Multiple doses of stem cells maintain urethral function in a model of neuromuscular injury resulting in stress urinary incontinence

Abstract

Stress urinary incontinence (SUI) is more prevalent among women who deliver vaginally than women who have had a cesarean section, suggesting that tissue repair after vaginal delivery is insufficient. A single dose of mesenchymal stem cells (MSCs) has been shown to partially restore urethral function in a model of SUI. The aim of the present study was to determine if increasing the number of doses of MSCs improves urethral and pudendal nerve function and anatomy. We hypothesized that increasing the number of MSC doses would accelerate recovery from SUI compared with vehicle treatment. Rats underwent pudendal nerve crush and vaginal distension or a sham injury and were treated intravenously with vehicle or one, two, or three doses of 2 × 10⁶ MSCs at 1 h, 7 days, and 14 days after injury. Urethral leak point pressure testing with simultaneous external urethral sphincter electromyography and pudendal nerve electroneurography were performed 21 days after injury, and the urethrovaginal complex and pudendal nerve were harvested for semiquantitative morphometry of the external urethral sphincter, urethral elastin, and pudendal nerve. Two and three doses of MSCs significantly improved peak pressure; however, a single dose of MSCs did not. Single, as well as repeated, MSC doses improved urethral integrity by restoring urethral connective tissue composition and neuromuscular structures. MSC treatment improved elastogenesis, prevented disruption of the external urethral sphincter, and enhanced pudendal nerve morphology. These results suggest that MSC therapy for postpartum incontinence and SUI can be enhanced with multiple doses.

INTRODUCTION

Stress urinary incontinence (SUI) is the involuntary loss of urine during effort or physical exertion, sneezing, or coughing (44). The prevalence of SUI among women is estimated at 48%, representing a major burden to the healthcare system (44). The risk of SUI is twice as high in women who deliver vaginally than in women who have had a cesarean section (34, 37), suggesting that innate tissue repair after vaginal delivery is insufficient in many women, resulting in SUI when compounded with the effects of aging (12, 32). Therefore, an appealing target for new treatment strategies is enhancement of tissue repair after pelvic floor trauma caused by vaginal delivery as a treatment for postpartum SUI and secondary prevention of SUI later in life.

Administration of various cell types has been shown to have a protective effect on the development of SUI in clinical trials (1, 15, 17, 31, 33) and after simulated childbirth injury in experimental animal models (2, 5, 6, 11, 13, 22). These experimental models are developed to simulate childbirth injury and cause damage to the pelvic floor, leading to symptoms mimicking SUI in women (18, 19, 26, 29). Simulated childbirth injury upregulates stem cell homing cytokines, attracting intravenously injected stem cells to injured pelvic organs (6, 43, 45). In addition, injection of the secretions produced by stem cells improves urethral recovery to a level comparable to that with mesenchymal stem cell (MSC) treatment, pinpointing the importance of secreted paracrine factors in the mechanism of action of stem cells (11, 13).

Recently, we demonstrated that a single systemic dose of MSCs partially restores urethral and nerve function in a dual simulated childbirth injury model of SUI consisting of pudendal nerve crush (PNC) and vaginal distension (VD) (11). MSCs also promoted elastogenesis and recovery of neuromuscular junctions and nerves in the urethra (11, 13). However, the recovery was only partial. Repeated injections of MSCs promoted tissue repair compared with a single dose in models of chronic kidney disease and perinatal cerebral hypoxia-ischemia (25, 41). Therefore, we hypothesized that increasing the number of MSC doses would accelerate recovery from SUI compared with vehicle treatment. In the present study, we investigated the effects of increasing the number of doses of MSCs on urethral and pudendal nerve function and morphology in a rat model of SUI created by dual PNC + VD.

MATERIALS AND METHODS

Mesenchymal stem cells.

