Agathe Gelot

Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors

Abdominal pain is common in the general population and, in patients with irritable bowel syndrome, is attributed to visceral hypersensitivity. We found that oral administration of specific Lactobacillus strains induced the expression of μ-opioid and cannabinoid receptors in intestinal epithelial cells, and mediated analgesic functions in the gut—similar to the effects of morphine. These results suggest that the microbiology of the intestinal tract influences our visceral perception, and suggest new approaches for the treament of abdominal pain and irritable bowel syndrome.

T-type calcium channels contribute to colonic hypersensitivity in a rat model of irritable bowel syndrome.

The symptoms of irritable bowel syndrome (IBS) include significant abdominal pain and bloating. Current treatments are empirical and often poorly efficacious, and there is a need for the development of new and efficient analgesics aimed at IBS patients. T-type calcium channels have previously been validated as a potential target to treat certain neuropathic pain pathologies. Here we report that T-type calcium channels encoded by the CaV3.2 isoform are expressed in colonic nociceptive primary afferent neurons and that they contribute to the exaggerated pain perception in a butyrate-mediated rodent model of IBS. Both the selective genetic inhibition of CaV3.2 channels and pharmacological blockade with calcium channel antagonists attenuates IBS-like painful symptoms. Mechanistically, butyrate acts to promote the increased insertion of CaV3.2 channels into primary sensory neuron membranes, likely via a posttranslational effect. The butyrate-mediated regulation can be recapitulated with recombinant CaV3.2 channels expressed in HEK cells and may provide a convenient in vitro screening system for the identification of T-type channel blockers relevant to visceral pain. These results implicate T-type calcium channels in the pathophysiology of chronic visceral pain and suggest CaV3.2 as a promising target for the development of efficient analgesics for the visceral discomfort and pain associated with IBS.

Interactive involvement of brain derived neurotrophic factor, nerve growth factor, and calcitonin gene related peptide in colonic hypersensitivity in the rat

Background and aims: Neutrophins are involved in somatic and visceral hypersensitivity. The action of nerve growth factor (NGF) on sensory neurones contributes to the development of referred colonic hypersensitivity induced by trinitrobenzene sulfonic acid (TNBS). Based on data on brain derived neurotrophic factor (BDNF) and calcitonin gene related peptide (CGRP) in pain, the aims of the present study were: (1) to investigate the involvement of BDNF and CGRP in this model of referred colonic hypersensitivity, (2) to test the effect of exogenous BDNF and CGRP on the colonic pain threshold, and (3) to investigate the relationship between BDNF, NGF, and CGRP by testing antineurotrophin antibodies or h-CGRP 8–37 (a CGRP antagonist) on bowel hypersensitivity induced by these peptides. Methods: Colonic sensitivity was assessed using a colonic distension procedure. Results: Anti-BDNF antibody and h-CGRP 8–37 reversed the induced decrease in colonic threshold (33.4 (2.1) and 40.3 (4.1) mm Hg, respectively, compared with a vehicle score of approximately 18 mm Hg; p<0.001). BDNF (1–100 ng/rat intraperitoneally) induced a significant dose dependent decrease in colonic reaction threshold in healthy rats. This effect was reversed by an anti-BDNF antibody and an anti-NGF antibody (33.4 (0.6) v 18.7 (0.7) mm Hg (p<0.001), anti-NGF v vehicle). NGF induced colonic hypersensitivity was reversed by h-CGRP 8–37 but not by the anti-BDNF antibody. Finally, antineurotrophin antibody could not reverse CGRP induced colonic hypersensitivity (at a dose of 1 µg/kg intraperitoneally). Conclusion: Systemic BDNF, NGF, and CGRP can induce visceral hypersensitivity alone and interactively. This cascade might be involved in TNBS induced referred colonic hypersensitivity in which each of these peptides is involved.

