Margaret A. Vizzard

Changes in urinary bladder neurotrophic factor mRNA and NGF protein following urinary bladder dysfunction.

Spinal cord injury and cyclophosphamide-induced cystitis dramatically alter lower urinary tract function and produce neurochemical, electrophysiological, and anatomical changes that may contribute to reorganization of the micturition reflex. Mechanisms underlying this neural plasticity may involve alterations in neurotrophic factors in the urinary bladder. These studies have determined neurotrophic factors in the urinary bladder that may contribute to reorganization of the micturition reflex following cystitis or spinal cord injury. A ribonuclease protection assay was used to measure changes in urinary bladder neurotrophic factor mRNA (betaNGF, BDNF, GDNF, CNTF, NT-3, and NT-4) following spinal cord injury (acute/chronic) or cyclophosphamide-induced cystitis (acute/chronic). The correlation between urinary bladder nerve growth factor mRNA and nerve growth factor protein expression was also determined. Each experimental paradigm resulted in significant (P </= 0.05-0.005) changes in urinary bladder neurotrophic factor mRNA, although the magnitude of the changes differed between paradigms. Urinary bladders from rats with acute spinal cord injury (4 days) exhibited the largest increase in neurotrophic factor mRNA levels (betaNGF, 21-fold increase; BDNF, 78-fold increase; GDNF, 11-fold increase; CNTF, 5.5-fold increase; NT-3, 10-fold increase; NT-4, 25-fold increase) relative to control urinary bladders. More modest but significant increases were demonstrated for urinary bladders from rats with chronic (4-6 weeks) spinal cord injury. Significant increases in urinary bladder neurotrophic factor mRNA levels of comparable magnitude were demonstrated following either acute or chronic cyclophosphamide-induced cystitis. Increased abundance of urinary bladder nerve growth factor mRNA was not always associated with increased total urinary bladder nerve growth factor. Total urinary bladder nerve growth factor decreased following acute or chronic cystitis despite increased abundance of nerve growth factor mRNA. Urinary bladder nerve growth factor mRNA correlates with protein measures 5-6 weeks following spinal cord injury but not earlier. The 5- to 6-week time point coincided with the reemergence of the spinal bladder-to-bladder reflex mechanisms following spinal cord injury. Discrepancies between two measures (mRNA and protein) may reflect retrograde axonal transport of nerve growth factor to the dorsal root ganglia (L6-S1). Retrogradely transported NGF may play a role in altered lower urinary tract function following spinal cord injury or cyclophosphamide-induced cystitis.

Alterations in neuropeptide expression in lumbosacral bladder pathways following chronic cystitis.

These studies examined changes in the expression of calcitonin gene-related peptide (CGRP) and substance P (SP) in lumbosacral (L6-S1) micturition reflex pathways, following chronic cystitis induced by cyclophosphamide (CYP). In control Wistar rats, CGRP- or SP-immunoreactivity (IR) was expressed in fibers in the superficial dorsal horn in all segmental levels examined (L4-S1). Bladder afferent cells in the dorsal root ganglia (DRG; L6, S1) from control animals also exhibited CGRP- (41-55%) or SP-IR (2-3%). Following chronic, CYP-induced cystitis, CGRP- and SP-IR were dramatically increased in spinal segments and DRG (L6, S1) involved in micturition reflexes. The density of CGRP- and SP-IR was increased in the superficial laminae (I-II) of the L6 and S1 spinal segments. No changes in CGRP- or SP-IR were observed in the L4-L5 segments. Staining was also dramatically increased in a fiber bundle extending ventrally from Lissauers tract in lamina I along the lateral edge of the DH to the sacral parasympathetic nucleus in the L6-S1 spinal segments. Following chronic cystitis, CGRP- and SP-IR in cells in the L6 and S1 DRG significantly (P< or =0.05) increased and the percentage of bladder afferent cells expressing CGRP- (76%) or SP-IR (11-18%) also significantly (P< or =0.001) increased. No changes were observed in the L4-L5 DRG. These studies suggest that the neuropeptides, CGRP and SP, may play a role in urinary bladder afferent pathways, following chronic urinary bladder inflammation. Changes in CGRP or SP expression following cystitis may contribute to the altered visceral sensation (allodynia) and/or urinary bladder hyperreflexia in the clinical syndrome, interstitial cystitis.

