Cell Tissue Res (2006) 324: 213–224 DOI 10.1007/s00441-005-0114-z
REGULAR A RTICLE
K. Zvarova . M. A. Vizzard
Changes in galanin immunoreactivity in rat micturition reflex pathways after cyclophosphamide-induced cystitis
Received: 1 July 2005 / Accepted: 31 October 2005 / Published online: 21 February 2006 # Springer-Verlag 2006
Abstract Alterations in the expression of the neuropeptide, galanin, were examined in micturition reflex pathways of rat after cyclophosphamide (CYP)-induced cystitis of variable duration: acute (4 h), intermediate (48 h), or chronic (10 days). In control animals, galanin expression was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure (DCM); (2) superficial dorsal horn; (3) the regions of the intermediolateral cell column (L1–L2) and the sacral parasympathetic nucleus (SPN, L6–S1); and (4) the lateral collateral pathway (LCP) in lumbosacral spinal segments. Densitometry analysis demonstrated significant decreases (P≤0.01) in galanin immunoreactivity (IR) in these regions of the L1–S1 spinal cord after acute or intermediate CYP-induced cystitis. In contrast, increases (P≤0.01) in galanin–IR were observed in the DCM, SPN, or LCP regions in the L6–S1 spinal segments in rats with chronic cystitis. No changes in the number of galanin–immunoreactive cells were observed in the L1–S1 dorsal root ganglia (DRG) after CYP-induced cystitis of any duration. A small percentage of bladder afferent cells (Fast-blue-labeled) in the DRG expressed galanin–IR in control rats; this was not altered with cystitis. Galanin–IR was observed encircling DRG cells after chronic cystitis. These changes may contribute to urinary bladder dysfunction, altered sensation, and referred somatic hyperalgesia after cystitis. Keywords Spinal cord . Micturition reflexes . Hyperalgesia . Cystitis . Rat (Wistar, female)
This work was supported in part through NIH grants DK051369, DK060481, DK065989, and NS040796. K. Zvarova . M. A. Vizzard (*) Departments of Neurology and Anatomy and Neurobiology, College of Medicine, University of Vermont, Burlington, VT 05405, USA e-mail:
[email protected] Tel.: 802-656-3209 Fax: 802-656-8704
Introduction Micturition is regulated by neural circuits in the brain and spinal cord; these circuits coordinate the activity of the smooth and striated muscles of the lower urinary tract (Morrison 1987; de Groat 1993; de Groat et al. 1998; de Groat and Araki 1999; Yoshimura 1999). Chronic pathological conditions inducing tissue irritation or inflammation can alter the properties of sensory pathways leading to a reduction in pain threshold (allodynia) and an amplification of painful sensations (hyperalgesia; Campbell and Meyer 1986). Peripheral sensitization of primary afferents or changes in central synapses can contribute to increased pain sensation (Ruda et al. 1988; Dubner and Ruda 1992; Dmitrieva and McMahon 1996). Experiments with a chemically (cyclophosphamide, CYP)-induced bladder inflammation (Cox 1979; Maggi et al. 1992;Lantéri-Minet et al. 1995) model have demonstrated alterations in neurochemical (Vizzard 2000c, 2001; Qiao and Vizzard 2002b, 2004), electrophysiological (Jennings and Vizzard 1999; Yoshimura and de Groat 1999), and organizational (Vizzard 1999, 2000a) properties of bladder afferent neurons in dorsal root ganglia (DRG) and spinal cord. Marked changes in bladder function have also been demonstrated after CYP-induced cystitis (Maggi et al. 1992, 1993; Hu et al. 2003). These changes suggest considerable reorganization of reflex connections in the spinal cord and marked changes in the properties of micturition reflexes with CYP treatment. The neuropeptide galanin has a widespread distribution in the central and peripheral nervous system and modulates sensory transmission in the spinal cord after peripheral nerve injury or inflammation (Wiesenfeld-Hallin et al. 1989; Zhang et al. 1998; Xu et al. 2000; Liu and Hökfelt 2002; Landry et al. 2003, 2005). Galanin is upregulated in DRG neurons (Villar et al. 1989; Zhang et al. 1998; Hökfelt et al. 1999; Xu et al. 2000; Liu and Hökfelt 2002; Landry et al. 2005), and galanin release is increased in the superficial dorsal horn (Colvin et al. 1997) after peripheral nerve injury. Additional roles for galanin in mediating survival or regeneration after neural injury have also been suggested (Liu and Hökfelt 2002; Mahoney et al. 2003; Landry et
214
al. 2005). Pain models for acute and chronic inflammation exhibit an initial decrease in galanin peptide and mRNA in DRG followed by an increase in galanin expression (Ji et al. 1995; Calza et al. 1998). Transgenic over-expression of galanin in the DRG modulates pain-related behaviors (Villar et al. 1989; Holmes et al. 2003; Hygge-Blakeman et al. 2004). Intrathecal injection of galanin results in a biphasic response with facilitation of the nociceptive flexor reflex at low doses and inhibition at high doses (WiesenfeldHallin et al. 1989). Thus, pronociceptive (Wiesenfeld-Hallin et al. 1989; Liu and Hökfelt 2002) and anti-nociceptive (Wiesenfeld-Hallin et al. 1989; 2005; Xu et al. 2000; Liu and Hökfelt 2002; Holmes et al. 2003; Hua et al. 2005) effects of galanin have been described, and differences have been attributed to the involvement of the various galanin receptors (Liu and Hökfelt 2002; Mahoney et al. 2003; Hua et al. 2005; Landry et al. 2005). A role for galanin in bladder–urethral function has been suggested based upon its anatomical distribution in lower urinary tract tissues (Bauer et al. 1986; Newton 1992a,b). Galanin expression has been documented in autonomic regions of the lumbosacral spinal cord, and a sexual dimorphism has been demonstrated in laminae VII and X (Newton 1992a,b). Recent studies (Zvarova et al. 2004) from our laboratory have demonstrated prominent upregulation of galanin in bladder afferent cells in the DRG and in the L2 and S1 spinal cord after central injury (i.e., spinal cord injury). However, decreases in galanin expression have also been observed in the L1 spinal cord nearest the spinal transection site (Zvarova et al. 2004). In the present study, we have investigated (1) the effects of CYP-induced cystitis (acute, intermediate, or chronic) on lumbosacral spinal cord and DRG galanin expression; (2) the topography of intensity of galanin immunostaining in specific regions of the lumbosacral spinal cord (dorsal horn, DH; lateral collateral pathway, LCP; dorsal commissure, DCM; sacral parasympathetic nucleus, SPN; intermediolateral cell column, IML); and (3) bladder afferent neuron expression of galanin in lumbosacral DRG in control and CYP-treated rats.
