Changes in pituitary adenylate cyclase activating polypeptide expression in urinary bladder pathways after spinal cord injury

Experimental Neurology 192 (2005) 46 – 59 www.elsevier.com/locate/yexnr

Changes in pituitary adenylate cyclase activating polypeptide expression in urinary bladder pathways after spinal cord injury Katarina Zvarovaa, J. Dana Dunleavya, Margaret A. Vizzarda,b,* a

Department of Neurology, University of Vermont, College of Medicine, D411 Given Building, Burlington, VT 05405, USA b Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, VT 05405, USA Received 6 May 2004; revised 29 September 2004; accepted 20 October 2004 Available online 13 January 2005

Abstract These studies examined changes in the pituitary adenylate cyclase activating polypeptide (PACAP) expression in micturition reflex pathways after spinal cord injury (SCI) of various durations. In spinal-intact animals, PACAP immunoreactivity (IR) was expressed in fibers in the superficial dorsal horn in all segmental levels examined (L1, L2, L4–S1). Bladder-afferent cells (35–45%) in the dorsal root ganglia (DRG; L1, L2, L6, S1) from spinal-intact animals also exhibited PACAP-IR. After SCI (6 weeks), PACAP-IR was dramatically increased in spinal segments and DRG (L1, L2, L6, S1) involved in micturition reflexes. The density of PACAP-IR was increased in the superficial laminae (I–II) of the L1, L2, L6, and S1 spinal segments. No changes in PACAP-IR were observed in the L4–L5 segments. Staining was also dramatically increased in a fiber bundle extending ventrally from Lissauer’s tract (LT) in lamina I along the lateral edge of the dorsal horn to the sacral parasympathetic nucleus (SPN) in the L6–S1 spinal segments (lateral collateral pathway of Lissauer, LCP). After SCI (range 48 h to 6 weeks), PACAP-IR in cells in the L1, L2, L6, and S1 DRG significantly ( P V 0.001) increased and the percentage of bladder-afferent cells expressing PACAP-IR also significantly ( P V 0.001) increased (70–92%). No changes were observed in the L4–L5 DRG. PACAP-IR was reduced throughout the urothelium and detrusor smooth muscle whole mounts after SCI. These studies demonstrate changes in PACAP expression in micturition reflex pathways after SCI that may contribute to urinary bladder dysfunction or reemergence of primitive voiding reflexes after SCI. D 2004 Elsevier Inc. All rights reserved. Keywords: Urinary bladder; Lateral collateral pathway; Lumbosacral spinal cord; Retrograde tracing; Urinary bladder

Introduction Due to the complexity of the neural mechanisms regulating the lower urinary tract (LUT), micturition is sensitive to a wide variety of injuries, diseases, and chemicals that affect the nervous system (de Groat and Kruse, 1993; de Groat et al., 1990, 1993, 1997; Kruse et al., 1993). The storage and efficient elimination of urine depend upon the coordinated activity of two functional units in the LUT: (1) a reservoir (the urinary bladder) and (2) an outlet, consisting of the bladder neck, the urethra, and the urethral

* Corresponding author. Fax: +1 802 656 8704. E-mail address: [email protected] (M.A. Vizzard). 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.10.017

sphincter (de Groat and Kruse, 1993; de Groat et al., 1990, 1997). Coordination between these organs is mediated by a complex neural control system located in the brain, spinal cord, and peripheral ganglia (de Groat and Kruse, 1993; de Groat et al., 1990, 1997). Complete transection of the spinal cord rostral to the lumbosacral level (upper motoneuron injury) eliminates voluntary supraspinal control of voiding (de Groat and Kruse, 1993; Kuru, 1965; Torrens and Morrison, 1987). This period of reflex suppression is followed by the emergence of automatic, involuntary reflex micturition with detrusor hyperreflexia and bladder–sphincter dyssynergia (de Groat and Kruse, 1993; de Groat et al., 1990, 1993, 1997; Kruse et al., 1993). Emergence of automatic micturition after spinal cord injury (SCI) may be depend-

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ent on multiple factors, including: (1) elimination of bulbospinal inhibitory pathways; (2) strengthening of existing synapses or formation of new synaptic connections due to axonal sprouting in the spinal cord; (3) changes in synthesis, release, or action of neurotransmitters; (4) alteration in afferent input from peripheral organs or (5) central (spinal cord) and peripheral (urinary bladder) changes in the expression of neurotrophic factors. A number of laboratories have demonstrated electrical (Yoshimura, 1999; Yoshimura and de Groat, 1993a; Yoshimura and de Groat, 1997), neurochemical (Vizzard, 1997, 1999, 2000b; Yoshimura et al., 1998), and organizational changes (Vizzard, 2000b) in central and peripheral micturition reflex pathways after SCI that may underlie the reemergence of the spinal micturition reflex with associated bladder dysfunction. Recent studies have suggested that the neuropeptide pituitary adenylate cyclase activation peptide (PACAP) may play a role in peripheral and central neurons after peripheral nerve injury (Sundler et al., 1996; Zhang et al., 1995a, 1996) or peripheral (Vizzard, 2000d; Zhang et al., 1998) or central inflammatory states (Hannibal et al., 1999). Studies have indicated that a subpopulation of PACAP-immunoreactive (IR) sensory neurons in the dorsal root ganglia (DRG) are colocalized with calcitonin generelated peptide and substance P and also exhibit capsaicin sensitivity (Moller et al., 1993). In addition, intrathecal administration of PACAP induces bladder overactivity in control rats (Ishizuka et al., 1995). We have previously demonstrated that PACAP expression is increased in micturition reflex pathways after cyclophosphamide (CYP)-induced cystitis and that PACAP antagonists reduce bladder overactivity induced by CYP-induced cystitis (Vizzard, 2003). The present study was performed to define changes in PACAP-IR in bladder-afferent cells in the DRG and in the corresponding spinal segments after SCI that are also characterized by urinary bladder hyperreflexia. In addition, PACAP expression in the urothelium and detrusor smooth muscle was also characterized in the context of SCI. Portions of these data were presented in abstracts (Vizzard et al., 2001; Vizzard et al., 2003).

