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Keywords:

  • Lactobacilli ;
  • migrating motor complex;
  • motility;
  • probiotics;
  • spatiotemporal map

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author Contributions
  10. References

Background  Commensal bacteria such as probiotics that are neuroactive acutely affect the amplitudes of intestinal migrating motor complexes (MMCs). What is lacking for an improved understanding of these motility effects are region specific measurements of velocity and frequency. We have combined intraluminal pressure recordings with spatiotemporal diameter maps to analyze more completely effects of different strains of beneficial bacteria on motility.

Methods  Intraluminal peak pressure (PPr) was measured and video recordings made of mouse ex vivo jejunum and colon segments before and after intraluminal applications of Lactobacillus rhamnosus (JB-1) or Lactobacillus reuteri (DSM 17938). Migrating motor complex frequency and velocity were calculated.

Key Results  JB-1 decreased jejunal frequencies by 56% and 34% in colon. Jejunal velocities increased 171%, but decreased 31% in colon. Jejunal PPr decreased by 55% and in colon by 21%. DSM 17938 increased jejunal frequencies 63% and in colon 75%; jejunal velocity decreased 57%, but increased in colon 146%; jejunal PPr was reduced 26% and 12% in colon. TRAM-34 decreased frequency by 71% and increased velocity 200% for jejunum, but increased frequency 46% and velocity 50% for colon; PPr was decreased 59% for jejunum and 39% for colon.

Conclusions & Inferences  The results show that probiotics and other beneficial bacteria have strain and region-specific actions on gut motility that can be successfully discriminated using spatiotemporal mapping of diameter changes. Effects are not necessarily the same in colon and jejunum. Further research is needed on the detailed effects of the strains on enteric neuron currents for each gut region.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author Contributions
  10. References

Ingestion of commensal and probiotic bacteria may modulate gut motility to benefit the host. In general, beneficial effects of ingestion may also include alterations in the composition of the microbiome,1 changes in rat enteric neuron function,2 alterations in mouse brain neurochemistry and behavior,3,4 reduction in blood pressure,5,6 increased anti-inflammatory and immunoregulatory activity,7 and modulation of motility with reductions in either diarrhea or constipation.8 The cellular mechanisms through which these effects are mediated are not clear. The influence of gastrointestinal commensals on motility is demonstrated from studies of germfree mice 9,10 and those whose normal bacterial community has been disrupted using antibiotics.11–13

Probiotics have been used therapeutically to help treat motility disturbances in humans and experimental animals. Meta-analysis of the research literature indicates that Lactobacillus species can be used to treat diarrhea in adults or infants.14

Probiotic organisms decreased colon contraction frequency and were used in double-blind controlled studies to treat infectious (Lactobacillus reuteri DSM 1793815) or functional (Bifidobacterium lactis HN01916) diarrhea in humans.15,16

DSM 17938 (DSM) gavage decreased rotavirus-induced diarrhea in mice.17 It also reduced infantile colic after 21 days oral administration18 in a randomized double-blind controlled trial. Infantile colic is a well-described common symptom in infants, but whether it is due to spasm of smooth muscle in the colon or jejunum is not known. It is plausible to suggest beneficial effects in the reduction in pain might have been due to decreased giant contractions of the small or large bowel.19

Functional and slow transit constipation can occur in infants or adulthood and is especially common in the institutionalized elderly.20 Probiotics may be useful in this condition. This would be particularly important because few long-term effective treatments for constipation exist, and those that are used often have significant side effects.21 DSM supplementation increased the frequency of bowel movements in chronically constipated infants.22Lactobacillus casei Lcr35 was as effective as magnesium oxide in treating children with chronic constipation,23 while Bifidobacterium breve decreased abdominal pain and fecal incontinence while increasing defecation frequency in chronically constipated children.24 In constipated but otherwise healthy human adults, ingestion of L. plantarum SN35N or SN13T strains increased defecation frequency compared with control bacteria (Lactococcus lactis A6 and Streptococcus thermophiles) in a randomized double-blind experimental design.25 A mixture of Lactobacillus, Bifidobacterium, and Streptococcus strains had a positive effect (reduced diarrhea, constipation, and improved nutritional status) on bowel movements among orthopedic rehabilitation in elderly (>80 years) patients.26

That microbiota can control or influence motility is evident from the disruption of intestinal myoelectric complexes of germfree rats.27 Conventionalization of such animals with normal feces or even a single bacterial strain restored adult type frequency of gut motility complexes. It is not immediately obvious how such studies add to the understanding of potential therapeutic probiotic modulation of gut motility. Probiotics do not need to colonize the gut to exert an effect on the host and their action is transient requiring regular repeated applications for the effect to be maintained.28 Because of this, we have devised experiments to study acute effects on motility, where the bacteria are placed into the gut lumen,29 although some investigators have added bacteria so that they contact the serosa (intraperitoneal) rather than the epithelial surface.30

To identify biomarkers for effective probiotics in a mouse model for human diarrhea or constipation,31 it would be desirable to differentiate between the effects of different probiotic strains on gut contractions. The motility effects of probiotics have been mainly measured using intraluminal pressure recordings from ex vivo gut segments in modified Trendelenburg preparations.2,29,32 In this way, the concentration of bacteria or derived compounds can be controlled and confounding influences of circulating hormones and supra-intestinal autonomic reflexes are removed. Nine day feeding of Lactobacillus rhamnosus (JB-1) to rats decreased colon migrating motor complex (MMC) amplitudes ex vivo,2 and this effect was reproduced within 15 min of introducing the same bacterium into the lumen of ex vivo segments of mouse jejunum.29 The dose responses obtained, suggest that, similar to many therapeutic compounds, those produced by, or emanating from JB-1, also have a dose dependent ‘drug-like’ action on peristalsis.

