Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / @xirurgi_2025 / @xirurgi_2025 - 1010 - файл
.pdf
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
5.2.4 Histological Evaluation
5.2.4.1 Immunohistochemical Staining
Tissue Preparation for Immunohistochemical Staining
Tissue was collected immediately after sacrifice. PE10 tubing (Polyethylene Tubing
Intramedic,Becton Dickinson, Sparks,MD) was inserted transurethrally,and the symphysis was cut to expose the pelvic floor,which was removed carefully to preserve the
original muscle orientation. The lower urinary tract was dissected and processed for
examination.
Immunohistochemical Staining
Paraffin-embedded urethra and bladder sections were stained with caveolin-1 and caveolin-3 antibodies (Transduction Laboratories, Lexington,KY) with an avidin-biotinenzyme complex (ABC) staining kit (Vectastain, Vector Laboratories, Burlingame,
CA). Briefly,tissue sections were incubated with 3% goat serum for 2 h and then with
1: 5,000 dilution of caveolin-1 and caveolin-3 antibody overnight. Tissue sections were
then incubated sequentially with biotinated secondary antibody (Vector Laboratories), peroxidase-conjugated avidin, and diaminobenzidine. Finally, these sections
were counterstained with hematoxylin (bluish staining of all cell nuclei), while positive (antigen-expressing) cells were stained with diaminobenzidine (brown color).
For control purposes, some sections were incubated as above,but without primary
antibody.No immunostaining was seen under this condition.
47
Immunostaining Results
With immunostaining,caveolin-1 was localized to the sarcolemma of the smooth muscle cells of the bladder and urethra; in contrast, caveolin-3 was localized in the sarcolemma of striated muscle cells in the intrinsic sphincter.
In the urethra of virgin rats, immunohistochemical staining for caveolin-1 revealed
densely packed smooth muscle cells with well-defined sarcolemma and centrally situated nuclei.In contrast,in pregnant rats expression of caveolin-1 was lower, with a concomitant loss of muscle cell architecture. Furthermore, in virgin rats, the smooth muscle cells appeared uniform in size with little intercellular space. In the 2-day postpartum rats, caveolin-1 staining was very dense. Many of the smooth muscle cells were
distorted, which could represent either distortion of the individual smooth muscle cell
or “internalization of the caveolae” as described by Thyberg et al. (1997). There was a
large amount of intercellular space among bundles of smooth muscle, suggestive of
interstitial edema shortly after delivery.In the urethra of 6-week postpartum rats, the
staining density was similar to that of the virgin rats. However, many of the smooth
muscle cells still appeared distorted, which again may represent distortion of the sarcolemma or internalization of the caveolae.In the bladder of pregnant and 2-day postpartum rats, immunohistochemical staining for caveolin-1 revealed that the smooth
muscle cells were arranged in small groups with larger intercellular spaces as opposed
to the virgin and 6-week postpartum rats.
5
48
Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
Caveolin-3 staining was seen only in the sarcolemma of the striated muscle of the
intrinsic urethral sphincter, and no significant difference in staining pattern among
the groups was noted.
5.2.4.2 Electron Microscopy
Tissue Preparation for Electron Microscopy
Tissue samples (bladder and mid-urethra) 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.
Electron Microscopy Results
As reported by others (Elbadawi 1995), the smooth muscle cells in the bladder are larger than those in the urethra. The morphology of bladder smooth muscle cells varies
from polygonal to cylindrical, depending on the plane of sectioning. In addition, they
pack together with minimal intercellular space (Fig. 5.1). The sarcolemma of the
smooth muscle cell contains thick electron-dense zones and thinner zones. The latter
contain varying numbers of sarcolemmal caveolae. In our study, after delivery, most
bladder smooth muscle cells showed increased mitochondrial aggregation and more
vacuoles than those in virgin rats (Table 5.2), and the number of sarcolemmal caveolae
was significantly less in pregnant and postpartum rats (Table 5.3).