Materials and methods CYP-induced cystitis Female Wistar rats were studied. Acute (n=14), intermediate (n=17–19), and chronic (n=16) CYP-induced cystitis rat models (Hu et al. 2003; Dattilio and Vizzard 2005) were examined. To induce chronic cystitis, rats received CYP (Sigma-Aldrich, St. Louis, Mo.) by injections (75 mg/kg, intraperitoneal, i.p.) every third day for 10 days. For acute CYP-induced cystitis, rats received a single injection (150 mg/kg, i.p.) and were allowed to survive for 4 h. For intermediate CYP-induced cystitis, rats received a single injection (150 mg/kg, i.p.) and were allowed to survive for 48 h. Control rats (n=15) received no treatment. All injections were performed under isoflurane (2%)
anesthesia. All experimental protocols involving animal use were approved by the University of Vermont Institutional Animal Care and Use Committee (IACUC no. 99– 059, 03–030). Animal care was under the supervision of the University of Vermont’s Office of Animal Care Management 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 label bladder afferent neurons retrogradely in control (n=8) and CYP-treated (n=6–10 for each group) rats. As previously described (Vizzard 2000c; Qiao and Vizzard 2002a,b), a total volume of 40 μl 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. Perfusion, tissue harvesting, and immunohistochemistry Tissue processing After CYP treatment (4 h, 48 h, or chronic), animals were deeply anesthetized with isoflurane (3%–4%) and then killed via intracardiac perfusion, first with oxygenated Krebs buffer (95% O2, 5% CO2) and then with 4% paraformaldehyde. Following perfusion, the spinal cord and DRG were quickly removed and postfixed for 3 h. Tissue was then rinsed in phosphate-buffered saline (PBS; 0.1 M NaCl, 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 was present after the last rib, and (2) the L6 vertebra was the last moveable vertebra followed by the fused sacral vertebrae. Another less precise criterion was the observation that the L6 DRG were the smallest ganglia following the largest, viz., L5 DRG. DRG and spinal cord segments (L1, L2, L4–S1) were sectioned parasagittally at a thickness of 20 μm or 40 μm, respectively, on a freezing microtome. Some DRG (L1, L2, L6, S1) were specifically chosen for analysis based upon the previously determined segmental representation of urinary bladder circuitry (Donovan et al. 1983; Keast and de Groat 1992; Nadelhaft and Vera 1995). Bladder afferents are not distributed within the L4–L5 DRG (Donovan et al. 1983; Keast and de Groat 1992) that contain only somatic afferents nor are neurons that are involved in urinary bladder function
215
observed in the L4–L5 spinal segments (Nadelhaft and Vera 1995). Thus, the L4–L5 DRG served as internal controls for these studies. Tissues from control animals with no treatment were handled in an identical manner to that described above. Galanin immunohistochemistry Spinal cord sections, detrusor, and urothelium for both control and CYP-treated rats were processed for galanin immunoreactivity (IR) by using a free–floating method (Zvarova et al. 2004). DRG were immunostained by an on– slide processing technique. Groups of control animals and experimental animals were processed simultaneously to decrease the possible incidence of variation in staining and background between tissues and between animals. Spinal cord and DRG sections, detrusor, or urothelium were incubated overnight at room temperature with rabbit anti– galanin antibody (1:3,000, Phoenix Pharmaceuticals) in 1% goat serum and 0.1 M KPBS (PBS with potassium), and then washed (3×10 min) with 0.1 M KPBS, pH 7.4. Tissue was then incubated with Cy3-conjugated goat anti– rabbit IgG (1:500; Jackson ImmunoResearch) for 2 h at room temperature. After several rinses with 0.1 M KPBS, tissues were mounted with Citifluor (Citifluor, London, UK) on slides and coverslipped. Control tissues incubated in the absence of primary or secondary antibody were also processed and evaluated for specificity or background staining levels. In the absence of primary antibody, no positive immunostaining was observed. 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 of 610–655 nm. In DRG from control and CYP-treated rats, galanin–immunoreactive cell profiles were counted in 10–15 sections of each selected DRG (L1, L2, L4–S1). Galanin–IR encircling DRG cells was also determined in 10–15 sections of each selected DRG (L1, L2, L4–S1) from control and CYP-treated groups. Only cell profiles with a nucleus were quantified. DRG sections with FB-labeled cells were viewed with a filter with an excitation wavelength of 340– 380 nm and an emission wavelength of 420 nm. Cells colabeled with FB and galanin–IR were similarly counted. Numbers of galanin–immunoreactive cell profiles per DRG section are presented (mean±SEM). The percentage of presumptive bladder afferent cells (FB-labeled) expressing galanin–IR in each DRG examined is also presented (mean±SEM). The results were not corrected for doublecounting. Comparisons between control and CYP-treated groups were made by using an analysis of variance
(ANOVA). 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 ANOVA. Thus, day was treated as a blocking effect in the model. Two variables were tested in the analysis: (1) experimental manipulation versus none (control), and (2) the effect of day (i.e., tissue from experimental and control groups of animals were processed on different days). When F ratios exceeded the critical value (P≤0.05), the Newman–Keuls test was used for multiple comparisons among means. Assessment of positive staining in spinal cord and DRG Staining observed in experimental tissue was compared with that observed from experiment-matched negative controls. Tissues exhibiting immunoreactivity that was greater than the background level observed in experimentmatched negative controls were considered positively stained. Spinal cord densitometry The density of galanin–IR in specific regions of spinal cord was determined by densitometry analysis (Image–Pro express, version 4.0, Media Cybernetics) as previously described (Vizzard 1999, 2000c; Zvarova et al. 2004). Spinal cord segments were sectioned entirely, from rostral to caudal. Every third tissue section was then processed for galanin–IR. Of these tissue sections, every fifth tissue section was then used for semi–quantitative analysis of galanin–IR. We did not select sections based upon staining intensity, and no sections were discarded from the 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 CYP-treated and control animals were analyzed: superficial, lateral DH (LDH), medial DH (MDH), and DCM regions, the region of the SPN (L6, S1), the region of the IML (L1, L2), and the region of the LCP (L6, S1; see Fig. 3e). Spinal cord sections were viewed with a 4× objective and captured through a video camera attachment to the microscope with the exposure time, brightness, and contrast being held constant. The image was converted into pixels on the computer monitor according to a gray scale that ranged 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, and LCP regions). The labeled area within the square was measured (see Fig. 3e). Transmittance (t) was calculated as t = (gray level + 1/256). Optical density (OD) was derived from OD=–log t. The background staining for each section was determined from the ventral horn and
216
subtracted from areas of interest. Comparisons among control and experimental groups were made by ANOVA. When F ratios exceeded the critical value (P≤0.05), Dunnett’s test was used to compare the control mean with the experimental mean.
acquiring images from control and CYP-treated animals processed. Images were analyzed on the same day; they were imported into Adobe Photoshop 7.0 (Adobe Systems Incorporated, San Jose, CA) where groups of images were assembled and labeled.
