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Keywords:

  • animal model;
  • barrier function;
  • sacral nerve stimulation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Assessment of the impact of SNS upon mucus expression in the mucosa
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References
  10. Supporting Information

Background  The mechanism of action of sacral nerve stimulation (SNS) remains largely elusive. The aims of this study were to develop a clinically relevant animal model for percutaneous SNS and to describe its effect on the epithelial barrier of the rectum.

Methods  Under general anesthesia and after percutaneous electrode placement for S3 nerve root stimulation, six pigs underwent unilateral stimulation and six bilateral stimulation. Animals were stimulated for 3 h using an external pulse generator (1–2.5 V; 14 Hz; 210 μs). Six animals underwent electrode implantation without stimulation and served as controls. Full-thickness rectal biopsies were performed prior to and after stimulation. Paracellular permeability was evaluated by measuring sulfonic acid flux across the rectal mucosa in Ussing chambers. Histological assessment of mucosal thickness, epithelial desquamation, and mucus expression were performed.

Key Results  Percutaneous stimulation resulted in successful anal contractions whose amplitude and uniformity was enhanced following bilateral compared with unilateral stimulation. In controls, paracellular permeability significantly increased during the stimulation period whereas it remained unchanged following unilateral stimulation. In contrast, permeability was significantly reduced by bilateral stimulation. This effect was associated with a concomitant reduction in mucosal thickness and a trend toward increased amount of mucus on surface epithelium compared with controls.

Conclusions & Inferences  The development of a porcine model of percutaneous SNS revealed the ability of neuromodulation to reinforce rectal epithelial barrier. Furthermore, our results suggest that SNS could be used for treatment of gastrointestinal pathologies with reduced rectal mucosal barrier functions


Abbreviations:
ENS

enteric nervous system

IEB

intestinal epithelial barrier

SNS

sacral nerve stimulation

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Assessment of the impact of SNS upon mucus expression in the mucosa
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References
  10. Supporting Information

Sacral nerve stimulation (SNS) has been shown to be effective in the treatment of severe fecal incontinence resulting in a significant improvement in continence and quality of life of affected individuals.1–3 Despite being one of the most significant developments in the management of fecal incontinence over the past decade, the precise mechanisms of action remains elusive and a matter of debate.4,5 Postulates include a direct increase in sphincter tone, modulatory effects on rectal and colonic motility, and central nervous system adaptation.6

Among other gut functions putatively targeted by SNS are the intestinal epithelial barrier (IEB) functions. In fact, recent studies have shown that electrical stimulation of the vagal nerve enhances IEB resistance and reduce paracellular permeability during gut insults.7,8 Interestingly, evidences suggest that SNS stimulates not only sympathetic but also presacral parasympathetic plexus.6 In addition, neuromediators such as acetylcholine or vasoactive intestinal peptide (VIP) have been shown to increase and decrease paracellular permeability, respectively.9,10 Furthermore, IEB lesions characterized by increased paracellular permeability have been identified in several gastrointestinal diseases associated with motility dysfunctions, such as Irritable Bowel Syndrome (IBS).11 In this context, reducing paracellular permeability reduces visceral hypersensitivity in animal model of IBS12 and even prevents the development of intestinal inflammation in IL10−/− mice.13 However, it remains currently unknown as to whether SNS can also modulate IEB functions, and in particular paracellular permeability, offering another potential explanation for its mechanism of action.6

Unraveling the precise mechanisms of action of SNS and the identification of novel therapeutic targets have been somewhat hampered over the years by the lack of feasible animal models on which to conduct relevant translational human trials. Animal models of SNS performed in rats, dog, and rabbits have identified possible mechanisms by which SNS affect gut motility, but the results have been conflicting at best, probably the result of interspecies differences and difficulties in assessing treatment outcomes, particularly with anal manometry.14 An alternative animal model that could be used to address the current shortfalls in existing animal models, both from a physiological and therapeutic standpoint, is the pig. Indeed, the porcine anatomy is more closely related to that of humans and thus more relevant than smaller animals models15 allowing more similar treatment parameters and protocols to be adapted for use in humans. In addition, pigs have also recently been used to better characterize the impact of SNS on voiding dysfunction. These studies have demonstrated a superior efficacy of bilateral stimulation compared with unilateral stimulation on bladder function.16 At present, however, all animal models for SNS have involved an invasive laminectomy for the placement of electrodes, which fails to replicate the percutaneous approach used in humans, which in itself could affect the true efficacy and interpretation of results gleaned from such trials as a result of the added morbidity of the laminectomy.