Materials and methods Experimental animals Adult female Wistar rats (Charles River, Canada; 150– 200 g; spinal cord intact (control; n = 6); spinal cord injury (SCI; 48 h, 1 week, 2 weeks, 6 weeks after SCI; n = 6 for each)) were used for anatomical studies. Spinal cord-intact rats had no surgery performed. For studies examining PACAP immunostaining in the urinary bladder, spinal cord-intact rats and those with SCI 5 days or 3 weeks prior to injury were used.

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Spinal cord transection Spinal cord transection was performed under isoflurane anesthesia (2–2.5%) 48 h to 6 weeks prior to intracardiac perfusion as previously described (Qiao and Vizzard, 2002b; Vizzard, 1997, 1999, 2000b). Briefly, the dorsal T7–T9 vertebrae were removed and the spinal cord was completely transected. The space between the retracted ends of the spinal cord was packed with Gelfoam (Upjohn Company of Canada, Ontario) and the incision sutured. Complete spinal transection was visually confirmed at the time of euthanasia and tissue dissection (see below). Following surgery, the animals were housed in Alpha-Dri (Shepherd Specialty Papers, Kalamazoo, MI)-lined cages and their bladders were manually expressed two to three times a day. An antibiotic (150 mg/kg ampicillin, sc) was administered prophylactically 1 day prior to surgery and for 3 days postoperatively. The analgesic buprenorphine (0.01 mg/kg, sc) was delivered postoperatively every 12 h for a total of four doses. Studies were restricted to female animals because manual expression of the bladder is more easily accomplished in the female rat because of the shorter and concomitantly smaller region occupied by the external urethral sphincter compared to male rats. Animals were studied 48 h to 6 weeks after spinal transection. All animals studied at 6 weeks after SCI had developed bladder-to-bladder and perineal-to-bladder reflexes and five out of six animals had developed these reflexes 2 weeks after SCI. All experimental protocols involving animal use were approved by the University of Vermont Institutional Animal Care and Use Committee (IACUC #00-114; 03-148). Animal care was under the supervision of the University of Vermont’s Animal Resource Center in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and National Institutes of Health guidelines. All efforts were made to minimize the potential for animal pain, stress, or distress. Retrograde labeling of bladder-afferent neurons Five to seven days prior to perfusion, Fast Blue (FB; 4%, weight/volume; Polyol, Gross Umstadt, Germany) was injected into the bladder to retrogradely label bladder-afferent neurons. As previously described (Qiao and Vizzard, 2002a,b; Vizzard, 2000d), a total volume of 40 Al divided into six to eight injections was injected into the dorsal surface of the bladder wall with particular care to avoid injections into the bladder lumen, major blood vessels, or overlying fascial layers. At each injection site, the needle was kept in place for several seconds after injection, and the site was washed with saline to minimize contamination of adjacent organs with FB. For rats surviving 48 h after SCI, FB was injected prior to spinal transection. For rats surviving 1 week after SCI, FB was injected at the time of the spinal transection procedure.

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Perfusion and tissue harvesting Tissue processing After spinalization (range 48 h to 6 weeks), animals were deeply anesthetized with isoflurane (3–4%) and then euthanized via intracardiac perfusion first with oxygenated Krebs buffer (95% O2, 5% CO2) followed by 4% paraformaldehyde. After perfusion, the spinal cord and DRG were quickly removed and postfixed for 6 h. Tissue was then rinsed in phosphate-buffered saline (PBS; 0.1 M NaCl, in phosphate buffer, pH 7.4) and placed in ascending concentrations of sucrose (10–30%) in 0.1 M PBS for cryoprotection. Spinal cord segments were identified based upon at least two criteria: (1) the T13 DRG exists after the last rib and (2) the L6 vertebra is the last moveable vertebra followed by the fused sacral vertebrae. Another less precise criterion is the observation that the L6 DRGs are the smallest ganglia following the largest, L5 DRG. DRG sections from the (L1, L2, L4– S1) spinal cord segments were sectioned parasagitally at a thickness of 20 Am on a freezing microtome. Some DRGs (L1, L2, L6, S1) were specifically chosen for analysis based upon the previously determined segmental representation of urinary bladder circuitry (de Groat et al., 1994; Donovan et al., 1983; Keast and de Groat, 1992; Nadelhaft and Vera, 1995). Bladder afferents are not distributed within the L4–L5 DRGs (Donovan et al., 1983; Keast and de Groat, 1992) that contain only somatic afferents nor are neurons that are involved in urinary bladder function observed in the L4–L5 spinal segments (de Groat et al., 1994; Nadelhaft and Vera, 1995). Thus, the L4–L5 DRG served as internal controls for these studies. Tissues from control animals with an intact spinal cord were handled in an identical manner to that described above. Whole mount bladder preparation Animals, with SCI performed 5 days or 3 weeks prior, were euthanized as described above. Prior to intracardiac perfusion with fixative, the urinary bladder was dissected and placed into Krebs solution (119.0 mmol NaCl, 4.7 mmol KCl, 24.0 mmol NaHCO3, 1.2 mmol KH2PO4, 1.2 mmol MgSO4.7H2O, 11.0 mmol glucose, 2.5 mmol CaCl2) (Zvarova et al., 2004). The bladder was cut open through the urethra in the midline and pinned flat on a sylgard-coated dish. After maximal stretch of the tissue, the bladder was incubated for 1.5 h at room temperature in cold fixative (2% paraformaldehyde + 0.2% picric acid) and the urothelium was removed. Urothelium and detrusor smooth muscle were examined separately for PACAP immunoreactivity (IR) by a free-floating technique. In some preparations, detrusor smooth muscle and urothelium stained for PACAP-IR (see below) were also immunostained for the capsaicin receptor (vanilloid receptor 1, VR1; 1:1000; Chemicon International, Temecula, CA).

