ANALYSIS
OF BLADDER HISTOLOGY AND URODYNAMICS IN FEMALE RATS SUBMITTED TO BLADDER
RECONSTRUCTION WITH RECTUS ABDOMINIS MUSCLE
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MAURÍCIO HACHUL, ANTÔNIO MACEDO JR., MIGUEL SROUGI
Division of Urology, Paulista School of Medicine, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil
ABSTRACT
Introduction:
Bladder histology and measures of maximal bladder pressure and maximal
bladder volume during urinary loss in female rats submitted to bladder
reconstruction with a myoperitoneal flap of rectus abdominis muscle were
studied.
Materials and Methods: Fifty female adult
Wistar rats were studied, separated in 5 groups: Group 1 (immediate):
submitted to laparotomy, cystostomy, and cystometry; Group 2 (sham): submitted
to laparotomy, anterior bladder wall incision, immediate suture and cystostomy;
Group 3 (cystectomy): laparotomy, partial cystectomy, cystostomy; Group
4 (cystoplasty): midline laparotomy, partial cystectomy, augmentation
with rectus abdominis muscle flap peritonized, cystostomy, total cystectomy,
the rats were sacrificed and an histological study of the augmented bladder
performed; Group 5 (cystostomy): midline laparotomy and cystostomy. Cystometry
was always performed in post-operative day 7, excepting for Group 1. Assessment
was comparative maximal bladder pressure and maximal bladder volume during
urinary loss among the different groups.
Results: In cystoplasty group, mean maximal
bladder pressure during urinary loss was lower than in immediate, sham,
and cystostomy groups. It was also observed that maximal bladder volume
during urinary loss presented mean and median values very close in each
group, and cystectomy group showed much lower values. The group submitted
to cystoplasty presented mean maximal bladder volume during urinary loss
higher than all groups analyzed. Histological analysis of myoperitoneal
flap augmented bladders showed partial and/or total epithelization in
the muscular flap interspersed region with transitional cells, squamous
metaplasia region and chronic inflammatory process.
Conclusion: The use of peritonized rectus
abdominis muscle flap to perform bladder augmentation was technically
viable in the animals, showing urothelial epithelization in the muscular
region of the flap, and a satisfactory gain of capacity and maintenance
of low bladder pressures.
Key words:
urinary bladder; reconstructive surgical procedures; rats; urodynamics;
histology
Int Braz J Urol. 2002; 28: 560-8
INTRODUCTION
Patients
with severe urinary bladder deformities, congenital or acquired, may present
a functional bladder capacity reduction, as well as alterations in bladder
complacence. In the pediatric population, neurogenic bladder due to meningomyelocele
and posterior urethral valve bladder are the most common examples of this
condition. It is known that whenever intravesical pressure during urinary
loss is superior to 40cm H20, there is risk of upper urinary tract impairment
and, therefore, initial clinical measures must be instituted (1). In the
occurrence of absence of response to clinical therapy, the requirement
for a reconstructive surgery should be considered, aiming at an organ
augmentation, favoring thus the urinary volume storage in a low intravesical
pressure.
Countless bladder augmentation techniques
using detubularized digestive tract segments were described during the
last decade, and are increasingly used as tissue source. However, the
existent surgical options may cause several troubles assessed during this
patients’ late follow-up (2-4).
Theoretically, the best way to treat these
cases would be to recommend a bladder reconstruction using an easily available
tissue, good blood supply conditions, and that would allow a sufficient
bladder capacity gain with adequate complacence. Büyükünal
et al. (5) initiated an experimental investigation about bladder reconstruction,
using a segment of abdominis rectus muscle of the rat aiming to assess
its technical feasibility and perform its histological study. Despite
being a pioneer study and relate more than one possible alternative for
bladder augmentations, this work has motivated new research that could
demonstrate the essential features for the success of a new bladder substitution
technique.
Recently, using an experimental model in
rabbits, the efficiency of bladder reconstruction technique with rectus
abdominis muscle use was assessed through radiological study in 15 animals,
and the results suggested its good viability (6).
Thusly, we begin to develop in our facility
a work for standardization of comparative analysis of surgical procedures
in rat bladder, through the creation of an experimental model defining
the features of urodynamics study performance, equipments, catheters,
and normal bladder histological pattern in rats (7).