Bone marrow-derived MSCs were obtained as previously described (13). Bone marrow was harvested from the femur and tibia of Sprague-Dawley rats and cultured under normoxic conditions in DMEM-12.5% FBS-1% antibiotic-antimycotic (100×) (Invitrogen, Carlsbad, CA). Cells were sorted for intracellular adhesion molecule 1 and transfected with a lentivirus to express green fluorescent protein and a lentivirus to express luciferase. Green fluorescent protein- and luciferase-positive cells were selected and cultured to passage 8–10. Cells were positive for CD29, CD54, and CD90 and negative for CD45 (11).

Animals.

Experiments were performed as approved by the Institutional Animal Care and Use Committee of the Cleveland Veterans Affairs Medical Center. Eighty-five age-matched virgin female Sprague-Dawley rats (225–275 g body wt, Harlan Laboratories, Indianapolis, IN) underwent dual simulated childbirth injury, consisting of PNC + VD, under 2–3% isoflurane inhalation anesthesia, as we have previously described (11). Briefly, for PNC, the pudendal nerve was isolated on both sides via a dorsal incision and crushed twice for 30 s with a Castroviejo needle holder. Immediately thereafter, the vagina was dilated using bougie à boule dilators (24- to 32-Fr), and a modified 10-Fr Foley catheter was inserted intravaginally and secured with a suture in preparation for VD. The balloon of the catheter was inflated with 3 ml water and left in place for 4 h in anesthetized rats. Thereafter, the Foley catheter was deflated and removed, and the suture securing the catheter was also removed. Sham-injured animals underwent dorsal skin and muscle incision only, the vagina was dilated using bougie à boule dilators, and a modified Foley catheter was inserted and secured with a suture for 4 h but not inflated. The analgesic carprofen (Rimadyl, 5 mg/kg) was administered subcutaneously once during surgery and once on the day after surgery.

At 1 h, 7 days, and 14 days after PNC + VD, 1 ml vehicle (saline) or 2 × 10⁶ MSCs suspended in 1 ml saline were infused into the lateral tail vein under isoflurane anesthesia. Rats were randomly divided into one of five experimental groups. Sham-operated rats underwent sham injury and received vehicle at all time points. All other rats underwent PNC + VD and received either vehicle at all time points, one dose of MSCs (1 h after injury) and two doses of vehicle (7 and 14 days after injury), two doses of MSCs (1 h and 7 days after injury) and one dose of vehicle (14 days after injury), or 3 doses of MSCs at all time points (Fig. 1). From a sample-size analysis based on our previous experience, we estimated that 11 rats/group would be needed to obtain statistically significant results compared with vehicle-treated animals. Leak point pressure (LPP) was the primary outcome, and once significance was achieved, the study was stopped. Mortality was observed in one rat shortly after injection of MSCs at 1 h after injury.

Fig. 1.

Experimental design. Rats underwent either pudendal nerve crush and vaginal distension (PNC + VD; yellow diamond) or sham injury (orange diamond) and received mesenchymal stem cells (MSCs; red triangle) or vehicle (VEH; blue triangle) at 1 h, 7 days, or 14 days after injury. At 21 days after injury, rats were either anesthetized for urethral and pudendal nerve (PN) function testing (n = 9–14 rats/group) or euthanized for morphometric testing (n = 6–7 rats/group).

In animals reserved for functional testing (Fig. 1), urethral and pudendal nerve testing was performed 21 days after PNC + VD or sham injury, and animals were euthanized with an intracardiac overdose of a pentobarbital (390 mg/ml)-phenytoin (10 mg/ml) solution (Euthasol). Additional animals in each experimental group were reserved for anatomic testing only (Fig. 1), since the dissection and electrode placement required for functional testing could affect urethral microstructure. These rats were euthanized 21 days after injury, as described above, under isoflurane anesthesia, and the urethral-anterior vaginal wall complex was harvested and cryopreserved for morphological analysis.

Functional testing.