The limitation of these approaches is that intraluminal pressure recordings sample a restricted information set. They cannot measure parameters such as MMC propagation velocity, or frequency when several MMCs occur simultaneously. Neither do they readily distinguish between MMCs and stationary contractions.33 Spatiotemporal maps constructed from video movies of the contracting gut can overcome these limitations because they do not record information from a single locus. This allows spatial and temporal patterns to be measured simultaneously.

For the present experiments, we have used ex vivo segments of small and large mouse intestine to study the effects of introducing intraluminal probiotic bacteria on motility. We used DSM, a probiotic strain with therapeutic effects in children and adults, and JB-1 whose effects on rodent myenteric intestinal primary afferent neurons have been previously studied.2Lactobacillus salivarius and L. reuteri PTA 6475 were used as comparators and controls. Spatiotemporal maps of motility were constructed from video recordings before, during and after luminal application of the bacteria, revealing motor patterns which are not readily detected using other methods.33

methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author Contributions
  10. References

We used adult male Swiss Webster mice (20–30 g) from Charles River Laboratories (Wilmington, MA, USA; http://http://www.criver.com). The mice were killed by cervical dislocation, in line with McMaster guidelines for the use and care of animals. All ensuing procedures were ex vivo.

Organ bath motility recordings

Four or more centimeter long jejunal or distal colon segments were excised and the contents emptied by flushing the segment with Krebs saline under a 2 hPa gravity pressure head. Each segment was mounted in a 20-mL organ bath chamber and submerged in oxygenated Krebs (Fig. 1A). Oral and anal ends were cannulated, and the lumen was gravity perfused with carbogen-gassed Krebs using several Mariotte bottles.34 The intraluminal compartment was perfused at 0.5 mL min−1 with room temperature buffer (19–22 °C) according to the precedent set by previous ex vivo gut segment preparations29,32,35,36 for which MMC can be recorded without rundown for at least 2 h. Pilot experiments have shown that raising the temperature to 34 °C decreases the duration for which steady-state MMCs could be recorded without rundown to 30 min; presumably because a higher mucosal metabolic demand and the lack of a blood supply.

image

Figure 1.  Experimental setup for video and pressure recordings. (A) Canulated intestinal segment was maintained in preheated oxygenated Krebs solution and additives are administered through Mariotte inflow tubes. A pressure sensor placed at the midpoint tracked the intraluminal pressure, while a camera placed atop captured videos of the intestinal movement; (B) The spatiotemporal map was produced via our StMap plugin which converts gut diameter into black and white hues for representation. Oral to anal black lines represent contractions, lighter areas are relaxations. The dotted line indicates the position of pressure sensor; (C) Diameter changes at the dotted line; (D) Pressure changes recorded at the dotted line.

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The organ chamber (serosal compartment) was perfused with prewarmed (34 °C), carbogen-gassed, Krebs solution at a rate of 5 mL min−1). Oxygenated Krebs buffer was of the following composition (mmol L−1): 118 NaCl, 4.8 KCl, 25 NaHCO3, 1.0 NaH2PO4, 1.2 MgSO4, 11.1 glucose, and 2.5 CaCl2 bubbled with carbogen gas (95% O2 and 5% CO2).At the beginning of the experiment, intraluminal pressure was adjusted to 3 hPa by adjusting the heights of inflow and outflow tubes and the recordings were made at this filling pressure. Bacteria were applied by switching the oral luminal inflow from Krebs to Krebs plus 8.0 log cfu mL−129 bacteria by closing and opening the appropriate stopcocks, as illustrated in Fig. 1A.

Intraluminal pressure changes were measured at the midpoint of the longitudinal axis of the gut segment using a Krebs-filled 0.58-mm external diameter non-distensible polyethylene tube as described in.32. The tube emerged from the anal and was attached to a COBE pressure transducer (Sorin Biomedical Inc., Irvine, CA, USA). The pressure signal was amplified, digitized, stored on a PC computer, and analyzed off-line using PClamp 9 software (Molecular Devices; Sunnyvale, CA, USA).32 Peak phasic intraluminal pressure increases (PPr) were identified and measured as described in.29

Images were recorded using a video camcorder (JVC Everio Hard Disk Camcorder Model GZ-MG155U) which was placed 10 cm above a gut segment (Fig. 1A). Recording was started in synchrony with the pressure recording using an 8–12 cm field of view for the duration of the experiment. The camera output was in raw video format (MOD) at 30 frames per second (fps). Ten-minute long video clips were excised from the MOD file using video editing software (Avidemux version 2.5.0; http://www.avidemux.org). The clips were then converted into the MOV format using a video converter (Zune converter version 1.1; http://ffmpeg.mplayerhq.hu). The final video clips were resampled to a resolution of 384 × 256 pixels and 25 fps.

