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Keywords:

  • anal continence;
  • anal sphincter;
  • external anal sphincter function;
  • length–tension characteristics

Abstract

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

Background  The length at which a muscle/sarcomere operates in vivo (operational length) and the length at which it generates maximal stress (optimal length) can be quite different. In a previous study, we found that the rabbit external anal sphincter (EAS) operates on the ascending limb of the length–tension curve, in other words at lengths shorter than its optimal length (short sarcomere length). In this study, we tested whether the human EAS muscle also operates at a short sarcomere length.

Methods  The length–tension relationship of the EAS muscle was studied in vivo in 10 healthy nullipara women. EAS muscle length was altered by anal distension using custom-designed probes of 5, 10, 15, and 20 mm diameter. Probes were equipped with a sleeve sensor to measure anal canal pressure. The EAS muscle electromyograph (EMG) was recorded using wire electrodes. Ultrasound images of anal canal were obtained to measure EAS muscle thickness and anal canal diameter. EAS muscle stress was calculated from the anal canal pressure, inner radius, and thickness of the EAS muscle.

Key Results  Rest and squeeze stress of the anal canal increased with the increase in probe size. Similarly, the change in anal canal stress, i.e. the difference between the rest and the squeeze, which represents the active contribution of EAS to the anal canal stress, increased with the increase in probe size. However, increase in probe size was not associated with an increase in the external anal sphincter EMG activity.

Conclusions & Inferences  Increase in EAS muscle stress with the increase in probe size, in the presence of constant EMG (neural input), demonstrates that the human EAS muscle operates on the ascending limb of the length–tension curve or at low sarcomere lengths. We propose that surgically adjusting EAS sarcomere length may represent a novel strategy to treat fecal incontinence in humans.


Introduction

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

Internal anal sphincter (IAS), external anal sphincter (EAS), and puborectalis muscle (PRM) are important components of the anal sphincter complex1–3 and together they contribute to generate the anal sphincter pressure. The latter is a key measure of the strength of the anal continence mechanism. It is generally believed that resting anal canal pressure is mostly related to the tonic contraction of IAS and that the increase in pressure with voluntary squeeze is due to the EAS. However, recent studies suggest that the voluntary contraction of PRM and EAS increases pressure in the proximal and distal parts of the anal canal, respectively.4,5

Skeletal muscles, such as the EAS, contract in response to central input from the cortical center and anterior horn cells of spinal cord. Neural impulses from cortical center impinge on the anterior horn cells of the spinal cord, which communicate to the skeletal muscle through peripheral nerves or through pudendal nerve in the case of EAS.6 Under physiological conditions, central mechanisms (neural drive to the muscle) and peripheral mechanisms (muscle length–tension curve) determine the ultimate force (stress) developed by the muscle during contraction.7 Sarcomere length is directly proportional to muscle length.8 Central drive or neural input to a muscle can be estimated by electromyographic (EMG) activity of muscle, and increase in anal canal pressure with voluntary squeeze can be used as a surrogate marker of the stress generated by the EAS muscle. Recent studies from our laboratory have focused on the active and passive length–tension characteristics of the EAS muscle in a rabbit model. Our results show that the EAS muscle operates at a short sarcomere length, which in turn suggests that it may be possible to gain EAS muscle function by increasing its sarcomere length.8,9 The goal of our studies was to determine if, similar to rabbit, the human EAS muscle also operates at the sarcomere length that is shorter than its optimal length, i.e. on the ascending limb of the length–tension curve.

Materials and Methods

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

The UCSD Institutional Review Board (Project #071326) approved the study protocol and each subject provided informed consent prior to participation in the study. Ten nullipara women (mean age: 33, range: 23–55) participated in the study protocol. Each subject completed the medical history and a urinary and fecal incontinence-scoring questionnaire to confirm the absence of urinary and fecal incontinence symptoms.

