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Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
Table 5.14. Acetylcholinesterase (ACEase)
77
Bladder EBM SM iM (%) oM (%) AD
Virgin 0.3:– 3.3:– 9.6:– 6.2:– 1.3:–
Group I 0.6:0 2:2 9:6.5 6.4:2.2 1:0
Group II 0.5: 0 2 : 1.6 7.7 :9.2 9 :11.4 0 : 0.6
Group III 0.6 :0 1.3 :0 9.7 : 6.7 6.8 :5.5 2 :0
Group IV 0 :0 2 : 1 12.6: 9.6 5.3:4.3 0: 0.5
Bladder neck EBM SM iM (%) oM (%) AD
Virgin 2.5:– 3:– 4.1:– 8.2:– 3:–
Group I 7: 3 2.6: 2 4.8 :4.1 7.2:2.8 3.6: 3
Group II 6: 5.3 2 : 2.3 2.8 :10.6 5.2:6.1 3 : 3.6
Group III 6.3 :7* 2.6:3 4.6 :14.1 4.3:10.6 3: 3.6
Group IV 5 :7.5* 2:3 7.8 :4.5 3.2 :4.9 5 :2.5
Mid-urethra EBM SM iM (%) oM (%) AD
Virgin 2.5:– 3:– 8.6:– 8.6:– 4.3:–
Group I 3: 2 4.6: 3 2.7 : 1.8* 1.4 : 3.4* 5.3 :5
Group II 1: 0.3 3.5 : 2 4 :2.5* 3 :4.1* 3.5 :3.3
Group III 1.3 :2 4.7±0.3 5.4:5.6* 3.7: 4.8* 4.6 : 3
Group IV 1 :1 2 : 3.5 2.7 :2.7* 3.5: 3* 6 :3.5
* p<0.05 compared with virgin.
For descriptions of the groups, see text.
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1995), and they suggested that this finding might result from hormonal changes rather
than from the obstructive effect of the uterus. Kostrzewska further suggested that hormones relax the smooth muscle of the urinary tract (Kostrzewska et al. 1993). In our
study, we observed increased bladder capacity and decreased modified leak-point
pressure (mLLP) on the day of delivery in all rats (Part II, Table 5.1). Except in group I,
both the bladder capacity and mLPP returned to levels similar to those in virgin rats
after 8 weeks. Why bladder capacity increased in group I rats is unknown. Although
mLPP here is a measurement at the time of overflow incontinence,it nevertheless provides a consistent method of continence assessment in addition to the stress/sneeze
test.Electrostimulation of the pelvic nerve and conscious voiding were not performed
because the study by Lin et al.showed damage to the pelvic ganglion in ballooned rats
and comparison between different groups would be difficult (Lin et al.1998).
After finding similar results in the female human and even being able to increase
the functional damage, as after heavy labor, the main change was expected to be in the
muscle cell. Therefore we investigated caveolae and its component caveolin as an imported cell organ at the cell membrane.Caveolae are 50- to 100-nm membrane microdomains representing a subcompartment of the plasma membrane (Lisanti et al.1994;
Yamada 1955). These microdomains can sequester membrane-bound ligands away
from the extracellular space and facilitate their delivery to the cytoplasm of the cell.
This process is called potocytosis (Anderson et al. 1992). What distinguishes it from
other endocytic pathways is the use of glycosylphosphatidylinositol (GPI)-anchored
membrane proteins to concentrate low-molecular-weight molecules and ions in the
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Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
Fig. 5.18a,b. A comparison between virgin (a) and group IV (delivered + ballooned + ovariecto-
mized) (b) urethral muscle layers (magnification 40×). The arrows mark the beginning of the
inner muscle layer (iM).The loss of ACEase is demonstrated in group IV
closed caveolae. Another potential function for potocytosis is to receive or transmit
various kinds of cellular signals such as signaling molecules derived from GPI-anchored membrane molecules.GPI-anchored membrane proteins have been implicated
as the source of inositolphosphoglycans,which act as second messengers for a variety
of hormones (Romero et al. 1988; Saltiel and Sorbara-Cazan 1987). In addition, 1,4,5triphosphate (IP3)-sensitive calcium channels and an adenosine triphosphate-dependent calcium pump have recently been localized to caveolae,which suggests a role for
potocytosis in calcium signaling (Fujimoto 1993). The present results showed a decreased number of sarcolemmal vacuoles in groups II–IV in both the bladder and urethral smooth muscles. By impairing calcium signaling, the decrease in caveolae may
contribute to a higher incontinence rate. Interestingly, in group III, the number of caveolae increased significantly in the bladder neck, corresponding to the lower rate of
incontinence in this group. This compensatory increase in caveolae in the ovariectomized rats warrants further investigation.