Therefore, the aims of our study was thus to firstly evaluate the feasibility of the percutanous approach for SNS in a porcine model and to subsequently determine the effects of SNS upon epithelial barrier functions in the rectum.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Assessment of the impact of SNS upon mucus expression in the mucosa
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References
  10. Supporting Information

Porcine model for percutanous SNS

A total of twenty pigs (weighing between 30 and 40 kgs) were used in this study. All pigs were 6 months old and had achieved sexual and brain maturity.17 Experiments were performed in a dedicated surgical facility of the Research Laboratory, LGA (Laboratoire des grands animaux, INSERM U 643), in accordance with French Veterinary Regulations and Ethics Committee standards (Agreement: E.44010). All procedures were performed under general anesthesia, with animals in the right lateral position, with induction by Isofluran 5% and nitrous-oxide 60% and thereafter maintained on Isofluran 2%. The SNS started at least after a mean 30 min of steady state anesthesia to minimize the influence of drugs of the induction phase on the central of peripheral nervous system.

After cleaning with an iodine-based solution and draping, percutaneous placement of electrodes was performed using the conventional peripheral nerve evaluation test kits for humans (Medtronic™number 041828-004; Medtronic inc. Minneapolis USA) and using surface landmarks predefined in preliminary experiments (Fig. 1A,B). Further confirmation of the accurate electrode placement was obtained with a laminectomy performed in the two-first stimulated animals (Fig. 1C,D).

image

Figure 1.  Electrode placement landmarks. (A) Percutanous placement of electrode for unilateral SNS using defined landmarks. (B) Percutaneous placement of electrodes for bilateral SNS. (C) Following laminectomy and dissection, S3 root is approximately 2 mm in diameter and clearly isolated. (D) Dissection after SNS revealed the close proximity between the electrode and the S3 nerve root.

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Twelve animals underwent percutaneous S3 stimulation, with six of them undergoing unilateral stimulation and the other six undergoing bilateral stimulation. The control group (sham stimulated) comprised six animals submitted to percutaneous puncture at the same landmarks without stimulation. Electrode placement and stimulation was deemed satisfactory when contractions of the anal sphincter were observed. Stimulation duration was set at 3 h and stimulation intensity was set at the minimum level required to obtain anal contraction (1–2.5 V; 14 Hz, 210 μs) using an external pulse generator (Medtronic™ Model 3625 Screener).

For each pig, two full-thickness rectal biopsies 3 cm above the dentate line were performed via a transanal approach before and after SNS. The rectal biopsy sites were primarily sutured for hemostasis. At the end of SNS procedure, the animals were sacrificed by intravenous injection of pentobarbital.

Ex-vivo assessment of rectal paracellular permeability

Each biopsy was placed in ice cold Krebs solution (NaH2PO42H2O 0.187 g L−1, NaCl 6.84 g L−1, KCl 0.35 g L−1, NaHCO3 2.10 g L−1, Glucose 1.98 g L−1, CaCl2.2H2O 0.368 g L−1, MgCl2.6H2O 0.244 g L−1) and sent to the laboratory for immediate processing and analysis. Each specimen was micro-dissected to separate the mucosa from the submucosa (in the plane of the submucosal blood vessels).

Specimens of rectal mucosa were then mounted in dedicated Ussing chambers (Transcellab, TBC, Paris, France) exposing a mean surface of 0.0314 cm2. Specimens were bathed on each side in 1.5 mL of Ham’s Nutrient Mixture (HAM/F12, Invitrogen, France). The media was continuously oxygenated and maintained at 37 °C by a gas flow of 95% O2 and 5% CO2. After an equilibration period of 30 min, 150 μL of apical medium was replaced by 150 μL of fluorescein–5.6 sulfonic acid (1 mg mL−1, 400 D, Invitrogen, France). The fluorescence level of basolateral aliquots of 150 μL was measured at 30 min intervals over a period of 180 min using a fluorimeter (Varioskan, Thermo SA, France). Paracellular permeability was determined by averaging the gradient of change in fluorescence intensity over time using a linear regression fit model measured in the specimens (GraphPad Prism 5, La Jolla, USA).

