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Bladder acellular matrix grafting regenerates urinary bladder in the spinal cord injury rat
BASIC SCIENCE
BLADDER ACELLULAR MATRIX GRAFTING REGENERATES URINARY BLADDER IN THE SPINAL CORD INJURY RAT TAKASHI OBARA, SHINOBU MATSUURA, SHINTARO NARITA, SHIGERU SATOH, NORIHIKO TSUCHIYA, AND TOMONORI HABUCHI
ABSTRACT Objectives. To assess the feasibility of bladder acellular matrix (BAM) grafting onto the bladder of rats with spinal cord injury (SCI). Methods. Female Wistar rats, weighing 100 to 150 g, were divided into four groups: neurologically intact groups with sham operation or BAM grafting and SCI rats with or without BAM grafting (grafted groups, n ⫽ 15 each; nongrafted groups, n ⫽ 5 each). The BAM was prepared from other normal rat bladder tissue. During BAM surgery, the rats underwent partial cystectomy, followed by BAM grafting as a bladder augmentation. The SCI was created by compressing the spinal cord at the 10th thoracic level. BAM grafting in SCI rats was performed 2 to 3 weeks after SCI. At 2, 4, and 12 weeks after grafting, cystometry was performed with the rats under pentobarbital anesthesia, and the bladders were subsequently harvested and immunostained with anti-PGP9.5, uroplakin III, and alpha-smooth muscle actin antibodies (n ⫽ 5 each time). For comparison, similar examinations were performed in the nongrafted groups (n ⫽ 5 each). Results. Regenerated urothelium, smooth muscles, and nerve fibers in the grafted BAM appeared at 2, 4, and 12 weeks, respectively, in both intact and SCI rats. Immunohistologic examination showed that these regenerated tissues inherited each characteristic of the host bladder tissue. The grafted BAM itself also showed the proper storage function of distensibility in the intact and SCI groups receiving BAM. Conclusions. Our data have indicated that BAM grafting is feasible, even in animals with spinal injury, suggesting that BAM may be one of the alternatives for patients with a neurogenic bladder who require augmentation enterocystoplasty in clinical situations. UROLOGY 68: 892–897, 2006. © 2006 Elsevier Inc.
A
ugmentation enterocystoplasty is suitable for selected patients with refractory overactive or end-stage bladder disorders that have failed to respond to conservative therapies.1–3 However, concerns regarding the potential surgical complications, dysbolism, stone formation, diarrhea, and cancer induction have limited its application.1–3 Recent animal experiments have shown promising outcomes after use of bladder acellular matrix (BAM) for urinary bladder regeneration,4 –7 suggesting that BAM could be one of the alternatives to augmentation enterocystoplasty. In clinical situations, neurogenic bladder has recently become the most common indication for From the Department of Urology, Akita University School of Medicine, Akita, Japan Reprint requests: Shinobu Matsuura, M.D., Department of Urology, Akita University School of Medicine, Hondo 1-1-1, Akita 010-8543, Japan. E-mail: [email protected] Submitted: December 14, 2005, accepted (with revisions): April 25, 2006 © 2006 ELSEVIER INC. 892
ALL RIGHTS RESERVED
augmentation surgery.1–3 However, no BAM experiments have been using animals with neurogenic bladder. The aim of this study was to examine the feasibility of BAM grafting in a rat with spinal cord injury (SCI). MATERIAL AND METHODS ANIMALS A total of 45 female Wister rats, weighing 100 to 150 g, were studied. Four rats died after the SCI surgery, and the remaining 40 rats were evaluated. The animals were divided into four groups: neurologically intact rats with sham-operation alone (intact group, n ⫽ 5) and BAM grafting (intact-BAM, n ⫽ 15) and two SCI groups with or without BAM grafting (SCI, n ⫽ 5 and SCI-BAM, n ⫽ 15, respectively).
BAM PREPARATION The BAM was basically prepared according to the method of Probst et al.7 In brief, whole bladder tissues were obtained from other intact adult female Wistar rats (weight 100 to 150 g) and were chemically treated with three solutions: sodium azide (0.1% in phosphate-buffered saline), deoxyribo0090-4295/06/$32.00 doi:10.1016/j.urology.2006.04.030
nuclease (2000 Kunitz units in 0.1 M NaCl), and sodium deoxycholate (4% in distilled water). The BAMs were stored in 10% neomycin sulfate at 4°C until grafting.