Electromyographic activity of the EAS was recorded using wire hook electrodes (Viasys Healthcare, Madison, WI, USA), which was placed transcutaneously 1–2 cm from the anal verge. The tip of the wire electrodes was placed at a depth of approximately 1.5 cm, at the 12 o’clock position. EMG signals were amplified using a custom-made EMG recorder. The bandwidth of the recorded signal was between 20 and 500 Hz. Raw EMG signal was integrated and a moving time (every 200 ms) average signal was recorded continuously during the entire study period and quantified in arbitrary units (AU). Electromyographic signal can be quantified either in microvolts (absolute values) or arbitrary units (to describe relative change). To measure in microvolts, system requires calibration with a device that can deliver a known amount of voltage to the amplifier. Arbitrary unit is a valid unit as long as one describes the relative changes in a given individual under different conditions, which was the case in our experiment. We have used the same methodology previously.10

Anal canal pressure was measured using a manometry catheter equipped with a 6 cm long reverse water-perfuse sleeve sensor.11,12 We developed this novel probe system to perform anal distension and at the same time measure anal canal pressure. The system consisted of placing a 5-mm diameter sleeve sensor in the custom-built probe holders of 10, 15, and 20 mm in diameter. The probe holders were made of a non-compliant material (propylene) and contained a groove for the placement of sleeve-sensor manometry catheter in the probe. The sleeve sensor was placed into the holder in such a way that the pressure-sensing surface of the sleeve faced outwards (Fig. 1). Sleeve sensor along with the holder was placed in the anal canal such that the pressure-sensing surface of the sleeve faced in the midline anterior direction of the anal canal. All signals, pressure, and EMG were recorded using a Medtronics Polygram (Medtronics, Minneapolis, MN, USA).

image

Figure 1.  Schematics of the manometry catheter and probe holder. Anal canal pressure is recorded using a 5 mm diameter manometry catheter, which is placed inside the probe holder of diameters of 10, 15, and 20 mm.

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Prior to the start of each study, subjects were trained to contract the EAS and pelvic floor muscles by the prompt: ‘squeeze as if you were trying to prevent the bowel movement or hold the stream of urine’. After each probe insertion, subjects were instructed to squeeze for 10 s and then relax for 30 s in between the squeezes to minimize EAS muscle fatigue.

Ultrasound images of the anal canal were obtained in each subject using a 3D ultrasound (US) system (Phillips Healthcare, Andover, MA, USA). We and others have described the details of the technique previously.5,13–18 Briefly, a 5–9 MHz US transducer was placed on the skin of the perineum with the transducer facing in the posterior direction. An US volume was captured to image the entire length of the anal canal. The entire cranio-caudal length of the EAS was identified in these US images, and a cross-sectional image of the anal canal at the mid location of the EAS was captured to measure the inner radius and thickness of the EAS muscle, as shown in Fig. 2. To determine the inner radius and the thickness of the EAS muscle for each probe size, the following calculations were made:

image

Figure 2.  Ultrasound image of the anal canal and measurement methods. Human anal canal US image with labels (left) and without labels (right). These measurements were used for the calculations of stress measurements as described in the Materials and Methods section. EASo and EASi denote EAS muscle outer and inner walls, respectively.

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  • 1
     Inner radius of EAS (ri) = √{(probe CSA + mucosal CSA) / π}.
  • 2
     EAS muscle thickness = √{(probe CSA + mucosal CSA + muscle CSA) / π} − inner EAS radius.
  • 3
     Mid EAS radius (rm) = inner EAS radius + ½ EAS muscle thickness.