Caveolin is a 21- to 24-kDa integral membrane protein and is an important structural and regulatory component of caveolae membranes that was first identified as a major-scr substrate in Rous sarcoma virus-transformed cells (Rothberg et al.1992). Cave-
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
79
Table 5.15. Tyrosine hydroxylase
Bladder EBM SM iM (%) oM (%) AD
Virgin –:– 4.3:– 8.0:– 13:– 9.3:–
Group I –:– 1.3:0* 3.3:1* 3:0* 2.3:0*
Group II –: – 1 :0.3* 0:0* 0 :0.7* 0 :1*
Group III –:– 2.5:3 0.5:1* 2:2* 1:2*
Group IV – :– 1 :1.5* 1: 1* 0:2* 2 :0.5*
Bladder neck EBM SM iM (%) oM (%) AD
Virgin – : – 13 :– 16.7 :– 11.7 :– 7:–
Group I –:– 2:1* 1:0* 1:0* 1.6:0*
Group II –: – 1.5 : 1.5* 0 :0* 1 :1* 1 :0.5
Group III – :– 4.5 :10 0.5: 1* 1/3* 1 :2
Group IV – :– 8 :3.5* 0: 1* 1 :2.5* 0 :3*
Mid-urethra EBM SM iM (%) oM (%) AD
Virgin –:– 1.7:– 44.7:– 4:– 7:–
Group I –:– 0.3:1 34.7:22 1:1 2.7:3*
Group II –: – 0 : 0* 19.5 : 18.7* 1 :1.7 0.5 :1.3*
Group III – :– 1.5 :1 36.5:23.5 2.5 :3 4.5 :1
Group IV –:– 1:1 17.5:14* 2.5:2 2:2*
* p<0.05 compared with virgin.
For descriptions of the groups, see text.
a
Although labor seemed to decrease the number of TH-positive nerves in the bladder and bladder neck, the main change was seen in the mid-urethra, where almost no TH-positive nerves
were observed after labor in the circular smooth muscle layer.
c:i c:i c:i c:i c:i
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a
olin may also act as a scaffolding protein within caveolae membranes and may represent an important structural protein for directing their formation (Fujimoto 1993).Caveolin copurifies with a number of lipid-modified cytoplasmic signaling molecules,
including G-protein,protein kinase Ca
2+
, scr-family tyrosine kinases and ras proteins
(Tang et al. 1997). Recently, the family of caveolin-related proteins grew: caveolin-1
(with the isoforms a and b), -2, and -3. Caveolin-1 is found in endothelial cells, fibroblasts, adipocytes and smooth muscles, while caveolin-3 is selectively expressed only
in heart and skeletal muscle tissues (Glenney 1992; Romero et al. 1988; Breton et al.
1998; Tang et al. 1996).
A change in the staining pattern of caveolin-1 and a decrease in immunoreactivity
was noted in urethral and bladder smooth muscle cells (in the urethra of groups I–IV
and bladder of groups II–IV). The decrease in both the caveolin-1 protein and the
number of caveolae in the above groups might be the reason for the decrease in muscle contraction force in the bladder and urethra. It should be noted that neither the
percentage of caveolin-1 stain nor the architecture and staining pattern of the smooth
muscle cells in the experimental rats returned to the condition of the virgin rats after
8 weeks.
