Mei Kuang

Lower urogenital tract anatomical and functional phenotype in lysyl oxidase like-1 knockout mice resembles female pelvic floor dysfunction in humans

Female pelvic floor dysfunction (FPFD) is a complex group of conditions that include urinary incontinence and pelvic organ prolapse (POP). In humans, elastin homeostasis has been implicated in the pathophysiology of FPFD. Lysyl oxidase-like 1 knockout (LOXL1-KO) mice demonstrate abnormal elastic fiber homeostasis and develop FPFD after parturition. We compared the lower urogenital tract (LUT) anatomy and function in LOXL1-KO mice with and without POP. LUT anatomy was assessed in LOXL1-KO mice over 28 wk. Pelvic visceral anatomy in LOXL1-KO was evaluated with a 7-Tesla magnetic resonance imaging (MRI) scanner. LUT function was assessed using conscious cystometry and leak point pressure (LPP) testing. Quantitative histological analysis of elastic fibers was performed on external urethral sphincter (EUS) cross sections. By 25 wk of age, 50% of parous LOXL1-KO mice developed POP. LOXL1-KO mice with POP had greater variability in the size and location of the bladder on MRI compared with mice without POP. Parity and POP were associated with lower LPP. Elastin clusters were significantly increased in the EUS of LOXL1-KO mice with POP. Because parity triggers POP in LOXL1-KO mice, LOXL1-KO mice with POP have variable internal pelvic anatomy, and both parity and POP are associated with a decrease in LPP, we conclude that LOXL1 LUT anatomical and functional phenotype resembles FPFD in humans. The increase in elastin clusters in the urethra of LOXL1-KO mice with POP suggests that elastin disorganization may lead to functional abnormalities. We conclude that LOXL1 warrants further investigation in the pathphysiology of FPFD.

Cytokine Expression After Vaginal Distention of Different Durations in Virgin Sprague-Dawley Rats

PURPOSE We investigated the effect of the duration of vaginal distention on the differential expression of stem cell homing, tissue repair cytokines and cytokine receptors to identify the factors most important for recovery from injury. MATERIALS AND METHODS A total of 20, 10-week-old virgin Sprague-Dawley rats were divided into 4 groups, including 1, 4 and 6-hour vaginal distention, and anesthetized sham operation. The vagina, bladder, urethra and rectum were harvested immediately after vaginal distention. Real-time polymerase chain reaction was used to determine the relative expression of cytokines and receptors of interest. Mixed models analysis was used to determine associations between expression levels and vaginal distention duration. RESULTS Positive associations between vaginal distention duration and the urethral expression level were found for 1 of the receptors of monocyte chemotactic protein-3, CCR1 (p = 0.0001) as well as for monocyte chemotactic protein-3 (p = 0.025), CCR5 (p = 0.032) and hypoxia inducible factor-1alpha (p = 0.023). A positive relationship between vaginal distention duration and monocyte chemotactic protein-3 expression was also observed in rectal tissue (p = 0.035). Urethral expression of CCR2, another receptor for monocyte chemotactic protein-3, approached significance (p = 0.066). An inverse relationship between vaginal distention duration and interleukin-8 expression was found in the bladder (p = 0.0008). No association was noted between vaginal distention duration and the expression of stromal derived factor-1, CXCR4, CCR3 and vascular endothelial growth factor in any pelvic organs. CONCLUSIONS These data support a relationship between vaginal distention duration and the subsequent expression of monocyte chemotactic protein-3 and 1 of its associated receptors, CCR1, in the urethra immediately following vaginal distention. The increase in hypoxia-inducible factor1alpha expression in the urethra with prolonged vaginal distention suggests a limited role of tissue ischemia in the immediate response of pelvic organs to vaginal distention.

Simulated childbirth injuries in an inbred rat strain

Vaginal distension (VD) in outbred rats has been shown to decrease urethral resistance, as well as increase the expression of the stem cell‐homing chemokine, monocyte chemotactic factor 3 (MCP‐3), but not stromal derived factor 1 (SDF‐1). The aim of this study was to determine if similar responses are induced by VD in an inbred rat strain.

