Measuring length-tension function of the anal sphincters and puborectalis muscle using the functional luminal imaging probe

Abstract

The functional luminal imaging probe (FLIP) has been used to measure the distensibility of the anal canal. We hypothesized that with increasing distension of the anal canal with FLIP there will be an increase in length of the anal sphincter muscle allowing measurement of the length-tension function of anal sphincter and puborectalis muscles (PRM). We studied 14 healthy nulliparous women. A custom-designed FLIP bag (30-mm diameter) was placed in the vagina and then in the anal canal, distended in 10-ml steps with volumes ranging from 30 to 90 ml. At each volume, subject performed maximal voluntary squeezes. Length-tension measurements were also made with a manometric probe system. Tension was calculated (pressure × radius) in Newtons per meter using a custom software program. Peak tensions at different FLIP volumes were compared with the manometric data. No change in the luminal CSA was noted at low fluid volumes; the sphincter muscles were able to fully collapse the FLIP bag within the anal canal/vagina even at rest. At larger volumes, with each squeeze there was an increase in the bag pressure and reduction in the cross-sectional area, which represents concentric contraction of the muscle. Both rest and squeeze tension increased with the increase in volume in the anal as well as vaginal canal indicating that the external anal sphincter and puborectalis muscles produce more tension when lengthened. FLIP device, which has been used to describe the distensibility of the anal canal can also provide information on the length-tension function of the anal sphincters and PRM.

NEW & NOTEWORTHY The functional luminal imaging probe (FLIP) has been used to describe the distensibility of the anal canal. This report is the first to describe the use of the FLIP in the vaginal canal and the anal canal to provide information on the length-tension function of the anal sphincter and puborectalis muscles, which may provide clinicians with additional information regarding the active components of muscle contraction involved in the anal closure function.

INTRODUCTION

Continence is maintained in part by the active and passive properties of internal anal sphincter (IAS) and external anal sphincter (EAS) and puborectalis muscles (PRM) along with the surrounding tissues of the anal canal (1, 24). While high-resolution anorectal manometry is the current “gold-standard” to measure anal sphincter function (18, 19), it does not measure the length-tension property of the anal sphincter muscles. The latter is an ideal measure of the function of any muscle, because it indicates the relationship between the amount of force a muscle can produce and its length. The length-tension measure is a function of the degree of actin-myosin cross bridging during contraction. Our earlier studies show that similar to myocardium, the EAS and PRM, under normal physiological conditions, operate at the short sarcomere or suboptimal length (16, 17, 21). The latter means that when stretched the EAS and PRM can generate more force. In our earlier studies, we devised manometry probes of different sizes to measure the length-tension function of EAS and PRM in normal subjects (16, 17) and found it to be impaired in patients with fecal incontinence (12).

The functional luminal imaging probe (FLIP) uses an impedance-based system to measure the pressure and cross-sectional area (CSA) of the balloon/bag, every 0.5 cm centimeter along the whole length of an 8-cm-long bag (15). It has been used primarily to study the distensibility/compliance of the lower esophageal sphincter and esophagus (4, 9). Several studies have also used FLIP to measure the distensibility of the anal canal in normal subjects and patients with anal incontinence (5, 26). The distensibility is a ratio of the CSA to pressure and anal canal distensibility; it is higher in patients with fecal incontinence compared with normal subjects. Distensibility/compliance generally implies passive properties of the material, but in the case of biological system like anal canal, it is related to both the passive and active properties of the anal sphincter muscles and surrounding tissue. Since with distension of the FLIP bag the anal canal diameter and therefore the length of the surrounding muscle will increase, it should be possible to study the length-tension function of the anal sphincter and PRM using the FLIP device.

We report the length-tension function of the anal sphincter and PRM using the FLIP device and compare it with the length-tension function measured by our previously described manometry probe system (16, 21).

