Lactobacillus farciminis treatment attenuates stress-induced overexpression of Fos protein in spinal and supraspinal sites after colorectal distension in rats

Authors


Dr Vassilia Theodorou, Neuro-Gastroenterology & Nutrition Unit, UMR 1054, INRA/EI-Purpan, 180 chemin de Tournefeuille, BP 3, 31931 Toulouse cedex 9, France.
Tel: +33 561 28 51 59; fax: +33 561 28 51 45;
e-mail: vtheodor@toulouse.inra.fr

Abstract

Abstract  Irritable bowel syndrome (IBS), frequently associated with psychological distress, is characterized by hypersensitivity to gut wall distension. Some probiotics are able to alleviate IBS symptoms and reduce visceromotor response to mechanical stimuli in animals. Moreover, we have previously shown that Lactobacillus farciminis treatment abolished the hyperalgesia to colorectal distension (CRD) induced by acute stress. The aims of the present study were to determine whether (i) stress-induced visceral hyperalgesia modifies the expression of Fos, a marker of general neuronal activation, induced by CRD, (ii) this activation can be modulated by L. farciminis treatment. Female rats were treated by L. farciminis and CRD was performed after partial restraint stress (PRS) or sham-PRS. The expression of Fos protein was measured by immunohistochemistry. After CRD or PRS, Fos expression was increased in spinal cord section (S1), nucleus tractus solitarius (NTS), paraventricular nucleus (PVN) of the hypothalamus, and in the medial nucleus of the amygdala (MeA). The combination of both stimuli, PRS and CRD, markedly increased this Fos overexpression in the sacral spinal cord section, PVN and MeA, but not in NTS. By contrast, a pretreatment with L. farciminis significantly reduced the number of Fos positive cells in these area. This study shows that PRS enhances Fos protein expression induced by CRD at the spinal and supraspinal levels in rats. Lactobacillus farciminis treatment inhibited this enhancing effect, suggesting that the antinociceptive effect of this probiotic strain results from a decrease of the stress-induced activation/sensitization of sensory neurons at the spinal and supraspinal level.

Abbreviations:
CRD

colorectal distension

CNS

central nervous system

IBS

irritable bowel syndrome

MeA

medial amygdala

NTS

nucleus tractus solitary

PVN

paraventricular nucleus of the hypothalamus

Introduction

Irritable bowel syndrome (IBS) is a highly prevalent functional gastrointestinal disorder affecting up to 3–15% of the general population in Western countries. It is defined by the coexistence of abdominal pain or discomfort and alteration in bowel habits.1 Various environmental factors such as stress or intestinal infection are known to exacerbate symptoms in IBS patients and may have indirect effects on visceral perception in the gut. As stressful life events trigger IBS symptoms,2 both acute and chronic stress paradigms were developed in animals to mimic changes in visceral sensitivity observed in IBS patients. In rats, restraint and passive avoidance stress are associated with gut hypersensitivity in response to rectal distension. This phenomenon involves both central and peripheral release of corticotropin releasing factor (CRF)3 and subsequent neuronal activation of spinal and supraspinal regions.4,5 While the exact pathophysiology of IBS remains unclear, altered intraluminal milieu, immune activation, enteric neuromuscular dysfunction and brain-gut axis dysregulation are currently explored hypotheses reported in IBS. Visceral hypersensitivity characterizing IBS patients results from complex interactions between altered gut mucosa physiology (low grade inflammation, immune activation and impaired intestinal barrier)6 and altered autonomic balance.7 Several observations suggest that indigenous gut flora plays a role in the pathogenesis of IBS. Gastrointestinal infection is known to alter gut flora8 and also to exacerbate or induce IBS.9 Changes in gut flora such as decrease in lactobacilli and bifidobacteria counts10 or development of small intestinal bacterial overgrowth11 have also been described in some IBS patients.

