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Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
Stress Test Results
In the stress/sneeze test,all virgin controls were continent.After delivery,29% were incontinent, whereas, at 8 weeks postpartum, the incontinence rates were as follows:
group I, 16%; group II, 58%; group III, 16%; and group IV, 72%. The percentages were
derived by using the mean of both anesthesia medications (one-half of rats under ketamine, one-half under pentobarbital). The increase in incontinence for groups II and
IV was statistically significant by the convergence tables. Additionally, under pentobarbital anesthesia, incontinence rates were higher in all four groups (results not
shown) (Table 5.5).
5.3.4 Histological Evaluation
5.3.4.1 Muscle Changes
Light Microscopy
Tissue Preparation. Tissue was prepared as described in Sect. 5.2.4.1.
Fixed Frozen Tissue. Specimens (pelvic floor, bladder, bladder neck and mid-ure-
thra) were fixed (2% formaldehyde, 0.2% saturated picric acid in 0.1 M phosphate buffer,pH 8.0) for 2 h before transfer to 15% sucrose in buffer for 24 h.The specimens were
embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek USA, Torrance, CA), frozen in liquid nitrogen, and stored at –80°C until use.
57
Tissue Preparation for Immunohistochemical Staining. Fixed frozen tissue specimens were cut at 8 µm, adhered to charged slides (SuperFrost Plus, Fisher Scientific,
Pittsburgh, PA), and air-dried. To block endogenous peroxidase activity, slides were
placed in 0.3% H2O2/methanol for 10 min.After rinsing, sections were treated with either 3% horse serum or 3% goat serum in PBS/0.3% Triton X-100 for at least 60 min to
eliminate nonspecific protein binding. Slides were incubated overnight at room temperature with mouse monoclonal antibodies to caveolin-1 or caveolin-3 (1 :5,000 dilution; Transduction Laboratories,Lexington, KY) or with rabbit polyclonal antibody to
nNOS (1: 5,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). After washing
with buffer, sections were immunostained according to the avidin-biotin peroxidase
method (Vectastain Elite Kit,Vector Laboratories,Burlingame, CA) with 3,3′-diamino-
benzidine plus hydrogen peroxide as the chromogen. Sections were counterstained
with hematoxylin, dehydrated to xylene, and mounted.
For control purposes, some sections were incubated as above,but without primary
antibody.No immunostaining was seen under this condition.
Statistical Analysis
The muscular structure was divided into two substructures (inner muscular layer and
outer muscular layer). For each substructure, four randomly chosen fields were analyzed at 400 ×light microscope magnification (Leica light microscope DM RB attached
5
58
Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
to a digital camera [Nikon N 90]). The changes of caveolin-1, caveolin-3 and nNOS
were quantified with Adobe Photoshop 4.0 (Adobe Systems Incorporated, Mountain
View, CA) installed on a Power Macintosh 7500/100 (Apple Computer, Inc. Cupertino,
CA). The ratio of the caveolin-1-, caveolin-3-, or nNOS-stained pixels to the total number of pixels yielded a percentage.
Immunostaining Results. Caveolin-1 is located mainly in the sarcolemma of
smooth muscle cells in the bladder, bladder neck and urethra as well as in smooth
muscle cells of blood vessels (Table 5.6).
In the bladder tissue of group I, caveolin-1 was decreased, but not significantly. In
groups II–IV caveolin-1 staining diminished significantly. The well-arrayed architecture of the muscle bundles seen on histological examination of the virgin controls was
lost in groups II–IV (Figures 5.8a, 5.8b).
In the urethra of virgin rats, densely packed smooth muscle cells with a well-defined sarcolemma and a centrally situated nucleus were seen (Fig. 5.8c, d). Caveolin-1
was significantly less in smooth muscle bundles of the urethra in groups I–IV than in
virgins (Fig. 5.8 e–h).
Regardless of location,smooth muscle cells of groups II–IV appeared to be less well
defined and unequal in size when compared with the virgin controls (Fig. 5.8d, f–h).
Ovariectomized animals (group III) had decreased smooth muscle cell size.