Considering this research line in bladder
reconstruction, we started to progress in our experimental investigation
in rats, comparatively analyzing urodynamic parameters and histological
studies in animals submitted to bladder reconstruction with rectus abdominis
muscle, which is the aim of this paper.
MATERIALS AND METHODS
Fifty
female adult rats, aged approximately 3 months, weight between 180 and
240 grams (mean=220 grams) were used. The experiment protocol was approved
by the Ethics Committee.
In the animal room, the rats were confined
in plastic cages with metal latticed cover, maintained in a room temperature
of approximately 22oC (71oF), and artificial light with fluorescent lamps.
A photoperiod light of 12 hours, intercalate by 12 hours dark was maintained.
The rats received Labina-Purina (São
Paulo, Brazil) rations and water ad libitum, and were maintained in the
animal room.
Anesthetics used to all animals were intraperitoneal,
using 50mg/kg thionembutal. The animals were operated with the aid of
2.5X magnification lenses and were distributed in 5 experimental groups,
with 10 female rats each: a)- Group 1 (immediate): submitted to midline
laparotomy, cystostomy and 2 catheters (3F) insertion in the bladder dome.
The catheters were sutured to the bladder for fixation and exteriorized
in dorsal region near the animal head through a subcutaneous route. In
this group, right after cystometric study the animals were sacrificed;
b)- Group 2 (sham): submitted to midline laparotomy, anterior bladder
wall incision, immediate suture, 2 catheters (3F) insertion in the bladder
dome; c)- Group 3 (cystectomy): midline laparotomy, half-bladder exeresis
and 2 catheters insertion after bladder immediate suture; d)- Group 4
(cystoplasty): midline laparotomy, half-bladder exeresis, and then augmentation
with myoperitoneal flap, using peritonized rectus abdominis muscle with
approximately 1.5cm length and 1.0cm width, with its blood supply by the
inferior epigastric artery (Figures-1 and 2), and also 2 catheters were
inserted and fixed in neobladder right superior region; e)- Group 5 (cystostomy):
submitted to midline laparotomy and 2 catheters insertion in bladder dome.
Only the animals in the first group were
immediately sacrificed. In the four remaining groups the animals were
kept alive for 7 days, with catheter exteriorization always in dorsal
region near the animal head.
The group submitted to myoperitoneal cystoplasty,
after this period, underwent a cystography (Figure-3), was sacrificed,
and the augmented bladders were submitted to histological analysis. For
all groups a measure of maximal bladder pressure and maximal bladder volume
during urinary loss was performed. In group 1, cystometry was immediately
done and in the remaining only at the 7th post-operative day. The equipment
used in the study was model Dynapack MPX616, computerized – RS 232.
An infusion pump, continuous type, with a 1mL syringe and 0.2mL/min saline
infusion rate was also used. The catheters were always 3F gauge with 20cm
length made by non-reactive polypropylene. The suture used for the bladder
was polyglycolic acid 6-0 (Vicryl™) and for the skin monofilament
nylon 4-0.
Urinary bladder sent to histological study
was fixed in formalin, processed in paraffin, sectioned, and stained with
hematoxilin-eosin. Histological study was performed in the pathology department.
Variance analysis was used to verify means
of maximal bladder pressure and maximal bladder volume during urinary
loss equivalence among the groups studied (8).
Subsequently, Fisher’s multiple comparison
test was used to detect where are the differences between the groups studied
(9), considering a global significance level >0.05 or 5%.
RESULTS
Maximal
Bladder Pressure during Urinary Loss
Observing Table-1 and Figure-4 we may verify
that, in all groups, the mean and the median were quite close, excepting
for the group who underwent cystoplasty type surgery, and presented a
diverse value for pressure measure.
When we analyze the variation coefficients
of the study groups, we observe that the group submitted to partial cystectomy
was the one presenting lower data variability, whilst the group submitted
to immediate technique presented the greatest amplitude.
There are evidences that the mean of the
group submitted to cystectomy type surgery was inferior to the mean of
all the other groups, whilst the group submitted to immediate technique
seems to have obtained the highest mean value of maximal bladder pressure
during urinary loss (Figure-4).
Bladder
Volume during Urinary Loss
Analyzing Table-2 and Figure-5 we may observe
that each group presents very close mean and median values. The group
submitted to cystectomy technique seems to have attained the lower mean
value of bladder volume during urinary loss, whereas the group submitted
to the cystoplasty technique seems to have attained the higher mean value.
