Xiaoyin Wu

Rifaximin Alters Intestinal Bacteria and Prevents Stress-Induced Gut Inflammation and Visceral Hyperalgesia in Rats

BACKGROUND & AIMS Rifaximin is used to treat patients with functional gastrointestinal disorders, but little is known about its therapeutic mechanism. We propose that rifaximin modulates the ileal bacterial community, reduces subclinical inflammation of the intestinal mucosa, and improves gut barrier function to reduce visceral hypersensitivity. METHODS We induced visceral hyperalgesia in rats, via chronic water avoidance or repeat restraint stressors, and investigated whether rifaximin altered the gut microbiota, prevented intestinal inflammation, and improved gut barrier function. Quantitative polymerase chain reaction (PCR) and 454 pyrosequencing were used to analyze bacterial 16S ribosomal RNA in ileal contents from the rats. Reverse transcription, immunoblot, and histologic analyses were used to evaluate levels of cytokines, the tight junction protein occludin, and mucosal inflammation, respectively. Intestinal permeability and rectal sensitivity were measured. RESULTS Water avoidance and repeat restraint stress each led to visceral hyperalgesia, accompanied by mucosal inflammation and impaired mucosal barrier function. Oral rifaximin altered the composition of bacterial communities in the ileum (Lactobacillus species became the most abundant) and prevented mucosal inflammation, impairment to intestinal barrier function, and visceral hyperalgesia in response to chronic stress. Neomycin also changed the composition of the ileal bacterial community (Proteobacteria became the most abundant species). Neomycin did not prevent intestinal inflammation or induction of visceral hyperalgesia induced by water avoidance stress. CONCLUSIONS Rifaximin alters the bacterial population in the ileum of rats, leading to a relative abundance of Lactobacillus. These changes prevent intestinal abnormalities and visceral hyperalgesia in response to chronic psychological stress.

Corticosterone Mediates Reciprocal Changes in CB 1 and TRPV1 Receptors in Primary Sensory Neurons in the Chronically Stressed Rat

BACKGROUND & AIMS Chronic stress is associated with visceral hyperalgesia in functional gastrointestinal disorders. We investigated whether corticosterone plays a role in chronic psychological stress-induced visceral hyperalgesia. METHODS Male rats were subjected to 1-hour water avoidance (WA) stress or subcutaneous corticosterone injection daily for 10 consecutive days in the presence or absence of corticoid-receptor antagonist RU-486 and cannabinoid-receptor agonist WIN55,212-2. The visceromotor response to colorectal distension was measured. Receptor protein levels were measured and whole-cell patch-clamp recordings were used to assess transient receptor potential vanilloid type 1 (TRPV1) currents in L6-S2 dorsal root ganglion (DRG) neurons. Mass spectrometry was used to measure endocannabinoid anandamide content. RESULTS Chronic WA stress was associated with visceral hyperalgesia in response to colorectal distension, increased stool output and reciprocal changes in cannabinoid receptor 1 (CB1) (decreased) and TRPV1 (increased) receptor expression and function. Treatment of WA stressed rats with RU-486 prevented these changes. Control rats treated with serial injections of corticosterone in situ showed a significant increase in serum corticosterone associated with visceral hyperalgesia, enhanced anandamide content, increased TRPV1, and decreased CB1 receptor protein levels, which were prevented by co-treatment with RU-486. Exposure of isolated control L6-S2 DRGs in vitro to corticosterone reproduced the changes in CB1 and TRPV1 receptors observed in situ, which was prevented by co-treatment with RU-486 or WIN55,212-2. CONCLUSIONS These results support a novel role for corticosterone to modulate CB1 and TRPV1-receptor pathways in L6-S2 DRGs in the chronic WA stressed rat, which contributes to visceral hyperalgesia observed in this model.

Enhanced responses of the anterior cingulate cortex neurones to colonic distension in viscerally hypersensitive rats

The anterior cingulate cortex (ACC) is critically involved in processing the affective component of pain sensation. Visceral hypersensitivity is a characteristic of irritable bowel syndrome. Electrophysiological activity of the ACC with regard to visceral sensitization has not been characterized. Single ACC neuronal activities in response to colorectal distension (CRD) were recorded in control, sham‐treated rats and viscerally hypersensitive (EA) rats (induced by chicken egg albumin injection, i.p). The ACC neurones of controls failed to respond to 10 or 30 mmHg CRD; only 22% were activated by 50 mmHg CRD. Among the latter, 16.4% exhibited an excitatory response to CRD and were labelled ‘CRD‐excited’ neurones. In contrast, CRD (10, 30 and 50 mmHg) markedly increased ACC neuronal responses of EA rats (10%, 28% and 47%, respectively). CRD produced greater pressure‐dependent increases in ACC spike firing rates in EA rats compared with controls. Splanchnicectomy combined with pelvic nerve section abolished ACC responses to CRD in EA rats. Spontaneous activity in CRD‐excited ACC neurones was significantly higher in EA rats than in controls. CRD‐excited ACC neurones in control and EA rats (7 of 16 (42%) and 8 of 20 (40%), respectively) were activated by transcutaneous electrical and thermal stimuli. However, ACC neuronal activity evoked by noxious cutaneous stimuli did not change significantly in EA rats. This study identifies CRD‐responsive neurones in the ACC and establishes for the first time that persistence of a heightened visceral afferent nociceptive input to the ACC induces ACC sensitization, characterized by increased spontaneous activity of CRD‐excited neurones, decreased CRD pressure threshold, and increased response magnitude. Enhanced ACC nociceptive transmission in viscerally hypersensitive rats is restricted to visceral afferent input.