Figure preparation Digital images were obtained by using a charge-coupled device camera (MagnaFire SP; Optronics, Optical Analysis, Nashua, N.H.) and an LG–3 frame grabber attached to an Olympus microscope (Optical Analysis). Exposure times, brightness, and contrast were held constant when
Fig. 1 Galanin immunoreactivity (IR) in the L1 spinal cord segment is decreased in specific regions after acute (4 h) cyclophosphamide (CYP) treatment (IML intermediolateral cell column, CC central canal). Fluorescence images of the L1 spinal segment from a control (a–c) and a CYP-treated (d–f) rat. Low-power fluorescence images of the L1 spinal segment from a control (a) and a 4-h CYP-treated (d) rat. In control groups, galanin–IR was present in the superficial dorsal horn (DH) and dorsal commissure region (DCM); however, little if any galanin–IR was observed in the ventral horn (VH). Higher power fluorescence images of the dorsolateral quadrant of the L1 spinal segment from control (b) or acute CYP-treated rats (e). With acute CYP treatment, galanin–IR decreased in the lateral and medial dorsal horn (LDH, MDH) regions (e vs. b). Galanin–IR in the DCM region also decreased in the DCM with acute CYP treatment (f vs. c). In both control and CYP-treated groups, galanin–IR was punctate, with no labeling of neuronal cell bodies being observed. Bar 320 μm (a, e), 95 μm (b, e), 40 μm (c, f)
Fig. 2 Galanin–IR in the L6 spinal segment is increased in specific regions with chronic CYP treatment. Fluorescence images of the L6 spinal segment from a control (a–d) and a chronic CYP-treated (d– h) rat. Low-power fluorescence images of the L6 spinal segment from a control (a) and a chronic CYP-treated (b) rat. In control groups, galanin–IR was present in the superficial dorsal horn (DH) and dorsal commissure region (DCM); however, little if any galanin–IR was observed in the ventral horn (VH). Higher power fluorescence images of the dorsolateral quadrant of the L6 spinal segment from control (b) or chronic CYP-treated (f) rats. With chronic CYP treatment, galanin–IR increased in the dorsal horn region (f vs. b). Galanin–IR in the DCM region also increased after chronic CYP treatment (g vs. cC). Increased galanin–IR was also observed in the region of the sacral parasympathetic nucleus (SPN) after chronic CYP treatment (h vs. d). In both control and CYPtreated groups, galanin–IR was punctate in nature with no labeling of neuronal cell bodies observed. Bar 320 μm (a, e), 95 μm (b, f, 40 μm (c, d, g, h)
217
Results Galanin–IR in spinal cord Distribution and general characteristics In control animals and at all segmental levels (L1, L2, L4– S1) examined, galanin–IR was expressed in distinct regions of the rat spinal cord as previously described. Briefly, galanin–IR was unique to specific segmental levels, i.e., rostral lumbar, L1–L2 (Fig. 1) and lumbosacral spinal cord, L6–S1 (Fig. 2), and other staining was similar in all segmental levels (L1, L2, L4–S1) examined. In all segmental levels examined in control animals, galanin–IR was expressed in the superficial DH, MDH, and LDH (laminae I–II; Figs. 1a,b, 2a,b, 3e), and in the region of the DCM (Figs. 1a,c, 2b,c, 3a,e). In the rostral lumbar (L1–L2; Figs. 1b, 3a) and caudal lumbosacral (L6–S1; Fig. 3e)
Fig. 3 Galanin–IR is altered in specific regions of the lumbosacral spinal cord (a–d L1, e–h L6). Fluorescence images of galanin–IR in L1 (a) and L6 (e) spinal segments in controls (a, e) or after CYP treatment (b acute, L1; f acute, L6; c intermediate, L1; g intermediate, L6; d chronic, L1; h chronic, L6). The image was converted to a gray scale ranging in intensity from 0 (white) to 255 (black) and was centered in the field. A standard size square (e, 1–12) was overlaid on the areas of interest (LDH lateral dorsal horn, MDH medial dorsal horn, DCM dorsal commissure, SPN sacral parasympathetic nucleus, LCP lateral collateral pathway), and the labeled area within the square was measured. Bar 150 μm
spinal cord, galanin–IR was also expressed in the region of the IML or SPN (Figs. 2b,d, 3e). Little if any galanin–IR was observed in the ventral horn of any segment examined (Figs. 1a, 2a). Some galanin–IR was observed in control animals in the LCP (Morgan et al. 1981; Steers and de Groat 1988; Steers et al. 1991b) in the L6–S1 segments (Fig. 3e). In the spinal cord, galanin–IR was not expressed in neuronal cell bodies but had a punctate appearance being present in nerve fibers (Figs. 1a–c, 2a–d). Changes in galanin–IR in the spinal cord following CYP-induced cystitis Following CYP treatment, galanin–IR was expressed in identical spinal cord regions as observed in the control, althouugh the intensity had decreased or increased in spinal cord segments and specific regions (see below; Figs. 1d–
218
Fig. 4 Histograms representing optical density (O.D.) measurements of galanin–IR in specific regions of the L1 (a), L2 (b), L4 (c), L5 (d), L6 (e), and S1 (f) spinal segments in control rats and rats treated with CYP (acute, 4 h; intermediate, 48 h; chronic, 10 days). Intensity of galanin–IR was determined in the medial dorsal horn (MDH), lateral dorsal horn (LDH), dorsal commissure (DCM), lateral collateral pathway (LCP), and in the region of the interme-
diolateral cell column (IML) or sacral parasympathetic nucleus (SPN). a Significant decreases (P≤0.01) in galanin–IR were demonstrated in the MDH, LDH, LCP, IML and SPN regions of spinal segments examined (L1–S1) with acute or intermediate CYP treatment (asterisks). e Significant increases (P≤0.01) in galanin–IR were detected in the DCM, SPN and LCP regions in the L6–S1 spinal segments with chronic CYP treatment (asterisks)
219
Fig. 5 Few bladder afferent cells in the lumbosacral dorsal root ganglia (DRG) express galanin (Gal)–IR in control (a1, a3) or CYPtreated rats (b1, b3). a1, b1 Fluorescence photographs of galanin– immunoreactive cells (arrows, arrowheads) in the L6 DRG from control (a1) and chronic CYP-treated (b1) rats. Bladder afferent cells in the DRG were labeled by retrograde transport of Fast blue (FB; a2, b2). Some bladder afferent cells express galanin–IR before
(a1, a2, arrowheads) and after CYP treatment (b1, b2, arrowheads). The majority of bladder afferent cells do not express galanin–IR in control or CYP-treated rats. a3, b3 Merged images demonstrating bladder afferent cells that also express galanin–IR (pink–purple, arrowheads) in control (a3) or after CYP treatment (b3). After CYP treatment, perineuronal galanin–IR is observed encircling small and medium-sized DRG cells (b1, b3, arrows). Bar 25 μm
f, 2e–h). As seen in control animals, galanin–IR was absent in neuronal cell bodies and was restricted to nerve fibers in the spinal cord after CYP treatment (Figs. 1d–f, 2e–h, 3b– d, f–h).
