Peripheral pacemakers and patterns of slow wavepropagation in the canine small intestine in vivo

Wim J.E.P. Lammers, Luc Ver Donck, Jan A.J. Schuurkes, and Betty Stephen

Abstract: In an anesthetized, open-abdomen, canine model, the propagation pattern of the slow wave and its direction,velocity, amplitude, and frequency were investigated in the small intestine of 8 dogs. Electrical recordings were madeusing a 240-electrode array from 5 different sites, spanning the length of the small intestine. The majority of slowwaves propagated uniformly and aborally (84%). In several cases, however, other patterns were found including propa-gation in the oral direction (11%) and propagation block (2%). In addition, in 69 cases (3%), a slow wave was initiatedat a local site beneath the electrode array. Such peripheral pacemakers were found throughout the entire intestine. Thefrequency, velocity, and amplitude of slow waves were highest in the duodenum and gradually declined along the intes-tine reaching lowest values in the distal ileum (from 17.4 ± 1.7 c/min to 12.2 ± 0.7 c/min; 10.5 ± 2.4 cm/s to 0.8 ±0.2 cm/s, and 1.20 ± 0.35 mV to 0.31 ± 0.10 mV, respectively; all p < 0.001). Consequently, the wavelength of the slowwave was strongly reduced from 36.4 ± 0.8 cm to 3.7 ± 0.1 cm (p < 0.001). We conclude that the patterns of slowwave propagation are usually, though not always, uniform in the canine small intestine and that the gradient in thewavelength will influence the patterns of local contractions.

Key words: slow waves, conduction velocity, peripheral pacemakers, wavelength.

Résumé : Dans un modèle canin à thorax ouvert, anesthésié, on a examiné le profil de propagation de l’onde lenteainsi que sa direction, sa vitesse, son amplitude et sa fréquence dans l’intestin grêle de 8 chiens. On a enregistrél’activité électrique au moyen d’une matrice de 240 électrodes à partir de 5 sites différents répartis sur toute la lon-gueur de l’intestin grêle. La majorité des ondes lentes se sont propagées de manière uniforme et en direction aborale(84 %). Dans plusieurs cas, toutefois, d’autres profils ont été observés, notamment une propagation en direction orale(11 %) et un blocage de la propagation (2 %). De plus, dans 69 cas (3 %), une onde lente a pris naissance au niveaud‘un site local sous la matrice d’électrodes. De tels pacemakers périphériques on été observés dans tout l’intestin. Lafréquence, la vitesse et l’amplitude des ondes lentes ont été maximales dans le duodénum et ont diminué graduellementle long de l’intestin, pour atteindre des valeurs minimales dans l’iléon distal (de 17,4 ± 1,7 c/min à 12,2 ± 0,7 c/min;10,5 ± 2,4 cm/s à 0,8 ± 0,2 cm/s, 1,20 ± 0,35 mV à 0,31 ± 0,10 mV respectivement; toutes p < 0,001). Par consé-quent, la longueur d’onde de l’onde lente a été fortement réduite, passant de 36,4 ± 0,8 cm à 3,7 ± 0,1 cm (p <0,001). Nous concluons que les profils de propagation de l’onde l’ente sont habituellement, mais pas toujours, unifor-mes dans l’intestin grêle canin et que le gradient de la longueur d’onde influera sur les profils des contractions locales.

Mots clés : ondes lentes, vitesse de conduction, pacemakers périphériques, longueur d’onde.

[Traduit par la Rédaction] Lammers et al. 1043

Introduction

The slow wave has been studied by many investigatorssince the early days of medical science (for a review seeSzurszewski 1997) and has been recognized as the drivingand organizing stimulus for many, if not all, intestinal con-tractions. The electrical slow wave has been studied in iso-

lated tissues, in anaesthetized animals, or, with implantedelectrodes, in conscious animals. Some information about theregional differences in slow wave propagation along the smallintestine is also available, especially related to its frequencygradient. There is, however, little information concerning thepattern of propagation of the slow wave throughout the smallintestine and only a few reports are available on the velocityprofile of the slow wave. Also, not much is known eitherabout the location of slow wave pacemakers or their distribu-tion along the small intestine.

We have reconstructed the propagation pattern of slowwave at various sites along the small intestine, using data re-corded from 8 anaesthetized and fasted dogs, using an arrayof 240 closely spaced extracellular recording electrodes. Inthe proximal part of the small intestine, slow wave conduc-tion was rapid with high amplitude signals, whereas conduc-tion velocity decreased strongly towards the distal parts ofthe small intestine with a concomitant decrease in amplitude.At all levels in the small intestine, occasional spontaneous

Can. J. Physiol. Pharmacol. 83: 1031–1043 (2005) doi: 10.1139/Y05-084 © 2005 NRC Canada

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Received 3 October 2004. Published on the NRC ResearchPress Web site at http://cjpp.nrc.ca on 23 December 2005.