Impact of Parturition on Chemokine Homing Factor Expression in the Vaginal Distention Model of Stress Urinary Incontinence

PURPOSE Human childbirth simulated by vaginal distention is known to increase the expression of chemokines and receptors involved in stem cell homing and tissue repair. We hypothesized that pregnancy and parturition in rats contributes to the expression of chemokines and receptors after vaginal distention. MATERIALS AND METHODS We used 72 age matched female Lewis rats, including virgin rats with and without vaginal distention, and delivered rats with and without vaginal distention. Each rat was sacrificed immediately, or 3 or 7 days after vaginal distention and/or parturition, and the urethra was harvested. Relative expression of chemokines and receptors was determined by real-time polymerase chain reaction. Mixed models were used with the Bonferroni correction for multiple comparisons. RESULTS Vaginal distention up-regulated urethral expression of CCL7 immediately after injury in virgin and postpartum rats. Hypoxia inducible factor-1α and vascular endothelial growth factor were up-regulated only in virgin rats immediately after vaginal distention. CD191 expression was immediately up-regulated in postpartum rats without vaginal distention compared to virgin rats without vaginal distention. CD195 was up-regulated in virgin rats 3 days after vaginal distention compared to virgin rats without vaginal distention. CD193 and CXCR4 showed delayed up-regulation in virgin rats 7 days after vaginal distention. CXCL12 was up-regulated in virgin rats 3 days after vaginal distention compared to immediately after vaginal distention. Interleukin-8 and CD192 showed no differential expression. CONCLUSIONS Vaginal distention results in up-regulation of the chemokines and receptors expressed during tissue injury, which may facilitate the spontaneous functional recovery previously noted. Pregnancy and delivery up-regulated CD191 and attenuated the expression of hypoxia inducible factor-1α and vascular endothelial growth factor in the setting of vaginal distention, likely by decreasing hypoxia.

The impact of cesarean delivery on pelvic floor dysfunction in lysyl oxidase like-1 knockout mice.

Objective: Lysyl oxidase like-1 (LOXL1) knockout mice have abnormal elastic fiber homeostasis and frequently develop pelvic floor dysfunction after pregnancy and delivery. The objective of this study was to test the hypothesis that tissue changes associated with vaginal delivery lead to pelvic floor dysfunction as a result of abnormal elastic fiber homeostasis. Methods: Female LOXL1 knockout mice delivered either spontaneously or by cesarean delivery. Mice were assessed weekly for pelvic organ prolapse (POP). At 12 weeks postpartum, lower urinary tract function was assessed by cystometry and leak-point pressure testing. Urethrovaginal cross-sections were analyzed using a histologic grading scale to assess elastin fiber disorganization. Results: A total of 39 mice delivered by spontaneous vaginal delivery and 36 by cesarean delivery. Twelve weeks after spontaneous vaginal delivery or cesarean delivery, 23 (59%) and 11 (31%) mice had developed POP, respectively. The mean time to develop POP was 7.2 weeks after spontaneous vaginal delivery and 10.5 weeks after cesarean delivery (log rank, P = 0.0008). The Cox proportional hazard ratio was 0.55 (95% confidence interval, 0.38–0.79). Mice with POP had increased frequency of bladder contractions not associated with voiding during cystometry (P = 0.02). POP, but not mode of delivery, was associated with increased elastic fiber disorganization. Conclusions: Cesarean delivery delays the development of POP in LOXL1 knockout mice. POP is associated with increased bladder contraction frequency and increased elastic fiber disorganization in the urethra and vagina. The mechanisms underlying these findings warrant further investigation.

Electrical Stimulation Followed by Mesenchymal Stem Cells Improves Anal Sphincter Anatomy and Function in a Rat Model at a Time Remote From Injury

BACKGROUND: We have explored cell-based therapy to aid anal sphincter repair, but a conditioning injury is required to direct stem cells to the site of injury because symptoms usually manifest at a time remote from injury. OBJECTIVE: We aimed to investigate the effect of local electrical stimulation followed by mesenchymal stem cell delivery on anal sphincter regeneration at a time remote from injury. DESIGN AND MAIN OUTCOME MEASURES: With the use of a rat model, electrical stimulation parameters and cell delivery route were selected based on in vivo cytokine expression and luciferase-labeled cell imaging of the anal sphincter complex. Three weeks after a partial anal sphincter excision, rats were randomly allocated to 4 groups based on different local interventions: no treatment, daily electrical stimulation for 3 days, daily stimulation for 3 days followed by stem cell injection on the third day, and daily electrical stimulation followed by stem cell injection on the first and third days. Histology-assessed anatomy and anal manometry evaluated physiology 4 weeks after intervention. RESULTS: The electrical stimulation parameters that significantly upregulated gene expression of homing cytokines also achieved mesenchymal stem cell retention when injected directly in the anal sphincter complex in comparison with intravascular and intraperitoneal injections. Four weeks after intervention, there was significantly more new muscle in the area of injury and significantly improved anal resting pressure in the group that received daily electrical stimulation for 3 days followed by a single injection of 1 million stem cells on the third day at the site of injury. LIMITATION: This was a pilot study and therefore was not powered for functional outcome. CONCLUSIONS: In this rat injury model with optimized parameters, electrical stimulation with a single local mesenchymal stem cell injection administered 3 weeks after injury significantly improved both new muscle formation in the area of injury and anal sphincter pressures.

