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Articles in PresS. Am J Physiol Regul Integr Comp Physiol (April 8, 2015). doi:10.1152/ajpregu.00445.2014 1 2 3 The effect of spinal cord injury on the neurochemical properties of 4 vagal sensory neurons 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 April N. Herritya,b, Jeffrey C. Petruskaa,b,c, David P. Stirlingb,c,d, Kristofer K. Raua,b,e and Charles H. Hubschera,b. a Department of Anatomical Sciences & Neurobiology, University of Louisville School of Medicine, Louisville, KY 40202 b Kentucky Spinal Cord Injury Research Center, University of Louisville, Louisville, KY 40202 c Department of Neurological Surgery, University of Louisville, Louisville, KY 40202 d Department of Microbiology & Immunology, University of Louisville School of Medicine, Louisville, KY 40202 e 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] 40 Copyright © 2015 by the American Physiological Society. 41 42 Abstract The vagus nerve is comprised primarily of non-myelinated sensory neurons 43 whose cell bodies are located in the nodose ganglion (NG). The vagus has widespread 44 projections that supply most visceral organs, including the bladder. Due to its non-spinal 45 route, the vagus nerve itself is not directly damaged from spinal cord injury (SCI). 46 Because most viscera, including bladder, are dually innervated by spinal and vagal 47 sensory neurons, an impact of SCI on the sensory component of vagal circuitry may 48 contribute to post-SCI visceral pathologies. To determine if SCI, in male Wistar rats, 49 might impact neurochemical characteristics of NG neurons, immunohistochemical 50 assessments were performed for P2X3 receptor expression, IB4 binding, and 51 Substance-P expression, three known injury-responsive markers in sensory neuronal 52 subpopulations. In addition to examining the overall population of NG neurons, those 53 innervating the urinary bladder also were assessed separately. All three of the 54 molecular markers were represented in the NG from non-injured animals, with the 55 majority of the neurons binding IB4. In the chronically injured rats, there was a 56 significant increase in the number of NG neurons expressing P2X3 and a significant 57 decrease in the number binding IB4 compared to non-injured animals, a finding that 58 held true also for the bladder-innervating population. Overall, these results indicate that 59 vagal afferents, including those innervating the bladder, display neurochemical plasticity 60 post-SCI that may have implications for visceral homeostatic mechanisms and 61 nociceptive signaling. 62 Keywords: Vagus nerve; Nodose ganglion; Spinal Cord Injury; Bladder; 63 Immunohistochemical phenotype 64 65 Introduction Spinal cord injury (SCI) results in deficits to sensorimotor systems and profoundly 66 affects the functionality of the autonomic nervous system. Basic research focusing on 67 improving pelvic-visceral outcomes following SCI is of great clinical importance, since 68 complications such as bladder, bowel and sexual dysfunction affect health and quality of 69 life for this population (4, 47, 67). Despite the direct immediate impact of injury to the 70 spinal-derived autonomic supply of the pelvic viscera, most of the body’s visceral 71 organs also are supplied by a non-spinal source through the vagus nerve. Since the 72 vagus nerve does not travel directly through the spinal cord, its neurocircuitry is often 73 considered intact following SCI. Nevertheless, there is some degree of indirect 74 involvement of both vagal afferents and efferents. For example, following SCI, 75 subsequent neuroplastic-responsive changes have been extensively described within 76 the dorsal vagal complex controlling gastric function (79). Gastrointestinal (GI) 77 alterations after human upper-thoracic SCI include conditions such as dysphagia (160), 78 esophagitis (133), peptic ulcerations (62, 138), gastroparesis and overall dysmotility (87, 79 123, 125, 159). Although the mechanisms of GI dysfunction in humans after SCI are not 80 thoroughly understood, experimental studies in rats suggest that many of the delays in 81 gastric emptying and transit may in part be attributed to vagally-mediated pathways (60, 82 61, 79, 140). In fact, subdiaphragmatic vagotomy has been shown to prevent much of 83 the SCI-induced GI sequelae (61). 