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Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
was used to compare the differences among groups with the Mann-Whitney U test
(GB-STAT, Dynamic Microsystems Inc., Silver Spring, MD). Values were considered
significant at p<0.05 (In Tables 8–15 significant results are marked by *p<0.05; – = no
animals in this subgroup,whereas 0 = no immune reactive (IR) nerve stain was seen).
Light Microscopy Staining Results: PGP 9.5.Virgin Controls. The urothelial surface
and the basal membrane were strongly stained in all specimens.Therefore, the staining pattern in the epithelium and basal membrane was not analyzed because it was impossible to recognize individually stained nerves (Table 5.8).
In all specimens, PGP 9.5-IR-positive nerves were seen surrounding small vessels,
and were present in higher numbers around larger veins in the bladder neck and midurethra.Within the smooth muscle,more nerves were noted in the mid-urethra, fewer
in the bladder neck, and fewer still in the bladder. A higher number of IR-positive
nerves was found in the inner muscle layer in the bladder neck and mid-urethra
and in the outer muscle layer of the bladder.In the mid-urethra, more PGP 9.5-IR-positive nerves were seen in the smooth muscle than in the striated muscle (Table 5.8, virgin).
Groups I–IV.In the bladder,a statistically significant decrease was noted in both inner and outer layers of the muscle in groups II, III and IV. Both ballooning (group II)
and ovariectomy (group III) decreased PGP 9.5-IR-positive nerves. The combination
of ballooning and ovariectomy (group IV) eliminated PGP 9.5 completely (Table 5.8a).
Table 5.8. Protein gene product 9.5
Bladder EBM
a
c:i c:i c:i c:i c:i
SM iM oM AD
67
Virgin 0:– 0:– 15:– 29:– 1:–
Group I 0: 0 1 : 1 16: 13.5 19:7 3:0
Group II 0: 0 0.5 :3.3 6 :7.3 6: 7.7 1 :0.3
Group III 0 :0 0: 0 6 : 4.5* 3.5 :6.5* 0 :1
Group IV 0:0 0:0 0:0* 0:0* 0:0
Bladder neck EBM SM iM oM AD
Virgin 0:– 5:– 37:– 14.5:– 6.0:–
Group I 0:0 0:0* 12.7:8* 20:8 4.7:4
Group II 0: 0 0 :0* 2 :2.3* 4 :7* 1:2*
Group III 0:0 0:0* 6.5:6* 7.5:8* 2.5:1*
Group IV 0:0 0:0* 3:0* 4:3* 1:1*
Mid-urethra EBM SM iM oM AD
Virgin 0:– 9.5:– 52:– 13.5:– 6:–
Group I 0:0 4.3:2* 40:12 8:10 7.3:0
Group II 0: 0 1.5: 4.3* 31.5 :32.6 8 :4.3 4 :3.3*
Group III 0 :0 2: –* 12.5 : 29* 10.5 :12 4.5: 4*
Group IV 0 :0 0: 3.5* 10 :21* 3 :3.5* 4 :3*
* p<0.05 compared with virgin.
For descriptions of the groups, see text.
a
Anti-PGP 9.5 immunoreactivity was strongly positive in the epithelium and basal membrane of
all three anatomic sections,although no nerves were observed.
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Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
In the bladder neck, PGP 9.5-IR-positive nerves were significantly decreased in the
submucosa and inner muscle layer in all groups.A significant change was also noted
in the outer muscle layer and adventitia of groups II, III and IV (Table 5.8b).
In the mid-urethra,there was a significant decrease in PGP 9.5-IR-positive nerve fibers in the submucosa of groups I–IV and a significant decrease in all layers in group
IV (Table 5.8C).
