Evaluation of different techniques to create chronic urinary incontinence in the rat

Authors


Jean-Jacques Wyndaele, Urology, University Hospital Antwerp, 10 Wilrijkstraat B 2650 Edegem, Belgium.
e-mail: wyndaelejj@skynet.be

Abstract

OBJECTIVE

To evaluate models for chronic urinary incontinence (UI) in the rat.

MATERIALS AND METHODS

Two models were fully evaluated: one of repeated dilatation of the vagina, simulating birth trauma, the vaginal dilatation (VD) group; the other, with surgical transposition of the urethra to a vertical position, the urethral transposition (UT) group. The VD rats were evaluated by the sneeze test. When negative, vaginal dilatation was repeated in a similar way. The UT group was evaluated by observation of continuous urine leakage. The leak-point pressure (LPP) was measured at study end in all the rats.

RESULTS

All the VD rats had occasional negative sneeze tests and all had to be dilated again. This resulted in persistent UI on sneeze testing for the entire period. In the UT group, 12 rats leaked continuously during the whole study period; in the other four UI became less at 4, 5, 6, and 7 weeks, respectively. The LPP in the rats with UI was significantly lower than in the respective control groups.

CONCLUSIONS

These models permit study of chronic stress UI and continuous UI in the rat. Spontaneous recovery of continence was seen mostly in the VD group.

Abbreviations
(S)UI

(stress) urinary incontinence

LPP

leak-point pressure

VD

vaginal dilatation

UT

urethral transposition.

INTRODUCTION

Genuine stress urinary incontinence (SUI) is defined as the involuntary leakage of urine on effort or exertion or on sneezing or coughing. SUI is a common problem in women but its pathophysiology is still not completely understood. The effect of more continuous UI on lower urinary tract function also remains greatly unknown.

Adequate animal models could give further insight. Animal models for SUI have been developed before [1,2]. However, the first observations showed a low rate of UI and a relatively short durability. Rodiguez et al. [3] decreased urethral resistance with urethrolysis, while Peng et al. [4] used pudendal nerve injury.

Childbirth by the vaginal route is a known factor in developing SUI and a model with vaginal balloon dilatation has been proposed [1,5].

We fully evaluated two models in the rat: a repeated vaginal dilatation model (VD) and a surgical urethral transposition model (UT).

MATERIALS AND METHODS

The institution’s Ethics Committee approved the study procedure. Female virgin Wistar rats were used (225–250 g). Four groups were included randomly: the VD group (13 rats), the control group for VD (six rats), the UT group (16 rats), and the control group for UT (six rats). After 8 weeks the rats were killed. For all surgical procedures the rats were anaesthetized with a mixture of i.p. ketamine (100 mg/kg) and xylazine (15 mg/kg). In all the rats, urinary retention was evaluated daily by digital palpation of the rats’ lower abdomen. All rats were able to eat and drink ad libidum.

For the VD model, a transurethral catheter (22 G) was inserted to avoid bladder over distension during the procedure. The rats were anaesthetized and an intravaginal balloon was inflated with 5 mL sterile water and kept in place for 3 h. Evaluation of UI was done at weekly intervals. A rat whisker was inserted into the nostril to induce sneezing [2]. The sneeze test was conducted twice consecutively and was considered positive if there was urinary leakage from the external meatus. If the sneeze test was negative, i.e. SUI no longer evident, intravaginal balloon dilatation was repeated under light anaesthesia (ketamine 20 mg/kg and xylazine 3 mg/kg). The rats were given analgesia (tramadol 5 mg/kg orally) for 1 day after dilatation. After 8 weeks SUI was evaluated finally by the sneeze test and the leak-point pressure (LPP) was measured. A control group underwent the same procedure with no inflation of the intravaginal balloon (six rats).

For the UT model, the rats (16) were anaesthetized and a transurethral catheter (22 G) was inserted. A suprapubic skin incision (1 cm) was made and the abdominal muscles were split to reach the urethra. The urethra together with the vagina were surgically freed and fixed to the abdominal skin in a vertical position. The incision was closed in two layers. In the sham-operated group (six rats) a transurethral catheter (22 G) was inserted, the suprapubic skin incision made and the abdominal muscles split. The abdomen was closed in two layers. Analgesia (tramadol 5 mg/kg orally) and antibiotics (enrofloxacin, 1 mL 5%, s.c.) were given for 5 days.

