Interdependency of stress relaxation and afferent nerve discharge in rat small intestine

Journal of Biomechanics 45 (2012) 1574–1579

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Interdependency of stress relaxation and afferent nerve discharge in rat small intestine Donghua Liao a,b,n, Xiao Lu b,1, Anthony J. Kirkup c, Wen Jiang c, David Grundy c, Hans Gregersen d a

Institute of Clinical Medicine, Aarhus University, Denmark Mech-Sense, Aalborg Hospital, Aarhus University, Denmark c Department of Biomedical Science, University of Sheffield, Sheffield, UK d GIOME and Sino-Danish Centre for Education and Research, Aarhus Denmark and Beijing China b

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 12 April 2012

Background and aims: To be able to characterize intestinal mechano-electrical transduction, i.e. the mechanoreceptor behaviour, quantitative nerve studies with controlled and quantified stimulus are needed. This study aimed to determine the relationship between mechanical stress relaxation and afferent discharge adaptation evoked by fast isovolumetric bag distensions in the rat jejunum. Methods: Multiunit afferent activity was recorded in vivo from jejunum afferents from five male Wistar rats. The jejunum was distended via a bag at a distension speed of 0.5 ml/s to volumes of 0.2, 0.25, 0.3 and 0.4 ml, respectively. The distension was stopped and the volume was kept constant for 2 min to induce stress relaxation. The pressure in the bag, the afferent discharge (spike rate) and the diameter of the segment during the relaxation time were recorded simultaneously. Results: The afferent discharge responses to distension showed a pattern with a peak during the sudden loading followed by decreasing activity with time. At distension volumes of 0.2, 0.25, 0.3 and 0.4 ml, the afferent discharge declined faster and to a greater extent (94%, 91%,96% and 87%) than the stress decreased (55%, 45%, 59% and 56%) during stress relaxation (p o 0.001). Both the stress and the afferent discharge during the constant volume distension were independent of the distension volumes (p 40.5). The stress and the afferent discharge during the distension can be described mathematically on the basis of the quasi-linear theory of viscoelasticity. The association between the stress and the afferent discharge during the constant volume distension is linear with the same slope under various distension volumes. Conclusions: Intestinal mechanoreceptors were sensitive to the stress stimulus and a linear association between the stress relaxation and afferent discharge adaptation was found. The quasi-linear theory of visco-elasticity can be transferred to analysis of mechanical stimulus evoked afferent discharge. & 2012 Elsevier Ltd. All rights reserved.

Keywords: Stress relaxation Afferent nerve discharge Distension

1. Introduction Activation of mechano-sensitive receptors in the gastrointestinal (GI) tract evokes spike discharges in afferent nerves (Lynn and Blackshaw, 1999; Grundy, 2004; Satchell and McLeod, 1984; Blackshaw and Grundy, 1990; Grundy, 1988; Blackshaw et al., 1987). These afferent nerve fibres play an important role in gastrointestinal reflexes, including the activation of secondary peristalsis and the induction of symptoms and pain (Rao et al., 1996). The electrical discharge can be recorded centrally or along the nerve

n Corresponding author at: Mech-Sense, Aalborg Hospital Science and Innovation Centre (AHSIC), Sdr.Skovvej 15, 9000 Aalborg, Denmark. Tel.: þ 45 99 326 907. E-mail addresses: [email protected], [email protected] (D. Liao). 1 Now at: Department of Biomedical Engineering, Purdue School of Engineering & Technology, Indiana University Purdue University Indianapolis, USA.