Urethral and pudendal nerve function were tested as previously described (11). Rats were anesthetized using both urethane (1.2 g/kg ip) and 1% isoflurane and placed in a supine position. A midline incision was made to expose the bladder dome, and a flared-tip polyethylene (PE-50) catheter was inserted and fixed into the bladder with a purse-string suture. The catheter was connected to a syringe pump (model 200, KD Scientific, New Hope, PA) and a pressure transducer (model PT300, Natus Neurology, Middleton, WI). Air pressure at the level of the bladder was used as a reference. The bladder was filled with saline via the suprapubic catheter (5 ml/h). After ligation of both inferior epigastric arteries and veins, the pubic bone was exposed, and the pubic symphysis was cut to reach the urethra. Bipolar parallel platinum-iridium electrodes (31-gauge blunt needles, 1 mm apart, FHC, Bowdoin, ME) were placed on the midurethra to record external urethral sphincter (EUS) electromyography (EMG) and connected to an amplifier (model P511) with band-pass frequencies of 3 Hz–3 kHz (model PT300, Natus Neurology) and an electrophysiological recording system with a 10-kHz sampling rate (PowerLab 8/35, ADInstruments, Colorado Springs, CO).

Isoflurane anesthesia, which negatively influences urethral function (3, 4), was discontinued before recordings were initiated. Urethral function was assessed under urethane anesthesia only via LPP testing with simultaneous recording of EUS EMG. At approximately half capacity (~0.4 ml), bladder pressure and simultaneous EUS EMG were recorded three to six times in each rat by manual elevation of bladder pressure and rapid removal of the externally applied pressure as soon as leakage was observed at the urethral meatus, as we have previously described (19). After LPP testing, the sensory branch of the pudendal nerve was separated from the surrounding connective tissue and hooked onto a bent bipolar electrode (31-gauge blunt needles, 0.8 mm apart), as we have previously described, to assess pudendal nerve function (11). Electroneurograms (ENGs) of the sensory branch of the pudendal nerve were recorded ∼5–10 times at rest and during brushing of the skin in the clitoral area with gauze.

Histology.

In animals reserved for anatomic investigation only, the urethra and anterior vaginal wall were harvested en bloc, and the pudendal nerve on both sides was isolated and harvested. Tissues were immediately fresh-frozen in OCT compound and stored at −80°C. Tissues were cryosectioned transversely (14 µm thickness). Adjacent urethral and anterior vaginal wall sections were fixed with 4% paraformaldehyde and stained with Hart’s stain and Masson’s trichrome to assess elastin as well as muscle and collagen, respectively. Additional near sections were fixed with acetone for immunofluorescence to assess neuromuscular junctions (NMJs) in the EUS and their innervation, as we have previously described (36). These sections were then incubated with mouse monoclonal anti-neurofilament 68 (Sigma-Aldrich, St. Louis, MO) and mouse monoclonal anti-neurofilament 200 (Sigma-Aldrich), combined in a 1:400 dilution, and then with the secondary antibody Alexa Fluor 488-conjungated donkey anti-mouse (1:400 dilution, Invitrogen, Carlsbad, CA). α-Bungarotoxin conjugated to rhodamine (1:400 dilution, Invitrogen) was used to label acetylcholine receptors of NMJs in the EUS. Alexa Fluor 350 conjugated to phalloidin (1:40 dilution, Invitrogen) was used to stain F-actin in the striated muscle of the EUS. The pudendal nerve was fixed with acetone and incubated with the same primary and secondary antibodies for neurofilament as the urethral sections to image the axons within the pudendal nerve, as we have previously described (11).

Data analysis.

LPP was calculated by subtraction of bladder pressure immediately before LPP testing (baseline pressure) from peak bladder pressure during LPP testing (LabChart 7, ADInstruments), as we have previously described (13). One-second segments of EUS EMG at baseline and peak bladder pressure during LPP testing were selected and used to calculate firing rate and amplitude using a fixed threshold, as we have previously described (11, 19). The increase in EUS EMG firing rate and amplitude between segments captured at baseline and peak bladder pressure during LPP testing was also calculated.

Similarly, at rest and during brushing of the clitoral area, 1-s ENG segments were selected and used to calculate firing rate and amplitude, as described above for the EMG data. The difference between ENG amplitude and firing rate during brushing and at rest was calculated. Mean values for each animal were calculated from three to six examples of LPP, EMG, and ENG in each animal, which were then used to create group means for statistical comparisons.

