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Keywords:

  • anal continence;
  • anal sphincter injury;
  • length–tension;
  • plication

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

Background  Our recent studies show that the external anal sphincter muscle (EAS) operates at a sarcomere length range which is below optimal. In this study, we tested the hypothesis that by surgically increasing sarcomere length and bringing it close to the optimal length, EAS muscle function and anal canal pressure can be enhanced.

Methods  Rabbits (n = 25) were anesthetized and subjected to either a sham or an EAS plication of different length by placing sutures at two locations, at a distance of 13%, 20%, 28%, or 35% of the circumferential length of the anal canal. Anal canal pressures were recorded before and after the plication. Anal canal was harvested and the EAS muscle sarcomere length was measured using laser diffraction.

Key Results  Electrical stimulation of the EAS muscle resulted in a stimulus-dependent increase in the anal canal pressure (mmHg) and EAS muscle stress (mN mm−2). A significant increase in maximal pressure (212 ± 13 after compared with 139 ± 22 before plication) as well as stress (166 ± 10 after as compared with 88 ± 14 before plication) was recorded at 20% plication length. Passive anal canal stress at 20% plication was not significantly different compared with the sham group. The mean sarcomere lengths with sham and 20% plication were 2.11 and 2.60 μm, respectively.

Conclusions & Inferences  EAS plication resulted in a length-dependent increase in EAS muscle sarcomere length with an optimal sarcomere length at 20% plication. These considerations may help guide repair of anal sphincter muscle defects in the humans.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

Smooth muscle of the internal anal sphincter (IAS), striated muscle of the external anal sphincter (EAS), and striated puborectalis muscle (PRM) play key roles in the fecal continence mechanism.1 Current understanding is that the resting anal canal pressure is mostly related to the IAS and the increase in anal canal pressure with voluntary squeeze is related to the contraction of striated EAS muscle.2–5 Pressure recording of the anal canal using side-hole manometry and pull-through techniques show that, both at rest and during voluntary squeeze, the highest pressure is located in the part of the anal canal where the IAS and EAS muscle overlap, suggesting that the EAS is most likely the strongest component of the anal continence mechanism.6 The EAS is also the most commonly affected muscle in women with fecal incontinence because it frequently gets damaged during childbirth secondary to trauma and episiotomy.7,8 Fecal incontinence secondary to EAS muscle dysfunction continues to present a difficult therapeutic challenge.4 Clinical studies show that disruption of EAS muscle fibers correlates with impaired function and symptoms of fecal incontinence. Anatomical data show defects of the EAS in 20–30% of multipara women (most probably related to the birth-related trauma)8 and functional data (based on the manometric studies) show poor squeeze pressure of the anal canal7,9 in significant number of patients with fecal incontinence. Overlapping sphincteroplasty of the EAS muscle is a commonly performed surgical procedure to treat fecal continence.10,11

The sarcomere length of a muscle determines the stress or the force it generates when stimulated; this is referred to as the length–tension property of a muscle12 At optimal sarcomere length, a muscle generates peak the highest force (optimal stress). The optimal muscle length is directly related to sarcomere length and it appears that the optimal muscle length may not be the length at which it operates in vivo. For example, cardiac muscles operate at suboptimal length (short sarcomere length) to increase ejection volume with ventricular filling.13 Multifidus, a skeletal muscle involved in locomotion and spine support also operates at short-sarcomere length.14 A recent study from our laboratory in rabbits show that the EAS muscle also operates at short sarcomere length,15 which suggests that it may be possible to gain EAS muscle function by increasing its sarcomere length. Thus, the goal of our study was to determine the effects of varying degrees of EAS muscle plication on sarcomere length, canal pressure, and the resulting EAS muscle stress.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

The institutional animal care and use committee at the VA San Diego Healthcare Systems approved the study protocol and all experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA). Adult New Zealand white female rabbits (n = 25; 4–5 kg) were anesthetized with an intramuscular injection of ketamine (35 mg kg−1) and xylazine (5 mg kg−1). An intravenous catheter was placed (ear vein) for administration of drugs.

