SP, 31.1 ± 10.3%; IB4 versus P2X3, 51.2
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± 7.9%). Overall, there were 8 different histochemical signatures represented in the NG
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(Figure 1B). The most prevalent combinations were neurons that were IB4+ only
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followed by the P2X3+ only and IB4+/P2X3+/SP- combinations (IB4+ only versus
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P2X3+ only, 33.3 ± 7.1% versus 15.0 ± 3.3%, p<.001; IB4+ only versus IB4+/P2X3+
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only, 33.3 ± 7.1% versus 15.0 ± 5.0%, p<.001). When considering specific co-
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localization patterns in the NG, about a third of all IB4 neurons contain P2X3 (30.9 ±
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9.2%) and about half of all P2X3 neurons contain IB4 (49.1 ± 8.6%). A typical example
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of the quadruple immunohistochemical staining in the NG is provided in Figure 2.
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Effect of SCI on NG neurons expressing P2X3, SP and binding IB4
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Following a chronic transection injury at T8, BBB locomotor assessments at the 6
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week time point for all transected animals revealed an average score of 0.9 (± 0.9).
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Only one animal had a BBB score greater than zero (score of 4.5: 6 on the left hindlimb
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and 3 on the right hindlimb). Post hoc histological assessments of the lesion site
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revealed that this same animal in the spinal cord-transected group had a small
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percentage of area spared within the lesion site (16.1% - located at the most ventral
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extent of the epicenter). The data from this incomplete transection rat is provided, but as
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a separate group with an n of 1. Although statistical analyses could not be performed
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with n=1, the immunohistochemical expression pattern in this SCI rat appeared to more
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closely resemble that of the naïve group (P2X3+ only, 11.5 ± 5.2%; IB4+ only, 44.0 ±
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2.2%). An example indicating a typical complete lesion is provided in Figure 3. Note that
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it was also found that the average cell diameter for NG neurons was 29.6 ± 5.7 µm. This
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morphological feature was not affected by spinal transection injury (28.5 ± 6.04 µm).
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In rats with a complete transection (n=3; 6 ganglia), there was a significant
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increase in the percentage of total NG neurons expressing P2X3 (p<.001) as well as a
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significant decrease in the percentage of total NG neurons binding IB4 (p<.05) relative
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to non-injured controls (Figure 4). There were no significant differences in SP or NeuN
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expression between groups. P2X3 expression and IB4 binding in the NG are
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demonstrated in Figure 5. Within the spinal cord-transected group, the percentage of
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neurons expressing P2X3 and the percentage of neurons binding IB4 were each
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significantly greater than the percentage of neurons expressing SP, which was
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unchanged from the non-injured group of animals (P2X3, 27.3 ± 4.8% versus SP, 6.5 ±
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2.8%, p<.01; IB4, 23.7 ± 6.5% versus SP, 6.5 ± 2.8%, p<.01; SP, 6.5 ± 2.8% versus SP-
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non-injured, 5.8 ± 3.8%). From the total population of NG neurons, SCI did not
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significantly impact the subset of NG neurons that co-expressed IB4 and P2X3 (Naïve,
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15.0 ± 5.0 versus TX, 13.5 ± 5.7, p>.05), nor did it impact the subset of all IB4 neurons
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that contained P2X3 (Naïve, 30.9 ± 9.2% versus TX, 35.9 ± 11.2%, p>.05). However,
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following SCI, the subset of all P2X3 neurons that contained IB4 was significantly lower
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compared to non-injured animals (Naïve, 49.1 ± 8.6% versus TX, 32.2 ± 10.5%, p<.01).
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With respect to the total number of labeled neurons within their individual
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populations (i.e. all P2X3+ neurons and all IB4+ neurons), the distribution of the
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P2X3+/IB4-/SP-only subset represents 50.7% of all P2X3+ neurons, while the
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IB4+/P2X3-/SP-only subset represents 42.6% of all IB4+ neurons. Overall, these
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populations of neurons comprised about half of the total population of NG neurons
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examined.
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Immunohistochemical profile of bladder non-injured and injured NG neurons
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The retrograde tracer DiI, was injected into the bladder wall in order to determine
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if the subsets of NG neurons affected by spinal cord transection include those that
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supply the bladder. Initially, we found that the percentage of NG neurons traced from
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the bladder in both groups in this study is similar to our previous study of spinally intact
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animals (22.2 ± 3.6% versus 21.4 ± 4.0%, (77)). Assessment of the superior cervical
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ganglion, which is located adjacent to the NG, did not reveal evidence of DiI tracer,
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indicating the tracer did not spread non-specifically (Figure 6).