0021-9290/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jbiomech.2012.04.013

fibres in animal experiments. To be able to characterize the mechano-electrical transduction, i.e. the mechanoreceptor behaviour, quantitative nerve studies with controlled and quantified stimuli are needed. Intestinal distension is often used as a stimulus to access bowel sensitivity. Use of bag distension at small volumes in the small intestine will allow us to accurately detect nerve discharge. The dynamic change in the discharge rate at distension volumes represents important afferent nerve signalling to the central nerve system conveying enhanced activity of the small intestine (Booth et al., 2008). It is well known that the mechanical behaviour of soft tissues is time-dependent (Fung, 1993). Furthermore, mechanical distension evoked afferent nerve discharge was also reported as a timedependent behaviour in oesophagus (Zagorodnyuk and Brookes, 2000; Zagorodnyuk et al., 2003) small intestine (Booth et al., 2008) and antrum (Peles et al., 2003). It is therefore important to study associations between the tissue mechanical behaviour and mechanoreceptor response history over time.

D. Liao et al. / Journal of Biomechanics 45 (2012) 1574–1579

The present study is devoted to study the relationship between the mechanical stress relaxation and afferent discharge adaptation from rat jejunum evoked by fast isovolumetric bag distension. The quasi-linear theory of viscoelasticity was used to describe the stress relaxation of the intestine and the distension evoked afferent nerve discharge. Since proper knowledge about the stimulus is essential for this kind of experiments, such information may be useful later for quantitative assessment of the discharge pattern in gastrointestinal diseases such as irritable bowel syndrome (IBS).

2. Materials and methods 2.1. Animals Experiments were conducted on five Sheffield-strain male Wistar rats (360–400 g). Care and termination of the rats followed principles of good laboratory practice in compliance with UK national laws and regulations. 2.2. Surgical procedures The procedures have been extensively described elsewhere (Grundy et al., 1998; Jiang and Grundy, 2000; Kirkup et al., 1999). Initially, general anaesthesia was produced with an i.p. injection of pentobarbitone sodium (60 mg kg  1) and sustained by intravenous (i.v.) infusion (0.5–1 mg kg  1 min  1). The trachea was intubated to facilitate spontaneous respiration. The right external jugular vein was cannulated with two saline-filled cannulae to facilitate maintenance anaesthesia and systemic administration of drugs. After this, the left common carotid artery was cannulated with a heparinised catheter (200 units ml  1 heparin in saline) to record blood pressure (Neurolog NL108, Digitimer, Welwyn Garden City, Hertfordshire, UK). The heart rate was obtained from the arterial pressure recording. Body temperature was monitored with a rectal thermistor and maintained at 37 1C by means of a heating blanket. On completion of an experiment, the rat was killed by an anaesthetic overdose followed by exsanguination. A midline laparotomy was done and the cecum was excised. A 10 cm loop of proximal jejunum was identified (typically 1–5 cm from the ligament of Treitz) and cannulated at the oral end with a balloon catheter passed through the right abdominal wall via a small incision. The bag catheter was 1.5 mm in diameter and contained separate channels for inflating the bag and for pressure measurement. The bag was 3 cm long and could be inflated with fluid up to a diameter of 1.2 cm without stretching the bag wall. The small incisions through which the intestinal cannulae passed were sewn up. The abdominal incision was sutured to a 5 cm steel ring to form a well that was subsequently filled with pre-warmed (37 1C) light liquid paraffin oil. 2.3. Nerve preparation and recording A mesenteric arcade was placed on a black Perspex platform in order to stabilise the preparation and a single nerve bundle was dissected from the surrounding tissue. This was severed and cleaned approximately 1–1.5 cm from the jejunal wall to eliminate efferent nerve activity and to make it satisfactory for recording. Then it was attached to one of the pair of platinum electrodes, with a strand of connective tissue wrapped around the other to act as a differential. The electrodes were connected to a Neurolog head stage (NL100) and the signal was amplified (NL104) and then filtered (NL125) with a bandwidth of 100–1000 Hz. This filtered nerve signal was displayed on a storage oscilloscope (Dual Beam Storage 5113, Tektronix, Guernsey, UK) and relayed, together with the signals from the arterial and intrajejunal pressure transducers, into a 1401 plus interface (Cambridge Electronic Design (CED), Cambridge, UK). These signals were sampled on-line by a PC running Spike 6.2 software (CED). 2.4. Experimental protocols After a 45–60 min stabilisation period, the sensitivity of the nerve bundle was assessed by gently touching the intestine. Nifedipine was injected into the jugular vein to relax the smooth muscle in the intestine to avoid that active stress confounded the measurements. The mechanical experiments were started 5 min later. Volume-controlled distensions of the jejunum were done using a syringe pump (Braun Perfusor Pump) delivering fluid at a rate of 0.5 ml/s during concomitant recording of afferent discharge, intraluminal pressure and digital video-imaging of the jejunal wall. The images provided data on the diameter of the jejunum during the distensions. The jejunum was distended with volumes of 0.2, 0.25, 0.3 and 0.4 ml. The volumes were selected according to a pilot study in order to keep the intraluminal pressure within the range 10–60 mmHg. Each volume