Table 5.6. Caveolin-1,-3 and neuronal nitric oxide synthase staining results
Virgin Group I Group II Group III Group IV
(n=10) (n=8) (n=8) (n=8) (n=8)
Cont.:incont. c:i c:i c:i c:i c:i
Caveolin-1
Urethra
Inner (%) 17.1:– 9.9 :8.3
a
8.2: 3.3
a
8.2: 3.3
a
9.3: 3.3
Outer (%)
Bladder neck
Inner (%) 11.6:– 10.1 :7.2 15.2 :11.2 14.44 :7.90 18.40 :8.63
Outer (%) 13.7: – 14.5 :13.8 13.3 :12.1 14.8 :12.6 15.7 :13.5
Bladder
Inner (%) 16.1:– 12.7 :12.2 12.7 :12.6
Outer (%) 21.7: – 18.7 :15.6 18.2 :9.9
a
a
8.7: 9.2
10.5: 5.5
a
a
4.4: 5.2
14.9: 6.3
Caveolin-3
Urethra
Inner (%)
Outer (%) 20.6: – 14.4 :12.3 14.8 :9.18
a
7.2: 3.0
a
8.9: 7.5
n-NOS
Urethra
Inner (%) 1.0: – 0.9 :n.i. n.i. n.i. n.i.
Outer (%) 5.5 :– 2.5: 0.5
a
Versus virgin.
a
1.7: n.i.
a
1.9: n.i
a
1.3: n.i.
p<0.05.
n.i. Non-immunoreactive.
a
a
a
a
a
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
59
Fig. 5.8a–h. Caveolin-1 staining of the bladder from a virgin (a) and a group IV rat (b) (delivered
+ ballooned + ovariectomized). The staining of group IV bladder muscle was significantly decreased. Caveolin-1 staining of the urethra revealed inner longitudinal and outer circular layers
with dense staining in the virgin (c and d). After ballooning (group II),a decreased intensity of
staining was noted (e and f). Further decrease in caveolin staining was noted in groups III (delivered + ovariectomized) (g) and IV (delivered + ballooned + ovariectomized) (h)
5
60
Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
Fig. 5.9a–f. In the virgin rat, a a completely circular striated muscle ring of the urethral outer
muscular layer is clearly seen after caveolin-3 staining; b the caveolin-3 staining is located predominantly in the sarcolemma.c The muscle layer after ballooning in a group II rat (
the symphysis,
sarcolemmal distribution in (b). e A further decrease in caveolin-3 staining and irregular arrangement of striated muscle were noted in rats from group III (delivered + ovariectomized)
and f group IV (delivered + ballooned + ovariectomized)
R toward the vagina); d distribution of caveolin-3 staining is even, rather than the
j towards
Caveolin-3,which stained the sarcolemma of the striated muscle of urethra and levator, was significantly decreased in groups II–IV (Fig. 5.9a, b). The architectural derangement in the smooth muscle layer was not seen in the striated muscle layer, where
the orientation was retained regardless of treatment (Fig.5.9b,d, e,g). There was no increase in connective tissue at the light microscopy level.
In treated rats, nNOS in the urethral striated muscle bundles showed a significant
decrease (Fig. 5.10a,b).
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
61
Fig. 5.10a, b. nNOS staining of the urethral striated muscle in (a) virgin and (b) group IV rats (delivered + ballooned + ovariectomized).Note the less well-defined individual muscle cells and the
decreased staining in (b)
Electron Microscopy
Tissue Preparation for Electron Microscopy. Tissue samples (bladder, bladder
neck, mid urethra and levator muscle) were immersion-fixed in 2.5% glutaraldehyde,
2.0% paraformaldehyde in 0.15 M sodium cacodylate buffer, pH 7.4. After postfixation
in 2% osmium tetroxide, the tissue was dehydrated in graded ethanol and propylene
oxide and subsequently embedded in Epon 812. Thick sections (1 µm) were cut on a
Sorvall MT 2-B microtome,stained with 1% methylene blue, and examined by Leitz Laborlux S light microscope (Leica Mikroskope und Systeme GmbH,Wetzlar,Germany).
Thin sections (~900 Å) were mounted on 200-mesh copper grids and stained with
10% uranyl acetate and lead citrate. Ultrastructural examination was performed with
a Zeiss transmission electron microscope Model 10.