MATERIALS AND METHODS

Fourteen nulliparous women (mean age: 34 yr; range: 22–50 yr) with no history of pelvic floor dysfunction were recruited for participation in this study. The protocol was approved by the Human Research Protection Program at University of California, San Diego, and all subjects signed a written informed consent before their participation in the study. Each participant completed a medical history questionnaire to confirm the absence of urinary and fecal incontinence symptoms.

Length-tension manometry was performed as described previously. Briefly, pressures were measured using a manometry catheter equipped with a 6-cm long, reverse-perfused sleeve sensor (25). The catheter is 5 mm in diameter (2.5-mm radius), and it can be placed in the custom built noncollapsible probes of 5-, 10-, and 15-mm radius to measure vaginal pressure (a marker of the PRM function) and 2.5-, 5-, 7.5-, and 10-mm radius to measure the anal sphincter function. For the vaginal and anal measurements, subjects were placed in the lithotomy position. Each size probe was placed sequentially, following 1–2 min of recordings at rest, subjects were instructed to “squeeze as if they were trying to hold a stream of urine or prevent bowel movement.” Participants performed three squeezes with each size probe.

The FLIP measurements were performed with the participant in the lithotomy position. A custom-designed 15-mm radius bag (30-mm diameter when fully distended) was used for these studies. The FLIP bag was first placed in the vagina and distended in steps of 10-ml increments, starting from 30 to 90 ml. The participants were asked to perform voluntary squeezes, three times at each volume, with the same cues as used for the manometry probes. The procedure was repeated with the FLIP bag in the anal canal.

Data analysis.

For manometry data, all pressures were measured in reference to atmospheric pressure. The mean rest pressure (just before each squeeze) and the peak pressure during squeeze were determined for each size probe to calculate the tension (pressure × radius). The FLIP data were exported in text file format, containing 16 dynamic CSA and pressure (single-channel) measurements for each subject during the entire protocol. Next, the mean values for each squeeze were obtained. The latter consisted of a continuous time signal, comprised of the transition from baseline to peak pressure and back to baseline. For each squeeze sequence, the channel with the minimum CSA was used for the tension calculations. Only those distension volumes that produced luminal diameters larger than the FLIP probe diameter (8.3 mm) were used for the tension calculations. At each time point during the sequence, tension was calculated (pressure × radius) in Newtons per meter using a custom developed software program, and peak tension was determined.

Statistical analysis.

Parametric data are presented as means ± SD and nonparametric data as medians (interquartiel range). Normality was tested using the Shapiro-Wilk test. A general linear model with repeated measures was performed. One-way repeated ANOVA was used for comparison of means between groups when differences were found. For nonparametric data, related samples Friedman’s two-way analysis was used. Post hoc Bonferroni correction was used for multiple comparisons. Statistical significance was defined as P < 0.05. All calculations were performed in Matlab 2017a (Mathworks, Natick, MA).

RESULTS

Figure 1 shows the FLIP images obtained at different volumes of bag distensions in the anal and vaginal canal. At lower bag volumes (<60 ml in the case of anal canal and <50 ml in the case of vaginal canal), an “hourglass” shape of the FLIP is noted with the bag fully collapsed in the center (minimal diameter 8.3 mm). On the other hand, at higher bag volume the anal canal is distended (above the minimal diameter recorded by the FLIP) at rest and fully collapsed with squeeze. At highest volumes the anal and vaginal canals are distended above the minimal radius dimension, both at rest and squeeze.

Fig. 1.

Images of the functional luminal imaging probe (FLIP) at rest (A and C) and contraction (B and D) in the anal (A and B) and vaginal (C and D) canal at different bag volumes noted at the bottom of squeeze images.

