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Small intestinal morphometric and biomechanical changes during physiological growth in rats
ARTICLE IN PRESS
Journal of Biomechanics 38 (2005) 417–426
Small intestinal morphometric and biomechanical changes during physiological growth in rats Xiao Lua,b, Jingbo Zhaoa,b, Hans Gregersena,b,c,* a
Center of Excellence in Visceral Biomechanics and Pain, Aalborg Hospital and Center of Sensory-Motor Interaction, Aalborg University, Hobrovej 18-22, Aalborg DK-9100, Denmark b Institute of Experimental Clinical Research, Aarhus University, Denmark c Haukeland Hospital and Bergen University, Bergen, Norway Accepted 27 April 2004
Abstract Changes in small intestinal geometry, residual strain and stress–strain properties during physiological growth were studied in rats ranging from 1 to 32 weeks of age. Small intestinal mass and dimensions increased many-fold with age, e.g. the weight per unit length increased five-fold with age and the wall cross-sectional area increased four-fold. The opening angle of duodenum obtained at zero-stress state was approximately 220 and 290 during the first and second week after birth and decreased to 170 at other ages (po0.005). The opening angle of ileum ranged between 120 and 150 . The residual strain of duodenum at the mucosal surface did not vary with age (p>0.05) whereas the residual strain of ileum at the mucosal surface decreased with age (po0.001). The circumferential and longitudinal stress–strain curves fitted well to a mono-exponential function. At a given circumferential stress, the corresponding strain values increased during the first 8 weeks of age (po0.05) where after no further change was observed. Hence, the small intestine became more compliant during early life. At a given longitudinal stress, the corresponding strains of ileum and duodenum became larger during the first 2–4 weeks of age (po0.05) where after no further change was observed. The small intestine was stiffer in longitudinal direction compared to the circumferential direction. In conclusion, pronounced morphometric and biomechanical changes were observed in the rat small intestine during physiological growth. Such data may prove useful in the understanding of the functional changes of the digestive tract during early life. r 2004 Elsevier Ltd. All rights reserved. Keywords: Small intestine; Morphometry; Residual strain; Opening angle; Maturation-related changes
1. Introduction The gastrointestinal (GI) tract primarily has a mechanical function. Contents received from the oesophagus and stomach are propelled further down the intestine and mixed with secreted fluids to digest and absorb the food constituents. The small intestine is a composite tube with muscle layers and mucosa-submucosal layers. The peristaltic transport of contents in the GI tract is a neuromuscular function affected by a number of factors (Christensen, 1987; Johnson et al., 1994; Kellow et al., 1986; Grundy, 1993). Maturationrelated alterations in intestinal function and structure *Corresponding author. Tel.: +45-9932-2064; fax: +45-9813-3060. E-mail address: [email protected] (H. Gregersen). 0021-9290/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2004.04.025
are important factors throughout the lifespan of the animal (Thomson and Keelan, 1986). For example, in physiological growth the small intestinal diameter and wall-thickness of mature animals increase manifold compared to the small intestinal diameter and wall thickness of the newborn animal. Studies have shown that the villus height and number, mucosal mass, protein, or deoxyribose nucleic acid, and the villus epithelium changed with age (Viguera et al., 1999; Holt et al., 1984). The age-related alterations in intestinal components and structure likely change the intestinal biomechanical properties. In biomechanical analysis, it is important to determine the stress-free state as the reference for the strain analysis. Residual stress and strain are the internal stress and strain that reside in the organ when external forces
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are removed (the no-load state). Recently, residual strain was demonstrated in the small intestine (Dou and Zhao, 2003; Gao et al., 2000; Gregersen and Kassab, 1996; Gregersen et al., 1997; Zhao et al., 2002). Residual stress can reduce transmural stress and strain variations at physiological loads in biological tissues and hence may optimise the mechanical function. Although the origin of residual stress in soft tissues remains uncertain, experimental and theoretical evidence suggest that residual stress can be altered by many factors, including tissue growth and remodelling (Dou et al., 2002; Zhao et al., 2003). In the rat aorta, changes in residual strain have been observed with age (Saini et al., 1995) and with hypertension induced by abdominal aortic banding (Fung and Liu, 1989). Omens et al. (1998) demonstrated pronounced geometric remodelling with age in the rat heart. Rodriguez et al. (1994) demonstrated by a theoretical analysis that volumetric tissue growth could alter residual stress. It was predicted that concentric hypertrophy, which increases the wall thickness-to-radius ratio, increased the opening angle and residual stress, and conversely eccentric hypertrophy decreased the opening angle. This prediction has not been tested in the small intestine during maturation but it has been shown in the rat abdominal aorta that changes in opening angle with age correlated with changes in wall thickness-to-radius ratio (Badreck-Amoudi et al., 1996). It is not yet known whether small intestinal geometric changes during physiological growth are associated with significant alterations in small intestinal residual strain and stress–strain properties. The goal of this study was to study the geometric and biomechanical changes during physiological growth in rats spanning from 1 to 32 weeks.
2. Materials and methods Forty-two Wistar rats of both sexes were used in this study. The rats were housed individually in metabolic cages under constant temperature and humidity conditions and with a 12 h dark/light circle. The animals were allocated to seven groups (n ¼ 6 in each group) based on the age when they were euthanised (1, 2, 4, 8, 16, 24, and 32 weeks old). At the time of termination each rat was anaesthetised with sodium pentobarbital (30 mg/kg) and weighed. The abdomen of the rats was opened in the mid-line to expose the small intestine and the abdominal aorta. Following an arteriotomy, an i.v. cannula (22 G/ 25 mm) was inserted into the abdominal aorta. The calcium antagonist papaverin (120 mg/kg) was injected into the aorta in order to abolish contractile activity in the small intestine. The duodenum was isolated from the pylorus to the ligament of Treitz. The distal ileum sample was taken from the ileo-caecal junction up-
stream. The length was approximately from 1.4 to 4.5 cm depending on the animal age. The mesentery was cut off. The duodenum and ileum samples were excised and transferred to a dish where after the rat was killed by an overdose of the anaesthetic drug. The lumen of the samples was washed with Krebs solution at temperature 4 C. Adjacent tissue was removed carefully with aid of a stereomicroscope. Both the duodenum and ileum samples were cut into two parts of equal length. The proximal samples were blotted dry, weighed and the length was measured. The distal samples were transferred to another container with Krebs solution containing EGTA 0.1 mg/ml and Dextran 60 mg/ml aerated with a gas mixture of 95% O2 and 5% CO2.
