Donald A. Simone - US grants

dorsal root; stimulus /response

? DESCRIPTION (provided by applicant): Sickle cell disease (SCD) SCD is an autosomal recessive disorder characterized by hemolytic anemia and systemic inflammation that afflicts millions of people worldwide. Chronic pain is a hallmark of SCD. Intense ongoing pain and episodic pain starts early in life and increase in severity with age. The primary treatment for pai is opioids which have a variety of undesirable side effects such as respiratory depression and tolerance, and may contribute to organ damage. Although the pathophysiology of SCD is well understood, little is known about the mechanisms that mediate the pain in SCD. We will use an established mouse model of SCD, Berkley (BERK) mice, to study peripheral mechanisms of pain in SCD. These mice offer a unique advantage because of their similarity to human genetic, hematologic and pathological disease, including ongoing pain and hyperalgesia. Earlier studies and preliminary data show that these mice exhibit increased levels of cyclooxygenase (COX)-2 in the spinal cord and dorsal root ganglia (DRG) and a decrease in tissue content of 2-arachidonoyl-sn-glycerol (2-AG). COX-2 may contribute to pain through the formation of prostaglandins as well as prostaglandin-glycerol esters (PG-Gs) generated from the oxidation of 2-AG by COX-2 (e.g., PGE2-G) that sensitize nociceptors. Our general hypothesis is that PGE2-G contributes to pain in SCD. A multidisciplinary approach incorporating parallel biochemical, behavioral, cellular and electrophysiological studies in mice with SCD will test this hypothesis. We will determine whether PGE2-G is elevated in DRG and peripheral tissue in sickle mice (Aim 1), whether deceased production of PGE2-G decreases hyperalgesia (Aim 2), and whether PGE2-G sensitizes nociceptors in control mice and contributes to nociceptor sensitization in sickle mice (Aim 3). These studies will provide new insights into the peripheral mechanisms underlying pain in SCD, as well as new information on the role of PGE2-G in nociceptor sensitization, and may help identify new approaches for treating the chronic, debilitating pain in SCD.

ABSTRACT The management of cancer pain remains a major challenge. Cancer pain is a complex pain state that includes inflammatory, neuropathic components and a unique set of cancer-specific components. Exosomes secreted by cancer cells is one of those cancer-specific factors. In preliminary studies, exosomes confirmed by size and expression of protein markers, induced acute mechanical and heat hyperalgesia following injection into the hind paw of both sexes of naïve C3H/HeN mice. This project will test the hypothesis that exosomes secreted by fibrosarcoma cells produce pain by sensitizing nociceptive primary afferent neurons via the autotaxin (ATX)-lysophosphatidic acid (LPA)-LPA1 receptor (R) pathway. Exosomes isolated from fibrosarcoma cell-conditioned media will be verified by size (Nanoparticle tracking analysis), expression of the exosome-specific markers, and activity of ATX. Electrophysiological studies in vivo will determine the contribution of ATX-LPA-LPA1R signaling to exosome-specific sensitization of nociceptors. At the cellular level, acute sensitization of small dorsal root ganglion (DRG) neurons from adult mice will be defined in a bioassay that measures the occurrence of a calcium transient in response to depolarization with 25 mM KCl in vitro with Indo-1. Whether the exosome-bound ATX-LPA complex released from fibrosarcoma cells sensitizes DRG neurons through activation of LPA1 receptors will be determined using pharmacological and molecular (siRNA) approaches. Resolvin D1 is proposed to attenuate exosome-evoked sensitization of nociceptors and hyperalgesia by interfering with ATX-LPA- LPA1R signaling. If exosome-mediated ATX-LPA-LPA1R signaling underlies hyperalgesia, it will provide insight into a new strategy for managing bone cancer pain. Future studies will further investigate the molecular, biochemical and electrophysiological mechanisms by which exosomes released from cancer cells contribute to cancer pain.

