Pradeep Kannampalli

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.

Probiotic Lactobacillus rhamnosus GG (LGG) and prebiotic prevent neonatal inflammation‐induced visceral hypersensitivity in adult rats

Increasing evidence indicates a positive effect of probiotics on the nervous system. The objective of this study was to determine if probiotic Lactobacillus rhamnosus GG (LGG) and/or prebiotics polydextrose/galactooligosaccharide (PDX/GOS) can alter the colonic sensitivity in a neonatal rat model of chronic visceral hyperalgesia and to determine whether altered sensitivity is associated with changes in neurotransmitter levels in the brain.

Analgesic effect of Minocycline in rat model of inflammation-induced visceral pain

The present study investigates the analgesic effect of minocycline, a semi-synthetic tetracycline antibiotic, in a rat model of inflammation-induced visceral pain. Inflammation was induced in male rats by intracolonic administration of tri-nitrobenzenesulphonic acid (TNBS). Visceral hyperalgesia was assessed by comparing the viscero-motor response (VMR) to graded colorectal distension (CRD) prior and post 7 days after TNBS treatment. Electrophysiology recordings from CRD-sensitive pelvic nerve afferents (PNA) and lumbo-sacral (LS) spinal neurons were performed in naïve and inflamed rats. Colonic inflammation produced visceral hyperalgesia characterized by increase in the VMRs to CRD accompanied with simultaneous activation of microglia in the spinal cord and satellite glial cells (SGCs) in the dorsal root ganglions (DRGs). Selectively inhibiting the glial activation following inflammation by araC (Arabinofuranosyl Cytidine) prevented the development of visceral hyperalgesia. Intrathecal minocycline significantly attenuated the VMR to CRD in inflamed rats, whereas systemic minocycline produced a delayed effect. In electrophysiology experiments, minocycline significantly attenuated the mechanotransduction of CRD-sensitive PNAs and the responses of CRD-sensitive LS spinal neurons in TNBS-treated rats. While the spinal effect of minocycline was observed within 5min of administration, systemic injection of the drug produced a delayed effect (60min) in inflamed rats. Interestingly, minocycline did not exhibit analgesic effect in naïve, non-inflamed rats. The results demonstrate that intrathecal injection of minocycline can effectively attenuate inflammation-induced visceral hyperalgesia. Minocycline might as well act on neuronal targets in the spinal cord of inflamed rats, in addition to the widely reported glial inhibitory action to produce analgesia.

Colonic butyrate- algesic or analgesic?

Irritable bowel syndrome (IBS) is a common health issue that is characterized by abdominal pain, abnormal bowel movements, and altered visceral perception. The complexity and variability in symptoms pose serious challenges in treating IBS. Current therapy for IBS is primarily focused on reducing the abdominal pain, thereby improving the quality of life to a significant extent. Although the use of fiber rich diet is widely recommended in treating IBS, some studies have questioned its use. Intra‐colonic butyrate, a short‐chain fatty acid, is primarily produced by the fermentation of dietary fibers in the colon. In the existing literature there are conflicting reports about the function of butyrate. In rats it is known to induce visceral hypersensitivity without altered pathology, whereas in humans it has been reported to reduce visceral pain. Understanding the molecular mechanisms responsible for this contrasting effect of butyrate is important before recommending fiber rich diet to IBS patients.

Visceral analgesic effect of 5-HT4 receptor agonist in rats involves the rostroventral medulla (RVM)