Galanin–IR in lumbosacral DRG
Galanin–IR is decreased in rostral lumbar (L1–L2) and caudal lumbosacral (L4–S1) spinal cord following acute (4 h) and intermediate (48 h) CYP treatment Following acute or intermediate CYP treatment, galanin– IR decreased in many regions in the L1–S1 spinal cord compared with the control (Figs. 1b versus e,c versus f, 3a– c, 4a–f). The density of galanin–IR was significantly decreased (P≤0.01) in the superficial laminae (I–II; ~two– fold) of the dorsal horn (MDH and LDH) in all segments examined (L1–S1; Figs. 1b,e, 4a–f), in the region of the dorsal commissure (1.5–fold to 2.3–fold) in all segments examined (L1–S1; Figs. 1c,f, 4a–f), in the IML region for L1–L2 spinal segments (~two–fold; Fig. 4a,b), and in the SPN region (2.3–fold) and LCP (1.3–1.7–fold) for L6–S1 spinal segments (Fig. 4e,f).
In contrast to galanin–IR in the spinal cord, galanin–IR in the DRG (L1–S1) was expressed by neuronal cell bodies and fibers throughout each DRG examined (Fig. 5a1,b1). In control animals, galanin–IR was present in small numbers of cells in the L1–S1 DRG (Figs. 5a1,b1, 6). The number of galanin–immunoreactive cells among the DRG examined was comparable (range: 12–30 galanin– immunoreactive cell profiles/section; Fig. 6). After acute, intermediate, or chronic CYP treatment, no changes in the number of galanin–immunoreactive DRG cells were observed in any DRG examined (Figs. 5a3,b3, 6). Both small (18.8±4.0 μm) and medium (24.5±2.5 μm)-sized DRG cells expressed galanin–IR in control animals and in those after CYP treatment. Although no changes in the
Galanin–IR is increased in L6–S1 spinal cord following chronic CYP treatment For the most part, galanin–IR returned to control levels with chronic CYP treatment (Fig. 4a–d); however, the MDH region of the L1 spinal segment (Fig. 4a) and the LDH region of the L5 spinal segment (Fig. 4d) remained significantly reduced compared with controls. In contrast, galanin–IR was significantly increased (P≤0.01) in the DCM region (1.25–fold), SPN region (1.4–fold), and LCP of the L6 (Fig. 4e) or S1 (Figs. 2e–h, 4f) spinal segments after chronic CYP treatment.
Fig. 6 Histogram depicting the number of galanin-immunoreactive (Gal-IR) DRG cells per section in DRG examined in control rats or those treated with CYP (acute, 4 h; intermediate, 48 h; chronic, 10 days). No changes in the numbers of galanin–immunoreactive cells in DRG examined (L1–S1) were observed between control and CYP–treated groups
220 Table 1 Percentage of bladder afferent cells (Fast-blue-labeled) that express galanin–IR in control and cyclophosphamide (CYP)-treated rats. No significant differences were observed CYP-induced cystitis
DRG (% of bladder afferent cells with galamin-IR) L1
Control Acute/4 h Intermediate/48 h Chronic
1.3±0.9 3.5±0.3 4.5±1.9 7.6±4.7
L2 0.7±0.3 5.5±2.4 7.4±5.3 9.9±8.0
L6 3.1±2.2 3.2±0.7 4.1±0.7 6.2±3.5
S1 3.9±3.0 4.7±1.0 6.2±1.2 10.5±4.9
number of DRG cells expressing galanin–IR were observed after CYP treatment, galanin–IR encircling DRG cells (i.e., perineuronal galanin–IR) was observed in DRG sections after chronic CYP treatment (Fig. 5a3,b3). Galanin–IR was observed encircling medium-sized DRG cells lacking galanin–IR after CYP treatment (Fig. 5a3,b3). Perineuronal galanin–IR in lumbosacral DRG was detected in all animals treated chronically with CYP (eight out of eight animals), whereas no perineuronal galanin–IR was detected in any DRG from a control animal (six out of six animals). The numbers of DRG cells encircled with galanin–IR varied (range: 10–30) from section to section, but perineuronal staining was observed in each DRG examined from chronic CYP-treated rats. Galanin–IR in bladder afferent cells in DRG is unchanged after CYP treatment To determine whether galanin–IR was expressed in bladder afferent cells, FB was injected into the urinary bladder to retrogradely label bladder afferent cells in the L1, L2, L6, and S1 DRG (Fig. 5a2,b2). In control animals, approximately 2.3% of bladder afferent cells in the L1, L2, L6 or S1 DRG exhibited galanin–IR (Fig. 5b2), similar to findings in our previous report (Zvarova et al. 2004). After CYP treatment of all durations examined, no changes in the percentage of bladder afferent cells exhibiting galanin–IR were observed (Table 1). The number of galanin–immunoreactive cell profiles/section in DRG (L1– S1) examined was similar with or without the presence of FB (data not shown).
Discussion The present studies demonstrate significant changes in galanin–IR in specific regions of the lumbosacral spinal cord after CYP-induced cystitis of variable duration. CYPinduced cystitis of an acute (4 h) or intermediate (48 h) duration resulted in decreases in galanin expression in a number of regions of the L1–S1 spinal cord. In contrast, chronic CYP-induced cystitis was associated with increases in galanin expression in specific regions of a limited number of spinal segments (L6–S1). Thus, CYPinduced cystitis resulted in a biphasic response in the
lumbosacral spinal cord with initial decreases in galanin– IR after acute CYP-induced cystitis and recovery or even increases in galanin–IR with chronic CYP-induced cystitis. The number of galanin–immunoreactive cells in any DRG examined (L1–S1) did not change with any duration of CYP-induced cystitis. No changes in the percentage of bladder afferent cells exhibiting galanin–IR were observed. Galanin–immunoreactive nerve fibers encircling DRG cells (i.e., perineuronal staining) were observed after chronic CYP treatment. Galanin is widely expressed in the lumbosacral spinal cord and a role in regulating pelvic function has been suggested. It has also been documented in autonomic regions of the lumbosacral spinal cord (Newton 1992a,b). In the present study, the expression of galanin has been determined in DCM, SPN, LCP, MDH, and LDH from female rats of unknown estrous cycle status. Sexual dimorphism has been demonstrated in the DCM and SPN regions (Newton 1992a,b), with male rats exhibiting significantly more galanin–IR than female rats. In addition, galanin–IR in these same regions (DCM and SPN) varies with the estrous cycle in females (Newton 1992a). Despite no knowledge of estrous cycle status, low variance in galanin–IR as determined with densitometry has been observed in all of spinal cord regions examined in this study, including the DCM and SPN regions. Thus, CYP-induced cystitis is thought to have a larger impact on the changes in galanin expression compared with the potential impact of estrous status, if any. Similarly, our previous study (Zvarova et al. 2004) has demonstrated significant changes with low variance in galanin expression in lower urinary tract tissues after spinal cord injury. These studies demonstrate spinal cord changes in galanin expression following CYP-induced cystitis; these changes may play a role in altered bladder function and sensory processing. However, no changes in galanin expression in bladder afferent cells in DRG have been demonstrated. The earliest time point after CYP-induced cystitis examined in the present study is 4 h; therefore, changes in galanin expression in DRG may have occurred prior to this time point but this seems unlikely in light of our previous studies (Qiao and Vizzard 2002a,b). In addition, we have demonstrated changes, in DRG, in the number of bladder afferent cells expressing various neuroactive compounds (Vizzard 1997, 2000c, 2001), including galanin (Zvarova et al. 2004), with urinary bladder dysfunction, by using identical techniques as those employed in the present study. However, the current finding does not preclude potential changes in galanin transcripts in lumbosacral DRG during CYPinduced cystitis. The present studies are consistent with previous studies examining acute and chronic inflammation pain models (Ji et al. 1995; Calza et al. 1998) but contrast with those of Callsen-Cencic and Mense (1997) who have demonstrated increases in galanin expression in bladder afferent cells in the L6 and S1 DRG 48 h after instillation of mustard oil (2%) into the urinary bladder. These differences may be attributed to the use of different models of bladder inflammation (CYP vs. mustard oil),