DESCRIPTION (provided by applicant): Peripheral neuropathy is a disabling complication of many conditions including diabetes, alcohol abuse, HIV infection, carpal tunnel syndrome, and chemotherapy for cancer. It is important to detect neuropathy early in the disease process, when potential for recovery is greatest and to initiate therapy to prevent or slow nerve degeneration. In neuropathy, sensory dysfunction almost always begins in the distal extremities (fingers or toes) where the sensation of touch is diminished or lost. Although quantitative sensory testing methods have been devised to assess cutaneous tactile and temperatures sensations objectively, there is no inexpensive, easy to use, and time efficient test to serve as a biomarker for early-stage peripheral neuropathy. The purpose of this proposal is to explore the utility of a novel approach to assess tactile sensation on the fingers in patients with peripheral neuropathy. We developed a new, objective, and sensitive device to detect impaired touch sensation early in nerve disease. In this test, referred to as the "Bumps" test, the subject searches for small objects (bumps) of varying heights that are randomly placed on a flat surface. This approach will be tested in patients taking chemotherapy for cancer because many patients develop chemotherapy-induced neuropathy and, unlike most neuropathies, baseline measures can be obtained before treatment. In addition, cutaneous innervation will be assessed by skin biopsy and immunostaining of peripheral nerve to determine the relationship between a decrease in tactile sensitivity and innervation by myelinated and unmyelinated nerve fibers in the finger pad. The first specific aim will determine bump detection thresholds (bump height) as a function of sex and age in healthy subjects. We will also determine changes in bump detection in patients undergoing chemotherapy. It is hypothesized that bump detection threshold will increase over time in patients receiving chemotherapy. In the second aim, biopsies will be obtained from the same finger pad used in the psychophysical test in patients at various times during chemotherapy and when bump thresholds increase. It is hypothesized that increased bump detection thresholds will correlate with a decrease in the number of Meissner corpuscles and their myelinated nerve fibers early in disease. Identifying patients with early onset neuropathy may lead to altered dosage or a change of therapeutic agent before the neuropathy becomes painful and intolerable, which often leads to the cessation of therapy. The Bumps test is an inexpensive, portable, and time efficient device for early detection of peripheral neuropathy. In the future, the Bumps test can be used in other disease states. PUBLIC HEALTH RELEVANCE: Peripheral neuropathy is a disabling complication of many conditions including diabetes, alcohol abuse, HIV infection, carpal tunnel syndrome, and chemotherapy for cancer. In neuropathy, sensory dysfunction almost always begins in the distal extremities (fingers or toes) where the sensation of touch is diminished or lost. It is important to detect neuropathy early in the disease process, when potential for recovery is greatest and to initiate therapy to prevent or slow nerve degeneration. We devised a new, objective, and sensitive device to detect impaired touch sensation on the finger pads early in nerve disease. In this test, referred to as the "Bumps" test, the subject searches for small objects (bumps) of varying heights that are randomly placed on a flat surface. This approach will be tested in patients taking chemotherapy for cancer because many patients develop chemotherapy- induced neuropathy and, unlike most neuropathies, baseline measures can be obtained before treatment. A skin biopsy will be obtained so we that can we can compare changes in touch sensitivity to the amount of nerve fibers in the skin. We hypothesize that a decrease in the ability to feel the bumps will correlate with a decrease in innervation of the skin. Identifying patients with early onset neuropathy may lead to altered dosage or a change of therapeutic agent before the neuropathy becomes painful and intolerable, which often leads to the cessation of therapy. If successful, the Bumps test will be an inexpensive, portable, and time efficient device for early detection of peripheral neuropathy. In the future, the Bumps test can be used to diagnose neuropathy in other disease states, such as diabetes, to track disease progression over time, and to assess therapeutic interventions.

DESCRIPTION (provided by applicant): Sickle cell disease (SCD) is accompanied by acute painful episodes (crises) superimposed on chronic pain. Opioids are the only therapy for severe pain, but high doses of opioids are required to treat pain in SCD. Chronic opioid use may lead to secondary adverse effects and opioid tolerance. The mechanisms underlying chronic pain in SCD remain unknown. Characterizing pain in SCD and defining the underlying mechanisms is required to develop novel analgesic therapies. Therefore, the goal of this proposal is to use a murine model of SCD to examine peripheral and spinal mechanisms that contribute to pain in this condition. Established mouse models of SCD offer a unique advantage because of their similarity to human genetic, hematologic and pathological disease. We hypothesize that persistent activation of nociceptors by inflammation, hypoxia- reperfusion injury and crises leads to central sensitization. Our preliminary data indicate that mice with SCD exhibit cutaneous (mechanical, heat and cold) and deep (decreased grip force) hyperalgesia similar to that observed in patients with SCD. We will employ a multidisciplinary approach with correlative behavioral, electrophysiological and neurochemical studies to examine the following hypotheses: (1) sickle mice exhibit cutaneous and deep hyperalgesia, which can be modulated by peripheral cannabinoids. We will examine cutaneous pain by measuring withdrawal responses to mechanical, heat and cold stimuli and deep/musculoskeletal pain using measurement of grip force. We hypothesize that intraplantar administration of cannabinoid receptor agonists reduce cutaneous hyperalgesia and this occurs through activation of CB1 and CB2 receptors. (2) SCD is accompanied by activation of peripheral nerve fibers and inflammatory mediators of pain in the skin and spinal cord. We will examine the activation of pro-inflammatory cells in the periphery and spinal cord that lead to neuronal activation and sensitization. (3) Pain in SCD involves sensitization of nociceptive spinal neurons. We will record electrophysiological responses of single, identified wide dynamic range and high threshold dorsal horn neurons in control and in SCD mice with hyperalgesia. In the proposed studies, we will use transgenic heterozygous BERK (hBERK) mice expressing sickle hemoglobin and age and sex matched control mice expressing normal human hemoglobin (HbA-BERK). Sickle hBERK mice have a mixed genetic background and carry a single copy of linked transgenes for human 1 and 2S globins. They are homozygous for knockout of murine 1 globin and heterozygous for knockout of murine 2 globin. HbA-BERK controls have an identical genetic background. We expect that sickle mice will show pain characteristics similar to pain in SCD, that activation of CB1 and C2 receptors by cannabinoids will attenuate hyperalgesia and that peripheral and central sensitization contributes to pain in sickle mice. Results of these studies will provide new information on the mechanisms underlying pain in SCD. Understanding the basic mechanisms of pain in SCD will lead to the development of novel and more effective approaches to treat pain in SCD. PUBLIC HEALTH RELEVANCE: Lifelong severe pain impairs quality of life in patients with SCD. High doses of opioids are the only therapy accompanied by secondary side effects. Our goal is to develop an understanding of mechanisms underlying pain in SCD to develop more effective and novel therapies to treat pain in SCD. If our hypotheses prove to be true, our results will provide an understanding that will facilitate treating pain in SCD and in developing novel and more effective analgesics.