Crohn's disease‐associated Escherichia coli LF82 aggravates colitis in injured mouse colon via signaling by flagellin

Background: Ileal lesions in Crohns disease patients are colonized by pathogenic adherent‐invasive Escherichia coli (AIEC) that harbor various virulence factors involved in adhesion to and invasion of intestinal epithelial cultured cells. We investigated in a mouse model of colonic inflammation the behavior of virulent AIEC reference bacteria LF82 compared to that of nonflagellated LF82 mutants. Methods: BALBc/J mice with intact or dextran sulfate sodium (DSS)‐injured colon were orally challenged daily with 108 bacteria. The severity of colitis was assessed by determining disease activity index, colonic histological score, and myeoloperoxidase activity. Flagellin receptor and cytokine expression was measured by reverse‐transcriptase polymerase chain reaction (RT‐PCR) in colonic tissue. Results: In contrast to nonpathogenic E. coli, virulent LF82 bacteria exacerbated colitis in DSS‐treated mice, substantially reducing survival rate, greatly lowering stool consistency, inducing marked weight loss and increased rectal bleeding, and significantly increasing erosive lesions and mucosal inflammation. Nonflagellated LF82 mutants behaved like nonpathogenic E. coli K‐12. Interestingly, oral infection with LF82 virulent bacteria, but not with a nonvirulent LF82 mutant, induced a 7.0‐fold increase in the levels of TLR5 and a 3.1‐fold increase in those of ipaf mRNA, which encode respectively membrane and cytosolic receptors involved in the recognition of flagellin. Hence, a 5.6‐fold increase in IL‐1&bgr; and a 5.3‐fold increase in mRNA of IL‐6 were observed in mice challenged with AIEC LF82 bacteria. Conclusions: Crohns disease‐associated virulent AIEC LF82 bacteria, via expression of flagella, are able to potentiate an inflammatory mucosal immune response involving increased expression of TLR5 and IPAF flagellin receptors.

State-dependent properties of a new T-type calcium channel blocker enhance CaV3.2 selectivity and support analgesic effects

Summary This study deciphers the mechanism of inhibition of T‐type calcium channels by TTA‐A2, demonstrating that TTA‐A2 affinity for CaV3.2‐inactivated state confers a preferential analgesic efficacy toward pathological pain. ABSTRACT T‐type calcium channels encoded by the CaV3.2 isoform are expressed in nociceptive primary afferent neurons where they contribute to hyperalgesia and thus are considered as a potential therapeutic target to treat pathological pain. Here we report that the small organic state‐dependent T‐type channel antagonist TTA‐A2 efficiently inhibits recombinant and native CaV3.2 currents. Although TTA‐A2 is a pan CaV3 blocker, it demonstrates a higher potency for CaV3.2 compared to CaV3.1. TTA‐A2 selectivity for T‐type currents was demonstrated in sensory neurons where it lowered cell excitability uniquely on neurons expressing T‐type channels. In vivo pharmacology in CaV3.2 knockout and wild type mice reveal that TTA‐A2‐mediated antinociception critically depends on CaV3.2 expression. The pathophysiology of irritable bowel syndrome (IBS) was recently demonstrated to involve CaV3.2 in a rat model of this disease. Oral administration of TTA‐A2 produced a dose‐dependent reduction of hypersensitivity in an IBS model, demonstrating its therapeutic potential for the treatment of pathological pain. Overall, our results suggest that the high potency of TTA‐A2 in the depolarized state strengthen its analgesic efficacy and selectivity toward pathological pain syndromes. This characteristic would be beneficial for the development of analgesics targeting T‐type channels, in particular for the treatment of pain associated with IBS.