2.1. Determination of no-load state and zero-stress state Five equatorial rings were cut at the proximal end from the samples of distal duodenum and ileum by a sharp blade as previously described (Gregersen et al., 1997). The rings were immersed separately in small organ baths containing the Krebs solution (EGTA 0.1 mg/ml, Dextran 60 mg/ml). The cross section of each ring in the no-load state was videotaped using a videostereomicroscopic system (Zeiss Stemi 2000-C and SONY CCD Camera). Three of the rings were cut radially opposite to the mesentery. The stress-free configuration was videotaped after 30 min equilibrium (Gregersen et al., 1997).
2.2. Stress–strain experiments The remaining part of distal duodenum and ileum were used for the stress–strain experiment. One end was mounted on a cannula that was connected to a hydrostatic pressure system containing the Krebs solution. The lumen was gently washed with the Krebs solution for 1 min by rising the container about 0.5 cm H2O relative to the level of the Krebs solution in the organ bath. The zero pressure level for the distension experiment was pre-set to the surface of the solution in the organ bath. The distal end of the samples was ligated using a suture and was movable. The step-wise pressure loading was carried out by increasing the container height corresponding to pressures at 0, 2, 5, 10, 15, and 20 cm H2O. Due to the passive conditions in this set-up, these pressures are supraphysiological. However, this is quite normal in biomechanical studies. Five minutes were awaited at each pressure level for equilibrium and the segment was videotaped for later measurement of length and diameter with the use of an image system (Optimas, ver. 5.2, Optimas Corp., USA). The measures were used in the computation of the circumferential and longitudinal stress–strain relations (see below).
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2.4. Measurements and data analysis The morphometric data were obtained from the digitised images of the photographs of segments in the zero-stress, no-load and pressurised states. The measurements were done with the use of dedicated software (Optimas ver. 5.2, Optimas Corp., USA). The following data were measured from each specimen: the circumferential length (C), the wall thickness (h), the wall area (A), and the opening angle at the zero-stress state (a). The villus and serosal boundaries were measured as the inner and outer circumferential length of small intestine. The mucosal radius was computed from mucosal circumferential length and then the wall thickness-toradius ratio was computed. The subscripts i; o; m; n; z and p refer to the inner (mucosal) surface, outer (serosal) surface, the mid-wall, the no-load state, zero-stress state and pressurised condition. The opening angle a was defined as the angle subtended by two radii drawn from the midpoint of the inner wall to the inner tips of two ends of the specimen (Fung and Liu, 1989). Furthermore, the outer diameter (D) and the length (Lp ) were measured from the images of the pressurised segments. The measured data was used for computation of the biomechanical parameters defined as Residual Green’s strain at the mucosal surface: ei ¼
ðCin =Ciz Þ2 1 : 2
ð1Þ
Residual Green’s strain at the serosal surface: ðCon =Coz Þ2 1 : eo ¼ 2
ð2Þ
The stress and strain of the small intestine in the pressurised state were determined under the assumption that the small intestinal wall is homogenous with circular cylindrical geometry. The calculations were based on knowing the no-load state and zero-stress state dimensions and the outer diameter and length of the specimen at varying pressures. We computed the longitudinal strains with reference to the length at noload state. It was based on the observation in pilot
Cmz ¼ ðciz þ coz Þ=2; the luminal radius, rip ¼ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi r2op ðAn =pll Þ; the wall thickness, hp ¼ rop rip ; the mucosal circumferential length, Cip ¼ 2prip ; and the serosal circumferential length, Cop ¼ 2prop ; respectively. Kirchhoff’s stress and Green’s strain at a given pressure were computed according to the following equations: The mean circumferential Kirchhoff’s stress: DPrip ty ¼ : ð3Þ hp l2y The mean longitudinal Kirchhoff’s stress: DPr2ip tz ¼ ; hp l2z ðrop þ rip Þ
ð4Þ
where DP is the transmural pressure difference. 600 500 Body Weight (g)
The sectors in zero-stress state, the two rings in no-load state, and pieces of tissue from the distended duodenum and ileum were fixed in 10% buffered Formalin over 24 h. Five-micrometer-thick transversal sections were cut and then stained with hematoxylin and eosin. The thickness of the mucosa, submucosa, circumferential muscle, longitudinal muscle, and villus height were measured. The ratios of the villus height-to-wall thickness ratio and mucosal and submucosal thickness-to-muscle layer thickness were computed. The same pathologist did the measurements in a blinded design.
experiments that the small intestinal mid-wall length at zero-stress state did not change comparing to the length at no-load state. We computed the longitudinal stretch ratio, ll ¼ Lp =Ln ; the mid-wall circumferential length, Cmp ¼ ðCip þ Cop Þ=2; the mid-wall circumferential stretch ratio, ly ¼ Cmp =Cmz where
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2.3. Histology
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100 80 60 duodenum ileum
40 20 0 0
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Fig. 1. Graphs of the body weight (top) and the weight per unit length in vitro of duodenum and ileum (bottom) as function of rat age. The body weight and the weight per unit length in vitro of both duodenum and ileum increased significantly with age (po0.001).
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The circumferential mid-wall Green’s strain: ey ¼
l2y 1 : 2
ð5Þ
The longitudinal Green’s strain: ez ¼
l2z 1 : 2
ð6Þ
To identify the distinctions of the stress–strain relationship at different ages, an exponential function (Eq. (7)) was used to fit the stress–strain data. Then, the corresponding circumferential and longitudinal strains at different ages were computed at the circumferential and longitudinal stress level of 2 kPa (close to the
2.5. Statistical analysis The data were representative of a normal distribution and accordingly the results are expressed as means7SD.
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ð7Þ
where t and e are stress and strain, respectively; a and b are constants; (e ; t ) represents one point on the stress– strain curve in the region of validity of the equation (Fung, 1993). The curve-fitting function in Sigmaplot (Jandel Scientific, Germany) was used in the calculation.
Duodenum
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Circumference (mm)
physiological state): t ¼ ðt þ bÞeaðee Þ b;
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10 8 6 4 2 0
0 0
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Fig. 2. The six graphs show the morphometric data (left for duodenum and right for ileum): circumferences (top), thickness (middle) and wall area (bottom) as the function of rat age. All parameters increased significantly with animal age (po0.05).
ARTICLE IN PRESS X. Lu et al. / Journal of Biomechanics 38 (2005) 417–426
The constants a and b from the above exponential function were used for the statistical evaluation of the stress–strain data. One-way analysis of variance was used to detect differences between the duodenum and ileum and age-related variations (Sigmastat 2.0t). Student’s t test was used to detect possible differences between duodenum and ileum. The results were regarded as significant when po0.05.
3. Results The rats gained weight rapidly during the first 15–20 weeks where after the weight increase levelled off at
approximately 450 g at weeks 16–32 (Fig. 1 top). The weight per unit length of both the duodenum and ileum increased (po0.001), especially during the first 8 weeks (Fig. 1, bottom).