Probiotics are defined as live microorganisms that exert a health benefit on the host when ingested in adequate amounts.12 In animal studies, probiotics can exert antibacterial effects,13 intestinal barrier enhancement,14 and anti-inflammatory activity15,16 but decrease the abdominal response to colorectal distension (CRD)14,17,18 providing a rationale for probiotics use in several gastrointestinal pathologies including IBS.19 Some recent data support an effect of probiotic treatments on visceral hypersensitivity.17Lactobacillus reuteri or its metabolites have been shown to inhibit the constitutive cardio-autonomic response to CRD in rats, probably through an effect on enteric nerves or their neuronal connections to the spinal cord.18Lactobacillus acidophilus has also been reported to reduce visceral perception in mice, putatively by acting at on opioid or canabinoid receptors.20 Despite these data, the effect of probiotics on spinal and brain regions related to the stress-induced alterations of visceral perception in response to gut distension remains to be investigated so far. As Lactobacillus farciminis (L. farciminis) suppresses stress-induced hypersensitivity in response to CRD, in this study we aimed at evaluating whether this antinociceptive effect is associated with changes in neuronal activation at spinal and supraspinal sites induced by L. farciminis strain. Neuronal activation was evaluated by measuring the expression of Fos protein. This protein is a marker of neuronal activation, which is rapidly and transiently expressed in neurons of the central nervous system in response to somatocutaneous or visceral sensory stimuli.21 Consequently, we measured first if stress-induced visceral hyperalgesia modifies Fos expression induced by CRD and second if this activation can be modulated by L. farciminis treatment.

Materials and Methods

Animals

Female Wistar rats (Janvier SA, Le Genest St Isle, France) weighing 200–225 g and housed individually in a temperature-controlled room (21 ± 1 °C) were used. They were allowed free access to water and fed standard pellets (SAFE, Augy, France). The local committee for animal use and care had approved all experimental protocols described in this study.

Bacteria preparation

Lactobacillus farciminis (CIP 103136, Institut Pasteur Collection, Paris, France) was grown at 37 °C in MRS broth (VWR international, Fontenay-sous-Bois, France). After 17 h incubation, cultures were harvested by centrifugation at 4500 g for 10 min. Strain extract was resuspended in 0.9 % NaCl and stored at −20 °C. Bacterial suspension was prepared daily in order to orally administer 1011 CFU per day per rat.

Partial restraint stress

All stress sessions were performed at the same period of the day (between 10:00 am and 12:00 pm) to minimize the influence of circadian rhythms, particularly on Fos expression. Stress effects were studied using the wrap partial restraint stress (PRS) model which is a mild and non-ulcerogenic stressor.22 Thus, animals were lightly anaesthetized with ethyl-ether, and their fore shoulders, upper forelimbs and thoracic trunk were wrapped in a confining harness of paper tape to restrict, but not to prevent, body movements. Later rats were placed in their home cage for 2 h. Rats recovered from ethyl-ether within 2–3 min and immediately moved around in their cage, although the restricted mobility of their forelimb prevented grooming behaviour. Sham-PRS rats considered as controls were anaesthetized as above, but were not wrapped and were allowed to move freely in their cages.

Colorectal distension

Rats were accustomed to be in polypropylene tunnels (diameter: 7 cm; length: 20 cm) for several days before colorectal distension (CRD) in order to minimize recording artefacts. The balloon used for CRD was 4 cm long and made from latex condom. It was fixed on a semi-rigid catheter (2 mm in diameter). The CRD was performed by insertion of the balloon in the rectum at 1 cm from the anus. Isobaric distensions of the colon were performed by connecting the balloon to a computerized barostat.23 Colonic pressure and balloon volume were continuously monitored on a potentiometric recorder (L6514, Linseis, Selb, Germany). Isobaric distensions were performed from 0 to 60 mmHg, each distension step lasting 5 min. The first distension was performed at a pressure of 15 mmHg and an increment of 15 mmHg was added at each following step, until a maximal pressure of 60 mmHg.

