Linda R. Watkins - US grants

It has long been recognized that there is a relationship between stressful life events and disease. For example, grief and bereavement appear to be associated with increased morbidity and mortality among the survivors. Depression, which often follows a loss or a separation is associated with a twofold increase in risk of cancer over a 17-year follow-up period. Similar, significant correlations, although retrospective, exist between stressful life events and the incidence or onset of major medical disorders, including AIDS. Given such correlations, it becomes important to examine what impact stress has on immune function. Dr. Linda Watkins will focus her research efforts on two inter- related questions which, taken together, should shed light on the underlying bases of immune responses to stress. First, a systematic examination will be performed to clearly define the temporal window within which exposure to stress can alter the immunologic response to antigenic challenge. Second, studies will be done aimed at defining mechanisms which allow these changes in immune function to occur. Standard plasma and tissue assays will be used.

DESCRIPTION (Applicant's Abstract): The proposed project is a request for a K02 award for the applicant to develop skills now required by new results in a programmatic investigation of cytokine-to-brain communication. Extensive evidence indicates that cytokines are critically involved in immune-to-brain communication. Indeed, cytokine-to-brain communication is of central importance in an organism's ability to respond to and survive immunological challenge. Alterations in neural function produced by cytokines such as IL1-beta may also be important for understanding stress and stress-related disorders such as depression. The candidate and colleagues have recently demonstrated (a) IL-1 binding sites on paraganglia which form afferent synapses with subdiaphragmatic vagal afferents and (b) that severing the subdiaphragmatic vagus blocks a variety of IL1-beta mediated responses. These data suggest that vagal afferents may provide a key neural pathway for cytokine signaling to brain. The research development and professional growth goals of the proposal involve a vigorous continuing education program for the PI, consisting of a number of academic courses, short courses, and workshops, as well as specialized conferences. In addition, the PI will intensively train with experts there at the University of Colorado, Boulder. The training will focus on molecular biology, immunohistochemistry and retrograde tracing, and electrophysiology. Additionally, the released time will foster further professional growth by yielding coherent blocks of time for concentrating on research and review projects.

DESCRIPTION:(adapted from applicant's abstract) The premise of this proposal is that activation of spinal cord microglia and astrocytes by immune system products can produce pain facilitation. These spinal cord glia recognize and become activated by foreign substances such as bacteria and viruses via specific receptor-mediated processes. Glia, but not neurons, recognize and become activated by HIV-1. Recognition by glia of HIV-1 is through receptor mediated binding of the HIV-1 envelope glycoprotein gp120. Such glial activation leads to the release of a variety of neuroactive substances, including proinflammatory cytokines (interleukin-1[IL1], IL6 & tumor necrosis factor), nerve growth factor nitric oxide and excitatory amino acids. These substances would be expected to lead to hyperalgesia and allodynia. This is potentially relevant to pain in AIDS as it suggests that: (a) pain of known peripheral origin in AIDS patients may be exaggerated by the ongoing HIV-1 induced spinal glial activation & (b) pain of unknown origin in AIDS patients may be created by spinal glial activation. This project will examine the role of spinal microglia and astrocytes in pain facilitation produced by intrathecal administration of gp120. A multidisciplinary approach will be used to examine a single intrathecal gp120 model. Using this model, the effects of gp120 on behavioral indices of pain response, on levels of presumptive glially produced pain enhancing endproducts and on immunohistochemical and mRNA expression for these same endproducts will be tested. This approach will be used to examine the potential mediators in pain facilitation known to be produced by intrathecal gp120: nerve growth factor, proinflammatory cytokines, nitric oxide and excitatory amino acids. In addition, it will be determined whether gp120 increases expression of activation markers in spinal glia, and whether disruption of glial function will block gp120-induced changes in pain response, end products and mRNA.

DESCRIPTION: (Adapted from the Investigator's Abstract) The core idea of this proposal is that, while mediators released by activated immune cells are adaptive when directed against microbes, these same mediators can be pathological when they act on neurons. It is clear from animal studies that "innocent bystander" damage can occur from immune activation near, but not directed at, peripheral nerves. Indeed, most human neuropathies are associated with immune activation rather than by physical (mechanical) trauma. These inflammatory neuropathies involve damage of peripheral nerves by immune cells and chronic pain. The overall goal of this proposal is to understand low-threshold mechanical allodynia (lowering of response threshold to mechanical stimuli) induced by immune activation and release of immune cells products in and around one healthy sciatic nerve (sciatic inflammatory neuritis (SIN). Unilateral and bilateral allodynia are rapidly induced by 4 and 160 ug peri-sciatic zymosan, respectively. We propose that SIN triggers a linear chain of events, resulting in allodynia: immune cells (activated by zymosan) release substances that alter peripheral nerve function, which in turn alters spinal cord function. Using a multidisciplinary approach, we will examine the basic elements of this chain of events & see how they interact to produce unilateral & bilateral allodynias. We will test peri-sciatically & intrathecally administered antagonists on behavioral indices of mechanical allodynia. We will then examine levels of SIN-induced pain enhancing endproducts from immune cells & spinal cord (measured by colorimetric assays & ELISAs), immunohistochemical expression of these endproducts in immune cells, sciatic nerve & spinal cord (by double-label immunohistochemistry & FACS flow cytometry), & changes in messenger RNA for these same endproducts in immune cells & spinal cord. Thus, this multidisciplinary approach will be used to examine the potential mediators of allodynia at the level of the immune cells (Specific Aim I), at the level of the sciatic nerve (Specific Aim II), & at the level of the spinal cord (Specific Aim III).