3.1. No-load and zero-stress state The geometric data obtained at the no-load and zerostress states are illustrated in Fig. 2. The inner and outer circumferences at no-load state as well as at zero-stress state increased (po0.001), especially during the first 5 weeks. The wall thickness of duodenum and ileum increased primarily during the first 8 weeks (po0.001) where after no further increase was observed. The wall 3
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duodenum
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Opening Angle (degrees)
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Fig. 3. The opening angles of both duodenum and ileum varied with age (top left). The wall thickness-to-radius of both duodenum and ileum is shown in the top right graph. The bottom graphs show the residual strain at inner and outer wall of duodenum (left) and ileum (right).
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thickness of duodenum was significant larger than that of ileum (po0.05). The lumen cross-sectional area showed the same tendency as wall thickness. The wall area increased during the whole study period (po0.001). The lumen cross-sectional area of ileum was significantly larger than that of duodenum from 4 weeks until 32 weeks (po0.05). The wall area did not differ between duodenum and ileum (p>0.1). The opening angle of duodenum was the largest in the 2nd week (approximately 300 ), decreased to a level of 130 at 4 weeks (po0.05) and then increased to a level of 180 (Fig. 3). The opening angles of ileum were relatively constant at 100–150 . The wall thickness-toradius ratio of duodenum and ileum decreased quickly during the first 4 weeks (po0.001) where after no further reduction was observed in ileum. The residual strains in
both duodenum and ileum were positive at the outer surface and negative at the inner surface. The residual strains at the outer surface in both duodenum and ileum varied with age. The residual strains at the inner surface of duodenum did not change from birth to 32 weeks (p>0.05). Then, the value of residual strains of ileum reduced quickly during 8 weeks (po0.001) where after no further reduction was observed (p>0.05). 3.2. Stress–strain data The circumferential and longitudinal stress–strain relation of both duodenum and ileum are shown in Fig. 4. At a circumferential stress level of 2 kPa, the corresponding strains in both duodenum and ileum became larger up to 8 weeks of age (po0.001) and no
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Fig. 4. The graphs show the stress–strain relations for circumferential (top) and longitudinal direction (bottom) of duodenum (left) and ileum (right) for 1–32 weeks.
ARTICLE IN PRESS X. Lu et al. / Journal of Biomechanics 38 (2005) 417–426 0.6 duo
ileum
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mucosal thickness in duodenum were significantly higher than that in ileum (po0.01). After 8 weeks, the duodenal submucosal thickness was larger than that in ileum. After 16 weeks, the thickness of duodenal muscle layer was larger than that in ileum (po0.05). The ratio of villus height-to-wall thickness of both the duodenum and ileum decreased in the first 4 weeks and then changed no further (Fig. 7, top). This ratio was significantly larger in duodenum than that in ileum (po0.01). Both the duodenal and ileal ratios of mucosal and submucosal thickness-to-muscle layer thickness were the highest at the first week (Fig. 7, bottom).
0.6
4. Discussion
Longitidinal Strain
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2
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Fig. 5. The strains in duodenum and ileum obtained at a circumferential stress level of 2 kPa shown in the top graph. The strains at a longitudinal stress level of 2 kPa shown in the bottom graph.
further change was observed after 8 weeks (Fig. 5, top graph), i.e. the duodenum and ileum became more compliant during the first 8 weeks of life. At a longitudinal stress level of 2 kPa, the corresponding strains values of duodenum and ileum became larger up to 4 weeks of age (po0.001) and 2 weeks of age (po0.001) where after no further change was observed (Fig. 5, bottom graph). Both duodenum and ileum were stiffer in longitudinal direction compared to the circumferential direction. We correlated the opening angle to the wall thicknessto-radius ratio in both duodenum and ileum. This analysis did not show a significant association between opening angles and the wall thickness-to-radius ratio of both duodenum and ileum (R2 ¼ 0:18; p>0.5 and R2 ¼ 0:28; p>0.3, respectively). The opening angles and residual strains at the outer surface was associated in both duodenum and ileum during the growth (R2 ¼ 0:95 and 0:58; respectively, po0.05). 3.3. Histology data The histological data are illustrated in Figs. 6 and 7. Most histological parameters increased rapidly in the first 8 weeks (po0.001) with no further increase except that the thickness of the duodenal muscle layer increased until 24 weeks (po0.01). The villus height and the
The main findings in this study were that pronounced morphometric and biomechanical changes occur during physiological growth. The small intestine becomes softer during maturation and the residual strain and opening angle also show variation with age. The largest changes of both the morphometric properties and biomechanics occur in the first 8 weeks. Furthermore, significant differences in these parameters were discovered between the two intestinal segments. Mechanical properties are important for cardiovascular (Fung, 1990; Dobrin, 1978; Mulvany, 1984) and urogenital function (Hansen and Gregersen, 1999; Van Duyl, 1984). The small intestine is similar to these organs in that it is functionally subjected to changes in wall stresses and strains. During the last decade, acquiring biomechanical information in the small intestine has increasingly called attention (Dou et al., 2002; Dou and Zhao, 2003; Gao et al., 2000; Gregersen et al., 1992, 1997, 2002; Gregersen and Kassab, 1996; Orvar et al., 1993; Zhao et al., 2002, 2003). The determination of the zero-stress state of GI tissue is the first step of the mechanical properties determination. The zero-stress state was used as the basic state to examine the remodelling of arterial tissue under stress (Fung and Liu, 1989, 1991; Liu and Fung, 1989) and theoretical analysis of structural components were made (Greenwald et al., 1997; Rachev et al., 1995). Arterial and left ventricular changes at zero-stress state were discovered during physiological growth (BadreckAmoudi et al., 1996; Omens et al., 1998). In this study an in vitro technique based on video imaging during loading and access of the no-load and zero-stress states allowed us to compute mechanical parameters for the duodenum and ileum obtained from rats of various ages. The present study aimed to describe the morphometry at the no-load and zero-stress states of the small intestine and to analyse the residual strains at the inner and outer wall and stress–strain relationship with reference to the zero-stress state in rats of different ages. This was done in order to study geometric and
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Submucosa Thickness (mm)
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Fig. 6. The thickness of the layers as function of rat age. All parameters increased significantly as function of age (po0.001).