Pituitary adenylate cyclase activating polypeptide (PACAP) immunohistochemistry Spinal cord sections, detrusor, and urothelium for both control and SCI animals were processed for PACAP-IR by using a free-floating method. PACAP staining in the spinal cord was evaluated in control (spinal intact) and SCI (6 weeks) rats when all SCI rats had developed spinal micturition reflexes. DRGs were immunostained using an on-slide processing technique. Groups of control animals and experimental animals were processed simultaneously to decrease the incidence of variation in staining and background that can occur between sections and between animals. Spinal cord sections, detrusor, and urothelium were incubated overnight at room temperature with PACAP antisera (mouse monoclonal antibody (code MabJH6F10); 1:20; gift from J. Fahrenkrug, University of Copenhagen, Copenhagen, Denmark) diluted in potassium PBS (0.01 M KPBS) with Triton X-100 (0.04%). Additional tissue sections were incubated 48 h at 48C with commercially available PACAP-27 or PACAP-38 antisera (rabbit antiPACAP-27 or -38; 1:3000; Phoenix Pharmaceuticals, Inc., Mountain View, CA). Tissue was rinsed in 0.1 M PBS (three rinses  10 min each). PACAP immunoreactivity in DRG (20 Am) was detected using an on-slide immunofluorescence technique. DRG sections were incubated for 48 h in a humidified box at 48C with PACAP antisera (monoclonal antibody; code MabJH6F10; 1:10; gift from J. Fahrenkrug, University of Copenhagen, Copenhagen, Denmark) diluted in KPBS plus 0.4% Triton X-100. Additional tissue sections were incubated 48 h at 48C with commercially available PACAP-27 or PACAP-38 antisera (rabbit anti-PACAP-27 or -38; 1:2000; Phoenix Pharmaceuticals, Inc.). Tissue was then incubated with Cy3-conjugated donkey anti-mouse IgG (1:400; Jackson ImmunoResearch Laboratories, West Grove, PA) or Cy3-conjugated goat antirabbit IgG (1:500; Jackson ImmunoResearch Laboratories) for 2 h at room temperature. Tissue was rinsed in PBS (three rinses  10 min each), mounted on gelatin-coated slides, and coverslipped with Citifluor (Citifluor Ltd., London, UK). Control sections in which primary antibody or secondary antibody was replaced with diluent (KPBS plus 0.4% Triton X-100) or with antibody preabsorbed with PACAP (20 Ag/ml) diluent were negative. The specification of the monoclonal antibody for PACAP has been characterized previously (Hannibal et al., 1995). Commercially available PACAP-27 and PACAP-38 antisera (Phoenix Pharmaceuticals, Inc.) do not exhibit cross-reactivity with vasoactive intestinal polypeptide as indicated by the manufacturer. Data analysis Tissues were examined under an Olympus fluorescence photomicroscope for visualization of Cy3 and FB. Cy3 was visualized with a filter with an excitation range of 560–596 nm and an emission range from 610 to 655 nm. In DRG

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from spinal-intact and SCI (range 48 h to 6 weeks SCI) rats, PACAP-IR cell profiles were counted in 10–15 sections of each selected DRG (L1, L2, L4–S1). Only cell profiles with a nucleus were quantified. DRG sections with FB-labeled cells were viewed with a filter with an excitation wavelength 340–380 nm and an emission wavelength of 420 nm. Cells colabeled with FB + PACAP-IR were similarly counted. Numbers of PACAP-IR cell profiles per DRG section are presented (mean F SEM). The percentage of presumptive bladder-afferent cells (FB labeled) expressing PACAP-IR in each DRG examined is also presented (mean F SEM). The results are not corrected for double counting. Comparisons between spinal-intact and SCI groups were made using analysis of variance. Percentage data were arcsin transformed to meet the requirements of this statistical test. Animals, processed and analyzed on the same day, were tested as a block in the analysis of variance. Two variables were being tested in the analysis: (1) experimental manipulation vs. control situation and (2) the effect of day (i.e., tissue from groups (experimental and control) of animals was processed on different days). When F ratios exceeded the critical value ( P V 0.05), Dunnett’s test was used to compare each experimental mean to the control mean. Assessment of positively stained DRG cells Staining observed in experimental tissue was compared to that observed from experiment-matched negative controls. DRG cells exhibiting immunoreactivity that was greater than the background level observed in experimentmatched negative controls were considered positively stained. Positively counted cells were not further divided into categories of different staining intensities.

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(LCP; L6, S1). Seven randomly chosen sections from each spinal segment examined were viewed with a 4 objective and captured through a video camera attachment to the microscope with exposure time, brightness, and contrast being held constant. The image was converted into pixels on the computer monitor according to a gray scale that ranges in intensity from 0 (white) to 255 (black). The spinal cord section was centered in the field and a standard size square was overlaid on the areas of interest (LDH, MDH, DCM, SPN, IML, LT, and LCP regions). The labeled area within the square was measured. Transmittance (t) was calculated as t = (gray level + 1/256). Optical density (OD) was derived from OD = log t. Comparisons among control and experimental groups were made using analysis of variance. When F ratios exceeded the critical value ( P V 0.05), Dunnett’s test was used to compare the control mean with the experimental mean. Figure preparation Digital images were obtained using a CCD camera (MagnaFire SP; Optronics; Optical Analysis Corp., Nashua, NH) and LG-3 frame grabber attached to an Olympus microscope (Optical Analysis Corp.). Exposure times, brightness, and contrast were held constant when acquiring images from spinal-intact and SCI animals processed and analyzed on the same day. Images were imported into Adobe Photoshop 7.0 (Adobe Systems Incorporated, San Jose, CA) where groups of images were assembled and labeled.