DESCRIPTION: (provided by applicant): Human pathological pain is a major unresolved problem. Recent data strongly support the argument that spinal cord glia (astrocytes and microglia) are critically involved in the creation and maintenance of diverse pathological pain states. Spinal cord glia create exaggerated pain states via the release of proinflammatory cytokines (PICs). Recognition of the key importance of spinal cord glia and glial PICs in pathological pain opens new avenues for pain control. There are various treatments available to control glial activation involved in enhanced pain. Interleukin-10 (IL10) is the most promising from a clinical point of view. IL10 is an excellent candidate for preventing and reversing PIC-driven pathological pain states. However, two practical problems need to be overcome. First, control of chronic pain requires chronic delivery of IL10. Second, IL10 cannot cross the blood-brain barrier, thus negating systemic administration. To resolve these issues, we have explored the feasibility of using prolonged spinal release of IL10 induced by gene therapy. Here, adenoviral vectors encoding IL10 are injected into the cerebrospinal fluid surrounding the spinal cord. Our preliminary data provide strong support for the possibility that spinal gene therapy with IL10 will prevent and reverse pathological pain. The aims of the present proposal are three-fold: (1) To determine the breadth of clinically relevant exaggerated pain states that can be prevented and/or reversed by gene therapy-induced IL10 in spinal CSF; (2) To construct a "gutless" adenoviral vector of IL10 which allows virally infected cells to avoid detection and destruction by the immune system; and (3) To characterize the patterns of IL10 release, characterize viral spread and clarity whether peripheral immune functions are impacted by this procedure. Together these studies will test the premise that intrathecal IL10 delivery via gene therapy is worthy of clinical development for controlling diverse pathological pain states. This approach to pain control represents a dramatic departure from all other available therapies.

DESCRIPTION (provided by applicant): The PI's preclinical research program seeks to understand how activation of peripheral immune cells and central nervous system microglia and astrocytes triggers a cascade of events leading to neuronal activation and pathological pain states. This current research focus is directly relevant to her long-term goals of understanding (a) immune-neural interactions and (b) endogenous pain modulation systems. The proposed project is a request for a K02 award for the PI to develop skills now required by new results in programmatic investigations of pathological pain states. The 2 animal models employed induce clinically relevant exaggerated pain states by: (a) peri-spinal administration of HIV-1 gp 120 and (b) sciatic inflammatory neuropathy. Extensive evidence indicates that peripheral immune cells and spinal immune-like glial cells play critical roles in the creation and maintenance of exaggerated pain phenomena. Of the substances released by these cells upon activation, the strongest evidence to date points to the proinflammatory cytokines tumor necrosis factor, interleukin-1, and interleukin-6. These signaling molecules are key spinal mediators of pathological pain induced by both peri-spinal gp120 and sciatic inflammatory neuropathy. Their release from peri-sciatic immune cells is also correlated with the induction and intensity of sciatic inflammatory neuropathy. The two parent R01 grants are aimed at clarifying the immune/glial mechanisms underlying these pain models using immunological, anatomical, molecular, pharmacological, and behavioral approaches. The PI seeks to gain further training in molecular biology techniques (RNase Protection Assays, in situ hybridization, and adenoviral vectors for gene therapy), to enroll in responsible conduct of research coursework, and to continue her education through project-relevant coursework and research forums. Additionally, the released time will foster further professional growth by yielding coherent blocks of time for concentrating on research and review projects.

DESCRIPTION (provided by applicant): There is growing recognition that dorsal spinal cord gila (astrocytes & microglia) contribute to the creation & maintenance of enhanced pain, including pathological pain. In addition, they may play important roles in the development of morphine tolerance & tolerance-associated pain facilitation. While currently available methods have provided initial insights into the role of gila in pain regulation, all are severely limited. For example, in vivo pharmacology & analyses of homogenated spinal cord & CSF cannot identify which cell type(s) is/are responsible for the observed effects. In situ hybridization & immunohistochemistry cannot assess whether the end-product is actually released, so the physiological relevance of the changes are unclear. Furthermore, none of these techniques can address whether glial interactions are involved, such as microglial-derived products stimulating astrocytes to release the key painmodulating substance(s). Such glial synergies/interactions are well documented elsewhere in the CNS but have not yet been examined in spinal cord. New methodologies are needed to understand how dorsal spinal cord astrocytes & microglia, singly & interactively, modulate pain. New methodologies are needed to enable rapid isolation of pure astrocytes 8, pure microglia from the dorsal spinal cords of adult rats to allow assessment of how manipulations of interest (intrathecal HIV-1 gp120, chronic neuropathy, chronic morphine, & other clinically relevant pain models) alter these cells in terms of their receptor, mRNA & endproduct expression. New methods are also needed to enable manipulation of dorsal spinal cord astrocytes microglia in culture to test their responses (cellular mRNA, cellular proteins, & released endproducts) to pain-relevant transmitters & modulators. With rare exception, such studies are not possible in vivo since neurons, astrocytes &/or microglia likely express receptors for these pain-relevant substances & can release many of the same neuroactive compounds, thus confounding interpretations of the results. Thus this Small Grant proposal is focused on developing & refining the new methods described above. These new methods will then undergo initial assessments, in terms of functional characterization & receptor expression of the isolated astrocytes & microglia.