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With respect to the total population of P2X3+ NG neurons after chronic SCI (50.7
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± 8.2%), bladder-innervating neurons (DiI+/P2X3+/IB4-) represented 32.8 ± 1.1%, while
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with respect to the total population of neurons that were IB4+ after injury (42.6 ± 5.1%),
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bladder-innervating neurons (DiI+/P2X3-/IB4+) represented 21.5 ± 7.4%. Overall, in
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these two distinct subsets of NG neurons, more than half of the neurons are traced from
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the bladder (Figure 7). Images of the DiI+/P2X3+ and DiI+/IB4+ subsets following
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transection are demonstrated in Figure 8. Note that in this study, the proportion of NG
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neurons traced from the bladder in spinally-intact rats (23.7 ± 3.6%) did not differ
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significantly from the proportion traced from the bladder after chronic spinal transection
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injury (20.2 ± 3.0%). Transection injury did however, result in a significant increase in
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bladder size (wet weight) compared to non-injured controls (.267 ± .118g, SCI vs .149 ±
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.35g, Naïve; p<.05), which can impact overall total bladder capacity and voiding
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efficiency, potentially leading to an alteration in bladder function.
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Discussion
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innervation from spinal and non-spinal (i.e., the vagus nerve) sources (28, 38, 57, 77,
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81, 84, 111). This study, using immunohistochemical techniques, examined the impact
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of SCI on the vagal component of that sensory innervation by assessing changes in the
Visceral organs including those in the pelvic region have a dual sensory
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presence of P2X3, IB4 and SP in NG neurons. Following spinal transection at T8,
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potential plasticity was evaluated in subsets of NG neurons which contain projections
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that bypass the spinal cord from visceral organs, including those projections that
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specifically supply the bladder. A major traumatic event to the nervous system, such as
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SCI, leads to dysfunction in multiple organ-systems, and ultimately influences the
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neurons that innervate these tissues. The findings of the current study indicate that
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vagal sensory cell bodies displayed an increase in P2X3 expression and a decrease in
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IB4 binding, which also held true for many neurons innervating the bladder. These
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results suggest that NG neurons, including the bladder subset, are sensitive to a spinal
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injury and are capable of responding by modifying their phenotype.
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Immunohistochemical phenotype of NG neurons
Overall, from the cellular markers examined in this study, the majority of NG
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neurons were IB4+. Isolectin B4 has been shown to label primarily a subpopulation of
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non-peptidergic spinal sensory afferents that are thought to be functionally distinct from
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peptidergic neurons or neurons that are IB4 negative (135). Even though IB4+ neurons,
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in general, are widely expressed within the NG (83, 98, 129, 169, 172), the meaning of
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IB4 binding in vagal afferents and whether or not these neurons share common
386
characteristics based on their IB4 binding is still unclear. Vagal afferents are largely
387
known for their involvement in conveying information about the physiologic state of the
388
viscera to the brain as part of homeostatic regulation (32, 66, 85). In the gastrointestinal
389
tract, vagal afferent fibers are responsive to stretch and tension as well as to locally
390
released hormones following the ingestion of food (16, 18, 119, 156). Although they are
391
typically not responders to visceral stimuli within the noxious range (112), previous data
392
from our lab and that of others suggest otherwise. For example, while spinal afferents
393
may be responsible for relaying mechanical nociceptive information, vagal afferents
394
may play more of a role in conveying chemical nociceptive stimuli, thus contributing to
395
disease-related conditions stemming from visceral hyperalgesia (44, 80, 86, 136).
396
Therefore, besides the known role of vagal afferents in relaying homeostatic information
397
to the brain, the population of vagal afferents that also are IB4+ may be involved in
398
visceral nociceptive processing (based on the putative role of the majority of DRG IB4
399
binding neurons (117, 151)).
