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Pressure-limited vesico-amniotic shunt tube for fetal obstructive uropathy
Journal of Pediatric Surgery (2006) 41, 2086 – 2089
www.elsevier.com/locate/jpedsurg
Pressure-limited vesico-amniotic shunt tube for fetal obstructive uropathy Hideki Nagaea, Hiroaki Kitagawaa,*, Kevin C. Pringleb, Junki Koikec, Jane Zuccollob, Yuriko Satoa, Yasuji Sekia, Munechika Wakisakaa, Koonosuke Nakadaa a
Division of Pediatric Surgery, St Marianna University School of Medicine, Miyamae-ku, Kawasaki 216-8511, Japan Department of Obstetrics and Gynecology, Wellington School of Medicine, Wellington, New Zealand c Department of Pathology, St Marianna University School of Medicine, Kawasaki, Japan b
Index words: Fetal surgery; Obstructive uropathy; Bladder; Fibrosis; Posterior urethral valves
Abstract Introduction: In utero shunting (vesico-amniotic shunt) of obstructive uropathy in fetal lambs produces a shrunken, noncompliant bladder. We hypothesized that using a ventriculo-peritoneal shunt for the vesico-amniotic shunt may preserve the filling/emptying cycle and thus normal bladder development. Materials and Methods: We created obstructive uropathy in 60-day gestation fetal lambs, ligating the urethra and urachus. Vesico-amniotic shunting was performed 21 days later using the valve end of a ventriculo-peritoneal shunt (valve shunt) or silastic tubing (nonvalve shunt). They were delivered at term (145 days), and the bladder volume was measured and compared to normal term fetuses. The lambs were sacrificed, and the kidneys and bladder removed for histology. Results: Twenty-seven lambs were shunted. Of 14 valve shunts, 8 were effective. Of 13 nonvalve shunts, 11 were effective. The mean bladder volume was 57 F 41 mL with a valve shunt and 8.8 F 4.7 mL with a nonvalve shunt ( P b .05) (normal term lambs, 65 F 18 mL, n = 5). Histology of the shunted bladders showed increased fibrosis in the submucosal and muscle layers. This was less obvious in lambs with a valve shunt. Conclusion: A pressure controlled shunt for fetal obstructive uropathy improves bladder volume but does not prevent bladder wall fibrosis. D 2006 Elsevier Inc. All rights reserved.
The earliest studies of obstructive uropathy emphasized the need to obstruct both the urachus and the urethra [1,2]. New fetal lamb models investigating obstructive uropathy were developed in the 1980s [3,4] when the availability of antenatal ultrasound renewed the interest in fetal obstructive
Presented at the 39th Annual Meeting of the Pacific Association of Pediatric Surgeons, May 14 – 18, 2006, Taipei, Taiwan. * Corresponding author. E-mail address: [email protected] (H. Kitagawa). 0022-3468/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2006.08.012
uropathy [5,6]. The effects of complete obstruction were found to depend on gestational age [7,8]. These experiments were extended to study the effects of fetal decompression of the urinary system to determine the efficacy of prenatal intervention. In 1999, we developed a reliable model for complete obstruction of the urethra in the female fetal lambs [9]. Our previous studies of the results of early vesicostomy in our fetal lamb obstructive uropathy model showed that the bladder developed dense fibrosis in the subepithelial connective tissue layer and between the muscle bundles [10]. These bladders had hypercontractility, low capacity,
Pressure-limited vesico-amniotic shunt tube for fetal obstructive uropathy Table 1 Comparison of the body weight, crown-rump length, bladder volume, and bladder wall thickness in valve shunt, nonvalve shunt, and control lambs
Body weight (g) Crown-to-rump length (cm) Bladder volume (mL) Bladder thickness (cm)
Valve shunt (n = 8)
Nonvalve shunt (n = 11)
Control (n = 5)
4150 F 753
4236 F 887
4870 F 626
46.6 F 4.0
47.6 F 3.6
49.1 F 3.2
57 F 41*
8.8 F 4.7*
65 F 18
1.6 F 0.5*
2.7 F 0.7*
0.4 F 0.2
There was a statistically significant difference between valve shunt and nonvalve shunt in bladder volume and control bladder wall thickness. * P b .01 (Student t test).
and very poor compliance. We hypothesized that preserving normal bladder physiology, including the normal storage and voiding cycle, is critical if normal bladder development is to be preserved after shunting fetal obstructive uropathy. We chose to use the valved end of a ventriculo-peritoneal (V-P) shunt as a vesico-amniotic (V-A) shunt in an attempt to preserve normal bladder function.