biomechanical remodelling with physiological growth. The analysis took into account large deformation theory and used the zero-stress state dimensions as reference for the strain analysis and is thus advantageous to previous studies of small intestinal mechanical function. As expected the small intestinal circumference and wall thickness increased during physiological growth. The largest morphological increase was in the first 8 weeks, at which time rats have reached sexual maturity. The histological data showed the similar growth progression as in vitro measures in no-load state. Similarly, biomechanical changes, including opening angles and residual strains and the stress–strain relationship, occurred in the puberty in the first 8 weeks. The morphology, histology, and biomechanics altered quite slowly and smoothly in the mature animal. We found that the small intestine became softer in both the circumferential and longitudinal direction in the time span studied. The intestine seems different from the vascular system where the aortic circumferential elastic modulus increased with age in rats (Berry et al., 1975). Collagen is an important determinant of the elasticity of tissue. The collagen-to-elastin ratio in blood
vessels of rat increased steadily with age and it correlated with the circumferential elastic modulus (Berry and Greenwald, 1976; Greenwald and Berry, 1978, 1980). Storkholm et al. (1998) found a positive relationship between the collagen content and the elastic in the small intestine. Although we did not measure the area fraction of collagen in small intestinal segments in this study, we found that the ratio of mucosa and submucosal thickness to muscle thickness decreased with age. Most collagen in the small rat intestine is found in the submucosal and mucosal layers. The decrease of mucosal and submucosal thickness-tomuscle thickness ratio may indicate that the ratio of collagen reduced in small intestinal wall with physiological growth. Hence, the softening observed in this study possibly correlated to the relative decrease in collagen. The small intestinal residual strains in the study were negative at mucosal layer and positive at serosal layer at all ages studied. This implied that the mucosa is under compression in the no-load state and at physiological conditions in the low-pressure range, whereas the muscle layers are in tension. The time course of the serosal residual strain seems to correlate better to the opening
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Villus Height to Wall Thickness Ratio
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Fig. 7. Graphs showing various ratios between layers’ thickness in the small intestine of rats aged 1–32 weeks.
angle than the mucosal residual strain does. The explanation for this is not clear. It has been demonstrated that residual stress reduces the stress concentration at the inner wall of the GI tract at no-load and homeostatic states (Gregersen et al., 1997; Gao et al., 2000). Thus, the compressed mucosa may be better protected against injury from the flow of luminal contents than an uncompressed mucosa. These protection mechanisms could be important when unphysiologically high pressures are reached, e.g. in mechanical obstruction. Gregersen et al. (2000) speculated that a positive correlation existed between the residual strain gradient and the gradient in the height of villi in small intestine. In this study the duodenal villus height-to-wall thickness ratio was larger than that of ileum in all age groups, correspondingly the duodenal opening angles were larger than those of the ileum were. The results also showed that the villus height-to-wall thickness ratio of both duodenum and ileum in the first 2 weeks were larger than that in other ages, and correspondingly the opening angles of duodenum and ileum in the first 2 weeks were much higher than that in other ages. These results support that the height of villi to some degree
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determines small intestinal opening angles in zero-stress state. The zero-stress state is very sensitive to remodelling by disease, growth or degeneration. The change of the opening angle of the zero-stress state is a result of nonuniform tissue remodelling of the organ wall. The opening angle increases when the inner layers have a higher growth rate than the outer layers or when the outer layers atrophy more than the inner layers and vice versa (Rodriguez et al., 1994). In other words, the opening angle at the zero-stress state do not change if equal growth rates occur in the inner and outer layers. Consistent with theoretical prediction (Rodriguez et al., 1994) and experimental studies on the aorta and left ventricular remodelling, the opening angle correlated significantly with the wall thickness-to-radius ratio (Badreck-Amoudi et al., 1996; Omens et al., 1998). Most studies conducted on the zero-stress state of the GI tract have found a correlation between the opening angle and the thickness-to-radius ratio (Gregersen et al., 1997; Gao et al., 2000). In this study, we computed the thickness-to-radius ratio of both duodenum and ileum. This ratio was large in the first 2 weeks and decreased to a stable level in the study span later. The changes showed a pattern similar to changes in opening angle with age and thus seem consistent with the theoretical prediction. The rather stable opening angle after 8 weeks of life implies that the small intestinal physiological growth was equal throughout the intestinal wall. The present study provides systematic data on the biomechanical and morphological variation of developing rat duodenum and ileum. The data illustrates a very pronounced morphometric and biomechanical remodelling during early life in rats. Such data may prove useful in the understanding of the functional changes of the digestive tract during early life.
Acknowledgements Karen Elise Jensens Foundation and the Danish Technical Research Council supported this work.
References Badreck-Amoudi, A., Patel, C.K., Kane, T.P.C., Greenwald, S.E., 1996. The effect of age on residual strain in the rat aorta. Journal of Biomechanical Engineering 118, 440–444. Berry, C.L., Greenwald, S.E., 1976. Effects of hypertension on the static mechanical properties and chemical composition of the rat aorta. Cardiovascular Research 10, 437–451. Berry, C.L., Greenwald, S.E., Rivett, J.F., 1975. Static mechanical properties of the developing and mature rat aorta. Cardiovascular Research 9, 669–678. Christensen, J., 1987. The oesophagus. In: Johnson, L.R., Christensen, J., Jackson, M.J., Jacobson, E.D., Walsh, J.H. (Eds.), Physiology
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Hansen, I., Gregersen, H., 1999. Morphometry and residual strain in the porcine ureter. Scandinavia Journal of Urological Nephrology 33, 10–16. Holt, P.R., Pascal, R.R., Kotler, D.P., 1984. Effect of aging upon small intestinal structure in the fischer rat. Journal of Gerontology 39, 642–647. Johnson, L.R., Christensen, J., Jackson, M.J., Jacobson, E.D., Walsh, J.H., 1994. In: Physiology of the Gastrointestinal Tract, 3rd edition. Raven Press, New York, pp. 109–289. Kellow, J.E., Borody, T.J., Phillips, S.F., Tucker, R.L., Haddad, R.C., 1986. Human interdigestive motility variations: in patterns from esophagus to colon. Gastroenterology 91, 386–395. Liu, S.Q., Fung, Y.C., 1989. Relationship between hypertension, hypertrophy, and opening angle of zero-stress state of arteries following aortic constriction. Journal of Biomechanical Engineering 111, 325–335. Mulvany, M.J., 1984. Resistance vessel abnormalities in spontaneously hypertensive rats. Journal of Cardiovascular Pharmacology 6 (Suppl. 4), S656–S665. Omens, J.H., Vaplon, S.M., Fazeli, B., McCulloch, A.D., 1998. Left ventricular geometric remodeling and residual stress in the rat heart. Journal of Biomechanical Engineering 120, 715–719. Orvar, K.B., Gregersen, H., Christensen, J., 1993. Biomechanical characteristics of the human esophagus. Digestive Diseases and Sciences 38, 197–205. Rachev, A., Greenwald, S., Kane, T., Moore Jr., J.E., Meister, J.J., 1995. Analysis of the strain and stress distribution in the wall of the developing and mature aorta. Journal of Biorheology 32, 473–485. Rodriguez, E.K., Hoger, A., McCulloch, A.D., 1994. Stress-dependent finite growth law in soft elastic tissue. Journal of Biomechanics 27, 455–467. Saini, A., Berry, C., Greenwald, S., 1995. Effects of age and sex on residual stress in the aorta. Journal of Vascular Research 32, 398–405. Storkholm, J.H., Villadsen, G.E., Jensen, S.L., Gregersen, H., 1998. Mechanical properties and collagen content differ between isolated guinea pig duodenum, jejunum, and distal ileum. Digestive Diseases and Sciences 43, 2034–2041. Thomson, A.B., Keelan, M.A., 1986. The aging gut. Canadian Journal of Physiology and Pharmacology 64, 30–38. Van Duyl, W.A., 1984. Theory of propagation of peristaltic waves along ureter and their simulation in electronic model. Urology 24, 511–520. Viguera, R.M., Rojas-Castaneda, J., Hernandez, R., Reyes, G., Alvarez, C., 1999. Histological characteristics of the intestinal mucosa of the rat during the first year of life. Laboratory Animal 33, 393–400. Zhao, J.B., Sha, H., Zhuang, F.Y., Gregersen, H., 2002. Morphological properties and residual strain along the small intestine in rats. World Journal of Gastroenterology 8, 312–317. Zhao, J.B., Yang, J., Gregersen, H., 2003. Biomechanical and morphometric intestinal remodeling during experimental diabetes in rats. Diabetologia 46, 1688–1697.