84 The vagus nerve, with sensory cell bodies primarily located in the nodose ganglia 85 (NG), provides innervation to the thoraco-abdominal structures. Despite the view that 86 the vagus nerve does not innervate viscera caudal to the transverse colon, numerous 87 experimental studies have demonstrated that it also provides sensory innervation to the 88 majority of the pelvic viscera (2, 28, 38, 57, 77, 81, 84, 111, 149). Even though the 89 vagus nerve has such widespread projections, SCI does not disconnect the anatomical 90 relationship the nerve has with the tissue it innervates. However, SCI does lead to 91 pathological changes and dysfunction of below-level target organs, such as the bladder 92 (45, 46, 94), and can thereby influence neuronal phenotype (122, 145, 158, 167, 176). 93 Furthermore, various classes of primary sensory neurons, including vagal afferents, 94 have been shown to alter their phenotype and the expression of different receptors in 95 response to nerve injury and tissue inflammation (11, 78, 103, 107, 155, 173). 96 In this experiment, P2X3 receptor and SP immunoreactivity (ir) as well as IB4 97 binding was examined in NG neurons. These particular markers were selected based 98 on their presence in the NG, involvement in the spinal and vagal circuitry, 99 responsiveness in other sensory neurons to injury and/or inflammation, and the 100 potential physiological role these markers may play in nociceptive signaling (11, 25, 30, 101 40, 42, 43, 83, 97, 103, 109, 146, 154, 163, 169, 172). In addition, anatomical evidence 102 that the vagus nerve provides sensory innervation to the bladder in male rats (77) and 103 the presence of these cellular markers in bladder tissue (6, 19, 100) adds to the 104 importance of understanding the relationship between target-organ tissue and its 105 innervating neurons. It is therefore hypothesized that following a spinal transection 106 injury which removes any potential sources of spinal input rostrally and isolates vagal 107 afferent fibers, the expression profile of known injury-responsive factors P2X3, IB4 and 108 SP would be altered in the NG in general and also for the subset of NG neurons 109 innervating the bladder. 110 111 Materials and Methods 112 Animals 113 All experimental procedures were carried out according to NIH guidelines and 114 protocols reviewed and approved by the Institutional Animal Care and Use Committee 115 at the University Of Louisville School Of Medicine. All adult male Wistar rats (n=16, 116 Harlan Sprague Dawley, Inc, Indianapolis, IN), approximately 250 grams in weight, were 117 individually housed in an animal room with a 12-hour light and dark cycle. They had ad 118 libitum access to water and food (Laboratory Rodent Diet). Groups were either naïve 119 (n=8) or spinal cord injured (n=8). Each group had a subset (n=4 each) which received 120 retrograde neural tracer injected into the bladder to enable identification of single NG 121 neurons which innervated the bladder. 122 123 124 Spinal cord injury Half of the animals (n=8) were anesthetized with a mixture of ketamine (80mg/kg) 125 and xylazine (10mg/kg), injected intraperitoneally, for spinal transection. All surgeries 126 were performed under aseptic conditions and the body temperature was maintained 127 within the range of 36-37ºC via a warm water recirculator (Gaymar T/Pump, Gaymar 128 Industries Inc, Orchard Park, NY) throughout the surgery and recovery period. Following 129 our previously published protocol (86), a dorsal longitudinal incision was made to 130 expose the T7 vertebra and a laminectomy was performed in order to expose the 131 underlying T8 spinal cord. The overlying dura was reflected laterally and the spinal cord 132 cut using a pair of surgical microdissecting scissors. Gentle suction