Back to normal: Optogenetic photoactivation of CRH neurons in the PMC reverses the abnormal voiding phenotype of cyclophosphamide-induced cystitis
Jason P. Van Batavia, MD. MSTR1, Stephan Butler, MS1, Joanna Fesi, BS1, Stefano Vicini, PhD2, Douglas Canning, MD1, Rita Valentino, PhD1, Stephen Zderic, MD1.
1The Children's Hospital of Philadelphia, Philadelphia, PA, USA, 2Georgetown University Medical Center, Washington DC, DC, USA.
Background Overactive bladder (OAB) is a common cause of lower urinary tract (LUT) dysfunction in children and is often characterized by increased frequency of smaller volume voids. Most basic and clinical research on OAB has focused on the bladder which is the target of almost all approved therapies, despite an increasing understanding of the role of the central nervous system, especially the pontine micturition center (PMC, aka Barrington’s nucleus) in micturition control. We have previously shown that PMC neurons that express corticotropin-releasing hormone (CRH) are inhibitory to volitional voiding. Here we utilize a well described rodent model of cyclophosphamide-induced cystitis (LaBerge et al., 2006; Bjorling et al., 2007; Guo et al., 2018) to generate mice with an altered voiding phenotype similar to that seen in OAB, and then describe the affect of optogenetic stimulation of the CRH-expressing neurons in the PMC of these mice. We hypothesized that stimulation of PMC-CRH neurons would reverse the altered voiding phenotype and result in more normal voiding in these cyclophosphamide exposed mice.
Methods: Female CRH-Cre mice were crossed with Cre-dependent light-activated channelrhodopsin-2 (ChR2) to generate CRH-Ch2R mice. ChR2 is an opsin that causes depolarization and activation of neurons when exposed to green light. CRH-ChR2 mice underwent stereotactic placement of a canula into the PMC. One week later they were placed in specialized metabolism chambers (UroVoid, Med Associates, St Albans, VT) and allowed a 24hr acclimation period. Voiding patterns were measured at baseline. Cystitis was induced by the ip injection of cyclophosphamide (150 mg/kg) in saline and 48hr later the voiding patterns were measured again. At this point, optogenetic photostimulation (470nm at 50Hz) was carried out for an additional 24 hours, and the voiding patterns were measured again. Voiding endpoints included urinary frequency, intermicturition interval, and voided volumes.
Results: Baseline voiding patterns were measured in metabolism chambers following a 24-hour acclimation period (Figure 1A). Voiding data collected 48h after cyclophosphamide injection revealed increased voiding frequency and diminished voided volumes (Figure 1B). Optogenetic photostimulation of CRH-PMC neurons was sufficient to reverse the effect of cyclophosphamide on the voiding patterns (Figure 1C). Repeated measures ANOVA revealed an effect of voiding condition (baseline, CPM cystitis, or optogenetic stimulation) on voiding frequency (F(2,5)=4.5, p=0.017), voided volumes (F(2,5)=4.9, p=0.01), and IMI (F(2,5)=6.2, p=0.02) (Figure 6D,E,F). Further analysis revealed statistical differences between baseline and CPM cystitis, and differences between CPM cystitis and optogenetic stimulation conditions, for all three parameters: voiding frequency, voided volumes, and IMI (Wilcoxon signed-rank test, all p<0.05). However, there was no difference observed between baseline parameters and those measured after optogenetic stimulation (Wilcoxon signed-rank test, all p=0.2-0.6).
Conclusions: Optogenetic PMC activation of CRH-expressing neurons reversed the overactive voiding phenotype associated with cyclophosphamide. The present findings have translational significance and implicate that targeting of CRH neurons in the PMC or the CRH pathway may be an effective novel therapeutic strategy for OAB. 
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