Journal of Biomechanics 38 (2005) 417–426
Small intestinal morphometric and biomechanical changes during physiological growth in rats Xiao Lua,b, Jingbo Zhaoa,b, Hans Gregersena,b,c,* a
Center of Excellence in Visceral Biomechanics and Pain, Aalborg Hospital and Center of Sensory-Motor Interaction, Aalborg University, Hobrovej 18-22, Aalborg DK-9100, Denmark b Institute of Experimental Clinical Research, Aarhus University, Denmark c Haukeland Hospital and Bergen University, Bergen, Norway Accepted 27 April 2004
Abstract Changes in small intestinal geometry, residual strain and stress–strain properties during physiological growth were studied in rats ranging from 1 to 32 weeks of age. Small intestinal mass and dimensions increased many-fold with age, e.g. the weight per unit length increased five-fold with age and the wall cross-sectional area increased four-fold. The opening angle of duodenum obtained at zero-stress state was approximately 220 and 290 during the first and second week after birth and decreased to 170 at other ages (po0.005). The opening angle of ileum ranged between 120 and 150 . The residual strain of duodenum at the mucosal surface did not vary with age (p>0.05) whereas the residual strain of ileum at the mucosal surface decreased with age (po0.001). The circumferential and longitudinal stress–strain curves fitted well to a mono-exponential function. At a given circumferential stress, the corresponding strain values increased during the first 8 weeks of age (po0.05) where after no further change was observed. Hence, the small intestine became more compliant during early life. At a given longitudinal stress, the corresponding strains of ileum and duodenum became larger during the first 2–4 weeks of age (po0.05) where after no further change was observed. The small intestine was stiffer in longitudinal direction compared to the circumferential direction. In conclusion, pronounced morphometric and biomechanical changes were observed in the rat small intestine during physiological growth. Such data may prove useful in the understanding of the functional changes of the digestive tract during early life. r 2004 Elsevier Ltd. All rights reserved. Keywords: Small intestine; Morphometry; Residual strain; Opening angle; Maturation-related changes
1. Introduction The gastrointestinal (GI) tract primarily has a mechanical function. Contents received from the oesophagus and stomach are propelled further down the intestine and mixed with secreted fluids to digest and absorb the food constituents. The small intestine is a composite tube with muscle layers and mucosa-submucosal layers. The peristaltic transport of contents in the GI tract is a neuromuscular function affected by a number of factors (Christensen, 1987; Johnson et al., 1994; Kellow et al., 1986; Grundy, 1993). Maturationrelated alterations in intestinal function and structure *Corresponding author. Tel.: +45-9932-2064; fax: +45-9813-3060. E-mail address: [email protected] (H. Gregersen). 0021-9290/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2004.04.025
are important factors throughout the lifespan of the animal (Thomson and Keelan, 1986). For example, in physiological growth the small intestinal diameter and wall-thickness of mature animals increase manifold compared to the small intestinal diameter and wall thickness of the newborn animal. Studies have shown that the villus height and number, mucosal mass, protein, or deoxyribose nucleic acid, and the villus epithelium changed with age (Viguera et al., 1999; Holt et al., 1984). The age-related alterations in intestinal components and structure likely change the intestinal biomechanical properties. In biomechanical analysis, it is important to determine the stress-free state as the reference for the strain analysis. Residual stress and strain are the internal stress and strain that reside in the organ when external forces
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are removed (the no-load state). Recently, residual strain was demonstrated in the small intestine (Dou and Zhao, 2003; Gao et al., 2000; Gregersen and Kassab, 1996; Gregersen et al., 1997; Zhao et al., 2002). Residual stress can reduce transmural stress and strain variations at physiological loads in biological tissues and hence may optimise the mechanical function. Although the origin of residual stress in soft tissues remains uncertain, experimental and theoretical evidence suggest that residual stress can be altered by many factors, including tissue growth and remodelling (Dou et al., 2002; Zhao et al., 2003). In the rat aorta, changes in residual strain have been observed with age (Saini et al., 1995) and with hypertension induced by abdominal aortic banding (Fung and Liu, 1989). Omens et al. (1998) demonstrated pronounced geometric remodelling with age in the rat heart. Rodriguez et al. (1994) demonstrated by a theoretical analysis that volumetric tissue growth could alter residual stress. It was predicted that concentric hypertrophy, which increases the wall thickness-to-radius ratio, increased the opening angle and residual stress, and conversely eccentric hypertrophy decreased the opening angle. This prediction has not been tested in the small intestine during maturation but it has been shown in the rat abdominal aorta that changes in opening angle with age correlated with changes in wall thickness-to-radius ratio (Badreck-Amoudi et al., 1996). It is not yet known whether small intestinal geometric changes during physiological growth are associated with significant alterations in small intestinal residual strain and stress–strain properties. The goal of this study was to study the geometric and biomechanical changes during physiological growth in rats spanning from 1 to 32 weeks.