Up-Regulation of Anterior Cingulate Cortex NR2B Receptors Contributes to Visceral Pain Responses in Rats

BACKGROUND & AIMS Electrophysiologic and behavioral studies have shown that increased N-methyl-D-aspartate (NMDA)-receptor activation of anterior cingulate cortex (ACC) neurons has a critical role in modulating visceral pain responses in viscerally hypersensitive (VH) rats. This study aimed to identify the NMDA receptor subtypes in perigenual ACC (pACC) neurons involved in the facilitation of visceral nociception. METHODS We performed in vivo electrophysiologic recordings of pACC neurons and examined the visceromotor response (VMR) to colorectal distention (CRD) in normal and VH rats induced by colonic anaphylaxis. The NR2A-subtype-receptor antagonist [(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) and the NR2B-receptor-antagonist Ro25-6981 were microinjected into the pACC. To down-regulate NR2B-receptor gene expression, an NR2B-specific small interfering RNA (siRNA) and a plasmid (pEGFP-N1) that expressed the green fluorescent protein were administered into ACC neurons by electroporation. RESULTS Reverse microdialysis of NVP-AAM077 had no effect on basal and CRD-induced ACC neuronal firing in VH and control groups. In VH rats, Ro25-6981 (500 micromol/L) inhibited ACC neuronal firing, evoked by 30 and 50 mm Hg CRD, by 98% and 52%, respectively. NVP-AAM077 did not affect the VMR in either group. Ro25-6981 significantly suppressed the VMR in VH but not normal rats. Immunoblot analysis showed increased NR2B-receptor expression in the pACC of VH rats. NR2B siRNA-treated VH rats showed a significant reduction in the VMR, compared with controls. CONCLUSIONS The NR2B subunit of the NMDA receptor has a critical role in the modulation of ACC sensitization and visceral pain responses in VH rats.

Anterior Cingulate Cortex Modulates Visceral Pain as Measured by Visceromotor Responses in Viscerally Hypersensitive Rats

BACKGROUND & AIMS We have identified that the anterior cingulate cortex (ACC) neurons are responsive to colorectal distention (CRD) and shown that sensitization of ACC neurons occurs in viscerally hypersensitive rats. However, the role of the ACC in pain response has not been clearly defined. We aimed to determine if ACC neuron activation enhances visceral pain in viscerally hypersensitive rats and to identify the receptor involved in facilitation of visceral pain. METHODS The nociceptive response (visceromotor response [VMR]) to CRD was recorded in normal and viscerally hypersensitive rats induced by colonic anaphylaxis. The ACC was stimulated electrically, and ACC lesions were generated with ibotenic acid. l-glutamate, alpha-amino-3-hydroxy-5-methyl-isoxozole propionic acid receptor antagonist cyanonitroquinoxaline dione, and N-methyl-d-aspartate receptor antagonist aminophosphonopentanoic acid were microinjected into the rostral ACC. RESULTS Electrical stimulation of the rostral ACC enhanced the VMR to CRD in normal rats. ACC lesions caused a decrease in the VMR in viscerally hypersensitive rats but had no effect in normal rats. ACC microinjection of 2 mmol/L glutamate increased the VMR to CRD (10 mm Hg) in viscerally hypersensitive rats, and 20 mmol/L glutamate induced a more potent VMR in viscerally hypersensitive than in normal rats. Cyanonitroquinoxaline dione did not affect the VMR in either group. Aminophosphonopentanoic acid significantly suppressed the VMR in viscerally hypersensitive rats but not in normal rats. CONCLUSIONS The ACC plays a critical role in the modulation of visceral pain responses in viscerally hypersensitive rats. This process appears to be mediated by enhanced activities of glutamate N-methyl-d-aspartate receptors.