Vaginal Expression of LOXL1 in Premenopausal and Postmenopausal Women With Pelvic Organ Prolapse.

Objectives This study aimed to compare cellular expression of lysyl oxidase-like 1 (LOXL1), a key enzyme in elastin metabolism, of premenopausal women with pelvic organ prolapse (POP) compared with premenopausal controls without POP and postmenopausal women with POP. In addition, we examined whether variation of LOXL1 expression was dependent on biopsy site. Methods A standardized protocol was utilized to obtain vaginal biopsies from 30 women (10 premenopausal POP, 10 postmenopausal POP, and 10 premenopausal non-POP). Expression levels of messenger RNA (mRNA) and protein of LOXL1 were determined using real-time quantitative polymerase chain reactions and enzyme-linked immunosorbant assays. Analysis was performed to determine if there were differences between group or biopsy site. Results Significant differences in LOXL1 mRNA expression were found between patient groups (P = 0.0033). LOXL1 mRNA expression (relative to 18S) was upregulated in the postmenopausal POP group (54.5 ± 14.7) compared with the premenopausal POP group (5.2 ± 14.7, P = 0.0034) and the premenopausal non-POP group (23 ± 18, P = 0.0359). No significant differences in LOXL1 protein expression (nanogram/milliliter per microgram total protein) were seen between groups (premenopausal POP, 3.2 × 10−3 ± 6.3 × 10−4; postmenopausal POP, 4.3 × 10−3 ± 6.3 × 10−4; premenopausal non-POP, 5.0 × 10−3 ± 7.7 × 10−4; P = 0.15). No differences in mRNA expression were seen between sites (P = 0.74), but significant variation was noted in protein expression (P = 0.001). Conclusions Premenopausal and postmenopausal women with POP exhibit differential expression of LOXL1 suggesting different pathways in the pathogenesis of POP. The role of biopsy location on LOXL1 expression requires further investigation.

Validation of genetically matched wild-type strain and lysyl oxidase-like 1 knockout mouse model of pelvic organ prolapse.

Objectives Lysyl oxidase–like 1 knockout (Loxl1−/−) mice demonstrate deficient elastin homeostasis associated with pelvic organ prolapse (POP). To further investigate the pathophysiology of POP in these animals, a genetically matched homozygous positive (Loxl1+/+) or wild-type strain is needed. This study sought to create and validate genetically matched Loxl1+/+ and Loxl1−/− strains. Methods Female Loxl1−/− mice were backcrossed with male wild-type mice. The resultant heterozygous mice were bred to produce Loxl1+/+ and Loxl1−/− mice, whose genotype was confirmed by polymerase chain reaction (PCR). Multiparous female Loxl1−/− (n = 7) and Loxl1+/+ (n = 9) mice were assessed for POP weekly for 12 weeks after their first vaginal delivery. Pelvic organ prolapse was compared between groups using a Kaplan-Meier survival curve with P of less than 0.05 indicating a significant difference. Vaginal connective tissue histologic finding was assessed qualitatively and quantitatively. Results There were no significant differences between the groups in age or parity. Of the 7 Loxl1−/− mice, 4 developed prolapse by 8 weeks and 6 by 12 weeks postpartum. No Loxl1+/+ mouse prolapsed. Loxl1−/− mice had significantly larger vaginas as determined by area within the lumen and total cross-sectional tissue area. Striated muscle fibers of the urethra in Loxl1−/− mice were less organized, shorter, and thinner than in Loxl1+/+ mice. Conclusions Genetically matched Loxl1−/− and Loxl1+/+ strains can be reliably created by a backcross method and differentiate in their prolapse phenotype. Loxl1−/− mice demonstrate pathology primarily characterized by enlargement of the vagina. Further studies are needed to elucidate the cause of this finding.

