Jyoti N. Sengupta

Differential c-Fos expression in the nucleus of the solitary tract and spinal cord following noxious gastric distention in the rat

c-Fos has been used as a marker for activity in the spinal cord following noxious somatic or visceral stimulation. Although the viscera receive dual afferent innervation, distention of hollow organs (i.e. esophagus, stomach, descending colon and rectum) induces significantly more c-Fos in second order neurons in the nucleus of the solitary tract and lumbosacral spinal cord, which receive parasympathetic afferent input (vagus, pelvic nerves), than the thoracolumbar spinal cord, which receives sympathetic afferent input (splanchnic nerves). The purpose of this study was to determine the contribution of sympathetic and parasympathetic afferent input to c-Fos expression in the nucleus of the solitary tract and spinal cord, and the influence of supraspinal pathways on Fos induction in the thoracolumbar spinal cord. Noxious gastric distention to 80 mmHg (gastric distension/80) was produced by repetitive inflation of a chronically implanted gastric balloon. Gastric distension/80 induced c-Fos throughout the nucleus of the solitary tract, with the densest labeling observed within 300 microns of the rostral pole of the area postrema. This area was analysed quantitatively following several manipulations. Gastric distension/80 induced a mean of 724 c-Fos-immunoreactive nuclei per section. Following subdiaphragmatic vagotomy plus distention (vagotomy/80), the induction of c-Fos-immunoreactive nuclei was reduced to 293 per section, while spinal transection at T2 plus distention (spinal transection/80) induced a mean of 581 nuclei per nucleus of the solitary tract section. Gastric distension/80 and vagotomy/80 induced minimal c-Fos in the T8-T10 spinal cord (50 nuclei/section), but spinal transection/80 induced 200 nuclei per section. Repetitive bolus injections of norepinephrine produced transient pressor responses mimicking the pressor response produced by gastric distension/80. This manipulation induced minimal c-Fos in the nucleus of the solitary tract and none in the spinal cord. It is concluded that noxious visceral input via parasympathetic vagal afferents, and to a lesser extent sympathetic afferents and the spinosolitary tract, contribute to gastric distention-induced c-Fos in the nucleus of the solitary tract. The induction of c-Fos in the nucleus of the solitary tract is significantly greater than in the viscerotopic segments of the spinal cord, which is partially under tonic descending inhibition, but is not subject to modulation by vagal gastric afferents. Distention pressures produced by noxious gastric distention are much greater than those produced during feeding, suggesting that c-Fos induction in the nucleus of the solitary tract to noxious distention is not associated with physiological mechanisms of feeding and satiety. The large vagal nerve-mediated induction of c-Fos in the nucleus of the solitary tract following gastric distension suggests that parasympathetic afferents contribute to the processing of noxious visceral stimuli, perhaps by contributing to the affective-emotional component of visceral pain.

Effects of kappa opioids in the inflamed rat colon

The objective of this study was to examine the antinociceptive effects of peripherally restricted kappa-opioid receptor agonists (ORAs) in a rat model of inflammatory bowel disease produced by intracolonic instillation of trinitrobenzine sulfonic acid (TNBS). Antinociceptive effects of mu-(morphine) and kappa-ORAs (EMD 61,753 and ICI 204,488) were evaluated in a behavioral model of visceral nociception. The effects of these agonists and a delta-ORA (SNC 80) on responses of pelvic nerve afferent fibers innervating the colon were also tested. In the behavioral study, systemic injections of morphine and both kappa-ORAs dose-dependently inhibited the visceromotor response to colorectal distension in rats with uninflamed or inflamed colons. The inhibitory effects of kappa-ORAs, but not morphine, were significantly greater in rats with colons inflamed 4 days previously by TNBS. A mu-receptor-selective dose (30 microg/kg) of naloxone methiodide (NLXM) blocked the inhibitory effect of morphine, but not of EMD 61,753. In the single-fiber study, neither morphine nor the delta-ORA SNC 80 attenuated the responses of pelvic nerve afferent fibers, whereas kappa-ORAs dose-dependently inhibited responses of pelvic nerve afferent fibers with significantly greater potency in the inflamed colon. Pretreatment with a non-opioid receptor-selective dose (2 mg/kg) of NLXM produced a rightward shift in the dose-response function of EMD 61,753. The greater potency of kappa-ORAs in the TNBS-inflamed condition suggests a peripheral upregulation of kappa-opioid receptors in colonic inflammation.

The role of transient receptor potential vanilloid 1 in mechanical and chemical visceral hyperalgesia following experimental colitis.

