The Effect of Partial Outlet Obstruction on Collagen and Smooth Muscle Myosin in the Rabbit Urinary Bladder
Connor M. Callaghan1, Catherine Schuler2, Nancy Gertzberg1,2, Arnie Johnson1, Robert E. Leggett2, Robert M. Levin1,2*
1Albany College
of Pharmacy and Health Sciences, New Scotland Ave. Albany, New York, USA
2Stratton VA Medical Center, Holland Ave. Albany, New York, USA
*Corresponding author: Robert M. Levin, Stratton VA Medical Center, Holland Ave. Albany, 12208 New York, USA.Tel: +15183690173; Email: Robert.levin2@va.gov
Received Date: 22 January,
2018; Accepted Date: 07 February, 2018; Published Date: 15 February,
2018
Citation: Callaghan CM, Schuler C, Gertzberg N, Johnson A, Leggett RE, et al. (2018) The Effect of Partial Outlet Obstruction on Collagen and Smooth Muscle Myosin in the Rabbit Urinary Bladder. Adv Biochem Biotechnol 2: 155. DOI: 10.29011/2574-7258.000055
Introduction: One of the major etiologies of obstructive bladder dysfunction
is tissue fibrosis.As obstructive bladder dysfunction progresses, the
replacement of functional smooth muscle with connective tissue results in
progressive bladder failure. In the current study, we quantitated the collagen
and smooth muscle myosin and analyzed them both by duration of obstruction and
severity of contractile dysfunction.
Materials and Methods: Four groups of eight rabbits each were subjected to Partial Bladder
Outlet Obstruction (PBOO) for 4, 8, and 12 weeks respectively. The control
group was comprised of eight sham surgical rabbits. At the end of the
experimental time period, the bladders from all rabbit groups were surgically
removed under general anesthesia and 2 full thickness strips of bladder body
were taken and fixed in formalin and embedded in paraffin for histology; and
three full thickness strips were taken for contractile studies. The balance of
the bladder body was separated into muscle and mucosal tissue, frozen and
stored at -80oC for biochemical analyses. Quantitative collagen and smooth
muscle myosin assays were conducted on bladder smooth muscle samples. Bladder
weight was used to categorize the rabbits into mild, intermediate, and severe
decompensation groups.
Results: Both collagen and smooth muscle myosin showed similar
correlations with both the severity of decompensation and the duration of
obstruction. Collagen levels increased significantly while smooth muscle myosin
levels decreased.
Discussion: The conversion of smooth muscle to collagen was shown to be an
important mechanism causing bladder decompensation.
1. Introduction
Recent studies have
focused on characterizing the relationship between partial bladder outlet
obstruction (PBOO) in the rabbit urinary bladder with several biomarkers and
enzymes. Nitrotyrosine (NT) and Dinitrophenyl (DNP) are markers of oxidative
stress [1,2]. The enzymes Superoxide Dismutase (SOD) and catalase are
endogenous antioxidants that are widespread in the body, and alterations in the
activities of these enzymes are also markers of oxidative stress[3,4].Calcium
activated enzymes such as calpain and phospholipase A2 (PLA2)
are biomarkers for intracellular calcium dysregulation resulting in increased
intracellular free calcium [5-7].
In recent publications, the data for these biomarkers were
grouped by both the duration and severity of bladder decompensation at the end
of the experiment. The duration of the obstruction was separated into4, 8, and
12 weeks obstruction; and the severity was separated into mild, intermediate,
and severe obstruction by the contractile responses to in-vitro stimulation
by field stimulation; carbachol; and KCl [8-10].
Perhaps the most novel and noteworthy finding from these studies
was that in mild decompensation, bladders did not show increased levels of
oxidative stress as shown by the nitrotyrosine and DNP levels, but the calpain
and PLA2 levels were elevated [8]. This suggested that
initial bladder decompensation may be mediated more by calcium dysregulation
than oxidative stress. The reason for this may be because the cell’s
antioxidant defense mechanisms were able to cope with oxidative stress whereas
the cell was less able to handle the increased cytosolic free calcium.
