- Email: [email protected]
Onlay foreskin flap anastomosed directly to the tunica albuginea: A short-term experimental study in rabbits
Accepted Manuscript Onlay foreskin flap anastomosed directly to the tunica albuginea: a short-term experimental study in rabbits H. Bacelar, A.V. Rondon, M.D., PhD, R. Mattos, J.G. Quitzan, B. Leslie, R. Delcelo, S.R. de Araújo, V. Ortiz, A. Macedo, Jr. PII:
S1477-5131(15)00203-X
DOI:
10.1016/j.jpurol.2015.04.027
Reference:
JPUROL 1951
To appear in:
Journal of Pediatric Urology
Received Date: 23 December 2014 Accepted Date: 27 April 2015
Please cite this article as: Bacelar H, Rondon AV, Mattos R, Quitzan JG, Leslie B, Delcelo R, de Araújo SR, Ortiz V, Macedo Jr A, Onlay foreskin flap anastomosed directly to the tunica albuginea: a short-term experimental study in rabbits, Journal of Pediatric Urology (2015), doi: 10.1016/j.jpurol.2015.04.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Onlay foreskin flap anastomosed directly to the tunica albuginea: a short-term experimental study in rabbits
de Araújoc, V. Ortiza, A. Macedo Jra
a
Department of Urology, Universidade Federal de São Paulo, São Paulo, Brazil
Small Animals Surgery Department, Pontifícia Universidade Católica do Paraná,
SC
b
RI PT
H. Bacelara, A.V. Rondona,*, R. Mattosa, J.G. Quitzanb, B. Lesliea, R. Delceloc, S.R.
São José dos Pinhais, Brazil
Department of Pathology, Universidade Federal de São Paulo, São Paulo, Brazil
Emails:
AC C
EP
TE D
[email protected]
M AN U
c
*Corresponding author: Atila Rondon, M.D., PhD Rua Maestro Cardim, 560/ 215 01323-000 São Paulo Brazil Email: [email protected] Tel +55 11 32870639 Fax +55 11 32873954
ACCEPTED MANUSCRIPT
Summary Introduction: In severe hypospadias, urethral plate division is necessary for curvature
RI PT
correction. To configure the new urethra, an approach has been described using a foreskin flap directly anastomosed in an ‘onlay’ fashion to the tunica albuginea of the corpora cavernosa. Results suggest that it is possible to use the corpus cavernosum
SC
albuginea as the posterior wall of the neourethra without the need of a dorsal graft. Objective: The present experimental study aimed to evaluate the histological
M AN U
characteristics and healing pattern of this procedure.
Study design: Sixteen New Zealand male rabbits were divided into two groups of eight animals. Eight animals underwent 1-cm longitudinal dorsal incision of the penile urethra and the edges were anastomosed to the tunica albuginea (Group 1). Eight other
TE D
animals underwent complete excision of 1.0 cm of penile urethra. Urethroplasty was performed using a foreskin flap directly anastomosed as an onlay to the albuginea, as shown in the figure (Group 2). Sacrifice and histological assessment was performed 2,
EP
4, 8 and 12 weeks postoperatively.
Results: In Group 1, a mild inflammatory process was noted that became almost
AC C
imperceptible at 12 weeks. Fibrosis was mild at all stages in this group. Over time, a regenerative epithelium covered the corpus cavernosum. Immunohistochemistry using specific CK-7 and CK-20 confirmed the presence of urothelium. No complications were microscopically detected in this group. Group 2 presented with a more intense inflammatory infiltrate, which also resolved over time. Fibrosis was slightly more intense in this group, especially in animals that had urethral strictures. Group 2 presented with three fistulas, two were associated with urethral stricture. Histological
1
ACCEPTED MANUSCRIPT evaluation showed the presence of epithelization over the albuginea, which turned out to be similar to the normal urothelium over time and was confirmed by immunohistochemistry. Non-keratinized stratified squamous epithelium of the foreskin
RI PT
flap showed good integration to the urethra. Discussion: Microscopic analysis showed that inflammation, fibrosis and complications were similar to previous studies. At 12 weeks there was a well-developed epithelium
SC
similar to normal urethra, which was confirmed by immunohistochemistry; this was similar to what occurs in the TIP technique, as previously demonstrated. It was
M AN U
hypothesized that the epithelium regeneration developed from the urethral edges, as demonstrated in other experimental studies.
Conclusion: The albuginea was covered by mature urothelium after 12 weeks, which presumably grew from the urethral edges. The foreskin flap onlay that was directly
neourethra.
TE D
anastomosed to the albuginea completely integrated and constituted the roof of the
AC C
EP
Keywords: Hypospadias; Surgical flaps; Urethra; Rabbits; Histology
2
ACCEPTED MANUSCRIPT
Introduction In recent decades, the treatment of proximal hypospadias has been an ongoing debate. Today, the consensus seems to be preservation of the urethral plate. However, in some
RI PT
patients with severe penile curvature, urethral plate division is necessary for curvature correction [1]. In these cases, a single procedure or staged strategy both remain
controversial [2-4]. Most surgeons choose the technique based on personal experience
SC
and not on technical feasibility or literature results [2, 4].
M AN U
The correction with a single-stage procedure has proven to be safe and has longterm success rates up to 70% [5, 6]. Two-stage techniques present complication rates of approximately 22%, but require at least three surgical procedures if a complication occurs [7].
Rigamonti and Castagnetti published an approach to configure the new urethra
TE D
using a foreskin flap directly anastomosed in an onlay fashion to the tunica albuginea of the corpora cavernosa, and found a complication rate around 21% in a mean follow-up
EP
of 7 months [8]. This is conceptually very similar to the Monseur urethroplasty principle (1968) and its several variants, which described focusing on the urothelial
AC C
lining of the tunica albuginea of the corpora cavernosa to complete the circumferential defect of the urethra [9]. These results suggest that it is possible to use the corpus cavernosum albuginea
as the posterior wall of the neourethra without the need of a dorsal graft. The present experimental study aimed to evaluate the histological characteristics and healing pattern of this procedure.
3
ACCEPTED MANUSCRIPT
Materials and Methods The Committee of Ethics in Research of the institution approved this study. A total of 16 New Zealand male rabbits were divided into two study groups of eight animals. The
RI PT
smallest estimated number of animals required to observe and analyze the results was used [10-15]. Animals were kept in a vivarium over a period of 3-7 days (induction period) prior to surgery.