Overexpression of NGF in mouse urothelium leads to neuronal hyperinnervation, pelvic sensitivity and changes in urinary bladder function

NGF has been suggested to play a role in urinary bladder dysfunction by mediating inflammation, as well as morphological and functional changes, in sensory and sympathetic neurons innervating the urinary bladder. To further explore the role of NGF in bladder sensory function, we generated a transgenic mouse model of chronic NGF overexpression in the bladder using the urothelium-specific uroplakin II (UPII) promoter. NGF mRNA and protein were expressed at higher levels in the bladders of NGF-overexpressing (NGF-OE) transgenic mice compared with wild-type littermate controls from postnatal day 7 through 12-16 wk of age. Overexpression of NGF led to urinary bladder enlargement characterized by marked nerve fiber hyperplasia in the submucosa and detrusor smooth muscle and elevated numbers of tissue mast cells. There was a marked increase in the density of CGRP- and substance P-positive C-fiber sensory afferents, neurofilament 200-positive myelinated sensory afferents, and tyrosine hydroxylase-positive sympathetic nerve fibers in the suburothelial nerve plexus. CGRP-positive ganglia were also present in the urinary bladders of transgenic mice. Transgenic mice had reduced urinary bladder capacity and an increase in the number and amplitude of nonvoiding bladder contractions under baseline conditions in conscious open-voiding cystometry. These changes in urinary bladder function were further associated with an increased referred somatic pelvic hypersensitivity. Thus, chronic urothelial NGF overexpression in transgenic mice leads to neuronal proliferation, focal increases in urinary bladder mast cells, increased urinary bladder reflex activity, and pelvic hypersensitivity. NGF-overexpressing mice may, therefore, provide a useful transgenic model for exploring the role of NGF in urinary bladder dysfunction.

Cystitis-induced upregulation of tyrosine kinase (TrkA, TrkB) receptor expression and phosphorylation in rat micturition pathways.

This study examined tyrosine kinase receptor (Trk) expression and phosphorylation in lumbosacral dorsal root ganglia (DRG) after acute (8 or 48 hours) or chronic (10 days) cyclophosphamide (CYP)–induced cystitis. Increases in the number of TrkA‐immunoreactive (IR) cell profiles were detected in the L1 and L6 DRG (four‐fold; P ≤ 0.01) and the S1 DRG (1.5‐fold; P ≤ 0.05) but not in the L2, L4, and L5 DRG with CYP‐induced cystitis of acute and chronic duration compared with control rats. The number of TrkB‐IR cell profiles increased in the L1 and L2 DRG (L1: 2.6‐fold; L2: 1.4‐fold; P ≤ 0.05) and in the L6 and S1 DRG (L6: 2.2‐fold; S1: 1.3‐fold; P ≤ 0.05) only after acute CYP treatment (8 hours). After CYP treatment, the percentage of bladder afferent cell profiles expressing TrkA‐IR (∼50%; P ≤ 0.05) increased in L1 and L6 DRG. The percentage of bladder afferent cell profiles expressing TrkB‐IR (∼45%; P ≤ 0.05) in L1, L2, L6, and S1 DRG also increased compared with control cell profiles. The increase in TrkA‐IR in bladder afferent cells occurred 8 hours after CYP treatment and was maintained in L1 DRG with chronic (10 days) CYP‐induced cystitis. However, the increase in bladder afferent cells expressing TrkB‐IR only occurred at the most acute time point examined (8 hours). TrkA‐IR and TrkB‐IR cell profiles also demonstrated phosphorylated Trk‐IR with acute and/or chronic CYP‐induced cystitis. These results demonstrated that CYP‐induced cystitis increases the expression and phosphorylation of Trk receptors in lumbosacral DRG. Expression of neurotrophic factors in the inflamed urinary bladder may contribute to this increased expression, and neurotrophic factor and Trk interactions may play unique roles in decreased urinary tract plasticity with CYP‐induced cystitis. J. Comp. Neurol. 454:200–211, 2002.