The rabbit model for anal canal function studies is an established model in our laboratory.15 For the in vivo plication studies, anal canal circumference was measured and an incision was made on the perianal skin to expose the EAS muscle. Two custom-designed copper wire, hook electrodes were placed in the EAS muscle for direct electrical stimulation. Anal canal pressure was measured using a manometric catheter equipped with 3-mm-diameter sleeve sensor (Dent Sleeve Inc, Ontario, ON, Canada). The catheter was placed in such a fashion that the pressure-sensing surface of the sleeve sensor faced the posterior midline direction. The sleeve sensor measures the highest pressure along its length and therefore the highest pressure along the length of the anal canal, irrespective of the muscle contributing to that pressure.16 Pressures were recorded at rest and then during electrical stimulation of EAS muscle. A pulse generator (Grass Technologies, West Warwick, RI, USA; Model S48) connected to a constant current unit (Grass Technologies, West Warwick, RI; Model CCU1A) with currents ranging from 1 to 6 mA, in steps of 1-mA increment (frequency 50 Hz, pulse duration of 0.2 ms) was used for electrical stimulation. Sodium nitroprusside (SNP; Sigma Chemicals, St. Louis, MO, USA) (1.5 μg kg−1) and pancuronium bromide (PB) (0.4 mg kg−1) were used during in vivo studies to define the relative contribution of IAS and EAS to the anal canal pressure.2,17 Animals were maintained on artificial ventilation (24 breaths min−1) after administration of PB using the Harvard Apparatus, dual phase control respiratory pump (Harvard Apparatus, Holliston, MA, USA; Model 613) via an endotracheal tube. Adequate anesthesia level was monitored by recording heart rate during surgery.

Figure 1 shows the schematic of surgical plication technique. Briefly, after skin incision the EAS muscle was exposed. Sutures (4.0-gauge polypropylene) were placed at two locations on the EAS muscle, at a distance of 13%, 20%, 28%, and 35% of the circumferential length of the anal canal for the animals in groups (n = 5 each) II, III, IV, and V, respectively. Group I (n = 5) represented control animals in which a skin incision was made, but no plication was performed. The two ends of the sutures were tied together (EAS plication) and the skin incision was closed. Studies were performed immediately after plication to evaluate the acute effect on anal canal pressure and muscle stress using electrical stimulation. Effect of EAS plication on the anal canal pressure during electrical stimulation of the EAS was also studied following administration of PB (0.4 mg kg−1) and SNP (1.5 μg kg−1) to define the passive anal canal stress.

image

Figure 1.  Schematic of surgical plication technique. Circumference of the anal canal was first measured and a skin incision was made to expose the external anal sphincter (EAS) muscle. Sutures (4.0-gauge polypropylene) were placed at two points on the EAS muscle, at a distance of 20% or 35% of the circumferential length of the anal canal. The two ends of the sutures are tied together (EAS plication) and the skin incision was closed. 1 = unplicated; 2 = plicated areas; ri is the inner radius; ro, the outer radius; and rm the mid-wall radius of the EAS muscle.

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Measurements of sarcomere length

At the end of recording period, the anal canal with intact sphincter was removed from control as well as plicated animals and fixed in 10% phosphate-buffered formalin solution for 24–48 h to fix the EAS muscle and other tissues. For analysis, the representative samples of EAS muscle were collected around the circumference at 3, 6, 9, and 12 o’clock positions of the anal canal. Samples were then rinsed in phosphate-buffered saline and placed in 15% sulfuric acid (8–12 h) to digest partially the connective tissues surrounding the muscle. After acid digestion, EAS muscle fibers were isolated by micro-dissection, mounted on microscopic glass slides and subjected to laser diffraction for sarcomere length determination.18 The muscle fiber bundle was trans-illuminated by a He–Ne laser (Model 05-LHR-171; Melles-Griot, Irvine, CA, USA) and sarcomere length was calculated using the grating equation: = d.sinθ, where n is the diffraction order (±1, ±2, ±3, etc.), λ is the laser wave length (0.632 μm), d is the grating spacing (which equals SL), and θ is the diffraction angle measured using a photodiode array.

Data analysis

All pressures were measured relative to atmospheric pressure. Resting pressure was determined as the average pressure recorded 30 s before EAS muscle stimulation. Maximum (total) pressure during EAS stimulation was defined as the peak pressure recorded during the 10-s stimulation period. The delta pressure was defined as the difference between maximum and rest pressure.