The urethra contains two layers of smooth muscle, an inner longitudinal and an
outer circular layer.In addition, an outer layer of striated muscle is present in the mid-
Table 5.2. Bladder changes on electron microscopy
Virgin Pregnant Postpartum Postpartum
Shape Irregular Polygonal Polygonal Polygonal
Vacuoles Rare Slight increase Moderate Moderate
Intercellular Space Small Slight increase Slight increase Slight increase
Mitochondria
Location Near nucleus Scattered Scattered Scattered
Swelling No Slight increase Moderate Moderate
Vacuolar deg. No Increase Moderate Moderate
Aggregation Scattered Increase increase increase
day 2 week 6
increase increase
increase increase
increase increase
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
49
Fig. 5.1a–d. Electron micrographs of (a) virgin, (b) pregnant,(c) 2nd-day,and (d) 6-weeks-postpartum rats’ bladder smooth muscle cells. Note decreased caveolae (arrows) in (b) and (c)
(20,000× magnification). Scale bar = 500 nm
Table 5.3. Number of caveolae in urethra and bladder smooth muscle cells
Virgin Pregnant Postpartum Postpartum
day 2 week 6
Urethra 22.1±2.7 15.3±3.8
Bladder 16.5±2.3 10.6±4.5
*
*
16±2.3
10.3±3.9
*
*
19.4±1.6
15.4±2.4
* p<0.05 compared with virgin.
5
50
Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
urethra. The urethral smooth muscle cells are arranged in small groups with large
intercellular spaces.The individual smooth muscle cell appears irregular in shape and
contains many finger-like processes (Fig. 5.2). In a separate pharmacological experiment (data not shown), phenylephrine pretreatment produced an irregular cell shape
and indented nucleus, while papaverine pretreatment produced a relatively smooth
cellular outline.Therefore, the irregular shape of the urethral smooth muscle cells in-
Fig. 5.2a–d. Electron micrographs of (a) virgin, (b) pregnant,(c) 2nd-day, and (d) 6-weeks-post-
partum rats’ urethral smooth muscle. Note the finger-like processes (arrows) and the numerous
caveolae in the plasma membrane of smooth muscle cells of (a).Note the round shape of smooth
muscle cells and decreased number of caveolae in the plasma membrane as well as increased
vacuoles in (c) (20,000× magnification). Scale bar = 500 nm
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
Table 5.4. Urethral changes on electron microscopy
51
Virgin Pregnant Postpartum Postpartum
Striated Mitochondria
muscle
Sarcolemmal Rare Moderate Moderate Moderate
accumulation
Giant Rare Slight Moderate Moderate
mitochondria increase increase increase
Vacuolar deg. No Slight Moderate Moderate
increase increase increase
Lipid droplets No Moderate Slight Slight
increase increase increase
Z-disk deg. No Few Moderate Moderate
Whole cell deg. No Few Few Moderate
Smooth Shape Irregular/ Polygonal/ Polygonal/ Polygonal/
muscle contracted cylindrical cylindrical cylindrical
Vacuolar deg. No Slight Moderate Moderate
increase increase increase
Mitochondria Rare Slight Slight Slight
accumulation increase increase increase
day 2 week 6
dicates that most of the urethral smooth muscles are contracted. In contrast, the bladder smooth muscles are in a relaxed state with smooth cellular perimeters.
The urethral smooth muscle cells in virgin rats were more irregular in shape
(Table 5.4) and possessed more sarcolemmal caveolae than in pregnant and postpartum rats (Table 5.3). In the latter, the numbers of finger-like processes and caveolae
markedly decreased. In addition, swelling and aggregation of mitochondria were observed, as well as vacuolar degeneration.Mitochondria were located near the nucleus
in virgin rats, but were scattered in the cytoplasm in pregnant and postpartum rats.
The interstitial connective tissue in the smooth muscle cells of the urethra and bladder was more abundant in pregnant and 2-day postpartum rats than in the virgin rats.
At 6 weeks after delivery, increased vacuoles were observed.