SPINAL INJURY After anesthesia with pentobarbital sodium (50 mg/kg intraperitoneally), laminectomy was applied at the 10th thoracic level to expose the spinal cord segment. SCI was created by compression with a 40-g rod, whose tip had an area of 3.0 ⫻ 2.2 mm, placed on the exposed dura for 30 minutes.8 Postoperatively, enrofloxacin (20 mg/kg subcutaneously) was prescribed for 2 days. After spinal injury, the bladder was compressed manually for urination twice daily until the rats were killed. Throughout this experiment, additional antibiotics were administered for a few days if urinary infection was confirmed.
BAM IMPLANTATION Under pentobarbital anesthesia, the bladder was exposed by way of a suprapubic midline incision. Partial cystectomy of the upper half of the bladder was performed without injury to the ureter. A BAM was firmly anastomosed to the host bladder with running and interlocking 7-0 absorbable sutures. Two 7-0 nylon sutures were placed as markers. Additionally, the anastomosis ends were marked with two titanium clips in 3 rats of each BAM-grafted group to check by radiography whether the grafted BAM was sufficiently expandable. Perigenital stimulation in the SCI rats encouraged reflex bladder emptying 2 to 3 weeks after spinal injury, and this phenomenon made urine evacuation easier.9 Next, the SCI-BAM rats received BAM grafting and the manual bladder compression was continued until death in all SCI rats. All surgical procedures were performed microscopically.
URODYNAMICS Cystometry was performed at 2, 4, and 12 weeks in the intact-BAM and SCI-BAM rats. After pentobarbital anesthesia, a polyethylene tube (PE-60, Clay Adams) with a sidearm for bladder infusion was gently placed transurethrally into the bladder. The tube was attached to pressure transducers (Statham P-23, Gulton Statham Transducers). The pressure signals were digitized with a measuring instrument (Menuet Compact, Dantec Medical A/S, Denmark). The bladder was constantly infused with room temperature 0.9% saline (0.1 mL/min). Administration of pentobarbital sodium (50 mg/kg intraperitoneally) induced sufficient anesthetization, and spontaneous micturition reflex was confirmed to have vanished, as previously reported.10 Therefore, we only checked the leak-point volume and pressure when saline leaked from the meatus around the tube. At 12 weeks, radiographic examinations were simultaneously performed in rats with titanium clipping.
HISTOLOGIC EXAMINATION After urodynamic evaluation, the rats were perfused using a transcardial approach with 0.01 M phosphate-buffered saline followed by 4% paraformaldehyde in phosphate-buffered saline. The bladder was removed, postfixed in the same fixative, and embedded in paraffin. A series of 4-m sections was obtained and stained with azan to assess microscopically. In addition, the sections were immunolabeled and visualized according to an avidin-biotin-complex method using avidin-biotin-complex and diaminobenzidine substrate kits (Vector Laboratories). Immunostaining was performed with the anti-protein gene product 9.5 (1:3000, PGP9.5, Ultraclone), uroplakin III (1:3000, Nichirei Bioscience), and alUROLOGY 68 (4), 2006
pha-smooth muscle actin (1:6000, Nichirei Bioscience) antibodies specific to nerves, urothelium, and smooth muscle cells, respectively. Images were captured by the video system connected by microscopy. The smooth muscle/connective tissue ratio within the bladder wall was quantitatively examined 12 weeks after BAM surgery using the Image, version J 1.34, software (National Institutes of Health, Bethesda, Md). Section slides immunostained with alpha-smooth muscle actin were selected (five fields for each group), and the number of pixels within the bladder wall muscle layer was counted. Pixels in the host and BAM-grafted regions of bladder were counted separately.
STATISTICAL ANALYSIS
The t test was used for statistical analysis, P ⬍0.05 was considered significant.