UI was evaluated weekly by objectifying continuous urine loss when running freely on an absorbing paper that colours in contact with urine for a couple of minutes during daylight.

The LLP was measured at 8 weeks and the rats were anaesthetized and positioned in dorsal decubitus. A 22 G transurethral catheter was inserted and bladder emptying was assisted with light manual pressure. Through one outlet of a pressure transducer (Hewlett Packard, 1290 C with HP sterile membrane transducer domes, 1925 A) the catheter was connected to a saline reservoir. The second outlet was connected to an oscilloscope (Hewlett-Packard 78342 A). Recording was done with an amplifier monitor (Hewlett-Packard 7834 A), an interface (1401 plus, Cambridge Electronic Design, CED) and the software program Spike2 (CED).

The bladder catheter was connected and calibrated. The bladder was filled with 0.2 mL saline. The LPP was measured as described previously [5]: manually increasing the abdominal pressure until leakage at the urethral meatus occurred. This procedure was performed twice.

Non-parametric tests (Mann–Whitney U) were used to compare the differences between the groups. Results are expressed as the mean (sem); P < 0.05 was considered to indicate statistical significance [6].

RESULTS

VD GROUP

During the total study period, all rats had occasional negative sneeze testing without a clear pattern (Table 1). All had to be dilated again at one time or another. The sneeze test was always negative 14 days after the previous dilatation. In the 8 week period, all rats were dilated five times. Repeated dilatation made SUI persist in all rats. None of the rats in the control group showed urinary leakage during any of the sneeze tests.

Table 1.  The experiment’s development for the VD (SUI) group. All rats were anaesthetized for 3 h at week 0 and the LPP was measured at week 8
 Week 0Week 1Week 2Week 3Week 4Week 5Week 6Week 7Week 8
  1. ST neg, sneeze test negative; ST pos, sneeze test positive.

VD group
rat 1VDST neg: VDST pos: no VDST neg: VDST pos: no VDST neg: VDST pos: no VDST neg: VDLPP
rat 2VDST neg: VDST pos: no VDST neg: VDST neg: VDST pos: no VDST neg: VDST pos: no VDLPP
rat 3VDST pos: no VDST neg: VDST pos: no VDST neg: VDST pos: no VDST neg: VDST neg: VDLPP
rat 4VDST neg: VDST pos: no VDST pos: no VDST neg: VDST neg: VDST pos: no VDST neg: VDLPP
rat 5VDST pos: no VDST neg: VDST neg: VDST pos: no VDST pos: no VDST neg: VDST neg: VDLPP
rat 6VDST pos: no VDST neg: VDST neg: VDST pos: no VDST neg: VDST pos: no VDST neg: VDLPP
rat 7VDST pos: no VDST neg: VDST pos: no VDST neg: VDST pos: no VDST neg: VDST neg: VDLPP
rat 8VDST neg: VDST pos: no VDST neg: VDST pos: no VDST neg: VDST pos: no VDST neg: VDLPP
rat 9VDST pos: no VDST neg: VDST pos: no VDST neg: VDST pos: no VDST neg: VDST neg: VDLPP
rat 10VDST pos: no VDST neg: VDST neg: VDST pos: no VDST neg: VDST neg: VDST pos: no VDLPP
rat 11VDST neg: VDST pos: no VDST neg: VDST pos: no VDST neg: VDST pos: no VDST neg: VDLPP
rat 12VDST pos: no VDST neg: VDST neg: VDST pos: no VDST neg: VDST neg: VDST pos: no VDLPP
rat 13VDST pos: no VDST neg: VDST pos: no VDST neg: VDST neg: VDST pos: no VDST neg: VDLPP
Control group SUI
rats 1–6No VDST neg: no VDST neg: no VDST neg: no VDST neg: no VDST neg: no VDST neg: no VDST neg: no VDLPP

The mean (sem) LPP in the VD rats was significantly lower than that in the control group, at 36.3 (4) cmH2O vs 64.3 (6) cmH2O (P = 0.008).

Urinary retention did not occur and all the rats survived anaesthesia and dilatation.

UT GROUP

Twelve rats leaked urine continuously during the entire study period (Table 2). In the other four rats UI became less at week 4, 5, 6, 7, respectively. The LPP in the UT rats was significant lower than in the control group, at 19.8 (6) cmH2O vs 61.8 (9) cmH2O (P = 0.001). Even the four rats that were less incontinent had a very low LPP (15, 17, 23, 35 cmH2O, respectively). The UT group had a significantly lower LPP than the VD group, at 19.8 (6) cmH2O vs 36.3 (4) cmH2O (P = 0.007).