In the external sphincter, striated muscle cells were mostly composed of slowtwitch myofibers showing more mitochondria, thin Z-disks and fewer glycogen particles (Fig. 5.3a).In pregnant and postpartum rats,lipid droplets and large vacuoles were
noticed, especially in the I-band and between the myofibers where the mitochondria
accumulate (Fig. 5.3b, c). Although lipid droplets were mostly recognized in striated
muscle cells of pregnant rats,vacuoles were mostly seen 6 weeks after delivery.Moreover, numerous subsarcolemmal mitochondria were observed in striated muscle cells
of pregnant and 2-day postpartum groups; whole cell degeneration and Z-disk degeneration were noticed in striated muscles of the rats 6 weeks after delivery (Table 5.4,
Fig. 5.3d).
5
52
Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
Fig. 5.3a–d. Electron micrographs of urethral striated muscle cells in (a) virgin, (b) pregnant,(c)
2nd-day, and (d) 6-weeks-postpartum rats. Note the subsarcolemmal mitochondria accumulation (arrows) and lipid droplets in (b) and (c) (arrowheads), and vacuolar degeneration (arrows)
in (d). Scale bar = 2.5 µm
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
5.2.5 RT-PCR
Western Blot Preparation
Freshly obtained samples from bladder and urethra were homogenized in ice protein
lysis buffer. Insoluble materials were removed by centrifugation. Protein concentration was determined by the BCA method (Pierce Chemical Company, Rockford, IL).
An equal amount (20 µg) from each sample was electrophoresed in SDS-PAGE. The
fractionated proteins were then transferred to PVDF membrane. The membrane was
stained with Ponceau S to verify the integrity of the transferred proteins and to monitor the unbiased transfer of all protein samples. The membrane was then subjected to
either the ECL (Amersham Life Sciences Inc., Arlington Heights, IL) or the AP procedure (Promega Corp., Madison, WI) for the detection of caveolin-1.A 1 : 1,000 dilution
of caveolin-1 and -3 antibodies was used for Western blotting.
Western Blot Results
Expression of caveolin-1 in the urethra and bladder was less in pregnant rats than in
virgin controls,but markedly increased in the 2-day postpartum rats.At 6 weeks postpartum, expression had returned to the level in the virgin rats (Fig. 5.4).No significant
difference was detected in caveolin-3 expression in the urethra of the four groups
(Fig. 5.5).
53
Fig. 5.4. Immunoblot shows caveolin-1 protein expression in the bladder (line A and C) and ure-
thra (line B and D) in virgin, pregnant,2nd-day-postpartum, and 6-weeks-postpartum rats.Note
high protein expression in 2nd-day-postpartum and low protein expression in pregnant rats’
bladder and urethra
54
Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
Fig. 5.5. Immunoblot shows caveolin-3 protein expression in the urethra of virgin (1, 2),pregnant
(3, 4), 2nd-day (5, 6), and 6-weeks-postpartum rats (7, 8 ). Rat muscle lysate was used as control
(C). No difference was noted among the groups
5
5.3 Part II
5.3.1 Experimental Set-up
Three-month-old female virgin (n=12; mean weight, 249.2 g) and primiparous preg-
nant Sprague-Dawley rats at gestational day 16 (n=48; mean weight,266.5 g) were obtained from the vendor. They were housed at 16°C constant room temperature and
47% humidity with a 12-h light–dark cycle and free access to standard laboratory chow
and tap water.
Immediately after parturition, cystometry and stress/sneeze testing were performed in all rats to serve as the baseline for future evaluations.Animals were separated into two groups, with half in each group undergoing balloon dilation. (Virgin rats
were also evaluated to establish control values.) One month after parturition, every
second animal of each group underwent ovariectomy,resulting in four groups (I = delivered, II = delivered and ballooned, III = delivered and ovariectomized, IV = delivered, ballooned and ovariectomized).
Approximately 9 weeks after the animals were obtained from the vender, - 2 months
after parturition – all animals were evaluated by transurethral urodynamic and
stress/sneeze test before sacrifice (Fig. 5.6). The lower urinary tract (bladder, bladder
Fig. 5.6. Illustration of the experimental protocol
Chapter 5 The Rodent Animal Model to Explain Stress Urinary Incontinence
neck, mid-urethra and levator muscle) was then removed and specimens were processed for light or electron microscopy.