Video recordings were analyzed using in-house image processing software (StMap) developed as a plug-in for NIH ImageJ (version 1.43c; NIH, Bethesda, MD, USA). The software converts the image (Fig. 1B) in each frame of the video into a black-and-white silhouette (Fig. 1C) and generates a spatiotemporal map using an edge detection routine. The routine first measures the diameter at each position along the gut and then represents the physical diameter at each position as a hue value (ranges from 0 to 255, black–white). As gut diameter decreases during contractions, the hue value is reduced toward 0 and will be shown as darker values. As the software reads through each 10-min clip, it generates a spatiotemporal map – a pattern of alternating bands of light and dark hues that contains three sets of information: position along the gut, time, and gut diameter. Using these variables, the spatiotemporal map becomes a motility ‘fingerprint’ whose sensitivity could be critically important in defining the detailed and perhaps nuanced effects that specific bacterial strains could have on motility.

As the StMap measures the diameter changes at each position, StMap can be interpreted as a stacking of numerous 2D diameters vs time graphs. In fact, for a spatiotemporal map, if the location of the pressure transducer were identified (dotted line in Fig. 1B) and were shown as a gray scale vs time graph (Fig. 1C), this graph would be in register with the simultaneously recorded pressure vs time recording at that locus (Fig. 1D). The diameter of the gut at each point was plotted as a color as previously described by Roberts et al.36; and, for ease of visualization, 3D maps were produced by adding a z-axis that plotted the diameter (Fig 2A).

image

Figure 2.  3D maps showing effect of TTX on jejunal motility. Hot areas (peaks) are relaxations and cold areas (blue) valleys are contractions. Slopes of troughs (mm s−1) give velocities of contractile wave. (A) Control Krebs jejunal activity; (B) Administration of TTX caused the abolition of migrating motor complexes (MMCs) and created a stationary mixing pattern as shown, this suggests that MMCs, and not the stationary contractions, depend on neuronal activity (5/6 see Results).

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Motility parameters were measured from the spatiotemporal map using the StMap plugin. Neurogenic, anally propagating, migrating motor complexes MCs of the type described in Wang et al.29 generated thick dark bands that slant diagonally from left to right. The propagation velocity (mm s−1) was measured from the slope of each band or trough in the each 3D map; the two measures are equivalent. For each treatment, slopes of 10–15 successive MMCs were averaged to calculate the propagation velocity. Migrating motor complex frequency (mHz) was calculated by counting the number of MMCs during a 10-min segment.

Bacteria and drugs

JB-1 bacteria were taken from in-house stock (see Bravo et al.4) DSM18 and L. reuteri PTA 6475 (PTA 6475) were donated by BioGaia AB, Stockholm, Sweden. L. salivarius was a gift from the Alimentary Pharmabiotic Centre, University College Cork.37

Cell numbers were determined optically,38 and viability was always checked by ability to grow after plating on growth medium agar plates. All other methods are as reported previously.2,38 Cells from frozen stocks were thawed and centrifuged at 500 g for 15 min, and the pellet was suspended in equal volume of Krebs buffer. Then, the suspension was washed by centrifugation at the same speed, and the cells were removed and resuspended in Krebs at the original concentration. Just prior to use, bacteria were diluted to a working concentration of 8 log cfu mL−1 in fresh Krebs buffer.

Krebs containing bacteria were fed to the intraluminal compartment while ion channel modulating drugs added to the Krebs buffer perfusing the serosal compartment. The time required for the drug solution to flow from the tap to the recording chamber was 30 s. The intermediate conductance calcium-dependent potassium (IKCa) ion channel blocker 1-[(2-chlorophenyl) diphenylmethyl]- 1H-pyrazole (TRAM-34) (Tocris Bioscience, Ellisville, MO, USA; http://www.tocris.com) distributed by Cedarlane Laboratories Ltd., Burlington, ON, Canada) was dissolved with pure dimethyl sulphoxide (DMSO) to make 10-mmol L−1 stock solutions, and these were diluted in oxygenated Krebs to make working concentrations 30 min before use.

Statistics

Statistics were calculated using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Descriptive statistics are given as means ± SD, but in concentration-response plots, sampling errors are displayed using SEM; the sample size is denoted by n. The statistically discernible difference for tests of significance was set at = 0.05; all tests were paired t-test and two tailed.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author Contributions
  10. References

Segments from 77 mice showed propagating MMC and recordings were made from 43 jejunal and 34 distal colonic segments taken from these animals. Migrating motor complexes propagated in the oral to anal direction spanning from 50% to 100% of the ex vivo segment. These contractions were in register with the pressure pulses recorded by the intraluminal probe when both were measured at the same locus (Fig. 1B). Similar to Roberts et al.36 we also observed rare colonic retrograde moving contractions, stationary or mixing motility patterns or motor complexes that only propagated for short distances (<50%); these were excluded from analysis as they did not represent full MMCs nor a repeatable and stable pattern.36 In no case (= 8) did application of TRAM-34 (serosally) or JB-1 (intraluminally) convert these irregular patterns to conventional MMCs as described by Roberts et al.36 Migrating motor complex frequencies ranged for 8–14 mHz which is comparable with Powell et al.39 who reported a MMC frequency of 8 mHz (1.5-min interevent interval) in a similar ex vivo mouse gut preparation. Four jejunal segments did not display anally propagating MMCs but exhibited multiple stationary contractions similar to those described by Gwynne et al.33 although only when Krebs buffer was in the lumen. To test whether stationary contractions are generated by the enteric nervous system, we added 0.6 μmol L−1 TTX to the Krebs buffer perfusing the serosal surface for six jejunal segments. In 5/6 experiments, propulsive MMCs were abolished and stationary, mixing-type contractions developed (Fig. 2), suggesting that, in mouse jejunum, MMCs, but not stationary contractions, are dependent on neural activity. Adding log 8 cfu mL−1 JB-1 to the lumen (n = 5) did not change the stationary contractions nor convert them to propagating ones. The stationary contractions were not examined further.