Results Comparison of PACAP antisera

Spinal cord densitometry The density of PACAP-IR in specific regions of spinal cord in control rats and those with a 6-week SCI was determined with densitometry analysis (Image-Pro express, version 4.0, Media Cybernetics, L.P.) as previously described (Vizzard, 1999, 2000d; Zvarova et al., 2004). Spinal cord segments were sectioned entirely from rostral to caudal. Every third and sixth tissue section was then processed for PACAP-IR. Of these tissue sections, every first and fifth tissue section was then used for semiquantitative analysis of PACAP-IR. We did not select sections based upon staining intensity and no sections were discarded from analysis because of low staining. Because stratification does not take the periodicity of the staining into account, it is random with respect to staining intensity. The following regions of spinal cord from both experimental and control animals were analyzed: superficial, lateral dorsal horn (LDH) and medial dorsal horn (MDH), dorsal commissure (DCM) region, the region of sacral parasympathetic nucleus (SPN; L6, S1), the region of the intermediolateral cell column (IML; L2), Lissauer’s tract (LT; L1, L2), and the region of lateral collateral pathway

Staining with the monoclonal PACAP antibody resulted in intense PACAP-IR in cell bodies in the lumbosacral DRG whereas PACAP-IR in fibers in the spinal cord segments examined was less intense. Staining with the PACAP-27 antibody resulted in very weak PACAP-IR in the spinal cord segments and DRG examined. In contrast, staining with the PACAP-38 antibody resulted in intense PACAP-IR in fibers in the spinal segments examined. However, background staining in DRG was much greater with the PACAP-38 antibody compared to the monoclonal antibody. Differences in staining between PACAP-27 and PACAP-38 observed in the present study are consistent with previous demonstrations indicating that PACAP-38 is the prevailing form in various tissues including the brain and that the amount of PACAP-27 in the rat brain is negligible compared to PACAP-38 (Arimura et al., 1991; Masuo et al., 1993). Due to the reduced background staining observed with the use of the monoclonal antibody, quantification of DRG sections (see below) was based solely upon the monoclonal antibody staining. Interpretation of PACAP-IR fiber staining in spinal cord

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segments and in the urinary bladder is based upon staining with the PACAP-38 antibody. These staining properties were identical to that from our previous study (Vizzard, 2000d). PACAP-IR in spinal cord Distribution and general characteristics In spinal-intact animals in all segmental levels (L1, L2, L4–S1) examined, PACAP-IR was expressed in distinct regions of the rat spinal cord. Some PACAP-IR was unique to specific segmental levels (i.e., rostral lumbar, L1–L2 or lumbosacral spinal cord, L6–S1) and other staining was similar in all segmental levels (L1, L2, L4– S1) examined. In all segmental levels examined in spinalintact animals, PACAP-IR was expressed in the superficial dorsal horn (medial and lateral laminae I–II) (Figs. 1A,B and 2A,C). Very faint PACAP-IR was occasionally observed in the region of the dorsal commissure located dorsal to the central canal (Fig. 2A). In the lumbosacral (L6–S1) spinal cord, faint PACAP-IR was also expressed in the region of the sacral parasympathetic nucleus (SPN)(Figs. 2A and C). Little if any PACAP-IR was observed in the ventral horn of any segmental level examined. PACAP-IR was expressed in nerve fibers in specific regions of the spinal cord and had a punctate staining quality. In the spinal cord, PACAP-IR was not expressed by neuronal cell bodies.

PACAP-IR in the lateral collateral pathway in the L6–S1 spinal cord Faint PACAP-IR fiber staining in the L6–S1 spinal cord of spinal-intact animals was apparent in a fiber bundle extending ventrally from Lissauer’s tract in lamina I along the lateral edge of the dorsal horn into the dorsal part of the sacral parasympathetic nucleus (SPN) (Figs. 2A and C). The general location of the PACAP-IR bundle in lamina I and its selective segmental distribution resembles the central projections of visceral afferents in the pelvic nerve that have been labeled in the rat and cat by axonal transport of horseradish peroxidase and designated the lateral collateral pathway of Lissauer’s tract (LCP) (Morgan et al., 1981; Steers and de Groat, 1988; Steers et al., 1991a, 1996). Changes in PACAP-IR in the spinal cord after SCI Rostral lumbar spinal cord (L1–L2). After SCI (6 weeks), the intensity and the overall distribution of PACAP staining were increased in specific spinal cord segments and regions. After SCI (6 weeks), PACAP-IR increased in several regions in the rostral lumbar L1–L2 spinal cord compared to spinal intact rats (Figs. 1B,D and 3). The density of PACAP-IR was increased in the superficial laminae (I–II) of the dorsal horn having a denser distribution throughout the entire medial (3.1-fold increase) to lateral (5.0-fold increase) extent of the laminae (Figs. 1B,D and 3). Increased (2.8-fold

Fig. 1. Fluorescence photographs of pituitary adenylate cyclase activating polypeptide (PACAP) immunoreactivity (IR) in the L1 spinal segment in control rats and in rats after spinal cord injury (SCI) for 6 weeks. PACAP-IR in a dorsolateral quadrant of the L1 spinal segment in control (spinal intact; A) or SCI animals (C). (B) Higher power photograph of the rectangular region in A of PACAP-IR in the superficial laminae (I–II) of the L1 spinal segment of control rats. In control animals, PACAP-IR was present in the superficial dorsal horn (DH) and sparse staining was present in Lissauer’s tract (LT). (C) After SCI, the density of PACAP-IR was increased in the superficial laminae of the DH. (D) Higher power photograph of the rectangular area in C showing increased density of PACAP-IR along the medial to lateral extent of the superficial laminae. In addition, PACAP-IR was increased in a fiber bundle extending from LT in lamina I laterally along the DH (arrows). Scale bar represents 125 Am in A and C; and 80 Am in B and D.