The 5-HT(4) receptor agonist tegaserod (TEG) has been reported to modulate visceral pain. However, the underlying mechanism remains unknown. The objective of the present study was to examine the analgesic mechanism and site of action of TEG. In male rats, visceral pain was assessed by measuring visceromotor response (VMR) to colorectal distension (CRD). Inflammation was induced by intracolonic injection of tri-nitrobenzene sulfonic acid (TNBS). The effect of TEG on the VMR was tested by injecting intraperitoneal (i.p.), intrathecal (i.t.), intracerebroventricular (i.c.v) or in the rostroventral medulla (RVM). The effect of the drug was also tested on responses of CRD-sensitive pelvic nerve afferents (PNA) and lumbo-sacral (LS) spinal neurons. Systemic injection of TEG attenuated VMR in naive and TNBS-treated rats. Similarly, supraspinal, but not spinal, injection of TEG attenuated the VMR. While GR113808, (selective 5-HT(4) antagonist) blocked the effect, naloxone (NLX) an opioid receptor antagonist reversed the effect of TEG. Although i.t. NLX did not block the inhibitory effect of TEG in VMR study, i.t. injection of α2-adrenergic receptor antagonist yohimbine blocked the effect of TEG when given systemically. While TEG had no effect on the responses of CRD-sensitive PNA, it inhibited the responses of CRD-sensitive LS neurons in spinal intact condition. This inhibition was blocked by GR113808, NLX and β-funaltrexamine (β-FNA) when injected into the RVM. Results indicate that TEG produces analgesia via activation of supraspinal 5-HT(4) receptors which triggers the release of opioids at supraspinal site, which activates descending noradrenergic pathways to the spinal cord to produce analgesia.

MicroRNA–mediated downregulation of potassium-chloride-cotransporter and vesicular γ-aminobutyric acid transporter expression in spinal cord contributes to neonatal cystitis–induced visceral pain in rats

Abstract Loss of GABAergic inhibition in pain pathways has been considered to be a key component in the development of chronic pain. In the present study, we intended to examine whether miR-92b–mediated posttranscriptional dysregulation of spinal potassium chloride cotransporter (KCC2) and vesicular &ggr;-aminobutyric acid transporter (VGAT) plays a major role in the development and maintenance of long-term visceral hyperalgesia in neonatal zymosan–treated rats. Neonatal cystitis was induced by transurethral zymosan administration from postnatal (P) days 14 to 16 (protocol 1). Two other zymosan protocols were also used: adult rechallenge on P57 to 59 following neonatal P14 to 16 exposures (protocol 2), and adult zymosan exposures on P57 to 59 (protocol 3). Both neonatal and adult bladder inflammation protocols demonstrated an increase in spinal miR-92b-3p expression and subsequent decrease in KCC2 and VGAT expression in spinal dorsal horn neurons. In situ hybridization demonstrated a significant upregulation of miR-92b-3p in the spinal dorsal horn neurons of neonatal cystitis rats compared with saline-treated controls. In dual in situ hybridization and immunohistochemistry studies, we further demonstrated coexpression of miR-92b-3p with targets KCC2 and VGAT in spinal dorsal horn neurons, emphasizing a possible regulatory role both at pre- and post-synaptic levels. Intrathecal administration of lentiviral pLSyn-miR-92b-3p sponge (miR-92b-3p inhibitor) upregulated KCC2 and VGAT expression in spinal dorsal horn neurons. In behavioral studies, intrathecal administration of lentiviral miR-92b-3p sponge attenuated an increase in visceromotor responses and referred viscerosomatic hypersensitivity following the induction of cystitis. These findings indicate that miR-92b-3p–mediated posttranscriptional regulation of spinal GABAergic system plays an important role in sensory pathophysiology of zymosan-induced cystitis.Loss of GABAergic inhibition in pain pathways has been considered to be a key component in the development of chronic pain. In the present study, we intended to examine if miR-92b-mediated post-transcriptional dysregulation of spinal potassium chloride cotransporter (KCC2) and vesicular GABA transporter (VGAT) plays a major role in the development and maintenance of long-term visceral hyperalgesia in neonatal zymosan-treated rats. Neonatal cystitis was induced by transurethral zymosan administration from postnatal (P) days 14-16 (protocol 1). Two other zymosan protocols were also used: adult re-challenge on P57-59 following neonatal P14-16 exposures (protocol 2), and adult zymosan exposures on P57-59 (protocol 3). Both neonatal and AC CE PT ED Copyright 2017 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited. 2 adult bladder inflammation protocols demonstrated an increase in spinal miR-92b-3p expression and subsequent decrease in KCC2 and VGAT expression in spinal dorsal horn neurons. In situ hybridization demonstrated a significant upregulation of miR-92b3p in the spinal dorsal horn neurons of neonatal cystitis rats compared with salinetreated controls. In dual in situ hybridization and immunohistochemistry studies, we further demonstrated co-expression of miR-92b-3p with targets KCC2 and VGAT in spinal dorsal horn neurons, emphasizing a possible regulatory role both at preand post-synaptic levels. Intrathecal administration of lentiviral pLSyn-miR-92b-3p sponge (miR-92b-3p inhibitor) upregulated KCC2 and VGAT expression in spinal dorsal horn neurons. In behavioral studies, intrathecal administration of lentiviral miR-92b-3p sponge attenuated an increase in visceromotor responses (VMRs) and referred viscerosomatic hypersensitivity following the induction of cystitis. These findings indicate that miR-92b-3p-mediated post-transcriptional regulation of spinal GABAergic system plays an important role in sensory pathophysiology of zymosan-induced cystitis.