W.J.E.P. Lammers1 and B. Stephen. Department ofPhysiology, Faculty of Medicine and Health Sciences, UnitedArab Emirates University, P.O.Box 17666, Al Ain, UnitedArab Emirates.L. Ver Donck and J.A.J. Schuurkes. Department ofInternal Medicine, Johnson & Johnson PharmaceuticalResearch and Development, Division of JanssenPharmaceutica NV, Beerse, Belgium.

1Corresponding author (e-mail: wlammers@smoothmap.org).

local slow wave pacemakers were found, which, transientlyor for longer periods of time, changed the nearby pattern ofslow wave propagation. In most cases, slow wave propaga-tion was aboral, but at any time and at any location, slowwave direction was found to change spontaneously.

Methods

Eight female beagles (12.6 ± 2.2 kg) that had been fastedthe day before the experiment were used in this study. Anes-thesia was induced with lofentanil (0.070 mg/kg body mass),a potent and long-acting analgesic and anesthetic at the doseused (Stanley et al. 1983), and tracheal intubation was facili-tated with the short acting anticholinergics scopolamine(0.015 mg/kg), and succinylcholine (1 mg/kg) (Perlstein et al.2002; Baldwin and Forney 1988). Anesthesia was maintainedthroughout the duration of the experiments (3–4 h) withetomidate (1.5 mg·kg–1·h–1) and fentanyl (0.025 mg·kg–1·h–1).All compounds were administered by intravenous route andwere used because of their minimal cardiovascular liabilityas opposed to other methods of sedation and anesthesia (Vande Water et al. 1996). The animals were ventilated throughthe tracheal tube and the left femoral artery was cannulatedto record systemic blood pressure. Vital signs such as heartrate and blood pressure were monitored continuously as pre-viously described (Lammers et al. 2003). Housing, care,type of anesthesia, and experimental procedures were ap-proved by the institutional ethics committee.

Following a median laparotomy, the abdominal walls werecarefully retracted and a loop of the small intestine was

identified (Fig. 1A). A wet cotton pad was positioned underthe loop and a 240-electrode assembly was gradually low-ered on the serosal surface until physical contact was madebetween tissue and the surface of the electrode assemblywithout exerting any undue pressure. As described in a pre-vious study (Lammers et al. 2003), the electrode tips wereflush with the recording surface of the assembly and therewere no protruding sharp edges that may have adversely af-fected the underlying tissue. A temperature probe was lo-cated alongside the intestine and a heat lamp kept theexperimental area at body temperature. After recording from1 area, another part of the intestine was chosen and the pro-cedure repeated. The order in which the areas were chosenwas different in every experiment. Figure 1C shows the lo-cation of all recording sites along the small intestine.

Following the positioning of the electrode assembly, thetissue was allowed to stabilize for 10 min before recordingfor 5 min. This procedure was then repeated in each dog at 4other sites along the length of the small intestine. The loca-tion of each recording site was marked with a loose ligaturelooped around the intestinal tube. At the end of the experi-ment, the animals were killed (pentobarbital, 200 mg/kg,i.v.) and the small intestine removed in toto. The total lengthof the small intestine and the distance of the recording sitesfrom the pylorus were measured. The average length of the 8canine small intestines was 293 ± 49 cm (range 223–366 cm).

Electrical recordings were unipolar with a subcutaneousneedle in the back right leg acting as the indifferent pole.Each of the 240 recording electrodes was connected throughshielded wires to 1 of 240 AC preamplifiers where the sig-

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Fig. 1. Experimental approach. (A) A photograph of the opened abdominal area of an anaesthetized dog with 1 intestinal loop posi-tioned on a wet cotton pad. An outline of the 240 multi-electrode array is superimposed on the photograph at the same scale. The 4outlined cylinders on top represent the shielded wires connecting the electrodes to the 240 amplifiers. (B) The dimension of the 24 ×10 recording array (inter-electrode distance of 2 mm) overlying the mapped segment of the small intestine. (C) A histogram showingthe frequency of all recording locations in 8 dogs (from Lammers et al. 2003, modified with permission of Am. J. Physiol. Vol. 285,pp. G1014–G1027, © 2003 The American Physiological Society).

nals were amplified (4000×), filtered (2–400 Hz), and digi-tized (1 kHz sampling rate) before being stored on the harddisk of a laptop.

Off-line analysis was performed by choosing a 16-s win-dow from the first, the third, and the fifth minute of each5 min recording. From each 16-s window, the propagation ofall slow waves was analyzed. Signals were digitally filtered

(20-point moving average) and displayed on screen in sets of20–24 electrograms at a time (Fig. 2, panel B). The local acti-vation time of a slow wave was identified by the moment ofmaximum negative slope (Specht 1976; Lammers et al. 1993)and marked with a cursor. All local activation times are re-lated to the timing of the first-detected slow wave in that cy-cle. The local activation times of all waveforms were then