The final part of these studies was analyzing the collagen and
smooth muscle myosin levels in the bladders that were exposed to PBOO. In
theory, the conversion of smooth muscle to collagen should directly correlate
with decreasing contractility and increasing bladder decompensation [11-13]. In
other words, the increasing oxidative stress and calcium activated enzymes
stimulate the conversion of smooth muscle to collagen thereby increasing the
rate of decompensation [14]. In addition to analyzing collagen using a
total collagen assay kit and smooth muscle myosin by Western blot analysis, we
also analyzed the bladder histologically into connective tissue and smooth
muscle compartments by digitally analyzing trichrome stained samples from each
bladder. These biochemical and histological studies were limited to the bladder
smooth muscle compartment only; the mucosa and serosa do not participate
actively in contraction and thus were not part of this study.
2. Materials and Methods
All studies were approved by the Institutional Animal Care and
Use Committee and the Research and Development Committee of the Stratton VA
Medical Center, Albany, NY.
2.1. Animal Model
For this study, thirty-two New Zealand white rabbits were divided
into four equal groups of eight rabbits. The first group was a control group
all of which underwent a sham obstruction that caused no significant bladder
decompensation. Partial bladder outlet obstructions were performed on the other
three groups by loosely tying a silk ligature around the catheterized urethras
of the anesthetized rabbits. The rabbits were obstructed for four, eight, and
twelve weeks respectively. At those times, the rabbits were again put under
anesthesia and the bladders were surgically removed. The bladder body and base
were separated at the level of the ureteral orifices. Two full thickness
samples of the bladder body were taken for histological studies, placed in
formalin for 8 hours, and then embedded in paraffin. Three full thickness
strips of bladder body were taken for in-vitro contractility
studies. The balance of the bladder body was separated between muscle and
mucosa by blunt dissection and stored in a freezer at -80º Celsius
for later biochemical analyses of collagen and smooth muscle myosin.
The categories of the decompensated bladders were determined by
their weight as published in previous studies [10,15]. The increase in bladder
mass correlated well with both bladder body hypertrophy and increasing bladder
decompensation [10,15]. Bladders weighing less than 6 grams were
considered mild, 6-20 grams were intermediate and over 20 grams were severely
decompensated.
2.2. Contractility Studies
Contractility studies were conducted by placing strips of
bladder tissue in baths containing warm, oxygenated Tyrode’s solution (37°C)
to observe maximal tension generation in response to various stimuli including Field
Stimulation (FS), carbachol, and Potassium chloride (KCl). The level of
contractile function (dysfunction) was calculated as the average percentage of
contraction of the control tissue; thus, the greater the dysfunction, the lower
the percentage of control.
2.3. Collagen Analysis
Collagen levels were determined on the muscle samples through
the use of a Quick Zyme Total Collagen Assay chemical kit. Tissue samples were
hydrolyzed at 100 mg/ mL in 6M HCl and incubated for 20 hours in a thermos block
at 95°C. Three fold dilutions were done on all tissue samples using
4M HCl. The standard curve was prepared from a stock of 1.2 mg/ mL in 12M HCl. The
curve created with dilutions in 4M HCl had values of 0.672, 0.448, 0.224,
0.112, 0.056, 0.028, 0.014 mg/ mL, and a blank. The kit required the use of
clear 96 well plates and a fluorescence plate reader (Spectra Max Plus by
Molecular Devices) set at 570nm.