Histological evaluation of the impact of SNS upon rectal mucosa

Following microdissection, specimens of mucosa were fixed in paraformaldehyde (4% in PBS). After tissue washing in PBS, they were dehydrated and embedded in paraffin. Sequential sections of mucosa (5 μm) were made. Bilateral stimulated and control tissues were analyzed by classical microscopy following haematoxylin-eosin staining. Each section was observed under an Olympus IX 50 microscope (Olympus Inc., Center Valley, PA, USA). Pictures were acquired using a digital camera DP71 (Olympus), connected to a computer through a frame grabber card (Cell B software, Olympus). Three parameters were analyzed to describe the morphology of the mucosa, as previously reported18: (i) mucosal thickness was determined by measuring the distance between the surface epithelium and the underlying muscularis mucosae.(ii) the height of the surface epithelium and (iii) assessment of the surface epithelium continuity (% of continuous epithelium along the mucosal surface), The mean values were therefore computed on a total of 10 fields (three values per field) examined per experiment and per condition. Investigators were blinded for histological observations.

Assessment of the impact of SNS upon mucus expression in the mucosa

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Assessment of the impact of SNS upon mucus expression in the mucosa
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References
  10. Supporting Information

Sections of mucosa (5 μm) of bilateral stimulated and control tissues were analyzed using classical microscopy following alcian blue staining staining. Evaluation of mucus expression on the surface epithelium and in the crypts was determined using a semi-quantitative standardized score defined as follows:

  • 0
     No mucus on the field
  • 1
     Small amount of mucus (<25% of the surface epithelium)
  • 2
     Average amount of mucus (25–75% of the surface epithelium)
  • 3
     Large amount of mucus (>75% of the surface epithelium)

Statistical analysis

Data are presented as mean ± standard deviation. Statistical analysis was performed using Prism (GraphPad Prism 5, La Jolla, CA, USA). To determine differences between two groups or between multiple groups, Wilcoxon test or Kruskall–Wallis followed by Dunn’s tests were used, respectively. Linear regression analysis was also performed. A P-value <0.05 was considered as statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Assessment of the impact of SNS upon mucus expression in the mucosa
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References
  10. Supporting Information

Percutaneous SNS in a porcine model

All 12 pigs undergoing stimulation had successful contractions of the anal sphincter. Clinical parameters (body core temperature, heart rate, and blood pressure) were monitored throughout the experiments in each group (Table 1). No significant variation was observed in heart rate and blood pressure either within or between the treatment groups. However, body temperature decreased significantly during the course of the experiments, but not differently between the three groups.

Table 1.   Clinical parameters monitored during the experiments
 TemperatureHeart rateOxygen saturation
StartEndStartEndStartEnd
  1. *P < 0.05 compared with the start of stimulation.

Mean ± SD37.1 ± 0.535.6 ± 1.2*88 ± 8107 ± 4295 ± 295 ± 2

The anatomical landmarks of electrode position were measured in eight animals at the end of the procedure. The electrodes were positioned at an average of 12.0 ± 2.5 cm (= 8) from the anal margin and 1 cm lateral to the vertebral midline, up to an average depth of 3.9 ± 0.2 cm (= 8).

Unilateral stimulation resulted in ipsilateral spastic contractions of the anus, whereas bilateral stimulation not only enhanced contraction amplitude, but also produced a more uniform contraction of the entire anal sphincter complex (Video S1). These contractions remained sustained during the whole stimulation procedure.

Effect of SNS upon rectal paracellular permeability

Paracellular permeability at the beginning of the experiment was similar in all three groups (= 0.31; = 18). In controls (sham stimulated), paracellular permeability increased by 24% (= 0.03; = 6) between the beginning and the end of the experimental procedure (Fig. 2A). During unilateral SNS, paracellular permeability remained unchanged (= 0.84; = 6) (Fig. 2B). In contrast, paracellular permeability was significantly reduced by 20% following bilateral stimulation (= 0.03; = 6) (Fig. 2C). Furthermore, compared with controls, changes in paracellular permeability induced during the course of SNS were significantly reduced following bilateral, but not unilateral SNS (Fig. 2D).