Light Microscopy Staining Results: CGRP. Virgin Controls. In the muscle layers the
number of PGP 9.5-IR-positive nerves,as noted above, was higher than the number of
CGRP-IR-positive nerves.However, directly beneath the basal membrane a large number of CGRP-IR-positive nerves were apparent in the three sections,as opposed to very
few PGP 9.5-IR-positive nerves or none. The nerve trunks, which demonstrated high
immunoreactive positive PGP 9.5 staining in the muscle layers and adventitia, had a
lower percentage of CGRP-IR-positive nerves (Table 5.9).
IR-positive nerves were mainly seen in the smooth muscle bundles, less frequently
around the striated muscles in the urethra and very infrequently around vessels.
When comparing the three tissue sections, more CGRP-IR-positive nerves were seen
in the bladder neck and mid-urethra than in the bladder (Table 5.9, virgin, Figs. 5.14a,
5.15a).
Groups I–IV. In the bladder, CGRP-IR-positive nerves were significantly decreased
in the inner muscle layer of all groups (I–IV) and in the outer muscle layer of groups
II–IV. IR-positive stained nerve trunks showed a decrease in CGRP. The submucosa
Table 5.9. Calcitonin gene-related peptide
Bladder EBM SM iM oM AD
Virgin 6.5:– 5:– 9:– 9:– 5:–
Group I 8.6:5 5.3:4 6:6* 10.6:6 4:2
Group II 4: 3.3 7 : 4.7 5 :4.7* 7 : 5 3.5 :3.3
Group III 9.3:3 2.3:2* 5:6* 4.7:4* 4.7:5
Group IV 5 :2 3: 2.5* 5: 3.5* 6: 5* 1 :2.5
Bladder neck EBM SM iM oM AD
Virgin 25.5:– 12:– 18.5:– 11:– 5.5:–
Group I 23.7:18 12.3:9 11.3:7* 8:5* 5:4
Group II 20: 23.3 5.5 :8.7 5: 5.3* 8.5 :7* 5.5 :5.7
Group III 26:20 11:11 6.5:6* 7.5:8* 3:3
Group IV 31 :7.5 11: 8 8 : 6* 7 :7 3 :5
Mid-urethra EBM SM iM oM AD
Virgin 20.5 : – 15.5 :– 13.5 : – 11.5 :– 6.0 :–
Group I 26.3:17 18.3:18 8.3:6* 10.3:8 11:4
Group II 11: 7.7* 11 :15 5.5 :5* 4 :4.7* 4.5:5.7
Group III 10.3 :15* 14.3 :18 3.7 :5* 2.3 :4* 1.3 :5
Group IV 10 :13.5* 14 :12.5 5 :4* 4 :2.5* 4 :5.5
*p<0.05 compared with virgin.
For descriptions of the groups, see text.
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Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
69
Fig. 5.14a, b. Basal membrane of the bladder comparing CGRP-positive nerves (arrowheads) of
virgin (a) and group II animals (delivered + ballooned) (b) where no difference is seen.(arrow-
heads point towards the nerves,magnification 63×)
demonstrated a decrease in CGRP-IR-positive nerves in groups III and IV, suggesting
an influence from ovariectomy (Table 5.9a, Fig. 5.14b).
In the bladder neck, there was a significant decrease in CGRP-IR-positive nerves in
the muscle layers of groups I–IV (Table 5.9b). Similar results were seen in the mid-urethra.Additionally, the number of IR-positive nerves was significantly reduced beneath
the basal membrane of groups II–IV (Table 5.9c, Fig. 15b).
Light Microscopy Staining Results: SP. Virgin Controls. SP-IR-positive nerves were
seen in similar locations as CGRP-IR-positive nerves, but in much lower numbers.
Higher numbers of SP-IR-positive nerves were counted in the muscle bundles of the
bladder neck than the mid-urethra (Table 5.10, virgin, Fig. 5.16a).
Groups I–IV.A statistically significant decrease in SP-IR-positive nerves was seen in
the inner muscle layers of the bladder in all groups.A significant decrease in SP directly beneath the basal membrane (Table 5.10), which was not demonstrable with the other stains, was noted in all four groups. Similar results were seen in the bladder neck
(Table 5.10).