The force of contraction of the EAS muscle (stress; Tm) was estimated using the following equation for thick-wall tube: Tm = Pri2/(ro2ri2) + Pri2ro2/[rm2(ro2ri2)], where P is intraluminal pressure, ri is the inner radius, ro the outer radius, and rm the mid-wall radius of the EAS muscle.19 Values for ri, ro, and rm were derived from the ultrasound images of the rabbit anal canal15 and confirmed by directly measuring these parameters in a freshly harvested specimen. Correction for changes in muscle thickness due to plication was applied based on the percent of muscle plicated, assuming a constant inner lumen radius (corresponding to 3-mm-diameter probe and mucosa). The outer muscle radius was calculated at each level of plication. For example at 35% plication, the active muscle area is 65% of the total muscle area and the inner EAS radius is the same (assuming no change in probe size or mucosa volume). From this area, we calculated the outer muscle radius, assuming a circular geometry (confirmed by US imaging—data not shown). The difference between the outer radius and inner EAS muscle radius was the muscle thickness. Mid-wall radius was estimated as the sum of inner radius and half of the muscle thickness. Total stress denotes maximum stress recorded for the stimulus and delta stress is the difference between the total and the resting or baseline stress. The EAS muscle stress expressed in units of mN cm−2 represents the average circumferential stress per unit area of the circular EAS muscle.20

Statistical analysis

Data are shown as mean ± standard error mean (SEM). For plication studies, mean values before and after surgery were compared using paired t-tests. For sarcomere length evaluations, one-way anova with post hoc Tukey test (SPSS, Chicago, IL, USA) was used for comparison. P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

Effect of plication on anal canal pressure/stress

Anal canal pressure was measured, both at rest and during electrical stimulation of the EAS, before and after EAS plication. Representative tracings of anal canal pressure (mmHg) before and after 20% plication surgery are shown in Fig. 2A. This record shows a significant and stimulus intensity-dependent increase in the anal canal pressure caused by EAS electrical stimulation, with 1–6 mA current intensities. These tracings also indicate that, for the same electrical stimulus, 20% EAS plication results in a significant increase in the anal canal pressure compared to no plication. On the other hand, with 35% plication, there was no noticeable increase in the anal canal pressure compared to no plication, for an equivalent electrical stimulus (Fig. 2B).

image

Figure 2.  Representative tracings show the effect of external anal sphincter (EAS) plication (20% and 35%) and electrical stimulation on anal canal pressure. Note a twofold increase in the anal pressure after 20% plication and with maximal electrical stimulus (6 mA) as compared to no plication (top). On the other hand, 35% plication did not cause greater increase in the anal canal pressure as compared to no plication.

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Effect of varying degrees of EAS plication on anal canal delta pressure (mmHg) and EAS muscle stress (mN mm−2) are summarized in Fig. 3. Luminal radius (mid-wall) in the unplicated (sham) and following 13%, 20%, 28%, and 35% plicated animal groups was 4.06, 4.02, 4.00, 3.98, and 3.96 mm, respectively. Wall thickness in the sham animals was 0.58 when compared to 0.51,0.47, 0.43, and 0.39 mm in 13%, 20%, 28% and 35% plicated animals, respectively. Electrical stimulation resulted in the stimulus intensity-dependent increase in the anal canal pressure and EAS muscle stress. A significant increase (P < 0.05) in the peak anal canal pressure was observed after 13% and 20% muscle plications, over the sham-operated animal for the 6-mA electrical stimulus. On the other hand, a significant increase in the peak stress was observed after 13%, 20%, 28%, and 35% muscle plication for the 6-mA electrical stimulus. The maximal or peak pressure was recorded at 20% plication length (212 ± 13 mmHg after plication compared with 139 ± 22 before plication). The corresponding peak stress was 166 ± 10 mN mm−2 after plication as compared with 88 ± 14 mN mm−2 before plication; which represents significant increase compared to the preplication value). Increases in plication length of more than 20% had no additional impact on the electrical stimulation-induced increase in the anal canal pressure (166 ± 29 mmHg after 35% plication in comparison with 152 ± 17 mmHg before plication) although the EAS muscle stress after 28% and 35% plication was significantly higher as compared with before plication. A similar trend in delta pressure and stress values was observed after varying degrees of plication (Fig. 3).

image

Figure 3.  Effect of external anal sphincter muscle plication and electrical stimulation on anal canal delta pressure (A) and stress (B). A significant increase (*P < 0.05) in the delta pressure was observed after 13% and 20% muscle plications, for the maximal electrical stimulus. On the other hand, a significant increase in the delta stress was observed after 13%, 20%, 28%, and 35% muscle plication for 6 mA electrical stimulus.