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Fig. 2. Analysis of slow wave propagation. (A) The 24 × 10 electrode array superimposed on the canine distal ileum (326 cm frompylorus). (B) A selection of 24 electrograms recorded from the electrodes located along the longitudinal axis of the segment (as indi-cated by corresponding numbers in A and D). The tracings show 2 slow wave cycles. Short vertical bars indicate the manual timemarkings of each local slow wave. During the second slow wave (SW2), electrograms 11–24 showed the spontaneous emergence of aslow wave from a local pacemaker site (close to electrode 14; indicated by a star). Slow wave intervals are plotted in 3 different trac-ings (italics in boxes). A weak cardiac signal is also visible in all leads as indicated by the open-arrow heads below tracing 24.(C) From the markings of the first slow wave cycle in panel B, a spatial plot of all slow wave activation times is constructed (the hori-zontal dimension of the array in this diagram has been expanded to accommodate the activation times). To visualize the pattern ofpropagation, isochrones were drawn in steps of 1000 ms. (D and E) Propagation maps of the first slow wave in D and the second in E(local activation times omitted). Isochrones and colors indicate the pattern of propagation; the arrows indicate the trajectory of propa-gation. In panel E, the location of the peripheral pacemaker halfway along the mapped segment is indicated by the star (close to elec-trode 14). (F) From the intervals between the 2 slow wave cycles, an interval map was created that shows that the intervals in thedistal part of the segment are shorter than those in the oral part owing to the peripheral slow wave. (G) and (H) Map of the conduc-tion directions and speed measured at different sites. The length of the arrows is proportional to the magnitude of the local conductionvelocity. Whereas panel G shows a relatively uniform aboral propagation of the slow wave, the pattern is disturbed in the next slowwave (H) due to the occurrence of a local pacemaker halfway the segment.

displayed on the grid of the original recording electrode array(24 × 10) (Fig. 2C). Isochrones were manually drawn aroundareas activated in steps of 1000 ms (Fig. 2C, 2D, and 2E).

After marking all local slow waves, the following addi-tional data were obtained: the amplitude of the slow waves;the intervals between successive slow waves (Fig. 2F); and,using the 4 neighboring activation times, the local conduc-tion velocities and direction. As an example, Fig. 2 shows 2successive slow waves in the distal ileum. Both cyclesshowed aboral propagation of the slow wave, whereas in thesecond cycle, another slow wave was initiated approximatelyhalfway along the mapped segment (Fig. 2E). As indicatedin panel B, this caused shorter intervals in the focal area,visible in the interval map plotted in panel F. In fact, this in-terval map shows that whereas the oral part of the segmenthad been activated after more than 6 s, the lower part wasactivated much earlier with values between 5 and 5.5 s.

A simple procedure was used to estimate slow wave con-duction velocity and direction in local regions using activationtimes measured at 4 neighboring electrodes. The approachchosen is shown in Fig. 3 and consists of calculating, fromthe local activation times, the time gradient components inthe X and Y direction, assuming uniform conduction in this2 mm × 2 mm area. The 2-gradients components provide auniform gradient of activation in this area from which theconduction velocity and direction were calculated. This proce-dure is repeated for all other sites throughout the activationmap and the results plotted as shown in Figs. 2G and 2H.

All pooled data are given as averages and standard devia-tions. Significance was tested by Student’s t test. The rela-tions between conduction velocity, amplitude, and frequencyof the slow wave to distance from the pylorus were modeledusing a 2- or 4-parameter logistic regression. The modelswere based on the inhibitory function of an indirect responsemodel (Gabrielsson and Weiner 2000) and were written asfollows:

MEASURE ~ A*(1 – IMAX*(DIST^G/(B^G + DIST^G))) for conduction velocity oramplitude; and

MEASURE ~ A –(DIST/(B + DIST)) forfrequency;

where MEASURE is conduction velocity, amplitude, or fre-quency; A is the value of the starting point (maximum) ofthe curve; DIST is the distance on the small intestine ex-pressed as % distance from the pylorus; and IMAX, B, andG are parameters describing the behavior of the function.IMAX describes the maximum inhibition of MEASURE(%), B is the distance at which 50% inhibition of MEA-SURE takes place (ID50) and G is a parameter to control thesteepness. The likelihood ratio test of a model includingonly 1 parameter A versus a model with all parameters is atest to evaluate whether there is a significant decrease of theresponse over the increasing distance. Since the measure-ments over the distance of the gut were taken in 8 dogs, thecorrelation structure of the repeated measures of distancewithin dogs had to be taken into account. For that purpose arandom effect was modeled for dog, thus considering eachdog as a random sample from a population of dogs. This fac-tor takes into account the dog-to-dog variability.

Results

In the small intestine, the most common pattern of slowwave propagation is a homogeneous aborally conductingwave front, moving like a “sheath” down the intestine (Basset al. 1961). Figure 4 shows 4 representative electrogramsand propagation maps obtained from duodenum, jejunum,proximal, and distal ileum. In the duodenum, the amplitudeand propagation velocity of the slow waves were both highand decrease progressively as they travel aborally, reachingvery low values in the distal ileum. Slow wave amplitudes,conduction velocities, and cycle lengths were measured fromsignals recorded at 37 sites along the small intestine in8 dogs and these values are plotted in Fig. 5. Along thelength of the small intestine, there is a significant decreasein amplitude, velocity, and cycle length (likelihood ratio test:

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Fig. 3. Procedure to estimate the local conduction velocity anddirection. From 4 adjacent electrodes (inter-electrode distance of2 mm), 4 activation times are used, grouped in 4 pairs (2 hori-zontal and 2 vertical). In the horizontal direction, the differencebetween the averages of the 2 pairs (divided by theinterelectrode distance) provides an estimate of the activationgradient in the X-direction. In a similar manner, the activationgradient in the Y-direction (vertical) is obtained. Given these ac-tivation gradient components, the conduction velocity and theconduction direction can be calculated as shown (outcomes1.26 cm/s and 24°, respectively).

p < 0.0001 for each measure). Modeling of the change ofeach slow wave measure relative to the distance from thepylorus showed that amplitude and conduction velocity de-creased with a similar slope, and that a half maximal reduc-tion (ID50) was reached at a distance of 26.3% ± 0.9% and20.4% ± 0.6%, respectively (Table 1). The maximal inhibi-tion of amplitude and conduction velocity in the distal ileumwas 76% and 92% of the initial values, respectively. The cy-cle length of the slow wave decreased with a much lowersteepness from the duodenum towards the ileum (2-parametermodeling); the ID50 would theoretically be reached at a dis-tance of 198%, indicating that there is a limited decline ofthis measure over the length of the small intestine (falling to70% of the initial value).

The homogeneous aborally propagating pattern of slowwave propagation was occasionally disturbed in 2 differentcircumstances. The first type of disturbance only occurred atthe beginning of the first 3 experiments, when the inductionanesthesia had not fully worn off and dissociated activitieswere sometimes visible (Sarna and Otterson 1990). An exam-ple of this is shown in Fig. 6. The left panel displayselectrograms and propagation maps during this dissociated

activity, whereas the right panel presents the situation at thesame site 10 min later. During dissociated activity, uniformand homogeneous propagation of a single slow wave isabsent. Instead, many wavelets are initiated at numerous dif-ferent locations, which propagate over a wide range of veryshort distances before dying out. There is no longer a constantand stable rhythm and activity is quite haphazard. Withinminutes however, this type of activity regularizes itself to thestable homogeneous and aborally propagating pattern seen inthe right panel (Fig. 2). Such dissociated activities were seenin the first 3 dogs during the initial recording epoch. Increaseof the control period to 60 min after induction of anesthesiaabolished such dysmyogenesia (Sarna and Otterson 1990).

The second and more common type of disturbance con-sisted of the sudden occurrence of variation in the directionof slow wave propagation. Such variations occurred occa-sionally throughout the course of the experiment anywherealong the small intestine. Several examples of this type ofaberrant slow wave propagation are illustrated in Fig. 7.Panel A shows an homogeneous aborally propagating slowwave that was followed by a slow wave propagating in theoral direction. Except for the change in direction, this orally

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Fig. 4. Aboral slow wave propagation in the canine (A) duodenum, (B) jejunum, (C) proximal, and (D) distal ileum. In each panel, aset of 22–24 electrograms recorded in the longitudinal direction displays the aborally propagating slow waves while the correspondingactivation map show the broad homogeneously propagating wave front. In the duodenum, slow wave conduction velocities are fast withhigh amplitudes. Both parameters decrease progressively to low values in the distal intestine. In addition, several spike discharges canbe seen at some locations following the leading slow wave. Time and amplitude scale, plotted in (D), applies to all panels. Isochronewidth is 100 ms in the duodenum and 500 ms in the other 3 panels.

directed slow wave propagated as uniformly and homoge-neously as the aboral wave that preceded it. Occasionally, itwas also possible to detect a collision between an oral andan aboral propagating slow wave (panel B). Another morecomplicated pattern was sometimes observed as shown inpanel C. This map shows that the initial slow wave propa-gating into this area was detected more or less simulta-neously along the 2 margins of the segment and not, as wasmore usual, across its complete width. As can be seen fromthe reconstructed pattern of slow wave propagation, the 2(marginal) wave fronts merged together to form a typical ab-orally propagating wave front. This pattern was most likelycaused by the initiation of the slow wave at the back and un-mapped part of the segmental tube, followed by the subse-quent radial propagation from this focus out into the frontmapped region. Panel D shows the occurrence of a conduc-tion block in which the slow wave did not propagate beyonda particular location. Such blocks were never permanent andafter a few cycles, as shown in the electrograms in panel D,an aborally propagating slow wave continued across theregion of temporary block, sometimes describing aWenckebach cycle.

To compare the frequency of occurrence of the differenttypes of propagation patterns, their occurrence was deter-mined in all 5 min recordings. Representative sets of 24electrograms such as those shown in Fig. 7 were manuallyscanned on screen and the following data were noted: num-ber of aboral or oral propagating slow waves; the number ofpacemakers; and the number of conduction blocks. The re-sult of such an analysis is shown in Fig. 8, reporting the di-rection of propagation of 83 consecutive slow waves during5 min of recording. Although the large majority of propaga-tion occurred in the aboral direction, there were instances oforal propagation, of collision between aborally and orallypropagating waves, and 1 case of a pacemaker discharge (cy-cle 62). The accumulated data from all 8 dogs, representinga total recording time of 185 min, is presented in Table 2.The vast majority of slow waves (84%) propagate aborally.However, this dominance is not absolute and there were nu-merous cases in which propagation was different, either asan orally propagating wave front (11%) or as block or spon-taneous pacemaker discharges. This distribution of propaga-tion patterns occurred with similar frequency in theduodenum, jejunum, or ileum (Table 2).