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Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
Fig. 5.15a, b. Basal membrane of the urethra comparing CGRP-positive nerves (arrowheads) of
virgin (a) and group II animals (delivered + ballooned) (b) where the number of nerves is significantly reduced (magnification 63×)
In the urethra, the number of SP-IR-positive nerves beneath the basal membrane
and in the submucosa decreased in groups II–IV, with the exception of the submucosa
of group III. The changes in the submucosa seem to be related to ballooning (groups II
and IV) (Table 5.10, Fig.5.16b).
Light Microscopy Staining Results: nNOS. Neuronal NOS-IR-positive nerves were
seen in the bladder neck and mid-urethra, but rarely in the bladder.
Virg in Controls. In the bladder neck, nNOS-IR-positive nerves were mainly observed in the smooth muscle layer as terminals accompanying the muscle bundles. In
addition to the high concentration in the inner smooth muscle layer of the mid-urethra, n-NOS-IR-positive nerves were detected around circular smooth muscle cells
and only a few around the striated muscle cells (Table 5.11, Fig. 5.17a).
Groups I–IV. In the bladder neck, nNOS-IR-positive nerves were decreased in all
four groups,except in group III. In group II only a few nNOS-IR-positive nerves were
observed (Table 5.11).
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
Table 5.10. Substance P
71
Bladder EBM SM iM oM AD
Virgin 7.3:– 3:– 1.6:– 5:– 2:–
Group I 1.6:0* 1.6:0 1.3:1* 3.3:2 0.7:0
Group II 0.5: 1.6* 0 :1.3 1:1.6* 1.3 :1.3 0 : 0.3
Group III 1:0* 0.6:0 1.6:0 1.6:0* 0:0
Group IV 3 :0.5* 2:0 2 :0.5* 3: 2 4:0
Bladder neck EBM SM iM oM AD
Virgin 14:– 5:– 6.3:– 13.3:– 1.3:–
Group I 8:5* 3.3:2 2:1* 5:3* 2.3:0
Group II 7: 6.6* 2.3 :2.3 1.2: 2.3* 3: 5* 1: 1.3
Group III 6.6:9* 2:2 1.6:3* 4.3:4* 1.6:1
Group IV 7 :7.5* 3 :3 3 :1* 6:4.5* 1: 2.5
Mid-urethra EBM SM iM oM AD
Virgin 15.6:– 7:– 3.3:– 2.6:– 0.3:–
Group I 9:5 7:2 3.6:3 5.3:2 2.6:0
Group II 2: 5.3* 1.5 :3.6* 0.5: 2.3 0:1.6 0 : 0.3
Group III 3 :5.3* 5.5:7 2.5 :3 1.5: 1 1.5 :2
Group IV 0 :1* 2 :1.5* 1 :1.5 1 : 1 0:1
*p<0.05 compared with virgin.
For descriptions of the groups, see text.
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c:i c:i c:i c:i c:i
The findings in the mid-urethra were similar. The decrease in nNOS was seen not
only in the muscle bundles but also around the arteries. The basal membrane did not
show any nNOS-IR-positive nerves in groups I–IV (Table 5.11, Fig.5.17 b).
Light Microscopy Staining Results: NPY. Virgin Controls. In the bladder, all sub-
structures demonstrated NPY immunoreactivity.The highest concentration was seen
in the muscle layers (equally in inner and outer layers).In the bladder neck and midurethra,the number of NPY-IR-positive nerves was higher than in the bladder,as seen
in most of the previously described neuropeptides. NPY terminals surrounded arteries in all specimens (bladder, bladder neck and urethra).The bladder neck, and especially the urethra, demonstrated large venous plexuses with terminal nerve endings.
The striated muscle of the outer muscular layer had far fewer NPY-IR-positive nerves
than did the smooth muscle bundles of this layer. Mainly NPY-IR-positive nerve
trunks of the adventitia passed through the outer muscle layer and some through the
inner muscle layer to innervate the submucosa and basal membrane (Table 5.12, virgin).