221
gender (female vs. male), or rat strain (Wistar vs. Sprague–Dawley). Previous studies have demonstrated both peripheral and central origins of galanin–IR in the spinal cord including brain, brainstem, DRG, and intrinsic spinal neurons (Tuchscherer and Seybold 1989; Zhang et al. 1995; Ohmachi et al. 1996). Thus, changes in galanin expression in the spinal cord with CYP-induced cystitis may stem from changes in these sources. Although we have not demonstrated changes in galanin in the DRG during cystitis, cystitis may stimulate the release of galanin from spinal cord primary afferent terminals, thereby resulting in galanin reduction in the superficial dorsal horn observed with acute and intermediate CYP-induced cystitis. Alternatively, changes in intraspinal (Ji et al. 1995; Landry et al. 2005) and supraspinal (Xu et al. 2005) galanin sources may also contribute to changes in galanin expression in the spinal cord observed during CYP-induced cystitis. Perineuronal baskets expressing galanin–IR have also been consistently observed in DRG after chronic CYPinduced cystitis. Similar perineuronal galanin–IR has been demonstrated in DRG after peripheral nerve lesions (Hu and McLachlan 2001) or in pelvic ganglia (Callsen– Cencic and Mense 1997) after bladder inflammation and has been suggested to contribute to altered sensory signals, neuropathic pain (Hu and McLachlan 2001), or altered postganglionic signaling (Callsen–Cencic and Mense 1997). Chronic pathological conditions inducing tissue irritation or inflammation can alter the properties of sensory pathways leading to a reduction in pain threshold (allodynia) and an amplification of painful sensations (hyperalgesia; for a review, see Campbell and Meyer 1986). Increased pain sensitivity can result from changes in peripheral nociceptor afferents (Perl et al. 1974; Beitel and Dubner 1976) or from changes in the central nervous system mechanisms that process nociceptive inputs (Coderre et al. 1993; Meller and Gebhart 1993). These changes have been linked with alterations in the gene expression and synthesis of neurotransmitters (Ruda et al. 1988; Donaldson et al. 1992; Smith et al. 1992; Hanesch et al. 1993). Recent experiments involving a chemically (CYP) induced urinary bladder inflammation model have demonstrated alterations in neurochemical (Vizzard 2000c; 2001; Qiao and Vizzard 2002b, 2004), electrophysiological (Jennings and Vizzard 1999; Yoshimura and de Groat 1999), and organizational (Vizzard 1999, 2000a) properties in micturition reflex pathways. Furthermore, CYP-induced cystitis is associated with increased frequency of voiding in awake rats and urinary bladder overactivity in anesthetized rats (Maggi et al. 1992; Lecci et al. 1994; Lantéri–Minet et al. 1995). In addition, referred hyperalgesia to the abdominal wall (McMahon and Abel 1987) and hindlimb (Jaggar et al. 1999; Guerios et al. 2005) has been demonstrated in rodent models of bladder inflammation. Therefore, these changes suggest considerable reorganization of reflex connections in the spinal cord, marked changes in the properties of micturition reflex pathways following CYPinduced cystitis, and referred hyperalgesia.
Interstitial cystitis (IC) is a chronic inflammatory bladder disease syndrome characterized by urinary frequency, urgency, and suprapubic and pelvic pain. Although the etiology and pathogenesis of IC are unknown, numerous theories including infection, autoimmune disorder, toxic urinary agents, deficiency in bladder wall lining, and neurogenic causes, have been proposed (Erickson and Davies 1998; Erickson 1999). Some patients with chronic pelvic pain, including IC, present with hyperalgesia associated with visceral pain (Wesselmann 2001; FitzGerald 2003). Hyperalgesia may involve referred visceral hyperalgesia to somatic structures and may involve reports of pain radiating to the perineal area, the thighs, the legs, abdomen, and back (Wesselmann 2001). The functional and sensory changes demonstrated with CYP-induced cystitis in rodents or in individuals with IC 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 (Vizzard 1997), pituitary adenylate cyclase–activating polypeptide (PACAP; Vizzard 2000c), substance P, and calcitonin gene-related peptide (CGRP) (Vizzard 2001) immunoreactivity in micturition reflex pathways after CYP-induced cystitis. Prior to examining changes in galanin expression during CYPinduced cystitis, all other neuropeptides examined have been shown significantly to increase in the lumbosacral spinal cord and DRG (Vizzard 2000c, 2001). We have previously suggested that increases in the expression of CGRP and PACAP contribute to urinary bladder overactivity during CYP-induced cystitis (Vizzard 1997, 2001; Vizzard et al. 2003). Earlier studies have demonstrated that galanin has potent neuromodulatory actions on isolated human detrusor and suppresses the cholinergic component of the detrusor response to electric field stimulation (Maggi et al. 1987). Thus, an inhibitory action for galanin on neurotransmitter release has been suggested in smooth muscle tissues and may also pertain to the urinary bladder (Maggi et al. 1987). In the present study, a reduction in galanin expression in the spinal cord of animals with acute CYP-induced cystitis could result in an excitatory shift (i.e., disinhibition) and may therefore be associated with increased efficacy in micturition pathways and increased bladder activity. This is consistent with the bladder overactivity observed during acute CYP treatment in rodents (Maggi et al. 1992, 1993; Hu et al. 2003). In addition, reductions in galanin expression in the spinal segments examined (L1–S1) in rats with acute CYP-induced cystitis may be pronociceptive (Wiesenfeld–Hallin et al. 1989) and contribute to referred somatic hyperalgesia observed in animal models of bladder inflammation (McMahon and Abel 1987; Jaggar et al. 1999; Guerios et al. 2005). In contrast, chronic CYP-induced cystitis is associated with significant increases in galanin expression in specific regions of the L6–S1 spinal cord. The chronic CYP-induced changes are far more restricted both in terms of spinal cord segments affected and in terms of regions affected. Thus, increases in galanin expression in L6–S1 spinal segments may offset changes in PACAP and substance P with known facilitatory effects on the lower urinary tract (Chien et al. 2003; Seki et al. 2005).