Experimental procedure

To reduce stress of environment and non-specific Fos expression, all animals were handled for 7 days prior to experiments. A total of 64 rats in eight groups (each groups, n = 8) were included in this study. All animals received orally during 14 days either saline (groups 1, 3, 5 and 7) or 1011 CFU per day of L. farciminis (groups 2, 4, 6 and 8). Rats were submitted to a partial restraint stress (groups 5–8) or sham-PRS (no stress; groups 1–4). Groups 4, 5, 7 and 8 were submitted to a progressive colorectal distension (CRD) performed 20 min after PRS or sham-PRS.

Fos immunohistochemistry

One hour after completion of the colorectal distension, rats were deeply anaesthetized with urethane (2 g kg−1 i.p.) and perfused transcardially with 100–150 mL isotonic saline (0.9% NaCl) followed by 400–500 mL of 4% paraformaldehyde. After fixation, lumbosacral segments (L6, S1 and S2) of the spinal cord and brain were dissected and removed, postfixed at 4 °C in 4% paraformaldehyde in PBS for 4 h and cryoprotected overnight in 30% sucrose at 4 °C. Frozen serial sections (35 μm-thick) were collected in phosphate-buffered saline (PBS), then rinsed twice. Sections were stained for Fos-like immunoreactivity using biotin–avidin–peroxidase complex. Sections were incubated at room temperature in a blocking solution of 2% normal goat serum in PBS with 0.25% Triton X-100 for 30 min and then incubated overnight at 4 °C with rabbit polyclonal Fos antibody diluted in blocking solution (1 : 10 000; Ab-5,AbCys, Paris, France) for 24 h at 4 °C. The incubated sections were washed twice and incubated with biotinylated goat antirabbit secondary antibody, diluted 1 : 1000 in blocking solution, and then incubated with the avidin–biotin complex (Vectastain Elite kit; Vector Laboratories, Paris, France). Peroxidase activity was revealed using diaminobenzidine as chromogene (DAB substrate kit, Vector Laboratories, France). The sections were then mounted on gelatin-coated slides, deshydrated and coverslipped with DePex. The presence of Fos immunoreactivity was detected as a dark brown reaction product in cell nuclei under a light microscope (90i Nikon, Nikon France, Champigny-sur-Marne, France).

Cells counting and statistical analysis

For quantitative assessment, the number of immunoreactive cells was counted bilaterally in 10–16 sections of selected nuclei using Lucia G4.8 software (Nikon France, Champigny–Sur-Marne, France). The planes of sections were standardized according to the Paxinos and Waston’s atlas24 coordinates (mm from bregma): −1.80 to −2.12 for the paraventricular nucleus of the hypothalamus (PVN); −1.80 to −2.56 for the medial nucleus of amygdala (MeA); −11.30 to −14.60 for the nucleus tractus solitarius (NTS). In lumbosacral segment, Fos immunoreactivity was counted in L6, S1 and S2. Quantification of Fos positive cells in the sacral spinal cord and brain areas mentioned above was performed in consecutive sections. Number of Fos positive cells per section in each nucleus was presented as means SEM of 7–8 rats for each group. Statistical comparisons between two groups were performed using one-way analysis of variance. Differences were considered significant when post student’s t-test (LSD) P < 0.05.

Results

Effect of stress on colorectal distension-induced Fos expression in the sacral spinal cord, brain stem and brain nuclei

In naïve rats (without CRD and without stress), the total number of Fos positive cells was very low in the different area investigated. In absence of stress (sham-stressed animals), CRD induced Fos expression in spinal cord sections (S1) (Fig. 1A) which are mainly located in the dorsal horns of the spinal gray matter, predominantly in laminae I–II and the area surrounding the central canal (area X) (Fig. 2B). Similar pattern of Fos expression were observed in L6 and S2 section segment of the spinal cord (data not shown). In the nucleus tractus solitary (NTS), colorectal distension also induced Fos expression (Fig. 1B).