DESCRIPTION (provided by applicant): The PI's preclinical research program seeks to understand how activation of peripheral immune cells and central nervous system microglia and astrocytes triggers a cascade of events leading to neuronal activation and pathological pain states. This current research focus is directly relevant to her long-term goals of understanding (a) immune-neural interactions and (b) endogenous pain modulation systems. The proposed project is a request for a K02 award for the PI to develop skills now required by new results in programmatic investigations of pathological pain states. The 2 animal models employed induce clinically relevant exaggerated pain states by: (a) peri-spinal administration of HIV-1 gp 120 and (b) sciatic inflammatory neuropathy. Extensive evidence indicates that peripheral immune cells and spinal immune-like glial cells play critical roles in the creation and maintenance of exaggerated pain phenomena. Of the substances released by these cells upon activation, the strongest evidence to date points to the proinflammatory cytokines tumor necrosis factor, interleukin-1, and interleukin-6. These signaling molecules are key spinal mediators of pathological pain induced by both peri-spinal gp120 and sciatic inflammatory neuropathy. Their release from peri-sciatic immune cells is also correlated with the induction and intensity of sciatic inflammatory neuropathy. The two parent R01 grants are aimed at clarifying the immune/glial mechanisms underlying these pain models using immunological, anatomical, molecular, pharmacological, and behavioral approaches. The PI seeks to gain further training in molecular biology techniques (RNase Protection Assays, in situ hybridization, and adenoviral vectors for gene therapy), to enroll in responsible conduct of research coursework, and to continue her education through project-relevant coursework and research forums. Additionally, the released time will foster further professional growth by yielding coherent blocks of time for concentrating on research and review projects.

The core idea of this proposal is that: (a) substances released by neurons trigger the activation of gila, & (b) this glial activation, in turn, leads to the release of glial products that induce & maintain pain facilitation. It is clear from prior studies that spinal cord gila (microglia and astrocytes) become activated in response to signaling by small diameter sensory afferents. It is also clear from prior studies that blocking either glial activation or the action of glial products prevents & reverses diverse enhanced pain states. What is not understood isare the signal(s) released by neurons which cause(s) such glial activation. The overall goal of this proposal is to understand this key step. We have recently discovered one putative neuron-to-gila signal so will focus on it. This signal is fractalkine, a protein tethered to the exterior surface of spinal cord neurons & sensory afferents. When neurons become strongly activated, fractalkine can break free to form a diffusible signal. We have shown in spinal cord that (a) microglia (& possibly astrocytes) express receptors for fractalkine; (b) injecting fractalkine induces thermal hyperalgesia & mechanical allodynia; & (c) early evidence suggests that fractalkine may potentially be an important signal for initiating & maintaining neuropathic pain. We propose to use a mulUdisciplinary approach to examine fractalkine-induced neuron-to-gila communication. First, we will define how critical fractalkine is for the induction & long-term maintenance of neuropathic pain by (a) testing the effect of a selective fractalkine receptor antagonist delivered prior to, or up to 3 mon after, nerve damage & (b) testing knockouts for both fractalkine and its receptor (CX3CR1). Second, we will define the effects of fractalkine on microglia versus astrocytes using a combination of approaches: pharmacological, immunohistochemical, in vitro & adoptive transfer (intrathecal injection of in vitro stimulated microglia & astrocytes). Last, as time permits, we will examine whether fractalkine creates pain facilitation via proinflammatory cytokine release &/or "priming" of gila. Together, this series of studies will provide new insights into how fractalkine in particular, & neuron-to-gila signaling in general, creates & maintains pain facilitation. Understanding this key step between neural activation & glial activation may have implications for developing new approaches for pain control.

[unreadable] DESCRIPTION (provided by applicant): Based entirely on rodent studies, there is growing acceptance that dorsal spinal cord glia (microglia & astrocytes) contribute to the creation & maintenance of enhanced pain, including pathological pain. Glial activation occurs in every rodent model of enhanced pain examined to date, & disruption of glial activation or glial proinflammatory cytokines blocks rodent enhanced pain responses. It has been proposed, based on such studies, that disruption of glial function should ameliorate human chronic pain. Whether it is valid to extrapolate these rodent glia data to humans has never been tested. It is critical to know whether there are, or are not, parallels between rodent & human dorsal spinal cord glial responses. This is not simply of theoretical importance, but of impending practical importance as well, as there is growing interest in the pharmaceutical/biotech sector in clinical approaches that target gila. Whether or not this is an approach to be encouraged depends on whether human glial responses mirror those of rodents. The purpose of the present small grant proposal is to provide an initial investigation of whether rodent data concerning glia involvement in pain reflect the human condition. Examining spinal cord glial activation & glial products in humans requires postmortem donor tissues. While this presents some constraints, these can be minimized by restricting analyses to tissues collected <4 hr of death. Sun Health Research Institute (SHRI) will provide chronic pain & control donor tissues for this project. Selected donors will provide brain, spinal cord, CSF, & plasma for protein, mRNA & immunohistochemical analyses of glial activation & glial products. SHRI will also assess the feasibility of using their donor population for a future prospective study of glial activation in chronic pain. The results from these studies will provide the first insights into whether glia are, or are not, activated in human chronic pain states. Either answer has major implications for the development of drug therapies for the control of clinical pain. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Chronic pain, including chronic orofacial pain, remains unsuccessfully treated in a large number of patients. Furthermore, the loss of analgesic efficacy with chronic administration of frontline analgesic drugs, such as morphine, severely limits their use. Recent data strongly suggest that spinal cord glia (astrocytes and microglia) oppose the analgesic effects of morphine, through the release of proinflammatory cytokines: tumor necrosis factor (TNF), interleukin-1 (IL1) &interleukin-6 (IL6). While as yet unexplored, this raises the possibility that glial activation by clinically relevant opioid analgesics may be broad in scope, rather than a phenomenon restricted to morphine. Therefore, (a) clinical pain control may currently be hindered by opioid-induced glial activation &, (b) if this is true, clinical pain control could be improved by finding ways to prevent or circumvent the effects of glial activation by opioid analgesics. Therefore, the aims of the proposal are to determine whether: (I) clinically relevant opioid analgesics, in general, induce proinflammatory cytokines in trigeminal nuclei, and also in spinal cord under normal (sham) &/or neuropathic (chronic constriction injury;CCI) pain conditions. Further, whether an anti-inflammatory cytokine will "unmask" analgesia following chronic opioid administration. The potential for chronically enhancing analgesic efficacy by chronic co-administration of an anti-inflammatory cytokine will also be explored. (II) the induction of spinal proinflammatory cytokines by morphine &other opioids is mediated, in part, via actions of their common, active metabolites (M6G or M3G);and (III) the elevated production/release of trigeminal and spinal proinflammatory cytokines induced by opioid pharmacotherapies is mediated via classical opioid receptors. Moreover, whether selective mu, delta &kappa receptor agonists mimic the effects of clinically relevant analgesics. Where feasible, sciatic CCI will be replaced by CCI of the infraorbital nerve &assessment of orofacial mechanical allodynia &thermal hyperalgesia. Together these studies will provide novel insights into the actions of opioid analgesics at both trigeminal &spinal sites, &will explore the potential for using anti-inflammatory cytokines as a means of potentiating the magnitude &duration of analgesia to relieve normal &neuropathic pain. If successful, these studies will lead to development of novel adjunct therapies for improving clinical pain control by controlling the negative consequences of opioid-induced glial activation.