with an air vacuum 133 was used to carefully elevate the cut stump in order to verify the completion of the 134 lesion. Gelfoam (Pharmacia & Upjohn Company, Kalamazoo, MI) soaked in topical 135 hemostat solution (Henry Schein Inc., Melville, NY) was placed in the lesion cavity. The 136 incision was closed using 4-0 nylon suture for the muscle layers and fascia and surgical 137 clips for the skin. Animals were given subcutaneous injections of ketoprofen (Ketofen, 138 2.5mg/kg, Fort Dodge Animal Health, Fort Dodge, IA) for analgesia twice a day for 2 139 days, 0.5ml of dual penicillin (Penicillin G coupled with Procaine, PenJect ®, The Butler 140 Company, Columbus, OH) in a single dose peri-operatively as a general prophylactic 141 and 5mg/kg gentamicin (GentaFuse®, Butler Schein, Dublin, OH) once per day for 5 142 days to prevent bladder infections. After surgery each animal was housed individually. 143 The urinary bladder was emptied by manual crede every 8 hours until the micturition 144 reflex occurred automatically, typically 6–12 days after surgery (82). Animals survived 145 for six weeks followed by euthanasia and tissue removal. 146 147 148 Retrograde tracer injection At five weeks post injury, four spinally-transected rats and four age-matched 149 naïve control rats were anesthetized with a mixture of ketamine (80mg/kg) and xylazine 150 (10mg/kg). They received a ventral/caudal midline peritoneal incision to expose the 151 urinary bladder which was subsequently manually voided by pressure. Using an 152 established protocol (77, 124), the fluorescent tracer FAST DiI™ oil (1,1’-dilinoleyl- 153 3,3,3’,3’,-tetramethylindo-carbocyanine perchlorate, 5mg dye dissolved in 0.1ml 154 methanol, Molecular Probes, Inc., Eugene, OR) was injected into the bladder wall with a 155 dye-dedicated 33-gauge needle coupled to a Hamilton microsyringe (Fisher Scientific, 156 Pittsburgh, PA). Note that the abbreviation DiI is used throughout the 157 text/figures/legends to refer to FAST DiI™ oil. Dye injections were made to the 158 circumference of the trigone, body and dome areas (10μl volume per animal divided into 159 10 injections of 1ul each; (77)). 160 Animal body temperature was maintained within the range of 36-37°C during 161 surgery via a warm water recirculator (Gaymar T/Pump, Gaymar Industries Inc, Orchard 162 Park, NY). After each injection, the needle was removed slowly and any dye-leakage 163 was removed by cotton-tipped applicators. After injections were completed, the exposed 164 viscera were hydrated as necessary with 5% Dextrose Lactated Ringers, the abdominal 165 musculature was sutured closed (Ethicon 4-0 non-absorbable surgical suture), the skin 166 closed with Michel clips (Fine Science Tools, Foster City, CA), and a topical antibiotic 167 (Bacitracin, Actavis Mid Atlantic LLC, Lincolnton, NC) applied. Following surgery, 168 animals were placed on a heating pad and core temperature monitored. Post-operative 169 medication was provided as per spinal transection surgery. All animals were monitored 170 daily to inspect the surgical incision and identify any changes in an animal’s general 171 condition. 172 173 Perfusion and Tissue Collection 174 All 16 animals were deeply anesthetized with a ketamine (80mg/kg body 175 weight)/xylazine (10mg/kg) mixture and transcardially exsanguinated with heparinized 176 saline, followed by 4% paraformaldehyde perfusion. Each vagus nerve was identified 177 adjacent to the carotid artery and gently separated from surrounding tissues and traced 178 rostrally to the NG, which was excised. The bladder was dissected free from the 179 prostate and extracted at the most distal aspect of the neck. Before weighing the 180 bladder, any residual intravesicular urine was allowed to flow out. Superior cervical 181 ganglia were identified on both sides at the bifurcation of the common carotid artery and 182 removed to be used as control tissue. For the transected group of rats, following a 183 dorsal spinal incision, removal of the spinal cord tissue 1cm above and below the 184 transection site was performed. All tissues were placed in individually-labeled tubes of 185 4% paraformaldehyde for at least 48h, followed by immersion in a cryoprotectant 186 solution of 30% sucrose/phosphate buffer solution with 1% sodium azide for at least 187 24h. Following removal from the cryoprotectant solution, NG’s were embedded in OCT® 188 compound (Baxter Scientific) and 12-14μm sections were cut on a cryostat (Leica CM 189 1850). During retrieval of the bladder, the abdominal cavity and surrounding viscera 190 were inspected for tracer leakage. 