SC
Anesthesia was induced by a licensed veterinarian (JQ) and was initiated by the
M AN U
intramuscular (IM) administration of a solution of acepromazine 1 mg/kg and fentanyl hydrochloride 3 ㎍/kg, and then a solution of ketamine 30 mg/kg with xylazine 5 mg/kg. Epidural block with lidocaine 2% and bupivacaine 0.5% with epinephrine was also performed. The surgical procedure was performed using magnification loupes of 3.5X.
TE D
Initially, the urethra was catheterized with an 8-F catheter and then the fold between the penis and the anus of the animal was sectioned to enable access to the
EP
urethra. The Buck´s fascia was laterally incised to the urethra and the corpus spongiosum and urethra were then released from the corpora cavernosa.
AC C
In animals from Group 1, a 1.0-cm longitudinal incision was made in the posterior surface of the urethra and the incised edges were laterally anastomosed to the corpus cavernosum, 0.4 cm apart from each other, with a continuous suture of 7-0 PDS®. The tunica albuginea of the corpus cavernosum then became the posterior wall of the neourethra (Fig. 2). In Group 2, after dissection of the urethra, a cross-sectional incision close to the edge of the inner foreskin of the penis was performed. To create the urethral defect the
4
ACCEPTED MANUSCRIPT urethral stumps were fixed to the tunica albuginea with 6-0 PDS® sutures, 1.0 cm apart, therefore simulating hypospadias with a need for urethral plate division and exposure of the tunica albuginea. A longitudinal foreskin flap of 1.0 cm x 0.4 cm was created on the
RI PT
ventral surface of the penis, and then rotated from distal to proximal, covering the urethral defect. The foreskin flap was sutured to the tunica albuginea with 7-0 PDS® sutures. In both groups, the urethral catheter was removed after the procedure.
SC
Each group of animals was then randomly divided into four subgroups of two rabbits to be sacrificed after 2, 4, 8, and 12 weeks of surgery. Immediately after
M AN U
sacrifice, a VCUG was performed and then the animal’s penis was removed and immediately fixed in 10% formaldehyde.
The pathology specimens were longitudinally fixed from the glans to base of the penis. Histological staining was performed with hematoxylin-eosin (HE) and Masson’s
TE D
trichrome (MT). An experienced pathologist in single analysis (DR) performed the histological evaluation. To confirm the presence of urothelium using specific CK-7 and CK-20 an experienced pathologist (SA) performed the immunohistochemistry. The
EP
pathologists used the same standardization of previous studies to assess the
AC C
inflammatory response related to the procedure [12-15].
Results
There were no deaths related to the procedure. The animals were sacrificed according to the previously established schedule. The animals in Group 1 showed no signs of urethral strictures on cystourethrography. In Group 2, there was difficulty in passing the catheter in three rabbits and urethral stricture was confirmed by urethrography. In two
5
ACCEPTED MANUSCRIPT of these animals, the presence of an associated fistula was identified. One rabbit in Group 2 presented with fistula alone. Histological analysis was performed with 10X, 40X, 100X and 200X
RI PT
magnifications on optical microscope. In Group 1, a mild inflammatory process with a predominance of lymphomononuclear cells was noted after 2 weeks, which became almost imperceptible at 12 weeks. Fibrosis was mild at all stages in this group. The
SC
epithelium covering the corpus cavernosum was initially an atrophic regenerative
epithelium, with only one layer of cells, but developed over time and at 12 weeks was
M AN U
composed of three or more layers. Immunohistochemistry using specific CK-7 and CK20 confirmed the presence of urothelium. No complications were microscopically detected in this group.
Group 2 presented with a more intense inflammatory infiltrate after 2 weeks,
TE D
with a predominance of lymphomononuclear cells, which also resolved over time and were very minimal in the 12-week group. Fibrosis was slightly more intense in this group, especially in animals that had urethral strictures; it remained mild to moderate up
EP
to 12 weeks.
The foreskin flap maintained its characteristic stratified squamous epithelium
AC C
and over time presented with acanthosis in some areas. The epithelium that covered the corpus cavernosum was initially atrophic and had an incomplete regenerative pattern, rendering some areas without any epithelium. In the 4-week group, the epithelium had fully covered the tunica albuginea. It developed over time and within 12 weeks presented as a robust epithelium with three or more layers of cells resembling the native epithelium of the urethra (Fig. 3). Immunohistochemistry using specific CK-7 and CK20 confirmed the presence of urothelium (Fig. 4). Complications were recorded in this
6
ACCEPTED MANUSCRIPT group. Microscopic analysis revealed two inclusion cysts, one foreign body granuloma and three previously macroscopically identified fistulas.
RI PT
Discussion
Some patients with proximal hypospadias and severe penile curvature require
SC
division of the urethral plate to correct the curvature [16]. In order to treat these patients in a single step, various tissues have been studied to replace the urethral plate when it
M AN U
needs to be sectioned. Among these are the peritoneum, bladder mucosa, tunica vaginalis, and buccal mucosa, with outcomes published in clinical and experimental studies over the years [12-15, 17-22]. Likewise, urethral healing has been experimentally studied for decades and more intensively over the past 10 years [12-15,
TE D
23-26].
The technique proposed by Rigamonti and Castagnetti, following the Monseur urethroplasty principle, assumes that it is unnecessary to apply any flap or graft at the
role [8, 9].
EP
dorsal component of the neourethra because the tunica albuginea itself could have that
AC C
In a clinical study, Macedo and Srougi proposed the 'three-in-one' technique and
were careful to reconstruct the urethral plate with a buccal mucosa graft, following the concept advocated by Barbagli [5, 27]. The possibility of simplifying this strategy prompted the present study to evaluate the histological aspect of Rigamonti’s procedure. Macroscopically, the rabbits of Group 1 had an uneventful outcome and presented with no fistula, stricture, or suture dehiscence. Microscopic analysis showed that inflammation, fibrosis, and complications were similar to previous studies. In an
7
ACCEPTED MANUSCRIPT experimental study with dogs, Bleustein et al. performed the TIP technique and postoperative histological analysis at day 21, which showed a sparse lymphocytic infiltrate [25]. Calado et al. and Souza et al. studied the integration of the tunica
RI PT
vaginalis and the buccal mucosa in an experimental model of urethroplasty, they also found inflammatory changes and fibrosis with temporal behavior similar to that found in the present study [12, 14].
