Dig Dis Sci (2008) 53:3145–3151 DOI 10.1007/s10620-008-0277-z
ORIGINAL ARTICLE
Phasic and Tonic Stress–Strain Data Obtained in Intact Intestinal Segment In Vitro Jingbo Zhao Æ Donghua Liao Æ Hans Gregersen
Received: 21 January 2008 / Accepted: 2 April 2008 / Published online: 7 May 2008 Ó Springer Science+Business Media, LLC 2008
Abstract The function of the small intestine is to a large degree mechanical, and it has the capability of deforming its shape by generating phasic (short-lasting) and tonic (sustained) contraction of the smooth muscle layers. The aim of this study was to obtain phasic and tonic stress– strain (normalized force–length) curves during distension of isolated rat jejunum and ileum (somewhat similar to the isometric length–tension diagram known from in vitro studies of muscle strips). We hypothesized that the circumferential stress–strain data depend on longitudinal stretch of the intestine. Intestinal segments were isolated from ten Wistar rats and put into an organ bath containing 37°C aerated Krebs solution. Ramp distension was done on active and passive intestinal segments at longitudinal stretch ratios of 0, 10, and 20%. Ramp pressures from 0 to 7.5 cmH2O were applied to the intestinal lumen at each longitudinal stretch ratio. Passive conditions were obtained by adding the calcium antagonist papaverine to the solution. Total and passive circumferential stress and strain were computed from the length, diameter and pressure data and from the zero-stress state geometry. The active stress was defined as the total stress minus the passive stress. The total and passive circumferential stresses increased
J. Zhao (&) D. Liao H. Gregersen Center of Excellence in Visceral Biomechanics and Pain, Aalborg Hospital Science and Innovation Center (AHSIC), Sdr. Skovvej 15, 9000 Aalborg, Denmark e-mail:
[email protected] H. Gregersen Center of Sensory-Motor Interaction, Aalborg University, 9220 Aalborg, Denmark H. Gregersen La Jolla Bioengineering Institute, La Jolla, CA, USA
exponentially as a function of the strain. The amplitude of both the total and passive stress was biggest in the jejunum. The total circumferential stress decreased whereas the passive circumferential stress increased when the intestine was stretched longitudinally. Consequently, longitudinal stretching caused the active circumferential stress to decrease. The passive circumferential stress during longitudinal stretching increased more in the jejunum than in the ileum. Therefore, the active circumferential stress decreased most in the jejunum. In conclusion, the circumferential active-passive stress and strain depend on the longitudinal stretch and differs between the jejunum and ileum. Keywords Intestine Rat Active stress Passive stress Length–tension diagram
Introduction Peristalsis in the gut causes mechanical deformation, i.e., stretch and compression. Therefore, the active and passive biomechanical properties of the intestine are important for achieving normal intestinal function [1]. Though the intestine serves primarily a mechanical function, the biomechanical properties of the normal and diseased intestine have only been studied to some extend [1–5]. Most studies have been done by obtaining isometric length–tension diagrams of phasic and tonic smooth muscle contraction in muscle strips in vitro [6]. This has provided data on the passive stress (force) and strain (deformation) as well as on the muscle dynamics. Recently, tools were developed for studying the active (phasic and tonic contractions) and passive length–tension behavior in the human gut in vivo using impedance planimetric distension [7–9]. By using
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similar methods in isolated intestinal segments in vitro, it will be possible to obtain the circumferential stress–strain relationship of the intestinal wall rather than merely the length–tension properties and how this relationship is affected by longitudinal stretch. The gastrointestinal tract may be exposed to longitudinal stretch under normal and pathophysiological circumstances, for example, shear stresses due to bolus flow, longitudinal muscle relaxation and distension due to obstructive diseases will elongate the intestine. It has been shown that longitudinal stretch increased the esophageal wall stiffness in the circumferential direction [10], evoked firing associated with contractile activity in the rectum of guinea pig [11] and changed activity of the circumferential muscle of colon [12]. In the present study we aimed to investigate the effect of longitudinal stretch on phasic and tonic stress–strain diagrams in intact intestinal segment of isolated rat jejunum and ileum. We hypothesized that the active and passive curves depend on the longitudinal stretch and that the properties differed between the jejunum and ileum.