2.4. Smooth Muscle Myosin
Frozen tissue of bladder muscle wall was homogenized on ice in
homogenization buffer (50 mM Tris, pH 7.5, 5% Tiron) containing the Halt
Protease Inhibitor Cocktail (Pierce, Rockford, IL) at 100 mg/ml. After addition
of SDS (final concentration, 1%),the sample was boiled for 4 min and
centrifuged at 10,000 rpm for 15 min. Protein concentration in the supernatant
was measured using the Pierce BCA protein assay kit. Membranes were blocked with
5% nonfat milk in 0.05% Tween 20 in PBS for 1 h at room temperature
and then incubated with primary antibody, monoclonal antibody to Smooth Muscle
Light Chain A; Smooth Muscle Light Chain B; and Smooth Muscle Heavy Chain. All
antibodies were obtained from Sigma-Aldrich [16,17]. After treatment with the
primary antibody, the membranes were washed and incubated with secondary
antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The substrate was
visualized by using ECL-Plus (Amersham Pharmacia Biotech, Buckinghamhire,
England) for 2 minutes and analyzed with a Kodak Image Station 440CF and
Kodak 1D image analysis software (Scientific Image System, Rochester, NY).
2.5. Histological Analysis
Each paraffin block was sectioned at 5 M and 3-4 sections were
placed on individual slides. Each section was de-paraffinized by graded ethanol
and stained with trichrome stain. Connective tissue stains blue while smooth
muscle stains red. Smooth muscle areas of each section (minus mucosa) were
digitally analyzed and the percentage of connective tissue and smooth muscle
were calculated and then normalized to 100% of the area under investigation.
3. Results
The bladder weights and severity levels for each duration are
given in Table1. Control rabbits had a mean bladder weight of 2.6 +/-
2 grams. The 4-week obstructed bladders had a mean weight of 10.5 +/- 5.8. The
8-week obstructed bladders had a mean weight of 16.4 +/- 4.8 grams. The 12-week
obstructed bladders had a mean weight of 27.2 +/- 6.5 grams. Control rabbits
had no decompensated bladders. The 4-week obstructed bladders had 3 mild, 3
intermediate, and 2 severely decompensated bladders; the 8-week obstructed
bladders had 2 mild, 4 intermediate, and 2 severely decompensated bladders. The
12-week obstructed bladders had 0 mild, 4 intermediate, and 4 severely
decompensated bladders.
The contractility studies from these groups have been published
previously [10].Although contractile responses decreased in
relation to both severity and duration; there was a closer
correlation with severity than with duration.
The collagen concentration in mg/ g of tissue was evaluated by
both severity of decompensation and duration of obstruction and presented
in Figure 1A. By duration, the collagen levels of both the 8weeks and
12 week groups were significantly higher than control. When analyzed by
severity, the mild and intermediate groups were near control levels. A major
increase was seen only in the severe group which was significantly higher than
both control and the other obstructed groups. In Figure 1B, the
collagen levels in mg/ bladder were compared by severity and duration. When
grouped by duration, the same general outline was seen as in Figure 1A.
However, after severity analysis a slightly different trend emerged. This time,
the collagen levels were the same in the control and mild groups, but a
significant increase was seen from both mild to intermediate and from
intermediate to severe decompensation.
The smooth muscle myosin heavy chain is presented in Figure
2by severity and duration. The data was nearly identical when comparing both
severity and duration with 4 weeks obstructed and mild decompensation being
equal to control,8 and 12-week obstructions; and Intermediate and severe
decompensations were equal to each other and significantly reduced from control
and 4 weeks obstructed.
Myosin light chains A and B (Figures 3A and 3B) showed very
similar results to those shown for myosin heavy chain except that for light
chain A, the mildly decompensated bladders showed significantly reduced
concentrations compared to control.
Figures 4-7 show representative trichrome-stained micrographs
for control, 4, 8, and 12-week obstructed bladders. For quantification of
connective tissue and smooth muscle, each histological section of each bladder
was separated by both duration and severity, and the ratio of smooth muscle/
collagen was calculated by digital analysis.
The percentages of Connective Tissue (CT) and Smooth Muscle (SM)
analyzed by histology for duration and severity are presented in Figures
8A and B. By duration, the percentage of SM was significantly higher than the
percentage of CT for control tissue. The percentages of CT for 4, 8, and
12-week obstructed rabbits were significantly higher than control while the
percentages of SM was significantly lower than control. There were similar
percentages of both SM and CT for all durations of obstruction (Figure 8A). Similar
results were observed for the analysis by severity, (Figure 8B) except
with mild decompensation there was an intermediate level of connective tissue
between control and intermediate levels of decompensation.