RESULTS Five rats that underwent SCI died of bladder rupture (n ⫽ 3) and presumed upper tract damages (n ⫽ 2) before BAM grafting. The remaining 40 rats were used for evaluations. BAM grafting itself did not cause fatal complications in this study. FUNCTIONAL EVALUATIONS Cystometry was performed at 2, 4, and 12 weeks postoperatively. Because pentobarbital anesthesia suppressed the neural circuit of the micturition reflex,10 the intravesical pressure rose gradually until the infused saline leaked from the meatus according to the bladder wall property. The leakpoint volume of the SCI rat was significantly larger than that of neurologically intact rats (Fig. 1A), and the leak-point pressure of the SCI rats was significantly lower that of the neurologically intact rats (Fig. 1B). These results indicated that the bladder wall tissue of the SCI rats became more compliant, and similar results have been previously documented.11 BAM grafting did not seem to influence the leak-point volume and pressure because no significant differences were found in the leak-point volume and pressure between the grafted and nongrafted bladders in each neurologically intact or SCI group (Fig. 1A,B). The radiographic examination at 12 weeks revealed that the grafted BAM obtained good distension in the intact-BAM and SCI-BAM rats (Fig. 1C,D); however, the BAMs in 4 rats macroscopically shrank (2 rats each in the intact-BAM and SCI-BAM groups), and all these rats had stones that almost entirely occupied the bladder lumen. Cystography of these rat bladders showed poor distensibility (Fig. 1E). These 4 rats presented with pyuria after BAM grafting, and the additional administration of antibiotics was required for several days. No other BAMgrafted rats showed urine infection or stone formation. 893
FIGURE 1. Leak-point (A) volume and (B) pressure of intact-BAM, SCI-BAM, and non-BAM grafted rats. Radiographic examinations of BAM-grafted bladders of (C) intact-BAM and (D,E) SCI-BAM rats. Note, leak-point volume and pressure of SCI rats was larger and lower, respectively, than that of neurologically intact rats, regardless of BAM grafting. Cystographies C and D show excellent distension of BAM-grafted region, and E shows stones in augmented BAM region. Arrows indicate titanium clips at anastomosis.
HISTOLOGIC EVALUATIONS The inside of the grafted BAM of intact-BAM and SCI-BAM rats was completely lined with urothelium within 2 weeks, and muscle cells migrating into the matrix in association with the new urothelium were observed within 4 weeks in both groups (Fig. 2). The regenerated urothelium and muscles in the grafted BAM were positively immunostained with anti-uroplakin III and alpha-smooth muscle actin antibodies, respectively (Fig. 3). Nerve fibers, immunostained with anti-PGP 9.5 antibody, appeared at 4 weeks (Fig. 3). These results have indicated that the regenerating process and pattern in grafted BAM may be similar between neurologically intact animals and those with spinal injury. The quantitative analysis showed that the BAM wall had a significantly smaller volume of smooth muscle compared with the host bladder wall of intact-BAM and SCI-BAM groups at 12 weeks 894
(Fig. 4). In contrast, significance was found in the ratio in the BAM-grafted region between the two groups (Fig. 4). COMMENT Our data have indicated that host cells can reconstitute novel bladder tissue into BAM grafted into the neurogenic bladder. The regenerated urothelium and smooth muscle/nerve fibers were observed at 2 and 4 weeks after BAM surgery, and the grafted BAM showed proper distension to store urine at 12 weeks. Clearly, these results compare favorably with those previously reported in normal rats.5,7 BAM is basically the acellular collagen matrix of the whole bladder and is made by the chemical digestion method, which removes all bladder com UROLOGY 68 (4), 2006
FIGURE 2. Azan staining of grafted BAM regions. Urothelium and muscle components were regenerated in (A) intact and (B) SCI rats at 12 weeks. Urothelium was completely lined inside grafted BAM (arrows) at 2 weeks (C), and muscle cells (asterisks) appeared at 4 weeks (D) and 12 weeks (E). Scale bar ⫽ 200 m.
ponents, except for collagen and elastin components, leaving their arrangement as is.12 BAM has not only been specifically used in urinary bladder regeneration, and excellent outcomes were obtained in other organs (eg, urethra and vaginal wall).13,14 Recently, several collagen-based biodegradable materials were applied to the reconstruction of several organs.15,16 These materials, such as small intestine submucosa and allogenic bladder submucosa, other than BAM, are made from a part of the organs.15,17 A few reports have raised some concerns that the implanted acellular matrix (eg, small intestine submucosa) could cause fibrosis, resulting in the atrophy of regenerated organs on a long-term basis.17 Presumably, retention of the UROLOGY 68 (4), 2006
whole bladder wall collagen arrangement, such as pore shape and size, contributes partly to the favorable biocompatibility and regenerating capability of BAM. However, the atrophy of grafted BAM, accompanied with stone formation, was confirmed in our 4 rats. These 4 rats had pyuria, and such an infection might have triggered calcification followed by the atrophic degeneration of BAM. We presumed that control of urinary infection was critical for successful regeneration after BAM grafting. It is hypothesized that epithelial-mesenchymal interactions are critical to regenerate tissues as an organ. In the bladder, signaling from the urothelium is thought to encourage the bladder mesen895
FIGURE 3. Representative immunostained sections with anti-uroplakin III, alpha-smooth muscle actin, and PGP9.5 antibodies of BAM-grafted (intact-BAM and SCI-BAM) and nongrafted bladders (intact and SCI). Note, regenerated urothelium, smooth muscles, and nerve fibers in grafted BAM appeared 2, 4, and 12 weeks, respectively, in intact and SCI rats. Scale bar ⫽ 50 m for anti-uroplakin III and SMA; scale bar ⫽ 100 m for anti-PGP9.5.