Observing Figure-5 we note that, in the
group submitted to technique sham, there was a possible “outlier”
(discordant value).
When we verify the variation coefficient,
we note that the group with greatest variability was the one submitted
to the immediate technique, while the group submitted to cystectomy technique
presented the lowest variation.
Inferential Analysis
Maximal
Bladder Pressure during Urinary Loss
Observing the descriptive levels (Table-3),
it is possible to note each surgical techniques presented significant
differences. Maximal bladder pressure during urinary loss for the mean
of study groups was lower in females rats submitted to cystoplasty compared
to groups sham, immediate, and cystostomy; however it was higher than
for the group submitted to partial cystectomy.
Bladder
Volume during Urinary Loss
Observing the descriptive levels (Table-4)
it is possible to verify which surgical techniques presented significant
differences. Maximal bladder volume during urinary loss for the mean of
study groups was higher in female rats submitted to cystoplasty compared
to all analyzed groups.
Histopathological
Analysis – Cystoplasty Group
Histological sections of the transition
zone between the bladder and the muscular flap interposition demonstrated
partial and/or total epithelization in the interspersed area of the muscular
graft. The epithelium thickness in the graft region varied from two to
six layers of transitional cells. In some cases, partial squamous metaplasia
of the transitional epithelium was observed. In the original bladder wall
a lining by the transitional epithelium of the smooth muscle tunic was
observed, contrasting with the graft region showing lining by the same
epithelium type, but presenting skeletal muscular tissue in the wall.
The majority of cases demonstrated in the transition zone ulceration points
covered by fibrin and neutrophiles, and chronic inflammatory process characterized
by presence of lymphocytes and plasmocytes (Figure-6).
DISCUSSION
The
search for an optimal tissue with the purpose of reconstructing the urinary
bladder motivated publishing about several experimental and clinical essays.
Despite the progress, primarily in the use
of detubularized gastrointestinal segments, one can recognize its limitations,
especially concerning its complications (2,10). The study of new bladder
reconstructive surgical alternatives requires experimental models to compare
variables involved in the method used. It is wise to warrant the viability
of an operative technique before its introduction in clinical practice.
On the other hand, the severity of especial conditions may force the adoption
of immediate and innovative solutions.
Skef et al. (1982) (11) performed surgical
treatment in a 14 months child with bladder extrophy, using a segment
of rectus abdominis muscle. The authors report the child developed hydronephrosis,
requiring posterior ureteral reimplantation, yet they observed a good
evolution of the grafted segment. One should remember that, when this
paper was published, little was known about bladder reconstruction with
detubularized bowel segments in children.
Only after seven years, Büyükünal
et al. (5) introduced in 1989 a pioneer experimental research, assessing
the use of rectus abdominis muscle to bladder augmentation in rats. This
study reported basically the possibility of grafted segment epithelization
from the second day post-operative on. Besides this evaluation, the authors
reported the possibility of a significant bladder capacity gain through
cystographies analysis.
Motivated by the new bladder reconstruction
alternative with peritonized rectus abdominis muscle, we opt to develop
in our milieu an experimental study methodology, using female rats for
a more detailed evaluation of this surgical technique, with an analysis
of the urodynamics study for these animals. This evaluation would allow
supporting this tissue viability to urinary bladder augmentation and its
substitution, as well as the occasional functional consequence to the
upper urinary tract.
In this case, aiming to define an efficient
methodology, we developed an initial study, using ten female adult rats
to standardize the urodynamics study performance in these animals and
to the knowledge of the normal bladder histologic analysis (7).
Albeit urodynamics study is an important
method to assess animals submitted to urinary reconstruction, we observed
the existence of few experimental studies in rats capable to define urodynamics
comparison parameters among different techniques.
Some authors tried to systemize the performance
of the urodynamics study in normal animals.
Initially, Saito et al. (1996) (12), with
the aim of studying regeneration and expansion of urinary tract in Sprague-Dawley
rats, weighting 350 to 400g, performed bladder pressure study with suprapubic
cystostomy, using a double lumen catheter and 0.05mL/min infusion rate.