4. Discussion
Figure 9shows a modification of our schematic on the etiology of
obstructive bladder dysfunction published in our review
article: Obstructive Bladder Dysfunction: Morphological, Biochemical and
Molecular Changes [18]. Based on our studies, the shift from
compensated to mild decompensation involves the left branch; i.e., calcium
dysregulation and the activation of calcium-dependent proteases and lipases. As
decompensation proceeds (from mild to severe) free radicals are generated by
the decreased blood flow, hypoxia, and ischemia due to bladder hypertrophy.
Simultaneous, collagen fibrosis occurs parallel to bladder hypertrophy which
adds to the severity of obstructive bladder dysfunction. Initially, the
fibrosis is reversible if the obstruction is relieved [19]. Interestingly,
the longer the obstruction lasts, independently of the severity the collagen
(connective tissue) develops cross bridges that resists reversal; thus, the
longer the duration, the greater the influence of fibrosis has on bladder
function. This is not the case with either calcium dysregulation or oxidative
stress where reversal depends on severity rather than on duration. These
differences are true for obstructive bladder dysfunction in humans where
obstructive bladder dysfunction can exist for years, and fibrosis has a greater
influence on bladder dysfunction than in rabbits [20,21].
In a study to determine the extent that collagen deposits can
influence contractile function, rabbits were divided into two groups: control
and 2-week obstructed. Each rabbit was anesthetized, and the bladder body was
excised and cut into equal width strips of 0.5, 1.0, and 2.0-cm lengths [22]. The contractile responses to field
stimulation, carbachol, and potassium chloride were determined. At the end of
the experiment, each strip was fixed in formalin and immune-stained for
collage. The contractile responses for the control were similar for all strip
lengths. In obstructed tissue, the shorter strip lengths generated
significantly more tension per cross-sectional area than did the longer strips.
The collagen density and distribution were significantly greater for the
obstructed bladder strips than in control strips. In addition, the obstructed
bladder strips had significantly increased collagen deposits between and within
the smooth muscle bundles and cells.
Because the relationship between strip size and contraction were
similar for field stimulation, carbachol, and potassium chloride, it was the
increased density of connective tissue within and between the muscle bundles
and fibers that interfered with contraction (i.e., the greater the strip
length, the greater the interference and the greater the contractile
dysfunction). Therefore, both functional and structural alterations in the
obstructed bladder participate in contractile dysfunction [22].
As expected, the bladder mass increased with increasing duration
of obstruction because of the associated increasing SM hypertrophy and mucosal
hyperplasia [23,24]. In regard to contraction, oxidative stress, and calcium
overload, the link is more direct between the severity of decompensation than
with the duration of obstruction [8,10]. Unlike the previous observations, the
increase in collagen and decrease in SM may be more closely related to duration
of obstruction than severity. When analyzed per bladder, the apparent
progressive increase in collagen is probably due to the progressive increase in
bladder mass, although there was a significant increase in collagen associated
with severe decompensation and not with mild or intermediate decompensation.
However, this trend was not observed in the histological study where all
durations and severities had approximately the same decrease in SM and increase
in CT. This is probably due to the fact that the histological analysis is a
qualitative analysis whereas the biochemical assays were quantitative.
The durations of obstruction were closely controlled while the
level of bladder decompensation was dependent on the response of the individual
rabbit to the presence of the obstruction. This observation has been made
previously in several models of obstruction [15,23,25]. This is similar to
the response in men to obstructive uropathy secondary to BPH. That is, the
severity of the obstruction is not related to the size of the prostate but to
the response of the individual to the presence of the constriction [26-28].