An investigator blinded to the experimental groups semiquantitatively assessed histological and immunofluorescence outcomes. Representative histological sections were selected for each animal and used for further analyses. Hart’s-stained slides were analyzed for the following parameters: organization, density, thickness, and length of the elastic fibers. For each individual slide, elastin fibers were scored as follows: aligned or disorganized, dense or sparse, thick or thin, and long or short. Masson’s trichrome-stained slides were analyzed by determining the number of myofibers comprising the EUS, whether the EUS was intact or disrupted, and whether collagen deposition was present or absent within the EUS. Neuromuscular morphology within the EUS was studied by assessing the presence of NMJs, shape of NMJs (dense or diffuse), presence or absence of axons innervating the NMJ, and presence or absence of collateral nerves. The morphology of the axons within the pudendal nerve was assessed by density and organization of the axons. For each individual image, axons were scored as follows: aligned or disorganized, dense or sparse, and thick or thin. Histological slides of individual animals were analyzed and then used to create group data for statistical comparisons.

Statistical analysis.

All urethral function data, including bladder pressure data, EUS EMG, and ENG data, were analyzed using one-way ANOVA with Bonferroni’s post hoc test with either vehicle or one dose of MSCs as reference groups for statistical comparison. Histological and immunofluorescence data were analyzed using a χ²-test, which, when outcomes indicated a significant difference, was followed by a Fisher’s exact test for individual group comparisons using Bonferroni’s correction (corrected P < 0.005), except for the number of myofibrils within the EUS, which were analyzed using a Kruskal-Wallis test (GraphPad Prism 6, GraphPad Software, La Jolla, CA, and IBM SPSS Statistics 21.0). Data are presented as means ± SE, medians, and ranges or numbers of animals. P < 0.05 was considered to indicate a statistically significant difference between groups in all comparisons.

RESULTS

A total of 85 rats underwent PNC + VD or sham injury and were treated with vehicle or one, two, or three doses of MSCs at 1 h, 7 days, and 14 days after injury (Fig. 1).

Multiple doses of MSCs improve urethral function.

Animals that underwent PNC + VD and vehicle treatment had significantly reduced urethral function at 21 days postinjury, since LPP and peak bladder pressure were significantly decreased in these animals compared with sham-injured animals (Figs. 2 and 3). After two doses of MSCs, there was a significant improvement in urethral function, represented by significantly increased peak bladder pressure and LPP compared with vehicle treatment (Fig. 3). Three doses of MSCs resulted in a significant increase in peak pressure, but not LPP, compared with vehicle treatment (Fig. 3). In contrast to these findings, there were no significant differences in peak pressure or LPP between a single dose of MSCs and vehicle treatment. There were also no significant differences in peak pressure and LPP between the groups that received two or three doses of MSCs and the group that received a single dose of MSCs. Although EUS EMG firing rate was decreased in vehicle-treated PNC + VD rats, this result was not significantly different from that in sham-injured animals, and there were no significant differences between any of the groups in EUS EMG firing rate or amplitude increase from baseline to peak pressure during LPP testing (Fig. 3).

Fig. 2.

Representative examples from sham-injured animals. A: leak point pressure (LPP) and simultaneous external urethral sphincter electromyogram (EUS EMG) recordings. B: pudendal nerve electroneurogram (ENG) recordings indicating when clitoral brushing began (brush) and ended (release).

Fig. 3.

Effects of multiple doses of mesenchymal stem cells (MSCs) on urethral function after pudendal nerve crush and vaginal distension (PNC + VD). A and B: urethral resistance to leakage was assessed by measuring peak bladder pressure and leak point pressure. C and D: external urethral sphincter electromyograms (EUS EMG) were performed to determine neuromuscular function of the EUS and quantified in terms of difference between baseline and peak bladder pressure during leak point pressure testing of EUS EMG firing rate (C) and amplitude (D) 21 days after PNC + VD or sham injury (sham). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with PNC + VD rats treated with vehicle (VEH). Each bar represents the mean ± SE of data from 14 animals in the sham group, 13 animals in the injury group, and 9 animals in the MSC groups.