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was maintained for 2 min. The interval time between distensions was 2 min. At the end of the experiment, the bag was removed and the jejunum was excised. Tissue rings were cut from both the site of distension and adjacent areas. The rings were immersed in physiologic saline solution and photographed in the no-load state where external forces are assumed not to influence the mechanical state of the tissue. The rings were then cut in a radial direction to obtain the zero-stress state where internal forces were released (Gregersen et al., 2000). The zero-stress state was used as the reference in the mechanical analysis. The no-load state data served for computation of the luminal radius during the distensions. There was no difference in the no-load and zero-stress state geometry of the rings between the rings taken from the distended section and adjacent areas.

2.5. Biomechanical analysis 2.5.1. Stress–strain analysis Circumferential stress and strain of the intestine during distension were determined under assumptions that the intestinal wall was homogenous and circular cylindrical. Calculation was done from knowing the zero-stress state and no-load state dimensions, the outer diameters and the length of the specimen at varying pressures, and by assuming incompressibility of the intestinal wall. Hence, the circumferential stress and strain are denoted as Kirchhoff stress,

Syy ¼

Pr ip

ð1Þ

2

hlyy

1 2 ðl 1Þ ð2Þ 2 yy qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi where r ip ¼ r op 2 ðRono load 2 Rino load 2 Þ, h ¼ro–p–ri–p and lyy ¼ p(ri–p þro–p)/ a(Ri  zero þRo–zero) are the inner radii at the pressurised state, wall thickness and the circumferential stretch ratio, respectively. P, ro–p, Ro–no load, Ri–no load, a, Ri–zero, and Ro–zero are the intraluminal pressure, outer radii at the pressurised state, inner and outer radii at the no-load state, opening angle and inner and outer radii at the zero-stress state, respectively. Green strain,

Eyy ¼

2.5.2. Stress relaxation analysis During a maintained isometric stretch or isovolumetric distension, afferent discharge showed adaptation following a similar pattern as stress relaxation, i.e., a peak response which adapts to a plateau (Sengupta et al., 1989; Zagorodnyuk and Brookes, 2000). Therefore, the afferent discharge evoked by isovolumetric distension can be described theoretically on the basis of existed mathematic descriptions on the stress relaxation. In this study, a quasi-linear viscoelasticity hypothesis (Fung, 1993) was employed for describing both the stress relaxation and afferent discharge adaptation behaviour. The stress and afferent discharge to a sudden infusion volume with time were denoted as GðtÞ ¼ TðtÞ=T max

Gð0Þ ¼ 1

ð3Þ

where G(t) is the reduced-relaxation function, which is assumed to be a function of time, T(t) is the stress or the afferent discharge at time t, Tmax is the amplitude at t¼ 0 corresponding to the maximum stress or the maximal afferent discharge. By using commercially available afferent discharge analysis software (Spike 6.2 in this study), afferent discharge within a given second was assumed as a constant. Consequently the afferent discharge during the first 2 s was calculated manually from the recorded afferent discharge with the time interval 0.01 s. A mathematical form of the G(t) in Eq. (3) was borrowed from previous cardiovascular studies as (Fung, 1993)        1 t t t2 GðtÞ ¼ 1 þ c E1 E1 1 þ c ln

t2

t1

t1

ð4Þ

where E1(z) is the exponential integral function, c, t1 and t2 are constants for theoretical G(t) definition, c is the log decay parameter, t1 is the fast time constant, and t2 is the slow time constant (Miller and Wong, 2000). The method for c, t1 and t2 acquirement were described in detail by Fung (1993) and Miller and Wong (2000).