Data analysis

Pressure transducers were calibrated prior to recording, and all pressures were measured referenced to atmospheric as the zero pressure. Rest pressure was determined as the highest pressure recorded 5 s before the subject was asked to squeeze. The squeeze pressure was the peak pressure recorded during the 10-s squeeze period. The ‘delta pressure’ is the difference between the squeeze and the rest pressure. The force of contraction of the EAS muscle (stress; Tm) was estimated using the equation for the thick-wall cylindrical structure because the ratio of radius to wall thickness for the anal canal is more than 10 : 1.19T= Pri2 / (rori2) + Pri2ro/ [rm2(ro− ri2)], where P is intraluminal pressure, ri is the inner radius, ro is the outer radius, and rm is the mid-wall radius of the EAS muscle. Values for ri,ro, and rm were derived from the ultrasound images of human EAS. External anal sphincter muscle stress represents the mean circumferential force per unit area of the muscle and was measured in mN cm−2. Rest and peak EMG activity was measured at the same time points as the rest and squeeze anal canal pressures.

Statistical analysis

Data are shown as means ± SEM. A two-way repeated measure anova with post hoc Tukey’s test (SPSS Inc., Chicago, IL, USA) was used to determine the effect of probe size on the anal canal pressure/stress and EMG activity. A Pearson correlation coefficient was used to determine if the EAS muscle stress was affected by the change in the anal probe size.

Results

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

Effect of probe size on the anal canal pressure

Sleeve sensor records the highest pressure along the length of anal canal, irrespective of the muscle contributing to the anal canal.20 A representative tracing of anal canal pressure and EMG activity with 5 and 20-mm diameter probes from one subject are shown in Fig. 3. This recording shows that, with voluntary squeeze, there is an increase in the anal canal pressure along with an increase in EAS muscle EMG activity. Furthermore, this recording also shows that with increase in probe size there is an increase in both the rest and the squeeze anal canal pressure. However, the increase in the probe size does not affect the amplitude of EMG activity of the EAS muscle.

image

Figure 3.  Representative records of the anal canal pressure and external anal sphincter (EAS) EMG with two different-sized anal probes. These tracings show that with the increase in the probe size, anal canal pressure (both rest and squeeze) increased without any increase in the EAS EMG activity. Arrows indicate onset of squeeze.

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In 10 subjects, the anal canal pressure with the 5-mm diameter sleeve sensor (without any probe holder) was 51 ± 8 mmHg and with squeeze it increased to 138 ± 17 mmHg. Rest, squeeze, and delta anal canal pressures increased with the increasing probe size. Rest and squeeze pressures with the 5-mm manometry catheter were significantly lower than the pressures recorded with other probe sizes (P < 0.001 and P < 0.01, respectively). Difference between the rest and the squeeze anal pressures for 5 and 10 mm probe size was statistically significant (222 mmHg with 10 mm probe vs 138 mmHg with 5 mm probe; P < 0.05); however, with the further increase in the probe size there was no significant increase in the squeeze and delta anal canal pressures (Fig. 4).

image

Figure 4.  Effect of probe size on the anal canal pressure at rest and squeeze. Asterisk denotes a significant increase (P < 0.05) in the rest and squeeze pressure when compared with 5 mm probe. The anal canal pressure is significantly higher when measured with 10-, 15-, and-20 mm diameter probes compared with 5-mm probe, but there is no difference between 10-, 15-, and 20-mm diameter probes. A two-way repeated-measures anova with post hoc Tukey’s test (SPSS Inc., Chicago, IL, USA) was used to determine the effect of probe size on the anal canal pressure.

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Effect of probe size on the anal canal stress

The stress measured from the pressure values at rest and squeeze reflects the total anal canal wall stress at rest and squeeze. Total anal canal stress is related to all anatomical structures that form the anal canal. On the other hand, the delta or the difference between the total and the rest anal canal stress reflect the contribution of the EAS to the total anal canal tension. Anal canal stress, rest, squeeze, and delta increased linearly with the increase in probe size (Fig. 5). The delta anal canal stress at squeeze for 5, 10, 15, and 20 mm probe was 2947 ± 285, 4509 ± 494, 5964 ± 701, and 8404 ± 868 mN cm−2 , respectively. Anal canal stress at maximal squeeze for 5, 10, 15, and 20 mm probe were 4651 ± 558, 9033 ± 1118, 12 520 ± 1129, and 16 590 ± 1824 mN cm−2, respectively. There was a linear (r= 0.99) increase in the anal canal delta stress with the increase in the probe size.

image

Figure 5.  Effect of probe size on anal canal stress at rest and squeeze. Note the probe size-dependent increase in anal canal stress at rest and squeeze. Asterisk denotes a significant probe size-dependent increase (P < 0.05) in the stress. In addition, note a linear (r= 0.99) increase in the anal canal delta stress with the increase in the probe size.