chyme to differentiate into smooth muscle cells.18 Several urothelium-originated growth factors such as fibroblast growth factor, keratinocyte growth factor, and transforming growth factor are believed to play an important role in organogenesis.18,19 In contrast, evidence has suggested that smooth muscle cells themselves may create an environment for differentiation.20 There are various conditions of neurogenic bladders, and some patients might have few tissues with good regenerative activity, especially among patients requiring aug896
mentation enterocystoplasty. In those patients, the addition of certain nutrition factors could be more beneficial to facilitate bladder regeneration when BAM is clinically applied in the future. CONCLUSIONS Our results have shown the feasibility of BAM transplantation in SCI animals, raising the future possibility that BAM grafting may be one of the UROLOGY 68 (4), 2006
FIGURE 4. Smooth muscle/connecting tissue ratio within host bladder and grafted BAM of intact-BAM and SCI-BAM rats at 12 weeks. Smooth muscle ratio was smaller in BAM than in host regions in both groups. Note, intact-BAM and SCI-BAM had a similar ratio in host or BAM-grafted regions with no significant difference.
clinical alternatives for patients with end-stage neurogenic bladder. REFERENCES 1. Blaivas JG, Weiss JP, Desai P, et al: Long-term followup of augmentation enterocystoplasty and continent diversion in patients with benign disease. J Urol 173: 1631–1634, 2005. 2. Gilbert SM, and Hensle TW: Metabolic consequences and long-term complications of enterocystoplasty in children: a review. J Urol 173: 1080 –1086, 2005. 3. Hasan ST, Marshall C, Robson WA, et al: Clinical outcome and quality of life following enterocystoplasty for idiopathic detrusor instability and neurogenic bladder dysfunction. Br J Urol 76: 551–557, 1995. 4. Dahms SE, Piechota HJ, Dahiya R, et al: Bladder acellular matrix graft in rats: its neurophysiologic properties and mRNA expression of growth factors TGF-alpha and TGF-beta. Neurourol Urodyn 17: 37–54, 1998. 5. Piechota HJ, Dahms SE, Nunes LS, et al: In vitro functional properties of the rat bladder regenerated by the bladder acellular matrix graft. J Urol 159: 1717–1724, 1998.
UROLOGY 68 (4), 2006
6. Piechota HJ, Dahms SE, Probst M, et al: Functional rat bladder regeneration through xenotransplantation of the bladder acellular matrix graft. Br J Urol 81: 548 –559, 1998. 7. Probst M, Piechota HJ, Dahiya R, et al: Homologous bladder augmentation in dog with the bladder acellular matrix graft. BJU Int 85: 362–371, 2000. 8. Ogawa Y, Sawamoto K, Miyata T, et al: Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats. J Neurosci Res 69: 925–933, 2002. 9. Kakizaki H, and de Groat WC: Reorganization of somato-urethral reflexes following spinal cord injury in the rat. J Urol 158: 1562–1567, 1997. 10. Matsuura S, and Downie JW: Effect of anesthetics on reflex micturition in the chronic cannula-implanted rat. Neurourol Urodyn 19: 87–99, 2000. 11. Gloeckner DC, Sacks MS, Fraser MO, et al: Passive biaxial mechanical properties of the rat bladder wall after spinal cord injury. J Urol 167: 2247–2252, 2002. 12. Sutherland RS, Baskin LS, Hayward SW, et al: Regeneration of bladder urothelium, smooth muscle, blood vessels and nerves into an acellular tissue matrix. J Urol 156: 571– 577, 1996. 13. Sievert KD, Wefer J, Bakircioglu ME, et al: Heterologous acellular matrix graft for reconstruction of the rabbit urethra: histological and functional evaluation. J Urol 165: 2096 –2102, 2001. 14. Wefer J, Sekido N, Sievert KD, et al: Homologous acellular matrix graft for vaginal repair in rats: a pilot study for a new reconstructive approach. World J Urol 20: 260 –263, 2002. 15. Kropp BP, Eppley BL, Prevel CD, et al: Experimental assessment of small intestinal submucosa as a bladder wall substitute. Urology 46: 396 – 400, 1995. 16. Yoo JJ, Meng J, Oberpenning F, et al: Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology 51: 221–225, 1998. 17. Kropp BP, Cheng EY, Lin HK, et al: Reliable and reproducible bladder regeneration using unseeded distal small intestinal submucosa. J Urol 172: 1710 –1713, 2004. 18. Baskin LS, Hayward SW, Sutherland RA, et al: Cellular signaling in the bladder. Front Biosci 2: d592– d595, 1997. 19. Kanematsu A, Yamamoto S, Noguchi T, et al: Bladder regeneration by bladder acellular matrix combined with sustained release of exogenous growth factor. J Urol 170: 1633– 1638, 2003. 20. Kanematsu A, Yamamoto S, Iwai-Kanai E, et al: Induction of smooth muscle cell-like phenotype in marrow-derived cells among regenerating urinary bladder smooth muscle cells. Am J Pathol 166: 565–573, 2005.