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Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
5
Table 5.7. Electron microscopy findings
a
Virgin Group I Group II Group III Group IV
(n=10) (n=8) (n=8) (n=8) (n=8)
Cont.:incont. c:i c:i c:i c:i c:i
Caveolae
b
Urethra 282.6 :– 301.0 :249.8 150.0 : 76.6
103.0: 77.0b41.5: 16.3
Bladder neck 79.0 :– 75.0 :60.5 81.0 :38.0 120.0 : 186.0b50.0: 39.3
Bladder 190.5 :– 150.0: 114.3
b
75.5: 65.3
b
61.5: 47.7* 23.5: 9.6
Cell Number
Urethra 17.7: – 15.0: 12.3 23.0 : 20.3 19.0: 15.0 18.0 : 19.3
Bladder neck 21.8 :– 19.0 :17.3 18.0 :13.7 30.0: 15.3 21.0 :15.0
Bladder 20.0: – 16.0 :13.67* 24.0 :15.3 20.0 :24.7 22.0 :33.3*
Connective Tissue
Urethra (%) 10.5 :– 11.0: 12.0 11.0 :14.5 9.9: 12.8 11.2 :14.7
Bladder neck (%) 6.8:– 7.2 : 6.7 8.1 :13.3 7.0: 7.83 10.0 :12.3
Bladder (%) 7.5 :– 7.4 :7.3 7.5 :9.1 6.6 :8.5 7.6 : 9.8
p<0.05.
a
The number of caveolae was already decreased in the bladder by labor. In the mid-urethra,the
influence of ballooning and/or ovariectomy was seen. The number of cells per field increased
with each additional treatment. The exception is the bladder neck with a decrease in smooth
muscle cells but an increase in connective tissue. The changes in cell number and connective
tissue were more obvious by further subdividing each group into continent (c) and incontinent
(i). The differences, when compared with the results of virgin controls, became obvious (data
not shown). However, because of the small numbers of incontinent/continent rats in each subgroup,statistical comparison with Mann-Whitney U test could not be performed.
b
Versus virgin,
b
b
b
Electron Microscopy Results. Smooth muscle cells were observed under electron
microscopy, confirming the results of caveolin-1 immunostaining. In the urethra, a decrease of caveolae in groups II–IV was seen (Fig. 5.11a, b).In the bladder, the number
of caveolae decreased significantly in groups I–IV when compared with virgin rats
(Table 5.7).
Bladder specimens of groups II and IV had a significant increase in smooth muscle
cells per field, suggestive of muscle atrophy (Fig. 5.11b); group I showed a significant
decrease, which may be indicative of cell swelling.
A significant increase in connective tissue between the smooth muscle bundles, in
relation to the entire scanned field,was observed only in the bladder neck of groups II
and IV.
In group I striated muscle cells, some levator muscle fibers showed thinner or deficient Z-bands (Fig.5.12b). Besides a decrease of T-tubules,large vacuoles were noted,
which, in virgin animals, were occupied by mitochondria. (Fig. 5.12a: for normal T-tubuli, Z-bands and mitochondria). Urethral striated muscle bundles showed slowtwitch myofibers. Group I specimens showed an increased number of swollen mitochondria (Fig. 5.13b) and normal muscle cells.In ovariectomized animals (group III),
hyperdense vacuoles (perhaps attributable to calcification) were seen (Fig.5.13c). A decrease in mitochondria in muscle cells was seen in groups II–IV (Fig.5.13).
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
63
Fig. 5.11a, b. Urethra from virgin rat (a) shows irregular and serrated smooth muscle cells with
abundant caveolae. Urethra from group IV (delivered + ballooned + ovariectomized) rat (b) re-
veals a significant change in the shape and number of caveolae.(12,550× magnification)
Statistical Analysis. The method, as described in “Statistical Analysis” above, was
also used to analyze the amount of intercellular tissue of muscle bundles by electronic microscopy (EM). These pictures were scanned using a ScanJet 3c (Hewlett Packard). The mean of four areas (area of 5 ×5cm
2
; EM-magnification, 4125×) was used to
compare the differences between the virgin and the various groups with the MannWhitney U test (GB-STAT, Dynamic Microsystems Inc., Silver Spring, MD). Values
were considered significant at p<0.05 and highly significant at p<0.005.
