- Email: [email protected]
Reduced Flow after Tubularized Incised Plate Urethroplasty—Increased Fibrogenesis, Elastin Fiber Loss or Neither?
Reduced Flow after Tubularized Incised Plate Urethroplastyd Increased Fibrogenesis, Elastin Fiber Loss or Neither? Lisieux Eyer Jesus,* Alberto Schanaider, Tyler Kirwan, Karen J. Aitken, Maria L. R. Caldas, ~o L. Pippi-Salle Elissa Fonseca, Alexander Marchenko, Darius J. Bagli and Joa From the Center for Experimental Surgery, Postgraduate Program in Surgical Sciences, Federal University of Rio de Janeiro (LEJ, AS), and Department of Pathology (MLRC, EF) and Department of Surgery, Division of Pediatric Surgery and Pediatric Urology (LEJ), Federal Fluminense University and Servidores do Estado Hospital, Rio de Janeiro, Brazil, and Division of Pediatric Urology (AM, DJB, JLP-S), and Division of Developmental and Stem Cell Biology (TK, KJA, DJB), Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
Abbreviations and Acronyms CC ¼ corpus cavernosum ECM ¼ extracellular matrix PCR ¼ polymerase chain reaction TIP ¼ tubularized incised plate urethroplasty TIPG ¼ tubularized incised plate urethroplasty with inlay preputial graft Accepted for publication November 25, 2013. Study received institutional animal care committee approval (REB 1000007883). Supported by grants from the Coordination of Improvement of Personnel of Superior Level (CAPES-Brazil) and the National Council for Scientific and Technological Development (CNPq-Brazil). * Correspondence: Hospital Universitario Ant^onio Pedro, Universidade Federal Fluminense, Rio de Janeiro, 52 Presidente Domiciano St., Apt. 801, Niteroi, Rio de Janeiro, Brazil CEP 24210-270 (e-mail: [email protected]).
Purpose: Low urinary flow rates are common after tubularized incised plate urethroplasty but the etiology remains unclear and may be related to low urethral compliance due to abnormal collagen concentrations and/or fewer elastic fibers in the healed urethral plate. We hypothesized that inserting a preputial mucosal graft over the dorsal raw area after the midline incision may avoid scarring and improve urethral compliance. Materials and Methods: Adult rabbits were submitted to tubularized incised plate urethroplasty with or without inlay preputial graft according to a previously described protocol. Tissular concentrations of collagens I, III, IV, VI, VIII and XIII were measured. Histomorphometric analysis was used to quantify elastic fibers in the urethra. Tubularized incised plate urethroplasty with and without inlay preputial graft was compared to normal rabbit urethras (controls). Results: mRNA concentrations for collagens I, II and XIII were similar between controls and operated rabbits. The proportions between collagens I and III were 1.05, 0.87 and 1.21, respectively, in controls and animals undergoing tubularized incised plate urethroplasty with and without inlay preputial graft. mRNA concentrations for collagen IV and collagens VI/VIII tended to be higher and lower, respectively, in the operated urethras, despite showing statistical significance only for collagen VIII in animals undergoing tubularized incised plate urethroplasty with inlay preputial graft vs controls (p ¼ 0.02). The operated animals did not demonstrate a reduced number of elastic fibers in the urethral tissues compared to controls. Conclusions: Elastic fiber number and distribution were similar between tubularized incised plate urethroplasty cases and controls, suggesting that decreased concentrations of elastic fibers do not explain the reduced urethral compliance after tubularized incised plate urethroplasty. The raw area determined by the dorsal urethral incision regenerated after standard tubularized incised plate urethroplasty, while cicatrization with fibrosis occurred in correspondence to the grafted areas after tubularized incised plate urethroplasty with inlay preputial graft. Key Words: collagen, elastin, hypospadias, skin transplantation, urethra
1856
j
www.jurology.com
0022-5347/14/1916-1856/0 THE JOURNAL OF UROLOGY® © 2014 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
http://dx.doi.org/10.1016/j.juro.2013.11.098 Vol. 191, 1856-1862, June 2014 Printed in U.S.A.
REDUCED URINARY FLOW AFTER TUBULARIZED INCISED PLATE URETHROPLASTY
TUBULARIZED incised plate urethroplasty is widely used for hypospadias repair.1 Abnormal urinary flow rates are common postoperatively, despite the absence of anatomical urethral obstruction in most cases.2 Previous experimental results indicate that tubularized incised plate urethroplasty does not induce extensive urethral fibrosis,3e5 but modifications of the collagen distribution or loss of elastic fibers in the operated area are still possible. We studied these aspects further in a previously described animal model,2 departing from the hypothesis of a loss of urethral compliance in TIP models, focusing instead on the possibility of fibrosis, differential collagen distribution or paucity of elastic fibers after cicatrization of the median incision. We also studied how the addition of a free graft over the raw dorsal area after the midline incision modified urethral cicatrization. Our objectives were to evaluate tissular concentration of different collagens, and to quantify and describe the distribution of elastic fibers in normal urethras and TIP and TIPG models.