Serotonin and cholecystokinin synergistically stimulate rat vagal primary afferent neurones

Recent studies indicate that cholecystokinin (CCK) and serotonin (5‐hydroxytryptamine, 5‐HT) act via vagal afferent fibres to mediate gastrointestinal functions. In the present study, we characterized the interaction between CCK and 5‐HT in the vagal primary afferent neurones. Single neuronal discharges of vagal primary afferent neurones innervating the duodenum were recorded from rat nodose ganglia. Two groups of nodose ganglia neurones were identified: group A neurones responded to intra‐arterial injection of low doses of cholecystokinin octapeptide (CCK‐8; 10–60 pmol); group B neurones responded only to high doses of CCK‐8 (120–240 pmol), and were also activated by duodenal distention. CCK‐JMV‐180, which acts as an agonist in high‐affinity states and as an antagonist in low‐affinity states, dose dependently stimulated group A neurones, but inhibited the effect of the high doses of CCK‐8 on group B neurones. Duodenal perfusion of 5‐HT evoked dose‐dependent increases in nodose neuronal discharges. Some neurones that responded to 5‐HT showed no response to either high or low doses of CCK‐8. A separate group of nodose neurones that possessed high‐affinity CCK type A (CCK‐A) receptors also responded to luminal infusion of 5‐HT. Further, a subthreshold dose of CCK‐8 (i.e. 5 pmol) produced no measurable electrophysiological effects but it augmented the neuronal responses to 5‐HT. This potentiation effect of CCK‐8 was eliminated by CR 1409. From these results we concluded that the vagal nodose ganglion contains neurones that may possess only high‐ or low‐affinity CCK‐A receptors or 5‐HT3 receptors. Some neurones that express high‐affinity CCK‐A receptors also express 5‐HT3 receptors. Pre‐exposure to luminal 5‐HT may augment the subsequent response to a subthreshold dose of CCK.

Role for NMDA receptors in visceral nociceptive transmission in the anterior cingulate cortex of viscerally hypersensitive rats

We have identified colorectal distension (CRD)-responsive neurons in the anterior cingulate cortex (ACC) and demonstrated that persistence of a heightened visceral afferent nociceptive input to the ACC induces ACC sensitization. In the present study, we confirmed that rostral ACC neurons of sensitized rats [induced by chicken egg albumin (EA)] exhibit enhanced spike responses to CRD. Simultaneous in vivo recording and reverse microdialysis of single ACC neurons showed that a low dose of glutamate (50 microM) did not change basal ACC neuronal firing in normal rats but increased ACC neuronal firing in EA rats from 18 +/- 2 to 32 +/- 3.8 impulses/10 s. A high dose of glutamate (500 microM) produced 1.95-fold and a 4.27-fold increases of ACC neuronal firing in sham-treated rats and in EA rats, respectively, suggesting enhanced glutamatergic transmission in the ACC neurons of EA rats. Reverse microdialysis of the 3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainite receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) reduced basal and abolished CRD-induced ACC neuronal firing in normal rats. In contrast, microdialysis of N-methyl-d-aspartate (NMDA) receptor antagonist AP5 had no effect on ACC neuronal firing in normal rats. However, AP5 produced 86% inhibition of ACC neuronal firing evoked by 50 mmHg CRD in the EA rats. In conclusion, ACC nociceptive transmissions are mediated by glutamate AMPA receptors in the control rats. ACC responses to CRD are enhanced in viscerally hypersensitive rats. The enhancement of excitatory glutamatergic transmission in the ACC appears to mediate this response. Furthermore, NMDA receptors mediate ACC synaptic responses after the induction of visceral hypersensitivity.

Hypothalamic regulation of pancreatic secretion is mediated by central cholinergic pathways in the rat

The vago‐vagal reflex plays an important role in mediating pancreatic secretion evoked by cholecystokinin and non‐cholecystokinin‐dependent luminal factors. We hypothesize that the vago‐vagal reflex mediating pancreatic secretion in the rat is under central control and regulated by cholinergic pathways in the hypothalamus. To test this hypothesis, we demonstrated that chronic decerebration decreased basal pancreatic enzyme secretion from 318 ± 12 to 233 ± 9 mg h−1 and reduced the net increase in pancreatic secretion stimulated by intraduodenal infusion of 5% peptone and hypertonic NaCl by 54% and 45%, respectively. Intracerebroventricular administration of methscopolamine (MSCP, 50 nmol (5 μl)−1), a blood‐brain barrier‐impermeant cholinergic muscarinic receptor antagonist, evoked results similar to those achieved by chronic decerebration. To localize the sites of action, we demonstrated that microinjection of MSCP (20 nmol) into the lateral hypothalamic nucleus or the paraventricular nucleus resulted in inhibition of both basal pancreatic protein secretion and luminally stimulated pancreatic secretion by 48% and 52%, respectively. Intracerebroventricular injection of hemicholinium‐3 at doses known to deplete the endogenous ACh store produced similar inhibitory results. In addition, microinjection of ACh (5 pmol) or the muscarinic M1 receptor agonist McN‐A‐343 (30 ng) into the lateral hypothalamic nucleus increased pancreatic secretion over basal levels by 46% and 40%, respectively. Selective lesions of lateral septal cholinergic neurons decreased basal pancreatic secretion and inhibited peptone‐induced pancreatic secretion by 30%. Destruction of the lateral parabrachial nucleus produced a 44% inhibition of peptone‐induced pancreatic section. Finally, microinjection of glutamate into the lateral septum or the lateral parabrachial nucleus stimulated vagal pancreatic efferent nerve firings from a basal level of 0 ± 0.5 impulses (30 s)−1 to 4.5 ± 0.5 and 14 ± 2 impulses (30 s)−1, respectively, and pancreatic protein output increased 50% and 84% over basal levels. Administration of MSCP to the paraventricular nucleus eliminated these effects. These observations suggest that cholinergic neurons of the lateral septum and lateral parabrachial nucleus regulate pancreatic secretion. Further, cholinergic input from the lateral parabrachial nucleus to the hypothalamus plays a major role in the modulation of vagal pancreatic efferent nerve activity and pancreatic secretion evoked by the vago‐vagal reflex.

