Sacral neuromodulation is a treatment option to alleviate lower urinary tract symptoms. However, the mechanism by which neuromodulation exerts beneficial effects on bladder symptoms is poorly understood as a chronic animal model has not been well established. We have previously described a murine model of chronic, repetitive neuromodulation using the technique of optogenetics. Optogenetics enables specific neurons to be activated by light through injection of target neurons with viral vectors carrying the opsin light-channel gene. Transdermal light can then stimulate targeted nerves multiple times noninvasively. We seek to evaluate the effect of chronic, repetitive neuromodulation on the diabetic cystopathy murine model using optogenetic neurostimulation.

In this study, 10 diabetic ovariectomized female mice were injected with a viral vector containing the channelrhodopsin gene (AAV6-hSyn-ChR2(H134R)-EYFP) into their left sciatic nerves. AAV6-hSyn-ChR2(H134R)-eYFP encodes an excitatory opsin, enabling light-inducible stimulation. Nine diabetic ovariectomized female underwent sham surgery and were injected with saline into their left sciatic nerves. Eight diabetic ovariectomized female mice and 6 non-diabetic ovariectomized female mice did not have injections of the sciatic nerve. Three weeks after injection, all mice were exposed to 30 min of blue 475nm light (1 mW/ mm2) per day in an LED chamber for 35 days. Voiding behavior before and after viral infection was monitored by 2-hour filter paper recordings twice weekly. Five rAAV6-hSyn-ChR2-EYFP–injected mice were perfused and sacrificed to stain for YFP fluorescence and c-fos immunohistochemistry and quantification. After 35 days of light exposure, the bladders were harvested for histologic staining. PCR and Western blot were performed to analyze collagen III, elastin, smoothelin, SMA, uroplakin, PGP9.5 and von Willebrand Factor. Voiding area and number of voids on filter paper were processed and calculated in Image J. SPSS was used to data management and statistical analysis. Linear regression and two-way ANOVA were used to evaluate significant relationships.

All 10 diabetic ovariectomized female mice underwent successfully surgery with injection of the AAV6-hSyn-ChR2(H134R)-eYFP virus into the epineurium of the sciatic nerve. rAAV6-hSyn-ChR2-EYFP–injected mice all flinched or engaged in prolonged paw-licking which are operant behaviors associated with pain. None of the other mice exhibit any flinching or paw-licking behavior. Opsin expression was confirmed by positive YFP fluorescence stain in the dorsal root ganglia of the sciatic nerve. C-fos immunohistochemistry and quantification confirmed neural activation (Figure 1). Initial analysis was performed on the filter paper of 5 diabetic rAAV6-hSyn-ChR2-EYFP–injected mice and 3 non-diabetic control mice. A two-way ANOVA of filter paper data showed no variability in the voiding volume between the three control mice and the five AAV6-hSyn-ChR2(H134R)-EYFP injected mice over time (p-value 0.318). A two-way ANOVA of filter paper data showed a significant increase in the number of voids over time (p-value 0.01) in the five AAV6-hSyn-ChR2(H134R)-EYFP injected mice compared to the thee control mice. Full voiding data on all four groups of mice, PCR, Western and histology are pending (Figure 2).

In this study, our initial analysis of the voiding studies of a small representative sample of two of our groups show an increase in the number of voids over time in the AAV6-hSyn-ChR2(H134R)-EYFP injected mice during light stimulation. Patients with late-stage diabetes present with an underactive bladder phenotype with urinary retention. Sacral neuromodulation has been shown to improve urinary retention associated with underactive bladder by increasing the number of voids. Histology staining, PCR and Western blot analysis of protein will help us to identify the myogenic and neurogenic changes in the bladder caused by neuromodulation that may improve voiding efficiency in this underactive bladder phenotype.

This study seeks to elucidate the mechanisms by which neuromodulation alters diabetic bladder function. Optogenetics allows specific, noninvasive, and chronic stimulation of the sciatic nerve, enabling us to understand the chronology of changes induced by neuromodulation in the diseased bladder.

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