2. Materials and methods Forty-two Wistar rats of both sexes were used in this study. The rats were housed individually in metabolic cages under constant temperature and humidity conditions and with a 12 h dark/light circle. The animals were allocated to seven groups (n ¼ 6 in each group) based on the age when they were euthanised (1, 2, 4, 8, 16, 24, and 32 weeks old). At the time of termination each rat was anaesthetised with sodium pentobarbital (30 mg/kg) and weighed. The abdomen of the rats was opened in the mid-line to expose the small intestine and the abdominal aorta. Following an arteriotomy, an i.v. cannula (22 G/ 25 mm) was inserted into the abdominal aorta. The calcium antagonist papaverin (120 mg/kg) was injected into the aorta in order to abolish contractile activity in the small intestine. The duodenum was isolated from the pylorus to the ligament of Treitz. The distal ileum sample was taken from the ileo-caecal junction up-
stream. The length was approximately from 1.4 to 4.5 cm depending on the animal age. The mesentery was cut off. The duodenum and ileum samples were excised and transferred to a dish where after the rat was killed by an overdose of the anaesthetic drug. The lumen of the samples was washed with Krebs solution at temperature 4 C. Adjacent tissue was removed carefully with aid of a stereomicroscope. Both the duodenum and ileum samples were cut into two parts of equal length. The proximal samples were blotted dry, weighed and the length was measured. The distal samples were transferred to another container with Krebs solution containing EGTA 0.1 mg/ml and Dextran 60 mg/ml aerated with a gas mixture of 95% O2 and 5% CO2.
2.1. Determination of no-load state and zero-stress state Five equatorial rings were cut at the proximal end from the samples of distal duodenum and ileum by a sharp blade as previously described (Gregersen et al., 1997). The rings were immersed separately in small organ baths containing the Krebs solution (EGTA 0.1 mg/ml, Dextran 60 mg/ml). The cross section of each ring in the no-load state was videotaped using a videostereomicroscopic system (Zeiss Stemi 2000-C and SONY CCD Camera). Three of the rings were cut radially opposite to the mesentery. The stress-free configuration was videotaped after 30 min equilibrium (Gregersen et al., 1997).
2.2. Stress–strain experiments The remaining part of distal duodenum and ileum were used for the stress–strain experiment. One end was mounted on a cannula that was connected to a hydrostatic pressure system containing the Krebs solution. The lumen was gently washed with the Krebs solution for 1 min by rising the container about 0.5 cm H2O relative to the level of the Krebs solution in the organ bath. The zero pressure level for the distension experiment was pre-set to the surface of the solution in the organ bath. The distal end of the samples was ligated using a suture and was movable. The step-wise pressure loading was carried out by increasing the container height corresponding to pressures at 0, 2, 5, 10, 15, and 20 cm H2O. Due to the passive conditions in this set-up, these pressures are supraphysiological. However, this is quite normal in biomechanical studies. Five minutes were awaited at each pressure level for equilibrium and the segment was videotaped for later measurement of length and diameter with the use of an image system (Optimas, ver. 5.2, Optimas Corp., USA). The measures were used in the computation of the circumferential and longitudinal stress–strain relations (see below).
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2.4. Measurements and data analysis The morphometric data were obtained from the digitised images of the photographs of segments in the zero-stress, no-load and pressurised states. The measurements were done with the use of dedicated software (Optimas ver. 5.2, Optimas Corp., USA). The following data were measured from each specimen: the circumferential length (C), the wall thickness (h), the wall area (A), and the opening angle at the zero-stress state (a). The villus and serosal boundaries were measured as the inner and outer circumferential length of small intestine. The mucosal radius was computed from mucosal circumferential length and then the wall thickness-toradius ratio was computed. The subscripts i; o; m; n; z and p refer to the inner (mucosal) surface, outer (serosal) surface, the mid-wall, the no-load state, zero-stress state and pressurised condition. The opening angle a was defined as the angle subtended by two radii drawn from the midpoint of the inner wall to the inner tips of two ends of the specimen (Fung and Liu, 1989). Furthermore, the outer diameter (D) and the length (Lp ) were measured from the images of the pressurised segments. The measured data was used for computation of the biomechanical parameters defined as Residual Green’s strain at the mucosal surface: ei ¼
ðCin =Ciz Þ2 1 : 2
ð1Þ
Residual Green’s strain at the serosal surface: ðCon =Coz Þ2 1 : eo ¼ 2
ð2Þ
The stress and strain of the small intestine in the pressurised state were determined under the assumption that the small intestinal wall is homogenous with circular cylindrical geometry. The calculations were based on knowing the no-load state and zero-stress state dimensions and the outer diameter and length of the specimen at varying pressures. We computed the longitudinal strains with reference to the length at noload state. It was based on the observation in pilot
Cmz ¼ ðciz þ coz Þ=2; the luminal radius, rip ¼ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi r2op ðAn =pll Þ; the wall thickness, hp ¼ rop rip ; the mucosal circumferential length, Cip ¼ 2prip ; and the serosal circumferential length, Cop ¼ 2prop ; respectively. Kirchhoff’s stress and Green’s strain at a given pressure were computed according to the following equations: The mean circumferential Kirchhoff’s stress: DPrip ty ¼ : ð3Þ hp l2y The mean longitudinal Kirchhoff’s stress: DPr2ip tz ¼ ; hp l2z ðrop þ rip Þ
ð4Þ
where DP is the transmural pressure difference. 600 500 Body Weight (g)
The sectors in zero-stress state, the two rings in no-load state, and pieces of tissue from the distended duodenum and ileum were fixed in 10% buffered Formalin over 24 h. Five-micrometer-thick transversal sections were cut and then stained with hematoxylin and eosin. The thickness of the mucosa, submucosa, circumferential muscle, longitudinal muscle, and villus height were measured. The ratios of the villus height-to-wall thickness ratio and mucosal and submucosal thickness-to-muscle layer thickness were computed. The same pathologist did the measurements in a blinded design.
experiments that the small intestinal mid-wall length at zero-stress state did not change comparing to the length at no-load state. We computed the longitudinal stretch ratio, ll ¼ Lp =Ln ; the mid-wall circumferential length, Cmp ¼ ðCip þ Cop Þ=2; the mid-wall circumferential stretch ratio, ly ¼ Cmp =Cmz where
400 300 200 100 0 0
4
8
12 16 20 Time (weeks)
24
28
32
120 Intestinal Weight Per Unit Length (mg/cm)
2.3. Histology
419
100 80 60 duodenum ileum
40 20 0 0
4
8
12 16 20 Time (weeks)
24
28
32
Fig. 1. Graphs of the body weight (top) and the weight per unit length in vitro of duodenum and ileum (bottom) as function of rat age. The body weight and the weight per unit length in vitro of both duodenum and ileum increased significantly with age (po0.001).
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The circumferential mid-wall Green’s strain: ey ¼
l2y 1 : 2
ð5Þ
The longitudinal Green’s strain: ez ¼
l2z 1 : 2
ð6Þ
To identify the distinctions of the stress–strain relationship at different ages, an exponential function (Eq. (7)) was used to fit the stress–strain data. Then, the corresponding circumferential and longitudinal strains at different ages were computed at the circumferential and longitudinal stress level of 2 kPa (close to the
2.5. Statistical analysis The data were representative of a normal distribution and accordingly the results are expressed as means7SD.