Increased Expression of Neuronal Nitric Oxide Synthase in Bladder Afferent and Spinal Neurons following Spinal Cord Injury

Changes in the distribution of neuronal nitric oxide synthase immunoreactivity (NOS-IR) after chronic (5-6 weeks) spinal cord injury (SCI) were examined in bladder afferent and spinal neurons in the region of the sacral parasympathetic nucleus (SPN) in the L6-S1 spinal segments. Bladder afferent neurons in the L1, L2, L6 and S1 dorsal root ganglia (DRG) were identified by retrograde axonal transport following injection of fluorogold (FG) into the urinary bladder. A differential distribution of NOS-IR was detected in DRG cells at different segmental levels of spinal cord intact animals with significantly greater numbers of NOS-IR cells present in thoracic (T8, T12; 30-40 NOS-IR cell profiles/section) and rostral lumbar (L1) DRGs (18 NOS-IR cell profiles/ section) compared to caudal lumbosacral (L5-S1) DRGs (0.2-0.4 NOS-IR cell profiles/section). A significant increase in the number of NOS-IR cells was detected in the L6-S1 DRG (p < or = 0.001; 12-20 NOS-IR cell profiles/section) and in the L1-L2 DRG (15-40 NOS-IR cell profiles/section) but not in the L5 DRG following SCI. In these ganglia, an average of 41.2 +/- 7.8% (L6) and 36.3 +/- 0.9% (S1) of FG-labeled bladder afferent neurons were NOS-IR. In contrast, in spinal cord intact animals, no FG-labeled bladder afferent neurons were NOS-IR. Following SCI, NOS-IR fibers were detected along the lateral edge of the dorsal horn extending from Lissauers tract to the region of the SPN (lateral collateral pathway of Lissauer) of the L6 and S1 spinal segments. These NOS-IR fibers were not detected in adjacent spinal segments (L5, S2). SCI also significantly (p < or = 0.001) increased the number of spinal neurons in the region of the SPN (presumptive preganglionic neurons) in the L6-S1 spinal segments exhibiting NOS-IR. These results indicate that NOS-IR in bladder afferent and spinal neurons is plastic and can be up-regulated by chronic SCI. Changes in the neurochemical properties of these neurons after SCI may be mediated by pathological changes in the target organ (i.e., urinary bladder) and/or spinal cord.

Neurochemical plasticity and the role of neurotrophic factors in bladder reflex pathways after spinal cord injury

Transection of the spinal cord that interrupts the spinobulbospinal micturition reflex pathway, abolishes voluntary voiding and initially produces an areflexic bladder with complete urinary retention. 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 animals with an intact spinal cord and can induce bladder hyperreflexia. However, the bladder does not empty efficiently due to a loss of bladder-sphincter coordination (bladder-sphincter dyssynergia). In contrast to normal animals in which the sphincter relaxes during voiding, animals with a spinal cord injury exhibit sphincter contractions during voiding, an increase in urethral outlet resistance, urinary retention, bladder hyperreflexia, bladder overdistension, and an increase in bladder afferent cell size. Changes in electrophysiological or neurochemical properties of bladder afferent cells in the dorsal root ganglia and of spinal pathways could contribute to the emergence of the spinal micturition reflex, bladder hyperreflexia and changes in the pharmacologic responses of reflex pathways in the lumbosacral spinal cord after spinal cord injury. Urinary bladder hyperreflexia after spinal cord injury may reflect a change in the balance of neuroactive compounds in bladder reflex pathways. This review will detail: (1) changes in the neurochemical phenotype of bladder afferent neurons and of spinal neurons mediating micturition reflexes after spinal cord injury, with an emphasis on three neuroactive compounds, neuronal nitric oxide synthase (nNOS), galanin, and pituitary adenylate cyclase activating polypeptide (PACAP); (2) possible functional consequences on bladder reflexes of changes in spinal cord neurochemistry after spinal cord injury, and (3) the potential role of neurotrophic factors expressed in the urinary bladder or spinal cord after spinal cord injury in mediating these neurochemical changes.

