**3. Establishing the causes of incontinence in animals**

#### **3.1. Causes of SUI**

The childbirth injury leads to SUI due to musculofascial and neurovascular damage causing weakness in pelvic floor support [24]. Rodents are used to establish the vaginal injury as a lead‐ ing cause of SUI which occur secondary to vaginal dilatation (VD) during childbirth in human. Several studies [25, 26] have demonstrated vaginal injury by VD which is induced by using a Foley catheter with cut tip and inflated with different fluid volumes from 2 to 4 ml. This creates pressure in vagina and iatrogenic injury to the urethra, bladder, vagina and levator muscles. Functionally, VD results in decreased urethral resistance, as evidenced by lowered leak point pressures on urodynamic testing done in most of the VD studies [27]. In a study by Lin et al., VD was created in mice by 0.1–0.3 ml balloon in comparison with sham distension. LPP was signifi‐ cantly lower in groups after VD with 0.2–0.3 ml as compared to sham [28]. Research has shown that this procedure has helped in understanding molecular factors like chemokines, neuro‐ regenerative agents and pharmacological agents that contribute to functional recovery includ‐ ing stem cell mobilization following injury [27, 29]. It has also helped in evaluation of the impact of contributing/decompensating factors in the pathophysiology and recovery of continence.

#### **3.2. Causes of UUI**

*2.2.2. Vertical tilt table LPP*

*2.2.3. Electrical stimulation LPP testing*

**2.3. Urethral closure pressure testing**

suppressing supraspinal continence control [22].

**3. Establishing the causes of incontinence in animals**

table test [21].

**3.1. Causes of SUI**

Rat is mounted on a vertical tilt table to keep the bladder erect during UDS, similar to human studies. A saline reservoir is connected to a suprapubic catheter to passively increase bladder

In this method, the spinal cord is often transected usually at T8–T9. This transaction elimi‐ nates the supraspinal reflex voiding but preserves the urethral reflexes induced by bladder distention, which are predominantly organized in the lumbosacral spinal cord [20]. Studies have shown comparable results of LPP with sneeze test, manual pressure test and vertical tilt

Electrical stimulation of abdominal muscles for 1 s induces sudden increase in both the intra‐ abdominal and the intravesical pressure. The lowest intravesical pressure that induced fluid leakage from the urethral orifice (leak point pressure) and the maximal intravesical pressure without urine leakage were recorded and were used to evaluate urethral resistance. However, like tilt table testing, electrical stimulation LPP testing also requires spinal cord transection,

Effects of stem cell transplantation in rats were evaluated through urodynamic testing, and morphologic changes of the urethra and surrounding tissues were studied [23] both before and after transplantation. The bladder catheter was used as an intraurethral pressure mea‐ surement catheter, connecting it to a three‐limb tube through a conversion joint. One end of that three‐limb tube was connected to the intraurethral pressure sensor, and the other end was connected to the micropump, maintaining the original intraurethral pressure measure‐ ment catheter. Pressure was set at 0, and infusion by micropump at rate of 0.25 ml/min was started. Urethral pressure profilometry (UPP) rod was used to pull the intraurethral pres‐ sure measurement catheter at 0.1 mm/s traction speed. Meanwhile, intraurethral pressure and intrabladder pressure were recorded. Maximum urethral closure pressure (MUCP) was intraurethral pressure minus intrabladder pressure. Functional urethra length (FUL) was also calculated. Transplantation of adipose‐derived stem cells significantly strengthened local ure‐ thral muscle layers and significantly improved the morphology and function of sphincters.

The childbirth injury leads to SUI due to musculofascial and neurovascular damage causing weakness in pelvic floor support [24]. Rodents are used to establish the vaginal injury as a lead‐ ing cause of SUI which occur secondary to vaginal dilatation (VD) during childbirth in human.

O) [19].

pressure by elevating it and maintaining it at a range of pressures (20, 40 and 60 cm H2

128 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

Urinary urge incontinence is observed among patient of overactive bladder (OAB) which is called wet OAB. There are many pathophysiological bases for its explanation including neuro‐ genic and myogenic theories. It has been established through animal studies that urge incon‐ tinence is predominantly due to a defect in bladder muscle [30]. In a study on pigs, unstable bladder contractions were produced against induced outflow obstruction, bladder distention and bladder transaction. In affected pigs, stimulation of the spinal roots could no longer alter detrusor contraction. Similarly, sectioning of the spinal roots in these animals did not elimi‐ nate the unstable pressure rise explaining myogenic basis of OAB [31]. These manipulations do not eliminate the possibility of increased neuronal firing at the ganglionic level. However, recently, it has been shown that both hexamethonium (which blocks ganglionic transmission) and tetrodotoxin (TTX, which abolishes all neuronal activity) inhibit micturition but do not abolish unstable contractions in the pigs or rats [32, 33], hence supporting myogenic theory. The majority of the structural changes seen were obtained with light microscopic techniques, and local detrusor changes were found similar to those among human with OAB.