1. Materials and methods After approval was obtained from the Wellington School of Medicine and Health Sciences Animal Ethics Committee (Application 8-03), timed gestation ewes (60 days’ gestation) were transported from the farm 24 to 48 hours before operation. They were examined by ultrasound to confirm the pregnancy and avoid unnecessary operations. The preoperative management and anesthetic techniques have been previously reported [9,11]. The surgical techniques used to create the obstruction have previously been described [9]. At the time of shunting, under general anesthesia, the uterus was reopened using the previous incision and the fetuses were exposed. The fetus’s lower extremities and abdomen were delivered through the uterus, and a small hole was made in the bladder. A purse string suture was placed in the bladder wall, and the peritoneal end of a Pudenz catheter (Integra NeuroSciences, Pudenz Peritoneal Catheter, Plainsboro, NJ, REF NL850-1380, low pressure 15-54 mm H2O) was inserted into the bladder. In the second group, either a vesicostomy was performed to create a bshuntQ [12] or a segment of peritoneal catheter tubing (without a valve) (Integra NeuroSciences) was inserted into the bladder. The fetuses were returned to the uterus, and the uterus and ewe’s abdominal wall were closed with Biosyn or Polysorb (US Surgical, Tyco
2087
Healthcare, Japan). Normal unoperated term lambs were used as controls. Most of these lambs were unoperated twins of experimental lambs. At term (145 days), the ewes were anesthetized and the fetuses were delivered by cesarean section. The initial bladder volume was measured by aspirating the urine through the catheter in fetuses with shunts, through the vesicostomy in fetuses with a vesicostomy or through the bladder wall in normal lambs. Bladder capacity was measured by infusing saline through the shunt or through a catheter until leakage occurred. The volume infused to that point was recorded as the bladder volume. The lambs were then sacrificed using pentobarbital injected into the umbilical vein. The lamb’s entire renal tract was removed en block and initially fixed in 10% formal saline. The bladders were fixed distended with formalin at a pressure of 30 cm H2O. Samples were taken from the kidneys, ureters, and the bladder wall. Histologic sections of the bladder wall were stained with hemotoxylin and eosin, Masson’s trichrome, and a-smooth muscle actin (a-SMA). The bladder wall thickness was measured on the histologic sections, using a micrometer. Data were compared with both groups and analyzed by Student t test or Welch’s test as appropriate.
2. Results Effective obstruction of both urethra and urachus was achieved in 27 lambs. Fourteen fetuses were shunted using a valve shunt, 8 of which were effective till term. Thirteen were shunted with no valve and 11 were effective till term. The difference between the 2 groups is explained by the fact that if the valve shunt was displaced, then it no longer functioned as a valve shunt and was therefore excluded,
Fig. 1 A-C, Direct visualization of the bladder wall (hematoxylin and eosin). The mean bladder thickness in valve shunt was 1.6 F 0.5 mm (n=8) compared with 2.7 F 0.7 (n = 11) in nonvalve shunt. There is a statistically significant difference between these groups ( P = .001). (Millimeter scales.)
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H. Nagae et al.
3. Discussion
Fig. 2 A-C, The immunohistochemical stain for a-SMA is positive in the muscle layers. In normal control bladders, there are 3 layers including the mucosal epithelium, a thin submucosal connective tissue layer, and the smooth muscle layer. In nonvalve shunt is markedly thickened compared to the control bladder. This is caused by submucosal connective tissue proliferation and thickening of muscle layer, and a significant increase of collagen within the muscle layer. The degree of positive a-SMA staining is minor in valve shunt compared to that in nonvalve shunt.