To demonstrate the strain specificity of the motor effects of specific bacteria, we tested additional bacteria or candidate probiotics. Log 8 cfu mL−1 of the bacteria were applied to the lumen for 30 min. The anti-inflammatory probiotic bacteria L. salivarius, which was without effect in a clinical trial in IBS,37 was also ineffective in previous jejunal motility experiments.29 Another L. reuteri, PTA 6475 with potent anti-inflammatory activity40 was also tested. In all, we tested five jejunal and five colonic segments for each of these bacterial strains. Neither L. salivarius nor PTA 6475 had any effect on MMC amplitudes, frequencies or velocities (data not shown).

Intraluminal JB-1 and DSM modulated motility. The onset latencies of the observed motor effects ranged from 10 to 20 min and plateaued at 20–30 min, and the effects were not reversible with 20 min Krebs washout.29

Nine jejunal and seven colonic segments were exposed to intraluminal JB-1 (Fig. 3): MMC frequencies decreased (Krebs vs JB-1) from 41 ± 18 to 18 ± 8 mHz (56%) (= 0.004) for jejunum and from 14 ± 4.5 to 9.3 ± 5.0 mHz (34%) (= 0.02) for colon. Migrating motor complex velocities for jejunum increased from 1.7 ± 0.9 to 4.6 ± 1.8 mm s−1 (171%) (= 0.004), but decreased from 1.6 ± 0.5 to 1.1 ± 0.4 mm s−1 for colon (31%) (= 0.02). Jejunal PPr decreased from 17 ± 4.1 to 7.6 ± 4.4 hPa (55%) (= 0.004), and also decreased from 42 ± 4.1 to 33 ± 6.5 hPa (21%) (= 0.03) for colon.

image

Figure 3.  Effects of JB1 on intestinal motility. (A) 3D map taken from jejunal recording; (B) The administration of JB1 altered the frequency and conduction velocity of the migrating motor complexes (MMCs); (C–H) Summaries of before and after experiments. (C–E) For jejunum JB1 reduced MMC frequency, increased MMC velocity, and reduced MC peak pressure. (F–H) for colon JB-1 reduced MMC frequency (F), reduced MMC velocity (G), and the MMC peak pressure (PPr) (H).

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Ten jejunal and seven colonic segments were exposed to DSM (Fig. 4). Migrating motor complex frequencies decreased from 51 ± 16 to 19 ± 16 mHz (63%) (= 0.008) for jejunum, but increased from 8.0 ± 4.7 to 14 ± 7.9 (75%) (= 0.02) for colon. DSM decreased jejunal MMC velocity from 2.0 ± 0.81 to 0.86 ± 1.5 mm s−1 (57%) (= 0.04), but increased velocity in colon from 1.3 ± 0.9 to 3.2 ± 1.6 mm s−1 (146%) (= 0.02). PPr decreased from 12 ± 0.7 to 8.9 ± 3.0 (26%) (= 0.08) hPa for jejunum and 42 ± 12 to 37 ± 6 hPa for colon (12%) (= 0.1).

image

Figure 4.  Effects of DSM on intestinal motility. (A–C) DSM reduced jejunal migrating motor complex (MMC) frequency, velocity, but not MMC peak pressure (PPr). (D–E) Representative 3D maps representing colon motility before (D) and 20 min after (E) adding DSM to lumen. (F–H) DSM increased colon MMC frequency and conduction velocity with no effect on PPr.

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We have previously shown that heat-killed JB-1 had no effect on mouse jejunum MMC PPr or frequency.29 However, we did not measure velocity in these experiments and thus cannot exclude the possibility that heat-killed bacteria somehow altered velocity without affecting the other parameters. We thus used heat-killed JB-1 (n = 5) and DSM (n = 5) to exclude this possibility. Migrating motor complex frequencies (mHz), velocities (mm s−1), and PPr (hPa) Krebs vs heat-killed JB-1 were 41 ± 10 vs 45 ± 13 (= 0.5), 1.5 ± 0.7 vs 1.3 ± 0.6 (= 0.7), and 15 ± 4 vs 18 ± 4 (= 0.3). The same parameters when DSM were applied to the jejunum were as follows: 44 ± 5 vs 44 ± 8 (= 0.9), 1.7 ± 0.5 vs 1.9 ± 0.6 (= 0.7), and 15 ± 2 vs 16 ± 4 (= 0.03).

