Stress Urinary Incontinence(SUI) is the leakage of urine due to an increase in abdominal pressure, which overcomes urethral resistance. It affects 30% of women over the age of 40. During vaginal delivery, when the child’s head passes through the birth canal, the pudendal nerve(PN) can be injured, as can the muscle it innervates, the external urethral sphincter(EUS). Women with postpartum SUI have been shown to have increased PN motor latency compared to continent women, indicating nerve function is important to maintaining continence, and suggesting that complete nerve regeneration after childbirth is also important[1]. Since post-partum SUI has a strong correlation to later development of SUI, a treatment that enhances nerve regeneration for postpartum incontinence could alleviate SUI symptoms and prevent later development of SUI[2]. However, no treatments are currently available to enhance nerve regeneration. 
A rat dual nerve and muscle simulated childbirth injury model has shown decreased nerve regeneration and increased time to functional recovery compared to either injury alone. The model demonstrates that brain derived neurotrophic factor(BDNF) is dysregulated following the dual injury, instead of being upregulated, which occurs after a nerve injury to facilitate nerve regeneration. Electrical stimulation(ES) of nerves accelerates nerve regeneration via a BDNF-mediated mechanism[3]. We have previously shown that such stimulation increases BDNF expression and improves outcomes following this simulated childbirth injury. To further optimize the stimulation protocol, we hypothesized that daily electrical stimulation will accelerate functional recovery and pudendal nerve regeneration beyond that of a less frequent stimulation protocol in this simulated childbirth injury model.

Thirty-five Sprague-Dawley rats were divided up into five groups: two groups received sham injuries, one with electrode implantation with sham stimulation and the other without electrode implantation. The other three groups received PN crush(PNC) and vaginal distension(VD) with electrode implantation either with sham stimulation, 4 times a week(4/week) stimulation, or daily stimulation. Two stainless-steel wires were hooked on the pudendal nerve, flanking the crush site or approximating the crush site in sham injured animals. PNC consisted of bilateral crush twice for 30 seconds, followed by VD for four hours with a modified Foley catheter filled with 3ml water.  Sham injury consisted of visualization of the PN without crush injury followed by insertion of the catheter without inflation for four hours. Electrodes were implanted on the PN, tunneled subcutaneously along the dorsal side, externalized at the neck, and were gently bent and sutured to the rat to prevent damage to the wires. Animals were placed under isoflurane anesthesia during stimulation or sham stimulation for one hour. Simulation parameters were 20Hz, 0.1ms, and 0.3mAmp. Stimulation and sham stimulation occurred for two weeks following the injury. 
Functional testing occurred 4 weeks after the injury and consisted of Leak Point Pressure(LPP) testing with simultaneous EUS electromyography(EMG), followed by pudendal nerve sensory branch potential testing(PMSBP). Animals were anesthetized with urethane(1.2g/kg) and a suprapubic bladder catheter(flared tip PE-50 tubing) was implanted in the dome of the bladder and was sutured in place with a 4-0 silk purse-string suture. The bladder was filled at a rate of 5ml/hr. Once the bladder was filled to half capacity, increasing pressure was applied to the abdomen until leakage was observed, this was then repeated three times. A parallel recording electrode was place on the EUS to record EMG. The clitoral region was brushed, while recordings were made from the PN sensory branch using hooked parallel recording electrodes. An ANOVA with Sudent-Newman-Keuls test was used to determine significant differences between groups(p< 0.05). Data presented as mean +/-standard error of the mean.  Urethra and pudendal nerves were harvested and flash frozen for qualitative assessment. EUS cross-sections (7 µm) were stained with Massons and a modified Harts stain for histological assessment of muscle, collagen & elastin. Additional cross-sections (14 µm) underwent immunofluorescence staining for neuromuscular junctions (NMJ:alpha-bungarotoxin), striated muscle (phalloidin), and innervating nerves (antibodies to neurofilament 68 and 200). Pudendal nerves were cross-sectioned and stained for neurofilament 68 and 200 to visualize axons for qualitative assessment.