SC
Epithelization of the tunica albuginea evolved from an atrophic, regenerative
epithelium with a single-cell layer to a more robust urothelium of two to three layers of
M AN U
cells at 12 weeks, which was confirmed by immunohistochemistry. Epithelialization probably occurs from the edges to the center, forming a ‘carpet’ of cells on the tunica albuginea that proliferate to form the new epithelium; this is similar to what occurs in the TIP technique, as previously experimentally demonstrated [24-26]. It seems that
TE D
Group 1 behaved like an extended TIP, in that healing was similar, even without the urethral plate underlying tissue, which is rich in blood vessels and connective tissue [28, 29].
EP
Group 2 presented with a macroscopic complication rate that reached 50%. Fistulae and strictures occurred in three and two animals, respectively (two animals with
AC C
strictures also had fistulas, thus, four animals had complications). Other experimental studies that used foreskin flaps found an incidence of fistulae of 12.5-25%, with no strictures reported in rabbits [15]. A rabbit´s urethra is very elastic and tends to avoid stricture formation. In the present study, there was no tissue associated with the foreskin besides the tunica albuginea; this theoretically makes the neourethra less complacent and therefore more prone to stricture formation.
8
ACCEPTED MANUSCRIPT Rigamonti and Castagnetti reported a complication rate of 21% (3/14 patients) with a fistula, a partial dehiscence and a dilation of the neourethra; there were no strictures. The initial results are encouraging because when treating these complex
RI PT
hypospadias, complication rates vary from 15-45% [7]. However, these results must be cautiously examined because the mean follow-up is very short (7 months), even though the study included patients up to 27 months of follow-up. Many complications in
SC
hypospadias surgery occur later on. Powell et al. evaluated 142 patients undergoing surgery for posterior hypospadias and reported 64% of fistulas appearing after 12
M AN U
months postoperatively (mean 23.9 months), 80% of meatal stenosis and 60% of proximal strictures appearing at 30.9 months of mean follow-up [30]. Therefore, in order to define the true rate of complications associated to this procedure it is imperative to await results with later follow-up.
TE D
Microscopic analysis showed that the behavior of the inflammatory process was similar to what happened in Group 1 and in previous experimental studies using an onlay foreskin flap [15]. Fibrosis was mild to moderate in most rabbits, which supports
EP
the findings of previous experimental studies, except in two rabbits that had strictures and in which more intense fibrosis was found [12, 14, 15, 22].
AC C
In the animals at 2 weeks of follow-up, a single-cell layer had already covered part of the corpora cavernosa. Some uncovered areas were surrounded by this same atrophic regenerative epithelium. In the other groups of later sacrifice, the whole surface of the corpus cavernosum was already covered by regenerative epithelium. At 12 weeks there was a well-developed epithelium similar to a normal urethra, which was confirmed by immunohistochemistry. It was hypothesized that the epithelium
9
ACCEPTED MANUSCRIPT regeneration developed from the urethral edges, as demonstrated in other experimental studies [12, 13, 15, 24-26]. In both groups, the gap between the urethral stumps was 1cm long and the lateral
RI PT
edges were 0.4 cm apart from each other. Therefore, the area of bare corpus cavernosum that was covered by an epithelium, which was incorporated into the new urethra to form the posterior wall, was 1.0 x 0.4 cm.
SC
It is worth reporting that in the clinical study of Rigamonti and Castagnetti, the authors performed a biopsy in one of their patients and found tissue similar to the
M AN U
foreskin flap in the dorsal wall of the neourethra, concluding that the healing in this area is made from the edges of the foreskin flap [8]. This finding contradicts the present observations in the experimental setting. Many hypotheses could explain this difference. One would be that the healing in humans occurs differently to rabbits, which seems to
TE D
be inconsistent. The second hypothesis could be that the biopsied site of the human urethra did not correspond to the location of the regenerated epithelium. In rabbits, the urothelium was practically normal after 12 weeks. In patients in the late postoperative
EP
period, it may be difficult to macroscopically differentiate a neo-formed mucosa from the internal foreskin, which also undergoes metaplasia over time.
AC C
The foreskin flap showed full integration to the neourethra, except in one rabbit with a fistula. Some areas of squamous metaplasia and acanthosis were observed. This finding is similar to that found by Leslie et al. who used a foreskin flap over the tunica vaginalis to form the neourethra. They found excellent integration of the foreskin within 2 weeks of monitoring and a transformation to a less-stratified epithelium over time up to 12 weeks [15].
10
ACCEPTED MANUSCRIPT Possible limitations of this study would include a short follow-up; most experimental studies using rabbits are designed to perform observations until week 16 [11-15, 22-24]. The number of animals used in an experimental study is always a
RI PT
concern of the Ethics Committee, so the minimum number was used to answer the questions that were proposed.
In experimental studies, any clinical conclusions should be examined very
SC
carefully. In the present study, there was no intention to compare macroscopic
complications that occur in experimental models with those found in clinical trials
M AN U
because the results couldn’t be generalized for human beings. From the beginning, according to the study design, no data were expected that could allow any conclusions to be made regarding surgical success or to recommend the use of this technique. The question was simple, and it was believed that it could be shown in rabbits that the
TE D
corpus cavernosum was covered by an epithelium similar to the native urethra, presumably growing from the urethral stump edges. In this animal model, urothelial regeneration happened, but with a high stricture rate.
EP
Therefore, the healing process of urethroplasty with a foreskin flap applied directly over the tunica albuginea of the corpus cavernosum was described. The
AC C
technical applicability for patients with hypospadias still requires further clinical studies with longer follow-up to assess its advantages in relation to already established procedures.
Conclusions In this experimental model, a foreskin flap directly anastomosed as an onlay to the tunica albuginea integrated properly and formed a neourethra.
11
ACCEPTED MANUSCRIPT The corpus cavernosum was initially covered by a regenerative epithelium, which over time turned into a mature epithelium similar to the native urethra, presumably growing from the urethral stump edges.
RI PT
In this short-term experimental study of rabbits, the complication rates, evidence of fibrosis and the inflammatory response were greater in the study group (Group 2).
AC C
EP
TE D
M AN U
SC
Conflict of Interest/Funding: None
12
ACCEPTED MANUSCRIPT
References [1] Baskin LS, Ebbers MB. Hypospadias: anatomy, etiology, and technique. J Pediatr Surg 2006;41:463-72.
RI PT
[2] Cook A, Khoury AE, Neville C, Bagli DJ, Farhat WA, Pippi Salle JL. A multicenter evaluation of technical preferences for primary hypospadias repair. J Urol 2005;174:2354-7, discussion 7. [3] Snodgrass W, Macedo A, Hoebeke P, Mouriquand PD. Hypospadias dilemmas: a round table. J Pediatr Urol 2011;7:145-57.