Groups I–IV.In the bladder,NPY-IR-positive nerves in the muscle layers decreased
significantly in all four groups (Table 5.12).
In the bladder neck and mid-urethra, a significant decrease in NPY-IR-positive
nerves was seen in the muscle layers and the submucosa,specifically around small vessels (Table 5.12).
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Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
Fig. 5.16a, b. SP-IR-positive nerves (arrowheads) beneath and in the basal membrane of the ure-
thra of virgin animals (a) were remarkably reduced by treatments, as shown in group IV (delivered + ballooned + ovariectomized) (b) (magnification 40×)
In the urethra, the NPY terminals were significantly reduced directly beneath the
basal membrane in groups I–IV (Table 5.12).
Light Microscopy Staining Results: VIP.Virgin Controls. Very few VIP-IR-positive
nerves were observed in the bladder and the bladder neck, which were small in diameter (Table 5.13, virgin). The main location was beneath the basal membrane and in the
muscle layer. In the mid-urethra, as in the bladder neck, a similar ratio between the
single substructures was demonstrated. The main VIP-IR-positive nerves were seen in
the wall of small arteries and around the smooth muscle bundles.Large veins around
the outer muscular layer did not demonstrate any nerve terminals. The numbers of
VIP-IR-positive nerves were lower than nNOS- and NPY-IR-positive nerves.
Groups I–IV.In the inner muscular layer of the urethra,delivery (group I) caused a
significant decrease in VIP with an additional slight decrease after ballooning (groups
II and IV). The ballooning decreased VIP-IR-positive nerves beneath the basal membrane and the submucosa,whereas in the outer muscular layer, a significant decrease
was recognized for groups II–IV. As noted in virgin animals, VIP nerves were rarely
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
Table 5.11. Neuronal nitric oxide synthase
73
Bladder EBM SM iM oM AD
Virgin 0:– 0:– 0:– 0:– 0:–
Group I 0:0 0:0 0:0 0:0 0:0
Group II 0:0 0:0 0:0 0:0 0:0
Group III 0:0 0:0 0:0 0:0 0:0
Group IV 0:0 0:0 0:0 0:0 0:0
Bladder neck EBM SM iM oM AD
Virgin 14.3 : – 5 :– 18: – 15.6 / - 4.6 : –
Group I 2 :0* 0 : 0* 4.5 / 6* 3.5 / 1* 0.5 / 2*
Group II 0:0 0:0 0:0 0:0 0:0
Group III 9 :10* 1 : 1* 2: 4* 5 :9 0 :0*
Group IV 4 :3.5* 0:0* 0 : 0* 2 :1* 3 :0*
Mid-urethra EBM SM iM oM AD
Virgin 12 : – 15.3 :– 29.3: – 33.6: – 5.6:–
Group I 0 :0* 2 :3* 2 :6* 6 :3.3* 1:2
Group II 0: 0* 0.5: 0.5* 3 :5* 1.5 :3.5* 1.5: 2*
Group III 0 :0* 1 :0* 3.6 :1* 1.6: 0* 1 : 1*
Group IV 0 :0* 1 :0.5* 0 :2.5* 1: 1.5* 0 : 1.5*
*p<0.05 compared with virgin.
For descriptions of the groups, see text.
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seen in all groups. When present, they were found especially around small arteries
(Table 5.13).
Light Microscopy Staining Results: ACEase. Virgin Controls. A large number of
ACEase-positive nerves was found, with similar distributions in the bladder, bladder
neck and mid-urethra. In the musculature,ACEase-positive nerves supplied the muscle bundles, equally in the smooth and striated muscle of the urethra, encircling small
arteries or running along the urothelium. ACEase-positive nerve trunks from the adventitia were seen traversing between the muscle bundles. The number of ACEasepositive nerves was similar to the number of PGP 9.5-IR-positive nerves, but they were
seen additionally as individual nerves directly beneath the basal membrane. Because
of the staining on the endplates in the muscular layers, which made comparison difficult, PhotoShop was used for analysis (Table 5.14, Fig.5.18a).