DESCRIPTION (provided by applicant): Pain from cancer, particularly cancer that metastasizes to bone, is often severe, difficult to manage, and contributes significantly to the patients'poor quality of life. Although opioids remain the primary treatment for managing severe cancer pain, the relative resistance of cancer pain to opioids requires increased doses, which limit their use because of the many undesirable side effects associated with actions in the central nervous system. It is important to understand the mechanisms by which opioids become less effective in treating cancer pain. The mechanisms that drive cancer pain are multi-factorial and include bone remodeling, inflammatory responses, neurodegeneration, and release of algesic substances from the cancer cells that excite or sensitize nociceptors on primary afferent fibers. ATP is one of the known algesic substances that cancer cells contain and release during tumor growth. ATP activates peripheral P2X receptors which are located on nociceptive nerve endings. Previous behavioral studies from our group indicated that ATP contributes to the development of hyperalgesia in a murine model of bone cancer. Moreover, opioids modulate signaling at P2X receptors. Opioids such as morphine decrease ATP-evoked currents that occur through P2X receptors. We hypothesize that this opioid modulation of signaling through the P2X receptor is diminished by cancer cells, and that this may contribute to the decreased effect of opioids in cancer pain. In the proposed project, we will use an in vivo and a new in vitro co-culture method to determine the effect of cancer cells on ATP-evoked responses of dorsal root ganglion (DRG) neurons, how those responses are modulated by 5- and 4-opioid receptor agonists, and whether cancer cells promote a change in the expression of opioid receptors in DRG neurons. We will determine the effect of fibrosarcoma cells on P2X- dependent activation of mouse DRG neurons, changes in opioid modulation of P2X currents produced by cancer cells, and alterations in expression of 5- and 4-opioid receptors. Also, we will compare changes that occur in vivo (in tumor-bearing mice) to changes that occur in our co-culture model in vitro. The proposed studies will be done in Ukraine (Kiev) at the Bogomoletz Institute of Physiology in collaboration with Dr. Oleg A. Krishtal, who is an expert in patch clamp electrophysiology and P2X receptor function. This study is an extension of our ongoing project, NIH Grant CA091007, in which we are examining the contribution of peripheral P2X and TRPV1 receptors in cancer pain and nociceptor sensitization Results from these studies will provide new information on the mechanisms by which ATP contributes to cancer pain, and how opioids modulate ATP-evoked responses during tumor growth. Understanding how opioids can modulate cancer-related changes in responses of sensory neurons may lead to the development of novel approaches for managing cancer pain. PUBLIC HEALTH RELEVANCE: It is estimated by the National Cancer Institute that more than 1.4 million new cases of cancer were diagnosed in the United States in 2007, and the World Health Organization estimates that up to 15 million new cases of cancer may be diagnosed world-wide in 2020. Approximately 85% of adult patients with terminal cancer report intolerable pain and up to 75% of children with cancer experience pain. Understanding the mechanisms that drive cancer pain so that new and improved therapeutic approaches for pain management can be developed is a relevant public health issue.