Dan Li Lin

Striated Muscle and Nerve Fascicle Distribution in the Female Rat Urethral Sphincter

The anatomical basis for urinary continence depends on a thorough understanding of the tissues in the urethra. The objective of this study was to evaluate the morphology and neuroanatomy of urethral striated muscle, called the rhabdosphincter or external urethral sphincter, in normal female rats. Urethras from 12 female rats were dissected from the bladder, fixed, embedded in paraffin or epon, and sectioned every 1 mm. Striated muscle content was taken as the ratio of the striated muscle area to net urethral area. Nerve fascicles containing myelinated axons near the rhabdosphincter were counted and mapped. Both striated muscle content and number of nerve fascicles peak in the proximal third of the urethra, with a secondary peak at the distal end of the urethra. This secondary peak may correspond to an analog of the combined compressor urethrae/urethrovaginal sphincter located in the distal urethra in human. The rhabdosphincter has a variable distribution along the length of the urethra. In the middle and distal thirds of the urethra, the dorsal striated muscle fibers between the urethra and vagina become more sparse. The majority of nerve fascicles are contained in the lateral quadrants of the urethra, similar to the lateral distribution of somatic nerves in humans. In conclusion, this study demonstrates the normal distribution of the striated musculature and neuroanatomy in the urethra, with similarities to the human. It thus supports and extends the usefulness of the rat as an experimental model for studying urinary incontinence. Anat Rec 290:145–154, 2007.

Short-term timecourse of bilateral pudendal nerve injury on leak-point pressure in female rats.

The pudendal nerve innervates the external urethral sphincter and, when injured, can contribute to incontinence development. This experiment was designed to study the time course of functional changes in the urethra after pudendal nerve crush in rats. Leak-point pressure (LPP) was measured 2, 4, 7, or 14 days after bilateral pudendal nerve crush and was compared to that of a control group. LPP at all four time points after nerve injury was significantly decreased compared to control values. A minimum was reached 4 days after injury, and LPP appeared to trend upward with increasing time after injury, suggesting that nerve function may begin to recover or compensatory changes in the urethra may occur. Pudendal nerve crush induces decreased LPP in female rats, mimicking the clinical symptoms of stress incontinence. When fully characterized, this model could be useful for preclinical testing of treatment and rehabilitation protocols.

Early structural effects of oestrogen on pudendal nerve regeneration in the rat.

To determine the early effects of oestrogen on the ultrastructure of the pudendal nerve and distal nerve fascicles near the external urethra sphincter (EUS) after a pudendal nerve crush injury. The pudendal nerve is one of the pelvic floor tissues injured during vaginal delivery, possibly contributing to the development of stress urinary incontinence (SUI) in women, the symptoms of which often do not appear until menopause, implicating hormonal factors.

Long-term effects of simulated childbirth injury on function and innervation of the urethra

Pudendal nerve and external urethral sphincter (EUS) injury during vaginal delivery are risk factors for stress urinary incontinence (SUI). Although most patients with short‐term postpartum SUI regain continence within 1 year, they have a higher predisposition to develop recurrent SUI years later, suggesting a possible mechanistic relationship. In contrast, animal models generally recover spontaneously and have not been studied much in the long term. The aim of this study was to investigate the long‐term effects of simulated childbirth injury in rats.

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.

MP26-11 THERAPEUTIC OUTCOMES OF INSULIN-LIKE GROWTH FACTOR-1 RELEASED FROM ALGINATE-GELATIN MICROBEADS ON STRESS URINARY INCONTINENCE IN RATS WITH SIMULATED CHILDBIRTH INJURY