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Experimental Setup The unloaded length of the intestinal segments was measured. The proximal end of the intestinal segment was tied on to a cannula mounted on the inside of the organ bath with silk threads and the cannula was connected via a tube to a syringe containing Krebs solution for distension. The distal end of the intestinal segment was tied by a silk thread on the three-way stopcock connected to the micromanipulator for stretching the intestinal segment in longitudinal direction. Ramp distensions were done by a pump (Genie Programmable Syringe Pump, World Precision Instrument) using an infusion rate of 0.66 ml/min up to a pressure of 7.5 cmH2O at longitudinal stretch ratios at 0, 10, and 20%. The pump was reversed at the maximum pressure and the infused volume was withdrawn. The pressure was measured at three locations with 0.5-cm interval using a 1.5mm three-lumen catheter with sideholes. The intestinal diameters at the locations corresponding to pressure measurements were videotaped using a CCD camera (Sony, Japan) through a stereomicroscope (Fig. 1). Determination of the No-Load and Zero-Stress States
Materials and Methods Animals Ten Male Wistar rats weighing 300 g were used in this study. The rats had access to water but were restricted from food intake from the last night before the experiments. Approval of the protocol was obtained from the Danish Committee for Animal Experimentation. Tissue Sampling The rats were anesthetized with Hypnorm 0.5 mg and Dormicum 0.25 mg per 100 g body weight (Hypnorm: Dormicum: sterile water = 1:1:2; subcutaneous injection). Anesthesia was maintained by injecting 0.6 ml of this solution every hour. Following laparotomy, 7 cm of jejunum from 5 cm distal to the ligament of Treitz, and 7 cm of ileum from 5 cm proximal to the ileo–cecal valve were excised. The residual contents in the lumen were gently cleared by perfusion with Krebs solution of the following composition (mmol/L): NaCl, 118; KCl, 4.7; NaHCO3, 25; NaH2PO4, 1.0; MgCl, 1.2; CaCl2–H2O, 2.5; glucose, 11; ascorbic acid, 0.11. The segment was transferred to the organ bath as fast as possible. The organ bath contained 37°C Krebs solution aerated with a gas mixture (95% O2 and 5% CO2 at pH 7.4). Thirty minutes equilibrating time was needed for recovery before the experiments started.
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The no-load and zero-stress states were assessed to obtain the correct reference state for the mechanical calculations [1–5]. Three rings of 1–2 mm in length were cut from the proximal end of the same intestinal segments that the distension experiment was done. The rings were immersed separately in small organ baths containing the aerated Krebs solution. The rings were photographed at the no-load state and then cut radially on the opposite side of the mesentery to obtain the zero-stress state. A 60-min-period was allowed for equilibration and the specimens were photographed again. The selection of this time period was based on previous experience. Mechanical Data Analysis Calculation was done from knowing the no-load state and zero-stress state geometry and the outer diameters of the specimen at varying pressures. The Kirchhoff’s stress and Green’s strain in the jejunal and ileal walls were computed at a given pressure and assuming circular geometry as: Circumferential Kirchhoff’s stress: Sh ¼
D Prip hp k2h
ð1Þ
Circumferential midwall Green’s strain: Eh ¼
k2h 1 2
ð2Þ
Dig Dis Sci (2008) 53:3145–3151
3147 VCR
Computer
Video Camera Micromanipulator
Pressure recorder
Inflation pump Intestinal segment
Fig. 1 Setup of experiment. The organ bath is composed of an inner small chamber and an outer larger chamber. The Krebs solution in the inner chamber was maintained constant at 37°C by circulating hot water in the outer chamber using a heater. The intestinal segment was placed in the Krebs solution in the inner chamber bath. Distension was
where DP is the transmural pressure difference, r is the luminal radius; h is the wall thickness; and kh is the circumferential stretch ratio. The total phasic stress and the total tonic stresses (composed of both active and passive tissue properties) were extracted from the top points during contraction and the baseline between the contractions during the distension, respectively (Fig. 2). The passive stress was extracted from the pressures during the distension after applying the calcium antagonist papaverine. The active phasic and tonic
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Pressure (cmH2O)
10 8 6 4 2 0 0
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done using a pump and the longitudinal stretch was controlled using a micromanipulator. A three-channel probe was used to measure the pressures. The diameter changes of the intestinal segments were videotaped through a stereomicroscope
stresses were defined as the total phasic and tonic stresses minus the passive stress. active phasic stress = total phasic stress passive stress; active tonic stress = total tonic stress passive stress Statistical Analysis
ð3Þ
The data were representative of a normal distribution and accordingly the results were expressed as means ± SEM. The circumferential stress–strain curve was fitted using the exponential function equation S ¼ ðS þ bÞeaðEE Þ b. S* and E* are the stress and strain at a physiological reference level [9]. The constants a and b obtained from applying the above exponential function were used for the statistical evaluation of the stress–strain data. Analysis of variance was used to detect possible differences of the various parameters in the two segments (Sigmastat 2.0TM). In case of significance, data were evaluated in pairs by a multiple comparison procedure (Student–Newman–Keuls method). If the normality test or the equal variance test failed, Kruskal–Wallis one-way ANOVA on ranks was used. The results were regarded as significant when P \ 0.05.
