Chronic spinal cord injury (SCI) rostral to the lumbosacral level induces both storage and voiding dysfunctions due to detrusor overactivity (DO) during the storage phase and detrusor-sphincter dyssynergia (DSD) during the voiding phase, respectively [1]. Glycine is one of major inhibitory neurotransmitters in the central nervous system (CNS), and activation of glycine receptors is known to induce a chloride ion influx to cause hyperpolarization in neuronal cells.  It has also been reported that the spinal glycinergic mechanism is downregulated in the lumbosacral spinal cord; thereby inducing DO after SCI in rats [2].  However, it is likely that activation of endogenous glycine receptors may lead to systemic adverse events because of their ubiquitous distribution in the CNS.  Therefore, this study aimed to develop a “druggable” approach targeting bladder afferent pathways by using gene delivery of the double mutant glycine (G2M) receptor, in which the receptor sensitivity to ivermectin, an anthelmintic used in filariasis, is increased with elimination of sensitivity to glycine [3].  For this purpose, we utilized replication-deficient herpes simplex virus (HSV) vectors because they have a natural property allowing transfection to sensory pathways following peripheral inoculation, which could offer an organ-specific treatment, and investigated whether ivermectin-induced activation of G2M receptors delivered by HSV vectors driven by non-selective cytomegalovirus (CMV) promoter to bladder afferent pathways improves storage and voiding dysfunctions in mice with SCI.

We used adult female C57BL/6 mice. The spinal cord was completely transected at the Th8/9 level. We prepared replication-deficient HSV vectors encoding G2M receptors driven by CMV promoter (CMVp-G2M; 1.10 x 10¹º plaque-forming units (PFU)/mL) and control HSV vectors encoding mCherry marker protein driven by CMV promoter (CMVp-Cont; 1.11 x 10¹º PFU/mL).  Two weeks after SCI, a total of 20 μl suspension of CMVp-G2M or CMVp-Cont vectors into the bladder wall of each mouse at four sites (5 μl per site) using a 31-guage Hamilton syringe.  Then, two weeks after HSV vector inoculation into the bladder wall, continuous and single-filling cystometrograms (CMG) were recorded under an awake condition to evaluate non-voiding contractions (NVCs) during the storage phase and voiding efficiency during the voiding phase before and after administration of IVM (2.5mg/kg) or vehicle (Veh) into the tail vein.  SCI mice were divided into 4 groups: (1)  the CMVp-G2M vector inoculation with intravenous (i.v.) IVM administration group (CMVp-G2M-IVM-iv group; n = 6), (2) the CMVp-G2M inoculation with i.v. vehicle administration group (CMVp-G2M-Veh-iv group; n = 6), (3) the CMVp-Cont inoculation with i.v. IVM administration group (CMVp-Cont-IVM-iv group; n = 6) and (4) the CMVp-Cont inoculation with i.v. vehicle administration group (CMVp-Cont-Veh-iv group; n = 6).

In SCI mice with control HSV vector inoculation into the bladder wall, mCherry marker protein expression was seen in L6 dorsal root ganglion (DRG) sections.   In the CMVp-G2M-IVM-iv group, the number of NVCs was significantly decreased from 1.24/min to 0.49/min after IVM administration. However, in other three groups, NVCs were not significantly decreased after IVM or vehicle administration (Figure 1).  Voiding efficiency was also significantly improved in the CMVp-G2M-IVM-iv group after IVM administration compared to other three groups (Figure 2).

It has been documented that SCI initially induces areflexic bladder and urinary retention, and then later develops DO and DSD during voiding due to the reorganization of reflex pathways in the spinal cord as well as alterations in the properties of bladder afferent neurons [1]. The results in this study indicate that ivermectin-induced activation of double mutant glycine (G2M) receptors expressed in bladder afferent neurons by bladder wall inoculation of HSV-G2M vectors driven by CMV promoter improved DO evident as a reduction of NVCs and inefficient voiding caused by DSD.  Thus, it seems likely that HSV vector-mediated gene delivery of G2M designer receptors to bladder afferent pathways could offer a new approach using an exogenously applied synthetic ligand, ivermectin, to improve SCI-induced storage and voiding dysfunctions.

Gene therapy with replication-deficient herpes simplex virus vectors encoding ivermectin-sensitive, mutant glycine receptors with exogenous ligand application could be a novel “druggable” treatment that can avoid systemic adverse events for neurogenic lower urinary tract dysfunction associated with SCI.

de Groat WC, Yoshimura N. Mechanisms underlying the recovery of lower urinary tract function following spinal cord injury. Prog Brain Res 152: 59–84, 2006.
Miyazato M, Sugaya K, Nishijima S, Ashitomi K, Hatano T, Ogawa Y. Inhibitory effect of intrathecal glycine on the micturition reflex in normal and spinal cord injury rats. Exp Neurol 183: 232–240, 2003.
Lynagh T, Lynch JW. An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations. J Biol Chem 285: 14890-14897, 2010.