METHODS The experimental protocol was approved by our institutional animal care committee (REB 1000007883). All handling and procedures were performed following the Canadian Council on Animal Care guidelines. A total of 25 adult male 3 to 3.5 kg New Zealand rabbits were kept in individual cages receiving a standard rabbit diet, water ad libitum and routine care, and preanesthetized with ketamine and acepromazine. Anesthesia was accomplished by a mixture of halothane, nitrous oxide and oxygen through a mask and penile block with 2% lidocaine. Animals were divided into 3 groups. Group 1 consisted of 9 nonoperated normal male adult rabbits (controls). Group 2 included 8 animals that underwent segmental TIP after resection of the ventrolateral portion of the urethral wall (not adherent to the CC), leaving approximately half of the urethral circumference, followed by a midline dorsal incision (limited to the adventitia of the CC) and ventral tubularization (single layered continuous
1857
polydioxanone 6-zero suture over a 10Fr catheter that was removed immediately after finishing the procedure). The operated segment was 2.5 cm long, beginning 1 cm proximal to the glans (fig. 1). Group 3 consisted of 8 animals undergoing the same procedure as group 2, except a mucosal preputial graft was placed over the incised area, sutured to the edges of the incision and quilted to the corporeal albuginea with interrupted 7-zero polyglactin sutures. The animals received routine care after recovering from anesthesia. Analgesics were not required postoperatively. All animals were observed for 6 weeks and then sacrificed using anesthetic overdose. Penile dissection and degloving was done immediately after death. Urethral compliance was evaluated by measuring tension in an isolated urethral segment after injections of specific volumes of air, as reported previously.6 A 3 cm urethra plus CC segment distal to the penopubic junction was isolated and sectioned in 3 equal parts, each containing a segment of operated urethra in groups 2 and 3. One segment was selected for histological studies, preserved in phosphate buffered formalin and sectioned (4 mm thick sections). Four sections for each animal were stained using hematoxylin and eosin (routine histological evaluation), Movat pentachrome (specific for elastic fibers), Masson trichrome and picrosirius red. The last two staining methods are specific to evaluating collagen deposition. A stained section for each rabbit was photographed using MIRAX Viewer software (Carl Zeiss, Thornwood, New York). Using 20 magnification, 4 sections were selected for elastin fiber counting and tissue description, starting from the mucosa inward, with section 1 being median dorsal, 2 and 4 lateral, and 3 median ventral (fig. 2). The selected areas were uploaded for manual counting of elastic fibers using a grid mask tool, spaced 40 40 (Image-ProÒ Plus software, version 4.5.0.29). Another urethral segment was used for PCR. The tissue was washed with saline, immediately frozen in dry ice and kept in a freezer at 8C. Measurement of collagen mRNA was done for collagens I, III, IV, VI, VIII and XIII. RNA corresponding to each collagen was obtained using oligo-dT primers and reverse transcriptase (InvitrogenÔ). The primers were custom designed and tested before use. DNA was induced with TRIzolÒ solution. Normalization of the results used the reference genes b actin and HPRT.
Figure 1. A, markings for urethral reduction (degloved penis). B, TIP model before tubularization. C, TIPG model before tubularization.
1858
REDUCED URINARY FLOW AFTER TUBULARIZED INCISED PLATE URETHROPLASTY
Figure 2. Areas selected for histological description and elastic fiber histomorphometry.
Amplification was counted using fluorescence microscopy (SYBRÒ Green), with 90% to 110% efficiency. The third segment of the specimen was preserved for future evaluations with other methods, if required. Correlation analyses were done with the Spearman test. Nonparametric variance analyses were also done, using Wilcoxon test between groups and Kruskal-Wallis test within groups and R, version 2.15.1 (R Foundation for Statistical Computing, Vienna, Austria). P values less than 0.05 were considered significant.
RESULTS All animals were operated on uneventfully, and there were no perioperative deaths. One animal in group 3 died on postoperative day 19 and was
excluded. Necropsy did not reveal any problems related to the urethral surgery. There were no urethral fistulas or stenoses in group 2 or 3. Three animals from group 1 and 2 from group 3 were excluded because of problems with identification and preservation of the specimens. One animal from group 1 and 1 from group 3 were also excluded because appropriate slides for elastic fiber counting could not be obtained (fig. 3). There was no significant difference in segmental urethral compliance between the groups.6 One animal was excluded from collagen XIII analysis based on technical criteria (outlier). mRNA expressions for collagens I, III and XIII were similar between the groups. Expressions for collagens VI and VIII were lower in the operated animals (greater in group 1 than in group 2, and greater in group 2 than in group 3). Collagen IV mRNA was greater in the operated animals (less in group 1 than in group 2, and less in group 2 than in group 3, fig. 4). The difference for collagen VIII between groups 1 and 3 was significant (p ¼ 0.02), while differences were not significant between groups 1 and 2 (p ¼ 0.33) or groups 2 and 3 (p ¼ 0.48). The proportions between collagens I and III differed between the groups (1.05, 1.21 and 0.87 for groups 1, 2 and 3, respectively). Urethras in groups 2 and 3 showed ventral fibrosis, and disorganization and dispersion of muscle fibers corresponding to the tubularization sutures. All grafts were taken in group 3 rabbits. Those animals demonstrated fibrosis corresponding to the area of the graft. Group 2 animals did not exhibit signs of cicatrization/fibrosis in the dorsal area, suggesting that regeneration occurs in the area of the dorsal incision in our TIP model (fig. 5). Animals in group 1 displayed more elastic fibers in the ventral than the dorsal part of the urethra
Figure 3. Description of cohort and exclusions. G, group.
REDUCED URINARY FLOW AFTER TUBULARIZED INCISED PLATE URETHROPLASTY
Figure 4. PCR results for collagen mRNAs (means). Statistically significant difference was observed only for collagen VIII when comparing group (G) 1 vs 3. Blue bars indicate group 1. Violet bars represent group 2. Yellow bars indicate group 3.
(normal pattern for species). Histomorphometric analysis revealed that the number of elastic fibers was comparable between the groups (p >0.05 for all comparisons, fig. 6).