DESCRIPTION (provided by applicant): The PI's preclinical research program seeks to understand how activation of peripheral immune cells and central nervous system glia (microglia and astrocytes) triggers a cascade of events leading to neuronal activation, pathological pain states, and dysregulation of the clinically relevant effects of analgesic drugs. The current research focus is directly relevant to her long-term goals of understanding (a) immune-neuralbidirectional interactions, and (b) pain modulation systems. The proposed project is a request for a K05 award for the PI to focus on programmatic investigations of pathological pain states. The currently funded projects include: (a) Immune/glial mediation of exaggerated pain states;NIDA K02 (Watkins PI;6/03-5/08;DA015642);(b) Pain control via spinal interleukin-10 gene therapy;NIDA CEBRA Phase II (R01;Milligan PI;Watkins coPI;09/04-09/09;DA018156);(c) Pain facilitation via neuron-to-glia signaling;NIDA R01 (Watkins PI;3/05-12/09;DA017670);(d) Opioid analgesics: modulation.of trigeminal &Spinal glial activation;NIDCR R01 (Watkins PI;7/06-4/11;DE017782);(e) Exploration of AV411, a blood brain barrier permeable glial activation inhibitor;Avigen (Watkins Pl;1/06-12/08);(f) Development of rat models to assess potential glial involvement in migraine;GlaxoSmithKline (Watkins PI;1/07-12/09);(g) Translation from rats to humans: are chronic pain states in humans associated with glial activation in spinal cord and/or brain?;American Fibromyalgia Syndrome Association (Watkins CD Boulder PI;open ended small grant);(h) Human spinal cord glial cytokines &chronic pain NIAMSD R01 (Lorton, PI;Watkins, coPI;7/07-6/12;AR054647);&(i) mRNA &protein analyses of spinal cord &CSF from spinal cord injury patients with vs. without chronic pain;Craig Spinal Cord Injury Treatment &Rehabilitation Hospital (Watkins CU Boulder PI;9/07-open ended small grant). In addition, projects under review include: (j) Development of opioid therapeutic approaches that fail to activate glia;Covidien (Watkins PI);(k) Exploring the potential of glia for regulating clinically relevant opioid actions, R01 proposal (Watkins PI);and (I) Exploiting viral mimicry to develop new therapies for treating neuropathic pain, R21 Proposal (Leinwand PI;Watkins coPI). Thus this is an active &diverse research program exploring multiple levels of analyses at the forefront of pain research. All of these projects are focused on understanding immune/glial regulation of pain and analgesic actions. Beyond this extensive research program, the PI is committed to education of the scientific and lay communities on these topics via review articles and chapters and invited talks, and the mentorship of young investigators.

Principal Investigator/Program Director (Last, first, middle): Watkins, Linda RESEARCH &RELATED Other Project Information 1. * Are Human Subjects Involved? m Yes l No 1.a. If YES to Human Subjects Is the IRB review Pending? m Yes m No IRB Approval Date: Exemption Number: 1 2 3 4 5 6 Human Subject Assurance Number 2. * Are Vertebrate Animals Used? l Yes m No 2.a. If YES to Vertebrate Animals Is the IACUC review Pending? l Yes m No IACUC Approval Date: Animal Welfare Assurance Number A3646-01 3. * Is proprietary/privileged information m Yes l No included in the application? 4.a.* Does this project have an actual or potential impact on m Yes l No the environment? 4.b. If yes, please explain: 4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or environmental impact statement (EIS) been performed? m Yes m No 4.d. If yes, please explain: 5.a.* Does this project involve activities outside the U.S. or m Yes l No partnership with International Collaborators? 5.b. If yes, identify countries: 5.c. Optional Explanation: 6. * Project Summary/Abstract 7854-Watkins.ProjectSummary.pdf Mime Type: application/pdf 7. * Project Narrative 176-Watkins.ProjectNarrative.pdf Mime Type: application/pdf 8. Bibliography &References Cited 1004-Watkins.LiteratureCited.pdf Mime Type: application/pdf 9. Facilities &Other Resources 8902-Watkins.Facilities.pdf Mime Type: application/pdf 10. Equipment 5753-Watkins.Equipment.pdf Mime Type: application/pdf Tracking Number: Other Information Page 5 OMB Number: 4040-0001 Expiration Date: 04/30/2008 Principal Investigator/Program Director (Last, first, middle): Watkins, Linda It has recently been discovered that glia become progressively more activated upon repeated exposure to morphine, &that this glial activation, in turn, modulates morphine's effects. This discovery was originally made in the context of studying the pain suppressive effects of morphine in spinal cord. The possibility that glia may be fundamentally important in determining the effects of opioids such as morphine is novel &important in its implications. Because of this, we propose to explore whether glia may profoundly alter the effects of repeated morphine in brain, as well. We believe that glia will prove to be powerfully involved in several phenomena currently thought to arise purely as a result of opioid effects on neurons;that is, dependence/withdrawal, reward &aversion. If this were true, it would provide evidence that glia are critically involved, not only in modulating the pain-suppressive effects of opioids, but also in

