Mechanisms and Management of Chronic Pain.

Chronic pain symptoms account up to half of all health care visits, afflicts >10% of US adults, at a higher prevalence in women with current analgesic drugs rarely provide enough efficacy in the absence of serious side effects. Chronic pain is also the root cause of the national opioid health crisis, which adds to health care costs and deaths. Thus, new pain therapies based on detailed understanding of nociceptive mechanisms are needed as alternatives to opioid analgesics and are of great societal importance. Chronic pain is one of the most prevalent human health problems that often is associated by the concomitant decline in cognitive and motor functions. Pain is strongly associated with other diseases that can lack of awareness to its pathology. Despite a successful reduction of pain with available medications, majority of treated patients were seeking professional help again. The average time duration between the onset of pain symptoms and the diagnosis is couple of years despite the fact that majority of patients with chronic pain suffer every day. Efficacious and reliable therapeutic intervention is still unavailable despite the tremendous economic burden imposed on healthcare to treat many diseases associated with chronic pain.

Peripheral modulation of chronic visceral pain.

Chronic visceral pain is a complex and often a serious burden on patients' life. It is strongly implicated in the etiology of many diseases, which often are complicated by co-morbid depression and other psychiatric disorders, all of which pose significant health risks. Understanding the mechanisms of nociception is an important step in treating pain-associated chronic diseases. The inflammatory process that is often associated with nociception produces a number of mediators, which activate nociceptors by interacting with ligand-gated ion channels, activation of different signal transduction pathways or by sensitizing primary afferent neurons located within the dorsal root ganglia (DRG). Primary afferents studied in vitro or in vivo are well-accepted models to examine various nociceptive and anti-nociceptive signals in peripheral nervous system. This review focuses on the recent work in the area of peripheral modulation of chronic pain at the level of visceral primary afferent neurons. Many studies intended to develop a coherent framework for a better understanding of heterogeneity of nociceptive neurons functioning as a gate for pain transmission and novel therapeutic tool for pain relief. Specifically, recent studies from the author's research group helped to define the role of ATP-sensitive purinergic and vanilloid-sensitive TRPV1 receptors in DRG-mediated nociceptive pathways. Tropic and physiological changes associated with chronic visceral pain indeed are mediated through different pathways; therefore, designing new and specific anti-nociceptive therapies will have a major impact on quality of life in patients by significantly reducing pharmacological and therapeutic interventions.

Physically disconnected non-diffusible cell-to-cell communication between neuroblastoma SH-SY5Y and DRG primary sensory neurons.

BACKGROUND: Cell-cell communication occurs via a variety of mechanisms, including long distances (hormonal), short distances (paracrine and synaptic) or direct coupling via gap junctions, antigen presentation, or ligand-receptor interactions. We evaluated the possibility of neuro-hormonal independent, non-diffusible, physically disconnected pathways for cell-cell communication using dorsal root ganglion (DRG) neurons. METHODS: We assessed intracellular calcium ([Ca(2+)]) in primary culture DRG neurons that express ATP-sensitive P2X3, capsaicinsensitive TRPV1 receptors modulated by estradiol. Physically disconnected (dish-in-dish system; inner chamber enclosed) mouse DRG were cultured for 12 hours near: a) media alone (control 1), b) mouse DRG (control 2), c) human neuroblastoma SHSY-5Y cells (cancer intervention), or d) mouse DRG treated with KCl (apoptosis intervention). RESULTS: Chemosensitive receptors [Ca(2+)](i) signaling did not differ between control 1 and 2. ATP (10 µM) and capsaicin (100nM) increased [Ca(2+)](i) transients to 425.86 + 49.5 nM, and 399.21 ± 44.5 nM, respectively. 17ß-estradiol (100 nM) exposure reduced ATP (171.17 ± 48.9 nM) and capsaicin (175.01±34.8 nM) [Ca(2+)](i) transients. The presence of cancer cells reduced ATP- and capsaicin-induced [Ca(2+)](i) by >50% (p<0.05) and abolished the 17ß-estradiol effect. By contrast, apoptotic DRG cells increased initial ATP-induced [Ca(2+)](i), flux four fold and abolished subsequent [Ca(2+)](i), responses to ATP stimulation (p<0.001). Capsaicin (100nM) induced [Ca(2+)](i) responses were totally abolished. CONCLUSION: The local presence of apoptotic DRG or human neuroblastoma cells induced differing abnormal ATP and capsaicin-mediated [Ca(2+)](i) fluxes in normal DRG. These findings support physically disconnected, non-diffusible cell-to-cell signaling. Further studies are needed to delineate the mechanism(s) of and model(s) of communication.