SC
[4] Springer A, Krois W, Horcher E. Trends in hypospadias surgery: results of a worldwide survey. Eur Urol 2011;60:1184-9.
M AN U
[5] Macedo A Jr., Srougi M. Onlay urethroplasty after sectioning of the urethral plate: early clinical experience with a new approach - the 'three-in-one' technique. BJU Int 2004;93:1107-9. [6] Macedo A Jr., Liguori R, Ottoni SL, Garrone G, Damazio E, Mattos RM, et al. Long-term results with a one-stage complex primary hypospadias repair strategy (the three-in-one technique). J Pediatr Urol 2011;7:299-304. [7] Castagnetti M, El-Ghoneimi A. Surgical management of primary severe hypospadias in children: systematic 20-year review. J Urol 2010;184:1469-74.
TE D
[8] Rigamonti W, Castagnetti M. Onlay on albuginea: modified onlay preputial island flap urethroplasty for single-stage repair of primary severe hypospadias requiring urethral plate division. Urology 2011;77:1498-502. [9] Monseur J. [Restoration of the urethral duct by means of supraurethral laminaie and the subcavernal grooves]. J Urol Nephrol (Paris) 1968;74:755-68.
EP
[10] Eckelman WC, Kilbourn MR, Joyal JL, Labiris R, Valliant JF. Justifying the number of animals for each experiment. Nucl Med Biol 2007;34:229-32.
AC C
[11] Hafez AT, Smith CR, McLorie GA, El-Ghoneimi A, Herz DB, Bagli DJ, et al. Tunica vaginalis for correcting penile chordee in a rabbit model: is there a difference in flap versus graft? J Urol 2001;166:1429-32. [12] Calado AA, Macedo A Jr., Delcelo R, de Figueiredo LF, Ortiz V, Srougi M. The tunica vaginalis dorsal graft urethroplasty: experimental study in rabbits. J Urol 2005;174:765-70. [13] Rosito TE, Pires JA, Delcelo R, Ortiz V, Macedo A Jr. Macroscopic and histological evaluation of tunica vaginalis dorsal grafting in the first stage of Bracka's urethroplasty: an experimental study in rabbits. BJU Int 2011;108:E17-22. [14] Souza GF, Calado AA, Delcelo R, Ortiz V, Macedo A Jr. Histopathological evaluation of urethroplasty with dorsal buccal mucosa: an experimental study in rabbits. Int Braz J Urol 2008;34:345-51; discussion 51-4.
13
ACCEPTED MANUSCRIPT [15] Leslie B, Barboza LL, Souza PO, Silva PS, Delcelo R, Ortiz V, et al. Dorsal tunica vaginalis graft plus onlay preputial island flap urethroplasty: experimental study in rabbits. J Pediatr Urol 2009;5:93-9. [16] Hollowell JG, Keating MA, Snyder HM 3rd, Duckett JW. Preservation of the urethral plate in hypospadias repair: extended applications and further experience with the onlay island flap urethroplasty. J Urol 1990;143:98-100; discussion -1.
RI PT
[17] Nanni L, Vallasciani S, Fadda G, Perrelli L. Free peritoneal grafts for patch urethroplasty in male rabbits. J Urol 2001;165:578-80.
[18] Mollard P, Mouriquand P, Bringeon G, Bugmann P. Repair of hypospadias using a bladder mucosal graft in 76 cases. J Urol 1989;142:1548-50.
SC
[19] Talja M, Kivisaari L, Makinen J, Lehtonen T. Free tunica vaginalis patch in urethroplasty. An experimental study. Eur Urol 1987;13:259-63.
M AN U
[20] Burger RA, Muller SC, el-Damanhoury H, Tschakaloff A, Riedmiller H, Hohenfellner R. The buccal mucosal graft for urethral reconstruction: a preliminary report. J Urol 1992;147:662-4. [21] Hill GA, Lewis AG, Sheldon CA. A rabbit model of free bladder mucosal grafting in a damaged urethral bed. J Urol 1994;152:983-6. [22] Oliva P, Delcelo R, Bacelar H, Rondon A, Barroso U Jr., Ortiz V, et al. The buccal mucosa fenestrated graft for Bracka first stage urethroplasty: experimental study in rabbits. Int Braz J Urol 2012;38:825-32. [23] Scherz HC, Kaplan GW, Boychuk DI, Landa HM, Haghighi P. Urethral healing in rabbits. J Urol 1992;148:708-10; discussion 11-3.
TE D
[24] Hafez AT, Herz D, Bagli D, Smith CR, McLorie G, Khoury AE. Healing of unstented tubularized incised plate urethroplasty: an experimental study in a rabbit model. BJU Int 2003;91:84-8.
EP
[25] Bleustein CB, Esposito MP, Soslow RA, Felsen D, Poppas DP. Mechanism of healing following the Snodgrass repair. J Urol 2001;165:277-9. [26] Lopes JF, Schned A, Ellsworth PI, Cendron M. Histological analysis of urethral healing after tubularized incised plate urethroplasty. J Urol 2001;166:1014-7.
AC C
[27] Barbagli G, Selli C, Tosto A, Palminteri E. Dorsal free graft urethroplasty. J Urol 1996;155:123-6. [28] Baskin LS, Erol A, Li YW, Cunha GR. Anatomical studies of hypospadias. J Urol 1998;160:1108-15; discussion 37. [29] Snodgrass W, Patterson K, Plaire JC, Grady R, Mitchell ME. Histology of the urethral plate: implications for hypospadias repair. J Urol 2000;164:988-9; discussion 990. [30] Powell CR, McAleer I, Alagiri M, Kaplan GW. Comparison of flaps versus grafts in proximal hypospadias surgery. J Urol 2000;163:1286-8; discussion 8-9.
14
ACCEPTED MANUSCRIPT Figure legends Summary figure legend Fig. 1. Procedure in Group 2. (A) Construction of 1.0-cm long urethral defect; (B) delimitation of foreskin flap on the ventral penile surface; (C) flap rotation from distal
RI PT
to proximal to cover the urethral defect; (D) flap sutured to the corpus cavernosum.
Manuscript figures legends
SC
Fig. 2. Procedure in Group 1. (A) 1.0-cm longitudinal incision in the urethral posterior surface; (B) suture of the urethral side edges to the corpus cavernosum; (C) final
M AN U
aspect of the urethra anastomosed to the corpus cavernosum.
Fig. 3. Group 2 epithelium evolution (HE 100X).
TE D
W = week; RE = regenerative epithelium; CC = corpus cavernosum.