The increase in firing rate and amplitude of pudendal nerve ENG during brushing of the clitoral area was significantly reduced in vehicle-treated PNC + VD compared with sham-injured animals, indicating impaired sensory function of the pudendal nerve (Fig. 4). Pudendal nerve function after PNC + VD was partially preserved with single and multiple doses of MSCs, since the increase in ENG firing rate with clitoral brushing was partially restored to sham-injured values and was not significantly different from either sham-injured or PNC + VD animals with vehicle treatment (Fig. 4A).

Fig. 4.

Effects of multiple doses of mesenchymal stem cells (MSCs) on the function of the sensory branch of the pudendal nerve after pudendal nerve crush and vaginal distension (PNC + VD). A and B: electroneurogram (ENG) of the sensory branch of the pudendal nerve was used to quantify pudendal nerve function in terms of firing rate and amplitude difference between rest and clitoral brushing 21 days after PNC + VD or sham injury (sham). *P < 0.05 and **P < 0.01 compared with PNC + VD rats treated with vehicle (VEH). Each bar represents the mean ± SE of data from 14 animals in the sham group, 13 animals in the injury group, and 9 animals in the MSC groups.

MSCs alter connective tissue composition in the urethra.

Urethral elastin was found mainly within the connective tissue and the urethral smooth muscle located between the urothelium and EUS (Fig. 5). PNC + VD caused disruption of elastic fibers, especially around and within urethral smooth muscle. The majority of elastin fibers were thin in PNC + VD rats compared with sham-injured rats, in which the majority of elastin fibers were thick (Fig. 5). MSC treatment resulted in significantly more thick elastin fibers within the urethra (P = 0.041; Fig. 5), suggesting that MSCs promote elastogenesis. No significant differences were observed between groups in elastic fiber length, elastin fiber density, or elastin organization, although there was a trend toward higher density of elastin fibers within the urethra in sham-injured and MSC-treated rats that received three doses than in PNC + VD rats (P = 0.053; Fig. 5).

Fig. 5.

Effects of multiple doses of mesenchymal stem cells (MSCs) on urethral elastogenesis after pudendal nerve crush and vaginal distension (PNC + VD). A–E: representative images of Hart’s-stained transverse urethral sections in which elastin is stained black (green arrows). A: sham-injured (sham) animals had well-organized short, thick elastic fibers located close to and perpendicular to the urothelium and long, thin elastic fibers along the length of the external urethral sphincter (EUS), indicated by the yellow hashtag. B: PNC + VD with vehicle treatment (VEH) caused disruption of elastic fibers, especially around the urothelium, where elastin was thinner than in sham-injured animals. C–E: administration of one, two, or three doses of MSCs resulted in thicker elastin fibers, which were aligned along the EUS. Bars represent data from 7 animals per group of animals treated with three doses of MSCs and 6 animals in all other groups. χ²-analysis indicated a significant difference in fiber thickness (P = 0.041) but not fiber length (P = 0.053), density (P = 0.135), or organization (P = 0.055). Fisher’s exact test comparing fiber thickness did not indicate a significant difference between individual groups.

In sham-injured animals, the EUS consisted of clearly defined, aligned striated muscle fibers with or without collagen disposition between the muscle fibers (Fig. 6). Semiquantitative analyses revealed significant disruption of the EUS in PNC + VD compared with sham-injured rats, which appeared to be prevented by one dose of MSCs (P = 0.017; Fig. 6). However, we did not observe this treatment effect in groups that were treated with two or three doses of MSCs. There were no significant differences in collagen infiltration into the EUS or the number of myofibrils with injury or MSC treatment.

Fig. 6.