Noncompensation in peptide/receptor gene expression and distinct behavioral phenotypes in VIP- and PACAP-deficient mice

Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are closely related neurotrophic peptides of the secretin/glucagon family. The two peptides are derived from a common ancestral gene and share many functional attributes in neuronal development/regeneration which occur not only from overlapping receptor subtype signaling but also through common mechanisms regulating their expression. Although PACAP or VIP null mice have been generated for study, it is unclear whether the expression of the complementary peptide or their receptor systems are altered in a compensatory manner during nervous system development. By radioimmunoassay and quantitative PCR measurements, we first show that PACAP and VIP have very different temporal patterns of expression in developing postnatal mouse brain. In wild‐type animals, PACAP transcript and peptide levels increased rapidly 2‐ and 5‐fold, respectively, within 1 week of age. These levels at 1 week of age were maintained through adulthood. VIP transcript and peptide levels, by contrast, increased 25‐ and 50‐fold, respectively, over a later time course. In parallel studies of development, there were no apparent compensatory increases in brain VIP expression in the PACAP knockout animals, PACAP expression in the VIP‐deficient animals, or receptor mRNA levels in either genotype. To the contrary, there was evidence for developmental delays in the expression of peptide and receptor transcripts in the knockout animals. A series of behavioral and neurological tests demonstrated differences between the knockout genotypes, revealing some functional distinctions between the two genes. These results suggest that the PACAP and VIP have evolved to possess distinct biological activities and intimate that the respective knockout phenotypes represent deficits unmitigated by the actions of the complementary related peptide.

Origin of pituitary adenylate cyclase‐activating polypeptide (PACAP)‐immunoreactive fibers innervating guinea pig parasympathetic cardiac ganglia

The present study investigated the origin of pituitary adenylate cyclase‐activating polypeptide (PACAP) ‐immunoreactive (IR) fibers innervating guinea pig cardiac ganglia. Immunohistochemistry was performed on whole‐mounts containing cardiac ganglia, and sections of stellate, nodose, and dorsal root ganglia (DRG, thoracic levels 1–4), and caudal medulla. In control preparations, only 4% of the cardiac neurons were PACAP‐IR, although most cardiac ganglion cells were surrounded by a network of PACAP‐IR fibers. After 3–7 days in explant culture, the number of PACAP‐IR cardiac neurons increased approximately eightfold. However, virtually all PACAP‐IR fibers surrounding the cardiac neurons had degenerated, demonstrating that the major source of the PACAP‐IR fibers was extrinsic to the cardiac ganglia preparation. PACAP‐ and choline acetyltransferase (ChAT) immunoreactivity were colocalized in fibers within the stellate ganglia but not within neuropeptide Y (NPY) ‐IR cell bodies and fibers. PACAP‐IR cells and fibers were present in the nodose ganglia. PACAP immunoreactivity also was present in fibers and primarily small neurons in thoracic DRGs. In situ hybridization demonstrated the presence of proPACAP mRNA within neurons in the region of the dorsal motor nucleus of the vagus and nucleus ambiguus. PACAP immunoreactivity was colocalized with ChAT immunoreactivity, but not with NPY immunoreactivity or SP immunoreactivity, in fibers surrounding neurons within cardiac ganglia. We conclude that PACAP‐containing fibers innervating the postganglionic parasympathetic neurons in guinea pig cardiac ganglia are primarily preganglionic parasympathetic axons. J. Comp. Neurol. 423:26–39, 2000.