The collagen levels did increase with increasing severity of
bladder decompensation but only in the severely decompensated group. This is
most likely because the collagen increase itself was directly involved with the
severe decompensation [22]. The smooth muscle myosin levels decreased similarly
with increasing bladder duration and severity of decompensation. The decreased
smooth muscle myosin was significant in the 8 and 12-week obstructed groups to
approximately the same degree as the intermediate and severe decompensation
groups. The decrease in SM was related to the increase in collagen because of
the conversion of SM to collagen in the presence of obstruction in both rabbits
and humans [29,30]. Since the SM allows the bladder to contract, the
decrease of SM subsequently mediated the bladder decompensation. Interestingly,
the smooth muscle myosin heavy chain concentration was nearly identical when
grouped by both severity and duration which was not seen in the collagen or
even in the smooth muscle light chains. This may be because the heavy chain
converts at a more stable rate than the light chains. The increased density of
the heavy chain may provide some means to allow the conversion to collagen to
be more gradual causing the graphs of both duration and severity to be similar.
It is also worth noting that light chain A seemed more
susceptible to PBOO than light chain B. The mildly decompensated group had much
lower smooth muscle myosin levels for light chain A than control, while for
light chain B, the mild group was the same as control. The interplay between
smooth muscle myosin heavy chain and light chain A and B could very well be
complex but also important to better understand the steps leading toward
decompensation. In any case, all three types of smooth muscle myosin were
significantly decreased by the time severe decompensation (or 12-week
obstruction) was reached while collagen levels were significantly increased.
The histological studies also would support the idea that duration is more
related to the ratio of SM to CT than severity.
5. Conclusions
The functional decrease in contractility following partial
bladder outlet obstruction is a multi-factorial process involving oxidative
stress, calcium overload, changes in SOD and catalase, and the conversion of SM
to collagen. Interestingly, oxidative stress and calcium overload appear to be
related to the severity of decompensation to a higher degree than to the
duration of obstruction, whereas the conversion of SM to collagen appears to be
more closely related to duration. This may well be a function of the conversion
of SM to collagen being a slow process whereas changes in oxidative stress and
intracellular calcium concentrations are relatively rapid processes.
6. Acknowledgement
This material is based upon work supported in part by the Office
of Research and Development Department of the Veterans Affairs and in part
by the Capital Region Medical Research Foundation.
Figure
1A: Collagen
concentration in mg/ g tissue following PBOO by duration and severity. Each bar
for duration is the mean +/- SEM of 8 individual rabbits. Each bar for severity
is the mean +/- SEM of between 5 and 11 rabbits. *
= significantly different from control, p < 0.05.
Figure
1B: Collagen
concentration in mg/ bladder following PBOO by duration and severity. Each bar
for duration is the mean +/- SEM of 8 individual rabbits. Each bar for severity
is the mean +/- SEM of between 5 and 11 rabbits. *=
significantly different from control, p < 0.05.
Figure
2: Smooth
muscle myosin heavy chain (optical density) by duration and severity. Each bar
for duration is the mean +/- SEM of 8 individual rabbits. Each bar for severity
is the mean +/- SEM of between 5 and 11 rabbits. *
= significantly different from control, p < 0.05.
Figure
3A: Smooth
muscle myosin light chain A (optical density) by duration and severity. Each
bar for duration is the mean +/- SEM of 8 individual rabbits. Each bar for
severity is the mean +/- SEM of between 5 and 11 rabbits. * = significantly different from control, p
< 0.05.
Figure
3B: Smooth
muscle myosin light chain B (optical density) by duration and severity. Each
bar for duration is the mean +/- SEM of 8 individual rabbits. Each bar for
severity is the mean +/- SEM of between 5 and 11 rabbits. * = significantly different from control, p
< 0.05.
Figure 4: Representative histological section from
a control rabbit.
Figure 5: Representative histological section from
a 4-week obstructed rabbit.
Figure 6: Representative histological section from
an8-week obstructed rabbit.
Figure 7: Representative histological section from
a 12-week obstructed rabbit.