Immunostaining demonstrates that caveolin-3 is localized to the sarcolemma of
striated muscles and coincides with the distribution of dystrophin (Song et al. 1996;
North et al. 1993). In the present study, caveolin-3 (analyzed in the mid-urethra) de-
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Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
Fig. 5.19a, b. A comparison between virgin (a) and group II (delivered+ballooned) (b) urethral
muscle layers (magnification 40×).The arrows mark the longitudinal and circular running layer
of the smooth muscle (b), demonstrating the loss of TH-IR-positive nerves in group II as a result
of the ballooning
creased significantly in groups II–IV, but not in group I.The significance of caveolin-1
and caveolin-3 in the continence mechanism is unknown. The demonstrated changes
may contribute to stress incontinence: a decrease in membrane caveolae, caveolin-1
and -3, and smooth muscle cells as well as an increase in collagen content in the bladder neck and urethra.
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
As found on immunostaining, the decrease in caveolae in the smooth muscle cells
was also demonstrable by electron microscopy.One of the most striking features of the
urethral smooth muscle of virgin rats was the rich presence of caveolae in the sarcolemma. Because the function of the caveolae is signal transduction and calcium transport (Fujimoto 1993; Tang et al. 1996; Sargiacomo et al.1995), the abundance of caveolae and caveolin-1 assures adequate intracellular concentration of calcium ions and
strong bladder and sphincter contraction.In pregnant rats,decreased caveolin-1 staining and protein expression may decrease the force of urethral muscle contraction and
render the urethra more compressible by the enlarged uterus.The increase in protein
expression and staining of caveolin-1 in the 2-day postpartum rats is intriguing. It may
represent either a tissue response to the relief of mechanical compression or a change
in hormonal environment.In the 6-week postpartum rats,although the caveolin staining and protein expression returned to the levels seen in virgin rats, the architecture of
the urethral wall remained somewhat distorted. In contrast, overall intercellular space
between smooth muscle groups returned to that found in virgin rats. Ultrastructural
analysis also showed that the urethral smooth muscle cells from virgin rats were irregular and serrated, with narrow intercellular spaces. Wider intercellular spaces in the
urethra of pregnant and postpartum rats may impair signal propagation and coordination among smooth muscle cells, which are important in the continence mechanism. Besides cell atrophy, an increase in connective tissue was demonstrated in
groups II–IV.
The striated muscle of the external sphincter showed lipid droplets in the I-band
and subsarcolemmal accumulation of mitochondria in the pregnant and 2-day postpartum rats. In one reported experimental study, the number and the size of subsarcolemmal mitochondria increased after 6–24 h of ischemia (Hanzlikova and Schiaffino
1977), and the authors concluded that an adaptation reaction of growth and multiplication occurs under extreme circumstances. The present results may represent a response to the stress of pregnancy and delivery.
Lipid droplets are not membrane-bound,and their number and size may vary considerably among different muscle types and in the same type of muscle in different areas of the body.They are frequently associated with mitochondria, and are sometimes
completely encircled by a mitochondrion (Jennekens et al. 1981). It has been shown
that experimental enzyme deficiencies in mitochondrial energy metabolism induce
accumulation of giant mitochondria and numerous lipid droplets (Jennekens et al.
1981). This has led to the suggestion that mitochondria may use lipids as a source of
energy for muscular contraction. The increased number of lipid droplets in the pregnant rats indicates that lipids may be an important energy source for striated muscle
during pregnancy. With additional manipulation, the damage to the striated muscle
cells became more obvious: e.g., a decrease in T-tubules and, in group IV, significantly
fewer mitochondria and almost no lipid droplets.
