Naoki Yoshimura

Plasticity of Na+ Channels in Afferent Neurones Innervating Rat Urinary Bladder Following Spinal Cord Injury

1 Whole‐cell patch‐clamp recordings in combination with axonal tracing techniques were used to investigate the effects of chronic spinal cord injury on the electrical properties of dorsal root ganglion neurones innervating the urinary bladder or colon of the adult rat. 2 In spinal intact animals, the majority (73–74%) of bladder and colon neurones which were small in size exhibited high‐threshold humped spikes mediated by tetrodotoxin (TTX)‐resistant Na+ channels, whereas large neurones had low‐threshold narrow spikes mediated by TTX‐sensitive Na+ channels. 3 In chronic spinal transected animals, 60% of bladder afferent neurones exhibited TTX‐sensitive low‐threshold spikes. The average diameter and input capacitance of the cells were significantly larger than those of cells obtained from spinal intact animals. 4 In bladder afferent neurones from chronic spinal transected rats, the density of TTX‐resistant Na+ currents significantly decreased from 60.5 to 17.9 pA pF−1, whereas that of TTX‐sensitive currents increased from 32.1 to 80.6 pA pF−1. 5 These changes in action potential and Na+ current characteristics were not detected in colon afferent neurones following spinal cord injury. 6 The results indicate that spinal cord injury increases bladder afferent neurone excitability by shifting the expression of Na+ channels from a high‐threshold TTX‐resistant type to a low‐threshold TTX‐sensitive type. This change in properties may occur in response to alterations in neurotrophic signals originating in the hypertrophied bladder.

Herpes simplex virus vector-mediated gene delivery of glutamic acid decarboxylase reduces detrusor overactivity in spinal cord injured rats

We examined whether replication-defective herpes simplex virus (HSV) vectors encoding the 67u2009kDa form of the glutamic acid decarboxylase (GAD67) gene product, the γ-aminobutyric acid (GABA) synthesis enzyme, can suppress detrusor overactivity (DO) in rats with spinal cord injury (SCI). One week after spinalization, HSV vectors expressing GAD and green fluorescent protein (GFP) (HSV-GAD) were injected into the bladder wall. Rats with SCI without HSV injection (HSV-untreated) and those injected with lacZ-encoding reporter gene HSV vectors (HSV-LacZ) were used as controls. Three weeks after viral injection, continuous cystometry was performed under awake conditions in all three groups. In the HSV-GAD group, the number and amplitude of non-voiding contractions (NVCs) were significantly decreased (40–45% and 38–40%, respectively) along with an increase in voiding efficiency, compared with HSV-untreated and HSV-LacZ groups, but micturition pressure was not different among the three groups. Intrathecal application of bicuculline partly reversed the decreased number and amplitude of NVCs, and decreased voiding efficiency in the HSV-GAD group. In the HSV-GAD group, GAD67 mRNA and protein levels were significantly increased in the L6-S1 dorsal root ganglia (DRG) compared with the HSV-LacZ group, while 57% of DRG cells were GFP-positive, and these neurons showed increased GAD67-like immunoreactivity compared with the HSV-LacZ group. These results indicate that GAD gene therapy effectively suppresses DO after SCI predominantly through the activation of spinal GABAA receptors. Thus, HSV-based GAD gene transfer to bladder afferent pathways may represent a novel approach for treatment of neurogenic DO.

Bladder hyperactivity and increased excitability of bladder afferent neurons associated with reduced expression of Kv1.4 α-subunit in rats with cystitis

Hyperexcitability of C-fiber bladder afferent pathways has been proposed to contribute to urinary frequency and bladder pain in chronic bladder inflammation including interstitial cystitis. However, the detailed mechanisms inducing afferent hyperexcitability after bladder inflammation are not fully understood. Thus, we investigated changes in the properties of bladder afferent neurons in rats with bladder inflammation induced by intravesical application of hydrochloric acid. Eight days after the treatment, bladder function and bladder sensation were analyzed using cystometry and an electrodiagnostic device of sensory function (Neurometer), respectively. Whole cell patch-clamp recordings and immunohistochemical staining were also performed in dissociated bladder afferent neurons identified by a retrograde tracing dye, Fast Blue, injected into the bladder wall. Cystitis rats showed urinary frequency that was inhibited by pretreatment with capsaicin and bladder hyperalgesia mediated by C-fibers. Capsaicin-sensitive bladder afferent neurons from sham rats exhibited high thresholds for spike activation and a phasic firing pattern, whereas those from cystitis rats showed lower thresholds for spike activation and a tonic firing pattern. Transient A-type K(+) current density in capsaicin-sensitive bladder afferent neurons was significantly smaller in cystitis rats than in sham rats, although sustained delayed-rectifier K(+) current density was not altered after cystitis. The expression of voltage-gated K(+) Kv1.4 alpha-subunits, which can form A-type K(+) channels, was reduced in bladder afferent neurons from cystitis rats. These data suggest that bladder inflammation increases bladder afferent neuron excitability by decreasing expression of Kv1.4 alpha-subunits. Similar changes in capsaicin-sensitive C-fiber afferent terminals may contribute to bladder hyperactivity and hyperalgesia due to acid-induced bladder inflammation.

