Journal Article Page
European UrologyVolume 62, issue 6, pages e95-e106, December 2012
From Lab to Clinic
Perioperative Betamethasone Treatment Reduces Signs of Bladder Dysfunction in a Rat Model for Neurapraxia in Female Urogenital Surgery
Accepted 10 April 2012, Published online 19 April 2012, pages 1076 - 1085
Information on autonomic neurapraxia in female urogenital surgery is scarce, and a model to study it is not available.
To develop a model to study the impact of autonomic neurapraxia on bladder function in female rats, as well as to assess the effects of corticosteroid therapy on the recovery of bladder function in this model.
Design, setting, and participants
Female Sprague-Dawley rats were subjected to bilateral pelvic nerve crush (PNC) and perioperatively treated with betamethasone or vehicle. Bladder function and morphology of bladder tissue were evaluated and compared with sham-operated rats.
Outcome measurements and statistical analysis
Western blot, immunohistochemistry, organ bath experiments, and cystometry.
Results and limitations
Sham-operated rats exhibited regular micturitions without nonvoiding contractions (NVCs). Crush of all nerve branches of the pelvic plexus or PNC resulted in overflow incontinence and/or NVCs. Betamethasone treatment improved recovery of regular micturitions (87.5% compared with 27% for vehicle; p < 0.05), reduced lowest bladder pressure (8 ± 2 cm H2O compared with 21 ± 5 cm H2O for vehicle; p < 0.05), and reduced the amplitude of NVCs but had no effect on NVC frequency in PNC rats. Compared with vehicle, betamethasone-treated PNC rats had less CD68 (a macrophage marker) in the pelvic plexus and bladder tissue. Isolated bladder from betamethasone-treated PNC rats exhibited better nerve-induced contractions, contained more cholinergic and sensory nerves, and expressed lower amounts of collagen III than bladder tissue from vehicle-treated rats.
PNC causes autonomic neurapraxia and functional and morphologic changes of isolated bladder tissue that can be recorded as bladder dysfunction during awake cystometry in female rats. Perioperative systemic betamethasone treatment reduced macrophage contents of the pelvic plexus and bladder, partially counteracted changes in the bladder tissue, and had protective effects on micturition function.
Carcinoma of the cervix is the third most common cancer worldwide and a main cause of cancer-related death in females . Radical hysterectomy (RH) is the standard treatment of early-stage cervical cancer but is associated with morbidities such as bladder dysfunction, anorectal disorders, and sexual dysfunction that affect a patient's quality of life . Sensory loss, storing and voiding dysfunction, urinary incontinence, and detrusor instability are the most common functional disorders of the lower urinary tract and are reported for ≤85% of patients following RH . Nerve-sparing RH (NSRH), which involves procedures that are suggested not to compromise oncologic radicality but to improve postoperative morbidity, including bladder dysfunction, has been introduced; however, large prospective randomized studies comparing RH with NSRH are missing . Experiences from NSRP suggest that despite refined surgical approaches, damage to the pelvic autonomic nerves occurs because of stretch, compression, thermal effects, ischemia, edema, and local inflammation . This damage leads to a temporary blockade of the signal function of the nerves (neurapraxia) and may also cause degeneration of nerve fibers and structural and functional alterations of penile erectile tissue  and . The rat model of cavernous nerve crush injury is widely used to simulate the neurapraxia associated with NSRP, and various approaches, such as pharmacotherapy, gene therapy, or stem cell therapy, have been investigated in this model to overcome neurapraxia, reduce nerve damage, and improve functional autonomic nerve recovery , , and . Similar models are also used for somatosensory nerves, and crush of the rat pudendal nerve may be used for the study of the external urethral sphincter  and .
To the best of our knowledge, models to simulate pelvic autonomic neurapraxia associated with female urogenital surgery are not established. We aimed to develop a model to study the impact of damage to pelvic autonomic nerves on bladder function in female rats. In addition, we aimed to study the effect of perioperative corticosteroid treatment on the recovery of bladder function in this model.
2. Material and methods
2.1. Ethical approval
The Ethics Committee of San Raffaele University, Milan, Italy, approved all procedures.
2.2. Design of investigation
To assess the extent of nerve damage needed to affect bladder function (study A), rats were subjected to bilateral pelvic nerve crush (PNC) (n = 11) or crush of the bilateral PNC, the hypogastric nerve, and the vesicogenital branches of the pelvic plexus supplying the urinary bladder, outflow region, and vagina (“clock nerve crush” [CNC]; n = 10) (Fig. 1). Comparisons were made with sham-operated rats (n = 10). Cystometry was performed on day 3 or day 10 after nerve injury. Based on findings in study A, rats (n = 22) were subjected to bilateral PNC (study B) and were randomly assigned to receive betamethasone (n = 10) or vehicle (n = 12) subcutaneously 2 d before, until 2 d after, nerve injury (Fig. 2). Eight sham-operated rats functioned as controls. On day 10, cystometry were performed. Bladders were harvested and processed for histologic and functional investigations  and .