Spinal cord plasticity and acid-sensing ion channels involvement in a rodent model of irritable bowel syndrome

Irritable bowel syndrome (IBS) is a common functional gastro‐intestinal disorder characterized by intractable chronic abdominal pain. In this study, we examined the possible spinal mechanisms underlying colonic hypersensitivity (CHS) using a non‐inflammatory rat model of IBS induced by rectal enemas of butyrate, a short‐chain fatty acid. We hypothesized that spinal plasticity could be responsible for CHS and that ASIC channels, which are known to support pain‐elicited currents in the spinal cord, could contribute to central sensitization in our model of IBS. First, in order to determine if visceral pain relies on changes in spinal activity, we analyzed Fos expression in the spinal cord of rats treated with butyrate following a challenge with repetitive noxious colorectal distension. We found that Fos immunoreactivity was increased in thoracic T10–11–12, lumbar L1–2–6 and sacral S1 spinal segments. In control rats treated with saline, noxious repetitive colorectal distensions evoked Fos expression only in L1–2–6 and S1 spinal segments. Secondly, intrathecal injection of PcTx1, a specific ASIC1A antagonist, in the lumbar spinal cord completely prevented the development of CHS induced by butyrate. ASIC1 and 2 mRNAs, especially ASIC1A, were upregulated in the lumbar spinal cord. ASIC1A could thus contribute to spinal sensitization in our model of IBS, as it is supported by spinal colocalization of ASIC1A and Fos proteins. The whole data pinpoint a potential critical role of thoracic spinal cord in non‐inflammatory pain states such as IBS and suggest that ASIC channels are part of the molecular effectors of central sensitization leading to visceral pain.Irritable bowel syndrome (IBS) is a common functional gastro-intestinal disorder characterized by intractable chronic abdominal pain. In this study, we examined the possible spinal mechanisms underlying colonic hypersensitivity (CHS) using a non-inflammatory rat model of IBS induced by rectal enemas of butyrate, a short-chain fatty acid. We hypothesized that spinal plasticity could be responsible for CHS and that ASIC channels, which are known to support pain-elicited currents in the spinal cord, could contribute to central sensitization in our model of IBS. First, in order to determine if visceral pain relies on changes in spinal activity, we analyzed Fos expression in the spinal cord of rats treated with butyrate following a challenge with repetitive noxious colorectal distension. We found that Fos immunoreactivity was increased in thoracic T10-11-12, lumbar L1-2-6 and sacral S1 spinal segments. In control rats treated with saline, noxious repetitive colorectal distensions evoked Fos expression only in L1-2-6 and S1 spinal segments. Secondly, intrathecal injection of PcTx1, a specific ASIC1A antagonist, in the lumbar spinal cord completely prevented the development of CHS induced by butyrate. ASIC1 and 2 mRNAs, especially ASIC1A, were upregulated in the lumbar spinal cord. ASIC1A could thus contribute to spinal sensitization in our model of IBS, as it is supported by spinal colocalization of ASIC1A and Fos proteins. The whole data pinpoint a potential critical role of thoracic spinal cord in non-inflammatory pain states such as IBS and suggest that ASIC channels are part of the molecular effectors of central sensitization leading to visceral pain.

Peripheral contribution of NGF and ASIC1a to colonic hypersensitivity in a rat model of irritable bowel syndrome

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder associated with idiopathic colonic hypersensitivity (CHS). However, recent studies suggest that low‐grade inflammation could underlie CHS in IBS. The pro‐inflammatory mediator nerve growth factor (NGF) plays a key role in the sensitization of peripheral pain pathways and several studies have reported its contribution to visceral pain development. NGF modulates the expression of Acid‐Sensing Ion Channels (ASICs), which are proton sensors involved in sensory neurons sensitization. This study examined the peripheral contribution of NGF and ASICs to IBS‐like CHS induced by butyrate enemas in the rat colon.