Figure
8A: Percent
connective tissue and smooth muscle normalized to 100% area and grouped by
duration for connective tissue and smooth muscle. Each bar for duration is the
mean +/- SEM of 8 individual rabbits .*
= significantly different from control, p < 0.05.
Figure
8B: Percent
connective tissue and smooth muscle normalized to 100% area and grouped by
severity for connective tissue and smooth muscle. Each bar for severity is the
mean +/- SEM of between 5 and 11 rabbits.*
= significantly different from control, p < 0.05.
Figure 9: Schematic of the etiology of
obstructive bladder dysfunction.
|
Bladder Weight gm |
Severity |
|
|
Number of Rabbits |
Control (8 rabbits) |
2.6 +/- 2.0 |
Mild0 |
Intermediate0 |
||
Severe0 |
||
4Week Obstructed |
10.5 +/- 5.8 * |
Mild3 |
Intermediate3 |
||
Severe2 |
||
8 Week Obstructed |
16.4 +/- 4.8 * |
Mild2 |
Intermediate4 |
||
Severe2 |
||
12 Week Obstructed |
27.2 +/- 6.5** |
Mild0 |
Intermediate4 |
||
Severe4 |
||
* = significantly different from control, p < 0.05 |
Table 1[10]: Bladder weight and distribution of severity groups.
- Juan YS, Lin WY, Kalorin C, Kogan BA, Levin RM et al. (2007) The effect of partial bladder outlet obstruction on carbonyl and nitrotyrosine distribution in rabbit bladder. Urology 70:1249-1253.
- Kalorin CM, Mannikarottu A, Neumann P, Leggett R, Weisbrot J, et al. (2008) Protein oxidation as a novel biomarker of bladder decompensation. BJU Int 102:495-499.
- Onal B, Levin RM, Kogan BA, Guven A, Leggett RE,et al. (2007) Novel alterations in superoxide dismutase and catalase activities in the female rabbit bladder subjected to hormonal manipulations. IntUrolNephrol 39:1049-1054.
- Lin AD, Mannikarottu A, Kogan BA, Whitbeck C, Leggett RE,et al. (2007) Effect of bilateral in vivo ischemia/reperfusion on the activities of superoxide dismutase and catalase: response to a standardized grape suspension. Mol Cell Biochem 296:11-16.
- Zhao Y, Levin SS, Wein AJ, Levin RM (1997) Correlation of ischemia/reperfusion or partial outlet obstruction-induced spectrin proteolysis by calpain with contractile dysfunction in rabbit bladder. Urology 49:293-300.
- O'Connor LJ, Nicholas T, Levin RM (1999) Subcellular distribution of free fatty acids, phospholipids, and endogenous lipase activity of rabbit urinary bladder smooth muscle and mucosa. Adv Exp Med Biol 462:265-273.
- O'Connor LJ, Goldner CW, Lau ST, Hass MA, Levin RM (1999) Effect of partial outflow obstruction on the distribution of free fatty acids and phospholipids in the rabbit bladder. World J Urol 17:261-265.
- Callaghan CM, Johnson A, Neumann P, Leggett RE, Schuler C,et al. (2013) The effect of partial outlet obstruction on calpain and phospholipase-2 activities: analyzed by severity and duration. Mol Cell Biochem 381:217-220.
- Callaghan CM, Schuler C, Leggett RE, Levin RM (2013) Effect of severity and duration of bladder outlet obstruction on catalase and superoxide dismutase activity. Int J Urol 20:1130-1135.
- Levin RM, Schuler C, Leggett RE, Callaghan C,Maknuru S (2013) Partial outlet obstruction in rabbits: duration versus severity. Int J Urol 20:107-114.
- Cortivo R, Pagano F, Passerini G, Abatangelo G, Castellani I (1981) Elastin and collagen in the normal and obstructed urinary bladder. Br J Urol 53:134-137.
- Kim JC, Yoon JY, Seo SI, Hwang TK,Park YH (2000) Effects of partial bladder outlet obstruction and its relief on types I and III collagen and detrusor contractility in the rat. NeurourolUrodyn 19:29-42.