Regenerating the Anal Sphincter: Cytokines, Stem Cells, or Both?

BACKGROUND: Healing of an anal sphincter defect at a time distant from injury is a challenge. OBJECTIVE: We aimed to investigate whether re-establishing stem cell homing at the site of an anal sphincter defect when cytokine expression has declined using a plasmid engineered to express stromal derived factor 1 with or without mesenchymal stem cells can improve anatomic and functional outcome. DESIGN: This was a randomized animal study. SETTINGS: Thirty-two female age- and weight-matched Sprague Dawley rats underwent 50% excision of the anal sphincter complex. Three weeks after injury, 4 interventions were randomly allocated (n = 8), including no intervention, 100-&mgr;g plasmid, plasmid and 800,000 cells, and plasmid with a gelatin scaffold mixed with cells. MAIN OUTCOME MEASURES: The differences in anal sphincter resting pressures just before and 4 weeks after intervention were used for functional analysis. Histology was analyzed using Masson staining. One-way ANOVA followed by the Tukey post hoc test was used for pressure and histological analysis. RESULTS: All 3 of the intervention groups had a significantly greater change in resting pressure (plasmid p = 0.009; plasmid + cells p = 0.047; plasmid + cells in scaffold p = 0.009) compared with the control group. The plasmid-with-cells group showed increased organization of muscle architecture and increased muscle percentage, whereas the control group showed disorganized architecture at the site of the defect. Histological quantification revealed significantly more muscle at the site of defect in the plasmid-plus-cells group compared with the control group, which had the least muscle. Quantification of connective tissue revealed significantly less fibrosis at the site of defect in the plasmid and plasmid-plus-cells groups compared with the control group. LIMITATIONS: Midterm evaluation and muscle morphology were not defined. CONCLUSIONS: At this midterm follow-up, local delivery of a stromal derived factor 1 plasmid with or without local mesenchymal stem cells enhanced anal sphincter muscle regeneration long after an anal sphincter injury, thereby improving functional outcome. See Video Abstract at http://links.lww.com/DCR/A324.

Stromal Cell-Derived Factor 1 Plasmid Regenerates Both Smooth and Skeletal Muscle After Anal Sphincter Injury in the Long Term

BACKGROUND: Regenerating muscle at a time remote from injury requires re-expression of cytokines to attract stem cells to start and sustain the process of repair. OBJECTIVE: We aimed to evaluate the sustainability of muscle regeneration after treatment with a nonviral plasmid expressing stromal cell-derived factor 1. DESIGN: This was a randomized study. SETTINGS: The study was conducted with animals in a single research facility. INTERVENTIONS: Fifty-six female age-/weight-matched Sprague–Dawley rats underwent excision of the ventral half of the anal sphincter complex. Three weeks later, rats were randomly allocated (n = 8) to one of the following groups: no treatment, 100 &mgr;g of plasmid encoding stromal cell-derived factor 1 injected locally, local injection of plasmid and 8 × 105 bone marrow–derived mesenchymal stem cells, and plasmid encoding stromal cell-derived factor 1 injected locally with injection of a gelatin scaffold mixed with bone marrow–derived mesenchymal stem cells. MAIN OUTCOME MEASURES: Anal manometry, histology, immunohistochemistrym and morphometry were performed 8 weeks after treatment. Protein expression of cytokines CXCR4 and Myf5 was investigated 1 week after treatment (n = 6 per group). ANOVA was used, with p < 0.0083 indicating significant differences for anal manometry and p < 0.05 for all other statistical analysis. RESULTS: Eight weeks after treatment, all of the groups receiving the plasmid had significantly higher anal pressures than controls and more organized muscle architecture in the region of the defect. Animals receiving plasmid alone had significantly greater muscle in the defect (p = 0.03) than either animals with injury alone (p = 0.02) or those receiving the plasmid, cells, and scaffold (p = 0.03). Both smooth and skeletal muscles were regenerated significantly more after plasmid treatment. There were no significant differences in the protein levels of CXCR4 or Myf5. LIMITATIONS: The study was limited by its small sample size and because stromal cell-derived factor 1 was not blocked. CONCLUSIONS: A plasmid expressing stromal cell-derived factor 1 may be sufficient to repair an injured anal sphincter even long after the injury and in the absence of mesenchymal stem cell or scaffold treatments. See Video Abstract at http://links.lww.com/DCR/A451.