The mean bladder pressures results in the
control group were 29.8cm H20, and bladder capacity was 0.4mL. The diversity
of values found by these authors compared to our study, that showed lower
values both in bladder capacity and pressure, is warranted by the use
in this work of animals of higher weight than in our study (180 to 240g).
On the other hand, Tillig & Constantinou
(1996) (13) developed an experimental method to investigate ureteral peristaltic
dynamics. They have submitted 27 female rats to cystometric analysis,
showing a bladder capacity similar to the one shown in our study, with
mean maximal bladder pressure slightly superior to our data (mean=30cm
H2O).
The animals were submitted to inhaled anesthesia
and the fluid infusion rate during cystometry was 0.2mL/min.
Regarding the method used to perform the
cystometry we shall remind that in our work the animals were submitted
to intraperitoneal anesthetics with thionembutal and that we used 2 suprapubic
catheters aiming to reduce the interference of pressure transmission during
fluid intravesical infusion. The infusion rate was also 0.2mL/min.
In other way, Miranda, D’Ancona et
al. (1998) (14), also aiming to assess the use of muscle flaps in bladder
reconstruction, performed a study in 24 animals, separating them in only
two groups to analyze urodynamics parameters. This work used another urodynamics
analysis methodology, for the rats were submitted to urethral catheterism
to verify pressure measurements.
One of this methodology’s embarrassments
is that presence of a catheter in the urethra of the rats may present
in fact a mechanism of infravesical obstruction, considering the diameter
of this animals’ urethra. All the same, the maintenance of a urethral
catheter, or even via suprapubic, to posterior urodynamics analysis is
not possible, since it is removed by the rats.
In our work we performed cystostomies with
catheter exteriorization in rats’ dorsal cervical region, making
impossible its removal by the animal. The catheters assumed a subcutaneous
route from bladder until the point of exteriorization.
Despite a distinct methodology, our study
proved a bladder capacity gain (Figure-6) and epithelization of the neobladder
for the mean of the animals evaluated.
Regarding histological analysis, our work
demonstrated the epithelization of transitional cells in all the animals,
varying from two to six layers, with absence of necrosis, indicating the
good vitality of the muscular flap. The presence of an inflammatory process
in most analysis possibly occurred due to local bacterial proliferation.
Moved by their experimental study, Büyükünal
(15) published in 1994 his clinical experience in the surgical treatment
of bladder extrophy in six patients using rectus abdominis muscle flap.
The author pointed out in his work that despite the low number of operated
children, there was technical viability in all cases, absence of mucus
production, that prevents urinary obstruction and also absence of electrolytic
disturbances by the epithelization of the interposed region by transitional
cells.
Also, regarding the benefits of the technique,
the author emphasized that it exempts manipulation of bowel loops, reducing
surgical morbidity and the possibility of future malignization.
Despite his evaluation of bladder capacity
and upper urinary tract consequences has been made solely by imaging exams,
such as cystography and urography, the author did not present a control
urodynamic analysis of the results in his study.
Our experimental work evidenced that rectus
abdominis muscle interposition resulted in a mean gain of 44% in bladder
capacity and a 37% decrease in both means compared to group sham. This
data favors using this method, during maximal urinary loss pressure, knowing
that probably a prejudicial pressure repercussion to upper urinary tract
does not occur, with good preservation of renal function.
Our study allows stimulating the investigation
about using rectus abdominis muscle in bladder reconstruction, but we
should reinforce some limitations of this work regarding the application
of this technique in future clinical series. The issue is the study is
done with small animals, and bladder and rectus abdominis muscle dimensions
are different than in humans. Standardization of urodynamics assessment
in rats is preliminary and was done in a limited number of cases. In our
experiment, data collection was performed in 7th post-operative day. In
this moment it was possible to obtain objective results consistent with
our wishes, but follow-up time is short and its behavior during a longer
follow-up is not known.
On the other hand, the study accomplished
its objectives and serves as a reference to new experiments using rectus
abdominalis muscle in bladder reconstruction. In this way we shall, in
a subsequent study, evaluate the results of this technique in bigger sized
animals.
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____________________
Received: July 29, 2002
Accepted after revision: November 29, 2002
_______________________
Correspondence address:
Dr. Maurício Hachul
Rua Vergueiro, 1855 / 114
São Paulo, SP, 04101-000, Brazil
Fax: + 55 11 5579-0008
E-mail: uromedical@uromedical.com.br