Fig. 4. Group 2 immunohistochemistry (40X) with CK-7 (A and B) and CK-20 (C and D). A and C: prepuce flap. B and D: epithelization similar to the normal
AC C
EP
urothelium over the albuginea.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S1477-5131(15)00203-X
DOI:
10.1016/j.jpurol.2015.04.027
Reference:
JPUROL 1951
To appear in:
Journal of Pediatric Urology
Received Date: 23 December 2014 Accepted Date: 27 April 2015
Please cite this article as: Bacelar H, Rondon AV, Mattos R, Quitzan JG, Leslie B, Delcelo R, de Araújo SR, Ortiz V, Macedo Jr A, Onlay foreskin flap anastomosed directly to the tunica albuginea: a short-term experimental study in rabbits, Journal of Pediatric Urology (2015), doi: 10.1016/j.jpurol.2015.04.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Onlay foreskin flap anastomosed directly to the tunica albuginea: a short-term experimental study in rabbits
de Araújoc, V. Ortiza, A. Macedo Jra
a
Department of Urology, Universidade Federal de São Paulo, São Paulo, Brazil
Small Animals Surgery Department, Pontifícia Universidade Católica do Paraná,
SC
b
RI PT
H. Bacelara, A.V. Rondona,*, R. Mattosa, J.G. Quitzanb, B. Lesliea, R. Delceloc, S.R.
São José dos Pinhais, Brazil
Department of Pathology, Universidade Federal de São Paulo, São Paulo, Brazil
Emails:
AC C
EP
TE D
[email protected]
M AN U
c
*Corresponding author: Atila Rondon, M.D., PhD Rua Maestro Cardim, 560/ 215 01323-000 São Paulo Brazil Email: [email protected] Tel +55 11 32870639 Fax +55 11 32873954
ACCEPTED MANUSCRIPT
Summary Introduction: In severe hypospadias, urethral plate division is necessary for curvature
RI PT
correction. To configure the new urethra, an approach has been described using a foreskin flap directly anastomosed in an ‘onlay’ fashion to the tunica albuginea of the corpora cavernosa. Results suggest that it is possible to use the corpus cavernosum
SC
albuginea as the posterior wall of the neourethra without the need of a dorsal graft. Objective: The present experimental study aimed to evaluate the histological
M AN U
characteristics and healing pattern of this procedure.
Study design: Sixteen New Zealand male rabbits were divided into two groups of eight animals. Eight animals underwent 1-cm longitudinal dorsal incision of the penile urethra and the edges were anastomosed to the tunica albuginea (Group 1). Eight other
TE D
animals underwent complete excision of 1.0 cm of penile urethra. Urethroplasty was performed using a foreskin flap directly anastomosed as an onlay to the albuginea, as shown in the figure (Group 2). Sacrifice and histological assessment was performed 2,
EP
4, 8 and 12 weeks postoperatively.
Results: In Group 1, a mild inflammatory process was noted that became almost
AC C
imperceptible at 12 weeks. Fibrosis was mild at all stages in this group. Over time, a regenerative epithelium covered the corpus cavernosum. Immunohistochemistry using specific CK-7 and CK-20 confirmed the presence of urothelium. No complications were microscopically detected in this group. Group 2 presented with a more intense inflammatory infiltrate, which also resolved over time. Fibrosis was slightly more intense in this group, especially in animals that had urethral strictures. Group 2 presented with three fistulas, two were associated with urethral stricture. Histological
1
ACCEPTED MANUSCRIPT evaluation showed the presence of epithelization over the albuginea, which turned out to be similar to the normal urothelium over time and was confirmed by immunohistochemistry. Non-keratinized stratified squamous epithelium of the foreskin
RI PT
flap showed good integration to the urethra. Discussion: Microscopic analysis showed that inflammation, fibrosis and complications were similar to previous studies. At 12 weeks there was a well-developed epithelium
SC
similar to normal urethra, which was confirmed by immunohistochemistry; this was similar to what occurs in the TIP technique, as previously demonstrated. It was
M AN U
hypothesized that the epithelium regeneration developed from the urethral edges, as demonstrated in other experimental studies.
Conclusion: The albuginea was covered by mature urothelium after 12 weeks, which presumably grew from the urethral edges. The foreskin flap onlay that was directly
neourethra.
TE D
anastomosed to the albuginea completely integrated and constituted the roof of the
AC C
EP
Keywords: Hypospadias; Surgical flaps; Urethra; Rabbits; Histology
2
ACCEPTED MANUSCRIPT
Introduction In recent decades, the treatment of proximal hypospadias has been an ongoing debate. Today, the consensus seems to be preservation of the urethral plate. However, in some
RI PT
patients with severe penile curvature, urethral plate division is necessary for curvature correction [1]. In these cases, a single procedure or staged strategy both remain
controversial [2-4]. Most surgeons choose the technique based on personal experience
SC
and not on technical feasibility or literature results [2, 4].
M AN U
The correction with a single-stage procedure has proven to be safe and has longterm success rates up to 70% [5, 6]. Two-stage techniques present complication rates of approximately 22%, but require at least three surgical procedures if a complication occurs [7].
Rigamonti and Castagnetti published an approach to configure the new urethra
TE D
using a foreskin flap directly anastomosed in an onlay fashion to the tunica albuginea of the corpora cavernosa, and found a complication rate around 21% in a mean follow-up
EP
of 7 months [8]. This is conceptually very similar to the Monseur urethroplasty principle (1968) and its several variants, which described focusing on the urothelial
AC C
lining of the tunica albuginea of the corpora cavernosa to complete the circumferential defect of the urethra [9]. These results suggest that it is possible to use the corpus cavernosum albuginea
as the posterior wall of the neourethra without the need of a dorsal graft. The present experimental study aimed to evaluate the histological characteristics and healing pattern of this procedure.
3
ACCEPTED MANUSCRIPT
Materials and Methods The Committee of Ethics in Research of the institution approved this study. A total of 16 New Zealand male rabbits were divided into two study groups of eight animals. The
RI PT
smallest estimated number of animals required to observe and analyze the results was used [10-15]. Animals were kept in a vivarium over a period of 3-7 days (induction period) prior to surgery.
SC
Anesthesia was induced by a licensed veterinarian (JQ) and was initiated by the
M AN U
intramuscular (IM) administration of a solution of acepromazine 1 mg/kg and fentanyl hydrochloride 3 ㎍/kg, and then a solution of ketamine 30 mg/kg with xylazine 5 mg/kg. Epidural block with lidocaine 2% and bupivacaine 0.5% with epinephrine was also performed. The surgical procedure was performed using magnification loupes of 3.5X.