Figure 2 shows a topographical plot of CSA over time measured by the FLIP in the anal and vaginal canal, with superimposed pressure, CSA, and tension lines. Note, with squeeze there is a decrease in the CSA of the anal and vaginal canal and an increase in the pressure and tension. The change in CSA occurs very quickly with the onset of squeeze and during sustained squeeze, the pressure and tension either remains constant or decreases with time, suggesting that the subject was not able to hold the strength of the squeeze during the test period. Figure 3 shows box plots with bag volumes of 70, 80, and 90 ml for the anal canal and 60–90 ml for the vaginal canal. Tension estimates are not shown for lower volumes, because the lumen was fully collapsed at these volumes, and therefore there was no change in the length of the muscle with bag distensions. The baseline and peak tension values increase with the increase in the bag volumes in both the anal and vaginal canal. The difference between baseline and peak tension, which reflects active tension during voluntary squeeze also increases with the increase in the bag volume.

Fig. 2.

Topographical plot of the luminal cross-sectional area (CSA) measured by the functional luminal imaging probe (FLIP) along the length of the anal (A) and vaginal (B) canal, with superimposed line graphs of the pressure, CSA, and tension. The top and bottom of the graph are the cranial and caudal ends of the bag, respectively. Arrows denote the onset and end of squeeze.

Fig. 3.

The average peak tension recorded by the functional luminal imaging probe (FLIP) system in the vaginal (A–C) and anal (D–F) canal at rest (A and D), squeeze (B and E), and the difference between the rest and squeeze (C and F); n = 14. See results for statistical differences.

Comparing rest values at different bag volumes in the anal canal, tension values for 90 ml were significantly higher than 80 and 70 ml (P < 0.05). Anal rest tension at 80 ml was not significantly different from that of 70 ml (P = 0.054). During squeeze, both 90- and 80-ml tensions were larger than their 70-ml counterpart (P < 0.05) but not from each other (P = 0.056). Finally, comparing the tension between rest and squeeze states, the differences were significant for all the volumes, with larger squeeze tensions compared with those of rest (P < 0.05).

For the FLIP system in the vaginal canal, estimated tension values from 90 ml at rest were significantly higher than all the other tensions at rest, using 60- to 90-ml volumes (P < 0.05). Moreover, tension using the 80-ml volume had larger values compared with the 60-ml probe (P < 0.05). The same trend also existed during squeeze. Finally, comparing the tension between rest and squeeze states, the differences were significant for all the volumes, with larger tensions at squeeze compared with rest (P < 0.05). Finally, comparing the tension between rest and squeeze states in the anal canal, all tensions during squeeze using different probes were significantly different from their rest counterparts (P < 0.05).

For the manometry probes (Fig. 4), the length-tension plots measured show an increase in the baseline and peak tension with the increase in probe size for both the anal and vaginal canals. The vaginal tension difference between rest and squeeze probes was significant for all probe sizes (P < 0.05). At rest, tension values for the 10-mm probe were significantly higher than all the other probes (P < 0.01). Moreover, the 7.5-mm probe had larger tension values than the 5- and 2.5-mm probe (P < 0.01). There was no difference between the 5- and 2.5-mm tension values (P = 0.16). During squeeze, tension values for all radii were statistically different from one another (P < 0.05).

Fig. 4.

Tension recorded by the manometric probe of different radii in the vaginal (A–C) anal (D–F) and canal at rest (A and D), peak of contraction (B and E), and the difference between the rest and squeeze (C and F). See results for statistical differences.

In the vagina using the manometry probes, the difference between rest and squeeze probes was significant (P < 0.01). At rest, tension values for the 15-mm probe was significantly higher than the 5- and 10-mm probes (P < 0.01). Although there was no difference between the 5- and 10-mm tension values (P = 0.16). During squeeze, tension values for the 15-mm probe was significantly higher than 5- and 10-mm probes (P < 0.01). Furthermore, tension values at 10 mm were also higher than the 5-mm probes (P < 0.01).

The comparison between the length-tension plots measured with manometric probes and FLIP is shown in Fig. 5. Slopes of both rest and squeeze states, in the vagina and anal canals, show that in the manometric probe system as well as in the FLIP system there is an increase in the tension slope going from rest to squeeze. The highest tension increase occurred in the vaginal canal in both systems. In general, the FLIP system has lower tensions than the manometry system due to the collapse of the FLIP bag. It was not possible to make a direct comparison between the manometry and FLIP system because the latter does not allow for an exact radius that would be comparable with the manometry system. No intersystem significance or agreement test was carried out due to relatively small number and uneven sampling points.