191 192 Histology of the lesion epicenter 193 The lesion cavity, coated in embedding media, was cut into 18µm sagittal 194 sections using a Leica CM 1850 cryostat and mounted onto gelatin-coated histological 195 slides (Azer Scientific, Morgantown, PA). The slides were then stained with both Luxol 196 fast blue and cresyl violet (Kluver-Barrera method) to observe myelin and Nissl 197 substance, respectively. Spot Advanced software (Diagnostic Instruments, Sterling 198 Heights, MI) and the Nikon E400 microscope were used to image the lesion cavity and 199 verify the completeness of the spinal transection (86). The percentage of spared white 200 matter from the transection lesion was calculated using Nikon Elements software. The 201 boundary between spared tissue at the ventral portion of the cord and the lesion cavity 202 was identified. The first anatomical region of interest (ROI) outlined was the portion of 203 spared tissue at the ventral aspect of the spinal cord and the second ROI outlined was 204 the entire lesion cavity, encompassing both spared and non-spared tissue and 205 extending from both the rostral and caudal cord stumps. Area was determined for each 206 ROI and the percentage of spared tissue was calculated by dividing these areas (95). 207 208 209 Hindlimb assessment for lesion completeness The Basso-Beattie-Bresnahan (BBB) scale (14), an open-field locomotor 210 assessment, was used to evaluate hindlimb function as an assessment of post-injury 211 spinal cord function. Each animal was placed in an open-field and tested for 4 minutes 212 by the same two scorers, who were presented with injured and non-injured animals in 213 random order. The 21-point BBB scale was used to assess hindlimb coordination and 214 rated parameters such as individual joint movements (0-7), weight support (8-13) and 215 paw placement (14-21). Intact animals should demonstrate a locomotor score of 21. 216 Animals that receive a completeT8 transection have been shown to exhibit BBB scores 217 of 3 (extensive movement of 2 joints) on average (13, 130). To prevent any functional 218 connections across the lesion site from potential spared tissue, gelfoam was placed 219 between the two cut stumps. It has been demonstrated that as little as 4-5% sparing 220 (primarily in the ventrolateral funiculi) was sufficient for attaining a BBB score of 7 221 following “complete” spinal transection (no gelfoam used across lesion) (53). 222 223 224 225 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). 226 Slide-mounted sections were incubated at room temperature for 2h in a solution of 2% 227 Triton X-100® in phosphate buffered saline (PBS). This pre-treatment step improves the 228 quality of P2X3-ir (116). The slides were rinsed in distilled water and then incubated for 229 30min in a solution of 10% normal donkey serum (Jackson Immuno Research, West 230 Grove, PA) in PBS with 0.3% Triton X-100 (MP Biomedicals, LLC, Solon, OH) to block 231 non-specific antibody binding. The immunohistochemical reagents and the labeling 232 procedures are summarized in Table 1. Incubations in primary antisera were performed 233 overnight (14–18 hours) at 4ºC. All steps were followed by multiple rinses with PBS. All 234 fluorescent secondary antisera were diluted 1:100 and incubations were 2 hours at 235 room temperature. The tyramide signal-amplification (TSA™) reagent kit (Sigma- 236 Aldrich, St. Louis, MO) (20-22) was used at 1:100 and incubations were for 4-5min 237 (124). Once all steps were completed, the slides were coverslipped with a glycerol- 238 based photobleach-protective medium (Fluoromount-G, Southern Biotech). 