Recent studies have shown that eNOS is associated with caveolin-1 in endothelial
cells and nNOS with skeletal muscle caveolin-3 (Venema et al. 1997; Michel and Feron
1997). A dynamic model of the NOS-caveolin/calmodulin cycle has been proposed by
Michel and Feron (1997): in the resting cell, the formation of the inhibitory NOS-caveolin complex suppresses NOS enzyme activity. After activation, the increase in intracellular calcium promotes calmodulin binding to NOS and dissociation of caveolin
from NOS. The activated NOS-calmodulin complex synthesizes NO until intracellular
calcium decreases to a point where calmodulin dissociates and the inhibitory NOS-caveolin complex reforms. The nNOS isoform is highly expressed in skeletal muscle, and
thus it appears to be involved in modulating contractile force. Neuronal NOS has also
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Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
been postulated as one of the neurotransmitters involved in the relaxation of urethral
sphincter muscle (Burnett 1995). The present study reveals a significant decrease in
nNOS-IR-staining (groups I–IV), which suggests a decrease in enzyme activity. The
nNOS isoform is highly expressed in skeletal muscle,and thus it appears to be involved
in modulating contractile force.The significance of caveolin-1 and -3 in the continence
mechanism is unknown. Theoretically, a decrease in caveolin would impair calcium
transport and enhance NO production – both of which decrease sphincter function –
and thus might contribute to urinary incontinence.
The present results of nerve locations for specific stains were similarly reported by
other authors (Persson et al. 1995;Alm et al. 1995; Gosling 1985; el-Badawi and Schenk
1966; Radziszewski et al. 1996). PGP 9.5 is a general cytoplasmic nerve marker, which
should be present in all types of efferent and afferent nerve fibers (Gulbenkian et al.
1987). In this study,PGP 9.5 was present in most nerve fibers, except beneath the basal
membrane where heavy staining in the urothelium may have masked it. These findings
are similar to the results of Alm et al. (1995). In addition, almost complete elimination
of PGP 9.5-IR-positive nerves was noted in the muscular layer of the bladder and bladder neck after both ballooning and ovariectomy, although staining was positive for
other neuropeptides.
The finding of nNOS-IR staining in the urethra and the bladder neck of virgin rats
is comparable to reports by others (Alm et al. 1995; Andersson and Persson 1995; Burnett et al. 1992).However, as opposed to their description of low NOS immunoreactivity (Alm et al. 1995), a high number of nNOS-IR-positive nerves was seen in the
smooth muscle bundles (inner and outer layers) of the urethra and bladder neck. In
the delivered rats, a strong decrease in nNOS was noted in the bladder neck and urethra. The significance of these findings is unknown because the role of NO in urethral
relaxation remains controversial (Werkstrom et al. 1998; Zhou and Ling 1999).
Comparison between ACEase and the immunostains was not useful because we analyzed ACEase content with Photoshop,which resulted in a percentage of the analyzed
picture in the muscle layers.
The results of the double stain for TH and NOS were not found to coincide in exactly the same nerves (Werkstrom et al. 1998;Vizzard et al. 1994). The ballooning caused
a significant decrease in TH-IR-positive nerves. In a previous report by others, treatment with 6-hydroxydopamine (6-OHDA) resulted in the complete absence of all THIR-positive nerves, mainly in the bladder base and urethra (Persson et al. 1997). The
changes demonstrated here, especially the almost complete loss of TH-IR-positive
nerves in the circular smooth muscle of the outer mid-urethral muscular layer,might
be strong contributory factors in the functional origins of incontinence.
In the elegant study of Alm et al.,the distribution of nitrergic, adrenergic, peptidergic, and cholinergic nerves in the lower urinary tract of the female rat is described in
detail, as are the changes after bilateral pelvic cryoganglionectomy, preganglionic decentralization and intravesical obstruction. They report an almost complete loss of all
nerves in the bladder and urethra after bilateral pelvic ganglionectomy and a selective
decrease in CGRP after preganglionic decentralization.Intravesical outlet obstruction
caused a significant decrease in PGP 9.5 and a nearly complete loss of NOS above the
obstruction (Alm et al. 1995).
In animal studies with intravesical outlet obstruction, the results of sensory neuropeptide (VIP, CGRP, SP and NPY) changes are not consistent. Chapple et al. (Chapple
et al. 1992) saw a reduction in the density of innervation of VIP, CGRP, and SP with no
changes for NPY, whereas Lasanen et al.(1992) reported an increase in VIP-, NPY-,and
SP-IR-positive nerves. As opposed to the obstructed animal model, no previous work
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
in an incontinence animal model has been described. Nevertheless, Gu et al.(1983) described a marked reduction of VIP in the bladder muscle layer of patients with idiopathic detrusor instability.