image

Figure 2.  Impact of sacral nerve stimulation (SNS) on paracellular permeability. (A) In controls (sham stimulated) paracellular permeability increased significantly between the beginning (t0) and the end of the procedure (t3). (B) During unilateral SNS, no significant change in paracellular permeability occurred. (C) Following bilateral SNS, paracellular permeability decreased significantly. Individual values represent the mean obtained from two segments per animal and per condition.*< 0.05 Wilcoxon test. (D) Impact of SNS upon the changes in paracellular permeability induced during the course of the experiments. Changes in paracellular permeability were significantly different between control and bilateral SNS *< 0.05 Kruskal–Wallis followed by Dunn’s test.

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Effects of SNS upon mucosal histology

We next focussed the study on bilateral SNS and controls, as it had more significant effects upon rectal barrier permeability than unilateral SNS. In controls, rectal mucosa thickness did not change between the beginning and the end of the procedure (93 ± 34 vs 87 ± 21 μm, respectively; = 0.31) (Fig. 3A,C). In contrast, mucosa thickness was significantly reduced between the beginning and the end of bilateral stimulation (101 ± 30 vs 75 ± 9 μm, respectively; = 0.03) (Fig. 3B,D). Concerning the surface epithelium continuity and height, no significant difference was observed between the control and the SNS group (data not shown).

image

Figure 3.  Impact of sacral nerve stimulation (SNS) on mucosal histology. Sections of mucosa from control (A) and bilateral SNS (B) stimulated pigs were stained with hematoxylin eosin and observed under a microscope. Mucosal thickness in the bilateral stimulated group (D) was significantly reduced at the end of the 3 h stimulation period (t3) compared with the beginning (t0). In contrast, no change in mucosal thickness was observed in the control group (C). Individual data represent the mean of three measures per field and 10 fields per animal and condition.*< 0.05 Wilcoxon test. Scale bar: 20 μm.

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Effects of SNS upon epithelial mucus distribution

Concerning bilateral SNS, the score reflecting the amount of mucus at the surface of the epithelium was increased at the end of the stimulation procedure compared with the beginning (0.30  ± 0.21 vs 0.15  ± 0.09, respectively; = 0.06; Fig. 4B,D). In contrast, in controls the mucus scores remained unchanged after 3 h (0.10 ± 0.08 vs 0.09 ± 0.06, respectively; = 0.47; Fig. 4A,C). No difference was measured in the mucus score of the crypts in both groups (Fig. 4E,F). However, interestingly, in stimulated pigs, the surface epithelium mucus score and the crypt mucus score were positively and linearly correlated (r2 = 0.88 and = 0.03).

image

Figure 4.  Effects of sacral nerve stimulation (SNS) upon epithelial mucus distribution. Sections of mucosa from control (A) and bilateral SNS (B) stimulated pigs were stained with alcian blue and observed under a microscope. In controls, the score reflecting the amount of mucus at the surface of the epithelium remained unchanged after 3 h (C). In the bilateral SNS group, the mucus score was increased at the end of the stimulation procedure compared with the beginning (= 0.06; Wilcoxon test) (D). No difference in mucus score was measured in the crypts of the control (E) and the bilateral SNS group (F). Scale bars: 10 μm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Assessment of the impact of SNS upon mucus expression in the mucosa
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References
  10. Supporting Information

In this study, we have first demonstrated the feasibility of a porcine model for percutaneous SNS, avoiding the morbidity of a laminectomy-based approach, providing therefore a suitable large animal model for SNS research. Secondly, to the best of our knowledge, this is also the first study showing that sustained SNS induces a significant reduction in mucosal paracellular permeability of the rectum, together with a concomitant reduction in mucosal thickness and increased surface mucus secretion.