239 240 Cell Quantification 241 To view labeled sections, imaging was performed using the Nikon Eclipse TiE 242 inverted microscope with NIS Elements software. Initially, images were captured using a 243 10x lens (APO DIC N1 10x/0.45 NA, Nikon) with consistent exposure times and 244 computationally stitched together to visualize whole ganglion sections. Imaging of 245 individual fluorophores was achieved with a mercury-arc lightsource and the following 246 filter sets: for Cy3 [543/22nm excitation, 593/40nm emission, 562nm dichroic, Semrock]; 247 for Alexa Fluor 488 [470/40nm excitation, 525/50nm emission, 495nm dichroic, Nikon]; 248 for Alexa Fluor 350 [350/50nm excitation, 460/50nm emission, 400nm dichroic, Nikon]; 249 for Cy5 [615/70nm excitation, 700/74nm emission, 660nm dichroic, Nikon]. 250 In order to obtain unbiased percentages of P2X3, SP and IB4 positive neurons in 251 the NG, the physical dissector method was applied (37, 113). Across the entire 252 ganglion, assembled by automated stitching, counts of all singly-, multi- as well as non- 253 labeled/other (collectively comprising total neuronal counts) NG neurons were made by 254 a scorer blinded to treatment groups. Starting with a random section, neurons with a 255 clearly visible nucleus and definable soma were counted only if they were not present in 256 an adjacent “look up” serial section. As an added measure to avoid double counting 257 single neurons, the counting-sections were at least 60 microns apart (every 5th section). 258 To differentiate background from foreground pixels, threshold values also were obtained 259 based off the image histogram for each marker and held constant for each image 260 quantified. Note that non-labeled/other cells (NeuN+ only) were quantified and 261 represented the total neuron population (59, 75, 105). Images of tissue without 262 application of the primary antibody were taken and utilized as baseline controls (images 263 not shown). Using Nikon Elements software, cell diameter between groups also was 264 determined by estimating the average of two cross-sectional diameters (longest and 265 shortest axis). 266 Positive neuron counts were expressed as a percentage of the total number of 267 neurons (NeuN+ only) from within the entire stitched ganglion as well as a percentage 268 of all labeled neurons. An Olympus 3 Laser scanning confocal microscope with 269 Fluoview 500 software (Mellville, NY) and a Nikon A1R MP+ confocal microscope with 270 Elements software were used to collect high resolution, serial optical sections of NG 271 neurons. 272 273 274 Statistics Analyses were performed using SPSS v19-20 (IBM, North Castle, NY). Levene’s 275 statistic was applied for homogeneity of variances and data are expressed as mean ± 276 standard deviation (SD). A one-way analysis of variance (ANOVA) with Tukey HSD post 277 hoc t-tests was performed for the assessment of all histochemical markers and NG 278 bladder labeling within groups. For the analysis of the histochemical markers between 279 groups, data were normalized as percentages of total NG cells and analyzed via a two- 280 way ANOVA with Tukey HSD post hoc t-tests. For all other analyses, two-tailed 281 Student's t-tests were performed assuming equal variance. Statistical significance was 282 defined as p≤.05. 283 284 Results 285 Immunohistochemical signature of NG neurons 286 IB4 binding in NG neurons was localized to the plasma and axonal membranes 287 as well as the Golgi complex. Immunoreactivity (-ir) for P2X3 and SP was present in the 288 cytoplasm and P2X3-ir also could be found in the plasma and axonal membranes. All of 289 the patterns of staining for these markers were consistent with previous studies in both 290 NG and DRG (8, 10, 83, 116, 151). When examining the total percentage of labeled NG 291 neurons, all of the markers were well represented (Figure 1A), with the majority of NG 292 neurons binding IB4 (IB4, 65.5 ± 6.2% versus SP, 31.1 ± 