Table 2.  The experiment’s development for the UT (continous UI) group. All rats were anaesthetised for UT or sham operation at week 0 and the LPP was measured at week 8
 Week 0Week 1Week 2Week 3Week 4Week 5Week 6Week 7Week 8
  1. SO, sham operated; test, running on absorbing paper to objectify UI.

UT group
rat 1UTtest: postest: postest: postest: postest: postest: postest: postest: pos, LPP
rat 2UTtest: postest: postest: postest: postest: postest: postest: postest: pos, LPP
rat 3UTtest: postest: postest: postest: postest: negtest:negtest: negtest: neg, LPP
rat 4UTtest: postest: postest: postest: postest: postest: postest: postest: pos, LPP
rat 5UTtest: postest: postest: postest: postest: postest: postest: postest: pos, LPP
rat 6UTtest: postest: postest: postest: postest: postest: postest: postest: pos, LPP
rat 7UTtest: postest: postest: postest: negtest: negtest: negtest: negtest: neg, LPP
rat 8UTtest: postest: postest: postest: postest: postest: postest: postest: pos, LPP
rat 9UTtest: postest: postest: postest: postest: postest: postest: negtest: neg, LPP
rat 10UTtest: postest: postest: postest: postest: postest: postest: postest: pos, LPP
rat 11UTtest: postest: postest: postest: postest: postest: postest: postest: pos, LPP
rat 12UTtest: postest: postest: postest: postest: postest: postest: postest: pos, LPP
rat 13UTtest: postest: postest: postest: postest: postest: postest: postest: pos, LPP
rat 14UTtest: postest: postest: postest: postest: postest: postest: postest: pos, LPP
rat 15UTtest: postest: postest: postest: postest: postest:negtest: negtest: neg, LPP
rat 16UTtest: postest: postest: postest: postest: postest: postest: postest: pos, LPP
Control group for UT
rats 1–6SOtest: negtest: negtest: negtest: negtest: negtest: negtest: negtest: neg, LPP

Urinary retention did not occur at any time. None of the sham-operated group had signs of UI or urinary retention. All rats survived anaesthesia and surgical manipulation. Weight gain in the 8 weeks was similar in the different groups.

DISCUSSION

Kim et al. [7] described the normal continence mechanism in female rats based on the external urethral sphincter. 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 analogue 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 fibres between the urethra and vagina become more sparse. Most nerve fascicles are contained in the lateral quadrants of the urethra, similar to the lateral distribution of somatic nerves in humans.

Kamo et al. [8] described the continence mechanism under a sneeze-induced stress condition in female rats. Their study showed that under stress conditions, such as sneezing, the rat urethra has active urethral closure mechanisms mediated by somatic nerve-induced reflex contractions of the external urethral sphincter and pelvic floor muscles, in addition to passive transmission of increased abdominal pressure: ‘the bladder-to-urethra reflex’. A passive increase of the intravesical pressure can elicit pelvic afferent nerve-mediated contractile reflexes in the proximal urethra mediated by activation of sympathetic and somatic nerves [9]. Also, Conway et al. [10] described the continence mechanism during the Valsalva manoeuvre including both passive and active closure of the urethra.

After birth trauma in rats, SUI is based on a decreased active closure mechanism at the mid-urethra without affecting the passive transmission of abdominal pressure in the proximal urethra [11].

The present VD rat model is based both on muscular and on neurological damage [12]. Hypoxia might cause an additional negative factor [13]. Lin et al. [1] reported a significant decrease in urethral wall musculature in UI rats compared with continent ones. Cannon et al. [5,14] also described disrupted muscle fibres around the urethra after balloon inflation. Lin et al. [1] reported that creatine phosphokinase and lactate dehydrogenase levels were increased after trauma, indicating muscular damage. C-Fos staining is used as a marker for nerve injury and Lin et al. [1] showed c-Fos immunoreactivity in neurones in the dorsal horn and around the central canal in spinal segments L6 and S1, indicating nerve injury or irritation in the area of simulated birth trauma.