222
A possible mechanism underlying the changes in galanin expression in micturition pathways may involve neurotrophic factors and/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, a recent study from our laboratory has demonstrated changes in mRNA and/or protein expression of neurotrophic factors in the urinary bladder after acute or chronic CYP-induced cystitis, including nerve growth factor (NGF), brain-derived neurotrophic factor, glial-derived neurotrophic factor, neurotrophin (NT)–3, and NT–4 (Vizzard 2000b). Excess NGF can increase the production of neuropeptides (i.e., substance P, CGRP, and PACAP) in sensory neurons (Donnerer et al. 1992; Donnerer and Stein 1992; Gary and Hargreaves 1992; Woolf et al. 1997). Intravesical administration of exogenous NGF in animals may facilitate afferent firing and induce bladder overactivity that is blocked by anti–NGF (Dmitrieva et al. 1997). In patients with IC, elevated levels of neurotrophic factors, including NGF have been demonstrated (Okragly et al. 1999). In addition, increased expression of NGF is present in bladder biopsies from women with IC (Lowe et al. 1997). Intravesical administration of NGF acutely induces bladder hyperactivity in rats (Chuang et al. 2001). Referred hyperalgesia after intravesical turpentine is also dependent on NGF (Jaggar et al. 1999). NGF has also been shown negatively to regulate the expression of galanin in DRG cells (Avelino et al. 2002). Thus, changes in the expression of neurotrophic factors after cystitis may result in changes in the neurochemical phenotype of bladder reflex pathways and contribute to urinary bladder dysfunction and to altered sensory processing from visceral and somatic sites. In summary, these studies demonstrate significant changes in galanin expression in the lumbosacral spinal cord during CYP-induced cystitis. Interestingly, significant decreases in galanin expression are observed after acute (4 h) or intermediate (48 h) CYP treatment in a number of regions in the L1–S1 spinal segments, whereas chronic CYP-induced cystitis induces significant increases in restricted regions of the L6–S1 spinal segments. No changes in galanin expression in lumbosacral DRG have been observed. Changes in spinal cord galanin expression may result from changes in galanin release from the endings of spinal cord primary afferents or from changes in intraspinal or supraspinal sources of galanin. Our studies suggest that galanin in CYP-induced cystitis may play different roles in different phases of CYP-induced cystitis. The exact function of galanin in lower urinary tract function needs to be determined but may include contractility effects on the urinary bladder and altered sensory processing from visceral and somatic sites.
References Avelino A, Cruz C, Cruz F (2002) Nerve growth factor regulates galanin and c-jun expression occurring in dorsal root ganglion cells after intravesical resiniferatoxin application. Brain Res 951:264–269 Beitel RE, Dubner (1976) Response of unmyelinated (C) polymodal nociceptors to thermal stimuli applied to monkey’s face. J Neurophysiol 39:1160–1175 Bauer FE, Christofides ND, Hacker GW, Blank MA, Polak JM, Bloom SR (1986) Distribution of galanin immunoreactivity in the genitourinary tract of man and rat.Peptides 7:5-10 Callsen-Cencic P, Mense S (1997) Expression of neuropeptides and nitric oxide synthase in neurones innervating the inflamed rat urinary bladder. J Auton Nerv Syst 65:33–44 Calza L, Pozza M, Zanni M, Manzini CU, Manzini E, Hökfelt T (1998) Peptide plasticity in primary sensory neurons and spinal cord during adjuvant-induced arthritis in the rat: an immunocytochemical and in situ hybridization study. Neuroscience 82:575–589 Campbell JN, Meyer RA (1986) Primary afferents and hyperalgesia. In: Yaksh T (ed) Spinal afferent processing. Plenum, New York, pp 59–81 Chien CT, Yu HJ, Lin TB, Lai MK, Hsu SM (2003) Substance P via NK1 receptor facilitates hyperactive bladder afferent signaling via action of ROS. Am J Physiol 284:F840–F851 Chuang YC, Fraser MO, Yu YB, Chancellor MB, Groat WC de, Yoshimura N (2001) The role of bladder afferent pathways in bladder hyperactivity induced by the intravesical administration of nerve growth factor. J Urol 165:975–979 Coderre TJ, Katx J, Vaccarino AL, Melzack R (1993) Contribution of central neuroplasticity to pathological pain: review of clinical and experimental evidence. Pain 52:259–285 Colvin LA, Mark MA, Duggan AW (1997) The effect of a peripheral mononeuropathy on immunoreactive (ir)-galanin release in the spinal cord of the rat. Brain Res 766:259–261 Cox PJ (1979) Cyclophosphamide cystitis—identification of acrolein as the causative agent. Biochem Pharmacol 28:2045 Dattilio A, Vizzard MA (2005) Up-regulation of protease activated receptors in bladder after cyclophosphamide induced cystitis and colocalization with capsaicin receptor (VR1) in bladder nerve fibers. J Urol 173:635–639 Dmitrieva N, McMahon SB (1996) Sensitisation of visceral afferents by nerve growth factor in the adult rat. Pain 66:87–97 Dmitrieva N, Shelton D, Rice ASC, McMahon SB (1997) The role of nerve growth factor in a model of visceral inflammation. Neuroscience 78:449–459 Donaldson LF, Harmar AJ, McQueen DS, Seckl JR (1992) Increased expression of preprotachykinin, calcitonin generelated peptide, but not vasoactive intestinal peptide messenger RNA in dorsal root ganglia during the development of adjuvant monoarthritis in the rat. Mol Brain Res 16:143–149 Donnerer J, Stein C (1992) Evidence for an increase in the release of CGRP and sensory nerves during inflammation. Ann N Y Acad Sci 657:505–506 Donnerer J, Schuligoi R, Stein C (1992) Increased content and transport of substance P and calcitonin gene-related peptide in sensory nerves innervating inflamed tissue: evidence for a regulatory function of nerve growth factor in vivo. Neuroscience 49:693–698 Donovan MK, Winternitz SR, Wyss JM (1983) An analysis of the sensory innervation of the urinary system of the rat. Brain Res Bull 11:321–324 Dubner R, Ruda MA (1992) Activity dependent neuronal plasticity following tissue injury and inflammation. Trends Neurosci 15:96–103