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Times (second)
Fig. 2 Illustration of the top points during contraction and the baseline between the contractions during distension. The closed symbols above the curve mark the phasic part (). The open symbols under the curve mark the tonic part (- - - -). The pressures from the phasic and tonic curves were used to compute the total phasic and tonic stress
Results Pressure–Diameter Curves Figures 3 and 4 show the pressures and diameters as function of time during the distension in the unstretched
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A 12
B 12
Without papaverine
Pressure (cmH2O)
Pressure (cmH2O)
Fig. 3 Illustration of ramp distension curves of the pressure and diameter of jejunum at stretch ratio 1.0. Waves of peristaltic contraction were clearly observed (a and b). The smooth muscle contraction was abolished by the calcium antagonist papaverine (c and d)
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10 8 6 4 2
With papaverine
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0 0
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0
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40 60 80 Time (seconds)
jejunum and ileum. Waves of peristaltic contractions were observed (Figs. 3a and 4a). The pressure increased and the diameter decreased at each phasic contraction wave (Figs. 3b and 4b). The filling time up to the pressure of 7.5 cmH2O was longer for the ileum (121 ± 23 s) than for the jejunum (67 ± 19 s) (P \ 0.05). Peristaltic contractions were not observed when papaverine was used (Figs. 3c, d and 4c, d). The filling time to the pressure of 7.5 cmH2O
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With papaverine
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The circumferential stress–strain diagrams for jejunum and ileum were decomposed into passive, phasic, and tonic stress (Fig. 5). The total and passive circumferential stress
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Tonic and Phasic Stress–Strain Curves
Without papaverine
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was much longer for the ileum (215 ± 24 s) than for the jejunum (110 ± 21 s) when using papaverine (P \ 0.01).
0
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Pressure (cmH2O)
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4 0
Fig. 4 Ramp distension curves of the pressure and diameter of ileum at stretch ratio 1.0. Waves of peristaltic contraction were observed (a and b). The smooth muscle contraction was abolished by papaverine (c and d)
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Time (seconds)
Time (seconds)
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Dig Dis Sci (2008) 53:3145–3151
Jejunum
Ileum
Stretch 0%
Stretch 0%
passive
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phasic-total
2
tonic total
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phasic active
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Stress (kPa)
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Fig. 5 The stress–strain diagrams with stress decomposed into passive, phasic and tonic stresses. Both for the jejunum (left panel) and ileum (right panel), the maximum point of the phasic and tonic active stress–strain curves shifted to the left when stretching the intestinal segments. The total tonic and phasic stresses as function of strain at different stretch ratios were significantly higher in the jejunum than in the ileum (P \ 0.01). The passive stress increased more in the jejunum than in the ileum during longitudinal stretching
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tonic active
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Green strain
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Stretch 10%
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Green strain
increased exponentially as function of strain. The total phasic and tonic circumferential stresses as function of strain decreased in response to longitudinal stretch whereas the passive circumferential stress increased in response to longitudinal stretching. Consequently, the active phasic and tonic circumferential stresses decreased. However, stretching the jejunum 10% caused the total phasic and tonic circumferential stress to increase. The maximum phasic and tonic active circumferential stress shifted to the left after stretching both intestinal segments (Table 1). The total tonic and phasic circumferential stresses as function of circumferential strain and of longitudinal stretch were significantly higher in the jejunum than in ileum (P \ 0.01). The passive circumferential stress increased more in the jejunum than in
0.6
0
0.2
0.4
0.6
Green strain
the ileum in response to longitudinal stretching. Therefore, the active phasic and tonic circumferential stress decreased most in the jejunum.