The transient receptor potential vanilloid 1 receptor (TRPV1) is an important nociceptor involved in neurogenic inflammation. We aimed to examine the role of TRPV1 in experimental colitis and in the development of visceral hypersensitivity to mechanical and chemical stimulation. Male Sprague-Dawley rats received a single dose of trinitrobenzenesulfonic acid (TNBS) in the distal colon. In the preemptive group, rats received the TRPV1 receptor antagonist JYL1421 (10 mumol/kg, i.v.) or vehicle 15 min prior to TNBS followed by daily doses for 7 days. In the post-inflammation group, rats received JYL1421 daily for 7 days starting on day 7 following TNBS. The visceromotor response (VMR) to colorectal distension (CRD), intraluminal capsaicin, capsaicin vehicle (pH 6.7) or acidic saline (pH 5.0) was assessed in all groups and compared with controls and naïve rats. Colon inflammation was evaluated with H&E staining and myeloperoxidase (MPO) activity. TRPV1 immunoreactivity was assessed in the thoraco-lumbar (TL) and lumbo-sacral (LS) dorsal root ganglia (DRG) neurons. In the preemptive vehicle group, TNBS resulted in a significant increase in the VMR to CRD, intraluminal capsaicin and acidic saline compared the JYL1421-treated group (P<0.05). Absence of microscopic colitis and significantly reduced MPO activity was also evident compared with vehicle-treated rats (P<0.05). TRPV1 immunoreactivity in the TL (69.1+/-4.6%) and LS (66.4+/-4.2%) DRG in vehicle-treated rats was increased following TNBS but significantly lower in the preemptive JYL1421-treated group (28.6+/-3.9 and 32.3+/-2.3 respectively, P<0.05). JYL1421 in the post-inflammation group improved microscopic colitis and significantly decreased the VMR to CRD compared with vehicle (P<0.05, >/=30 mm Hg) but had no effect on the VMR to chemical stimulation. TRPV1 immunoreactivity in the TL and LS DRG was no different from vehicle or naïve controls. These results suggest an important role for TRPV1 channel in the development of inflammation and subsequent mechanical and chemical visceral hyperalgesia.

Kappa, but not mu or delta, opioids attenuate responses to distention of afferent fibers innervating the rat colon

BACKGROUND & AIMS Discomfort and pain are the principal conscious sensations that arise from the viscera, and both are increased in frequency and intensity in patients with a functional bowel disorder. Visceral receptors, perhaps sensitized, may contribute to these altered sensations. The aim of this study was to evaluate the effects of opioid receptor-selective agonists on afferent fibers innervating the colon. METHODS Mechanosensitive pelvic nerve afferent fibers were recorded from the decentralized S1 dorsal root in anesthetized rats. The effects of opioid agonists, given intra-arterially, were studied based on the fibers responses to noxious colorectal distention (CRD) (80 mm Hg, 30 seconds). RESULTS A total of 115 distention-sensitive fibers innervating the colon were studied, including 32 that were studied after colonic inflammation with 2.5% acetic acid. Neither mu-(morphine and fentanyl) nor delta- ([D-Pen2, D-Pen5]enkephalin- and SNC-80) opioid receptor agonists affected responses to CRD. In contrast, kappa- (U-50,488 and fedotozine) opioid receptor agonists dose-dependently attenuated responses to CRD. Acetic acid sensitized about half of the fibers studied, but neither the potency nor the efficacy of U-50, 488 or FDZ were changed after colonic inflammation. CONCLUSIONS These results suggest a role for peripheral kappa-opioid receptors in the modulation of visceral nociception.

Visceral Pain: The Neurophysiological Mechanism

The mechanism of visceral pain is still less understood compared with that of somatic pain. This is primarily due to the diverse nature of visceral pain compounded by multiple factors such as sexual dimorphism, psychological stress, genetic trait, and the nature of predisposed disease. Due to multiple contributing factors there is an enormous challenge to develop animal models that ideally mimic the exact disease condition. In spite of that, it is well recognized that visceral hypersensitivity can occur due to (1) sensitization of primary sensory afferents innervating the viscera, (2) hyperexcitability of spinal ascending neurons (central sensitization) receiving synaptic input from the viscera, and (3) dysregulation of descending pathways that modulate spinal nociceptive transmission. Depending on the type of stimulus condition, different neural pathways are involved in chronic pain. In early-life psychological stress such as maternal separation, chronic pain occurs later in life due to dysregulation of the hypothalamic-pituitary-adrenal axis and significant increase in corticotrophin releasing factor (CRF) secretion. In contrast, in early-life inflammatory conditions such as colitis and cystitis, there is dysregulation of the descending opioidergic system that results excessive pain perception (i.e., visceral hyperalgesia). Functional bowel disorders and chronic pelvic pain represent unexplained pain that is not associated with identifiable organic diseases. Often pain overlaps between two organs and approximately 35% of patients with chronic pelvic pain showed significant improvement when treated for functional bowel disorders. Animal studies have documented that two main components such as (1) dichotomy of primary afferent fibers innervating two pelvic organs and (2) common convergence of two afferent fibers onto a spinal dorsal horn are contributing factors for organ-to-organ pain overlap. With reports emerging about the varieties of peptide molecules involved in the pathological conditions of visceral pain, it is expected that better therapy will be achieved relatively soon to manage chronic visceral pain.