DESCRIPTION (provided by applicant): NIDCR Proposal: Models and mechanisms for the transition of acute-to-chronic orofacial pain Project Summary/Abstract This application addresses broad Challenge Area (15) Translational Science &specific Challenge Topic 15- DE-102*: New Models and Measures in Pre-Clinical Chronic Pain Research. The critical features that predict the transition from acute to chronic pain remain unresolved. The present proposal explores whether microglial "priming" may be of particular importance in explaining the progression from acute to chronic pain. Activation of microglia &astrocytes mediates diverse enhanced pain states. One important aspect of glial functioning that has not been explored in the context of pain is the effect of a sensitized, or "primed", microglial response. Research outside of the field of pain indicates that the past history of microglial activation can greatly alter their response to new challenges. Microglia can reach a primed state via prior stress, pain, trauma &inflammation, &exposure to opioids, which strikingly are known co-morbidities for the transition of acute to chronic pain in the trigeminal system. While in such a primed state, microglia now dramatically over-respond to new challenges, stronger &longer than before. We believe such prior microglial priming can set the stage for the transition of acute to chronic pain in temporomandibular joint (TMJ) disorders &other orofacial pain disorders. Re-activation of primed spinal microglia may lead to a transition from acute pain to chronic pain as a result of a neuroinflammatory response that is greatly amplified in both magnitude &duration. This proposal aims to develop new rat models for the study of the transition from acute to chronic orofacial pain, based on the premise that a first challenge (prior pain, stress, trauma/inflammation, opioids) will markedly enhance pain induced by a subsequent challenge to the trigeminal system (facial allodynia induced by inflammation of either the TMJ or dura). Once robust models are defined &refined, an initial exploration of potential glial cell influence on the transition from acute to chronic pain will be undertaken. This is, by necessity of time constraints, meant as simply the first step toward a thorough investigation to be undertaken in a future proposal based on the data generated by this project. Here, the most robust models will be determined for study using the two blood brain barrier permeable glial activation inhibitors now approved by the FDA for clinical trials aimed at treating neuropathic pain: ibudilast (AV411) &propentofylline (SLC022). These non-opioid, non-addictive drugs will be tested in an initial screen to determine whether either or both compounds may be able to prevent the transition of acute to chronic pain. If they do, as expected, this would suggest that preventing or suppressing glial priming may provide a significant advance in our basic science understanding of how acute pain becomes chronic, as well as provide a clinically testable means by which to prevent &reverse the transition to chronicity. Exploring how known co-morbidities set the stage for the transition from acute to chronic pain by inducing microglia to enter into an over-reactive primed state is a topic never before explored &exciting in its potential practical &theoretical applications. PUBLIC HEALTH RELEVANCE: This proposal aims to develop new rat models for the study of the transition from acute to chronic orofacial pain, based on the premise that a first challenge ("Hit 1": prior pain, stress, trauma/inflammation, opioids) will markedly enhance pain induced by a subsequent challenge to the trigeminal system ("Hit 2": inflammation of either the TMJ or dura). We believe that this transition to chronic pain will be due to sensitization of glia by Hit 1, causing them to massively over respond in response to Hit 2, and that treatment with clinically-relevant glial activation inhibitors will prevent the transition to chronic pain.