Determination of the direction of propagation allowed usto assess whether or not this had any effect on the amplitudeor velocity of the slow wave. As shown in Tables 3 and 4,there were no significant differences in velocity and ampli-tude of orally or aborally conducted slow wave in the duode-num, jejunum, or ileum.

Further analysis of the spatial and temporal characteristicsof peripheral (local) pacemakers was performed (Fig. 9). Inthis figure, 3 examples of a sudden and spontaneous emer-gence of a local pacemaker is shown in the duodenum, jeju-num, and ileum. In the duodenum, the pacemaker had a verylimited and transient effect of advancing the descendingwave front by approximately 100 ms. Furthermore, subse-quent cycles (cycles +1 and +2) showed no residual signs ofthis single event since propagation patterns in these cyclesare identical to those before the occurrence of the pacemaker(cycles –2 and –1). Something similar also happened in theileum (right panels) although in this case the wave front wasadvanced by approximately 1 s. In the jejunum, the situationwas slightly more complicated as the initial propagation hadbeen in the oral direction. In the third map, the sudden oc-currence of a peripheral pacemaker led to a collision withthe orally conducting slow wave. In the next cycle (+1), boththe incoming distal and the local pacemaker no longer dis-charged and the slow wave came from oral. The bottom ofFig. 9 shows diagrammatically the location of 15 local pace-

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Fig. 5. Plot of the slow wave amplitude, conduction velocity, andcycle length as measured along the small intestine at 37 sites in8 dogs. Individual dots display values measured in individual slowwave maps. The curved lines describe the (A and B) 4- or (C) 2-parameter logistic regression.

Measure

Amplitude(mV)

Conduction velocity(cm/s)

Frequency(min–1)

A 1.3±0.10 10.4±0.71 17.8±0.28B (%) 26.3±0.90 20.4±0.57 198.5±12.51G 3.4±0.37 3.8±0.26 NAIMAX (%) 76.1±0.02 92.0±0.01 NA

Note: A, initial value; B, ID50; G, slope; IMAX, maximum inhibition ofthe measure; NA, not applicable.

Table 1. Values of the parameters in the modeling of slow waveamplitude, conduction velocity, and frequency.

makers, illustrating that such pacemakers may occur any-where along the length of the small intestine.

An attempt was made to determine whether or not theemergence of a peripheral pacemaker was associated withany significant changes in the time of arrival of the preced-

ing slow wave. A delay in arrival of the usually descendingslow wave, for instance, may have enabled the peripheralpacemaker to emerge. The analysis was done by measuringthe intervals of 4–5 cycles before and after the pacemakerdischarge using an electrogram recorded as close as possible

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Fig. 6. Dissociated activity (duodenum; 7 cm from pylorus). Activation maps of a first recording soon after the initiation of anesthesia(left panels) and 10 min later at the same location. Top panels show samples of 24 electrograms recorded from electrodes oriented inthe longitudinal direction (double circles in maps) and the lower panels the corresponding activation maps. Dissociated activity wascharacterized by slow waves that originated chaotically at many different sites and times and that propagated for varying but short dis-tances before dying out. No temporal or spatial order was discernible. Subsequently, this dissociated pattern gradually changed until aregular and homogeneous pattern of aboral propagation was restored (right panels). In both situations, spikes may also occur as indi-cated by the ovals in the electrograms.

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Fig. 7. Aberrant patterns of slow wave propagation. (A) Electrograms and activation map of an orally propagating slow wave. As visi-ble from the electrograms, this cycle was preceded by an aborally propagating slow wave. (B) A collision between an aboral and anoral propagating slow wave. As may be seen in the electrograms, this event followed an orally propagating slow wave. (C) 2 areas(close to electrode locations 1 and 9) were simultaneously activated along both borders of the investigated segment. As indicated bythe arrows, this is most likely to have been caused by the discharge of a peripheral pacemaker located at the back of the mapped areawith subsequent emergence of 2 wave fronts in the recording area, which merged into an aborally propagating slow wave. (D) Occur-rence of conduction block. This propagation block was followed by a second (SW2), which also died-out in the same area. During thethird and fourth cycles, conduction was restored.

Aboral Oral Pacemaker Block

Duodenum 348 (81%) 45 (11%) 18 (4%) 14 (3%)Jejunum 542 (91%) 31 (5%) 10 (2%) 11 (3%)Ileum 1177 (82%) 186 (13%) 41 (3%) 35 (2%)Total 2067 (84%) 262 (11%) 69 (3%) 60 (2%)

Table 2. Patterns of slow wave propagation.

Aboral (n = 201) Oral (n = 73)

Duodenum 1.16±0.36 1.28±0.27 (ns)Jejunum 1.00±0.16 1.24±0.24 (ns)Ileum 0.45±0.20 0.44±0.11 (ns)

Note: ns, not significant.