TRAM-34 was added to the Krebs buffer perfusing the serosal surface of eight jejunal and ten colonic segments (Fig. 5). TRAM-34 changed rat jejunal patterns of intraluminal pressure waves so that they were less evenly spaced than for Krebs controls. The contractions appeared to occur more in clusters with longer intervals between clusters.41 Consistent with these former observations, we found that intervals between clustered jejunal MMC lengthened (Fig. 5A,B) so that the average frequency of MMCs decreased from 34 ± 24 to 10 ± 6 mHz (71%) (= 0.008). For colon the MMC pattern remained the same after adding TRAM-34, but there was an increase in frequency from 5.9 ± 4.3 to 8.6 ± 3.1 (46%) (= 0.03). MMC velocity was increased from 1.5 ± 0.7 to 4.5 ± 3.6 (200%) (= 0.04) for jejunum, and from 1.0 ± 0.3 to 1.5 ± 0.04 mm s−1 (50%) (= 0.03) for colon. TRAM-34 reduced PPr for jejunum and colon, the respective reductions being from 14 ± 5.5 to 5.8 ± 2.4 (59%) (= 0.02) and from 54 ± 7.9 to 33 ± 8.3 hPa (39%) (= 0.002).

image

Figure 5.  Effects of TRAM-34 on intestinal motility. (A–C) TRAM-34 decreased jejunal migrating motor complex (MMC) frequency, increased MMC velocity, and decreased intraluminal peak pressure (PPr). (D–F) Colonic MMC frequency and velocity were increased by TRAM-34, but PPr decreased.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author Contributions
  10. References

Evidence is accumulating that certain probiotic strains have acute actions in vivo5 and ex vivo,29 on the host’s autonomic reflexes. These effects can occur within minutes suggesting that the inter-kingdom signaling responsible for them would not rely on colonization, alteration in the microbiome composition or other longer term adjustments.

Our experiments utilized established ex vivo mouse jejunal29 or colon 36,39 perfusion models for which MMCs have been shown to be generated by the enteric nervous system because they are abolished by silencing the neurons using TTX.29,32,36 We applied the bacteria intraluminally, a technique that has been used for probiotics5 or 5-HT,42 and used the methods of intraluminal pressure recording and spatiotemporal mapping36,43–45 to measure MMC parameters as previously described.46 The use of these substantiated techniques to compare probiotics strains in colon vs jejunal segments has not previously been attempted.

Our results (Table 1) clearly show that such discrimination was indeed possible for the Lactobacillus strains tested and TRAM-34 in terms of the effect on motor behavior of both small and large intestine. In this regard, we had the surprising result that DSM and JB-1 acted differently on the small compared with the large intestine. There is at present no theory of enteric neurophysiology that might explain such region-specific differences, highlighting the scantiness of basic research data in this area. The finding that L. salivarius which was ineffective in vivo in an IBS clinical trial, was also without effect on MMCs in jejunum29 and in the present experiments on frequencies or velocity in the colon, support the specificity of the results. In addition, heat-killed JB-1 had no effect on jejunal MMC velocity, extending the previous findings that killed JB-1 do not alter PPr or frequency.29 Our findings may have important clinical ramifications as the motor disturbances of small or large intestines could give rise to different clinical signs and symptoms which then may require region-specific treatments.

Table 1. Table 1 Summary table for the effects of JB1, DSM, and TRAM-34 on jejunum and colon [UPWARDS ARROW] indicates an increase, [DOWNWARDS ARROW] a decrease, and ○ no change in MMC parameters measured
 JejunumColon
JB-1
 Frequency[DOWNWARDS ARROW][DOWNWARDS ARROW]
 Velocity[UPWARDS ARROW][DOWNWARDS ARROW]
 Peak pressure[DOWNWARDS ARROW][DOWNWARDS ARROW]
DSM
 Frequency[DOWNWARDS ARROW][UPWARDS ARROW]
 Velocity[DOWNWARDS ARROW][UPWARDS ARROW]
 Peak pressure
TRAM-34
 Frequency[DOWNWARDS ARROW][UPWARDS ARROW]
 Velocity[UPWARDS ARROW][UPWARDS ARROW]
 Peak pressure[DOWNWARDS ARROW][DOWNWARDS ARROW]

TRAM-34 is an IKCa channel blocker with a high degree of specificity for this channel.47 The blocker is thought to selectively act on intrinsic primary afferent neurons-IPANs (AH cells), increasing their excitability by reducing the postspike slow afterhyperpolarization (sAHP), as only these neurons functionally express IKCa channels when there is no inflammation.32,48,49

In healthy guinea pig distal colon 10 μmol L−1 TRAM-34 applied serosally reduced pellet velocity.50,51 Conversely, in guinea pig jejunum 1 μmol L−1 TRAM-34 was reported to not alter the number or velocity of long distance propagating contractions.52 In rat colon, there was no change in MMC frequency for 3 μmol L−1 TRAM-34,32 and in mouse jejunum with 5 μmol L−1 TRAM-34 there was a non-significant trend for reduction in MMC frequency of 4.9–4.1 mHz.29In vivo rat jejunum TRAM-34 1 mg kg−1 clustered MMCs intervals so that MMCs within each cluster may have increased in frequency, but there appeared to be longer intervals between the clusters.41Thus, there is at present no current consensus on species-specific effects of TRAM-34 on propagated propulsive contractions in small vs large intestine.

JB-1 has previously been studied in terms of action on the gut’s neuromuscular machinery. We have shown that when myenteric neurons have been silenced by adding TTX to the superfusate, small and large intestinal MMCs vanish and the bacterium does not modulate remaining neurally independent contractile activity in either rat or mouse.29,32 Furthermore, intraluminal application decreased the amplitude of anally propagated MMCs. Repeated daily ingestion or acute epithelial application of JB-1 reduced the magnitude and duration of the IPAN sAHP. The sAHP is generated by IKCa channel opening and TRAM-34 similarly decreases IPAN sAHP. Arguing by analogy,29,32 we proposed that JB-1 exerted its effects on IPAN excitability, and therefore motility, by reducing IKCa channel opening and thus the sAHP.