400
In the current study, it was shown that many vagal visceral afferents innervating
401
the bladder also were IB4+. In other NG visceral afferents and in line with the results
402
here, labeled vagal afferents from the stomach and duodenum demonstrated a
403
substantial percentage of IB4 binding (172). Furthermore, numerous studies identify a
404
low percentage of calcitonin gene related peptide (CGRP)-ir or peptide-containing NG
405
neurons projecting to thoraco-abdominal viscera (63, 175). Despite the fact that a large
406
proportion of visceral NG neurons appear to be IB4+, the functional significance of
407
these specific subsets also has yet to be determined, as evidence of particular markers
408
for the coding of vagal afferent subtypes are limited (17). One exception may be
409
calretinin (calcium binding protein), which appears to be expressed specifically by
410
cervical esophageal vagal afferents (49).
411
Similar to other studies reporting P2X3 receptor expression in vagal sensory cell
412
bodies using immunohistochemical techniques, P2X3 receptors were highly prevalent
413
and distributed throughout the NG in the current study (11, 154). These findings suggest
414
that large populations of vagal afferents are sensitive to ATP and thus, vagal pathways
415
may be activated through purinergic signaling mechanisms. When considering
416
individual subsets of neurons based on the cellular markers examined in this study,
417
P2X3 receptor expression was present in about half of the IB4+ NG neurons, a co-
418
expression subset previously reported by our group in the NG (83) and others in the
419
DRG (25, 150, 152, 153, 164) and trigeminal ganglion (TG) (131) . However, while the
420
percentage of overlap of IB4+ neurons expressing P2X3 is relatively higher in the DRG
421
(67.5%, (25)), this co-expression pattern was similar in the TG (32%, (131)) to our
422
findings in the NG (31%). Furthermore, the percentages for all P2X3+ neurons
423
containing IB4 are both greater in the DRG (98%) and TG (64%) compared to our
424
findings in the NG (49%). The lower percentage of overlap between IB4 and P2X3
425
overall in the NG compared to other sensory ganglia may be attributed to differences in
426
embryological origin (NG, epibranchial placode derived vs. DRG, neural crest derived
427
vs. TG, both placode and neural crest), which appears to influence the neurochemical
428
phenotype of visceral afferents. For instance, DRG afferents innervating different
429
viscera such as the stomach (63, 64, 128, 172), duodenum (172) and pancreas (54,
430
128) are primarily peptidergic, expressing TRPV1, CGRP or SP, whereas NG afferents
431
innervating those same tissues display limited expression of these peptidergic markers.
432
In addition, DRG afferents innervating bladder (168), colon (35) and gastroduodenal
433
(172) tissue exhibit low IB4 binding, which is in contrast to the substantial degree of
434
bladder-innervating IB4+ neurons reported here in the NG and the significantly greater
435
percentage of IB4+ NG afferents innervating the stomach and duodenum reported
436
previously (172).
437
In regards to the overall population of NG neurons, the percentage of SP+ only
438
neurons we found was similar to an earlier report, around 30% (161). While it is noted
439
that SP+ neurons are abundant in the NG (175), their distribution has been reported to
440
be located near the rostral pole of the ganglion (72, 161, 175). We and others have
441
previously found a homogenous distribution of visceral labeling throughout the NG (1,
442
77, 127, 172). Although we did not assess the existence of an organotypic distribution of
443
labeling for the histological markers of interest within the NG in this study, the presence
444
of many SP-ir neurons in the rostral region may be anticipated due to the fact that it is
445
anatomically close to the jugular ganglion, containing neuropeptide-rich neurons that
446
primarily project to rostrally located viscera such as the esophagus and lungs (120, 143,
447
157) and is embryologically distinct (neural crest-derived) from the NG (placode-
448
derived) (9). The proximity of neurons with similar neurochemical phenotypes may be
449
important for performing like functions and even for sensory afferent integration (26).
450
Furthermore, given the embryological distinction between the vagal ganglia, the
451
phenotype of vagal afferents also may be influenced by whether or not the cell body is
452
located in either the nodose or jugular ganglion (170).
453
454
455
Effect of SCI on P2X3-ir in NG neurons
Following chronic SCI, two significant changes were observed in subsets of NG
456
neurons. The first finding was a significant increase in the number of neurons
457
expressing P2X3-ir in the spinal-transected group relative to non-injured controls. In the
458
somatosensory system, alterations in P2X3 expression following nerve injury have been
459
mixed. Both down-regulation (25) and up-regulation (50, 109) of the receptor have been
460
documented in various peripheral nerve injury models such as axotomy, ligation and
461
chronic constriction. In both studies where there were increases in P2X3 expression,
462
the injury model used resulted in some neurons that would be potentially “uninjured”. To
463
assess these differences, activating transcription factor 3 (ATF3), a marker of peripheral
464
nerve injury and absent from intact neurons (141), identified decreased P2X3 (mRNA)
465
in ATF3-ir neurons, while the increased expression was evident in the intact subset of
466
neurons (142). The significant increase in NG P2X3-ir found in the present study was
467
consistent with the ATF3-ir findings (142), since the vagal afferents are likely not directly
468
injured given they by-pass the SCI, though this has not yet been directly tested.