Table 3. Slow wave amplitude (mV) and direction of slow wavepropagation.

Initial 30 min

Amplitude (mV) 0.47±0.21 0.51±0.23 (ns)Conduction velocity (cm/s) 2.6±3.6 2.9±3.5 (ns)Cycle length (c/min) 14.4±2.1 14.2±2.3 (ns)

Note: ns, not significant.

Table 5. Slow wave propagation parameters during initial record-ings and after 30 min.

Aboral (n = 201) Oral (n = 73)

Duodenum 10.00±2.23 11.9±1.73 (ns)Jejunum 7.11±3.19 6.17±2.6 (ns)Ileum 1.22±0.68 0.99±0.3 (ns)

Note: ns, not significant.

Table 4. Conduction velocity (cm/s) and direction of slow wavepropagation.

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Fig. 8. Diagram showing an example of the prevalence of different slow wave propagation patterns during a 5 min recording session(canine duodenum). The direction of slow wave propagation during 83 successive slow wave cycles is shown. A set of 24 longitudi-nally oriented electrograms was scanned throughout a 5 min recording period and, for each cycle, the main direction of propagationwas determined. A, aboral; O, oral; C, collision; P, pacemaker. The propagation maps of cycles 60–64 are shown in Fig. 9.

Fig. 9. Examples of patterns of propagation before, during, and after a peripheral pacemaker discharge in the duodenum, jejunum, andileum. The propagation maps of 5 successive slow waves are shown; 2 before and 2 after the occurrence (cycles –2 to +2). The emer-gence of a peripheral pacemaker in the duodenum and ileum had a very limited effect on the slow wave propagation, essentially ad-vancing the propagating wave front slightly by 100 ms and 1 s in duodenum and ileum, respectively. The sequence of events in thejejunum also showed a gradual transition from oral propagation to aboral propagation during these 5 cycles. The lower panel depictsthe location of 15 observed peripheral pacemakers along the normalized length of the small intestine, showing that peripheral pacemak-ers occurred in all areas of the small intestine.

to the site of the pacemaker. The data plotted in Fig. 10shows the individual intervals before and after the emer-gence of 13 individual pacemakers, normalized to the aver-age interval of each sequence. In general, there was not aclear trend for longer intervals before the occurrence of aperipheral pacemaker.

As the recordings sessions at any 1 site were limited to5 min, an attempt was made to determine whether theparameters of the slow wave propagation during these 5 minwere stable over time. To this end, in 5 experiments, theelectrode assembly was left at the same location for 30 minand a second recording was made from that same site. Sub-sequently, activation maps were constructed from each ofthese recordings, analyzed, and the results were displayed inTable 5. As can be seen from the average slow wave ampli-tudes, conduction velocities, and cycle lengths, there wereno significant differences in any of these parameters.

Discussion

It has been known for many years that isolated pieces ofthe small intestine are always spontaneously active (Alvarez1915; Ambache 1947; Bortoff 1961; Connor et al. 1979;Specht and Bortoff 1972) inferring that an intrinsic ability toinduce slow waves is located at all levels of the intestine. Inaddition, in the intact intestine, local hypoxia (Szurszewskiand Steggerda 1968), cooling (Hasselbrack and Thomas1961), ischaemia (Lammers et al. 1997), ligation (Bass andWiley 1965), or transection (Code and Szurszewski 1970;Diamant and Bortoff 1969a) have all been used to demon-strate the emergence of slow wave pacemakers in or distal tothe insult. In such situations, retrograde propagation wasnoted, suggesting the presence of distal pacemakers (Bassand Wiley 1965; Diamant and Bortoff 1969a). Diamant andBortoff (1969b) proposed the presence of multiple pacemak-ers to explain the stepwise slow wave frequency gradient.Hasselbrack and Thomas (1961), in the intact dog, some-times recorded reversal of the frequency gradient in contrac-tions in the terminal ileum, suggesting distally locatedpacemakers. A similar phenomenon was also seen electrically

by Szurszewski and colleagues (Szurszewski et al. 1970).The current study now documents the presence of slow wavepacemakers at all levels in the intact small intestine.

Our recorded occurrence of peripheral pacemakers wassmall but certainly underestimated for the following reasons.Each recording was made from a very short length of the in-testine (5 cm from an average length of 293 cm) and overshort time periods. Peripheral pacemakers were either re-vealed in the randomly chosen maps that were analyzed orby scanning sets of 24 electrograms (10% of the total) fromthe 5-min recordings. In both cases, other pacemakers mighteasily have been missed. An indication of our under-estimation is the much larger incidence of orally directedpropagating slow waves (11%). Such retrograde propagatingslow waves must have originated from aboral pacemakersites.

A reason for the emergence of ectopic pacemakers couldbe due to the anesthesia used. Sarna and Otterson (1990)have shown that opioids induce dysmyogenesia. Indeed, inour own experiments, we saw aberrant slow wave initiationand propagation in those cases in which not enough time hadbeen allowed to stabilize the animal. The activation maps ofthese events however (Fig. 6) are very different from that ofperipheral pacemaking (Fig. 9), suggesting different mecha-nisms.