DESCRIPTION (provided by applicant): : Spinal adenosine modulator: enduring anti-inflammatory action in neuropathic pain Project Summary/Abstract This application addresses broad Challenge Area (15) Translational Science &specific Challenge Topic 15- NS-103 Demonstration of "proof-of-concept" for a new therapeutic approach in a neurological disease. Neuropathic pain remains intractable despite treatment with currently available therapeutic agents. Therefore, it is necessary to identify novel pharmacotherapeutics that can effectively attenuate neuropathic pain. Activation of microglia and astrocytes (glial cells) plays a crucial role in chronic pain states, including neuropathic pain, by chronically inducing the release of neuroexcitatory substances such as pro-inflammatory cytokines and chemokines. This pro-inflammatory milieu surrounds neurons, helping to maintain the hyperexcitable state of neurons associated with chronic pain states. An optimal therapeutic would "reset" these chronically activated glial cells to an overtly anti-inflammatory state, thus returning the neuronal excitation to basal levels. Such a drug, were it to exist, would be predicted to reduce neuropathic pain by removing the tonic "drive" to the pain system provided by glial activation. We believe that we have identified such agents;namely, adenosine 2A (A2A) agonists. Mechanistically, these are agonists at one adenosine receptor subtype. Adenosine is a purine that exerts its effects via four subtypes of G-protein coupled adenosine receptors (A1, A2A, A2B and A3). Adenosine 2A receptors are found on most tissues in the body including spinal cord neurons, microglia, astrocytes, endothelial cells and oligodendrocytes. The selective activation of the A2A receptor subtype is immunosuppressive, decreasing pro-inflammatory cytokines and increasing the powerful anti-inflammatory cytokine, interleukin-10 (IL-10). In addition, activation of A2A receptors reduces NMDA activation in primary sensory neurons. Preliminary data, using the chronic constriction injury (CCI) model, support that a single intrathecal injection of A2A agonists produces a remarkably enduring reversal of neuropathic pain of at least several weeks. In order to fully understand the impact of A2A agonists on chronic pain conditions, we are proposing to 1) thoroughly characterize the reversal in established neuropathic pain following a single versus multiple injections to ascertain if tolerance to the agonist's pain suppressive effects develops;and, 2) begin to characterize the cell types involved in the agonist's pain suppression. While the mechanism of action most likely includes a neuron-glia interaction, given the known role of glia in pain development and maintenance, our focus will be on glial changes, with neuronal changes assessed concurrently as time and funds allow. In addition, we will assess whether the persistent effect of the A2A agonist is specific to the A2A adenosine receptor or if modulating adenosine regulation via other adenosine receptors will produce the same effect. PUBLIC HEALTH RELEVANCE: NINDS Proposal: Spinal adenosine modulator: enduring anti-inflammatory action in neuropathic pain Project Narrative This proposal builds from our discovery that a single intrathecal administration of adenosine 2A (A2A) agonists produces a remarkably enduring reversal of neuropathic pain of at least several weeks, with evidence to date suggestive that such drugs may "reset" chronically activated spinal glial cells to an overtly anti-inflammatory state that suppresses pain. This project is aimed at providing the "proof-of-concept" for using A2A agonists as a new therapeutic approach for chronic pain.

DESCRIPTION (provided by applicant): While there has been increasing recognition of the importance of microglial and astrocyte activation in the creation & maintenance of diverse enhanced pain states, an important aspect of glial functioning that has not yet been explored in the context of pain enhancement is the effect of a sensitized, or primed, microglial response. Evidence has accrued from outside of the pain field that the past history of microglial activation can dramatically alter their response to new challenges. Microglia can reach a primed state via a variety of challenges, including peripheral or central trauma/inflammation, stress, prior pain, and exposure to opioids, which strikingly are known co-morbidities for the transition of acute to chronic pain, including neuropathic pain. While in such a primed state, microglia now dramatically over-respond to new challenges, stronger and longer than before. We believe such prior challenges that result in glial priming can set the stage for the transition of acute to chronic pain following peripheral & central neural damage, resulting in chronic neuropathic pain. Re-activation of primed microglia may lead to a transition from acute pain to chronic pain as a result of a neuroinflammatory response that is greatly amplified in both magnitude and duration. Goals. (1) In accordance with the specified goals of this RFA, develop new rat models to study the transition from acute to chronic neuropathic pain, based on the premise that a first challenge (Hit 1: peripheral or central trauma/inflammation, stress, prior pain, exposure to opioids) will markedly enhance pain induced by a subsequent (second) challenge (Hit 2: peripheral or central neural inflammation/injury). (2) Utilize the refined robust models to test the potential of non-opioid, non-addictive blood-brain barrier permeable glial activation inhibitors & resolvins to prevent the transition of acute to chronic pain. (3) Given the remarkably powerful positive effects produced by chronic voluntary exercise in creating resiliency to a multitude of negative outcomes (including constraining glial/immune reactivity), chronic voluntary exercise will also be tested for its ability to prevent the transition from acute to chronic pain, an approach enabled by teaming with an expert from outside the pain field (M. Fleshner). (4) Discover intracellular changes that differentiate rats which do vs. do not transition from acute to chronic neuropathic pain, & define how these potential cellular markers of impending chronic pain are affected by successful interventions (glial inhibitors, voluntary exercise). This will lay the groundwork for identifying and targeting changes reliably predictive of the transition of acute to chronic neuropathic pain.

The Pi'spreclinical research program seeks to understand how activation of peripheral immune cells and central nervous system glia (microglia and astrocytes) triggers a cascade of events leading to neuronal activation, pathological pain states, and dysregulation of the clinically relevant effects of analgesic drugs. The current research focus is directly relevant to her long-term goals of understanding (a) immune-neural bi- directional interactions, and (b) pain modulation systems. The proposed project is a request for a K05 award for the PI to focus on programmatic investigations of pathological pain states. The currently funded projects include: (a) Immune/glial mediation of exaggerated pain states; NIDA K02 (Watkins PI; 6/03-5/08; DA015642); (b) Pain control via spinal interleukin-10 gene therapy; NIDA CEBRA Phase II (R01; Milligan PI; Watkins coPI; 09/04- 09/09; DA018156); (c) Pain facilitation via neuron-to-glia signaling; NIDA R01 (Watkins PI; 3/05-12/09; DA017670); (d) Opioid analgesics: modulation.of trigeminal & spinal glial activation; NIDCR R01 (Watkins PI; 7/06-4/11; DE017782); (e) Exploration of AV411, a blood brain barrier permeable glial activation inhibitor; Avigen (Watkins Pl;1/06-12/08); (f) Development of rat models to assess potential glial involvement in migraine; GlaxoSmithKline (Watkins PI; 1/07-12/09); (g) Translation from rats to humans: are chronic pain states in humans associated with glial activation in spinal cord and/or brain?; American Fibromyalgia Syndrome Association (Watkins CD Boulder PI; open ended small grant); (h) Human spinal cord glial cytokines & chronic pain NIAMSD R01 (Lorton, PI; Watkins, coPI; 7/07-6/12; AR054647); & (i) mRNA & protein analyses of spinal cord & CSF from spinal cord injury patients with vs without chronic pain; Craig Spinal Cord Injury Treatment & Rehabilitation Hospital (Watkins CU Boulder PI; 9/07-open ended small grant). In addition, projects under review include: (j) Development of opioid therapeutic approaches that fail to activate glia; Covidien (Watkins PI);(k) Exploring the potential of glia for regulating clinically relevant opioid actions, R01 proposal (Watkins PI);and (I) Exploiting viral mimicry to develop new therapies for treating neuropathic pain, R21 proposal (Leinwand PI; Watkins coPI). Thus this is an active & diverse research program exploring multiple levels of analyses at the forefront of pain research. All of these projects are focused on understanding immune/glial regulation of pain and analgesic actions. Beyond this extensive research program, the PI is committed to education of the scientific and lay communities on these topics via review articles and chapters and invited talks, and the mentorship of young investigators.