DISCUSSION Urethral healing after TIP seems to occur by centripetal progressive epithelialization and is not related to higher total concentrations of collagen or discernible fibrosis in various hypospadias models by histological, histomorphometric and/or biochemical methods.3e5 Our group has also shown the absence of significant fibrosis after TIP in the same model used in this study.2 Our findings demonstrated that acute postoperative tissue responses subsided after 6 weeks, which is the basis of our choice of this interval for urethral harvest. The absence of fibrosis in TIP models suggests similar concentrations of structural collagens (I/III)
1859
compared to normal controls, although different concentrations of other collagens, which have diverse functions and present in much lower tissular concentrations, may be present. Different concentrations of nonstructural collagens could explain different tissular biophysical characteristics by modulation of ECM components. Alternatively varying proportions between collagens I and III may modify elastic properties and explain divergent biophysical tissular responses. Specific collagens have distinct biological functions related to modulation of tissue metabolism, reconstruction, angiogenesis, healing and remodeling processes, programmed by complex interactions with cell receptors and ECM components, including biophysical signaling. Besides the concentration of the protein per se, the 3-dimensional collagen architecture contributes to mechanical tissue properties. We chose to study collagens related to tissue structure (fibril forming in collagens I and III, microfibrilar in VI and short chain in VIII), basal membrane (IV) and transmembrane regulator (XIII). Collagens I and III directly relate to tissue biophysical properties, VI and VIII are thought to be fibrogenic through fibroblast recruiting, IV is the most important structural component of basal membranes, and XIII regulates growth and regenerative tissular processes.7e9 Collagen I/III proportion increases in the initial phases of urethral healing.4 Collagen III is abundant in elastic tissues and visceral reticular fibers, possibly relating to elasticity and organization of elastic fibers.7,10 Baskin et al suggest that the
Figure 5. Panoramic views of urethras. Arrows indicate fibrotic areas on dorsal urethra corresponding to areas where grafts were inserted. CB, cavernosum body. G, group. HE, hematoxylin and eosin. VU, ventral urethra. Reduced from 20.
1860
REDUCED URINARY FLOW AFTER TUBULARIZED INCISED PLATE URETHROPLASTY
Figure 6. Elastic fiber counts. Blue bars represent group (G) 1. Violet bars indicate group 2. Yellow bars represent group 3.
normal human urethra is composed of 75% collagen I and 25% collagen III, while in strictured urethras the proportions are 83% and 16%.11 In our model involving animals without stenoses in a late phase of healing the mean proportions of mRNA for collagen I/III were 1.05 (controls), 1.21 (TIP) and 0.87 (TIPG), suggesting that TIP may increase and TIPG may decrease the collagen I/III proportion. By contrast, Hayashi et al assert that collagen III is absent in the urethral plates.12 Interpreting these disparate results is difficult, as their study evaluated urethral plates in human children and used immunohistochemical analysis to map collagen distribution, while our study deals with normal urethras in rabbits and estimates collagen concentration with PCR, without considering tissular architectural distribution of the protein. Collagen IV RNA was increased in the experimental groups, predominating in TIPG animals. Hayashi et al studied collagen IV distribution in biopsies taken from the tissue between the urethral plate and adventitia of the corpora in human children and suggest that collagen IV is restricted to basal membranes of the capillaries.12 However, collagen IV has been described in the basement membrane of the urothelium and detrusor muscle by others, who relate focal losses of collagen IV reactivity to inflammation, dysplasia and noninvasive bladder cancer.13,14 It is possible that the restricted distribution of collagen IV described by Hayashi et al is due to the restricted biopsies taken, which did not include urethral epithelium.12 In our research collagen VI mRNA expression tended to be decreased in the experimental groups. This collagen is ubiquitous and forms networks linking either cells and basement membranes to the matrix or collagen I and III fibrils to the basement membranes, suggesting an important role in the regulation of tissular architecture. In humans collagen VI mRNA expression stimulates fibroblast proliferation and is up-regulated in the skin from 3 days after tissue trauma, persisting during the late healing phase, possibly with an active role in fibrogenesis.15
Collagen VIII may be related to ECM stabilization, active remodeling, myocyte migration and dedifferentiation into fibroblasts.16e18 Data suggest that collagen VIII is actively secreted by myocytes in the remodeling processes of biological tubes (vessels and bronchi) exposed to intermittent continued mechanical stresses and in tissue trauma.18,19 We did not expect this collagen to be decreased in the experimental groups, especially in TIPG models, considering its role in remodeling and fibroblast recruiting. Reduced synthesis of type VIII collagen may be due to a decreased number of myocytes in our model, which could modify tissue remodeling after trauma. Assessing the differential cell populations in the wounds may help to determine an explanation for the reduced levels of collagen VIII in the operated animals in the near future. Concerning elastic fibers, our intention was to estimate the number and distribution of mature fibers, reflecting the effective elasticity of the tissue and excluding nonfunctional elastin (elastosis). This is why we chose direct counting of the fibers instead of measurement of elastin. We opted for manual grid counting to eliminate potential errors due to mistaken automatic counting of other deep, dark structures (such as cell nuclei) by automatic system software after the Movat dyeing method. Tissular compliance combines resilience, deformability and morphological stability, and involves interactions between elastin and collagen, as well as their tridimensional distribution. The system is dynamic, and interactions between ECM, cells and mechanical stimuli modulate tissue conformation. Elastic fibers are essential in organs that undergo periodic changes in shape, such as the male urethra, which is periodically distended and elongated during voiding and erections. Urethral connective tissue contains abundant elastin fibers distributed in the ECM that form a net. The urethral submucosa displays the highest volumetric proportion of elastic fibers/tissue among all human tissues.20 Elastic fibers are long, numerous and tortuous, distributing mainly in the spongiosum and immediately beneath the basement membrane.21,22 Human fetuses exhibit progressive accumulation of elastin fibers from the second trimester to term, with the number, size and thickness of elastic fibers increasing almost 4 times from the fetal second trimester to adulthood. Interestingly the concentration of elastic fibers in the adult is 1.3 times greater than in term fetuses, implying that some effective elastogenesis occurs after birth.21 Studies about elastic fiber distribution in the urethra are rare. Most of the information available concerns the cavernosum and spongiosum bodies and deals with erection mechanisms. To our knowledge
REDUCED URINARY FLOW AFTER TUBULARIZED INCISED PLATE URETHROPLASTY
there are no published reports concerning the relationship between elastin fibers and voiding. Our results confirm a decreased concentration of elastin fibers in the dorsal urethra of normal rabbits. Elastin fiber counts were comparable between the groups, implying that elastic fiber loss may not explain the postoperative biomechanical problems. The absence of spongiosum influences the determination of the biomechanical properties of neourethras constructed to treat hypospadias. However, we were unable to find data comparing urethral function with and without spongioplasty. Our model does not consider this specific aspect, as it departs from normal urethras that are not devoid of spongiosum. Our study has several limitations. First, our experimental model, despite being well accepted for hypospadias surgery, is performed in adult rabbits, whose urethras are anatomically different from human counterparts and were exposed to androgenic stimuli. The reconstructions were done in urethral remnants, which are not real hypospadiac urethral plates. These limitations are also present in other experimental series dealing with hypospadias, as there is still no easily available and predictable animal model of congenital hypospadias. Another limitation is that assessment of elastin fibers in a unidimensional frame and measurement of collagen concentrations do not reveal the tissular tridimensional structure, which may be important in biomechanical functions. Furthermore, although there are no published data specifically concerning the urethra, the collagen structure of the penis changes with maturation and age.23 It is possible that some of our findings would be different in
1861
immature animals, especially concerning collagen XIII, which is over expressed in immature and growing tissues.9 Finally, our study analyzed quantitative mRNA expression for collagen, which may differ from final protein expression, despite a direct relationship between mRNA and final protein concentrations being expected. Future research regarding specific collagen tissular distribution by immunohistochemical analysis would be a further step in explaining the relationship between tissular proteins, collagen and biophysical properties of the virgin and postoperative urethra, by showing the architectural pattern of distribution of different types of collagen. Also studying the cell populations in each model, especially myofibroblasts and myocytes, could help us to understand different circumstantial patterns of collagen secretion.