#### **3.3. Causes of DI**

The innervation of the external urethral sphincter (EUS) from the pudendal nerve is similar between rats and humans [34]. In female rats, the motor pudendal nerve bifurcates within Alcock's canal into separate fascicles that innervate the external anal sphincter (EAS) and EUS. The pudendal nerve controls EUS activity, including tonic activity during continence, and acti‐ vates to strengthen the guarding response to prevent urinary leakage [35]. It can be trapped and injured during vaginal childbirth because it passes through Alcock's canal in the ischiorec‐ tal fossa, especially between the sacrospinous and the sacrotuberous ligaments [36]. Pudendal nerve crush (PNC) injury was induced in rats simulating childbirth injury, leading to deficiency of EUS and causing SUI [37]. Another rat study demonstrated the Pudendal nerve injury effects on external anal sphincter similar to injury during child birth in human affecting EAS and causing FI. In Healy et al.'s study [38], one group of rats used for the experiment had induced bilateral inferior rectal nerve crush (Group A) injury which then acted as a positive control and was observed for EAS effects. In another group (Group B), an intrapelvic retro‐uterine balloon inflation was performed, mimicking the pressure effects of child birth on the pelvic side wall and pelvic floor. Both groups of rats showed signs of EAS muscle atrophy and denervation, leading to FI. However, EMG signs of re‐innervation were seen in both groups and recovery of muscle mass at 4 weeks, mimicking human pathophysiology of fecal incontinence.

*4.1.1. Selective norepinephrine reuptake inhibitor*

models [41].

results [45].

*4.1.3. Stem cell therapy*

Venlafaxine is a selective norepinephrine (NE) reuptake inhibitor, and it significantly decreases the contraction of bladder muscle and increases urethral resistance. This was ini‐ tially tested on rabbits and rodents. Bladder and proximal urethral muscle strips were electri‐ cally stimulated, and their contractile responses were measured both pre‐ and posttreatment with venlafaxine. It was observed that it significantly increased the contraction of urethral

Animal Models of Double Incontinence: "Fecal and Urinary"

http://dx.doi.org/10.5772/intechopen.69962

131

Duloxetine, a norepinephrine (NE) and serotonin (5‐HT) reuptake inhibitor, can prevent SUI by facilitating noradrenergic and serotonergic systems in the spinal cord at S3 level (nucleus of Onuf) to enhance the sneeze‐induced active urethral closure mechanism. Based on this mechanism, duloxetine is currently being used in humans for conservative manage‐ ment of SUI. Before the human trials, it was tested on cat sphincter [40] and in rat models. Duloxetine caused urethral closing contractions and increased the urethral resistance (leak point pressure) measured using a microtip transducer catheter in the middle urethra of rat

One of the SUI causes includes urethral sphincter deficiency which is called type III SUI or intrinsic sphincter deficiency (ISD). This occurs usually due to inherent defects in the collagen and elastin of urethral sphincter. Many preclinical trials have investigated whether trans‐ plantation of patient's own skeletal muscle‐derived cells (SkMDCs) can restore the sphincter musculature. The specific cell type of SkMDCs is myoblasts, satellite cells, muscle progenitor cells, or muscle‐derived stem cells. The other stem cell (SC) types used for urethral defects include those from the bone marrow, umbilical cord blood and adipose tissue. These cells are injected as periurethral injections. Herrera‐Imbroda et al. used rat models for SC injection, and rats were assessed by LPP testing for therapeutic efficacy of SC treatment [42]. The study also used histological assessment, which revealed the sphincter muscle content, existence of

Rodents were also used to explore the feasibility, safety and efficacy of cellular regi‐ men to treat SUI. SUI was induced by vaginal dilatation (VD), and cystoscopic urethral injections of bone marrow or adipose tissue‐derived mesenchymal stromal cells (BMSC/ ADSC) were given to rats. It was observed that MSCs restored the continence mecha‐ nism by improving vascular and connective tissue status of urethral tissues after VD [43]. In another study, human mesenchymal stromal cells were isolated, expanded and char‐ acterized. These cells were injected trans‐urethrally in immune‐suppressed Göttingen Minipigs. The study found this cellular sphincter therapy in Göttingen Minipigs as very safe and effective against SUI [44]. Some animal studies employed dogs with induced SUI and injected SCs therapy to test safety and efficacy for SUI treatment and found similar

strips (*P* = 0.008) tested by urethral pressure profilometry (UPP) [39].

*4.1.2. Norepinephrine (NE) and serotonin (5‐HT) reuptake inhibitor*

transplanted SCs and possible differentiation of these SCs.