TE D
Initially, the urethra was catheterized with an 8-F catheter and then the fold between the penis and the anus of the animal was sectioned to enable access to the
EP
urethra. The Buck´s fascia was laterally incised to the urethra and the corpus spongiosum and urethra were then released from the corpora cavernosa.
AC C
In animals from Group 1, a 1.0-cm longitudinal incision was made in the posterior surface of the urethra and the incised edges were laterally anastomosed to the corpus cavernosum, 0.4 cm apart from each other, with a continuous suture of 7-0 PDS®. The tunica albuginea of the corpus cavernosum then became the posterior wall of the neourethra (Fig. 2). In Group 2, after dissection of the urethra, a cross-sectional incision close to the edge of the inner foreskin of the penis was performed. To create the urethral defect the
4
ACCEPTED MANUSCRIPT urethral stumps were fixed to the tunica albuginea with 6-0 PDS® sutures, 1.0 cm apart, therefore simulating hypospadias with a need for urethral plate division and exposure of the tunica albuginea. A longitudinal foreskin flap of 1.0 cm x 0.4 cm was created on the
RI PT
ventral surface of the penis, and then rotated from distal to proximal, covering the urethral defect. The foreskin flap was sutured to the tunica albuginea with 7-0 PDS® sutures. In both groups, the urethral catheter was removed after the procedure.
SC
Each group of animals was then randomly divided into four subgroups of two rabbits to be sacrificed after 2, 4, 8, and 12 weeks of surgery. Immediately after
M AN U
sacrifice, a VCUG was performed and then the animal’s penis was removed and immediately fixed in 10% formaldehyde.
The pathology specimens were longitudinally fixed from the glans to base of the penis. Histological staining was performed with hematoxylin-eosin (HE) and Masson’s
TE D
trichrome (MT). An experienced pathologist in single analysis (DR) performed the histological evaluation. To confirm the presence of urothelium using specific CK-7 and CK-20 an experienced pathologist (SA) performed the immunohistochemistry. The
EP
pathologists used the same standardization of previous studies to assess the
AC C
inflammatory response related to the procedure [12-15].
Results
There were no deaths related to the procedure. The animals were sacrificed according to the previously established schedule. The animals in Group 1 showed no signs of urethral strictures on cystourethrography. In Group 2, there was difficulty in passing the catheter in three rabbits and urethral stricture was confirmed by urethrography. In two
5
ACCEPTED MANUSCRIPT of these animals, the presence of an associated fistula was identified. One rabbit in Group 2 presented with fistula alone. Histological analysis was performed with 10X, 40X, 100X and 200X
RI PT
magnifications on optical microscope. In Group 1, a mild inflammatory process with a predominance of lymphomononuclear cells was noted after 2 weeks, which became almost imperceptible at 12 weeks. Fibrosis was mild at all stages in this group. The
SC
epithelium covering the corpus cavernosum was initially an atrophic regenerative
epithelium, with only one layer of cells, but developed over time and at 12 weeks was
M AN U
composed of three or more layers. Immunohistochemistry using specific CK-7 and CK20 confirmed the presence of urothelium. No complications were microscopically detected in this group.
Group 2 presented with a more intense inflammatory infiltrate after 2 weeks,
TE D
with a predominance of lymphomononuclear cells, which also resolved over time and were very minimal in the 12-week group. Fibrosis was slightly more intense in this group, especially in animals that had urethral strictures; it remained mild to moderate up
EP
to 12 weeks.
The foreskin flap maintained its characteristic stratified squamous epithelium
AC C
and over time presented with acanthosis in some areas. The epithelium that covered the corpus cavernosum was initially atrophic and had an incomplete regenerative pattern, rendering some areas without any epithelium. In the 4-week group, the epithelium had fully covered the tunica albuginea. It developed over time and within 12 weeks presented as a robust epithelium with three or more layers of cells resembling the native epithelium of the urethra (Fig. 3). Immunohistochemistry using specific CK-7 and CK20 confirmed the presence of urothelium (Fig. 4). Complications were recorded in this
6
ACCEPTED MANUSCRIPT group. Microscopic analysis revealed two inclusion cysts, one foreign body granuloma and three previously macroscopically identified fistulas.
RI PT
Discussion
Some patients with proximal hypospadias and severe penile curvature require
SC
division of the urethral plate to correct the curvature [16]. In order to treat these patients in a single step, various tissues have been studied to replace the urethral plate when it
M AN U
needs to be sectioned. Among these are the peritoneum, bladder mucosa, tunica vaginalis, and buccal mucosa, with outcomes published in clinical and experimental studies over the years [12-15, 17-22]. Likewise, urethral healing has been experimentally studied for decades and more intensively over the past 10 years [12-15,
TE D
23-26].
The technique proposed by Rigamonti and Castagnetti, following the Monseur urethroplasty principle, assumes that it is unnecessary to apply any flap or graft at the
role [8, 9].
EP
dorsal component of the neourethra because the tunica albuginea itself could have that
AC C
In a clinical study, Macedo and Srougi proposed the 'three-in-one' technique and
were careful to reconstruct the urethral plate with a buccal mucosa graft, following the concept advocated by Barbagli [5, 27]. The possibility of simplifying this strategy prompted the present study to evaluate the histological aspect of Rigamonti’s procedure. Macroscopically, the rabbits of Group 1 had an uneventful outcome and presented with no fistula, stricture, or suture dehiscence. Microscopic analysis showed that inflammation, fibrosis, and complications were similar to previous studies. In an
7
ACCEPTED MANUSCRIPT experimental study with dogs, Bleustein et al. performed the TIP technique and postoperative histological analysis at day 21, which showed a sparse lymphocytic infiltrate [25]. Calado et al. and Souza et al. studied the integration of the tunica
RI PT
vaginalis and the buccal mucosa in an experimental model of urethroplasty, they also found inflammatory changes and fibrosis with temporal behavior similar to that found in the present study [12, 14].
SC
Epithelization of the tunica albuginea evolved from an atrophic, regenerative
epithelium with a single-cell layer to a more robust urothelium of two to three layers of
M AN U
cells at 12 weeks, which was confirmed by immunohistochemistry. Epithelialization probably occurs from the edges to the center, forming a ‘carpet’ of cells on the tunica albuginea that proliferate to form the new epithelium; this is similar to what occurs in the TIP technique, as previously experimentally demonstrated [24-26]. It seems that
TE D
Group 1 behaved like an extended TIP, in that healing was similar, even without the urethral plate underlying tissue, which is rich in blood vessels and connective tissue [28, 29].