CONCLUSIONS Decreased concentration of elastic fibers does not explain reduced urethral compliance after TIP in this model. The dorsal urethral segment regenerated after TIP incision but healing with fibrosis occurred in the ventral sutured areas in the operated animals and grafted areas after TIPG.
ACKNOWLEDGMENTS Paula Pflugfelder and Marvin Estrada, Experimental Surgery Department, and Greg Patterson, engineering technician, Hospital for Sick Children, Toronto University, Toronto, Ontario, Canada, assisted with the study. Alexandre Santos Aguiar conducted biostatistical analysis.
REFERENCES 1. Snodgrass W: Tubularized, incised plate urethroplasty for distal hypospadias. J Urol 1994; 151: 464. 2. Leslie B, Jesus LE, El-Hout Y et al: Comparative histological and functional controlled analysis of tubularized incised plate urethroplasty with and without dorsal inlay graft: a preliminary experimental study in rabbits. J Urol, suppl., 2011; 186: 1631.
the urethral wound: an experimental study. J Pediatr Urol 2006; 2: 182. 6. Jesus LE, Schanaider A, Patterson G et al: Urethral compliance in hypospadias operated by tubularized incised urethral plate (TIP) with and without a dorsal inlay graft: an experimental controlled study. World J Urol 2013; 31: 971. 7. Gelse K, P€oschl E and Aigner T: Collagensdstructure, function, and biosynthesis. Adv Drug Deliv Rev 2003; 55: 1531.
3. Bleustein CB, Esposito MP, Soslow RA et al: Mechanism of healing following the Snodgrass repair. J Urol 2001; 165: 277.
8. Gordon MK and Hahn RA: Collagens. Cell Tissue Res 2010; 339: 247.
4. Taneli F, Ulman C, Genc A et al: Biochemical analysis of urethral collagen after tubularized incised plate urethroplasty: an experimental study in rabbits. Urol Res 2004; 32: 219.
9. Hiekkinen A, Tu H and Pihlajaniemi T: Collagen XIII: a type II transmembrane protein with relevance to musculoskeletal tissues, microvessels and inflammation. Int J Biochem Cell Biol 2012; 44: 714.
5. Somuncu S, Caglayan O, Cakmak M et al: The effect of indwelling catheter on OH-proline in
10. von der Mark K: Localization of collagen types in tissues. Int Rev Connect Tissue Res 1981; 9: 265.
11. Baskin LS, Constantinescu SC, Howard PS et al: Biochemical characterization and quantitation of the collagenous components of urethral stricture tissue. J Urol 1993; 150: 642. 12. Hayashi Y, Mizuno K, Kojuma Y et al: Characterization of the urethral plate and the underlying tissue defined by expression of collagen subtypes and microarchitecture in hypospadias. Int J Urol 2011; 18: 217. 13. Borza DB, Bondar O, Ninomiya Y et al: The NC1 domain of collagen IV encodes a novel network composed of the alpha 1, alpha 2, alpha 5 and alpha 6 chains in smooth muscle basement membranes. J Biol Chem 2001; 276: 28532. 14. Deen S and Ball RY: Basement membrane and extracellular interstitial matrix components in bladder neoplasiadevidence of angiogenesis. Histopathology 1994; 25: 475.
1862
REDUCED URINARY FLOW AFTER TUBULARIZED INCISED PLATE URETHROPLASTY
15. Oono T, Specks U, Eckes B et al: Expression of type VI collagen mRNA during wound healing. J Invest Dermatol 1993; 100: 329. 16. MacBeath JR, Kielty CM and Shuttleworth CA: Type VIII collagen is a product of vascular smooth-muscle cells in development and disease. Biochem J 1996; 319: 993. 17. Cherepanova OA, Pidkovka NA, Sarmento OF et al: Oxidized phospholipids induce type VIII collagen expression and vascular smooth cell migration. Circ Res 2009; 104: 609.
18. Hou G, Mulholland D, Gronska MA et al: Type VIII collagen stimulates smooth muscle cell migration and matrix metalloproteinase synthesis after arterial injury. Am J Pathol 2000; 156: 467. 19. Hasaneen NA, Zucker S, Lin RZ et al: Angiogenesis is induced by airway smooth muscle strain. Am J Physiol Lung Cell Mol Physiol 2007; 293: L1059. 20. Testut L: Treatise on Human Anatomy, 16th ed. Paris: Doin 1912; vol 4, pp 507e534 and 627e630.