Changes in galanin immunoreactivity in rat lumbosacral spinal cord and dorsal root ganglia after spinal cord injury

Alterations in the expression of the neuropeptide galanin were examined in micturition reflex pathways 6 weeks after complete spinal cord transection (T8). 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; (2) the superficial dorsal horn; (3) the regions of the intermediolateral cell column (L1–L2) and the sacral parasympathetic nucleus (L6–S1); and (4) the lateral collateral pathway in lumbosacral spinal segments. Densitometry analysis demonstrated significant increases (P ≤ 0.001) in galanin immunoreactivity (IR) in these regions of the S1 spinal cord after spinal cord injury (SCI). Changes in galanin‐IR were not observed at the L4–L6 segments except for an increase in galanin‐IR in the dorsal commissure in the L4 segment. In contrast, decreases in galanin‐IR were observed in the L1 segment. The number of galanin‐IR cells increased (P ≤ 0.001) in the L1 and S1 dorsal root ganglia (DRG) after SCI. In all DRG examined (L1, L2, L6, and S1), the percentage of bladder afferent cells expressing galanin‐IR significantly increased (4–19‐fold) after chronic SCI. In contrast, galanin expression in nerve fibers in the urinary bladder detrusor and urothelium was decreased or eliminated after SCI. Expression of the neurotrophic factors nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) was altered in the spinal cord after SCI. A significant increase in BDNF expression was present in spinal cord segments after SCI. In contrast, NGF expression was only increased in the spinal segments adjacent and rostral to the transection site (T7–T8), whereas spinal segments (T13–L1; L6–S1), distal to the transection site exhibited decreased NGF expression. Changes in galanin expression in micturition pathways after SCI may be mediated by changing neurotrophic factor expression, particularly BDNF. These changes may contribute to urinary bladder dysfunction after SCI. J. Comp. Neurol. 475:590–603, 2004.

Up-regulation of tyrosine kinase (Trka, Trkb) receptor expression and phosphorylation in lumbosacral dorsal root ganglia after chronic spinal cord (T8–T10) injury

Previous studies have demonstrated changes in urinary bladder neurotrophic factors after bladder dysfunction. We have hypothesized that retrograde transport of neurotrophin(s) from the bladder to lumbosacral dorsal root ganglia (DRG) may play a role in bladder reflex reorganization after spinal cord injury (SCI). In this study, we determined whether the expression of tyrosine kinase receptors (TrkA, TrkB) is altered in lumbosacral DRG after SCI through immunofluorescence techniques. Complete transection of the spinal cord (T8–T10) was performed in female Wistar rats (120–150 g), and animals were studied 5–6 weeks after SCI. One week before killing, Fast Blue (FB) was injected into the bladder to label bladder afferent cells in the L1, L2, L6, and S1 DRG. After SCI, a significant increase in the number of TrkA‐immunoreactive (IR) positive cells was detected in the L6‐S1 DRG (L6: 1.9‐fold, P ≤ 0.01; S1: 1.7‐fold, P ≤ 0.05) and in the L1 DRG (3.0‐fold; P ≤ 0.01) but not in the L4‐L5 DRG compared with spinal‐intact (control) rats. After SCI, a significant increase in the number of TrkB‐IR cells was also detected in the L6‐S1 DRG (L6: 2.2‐fold, P ≤ 0.01; S1: 1.5‐fold, P ≤ 0.05) and in the L1‐L2 DRG (L1: 1.5‐fold, P ≤ 0.01; L2: 1.3‐fold, P ≤ 0.05) but not in the L4‐L5 DRG compared with control rats. After SCI, the percentage of FB‐labeled cells expressing TrkA immunoreactivity (∼68%) or TrkB immunoreactivity (∼65%) in L1 and L6 DRG significantly (P ≤ 0.01) increased compared with control (20–30%) DRG. After SCI, the percentage of TrkA‐IR cells expressing phosphorylated (p)‐Trk immunoreactivity significantly increased (1.5‐ to 2.3‐fold increase) in the L1, L6, and S1 DRG. The percentage of TrkB‐IR cells expressing p‐Trk immunoreactivity after SCI also increased (1.3‐fold increase) in the L1 and L6 DRG. These results demonstrate that (1) TrkA and TrkB immunoreactivity is increased in bladder afferent cells after SCI and (2) TrkA and TrkB receptors are phosphorylated in DRG after SCI. Neuroplasticity of lower urinary tract reflexes after SCI may be mediated by both nerve growth factor and brain‐derived neurotrophic factor. J. Comp. Neurol. 449:217–230, 2002.