It is interesting that the present results are similar to those of Alm et al. (1995). As
with bilateral pelvic cryoganglionectomy, a decrease was apparent in the staining of
many of the neuropeptides studied. Because they did not describe the results in the
substructures, a comparison is difficult to make. From the present results, it is apparent that delivery, ballooning and ovariectomy decrease the number of nerves in the
bladder,bladder neck and urethra. Delivery and ballooning seem to have a damaging
effect similar to that of pelvic cryoganglionectomy, but not as severe. In the previous
study in virgin rats of Lin et al., the number of ganglion cells in the neural plexus posterolateral to the vagina was significantly decreased after ballooning (Lin et al. 1998).
This may explain the similarity between the present results and those after pelvic
cryoganglionectomy in the report of Alm et al.(1995).
Surprisingly, even a normal delivery (group I) altered the staining patterns both
quantitatively and qualitatively. The effect was more pronounced in the mid-urethra
and bladder neck, especially in the inner muscular layer where almost all the nitrergic
and peptidergic nerve fibers were affected (PGP 9.5, CGRP, SP, nNOS and NPY in the
bladder neck and CGRP, nNOS,NPY, VIP and cholinergic in the mid-urethra). Because
the staining pattern in pregnant rats was not studied,one may argue that these changes may be attributed to pregnancy alone – and this cannot be totally excluded. However,in the previous study of Bakircioglu et al. (2000),no damage to the pelvic ganglion
was noted during pregnancy. In addition, since similar decreases in IR-staining patterns for PGP 9.5, CGRP, SP, nNOS and NPY were noted after ballooning (group II),one
may postulate that a normal delivery has a mini-ballooning effect on the continence
mechanism, which explains the high incidence of incontinence immediately after
birth in female rats and humans.
The present results fail to show a specific pattern of neuronal change associated
with pregnancy, delivery, ovariectomy or a combination.Although a decrease in SP in
the submucosa and a decrease in VIP in the basal membrane of the mid-urethra seem
to correlate with ballooning, and a decrease in CGRP in the bladder submucosa suggests an influence from ovariectomy, these findings may be simply coincidental.Nevertheless, the most striking effect is the significant decrease in nNOS in all tissue layers
in the bladder neck and mid-urethra in groups I–IV, making it the most sensitive indicator of tissue or nerve injury. The almost complete loss of TH-IR-positive nerves in
the circular layer of the mid-urethra could also be contributory.
In summary, functional studies in a pregnant rat model demonstrated a large increase in the rate of incontinence in group II (delivery and ballooning) and group IV
(delivery, ballooning and ovariectomy) animals. Ovariectomy appeared to have no effect when combined with delivery alone, but significantly increased the incontinence
rate in rats that underwent delivery and ballooning. Ultrastructural and immunohistochemical studies revealed various degrees of changes in plasma membrane caveolae,
caveolin-1 and -3 and nNOS. The increase of stress test-positive animals and the decrease of caveolin-1 and -3 – hypothesized to be important for the contractility of muscle cells (at least as it relates to calcium channels) – and the demonstrable increase in
connective tissue caused by heavy birth trauma may explain the lack of necessity for
further muscle relaxation.This may likewise explain the decrease in nNOS, i.e.,a reaction to the caveolin decrease,and not a direct result of additional treatment(s).
The functional changes observed should not be directly attributed to the changes in
immunostaining, which only demonstrate the quantitative changes in the nerve fiber
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Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
containing a particular neuropeptide. Given the complexity of the innervation of the
lower urinary tract as well as the co-localization, co-transmission,cross-talk, and preand postsynaptic modulation of these neuropeptides, it is short-sighted to infer definitive functional significance. Nevertheless, this study may provide a stepping-stone for
further exploration of the significance of neuropeptides in urinary incontinence.
Acknowledgements
This study was supported in part by the “Deutsche Forschungsgemeinschaft” (grant
Si 679/1–1) and the National Institutes of Health (grant 2 R01 DK 51374) and a grant
from the Montgomery Street Foundation.
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