897
BLADDER ACELLULAR MATRIX GRAFTING REGENERATES URINARY BLADDER IN THE SPINAL CORD INJURY RAT TAKASHI OBARA, SHINOBU MATSUURA, SHINTARO NARITA, SHIGERU SATOH, NORIHIKO TSUCHIYA, AND TOMONORI HABUCHI
ABSTRACT Objectives. To assess the feasibility of bladder acellular matrix (BAM) grafting onto the bladder of rats with spinal cord injury (SCI). Methods. Female Wistar rats, weighing 100 to 150 g, were divided into four groups: neurologically intact groups with sham operation or BAM grafting and SCI rats with or without BAM grafting (grafted groups, n ⫽ 15 each; nongrafted groups, n ⫽ 5 each). The BAM was prepared from other normal rat bladder tissue. During BAM surgery, the rats underwent partial cystectomy, followed by BAM grafting as a bladder augmentation. The SCI was created by compressing the spinal cord at the 10th thoracic level. BAM grafting in SCI rats was performed 2 to 3 weeks after SCI. At 2, 4, and 12 weeks after grafting, cystometry was performed with the rats under pentobarbital anesthesia, and the bladders were subsequently harvested and immunostained with anti-PGP9.5, uroplakin III, and alpha-smooth muscle actin antibodies (n ⫽ 5 each time). For comparison, similar examinations were performed in the nongrafted groups (n ⫽ 5 each). Results. Regenerated urothelium, smooth muscles, and nerve fibers in the grafted BAM appeared at 2, 4, and 12 weeks, respectively, in both intact and SCI rats. Immunohistologic examination showed that these regenerated tissues inherited each characteristic of the host bladder tissue. The grafted BAM itself also showed the proper storage function of distensibility in the intact and SCI groups receiving BAM. Conclusions. Our data have indicated that BAM grafting is feasible, even in animals with spinal injury, suggesting that BAM may be one of the alternatives for patients with a neurogenic bladder who require augmentation enterocystoplasty in clinical situations. UROLOGY 68: 892–897, 2006. © 2006 Elsevier Inc.
A
ugmentation enterocystoplasty is suitable for selected patients with refractory overactive or end-stage bladder disorders that have failed to respond to conservative therapies.1–3 However, concerns regarding the potential surgical complications, dysbolism, stone formation, diarrhea, and cancer induction have limited its application.1–3 Recent animal experiments have shown promising outcomes after use of bladder acellular matrix (BAM) for urinary bladder regeneration,4 –7 suggesting that BAM could be one of the alternatives to augmentation enterocystoplasty. In clinical situations, neurogenic bladder has recently become the most common indication for From the Department of Urology, Akita University School of Medicine, Akita, Japan Reprint requests: Shinobu Matsuura, M.D., Department of Urology, Akita University School of Medicine, Hondo 1-1-1, Akita 010-8543, Japan. E-mail: [email protected] Submitted: December 14, 2005, accepted (with revisions): April 25, 2006 © 2006 ELSEVIER INC. 892
ALL RIGHTS RESERVED
augmentation surgery.1–3 However, no BAM experiments have been using animals with neurogenic bladder. The aim of this study was to examine the feasibility of BAM grafting in a rat with spinal cord injury (SCI). MATERIAL AND METHODS ANIMALS A total of 45 female Wister rats, weighing 100 to 150 g, were studied. Four rats died after the SCI surgery, and the remaining 40 rats were evaluated. The animals were divided into four groups: neurologically intact rats with sham-operation alone (intact group, n ⫽ 5) and BAM grafting (intact-BAM, n ⫽ 15) and two SCI groups with or without BAM grafting (SCI, n ⫽ 5 and SCI-BAM, n ⫽ 15, respectively).