469
Even though contact between the vagus nerve and its peripheral targets has not
470
been severed, there is an overall effect of injury on the vagal afferent neurocircuitry (81,
471
86) and our discovery of the increased P2X3 expression seen in the NG here may help
472
to improve our understanding of the indirect effect on the vagal system after injury. For
473
instance, the increased P2X3-ir following SCI may be attributed to an inflammatory
474
reaction of the system due to the nature of the injury itself. Acutely, SCI triggers an
475
inflammatory response characterized by various resident (i.e. CNS origin) (55, 90, 121)
476
and blood-derived (134) cellular events such as the synthesis of cytokines, chemokines
477
and the infiltration of leukocytes, neutrophils and monocytes, which, over time,
478
systemically may affect tissues outside the central nervous system leading to organ
479
dysfunction. Released inflammatory cells from the blood stream can impact the
480
functionality of different viscera due to the intimate relationship these organs have with
481
the vascular system (12, 31, 65). In addition, both acute and chronic SCI induce
482
significant changes in organs with spinal innervation from segments below the lesion-
483
level. Organs such as the bladder experience substantial stress and histopathology,
484
which can lead to alterations in the integrity of the lining of the bladder (5) making the
485
bladder more susceptible to chronic inflammation (76).
486
Structural changes after SCI also include bladder (detrusor muscle) hypertrophy,
487
which triggers a release of neurotrophic factors such as NGF from the urothelial lining
488
(56, 126, 147, 148). Increases in NGF following SCI (147, 158, 165) or inflammation
489
(110, 132) as well as other excitatory neurotransmitters such as ATP (137), play a major
490
role in neuro-epithelial interactions. For example, in a migraine headache model,
491
retrograde transport of NGF from the periphery to the trigeminal ganglion or exposure of
492
trigeminal afferents to NGF led to an upregulation of P2X3 receptor protein in the cell
493
bodies (41, 58). Given that the vagus nerve provides a substantial degree of innervation
494
to the bladder (77), the fact that we found many co-labeled DiI+/P2X3+ NG neurons
495
after injury in this study, the presence of the high affinity receptor for NGF (TrkA) (70,
496
96, 174) and low affinity (p75) receptor (144, 175) and that vagal afferents have the
497
capability to transport NGF (70), the phenotypic changes with respect to P2X3 -ir in the
498
bladder-innervating NG neurons have the potential to be mediated through the actions
499
of NGF. Importantly, in a manner distinct from the actions of NGF (41), CGRP-mediated
500
insertion of P2X3 into the cell-surface membrane is an alternative mechanism, which
501
has been demonstrated in sensitized trigeminal ganglion neurons (52). However, since
502
the majority of CGRP expressing neurons appear to reside in other cranial ganglia
503
(petrosal, trigeminal, glossopharyngeal and jugular) compared to the NG (68, 69, 71,
504
72) this molecular mechanism may indirectly affect NG neurons, perhaps acting at a
505
distance through en passant synaptic contact (91).
506
The P2X3 receptor, predominantly expressed on sensory afferents (33, 97),
507
including vagal fibers (88), also can be separately retrogradely transported from the
508
periphery to the cell body via endosomes (34). This retrograde transport is thought be
509
important for maintaining neuronal activity and cell excitability through activation of
510
transcription factors (34). In disease states, such as SCI, the extracellular milieu of ATP
511
may be relatively high compared to healthy states, where excesses are rapidly
512
hydrolyzed (29, 89, 108). Large amounts of ATP (likely released from damaged tissue
513
(39)), can signal through P2X3 receptors and may show that P2X3 has a more
514
extensive role in the NG besides normal visceral afferent transduction, perhaps
515
contributing to nociceptive signaling following injury or tissue inflammation.