Using the technique of VD to simulate birth trauma in the present study, all the rats become continent again after 1 or 2 weeks. To permit a longer study of SUI one dilatation is not enough and repetition of the dilatation is needed. This proved feasible and successful in the present study. A sneeze test can help to indicate when a further dilatation is required thus preventing unnecessary procedures and anaesthesia. This approach allows SUI to be maintained for many weeks.

The UT model used in the present study is based on the supportive ‘hammock hypothesis’[15]. In rats both the anterior vaginal wall and the surrounding pelvic floor muscles contribute to the continence mechanism [16]. Mobilizing both the urethra and vagina neutralizes this continence mechanism and induces UI. In the present study, total UI occurred for at least 4 weeks in all the UT rats, but later some developed SUI. One reason for the decrease of grade of UI in these rats may be the more pervious development of connective tissue around the urethra and vagina, creating a hammock-like support. Such an effect after surgery will probably differ in each rat.

The LPP was strongly reduced in both models and significantly more in the UT than in the VD group. The reason why the LPP is higher in the present study compared with data published by Cannon et al.[5,14] is most probably because of the transurethral catheter we used, creating a partial obstruction. This does not limit significantly, in our opinion, the conclusions, as the measurement technique was identical in both the study and control groups.

The present study shows that regular control of UI is necessary in both models. Both procedures seem to have little effect on the general condition of the rats. All continued drinking and eating normally, and all gained weight equally during the study period.

During the present experiments, we also tried other UI models. First, we dilated the urethra by inserting a balloon catheter, as used in percutaneous transluminal angioplasty, and inflated the balloon with increasing diameter up to 3.5 mm. During the procedure urine leakage occurred. But subsequently urinary retention occurred due to urethral oedema, with bladder overdistension and therefore possible dysfunction. Later we tried to create UI by inserting a transurethral stent, as used in percutaneous transluminal angioplasty. Unfortunately, UI persisted only for a mean of 9 days. The procedure could not be repeated. The occlusion of the stent might occur from tissue overgrowth or by stone formation, adherent on the stent. Next, we performed the transposition of the urethra alone using a pernieal approach, to a vertical position, without the vaginal dissection. However, the urethral tissue was too frail and necrosis occurred. None of these techniques proved successful on a chronic basis and have therefore not been described in more detail here.

CONFLICT OF INTEREST

None declared.

REFERENCES

EDITORIAL COMMENT

Rodent animal models for SUI have greatly added to our understanding of pathophysiology of this prevalent disease, since it was first described 10 years ago when a Foley balloon was distended in a rat’s vagina to simulate birth trauma [1]. Variations of this model have emerged in the past decade, each utilizing different methods of injury to the continence mechanism: spinal cord injury [2], bilateral pudendal nerve crush [3], direct electrocautery destruction [4], and complete transabdominal urethrolysis [5]. Each model offers distinctive advantages over the others in studying UI (i.e. some are better suited to study neurogenic UI, while others mimic intrinsic sphincter deficiency) as well as disadvantages, mainly due to the varying degrees of reproducibility and durability of UI.

In this paper, Pauwels et al. address the clear need to compare the various previously described SUI animal models. The authors conclude that rats which undergo vaginal distension eventually recover in 1–2 weeks, thereby allowing us to study the acute tissue injury/recovery phase after simulated birth trauma. On the other hand, the urethrolysis technique produced a more prolonged and severe UI in 12 of the 16 rats. This comparison is valuable in highlighting the differences between both models. Unfortunately, some of the pertinent data is not presented such as assessment of bladder capacity, as testing at different degrees of filling can affect the interpretation of the results. It would have been helpful to see the raw LPP data of all groups to better assess the results and outcomes of the various surgical techniques. Furthermore, a study design incorporating histological analysis of the sphincteric unit from each of the study groups would have allowed the reader to differentiate the level of injury incurred in the incontinent, continent, and control rats.

Lastly, although all the previously described animal models have allowed investigators to assess aspects contributing to the pathophysiology of SUI and the impact of developing therapies, there is no ideal model that truly mimics the likely multifactorial aspect of the pathophysiology of this condition. In addition, the modelling of SUI that occurs in a biped erect human is at best poorly represented by injury models of quadruped animals.

Larissa V. Rodriguez

Associate Professor of Urology, Co-Director Division of Female Urology, Reconstructive Surgery and Urodynamics, Director Female Urology Research, Department of Urology, The Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, CA, USA

REFERENCES

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