Normal female Sprague-Dawley rats (n = 61; 250 g) were used. Intraperitoneal ketamine (75 mg/kg) and xylazine (50 mg/kg) were used for anesthesia for surgical procedures. Rats were sacrificed by carbon dioxide asphyxia.
2.4. Nerve crush damage
A polyethylene catheter was implanted in the bladder dome, and 3 d later, conscious rats underwent cystometry . The following parameters were recorded: (1) lowest intravesical pressure, (2) highest intravesical pressure, and (3) area under the curve (AUC). The incidence of overflow incontinence and nonvoiding contractions (NVCs) was evaluated, as well as the frequency and amplitude of NVCs, when convenient.
2.6. Functional studies of isolated tissue
Detrusor preparations (2 × 1 × 6 mm) were dissected. Experiments were performed in aerated tissue baths (37 °C, pH 7.4), and activation of nerves was performed transmurally by electrical field stimulation with a Grass S48 stimulator (Grass Instruments, USA) .
Immunofluorescence stainings for calcitonin gene-related peptide (CGRP) (1:1000; Euro-Diagnostica, Sweden), protein gene product (PGP) 9.5 (1:2000; Ultraclone, UK), vesicular acetylcholine transporter protein (VAChT) (1:2000; Phoenix Pharmaceuticals, USA), smooth muscle α-actin (1:2000; Sigma-Aldrich, USA), and CD68 (a marker for macrophages) (1:200; Abcam, UK) were evaluated . Sections were processed in a Leica image system (Leica Microsystems, Italy). The percent area of the bladder wall or pelvic plexus (×100 magnification) occupied by stainings was evaluated in Image J, v.1.45 s (Wayne Rasband, USA) . The ratio of VAChT cell bodies to the total number of cell bodies of the pelvic plexus was analyzed.
2.8. Western blot
Amounts of early collagen deposits (collagen type III) (1:5000; Abcam) in bladder tissue were evaluated as previously described .
Incidences are given as percentages, whereas other values are given as mean plus or minus standard error of the mean. For comparisons among groups, a Fischer exact test, an analysis of variance (Student-Newman-Keuls post hoc test), an extra sum square F test, and an unpaired t test were used. All calculations are based on the number of individual animals.
3.1. Study A: cystometry
Whereas sham-operated controls displayed regular micturition cycles without NVCs, animals subjected to nerve crush exhibited, at 3 d, overflow incontinence and/or NVCs, without regular micturition cycles (Table 1). At 10 d after nerve crush, overflow incontinence and NVCs were more common in PNC and CNC rats compared with sham-operated rats (Table 2). At 3 or 10 d (Table 1 and Table 2), rats with PNC or CNC exhibited mean lowest intravesical pressures that were 5.8–7.3 cm H2O (59–100%) and 6.3–7.7 cm H2O (58–107%) higher than sham-operated rats, and mean highest intravesical pressures that were 37–44 cm H2O (45–55%) and 41–44 cm H2O (51–54%) lower than sham-operated rats. The AUC was 10.7–11.6 cm H2O/s (84–112%) and 10.1–11.9 cm H2O/s (79–115%) larger in PNC and CNC rats compared with sham-operated rats.
|Urodynamic parameters 3 d after nerve crush/sham operation||Overflow incontinence, incidence, %||Nonvoiding contractions, incidence, %||Lowest intravesical pressure, cm H2O||Highest intravesical pressure, cm H2O||Area under the curve, cm H2O/s|
|Sham, n = 5||0||0||7.3 ± 0.8||74.3 ± 5.9||10.4 ± 0.9|
|Pelvic nerve crush, n = 5||100§§||100§||14.6 ± 1.8*||35.3 ± 3.3*||22.0 ± 1.9*|
|Clock nerve crush, n = 5||100§§||100§||15.0 ± 2.0*||38.2 ± 5.0*||22.3 ± 2.0*|
Overflow incontinence is leakage of bladder content without any regular micturition cycles. Nonvoiding contractions were defined as continuously occurring increases (>2 cm H2O) in intravesical pressure. Lowest intravesical pressure is the lowest recorded pressure in the bladder, which in sham-operated rats is equal to basal pressure. Highest intravesical pressure is the highest recorded pressure in the bladder, which in sham-operated rats is equal to maximum micturition pressure. The area under the curve was analyzed during a 20-min period at any time in nerve crush rats and in between micturitions in sham-operated rats during a similar period. Incidence values are given as percentages, and pressure parameters are given as mean plus or minus standard error of the mean.