PROJECT SUMMARY Multiple sclerosis (MS) is a life-long, debilitating disease in both males and females. Symptoms include loss of motor function, neuropathic pain, cognitive impairments, and impaired social interaction. MS is furthermore associated with elevations in circulating and central (spinal cord and brain) levels of pro- inflammatory cytokines and decreased levels of anti-inflammatory cytokines, suggesting a dysregulation of immune and glial processes resulting in chronic inflammation. This ongoing inflammation is important in demyelination, chronic glial activation, and neuronal death characteristic of the disease. Targeted suppression of spinal cord neuroinflammation using anti-inflammatory strategies dramatically improves symptoms of experimental autoimmune encephalomyelitis (EAE), a rat model of MS. Looking forward toward translation, what is needed is not our current approaches that are injected intrathecally, but rather a means to effectively treat EAE/MS via a clinically relevant, orally available, blood-brain barrier permeable small molecule that targets EAE/MS pathology driven by neuroinflammation. We have discovered, and extensively characterized, such a small molecule. This drug (the non-opioid (+)-isomer of naltrexone; (+)-naltrexone) is rapidly moving toward FDA application for Investigational New Drug status. This is a selective antagonist at toll-like receptor 4 (TLR4) and TLR2. As it fails to bind classical opioid receptors, (+)-naltrexone does not interfere with the efficacy of opioids for pain control or normal functioning of opioid receptor systems. Targeting TLR2 and TLR4 arises from an extensive literature demonstrating the importance of these receptors in the neuroinflammatory processes and EAE/MS symptoms to be studied here. A complimentary series of behavioral and immunohistochemistry studies are proposed in males and females which will explore the ability of (+)-naltrexone to suppress EAE-induced (a) paresis/paralysis, (b) neuropathic pain, (c) cognitive impairment, and (d) social interaction impairment, as well as (e) improve survival, when (+)-naltrexone is systemically administered across days, comparing dosing early vs. late in the EAE timecourse. The IHC studies will analyze brain and spinal cord tissues collected after early vs. late (+)- naltrexone treatment (mapping onto the behavioral studies) to define whether (+)-naltrexone suppresses glial activation, neuronal cell death, and demyelination, as well as stimulates remyelination, as predicted. These studies create two complimentary Aims. Aim 1 explores a novel means of positively intervening in EAE-induced motor dysfunction, neuropathic pain, deficits in social interaction and cognition, and loss of life by targeting TLR4/TLR2 by systemic administration of (+)-naltrexone. Aim 2 transitions to an initial exploration of potential mechanisms underlying pathophysiology, focusing on the impact of TLR4/TLR2 blockade on demyelination and remyelination, neuronal cell death, and neuroinflammation. These studies provide a thorough investigation of the role of TLR4/TLR2 in major MS-relevant symptoms of EAE.

PROJECT SUMMARY Neuropathic pain occurs in epidemic proportions worldwide and none of the currently available therapeutics provides adequate pain relief and all have significant side effects. None are ?disease modifying? as all are simply palliative in targeting symptoms, not cause. There must be a better approach to pain control. What we have discovered could potentially revolutionize the clinical treatment of trauma and surgical pa- tients, all of whom are at marked risk (~50-70%) for developing neuropathic pain. Our discovery is that 6 wk of moderate voluntary exercise that ceases at the time of nerve trauma appears to permanently suppress the lat- er development of neuropathic pain. Such an effect has never been previously reported. The core thesis of this proposal is that novel, superior pharmacological and/or herbalism treatment strate- gies will arise from understanding how non-pharmacological voluntary exercise produces dramatic prevention of chronic pain. The critical first step is to understand how VWR prevents chronic pain. This enlightens target- ed, evidence-based steps toward achieving the same dramatic prevention of pain via pharmacological and/or herbal medicine approaches. The mechanisms explored in the present proposal are unique from those of any currently available pain therapeutic. If we can understand and harness prevention of neuropathic pain, this should lead to early drug interventions of broad practical importance. How 6 wk voluntary wheel running (VWR) could profoundly influence the cascade of neuroimmunological and neuropathological events set into motion by later nerve injury has never been explored. This is critical to understand at a mechanistic level, as prior VWR appears to be the first ?disease modifying? approach to con- trolling whether chronic pain develops. Understanding how this occurs will enable development of novel drug regimens to pharmacologically duplicate these effects without the necessity of exercise regimens that few peo- ple will follow. We predict that understanding how prior voluntary exercise (VWR) exerts such powerful, and seemingly permanent suppression of neuropathic pain is a tractable research goal, addressable by the multi-disciplinary approach proposed. This would first seek to understand the behavioral, immunological, neuroimmunological, and functional effects of: (a) VWR, (b) nerve injury (classic sciatic chronic constriction injury [CCI] model in male and female rats), and (c) their interaction on the aftermath of nerve injury. Based on these findings, mechanistic studies are proposed so to begin to explore how prior VWR could exert such a positive, pain- preventative effect on later nerve injury. Toward this goal, studies that seek to inhibit and to recapitulate the effects of VWR are both proposed. The long term aim is to capitalize on the understanding of how prior VWR creates such long-lasting suppression of neuropathic pain so to identify clinically relevant approaches to neu- ropathic pain prevention, thereby providing a far superior approach to pain control than currently available.