Effect of reflux-induced inflammation on transient receptor potential vanilloid one (TRPV1) expression in primary sensory neurons innervating the oesophagus of rats

Abstract  A possible mechanism of oesophageal hypersensitivity is the acid‐induced activation of transient receptor potential vanilloid receptor 1 (TRPV1) in the primary sensory neurons. We investigated TRPV1 expression and its colocalization with substance P (SP) and isolectin B4 (IB4)‐positive cells in the thoracic dorsal root ganglia (DRGs) and nodose ganglia (NGs) of rats with reflux‐induced oesophagitis (RO). RO was developed by fundus ligation and partial obstruction of the pylorus of Sprague‐Dawley rats. Four groups of rats were used; fundus ligated acute (RO 48 h), chronic 7 days (RO 7D), RO 7D + omeprazole (7D + Omz, 40 mg kg−1, i.p.) and sham‐operated controls. Immunohistochemical analysis of TRPV1, SP and IB4 expression were carried out in spinal cord (SC), DRGs and NGs. RO rats exhibited significant inflammation and increase in TRPV1‐ir and SP‐ir expressions in the SC, DRGs and NGs. The maximum colocalization of TRPV1 and SP was observed in RO 7D rats, but Omz prevented inflammation and over expression of TRPV1 and SP. TRPV1‐ir significantly increased in IB4‐positive cells in DRGs and SC, but not in the NGs. Results document that acid‐induced oesophagitis increases TRPV1 expression in both SP‐ and IB4‐positive sensory neurons. The over expression of TRPV1 may contribute to oesophageal hypersensitivity observed in gastro‐oesophageal reflux disease (GORD).

An overview of esophageal sensory receptors

The neurophysiological basis of esophageal pain and discomfort is not well known. Functional disorder, such as noncardiac chest pain, is thought to be associated with hypersensitivity of primary afferents innervating the esophagus and/or sensitization of spinal dorsal horn cells receiving input from the organ. Although we have accumulated a large body of information about the morphologic structure and neuropeptide contents of the extrinsic primary afferents, we lack a full understanding of its integrative function in esophageal pain. The esophagus is innervated dually by vagus and spinal nerves. The majority of sensory afferents in the vagal and spinal pathway are pseudounipolar cells, with their cell bodies (soma) located in the nodose and dorsal root ganglia, respectively. These afferent fibers innervate serosa (adventitia), longitudinal and circular muscles, and mucosa of the esophagus. Afferents innervating the muscle are sensitive to intraluminal distension. In the vagus, these afferents exhibit low threshold for response, whereas the spinal afferents, including the splanchnic nerve afferents, have either low or high thresholds for response. In addition, these afferents are chemosensitive. Both vagal and spinal afferents also innervate the mucosa of the esophagus. These fibers are exquisitely sensitive to light touch of the mucosa and are sensitive to pH and chemicals. The spinal afferents, including splanchnic nerve afferents, project to the spinal cord, spanning from upper cervical (C1) to upper lumbar (L2) segments. A majority of the spinal dorsal horn neurons receiving input from the esophageal spinal afferents also receives somatic converging input. The somatic receptive fields are distributed mainly ipsilaterally over the chest and forearm area. These spinal dorsal horn neurons exhibit either excitatory, inhibitory, or biphasic (i.e., excitation followed by inhibition) responses to esophageal distension.

MicroRNA-mediated GABAAα-1 receptor subunit down-regulation in adult spinal cord following neonatal cystitis-induced chronic visceral pain in rats