Groups I–IV.No significant change was noted in the bladder (Table 5.14a). A significant increase directly beneath the basal membrane of groups III and IV (Table 5.14b)
was noted in the bladder neck.In the mid-urethra, both muscle layers showed a significant decrease in ACEase in groups I–IV (Table 5.14, Fig. 5.18b).
Light Microscopy Staining Results: TH. Virgin Controls. TH-IR-positive nerves
were mainly seen within the smooth muscle bundles and vessel wall. They were occasionally seen around the striated muscles in the mid-urethra. More TH-IR-positive
5
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Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
Fig. 5.17a, b. The treatment of group IV (delivered + ballooned + ovariectomized) (b) caused
nearly a complete loss of nNOS-IR-positive nerves (arrowheads), which were demonstrated in
the urethra of virgin animals (a). The arrows mark the direction through the outer muscle layer
(oM) (magnification 63×)
nerves were seen in the mid-urethra (Table 5.15, virgin, Fig. 5.19a) and bladder neck
than in the bladder.
Groups I–IV. In the bladder, TH-IR-positive nerves were significantly decreased in
both muscle layers of all groups (I–IV). In the submucosa, a decrease in TH-IR-positive nerves was noted in groups I,II and IV (Table 5.15).
In the bladder neck,there was a significant decrease in TH-IR staining in both muscle layers of groups I–IV (Table 5.15).
In mid-urethra, the main change was seen in the smooth muscle cells of groups II
and IV as the result of the ballooning. The most striking change was the almost complete loss of TH-IR-positive nerves in the circular smooth muscle layer (Table 5.15,
Fig. 5.19b).
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
75
Table 5.12. Neuropeptide Y
Bladder EBM SM iM oM AD
Virgin 4.3:– 0.6:– 31:– 27:– 2:–
Group I 1:0 1.6:1 3.3:2* 4.6:3* 3.3:0
Group II 0: 0.3 0.5 : 1.3 2 :3.6* 1 :6* 0 :0.3
Group III 0.6:0 1.6:1 3.6:2* 4:5* 1.6:0
Group IV 1 :0 1 : 1 2 :2.5* 2 :2* 0 :1.5
Bladder neck EBM SM iM oM AD
Virgin 4:– 4:– 40.3:– 14.6:– 3.6:–
Group I 3:2 1.3:1* 5:5* 7.3:10* 2.6:4
Group II 1: 2 0.5 : 0.5* 2.5 :2.5* 8:8* 0.5 :1.5
Group III 4 :1 1.3 : 1* 3.3 :4* 7.3: 10* 2.6: 2
Group IV 3 :3 1: 1.5* 4:5* 7 :7.5* 2: 2.5
Mid-urethra EBM SM iM oM AD
Virgin 7:– 9.3:– 53.6:– 7.3:– 3.6:–
Group I 3.6: 1* 1.6: 1* 22.6 :17* 1.6: 3 3.3 :3
Group II 3.5: 3 1.5 : 1* 21: 18.5* 2.5: 1.5 1: 1.5*
Group III 1 :2* 1 :3* 20.6 :21* 0.6: 2* 1.3 :2
Group IV 3 :2.5* 1:2* 18 : 18.5* 2 :1* 0 :2.5
* p<0.05 compared with virgin.
For descriptions of the groups, see text.
a
NPY, mainly seen in the muscle layers of all three tissue-sections, was significantly decreased
by labor (group I). In the urethra, results became significant after additional of ovariectomy
(group III). Fewer NPY-positive nerves around vessels caused the changes in the submucosa.
a
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5.4 Discussion
A rat model to study stress urinary incontinence was first reported by Lin et al. (1998).