2.6. Statistics Data are expressed as means7 SD. Two-way analysis of variance was used to detect relaxation function parameters difference between the stress relaxation and the afferent discharge in four distension volumes. For post hoc analysis, the Tukey test was used. Differences were considered statistically significant if po 0.05. All analyses were done by using the software package Sigma Stat 2.0 (SPSS Inc.).

D. Liao et al. / Journal of Biomechanics 45 (2012) 1574–1579

Afferent discharge (spike.s-1)

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250 200 150 100 50 0

0

100

200

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600

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1000

1100

1000

1100

Luminal Pressure (mmHg)

40 0.4 ml

30 20 10

0.3 ml

0.25 ml 0.2 ml

0 -10

0

100

200

300

400

500 600 Time (sec)

700

800

900

Fig. 1. A representative afferent discharge (A) and intraluminal pressure (B) recordings during an experiment. Afferent discharge is shown as a sequential rate histogram in 1 s bin during step distension volumes of 0.2, 0.25, 0.3 and 0.4 ml.

Afferent Discharge (spike.s-1)

40

Stress (kPa)

30 20 10 0 20

40

60 Time (sec)

80

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200 150 100 50 0

120

0

1

1

0.8

0.8

Discharge G (t)

Stress G (t)

0

250

0.6 0.4 0.2 0 0.01

20

40

60 Time (sec)

80

100

120

0.6 0.4 0.2

0.1

1 10 Time (sec)

100

1000

0 0.01

0.1

1

10

Time (sec)

Fig. 2. A typical stress relaxation (A) and afferent discharge adaptation (C) during the relaxation time at a distension volume of 0.25 ml. Both curves showed a high initial peak followed by a plateau. The normalised stress relaxation function G(t) (B) and the corresponding normalised afferent discharge adaptation function G(t) (D) during the first 5 s indicating the faster and greater decay for the afferent discharge after the sudden step distension. The abscissas in (B) and (D) are logarithmic scales. The solid line in (D) is afferent discharge fitted by an exponential decay function.

D. Liao et al. / Journal of Biomechanics 45 (2012) 1574–1579

3. Results 3.1. Multiunit nerve activity during stress relaxation Data from one animal were excluded due to insufficient distension in the relaxation test. The afferent discharge to distension volumes of 0.2, 0.25, 0.3 and 0.4 ml are shown in Fig. 1 together with a representative trace of the pressure during the constant volume distensions. The sudden isovolumetric distension produced a dynamic response of the afferent nerve followed by a faster decline in afferent discharge simultaneous to decrease of the luminal pressure (Fig. 1A and B). At distension volumes of 0.2, 0.25, 0.3 and 0.4 ml, the afferent discharge declined faster and to a greater extent (94%70.06, 91%70.09, 96%70.04 and 87%70.07) than the circumferential stress did (55%70.04, 45%70.03, 59%70.06 and 56%70.09) during the relaxation (F¼186.6, po0.001). However, both the adapted afferent discharge and the relaxed stress after the