Summary In an experimental model of neonatal cystitis, microRNA‐mediated post‐transcriptional deregulation of spinal GABAergic system is involved in long‐lasting visceral hyperalgesia. Abstract The nociceptive transmission under pathological chronic pain conditions involves transcriptional and/or translational alteration in spinal neurotransmitters, receptor expressions, and modification of neuronal functions. Studies indicate the involvement of microRNA (miRNA) – mediated transcriptional deregulation in the pathophysiology of acute and chronic pain. In the present study, we tested the hypothesis that long‐term cross‐organ colonic hypersensitivity in neonatal zymosan‐induced cystitis is due to miRNA‐mediated posttranscriptional suppression of the developing spinal GABAergic system. Cystitis was produced by intravesicular injection of zymosan (1% in saline) into the bladder during postnatal (P) days P14 through P16 and spinal dorsal horns (L6–S1) were collected either on P60 (unchallenged groups) or on P30 after a zymosan re‐challenge on P29 (re‐challenged groups). miRNA arrays and real‐time reverse transcription–polymerase chain reaction (RT‐PCR) revealed significant, but differential, up‐regulation of mature miR‐181a in the L6–S1 spinal dorsal horns from zymosan‐treated rats compared with saline‐treated controls in both the unchallenged and re‐challenged groups. The target gene analysis demonstrated multiple complementary binding sites in miR‐181a for GABAA receptor subunit GABAAα‐1 gene with a miRSVR score of −1.83. An increase in miR‐181a concomitantly resulted in significant down‐regulation of GABAAα‐1 receptor subunit gene and protein expression in adult spinal cords from rats with neonatal cystitis. Intrathecal administration of the GABAA receptor agonist muscimol failed to attenuate the viscero‐motor response (VMR) to colon distension in rats with neonatal cystitis, whereas in adult zymosan‐treated rats the drug produced significant decrease in VMR. These results support an integral role for miRNA‐mediated transcriptional deregulation of the GABAergic system in neonatal cystitis‐induced chronic pelvic pain.

Acute nociceptive somatic stimulus sensitizes neurones in the spinal cord to colonic distension in the rat

The common co‐existence of fibromyalgia and chronic abdominal pain could be due to sensitization of spinal neurones (SNs), as a result of viscero‐somatic convergence. The objective of this study is to explore the influence of acute nociceptive somatic stimulation in the form of acid injections, into the ipsilateral somatic receptive field of neurones responsive to colorectal distension (CRD), and the potential role of ionotropic glutamate receptors on sensitization. Action potentials of CRD‐sensitive SNs were recorded extracellularly from the lumbar (L2–L5) spinal cord. Stimulus–response functions (SRFs) to graded CRD (10–80 mmHg, 30 s) were constructed before and 30 min after ipsilateral injection of low pH (4.0, 100 μl) saline into the somatic receptive fields. In some experiments, cervical (C1–C2) spinalization was performed to eliminate supraspinal influence. The selective NMDA receptor antagonist CGS 19755 and AMPA receptor antagonist NBQX were injected (25 μmol kg−1, i.v.) to examine their influence on sensitization. Three types of neurones were characterized as short‐latency abrupt (SLA, n= 24), short latency sustained (SLS, n= 12), and long‐latency (LL, n= 6) to CRD. Ipsilateral injection of low pH (4.0) in the somatic receptive field, but not the contralateral gastrocnemius (GN) or front leg muscles, sensitized responses of these neurones to CRD. Spinalization had no influence on the development of low pH‐induced sensitization. Both CGS 19755 and NBQX significantly attenuated the sensitized response to CRD in intact and spinalized animals. Acute nociceptive somatic stimulus sensitizes CRD‐sensitive SNs receiving viscero‐somatic convergence. The sensitization occurs at the spinal level and is independent of supraspinal influence. Ionotropic glutamate receptors in the spinal cord are involved in sensitization.

Antinociceptive effects of melatonin in a rat model of post-inflammatory visceral hyperalgesia: a centrally mediated process.

&NA; Previous reports suggest that melatonin may play an important role in visceral nociception and neurogenic inflammation. We aimed to examine the role of melatonin on visceral hypersensitivity and to explore the site of action using a rat model of post‐inflammatory visceral hyperalgesia. In all rats, a baseline viscero‐motor response (VMR) to graded colorectal distension (CRD; 10–60 mmHg) was recorded prior and 1 week following tri‐nitrobenzenesulfonic acid (TNBS) induced colonic inflammation. Melatonin (30, 45 or 60 mg/kg, ip) was given 20 min before testing the VMR in naïve and TNBS‐treated rats. Extracellular single‐unit recordings were made from CRD‐sensitive pelvic nerve afferent (PNA) fibers and lumbosacral (LS) spinal neurons in TNBS‐treated animals. The effect of melatonin (60 mg/kg) was examined on responses of PNAs and spinal neurons to graded CRD. In separate experiments, luzindole (non‐specific MT1/MT2 receptor antagonist) or naltrexone (non‐specific opiod receptor antagonist) was injected prior to melatonin. Following TNBS, there was a significant increase in the VMR to CRD compared to baseline. This increase was attenuated by melatonin (60 mg/kg) at pressures >20 mmHg. The same dose of melatonin had no effect on the VMR in naïve animals. In TNBS‐treated rats, melatonin significantly attenuated the responses of CRD‐sensitive spinal neurons to CRD, but had no effect in spinal transected rats or PNA fibers. Both luzindole and naltrexone blocked melatonins effect on the VMR and LS spinal neurons. Results indicate melatonins antinociceptive effects are not via a peripheral site of action but rather a supra‐spinal process linked to the central opioidergic system.