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Effect of probe size on the external anal sphincter EMG activity

The EAS muscle demonstrated continuous tonic EMG activity at rest and with voluntary squeeze, a two-to three-fold increase in the activity (322 vs 836 AU). With the increase in probe size there was small increase in the rest EMG activity in some individuals, but the mean value at rest was not affected by the increase in probe size. Similarly, the squeeze EMG activity of EAS was not affected by the increase in probe size (Fig. 6).

image

Figure 6.  Electromyographic activity of EAS muscle at rest and squeeze. Raw EMG signal was integrated and a moving time (every 200 ms) average signal was recorded continuously during the entire study period and quantified in arbitrary units (AU). Note that there is no increase in the EMG activity, neither at rest nor at squeeze, with the increase in probe size.

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Discussion

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

The purpose of this study was to understand the physiological function of the EAS. Our data demonstrate that distension of the anal canal increases resting as well as the squeeze pressures and anal canal wall stress. Furthermore, the delta or the change in the anal canal pressure associated with voluntary contraction increases with the increase in probe size. The EAS muscle stress increases in a linear fashion with the increase in probe size and this increase is not associated with the increase in EAS muscle EMG activity. As reasoned in the following paragraphs, these observations support our assertion that the human EAS muscle operates on the ascending limb of its length–tension relationship or in other words at short sarcomere lengths.

Recently, we performed studies to evaluate the length–tension relationship of the rabbit EAS muscle.8 The goal of those studies was to determine the in vivo operational length of the EAS muscle. Similar to the probe system used in this study, we used various size probe to alter EAS muscle length and thus the EAS sarcomere length in the rabbit. We found that with the increase in the probe size there was a linear increase in sarcomere length. The latter was measured using laser diffraction method. Optimal EAS sarcomere length was determined from the thin filament (actin) length, which was measured using the distributed deconvolution method of immunohistochemistry images.21 According to the sliding filament theory,7 optimal sarcomere length is equal to twice the thin filament length plus the Z band width. Using above techniques, we found that the operational and the optimal lengths of the EAS muscle in the rabbit are approximately 2.1 and 2.6 μm, respectively.8 In other words, the EAS muscle length at which it operates in vivo is significantly (approximately 20%) shorter than its optimal length.

In this study, we did not measure sarcomere length and thin filament length, then how does our data prove that the EAS muscle operates on the ascending limb of the length–tension curve in humans? We show that with the increase in the probe size there is a linear increase in anal canal stress, both at rest and during squeeze. Anal canal pressure at rest is due to contributions from the IAS, EAS, and PRM.2,5 On the other hand, the change in anal canal pressure with voluntary squeeze can be due to the PRM and EAS muscles.5 Our studies also show that the PRM and EAS contraction increases pressure in the proximal and distal segments of the anal canal, respectively. Furthermore, the EAS-related increase in anal canal pressure is significantly larger than the PRM-related increase in pressure.4,5 As sleeve sensor measures the highest pressure along the anal canal length,10 we suggest that the squeeze-related increase in anal canal pressure (delta pressure) is entirely due to the EAS muscle. Our data show that the anal canal pressures are significantly higher with a 10-mm probe as compared with the 5-mm probe, but they do not increase in a linear fashion with the increase in probe size. On the other hand, the EAS muscle stress values show a linear increase with the increase in probe size. The latter is because, with the increase in the probe size there is an increase in the EAS muscle length and a decrease in the EAS muscle thickness, which when computed to determine EAS muscle tension, shows a linear increase in the EAS muscle stress with increasing probe size.