Fig. 5.

Comparison of the rest (A and C) and squeeze tension (B and D) recorded by different probe sizes in the case of manometry probes and different anal (A and B) and vaginal (C and D) radii in the case of functional luminal imaging probe (FLIP). The data in blue and red are with manometry probes and FLIP, respectively.

DISCUSSION

What does tension value represent in the anal and vaginal canal? The pressure measured in the anal canal is due to the active and passive properties of structures surrounding the anal canal that include IAS, EAS, and PRM. Along the same lines, the vaginal pressure is generated by the active and passive tissues of the vagina and surrounding muscle, i.e., levator-ani or pelvic floor muscles. Our studies show that the PRM is the major contributor to the genesis of both baseline as well as peak pressure during voluntary squeeze in the vaginal canal. The force generated by a given muscle in the body is related to the length of the muscle, known as the length-tension property. In general, the force-tension increases with the increase in muscle length. At optimal length, all muscles generate maximum force. Intuitively, one would think that under normal physiological conditions muscles in the body must be placed at an optimal length so that with contraction they generate maximal force, which is actually not the case. Similar to myocardium, the EAS (16) and PRM (17) operate at the suboptimal length under normal physiological conditions, i.e., when they are stretched using different size probes, the force generated increases with the increase in the probe size.

Our data show that similar to the manometric probes, the FLIP system records an increase in the baseline as well as squeeze tension with increase in the dimension of anal and vaginal canal. Our study is the first to use FLIP system in the vaginal canal and to describe its use as a measure of the muscle function. The increase in tension with manometric probes appears to be bigger as compared with the FLIP, especially at higher degrees of distension. The difference between manometric and FLIP system is that the former measure isometric contraction of the muscle, i.e., the manometric probe size does not change during active contraction. On the other hand, the FLIP measures concentric muscle contraction, which implies that with the balloon volumes that show baseline CSA values higher than the minimal size measurable by the FLIP, there is reduction in the size of the FLIP bag/luminal collapse with voluntary contraction. In other words, the length of the muscles decreases during contraction when measured with the FLIP system. It is for the latter reason we developed tension-time plots for each bag volume and used peak tension to compare between the FLIP and manometric probe measurements. The peak tensions are smaller during concentric contraction as compared with isometric contraction.

In general, it is believed that the resting pressure in the anal canal is due to IAS and the increase with voluntary contraction is due to EAS. Since the IAS, EAS, and PRM are anatomically overlapping structures, the contribution of each muscle to the anal pressure along the length of the anal canal may vary, e.g., in the proximal part the pressures are related to IAS and PRM, in the middle part to IAS and EAS, and in the most distal/caudal part to EAS only. The manometric probes that we use for our study utilizes a sleeve sensor that measures the highest pressure along its length (13) in the anal canal. Concurrent manometry and three-dimensional ultrasound or magnetic resonance imaging recordings reveal that the highest pressure at rest is located where the IAS and EAS overlap, i.e., in the distal part of anal canal (3, 11). During squeeze also the highest pressure is located in the part of the anal canal surrounded by the EAS (14). Therefore, most likely EAS is the major contributor to anal canal pressure recorded by the sleeve sensor, especially during squeeze. The hour glass shape of the FLIP bag is related to pressure profile or the pressure distribution along the length of the anal canal, with the smallest CSA at the site of highest anal canal pressure. Since we used the minimal CSA values for our tension calculations, we believe that the tension values measured by manometry probes and FLIP are related to the same structure, i.e., EAS, especially during voluntary squeeze. On the other hand, in the rest state, we cannot be certain whether the increase in tension with the increase in the probe size is due to active contraction of the IAS, EAS or passive properties of the tissues of anal canal that include IAS, EAS, and PRM. However, based on our studies in the rabbit, we found that the contribution of passive properties of tissues of the anal canal is much smaller than the active contraction of the muscles (21). Similarly, in the case of vaginal pressure, one cannot say with one hundred percent certainty, but our studies show that the vaginal pressures are mostly related to the PRM contraction (8, 10, 23), both at rest and squeeze. Therefore, the vaginal length-tension measurements we report are most likely related to the PRM.