Herpes Simplex Virus Vector Mediated Gene Therapy of Tumor Necrosis Factor-α Blockade for Bladder Overactivity and Nociception in Rats

PURPOSEnWe examined the effects of tumor necrosis factor-α blockade on bladder overactivity and nociception using replication defective HSV vectors expressing tumor necrosis factor-α soluble receptor.nnnMATERIALS AND METHODSnHSV vectors expressing tumor necrosis factor-α soluble receptor or β-galactosidase/green fluorescent protein as the control were injected into the bladder wall of female Sprague-Dawley® rats. Green fluorescent protein was observed with fluorescent microscopy in the bladder and L6 dorsal root ganglia. mRNA and protein expression of tumor necrosis factor-α, and interleukin-1β and 6 as well as myeloperoxidase activity in the bladder were determined by quantitative reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay 4 hours after intravesical resiniferatoxin administration. c-Fos positive neurons were counted in the L6 spinal dorsal horn. Cystometry and behavioral analyses were also performed.nnnRESULTSnGreen fluorescent protein expression was confirmed in the bladder and L6 dorsal root ganglia. Resiniferatoxin administration significantly increased tumor necrosis factor-α mRNA and protein levels in the bladder in controls. Tumor necrosis factor-α mRNA was also increased in the tumor necrosis factor-α soluble receptor group, although tumor necrosis factor-α protein up-regulation was suppressed. The up-regulation of interleukin-1β and 6 mRNA and protein levels, and the myeloperoxidase activity seen in controls were suppressed in the tumor necrosis factor-α soluble receptor group. c-Fos positive cells in the L6 spinal dorsal horn were less prominent in the tumor necrosis factor-α soluble receptor group than in controls. On cystometry the significant decrease in intercontraction intervals after resiniferatoxin infusion detected in controls was not seen in the tumor necrosis factor-α soluble receptor group. On behavioral analyses freezing behavior was significantly decreased in the tumor necrosis factor-α soluble receptor group without affecting licking behavior.nnnCONCLUSIONSnHSV vector mediated tumor necrosis factor-α blockade gene therapy in the bladder and bladder afferent pathways decreases the bladder pain and overactivity induced by nociceptive bladder stimuli.

Upregulation of Androgen-Responsive Genes and Transforming Growth Factor-β1 Cascade Genes in a Rat Model of Non-bacterial Prostatic Inflammation

Prostatic inflammation is associated with the development of prostatic hyperplasia. We investigated the effects of prostatic inflammation on expression levels of androgen‐responsive genes and growth factors in the prostate.

Influence of E. coli-induced prostatic inflammation on expression of androgen-responsive genes and transforming growth factor beta 1 cascade genes in rats

Prostatic inflammation is reportedly associated with the development of prostatic hyperplasia. We investigated the effects of prostatic inflammation on expression levels of androgen‐responsive genes and growth factors in the rat prostate.

Differential contribution of Kv4-containing channels to A-type, voltage-gated potassium currents in somatic and visceral dorsal root ganglion neurons

Little is known about electrophysiological differences of A-type transient K(+) (KA) currents in nociceptive afferent neurons that innervate somatic and visceral tissues. Staining with isolectin B4 (IB4)-FITC classifies L6-S1 dorsal root ganglion (DRG) neurons into three populations with distinct staining intensities: negative to weak, moderate, and intense fluorescence signals. All IB4 intensely stained cells are negative for a fluorescent dye, Fast Blue (FB), injected into the bladder wall, whereas a fraction of somatic neurons labeled by FB, injected to the external urethral dermis, is intensely stained with IB4. In whole-cell, patch-clamp recordings, phrixotoxin 2 (PaTx2), a voltage-gated K(+) (Kv)4 channel blocker, exhibits voltage-independent inhibition of the KA current in IB4 intensely stained cells but not the one in bladder-innervating cells. The toxin also shows voltage-independent inhibition of heterologously expressed Kv4.1 current, whereas its inhibition of Kv4.2 and Kv4.3 currents is voltage dependent. The swapping of four amino acids at the carboxyl portion of the S3 region between Kv4.1 and Kv4.2 transfers this characteristic. RT-PCRs detected Kv4.1 and the long isoform of Kv4.3 mRNAs without significant Kv4.2 mRNA in L6-S1 DRGs. Kv4.1 and Kv4.3 mRNA levels were higher in laser-captured, IB4-stained neurons than in bladder afferent neurons. These results indicate that PaTx2 acts differently on channels in the Kv4 family and that Kv4.1 and possibly Kv4.3 subunits functionally participate in the formation of KA channels in a subpopulation of somatic C-fiber neurons but not in visceral C-fiber neurons innervating the bladder.