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Fig. 2. PACAP-IR in the L6 (A and B) and S1 (C and D) spinal segments in control animals and after spinal cord injury (SCI, 6 weeks). (A) Fluorescence photograph showing PACAP-IR in the dorsolateral quadrant of the L6 spinal segment of a control animal. (B) Fluorescence photograph showing PACAP-IR in the L6 spinal segment after SCI. (C) Fluorescence photograph showing PACAP-IR in the dorsolateral quadrant of the S1 spinal segment of a control animal. (D) Fluorescence photograph showing PACAP-IR in the S1 spinal segment after SCI. Increased density of PACAP-IR was observed in the medial to lateral extent of the superficial laminae (I–II) of the dorsal horn (DH) following SCI (A vs. B). Increased PACAP-IR was present in a fiber bundle (B) extending from Lissauer’s tract in lamina I along the lateral edge of the DH to the region of the sacral parasympathetic nucleus (SPN) (lateral collateral pathway of Lissauer, LCP). Although this fiber bundle was present in control tissue sections, the staining was less intense (C) and was less frequently observed in transverse sections compared to that after SCI. Faint PACAP-IR was present in the region of the SPN in control sections (A and C) that was increased after SCI (B and D). Some PACAP-IR fibers in the LCP appeared to terminate in the region of the SPN whereas others projected medially toward the central canal (D, arrows). CC, central canal; DCM, dorsal commissure. Scale bar represents 125 Am.

Fig. 3. Histogram summarizing changes in PACAP staining density in specific regions of the L1 spinal cord after spinal cord injury (SCI, 6 weeks). The spinal cord inset depicts the areas analyzed: medial dorsal horn (MDH), lateral dorsal horn (LDH), Lissauer’s tract (LT), and ventral horn (VH). The density of PACAP-IR was significantly increased in the LDH, MDH, and LT of the L1 spinal cord segment. Similar changes were observed in the L2 spinal cord segment. CC, central canal. *P V 0.001.

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increase) PACAP-IR fiber staining was also present after SCI in a small fiber bundle extending laterally from Lissauer’s tract (LT) in lamina I into the dorsolateral funiculus (Figs. 1D and 3). No dramatic changes in PACAP-IR were observed in the region of the IML following SCI. Similar changes were observed in the L2 spinal segment after SCI. Lumbosacral spinal cord (L4–S1). PACAP-IR was unchanged in the L4–L5 segments after SCI in any region examined: dorsal horn, dorsal commissure, or lateral horn regions. In contrast, significant changes in PACAP-IR were detected in the L6–S1 spinal cord after SCI (Figs. 2B,D and 4). In the L6 spinal segment, PACAP-IR was dramatically increased in the dorsal horn (1.4–7.4-fold increase), dorsal commissure (7.0-fold increase), SPN (7.8-fold increase), and LCP (9.0-fold increase) (Figs. 2B,D and 4). Similarly, changes in PACAP-IR in the S1 segment were comparable to those in the L6 segment after SCI (Fig. 2D). In some transverse sections of the L6–S1 spinal cord, PACAP-IR axons in the LCP terminated at the base of the dorsal horn (Fig. 2B) whereas in others they extended medially toward the central canal in distinct bundles through laminae V, VI, and VII (Fig. 2D). PACAP-IR in lumbosacral dorsal root ganglia (DRG) In contrast to PACAP-IR in the spinal cord, PACAP-IR in the DRG (L1–S1) was expressed by neuronal cell bodies and fibers throughout each DRG examined. In control animals, PACAP-IR was present in small numbers of cells in the L1–S1 DRG (Fig. 5). The number of PACAP-IR cells among the DRG examined was comparable (range 20–24 PACAP-IR cell profiles/section). After SCI of various durations (48 h to 6 weeks), PACAP-IR was significantly

( P V 0.001) increased in the rostral lumbar (L1–L2) and lumbosacral (L6–S1) DRG (Fig. 5). Both small (16.8 F 3.5 Am)- and medium (24.0 F 2.0 Am)-sized DRG cells expressed PACAP-IR in spinal-intact animals and SCI. PACAP-IR was occasionally observed in larger (=30 Am)sized DRG cells. No change in numbers of cells expressing PACAP-IR was observed in the L4–L5 DRG after SCI of any duration (Fig. 5). PACAP-IR in bladder-afferent cells in control animals and after SCI To determine if PACAP-IR was expressed in bladderafferent cells, Fast Blue (FB) was injected into the urinary bladder to retrogradely label bladder-afferent cells in the L1, L2, L6, S1 DRG (Fig. 6A). In spinal-intact animals, approximately 45% of bladder-afferent cells in the L6–S1 DRG exhibited PACAP-IR (Fig. 6D). A similar percentage (40%) of bladder-afferent cells in rostral lumbar DRG (L1–L2) of control animals also exhibited PACAP-IR. After SCI (6 weeks), the percentage of bladder-afferent cells exhibiting PACAP-IR significantly ( P V 0.001) increased in the L6 (88.8 F 2.2%) and S1 DRG (80.2 F 2.5%) and in the L1–L2 DRG (L1, 74.8 F 3.5%; L2, 69.5 F 3.2%) (Figs. 6C and D). Not all bladder-afferent cells expressed PACAP-IR either in spinal-intact rats or after SCI nor were all PACAP-IR cells in the DRG accounted for by FB-labeled bladderafferent cells (Figs. 6A, B, and C). Increases in the percentage of bladder-afferent cells expressing PACAP-IR after SCI were observed at the earliest time point after SCI (48 h) and were maintained up to 6 weeks after SCI with only modest changes in the percentage of bladderafferent cells expressing PACAP (Fig. 6D). The numbers of PACAP-IR cell profiles per section in DRG (L1–S1)

Fig. 4. Histogram summarizing changes in PACAP staining density in specific regions of the L6 spinal cord after spinal cord injury (SCI, 6 weeks). The spinal cord inset depicts the areas analyzed: medial dorsal horn (MDH), lateral dorsal horn (LDH), lateral collateral pathway of Lissauer (LCP), sacral parasympathetic nucleus (SPN), dorsal commissure (DCM), and ventral horn (VH). The density of PACAP-IR was significantly increased in the LDH, MDH, SPN, DCM, and LCP of the L6 spinal cord segment. Similar changes were observed in the S1 spinal cord segment. CC, central canal. *P V 0.001.