The present results (Table 1) for JB-1 and DSM and TRAM-34 demonstrate that a decrease in the IKCa channel opening is insufficient to explain all the temporal changes evoked in jejunal and colonic MMCs. Somatic ion channels other than IKCa, or synaptic transmission between IPANs or from IPANs to inter-or motor neurons might also be modulated by the probiotics to achieve the overall motor effects. It is also possible, but less likely, as they do not innervate the mucosa, that inter-or motor neurons might have their firing patterns altered by the bacteria.

As IPANs are thought to transmit to excitatory motor neurons by a fast neurotransmission53 and inhibitory in the neurons predominantly via slow transmission,52,54 it has been postulated that the timing of muscle contractile patterns depends on a complex interaction between these neural subcircuits and sensory feedback from contracting muscle fibers.52 In this schema, the degree of suppression of the IPAN sAHP (TRAM-34 dose) may be critical in determining final form and timing of the overall motor pattern.55

DSM has been shown to be effective in well-conducted trials in human infantile colic18 and also on functional gastric disturbances, thought to be due to disordered gastric motility.56 Our results suggest that in terms of infantile colic the beneficial effects of the probiotic may be due to effects on jejunum as much as the colon as jejunal MMC frequency and velocity were decreased in our model (Table 1). In this respect, the use of the mouse colon as a surrogate for human pharmacologic effectiveness of bioactive molecules is strongly supported by other data.31 Thus, the demonstration that another L. reuteri (PTA 6475), with strong anti-inflammatory activity had no effects on either small or large intestinal segments predicts that it would have little to no effect in infantile colic. Furthermore, DSM has been used clinically to help treat pediatric functional constipation.22 In our system, DSM increased both colonic MMC frequency and velocity (Table 1), an outcome which is again in register with the idea that the recording of mouse colon MMCs can be used to screen for positive effects of candidate bacteria or products. We tentatively predict that based on the effects of DSM on adult mouse colon, DSM might have therapeutic potential in slow transit constipation in elderly adults.

In conclusion, we have presented a model system that can effectively differentiate between Lactobacillus strains in terms of several key parameters of mouse gut MMCs, and between the actions they have on the small vs large intestine. This approach may help to screen and identify potential therapeutic effects of currently used or newly identified probiotic strains, and help in correlating the specific effects of such bacteria on the enteric nervous system with their actions on gut motility.

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author Contributions
  10. References

This study was supported by a grant from the Natural Sciences and Engineering Council of Canada (371955-2009), the Guglietti Family Foundation, and an unrestricted grant from BioGaia AB.

Author Contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author Contributions
  10. References