516
517
Effect of SCI on IB4 binding in NG neurons
518
The second change following chronic transection injury was a decrease in NG
519
IB4 binding relative to controls. This finding is similar to that of others in cases where
520
decreases in the total number of IB4 binding DRG neurons on the contralateral side
521
(uninjured side) also have been demonstrated following L5 spinal nerve transection
522
(99). Importantly, the numbers remain reduced at the chronic time point (5 weeks post-
523
injury), suggesting the effect was not transient. Since many neurons in general in the
524
NG were found to bind IB4, the significant post-SCI decrease in the co-expression
525
subset of P2X3 neurons containing IB4 may be attributed to the overall decrease in this
526
relatively large population of IB4 neurons.
527
528
An explanation for the decrease in IB4 binding may be attributed to a stress
response to the system following transection. Since glial cell-line derived neurotrophic
529
factor (GDNF) supports and aids in the regulation of IB4 neurons post-natally (104),
530
perhaps some disruption to its availability or receptor complex as well as alteration to
531
the IB4 binding glycoconjugate could explain the observed decrease (15, 118).
532
However, in response to peripheral nerve injury, spared IB4 neurons also demonstrate
533
the capability to sprout, forming perineuronal nets with both satellite and adjacent cells
534
within the ganglion (99). It has been suggested that a mechanism behind this sprouting
535
in response to nerve injury may involve inflammatory environmental changes that create
536
a chemotactic gradient, attracting various chemokines (23). This communication
537
between injured and non-injured “neighbors” within the ganglion may serve as a basis
538
for cross-excitation and could eventually induce hyperalgesia or allodynia (3, 24). Even
539
though the injury model used in this study does not directly injure vagal neurons, they
540
could be considered “spared” neurons that also demonstrate a phenotypic switch in
541
response to CNS damage and have the potential to drive visceral nociceptive signaling.
542
543
544
Effect of SCI on SP expression in NG neurons
SP is one of the main neuropeptides released from a proportion of primary
545
afferent terminal endings that express SP, in response to irritation or inflammation (7,
546
27, 48) and is present in NG neurons (175). No significant differences in SP-ir were
547
present in this study between transected and non-injured groups. A previous report
548
assessing changes in NG neurotransmitters found that SP-ir was unaffected by vagal
549
axotomy (74). The lack of changes in the NG with respect to SP-ir following injury does
550
not preclude any particular alterations at terminal endings, either peripherally in target
551
organs or centrally (solitary nucleus). For instance, there is a high concentration of SP
552
afferent terminals, primarily of vagal origin, present in solitary nucleus (73, 102, 175).
553
Alternatively, there may be molecular pathway alterations involved in the release of SP
554
and translation at the cell body (114, 139). An acute SCI or direct tissue inflammation
555
model (such as acetic acid instillation into the bladder) may provide more insight to
556
vagal SP expression in the rat (10, 114).
557
558
559
Alterations to bladder NG neurons following SCI
SCI did not result in differences compared to non-injured controls in the number
560
of NG neurons labeled from bladder, confirming these vagal afferents remain intact after
561
cord transection. Although vagal afferents exhibit a high degree of neurochemical and
562
electrophysiological plasticity in response to trauma and inflammation (26, 106, 115,
563
171), it is likely that the observed neurochemical changes in this study are a result of
564
interactions with the target organs these vagal fibers innervate rather than direct neural
565
damage. It should be noted, however, that other, extrinsic sources of ATP can reach
566
P2X3 receptors through release from sympathetic neurons, tumor cells or from vascular
567
endothelial cells associated with ischemia (30).
568
Plasticity related changes in bladder vagal afferents falls in line with evidence
569
from the spinal system after SCI. Spinal sensory neurons innervating the bladder exhibit
570
both morphological and physiological changes after SCI (93, 166). Given the important
571
transduction role of P2X3 receptors in spinal bladder afferents (36) and the fact that
572
many vagal neurons traced from the bladder expressed P2X3 suggests that the vagus
573
nerve may participate in the sensory portion of micturition function. Our collective recent
574
data indicating extensive vagal afferent innervation of mammalian urinary bladder (77)
575
and SCI-induced changes in a transduction channel like P2X3, may have important
576
clinical applications. Such changes could contribute to whatever plasticity underlying
577
reports of altered sensations stemming from the below level viscera, such as sensations
578
of bladder filling or fullness in clinically complete SCI patients above T10 (51, 92, 162).
579
A large proportion of the IB4 neurons were traced from the bladder which is
580
complementary to an earlier study showing that IB4 binds different types of visceral
581
afferents in the NG (172). Although the distal urethra was not examined in the current
582
study, a large proportion of spinal neurons which innervate this region of the lower
583
urinary tract include IB4+ afferents (168).