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Fig. 5. Histogram showing the number of PACAP-IR cell profiles/section in dorsal root ganglia (DRG; L1–S1) examined in control rats and after SCI of various durations (48 h, 1 week, 2 weeks, 6 weeks). In control animals, the numbers of PACAP-IR cells/section among the DRG examined were comparable. After SCI, significant (*P V 0.001) increases in PACAP-IR cell profiles/section were observed in the L1, L2, L6, and S1 DRG. No changes were observed in the L4–L5 DRG.

Fig. 6. PACAP-IR in the L6 DRG after SCI (A and B). (A) Fast Blue (FB)-labeled bladder-afferent cells in a L6 DRG section after SCI. (B) Same L6 DRG section shown in A immunostained for PACAP-IR. PACAP-IR was primarily located in small- and medium-sized DRG cells. Bladder-afferent cells expressing PACAP-IR are indicated by white arrows (A and B). (C) Merged image of panels A and B with FB cells pseudocolored blue and PACAP-IR cells pseudocolored red. FB cells (presumptive bladder afferents) expressing PACAP-IR appear pinkish-purple (white arrows). Some PACAP-IR cells do not show FB (red cells, yellow arrows). (D) After SCI, a significantly greater percentage (~85%) of FB-labeled bladder-afferent cells expressed PACAP-IR at all time points examined; however, not all bladder-afferent cells expressed PACAP after SCI. In addition, not all PACAP-IR in the L6 DRG is accounted for by bladderafferent cells (B and C, yellow arrows). Scale bar represents 40 Am in A, B, and C. *P V 0.001.

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Fig. 7. PACAP-IR in urothelium (U) and detrusor smooth muscle (M) from control (spinal intact) or SCI rats of various durations. (A and B) In control rats, PACAP-IR was densely distributed in nerve fibers throughout the U whole mount. (B) Higher power photograph of PACAP-IR nerve fibers in the U. (C) In control rats, thick PACAP-IR nerve trunks were present in the trigone and bladder neck region. Nerve trunks were also present at the point of ureteral insertion and extended dorsally toward the bladder dome. SCI of 5-day or 3-week duration resulted in a significant reduction in PACAP-IR in the U (D and E) and M (F and I). PACAP-IR nerve fibers in the U (G) were also immunoreactive for the capsaicin receptor (VR-1; H), suggesting that PACAP is expressed in C-fiber bladder afferents. Scale bar represents 100 Am.

examined from spinal-intact animals and SCI animals with or without FB were not different (data not shown). PACAP-IR in the detrusor muscle and urothelium in control and SCI rats In spinal-intact animals, PACAP-IR nerve fibers were present in the detrusor and urothelium whole mounts (Figs. 7A, B, and C). In the detrusor, PACAP-IR was present in thick nerve trunks just dorsal to the point of ureter insertion (Fig. 7C). These fibers extended rostrally to the dome of the bladder but were of a finer caliber in the dome. PACAP-IR fibers were also present throughout the urothelium (Figs. 7A, B, and G) facing the detrusor. After 5 days (Figs. 7D and F) or 3 weeks after SCI (Figs. 7E and I), PACAP-IR was decreased throughout the detrusor including the region dorsal to ureter insertion and in the urothelium. No PACAP-IR fibers were present on the adventitia portion of the detrusor. PACAP-IR nerve fibers in the detrusor or urothelium were colocalized with immunostaining for the capsaicin receptor (vanilloid receptor, VR1) (Figs. 7G and H).

Discussion These studies examined changes in the expression of pituitary adenylate cyclase activating polypeptide (PACAP)

in micturition reflex pathways after spinal cord injury (SCI) of various durations. In spinal-intact animals, PACAP immunoreactivity (IR) was expressed in fibers in the superficial dorsal horn in all segmental levels examined (L1, L2, L4–S1). Bladder-afferent cells (35–45%) in the dorsal root ganglia (DRG; L1, L2, L6, S1) from spinal-intact animals also exhibited PACAP-IR. After SCI (6 weeks), PACAP-IR was dramatically increased in spinal segments and DRG (L1, L2, L6, S1) involved in micturition reflexes. The density of PACAP-IR was increased in the superficial laminae (I–II) of the L1, L2, L6, and S1 spinal segments. No changes in PACAP-IR were observed in the L4–L5 segments. Staining was also dramatically increased in a fiber bundle extending ventrally from Lissauer’s tract in lamina I along the lateral edge of the dorsal horn to the SPN in the L6–S1 spinal segments (lateral collateral pathway of Lissauer, LCP). After SCI (range 48 h to 6 weeks), PACAP-IR in cells in the L1, L2, L6, and S1 DRG significantly increased and the percentage of bladder-afferent cells expressing PACAP-IR also significantly increased (70–92%). No changes were observed in the L4–L5 DRG. PACAP-IR was reduced throughout the urothelium and detrusor smooth muscle whole mounts after SCI. These studies demonstrate changes in PACAP expression in micturition reflex pathways after SCI that may contribute to urinary bladder dysfunction or reemergence of primitive voiding reflexes after SCI.