Figure 1.

 Number of Fos positive cells was quantified in the (A) sacral of spinal cord (S1), (B) nucleus tractus solitary (NTS), (C) paraventricular nucleus of hypothalamus (PVN) and (D) medial amygdaloid nucleus (MeA) in treated rats with Lactobacillus farciminis (gray bars) or saline (white bars) after sham-stress and stress in response to CRD or not. Each column represents the mean ± SEM of mean number of Fos positive cells generated from 10 to 16 sections cell count/animal. *P < 0.05 compared with stressed animals with CRD; # P < 0.05 compared with sham-stress submitted to CRD. Sham-stress groups treated or not with L. farciminis were significant from other groups.

Figure 2.

 Photomicrographs of representative sections in different regions of sham-stress (first column), stress + CRD (second column) and stress + CRD + Lactobacillus farciminis (third column) rats. (A, B and C) sacral spinal cord sections; (D, E and F) paraventricular nucleus of hypothalamus; (G, H and I) medial amygdaloid nucleus. Scare bars : 100 μm. III = third ventricule; PVN subdivision: dp = dorsal parvocellular; mp = medial parvocellular; pm = posterior magnocellular; pv = periventricullar subdivision.

Fos expression was also induced in the brain areas: the paraventricular nucleus (PVN) of hypothalamus (Fig. 1C), mainly in the medial subdivision of its parvocellular part (Fig. 2E), and in the medial nucleus of the amygdala (MeA) (Figs 1D and 2H).

Partial restraint stress for 2 h induced Fos expression in the same areas than that activated by colorectal distension (Fig. 1). When partial restraint stress was applied before CRD, the number of Fos positive cells was significantly increased (P < 0.05); when compared with CRD alone, in the sacral spinal cord (1.7-fold), the PVN (2.5-fold) and the MeA (1.8-fold) (Fig. 1A, C, D). On the contrary, it was significantly reduced in the NTS (1.5-fold) (Fig. 1B).

Effect of Lactobacillus farciminis treatment on stress induced overexpression of Fos protein after colorectal distension in spinal and supraspinal level

After a 2-week treatment with L. farciminis (1011 CFU per day), the number of Fos positive neurons observed after colorectal distension in absence of stress was not significantly modified in the sacral spinal cord, the NTS and the PVN, in comparison with rats receiving saline. It was significantly reduced only in the MeA (Fig. 1). In rats submitted to partial restraint stress in absence of CRD, L. farciminis treatment did not modify Fos expression in the four areas investigated (Fig. 1). On the contrary, the overexpression of Fos protein observed in the sacral spinal cord, the PVN and the MeA when CRD was preceded by partial restraint stress, was significantly reduced (P < 0.05) by L. farciminis treatment (Fig. 1A, C and D). No effect was observed at NTS level, where no overexpression of Fos protein appeared after the combination of stress and CRD (Fig. 1B). Moreover, L. farciminis had not effect per se on Fos protein expression in the basal condition (no stress and no CRD) (Fig. 1). Representative micrographs of section at S1, PVN and MeA after treatment with saline or L. farciminis are shown on Fig. 2.

Discussion

This study shows that acute stress exacerbates Fos protein expression induced by colorectal distension in the lumbosacral section of spinal cord (L6-S1), in the PVN and MeA and L. farciminis treatment reduces this enhanced neuronal activation.