The FLIP recordings are gaining popularity for the assessment of biomechanical properties of the esophagus, lower esophageal sphincter, and anal sphincter (4). The inventor of the FLIP system, Hans Gregersen, would like the users to pay attention, and very appropriately so, to what exactly they are measuring with the FLIP (7). The majority of the investigators have used distensibility index parameter, which is a ratio of the CSA area and pressure; it can also be thought of as tissue compliance. The latter is generally denoted to measure the mechanical properties of the material and in the case of live tissue the biomechanical property of tissue. The latter may be related to 1) passive (visco-elastic), 2) active (muscle contraction), or 3) a combination of the two, properties in the case of live muscle. The distensibility index does not reveal the contribution of active vs. passive property of the muscle. Therefore, one has to use other methods/maneuvers to differentiate between the active vs. the passive component, e.g., increase in tension with voluntary contraction is all related to the active muscle contraction. On the other hand, one cannot be certain of the contribution of active vs. passive components in the resting tension values. In the animal experiments, one can use pharmacologic agents to paralyze skeletal and smooth muscles to abolish active muscle contraction, which is somewhat difficult but can be done in the human setting (2).

While our study is the first to describe the use of the FLIP to measure length-tension function of the anal sphincter and PRM, there are limitations to be considered in this preliminary work. First, we report on a small sample of nulliparous women. We chose to include women only as we wanted to report on the use of the FLIP in the vagina and measure PRM separately from the anal sphincter muscles. These tests need to be repeated in men to determine if these findings are generalizable. Second, we studied a wide age range of nulliparous women (22–50 yr). While the average age of menopause is 52, it is possible that older participants could have been perimenopausal. Future studies should investigate the impact of age and menopausal status on the length-tension function in the vaginal and anal canal using FLIP. Several investigators have measured distensibility index of the anal canal at rest and squeeze and found it to be higher in patients with anal incontinence (5, 26). If we could distinguish the actual property of the tissue being measured by FLIP, passive or active, we may be able to make significant advances in our understanding of the biological systems, i.e., pathophysiology of the disease processes and possibly better use of the FLIP system for diagnostic purposes. These findings are preliminary and should be confirmed in a larger population. Using the manometric probe system, we found that patients with fecal incontinence have lower length-tension values of the anal and vaginal canal compared with normal subject indicating impaired EAS and PRM function (12). With the use of length-tension principles, it is possible to adjust the EAS length to increase the anal canal pressure (20, 22). The FLIP system allows one to measure the length-tension properties of the EAS and PRM, and therefore, it could be an important tool to identify patients who may be candidates for possible EAS and PRM length adjustment surgery to treat anal incontinence. Injection or implant therapy is an Food and Drug Administration-approved treatment for the treatment of fecal incontinence (6); the mechanism by which it works is not clear. It is very possible that implanting nonabsorbable material in the anal canal increases anal sphincter length and thus improves muscle function by increasing its length.

GRANTS

This work was supported by National Institute of Child Health and Human Development Grant 1R01-HD-088688.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

L.J.T. and R.K.M. conceived and designed research; L.J.T., J.S., S.R., and R.K.M. performed experiments; L.J.T., A.Z., C.S., J.S., and R.K.M. analyzed data; L.J.T. and R.K.M. interpreted results of experiments; L.J.T. and R.K.M. drafted manuscript; L.J.T., A.Z., and R.K.M. edited and revised manuscript; L.J.T., A.Z., and R.K.M. approved final version of manuscript; A.Z. prepared figures.