BAM PREPARATION The BAM was basically prepared according to the method of Probst et al.7 In brief, whole bladder tissues were obtained from other intact adult female Wistar rats (weight 100 to 150 g) and were chemically treated with three solutions: sodium azide (0.1% in phosphate-buffered saline), deoxyribo0090-4295/06/$32.00 doi:10.1016/j.urology.2006.04.030
nuclease (2000 Kunitz units in 0.1 M NaCl), and sodium deoxycholate (4% in distilled water). The BAMs were stored in 10% neomycin sulfate at 4°C until grafting.
SPINAL INJURY After anesthesia with pentobarbital sodium (50 mg/kg intraperitoneally), laminectomy was applied at the 10th thoracic level to expose the spinal cord segment. SCI was created by compression with a 40-g rod, whose tip had an area of 3.0 ⫻ 2.2 mm, placed on the exposed dura for 30 minutes.8 Postoperatively, enrofloxacin (20 mg/kg subcutaneously) was prescribed for 2 days. After spinal injury, the bladder was compressed manually for urination twice daily until the rats were killed. Throughout this experiment, additional antibiotics were administered for a few days if urinary infection was confirmed.
BAM IMPLANTATION Under pentobarbital anesthesia, the bladder was exposed by way of a suprapubic midline incision. Partial cystectomy of the upper half of the bladder was performed without injury to the ureter. A BAM was firmly anastomosed to the host bladder with running and interlocking 7-0 absorbable sutures. Two 7-0 nylon sutures were placed as markers. Additionally, the anastomosis ends were marked with two titanium clips in 3 rats of each BAM-grafted group to check by radiography whether the grafted BAM was sufficiently expandable. Perigenital stimulation in the SCI rats encouraged reflex bladder emptying 2 to 3 weeks after spinal injury, and this phenomenon made urine evacuation easier.9 Next, the SCI-BAM rats received BAM grafting and the manual bladder compression was continued until death in all SCI rats. All surgical procedures were performed microscopically.
URODYNAMICS Cystometry was performed at 2, 4, and 12 weeks in the intact-BAM and SCI-BAM rats. After pentobarbital anesthesia, a polyethylene tube (PE-60, Clay Adams) with a sidearm for bladder infusion was gently placed transurethrally into the bladder. The tube was attached to pressure transducers (Statham P-23, Gulton Statham Transducers). The pressure signals were digitized with a measuring instrument (Menuet Compact, Dantec Medical A/S, Denmark). The bladder was constantly infused with room temperature 0.9% saline (0.1 mL/min). Administration of pentobarbital sodium (50 mg/kg intraperitoneally) induced sufficient anesthetization, and spontaneous micturition reflex was confirmed to have vanished, as previously reported.10 Therefore, we only checked the leak-point volume and pressure when saline leaked from the meatus around the tube. At 12 weeks, radiographic examinations were simultaneously performed in rats with titanium clipping.
HISTOLOGIC EXAMINATION After urodynamic evaluation, the rats were perfused using a transcardial approach with 0.01 M phosphate-buffered saline followed by 4% paraformaldehyde in phosphate-buffered saline. The bladder was removed, postfixed in the same fixative, and embedded in paraffin. A series of 4-m sections was obtained and stained with azan to assess microscopically. In addition, the sections were immunolabeled and visualized according to an avidin-biotin-complex method using avidin-biotin-complex and diaminobenzidine substrate kits (Vector Laboratories). Immunostaining was performed with the anti-protein gene product 9.5 (1:3000, PGP9.5, Ultraclone), uroplakin III (1:3000, Nichirei Bioscience), and alUROLOGY 68 (4), 2006
pha-smooth muscle actin (1:6000, Nichirei Bioscience) antibodies specific to nerves, urothelium, and smooth muscle cells, respectively. Images were captured by the video system connected by microscopy. The smooth muscle/connective tissue ratio within the bladder wall was quantitatively examined 12 weeks after BAM surgery using the Image, version J 1.34, software (National Institutes of Health, Bethesda, Md). Section slides immunostained with alpha-smooth muscle actin were selected (five fields for each group), and the number of pixels within the bladder wall muscle layer was counted. Pixels in the host and BAM-grafted regions of bladder were counted separately.