Anti-nociceptive effect of Faecalibacterium prausnitzii in non-inflammatory IBS-like models

Visceral pain and intestinal dysbiosis are associated with Irritable Bowel Syndrome (IBS), a common functional gastrointestinal disorder without available efficient therapies. In this study, a decrease of Faecalibacterium prausnitzii presence has been observed in an IBS-like rodent model induced by a neonatal maternal separation (NMS) stress. Moreover, it was investigated whether F. prausnitzii may have an impact on colonic sensitivity. The A2-165 reference strain, but not its supernatant, significantly decreased colonic hypersensitivity induced by either NMS in mice or partial restraint stress in rats. This effect was associated with a reinforcement of intestinal epithelial barrier. Thus, F. prausnitzii exhibits anti-nociceptive properties, indicating its potential to treat abdominal pain in IBS patients.

Colonic overexpression of the T-type calcium channel Cav3.2 in a mouse model of visceral hypersensitivity and in irritable bowel syndrome patients

Among the different mechanisms involved in irritable bowel syndrome (IBS) physiopathology, visceral hypersensitivity seems to play a key role. It involves sensitization of the colonic primary afferent fibers, especially through an overexpression of ion channels. The aims of this translational study were to investigate the colonic expression of Cav3.2 calcium channels and their involvement in an animal model of colonic hypersensitivity, and to assess their expression in the colonic mucosa of symptomatic IBS patients.

Comparative effects of α2δ-1 ligands in mouse models of colonic hypersensitivity

AIM To investigate anti-hypersensitive effects of α2δ-1 ligands in non-inflammatory and inflammation-associated colonic hypersensitivity (CHS) mouse models. METHODS To induce an inflammation-associated CHS, 1% dextran sulfate sodium (DSS) was administered to C57Bl/6J male mice, in drinking water, for 14 d. Regarding the non-inflammatory neonatal maternal separation (NMS) -induced CHS model, wild-type C57BI/6J pups were isolated from their mother from day 2 to day 14 (P2 to P14), three hours per day (from 9:00 a.m. to 12:00 p.m.). Colorectal distension was performed by inflating distension probe from 20 μL to 100 μL by 20 μL increment step every 10 s. After a first colorectal distension (CRD), drugs were administered subcutaneously, in a cumulative manner, (Gabapentin at 30 mg/kg and 100 mg/kg; Pregabalin at 10 mg/kg and 30 mg/kg; Carbamazepine at 10 mg/kg and 30 mg/kg) and a second CRD was performed one hour after each injection. RESULTS The visceromotor response (VMR) to CRD was increased by our NMS paradigm protocol in comparison to non-handled (NH) mice, considering the highest distension volumes (80 μL: 0.783 ± 0.056 mV/s vs 0.531 ± 0.034 mV/s, P < 0.05 and 100 μL: 1.087 ± 0.056 mV/s vs 0.634 ± 0.038 mV/s, P < 0.05 for NMS and NH mice, respectively). In the inflammation-associated CHS, DSS-treated mice showed a dramatic and significant increase in VMR at 60 and 80 μL distension volumes when compared to control mice (60 μL: 0.920 ± 0.079 mV/s vs 0.426 ± 0.100 mV/s P < 0.05 and 80 μL: 1.193 ± 0.097 mV/s vs 0.681 ± 0.094 mV/s P < 0.05 for DSS- and Water-treated mice, respectively). Carbamazepine failed to significantly reduce CHS in both models. Gabapentin significantly reduced CHS in the DSS-induced model for both subcutaneous injections at 30 or 100 mg/kg. Pregabalin significantly reduced VMR to CRD in the non-inflammatory NMS-induced CHS model for the acute subcutaneous administration of the highest cumulative dose (30 mg/kg) and significantly reduced CHS in low-dose DSS-treated mice in a dose-dependent manner. Finally, the percent decrease of AUC induced by acute GBP or Pregabalin treatment were higher in the inflammatory DSS-induced CHS model in comparison to the non-inflammatory NMS-induced CHS model. CONCLUSION This preclinical study demonstrates α2δ-1 ligands efficacy on inflammation-associated CHS, highlighting their potential clinical interest in patients with chronic abdominal pain and moderate intestinal inflammation.