Many terms such as ectopic, distal, deviant, and aberranthave been used to describe the emergence of slow waves dis-tal to the dominant pacemaker, but our present data suggeststhat these distal pacemakers occur under normal conditionsas part of the normal physiology of the small intestine. Forthis reason we suggest that terminology implying abnormalphysiology (including ectopic and abberant) should beavoided. We do not prefer the terms distal or aboral sincethey do not distinguish the lower incidence or impact ofthese pacemakers from the dominant oral pacemaker, and sowould like to suggest the term ‘peripheral’ for these normalbut relatively uncommon events.

The lack of a significant increase or decrease of the inter-val preceding a peripheral pacemaker discharge was surpris-ing. Specht (1976) was able to measure pacemaker discharge

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Fig. 10. Rhythm of slow waves before and after the discharge of a peripheral pacemaker. In each of 13 peripheral pacemaker events,the intervals between slow waves before the emergence of the peripheral pacemaker (–5 to –1) and those following the peripheralpacemaker (+1 to +5) were measured and plotted on a normalized scale. Slow wave intervals, in the absence of a peripheral discharge,show spontaneous variations of up to 20%. Nevertheless, it is clear that there was no change in the slow wave interval before theemergence of the peripheral pacemaker.

after external stimulation in isolated strips from the cat andshowed that such pacemakers were reset by the previouswave front. Other studies, to our knowledge, have not beenperformed in the small intestine and our results seem to indi-cate that the dynamics of peripheral pacemakers are morecomplex. Nevertheless, what was striking was that changesin slow wave conduction direction, caused by the emergenceof peripheral pacemakers, could occur suddenly from 1 beatto the next (Figs. 7, 8, and 9). Recently, an interesting pro-posal was made to explain slow wave propagation by linkingintracellular events such as Ca2+ waves by an electrical con-nection to cells located further away (van Helden and Imtiaz2003); reminiscing the coupled oscillator model proposedmany years ago. Although this model may be used to ex-plain some of the aspects of slow wave entrainment, itwould seem difficult to explain the sudden emergence of pe-ripheral pacemakers found in this study.

In addition to the demonstration of peripheral slow wavepacemaking, this study has also quantified the intestinal gra-dient in slow wave amplitude, velocity, and frequency. Thedecrease in slow wave frequency along the intestine hasbeen reported many times, and our values correspond verywell with these previous reports measured from dogs in situ(Diamant and Bortoff 1969b; Szurszewski et al. 1970;Armstrong et al. 1956). Fewer studies have measured theprofile of the conduction velocity. Armstrong et al. (1956),by exteriorizing different parts of the small intestine in sev-eral dogs, measured a drop in velocity from 13.9 cm/s in theduodenum, to 1.8 and 0.5 cm/s in jejunum and ileum, re-spectively. McCoy and Baker (1969) measured a drop in ve-locity from 15 cm/s in the duodenum to 3 cm/s in thejejunum in conscious dogs with implanted electrodes. Wehave extended these observations to the whole small intes-tine together with a concomitant decrease in the amplitudeof the slow wave.

The decrease in conduction velocity and amplitude downthe intestine is quite marked, reaching values in the distal il-eum of only 24% (amplitude) and 8% (velocity) of the val-ues in the duodenum. In cardiac electrophysiology, theintimate relationship between the conduction velocity andthe amplitude of the extracellular electrogram has been thor-oughly evaluated (Spach 1983). A similar relationship be-tween the extracellular amplitude of the slow wave and itsvelocity, as determined in this study, suggests an electricalcontribution to its propagation. However, there does notseem to be a universal explanation for the decrease in veloc-ity. Bortoff (1976) suggested changes in tissue impedancealong the intestine, at least between the duodenum and jeju-num. Gabella and Blundell (1981) reported that there aremany more nexuses in the duodenum than in jejunum and il-eum. Furthermore, in the guinea pig, a change in ratio thick-ness was demonstrated between the longitudinal and circularmuscle layers from 4.6:1 in the duodenum to 2:1 in the ileum(Gabella 1981). These factors seem to point to an involve-ment of muscle cells in the propagation of the slow wave.Recently, however, studies of the generation of slow wavesin the mice antrum suggest that the interplay of interstialcells of Cajal of the myenteric plexus (ICC-MY) andinterstial cells of Cajal in smooth muscle bundles (ICC-IM)could be responsible for propagation with little contributionfrom the muscle cells themselves (Dickens et al. 2001). A

different morphological distribution of the ICC-system alongthe intestine could also play a role in explaining the velocitygradient.

Many studies have shown that the rhythm of the slowwave has an effect on the rate of certain types of contrac-tions (Bass et al. 1961; Berkson et al. 1932; Bortoff andGhalib 1972; Christensen et al. 1971; Daniel and Chapman1963; Ehrlein et al. 1987). A fundamental question nowraised by this study is whether or not the direction of theslow wave propagation has a role to play in contractions.