§§p < 0.01 (Fischer exact test compared with sham).
§p < 0.05.
For comparisons between all groups, * p < 0.05 (analysis of variance).
|Urodynamic parameters 10 d after nerve crush/sham operation||Overflow incontinence, incidence, %||Nonvoiding contractions, incidence, %||Lowest intravesical pressure, cm H2O||Highest intravesical pressure, cm H2O||Area under the curve, cm H2O/s|
|Sham, n = 5||0||0||8.8 ± 1.2||79.8 ± 9.8||11.5 ± 0.7|
|Pelvic nerve crush, n = 6||83§||100§§||15.5 ± 1.4*||44.9 ± 7.3*||23.5 ± 4.3*|
|Clock nerve crush, n = 5||75§||100§||16.0 ± 3.2*||38.0 ± 5.6*||22.9 ± 6.0*|
Overflow incontinence is leakage of bladder content without any regular micturition cycles. Nonvoiding contractions were defined as continuously occurring increases (>2 cm H2O) in intravesical pressure. Lowest intravesical pressure is the lowest recorded pressure in the bladder, which in rats with regular micturitions is equal to basal pressure. Highest intravesical pressure is the highest recorded pressure in the bladder, which in sham-operated rats is equal to maximum micturition pressure. The area under the curve was analyzed during a 20-min period at any time in nerve crush rats and in between micturitions in sham-operated rats during a similar period. Incidence values are given as percentages, and pressure parameters are given as mean plus or minus standard error of the mean.
§§p < 0.01 (Fischer exact test compared with sham).
§p < 0.05.
For comparisons between all groups, * p < 0.05 (analysis of variance).
3.2. Study B
Two betamethasone-treated rats and one vehicle-treated rat died during the study. Body weights were 262 ± 7 g (sham; n = 8), 276 ± 4 g (PNC vehicle; n = 11), and 277 ± 2 g (PNC betamethasone; n = 8). Bladder weights amounted to 135 ± 12 mg (sham), 486 ± 10 mg (PNC vehicle; p < 0.01 compared with sham), and 285 ± 4 mg (PNC betamethasone; p < 0.05 compared with sham and PNC vehicle).
Sham-operated rats exhibited regular micturitions without NVCs. Eight of 11 vehicle-treated PNC rats exhibited overflow incontinence. Among PNC rats treated with betamethasone, only one rat exhibited overflow incontinence, whereas the other seven rats exhibited regular micturitions (Table 3; Fig. 3).
|Urodynamic parameters 10 d after nerve crush||Overflow incontinence, incidence, %||Nonvoiding contractions, incidence, %||Nonvoiding contractions, no./min||Nonvoiding contractions, amplitude, cm H2O||Lowest intravesical pressure, cm H2O||Highest intravesical pressure, cm H2O||Area under the curve, cm H2O/s|
|Sham, n = 8||0||0||–||–||9.2 ± 1.1||86.1 ± 4.1||11.9 ± 0.9|
|Vehicle, n = 11||73§||100§||6.1 ± 0.5||11.3 ± 2.0||20.8 ± 5.5*||55.6 ± 8.5*||29.6 ± 6.5*|
|Betamethasone, n = 8||12.5||75§||6.2 ± 1.4||4.3 ± 1.6ϒ||8.1 ± 1.9||45.2 ± 6.7*||15.6 ± 3.4|
Overflow incontinence is leakage of bladder content without any regular micturition cycle. Nonvoiding contractions were defined as continuously occurring increases (>2 cm H2O) in intravesical pressure. Lowest intravesical pressure is the lowest recorded pressure in the bladder, which in rats with regular micturitions is equal to basal pressure. Highest intravesical pressure is the highest recorded pressure in the bladder, which in rats with regular micturitions is equal to maximum micturition pressure. The area under the curve was analyzed during a 20-min period at any time in rats with dribbling incontinence and between micturitions during a similar period in rats with regular micturition. Incidence values are given as percentages, and pressure parameters are given as mean plus or minus standard error of the mean.
§p < 0.05 (Fischer exact test compared with sham).
For comparisons between all groups, * p < 0.05 (analysis of variance).
ϒp < 0.05 (t test compared with vehicle).