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Transection of the spinal cord interrupts the spinobulbospinal micturition reflex pathway, blocks voluntary voiding, and initially produces an areflexic bladder with complete urinary retention (de Groat et al., 1993; Kuru, 1965; Torrens and Morrison, 1987). However, depending upon the species, reflex bladder activity slowly recovers over the course of weeks or months. In chronic spinal animals, reflex mechanisms in the lumbosacral spinal cord are capable of duplicating many of the functions performed by reflex pathways in the spinal cord-intact animal and can induce bladder hyperreflexia (de Groat, 1975; de Groat and Ryall, 1969). However, the bladder does not empty efficiently due to a loss of bladder– sphincter coordination (de Groat and Kruse, 1993; de Groat et al., 1993; Kruse et al., 1995; McGuire and Brady, 1979; Morrison, 1987; Wyndaele, 1987). In contrast to normal animals in which the sphincter relaxes during voiding, SCI animals exhibit sphincter contractions (bladder–sphincter dyssynergia) during voiding, an increase in urethral outlet resistance, urinary retention, bladder hyperreflexia, bladder overdistension, and an increase in bladder-afferent cell size (de Groat and Kruse, 1993; de Groat et al., 1993; Kruse et al., 1995; McGuire and Brady, 1979; Morrison, 1987; Wyndaele, 1987). Changes in electrophysiological (Yoshimura, 1999; Yoshimura and de Groat, 1993a, 1997) or neurochemical properties of bladder-afferent cells in the DRG could contribute to the emergence of the spinal micturition reflex (de Groat and Kruse, 1993; de Groat et al., 1993), bladder hyperreflexia, or changes in the pharmacologic responses of reflex pathways (Yoshimura, 1999; Yoshimura and de Groat, 1993a, 1997) in the lumbosacral spinal cord after SCI. Urinary bladder hyperreflexia after SCI may reflect a change in the balance of neuroactive compounds in bladder reflex pathways. Our previous studies have examined changes in neuronal nitric oxide synthase (nNOS) (Vizzard, 1997) and galanin immunoreactivity (IR) (Zvarova et al., 2004) in micturition reflex pathways after SCI. After chronic SCI, bladder-afferent neurons in the DRG significantly increased expression of both nNOS and galanin-IR (Vizzard, 1997; Zvarova et al., 2004). We have previously suggested that increases in the expression of these two neuroactive compounds contribute to urinary bladder hyperreflexia after SCI (Vizzard, 1997; Zvarova et al., 2004). The changes that occur in spinal voiding reflexes following SCI (upper motoneuron injury) appear to be similar in humans and experimental animals and are beginning to provide important insights into a variety of neurogenic disorders of the lower urinary tract (LUT) (de Groat and Kruse, 1993; de Groat et al., 1993). A major breakthrough has been the recognition that C-fiber bladder afferents can trigger bladder hyperactivity (de Groat et al., 1981, 1990, 1993; Fall et al., 1990). In spinalized cats, the properties of C-fiber bladder afferents are altered so that they become mechanosensitive and now respond to bladder distension (de Groat et al., 1981, 1990, 1993; Fall et al.,

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1990). In chronic SCI, C-fiber afferent evoked bladder reflexes emerge; however, in cats with an intact spinal cord, myelinated (A-?) afferents activate the micturition reflex (de Groat, 1975; de Groat and Ryall, 1969; de Groat et al., 1993). de Groat and Kruse (1993) and de Groat et al. (1990, 1993) have demonstrated that systemically administered capsaicin, a C-fiber neurotoxin, blocked bladder hyperreflexia in the chronic paraplegic cat but was without effect in spinal-intact cats. In the rat, both the spinal and supraspinal micturition reflexes are activated by capsaicinresistant A? afferents (Mallory et al., 1989); however, capsaicin-sensitive afferents do appear to modulate micturition under certain conditions (Maggi, 1991, 1993). In the present study, we confirm, by immunohistochemistry, that PACAP is expressed in C-fiber bladder afferents (Fahrenkrug and Hannibal, 1998). Although numerous studies have demonstrated changes in PACAP expression in sensory neurons following nerve injury (i.e., axotomy) (Larsen et al., 1997; Moller et al., 1997; Zhang et al., 1995a,b, 1996), this, we believe, is the first study examining PACAP expression after a central injury. The question arises as to the possible functional significance of an upregulation of PACAP in bladder pathways after SCI. One report (Ishizuka et al., 1995) suggests that PACAP is involved in the facilitation of spontaneous bladder contractions in control animals. SCI results in urinary bladder hyperreflexia and urinary bladder– sphincter dyssynergia. Thus, it is possible that increased expression of PACAP-IR in bladder-afferent cells and projections could contribute to this hyperreflexia. This suggestion is the subject of an ongoing study. Additional roles for PACAP in micturition reflex pathways after SCI include modulation of nociceptive transmission through interaction with NMDA receptors (Ohsawa et al., 2002) or modulation of inflammatory responses that follow SCI (Kim et al., 2000). It has been suggested that neuropeptidemediated downregulation of inflammatory cytokines including tumor necrosis factor-alpha (TNF-a) may be a valuable therapy in the context of SCI (Kim et al., 2000). Previous studies have demonstrated a reduction in a number of neuroactive compounds in the human detrusor and suburothelial plexus after SCI (Drake et al., 2000). The present studies also demonstrate a dramatic reduction or disappearance of PACAP expression in nerve fibers in rat detrusor and urothelium 5 days or 3 weeks after SCI. Although bladder-afferent cells in the lumbosacral DRG increase expression of PACAP after SCI, there is no corresponding increase in PACAP expression in peripheral nerve fibers in the urinary bladder. The reasons for this apparent contrast in PACAP expression are not known, but it should be stated that peripheral nerve fibers in the urinary bladder can represent both the peripheral arbors of primary afferent cells in DRG or postganglionic efferent fibers projecting from the pelvic ganglia. We do know that postganglionic cells in the pelvic ganglia express PACAPIR (Vizzard, unpublished observations). Thus, differences in