Expression of P2X3 and TRPV1 receptors in primary sensory neurons from estrogen receptors-α and estrogen receptor-ß knockout mice.

In women, pain symptoms and nociceptive thresholds vary with the reproductive cycle, suggesting the role of estrogen receptors (ERs) in modulating nociception. Our previous data strongly suggest an interaction between ERs and ATP-induced purinergic (P2X3) as well as ERs and capsaicin-induced vanilloid (TRPV1) receptors at the level of dorsal root ganglion (DRG) neurons. In this study, we investigated the expression of P2X3 and TRPV1 receptors by western blotting and immunohistochemistry in lumbosacral DRGs from wild type, ERα, and ERß knockout mice. We found a significant decrease for both P2X3 and TRPV1 in ERαKO and ERßKO. This phenomenon was visualized in L1, L2, L4, and L6 levels for P2X3 receptors and in L1, L2, and S2 levels for TRPV1 receptors. This tan interaction between P2X3/TRPV1 and ERs expression in sensory neurons may represent a novel mechanism that can explain the sex differences in nociception observed in clinical practice. The DRG is an important site of visceral afferent convergence and cross-sensitization and a potential target for designing new anti-nociceptive therapies.

Peripheral sensitization of sensory neurons.

INTRODUCTION: Defining the sites and mechanisms of nociception is an important step in understanding and treating pain. During inflammation, increased nociceptive input from an inflamed organ can sensitize neurons that receive convergent input from an unaffected organ, but the site of visceral cross-sensitivity is unknown. This study examined the cellular responses to ATP and substance P stimulation in sensory neurons innervating visceral organs. METHODS: Lumbosacral dorsal root ganglia (L6-S1) were cut into slices and processed for substance P receptor expression using immunocytochemistry. Primary culture of dorsal root ganglion (DRG) neurons was used for [Ca2+]i measurement by videomicroscopy. RESULTS: DRG neurons express substance P receptors. Both brief addition of low dose adenosine triphosphate (ATP, 5 microM) and substance P (10 microM) significantly increased subsequent ATP stimulation at the same neuron. DISCUSSION: Sensitization of the DRG neurons innervating the different organs may be through the release of nociceptive transmitters such as ATP and/or substance P within the ganglion. Together, these experiments will increase our understanding of the important modulatory role of peripheral sensitization in nociceptive transmission and suggest potential periphepheral sites for therapeutic intervention.

Visceral sensory neurons that innervate both uterus and colon express nociceptive TRPv1 and P2X3 receptors in rats.

In women, clinical studies suggest that functional pain syndromes such as irritable bowel syndrome, interstitial cystitis, and fibromyalgia, are co-morbid with endometriosis, chronic pelvic pain, and others diseases. One of the possible explanations for this phenomenon is visceral cross-sensitization in which increased nociceptive input from inflamed reproductive system organs sensitize neurons that receive convergent input from an unaffected visceral organ to the same dorsal root ganglion (DRG). The purpose of this study was to determine whether primary sensory neurons that innervate both visceral organs--the uterus and the colon--express nociceptive ATP-sensitive purinergic (P2X3) and capsaicin-sensitive vanilloid (TRPV1) receptors. To test this hypothesis, cell bodies of colonic and uterine DRG were retrogradely labeled with fluorescent tracer dyes micro-injected into the colon/rectum and uterus of rats. Ganglia were harvested, cryo-protected, and cut in 20-microm slices for fluorescent microscopy to identify positively stained cells. Up to 5% neurons were colon-specific or uterus-specific, and 10%-15% of labeled DRG neurons innervate both viscera in the lumbosacral neurons (L1-S3 levels). We found that viscerally labeled DRGs express nociceptive P2X3 and TRPV1 receptors. Our results suggest a novel form of visceral sensory integration in the DRG that may underlie co-morbidity of many functional pain syndromes.