EP
Group 2 presented with a macroscopic complication rate that reached 50%. Fistulae and strictures occurred in three and two animals, respectively (two animals with
AC C
strictures also had fistulas, thus, four animals had complications). Other experimental studies that used foreskin flaps found an incidence of fistulae of 12.5-25%, with no strictures reported in rabbits [15]. A rabbit´s urethra is very elastic and tends to avoid stricture formation. In the present study, there was no tissue associated with the foreskin besides the tunica albuginea; this theoretically makes the neourethra less complacent and therefore more prone to stricture formation.
8
ACCEPTED MANUSCRIPT Rigamonti and Castagnetti reported a complication rate of 21% (3/14 patients) with a fistula, a partial dehiscence and a dilation of the neourethra; there were no strictures. The initial results are encouraging because when treating these complex
RI PT
hypospadias, complication rates vary from 15-45% [7]. However, these results must be cautiously examined because the mean follow-up is very short (7 months), even though the study included patients up to 27 months of follow-up. Many complications in
SC
hypospadias surgery occur later on. Powell et al. evaluated 142 patients undergoing surgery for posterior hypospadias and reported 64% of fistulas appearing after 12
M AN U
months postoperatively (mean 23.9 months), 80% of meatal stenosis and 60% of proximal strictures appearing at 30.9 months of mean follow-up [30]. Therefore, in order to define the true rate of complications associated to this procedure it is imperative to await results with later follow-up.
TE D
Microscopic analysis showed that the behavior of the inflammatory process was similar to what happened in Group 1 and in previous experimental studies using an onlay foreskin flap [15]. Fibrosis was mild to moderate in most rabbits, which supports
EP
the findings of previous experimental studies, except in two rabbits that had strictures and in which more intense fibrosis was found [12, 14, 15, 22].
AC C
In the animals at 2 weeks of follow-up, a single-cell layer had already covered part of the corpora cavernosa. Some uncovered areas were surrounded by this same atrophic regenerative epithelium. In the other groups of later sacrifice, the whole surface of the corpus cavernosum was already covered by regenerative epithelium. At 12 weeks there was a well-developed epithelium similar to a normal urethra, which was confirmed by immunohistochemistry. It was hypothesized that the epithelium
9
ACCEPTED MANUSCRIPT regeneration developed from the urethral edges, as demonstrated in other experimental studies [12, 13, 15, 24-26]. In both groups, the gap between the urethral stumps was 1cm long and the lateral
RI PT
edges were 0.4 cm apart from each other. Therefore, the area of bare corpus cavernosum that was covered by an epithelium, which was incorporated into the new urethra to form the posterior wall, was 1.0 x 0.4 cm.
SC
It is worth reporting that in the clinical study of Rigamonti and Castagnetti, the authors performed a biopsy in one of their patients and found tissue similar to the
M AN U
foreskin flap in the dorsal wall of the neourethra, concluding that the healing in this area is made from the edges of the foreskin flap [8]. This finding contradicts the present observations in the experimental setting. Many hypotheses could explain this difference. One would be that the healing in humans occurs differently to rabbits, which seems to
TE D
be inconsistent. The second hypothesis could be that the biopsied site of the human urethra did not correspond to the location of the regenerated epithelium. In rabbits, the urothelium was practically normal after 12 weeks. In patients in the late postoperative
EP
period, it may be difficult to macroscopically differentiate a neo-formed mucosa from the internal foreskin, which also undergoes metaplasia over time.
AC C
The foreskin flap showed full integration to the neourethra, except in one rabbit with a fistula. Some areas of squamous metaplasia and acanthosis were observed. This finding is similar to that found by Leslie et al. who used a foreskin flap over the tunica vaginalis to form the neourethra. They found excellent integration of the foreskin within 2 weeks of monitoring and a transformation to a less-stratified epithelium over time up to 12 weeks [15].
10
ACCEPTED MANUSCRIPT Possible limitations of this study would include a short follow-up; most experimental studies using rabbits are designed to perform observations until week 16 [11-15, 22-24]. The number of animals used in an experimental study is always a
RI PT
concern of the Ethics Committee, so the minimum number was used to answer the questions that were proposed.
In experimental studies, any clinical conclusions should be examined very
SC
carefully. In the present study, there was no intention to compare macroscopic
complications that occur in experimental models with those found in clinical trials
M AN U
because the results couldn’t be generalized for human beings. From the beginning, according to the study design, no data were expected that could allow any conclusions to be made regarding surgical success or to recommend the use of this technique. The question was simple, and it was believed that it could be shown in rabbits that the
TE D
corpus cavernosum was covered by an epithelium similar to the native urethra, presumably growing from the urethral stump edges. In this animal model, urothelial regeneration happened, but with a high stricture rate.
EP
Therefore, the healing process of urethroplasty with a foreskin flap applied directly over the tunica albuginea of the corpus cavernosum was described. The
AC C
technical applicability for patients with hypospadias still requires further clinical studies with longer follow-up to assess its advantages in relation to already established procedures.
Conclusions In this experimental model, a foreskin flap directly anastomosed as an onlay to the tunica albuginea integrated properly and formed a neourethra.
11
ACCEPTED MANUSCRIPT The corpus cavernosum was initially covered by a regenerative epithelium, which over time turned into a mature epithelium similar to the native urethra, presumably growing from the urethral stump edges.
RI PT
In this short-term experimental study of rabbits, the complication rates, evidence of fibrosis and the inflammatory response were greater in the study group (Group 2).
AC C
EP
TE D
M AN U
SC
Conflict of Interest/Funding: None
12
ACCEPTED MANUSCRIPT
References [1] Baskin LS, Ebbers MB. Hypospadias: anatomy, etiology, and technique. J Pediatr Surg 2006;41:463-72.
RI PT
[2] Cook A, Khoury AE, Neville C, Bagli DJ, Farhat WA, Pippi Salle JL. A multicenter evaluation of technical preferences for primary hypospadias repair. J Urol 2005;174:2354-7, discussion 7. [3] Snodgrass W, Macedo A, Hoebeke P, Mouriquand PD. Hypospadias dilemmas: a round table. J Pediatr Urol 2011;7:145-57.
SC
[4] Springer A, Krois W, Horcher E. Trends in hypospadias surgery: results of a worldwide survey. Eur Urol 2011;60:1184-9.