whereas if a nonvalve shunt was displaced it still functioned as a shunt. There was no significant difference between the body weight or the crown-to-rump length between the 2 groups or normal control lambs (Table 1). The mean bladder volume in the valve shunt group was not significantly different to that in control lambs, but did significantly differ from nonvalve shunt bladder volume. The thickness of the bladder wall is demonstrated in Fig. 1 (whole mounts). The mean thickness of the bladder wall in the valve shunted group was significantly less than that of the nonvalve shunt group, but was significantly thicker than normal bladders ( P b .001) (Table 1). Histologic examination of the shunted bladders showed that the bladders from both valve and nonvalve shunt group had increased fibrosis in the submucosal and muscle layers compared with control bladders. However, there is an impression that this is less marked in the bladders from valve shunt lambs. After staining with Masson’s trichrome stain, the bladder wall was compared in these lambs. In the nonvalve shunt there is marked muscle hypertrophy and interstitial fibrosis. Conversely, there is only mild muscle hypertrophy and minimal interstitial fibrosis in the valve shunt group. Immunohistochemical staining for a-SMA was also positive in the muscle layers but not in the submucosal area in the bladder walls of control lambs. In the nonvalve shunt group, there was marked bladder wall thickening with dense fibrosis extending from the submucosal layer into the muscular layers. This is much less obvious in the valve shunt group (Fig. 2).
Fetal therapy for obstructive uropathy was first reported in 1982 by Harrison et al [6]. After an initial flurry of enthusiasm for fetal treatment, long-term follow-up studies started to raise concerns about bladder function. Freedman et al [13] investigated the short-term and long-term outcome of 55 sequential fetuses evaluated in a single center and shunted for presumed obstructive uropathy. Survival was 61%. They reported the long-term results of 14 children who underwent vesico-amniotic shunting who survived to more than 2 years of age. Eight children void spontaneously, 4 children catheterize in addition to voiding, and 2 children catheterize only. Of the 4 children with posterior urethral valves, 3 have undergone bladder augmentation. Our fetal lamb model of obstructive uropathy produced results very similar to posterior urethral valve in humans. We shunted 22 fetuses and 11 had a successful shunt that persisted till term [14]. Six (55%) had normal nephrogenesis, but, interestingly, this entire group had a small bladder (8 F 5 mL) compared with controls (71 F 19 mL). These bladders not only had a very small volume, but they also had a very poor compliance [14]. We hypothesized that the severely dilated fetal bladder resulted in increased connective tissue in the submucosal layer, extending into the intramuscular region. Peters et al [15], using a fetal lamb model, reported that obstruction of the developing bladder caused a 4.6 times increase in bladder weight at term, reflecting a 5.8 times increase in smooth muscle mass. Congenital bladder obstruction produces a variety of structural, biochemical, and functional changes in the developing bladder indicative of alterations in the regulation of growth and differentiation. Early shunting may prevent this phenomenon, but, more importantly, it may also preserve normal bladder physiology. The use of a shunt with a pressure-limited valve to bypass the obstructive lesion maintained the normal storage and voiding bladder cycle, which has been reported to play a critical role in bladder development [16]. Our shunt tube is designed to maintain a constant pressure in the bladder and allow the bladder to empty against a resistance, providing a continuous resistance of approximately 15 to 54 mm H2O. Our current hypothesis is that the overdistended fetal bladder results in the disruption of the developing muscle layers. When this bladder is shunted and the volume is not at least partially maintained (vesicostomy or a shunt with no valve), then the damaged bladder collapses and the damaged muscle is able to bhealQ with the development of considerable fibrosis. Conversely, when the distended bladder is shunted using a shunt with a valve, then the bladder continues to expand and contract, preserving normal muscle development and reducing the amount of fibrosis. This is reflected in the differences observed in the sections stained for a-SMA. Bagli et al [17] recently described a receptor for hyaluronic acid–mediated motility which is a hyaluronic acid–binding protein known to influence
Pressure-limited vesico-amniotic shunt tube for fetal obstructive uropathy multiple types of cell–extracellular matrix interactions in development. We speculate that the differences in the degree of muscle hypertrophy that we have observed in our 2 shunt models might be related to different expressions of this receptor. Capolicchio et al [18] reported that, in 30-day-old female rats, distending the bladder using a pressure of 40 cm H2O creates transient clinical injury to the rat bladder confirmed by the presence of gross hematuria. This would suggest that our low-pressure V-P shunt tube (15-54 mm H2O) is a reasonable pressure to use for the fetal sheep bladder. We conclude that fetal obstructive uropathy in this fetal lamb model causes a bladder stretch injury, but that a pressurecontrolled V-P shunt tube is able to decompress the bladder while also preserving its volume and reduce both muscle hypertrophy and interstitial fibrosis.
Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research (C) from the Japanese Society for Grant in Aid for Scientific Research. Suture materials were supplied by Tyco Healthcare (Tokyo, Japan). Tokibo Co, Ltd (Tokyo, Japan) supplied part of the V-P shunt tube. All animals are consistently bred to timing of less than 24 hours by Doug Jensen of the Wallaceville Animal Research Facility.
References [1] Tanagho EA. Surgically induced partial urinary obstruction in the fetal lamb: I. Technique. Invest Urol 1972;10:19 - 24. [2] Tanagho EA. Surgically induced partial urinary obstruction in the fetal lamb: II. Ureteral obstruction. Invest Urol 1972;10:35 - 52. [3] Harrison MR, Nakayama DK, Noall R, et al. Correction of congenital hydronephrosis in utero: II. Decompression reverses the effect of obstruction on the fetal lung and urinary tract. J Pediatr Surg 1982; 17:965 - 74.
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[4] Glick PL, Harrison MR, Adzic NS, et al. Correction of congenital hydronephrosis in utero: IV. In utero decompression prevents renal dysplasia. J Pediatr Surg 1984;19:649 - 57. [5] Golbus MS, Harrison MR, Filly RA, et al. In utero treatment of urinary tract obstruction. Am J Obstet Gynecol 1982;142:383 - 8. [6] Harrison MR, Golbus MS, Filly RA, et al. Fetal surgery for congenital hydronephrosis. N Engl J Med 1982;306:591 - 3. [7] Harrison MR, Filly RA. The fetus with obstructive uropathy: pathophysiology, natural history, selection, and treatment. In: Harrison MR, Golbus MS, Filly RA, editors. The unborn patient: prenatal diagnosis and treatment. Philadelphia7 WB Saunders; 1991. p. 328 - 45. [8] Kitagawa H, Pringle KC, Koike J, et al. Different phenotypes of dysplastic kidney in obstructive uropathy in fetal lambs. J Pediatr Surg 2001;36:1698 - 703. [9] Kitagawa H, Pringle KC, Zuccollo J, et al. The pathogenesis of dysplastic kidney in a urinary tract obstruction in the female fetal lamb. J Pediatr Surg 1999;34:1678 - 83. [10] Sato Y, Kitagawa H, Pringle KC, et al. Effect of early vesicostomy in obstructive uropathy on bladder development. J Pediatr Surg 2004; 39:1849 - 52. [11] Pringle KC. Fetal lamb and fetal lung growth following creation and repair of a diaphragmatic hernia. In: Nathanielsz PW, editor. Animal models in fetal medicine, vol. IV. Ithaca (NY)7 Perinatology Press; 1984. p. 109 - 48. [12] Kitagawa H, Pringle KC, Koike J, et al. Fetal hydrops in experimental obstructive uropathy resolves after vesicostomy formation: is this cause and effect? Pediatr Surg Int 2005;21:25 - 8. [13] Freedman AL, Johnson MP, Smith CA, et al. Long-term outcome in children after antenatal intervention for obstructive uropathy. Lancet 1999;354:374 - 7. [14] Kitagawa H, Pringle KC, Koike J, et al. Vesicoamniotic shunt for complete urinary tract obstruction is partially effective. J Pediatr Surg 2006;41:394 - 402. [15] Peters CA, Vasavada S, Dator D, et al. The effect of obstruction on the developing bladder. J Urol 1992;148:491 - 6. [16] Hoyes AD, Ramus NI, Martin BG. Ultrastructural aspects of the development of the innervation of the vesical musculature in the early human fetus. Invest Urol 1973;10:307 - 12. [17] Bagli DJ, Joyner BD, Mahoney SR, et al. The hyaluronic acid receptor is induced by stretch injury of rat bladder in vivo and influences smooth muscle cell contraction in vitro. J Urol 1999;162:832 - 40. [18] Capolicchio G, Atken KJ, Gu J, et al. Extracellular matrix gene responses in a novel ex vivo model of bladder stretch injury. J Urol 2001;165:2235 - 40.