Discussion The well-known active-passive length–tension diagrams known from physiological and pharmacological studies of smooth muscle strips in vitro [5, 10] can be reproduced in intact segment of intestine in vitro as shown in this study. Previous human studies have shown similar relationships [13]. In the present study in vitro we were able to control the longitudinal stretch and obtain measures for computation of
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3150 Table 1 The maximum phasic and tonic active stresses and corresponding strain (Lmax) (data also displayed in Fig. 5)
Dig Dis Sci (2008) 53:3145–3151
Segments Jejunum
Ileum * Comparing to the stretch ratio 20%: P \ 0.05
Longitudinal stretch ratio (%)
Phasic active strain
Tonic active stress
Tonic active strain
0
1.01 ± 0.21
0.38
0.43 ± 0.19
0.14
10
1.29 ± 0.41*
0.34
1.00 ± 0.29*
0.24
20
1.04 ± 0.18
0.18
0.39 ± 0.08
0.18
0
1.48 ± 0.26*
0.49
1.06 ± 0.24*
0.48
10
1.33 ± 0.37*
0.49
0.75 ± 0.20*
0.21
20
0.64 ± 0.22
0.20
0.40 ± 0.08
0.18
stresses and strains. We succeeded in differentiating passive and active stress and to decompose the active stress into tonic and phasic components. The amplitude of both total and passive circumferential stress as function of strain was bigger in the jejunum than in the ileum. The circumferential active and passive properties were clearly influenced by longitudinal stretching. Significance of Stress–Strain Data The mechanical properties of the small intestine can be divided into properties arising from (1) a ‘‘passive’’ or connective tissue element, (2) an active (‘‘tonic’’) element, reflecting baseline muscle activity, and (3) an active (‘‘phasic’’) element, reflecting the effects of distensioninduced neural reflexes. The passive and the active stress– strain curves depend on the wall structure, the wall mechanical properties, and the smooth muscle contractile properties. Thus, the stress–strain data are important for understanding the muscle mechanical function in the intestine. It has been possible to derive length–tension diagrams in the human gastric antrum and duodenum [9, 14], however, with the limitation that butylscopolamine may not abolish all phasic activity. In the present in vitro study, the smooth muscle activity was completely abolished by papaverine. Another factor of importance is that it is possible in vitro to measure the wall thickness. Consequently, stress and strain can be computed rather than the length–tension diagram. Active–Passive Stress–Strain Curves and Longitudinal Stretch From this study it is evident that the active stresses contribute to the total stress at low distension loads whereas the passive stress is predominant at higher loads. Thus, the contractile activity primarily takes place within the physiological load range. The maximum active stress is presumably reached at a level of optimum overlap between the sliding filaments in the intestinal muscle cells [15, 16]. The exponential behavior of the passive stress is a mechanism to protect the tissue against overstretch and damage at high luminal pressure loads.
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Phasic active stress
It is well known that circumferential stretch affects the smooth muscle contraction through a mechanoreceptor mechanism [17–22]. However, the effect of longitudinal stretch on intestinal motility and mechanical properties has rarely been reported. The contraction of circumferential and longitudinal layers occurs together most of the time [22]. Therefore, longitudinal stretch may likely also affect the muscle activity. Evidence exists that longitudinal stretch evokes nerve activity associated with contractile activity in the rectum of guinea pig [11] and altered colonic muscle contractility [12]. The present study showed that longitudinal stretch influenced the circumferential active and passive properties. Longitudinal stretch decreased the circumferential muscle contractility. Such information may be relevant because lengthening can be caused by shear stresses induced by chyme flow [10, 23], longitudinal muscle relaxation [24], and distension due to obstructive diseases [25, 26]. The 20% elongation used in this study is probably in the same range as such physiological length changes whereas the deformation used in muscle strips studies often are much larger. The cause of the reduced circumferential stress during longitudinal stretch may be attributed to increased tissue stiffness or alterations in the length–tension properties of the muscle. Longitudinal stretch increased the passive stress, i.e., lengthening changes the material properties. This change is likely due to changes in the geometry of tissue components such as collagen. Increased wall stiffness will result in decreased active stress because the muscle has to work against the resistance of the stiff tissue. In the rat esophagus, longitudinal stretch increased the oesophageal wall stiffness in the circumferential direction [10]. This present study shows the same phenomena. Differences Between Jejunum and Ileum We have previously demonstrated that the jejunum was stiffer than the ileum in circumferential and longitudinal direction [27, 28]. This study confirms these findings. It is well known that the structure and physiological function of proximal and distal segments of the small intestine are different. The differences in biomechanical properties are likely associated with the specialized functions of the
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proximal and distal segments of the small intestine [2, 4]. The jejunum and proximal ileum are the main sites of digestion and absorption, whereas the distal ileum acts mainly as a reservoir. Chyme will be slowed to a lesser degree and mixed with digestive fluid to a larger degree in the jejunum where the amplitude of both total and passive stress was bigger. In contrast, the compliant ileum bulges, which leads to pooling of luminal contents and decreased flow. Conclusions The circumferential active–passive stress–strain relation depends on the longitudinal stretch. The active–passive stress–strain relations referenced to the different longitudinal stretch differ between the jejunum and ileums. Acknowledgments This study were partly financially supported by a grant from Karen Elise Jensen’s Foundation for Jingbo Zhao and NIH grant 1RO1DK072616-01A2. The technicians Ole Sørensen, Torben Madsen, and Jens Sørensen are thanked for handling the animals.
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