Estrogen receptor-alpha mediates estradiol attenuation of ATP-induced Ca2+ signaling in mouse dorsal root ganglion neurons.

A mechanism underlying gender-related differences in pain perception may be estrogen modulation of nociceptive signaling in the peripheral nervous system. In rat, dorsal root ganglion (DRG) neurons express estrogen receptors (ERs) and estrogen rapidly attenuates ATP-induced Ca2+ signaling. To determine which estrogen receptor mediates rapid actions of estrogen, we showed ERalpha and ERbeta expression in DRG neurons from wild-type (WT) female mice by RT-PCR. To study whether ERalpha or ERbeta mediates this response, we compared estradiol action mediating Ca2+ signaling in DRG neurons from WT, ERalpha knockout (ERalphaKO), and ERbetaKO mice in vitro. ATP, an algesic agent, induced [Ca2+]i transients in 48% of small DRG neurons from WT mice. 17beta-Estradiol (E2) inhibited ATP-induced intracellular Ca2+ concentration ([Ca2+]i) with an IC50 of 27 nM. The effect of E2 was rapid (5-min exposure) and stereo specific; 17alpha-estradiol had no effect. E2 action was blocked by the ER antagonist ICI 182,780 (1 microM) in WT mouse. Estradiol coupled to bovine serum albumin (E-6-BSA), which does not penetrate the plasma membrane, had the same effect as E2 did, suggesting that a membrane-associated ER mediated the response. In DRG neurons from ERbetaKO mice, E2 attenuated the ATP-induced [Ca2+]i flux as it did in WT mice, but in DRG neurons from ERalphaKO mice, E2 failed to inhibit the ATP-induced [Ca2+]i increase. These results show that mouse DRG neurons express ERs and the rapid attenuation of ATP-induced [Ca2+]i signaling is mediated by membrane-associated ERalpha.

A membrane estrogen receptor mediates intracellular calcium release in astrocytes.

Appreciating the physiology of astrocytes and their role in brain functions requires an understanding of molecules that activate these cells. Estradiol may influence astrocyte functions. We now report that estrogen altered intracellular calcium concentration ([Ca(2+)](i)) in neonatal astrocytes that expressed estrogen receptor (ER) mRNA in vitro. Western blotting revealed both ERalpha and ERbeta proteins in both the nuclear fractions and plasma-membrane fractions. Application of 17beta-estradiol (20 nm) to fura 2-loaded astrocytes in vitro stimulated [Ca(2+)](i) in 75% of astrocytes with an EC(50) of 12.7 +/- 3.1 nm. This rapid action of estradiol was blocked by the ER antagonist, ICI 182,780. The membrane-impermeable estradiol-BSA induced a [Ca(2+)](i) flux that was statistically similar to estradiol. Removal of extracellular Ca(2+) did not alter the effect of estradiol, but phospholipase C inhibitor U73122 (10 microm) and 2-aminoethoxydiphenyl borate (5 microm), an inhibitor of the inositol-1,4,5,-trisphosphate-gated intracellular Ca(2+) channel, significantly decreased the estradiol-induced [Ca(2+)](i) flux. Estradiol was unable to induce [Ca(2+)](i) flux in thapsigargin-depleted cells. These results indicate that estradiol mediates [Ca(2+)](i) flux in astrocytes through a membrane-associated ER that activates the phospholipase C pathway.