www.elsevier.com/locate/jpedsurg
Pressure-limited vesico-amniotic shunt tube for fetal obstructive uropathy Hideki Nagaea, Hiroaki Kitagawaa,*, Kevin C. Pringleb, Junki Koikec, Jane Zuccollob, Yuriko Satoa, Yasuji Sekia, Munechika Wakisakaa, Koonosuke Nakadaa a
Division of Pediatric Surgery, St Marianna University School of Medicine, Miyamae-ku, Kawasaki 216-8511, Japan Department of Obstetrics and Gynecology, Wellington School of Medicine, Wellington, New Zealand c Department of Pathology, St Marianna University School of Medicine, Kawasaki, Japan b
Index words: Fetal surgery; Obstructive uropathy; Bladder; Fibrosis; Posterior urethral valves
Abstract Introduction: In utero shunting (vesico-amniotic shunt) of obstructive uropathy in fetal lambs produces a shrunken, noncompliant bladder. We hypothesized that using a ventriculo-peritoneal shunt for the vesico-amniotic shunt may preserve the filling/emptying cycle and thus normal bladder development. Materials and Methods: We created obstructive uropathy in 60-day gestation fetal lambs, ligating the urethra and urachus. Vesico-amniotic shunting was performed 21 days later using the valve end of a ventriculo-peritoneal shunt (valve shunt) or silastic tubing (nonvalve shunt). They were delivered at term (145 days), and the bladder volume was measured and compared to normal term fetuses. The lambs were sacrificed, and the kidneys and bladder removed for histology. Results: Twenty-seven lambs were shunted. Of 14 valve shunts, 8 were effective. Of 13 nonvalve shunts, 11 were effective. The mean bladder volume was 57 F 41 mL with a valve shunt and 8.8 F 4.7 mL with a nonvalve shunt ( P b .05) (normal term lambs, 65 F 18 mL, n = 5). Histology of the shunted bladders showed increased fibrosis in the submucosal and muscle layers. This was less obvious in lambs with a valve shunt. Conclusion: A pressure controlled shunt for fetal obstructive uropathy improves bladder volume but does not prevent bladder wall fibrosis. D 2006 Elsevier Inc. All rights reserved.
The earliest studies of obstructive uropathy emphasized the need to obstruct both the urachus and the urethra [1,2]. New fetal lamb models investigating obstructive uropathy were developed in the 1980s [3,4] when the availability of antenatal ultrasound renewed the interest in fetal obstructive
Presented at the 39th Annual Meeting of the Pacific Association of Pediatric Surgeons, May 14 – 18, 2006, Taipei, Taiwan. * Corresponding author. E-mail address: [email protected] (H. Kitagawa). 0022-3468/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2006.08.012
uropathy [5,6]. The effects of complete obstruction were found to depend on gestational age [7,8]. These experiments were extended to study the effects of fetal decompression of the urinary system to determine the efficacy of prenatal intervention. In 1999, we developed a reliable model for complete obstruction of the urethra in the female fetal lambs [9]. Our previous studies of the results of early vesicostomy in our fetal lamb obstructive uropathy model showed that the bladder developed dense fibrosis in the subepithelial connective tissue layer and between the muscle bundles [10]. These bladders had hypercontractility, low capacity,
Pressure-limited vesico-amniotic shunt tube for fetal obstructive uropathy Table 1 Comparison of the body weight, crown-rump length, bladder volume, and bladder wall thickness in valve shunt, nonvalve shunt, and control lambs
Body weight (g) Crown-to-rump length (cm) Bladder volume (mL) Bladder thickness (cm)
Valve shunt (n = 8)
Nonvalve shunt (n = 11)
Control (n = 5)
4150 F 753
4236 F 887
4870 F 626
46.6 F 4.0
47.6 F 3.6
49.1 F 3.2
57 F 41*
8.8 F 4.7*
65 F 18
1.6 F 0.5*
2.7 F 0.7*
0.4 F 0.2
There was a statistically significant difference between valve shunt and nonvalve shunt in bladder volume and control bladder wall thickness. * P b .01 (Student t test).
and very poor compliance. We hypothesized that preserving normal bladder physiology, including the normal storage and voiding cycle, is critical if normal bladder development is to be preserved after shunting fetal obstructive uropathy. We chose to use the valved end of a ventriculo-peritoneal (V-P) shunt as a vesico-amniotic (V-A) shunt in an attempt to preserve normal bladder function.