Role of principal ionotropic and metabotropic receptors in visceral pain.

Visceral pain is the most common form of pain caused by varied diseases and a major reason for patients to seek medical consultation. It also leads to a significant economic burden due to workdays lost and reduced productivity. Further, long-term use of non-specific medications is also associated with side effects affecting the quality of life. Despite years of extensive research and the availability of several therapeutic options, management of patients with chronic visceral pain is often inadequate, resulting in frustration for both patients and physicians. This is, most likely, because the mechanisms associated with chronic visceral pain are different from those of acute pain. Accumulating evidence from years of research implicates several receptors and ion channels in the induction and maintenance of central and peripheral sensitization during chronic pain states. Understanding the specific role of these receptors will facilitate to capitalize on their unique properties to augment the therapeutic efficacy while at the same time minimizing unwanted side effects. The aim of this review is to provide a concise review of the recent literature that reports on the role of principal ionotropic receptors and metabotropic receptors in the modulation visceral pain. We also include an overview of the possibility of these receptors as potential new targets for the treatment of chronic visceral pain conditions.

NMDA receptor mediates chronic visceral pain induced by neonatal noxious somatic stimulation.

NMDA receptors (NMDAR) are important in the development and maintenance of central sensitization. Our objective was to investigate the role of spinal neurons and NMDAR in the maintenance of chronic visceral pain. Neonatal rats were injected with acidic saline adjusted to pH 4.0 in the gastrocnemius muscle every other day for 12 days. In adult rats, NR1 and NR2B subunits were examined in the lumbo-sacral (LS) spinal cord. A baseline, visceromotor response (VMR) to graded colorectal distension (CRD) was recorded before and after administration of the NMDA antagonist, CGS-19755. Extracellular recordings were performed from CRD-sensitive LS spinal neurons and pelvic nerve afferents (PNA) before and after CGS-19755. Rats that received pH 4.0 saline injections demonstrated a significant increase in the expression NR2B subunits and VMR response to CRD>20 mmHg. CGS-19755 (i.v. or i.t.) had no effect in naïve rats, but significantly decreased the response to CRD in pH 4.0 saline injected rats. CGS-19755 had no effect on the spontaneous firing of SL-A, but decreased that of SL-S. Similarly, CGS-19755 attenuates the responses of SL-S neurons to CRD, but had no effect on SL-A neurons or on the response characteristics of PNA fibers. Neonatal noxious somatic stimulation results in chronic visceral hyperalgesia and sensitizes a specific subpopulation of CRD-sensitive spinal neurons. The sensitization of these SL-S spinal neurons is attenuated by the NMDAR antagonist. The results of this study suggest that spinal NMDARs play an important role in the development of hyperalgesia early in life.