100

Stress G (t)%

relaxation showed no difference at any of the distension volumes (p40.5). A typical stress with corresponding reduced relaxation function G(t) (stress G(t)) obtained at a volume of 0.25 ml showed that the stress declined during the isovolumetric distension. The stress decreased continuously for 120 s (Fig. 2A and B). Simultaneous recording of the afferent discharge and the reduced adaptation function G(t) (discharge G(t)) showed that the afferent discharge had a high initial peak followed by a plateau after a few seconds (‘‘rapid adaptation’’; Fig. 2C and D). The afferent discharge adapted more than 75% of the initial value in the first 5 s. Hence, afferent discharge in the first 5 s of the distension was used to represent the afferent discharge adaptation in this study. The adaptation history in the first 5 s was assumed to obey the exponential decay function y ¼ y0 þA1 expðt=t 1 Þ where y0, A1 and t1 are the constants. The curve fitted data were then used for the afferent discharge adaptation analysis. The stress G(t) and the afferent discharge G(t) in the first 5 s showed no significant differences at any of the distension volumes (p40.5) (Fig. 3). The constants c, t1 and t2 in Eq. (4) between the

Volume=0.2 ml Volume=0.25 ml Volume=0.3 ml Volume=0.4 ml

90

1 0.8 Stress G (t)

80

70

60 0.01

0.1

1

0.4 Theoretical Experimental

0 0.01

100 80 Discharge G (t)%

0.6

0.2

10

Time (sec)

0.1

1 10 Times (sec)

100

1000

1 Discharge G (t)

60 40 20 0 0.01

1577

0.1

1

0.8 0.6 0.4 Theoretical Experimental

0.2

10

0 0.01

Time (sec) Fig. 3. Percentage change of the averaged stress G(t) (n¼ 4) (A) and the averaged afferent discharge G(t) (n¼ 4) (B) during the first 5 s of step distensions at volumes of 0.2 ml, 0.25 ml, 0.3 ml and 0.4 ml. The afferent discharge was fitted from the exponential decay function. It showed that the afferent discharge declined faster and to a greater extent (92%7 0.04, averaged from all distension volumes) than the circumferential stress (36%7 0.03, averaged from all distension volumes) after the first 5 s (F¼ 97.6, po 0.001).

0.1

1

10

Time (sec) Fig. 4. Representative comparisons between the theoretical analysis and experimental data on the stress G(t) (A) and afferent discharge G(t) (B). The theoretical G(t) was calculated from Eq. (4) with parameters presented in Table 1 and the experimental data were averaged from Fig. 3A for stress G(t) and Fig. 3B for afferent discharge G(t).

Table 1 Constants for stress relaxation and afferent discharge adaptation. Stress relaxationa Volume (ml) C t2 (s) t1 (s)

Afferent discharge adaptationb 0.2 0.147 0.03 52.6 7 13.8 2.53 7 1.33

0.25 0.18 7 0.08 50.1 7 12.7 2.72 7 1.26

0.3 0.14 70.04 53.8 718.5 2.64 71.44

0.4 0.157 0.02 81.15 7 2.21 4.337 0.64

0.2 4.86 7 0.63 2.32 7 1.35 0.117 0.08

C is the log decay parameter, t1 is the fast time constant, and t2 is the slow time constant. a b

No differences were found for all the three parameters at any of the four volumes (Fo 3.7, p 40.06). No differences were found for all the three parameters at any of the four volumes (Fo 2.8, p 40.1).

0.25 4.00 7 0.96 4.78 7 2.38 0.17 7 0.15

0.3 5.43 71.89 3.38 71.07 0.14 70.06

0.4 2.55 7 0.53 2.99 7 1.21 0.137 0.04

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D. Liao et al. / Journal of Biomechanics 45 (2012) 1574–1579

Afferent discharge (spike.s-1)

600 500

Volume=0.2ml Volume=0.25ml Volume=0.3ml Volume=0.4ml

400 300 200 100 0 0

5

10

15

20

25

30

Circumferential Stress (kPa) Fig. 5. Relationships between the circumferential stress and afferent discharge during the first 5 s during one experiment with step distension volumes of 0.2, 0.25, 0.3 and 0.4 ml. The scatters are experimental data and the lines are linear regression results. The afferent discharge declined 69% 7 0.08 in the first second.