The autonomic pathways are involved in visceral pain response and changes in autonomic balance may alter visceral perception.7 Experimental animal models have been established to gain insight into the pathogenesis and the mechanisms of visceral hyperalgesia. Colorectal distension (CRD) is a reproducible technique largely used to deliver noxious visceral stimuli to rats.25 Induction of Fos expression is a well established marker of neuronal activation and immunohistological detection of this protein allows a mapping of activated brain nuclei at a single cell level.26

In our study increased Fos protein expression was observed after a CRD session in the lumbosacral section of the spinal cord and in the NTS, the PVN and the limbic brain structure, MeA. In the rat, the distal colon and rectum are innervated by primary afferent fibers projecting in the pelvic nerves to the lumbosacral spinal cord segments. The increased expression of Fos observed in the dorsal horn of L6-S1 segments and more particularly in the lamina I and II layers by CRD, is in agreement with previous findings showing increased activation in this structure in response to CRD.27 The lamina I and II layers of the lumbosacral spinal cord correspond to the endings of primary visceral sensory afferents and this is consistent with the activation of pain related circuits.28 Further, neurons responding to CRD in the lumbosacral spinal cord show higher activity compared with those in the thoracolumbar section regarding the percentage of excited neurons, the mean magnitude and the threshold of the response.29 The CRD applied in this study concerns distal parts of the colon. As the descending colon of the rat also receives vagal innervation30 from the celiac branch, the stimulation of mechanosensitive vagal afferents might also contribute directly to the pattern of Fos expression in the brain. Further, increased Fos expression in the NTS may result from spino-NTS activated pathway. Consistent with such a pathway, we have found increased Fos expression in the NTS, the site of entrance of visceral vagal afferents in the brain.31

Hypothalamic structures are interconnected by abundant afferent and efferent projections with numerous brain nuclei, including the PVN.32 Thus, colon distension-induced neuronal activation in these areas might result in hypothalamic activation by painful stimuli. As a consequence, in this study we have found an increased Fos expression in the PVN induced by CRD as already observed by others.33,34

Higher cognitive functions related to visceral pain perception also depend on many limbic brain structures. Previous studies have shown that noxious visceral stimuli, for example CRD result in Fos expression in limbic structures, such as medial amygdala.35 Our study confirms colorectal distension-induced Fos expression in this limbic area.

The number of Fos positive cells significantly increased after restraint stress session (in absence of CRD) in the sacral section of the spinal cord and in the NTS, the PVN and MeA. On the other hand, stress is recognized to affect the neuroendocrine system and the production of neuropeptides in many brain regions. Activation of Fos expression after stress, may be related to the production of these neuromediators. For example the parvocellular parts of PVN, a subregion of PVN where CRF producing neurons are located, is characterized by a pronounced Fos expression induced by stress.36 In order to confirm positive CRF-containing neurons in the PVN further investigations using double immunostanning (Fos/CRF) are needed.

The amygdala is widely considered as the brain region involved in both emotion of fear and stress as damage in this area has been shown to reduce hypothalamic–pituitary–adrenal (HPA) responses to various stressors including restraint stress.37 In a study using the restraint stress model, authors have shown a linear increase in neuronal cells activity in the medial amygdala, and NTS with increasing durations of restraint.4 Findings in present study indicates an increased number of Fos positive cells in MeA and NTS induced by stress are indeed consistent with the literature.

The increased Fos immunostaining in the lumbosacral section L6, S1 and S2 in stressed rats in absence of CRD may depend on a painful component of restraint rather than stress itself.

However, Fos expression induced by CRD was exacerbated by acute stress in the dorsal horn of the lumbosacral section L6-S1. This is in agreement with several studies showing that restraint stress induces visceral hypersensitivity in female rats.14,38 In a recent study conducted in humans it has been shown that children who underwent the stress tasks before the pain task exhibited lower levels of pain tolerance than those who received the pain task first,39 suggesting that stress facilitates nociception. Similarly, in a model of maternal deprivation, increased nociception has been observed in prenatally stressed pups associated with a decrease in the intensity of serotonin-like immunoreactivity and density of serotonergic cells in the lumbar spinal cord dorsal horn40, and in adult rats correlated with an increased NGF expression.41