Pronociceptive effect of 5-HT1A receptor agonist on visceral pain involves spinal N-methyl-d-aspartate (NMDA) receptor

The functional role of serotonergic 5-HT(1A) receptors in the modulation of visceral pain is controversial. The objective of this study was to systematically examine the mechanism and site of action of a selective 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (DPAT) on visceral pain. In the behavioral model of visceral pain, systemic injection (5-250 μg/kg) of DPAT produced a significant increase in the viscero-motor response (VMR) to colorectal distension (CRD) and this effect was blocked by the selective 5-HT(1A) receptor antagonist WAY-100135 (5 mg/kg, s.c.). Similarly, intrathecal (i.t.) injection (5 μmol) of DPAT into the lumbo-sacral (L6-S1) spinal cord produced a significant increase in VMR. The administration of N-methyl D-aspartate (NMDA) receptor antagonist AP5 (50 μg/kg) prior to DPAT injection completely blocked the pronociceptive effect of DPAT. Similarly, DPAT failed to increase VMR in rats chronically treated with NR1 subunit-targeted antisense oligonucleotide (ON), whereas the drug increased VMR in rats treated with mismatched-ON. Chronic i.t. injection of allylglycine (AG), a γ-amino decarboxylase (GAD) enzyme inhibitor, produced significant increase in VMRs, suggesting that the inhibition of GABA synthesis produces pronociception. In AG-treated rats, i.t. injection of DPAT failed to further increase in VMR, suggesting that the DPAT action is linked to GABA release. Similarly, WAY-100135 failed to attenuate VMR in AG-treated rats, suggesting that unlike DPAT, AG action is not via the activation of 5-HT(1A) receptors. In electrophysiology experiments, DPAT (50 μg/kg) significantly increased the responses of spinal neurons to CRD, but did not influence the mechanotransduction property of CRD-sensitive pelvic nerve afferent fibers. The effect of DPAT on spinal neurons remained unaffected when tested in spinal-transected (C1-C2) rats. These results indicate that the 5-HT(1A) receptor agonist DPAT produces pronociceptive effects, primarily via the activation of presynaptic 5-HT(1A) receptors in GABAergic neuron to restrict GABA release and thereby disinhibits the excitatory glutamatergic neurons in the spinal cord.

Neonatal bladder inflammation induces long-term visceral pain and altered responses of spinal neurons in adult rats

Painful events early in life have been shown to increase the incidence of interstitial cystitis/painful bladder syndrome in adulthood. However, the intrinsic mechanism is not well studied. We previously reported that neonatal bladder inflammation causes chronic visceral hypersensitivity along with molecular disruption of spinal GABAergic system in rats. The present study investigates whether these molecular changes affect the integrative function and responses of bladder-sensitive primary afferent and spinal neurons. Neonatal bladder inflammation was induced by intravesicular injection of zymosan during postnatal (P) days 14-16. In adulthood (P60), the viscero-motor response (VMR) to visceral stimuli was significantly inhibited by intrathecal (i.t) HZ166 (GABAAα-2 agonist) only in neonatally saline-treated, but not in neonatally zymosan-treated rats. HZ166 significantly inhibited the responses of bladder-responsive lumbosacral (LS) spinal neurons to urinary bladder distension (UBD) and slow infusion (SI) in neonatally saline-treated rats. Similar results were also observed in naïve adult rats where HZ166 produced significant inhibition of bladder-responsive spinal neurons. However, HZ166 did not inhibit responses of UBD-responsive spinal neurons from neonatally zymosan-treated rats. The drug did not attenuate the responses of UBD-sensitive pelvic nerve afferent (PNA) fibers to UBD and SI in either group of rats tested. Immunohistochemical studies showed a significantly lower level of GABAAα-2 receptor expression in the LS spinal cord of neonatally zymosan-treated rats compared to saline-treated rats. These findings indicate that neonatal bladder inflammation leads to functional and molecular alteration of spinal GABAAα-2 receptor subtypes, which may result in chronic visceral hyperalgesia in adulthood.