10.3%; IB4 versus P2X3, 51.2 293 ± 7.9%). Overall, there were 8 different histochemical signatures represented in the NG 294 (Figure 1B). The most prevalent combinations were neurons that were IB4+ only 295 followed by the P2X3+ only and IB4+/P2X3+/SP- combinations (IB4+ only versus 296 P2X3+ only, 33.3 ± 7.1% versus 15.0 ± 3.3%, p<.001; IB4+ only versus IB4+/P2X3+ 297 only, 33.3 ± 7.1% versus 15.0 ± 5.0%, p<.001). When considering specific co- 298 localization patterns in the NG, about a third of all IB4 neurons contain P2X3 (30.9 ± 299 9.2%) and about half of all P2X3 neurons contain IB4 (49.1 ± 8.6%). A typical example 300 of the quadruple immunohistochemical staining in the NG is provided in Figure 2. 301 302 Effect of SCI on NG neurons expressing P2X3, SP and binding IB4 303 Following a chronic transection injury at T8, BBB locomotor assessments at the 6 304 week time point for all transected animals revealed an average score of 0.9 (± 0.9). 305 Only one animal had a BBB score greater than zero (score of 4.5: 6 on the left hindlimb 306 and 3 on the right hindlimb). Post hoc histological assessments of the lesion site 307 revealed that this same animal in the spinal cord-transected group had a small 308 percentage of area spared within the lesion site (16.1% - located at the most ventral 309 extent of the epicenter). The data from this incomplete transection rat is provided, but as 310 a separate group with an n of 1. Although statistical analyses could not be performed 311 with n=1, the immunohistochemical expression pattern in this SCI rat appeared to more 312 closely resemble that of the naïve group (P2X3+ only, 11.5 ± 5.2%; IB4+ only, 44.0 ± 313 2.2%). An example indicating a typical complete lesion is provided in Figure 3. Note that 314 it was also found that the average cell diameter for NG neurons was 29.6 ± 5.7 µm. This 315 morphological feature was not affected by spinal transection injury (28.5 ± 6.04 µm). 316 In rats with a complete transection (n=3; 6 ganglia), there was a significant 317 increase in the percentage of total NG neurons expressing P2X3 (p<.001) as well as a 318 significant decrease in the percentage of total NG neurons binding IB4 (p<.05) relative 319 to non-injured controls (Figure 4). There were no significant differences in SP or NeuN 320 expression between groups. P2X3 expression and IB4 binding in the NG are 321 demonstrated in Figure 5. Within the spinal cord-transected group, the percentage of 322 neurons expressing P2X3 and the percentage of neurons binding IB4 were each 323 significantly greater than the percentage of neurons expressing SP, which was 324 unchanged from the non-injured group of animals (P2X3, 27.3 ± 4.8% versus SP, 6.5 ± 325 2.8%, p<.01; IB4, 23.7 ± 6.5% versus SP, 6.5 ± 2.8%, p<.01; SP, 6.5 ± 2.8% versus SP- 326 non-injured, 5.8 ± 3.8%). From the total population of NG neurons, SCI did not 327 significantly impact the subset of NG neurons that co-expressed IB4 and P2X3 (Naïve, 328 15.0 ± 5.0 versus TX, 13.5 ± 5.7, p>.05), nor did it impact the subset of all IB4 neurons 329 that contained P2X3 (Naïve, 30.9 ± 9.2% versus TX, 35.9 ± 11.2%, p>.05). However, 330 following SCI, the subset of all P2X3 neurons that contained IB4 was significantly lower 331 compared to non-injured animals (Naïve, 49.1 ± 8.6% versus TX, 32.2 ± 10.5%, p<.01). 332 With respect to the total number of labeled neurons within their individual 333 populations (i.e. all P2X3+ neurons and all IB4+ neurons), the distribution of the 334 P2X3+/IB4-/SP-only subset represents 50.7% of all P2X3+ neurons, while the 335 IB4+/P2X3-/SP-only subset represents 42.6% of all IB4+ neurons. Overall, these 336 populations of neurons comprised about half of the total population of NG neurons 337 examined. 