Effects of multiple doses of mesenchymal stem cells (MSCs) on external urethral sphincter (EUS) and collagen regeneration after pudendal nerve crush and vaginal distension (PNC + VD). A–E: representative images of Masson’s trichrome-stained transverse urethral sections. Pink arrowheads indicate collagen, yellow triangles indicate striated muscle, and green arrows indicate smooth muscle. PNC + VD with vehicle (VEH) treatment only (B) caused significantly more disruption of the EUS than in the sham-injured group (A) or any of the MSC-treated groups (C–E). Bars represent data from 6 animals in the sham and single-dose group, 5 animals in the two- and three-dose groups, and 4 animals in the injury group. χ²-analysis indicated a significant difference in EUS intactness (P = 0.017) but not collagen infiltration into the EUS (P = 0.54) or number of myofibrils in the EUS (P = 0.75). Fisher’s exact test comparing EUS intactness did not indicate a significant difference between individual groups.

MSCs improve nerve and NMJ morphology.

The EUS of sham-injured animals contained compact NMJs innervated by one or more axons (Fig. 7). There was a trend toward signs of NMJ denervation after PNC + VD (Fig. 7) and a trend toward increased NMJ innervation after two doses of MSCs (P = 0.065; Fig. 7). There were no significant differences between groups with regard to whether a NMJ was present in the tissue sections, whether the NMJ was innervated by a nerve, or whether collateral nerves were present.

Fig. 7.

Effects of multiple doses of mesenchymal stem cells (MSCs) on urethral neuromuscular regeneration after pudendal nerve crush and vaginal distension (PNC + VD). A–E: representative images of immunofluorescently labeled neurofilaments (green), ACh receptor in neuromuscular junctions (NMJs; red), and F-actin in the striated muscle fibers of the EUS (blue) in urethral transverse sections. A: sham-injured animals had well-defined and compact NMJs innervated by one or more axons. B: PNC + VD changed the morphology of NMJs, which became less defined and more diffuse, indicative of denervation. C–E: administration of one, two, or three doses of MSCs began the process of reinnervation of the NMJs by one or more axons. Bars represent data from 6 animals/group. χ²-analysis did not indicate a significant difference in the presence of NMJs (P = 0.13), neuromuscular junction shape (P = 0.065), innervation of NMJs (P = 0.58), or the presence of collateral axons (P = 0.42).

The pudendal nerve of sham-injured animals demonstrated well-organized, dense axons on immunofluorescence (Fig. 8). There was a significant loss of axon organization after PNC + VD compared with sham injury (Fig. 8) and improved organization after administration of one dose of MSCs (P = 0.020; Fig. 8). However, this improvement in axon organization was not observed after two or three doses of MSCs. There were no significant differences in axon thickness and density within the pudendal nerve between experimental groups.

Fig. 8.

Effects of multiple doses of mesenchymal stem cells (MSCs) on pudendal nerve regeneration after pudendal nerve crush and vaginal distension (PNC + VD). A–E: representative images of immunofluorescently labeled neurofilament (green) of pudendal nerve cross sections. A and B: there was a trend toward loss of axon organization after PNC + VD (B) compared with sham-injured rats (A). C–E: there was an improved organization of axons after administration of one, two, or three doses of MSCs. Bars represent data from 6 animals/group. χ²-analysis indicated a significant difference in organization of axons (P = 0.020) but not axon density (P = 0.91) or axon thickness (P = 0.29). Fisher’s exact test of organization of axons did not indicate significant differences between individual groups.

DISCUSSION

In women, vaginal delivery is a major risk factor for developing SUI. Trauma to the pelvic floor induced by childbirth includes physical injury to pelvic organs, muscles, nerves, and connective tissue, leading to loss of bladder neck support, decreased innervation of the urethra, and EUS dysfunction (8, 36). Several animal models based on these pathophysiological mechanisms of SUI have been developed (14). The loss of bladder neck support by urethrolysis or pubourethral ligament injury has also been shown to decrease LPP in female rats (21). A disadvantage of these models is that collateral damage to the surrounding tissues is almost unavoidable when animals are subjected to these injuries. Multiple intrinsic urethral sphincter deficiency models, for example, pudendal nerve transection and urethral cauterization, have been studied (16). Although these models cause SUI in female rats, they do not represent the clinical injuries that lead to SUI in women (30). The VD model causes neuromuscular and connective tissue injury and, paired with PNC, which simulates pudendal nerve compression in Alcock’s canal during human vaginal delivery, produces an injury model that better represents the injury to women during childbirth.