All vehicle-treated rats and most of the betamethasone-treated rats displayed NVCs. The frequency of NVCs was similar in PNC rats, but the amplitude of the NVCs of vehicle-treated rats was larger than for betamethasone-treated rats (Table 3). The highest intravesical pressures were lower in PNC rats compared with sham-operated rats, whereas the lowest intravesical pressure and the AUC were higher in vehicle-treated rats compared with sham-operated rats and betamethasone-treated PNC rats (Table 3).
3.2.3. Functional studies
Carbachol produced larger contractions of bladder preparations from PNC rats treated with vehicle or betamethasone than of bladder preparations from sham-operated rats (Fig. 4). Contractions to activation of nerves were lower in bladder preparations from PNC rats treated with vehicle compared with bladder preparations from sham-operated rats or PNC rats treated with betamethasone (Fig. 4).
3.2.4. Morphology and Western blot
All PNC rats exhibited more CD68 immunoreactivity (IR) in the pelvic plexus compared with sham-operated rats. No difference in CD68 content of bladder tissue was observed between betamethasone-treated PNC rats and sham-operated rats. Vehicle-treated PNC rats had larger amounts of CD68-IR in the pelvic plexus and bladders than betamethasone-treated rats (Fig. 5). Vehicle-treated PNC rats had fewer VAChT-IR cell bodies in the pelvic plexus than betamethasone-treated or sham-operated rats (Fig. 5). CD68-IR was located to 4′,6-diamidino-2-phenylindole–positive cells of the pelvic plexus (Fig. 6) and the bladder (data not shown). By Western blot, larger amounts of collagen were detected in bladder specimens from vehicle-treated rats than in bladders from betamethasone-treated rats (Fig. 7).
Whereas vehicle-treated PNC rats had lower amounts of PGP-IR in the bladder, no difference in PGP-IR content was observed for sham-operated and betamethasone-treated PNC rats (Fig. 8). Compared with sham-operated rats, all PNC rats expressed lower amounts of VAChT-IR and CGRP-IR nerves in the bladders, but betamethasone-treated rats had larger amounts of these nerves than vehicle-treated rats (Fig. 8). The distribution of nerves in bladders of rats treated with betamethasone appeared more homogeneous compared with vehicle-treated PNC rats, which exhibited areas that were devoid of nerves (Fig. 8).
No difference in the areas of the bladder sections that were occupied by smooth muscle α-actin–IR was detected between sham-operated rats (35.8 ± 2.5%) or PNC rats treated with vehicle (27.8 ± 3.0%) or betamethasone (33.2 ± 3.2%).
This study characterizes a model for autonomic neurapraxia in female rats with related bladder dysfunction during awake cystometry. It also demonstrates that perioperative systemic treatment with betamethasone ameliorates bladder dysfunction in rats with PNC. In addition, we present evidence that betamethasone depresses the immunologic responses in the pelvic plexus and the bladder to the nerve damage, and that this action protects the integrity and function of the bladder tissue and improves recovery of micturition.
After conventional RH, an initial hyperreflexic bladder dysfunction occurs, after which a hypotonic bladder may develop . Various nerve-sparing procedures are reported to reduce early postoperative bladder dysfunction after RH as assessed by improved recovery of spontaneous voiding or bladder sensation, less postoperative need for catheterization, and less time to achieve low postvoid residual volumes , , , and .
In rats, ablation of the pelvic nerve has been shown to cause persistent bladder dysfunction and overflow incontinence in studies ≤6 wk after nerve transections  and . The current investigation describes early overflow incontinence of rats subjected to crush to all nerve branches of the pelvic plexus or PNC alone, and it found that recovery at 10 d of regular micturition was similar (17–25%) for either procedure. We also noted reduced maximal intravesical pressures for all nerve crush rats that may be comparable to findings in humans after NSRH . Both crush procedures seem to cause changes in bladder function that may be used to study the impact of partial nerve injury on bladder function in a female rat model, but PNC is technically more convenient.
The nerve crush injury damages the myelin sheath and the axon, but it leaves the basal lamina and Schwann cell tubes intact . This situation causes proliferation of Schwann cells, rapid upregulation of cytokines and chemokines, and recruitment of circulating macrophages . The initial inflammation and actions by invading immune cells that are necessary for nerve regeneration also have negative effects associated with denervation, reorganization of the extracellular matrix, delayed functional recovery, or development of nerve dysfunction  and . Recent studies also identify macrophages as important activators of fibroblasts . As seen in PNC rats, the contents of cholinergic motor nerves and sensory nerves of the bladder were reduced and collagen contents increased. Concomitantly, nerve-mediated contractile responses were lower in isolated bladder tissue from these animals that also exhibited bladder dysfunction in vivo. Our findings are comparable to morphologic changes in, and dysfunction of, isolated erectile tissue from rats that exhibit erectile dysfunction due to crush of the cavernous nerve  and .