There are two main explanations as to why EAS muscle stress could increase with increasing probe size: (i) an increase in the neural drive to the EAS muscle (central mechanism), or (ii) an increase in EAS muscle sarcomere length if the EAS were to operate on the ascending limb of the length–tension curve (peripheral mechanism).8,22 Electromyograph activity of the EAS is a surrogate of the neural drive to the muscle. We observed that with an increase in probe size, there was no increase in the EMG activity of the EAS at rest or with voluntary contraction. The increase in the anal probe size should result in an increase in the EAS muscle sarcomere length, similar to what we found in our rabbit study.8 Therefore, we propose that the increase in EAS muscle stress with increase in probe size results from the increase in EAS sarcomere length. The latter is only possible if the EAS muscle were to operate in vivo at relatively short sarcomere length. On the other hand, if the EAS muscle were to operate at optimal or supra-optimal length in vivo; based on the length–tension relation of a muscle,6 one would expect a decrease in the EAS muscle stress with an increase in anal probe size. Our data also show that the EAS muscle tension with a 20-mm diameter probe was larger than the one with a 15-mm probe, which means that we did not achieve optimal tension even with a 20-mm diameter probe. As probes larger than 20-mm diameter were likely to cause discomfort to study subjects, we did not use bigger probes.

What is the advantage for the EAS muscle to work at short sarcomere length? Sphincters in general provide a barrier function for the movements of contents from one cavity to the other by remaining tonically contracted, and they open to allow movement of contents from one cavity to another during relaxation. Therefore, by operating at short sarcomere lengths, the sphincter muscle offers minimal passive resistance to the anal canal opening. It is not known if all the sphincters in the body follow similar principles; our own observation with the puborectalis muscle, which also provides a ‘sphincter-like’ function to the anal canal shows that indeed it also operates at the short sarcomere length (unpublished data).

One of the limitations of this study is the lack of direct measurement of sarcomere lengths in the human EAS, which would require harvesting the EAS muscle at each probe size, similar to what we did in the rabbits. Such tissue harvesting and measurements are impractical in studies involving the human subjects. However, we feel confident in our assumption that similar to rabbit, increase in anal probe size will increase the EAS muscle and sarcomere length.

Our finding of in vivo operational length of the EAS muscle being significantly shorter than its optimal length has considerable clinical relevance for surgical reconstruction of the EAS muscle. Overlapping sphincteroplasty of the EAS muscle is a commonly performed surgical procedure to treat fecal continence.23 The degree of muscle overlap during sphincteroplasty is likely to be the major determinant of sarcomere length after operation. Future studies should explore the ideal degree of muscle overlap or the optimal sarcomere length required to achieve maximum gain in EAS muscle function after overlapping sphincteroplasty. A recent study from our laboratory also shows that the plication of the normal EAS muscle in rabbit increases the EAS muscle sarcomere length and EAS muscle stress.16 A 20% plication of the EAS results in an approximately 70–80% increase in the EAS contraction-related anal canal pressure.23 Therefore, it is possible to gain anal canal closure function in humans by surgical adjustments of the EAS sarcomere length even in the setting of an anatomically intact EAS muscle. We propose that the above strategy could be important in patients with fecal incontinence with a poorly contracting EAS muscle as a result of either partial pudendal nerve injury or muscle atrophy.

Acknowledgments

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

This work was supported by Veterans Administration MERIT Grant and a grant from the American Society of Colon and Rectal Surgeons Research Foundation.

Author Contribution

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

RKM, GS, and RL contributed to the conception and design of the study; GS and BP performed the acquisition of data; RKM, MRR, and RL participated in the analysis and interpretation of data, drafting of the article, and revising it critically for intellectual content; RKM gave the final approval for the version.

References

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