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PACAP expression in DRG cells and nerve fibers in the urinary bladder may be accounted for by the type (afferent vs. efferent) of nerve fiber being evaluated. In a previous study (Zvarova et al., 2004), we also demonstrated significant decreases in galanin expression in the detrusor smooth muscle and urothelium after SCI. It has been suggested (Drake et al., 2000) that the suburothelial denervation observed after SCI decreases the likelihood that nonvoiding bladder contractions originate from exaggerated bladder afferent activity after SCI. This suggestion assumes that the actions of nerve fibers in the urinary bladder are excitatory and that this action does not change after injury or inflammatory conditions. However, PACAPinduced relaxations of the pig intravesical ureter have been demonstrated (Hernandez et al., 2004). The actions of PACAP at the level of the urinary bladder are the subject of an ongoing investigation. A large number of studies have demonstrated that pathological changes in a target organ after SCI can alter the neurochemical (Vizzard, 1997, 1999, 2001; Vizzard et al., 2003; Yoshimura, 1999; Yoshimura et al., 1998), electrical (Yoshimura, 1999; Yoshimura and de Groat, 1993a,b, 1997), and organizational (Vizzard, 2000b) properties of micturition reflex pathways. A possible mechanism underlying these changes may involve neurotrophic factors or neural activity arising in the bladder. Previous experiments have demonstrated target organ to neuron interactions in the adult animal (Steers and de Groat, 1988; Steers et al., 1991a,b, 1996; Tuttle et al., 1994; Zvara et al., 2002). Furthermore, we have previously demonstrated changes in mRNA or protein expression of neurotrophic factors in the urinary bladder after complete SCI including, hNGF, BDNF, glial-derived neurotrophic factor (GDNF), NT-3, and NT-4 (Vizzard, 2000a). Both acute and chronic SCI result in significant increases in NGF, BDNF, GDNF, NT-3, and NT-4 transcript expression as well as increased NGF protein expression in urinary bladder 4–6 weeks after SCI (Vizzard, 2000a). It has also been reported that NGF levels modestly increase in the transected spinal cord (Frisen et al., 1992; Krenz and Weaver, 2000) after SCI. Our recent studies have also demonstrated a significant increase in NGF content in spinal segments adjacent to the transection site (Zvarova et al., 2004). However, other spinal segments, distal to the transection site, exhibited decreased NGF protein content with acute or chronic SCI. In contrast, BDNF protein content significantly increased in the majority of spinal segments examined (Zvarova et al., 2004). Thus, bladder-afferent neurons may have at least two potential sources of increased neurotrophic factor production following SCI: (1) central terminals in the spinal cord (Zvarova et al., 2004) and (2) peripheral terminals in the urinary bladder (Vizzard, 2000a). Changes in neurotrophic factor expression in LUT tissues after SCI may be associated with changes in the neurochemical properties of LUT tissues. At sites of tissue injury, inflammation, or target organ hypertrophy, cytokines and

growth factors are upregulated that can result in the upregulation of NGF (Dray, 1995; Lewin and Mendell, 1993; Lindholm et al., 1987; Meller et al., 1994; Woolf et al., 1997). NGF activates TrkA receptors on axon terminals in the urinary bladder or spinal cord resulting in internalization and retrograde transport of activated TrkA (Kuruvilla et al., 2004) to afferent cells in the DRG. Alternative retrograde signaling mechanisms have also been proposed (Campenot and MacInnis, 2004). Recent studies (Qiao and Vizzard, 2002b) from this laboratory have demonstrated significant increases in the percentage of bladder-afferent neurons in the DRG that express tyrosine kinase membrane receptors, TrkA or TrkB, after SCI. Excess NGF within the DRG may induce increased production of neuropeptides (i.e., substance P, CGRP, and PACAP) in sensory neurons (Donnerer and Stein, 1992; Donnerer et al., 1992; Gary and Hargreaves, 1992; Woolf et al., 1997). An increase in the levels of neuroactive compounds (e.g., enkephalin (Lewin and Mendell, 1993), dynorphin (Ruda et al., 1988), CGRP (Donnerer and Stein, 1992; Gary and Hargreaves, 1992; Vizzard, 2001; Woolf et al., 1997), substance P (Gary and Hargreaves, 1992; Lewin and Mendell, 1993; Ruda et al., 1988; Vizzard, 2001), neuropeptide Y (Lewin and Mendell, 1993), nNOS (Vizzard, 1997; Vizzard and de Groat, 1996; Vizzard et al., 1995), and PACAP (Jongsma et al., 2000; Vizzard, 2000d)) following noxious peripheral stimulation or cyclophosphamideinduced cystitis (Vizzard, 2000c,d, 2001; Vizzard and de Groat, 1996) has also been demonstrated in DRG cells as well as in spinal cord neurons. In the present study, we demonstrate increases in PACAP expression in bladderafferent cells in DRG and lumbosacral spinal cord after SCI. This upregulation of PACAP expression in the spinal cord may represent induction of PACAP in a novel population of bladder-afferent cells and their central projections or may represent an upregulation of PACAP in the original population of PACAP-IR DRG cells. Immunocytochemistry studies in combination with studies evaluating PACAP mRNA expression in DRG are needed to discriminate between these possibilities. Increased PACAP expression in lumbosacral spinal cord after SCI is consistent with reports of increased growth-associated protein (GAP)-43 expression in nerve fibers in spinal cord segments caudal to SCI (Ondarza et al., 2003; Vizzard, 1999). In the present study, PACAP-IR fibers in the spinal cord may represent a subpopulation of the GAP-43-IR fibers previously observed after SCI. It has been demonstrated that NGF upregulates PACAP expression in intact and injured DRG cells expressing TrkAIR (Jongsma Wallin et al., 2001). Anti-NGF treatment prevents PACAP upregulation in DRG cells after adjuvantinduced peripheral inflammation (Jongsma Wallin et al., 2003). Interestingly, a recent report (Lee et al., 2002) indicates the activation of Trk receptors by PACAP. Thus, it is suggested that PACAP may exert some of its effects through a mechanism involving Trk receptors and associated tyrosine kinase signaling. The actions of PACAP mediated by Trk receptors may be in addition to those

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mediated by PAC1, VPAC1, and VPAC2 receptors (Zhou et al., 2002), thereby adding to the wide variety of effects for PACAP in the nervous system in health as well as after injury or disease.

Acknowledgments This work was aided by grants DK051369, NS040796, DK065989, DK060481. The authors thank Dr. Jan Fahrenkrug, University of Copenhagen, Copenhagen, Denmark, for his generosity in supplying the monoclonal PACAP antisera.

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