Inflammasomes are important mediators of prostatic inflammation associated with BPH

BackgroundThere is mounting evidence to support the role of inflammation in benign prostate hyperplasia (BPH), and a recent study reported expression of inflammasome derived cytokine IL-18 in prostate biopsy of BPH patients. Here we examined the expression of inflammasome-derived cytokines and activation of nucleotide-binding oligomerization domain-like receptor with pyrin domain protein 1 (NLRP) 1 inflammasome in a rat model of prostatic inflammation relevant to BPH.MethodsProstatic inflammation was experimentally induced in three-month-old male Sprague–Dawley rats by intraprostatic injection (50xa0μL) of either 5xa0% formalin or saline (sham) into the ventral lobes of prostate. 7xa0days later, prostate and bladder tissue was harvested for analysis of inflammasome by Western blot, immunohistochemistry and downstream cytokine production by Milliplex.ResultsExpression of interleukins, CXC and CC chemokines were elevated 2-15 fold in formalin injected prostate relative to sham. Significant expression of NLRP1 inflammasome components and caspase-1 in prostate were associated with significant elevation of pro and cleaved forms of IL-1β (25.50u2009±u20091.16 vs 3.05u2009±u20090.65xa0pg/mg of protein) and IL-18 (1646.15u2009±u2009182.61 vs 304.67u2009±u2009103.95xa0pg/mg of protein). Relative to prostate tissue, the cytokine expression in bladder tissue was much lower and did not involve inflammasome activation.ConclusionsSignificant upregulation of NLRP1, caspase-1 and downstream cytokines (IL-18 and IL-1β) suggests that a NLRP1 inflammasome is assembled and activated in prostate tissue of this rat model. Recapitulation of findings from human BPH specimens suggests that the inflammasome may perpetuate the inflammatory state associated with BPH. Further clarification of these pathways may offer innovative therapeutic targets for BPH-related inflammation.

Laser‑capture microdissection for analysis of cell type‑specific gene expression of muscarinic receptor subtypes in the rat bladder with cyclophosphamide‑induced cystitis

AbstractPurposenThis study examined whether the laser-capture microdissection (LCM) method can achieve separation of urothelial cells from detrusor cells or superficial urothelial cells from intermediate/basal urothelial cells, using α-smooth muscle actin (SMA) and cytokeratin 20 (CK20). In addition, we investigated the changes in expression of muscarinic receptors in laser-captured urothelial and detrusor cells in rats with chronic cystitis.MethodsFemale SD rats were injected with cyclophosphamide (75xa0mg/kg) intraperitoneally at day 1, 4, 7 and 10 to induce chronic cystitis. Saline was injected in the same protocol for controls. Bladder specimens were cut at 8xa0μm thickness, fixed in 70xa0% ethanol and lightly stained by hematoxylin and eosin, and then superficial urothelium, intermediate/basal urothelium and detrusor muscles were laser-captured separately. Real-time PCR was performed to examine expressions of α-SMA, CK20, muscarinic 2 receptors (M2R) and muscarinic 3 receptors (M3R).ResultsThe expression of α-SMA mRNA in detrusor muscle cells was 200 times higher than that in urothelial cells in controls. CK20 mRNA expression in apical urothelial cells was 55 times more than that in detrusor muscle and four times more than that in intermediate/basal urothelial cells. Expressions of M2R and M3R mRNA were increased in urothelial cells and decreased in detrusor muscles following chronic cystitis.ConclusionsThe LCM could be useful for tissue collection of detrusor muscle and different layers of urothelial cells with minimal contamination of other cell types, and cell type-specific changes in molecular expression could accurately be analyzed. Increased expression of urothelial MR might enhance urothelial–afferent interactions to induce bladder overactivity/pain conditions associated with bladder inflammation.