Project Summary Activation of microglia and astrocytes (glia) plays a key role in chronic pain, including neuropathic pain, by chronic release of neuroexcitatory substances such as pro-inflammatory cytokines. This pro-inflammatory milieu surrounds neurons, helping to maintain neuronal hyperexcitability that underlies chronic pain. An optimal therapeutic strategy would be to ?reset? these chronically activated proinflammatory glia to a persistent anti-inflammatory state, thus restoring normal neuronal tone. We believe that we have identified such a therapeutic; namely, adenosine 2a receptor (A2aR) agonists. Our studies, across multiple pain models, support that a single intrathecal injection of an A2aR agonist produces a remarkably enduring reversal of neuropathic pain across weeks. This efficacy is observed under conditions of pre-existing neuroinflammation, known to upregulate the expression of A2aR. The profoundly enduring resolution of neuropathic pain is a unique and striking finding, not duplicated by any therapeutic short of gene therapy. The breadth and consistency of effect across multiple models of peripheral and central neuropathic pain predicts broad clinical utility of such agents, and supports the importance of understanding how this sustained pain reversal occurs. Discovering how A2aR agonists create multi-week resolution of pain after a single dose would provide a fundamental paradigm shift in our understanding of pain regulation and redirect strategies for development of pain therapeutics. What is clear regarding mechanism is that A2aR agonists set into motion a cascade of events resulting in the suppression of ongoing neuroinflammation via the persistent, multi-week release of the potent anti- inflammatory cytokine, interleukin-10 (IL10). This creates enduring resolution of neuropathic pain, suppression of spinal proinflammatory cytokines, and suppression of spinal glial activation. From all regards, this is an unprecedented phenomenon worthy of understanding and harnessing for improving clinical pain control in males and females and, likely, more broadly for controlling negative sequelae arising from other persistent neuroinflammatory states as well. We propose mRNA and microRNA RNA-seq and epigenetic analyses in male and female rats to identify high probability mediators and candidate signaling pathways by which A2aR agonists create their enduring pain suppressive effects. Given the uniqueness of this phenomenon, we predict that the results will yield novel mediators not currently known to the pain field. By this high risk/high reward approach, these RNA-seq and epigenetic results will define the goals to be pursued in subsequent grant projects by discovering unique targets and pathways. This is a hypothesis-generating proposal seeking to understand a completely novel finding. We predict that A2aR agonism alters the transcriptional program of glial cells by creating lasting epigenetic modifications.

Project Summary Opioids are widely used to treat pain after trauma. Opioid use for pain management has dramatically in- creased, with little assessment of potential negative consequences for ongoing pain. Recent reports are critical of the lack of controlled, long-term studies to support the dramatic escalation of opioid treatment for chronic pain over the past decade. While one long-term concern is that there may be no benefit, another is that opioids could have negative consequences for pain. There would be major implications were opioid treatment to pro- long the course of pain long after opioid cessation. As described in this proposal, robust opioid-induced chroni- fication of pain does indeed occur, making this a phenomenon critical to understand. Disturbingly, we have discovered that opioids given around the time of trauma may be contraindicated: a brief course of treatment with morphine (5 mg/kg b.i.d. for 5-7 days) can amplify the magnitude and duration of neuropathic pain for months thereafter. Strikingly, this deleterious opioid effect occurs across all models tested to date: inflammatory pain, peripheral and central neuropathic pain, and post-operative pain, supportive that this is a widespread phenomenon worthy of study. This unanticipated effect of morphine across time and di- verse pain models had not been previously reported. Beyond our initial studies, nothing is known regard- ing the spinal mechanistic underpinnings of this multi-month exaggeration of neuropathic pain by a brief exposure to morphine restricted to the early post-trauma period. Three Aims are proposed. All studies are undertaken in both sexes, given that documented male/female differences in immune and glial function, neuropathic pain, and responses to opioids, suggest that distinct un- derlying mechanisms will likely be found across sexes. The first Aim examines how a short course of morphine in the early post-trauma period functionally modifies the neuroimmunology of the ipsilateral lumbar dorsal spi- nal cord and discovers which of these changes mediate pain enhancement. The second Aim utilizes state-of- the-art Robust Activity Marking (RAM) technologies in spinal cord to address how identified mediators of mor- phine-induced pain enhancement align with retrogradely labeled spinothalamic neurons with defined activation state. The third Aim examines supraspinal mechanisms contributing to morphine-induced chronification of neu- ropathic pain. Aim 3 utilizes state-of-the-art DREADD reversible inactivation of microglia vs. excitatory neurons to define the role of the caudal granular insular cortex (CGIC), which we have previously shown (in the ab- sence of early post-trauma morphine) to be critical to chronic pain maintenance. Here we will reversibly inhibit, in a cell-type targeted fashion, either microglia or excitatory neurons in CGIC either only during morphine dos- ing or only during the period of morphine-induced chronification of pain to define CGIC involvement in induction versus maintenance of this enhanced neuropathic pain state.