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.

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 γ-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.

Analgesic effect of ADX71441, a positive allosteric modulator (PAM) of GABAB receptor in a rat model of bladder pain.

Therapeutic use of GABAB receptor agonists for conditions like chronic abdominal pain, overactive bladder (OAB) and gastroesophageal reflux disease (GERD) is severely affected by poor blood-brain barrier permeability and potential side effects. ADX71441 is a novel positive allosteric modulator (PAM) of the GABAB receptor that has shown encouraging results in pre-clinical models of anxiety, pain, OAB and alcohol addiction. The present study investigates the analgesic effect of ADX71441 to noxious stimulation of the urinary bladder and colon in rats. In female Sprague-Dawley rats, systemic (i.p), but not intrathecal (i.t), administration of ADX71441 produced a dose-dependent decrease in viscero-motor response (VMR) to graded urinary bladder distension (UBD) and colorectal distension (CRD). Additionally, intra-cerebroventricular (i.c.v.) administration of ADX71441 significantly decreased the VMRs to noxious UBD. In electrophysiology experiments, the drug did not attenuate the responses of UBD-sensitive pelvic nerve afferent (PNA) fibers to UBD. In contrast, ADX71441 significantly decreased the responses of UBD-responsive lumbosacral (LS) spinal neurons in spinal intact rats. However, ADX71441 did not attenuate these LS neurons in cervical (C1-C2) spinal transected rats. During cystometrogram (CMG) recordings, ADX71441 (i.p.) significantly decreased the VMR to slow infusion without affecting the number of voiding contraction. These results indicate that ADX71441 modulate bladder nociception via its effect at the supra-spinal sites without affecting the normal bladder motility and micturition reflex in naïve adult rats.

Percutaneous electrical nerve field stimulation modulates central pain pathways and attenuates post-inflammatory visceral and somatic hyperalgesia in rats.

A non-invasive, auricular percutaneous electrical nerve field stimulation (PENFS) has been suggested to modulate central pain pathways. We investigated the effects of BRIDGE® device on the responses of amygdala and lumbar spinal neurons and the development of post-colitis hyperalgesia. Male Sprague-Dawley rats received intracolonic trinitrobenzene sulfonic acid (TNBS) and PENFS on the same day. Control rats had sham devices. The visceromotor response (VMR) to colon distension and paw withdrawal threshold (PWT) was recorded after 7days. A different group of rats had VMR and PWT at baseline, after TNBS and following PENFS. Extracellular recordings were made from neurons in central nucleus of the amygdala (CeA) or lumbar spinal cord. Baseline firing and responses to compression of the paw were recorded before and after PENFS. Sham-treated rats exhibited a much higher VMR (>30mmHg) and lower PWT compared to PENFS-treated rats (p<0.05). PENFS decreased the VMR to colon distension and increased the PWT compared to pre-stimulation (p<0.05). PENFS resulted in a 57% decrease in spontaneous firing of the CeA neurons (0.59±0.16 vs control: 1.71±0.32imp/s). Similarly, the response to somatic stimulation was decreased by 56% (3.6±0.52 vs control: 1.71±0.32 imps/s, p<0.05). Spinal neurons showed a 47% decrease in mean spontaneous firing (4.05±0.65 vs control: 7.7±0.87imp/s) and response to somatic stimulation (7.62±1.7 vs control: 14.8±2.28imp/s, p<0.05). PENFS attenuated baseline firing of CeA and spinal neurons which may account for the modulation of pain responses in this model of post-inflammatory visceral and somatic hyperalgesia.

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.

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 re-search and the availability of several therapeutic options, management of patients with chronic visceral pain is often in-adequate, 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 ther-apeutic 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.

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.

Visceral analgesic effect of 5-HT(4) 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 ß-funaltrexamine (ß-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 GABA Aα-1 receptor subunit down-regulation in adult spinal cord following neonatal cystitis-induced chronic visceral pain in rats.

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 GABA(A) receptor subunit GABA(Aα-1) gene with a miRSVR score of -1.83. An increase in miR-181a concomitantly resulted in significant down-regulation of GABA(Aα-1) receptor subunit gene and protein expression in adult spinal cords from rats with neonatal cystitis. Intrathecal administration of the GABA(A) 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.