1. Materials and methods After approval was obtained from the Wellington School of Medicine and Health Sciences Animal Ethics Committee (Application 8-03), timed gestation ewes (60 days’ gestation) were transported from the farm 24 to 48 hours before operation. They were examined by ultrasound to confirm the pregnancy and avoid unnecessary operations. The preoperative management and anesthetic techniques have been previously reported [9,11]. The surgical techniques used to create the obstruction have previously been described [9]. At the time of shunting, under general anesthesia, the uterus was reopened using the previous incision and the fetuses were exposed. The fetus’s lower extremities and abdomen were delivered through the uterus, and a small hole was made in the bladder. A purse string suture was placed in the bladder wall, and the peritoneal end of a Pudenz catheter (Integra NeuroSciences, Pudenz Peritoneal Catheter, Plainsboro, NJ, REF NL850-1380, low pressure 15-54 mm H2O) was inserted into the bladder. In the second group, either a vesicostomy was performed to create a bshuntQ [12] or a segment of peritoneal catheter tubing (without a valve) (Integra NeuroSciences) was inserted into the bladder. The fetuses were returned to the uterus, and the uterus and ewe’s abdominal wall were closed with Biosyn or Polysorb (US Surgical, Tyco
2087
Healthcare, Japan). Normal unoperated term lambs were used as controls. Most of these lambs were unoperated twins of experimental lambs. At term (145 days), the ewes were anesthetized and the fetuses were delivered by cesarean section. The initial bladder volume was measured by aspirating the urine through the catheter in fetuses with shunts, through the vesicostomy in fetuses with a vesicostomy or through the bladder wall in normal lambs. Bladder capacity was measured by infusing saline through the shunt or through a catheter until leakage occurred. The volume infused to that point was recorded as the bladder volume. The lambs were then sacrificed using pentobarbital injected into the umbilical vein. The lamb’s entire renal tract was removed en block and initially fixed in 10% formal saline. The bladders were fixed distended with formalin at a pressure of 30 cm H2O. Samples were taken from the kidneys, ureters, and the bladder wall. Histologic sections of the bladder wall were stained with hemotoxylin and eosin, Masson’s trichrome, and a-smooth muscle actin (a-SMA). The bladder wall thickness was measured on the histologic sections, using a micrometer. Data were compared with both groups and analyzed by Student t test or Welch’s test as appropriate.
2. Results Effective obstruction of both urethra and urachus was achieved in 27 lambs. Fourteen fetuses were shunted using a valve shunt, 8 of which were effective till term. Thirteen were shunted with no valve and 11 were effective till term. The difference between the 2 groups is explained by the fact that if the valve shunt was displaced, then it no longer functioned as a valve shunt and was therefore excluded,
Fig. 1 A-C, Direct visualization of the bladder wall (hematoxylin and eosin). The mean bladder thickness in valve shunt was 1.6 F 0.5 mm (n=8) compared with 2.7 F 0.7 (n = 11) in nonvalve shunt. There is a statistically significant difference between these groups ( P = .001). (Millimeter scales.)
2088
H. Nagae et al.
3. Discussion
Fig. 2 A-C, The immunohistochemical stain for a-SMA is positive in the muscle layers. In normal control bladders, there are 3 layers including the mucosal epithelium, a thin submucosal connective tissue layer, and the smooth muscle layer. In nonvalve shunt is markedly thickened compared to the control bladder. This is caused by submucosal connective tissue proliferation and thickening of muscle layer, and a significant increase of collagen within the muscle layer. The degree of positive a-SMA staining is minor in valve shunt compared to that in nonvalve shunt.
