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Quantitative analysis of sacral parasympathetic nucleus innervating the rectum in rats with anorectal malformation
Journal of Pediatric Surgery (2007) 42, 1544–1548
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Quantitative analysis of sacral parasympathetic nucleus innervating the rectum in rats with anorectal malformation Huimin Jia, Keren Zhang, Shucheng Zhang, Zhengwei Yuan, Yuzuo Bai, Weilin Wang⁎ Department of Paediatric Surgery, The 2nd Clinical College of China Medical University, Shenyang 110004, PR China
Index words: Anorectal malformation; Rectum; Sacral parasympathetic neurons
Abstract Background/Purpose: The purpose of this experiment was to identify the neurons in the lumbosacral spinal cord involved in colon-rectal function and to compare normal and anorectal malformation of fetal rats. Methods: The authors quantified the sacral parasympathetic nucleus (SPN) innervation of the rectum by Fluorogold (FG) (Fluorochrome, Englewood, CO) retrograde tracing experiment in fetal rats with normal and anorectal malformation. Anorectal malformation was induced in rat fetuses by ethylenethiourea (ETU). The number of FG-labeled SPNs was scored and compared between male fetuses with or without malformation in the ETU-fed group and control groups. Results: The number of FG-labeled SPNs in the fetuses without a defect, with ETU injected but without any defects of the anorectum or neural tube, with low-type deformity, and with high-type deformity were (mean ± SEM) 47.3 ± 2.9, 45.6 ± 3.2, 24.2 ± 3.8, and 8.5 ± 2.5, respectively. Fluorogold-labeled SPNs in the fetuses with high-type deformity were significantly fewer than those in fetuses without defects (P b .05) and in controls (P b .05). Conclusions: These findings suggest that defective SPN innervation to the rectum is a primary anomaly that coexists with the alimentary tract anomaly in anorectal malformation during fetal development. The intrinsic neural deficiency is an important factor likely to contribute to poor postoperative anorectal function despite surgical correction of the malformation. © 2007 Elsevier Inc. All rights reserved.
Anorectal malformation (ARM) is a common surgical problem affecting 1 in 1500 to 1 in 5000 live births. Posterior sagittal anorectoplasty (PSARP) as proposed by De Vries and Peña has been a popular procedure used to correct ARMs since the 1980s. The advantage of this procedure is the extensive exposure allowing meticulous anatomical repair of all the muscular structures present to achieve optimum continence [1]. There is evidence indicating that PSARP is equivocal in achieving good postoperative continence [2-5] ⁎ Corresponding author. Fax: +86 24 23903396. E-mail address: [email protected] (W. Wang). 0022-3468/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2007.04.034
and is associated with a tendency toward constipation, particularly in high or intermediate anomalies [6]. Severe constipation associated with megarectum has been noted sporadically after conventional anorectoplasty (ARP) in either low or high anomalies [7,8]. The causes of post-ARP constipation are still obscure and have been attributed to sacral anomalies [6], rectal inertia [8-10], Hirschsprung disease [11,12], and neuromuscular or sensory damage to the neorectum [11] as well as the well-preserved bowel end with intact sphincter muscle [1]. The lower urinary tract colonrectum share 2 common functions: storage and periodic elimination of body waste. These functions are regulated by
Quantitative analysis of sacral parasympathetic nucleus
1545
neural pathways located in the brain, spinal cord, peripheral ganglia, and enteric nervous system. We suspect that the postoperative effect is related to the rectal function. When the parasympathetic nerves are excited, the motility of the colon and rectum will be increased. We hypothesized that the development of SPNs is related to constipation. Results of our previous histopathologic study showed that there were fewer motor neurons in the sacral spinal ventral horn in patients with imperforate anus [13]. Yuan et al [14] quantified how the motor neurons innervating the levator ani muscle (rat equivalent of the puborectalis muscle in the human) were decreased, which plays a important role in fecal continence. They used a retrograde tract-tracing method in a rat model of ARM. To evaluate our hypothesis, we followed it using a microinjection method during fetal surgery to locate and quantify the parasympathetic nucleus in the lumbosacral spinal cord innervating the rectum. These studies have established that the Fluorogold (FG) is a reliable transsynaptic retrograde tracing agent that passes between neurons only at regions of synaptic contact [14,15].
anesthesia within 1 hour and were returned to their original cages to allow transport of the neuronal tracer.
1. Materials and methods 1.1. Experimental animals Twenty pregnant time-mated Wistar rats (250-300 g) were randomly assigned into 2 groups: 15 were gavage-fed with a single dose of 125 mg/kg of 1% ETU (ethylenethiourea [2-imidazolidinethione, 98%], C3H6N2S), and 5 of them were gavage-fed with an equal dose of physiologic saline on gestation day 10 (gestation day 0: sperm in vaginal smear after overnight mating) at the same time as the control group. All of the animal experiments were performed with an approval obtained from the appropriate local ethics committee.
1.3. Perfusion and histology After 24 hours postoperatively, the dams were reanesthetized with an overdose of pentobarbitone sodium, and the fetuses that had been injected with FG were harvested by cesarean delivery. Fetuses were perfused transcardially with 15 mL of physiologic saline followed by 25 mL 4% paraformaldehyde (wt/vol in 0.1 mol/L phosphate buffer). The lumbosacral spinal cord was dissected and postfixed in the same fixative for 24 hours at 4°C for 18 hours' incubation in 20% sucrose in 0.1 mol/L phosphate buffer. The spinal cords were sectioned on a cryotome into 45-μm serial transverse sections and mounted onto clean gelatinized microscopic glass slides. The series of transverse sections was used to locate the labeled SPNs in different segments of spinal cord and count the number of labeled SPNs. The FGlabeled sections were examined by a fluorescent microscope equipped with a digital charge-coupled device camera.
1.4. Microscopic and statistical analysis After fluorescent microscopic analysis, the labeled SPNs on each section were mounted, and an average was obtained. The results were analyzed using Student's t test. Significant differences were considered when the P value was less than .05. Data are presented as the mean ± SEM.
2. Results 2.1. General characteristics of experimental animals
1.2. Fetal surgery and injection of FG tracing dye The pregnant rats were anesthetized with ethyl carbamate (7 mL/kg body weight) on gestation day 20. An incision was made in the abdominal wall, and the uterine horn was exteriorized; then the uterus was covered by wet gauze immersed with warm physiologic saline. The uterus was opened close to the fetal tail, then the rectum or the rectal pouch was exposed under a dissection microscope. Five percent FG was injected into the wall of the rectum or the blind end of the rectum using a 30-gauge needle attached to a Hamilton syringe. The injection volume of FG at each point was 1 μL (at 4 sites: 3, 6, 9, 12 o'clock position). At each injection site, the needle was kept in place for several seconds after the injection while applying slight pressure to reduce leakage. The injected rectum or the blind end was thoroughly rinsed with physiologic saline to remove excess FG that might have leaked from the injection site. The fetuses were returned to the uterus, and the uterine wound was closed with 7-0 sterile silk suture. The pregnant rats recovered from the
In the ETU-injected group, 135 (58 females, 77 males) live fetuses were harvested from 15 dams. In external morphological inspection, 100% (135/135) of fetuses had a short or absent tail, 62.2% (84/135) of fetuses had an ARM, 57.2% (77/135) showed anomalies of the neural tube (including encephalocele, meningocele, or spina bifida), and 27.5% (37/135) showed anomalies of hind limbs. In this group, 39 fetuses were injected with FG. In the control group, 52 (20 females, 32 males) fetuses were obtained from 5 dams. None of them displayed any anomalies noted previously, and 10 fetuses were injected with FG. One dam died as a result of unsuccessful anesthesia, and 5 fetuses died of a long duration of injection procedure or blood loss.
2.2. Normal location and quantification of FG-labeled SPNs [16,17] In the control group, the FG-labeled rectal SPNs were seen from the L6 to S1 segments (Figs. 1 and 2).
1546
H. Jia et al. Table 1 The numbers of SPNs in different groups with congenital malformations Group 1 (10) Labeled SPNs
Group 2 (10)
Group 3 (8)
Group 4 (16)
47.3 ± 2.9 45.6 ± 3.2 24.2 ± 3.8*,† 8.5 ± 2.5*,†
Values within parentheses indicate the number of fetuses. Group 1 represents controls; group 2, fetuses with FG injected but without any defects of anorectum or neural tube; group 3, fetuses with low-type deformity; group 4, fetuses with high-type deformity. ⁎ P b .05 from control group. † P b .05 from group 2.
Fig. 1 Representative photomicrograph of transverse section from lumbosacral spinal cord of normal fetal rat (original magnification×100).
The location and quantification of FG-labeled SPNs in ARM was as follows: in the ETU-injection group, 39 fetuses were injected with FG. When controls and ETU-injected fetuses without any anomalies were compared, SPNs were fewer and smaller in the spinal cord in fetuses with imperforate anus. When the blind end of rectum was found below the levator ani muscle, it was defined as low-type deformity and as high-type deformity otherwise. (Table 1, Figs. 3 and 4). The number of labeled SPNs in abnormality group was fewer than that in the normal group. The number of labeled SPNs in the high-type deformity group was significantly fewer than that in the low-type group.
3. Discussion The pathophysiologic characteristics of anorectal function is complicated, and there has been no consensus regarding
Fig. 2 The region was magnified as shown in Fig. 1 (original magnification ×400).
the essential factors that control anal continence or the etiology of constipation. Schärli and Kiesewetter [12] proposed that anal continence after ARP for imperforate anus depends on 3 elements: adequate length of the anal canal, 25 mm Hg or more of resting anal pressure, and adequate action of the external and internal sphincters together with an intact puborectalis sling. Arhan et al [18] suggested 2 additional factors, stressing the importance of sensitivity of the anal skin and the presence of the rectoanal sphincter inhibitory reflex, which was supported by Taylor et al [19]. The suggested causes of constipation are much more complex and varied, including rectal inertia or ectasia, which may be congenital [6,8,10,20] or acquired [21], reduced rectal sensitivity [6,20], an abnormal internal anal sphincter that fails to relax on rectal distension [21], an intact or hyperactive internal sphincter [6,20], an abnormal rectoanal sphincter inhibitory reflex and increased sphincter pressure [22], and Hirschsprung disease [6]. With the conventional abdominoperineal pull-through and perineal anoplasty for ARM, constipation was not a significant problem after ARP, although there were sporadic reports of constipation after repair, particularly in low anomalies [7,8,10,21]. Peña [9] reported an especially high incidence (70%) of constipation in instances of anovestibular fistula,
Fig. 3 Fluorogold-labeled SPNs were fewer and smaller in the lumbosacral spinal cord of fetal rat with low-type imperforate anus (original magnification ×100).
Quantitative analysis of sacral parasympathetic nucleus
Fig. 4 Fluorogold-labeled SPNs were fewest and smallest in the lumbosacral spinal cord of fetal rat with high-type imperforate anus (original magnification ×100).
with an overall constipation rate of 30% after repair among 131 cases of ARM without other complex malformations that were followed for more than 3 years after PSARP. Whereas most of the reported constipation was encountered in low anomalies, Rintala et al, in contrast, noticed a high incidence of constipation in high or intermediate anomalies undergoing PSARP, similar to our clinical experience. They reported constipation in 70% and 28% in high anomalies undergoing PSARP with or without salvage of the internal sphincter, respectively, suggesting an intact internal anal sphincter as one of the contributory factors in post-ARP constipation [6,20]. Chen reported that the incidence of constipation after ARP was related to the extent of pelvic dissection. During PSARP, the endopelvic fascia has to be dissected away from the terminal bowel and its fistula; and thus, the autonomic nervous system, especially the parasympathetic inferior hypogastric plexus, will inevitably be more or less damaged. However, clinical evaluations demonstrate that a higher incidence of constipation occurs in patients with lower malformations who had less pelvic dissection [23]. Therefore, we hypothesized that either abnormalities of the nerve ending or the central nervous system would affect continence. We hypothesized that the number of the SPNs may be related to constipation. The results of this study confirmed our hypothesis that there was a deficiency of SPNs in the spinal cord innervating the rectum in an animal model with ARM. The number of labeled SPNs in the high-type deformity group was significantly fewer than that in the low-type group. Because the parasympathetic nerves are motor to the rectal wall muscle and inhibitory to the internal sphincter, their damage will affect the motor activity of the anorectum, resulting in a reduced resting rectal pressure, an increased resting anal pressure, and subsequent increase in ano-rectal pressure gradient and constipation. Dysplasia of rectum may be an additional possibility that induces the deficiency of SPNs. Rectal dysplasia can be affected by the type of colostomy chosen (transverse and loop stomas may
1547 result in megarectum) and by how long the colostomy was left in place before closure. Unfortunately, we did not find any abnormality of the rectum in this study, so we could not determine the major cause of the defects in the developing spinal cord in imperforate anus. We hope to answer this question in our further studies. In evaluating evidence of earlier studies, the ETU appears to have various targets in the rat embryo; differentiation of the mesoblast of the different organs is inhibited by ETU resulting in malformations. However, ETU toxicity did not directly affect the survival of SPNs because the number of labeled SPNs in the ETU-injected fetus without imperforate anus or other anomalies was similar to those in controls. In the future, we plan to apply gene therapy using neurotrophic factors to rescue the SPNs (alter the deficiency) in this malformation. This study confirms our hypothesis that there was defect of SPNs in the spinal cord innervating the rectum in an animal model with ARM. Our findings suggest that defective SPN innervation to the rectum mechanism may coexist with the alimentary tract anomaly in ARM. The intrinsic neural deficiency is an important factor likely to contribute to poor postoperative anorectal function despite surgical correction of ARM.
References [1] De Vries PE, Peña A. Posterior sagittal anorectoplasty. J Pediatr Surg 1982;17:638-43. [2] Langemeijer RATM, Molenaar JC. Continence after posterior sagittal anorectoplasty. J Pediatr Surg 1991;26:587-90. [3] Cahill JL, Christie DL. Results after posterior sagittal anorectoplasty: a new approach to high imperforate anus. Am J Surg 1985;149:629-31. [4] Peña A. Posterior sagittal approach for the correction of anorectal malformations. Adv Surg 1986;19:69-99. [5] Smith D. The bath water needs changing, but don't throw out the baby: an overview of anorectal anomalies. J Pediatr Surg 1987;22:335-48. [6] Rintala R. Postoperative internal sphincter function in anorectal malformation: a manometric study. Pediatr Surg Int 1990;5:127-30. [7] Cheu HW, Grosfeld JL. The atonic baggy rectum: a case of intractable obstipation after imperforate anus repair. J Pediatr Surg 1992;27: 1071-4. [8] Powell RW, Sherman JO, Raffensperger JG. Megarectum: a rare complication of imperforate anus repair and its surgical correction by endorectal pull through. J Pediatr Surg 1982;17:786-95. [9] Peña A. Posterior sagittal anorectoplasty: result in management of 332 cases of anorectal malformations. Pediatr Surg Int 1988;3:94-104. [10] Brent L, Stephens FD. Primary rectal ectasia: a quantitative study of smooth muscle cells in normal and hypertrophied human bowel. Prog Pediatr Surg 1976;9:41-62. [11] Kiesewetter WB, Sukanochana K, Sieber WK. The frequency of aganglionosis associated with imperforate anus. Surgery 1977;58: 877-90. [12] Schärli AF, Kiesewetter WB. Imperforate anus: anorectosigmoid pressure studies as a quantitative evaluation of postoperative continence. J Pediatr Surg 1969;4:694-5. [13] Li L, Li Z, Wang LY, et al. Anorectal anomaly: neuropathological change in the sacral spinal cord. J Pediatr Surg 1993;28:880-5. [14] Yuan ZW, Lui VCH, Tam PKH. Deficient motor innervation to the sphincter mechanism in fetal rats with anorectal malformation: a quantitative study by Fluorogold retrograde tracing. J Pediatr Surg 2003;38:1383-8.
1548 [15] Abdel-Majid RM, Tremblay F, Baldridge WH. Tracer coupling of neurons in the brain retina inner nuclear layer labeled by Fluorogold. Brain Res 2005;1063:114-20. [16] Andrew LK, Blackshaw LA. Colonic mechanoreceptor inputs to rat lumbo-sacral dorsal horn neurones: distribution, thresholds and chemosensory modulation. Neurogastroenterol Motil 2001;13: 333-7. [17] Martinez V, Wang LX, Mayer E, et al. Proximal colon distention increase Fos expression in the lumbosacral spinal cord and activates sacral parasympathetic NADPHd-positive neurons in rats. J Comp Neurol 1998;390:311-21. [18] Arhan P, Faverdin C, Devroede G, et al. Manometric assessment of continence after surgery for imperforate anus. J Pediatr Surg 1976;11: 157-67.
H. Jia et al. [19] Taylor I, Duthie HL, Nachary RB. Anal continence following surgery for imperforate anus. J Pediatr Surg 1973;8:497-503. [20] Rintala R, Lindahl H, Marttinen, et al. Constipation is a major functional complication after internal sphincter saving posterior sagittal anorectoplasty for high and intermediate anorectal malformations. J Pediatr Surg 1993;28:1054-8. [21] Nixon HH, Puri P. The results of treatment of anorectal anomalies: a thirteen to twenty year follow-up. J Pediatr Surg 1977;12:27-37. [22] Verma JS, Smith AN. Neurophysiological dysfunction in young woman with intractable constipation. Gut 1988;29:963-8. [23] Peña A, Amroch D, Baeza C, et al. The effects of the posterior sagittal approach on rectal function (experimental study). J Pediatr Surg 1993;28:773-8.
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Quantitative analysis of sacral parasympathetic nucleus innervating the rectum in rats with anorectal malformation Huimin Jia, Keren Zhang, Shucheng Zhang, Zhengwei Yuan, Yuzuo Bai, Weilin Wang⁎ Department of Paediatric Surgery, The 2nd Clinical College of China Medical University, Shenyang 110004, PR China
Index words: Anorectal malformation; Rectum; Sacral parasympathetic neurons
Abstract Background/Purpose: The purpose of this experiment was to identify the neurons in the lumbosacral spinal cord involved in colon-rectal function and to compare normal and anorectal malformation of fetal rats. Methods: The authors quantified the sacral parasympathetic nucleus (SPN) innervation of the rectum by Fluorogold (FG) (Fluorochrome, Englewood, CO) retrograde tracing experiment in fetal rats with normal and anorectal malformation. Anorectal malformation was induced in rat fetuses by ethylenethiourea (ETU). The number of FG-labeled SPNs was scored and compared between male fetuses with or without malformation in the ETU-fed group and control groups. Results: The number of FG-labeled SPNs in the fetuses without a defect, with ETU injected but without any defects of the anorectum or neural tube, with low-type deformity, and with high-type deformity were (mean ± SEM) 47.3 ± 2.9, 45.6 ± 3.2, 24.2 ± 3.8, and 8.5 ± 2.5, respectively. Fluorogold-labeled SPNs in the fetuses with high-type deformity were significantly fewer than those in fetuses without defects (P b .05) and in controls (P b .05). Conclusions: These findings suggest that defective SPN innervation to the rectum is a primary anomaly that coexists with the alimentary tract anomaly in anorectal malformation during fetal development. The intrinsic neural deficiency is an important factor likely to contribute to poor postoperative anorectal function despite surgical correction of the malformation. © 2007 Elsevier Inc. All rights reserved.
Anorectal malformation (ARM) is a common surgical problem affecting 1 in 1500 to 1 in 5000 live births. Posterior sagittal anorectoplasty (PSARP) as proposed by De Vries and Peña has been a popular procedure used to correct ARMs since the 1980s. The advantage of this procedure is the extensive exposure allowing meticulous anatomical repair of all the muscular structures present to achieve optimum continence [1]. There is evidence indicating that PSARP is equivocal in achieving good postoperative continence [2-5] ⁎ Corresponding author. Fax: +86 24 23903396. E-mail address: [email protected] (W. Wang). 0022-3468/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2007.04.034
and is associated with a tendency toward constipation, particularly in high or intermediate anomalies [6]. Severe constipation associated with megarectum has been noted sporadically after conventional anorectoplasty (ARP) in either low or high anomalies [7,8]. The causes of post-ARP constipation are still obscure and have been attributed to sacral anomalies [6], rectal inertia [8-10], Hirschsprung disease [11,12], and neuromuscular or sensory damage to the neorectum [11] as well as the well-preserved bowel end with intact sphincter muscle [1]. The lower urinary tract colonrectum share 2 common functions: storage and periodic elimination of body waste. These functions are regulated by
Quantitative analysis of sacral parasympathetic nucleus
1545
neural pathways located in the brain, spinal cord, peripheral ganglia, and enteric nervous system. We suspect that the postoperative effect is related to the rectal function. When the parasympathetic nerves are excited, the motility of the colon and rectum will be increased. We hypothesized that the development of SPNs is related to constipation. Results of our previous histopathologic study showed that there were fewer motor neurons in the sacral spinal ventral horn in patients with imperforate anus [13]. Yuan et al [14] quantified how the motor neurons innervating the levator ani muscle (rat equivalent of the puborectalis muscle in the human) were decreased, which plays a important role in fecal continence. They used a retrograde tract-tracing method in a rat model of ARM. To evaluate our hypothesis, we followed it using a microinjection method during fetal surgery to locate and quantify the parasympathetic nucleus in the lumbosacral spinal cord innervating the rectum. These studies have established that the Fluorogold (FG) is a reliable transsynaptic retrograde tracing agent that passes between neurons only at regions of synaptic contact [14,15].
anesthesia within 1 hour and were returned to their original cages to allow transport of the neuronal tracer.
1. Materials and methods 1.1. Experimental animals Twenty pregnant time-mated Wistar rats (250-300 g) were randomly assigned into 2 groups: 15 were gavage-fed with a single dose of 125 mg/kg of 1% ETU (ethylenethiourea [2-imidazolidinethione, 98%], C3H6N2S), and 5 of them were gavage-fed with an equal dose of physiologic saline on gestation day 10 (gestation day 0: sperm in vaginal smear after overnight mating) at the same time as the control group. All of the animal experiments were performed with an approval obtained from the appropriate local ethics committee.
1.3. Perfusion and histology After 24 hours postoperatively, the dams were reanesthetized with an overdose of pentobarbitone sodium, and the fetuses that had been injected with FG were harvested by cesarean delivery. Fetuses were perfused transcardially with 15 mL of physiologic saline followed by 25 mL 4% paraformaldehyde (wt/vol in 0.1 mol/L phosphate buffer). The lumbosacral spinal cord was dissected and postfixed in the same fixative for 24 hours at 4°C for 18 hours' incubation in 20% sucrose in 0.1 mol/L phosphate buffer. The spinal cords were sectioned on a cryotome into 45-μm serial transverse sections and mounted onto clean gelatinized microscopic glass slides. The series of transverse sections was used to locate the labeled SPNs in different segments of spinal cord and count the number of labeled SPNs. The FGlabeled sections were examined by a fluorescent microscope equipped with a digital charge-coupled device camera.
1.4. Microscopic and statistical analysis After fluorescent microscopic analysis, the labeled SPNs on each section were mounted, and an average was obtained. The results were analyzed using Student's t test. Significant differences were considered when the P value was less than .05. Data are presented as the mean ± SEM.
2. Results 2.1. General characteristics of experimental animals
1.2. Fetal surgery and injection of FG tracing dye The pregnant rats were anesthetized with ethyl carbamate (7 mL/kg body weight) on gestation day 20. An incision was made in the abdominal wall, and the uterine horn was exteriorized; then the uterus was covered by wet gauze immersed with warm physiologic saline. The uterus was opened close to the fetal tail, then the rectum or the rectal pouch was exposed under a dissection microscope. Five percent FG was injected into the wall of the rectum or the blind end of the rectum using a 30-gauge needle attached to a Hamilton syringe. The injection volume of FG at each point was 1 μL (at 4 sites: 3, 6, 9, 12 o'clock position). At each injection site, the needle was kept in place for several seconds after the injection while applying slight pressure to reduce leakage. The injected rectum or the blind end was thoroughly rinsed with physiologic saline to remove excess FG that might have leaked from the injection site. The fetuses were returned to the uterus, and the uterine wound was closed with 7-0 sterile silk suture. The pregnant rats recovered from the
In the ETU-injected group, 135 (58 females, 77 males) live fetuses were harvested from 15 dams. In external morphological inspection, 100% (135/135) of fetuses had a short or absent tail, 62.2% (84/135) of fetuses had an ARM, 57.2% (77/135) showed anomalies of the neural tube (including encephalocele, meningocele, or spina bifida), and 27.5% (37/135) showed anomalies of hind limbs. In this group, 39 fetuses were injected with FG. In the control group, 52 (20 females, 32 males) fetuses were obtained from 5 dams. None of them displayed any anomalies noted previously, and 10 fetuses were injected with FG. One dam died as a result of unsuccessful anesthesia, and 5 fetuses died of a long duration of injection procedure or blood loss.
2.2. Normal location and quantification of FG-labeled SPNs [16,17] In the control group, the FG-labeled rectal SPNs were seen from the L6 to S1 segments (Figs. 1 and 2).
1546
H. Jia et al. Table 1 The numbers of SPNs in different groups with congenital malformations Group 1 (10) Labeled SPNs
Group 2 (10)
Group 3 (8)
Group 4 (16)
47.3 ± 2.9 45.6 ± 3.2 24.2 ± 3.8*,† 8.5 ± 2.5*,†
Values within parentheses indicate the number of fetuses. Group 1 represents controls; group 2, fetuses with FG injected but without any defects of anorectum or neural tube; group 3, fetuses with low-type deformity; group 4, fetuses with high-type deformity. ⁎ P b .05 from control group. † P b .05 from group 2.
Fig. 1 Representative photomicrograph of transverse section from lumbosacral spinal cord of normal fetal rat (original magnification×100).
The location and quantification of FG-labeled SPNs in ARM was as follows: in the ETU-injection group, 39 fetuses were injected with FG. When controls and ETU-injected fetuses without any anomalies were compared, SPNs were fewer and smaller in the spinal cord in fetuses with imperforate anus. When the blind end of rectum was found below the levator ani muscle, it was defined as low-type deformity and as high-type deformity otherwise. (Table 1, Figs. 3 and 4). The number of labeled SPNs in abnormality group was fewer than that in the normal group. The number of labeled SPNs in the high-type deformity group was significantly fewer than that in the low-type group.
3. Discussion The pathophysiologic characteristics of anorectal function is complicated, and there has been no consensus regarding
Fig. 2 The region was magnified as shown in Fig. 1 (original magnification ×400).
the essential factors that control anal continence or the etiology of constipation. Schärli and Kiesewetter [12] proposed that anal continence after ARP for imperforate anus depends on 3 elements: adequate length of the anal canal, 25 mm Hg or more of resting anal pressure, and adequate action of the external and internal sphincters together with an intact puborectalis sling. Arhan et al [18] suggested 2 additional factors, stressing the importance of sensitivity of the anal skin and the presence of the rectoanal sphincter inhibitory reflex, which was supported by Taylor et al [19]. The suggested causes of constipation are much more complex and varied, including rectal inertia or ectasia, which may be congenital [6,8,10,20] or acquired [21], reduced rectal sensitivity [6,20], an abnormal internal anal sphincter that fails to relax on rectal distension [21], an intact or hyperactive internal sphincter [6,20], an abnormal rectoanal sphincter inhibitory reflex and increased sphincter pressure [22], and Hirschsprung disease [6]. With the conventional abdominoperineal pull-through and perineal anoplasty for ARM, constipation was not a significant problem after ARP, although there were sporadic reports of constipation after repair, particularly in low anomalies [7,8,10,21]. Peña [9] reported an especially high incidence (70%) of constipation in instances of anovestibular fistula,
Fig. 3 Fluorogold-labeled SPNs were fewer and smaller in the lumbosacral spinal cord of fetal rat with low-type imperforate anus (original magnification ×100).
Quantitative analysis of sacral parasympathetic nucleus
Fig. 4 Fluorogold-labeled SPNs were fewest and smallest in the lumbosacral spinal cord of fetal rat with high-type imperforate anus (original magnification ×100).
with an overall constipation rate of 30% after repair among 131 cases of ARM without other complex malformations that were followed for more than 3 years after PSARP. Whereas most of the reported constipation was encountered in low anomalies, Rintala et al, in contrast, noticed a high incidence of constipation in high or intermediate anomalies undergoing PSARP, similar to our clinical experience. They reported constipation in 70% and 28% in high anomalies undergoing PSARP with or without salvage of the internal sphincter, respectively, suggesting an intact internal anal sphincter as one of the contributory factors in post-ARP constipation [6,20]. Chen reported that the incidence of constipation after ARP was related to the extent of pelvic dissection. During PSARP, the endopelvic fascia has to be dissected away from the terminal bowel and its fistula; and thus, the autonomic nervous system, especially the parasympathetic inferior hypogastric plexus, will inevitably be more or less damaged. However, clinical evaluations demonstrate that a higher incidence of constipation occurs in patients with lower malformations who had less pelvic dissection [23]. Therefore, we hypothesized that either abnormalities of the nerve ending or the central nervous system would affect continence. We hypothesized that the number of the SPNs may be related to constipation. The results of this study confirmed our hypothesis that there was a deficiency of SPNs in the spinal cord innervating the rectum in an animal model with ARM. The number of labeled SPNs in the high-type deformity group was significantly fewer than that in the low-type group. Because the parasympathetic nerves are motor to the rectal wall muscle and inhibitory to the internal sphincter, their damage will affect the motor activity of the anorectum, resulting in a reduced resting rectal pressure, an increased resting anal pressure, and subsequent increase in ano-rectal pressure gradient and constipation. Dysplasia of rectum may be an additional possibility that induces the deficiency of SPNs. Rectal dysplasia can be affected by the type of colostomy chosen (transverse and loop stomas may
1547 result in megarectum) and by how long the colostomy was left in place before closure. Unfortunately, we did not find any abnormality of the rectum in this study, so we could not determine the major cause of the defects in the developing spinal cord in imperforate anus. We hope to answer this question in our further studies. In evaluating evidence of earlier studies, the ETU appears to have various targets in the rat embryo; differentiation of the mesoblast of the different organs is inhibited by ETU resulting in malformations. However, ETU toxicity did not directly affect the survival of SPNs because the number of labeled SPNs in the ETU-injected fetus without imperforate anus or other anomalies was similar to those in controls. In the future, we plan to apply gene therapy using neurotrophic factors to rescue the SPNs (alter the deficiency) in this malformation. This study confirms our hypothesis that there was defect of SPNs in the spinal cord innervating the rectum in an animal model with ARM. Our findings suggest that defective SPN innervation to the rectum mechanism may coexist with the alimentary tract anomaly in ARM. The intrinsic neural deficiency is an important factor likely to contribute to poor postoperative anorectal function despite surgical correction of ARM.
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