Stress urinary incontinence (SUI) is the complaint of involuntary loss of urine on effort or physical exertion (e.g., exercise) or on sneezing or coughing, and the primary risk factor is childbirth.1 While passing through the birth canal, the baby's head injures the pudendal nerve (PN) and the muscle it innervates, external urethral sphincter (EUS). Post-partum SUI patients have decreased leak point pressure (LPP) and increased PN latency compared to continent women, suggesting that PN function and regeneration is vital to maintaining continence. Since post-partum SUI has a strong correlation to later development of SUI, a treatment that enhances nerve regeneration could alleviate post-partum SUI symptoms and prevent later development of SUI. However, there are no current therapeutic options available to enhance nerve regeneration. 

A dual nerve and muscle childbirth injury model, consisting of PN crush and vaginal distension, results in delayed functional recovery, attenuated nerve regeneration, and dysregulated expression of brain derived neurotrophic factor (BDNF). Electrical stimulation (ES) of peripheral nerves upregulates neuronal BDNF, resulting in accelerated nerve regeneration and functional recovery. The aim of this study was to test the hypothesis that BDNF is necessary for accelerated functional recovery via ES of the PN, in a dual nerve and muscle childbirth injury model. Previous studies have used a fusion protein of tyrosine kinase B (TrkB), the primary receptor for BDNF to bind free BDNF and inhibit the BDNF pathway.3 To test the hypothesis, we inhibited the BDNF pathway with TrkB with the expectation that this would slow functional recovery with ES in the dual injury SUI model.

Thirty-nine age-matched female Sprague-Dawley rats (225-250g) were divided into four groups: the first group received a sham injury with no electrode implantation and saline treatment via osmotic pumps (sham injury) and served as the positive control for the study. The other three groups underwent PN crush + vaginal distension (dual injury) with electrode implantation and osmotic pump implantation for: sham stimulation with saline treatment (dual injury + sham), ES with saline treatment (dual injury + ES), or ES with TrkB treatment (dual injury + ES + TrkB). Dual injury consisted of bilateral PN crush injury, twice for 30 seconds, followed by a VD for four hours with a modified Foley catheter filled with 3 ml water under isoflurane anesthesia. Sham injury consisted of visualization of the PN without crush injury followed by insertion of the catheter in the vagina without inflation for four hours. Electrode implantation was done immediately after VD while the rat was under anesthesia by looping 2 stainless steel wires around the PN proximal to the crush site. The wires were tunneled subcutaneously along the dorsal side of the animals and externalized at the neck, then gently bent and sutured to the skin to prevent the animal from damaging the wires. Animals were placed under isoflurane anesthesia during ES or sham stimulation for one hour. ES parameters were 20 Hz, 0.1 ms, and 0.3 mA, four times a week for two weeks following the injury. The first ES was done 1 hour after electrode implantation while the animal was still under anesthesia.

Four weeks after the injury, the animals underwent functional testing consisting of leak point pressure (LPP) testing with simultaneous EUS electromyography (EMG). 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 5 ml/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 placed on the EUS to record EMG. Data is presented as mean  standard error of the mean. An ANOVA with Holm-Sidak posthoc test was used to determine statistically significant differences between groups (p< 0.05). The urethra and pudendal nerves were harvested and flash-frozen for qualitative histological and immunoflurescence. EUS cross-sections (7 µm) were stained with Masson's Trichrome for evaluation 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 in the dual injury + sham group (18.7 +/- 2.6 cm H2O) was significantly decreased compared to the sham injury group (29.3  +/- 3.0 cm H2O). In contrast, LPP after the dual injury + ES group (29.5 +/- 3.3 cm H2O) was significantly increased compared to both the dual injury + sham group and the dual injury + ES + TrkB group (17.3 +/- 2.0 cm H2O), suggesting impaired recovery with inhibition of the BDNF pathway. EUS EMG firing rate of the dual injury + sham group (71.5  +/-  25.2 Hz) was significantly decreased compared to that of the sham injury group (388.8 +/-  66.4 Hz). EUS EMG firing rate of the dual injury + ES group (325.2  +/- 90.0 Hz) was significantly increased compared to that of the dual injury + sham and dual injury + ES + TrkB (62.6 +/-  47.9 Hz) groups. 
 
EUS striated muscle fibers were disrupted after dual injury + sham compared to sham injury. In contrast, EUS morphology after dual injury + ES was similar to that of the sham injury group, but the EUS was disrupted in the dual injury + ES + TrkB group, suggesting impaired recovery when the BDNF pathway is blocked. Similarly, the dual injury + sham group had fewer innervated NMJ than the sham injury group. in contrast, dual injury + ES resulted in a greater number of innervated NMJs than either dual injury + sham or dual injury + ES + TrkB. Neurofilament staining was significantly decreased after dual injury + sham compared to the sham injury group. The dual injury + ES group had improved nerve morphology compared to dual injury + sham and dual injury + ES + TrkB, suggesting impaired neuroregeneration when the BDNF pathway is inhibited.

The dual injury model of SUI results in decreased LPP, while ES accelerated LPP recovery; however, treatment with TrkB inhibited LPP recovery, demonstrating that BDNF is important to the accelerated functional recovery via ES. EUS EMG firing rate as well as EUS morphology,  NMJ staining, and neurofilament staining results support these findings.

This study demonstrates that ES of the PN accelerates functional recovery in part via a BDNF-mediated pathway after a dual injury SUI model. These findings suggest ES of the PN could serve as a possible regenerative therapy for women with post-partum SUI and may prevent later development of SUI.

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