STATISTICAL ANALYSIS
The t test was used for statistical analysis, P ⬍0.05 was considered significant.
RESULTS Five rats that underwent SCI died of bladder rupture (n ⫽ 3) and presumed upper tract damages (n ⫽ 2) before BAM grafting. The remaining 40 rats were used for evaluations. BAM grafting itself did not cause fatal complications in this study. FUNCTIONAL EVALUATIONS Cystometry was performed at 2, 4, and 12 weeks postoperatively. Because pentobarbital anesthesia suppressed the neural circuit of the micturition reflex,10 the intravesical pressure rose gradually until the infused saline leaked from the meatus according to the bladder wall property. The leakpoint volume of the SCI rat was significantly larger than that of neurologically intact rats (Fig. 1A), and the leak-point pressure of the SCI rats was significantly lower that of the neurologically intact rats (Fig. 1B). These results indicated that the bladder wall tissue of the SCI rats became more compliant, and similar results have been previously documented.11 BAM grafting did not seem to influence the leak-point volume and pressure because no significant differences were found in the leak-point volume and pressure between the grafted and nongrafted bladders in each neurologically intact or SCI group (Fig. 1A,B). The radiographic examination at 12 weeks revealed that the grafted BAM obtained good distension in the intact-BAM and SCI-BAM rats (Fig. 1C,D); however, the BAMs in 4 rats macroscopically shrank (2 rats each in the intact-BAM and SCI-BAM groups), and all these rats had stones that almost entirely occupied the bladder lumen. Cystography of these rat bladders showed poor distensibility (Fig. 1E). These 4 rats presented with pyuria after BAM grafting, and the additional administration of antibiotics was required for several days. No other BAMgrafted rats showed urine infection or stone formation. 893
FIGURE 1. Leak-point (A) volume and (B) pressure of intact-BAM, SCI-BAM, and non-BAM grafted rats. Radiographic examinations of BAM-grafted bladders of (C) intact-BAM and (D,E) SCI-BAM rats. Note, leak-point volume and pressure of SCI rats was larger and lower, respectively, than that of neurologically intact rats, regardless of BAM grafting. Cystographies C and D show excellent distension of BAM-grafted region, and E shows stones in augmented BAM region. Arrows indicate titanium clips at anastomosis.
HISTOLOGIC EVALUATIONS The inside of the grafted BAM of intact-BAM and SCI-BAM rats was completely lined with urothelium within 2 weeks, and muscle cells migrating into the matrix in association with the new urothelium were observed within 4 weeks in both groups (Fig. 2). The regenerated urothelium and muscles in the grafted BAM were positively immunostained with anti-uroplakin III and alpha-smooth muscle actin antibodies, respectively (Fig. 3). Nerve fibers, immunostained with anti-PGP 9.5 antibody, appeared at 4 weeks (Fig. 3). These results have indicated that the regenerating process and pattern in grafted BAM may be similar between neurologically intact animals and those with spinal injury. The quantitative analysis showed that the BAM wall had a significantly smaller volume of smooth muscle compared with the host bladder wall of intact-BAM and SCI-BAM groups at 12 weeks 894
(Fig. 4). In contrast, significance was found in the ratio in the BAM-grafted region between the two groups (Fig. 4). COMMENT Our data have indicated that host cells can reconstitute novel bladder tissue into BAM grafted into the neurogenic bladder. The regenerated urothelium and smooth muscle/nerve fibers were observed at 2 and 4 weeks after BAM surgery, and the grafted BAM showed proper distension to store urine at 12 weeks. Clearly, these results compare favorably with those previously reported in normal rats.5,7 BAM is basically the acellular collagen matrix of the whole bladder and is made by the chemical digestion method, which removes all bladder com UROLOGY 68 (4), 2006
FIGURE 2. Azan staining of grafted BAM regions. Urothelium and muscle components were regenerated in (A) intact and (B) SCI rats at 12 weeks. Urothelium was completely lined inside grafted BAM (arrows) at 2 weeks (C), and muscle cells (asterisks) appeared at 4 weeks (D) and 12 weeks (E). Scale bar ⫽ 200 m.