Low-affinity CCK-A receptors are coexpressed with leptin receptors in rat nodose ganglia: implications for leptin as a regulator of short-term satiety.

The paradigm for the control of feeding behavior has changed significantly. Research has shown that leptin, in the presence of CCK, may mediate the control of short-term food intake. This interaction between CCK and leptin occurs at the vagus nerve. In the present study, we aimed to characterize the interaction between CCK and leptin in the vagal primary afferent neurons. Single neuronal discharges of vagal primary afferent neurons innervating the gastrointestinal tract were recorded from rat nodose ganglia. Three groups of nodose ganglia neurons were identified: group 1 responded to CCK-8 but not leptin; group 2 responded to leptin but not CCK-8; group 3 responded to high-dose CCK-8 and leptin. In fact, the neurons in group 3 showed CCK-8 and leptin potentiation, and they responded to gastric distention. To identify the CCK-A receptor (CCKAR) affinity states that colocalize with the leptin receptor OB-Rb, we used CCK-JMV-180, a high-affinity CCKAR agonist and low-affinity CCKAR antagonist. As expected, immunohistochemical studies showed that CCK-8 administration significantly potentiated the increase in the number of c-Fos-positive neurons stimulated by leptin in vagal nodose ganglia. Administration of CCK-JMV-180 eliminated the synergistic interaction between CCK-8 and leptin. We conclude that both low- and high-affinity CCKAR are expressed in nodose ganglia. Many nodose neurons bearing low-affinity CCKAR express OB-Rb. These neurons also respond to mechanical distention. An interaction between CCKAR and OB-Rb in these neurons likely facilitates leptin mediation of short-term satiety.

Diabetic Visceral Hypersensitivity Is Associated With Activation of Mitogen-Activated Kinase in Rat Dorsal Root Ganglia

OBJECTIVE Diabetic patients often experience visceral hypersensitivity and anorectal dysfunction. We hypothesize that the enhanced excitability of colon projecting dorsal root ganglia (DRG) neurons observed in diabetes is caused by a decrease in the amplitude of the transient A-type K+ (IA) currents resulting from increased phosphorylation of mitogen-activated protein kinases (MAPK) and reduced opening of Kv4.2 channels. RESEARCH DESIGN AND METHODS We performed patch-clamp recordings of colon projecting DRG neurons from control and streptozotocin-induced diabetic (STZ-D) rats. Western blot analyses and immunocytochemistry studies were used to elucidate the intracellular signaling pathways that modulate the IA current. In vivo studies were performed to demonstrate that abnormal MAPK signaling is responsible for the enhanced visceromotor response to colorectal distention in STZ-D rats. RESULTS Patch-clamp studies demonstrated that IA current was diminished in the colon projecting DRG neurons of STZ-D rats. Western blot analysis of STZ-D DRG neurons revealed increases in phosphorylated MAPK and KV4.2. In diabetic DRG neurons, increased intracellular Ca2+ ([Ca2+]i), protein kinase C (PKC), and MAPK were involved in the regulation of IA current through modulation of Kv4.2. Hypersensitive visceromotor responses to colorectal distention in STZ-D rats were normalized by administration of MAPK inhibitor U0126. CONCLUSIONS We demonstrated that reduction of the IA current in STZ-D DRG neurons is triggered by impaired [Ca2+]i ion homeostasis, and this in turn activates the PKC-MAPK pathways, resulting in decreased opening of the Kv4.2 channels. Hence, the PKC-MAPK–Kv4.2 pathways represent a potential therapeutic target for treating visceral hypersensitivity in diabetes.