584
585
586
Perspectives and Significance
Due to the chronic extent of multi-system functional impairment and disability,
587
SCI presents a significant economic burden for the patient, family unit and society
588
overall with high direct and indirect costs estimated in the billions (101). Apart from
589
paralysis, some of the major complications of SCI affecting quality of life, include deficits
590
to urological function (4, 47, 67). The vagus nerve, with the majority of its cell bodies
591
located in the NG, is an extraspinal pathway through which information from regions
592
below the level of a spinal lesion can directly travel to the brainstem, bypassing the
593
spinal cord entirely. Work from our lab previously identified an anatomical connection to
594
the male rat urinary bladder through the vagus nerve (77). The current study examined
595
the immunohistochemical phenotype of vagal sensory neurons overall as well as in
596
those that innervate the bladder. The results from this study demonstrate that vagal
597
afferents are responsive to spinal injury and further assessments of their functional
598
nature may provide insight on how to take advantage of this route that by-passes the
599
spinal cord in order to improve therapeutic interventions for SCI patients.
600
601
602
603
Conclusion
The present study demonstrated neurochemical changes in the NG, a site
604
remote from the injured spinal cord. Through target-organ neural interactions, vagal
605
afferent fibers are influenced by their connections to the viscera. Therefore, overall
606
changes in these organs following SCI could impact the neurochemical properties of
607
vagal afferents innervating them. The increased expression of P2X3 and the decreased
608
binding of IB4 in the sensory cell bodies of vagal afferents post-SCI indicates an indirect
609
effect of injury on the vagal neurocircuitry. A majority of neurons in the P2X3 subset
610
after spinal transection were DiI+ indicating many NG bladder afferents have the
611
potential to respond to alterations in ATP, perhaps even playing a role in generating
612
specific sensations associated with the bladder such as fullness. In addition, the
613
considerable proportion of bladder IB4+ NG neurons demonstrates that vagal afferents
614
may participate in visceral nociceptive processing. Whether or not SCI induces changes
615
in NG neuron function will require an examination of their electrophysiological properties
616
in bladder populations.
617
618
619
620
621
622
Role of the Authors
All authors had full access to all the data in the study and take responsibility for
623
624
the integrity of the data and the accuracy of the data analysis. Study concept and
625
design: A.H., J.P., K.R., C.H. Acquisition of data: A.H., C.H. Analysis and interpretation
626
of data: A.H., J.P., D.S., K.R., C.H. Manuscript editing and approval: A.H., J.P., D.S.,
627
K.R., C.H. The authors report no conflict of interest with this project.
628
629
Acknowledgements
The authors thank Jim Armstrong, Jason Fell, Patricia Ward, Ann-Claude
630
631
Rakotoniaina, Arkadiusz Slusarczyk, Jason Beare, Shelia Arnold and Amanda
632
Pocratsky for technical assistance. The authors also thank Drs. Scott Whittemore,
633
Martha Bickford and Theo Hagg for antibody donations and Darlene Burke for statistical
634
assistance. Supported by NIH NINDS Grant F31NS077750 (A.H.) and Intramural
635
Research Incentive Grant from the University of Louisville (C.H.). This project also
636
utilized KSCIRC Neuroscience Core facilities that are supported by the NIH/NCRR P30
637
8P30GM103507 grant.
638
639
640
1
Abbreviations
641
1
SCI, Spinal cord injury; NG, Nodose ganglion; DRG, Dorsal root ganglion; IB4, Isolectin B4; SP, Substance P; -IR,
Immunoreactivity; GI, Gastrointestinal; BBB, Basso Beattie Bresnahan; FAST DiI oil/DiI, 1,1’-dilinoleyl-3,3,3’,3’,tetramethylindo-carbocyanine perchlorate; ATP, Adenosine tri-phosphate; GDNF, Glial cell-line derived
neurotrophic factor; NGF, Nerve growth factor; CGRP, Calcitonin gene-related peptide;
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
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Table 1. Immunohistochemical Reagents
Target
P2X3
Primary
Detection
Dilution/Vendor/
GP-anti P2X3
1:1000/Neuromics/ Dky anti-GPAF®488
GP10108
1:100/Jackson/
1:500/SigmaAldrich/L2140
1:100/Molecular
Probes/T20937
Biotinylated
Lectin from
Bandeiraea
Simplicifolia
Isolectin B4 (IB4)
HRP-SA
SP
RBT-anti SP
Catalog #
1:1000/Abcam/
MS-anti NeuN
1:1000/Chemicon/
MAB377
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1073
1074
1075
TyramideAF®350
Dilution/Vendor/
Catalog #
706-545-148
TSA™ Kit#27
ab67006
NeuN
Secondary
Detection
Dky anti-RBTCy™3
1:100/Jackson/
711-165-152
Dky anti-MSCy™5
1:100/Jackson/
715-175-151
AF, Alexa Fluor; Dky, Donkey; GP, Guinea Pig; HRP, Horseradish peroxidase; MS,
Mouse; RBT, rabbit; SA, Strepavidin; SP, Substance P; TSA, Tyramide Signal
Amplification
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Figure 1. Immunohistochemical representation of P2X3, IB4 and SP in NG
neurons.