223 Erickson DR (1999) Interstitial cystitis: update on etiologies and therapeutic options. J Womens Health Gender-Based Med 8:745–758 Erickson DR, Davies MF (1998) Interstitial cystitis. Int Urogynecol J 9:174–183 FitzGerald MP (2003) Chronic pelvic pain. Curr Womens Health Rep 3:327–333 Gary MB, Hargreaves KM (1992) Enhanced release of immunoreactive CGRP and substance P from spinal dorsal horn slices occurs during carrageenan inflammation. Brain Res 582:139–142 Groat WC de (1993) Anatomy and physiology of the lower urinary tract. Urol Clin North Am 20:383–401 Groat WC de, Araki I (1999) Maturation of bladder reflex pathways during postnatal development. Adv Exp Med Biol 462:253– 263 (discussion 311–220) Groat WC de, Araki I, Vizzard MA, Yoshiyama M, Yoshimura N, Sugaya K, Tai C, Roppolo JR (1998) Developmental and injury induced plasticity in the micturition reflex pathway. Behav Brain Res 92:127–140 Guerios SD, Wang Z-Y, Bjorling DE (2005) Cyclophosphamideinduced peripheral hypersensitivity is mediated by nerve growth factor. Urothelial Cell Physiol Normal Dis States p52 Hanesch U, Pfrommer U, Grubb BD, Schaible H-G (1993) Acute and chronic phases of unilateral inflammation in rat’s ankle are associated with an increase in the proportion of calcitonin generelated peptide-immunoreactive dorsal root ganglion cells. Eur J Neurosci 5:154–161 Hökfelt T, Broberger C, Diez M, Xu ZQ, Shi T, Kopp J, Zhang X, Holmberg K, Landry M, Koistinaho J (1999) Galanin and NPY, two peptides with multiple putative roles in the nervous system. Hormone Metab Res 31:330–334 Holmes FE, Bacon A, Pope RJ, Vanderplank PA, Kerr NC, Sukumaran M, Pachnis V, Wynick D (2003) Transgenic overexpression of galanin in the dorsal root ganglia modulates pain-related behavior. Proc Natl Acad Sci USA 100:6180–6185 Hu P, McLachlan EM (2001) Long-term changes in the distribution of galanin in dorsal root ganglia after sciatic or spinal nerve transection in rats. Neuroscience 103:1059–1071 Hu VY, Malley S, Dattilio A, Folsom JB, Zvara P, Vizzard MA (2003) COX-2 and prostanoid expression in micturition pathways after cyclophosphamide-induced cystitis in the rat. Am J Physiol 284:R574–R585 Hua X-Y, Salgado KF, Gu G, Fitzsimmons B, Kondo I, Bartfai T, Yaksh TL (2005) Mechanisms of antinociception of spinal galanin: how does galanin inhibit spinal sensitizations? Neuropeptides 39:211–216 Hygge-Blakeman K, Brumovsky P, Hao J-X, Wu X-J, Hökfelt T, Crawley JN, Wiesenfeld-Hallin Z (2004) Galanin over-expression decreases the development of neuropathic pain-like behaviors in mice after partial sciatic nerve injury. Brain Res 1025:152–158 Jaggar SI, Scott HC, Rice AS (1999) Inflammation of the rat urinary bladder is associated with a referred thermal hyperalgesia which is nerve growth factor dependent. Br J Anaesth 83:442–448 Jennings LJ, Vizzard MA (1999) Cyclophosphamide-induced inflammation of the urinary bladder alters electrical properties of small diameter afferent neurons from dorsal root ganglia. FASEB J 13:A57 Ji R-R, Zhang X, Zhang Q, Dagerlind Å, Nilsson S, WiesenfeldHallin Z, Hökfelt T (1995) Central and peripheral expression of galanin in response to inflammation. Neuroscience 68:563–576 Keast JR, Groat WC de (1992) Segmental distribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male rats. J Comp Neurol 19:615–623 Landry M, Liu HX, Shi TJ, Brumovsky P, Nagy F, Hökfelt T (2005) Galaninergic mechanisms at the spinal level: focus on histochemical phenotyping. Neuropeptides 39:223–231 Landry M, Aman K, Dostrovsky J, Lozano AM, Carlstedt T, Spenger C, Josephson A, Wiesenfeld-Hallin Z, Hökfelt T (2003) Galanin expression in adult human dorsal root ganglion neurons: initial observations. Neuroscience 117:795–809
Lantéri-Minet M, Bon K, Pommery J de, Michiels JF, Menétrey D (1995) Cyclophosphamide cystitis as a model of visceral pain in rats: model elaboration and spinal structures involved as revealed by the expression of c-Fos and Krox-24 proteins. Exp Brain Res 105:220–232 Lecci A, Giulani S, Santiciolo P, Maggi CA (1994) Involvement of spinal tachykinin NK1 and NK2 receptors in detrusor hyperreflexia during chemical cystitis in anaesthetized rats. Eur J Pharm 259:129–135 Liu HX, Hökfelt T (2002) The participation of galanin in pain processing at the spinal level. Trends Pharmacol Sci 23:468–474 Lowe EM, Anand P, Terenghi G, Willimans-Chestnut RE, Sinicropi DV (1997) Increased nerve growth factor levels in the urinary bladder of women with idiopathic sensory urgency and interstitial cystitis. Br J Urol 79:572–577 Maggi CA, Santicioli P, Patacchini R, Turini D, Barbanti G, Beneforti P, Giuliani S, Meli A (1987) Galanin: a potent modulator of excitatory neurotransmission in the human urinary bladder. Eur J Pharmacol 143:135–137 Maggi CA, Lecci A, Santiciolo P, Del Biance E, Giuliani S (1992) Cyclophosphamide cystitis in rats: involvement of capsaicinsensitive primary afferents. J Auton Nerv Syst 38:201–208 Maggi CA, Lecci A, Santicioli P, Del Bianco E, Giuliani S (1993) Cyclophosphamide-induced cystitis in rats: involvement of capsaicin-sensitive primary afferents. Agents Actions 38: C28–C30 Mahoney SA, Hosking R, Farrant S, Holmes FE, Jacoby AS, Shine J, Iismaa TP, Scott MK, Schmidt R, Wynick D (2003) The second galanin receptor GalR2 plays a key role in neurite outgrowth from adult sensory neurons. J Neurosci 23: 416–421 McMahon SB, Abel C (1987) A model for the study of visceral pain states: chronic inflammation of the chronic decerebrate rat urinary bladder by irritant chemicals. Pain 28:109–127 Meller ST, Gebhart GF (1993) Nitric oxide (NO) and nociceptive processing in the spinal cord. Pain 52:127–136 Morgan C, Nadelhaft I, Groat WC de (1981) The distribution of visceral primary afferents from the pelvic nerve within Lissauer’s tract and the spinal gray matter and its relationship to the sacral parasympathetic nucleus. J Comp Neurol 201:415–440 Morrison JFB (1987) Reflex control of the lower urinary tract. In: Torrens M, Morrison JFB (eds) The physiology of the lower urinary tract. Springer, Berlin Heidelberg New York, pp 53–132 Nadelhaft I, Vera PL (1995) Central nervous system neurons infected by pseudorabies virus injected into the rat urinary bladder following unilateral transection of the pelvic nerve. J Comp Neurol 359:443–456 Newton BW (1992a) Galanin-like immunoreactivity in autonomic regions of the rat lumbosacral spinal cord is sexually dimorphic and varies with the estrous cycle. Brain Res 589:69–83 Newton BW (1992b) A sexually dimorphic population of galaninlike neurons in the rat lumbar spinal cord: functional implications. Neurosci Lett 137:119–122 Ohmachi T, Nakamura T, Zhang FZ, Tani I, Takagi H (1996) Morphological analyses of galaninergic inputs to the rat spinal parasympathetic nucleus. Exp Brain Res 109:399–406 Okragly AJ, Niles AL, Saban R, Schmidt D, Hoffman RL, Warner TF, Moon TD, Uehling DT, Haak-Frendscho M (1999) Elevated tryptase, nerve growth factor, neurotrophin-3 and glial cell linederived neurotrophic factor levels in the urine of interstitial cystitis and bladder cancer patients. J Urol 161:438–442 Perl ER, Kumuzawa T, Lynn B, Kenins P (1974) Sensitization of high threshold receptors with unmyelinated (C) afferent fibres. In: Iggo A, Ilynsky I (eds) Somatosensory and visceral receptor mechanisms: progress in brain research. Elsevier, Amsterdam, pp 263–278 Qiao LY, Vizzard MA (2002a) Up-regulation of tyrosine kinase (Trka, Trkb) receptor expression and phosphorylation in lumbosacral dorsal root ganglia after chronic spinal cord (T8– T10) injury. J Comp Neurol 449:217–230
