Pelvic floor disorders, including stress urinary incontinence (SUI) and pelvic organ prolapse (POP), reduce quality of life. The etiology of SUI and POP in women is multifactorial, and the histories of vaginal childbirth, aging, estrogen deficiency, and connective tissue deficiencies have previously been reported to contribute significantly to the emergence of both SUI and POP. The tensile strength and elasticity of the pelvic floor, which holds the pelvic organs in place, is provided by the connective tissues, two important components of which are the proteins collagen and elastin. Elastin attains its tensile strength and resilience by forming a cross-linked polymer. The lysyl oxidase-like 1 (Loxl1) gene is involved in forming elastin polymers: It encodes a copper-dependent monoamine oxidase that catalyzes the deamination of a lysine residue on tropoelastin monomers during the formation of crosslinks between monomers.[1] Mice lacking the protein Loxl1 do not deposit normal elastic fibers in the uterine tract postpartum and develop POP; enlarged airspaces in the lung; loose skin; and lower urinary tract dysfunction, with decreased bladder capacity and voiding pressure.[1]  Although elastin might have important roles in SUI and POP, the details of its function in the lower urinary tract are still unclear.
On the basis of the above, we hypothesized that a rat model lacking the protein Loxl1 could be useful to examine bladder and urethral functions and the roles of elastin in the urethral continence function.

For this study, a genetically modified rat line with a Loxl1 gene knockout was developed from outbred Sprague-Dawley rats by CRISPR/Cas9 technology. Female nulliparous rats of Loxl1−/− (n = 26)　or age-matched wild type (WT) rats (n = 28)　had leak-point pressure (LPP) testing, cystometry, and histopathological analyses of lower urinary tract.
<Measurement of leak point pressure by electrical stimulation of abdominal muscle>
After anesthetizing rats (Loxl1−/−: n = 8; WT: n = 8) with urethane, We repeated electrical stimulation every 10 s and gradually increased the stimulus intensity from 2 V to a maximum of 25 V to increase the intravesical pressure in a stepwise manner. We defined leak point pressure (LPP) as the intravesical pressure at which we observed fluid leakage from the urethral orifice.
<Continuous cystometry analysis>
We anesthetized a separate group of rats (Loxl1−/−: n=8 and WT: n=8) with urethane and monitored bladder activity such as the intercontraction interval (ICI), maximum contraction pressure during voiding (MCP), intravesical baseline pressure (IVBP), voided volume, residual volume and bladder capacity. 
<Histological analyses>
The vagina, bladder and urethra of female nulliparous rats (Loxl1−/−: n = 3; WT: n = 3) were stained with elastica van Gieson stain for quantitative assessment of elastic fibers.

<Observation of external genitalia>
The Loxl1−/− male and female rats showed increased looseness and redundancy of the skin when compared with the WT rats. Although, some of the Loxl1−/− male rats showed rectal prolapse, the Loxl1−/− female rats showed no POP or descent of the pelvic floor, even after giving birth. 
<Leak point pressure measurements>
The LPP was significantly lower in the Loxl1−/− rats (32.62 ± 2.61 cm H2O) than in the WT rats (77.39 ± 2.17 cm H2O). (Fig. 1)
<Continuous cystometry analyses>
Cystometry analyses showed a significantly lower ICI and voided volume in the Loxl1−/− group than in the WT group. The increase in residual urine volume and decrease in bladder capacity tended to be lower in the Loxl1−/− rats than in the WT rats.
<Histological analyses>
On elastica van Gieson staining, massive elastic fibers were seen in the lamina propria and muscle layer of the urethra, bladder and vagina in the WT rats. In particular, the median sagittal section of the proximal urethra revealed thick and long elastic fibers in the lamina propria and between the longitudinal smooth muscle bundles.

We investigated the roles of elastin in urethral continence function in a rat model lacking the protein Loxl1. We found the following in the Loxl1−/− rats compared with the WT rats: (1) male and female rats had loosening and redundancy of the skin, and some male rats showed rectal prolapse, but no female rats did; (2) the ICI and voided volume were significantly lower, (3) the LPP was significantly lower; and (4) elastic fibers of the bladder, urethra, and vagina were thinner and shorter. 
The present study showed that urethral continence function was inefficient in the Loxl1−/− rats, as evidenced by the lower LPP compared with the WT animals. The mechanism of resting urethral closure pressure is well established and is important for preserving continence. Continence is preserved by the integrity of the vesicourethral junction, the urethral and pelvic floor musculature, the hydrostatic pressure in the submucosal blood vessels, and the tension of urethral fibroelastic elements.[2] The submucosal fibroelastic tissue has been assumed to be a major factor in maintaining resting urethral closure pressure.[3] Our histopathological analyses, especially in the urethra, showed that the elastic fibers was shorter and thinner in the Loxl1−/− rats than in the WT rats. Deficient elastin fiber might reduce the resting urethral closure pressure and induce the lower LPP in the Loxl1−/− rats compared with the WT rats.
In the cystometry analysis, the Loxl1−/− rats showed a significantly lower ICI and voided volume than the WT rats, as well as a tendency towards a higher residual urine volume and lower bladder capacity. We hypothesize that, because elastin is one of the main components that provides tissue with elasticity, the shorter and thinner elastin fiber in the Loxl1-/- rats may decrease the elasticity of the bladder wall, resulting in a reduced ICI and voided volume. Our findings of insufficient urethral closure pressure (shown by the low LPP and deficient elastic fibers in the bladder wall) in Loxl1−/− rats indicate that insufficient elastin synthesis might have a negative influence on bladder activity.

In the Loxl1−/− rats impaired elastin synthesis in the submucosa of the urethra might reduce the resting urethral closure pressure, and a diminished cholinergic contractile response of detrusor smooth muscle might be involved in bladder activity.

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