Intravaginal Balloon Inflation
Immediately after parturition, half of the animals were anaesthetized with 18–20 mg/
kg intraperitoneal pentobarbital (groups II and IV). The bladder was emptied via a
transurethrally placed 22G Intracath(Becton Dickinson, Sandy, UT). A modified 22°F
Foley catheter (entire weight, 130 g) was inserted intravaginally and inflated with 5 ml
distilled water.The rat was placed in a fixed prone position,the symphysis at the table
edge for 3 h.The symphysis became a fulcrum for the catheter,which hung freely without touching the table (Fig. 5.7). (The pulling directed the pressure downward to simulate the pressure of a large fetus on the pelvic floor and lower urinary tract during a
prolonged second stage of labor.)
5.3.2 Statistical Analysis
Functional evaluation of bladder capacity, modified leak-point pressure (mLPP),
bladder pressure,and residual volume was repeated at least three times and the mean
was used for further analysis. The values of different groups were compared with those
of the virgin rats. The Mann-Whitney U test was used for statistical analysis. The
stress/ sneeze test, used as the indicator for incontinence, was verified by convergence
tables.
55
Fig. 5.7. Intravaginally placed Foley catheter. The force of the balloon is directed to the pelvic
floor to simulate human labor
56
Karl-Dietrich Sievert, Emer Bakircioglu,Lora Nunes, Tony Tsai,Tom F. Lue
5.3.3 Functional Evaluation
Cystometry and Leak-point Pressure Measurement
Cystometry and leak-point pressure measurements were performed as described in
Sect. 5.2.3.
Urodynamic Results
5
Bladder capacity on the day of delivery was significantly increased (mean bladder capacity 2.1±0.2 ml [delivered] vs 1.3±0.2 ml [virgin], p<0.005). In group I, the bladder
capacity remained the same 8 weeks after delivery,but a decrease was noted in groups
II–IV. The mLPP decreased significantly at the day of delivery (28.9±1.3 cm H
returned to the level of virgin rats (49.7±2.4 cm H
O) after 8 weeks. Animals under
2
O) and
2
ketamine anesthesia had higher mLPPs than did those under pentobarbital anesthesia
(data not shown). There was an increase in residual volume in group I rats (mean,
1.1±0.4 ml) when compared with virgin rats (mean, 0.3±0.1 ml) (p<0.05) (Table 5.5).
Stress/Sneeze Test
After cystometry, the bladder was filled to half capacity with saline solution (0.9%;
37°C) mixed with one drop of methylene blue to facilitate the determination of urinary
leakage. After removal of the catheter, a rat whisker was inserted into the nostril and
moved to induce sneezing, and the urethral meatus was observed for urinary leakage.
The test was repeated twice to verify the findings.
Table 5.5. Urodynamic results
Virgin Day 0 Group I Group II Group III Group IV
n=10 n=48 n=12 n=12 n=12 n=11
Bladder capacity c=1.4 c=2.5* c=2.0* c=2.1* c=1.1 c=1. 3
(ml) i=2.5* i=1.6 i=1.5 i =1.6 i =1.6
Delivery 8 weeks after delivery
mLPP (cmH
(ketamine)
Residual volume c=0.3 c=0.3 c=1.2* c=0.8 c=0.7 c=0.7
(ml) i=0.8 i=0.8 i=0.5 i=0.8 i =0.6
Incontinent 0 29.2* 16.7* 58.3* 16.7* 72.7*
stress test + (%)
c continent rats,i incontinent rats.
* p<0.05 compared with virgin.
a
The mLPP results under pentobarbital sedation showed a similar trend among groups, although with a lower pressure (virgin=35.0±2.2, group I=42.5±4.4, II=36.2±4.4, III=36.8±3.6
and IV=33.8±2.1; mean values of the whole group).
O) c=49.7 c=27.3* c=48.3 c=43.0 c=49.6 c=59.0
2
a
i=23.2* i=46.5 i =38.3 i =42.0 i =38.3
Соседние файлы в папке @xirurgi_2025