Treatment options for women with SUI, such as surgery and physical therapy, are aimed at improving pelvic floor muscular function and bladder neck stability. Surgery is more effective than physical therapy (24), but implantation of midurethral slings can result in complications, such as erosion, voiding dysfunction, urgency, and chronic pain (7). There is no treatment to improve neuromuscular and connective tissue function within the urethra. Therefore, there is a need for additional treatment options, which may be provided by cell-based therapies (42). Multiple animal and clinical studies have demonstrated that cell therapy can restore urethral function (1, 2, 5, 11, 13, 15, 22, 31, 33).

In the present study, we demonstrated that two and three doses of MSCs restore urethral function in rats. We did not observe a statistically significant difference in LPP and peak pressure between multiple doses and a single dose of MSCs. Our data support findings from a previous study (11) showing that a single dose of MSCs leads to partial restoration of function and regeneration of the urethra after PNC + VD. Deng et al. (11) demonstrated enhanced elastogenesis, as well as NMJ and neuromuscular regeneration, 3 wk after PNC + VD. Although we did not demonstrate statistical significance in the current study, a single dose of MSCs showed an increase in LPP similar to that previously reported by Deng et al. (11). A possible explanation for these different findings could be the statistical analysis using multiple comparisons in the present study in combination with greater variability and a smaller number of animals in each group. Therefore, two doses are likely needed in future studies to demonstrate a functional effect.

Our findings regarding LPP data are comparable to those reported previously. Li et al. (27) reported a mean LPP of 50 cmH₂O in control rats, with a decrease of urethral function after VD to a mean LPP of 30 cmH₂O. In our study, mean LPP was 39 cmH₂O in sham-injured animals and decreased to 27 cmH₂O after PNC + VD. Possible explanations for these differences are variability in individual experimental conditions, including variability in Sprague-Dawley rats, an outbred strain.

We demonstrated significantly increased peak pressure and LPP after two and three doses of MSCs but did not demonstrate significant effects of this treatment on EUS EMG or ENG of the pudendal nerve. Nonetheless, our histological data support neuromuscular recovery within the EUS, since we demonstrated that MSCs prevented disruption of the EUS muscle fibers after injury. Jiang et al. (20) demonstrated that while the EUS is the largest single contributor to LPP measurement, it accounts for only 30–40% of LPP. In our study, we found evidence of restored connective tissue within the urethra after MSC treatment, which likely contributes to improvement of urethral function, even in the absence of neuromuscular recovery. Later time points would likely demonstrate greater neuromuscular recovery (36).

Local, as well as systemic, MSC treatment has shown beneficial effects on urethral function after simulated childbirth injury (11, 13, 35). Since local injection could cause bleeding and microvascular damage to the connective tissue around the urethra and possibly influence functional and morphometric results (13), we chose to administer MSCs systemically. Stem cells can act through homing and differentiation into target tissues as well as by secretion of paracrine factors (40). Woo et al. (43) showed an increase in stem cell homing cytokine (C-C motif) ligand (CCL7; MCP-3) expression in the rat urethra and vagina after VD, which decreased 24 h later (43). CCL7 receptors chemokine (C-C motif) receptors 1 and 5 were also upregulated in the urethra until ≥6 h after VD in rats (45). Intravenously injected stem cells can be expected to home to the pelvic area after simulated childbirth injury when injected 1 h after injury (6). Nonetheless, we did not investigate the presence of MSCs within the urethra, since others have found a short lifespan of MSCs after intravenous injection (11).