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Fig. 11. (A) Change in magnitude of conduction velocity, inter-val, and wavelength of the slow wave along the length of the ca-nine small intestine. The wavelength (velocity × interval) showsa large decrease from 36.4 cm in the duodenum to 3.7 cm in thedistal ileum. (B) A snapshot of a 50-cm segment of duodenumand ileum illustrating the difference in the number and wave-length of slow waves in the 2 regions. (C) An activation mapfrom the distal ileum displaying the presence of 2 wave frontssimultaneously present in this short 5-cm long segment.

Normal peristaltic contractions always seem to be polarized,although on occasion, antiperistalsis seems to occur. In the1 case that peristaltic activity was recorded together with theslow wave, the direction of peristalsis was independent ofthat of the preceding slow wave (Lammers et al. 2002), al-though their timings were coupled. Segmental or standingcontractions are probably not influenced by the direction ofthe slow wave. Ehrlein et al. (1987) have recorded propagat-ing contractions in antegrade and retrograde directions inconscious dogs, which could suggest a role in slow wave di-rection. In addition, their values of the propagation veloci-ties of the contraction waves are very similar to the slowwave velocities measured in this study. Pendular contrac-tions also occur in the rhythm of the slow wave, and a recentstudy has suggested that the direction of the pendular swingis determined by the direction of the slow wave (Lammers2005). Interestingly, in humans, high-resolution analysis ofpressure waves during migrating motor complex (MMC)phase 3 revealed a susbtantial retrograde component in theduodenum at a cycle length similar to the expected slow wave(Castedal and Abrahamsson 2001). In summary, althoughthere is little data available, it is possible that slow wave di-rection could have an effect on direction of certain types ofcontractions. Retrograde slow wave propagation, as shownin this study, may therefore have an impact on local contrac-tions.

The conduction velocity of the slow wave may also play arole in determining the duration of intestinal contractions.Several authors have pointed out that because of this veloc-ity gradient, the wavelength of contraction should signifi-cantly differ along the small intestine (Code et al. 1968;Siegle and Ehrlein 1988; Weems 1981). This influence ofthe slow wavelength is also reflected in the spatial spread ofspike patches that accompany the slow wave. The spread ofspike patches is much larger in the duodenum than in the il-eum (Lammers et al. 2003). Ehrlein and collaboratorsshowed, in the dog, that the length of individual propagatedcontractions is much shorter in the ileum than in the jejunum(Siegle and Ehrlein 1988; Schemann and Ehrlein 1986).Similar findings were also reported by Johnson et al. (1997)with the addition that intestinal transit in the ileum wasmuch slower than in the jejunum. Our data now allow us toplot the slow wave velocity, period, and wave length as afunction of distance along the small intestine. The datashown in Fig. 11 correspond rather well with what was sug-gested earlier by Code and colleagues (Code et al. 1968).The diagram in panel B illustrates the meaning of this largedifference in wavelength by showing the number of slowwaves (or wave crests) that are simultaneously present in anarbitrary 50-cm stretch in the duodenum and in the ileum. Itcan be seen that there are 10 times more waves in the ileumthan in the duodenum. The length of our electrode assembly,5 cm, is too short to measure the duodenal wavelength butlarge enough to register the common occurrence of 2 slowwaves simultaneously present within this length in the ileum(Fig. 11, panel C)

It should be remembered however that the wavelength doesnot equate directly to the length of the contraction wave. AsDaniel and Chapman pointed out (1963), the length of the ex-citability wave is less than the wavelength because spikes arelocked to specific parts of the slow wave cycle (Sancholuz

et al. 1975). Therefore, the length of the excitable wave isless but probably runs parallel to that of the wavelength.

The limitations of this study must be clear. Although thesmall intestines were studied in situ, the animals were anes-thetized and the intestines were empty. In addition, the fre-quency of peripheral pacemakers might have been affectedor modulated by the opioids used. Most importantly, our re-cordings were restricted to a relatively small area and brief5-minute recording periods.

Nevertheless, this study has confirmed the existence ofperipheral slow wave pacemakers in the small intestine. Italso shows the disturbances in slow wave propagation in-duced by the slow waves that emerge from such foci. Theseslow wave disturbances must have disturbing effects on theon-going local intestinal contractions. In addition, the steepchange in the wavelength of the slow wave along the lengthof the intestine may play an important role on propagatingcontractions. All of these factors will ultimately have an im-pact on the organization and the pattern of contractions inthe small intestine.

Acknowledgements

We thank all those involved in Beerse and in Al Ain fortheir contribution to this work and especially Jan Eelen, Pat-rick Van Bergen, Dirk Smets, and Diane Verkuringen. Inaddition, we thank Dr. Arno Muijtjens, Department of Educa-tional Development and Research, University of Maastricht,the Netherlands, for the algorithm to calculate conduction ve-locities, Mr. S. Dhanasekaran for constructing the electrodearray, Professor J.R. Slack for his editorial help, and Dr. LucBijnens and Filip De Ridder, from the Department of Bio-metrics and Reporting, Johnson & Johnson PharmaceuticalResearch and Development, in Beerse, for the statistical anal-ysis. This work was supported by Johnson & Johnson Phar-maceutical Research and Development, a Division of JanssenPharmaceutica N.V., Beerse, Belgium.

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