Ivermectin-induced activation of mutant glycine receptors delivered to Aδ-fiber bladder afferents by herpes simplex virus vectors driven by a subpopulation-specific neurofilament promoter improves detrusor-sphincter dyssynergia and inefficient voiding in mice with spinal cord injury

Saito T1, Shimizu N1, Goins W1, Suzuki T1, Shimizu T1, Wada N1, Ishizuka O2, Glorioso J1, Yoshimura N1

Research Type

Basic Science / Translational

Abstract Category

Neurourology

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PRIZE AWARD: Best Non-Clinical Abstract
Abstract 272
Best Basic Science
Scientific Podium Session 16
Thursday 5th September 2019
09:30 - 09:45
Hall G3
Basic Science Spinal Cord Injury Voiding Dysfunction
1.University of Pittsburgh, 2.Shinshu University
Presenter
T

Tetsuichi Saito

Links

Abstract

Hypothesis / aims of study
Neurogenic lower urinary tract dysfunction induced by spinal cord injury (SCI) rostral to the lumbosacral level includes detrusor overactivity (DO) and detrusor sphincter dyssynergia (DSD), and the latter leads to inefficient voiding with high post-void residual volume.  Previous studies have reported that treatments targeting C-fiber afferent pathways using antibodies against nerve growth factor or capsaicin pretreatment improved DO, but not DSD [Refs 1, 2].  We therefore hypothesized that another population of bladder afferent pathway; i.e., neurofilament (NF)-positive Aδ-fiber afferents, contributes to DSD inducing hyperactivity of the external urinary sphincter (EUS) during voiding. Thus, this study utilized a chemogenetic approach using the double mutant glycine (G2M) receptor with increased sensitivity to ivermectin, activation of which induces a chloride ion influx and causes hyperpolarization in neuronal cells [Ref 3], in combination with replication-defective herpes simplex virus (HSV) vector-mediated gene delivery methods.  By inoculating HSV vectors driven by an NF200 promoter (NF200p) to the bladder wall, we assessed the morphological changes of bladder afferent neurons after SCI and examined the effect of ivermectin-induced activation of G2M receptors expressed in Aδ-fiber bladder afferent pathways on DSD and inefficient voiding in mice with SCI.
Study design, materials and methods
All experiments were conducted in accordance with institutional guidelines and was approved by the institutional animal care and use committee. Thirty 8 to 9 weeks-old female C57BL/6N mice were used and divided into 4 groups; (1) spinal intact (SI) mice+NF200p-driven HSV vectors encoding a marker gene, mCherry, (2) SCI mice+NF200p-HSV vectors encoding mCherry, (3) SCI mice+NF200p-HSV vectors encoding G2M receptors with vehicle administration, (4) SCI mice+NF200p-HSV-G2M vectors with ivermectin administration.  The SCI groups (2), (3) and (4) underwent complete transection of Th8/9 spinal cord under isoflurane anesthesia.  Thereafter, the bladder of SCI mice was emptied by perineal stimulation and bladder compression daily for 4 weeks post SCI.  In SI mice and 2-weeks SCI mice, a total of 20 μl suspension containing 3x107 plaque-forming units of NF200p-driven HSV vectors expressing either mCherry marker protein or G2M receptors were inoculated into the bladder wall at four sites (5 μl per site) using a 31-guage Hamilton syringe.  At 2 weeks after HSV vector inoculation, L1 and L6 dorsal root ganglia (DRG) of the groups (1) and (2) were harvested, and histological analyses were preformed to evaluate SCI-induced morphological changes in Aδ-fiber bladder afferent neurons identified by mCherry expression.  In HSV-G2M-inoculated SCI groups (3) and (4), ivermectin (50 μl of 1μM solution) or vehicle (DMSO 50 μl) was intra-peritoneally injected daily for 7 days from 1 week after vector inoculation.  After 7 days of intra-peritoneal injection, the SCI groups (3) and (4) underwent single-filling cystometry (CMG) and external urethral sphincter (EUS) electromyogram (EMG) under an awake condition. In the single CMG recordings, the number of non-voiding contractions (NVC), micturition pressure, post-void residual volume and voiding efficiency was calculated. In EUS-EMG recordings, voiding contraction time, reduced EMG activity time and the ratio of reduced EMG activity time to voiding contraction time were calculated to evaluate DSD. To calculate the EMG activity time, we evaluated the EMG and CMG charts in parallel and recorded the EMG reduction time when the reduced EUS-EMG activity was associated with the oscillatory reduction of bladder pressures and fluid elimination from the urethra (Fig. 1-a).
Results
Histological evaluation showed that the number of mCherry-positive, Aδ-fiber bladder afferent neurons in L1 and L6 DRG sections from SCI mice were significantly smaller than those from SI mice. The median cell size of the NF200 promoter vector-labeled, Aδ-fiber bladder afferent neurons in the L1 DRG shifted to a larger area size from 281.2 microm² in SI mice to 326.6 microm² in SCI mice.  In SCI mice inoculated with NF200 promoter vectors expressing G2M receptors, ivermectin treatment for 7 days improved the EMG activity reduction time during voiding bladder contraction and increased voiding efficiency compared to vehicle treatment; however, no improvement was seen in the number of NVC during the storage phase (Fig. 1-b, 1-c).
Interpretation of results
These results indicate that ivermectin-induced activation of double mutant glycine (G2M) receptors expressed in Aδ-fiber bladder afferent neurons by bladder wall inoculation of HSV-G2M vectors driven by NF200 promoter improved DSD evident as increased voiding efficiency and EMG activity reduction time during voiding bladder contraction. Thus, it seems likely that Aδ-fiber bladder afferent pathways play an important role in SCI-induced DSD and inefficient voiding. Furthermore, in the histological study, although the number of Aδ-fiber bladder afferent neurons were decreased, their neuronal cell size was increased in SCI mice, suggesting that cell hypertrophy of Aδ-fiber bladder afferent neurons could contribute to the enhancement of bladder-to-EUS reflex activity resulting in DSD after SCI.
Concluding message
Gene delivery of G2M designer receptors to Aδ-fiber bladder afferent neurons using HSV vectors with subpopulation-specific promoters such as NF200p enables to develop a “druggable” approach using an endogenously applied synthetic ligand, ivermectin, to suppress Aδ-fiber bladder afferent activity, which is enhanced to induce DSD and inefficient voiding in SCI.
Figure 1
References
  1. Wada N, et al. The effect of neutralization of nerve growth factor (NGF) on bladder and urethral dysfunction in mice with spinal cord injury. Neurourol Urodyn (2018);37:1889-1896.
  2. Kadekawa K, et al. The role of capsaicin-sensitive C-fiber afferent pathways in the control of micturition in spinal-intact and spinal cord-injured mice. Am J Physiol Renal Physiol (2017);313:F796-F804.
  3. Lynagh T, et al. An Improved Ivermectin-activated Chloride Channel Receptor for Inhibiting Electrical Activity in Defined Neuronal Populations. J Biol Chem (2010);285:14890-7.
Disclosures
<span class="text-strong">Funding</span> DOD W81XWH-17-1-0403, NIH P01DK093424 <span class="text-strong">Clinical Trial</span> No <span class="text-strong">Subjects</span> Animal <span class="text-strong">Species</span> mouse <span class="text-strong">Ethics Committee</span> University of Pittsburgh Institutional Animal Care and Use Commitee