A first result of this pilot study was the observation that bilateral SNS significantly reduced paracellular permeability of the rectal mucosa. Although unilateral did not decrease paracellular permeability, it prevented the increase in paracellular permeability observed in sham animals. These results performed on small-sized groups need to be confirmed in the future on larger animal samples. Previous studies have shown that vagal stimulation can enhance IEB resistance and reduce paracellular permeability in the small intestine.7,8 The effects of vagal stimulation could be ultimately mediated by enteric neurons and/or glial cells, as vagal stimulation increases enteric neuronal activity.19,20 Consistently, direct electrostimulation of the human enteric nervous system has also been shown to reduce paracellular permeability of intestinal epithelial monolayers.10 Our results obtained with SNS extend these findings to regions of the digestive system that are not under direct vagal control (i.e. distal colon and rectum). The SNS could exert its effects upon the rectal barrier through stimulation of parasympathetic presacral plexus, which has been shown to be activated during SNS stimulation.6 However, to date, the mediators responsible for the barrier reinforcing effects of SNS remain to be identified. Among putative candidates, vasoactive intestinal peptide or S-Nitrosoglutathione have been identified as enteric neuro-glial mediators enhancing barrier functions.21,22 Interestingly, these mediators can reduce paracellular permeability over short-term periods similar to the duration of SNS used in our study. The ability of bilateral SNS, but not unilateral SNS to reduce paracellular permeability could be due to the increased release of mediators by bilateral SNS compared with unilateral one. Interestingly, improved results of bilateral SNS compared with unilateral stimulation have been reported in the management of voiding dysfunction, 16 but also in the treatment of fecal incontinence.23

We have further demonstrated that SNS increased epithelial surface mucus amount. This could be due to an increase in mucus secretion induced by SNS. Indeed, previous studies have shown that neuromediators or activation of the ENS can induce mucus secretion.24,25 Consistently, this increase in surface epithelium mucus amount was correlated with the amount of mucus observed in the crypts, suggesting a secretory process. Altogether, increased mucus amount on surface epithelium could, besides reduced paracellular permeability, also enhance barrier protection by SNS.

The exact mechanism of action of SNS in treating fecal incontinence remains still largely unknown.6 Apart from a direct effect on sphincter tone, SNS has been shown to modify colonic motility,26,27 probably via the activation of central and peripheral nervous pathways.28,29 Our findings offer new insights into the possible mechanism of action, in which SNS reinforces the rectal mucosa barrier. In particular, the effects of SNS upon mucosal barrier functions could be involved in the success of SNS reported in patients with IBS, chronic constipation and chronic pelvic pain.29–32 In addition, recent reports highlight the need for a critical appraisal of the criteria for selecting patients for SNS.4,5 In this context, our findings set the basis for evaluating the effects of SNS upon paracellular permeability in patients treated for fecal incontinence. If correlated to improvement of anorectal functions, these changes in paracellular permeability induced by SNS could be used as future new marker of response to SNS. Therefore, such a parameter could improve the early selection of responding patients during the peripheral nerve evaluation phase and the subsequent definitive pacemaker implantation. Provided reinforcement of the rectal barrier by SNS is confirmed in humans, its potential benefits in gastrointestinal pathologies with impaired mucosal barrier should be evaluated.

In conclusion, this study describes the first porcine model for percutanous SNS that best replicates the technique routinely employed in humans for the treatment of severe fecal incontinence. We further demonstrate that SNS can reduce paracellular permeability of the rectal mucosa. These findings offer new insights into the mechanisms of action of SNS on anorectal functions. Furthermore, SNS could offer new therapeutical perspectives for treatment of gastrointestinal pathologies with reduced mucosal barrier functions

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Assessment of the impact of SNS upon mucus expression in the mucosa
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References
  10. Supporting Information

The authors thank David Minault, Jeremy Hervouet, and Pr Gilles Blancho for their expertise and help during the experiments. The authors also thank Julien Chevalier and Maxime Mahe for their technical assistance. The present study received supports by grants from ‘La Fondation Benoit Malassagne’ and ‘La Fondation de l’Avenir’ attributed to CB. Devices required for the experiments (pulse generator and PNE test-kit) were provided by Medtronic Inc.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Assessment of the impact of SNS upon mucus expression in the mucosa
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Assessment of the impact of SNS upon mucus expression in the mucosa
  6. Results
  7. Discussion
  8. Acknowledgments
  9. References
  10. Supporting Information

Video S1. Assessment of anal contraction during bilateral SNS in pig. Right and left S3 stimulation lead to right side (first arrow) and left side (second arrow) anal sphincter contraction, respectively. Bilateral stimulation causes a full sphincter contraction.

FilenameFormatSizeDescription
NMO_1839_sm_videoS1.avi2822KSupporting info item

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