338 339 Immunohistochemical profile of bladder non-injured and injured NG neurons 340 The retrograde tracer DiI, was injected into the bladder wall in order to determine 341 if the subsets of NG neurons affected by spinal cord transection include those that 342 supply the bladder. Initially, we found that the percentage of NG neurons traced from 343 the bladder in both groups in this study is similar to our previous study of spinally intact 344 animals (22.2 ± 3.6% versus 21.4 ± 4.0%, (77)). Assessment of the superior cervical 345 ganglion, which is located adjacent to the NG, did not reveal evidence of DiI tracer, 346 indicating the tracer did not spread non-specifically (Figure 6). 347 With respect to the total population of P2X3+ NG neurons after chronic SCI (50.7 348 ± 8.2%), bladder-innervating neurons (DiI+/P2X3+/IB4-) represented 32.8 ± 1.1%, while 349 with respect to the total population of neurons that were IB4+ after injury (42.6 ± 5.1%), 350 bladder-innervating neurons (DiI+/P2X3-/IB4+) represented 21.5 ± 7.4%. Overall, in 351 these two distinct subsets of NG neurons, more than half of the neurons are traced from 352 the bladder (Figure 7). Images of the DiI+/P2X3+ and DiI+/IB4+ subsets following 353 transection are demonstrated in Figure 8. Note that in this study, the proportion of NG 354 neurons traced from the bladder in spinally-intact rats (23.7 ± 3.6%) did not differ 355 significantly from the proportion traced from the bladder after chronic spinal transection 356 injury (20.2 ± 3.0%). Transection injury did however, result in a significant increase in 357 bladder size (wet weight) compared to non-injured controls (.267 ± .118g, SCI vs .149 ± 358 .35g, Naïve; p<.05), which can impact overall total bladder capacity and voiding 359 efficiency, potentially leading to an alteration in bladder function. 360 361 362 363 Discussion 364 innervation from spinal and non-spinal (i.e., the vagus nerve) sources (28, 38, 57, 77, 365 81, 84, 111). This study, using immunohistochemical techniques, examined the impact 366 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 367 presence of P2X3, IB4 and SP in NG neurons. Following spinal transection at T8, 368 potential plasticity was evaluated in subsets of NG neurons which contain projections 369 that bypass the spinal cord from visceral organs, including those projections that 370 specifically supply the bladder. A major traumatic event to the nervous system, such as 371 SCI, leads to dysfunction in multiple organ-systems, and ultimately influences the 372 neurons that innervate these tissues. The findings of the current study indicate that 373 vagal sensory cell bodies displayed an increase in P2X3 expression and a decrease in 374 IB4 binding, which also held true for many neurons innervating the bladder. These 375 results suggest that NG neurons, including the bladder subset, are sensitive to a spinal 376 injury and are capable of responding by modifying their phenotype. 377 378 379 380 Immunohistochemical phenotype of NG neurons Overall, from the cellular markers examined in this study, the majority of NG 381 neurons were IB4+. Isolectin B4 has been shown to label primarily a subpopulation of 382 non-peptidergic spinal sensory afferents that are thought to be functionally distinct from 383 peptidergic neurons or neurons that are IB4 negative (135). Even though IB4+ neurons, 384 in general, are widely expressed within the NG (83, 98, 129, 169, 172), the meaning of 385 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 References 1. Altschuler SM, Bao XM, Bieger D, Hopkins DA, and Miselis RR. Viscerotopic representation of the upper alimentary tract in the rat: sensory ganglia and nuclei of the solitary and spinal trigeminal tracts. 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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 1072 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 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 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). 1099 Figure 3. Spinal transection injury at T8 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 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) 1121 1122 1123 1124 1125 1126 1127 1128 1129 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). 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 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). .