The stress-induced increased Fos immunostaining induced by colorectal distension in the PVN and MeA can be explained by the facilitation nociceptive pathways provoked by stress. Indeed, these regions can be activated by noxious visceral stimuli and have been shown to be involved in stress-induced hypersensitivity.36 The absence of stress-induced exacerbated Fos expression induced by colorectal distension in the NTS argues against vagal involvement in the stress nociceptive facilitation. This is in agreement with previous findings showing that the development of delayed stress-induced hyperalgesia is unaffected by afferent vagotomy suggesting that vagal afferents are not a required for the modulatory effect of stress on visceral sensitivity.42 However, Chen et al. have recently shown that low intensity electrical vagal stimulation activating vagal afferences is able to reduce the visceromotor response to CRD.43

The most interesting finding of this study is that L. farciminis treatment decreases stress-induced Fos overexpression in spinal (L6, S1 and S2) and supraspinal sites (PVN and MeA) after CRD. Furthermore, L farciminis did not affect changes in Fos immunostaining neither in sham stressed animals submitted to CRD, except in the MeA, nor in stressed animals in absence of CRD. Taken together, these observations suggest that the antinociceptive effect of L. farciminis on stress-induced hypersensitivity results from a decrease in the stress-induced activation/sensitization of sensory neurons at the spinal cord level and subsequent nociceptive facilitation at supraspinal sites. We have no explanations for the decrease in Fos protein expression in the MeA observed after CRD in absence of stress in L. farciminis-treated rats.

One hypothesis is that of the L. farciminis effect on Fos immunostaining in stressed animals submitted to CRD is indirect, linked to its protective effect on the intestinal barrier. In rats, restraint stress triggers alterations of the intestinal barrier characterized by an increased permeability to macromolecules, enhancing thus mast cells degranualtion,44 peripheral CRF-related mechanisms45 and results from epithelial cell cytoskeleton contraction.46 In our previous study, L. farciminis treatment prevents stress-induced increase in colonic paracellular permeability, and colonocyte MLC phosphorylation leading to cytoskeleton contraction inhibition and subsequent decrease in tight junction opening.14 Blockade of stress-induced increase in gut permeability prevents visceral hypersensitivity evidencing a cause-effect relationship between permeability and sensitivity in the gut.46 Therefore, we hypothesize that L. farciminis prevents excessive uptake of endoluminal factors (microbial antigens and bacterial products) by decreasing stress-induced changes in permeability. This process activates mucosal immunity and provokes mediators release which in turn may sensitize sensory afferents. Similar sensitization occurs in response to gut tissue injury in models of CRD.47

Even if not investigated in this study, a modulation in the neurochemistry of the autonomic nervous system by L. farciminis cannot be excluded. Another probiotic, L. paracasei inhibits the visceral hypersensitivity associated with inflammation in healthy mice in which the bacterial microbiota has been disturbed by antibiotics.17 This latter study shows an anti-inflammatory effect and also an inhibition of substance P-staining, a marker of afferent pain pathways in the myenteric and submucous plexus, while it was increased after the antibiotic treatment. Direct actions of bacteria on the enteric nervous system are possible as pathogenic bacteria can upregulate the substance P-receptor in infected tissue48 and as expression of genes encoding enteric neural components are different in germ-free and colonized mice.49 Kamm et al. have shown that among several markers tested only calbindin positive myenteric neurons decrease after a Saccharomyces boulardii treatment in pigs.50 In unstimulated conditions, L. reuteri inhibits the constitutive cardio-autonomic response to CRD as shown by decreased dorsal root ganglion single unit activity to distension.18

Finally, our results may support recent findings showing that, in human volunteers, a probiotics treatment significantly reduced stress-induced gastrointestinal symptoms such as nausea and abdominal pain.51

In conclusion, in this study we report for the first time that a probiotic suppresses stress-induced visceral hypersensitivity in rats throught visceral nociceptive processing at spinal and supraspinal sites. Considering that visceral hypersensitivity and disturbed autonomic function are key features of IBS, this study supports rationale for selected probiotic strains use in the IBS management.

Competing interest

The authors have no competing interests.

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