alteration of histopathology and expression of TLR4 and NLRP3 inflammasome-related molecules in the bladder using spontaneously hypertensive rats (SHRs) as an OAB model. METHODS: Twenty-weeks-old male SHRs and Wistar Kyoto rats (control) were used. After voiding function was analyzed by using metabolic cages, the bladder was excised for analysis of histopathology and mRNA expression. Hematoxylin eosin and Masson0s trichrome stain were performed to analyze bladder inflammatory condition and fibrosis. Immunohistostaining for NLRP3, TLR4 was also performed. Expression levels of NLRP3, IL1b, IL-18, IL6, IL8 and TGFb mRNA in the bladder were investigated by real-time PCR. Statistical analysis was performed using Mann-Whitney U test. P value less than 0.05 was considered statistically significant. RESULTS: In voiding function analyses, single urine volume was significantly decreased and voiding frequency was significantly increased in SHRs compared to control rats. In histological evaluation, suburothelial fibrosis was shown in SHRs compared to controls. Furthermore, immunohistostaining showed localized expression of NLRP3 and TLR4 in the bladder urothelium in both groups. In RT-qPCR analyses, mRNA expression levels of NLRP3, TLR4, IL1b, IL-18, IL6, IL8 and TGFb were significantly increased in SHRs in the bladder compared to controls. CONCLUSIONS: These results suggest that activation of TLR4 associated with oxidative stress is implicated in bladder chronic inflammation, which leads to frequent urination through NLRP3 inflammasome pathways. Therefore, further clarification of interactions between TLR4 and inflammasome pathways may offer new therapeutic targets for OAB associated with chronic inflammation.

AB303. SPR-30 Neurotrophin dysregulation after dual injury childbirth model is corrected via electrical stimulation of the pudendal nerve in a rat model

Objective Stress urinary incontinence (SUI), the leakage of urine due to an increase in abdominal pressure, affects 30% of women over the age of 40. Childbirth is the primary risk factor for SUI since the child’s head passing through the birth canal can injure the EUS and the nerve that innervates it, the pudendal nerve (PN). Animal models with combined EUS [vaginal distension (VD)] and PN crush (PNC) injuries have shown that the nerve does not recover as well as with PNC alone and the animals have delayed return of function. Typically after a nerve injury, the neuron upregulates neurotrophin factors [i.e., brain derived neurotrophin factor (BDNF)] and their receptors [i.e., tyrosine kinase B (Trk B) and p75], and Schwann cells also upregulate neurotrophin factors [i.e., glial derived neurotrophin factor (GDNF) and NT4] as part of the regeneration process. The impaired nerve regeneration in the dual injury model was shown to be due to a dysregulation of BDNF in the PN motoneuron, so any potential treatment would need to upregulate BDNF. Electrical stimulation (ES) has been shown to upregulate BDNF and Trk B in neurons and GDNF in Schwann cells. Our previous work has shown daily and 4 times per week stimulation for 2 weeks accelerates functional recovery after PNC + VD. The aim of the study was to determine if daily stimulation causes an upregulation of neurotrophin factor and their receptors in motoneurons of the PN after injury. We hypothesize that the accelerated recovery from ES is mediated by an upregulation of BDNF and NT4 and their receptor Trk B, as well as GFRα1, the receptor of GDNF. Methods Animals were divided into five groups: Sham PNC, PNC, PNC + VD, PNC + VD with sham stimulation (SS) or PNC + VD + ES. ES (20 Hz, 0.3 mA, 0.1 ms) was conducted daily for 1 or 2 weeks after injury, when the spinal cord was harvested from L4–S3. The spinal cord was sectioned and Onuf’s nucleus, the motoneuron nucleus for the PN, was harvested via laser capture microscopy. RNA was then isolated and preamplified before running PCR and compared to GAPDH expression. A one way Anova was used to determine statistically significant differences between groups. Results One week after PNC + VD, BDNF was not upregulated as previously shown. Neither were p75 or GFRα1, as expected. NT4 was not downregulated after PNC + VD, as expected. P75 was significantly increased in the PNC + VD + ES group compared to the PNC + VD group 2 weeks after injury. BDNF was also upregulated 2 weeks after PNC + VD + ES compared to PNC + VD. Conclusions P75, NT4 and GFRα1 expression data suggest that PNC + VD causes dysregulation of other regenerative proteins other than BDNF. BDNF was upregulated 2 weeks after PNC + VD with ES. Future research will be designed to investigate the mechanism of this effect. Funding Source(s) VA Merit A1262-R and the RR&D Service of the VA