- Tekgul S, Yoshino K, Bagli D, Carr MC, Mitchell ME,et al. (1996) Collagen types I and III localization by in situ hybridization and immunohistochemistry in the partially obstructed young rabbit bladder. J Urol 156:582-586.
- Lee BR, Perlman EJ, Partin AW, Jeffs RD, Gearhart JP (1996) Evaluation of smooth muscle and collagen subtypes in normal newborns and those with bladder exstrophy. J Urol 156:2034-2036.
- Kato K, Monson FC, Longhurst PA, Wein AJ, Haugaard N, et al. (1990) The functional effects of long-term outlet obstruction on the rabbit urinary bladder. J Urol 143:600-606.
- Samuel M, Kim Y, Horiuchi KY, Levin RM, Chacko S (1992) Smooth muscle myosin isoform distribution and myosin ATPase in hypertrophied urinary bladder. BiochemInt 26:645-652.
- Wang ZE, Gopalakurup SK, Levin RM, Chacko S (1995) Expression of smooth muscle myosin isoforms in urinary bladder smooth muscle during hypertrophy and regression. Lab Invest 73:244-251.
- Levin RM, Chichester P, Hass MA, Gosling JA, Buttyan R (2002) Obstructive bladder dysfunction: Morphological, biochemical, and molecular changes. European Urology Supplements 1:14-20.
- Jock M, Leggett RE, Schuler C, Callaghan C, Levin RM (2014) Effect of partial bladder outlet obstruction and reversal on rabbit bladder physiology and biochemistry: duration of recovery period and severity of function. BJU Int 114:946-954.
- Collado A, Batista E, Gelabert-Mas A, Corominas JM, Arano P, et al. (2006) Detrusor quantitative morphometry in obstructed males and controls. J Urol 176:2722-2728.
- Lepor H, Sunaryadi I, Hartanto V,Shapiro E (1992) Quantitative morphometry of the adult human bladder. J Urol 148:414-417.
- Levin RM, Reed TP, Whitbeck C, Chichester P, Damaser M (2005) Effect of strip length on the contractile dysfunction of bladder smooth muscle after partial outlet obstruction. Urology 66:659-664.
- Levin RM, Brading AF, Mills IW, Longhust PA (1999) Experimental Models of Bladder Obstruction. In: Lepor H (ed) Prostatic Disease, W.B. Saunders Co, Philadelphia169-196
- Levin RM, Longhurst PA, Monson FC, Kato K, Wein AJ (1990) Effect of bladder outlet obstruction on the morphology, physiology, and pharmacology of the bladder. Prostate Suppl 3:9-26.
- Kato K, Wein AJ, Radzinski C, Longhurst PA, McGuire EJ, et al. (1990) Short term functional effects of bladder outlet obstruction in the cat. J Urol 143:1020-1025.
- Levin RM, Haugaard N, O'Connor L, Buttyan R, Das A, et al. (2000) Obstructive response of human bladder to BPH vs. rabbit bladder response to partial outlet obstruction: a direct comparison. NeurourolUrodyn 19:609-629.
- Mirone V, Imbimbo C, Longo N, Fusco F (2007) The detrusor muscle: an innocent victim of bladder outlet obstruction. EurUrol 51:57-66.
- Steers WD, Zorn B (1995) Benign prostatic hyperplasia. Dis Mon 41:437-497.
- Polido Junior A, Costa JM, Munhoz T, Sampaio FJ, Cardoso LE, et al. (2010) Intravesical oxybutynin protects the vesical wall against functional and smooth muscle changes in rabbits with detrusor overactivity. IntUrogynecol J 21:1539-1544.
- Lee SD, Akbal C, Miseeri R, Jung C, Rink R, et al. (2006) Collagen prolyl 4-hydroxylase is up-regulated in an acute bladder outlet obstruction. J PediatrUrol 2:225-232.