whereas if a nonvalve shunt was displaced it still functioned as a shunt. There was no significant difference between the body weight or the crown-to-rump length between the 2 groups or normal control lambs (Table 1). The mean bladder volume in the valve shunt group was not significantly different to that in control lambs, but did significantly differ from nonvalve shunt bladder volume. The thickness of the bladder wall is demonstrated in Fig. 1 (whole mounts). The mean thickness of the bladder wall in the valve shunted group was significantly less than that of the nonvalve shunt group, but was significantly thicker than normal bladders ( P b .001) (Table 1). Histologic examination of the shunted bladders showed that the bladders from both valve and nonvalve shunt group had increased fibrosis in the submucosal and muscle layers compared with control bladders. However, there is an impression that this is less marked in the bladders from valve shunt lambs. After staining with Masson’s trichrome stain, the bladder wall was compared in these lambs. In the nonvalve shunt there is marked muscle hypertrophy and interstitial fibrosis. Conversely, there is only mild muscle hypertrophy and minimal interstitial fibrosis in the valve shunt group. Immunohistochemical staining for a-SMA was also positive in the muscle layers but not in the submucosal area in the bladder walls of control lambs. In the nonvalve shunt group, there was marked bladder wall thickening with dense fibrosis extending from the submucosal layer into the muscular layers. This is much less obvious in the valve shunt group (Fig. 2).
Fetal therapy for obstructive uropathy was first reported in 1982 by Harrison et al [6]. After an initial flurry of enthusiasm for fetal treatment, long-term follow-up studies started to raise concerns about bladder function. Freedman et al [13] investigated the short-term and long-term outcome of 55 sequential fetuses evaluated in a single center and shunted for presumed obstructive uropathy. Survival was 61%. They reported the long-term results of 14 children who underwent vesico-amniotic shunting who survived to more than 2 years of age. Eight children void spontaneously, 4 children catheterize in addition to voiding, and 2 children catheterize only. Of the 4 children with posterior urethral valves, 3 have undergone bladder augmentation. Our fetal lamb model of obstructive uropathy produced results very similar to posterior urethral valve in humans. We shunted 22 fetuses and 11 had a successful shunt that persisted till term [14]. Six (55%) had normal nephrogenesis, but, interestingly, this entire group had a small bladder (8 F 5 mL) compared with controls (71 F 19 mL). These bladders not only had a very small volume, but they also had a very poor compliance [14]. We hypothesized that the severely dilated fetal bladder resulted in increased connective tissue in the submucosal layer, extending into the intramuscular region. Peters et al [15], using a fetal lamb model, reported that obstruction of the developing bladder caused a 4.6 times increase in bladder weight at term, reflecting a 5.8 times increase in smooth muscle mass. Congenital bladder obstruction produces a variety of structural, biochemical, and functional changes in the developing bladder indicative of alterations in the regulation of growth and differentiation. Early shunting may prevent this phenomenon, but, more importantly, it may also preserve normal bladder physiology. The use of a shunt with a pressure-limited valve to bypass the obstructive lesion maintained the normal storage and voiding bladder cycle, which has been reported to play a critical role in bladder development [16]. Our shunt tube is designed to maintain a constant pressure in the bladder and allow the bladder to empty against a resistance, providing a continuous resistance of approximately 15 to 54 mm H2O. Our current hypothesis is that the overdistended fetal bladder results in the disruption of the developing muscle layers. When this bladder is shunted and the volume is not at least partially maintained (vesicostomy or a shunt with no valve), then the damaged bladder collapses and the damaged muscle is able to bhealQ with the development of considerable fibrosis. Conversely, when the distended bladder is shunted using a shunt with a valve, then the bladder continues to expand and contract, preserving normal muscle development and reducing the amount of fibrosis. This is reflected in the differences observed in the sections stained for a-SMA. Bagli et al [17] recently described a receptor for hyaluronic acid–mediated motility which is a hyaluronic acid–binding protein known to influence
Pressure-limited vesico-amniotic shunt tube for fetal obstructive uropathy multiple types of cell–extracellular matrix interactions in development. We speculate that the differences in the degree of muscle hypertrophy that we have observed in our 2 shunt models might be related to different expressions of this receptor. Capolicchio et al [18] reported that, in 30-day-old female rats, distending the bladder using a pressure of 40 cm H2O creates transient clinical injury to the rat bladder confirmed by the presence of gross hematuria. This would suggest that our low-pressure V-P shunt tube (15-54 mm H2O) is a reasonable pressure to use for the fetal sheep bladder. We conclude that fetal obstructive uropathy in this fetal lamb model causes a bladder stretch injury, but that a pressurecontrolled V-P shunt tube is able to decompress the bladder while also preserving its volume and reduce both muscle hypertrophy and interstitial fibrosis.
Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research (C) from the Japanese Society for Grant in Aid for Scientific Research. Suture materials were supplied by Tyco Healthcare (Tokyo, Japan). Tokibo Co, Ltd (Tokyo, Japan) supplied part of the V-P shunt tube. All animals are consistently bred to timing of less than 24 hours by Doug Jensen of the Wallaceville Animal Research Facility.
References [1] Tanagho EA. Surgically induced partial urinary obstruction in the fetal lamb: I. Technique. Invest Urol 1972;10:19 - 24. [2] Tanagho EA. Surgically induced partial urinary obstruction in the fetal lamb: II. Ureteral obstruction. Invest Urol 1972;10:35 - 52. [3] Harrison MR, Nakayama DK, Noall R, et al. Correction of congenital hydronephrosis in utero: II. Decompression reverses the effect of obstruction on the fetal lung and urinary tract. J Pediatr Surg 1982; 17:965 - 74.
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[4] Glick PL, Harrison MR, Adzic NS, et al. Correction of congenital hydronephrosis in utero: IV. In utero decompression prevents renal dysplasia. J Pediatr Surg 1984;19:649 - 57. [5] Golbus MS, Harrison MR, Filly RA, et al. In utero treatment of urinary tract obstruction. Am J Obstet Gynecol 1982;142:383 - 8. [6] Harrison MR, Golbus MS, Filly RA, et al. Fetal surgery for congenital hydronephrosis. N Engl J Med 1982;306:591 - 3. [7] Harrison MR, Filly RA. The fetus with obstructive uropathy: pathophysiology, natural history, selection, and treatment. In: Harrison MR, Golbus MS, Filly RA, editors. The unborn patient: prenatal diagnosis and treatment. Philadelphia7 WB Saunders; 1991. p. 328 - 45. [8] Kitagawa H, Pringle KC, Koike J, et al. Different phenotypes of dysplastic kidney in obstructive uropathy in fetal lambs. J Pediatr Surg 2001;36:1698 - 703. [9] Kitagawa H, Pringle KC, Zuccollo J, et al. The pathogenesis of dysplastic kidney in a urinary tract obstruction in the female fetal lamb. J Pediatr Surg 1999;34:1678 - 83. [10] Sato Y, Kitagawa H, Pringle KC, et al. Effect of early vesicostomy in obstructive uropathy on bladder development. J Pediatr Surg 2004; 39:1849 - 52. [11] Pringle KC. Fetal lamb and fetal lung growth following creation and repair of a diaphragmatic hernia. In: Nathanielsz PW, editor. Animal models in fetal medicine, vol. IV. Ithaca (NY)7 Perinatology Press; 1984. p. 109 - 48. [12] Kitagawa H, Pringle KC, Koike J, et al. Fetal hydrops in experimental obstructive uropathy resolves after vesicostomy formation: is this cause and effect? Pediatr Surg Int 2005;21:25 - 8. [13] Freedman AL, Johnson MP, Smith CA, et al. Long-term outcome in children after antenatal intervention for obstructive uropathy. Lancet 1999;354:374 - 7. [14] Kitagawa H, Pringle KC, Koike J, et al. Vesicoamniotic shunt for complete urinary tract obstruction is partially effective. J Pediatr Surg 2006;41:394 - 402. [15] Peters CA, Vasavada S, Dator D, et al. The effect of obstruction on the developing bladder. J Urol 1992;148:491 - 6. [16] Hoyes AD, Ramus NI, Martin BG. Ultrastructural aspects of the development of the innervation of the vesical musculature in the early human fetus. Invest Urol 1973;10:307 - 12. [17] Bagli DJ, Joyner BD, Mahoney SR, et al. The hyaluronic acid receptor is induced by stretch injury of rat bladder in vivo and influences smooth muscle cell contraction in vitro. J Urol 1999;162:832 - 40. [18] Capolicchio G, Atken KJ, Gu J, et al. Extracellular matrix gene responses in a novel ex vivo model of bladder stretch injury. J Urol 2001;165:2235 - 40.