14 Circumference (mm)
12 10 8 6 4
12 10 8 6 4 2
2
0
0 0
4
8
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24
28
32
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1.4
1.2
1.2
Wall Thickness (mm)
Wall Thickness (mm)
Ileum
16
inner at no-load state outer at no-load state inner at zero-stress state outer at zero-stress state
14
1 0.8 0.6 0.4 0.2 0
0
4
8
0
4
8
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4
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20
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20
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24
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32
12
Lumen 2
wall
10
12
Time (weeks)
Cross-Sectional Area (mm )
2
12 16 20 Time (weeks)
0 0
Cross-Sectional Area (mm )
ð7Þ
where t and e are stress and strain, respectively; a and b are constants; (e ; t ) represents one point on the stress– strain curve in the region of validity of the equation (Fung, 1993). The curve-fitting function in Sigmaplot (Jandel Scientific, Germany) was used in the calculation.
Duodenum
16
Circumference (mm)
physiological state): t ¼ ðt þ bÞeaðee Þ b;
8 6 4 2
10 8 6 4 2 0
0 0
4
8
12
16
20
Time (weeks)
24
28
32
0
4
8
12 16 20 Time (weeks)
Fig. 2. The six graphs show the morphometric data (left for duodenum and right for ileum): circumferences (top), thickness (middle) and wall area (bottom) as the function of rat age. All parameters increased significantly with animal age (po0.05).
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The constants a and b from the above exponential function were used for the statistical evaluation of the stress–strain data. One-way analysis of variance was used to detect differences between the duodenum and ileum and age-related variations (Sigmastat 2.0t). Student’s t test was used to detect possible differences between duodenum and ileum. The results were regarded as significant when po0.05.
3. Results The rats gained weight rapidly during the first 15–20 weeks where after the weight increase levelled off at
approximately 450 g at weeks 16–32 (Fig. 1 top). The weight per unit length of both the duodenum and ileum increased (po0.001), especially during the first 8 weeks (Fig. 1, bottom).
3.1. No-load and zero-stress state The geometric data obtained at the no-load and zerostress states are illustrated in Fig. 2. The inner and outer circumferences at no-load state as well as at zero-stress state increased (po0.001), especially during the first 5 weeks. The wall thickness of duodenum and ileum increased primarily during the first 8 weeks (po0.001) where after no further increase was observed. The wall 3
400 350
duodenum
duodenum
2.5
ileum
Ileum
Thickness-to-Radius Ratio
Opening Angle (degrees)
421
300 250 200 150 100
2
1.5
1
0.5
50 0
0
0
4
8
12
16
20
24
28
0
32
4
8
16
20
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32
24
28
32
Time (weeks)
Time (weeks) 1.2
1.2
inner wall
inner wall
outer wall
outer wall
0.8
0.8 Ileum Residual strain
Duodenum Residual Strain
12
0.4
0
0.4
0
-0.4
-0.4
-0.8
-0.8 0
4
8
12
16
20
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24
28
32
0
4
8
12
16
20
Time (weeks)
Fig. 3. The opening angles of both duodenum and ileum varied with age (top left). The wall thickness-to-radius of both duodenum and ileum is shown in the top right graph. The bottom graphs show the residual strain at inner and outer wall of duodenum (left) and ileum (right).
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422
thickness of duodenum was significant larger than that of ileum (po0.05). The lumen cross-sectional area showed the same tendency as wall thickness. The wall area increased during the whole study period (po0.001). The lumen cross-sectional area of ileum was significantly larger than that of duodenum from 4 weeks until 32 weeks (po0.05). The wall area did not differ between duodenum and ileum (p>0.1). The opening angle of duodenum was the largest in the 2nd week (approximately 300 ), decreased to a level of 130 at 4 weeks (po0.05) and then increased to a level of 180 (Fig. 3). The opening angles of ileum were relatively constant at 100–150 . The wall thickness-toradius ratio of duodenum and ileum decreased quickly during the first 4 weeks (po0.001) where after no further reduction was observed in ileum. The residual strains in
both duodenum and ileum were positive at the outer surface and negative at the inner surface. The residual strains at the outer surface in both duodenum and ileum varied with age. The residual strains at the inner surface of duodenum did not change from birth to 32 weeks (p>0.05). Then, the value of residual strains of ileum reduced quickly during 8 weeks (po0.001) where after no further reduction was observed (p>0.05). 3.2. Stress–strain data The circumferential and longitudinal stress–strain relation of both duodenum and ileum are shown in Fig. 4. At a circumferential stress level of 2 kPa, the corresponding strains in both duodenum and ileum became larger up to 8 weeks of age (po0.001) and no
12
12
10 Circumferential Stress (kPa)
Circumferential Stress (kPa)
Duodenum
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6
4
2
0
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0
0.2
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24 week 32 week
4
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2
0
0 0
0.2
0.4 Longitudinal Strain
0.6
0.8
0
0.2
0.4
0.6
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Longitudinal Strain
Fig. 4. The graphs show the stress–strain relations for circumferential (top) and longitudinal direction (bottom) of duodenum (left) and ileum (right) for 1–32 weeks.
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ileum
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0.4
0.3
0.2
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0 1
2
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mucosal thickness in duodenum were significantly higher than that in ileum (po0.01). After 8 weeks, the duodenal submucosal thickness was larger than that in ileum. After 16 weeks, the thickness of duodenal muscle layer was larger than that in ileum (po0.05). The ratio of villus height-to-wall thickness of both the duodenum and ileum decreased in the first 4 weeks and then changed no further (Fig. 7, top). This ratio was significantly larger in duodenum than that in ileum (po0.01). Both the duodenal and ileal ratios of mucosal and submucosal thickness-to-muscle layer thickness were the highest at the first week (Fig. 7, bottom).
0.6
4. Discussion
Longitidinal Strain
0.5 0.4 0.3 0.2 0.1 0 1
2
4
8
16
24
32
Time (weeks)
Fig. 5. The strains in duodenum and ileum obtained at a circumferential stress level of 2 kPa shown in the top graph. The strains at a longitudinal stress level of 2 kPa shown in the bottom graph.