However, the model has been criticized because the study was performed in virgin rats
and balloon dilation was not modified: i.e., the direction of the force of the balloon in
a quadruped might reasonably be expected to differ from the force a fetus exerts in a
woman. In the present study, pregnant rats were used for the experiments and the balloon was attached to weighted traction to direct the force to the pelvic floor.To permit
normal delivery,the balloon dilation was performed immediately after birth when the
tissue in the pelvis was still relaxed. One may argue that this differs from a prolonged
second stage of labor that occurs in humans. However, we believe this modified model is adequate for studying the effect of delivery, difficult labor,and ovariectomy on the
continence mechanism.
The effect of birth trauma and menopause on the urinary tract is well known. Clinically,symptoms such as frequency,urgency, retention, and incomplete voiding occur
often.After vaginal delivery,a large percentage of women experience stress urinary incontinence; in some cases,this persists even after 12 months.
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Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
Table 5.13. Vasoactive intestinal polypeptide
5
Bladder EBM SM iM oM AD
c:i c:i c:i c:i c:i
Virgin 0:– 0:– 0.6:– 1:–* 0:–
Group I 0:0 0:0 0:0 0:0 0:0
Group II 0:0 0:0 0:0 0:0 0:0
Group III 0:0 0:0 0:0 0:0 0:0
Group IV 0:0 0:0 0:0 0:0 0:0
Bladder neck EBM SM iM oM AD
c:i c:i c:i c:i c:i
Virgin 3.3:– 0.6:– 2:– 0.3:– 0:–
Group I 0:0 0:0 0:0 0:0 0:0
Group II 0:0 0:0 0:0 0:0 0:0
Group III 0:0 0:0 0:0 0:0 0:0
Group IV 0:0 0:0 0:0 0:0 0:0
Mid-urethra EBM SM iM oM AD
c:i c:i c:i c:i c:i
Virgin 8:– 4.3:– 20.6:– 2.6:– 0:–
Group I 4.3:3 4.5:4 2.5:3 3:0
Group II 1: 0.5 2 : 1.5 4 :2.5* 1 :1* 1 :1
Group III 4.3:6 3.3:5 4.3:4* 0.3:1* 1:1
Group IV 1 :2.5* 0:2.5 2 :4* 1 :0.5* 0 :1.5
* p<0.05 compared with virgin.
For descriptions of the groups, see text.
In Part I of this study,increased bladder capacity, decreased micturition pressure,
and higher residual volume were observed during pregnancy. On the other hand, residual volume was decreased in 2-day postpartum rats. In Part II, incontinence was
noted in 29% of rats on the day of delivery, and this decreased to 16% at 8 weeks postpartum. However, in group II (delivery and ballooning) and group IV (delivery, ballooning, and ovariectomy),the rate of incontinence increased to 58% and 71%, respectively (Part II, Table 5.5).One might hypothesize that, although the injury to the continence mechanism is relatively minor after a normal delivery, the ischemia caused by
prolonged pressure and intravaginal ballooning can aggravate the injury and cause irreversible damage to the urethra and levator. Interestingly, delivery and ovariectomy
did not result in a higher incontinence rate than did delivery alone.
The effect of hormone deficiency is intriguing. It is possible that a relatively healthy
continence mechanism can be compensatory. However, if tissue damage is severe
enough, the added hormone deficiency may significantly impact the continence
mechanism. This may explain some of the inconsistent reports in women given hormone therapy for incontinence. Nevertheless, the exact mechanism requires further
investigation.
In 20 patients,in whom Kerr-Wilson et al.performed cystoscopy and cystometry at
48 h postpartum (Kerr-Wilson et al.1984), those who had undergone vaginal delivery
had greater bladder capacity and lesser bladder tone than those who had undergone
cesarean section. In an experimental study, Hsia et al. found that pregnant rats had
higher bladder compliance and capacity than non-pregnant rats (Hsia and Shortliffe
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