224 Qiao LY, Vizzard MA (2002b) Cystitis-induced upregulation of tyrosine kinase (TrkA, TrkB) receptor expression and phosphorylation in rat micturition pathways. J Comp Neurol 454:200–211 Qiao LY, Vizzard MA (2004) Up-regulation of phosphorylated CREB but not c-Jun in bladder afferent neurons in dorsal root ganglia after cystitis. J Comp Neurol 469:262–274 Ruda MA, Iadarola MJ, Cohen LV, Young II WS (1988) In situ hybridization, histochemistry and immunocyctochemistry reveal an increase in spinal dynorphin biosynthesis in a rat model of peripheral inflammation and hyperalgesia. Proc Natl Acad Sci USA 85:622–626 Seki S, Erickson KA, Seki M, Nishizawa O, Igawa Y, Ogawa T, Groat WC de, Chancellor MB, Yoshimura N (2005) Elimination of rat spinal neurons expressing neurokinin 1 receptors reduces bladder overactivity and spinal c-fos expression induced by bladder irritation. Am J Physiol Renal 288:F466–F473 Smith GD, Harmar AJ, McQueen DS, Seckl JR (1992) Increase in substance P and CGRP, but not somatostatin content of innervating dorsal root ganglia in adjuvant monoarthritis in the rat. Neurosci Lett 137:257–260 Steers WD, Groat WC de (1988) Effect of bladder outlet obstruction on micturition reflex pathways in the rat. J Urol 140:864–871 Steers WD, Kolbeck S, Creedon D, Tuttle JB (1991a) Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function. J Clin Invest 88:1709–1715 Steers WD, Ciambotti J, Etzel B, Erdman S, Groat WC de (1991b) Alterations in afferent pathways from the urinary bladder of the rat in response to partial urethral obstruction. J Comp Neurol 310:1–10 Steers WD, Creedon DJ, Tuttle JB (1996) Immunity to nerve growth factor prevents afferent plasticity following urinary bladder hypertrophy. J Urol 155:379–385 Tuchscherer MM, Seybold VS (1989) A quantitative study of the coexistence of peptides in varicosities within the superficial laminae of the dorsal horn of the rat spinal cord. J Neurosci 9:195–205 Tuttle JB, Steers WD, Albo M, Nataluk E (1994) Neural input regulates tissue NGF and growth of the adult rat urinary bladder. J Auton Nerv Syst 49:147–158 Villar MJ, Cortes R, Theodorsson E, Wiesenfeld-Hallin Z, Schalling M, Fahrenkrug J, Emson PC, Hökfelt T (1989) Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin. Neuroscience 33:587–604 Vizzard MA (1997) Increased expression of neuronal nitric oxide synthase in bladder afferent and spinal neurons following spinal cord injury. Dev Neurosci 19:232–246 Vizzard MA (1999) Alterations in growth-associated protein (GAP43) expression in lower urinary tract pathways following chronic spinal cord injury. Somatosens Motor Res 16:369–381 Vizzard MA (2000a) Increased expression of spinal Fos protein in lower urinary tract pathways induced by bladder distension following chronic cystitis. Am J Physiol 279:R295–R305
Vizzard MA (2000b) Changes in urinary bladder neurotrophic factor mRNA and NGF protein following urinary bladder dysfunction. Exp Neurol 161:273–284 Vizzard MA (2000c) Up-regulation of pituitary adenylate cyclaseactivating polypeptide in urinary bladder pathways after chronic cystitis. J Comp Neurol 420:335–348 Vizzard MA (2001) Alterations in neuropeptide expression in lumbosacral bladder pathways following chronic cystitis. J Chem Neuroanat 21:125–138 Vizzard MA, Qiao L, Zvara P (2003) Neurotrophic mechanisms in bladder overactivity after spinal cord injury. Auton Neurosci 106:14 Wesselmann U (2001) Neurogenic inflammation and chronic pelvic pain. World J Urol 19:180–185 Wiesenfeld-Hallin Z, Xu XJ, Villar MJ, Hökfelt T (1989) The effect of intrathecal galanin on the flexor reflex in rat: increased depression after sciatic nerve section. Neurosci Lett 105:149–154 Wisenfeld-Hallin Z, Xu X-J, Crawley JN, Hökfelt T (2005) Galanin and spinal nociceptive mechanisms: recent results from transgenic and knock-out models. Neuropeptides 39:207–210 Woolf CJ, Allchorne A, Safieh-Garabedian B, Poole S (1997) Cytokines, nerve growth factor and inflammatory hyperalgesia: the contribution of tumour necrosis factor. Br J Pharm 121:417–424 Xu XJ, Hökfelt T, Bartfai T, WiesenfeldHallin Z (2000) Galanin and spinal nociceptive mechanisms: recent advances and therapeutic implications. Neuropeptides 34:137–147 Xu ZQ, Zheng K, Hökfelt T (2005) Electrophysiological studies on galanin effects in brain—progress during the last six years. Neuropeptides 39:269–275 Yoshimura N (1999) Bladder afferent pathways and spinal cord injury: possible mechanisms inducing hyperreflexia of the urinary bladder. Prog Neurobiol 57:583–606 Yoshimura N, Groat WC de (1999) Increased excitability of afferent neurons innervating rat urinary bladder following chronic bladder inflammation. J Neurosci 19:4644–4653 Zhang X, Bean AJ, Wiesenfeld-Hallin Z, Hökfelt T (1995) Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord-IV. Effects of peripheral axotomy with special reference to neuropeptide Y and vasoactive intestinal polypeptide/peptide histidine isoleucine. Neuroscience 64:917–941 Zhang X, Xu ZO, Shi TJ, Landry M, Holmberg K, Ju G, Tong YG, Bao L, Cheng XP, Wiesenfeld-Hallin Z, Lozano A, Dostrovsky J, Hökfelt T (1998) Regulation of expression of galanin and galanin receptors in dorsal root ganglia and spinal cord after axotomy and inflammation. Ann N Y Acad Sci 863:402–413 Zvara P, Kliment J, DeRoss AL, Irwin BH, Malley SE, Plante MK, Vizzard MA (2002) Differential expression of bladder neurotrophic factor mRNA in male and female rats after bladder outflow obstruction. J Urol 168:2682–2688 Zvarova K, Murray E, Vizzard MA (2004) Changes in galanin immunoreactivity in rat lumbosacral spinal cord and dorsal root ganglia after spinal cord injury. J Comp Neurol 475:590–603