Based on the timing of expression of homing cytokines, one could hypothesize that multiple doses of MSCs administered systemically >24 h after injury will not have an additional effect on tissue regeneration. However, others have shown beneficial effects of repeated MSC treatments compared with a single dose. For example, van Velthoven et al. (41) showed that while a single dose of MSCs in the ipsilateral hemisphere after hypoxic-ischemic brain injury improved sensorimotor function, a second dose of MSCs further enhanced sensorimotor function. In a rat model of chronic kidney disease, Lee et al. (25) demonstrated that four weekly intravenous injections of MSCs had a beneficial effect on kidney function 5 wk after partial nephrectomy. A single dose of MSCs did not have these beneficial effects on functional outcomes but did have significant effects on histological outcomes (25). Our data show a similar trend: although a single dose of MSCs did not significantly improve functional outcomes, it improved histological outcomes, and additional doses did not further enhance the histological results.

Possible explanations for different effects of single versus multiple doses of MSC are a change in the microenvironment by MSCs and alterations in cytokine expression shortly after tissue damage, as stated above (43, 45). It is likely that effects of multiple doses of stem cells are mediated by paracrine effects, resulting in decreased fibrosis and prolonged proliferative response in damaged tissues (38).

This study is limited by the use of an animal model for SUI, which only partly mimics SUI after childbirth injury in women. The mechanism of SUI in this quadruped model is different from that in humans. The rodent bladder is supported by the abdominal wall, and urethral hypermobility does not likely play a role in SUI development (21, 23). The human bladder, on the other hand, is supported by the muscles of the pelvic floor, which play an important role in continence due to stabilization of the urethra, preventing urethral hypermobility (9). SUI in women has a multifactorial etiology. Risk factors for SUI, such as high body mass index, hysterectomy, aging, and parity (44), were not investigated in this study. Furthermore, we studied only short-term effects of MSC administration. Future studies will be aimed at long-term outcomes and investigation of delayed treatment, as well as investigation of the mechanism of action of repeated doses of MSCs, on urethral function. Finally, the fact that we did not observe significant differences in urethral function measures after multiple doses compared with a single dose of MSCs could be due to a lack of power because of multiple comparison and relatively small number of animals per group. These factors, in combination with animal variability, may have resulted in the lack of a significant increase in LPP after three doses of MSCs compared with vehicle, although three doses of MSCs significantly increased peak pressure, an alternate measure of urethral competency.

Notably, we found that MSC treatment improved NMJ innervation and EUS integrity; however, we did not demonstrate an increase in EUS function as measured with EUS EMG firing rate and amplitude during LPP testing. Likewise, we showed a trend toward improved pudendal nerve morphology with MSC treatment, but our data did not show an increase in pudendal nerve function, as measured with ENG, after MSC treatment. We hypothesize that this is explained by the ongoing restoration process, resulting in morphological, but not yet functional, regeneration. In support of this hypothesis, it has been demonstrated that morphological parameters of peripheral nerve regeneration do not always correlate with functional improvement (10, 28).

In conclusion, in the present study, we show that two or three doses of MSCs after simulated childbirth injury significantly improve urethral function. Moreover, we show that single, as well as repeated, treatments with MSCs improve urethral integrity by restoring urethral connective tissue composition and neuromuscular structures. MSC treatment improved elastogenesis, prevented disruption of the EUS, and enhanced pudendal nerve morphology. There was a trend toward improved reinnervation of NMJs within the EUS. Based on these data, we conclude that single and multiple doses of MSCs regenerate urethral integrity and that the efficacy of MSC treatment to prevent SUI by regenerating urethral function can be improved with multiple doses.

GRANTS

This work was supported by Cleveland Clinic and Rehabilitation Research and Development Service of the Department of Veterans Affairs Merit Review Award I01 RX000228-01A.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

K.J., C.H.v.d.V, and M.S.D. conceived and designed research; K.J., D.L.L., B.H., K.D., and B.M.B. performed experiments; K.J., B.H., K.D., B.M.B., and M.S.D. analyzed data; K.J., B.H., K.D., B.M.B., and M.S.D. interpreted results of experiments; K.J., B.M.B., and M.S.D. prepared figures; K.J., B.M.B., and C.H.v.d.V. drafted manuscript; K.J., D.L.L., B.H., K.D., B.M.B., C.H.v.d.V., and M.S.D. edited and revised manuscript; K.J., B.H., K.D., B.M.B., C.H.v.d.V., and M.S.D. approved final version of manuscript.