WAK and RYW designed the research and wrote the manuscript and PF helped with the design; MP, BW, YM, PS, and AMS performed the experiments; RYW and MP analyzed the data; JB contributed to drafting and editing the manuscript. All authors approved the manuscript for publication. Experiments were conducted in the McMaster Brain-Body Institute at St Joseph’s Healthcare Hamilton.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author Contributions
  10. References
  • 1
    Riboulet-Bisson E, Sturme MH, Jeffery IB et al. Effect of Lactobacillus salivarius bacteriocin Abp118 on the mouse and pig intestinal microbiota. PLoS ONE2012; 7: e31113.
  • 2
    Kunze WA, Mao YK, Wang B et al. Lactobacillus reuteri enhances excitability of colonic AH neurons by inhibiting calcium-dependent potassium channel opening. J Cell Mol Med2009; 13: 226170.
  • 3
    Forsythe P, Kunze WA. Voices from within: gut microbes and the CNS. Cell Mol Life Sci2012. [Epub ahead of print]. doi: 10.1007/s00018-012-1028-z.
  • 4
    Bravo JA, Forsythe P, Chew MV et al. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci USA2011; 108: 160505.
  • 5
    Tanida M, Yamano T, Maeda K, Okumura N, Fukushima Y, Nagai K. Effects of intraduodenal injection of Lactobacillus johnsonii La1 on renal sympathetic nerve activity and blood pressure in urethane-anesthetized rats. Neurosci Lett2005; 389: 10914.
  • 6
    Jauhiainen T, Vapaatalo H, Poussa T, Kyronpalo S, Rasmussen M, Korpela R. Lactobacillus helveticus fermented milk lowers blood pressure in hypertensive subjects in 24-h ambulatory blood pressure measurement[ast]. Am J Hypertens2005; 18: 16005.
  • 7
    Quigley EM. Probiotics in the management of colonic disorders. Curr Gastroenterol Rep2007; 9: 43440.
  • 8
    Moayyedi P, Ford A, Talley N et al. The efficacy of probiotics in the treatment of irritable bowel syndrome: a systematic review. Gut2010; 59: 32532.
  • 9
    Caenepeel P, Janssens J, Vantrappen G, Eyssen H, Coremans G. Interdigestive myoelectric complex in germ-free rats. Dig Dis Sci1989; 34: 11804.
  • 10
    Husebye E, Hellstrom PM, Midtvedt T. Intestinal microflora stimulates myoelectric activity of rat small intestine by promoting cyclic initiation and aboral propagation of migrating myoelectric complex. Dig Dis Sci1994; 39: 94656.
  • 11
    Barbara G, Stanghellini V, Brandi G et al. Interactions between commensal bacteria and gut sensorimotor function in health and disease. Am J Gastroenterol2005; 100: 25608.
    Direct Link:
  • 12
    Abraham BP, Sellin JH. Drug-induced diarrhea. In: Guandalini S, Vaziri H, eds. Diarrhea. Chpt. 23 in Clinical Gastroenterology, New York: Humana Press, 2011: 393423.
  • 13
    Verdu E, Bercik P, Collins S. Effect of probiotics on gastrointestinal function: evidence from animal models. Therap Adv Gastroenterol2009; 2: 315.
  • 14
    Ritchie ML, Romanuk TN. A meta-analysis of probiotic efficacy for gastrointestinal diseases. PLoS ONE2012; 7: e34938.
  • 15
    Francavilla R, Lionetti E, Castellaneta S et al. Randomised clinical trial: Lactobacillus reuteri DSM 17938 vs. placebo in children with acute diarrhoea – a double-blind study. Aliment Pharmacol Ther2012; 36: 3639.
  • 16
    Waller PA, Gopal PK, Leyer GJ et al. Dose-response effect of Bifidobacterium lactis HN019 on whole gut transit time and functional gastrointestinal symptoms in adults. Scand J Gastroenterol2011; 46: 105764.
  • 17
    Preidis GA, Saulnier DM, Blutt SE et al. Host response to probiotics determined by nutritional status of rotavirus-infected neonatal mice. J Pediatr Gastroenterol Nutr2012; 55: 299307.
  • 18
    Savino F, Cordisco L, Tarasco V et al. Lactobacillus reuteri DSM 17938 in infantile colic: a randomized, double-blind, placebo-controlled trial. Pediatrics2010; 126: e52633.
  • 19
    Sarna S. Colonic Motility: From Bench Side to Bedside. San Rafael (CA): Morgan & Claypool Life Sciences, 2010.
  • 20
    Gallegos-Orozco JF, Foxx-Orenstein AE, Sterler SM, Stoa JM. Chronic constipation in the elderly. Am J Gastroenterol2012; 107: 1825.
  • 21
    Tack J, Muller-Lissner S, Stanghellini V et al. Diagnosis and treatment of chronic constipation – a European perspective. Neurogastroenterol Motil2011; 23: 697710.
  • 22
    Coccorullo P, Strisciuglio C, Martinelli M, Miele E, Greco L, Staiano A. Lactobacillus reuteri (DSM 17938) in infants with functional chronic constipation: a double-blind, randomized, placebo-controlled study. J Pediatr2010; 157: 598602.
  • 23
    Bu LN, Chang MH, Ni YH, Chen HL, Cheng CC. Lactobacillus casei rhamnosus Lcr35 in children with chronic constipation. Pediatr Int: Official J Japan Pediatr Soc2007; 49: 48590.
  • 24
    Tabbers MM, de Milliano I, Roseboom MG, Benninga MA. Is Bifidobacterium breve effective in the treatment of childhood constipation? Results from a pilot studyNutr J2011; 10: 1924.
  • 25
    Higashikawa F, Noda M, Awaya T, Nomura K, Oku H, Sugiyama M. Improvement of constipation and liver function by plant-derived lactic acid bacteria: a double-blind, randomized trial. Nutrition2010; 26: 36774.
  • 26
    Zaharoni H, Rimon E, Vardi H, Friger M, Bolotin A, Shahar DR. Probiotics improve bowel movements in hospitalized elderly patients – the PROAGE study. J Nutr Health Aging2011; 15: 21520.
  • 27
    Husebye E, Hellstrom PM, Sundler F, Chen J, Midtvedt T. Influence of microbial species on small intestinal myoelectric activity and transit in germ-free rats. Am J Physiol Gastrointest Liver Physiol2001; 280: G36880.
  • 28
    Bourlioux P, Koletzko B, Guarner F, Braesco V. The intestine and its microflora are partners for the protection of the host: report on the Danone Symposium “The Intelligent Intestine,” held in Paris, June 14, 2002. Am J Clin Nutr2003; 78: 67583.