Figure 1. A. Following staining of the immunohistochemical markers P2X3, SP and IB4,
the bar graph demonstrates that all three were well represented in the NG, with
the majority being IB4+ (IB4 vs P2X3, #p<.05; IB4 vs SP, **p<.001; P2X3 vs SP,
*p<.01). B. A pie graph depicting all possible combinations of the molecular
targets in the NG. Note that neurons that were IB4+ only were the most prevalent
and most NG neurons were labeled with at least one of the three cellular targets
examined. (One-way ANOVA with Tukey post hoc t-tests, n=3, 6 ganglia, values
are expressed as mean ± S.D.)
Figure 2. Quadruple immunohistochemical staining in the NG
Figure 2. A confocal image displays the typical staining within the NG. NeuN was used
to label all neurons. Different histochemical combinations include neurons that
were IB4+, P2X3+, but SP- (white arrows) and neurons that were IB4-, P2X3+
and SP- (yellow arrows). Scale bar indicates 25µm (20X objective).
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Figure 3. Spinal transection injury at T8
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Figure 3. An 18µm thick sagittal section stained with the Kluver-Barrera method
illustrates a complete spinal transection at T8. Gelfoam is placed in the lesion
cavity to prevent contact from between rostral and caudal spinal cord tissue. The
scale bar indicates 500 µm (4X objective).
Figure 4. The effect of SCI on number of NG neurons expressing the individual
markers
Figure 4. Following SCI, there was an increase in P2X3-ir in the transected group
relative to intact/normal (SCI, 27.3 ± 4.8% versus non-injured, 15.0 ± 3.3%,
*p<.001) and a decrease in IB4 binding in the transected group relative to
intact/normal (SCI, 23.7 ± 6.5% versus non-injured, 33.3 ± 7.1%, #p<.05). No
changes were apparent for SP. The “Other” category represents neurons that
were NeuN+, but did not express or bind any of the markers. (N=6, 12 ganglia)
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Figure 5. The effect of SCI on P2X3 and IB4 in the NG
Figure 5. An example displaying P2X3-ir (A) and IB4 binding (C) in the NG and
following chronic spinal cord transection injury at T8 (B and D, respectively). Note the
presence of increased P2X3-ir and decreased IB4 binding post-SCI. Images of sections
from both SCI and non-injured animals were stained and captured with the same
protocols and at the same time (10X objective).
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Figure 6. Superior Cervical Ganglion
Figure 6. There was no evidence of DiI punctate labeling present in the SCG. The scale
bar represents 20µm (20X objective).
Figure 7. Effect of SCI on bladder-traced NG neurons
Figure 7. Demonstrating that out of the total percentage of either P2X3 or IB4 subsets
after injury, more than half of the neurons were traced from bladder. Bladder
innervating neurons in the P2X3+ subset represent (32.8 ± 1.1%) while in the
IB4+ subset, they represent (42.6 ± 5.1%). (N=3, 6 ganglia)
Figure 8. P2X3-ir and IB4 binding in bladder-traced NG neurons after transection
Figure 8. A confocal image illustrating a DiI+ neuron in panel A that is also IR for P2X3
in panel B (white arrows). Panel C demonstrates the overlay. An image from the
inverted Nikon microscope illustrating a DiI+ neuron in panel D that also binds
IB4 in panel E (white arrowhead). Panel F demonstrates the overlay. One
example of each is displayed. In both images, the scale bar indicates 25 µm (20X
objective).
.