Corticosteroids have beneficial effects on functional recovery after spinal cord injuries and are known to inhibit the vasodilation, vascular permeability, and edema that occur in inflammation  and . Actions by corticosteroids include transcriptional repression of immune progenitor cells, reversal of cytokine responses toward anti-inflammation, reduction of macrophage migration, and inhibition of lymphocytes  and . In models for sciatic or cochlear nerve crush injury, systemic perioperative treatment with corticosteroids is reported to decrease nerve damage or degeneration, with concomitant reductions of the early recruitment of macrophages , , and . Similarly, we demonstrate less CD68 (a macrophage marker) in bladder tissue and the pelvic plexus and more nerves and less collagen in bladder tissue from PNC rats treated with betamethasone compared with vehicle. Nerve function of isolated bladders was better in PNC rats treated with betamethasone than with vehicle. However, isolated bladders from all PNC rats exhibited muscarinic agonist supersensitivity. Even if betamethasone-treated rats had more VAChT nerves than vehicle-treated rats, compensatory upregulation of postjunctional muscarinic receptors may be expected as a result of partial loss of bladder motor neurons in both groups compared with sham-operated rats. The idea that this contributed to preserved nerve-induced contractions of bladder preparations from betamethasone PNC rats cannot be excluded.
In agreement with the effects of betamethasone on the morphology and function of isolated bladders from PNC rats, the main finding of this study was that 87.5% of the betamethasone-treated rats, and only 27% of the vehicle-treated rats, exhibited regular micturitions. We also recorded similar lowest intravesical pressure (basal pressure) and AUC in betamethasone-treated PNC rats and sham-operated rats. Probably because of a combination of partial loss of motor nerves and change in viscous properties of the bladder due to increase in collagen, the capacity of PNC rats to generate higher pressures was reduced. This characteristic was not reversed by betamethasone at 10 d.
In contrast to our findings, studies of the effect of corticosteroids on recovery of sexual function in men after NSRP have previously not shown promising results . In these studies, systemic corticosteroid treatment was initiated on postoperative day 1 or corticosteroids were applied locally on the neurovascular bundle during surgery . Considering that the inflammatory response is already active within hours after nerve injury and that recruitment of circulating immune cells occurs in the degeneration process, the timing and route of administration of corticosteroids is of importance in the anti-inflammatory effect  and . In the current study, we initiated betamethasone treatment 2 d before nerve crush to ensure optimal tissue concentrations and to try to improve effects on gene repression of immune progenitor cells at the time of injury.
PNC causes autonomic neurapraxia that can be recorded as bladder dysfunction during awake cystometry in female rats. Perioperative systemic betamethasone treatment reduced macrophage contents of the pelvic plexus and bladder, partially counteracted functional and morphologic changes of isolated bladder tissue, and improved recovery of micturition function in these rats.
Author contributions: Petter Hedlund had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Castiglione, Hedlund.
Acquisition of data: Castiglione, Bergamini, Strittmatter, Gandaglia, Bettiga, Hedlund.
Analysis and interpretation of data: Castiglione, Bergamini, Bettiga, Benigni, Hedlund.
Drafting of the manuscript: Castiglione, Bergamini, Bivalacqua, Hedlund.
Critical revision of the manuscript for important intellectual content: Benigni, Bivalacqua, Hedlund.
Statistical analysis: Castiglione, Bettiga, Hedlund.
Obtaining funding: Montorsi, Rigatti, Hedlund.
Administrative, technical, or material support: Benigni, Bivalacqua.
Supervision: Benigni, Bivalacqua, Hedlund
Other (specify): None.
Financial disclosures: Petter Hedlund certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: None.
Funding/Support and role of the sponsor: The production of the manuscript was supported by the Swedish Medical Research Council, the Gester Foundation, and the Urological Research Institute. The funding organizations have only supported the project financially and have not been involved in the design, production, or handling of the intellectual content of the manuscript.
Acknowledgment statement: We are grateful to Mrs. Giorgia Colciago for technical assistance during the production of this manuscript.
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a Urological Research Institute, San Raffaele University, Milan, Italy
b Department of Clinical and Experimental Pharmacology, Lund, Sweden
c Department of Urology, Johns Hopkins University, Baltimore, MD, USA
d Department of Urology, Munich University, Munich, Germany
e Department of Clinical Pharmacology, Linköping, Sweden
© 2012 Published by Elsevier B.V.
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