stress relaxation and afferent discharge adaptation differs significantly due to the slower stress relaxation and the faster afferent discharge adaptation (F464.4, po0.001). However, no significant difference was found for the parameters in Table 1 at any of the distension volumes. The theoretical G(t) calculated from Eq. (4) and Table 1 agreed well with the experimental data for both the stress relaxation curve and the afferent discharge adaptation (Fig. 4). 3.2. Relationship between the stress relaxation and afferent discharge The afferent discharge was linear as function of the circumferential stress at all distension volumes (Fig. 5). The linear relationship can be expressed as stress¼ andischargeþ b in which a is the slope of the straight line and b is a constant term. The slopes did not differ at any of the distension volumes (p40.5).

the distension. For example, at the distension volume of 0.2 ml, the afferent discharge of 387.67160.9 spikes s  1 at 0.01 s was about two times higher than the programme obtained (spike 6.2) afferent discharge of 182.2780.9 spikes s  1 during the first second. The detailed data on the dynamic response in this paper made it possible to determine the time-dependent behaviour of the afferent discharge during the onset of the isovolumetric distension. It appeared in this study that the rapid decay of the afferent discharge can be described theoretically by quasi-linear viscoelastic equations and that the dependency between the afferent discharge and stress relaxation was linear. These findings appeared consistent with a previous study on the trachea where the adaptive processes of the mechanosensory receptors in the trachea were essentially dependent on the viscoelastic properties of the trachealis muscle (Davenport et al., 1981). Only a limited number of experiments were included in this study. However, the data were thoroughly analysed for demonstrating the afferent discharge adaptation processing and the linear relationship between the afferent discharge and stress relaxation. For better understanding the association between the mechanical stimuli and the afferent nerve response, further experiments on healthy and diseased animal models are needed. In conclusion, isovolumetric distension evoked an increase in whole nerve afferent discharge. The response consisted of an initial peak response that rapidly adapted to a second plateau phase. The peak component decreased linearly with the relaxation of the stress and the discharge decline can be described mathematically by quasi-linear theory of visco-elasticity. Understanding the association between the mechanical stimuli and the afferent nerve response may have implications for our understanding of the hypersensitivity caused by distension in functional or organic intestinal disorders.

Conflict of interest 4. Discussion The major findings of this study were that the relationship between the stress relaxation and afferent discharge adaptation was linear during isovolumetric distension and the linearity was independent of the distension volume. The jejunum afferent discharge evoked by distension showed an initial peak response that rapidly adapted to a plateau phase simultaneous with luminal pressure decrease. Detailed data on afferent discharge adaptation was obtained and was described mathematically by using quasi-linear viscoelastic theory. Afferent discharge evoked by phasic distension (isovolumetric or isotonic) have been reported as two phases reflecting a dynamic response (initial burst) which adapts to a plateau (Zagorodnyuk and Brookes, 2000; Zagorodnyuk et al., 2003; Booth et al., 2008; Peles et al., 2003). The dynamic response consists of a burst of discharges at the onset of phasic distension. This high-frequency discharge is likely due to an initial stretch that develops during active resistance offered by smooth muscle and local excitatory reflexes produced by intrinsic nerves (Ozaki et al., 1999). In this study, we moved a step further compared to previous studies by obtaining the detailed afferent discharge frequency during the initial state of the distension. The discharge change during the beginning of the distension is the most important in the discharge adaptation process since the afferent discharge can be adapted as fast as in approximately 3 s (Zagorodnyuk and Brookes, 2000; Zagorodnyuk et al., 2003; Booth et al., 2008). Consequently the commonly used afferent discharge, always calculated under the assumption that afferent discharge is constant within a given second, is not accurate enough to describe the dramatic decay in the discharge frequency at the beginning of

We declare that we have no proprietary, financial, professional or other personal interest of any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitle ‘‘Interdependency of stress relaxation and afferent nerve discharge in rat small intestine’’.

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