LPP after sham injury with sham stimulation(39.9+/-2.1cmH2O) was not significantly different from sham injury + no electrodes(NE) (40.6+/-3.2cmH2O). In contrast, LPP after PNC+VD with sham stimulation(19.0+/-2.4cmH2O) was significantly decreased compared to sham injury + sham stimulation, sham injury + NE, and PNC+VD with 4/week stimulation(39.9+/-3.6cmH2O). LPP after PNC+VD with daily stimulation(26.5+/-1.8cmH2O) was significantly increased compared to PNC+VD with sham stimulation, but significantly decreased compared to sham injury + sham stimulation, sham injury + NE, and PNC+VD with 4/week stimulation. 
EUS-EMG amplitude and firing rate were not significantly different between sham injury + NE(13.5+/-3.8V; 868.8+/-38.5Hz) and sham injury + sham stimulation(13.7+/-3.7 V; 377.8 +/-71.7 Hz). EUS-EMG amplitudes for PNC+VD with sham stimulation(1.5+/-2.3V) and PNC+VD with daily stimulation(1.2+/-0.5V) were significantly decreased compared to sham injury groups, but PNC+VD with 4/week stimulation(5.8+/-1.8V) was not significantly different compared to sham injured groups. EUS-EMG firing rate was significantly decreased only after PNC+VD with sham stimulation(164.6+/-43.0Hz) compared to sham injured rats. Firing rate after PNC+VD with 4/week stimulation(288.4+/-63.4Hz) or daily stimulation(285.3+/-78.8Hz) were not significantly different from sham injured groups. PNSBP amplitudes and firing rates for sham injury + electrodes(0.2+/-0.1V; 406.7+/-149.3Hz), PNC+VD with sham stimulation(0.1+/-0.0V; 101.0+/-32.3Hz), PNC+VD with 4/week stimulation(0.2+/-0.1V; 160.0+/-90.2Hz), and PNC+VD with daily stimulation(0.1+/-0.0V; 139.8+/-45.2Hz) were significantly decreased compared to sham injury + NE(0.6+/-0.1V; 868.8+/-38.5Hz). 
The EUS was disrupted following PNC+VD with sham stimulation compared to sham injured groups. In contrast, EUS morphology after PNC+VD with 4/week stimulation was similar to that of sham injured rats. EUS morphology after PNC+VD with daily stimulation was not as well recovered as with 4/week stimulation. There were fewer elastic fibers in the EUS after PNC+VD with sham stimulation than after sham injury. In contrast, there were more elastic fibers in the EUS after PNC+VD with 4/week stimulation than after daily stimulation, while both showed an increase in elastic fibers compared to PNC+VD with sham stimulation. There were fewer EUS NMJs after PNC+VD with sham stimulation than in sham injured rats; whereas there was increased NMJ staining after PNC+VD with 4/week stimulation compared to sham stimulation. Neurofilament staining was significantly decreased after PNC+VD with sham stimulation compared to sham injured groups. PNC+VD with 4/week stimulation and PNC+VD with daily stimulation had similar staining intensity, but staining was decreased compared to sham injured groups.

Electrode implantation does not affect LPP, as shown by the sham injured groups. PNC+VD significantly decreased LPP, while 4/week stimulation improved LPP to a greater extent than daily stimulation, suggesting that ES accelerates recovery in this model. EUS-EMG firing rate support these results, while EUS-EMG amplitude suggests that 4/week stimulation is more effective than daily stimulation. Chronic implantation of electrodes affected nerve signaling and ES did not attenuate these effects. Stimulation 4/week improved EUS morphology and increased NMJ staining compared to daily stimulation, supporting the LPP findings. Neurofilament staining supports the findings that ES accelerates recovery in this model.

Four/week stimulation and daily stimulation improve functional recovery after PNC+VD, demonstrating that ES can accelerate recovery. Four/week stimulation was more effective then daily stimulation. The nerve potential recordings suggest investigation into non-invasive techniques of nerve stimulation would be beneficial for clinical applications, since electrode implantation affects nerve signaling. This would allow for translation of ES of nerves for the treatment of post-partum SUI and possible prevention or attenuation of later development of SUI.

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