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Mean anal canal rest pressure in the unplicated (sham) and following 13%, 20%, 28%, and 35% plicated animal groups was 11 ± 4, 12 ± ,1, 14 ± 1, 15 ± 2, and 12 ± 3 mmHg, respectively (Fig. 4A). The corresponding EAS muscle resting stress in these groups was 6.83 ± 2, 7.43 ± 0.6, 9.07 ± 4, 9.61 ± 2, and 7.61 ± 2 mN mm−2, respectively (Fig. 4B). Mean anal canal passive pressure (rest pressure measured after the administration of PB + SNP) in sham, 13%, 20%, 28%, and 35% plicated animals were fairly low, 4 ± 2, 5 ± 0.5, 8 ± 2.3, 5 ± 0.7, and 4 ± 0.4 mmHg and these values did not increase with plication (Fig. 4A). The corresponding passive stress in these groups was 2.54 ± 1, 3.61 ± 0.4, 6.26 ± 2, 4.33 ± 0.6, and 3.82 ± 0.4 mN mm−2, respectively. These data demonstrate that plication had no significant effect on the passive anal canal stress (Fig. 4B).

image

Figure 4.  Effect of external anal sphincter (EAS) muscle plication on the anal canal pressure (A) and stress (B) at rest and following administration of pancuronium bromide (PB) and sodium nitroprusside (SNP), which represents the passive anal canal stress. Note, that the EAS plication had no significant effects on the passive anal canal pressure or stress.

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Sarcomere length measurement

Figure 5 shows the effect of EAS plication on the EAS sarcomere length. In sham animals, mean sarcomere length of the EAS muscle was 2.11 ± 0.09 μm. EAS muscle plication resulted in the length-dependent increase in the EAS sarcomere length (Fig. 5). The mean sarcomere length in sham, 13%, 20%, 28%, and 35% plication was 2.11 ± 0.09, 2.24 ± 0.03, 2.60 ± 0.03, 2.69 ± 0.03, and 2.80 ± 0.04 μm, respectively. Sarcomere length recorded from the excluded part of the EAS was 2.06 ± 0.07 which was not significantly different from the sham animals. There was no significant difference in sarcomere length from the muscle fiber samples collected around the circumference of the anal canal at 3, 6, 9, and 12 o’clock positions of the anal canal (data not shown). Optimal sarcomere length for the EAS muscle, based on our previous study, where we measured thin filament (actin) length, using immunofluorescence technique, is 2.59 μm.15 Therefore, the sarcomere length at 20% plication (2.60 μm), measured by laser diffraction is identical to the optimal EAS sarcomere length measured by the thin filament length using immunofluorescence technique.

image

Figure 5.  Effect of plication on the external anal sphincter (EAS) muscle sarcomere length. Anal canal was harvested from sham operated and after EAS plication of varying lengths and immersion fixed in the buffered formalin. EAS muscle fibers were dissected, fixed on microscopic slides and subjected to laser diffraction. Bar graph shows plication length-dependent increase in the EAS sarcomere length. (*P <  0.05).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

The results of this study demonstrate the following: (1) Surgical plication of the EAS muscle results in an increase in the sarcomere length. (2) The effects of EAS muscle plication are dependent on the length of plicated EAS muscle. Optimal sarcomere length, optimal EAS muscle stress and maximal anal canal pressure were observed at 20% plication. (3) Passive anal canal pressure/stress was not affected by the EAS muscle plication. Our data demonstrate that the EAS muscle operates at a short sarcomere length and it is possible to enhance the anal canal pressure/EAS muscle stress by surgically adjusting the EAS sarcomere length to its optimal length.

The length–tension curve of skeletal muscle shows that at the optimal sarcomere length an optimal tension is generated.12 Increase in the muscle length beyond its optimal length decreases active muscle contraction and increases passive tension. Results of this study corroborate our earlier findings15 that the EAS muscle operates at relatively short sarcomere lengths, or in other words, on the ascending limb of the length–tension curve. These results also prove that the EAS function can be enhanced by surgically increasing the EAS sarcomere length to its optimal length. In fact, EAS function should increase linearly with increased EAS muscle length and the current results could be used to define precisely any tension desired. Our results show that up to 20% plication there was plication-dependent increase in the anal canal pressure, EAS muscle stress, and sarcomere length without affecting the resting stress. At 28% and 35% plications, the effects on EAS muscle stress were more pronounced than anal canal pressure because of the contribution of muscle thickness in the stress calculations. Wall thickness is a denominator in the stress calculations and there was a plication-dependent decrease in the EAS muscle thickness which contributed to the observed stress values.