  • 29
    Wang B, Mao YK, Diorio C et al. Luminal administration ex vivo of a live Lactobacillus species moderates mouse jejunal motility within minutes. FASEB J2010; 24: 407888.
  • 30
    Massi M, Ioan P, Budriesi R et al. Effects of probiotic bacteria on gastrointestinal motility in guinea-pig isolated tissue. World J Gastroenterol2006; 12: 598794.
  • 31
    Keating C, Martinez V, Ewart L et al. The validation of an in vitro colonic motility assay as a biomarker for gastrointestinal adverse drug reactions. Toxicol Appl Pharmacol2010; 245: 299309.
  • 32
    Wang B, Mao YK, Diorio C et al. Lactobacillus reuteri ingestion and IK(Ca) channel blockade have similar effects on rat colon motility and myenteric neurones. Neurogastroenterol Motil2010; 22: 98107.
  • 33
    Gwynne RM, Thomas EA, Goh SM, Sjovall H, Bornstein JC. Segmentation induced by intraluminal fatty acid in isolated guinea-pig duodenum and jejunum. J Physiol2004; 556: 55769.
  • 34
    McCarthy EL. Mariotte’s bottle. Science1934; 80: 100.
  • 35
    Huizinga JD, Ambrous K, Der-Silaphet T. Co-operation between neural and myogenic mechanisms in the control of distension-induced peristalsis in the mouse small intestine. J Physiol1998; 506: 84356.
  • 36
    Roberts RR, Murphy JF, Young HM, Bornstein JC. Development of colonic motility in the neonatal mouse-studies using spatiotemporal maps. Am J Physiol Gastrointest Liver Physiol2007; 292: G9308.
  • 37
    O’Mahony L, McCarthy J, Kelly P et al. Lactobacillus and Bifidobacterium in irritable bowel syndrome: symptom responses and relationship to cytokine profiles. Gastroenterology2005; 128: 54151.
  • 38
    Kamiya T, Wang L, Forsythe P et al. Inhibitory effects of Lactobacillus reuteri on visceral pain induced by colorectal distension in Sprague-Dawley rats. Gut2006; 55: 1916.
  • 39
    Powell AK, Fida R, Bywater RA. Motility in the isolated mouse colon: migrating motor complexes, myoelectric complexes and pressure waves. Neurogastroenterol Motil2003; 15: 25766.
  • 40
    Liu Y, Fatheree NY, Mangalat N, Rhoads JM. Human-derived probiotic Lactobacillus reuteri strains differentially reduce intestinal inflammation. Am J Physiol Gastrointest Liver Physiol2010; 299: G108796.
  • 41
    Ferens D, Baell J, Lessene G, Smith JE, Furness JB. Effects of modulators of Ca(2 + )-activated, intermediate-conductance potassium channels on motility of the rat small intestine, in vivo. Neurogastroenterol Motil2007; 19: 3839.
  • 42
    Bulbring E, Lin RC. The effect of intraluminal application of 5-hydroxytryptamine and 5-hydroxytryptophan on peristalsis; the local production of 5-HT and its release in relation to intraluminal pressure and propulsive activity. J Physiol1958; 140: 381407.
  • 43
    Hennig GW, Costa M, Chen BN, Brookes SJ. Quantitative analysis of peristalsis in the guinea-pig small intestine using spatio-temporal maps. J Physiol1999; 517(Pt 2): 57590.
  • 44
    Bercik P, Bouley L, Dutoit P, Blum A, Kucera P. Quantitative analysis of intestinal motor patterns: spatiotemporal organization of nonneural pacemaker sites in the rat ileum. Gastroenterology2000; 119: 38694.
  • 45
    Seerden TC, Lammers WJ, De Winter BY, De Man JG, Pelckmans PA. Spatiotemporal electrical and motility mapping of distension-induced propagating oscillations in the murine small intestine. Am J Physiol Gastrointest Liver Physiol2005; 289: G104351.
  • 46
    Lammers WJ, Cheng LK. Simulation and analysis of spatio-temporal maps of gastrointestinal motility. Biomed Eng Online2008; 7: 2.
  • 47
    Wulff H, Miller MJ, Hansel W, Grissmer S, Cahalan MD, Chandy KG. Design of a potent and selective inhibitor of the intermediate-conductance Ca2 + -activated K+ channel, IKCa1: a potential immunosuppressant. Proc Natl Acad Sci USA2000; 97: 81516.
  • 48
    Nurgali K, Nguyen TV, Matsuyama H, Thacker M, Robbins HL, Furness JB. Phenotypic changes of morphologically identified guinea-pig myenteric neurons following intestinal inflammation. J Physiol2007; 583: 593609.
  • 49
    Neylon CB, Nurgali K, Hunne B et al. Intermediate-conductance calcium-activated potassium channels in enteric neurones of the mouse: pharmacological, molecular and immunochemical evidence for their role in mediating the slow afterhyperpolarization. J Neurochem2004; 90: 141422.
  • 50
    Hoffman J, McKnight N, Sharkey K, Mawe GM. The relationship between inflammation-induced neuronal excitability and disrupted motor activity in the guinea pig distal colon. Neurogastroenterol Motil2011; 23: 673e279.
  • 51
    Strong DS, Sharkey KA, Mawe GM. Relationship between AH neuron excitability and peristalsis in normal versus inflamed guinea pig distal colon. Neurogastroenterol Motil2006; 18: 695: A95.
  • 52
    Chambers JD, Bornstein JC, Thomas EA. Multiple neural oscillators and muscle feedback are required for the intestinal fed state motor program. PLoS ONE2011; 6: e19597.
  • 53
    Stebbing MJ, Bornstein JC. Electrophysiological mapping of fast excitatory synaptic inputs to morphologically and chemically characterized myenteric neurons of guinea-pig small intestine. Neuroscience1996; 73: 101728.
  • 54
    Kunze WA, Furness JB, Bornstein JC. Simultaneous intracellular recordings from enteric neurons reveal that myenteric AH neurons transmit via slow excitatory postsynaptic potentials. Neuroscience1993; 55: 68594.
  • 55
    Gwynne RM, Bornstein JC. Mechanisms underlying nutrient-induced segmentation in isolated guinea pig small intestine. Am J Physiol Gastrointest Liver Physiol2007; 292: G116272.
  • 56
    Indrio F, Riezzo G, Raimondi F et al. Lactobacillus reuteri accelerates gastric emptying and improves regurgitation in infants. Eur J Clin Invest2011; 41: 41722.