21. Bastos AL, Silva EA, Costa WS et al: Concentration of elastic fibers in the male urethra during human fetal development. BJU Int 2004; 94: 620. 22. Hsu GL, Brock A, Von Heyden B et al: The distribution of elastic fibrous elements within the human penis. Br J Urol 1994; 73: 566. 23. Mersdorf A, Goldsmith PC, Diederichs W et al: Ultrastructural changes in impotent penile tissue: comparison of 65 patients. J Urol 1991; 145: 749.
Abbreviations and Acronyms CC ¼ corpus cavernosum ECM ¼ extracellular matrix PCR ¼ polymerase chain reaction TIP ¼ tubularized incised plate urethroplasty TIPG ¼ tubularized incised plate urethroplasty with inlay preputial graft Accepted for publication November 25, 2013. Study received institutional animal care committee approval (REB 1000007883). Supported by grants from the Coordination of Improvement of Personnel of Superior Level (CAPES-Brazil) and the National Council for Scientific and Technological Development (CNPq-Brazil). * Correspondence: Hospital Universitario Ant^onio Pedro, Universidade Federal Fluminense, Rio de Janeiro, 52 Presidente Domiciano St., Apt. 801, Niteroi, Rio de Janeiro, Brazil CEP 24210-270 (e-mail: [email protected]).
Purpose: Low urinary flow rates are common after tubularized incised plate urethroplasty but the etiology remains unclear and may be related to low urethral compliance due to abnormal collagen concentrations and/or fewer elastic fibers in the healed urethral plate. We hypothesized that inserting a preputial mucosal graft over the dorsal raw area after the midline incision may avoid scarring and improve urethral compliance. Materials and Methods: Adult rabbits were submitted to tubularized incised plate urethroplasty with or without inlay preputial graft according to a previously described protocol. Tissular concentrations of collagens I, III, IV, VI, VIII and XIII were measured. Histomorphometric analysis was used to quantify elastic fibers in the urethra. Tubularized incised plate urethroplasty with and without inlay preputial graft was compared to normal rabbit urethras (controls). Results: mRNA concentrations for collagens I, II and XIII were similar between controls and operated rabbits. The proportions between collagens I and III were 1.05, 0.87 and 1.21, respectively, in controls and animals undergoing tubularized incised plate urethroplasty with and without inlay preputial graft. mRNA concentrations for collagen IV and collagens VI/VIII tended to be higher and lower, respectively, in the operated urethras, despite showing statistical significance only for collagen VIII in animals undergoing tubularized incised plate urethroplasty with inlay preputial graft vs controls (p ¼ 0.02). The operated animals did not demonstrate a reduced number of elastic fibers in the urethral tissues compared to controls. Conclusions: Elastic fiber number and distribution were similar between tubularized incised plate urethroplasty cases and controls, suggesting that decreased concentrations of elastic fibers do not explain the reduced urethral compliance after tubularized incised plate urethroplasty. The raw area determined by the dorsal urethral incision regenerated after standard tubularized incised plate urethroplasty, while cicatrization with fibrosis occurred in correspondence to the grafted areas after tubularized incised plate urethroplasty with inlay preputial graft. Key Words: collagen, elastin, hypospadias, skin transplantation, urethra
1856
j
www.jurology.com
0022-5347/14/1916-1856/0 THE JOURNAL OF UROLOGY® © 2014 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
http://dx.doi.org/10.1016/j.juro.2013.11.098 Vol. 191, 1856-1862, June 2014 Printed in U.S.A.
REDUCED URINARY FLOW AFTER TUBULARIZED INCISED PLATE URETHROPLASTY
TUBULARIZED incised plate urethroplasty is widely used for hypospadias repair.1 Abnormal urinary flow rates are common postoperatively, despite the absence of anatomical urethral obstruction in most cases.2 Previous experimental results indicate that tubularized incised plate urethroplasty does not induce extensive urethral fibrosis,3e5 but modifications of the collagen distribution or loss of elastic fibers in the operated area are still possible. We studied these aspects further in a previously described animal model,2 departing from the hypothesis of a loss of urethral compliance in TIP models, focusing instead on the possibility of fibrosis, differential collagen distribution or paucity of elastic fibers after cicatrization of the median incision. We also studied how the addition of a free graft over the raw dorsal area after the midline incision modified urethral cicatrization. Our objectives were to evaluate tissular concentration of different collagens, and to quantify and describe the distribution of elastic fibers in normal urethras and TIP and TIPG models.
METHODS The experimental protocol was approved by our institutional animal care committee (REB 1000007883). All handling and procedures were performed following the Canadian Council on Animal Care guidelines. A total of 25 adult male 3 to 3.5 kg New Zealand rabbits were kept in individual cages receiving a standard rabbit diet, water ad libitum and routine care, and preanesthetized with ketamine and acepromazine. Anesthesia was accomplished by a mixture of halothane, nitrous oxide and oxygen through a mask and penile block with 2% lidocaine. Animals were divided into 3 groups. Group 1 consisted of 9 nonoperated normal male adult rabbits (controls). Group 2 included 8 animals that underwent segmental TIP after resection of the ventrolateral portion of the urethral wall (not adherent to the CC), leaving approximately half of the urethral circumference, followed by a midline dorsal incision (limited to the adventitia of the CC) and ventral tubularization (single layered continuous
1857
polydioxanone 6-zero suture over a 10Fr catheter that was removed immediately after finishing the procedure). The operated segment was 2.5 cm long, beginning 1 cm proximal to the glans (fig. 1). Group 3 consisted of 8 animals undergoing the same procedure as group 2, except a mucosal preputial graft was placed over the incised area, sutured to the edges of the incision and quilted to the corporeal albuginea with interrupted 7-zero polyglactin sutures. The animals received routine care after recovering from anesthesia. Analgesics were not required postoperatively. All animals were observed for 6 weeks and then sacrificed using anesthetic overdose. Penile dissection and degloving was done immediately after death. Urethral compliance was evaluated by measuring tension in an isolated urethral segment after injections of specific volumes of air, as reported previously.6 A 3 cm urethra plus CC segment distal to the penopubic junction was isolated and sectioned in 3 equal parts, each containing a segment of operated urethra in groups 2 and 3. One segment was selected for histological studies, preserved in phosphate buffered formalin and sectioned (4 mm thick sections). Four sections for each animal were stained using hematoxylin and eosin (routine histological evaluation), Movat pentachrome (specific for elastic fibers), Masson trichrome and picrosirius red. The last two staining methods are specific to evaluating collagen deposition. A stained section for each rabbit was photographed using MIRAX Viewer software (Carl Zeiss, Thornwood, New York). Using 20 magnification, 4 sections were selected for elastin fiber counting and tissue description, starting from the mucosa inward, with section 1 being median dorsal, 2 and 4 lateral, and 3 median ventral (fig. 2). The selected areas were uploaded for manual counting of elastic fibers using a grid mask tool, spaced 40 40 (Image-ProÒ Plus software, version 4.5.0.29). Another urethral segment was used for PCR. The tissue was washed with saline, immediately frozen in dry ice and kept in a freezer at 8C. Measurement of collagen mRNA was done for collagens I, III, IV, VI, VIII and XIII. RNA corresponding to each collagen was obtained using oligo-dT primers and reverse transcriptase (InvitrogenÔ). The primers were custom designed and tested before use. DNA was induced with TRIzolÒ solution. Normalization of the results used the reference genes b actin and HPRT.
Figure 1. A, markings for urethral reduction (degloved penis). B, TIP model before tubularization. C, TIPG model before tubularization.
1858
REDUCED URINARY FLOW AFTER TUBULARIZED INCISED PLATE URETHROPLASTY
Figure 2. Areas selected for histological description and elastic fiber histomorphometry.
Amplification was counted using fluorescence microscopy (SYBRÒ Green), with 90% to 110% efficiency. The third segment of the specimen was preserved for future evaluations with other methods, if required. Correlation analyses were done with the Spearman test. Nonparametric variance analyses were also done, using Wilcoxon test between groups and Kruskal-Wallis test within groups and R, version 2.15.1 (R Foundation for Statistical Computing, Vienna, Austria). P values less than 0.05 were considered significant.
RESULTS All animals were operated on uneventfully, and there were no perioperative deaths. One animal in group 3 died on postoperative day 19 and was
excluded. Necropsy did not reveal any problems related to the urethral surgery. There were no urethral fistulas or stenoses in group 2 or 3. Three animals from group 1 and 2 from group 3 were excluded because of problems with identification and preservation of the specimens. One animal from group 1 and 1 from group 3 were also excluded because appropriate slides for elastic fiber counting could not be obtained (fig. 3). There was no significant difference in segmental urethral compliance between the groups.6 One animal was excluded from collagen XIII analysis based on technical criteria (outlier). mRNA expressions for collagens I, III and XIII were similar between the groups. Expressions for collagens VI and VIII were lower in the operated animals (greater in group 1 than in group 2, and greater in group 2 than in group 3). Collagen IV mRNA was greater in the operated animals (less in group 1 than in group 2, and less in group 2 than in group 3, fig. 4). The difference for collagen VIII between groups 1 and 3 was significant (p ¼ 0.02), while differences were not significant between groups 1 and 2 (p ¼ 0.33) or groups 2 and 3 (p ¼ 0.48). The proportions between collagens I and III differed between the groups (1.05, 1.21 and 0.87 for groups 1, 2 and 3, respectively). Urethras in groups 2 and 3 showed ventral fibrosis, and disorganization and dispersion of muscle fibers corresponding to the tubularization sutures. All grafts were taken in group 3 rabbits. Those animals demonstrated fibrosis corresponding to the area of the graft. Group 2 animals did not exhibit signs of cicatrization/fibrosis in the dorsal area, suggesting that regeneration occurs in the area of the dorsal incision in our TIP model (fig. 5). Animals in group 1 displayed more elastic fibers in the ventral than the dorsal part of the urethra
Figure 3. Description of cohort and exclusions. G, group.
REDUCED URINARY FLOW AFTER TUBULARIZED INCISED PLATE URETHROPLASTY
Figure 4. PCR results for collagen mRNAs (means). Statistically significant difference was observed only for collagen VIII when comparing group (G) 1 vs 3. Blue bars indicate group 1. Violet bars represent group 2. Yellow bars indicate group 3.
(normal pattern for species). Histomorphometric analysis revealed that the number of elastic fibers was comparable between the groups (p >0.05 for all comparisons, fig. 6).
DISCUSSION Urethral healing after TIP seems to occur by centripetal progressive epithelialization and is not related to higher total concentrations of collagen or discernible fibrosis in various hypospadias models by histological, histomorphometric and/or biochemical methods.3e5 Our group has also shown the absence of significant fibrosis after TIP in the same model used in this study.2 Our findings demonstrated that acute postoperative tissue responses subsided after 6 weeks, which is the basis of our choice of this interval for urethral harvest. The absence of fibrosis in TIP models suggests similar concentrations of structural collagens (I/III)
1859
compared to normal controls, although different concentrations of other collagens, which have diverse functions and present in much lower tissular concentrations, may be present. Different concentrations of nonstructural collagens could explain different tissular biophysical characteristics by modulation of ECM components. Alternatively varying proportions between collagens I and III may modify elastic properties and explain divergent biophysical tissular responses. Specific collagens have distinct biological functions related to modulation of tissue metabolism, reconstruction, angiogenesis, healing and remodeling processes, programmed by complex interactions with cell receptors and ECM components, including biophysical signaling. Besides the concentration of the protein per se, the 3-dimensional collagen architecture contributes to mechanical tissue properties. We chose to study collagens related to tissue structure (fibril forming in collagens I and III, microfibrilar in VI and short chain in VIII), basal membrane (IV) and transmembrane regulator (XIII). Collagens I and III directly relate to tissue biophysical properties, VI and VIII are thought to be fibrogenic through fibroblast recruiting, IV is the most important structural component of basal membranes, and XIII regulates growth and regenerative tissular processes.7e9 Collagen I/III proportion increases in the initial phases of urethral healing.4 Collagen III is abundant in elastic tissues and visceral reticular fibers, possibly relating to elasticity and organization of elastic fibers.7,10 Baskin et al suggest that the
Figure 5. Panoramic views of urethras. Arrows indicate fibrotic areas on dorsal urethra corresponding to areas where grafts were inserted. CB, cavernosum body. G, group. HE, hematoxylin and eosin. VU, ventral urethra. Reduced from 20.
1860
REDUCED URINARY FLOW AFTER TUBULARIZED INCISED PLATE URETHROPLASTY
Figure 6. Elastic fiber counts. Blue bars represent group (G) 1. Violet bars indicate group 2. Yellow bars represent group 3.
normal human urethra is composed of 75% collagen I and 25% collagen III, while in strictured urethras the proportions are 83% and 16%.11 In our model involving animals without stenoses in a late phase of healing the mean proportions of mRNA for collagen I/III were 1.05 (controls), 1.21 (TIP) and 0.87 (TIPG), suggesting that TIP may increase and TIPG may decrease the collagen I/III proportion. By contrast, Hayashi et al assert that collagen III is absent in the urethral plates.12 Interpreting these disparate results is difficult, as their study evaluated urethral plates in human children and used immunohistochemical analysis to map collagen distribution, while our study deals with normal urethras in rabbits and estimates collagen concentration with PCR, without considering tissular architectural distribution of the protein. Collagen IV RNA was increased in the experimental groups, predominating in TIPG animals. Hayashi et al studied collagen IV distribution in biopsies taken from the tissue between the urethral plate and adventitia of the corpora in human children and suggest that collagen IV is restricted to basal membranes of the capillaries.12 However, collagen IV has been described in the basement membrane of the urothelium and detrusor muscle by others, who relate focal losses of collagen IV reactivity to inflammation, dysplasia and noninvasive bladder cancer.13,14 It is possible that the restricted distribution of collagen IV described by Hayashi et al is due to the restricted biopsies taken, which did not include urethral epithelium.12 In our research collagen VI mRNA expression tended to be decreased in the experimental groups. This collagen is ubiquitous and forms networks linking either cells and basement membranes to the matrix or collagen I and III fibrils to the basement membranes, suggesting an important role in the regulation of tissular architecture. In humans collagen VI mRNA expression stimulates fibroblast proliferation and is up-regulated in the skin from 3 days after tissue trauma, persisting during the late healing phase, possibly with an active role in fibrogenesis.15
Collagen VIII may be related to ECM stabilization, active remodeling, myocyte migration and dedifferentiation into fibroblasts.16e18 Data suggest that collagen VIII is actively secreted by myocytes in the remodeling processes of biological tubes (vessels and bronchi) exposed to intermittent continued mechanical stresses and in tissue trauma.18,19 We did not expect this collagen to be decreased in the experimental groups, especially in TIPG models, considering its role in remodeling and fibroblast recruiting. Reduced synthesis of type VIII collagen may be due to a decreased number of myocytes in our model, which could modify tissue remodeling after trauma. Assessing the differential cell populations in the wounds may help to determine an explanation for the reduced levels of collagen VIII in the operated animals in the near future. Concerning elastic fibers, our intention was to estimate the number and distribution of mature fibers, reflecting the effective elasticity of the tissue and excluding nonfunctional elastin (elastosis). This is why we chose direct counting of the fibers instead of measurement of elastin. We opted for manual grid counting to eliminate potential errors due to mistaken automatic counting of other deep, dark structures (such as cell nuclei) by automatic system software after the Movat dyeing method. Tissular compliance combines resilience, deformability and morphological stability, and involves interactions between elastin and collagen, as well as their tridimensional distribution. The system is dynamic, and interactions between ECM, cells and mechanical stimuli modulate tissue conformation. Elastic fibers are essential in organs that undergo periodic changes in shape, such as the male urethra, which is periodically distended and elongated during voiding and erections. Urethral connective tissue contains abundant elastin fibers distributed in the ECM that form a net. The urethral submucosa displays the highest volumetric proportion of elastic fibers/tissue among all human tissues.20 Elastic fibers are long, numerous and tortuous, distributing mainly in the spongiosum and immediately beneath the basement membrane.21,22 Human fetuses exhibit progressive accumulation of elastin fibers from the second trimester to term, with the number, size and thickness of elastic fibers increasing almost 4 times from the fetal second trimester to adulthood. Interestingly the concentration of elastic fibers in the adult is 1.3 times greater than in term fetuses, implying that some effective elastogenesis occurs after birth.21 Studies about elastic fiber distribution in the urethra are rare. Most of the information available concerns the cavernosum and spongiosum bodies and deals with erection mechanisms. To our knowledge
REDUCED URINARY FLOW AFTER TUBULARIZED INCISED PLATE URETHROPLASTY
there are no published reports concerning the relationship between elastin fibers and voiding. Our results confirm a decreased concentration of elastin fibers in the dorsal urethra of normal rabbits. Elastin fiber counts were comparable between the groups, implying that elastic fiber loss may not explain the postoperative biomechanical problems. The absence of spongiosum influences the determination of the biomechanical properties of neourethras constructed to treat hypospadias. However, we were unable to find data comparing urethral function with and without spongioplasty. Our model does not consider this specific aspect, as it departs from normal urethras that are not devoid of spongiosum. Our study has several limitations. First, our experimental model, despite being well accepted for hypospadias surgery, is performed in adult rabbits, whose urethras are anatomically different from human counterparts and were exposed to androgenic stimuli. The reconstructions were done in urethral remnants, which are not real hypospadiac urethral plates. These limitations are also present in other experimental series dealing with hypospadias, as there is still no easily available and predictable animal model of congenital hypospadias. Another limitation is that assessment of elastin fibers in a unidimensional frame and measurement of collagen concentrations do not reveal the tissular tridimensional structure, which may be important in biomechanical functions. Furthermore, although there are no published data specifically concerning the urethra, the collagen structure of the penis changes with maturation and age.23 It is possible that some of our findings would be different in
1861
immature animals, especially concerning collagen XIII, which is over expressed in immature and growing tissues.9 Finally, our study analyzed quantitative mRNA expression for collagen, which may differ from final protein expression, despite a direct relationship between mRNA and final protein concentrations being expected. Future research regarding specific collagen tissular distribution by immunohistochemical analysis would be a further step in explaining the relationship between tissular proteins, collagen and biophysical properties of the virgin and postoperative urethra, by showing the architectural pattern of distribution of different types of collagen. Also studying the cell populations in each model, especially myofibroblasts and myocytes, could help us to understand different circumstantial patterns of collagen secretion.
CONCLUSIONS Decreased concentration of elastic fibers does not explain reduced urethral compliance after TIP in this model. The dorsal urethral segment regenerated after TIP incision but healing with fibrosis occurred in the ventral sutured areas in the operated animals and grafted areas after TIPG.
ACKNOWLEDGMENTS Paula Pflugfelder and Marvin Estrada, Experimental Surgery Department, and Greg Patterson, engineering technician, Hospital for Sick Children, Toronto University, Toronto, Ontario, Canada, assisted with the study. Alexandre Santos Aguiar conducted biostatistical analysis.
REFERENCES 1. Snodgrass W: Tubularized, incised plate urethroplasty for distal hypospadias. J Urol 1994; 151: 464. 2. Leslie B, Jesus LE, El-Hout Y et al: Comparative histological and functional controlled analysis of tubularized incised plate urethroplasty with and without dorsal inlay graft: a preliminary experimental study in rabbits. J Urol, suppl., 2011; 186: 1631.
the urethral wound: an experimental study. J Pediatr Urol 2006; 2: 182. 6. Jesus LE, Schanaider A, Patterson G et al: Urethral compliance in hypospadias operated by tubularized incised urethral plate (TIP) with and without a dorsal inlay graft: an experimental controlled study. World J Urol 2013; 31: 971. 7. Gelse K, P€oschl E and Aigner T: Collagensdstructure, function, and biosynthesis. Adv Drug Deliv Rev 2003; 55: 1531.
3. Bleustein CB, Esposito MP, Soslow RA et al: Mechanism of healing following the Snodgrass repair. J Urol 2001; 165: 277.
8. Gordon MK and Hahn RA: Collagens. Cell Tissue Res 2010; 339: 247.
4. Taneli F, Ulman C, Genc A et al: Biochemical analysis of urethral collagen after tubularized incised plate urethroplasty: an experimental study in rabbits. Urol Res 2004; 32: 219.
9. Hiekkinen A, Tu H and Pihlajaniemi T: Collagen XIII: a type II transmembrane protein with relevance to musculoskeletal tissues, microvessels and inflammation. Int J Biochem Cell Biol 2012; 44: 714.
5. Somuncu S, Caglayan O, Cakmak M et al: The effect of indwelling catheter on OH-proline in
10. von der Mark K: Localization of collagen types in tissues. Int Rev Connect Tissue Res 1981; 9: 265.
11. Baskin LS, Constantinescu SC, Howard PS et al: Biochemical characterization and quantitation of the collagenous components of urethral stricture tissue. J Urol 1993; 150: 642. 12. Hayashi Y, Mizuno K, Kojuma Y et al: Characterization of the urethral plate and the underlying tissue defined by expression of collagen subtypes and microarchitecture in hypospadias. Int J Urol 2011; 18: 217. 13. Borza DB, Bondar O, Ninomiya Y et al: The NC1 domain of collagen IV encodes a novel network composed of the alpha 1, alpha 2, alpha 5 and alpha 6 chains in smooth muscle basement membranes. J Biol Chem 2001; 276: 28532. 14. Deen S and Ball RY: Basement membrane and extracellular interstitial matrix components in bladder neoplasiadevidence of angiogenesis. Histopathology 1994; 25: 475.
1862
REDUCED URINARY FLOW AFTER TUBULARIZED INCISED PLATE URETHROPLASTY
15. Oono T, Specks U, Eckes B et al: Expression of type VI collagen mRNA during wound healing. J Invest Dermatol 1993; 100: 329. 16. MacBeath JR, Kielty CM and Shuttleworth CA: Type VIII collagen is a product of vascular smooth-muscle cells in development and disease. Biochem J 1996; 319: 993. 17. Cherepanova OA, Pidkovka NA, Sarmento OF et al: Oxidized phospholipids induce type VIII collagen expression and vascular smooth cell migration. Circ Res 2009; 104: 609.
18. Hou G, Mulholland D, Gronska MA et al: Type VIII collagen stimulates smooth muscle cell migration and matrix metalloproteinase synthesis after arterial injury. Am J Pathol 2000; 156: 467. 19. Hasaneen NA, Zucker S, Lin RZ et al: Angiogenesis is induced by airway smooth muscle strain. Am J Physiol Lung Cell Mol Physiol 2007; 293: L1059. 20. Testut L: Treatise on Human Anatomy, 16th ed. Paris: Doin 1912; vol 4, pp 507e534 and 627e630.
21. Bastos AL, Silva EA, Costa WS et al: Concentration of elastic fibers in the male urethra during human fetal development. BJU Int 2004; 94: 620. 22. Hsu GL, Brock A, Von Heyden B et al: The distribution of elastic fibrous elements within the human penis. Br J Urol 1994; 73: 566. 23. Mersdorf A, Goldsmith PC, Diederichs W et al: Ultrastructural changes in impotent penile tissue: comparison of 65 patients. J Urol 1991; 145: 749.