A 20% plication of the EAS muscle resulted in an optimal EAS muscle stress and optimal sarcomere length of 2.6 μm. It is important to note that the optimal sarcomere length measured by laser diffraction in this study is identical to the optimal sarcomere length calculated using the immunofluorescence technique in our previous study.15 Optimal sarcomere length is equal to twice the length of thin (actin) filament length (1.22 μm) + the width of the Z band (0.15 μm). The width of Z band is constant in all muscles and therefore one can calculate optimal sarcomere length by determining the thin (actin) filament length using immunofluorescence technique.15

What is the advantage for the EAS muscle to operate at a short sarcomere length in vivo? Based on the length–tension property of the muscle, passive tension generated by a muscle increases as its length increases, especially beyond its optimal length. Sphincter muscles in general have two functions, they maintain closure by muscle contraction and open after muscle relaxation to allow passage of contents. The EAS muscle must increase in length during anal canal distension to allow passage of contents, fecal pellets in the case of the anal sphincter. We propose that by operating at short sarcomere lengths, although the EAS muscle does not generate optimal stress, it does not generate significant passive stress when stretched. Low stress during distension will allow opening of the anal canal with minimal resistance thus permitting passage of reasonable size fecal pellets. It is surprising although that in our study, even with 28% and 35% plication there was no significant increase in the passive anal canal stress. Previously, Krier et al.21 studied the length–tension characteristics of the cat EAS muscle strips in vitro and major finding of their study was that at optimal length (Lo) the passive tension generated by the EAS muscle accounted for a significant, i.e., 40% of the total tension. In our length–tension experiments of the EAS muscle, we observed that passive tension accounted for 21% of the total tension at Lo.15 If 20% plication represents optimal EAS muscle length, any increase in the muscle length beyond optimal muscle length should result in an increase in the passive muscle tension, which was not the case in our study. We measured EAS muscle tension from the anal canal pressure values and these pressures were quite low after administration of PB and SNP, and did not increase with plication length. The passive tension that we measured in our experiments is the total anal canal tension and not just the EAS muscle tension. The anal canal tension in our experiment is calculated from the anal canal pressure which is the sum of passive tensions generated by all the structure of anal canal that include mucosa, IAS, EAS, subcutaneous tissue, and skin. It may be that the contribution of EAS muscle to the total passive anal tension is relatively small. On the other hand, active EAS muscle tension in our experiments was calculated from the increase in anal canal pressure with electrical stimulation of the EAS, which represents true contribution of the EAS muscle to anal canal pressure.

EAS muscle injury related to birth trauma in women is probably the most common cause of fecal incontinence and overlapping sphincteroplasty is the most commonly performed operation for the treatment of fecal incontinence (when the EAS muscle is anatomically disrupted).22,23 Principles of overlapping sphincteroplasty procedure include the preservation of scar tissue to anchor the sutures and overlapping of the fibro-muscular divided ends.24,25 Following initial enthusiasm26 with the success of operation, in the 1980s and 1990s, the follow-up studies show that overlapping sphincteroplasty does not provide relief from fecal incontinence in significant number of patients.27,28 There are several hypotheses as to why sphincteroplasty may fail, (i) breakdown of repair, (ii) aging, (iii) scarring, (iv) progressive pudendal neuropathy related either to the initial injury or subsequent repair, or (v) progressive changes in the muscle due to a large time interval between the time of trauma (usually at the time of childbirth) and surgery (usually 20–30 years later).28 Based on the current surgical practice, the degree of muscle overlap during sphincteroplasty operation is arbitrary. However, late failure after overlapping sphincteroplasty cannot be explained by the length of plicated muscle. Without the understanding of relationship between sarcomere length and muscle function, overstretch or under stretch of the EAS muscle may easily occur during overlapping sphincteroplasty and could lead to early failure of sphincteroplasty operation.

In conclusion, we tested a novel hypothesis; increasing sarcomere length surgically increases EAS muscle tension and anal canal pressure. Our findings have relevance to improve the surgical technique for the overlapping sphincteroplasty operation. In addition, our findings demonstrate that the EAS muscle force generation can be increased, even with an anatomically intact and normal functioning EAS muscle. The EAS muscle contraction may be weak in patients with partial pudendal nerve injury.10,29–32 Furthermore, studies suggest that the EAS muscle strength decreases with age.33–35 We propose that EAS muscle plication can improve the EAS muscle tension and anal canal function in the above settings as well. Therefore, it may be possible to treat fecal incontinence in the elderly and patients with partial pudendal nerve injury using EAS plication technique even when the EAS muscle is anatomically intact. Future studies should investigate if the gain in the anal canal function following EAS plication is maintained over extended time periods.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

This research was supported by a VA Rehabilitation Research & Development Service Merit Grant.

Author contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References

MRR performed the research, analyzed the data, wrote the paper; YJ performed research; VB analyzed data; RLL contributed to research design, provided essential tools; RKM designed the research study, provided essential tools, wrote the paper.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. References