ponents, except for collagen and elastin components, leaving their arrangement as is.12 BAM has not only been specifically used in urinary bladder regeneration, and excellent outcomes were obtained in other organs (eg, urethra and vaginal wall).13,14 Recently, several collagen-based biodegradable materials were applied to the reconstruction of several organs.15,16 These materials, such as small intestine submucosa and allogenic bladder submucosa, other than BAM, are made from a part of the organs.15,17 A few reports have raised some concerns that the implanted acellular matrix (eg, small intestine submucosa) could cause fibrosis, resulting in the atrophy of regenerated organs on a long-term basis.17 Presumably, retention of the UROLOGY 68 (4), 2006
whole bladder wall collagen arrangement, such as pore shape and size, contributes partly to the favorable biocompatibility and regenerating capability of BAM. However, the atrophy of grafted BAM, accompanied with stone formation, was confirmed in our 4 rats. These 4 rats had pyuria, and such an infection might have triggered calcification followed by the atrophic degeneration of BAM. We presumed that control of urinary infection was critical for successful regeneration after BAM grafting. It is hypothesized that epithelial-mesenchymal interactions are critical to regenerate tissues as an organ. In the bladder, signaling from the urothelium is thought to encourage the bladder mesen895
FIGURE 3. Representative immunostained sections with anti-uroplakin III, alpha-smooth muscle actin, and PGP9.5 antibodies of BAM-grafted (intact-BAM and SCI-BAM) and nongrafted bladders (intact and SCI). Note, regenerated urothelium, smooth muscles, and nerve fibers in grafted BAM appeared 2, 4, and 12 weeks, respectively, in intact and SCI rats. Scale bar ⫽ 50 m for anti-uroplakin III and SMA; scale bar ⫽ 100 m for anti-PGP9.5.
chyme to differentiate into smooth muscle cells.18 Several urothelium-originated growth factors such as fibroblast growth factor, keratinocyte growth factor, and transforming growth factor are believed to play an important role in organogenesis.18,19 In contrast, evidence has suggested that smooth muscle cells themselves may create an environment for differentiation.20 There are various conditions of neurogenic bladders, and some patients might have few tissues with good regenerative activity, especially among patients requiring aug896
mentation enterocystoplasty. In those patients, the addition of certain nutrition factors could be more beneficial to facilitate bladder regeneration when BAM is clinically applied in the future. CONCLUSIONS Our results have shown the feasibility of BAM transplantation in SCI animals, raising the future possibility that BAM grafting may be one of the UROLOGY 68 (4), 2006
FIGURE 4. Smooth muscle/connecting tissue ratio within host bladder and grafted BAM of intact-BAM and SCI-BAM rats at 12 weeks. Smooth muscle ratio was smaller in BAM than in host regions in both groups. Note, intact-BAM and SCI-BAM had a similar ratio in host or BAM-grafted regions with no significant difference.
clinical alternatives for patients with end-stage neurogenic bladder. REFERENCES 1. Blaivas JG, Weiss JP, Desai P, et al: Long-term followup of augmentation enterocystoplasty and continent diversion in patients with benign disease. J Urol 173: 1631–1634, 2005. 2. Gilbert SM, and Hensle TW: Metabolic consequences and long-term complications of enterocystoplasty in children: a review. J Urol 173: 1080 –1086, 2005. 3. Hasan ST, Marshall C, Robson WA, et al: Clinical outcome and quality of life following enterocystoplasty for idiopathic detrusor instability and neurogenic bladder dysfunction. Br J Urol 76: 551–557, 1995. 4. Dahms SE, Piechota HJ, Dahiya R, et al: Bladder acellular matrix graft in rats: its neurophysiologic properties and mRNA expression of growth factors TGF-alpha and TGF-beta. Neurourol Urodyn 17: 37–54, 1998. 5. Piechota HJ, Dahms SE, Nunes LS, et al: In vitro functional properties of the rat bladder regenerated by the bladder acellular matrix graft. J Urol 159: 1717–1724, 1998.
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