M AN U
[5] Macedo A Jr., Srougi M. Onlay urethroplasty after sectioning of the urethral plate: early clinical experience with a new approach - the 'three-in-one' technique. BJU Int 2004;93:1107-9. [6] Macedo A Jr., Liguori R, Ottoni SL, Garrone G, Damazio E, Mattos RM, et al. Long-term results with a one-stage complex primary hypospadias repair strategy (the three-in-one technique). J Pediatr Urol 2011;7:299-304. [7] Castagnetti M, El-Ghoneimi A. Surgical management of primary severe hypospadias in children: systematic 20-year review. J Urol 2010;184:1469-74.
TE D
[8] Rigamonti W, Castagnetti M. Onlay on albuginea: modified onlay preputial island flap urethroplasty for single-stage repair of primary severe hypospadias requiring urethral plate division. Urology 2011;77:1498-502. [9] Monseur J. [Restoration of the urethral duct by means of supraurethral laminaie and the subcavernal grooves]. J Urol Nephrol (Paris) 1968;74:755-68.
EP
[10] Eckelman WC, Kilbourn MR, Joyal JL, Labiris R, Valliant JF. Justifying the number of animals for each experiment. Nucl Med Biol 2007;34:229-32.
AC C
[11] Hafez AT, Smith CR, McLorie GA, El-Ghoneimi A, Herz DB, Bagli DJ, et al. Tunica vaginalis for correcting penile chordee in a rabbit model: is there a difference in flap versus graft? J Urol 2001;166:1429-32. [12] Calado AA, Macedo A Jr., Delcelo R, de Figueiredo LF, Ortiz V, Srougi M. The tunica vaginalis dorsal graft urethroplasty: experimental study in rabbits. J Urol 2005;174:765-70. [13] Rosito TE, Pires JA, Delcelo R, Ortiz V, Macedo A Jr. Macroscopic and histological evaluation of tunica vaginalis dorsal grafting in the first stage of Bracka's urethroplasty: an experimental study in rabbits. BJU Int 2011;108:E17-22. [14] Souza GF, Calado AA, Delcelo R, Ortiz V, Macedo A Jr. Histopathological evaluation of urethroplasty with dorsal buccal mucosa: an experimental study in rabbits. Int Braz J Urol 2008;34:345-51; discussion 51-4.
13
ACCEPTED MANUSCRIPT [15] Leslie B, Barboza LL, Souza PO, Silva PS, Delcelo R, Ortiz V, et al. Dorsal tunica vaginalis graft plus onlay preputial island flap urethroplasty: experimental study in rabbits. J Pediatr Urol 2009;5:93-9. [16] Hollowell JG, Keating MA, Snyder HM 3rd, Duckett JW. Preservation of the urethral plate in hypospadias repair: extended applications and further experience with the onlay island flap urethroplasty. J Urol 1990;143:98-100; discussion -1.
RI PT
[17] Nanni L, Vallasciani S, Fadda G, Perrelli L. Free peritoneal grafts for patch urethroplasty in male rabbits. J Urol 2001;165:578-80.
[18] Mollard P, Mouriquand P, Bringeon G, Bugmann P. Repair of hypospadias using a bladder mucosal graft in 76 cases. J Urol 1989;142:1548-50.
SC
[19] Talja M, Kivisaari L, Makinen J, Lehtonen T. Free tunica vaginalis patch in urethroplasty. An experimental study. Eur Urol 1987;13:259-63.
M AN U
[20] Burger RA, Muller SC, el-Damanhoury H, Tschakaloff A, Riedmiller H, Hohenfellner R. The buccal mucosal graft for urethral reconstruction: a preliminary report. J Urol 1992;147:662-4. [21] Hill GA, Lewis AG, Sheldon CA. A rabbit model of free bladder mucosal grafting in a damaged urethral bed. J Urol 1994;152:983-6. [22] Oliva P, Delcelo R, Bacelar H, Rondon A, Barroso U Jr., Ortiz V, et al. The buccal mucosa fenestrated graft for Bracka first stage urethroplasty: experimental study in rabbits. Int Braz J Urol 2012;38:825-32. [23] Scherz HC, Kaplan GW, Boychuk DI, Landa HM, Haghighi P. Urethral healing in rabbits. J Urol 1992;148:708-10; discussion 11-3.
TE D
[24] Hafez AT, Herz D, Bagli D, Smith CR, McLorie G, Khoury AE. Healing of unstented tubularized incised plate urethroplasty: an experimental study in a rabbit model. BJU Int 2003;91:84-8.
EP
[25] Bleustein CB, Esposito MP, Soslow RA, Felsen D, Poppas DP. Mechanism of healing following the Snodgrass repair. J Urol 2001;165:277-9. [26] Lopes JF, Schned A, Ellsworth PI, Cendron M. Histological analysis of urethral healing after tubularized incised plate urethroplasty. J Urol 2001;166:1014-7.
AC C
[27] Barbagli G, Selli C, Tosto A, Palminteri E. Dorsal free graft urethroplasty. J Urol 1996;155:123-6. [28] Baskin LS, Erol A, Li YW, Cunha GR. Anatomical studies of hypospadias. J Urol 1998;160:1108-15; discussion 37. [29] Snodgrass W, Patterson K, Plaire JC, Grady R, Mitchell ME. Histology of the urethral plate: implications for hypospadias repair. J Urol 2000;164:988-9; discussion 990. [30] Powell CR, McAleer I, Alagiri M, Kaplan GW. Comparison of flaps versus grafts in proximal hypospadias surgery. J Urol 2000;163:1286-8; discussion 8-9.
14
ACCEPTED MANUSCRIPT Figure legends Summary figure legend Fig. 1. Procedure in Group 2. (A) Construction of 1.0-cm long urethral defect; (B) delimitation of foreskin flap on the ventral penile surface; (C) flap rotation from distal
RI PT
to proximal to cover the urethral defect; (D) flap sutured to the corpus cavernosum.
Manuscript figures legends
SC
Fig. 2. Procedure in Group 1. (A) 1.0-cm longitudinal incision in the urethral posterior surface; (B) suture of the urethral side edges to the corpus cavernosum; (C) final
M AN U
aspect of the urethra anastomosed to the corpus cavernosum.
Fig. 3. Group 2 epithelium evolution (HE 100X).
TE D
W = week; RE = regenerative epithelium; CC = corpus cavernosum.
Fig. 4. Group 2 immunohistochemistry (40X) with CK-7 (A and B) and CK-20 (C and D). A and C: prepuce flap. B and D: epithelization similar to the normal
AC C
EP
urothelium over the albuginea.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT