OBJECTIVE: To estimate the effect of prolonged vaginal distention and anal sphincter transection on contractile properties of the external anal sphincter.
METHODS: Young female virgin rats were randomly assigned to four treatment groups (sham, vaginal distention, transection of anal sphincter plus repair, or combined distention and transection plus repair). After 3 weeks, the anal sphincter complex was analyzed for twitch tension, maximal tetanic force, fatigue, and maximal responses to electrical field stimulation. Each sphincter was serially cross-sectioned and stained with hematoxylin and eosin to evaluate sphincter integrity. Statistical evaluation was performed using analysis of variance (Student-Newman-Keuls) and χ2 analysis.
RESULTS: The function of a transected and repaired anal sphincter is compromised significantly after 3 weeks. Vaginal distention alone did not alter function of the sphincter. Further, prolonged vaginal distention together with sphincter transection did not result in statistically significant differences in sphincter function compared with transection alone.
CONCLUSION: In an animal model, there is a major effect on external anal sphincter function in vitro 3 weeks after laceration with repair. Although prolonged vaginal distention had no significant effect alone and minimal adverse effects on the lacerated anal sphincter, laceration of the sphincter has major adverse effects on its morphology and function.
LEVEL OF EVIDENCE: II
In an animal model, there is major effect on external anal sphincter function in vitro 3 weeks after laceration with repair.
From the Divisions of 1Urogynecology and Reconstructive Pelvic Surgery and 3Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas; and 2ProPath Laboratories, Inc., Dallas, Texas.
Supported by a research grant from The American Urogynecologic Society/June Allyson Foundation.
Corresponding author: Clifford Y. Wai, MD, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9032; e-mail: Clifford.Wai@UTSouthwestern.edu.
Financial Disclosure The authors have no potential conflicts of interest to disclose.
Anal incontinence is a debilitating condition that has psychosocial and economic consequences. The rate of postpartum anal incontinence ranges from 6–24%.1–4 Research indicates that direct injury of the anal sphincter mechanism is associated with postpartum anal incontinence.1–17 The exact mechanism is unclear. It is unknown whether prolonged vaginal distension by the fetal head alters the effect of anal sphincter injury on the continence mechanism, whether the muscular injury and defective repair process cause anal incontinence, or whether the circumstances leading to anal sphincter laceration during childbirth (ie, denervation and hypoxia caused by prolonged vaginal distention) contribute to this disorder. Finally, the effect of prolonged vaginal distention on healing of the surgically repaired anal sphincter complex is not defined. It is possible that neuronal injury occurs before episiotomy, and the combination of neuronal injury (or stretch) and fourth-degree laceration worsens anal sphincter function.
In the current study, we used the rat as an animal model to begin to understand the effect of prolonged vaginal distention on function of the external anal sphincter and to estimate the effect of prolonged vaginal distention and anal sphincter laceration (with repair) on physiologic function of the external anal sphincter.
MATERIALS AND METHODS
All procedures were approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center. After anesthesia with ketamine (40 mg/kg), acepromazine (0.2 mg/kg), and xylazine (10 mg/kg), young (2–3 months) virgin female Sprague-Dawley rats (200–300 g) were randomly assigned to one of four treatment groups using a random number table: a proctoepisiotomy, vaginal balloon distention, combined vaginal balloon distention and proctoepisiotomy, or sham operation (10–13 animals in each group). To control for the potential effects of suture and repair, a superficial vaginal incision (not involving the anal sphincter or rectum) was performed in all rats (including sham). The superficial incision was closed with 4-0 braided polyglactin suture (Vicryl, Ethicon, Inc, Piscataway, NJ).
For sham-operated animals, in addition to a superficial vaginal incision, a deflated 5-mL, 12-F Foley catheter (Bardex I. C., C. R. Bard, Inc., Covington, GA) (with the tip cut off approximately 2 cm to ensure the balloon portion of the catheter was positioned in the vaginal canal) was placed in the vagina of sham-operated controls for 1 hour. For the proctoepisiotomy group, a 7-mm incision was made with dissecting scissors through the anal sphincter complex. This incision extended beyond the superior margins of the external anal sphincter, which is approximately 3–4 mm in longitudinal length. The rectal mucosa was reapproximated with single interrupted stitches (1 mm apart) of 5-0 braided polyglactin suture. A second layer of single interrupted stitches (1 mm apart) of the same suture and caliber was used as a reinforcing layer. The external anal sphincter was reapproximated with two single interrupted stitches of 5-0 braided polyglactin suture. For simulated prolonged vaginal delivery, a modification of a previously described animal model was used.18 The 12-F Foley catheter was inserted into the vagina and secured in place with a pursestring suture of 4-0 braided polyglactin, with special care made to avoid the vicinity of the anal sphincter. The Foley balloon was inflated with 2.0 mL of water and left in place for 1 hour. A 50-g weight was attached to the catheter and suspended over the edge of the operating table. For combination balloon distention and proctoepisiotomy, rats underwent vaginal distention as described above. After deflation of the Foley catheter, a proctoepisiotomy was performed and repaired as described.
Three weeks after either proctoepisiotomy, vaginal balloon distention, combined proctoepisiotomy–balloon distention, or sham, rats were killed with pentobarbital (50 mg/kg intraperitoneally). Animals were weighed, and the anal sphincter complex was dissected and removed. A circumferential incision was made approximately 5 mm from the external anal orifice. Gentle traction of the perianal skin was used to facilitate sharp dissection of the anal complex from the connective tissue in the ischiorectal fossa laterally and the vagina anteriorly. The anal complex was removed intact after transecting the rectum 1.5–2 cm cephalad from the external anal orifice. Thereafter, a microscope was used to dissect the anal complex free of perianal fat and most of the perianal skin. To preserve integrity of the external anal sphincter, however, perianal tissue that was immediately adjacent to the sphincter was not teased away. The striated muscle of the external anal sphincter was identified, and the lower rectum was transected 1–2 mm above the sphincter. Tissues were mounted as a ring between two stainless steel wires in water-jacketed baths for assessment of contractile function, as previously described.19 Muscles were stretched to optimal length for force development (ie, 1.2–1.8-g resting tension). Optimal resting tension was maintained throughout the course of the experiment. Tissue dissection and experimental protocols were performed in physiological salt solution of the following composition: NaCl (120.5 mmol/L), KCl (4.8 mmol/L), magnesium sulfate (1.2 mmol/L), NaH2PO4 (1.2 mmol/L), NaHCO3 (20.4 mmol/L), CaCl2 (1.6 mmol/L), D-glucose (10 mmol/L), pyruvate (1 mmol/L), at a pH of 7.4. The solution was gassed with 95% oxygen and 5% carbon dioxide.
For electrical stimulation, platinum wire electrodes were mounted parallel to the suspended tissues. Electrodes were connected to an isolated pulse stimulator and a Grass stimulator (Grass Model S48, Grass Instrument Co., Quincy, MA) through a current-boosting amplifier (Bipolar Power Supply/Amplifier, Model 6826A, Agilent Technologies, Santa Clara, CA). Stimulation of each tissue was also controlled by driver and signal conditioning/amplifier units (Grass DC driver amplifiers 7DAE or 7DAF, Grass low-level DC Amplifiers 7PIF). Analog force signals were captured with a multi-channel analog-to-digital computer interface (National Instruments PCI-6032E, Austin, TX). Data were acquired at 100 Hz through the computerized interface. Data were collected before, during, and after initiation of field stimulation. Stimulation patterns were controlled by a computer program interfaced to the stimulator voltage source using a computer interface (National Instruments PC-DIO-24 Digital I/O Board controlling a specially designed circuit). A central computerized controller delivered precise stimulation duration and frequencies. Voltage and pulse duration (in milliseconds) was determined manually through settings on the stimulator unit. Voltage settings, duration and frequency were varied to obtain force–frequency and force–voltage response curves. Peak force production, defined as the maximal force produced during a contraction, was determined.
To determine twitch tension, tetanic force generation, and fatigue, experiments were conducted at 30°C. Twitch tension was determined after stimulation with one square 0.4 millisecond pulse of 50 volts. For tetanic force generation, the muscle was allowed to recover and then force–frequency curves were determined by stimulation at 10–120 Hz, 50 V, 0.4 millisecond pulse duration, for 300 milliseconds. After each frequency, muscles were stimulated with maximal stimulation of 50 V, 150 Hz. Muscles were allowed to recover for a minimum of 120 seconds before the next increase in frequency. Forces obtained at each frequency were compared with forces obtained with maximal stimulation immediately thereafter. The ratio of single twitch tension to maximal tetanic tension was determined for each sphincter. Fatigue was determined by maximally stimulating the muscle for 30 seconds at 50 V, 150 Hz. Force generation at 30 seconds was expressed relative to initial maximal force generation. Maximal responses to electrical field stimulation were then determined at 37°C. All experimental procedures before tetrodotoxin incubation were completed within 90 minutes, and we confirmed that sphincter function was maintained at maximal levels throughout the experimental time frame.
Atropine resistance of field-stimulated contractions was determined at 37°C by stimulation of the external anal sphincter tissues 20 minutes after treatment with atropine (10−6 mmol/L). After atropine-independent contractions were determined, tetrodotoxin (5×10−7 mmol/L) was added and, after 10 minutes, a final stimulation was conducted to assess nerve-independent contractile force.
Tissues were fixed at optimal length in neutral buffer formalin (10%) for 24 hours. Five micrometer cross-sections of formalin-fixed, paraffin-embedded tissues were obtained at 100-μm intervals throughout the entire complex and analyzed for possible disruption. Multiple cuts through the sphincter were necessary to control for artifacts imposed by a single plane of cross-section. Tissue sections were stained with hematoxylin and eosin and analyzed with a Nikon Eclipse E1000N microscope (Nikon Inc., New York, NY).
Statistical comparisons between groups were conducted by analysis of variance followed by Student-Newman-Keuls post-hoc testing. We used χ2 analysis and Fisher exact tests to compare the categorical variables of disruption or preservation of sphincter integrity, or impaired with nonimpaired functions. A nonimpaired sphincter was defined as values within 95% confidence interval (CI) of sham-operated controls. Data from previous experiments were used to predict sample sizes. To detect a 60% change in force, with a power of 0.8 at an alpha of 0.05, in a study involving four treatment groups, a sample size of 11 animals per treatment group was determined. P≤.05 was considered significant.
In this study, external anal sphincter function and morphology was analyzed 3 weeks after surgery. This time frame was chosen because, in the rat model, recovery from parturition seems to be complete by this time point, and other studies indicate that, in the rat, striated muscles exhibit complete resolution from injury by 3 weeks.20–22 Electrical field stimulation for 5 seconds at optimal characteristics and maximal contractile force resulted in prompt force development, which was maintained during 5 seconds of stimulation, followed by rapid relaxation after cessation of electrical field stimulation (Fig. 1A). There was no statistical difference in contractile force generation in sphincters between sham-operated animals and those treated with balloon distention (Fig. 1B). However, maximal force generation was significantly impaired in sphincters that were either transected and repaired alone or treated with combined distention plus transection/repair (Fig. 1, Table 1). Using the 5% CI of sham-operated controls as the lower limit of normal, none of the shams exhibited impaired responses to field stimulation, whereas three of 12 (25%) sphincters in the vaginal distention group were impaired (P=.12). Five of 13 (38%) and five of 10 (50%) sphincters were impaired in the transection of the anal sphincter with repair and transection of the anal sphincter with repair plus vaginal distention groups, respectively.
To further define responses to electrical field stimulation, force–frequency and force–voltage response curves were conducted in the external anal sphincters from all animals (Fig. 2). Frequency- and voltage-induced force generation in vaginal distention alone animals was similar to that of sphincters from control animals. Field-stimulated contractions of the sphincter transection group were decreased, attaining statistical significance at 30 V and 40 V, and a P of .052 at 50 V (Fig. 2). Field-stimulated contractions were significantly impaired after combined vaginal distention plus anal sphincter laceration/repair regardless of frequency or voltage (Fig. 2).
Additional characteristics of striated muscle function (twitch tension, tetanic force–frequency relationships, and fatigability) of the external anal sphincter were evaluated for each treatment group (Figs. 3 and 4). Force production during twitch contraction was unaffected by either vaginal distention or sphincter transection alone. However, combination distention with sphincter transection/repair resulted in significant impairment of twitch tension (P=.026, Fig. 3A) and maximal tetanic force generation (Fig. 3B). The twitch tension/tetanic tension ratio (Fig. 3C), tetanic force generation during the force–frequency protocol (Fig. 3D), or fatigability (Fig. 4) were not significantly different among groups.
To examine the effect of episiotomy and vaginal distention on cholinergic nerve-mediated contractions, maximal force of contraction was measured before and after treatment with the nonspecific muscarinic receptor antagonist, atropine. Field-stimulated contractions were inhibited approximately 30% by atropine in sphincters from sham-operated controls (Table 1). Incubation with atropine resulted in similar inhibition of contraction in sphincters from the other treatment groups (from 29–44%, Table 1), suggesting that the percentage of cholinergic nerves innervating the external anal sphincter is not affected by vaginal distention or proctoepisiotomy repair.
The normal morphology of the rat anal sphincter complex is illustrated in Fig. 5. The external anal sphincter is embedded in loose areolar connective tissue surrounding the perianal skin and the vaginal wall. The external anal sphincter circumscribes and is intimately associated with the internal anal sphincter (ie, the circular smooth muscle of the lower rectum). Some striated muscle fibers from the posterior aspect of the external anal sphincter are inserted on the ventral surface of the tail. The internal anal sphincter surrounds the lamina propria and epithelial cell layer of the rectal mucosa. Perianal glands are present within the lamina propria of the rectal wall.
The integrity and histomorphology of sphincter complexes from sham-operated controls, balloon distention, proctoepisiotomy repair, and combination of balloon distention plus proctoepisiotomy repair were analyzed. To control for possible error in interpretation due to oblique or imprecise sectioning, sphincter complexes were cross-sectioned at 100 μm intervals beginning at the perianal skin. Of 46 tissues, successful orientation and serial sectioning was conducted in 10 shams, 10 animals with balloon distention, 11 animals with proctoepisiotomy repair, and nine animals with distention plus proctoepisiotomy repair. The striated muscle of the external anal sphincter was identified in sections and noted as intact or disrupted in each section (Fig. 5). The sphincter was considered disrupted if an intact ring of striated muscle could not be visualized in any of the sections or if it appeared disrupted in all sections but attenuated in only one section. As expected, the external anal sphincter was intact in all sham-operated animals. In contrast, the anal sphincter was disrupted in seven of 11 (63%) with proctoepisiotomy repair and seven of nine (78%) with combined balloon distention and anal sphincter transection/repair. The relationship between disruption of the external anal sphincter and maximal force generation of the sphincter is illustrated in Figure 6. Although sphincter function tended to be decreased in disrupted or attenuated external anal sphincter muscles, similar histologic findings were observed in some tissues with excellent force of contraction. In animals with combined proctoepisiotomy repair plus balloon distention, force of contraction was uniformly decreased whether or not the sphincter was intact (Fig. 6). Fibroblast ingrowth, matrix deposition, and inflammatory infiltration accompanied disruption of the anal sphincter (Fig. 7). As expected, the site of disruption always occurred at the site of transection, and inflammatory cell aggregates (comprised predominantly of neutrophils, monocytes, eosinophils, and macrophages) were concentrated at this site. Striated muscle fibers did not span the site of transection, but were dispersed toward the vaginal epithelium and appeared disoriented (Fig. 7).
Overall, data obtained in this study indicate that, in an animal model, proctoepisiotomy with repair results in significant impairment of external anal sphincter function and disruption of the sphincter despite microscopic repair under experimental laboratory conditions. Further, the results indicate that although vaginal distention alone did not affect sphincter function, vaginal distention also had minimal effects on the impaired function induced by proctoepisiotomy with repair.
The relationship between pudendal denervation and anal incontinence has been previously demonstrated.9,23 Evidence of pudendal denervation is present in 15% to 42% of women after vaginal delivery.2,4,8 Anal incontinence, however, is present in a minority of these women. Thus, factors other than pudendal nerve injury seem to affect external anal sphincter function after parturition.
The external anal sphincter of the rat is comprised of both slow- and fast-twitch fibers. In general, after denervation, the proportion of fast-twitch fibers increase in slow-twitch muscles.24 Fatigability of fast-twitch skeletal muscle is more pronounced and the twitch-to-tetany ratio is decreased.25 Therefore, denervated slow-twitch muscles exhibit increased fatigability and increases in the twitch-to-tetany ratio as the muscle adapts to denervation and transitions from the slow- to fast-twitch phenotype.24,25 Herein, we found that the force-generating capacity of the external anal sphincter was significantly reduced in animals with proctoepisiotomy repair with or without prolonged vaginal distention. Fatigability and the twitch-to-tetany ratio, however, were not altered, and cholinergic nerves were not preferentially affected by episiotomy or vaginal distention. These findings suggest that the primary cause of the impaired function of the sphincter under these experimental conditions was not denervation. Further, reinnervation of a denervated muscle is not likely with a normal twitch-to-tetany ratio.22
The absence of denervation properties in the external anal sphincter 3 weeks after injury suggests that the sphincter may be mechanically disrupted. Sphincteric skeletal muscles must contract as a synchronous unit. Mature skeletal muscle fibers are fully differentiated and do not replicate in response to injury. Mature muscle fibers are not able to proliferate to bridge a defect in the transected sphincter. Instead, the sphincter contracts using the repaired connective tissue as an insertion point. Mechanical disruption was found in seven of nine (78%) sphincters from animals with balloon distention plus sphincter transection/repair. Force generation, however, was compromised in all animals in this group. Sphincter disruption was identified in seven of 11 (63%) sphincters from animals with proctoepisiotomy alone. Although force generation was decreased significantly in this subset of disrupted sphincters, sphincter impairment was also noted in some animals with an intact sphincter histologically. Collectively, these data indicate that although sphincter integrity is important in the overall function of the external anal sphincter, other factors also contribute to return of normal sphincter function after proctoepisiotomy repair. Combined vaginal distention and proctoepisiotomy repair resulted in impaired sphincter function in all animals (nine of nine) regardless of sphincter integrity. Proctoepisiotomy alone, however, resulted in impaired function in seven of 11 animals, a value not significantly different from the combination injury. Thus, although we were not able to demonstrate a significant adverse effect of vaginal distention on sphincter function after proctoepisiotomy with this sample size, the finding that all characteristics of sphincter function were decreased in the combination group relative to the proctoepisiotomy alone group suggests that vaginal distention may compound the effect of proctoepisiotomy on sphincter function if more animals were included in the analysis. Nevertheless, the data indicate that the major determinant for adverse function of the anal sphincter 3 weeks after injury is anal laceration, not prolonged vaginal distention.
The effects of episiotomy and vaginal distention on external anal sphincter function reported herein with nonpregnant rats may be different in pregnant animals. During pregnancy, vascularization of the perineum and pelvic organs is increased dramatically, and the vaginal wall undergoes significant remodelling, including recruitment of inflammatory cells. The effect of these changes on repair of an injured external anal sphincter is not known. Studies are ongoing to address this issue. It should be emphasized that external anal sphincter function is only one component of the continence mechanism. Studies involving women with cloacae and investigations with women and other species indicate that the puborectalis and internal anal sphincter also play important roles in maintaining anal continence.26 In this regard, we found that the external anal sphincter of the rat exhibited increased passive tension relative to nonsphincteric skeletal muscle (data not shown). This is in agreement with studies reported in the cat in which the increased amounts of connective tissue between myocytes of the external anal sphincter are believed to contribute to increased passive tension.27
In conclusion, data from this investigation indicate that function of the external anal sphincter is significantly impaired after proctoepisiotomy repair. Prolonged vaginal distention alone seems to have little effect on function of the sphincter. Although prolonged vaginal distention may compound the effect of proctoepisiotomy repair, mechanical trauma is the primary determinant for impaired function of the sphincter 3 weeks after injury. External anal sphincter function was severely compromised in all animals that underwent combined vaginal distention and proctoepisiotomy repair regardless of sphincter integrity. These data suggest that mechanical injury of the external anal sphincter should be avoided and that, although sphincter integrity is important for function of the sphincter, other factors are also important for full recovery of sphincter function after muscle injury.
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