further change was observed after 8 weeks (Fig. 5, top graph), i.e. the duodenum and ileum became more compliant during the first 8 weeks of life. At a longitudinal stress level of 2 kPa, the corresponding strains values of duodenum and ileum became larger up to 4 weeks of age (po0.001) and 2 weeks of age (po0.001) where after no further change was observed (Fig. 5, bottom graph). Both duodenum and ileum were stiffer in longitudinal direction compared to the circumferential direction. We correlated the opening angle to the wall thicknessto-radius ratio in both duodenum and ileum. This analysis did not show a significant association between opening angles and the wall thickness-to-radius ratio of both duodenum and ileum (R2 ¼ 0:18; p>0.5 and R2 ¼ 0:28; p>0.3, respectively). The opening angles and residual strains at the outer surface was associated in both duodenum and ileum during the growth (R2 ¼ 0:95 and 0:58; respectively, po0.05). 3.3. Histology data The histological data are illustrated in Figs. 6 and 7. Most histological parameters increased rapidly in the first 8 weeks (po0.001) with no further increase except that the thickness of the duodenal muscle layer increased until 24 weeks (po0.01). The villus height and the
The main findings in this study were that pronounced morphometric and biomechanical changes occur during physiological growth. The small intestine becomes softer during maturation and the residual strain and opening angle also show variation with age. The largest changes of both the morphometric properties and biomechanics occur in the first 8 weeks. Furthermore, significant differences in these parameters were discovered between the two intestinal segments. Mechanical properties are important for cardiovascular (Fung, 1990; Dobrin, 1978; Mulvany, 1984) and urogenital function (Hansen and Gregersen, 1999; Van Duyl, 1984). The small intestine is similar to these organs in that it is functionally subjected to changes in wall stresses and strains. During the last decade, acquiring biomechanical information in the small intestine has increasingly called attention (Dou et al., 2002; Dou and Zhao, 2003; Gao et al., 2000; Gregersen et al., 1992, 1997, 2002; Gregersen and Kassab, 1996; Orvar et al., 1993; Zhao et al., 2002, 2003). The determination of the zero-stress state of GI tissue is the first step of the mechanical properties determination. The zero-stress state was used as the basic state to examine the remodelling of arterial tissue under stress (Fung and Liu, 1989, 1991; Liu and Fung, 1989) and theoretical analysis of structural components were made (Greenwald et al., 1997; Rachev et al., 1995). Arterial and left ventricular changes at zero-stress state were discovered during physiological growth (BadreckAmoudi et al., 1996; Omens et al., 1998). In this study an in vitro technique based on video imaging during loading and access of the no-load and zero-stress states allowed us to compute mechanical parameters for the duodenum and ileum obtained from rats of various ages. The present study aimed to describe the morphometry at the no-load and zero-stress states of the small intestine and to analyse the residual strains at the inner and outer wall and stress–strain relationship with reference to the zero-stress state in rats of different ages. This was done in order to study geometric and
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biomechanical remodelling with physiological growth. The analysis took into account large deformation theory and used the zero-stress state dimensions as reference for the strain analysis and is thus advantageous to previous studies of small intestinal mechanical function. As expected the small intestinal circumference and wall thickness increased during physiological growth. The largest morphological increase was in the first 8 weeks, at which time rats have reached sexual maturity. The histological data showed the similar growth progression as in vitro measures in no-load state. Similarly, biomechanical changes, including opening angles and residual strains and the stress–strain relationship, occurred in the puberty in the first 8 weeks. The morphology, histology, and biomechanics altered quite slowly and smoothly in the mature animal. We found that the small intestine became softer in both the circumferential and longitudinal direction in the time span studied. The intestine seems different from the vascular system where the aortic circumferential elastic modulus increased with age in rats (Berry et al., 1975). Collagen is an important determinant of the elasticity of tissue. The collagen-to-elastin ratio in blood
vessels of rat increased steadily with age and it correlated with the circumferential elastic modulus (Berry and Greenwald, 1976; Greenwald and Berry, 1978, 1980). Storkholm et al. (1998) found a positive relationship between the collagen content and the elastic in the small intestine. Although we did not measure the area fraction of collagen in small intestinal segments in this study, we found that the ratio of mucosa and submucosal thickness to muscle thickness decreased with age. Most collagen in the small rat intestine is found in the submucosal and mucosal layers. The decrease of mucosal and submucosal thickness-tomuscle thickness ratio may indicate that the ratio of collagen reduced in small intestinal wall with physiological growth. Hence, the softening observed in this study possibly correlated to the relative decrease in collagen. The small intestinal residual strains in the study were negative at mucosal layer and positive at serosal layer at all ages studied. This implied that the mucosa is under compression in the no-load state and at physiological conditions in the low-pressure range, whereas the muscle layers are in tension. The time course of the serosal residual strain seems to correlate better to the opening
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Fig. 7. Graphs showing various ratios between layers’ thickness in the small intestine of rats aged 1–32 weeks.
angle than the mucosal residual strain does. The explanation for this is not clear. It has been demonstrated that residual stress reduces the stress concentration at the inner wall of the GI tract at no-load and homeostatic states (Gregersen et al., 1997; Gao et al., 2000). Thus, the compressed mucosa may be better protected against injury from the flow of luminal contents than an uncompressed mucosa. These protection mechanisms could be important when unphysiologically high pressures are reached, e.g. in mechanical obstruction. Gregersen et al. (2000) speculated that a positive correlation existed between the residual strain gradient and the gradient in the height of villi in small intestine. In this study the duodenal villus height-to-wall thickness ratio was larger than that of ileum in all age groups, correspondingly the duodenal opening angles were larger than those of the ileum were. The results also showed that the villus height-to-wall thickness ratio of both duodenum and ileum in the first 2 weeks were larger than that in other ages, and correspondingly the opening angles of duodenum and ileum in the first 2 weeks were much higher than that in other ages. These results support that the height of villi to some degree
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determines small intestinal opening angles in zero-stress state. The zero-stress state is very sensitive to remodelling by disease, growth or degeneration. The change of the opening angle of the zero-stress state is a result of nonuniform tissue remodelling of the organ wall. The opening angle increases when the inner layers have a higher growth rate than the outer layers or when the outer layers atrophy more than the inner layers and vice versa (Rodriguez et al., 1994). In other words, the opening angle at the zero-stress state do not change if equal growth rates occur in the inner and outer layers. Consistent with theoretical prediction (Rodriguez et al., 1994) and experimental studies on the aorta and left ventricular remodelling, the opening angle correlated significantly with the wall thickness-to-radius ratio (Badreck-Amoudi et al., 1996; Omens et al., 1998). Most studies conducted on the zero-stress state of the GI tract have found a correlation between the opening angle and the thickness-to-radius ratio (Gregersen et al., 1997; Gao et al., 2000). In this study, we computed the thickness-to-radius ratio of both duodenum and ileum. This ratio was large in the first 2 weeks and decreased to a stable level in the study span later. The changes showed a pattern similar to changes in opening angle with age and thus seem consistent with the theoretical prediction. The rather stable opening angle after 8 weeks of life implies that the small intestinal physiological growth was equal throughout the intestinal wall. The present study provides systematic data on the biomechanical and morphological variation of developing rat duodenum and ileum. The data illustrates a very pronounced morphometric and biomechanical remodelling during early life in rats. Such data may prove useful in the understanding of the functional changes of the digestive tract during early life.
Acknowledgements Karen Elise Jensens Foundation and the Danish Technical Research Council supported this work.
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