5
64
Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
Fig. 5.12a–c. Striated muscle of the levator from (a) virgin rats. Note the T-tubules (arrowheads),
Z bands (arrows) and mitochondria (asterisks). b Group I (delivered) rats: large vacuoles, a de-
crease in mitochondria and glycogen, and deficient Z-bands (arrows) are noted. c In group IV
(delivered + ballooned + ovariectomized) rats, there was a marked loss of mitochondria and a
reduction in the diameter of individual muscle cells. This phenomenon is seen only in ovariectomized rats (12,550 × magnification)
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
65
Fig. 5.13a, b. Striated muscle of mid-urethra in rats from a virgin, b group II (delivered + ballooned) and C group IV (delivered + ballooned + ovariectomized).In group II, large vacuoles replacing mitochondria were noted and the number of mitochondria was significantly decreased.
Further decrease in the number of mitochondria and calcifications (black dots) are seen in group
IV (12,550 × magnification)
66
Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
5.3.4.2 Neural Changes
Light Microscopy
Tissue Preparation for Light Microscopy. Tissues were prepared as described in
“Electron Microscopy”above.
Fixed Frozen Tissue. Frozen tissue samples were prepared as described in “Fixed
Frozen Tissue” above.
5
Staining. Fixed frozen tissue specimens were cut at 8 µm,adhered to charged slides
(SuperFrost Plus Fisher Scientific, Pittsburgh, PA) and air-dried. For immunohistochemistry, endogenous peroxidase activity was blocked by placing slides in 0.3%
H2O2/methanol for 10 min.After rinsing, sections were treated with 3% goat serum in
PBS/0.3% Triton X-100 for at least 60 min to eliminate nonspecific protein binding.
Slides were incubated with specific antibodies (anti-protein gene product 9.5 [PGP
9.5], 1:8,000 [Accurate Chemical & Scientific Corporation,Westbury, NY]; anti-calcitonin-gene-related-peptide [CGRP], 1: 1,800; anti-substance P [SP], 1 :8,000; anti-neuropeptide Y [NPY], 1:6,000; anti-vasoactive intestinal polypeptide [VIP], 1: 6,000 [Peninsula Laboratories Inc., Belmont, CA]; anti-neuronal nitric oxide synthase [nNOS],
1: 5,000 [Santa Cruz Biotechnology,Santa Cruz, CA], anti-tyrosine hydroxylase [TH],
1: 20, [Newcastle-Upon-Tyne, UK]). All sections were incubated at room temperature
overnight, except for CGRP for which sections were incubated for 60 min.After washing with buffer, sections were immunostained according to the avidin-biotin peroxidase method (Vectastain Elite Kit,Vector Laboratories, Burlingame, CA) with 3,3′-dia-
minobenzidine with hydrogen peroxide as the chromogen. Sections were counterstained with hematoxylin, dehydrated to xylene, and mounted.
For control purposes, some sections were incubated as above without primary antibody.No immunostaining was seen under this condition.
Acetylcholinesterase (ACEase) staining was performed according to the method of
Goto et al.(1984). Tissue sections were incubated for 60 min with 0.065 M maleate buffer,pH 6.0, containing sodium citrate (5 mM),copper sulfate (3 mM), potassium ferricyanide (0.5 mM) and acetylthiocholine iodine (1.7 mM). To serve as control,some sections were incubated in a mixture without acetylthiocholine iodine. Sections were
briefly rinsed with water, then incubated for 2–10 min with a mixture of 8% sodium
acetate and 0.02% rubeanic acid.Tissues were lightly counterstained with eosin, dehydrated, and cover-slipped. Acetylcholinesterase-stained nerves appeared as black fibers against the eosin background.
Statistical Analysis. Specimens were divided into five structural categories: epithelium and basal membrane (EBM); submucosa (SM); inner muscular layer (iM); outer
muscular layer (oM); and adventitia (AD). For each substructure, four randomly chosen fields (one main field is subdivided in 5 × 5 subfields = 25 subfields) were counted
at 400 × magnification (Leica light microscope DM RB attached to a digital camera
[Nikon N 90]).Because the amount of ACEase positive staining was not countable, as
the other nerve quantities, the ACEase stain changes were quantified with Adobe Photoshop 4.0 (Adobe Systems Incorporated, Mountain View, CA) installed on a Power
Macintosh 7500/100 (Apple Computer,Inc. Cupertino,CA). The ratio of ACEase-positive pixels to the total number of pixels yielded a percentage. The mean of four areas
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