Donna L. Hammond - US grants

Previous studies of the pharmacologic and physiologic mechanisms of antinociception have focused primarily on the involvement of opioid peptides and monoamines. Although several lines of evidence implicate GABA in the modulation of nociception, limitations in our understanding of the pharmacology of GABA receptors and a paucity of drugs with which to manipulate these receptors have severely hampered studies of the role of GABA to date. However, recent advances in the pharmacology of both GABAA and GABAB receptors now provide the tools with which to more thoroughly characterize the role of GABA in antinociception. This proposal will systematically characterize involvement of GABAA and GABAB receptors in the modulation of nociception by neurons in three regions of the CNS for which the pharmacologic and physiologic mechanisms of antinociception have been best characterized: the spinal cord, n. raphe magnus and n. reticularis gigantocullaris pars alpha. These studies will first assess the effects of GABAA and GABAB agonists and antagonists on nociceptive sensitivity and motor function following intracerebral microinjection and intrathecal injection. The pharmacologic specificity of these effects will then be determined. With respect to the GABAA receptor, these studies will determine whether benzodiazepine agonists in a manner consonant with GABAA receptor pharmacology. Of particular interest will be the determination of (1) whether GABAA receptors involved in nociception are linked to a benzodiazepine modulatory site as it now appears that not all GABAA sites are linked to benzodiazepine sites and (2) whether a specific subtype of the GABAA receptor mediates antinociception. With respect to the GABAB receptor, these studies will determine, using the newly introduced GABAB antagonists, whether the antinociception produce by baclofen is truly mediated by a GABAB receptor or by a "baclofen" receptor. Finally, this proposal will also examine the pharmacology of the neuronal pathways that mediate the effects of GABAA and GABAB receptor agonists and antagonists on nociception. Using a multidisciplinary approach, these studies will determine whether GABAergic modulation of nociception is mediated by activation or inhibition of a bulbospinal serotonergic projection originating in the n. raphe magnus. The results of these studies will provide new insights into non-opioid mechanisms of antinociception.

DESCRIPTION (provided by applicant): By comparison to the primary afferent and dorsal horn neurons, our understanding of the mechanisms by which peripheral inflammatory injury leads to sustained changes in the function and properties of brainstem neurons that modulate nociception remains rudimentary. This laboratory was the first to identify that persistent inflammatory nociception enhances the antinociceptive and anti-hyperalgesic effects of MOR and DOR agon- ists in the brainstem, providing early direct evidence that persistent inflammatory nociception alters the phar- macology and physiology of bulbospinal pain modulatory neurons. Whole-cell patch-clamp recordings from RVM neurons have now identified two populations of spinally-projecting RVM neurons, one serotonergic and the other non-serotonergic, in which the postsynaptic inhibitory effect of a MOR agonist is enhanced in CFA- treated rats. These neurons also exhibit alterations in their passive membrane properties or spontaneous ac- tivity, and the strength of glutamatergic inputs to the non-serotonergic neurons is greatly increased in CFA- treated rats. We hypothesize that these neurons correspond to two populations of pain facilitatory neurons. We now propose to investigate the presynaptic mechanisms by which MOR and DOR agonists act in the RVM to produce anti-hyperalgesia. The first specific aim will use whole-cell patch clamp recording from retrogradely labeled, immunohistochemically identified RVM neurons to test three hypotheses: 1) that persistent inflamma- tory nociception increases excitatory drive to specific populations of spinally-projecting RVM neurons; 2) that it enhances the ability of MOR, and possibly DOR, agonists to inhibit excitatory drive to these neurons; and 3) that the mechanism of opioid enhancement entails an upregulation of MOR, but not DOR, on glutamatergic afferents to these neurons. The second specific aim will also use whole-cell patch clamp recording to test three complementary hypotheses: 1) that persistent inflammatory nociception differentially alters inhibitory drive to specific populations of spinally-projecting RVM neurons, 2) that it increases the ability of MOR and DOR agon- ists to inhibit inhibitory drive to these same neurons; and 3) that the mechanism of enhancement entails a dif- ferential upregulation of MOR and DOR on GABAergic terminals to these neurons. Immunohistochemistry will be used to quantitate colocalization of MOR or DOR immunoreactivity with vGLUT, a marker of glutamatergic terminals, or with vGAT, a marker of GABAergic terminals, in the RVM of saline- and CFA-treated rats to ob- tain complementary anatomical data to support or refute these hypotheses. The outcome of these experiments will be a mechanistic framework for the antinociceptive and anti-hyperalgesic effects of opioids in the RVM. In turn, through studies of this class of analgesic we will be better able to identify the function of the different types of RVM neurons that are critically involved in the nociception. These data will advance our understanding of how peripheral inflammatory injury alters the responses and function of critical brainstem pain modulatory systems and inform a more rationale development of centrally-acting analgesics for the relief of persistent pain. PUBLIC HEALTH RELEVANCE Persistent pain of an inflammatory nature, such as that associated with arthritis or soft tissue injury, exacts a significant financial, emotional and physical toll on its sufferers. The results of these studies will identify how persistent pain changes the function of brainstem pathways that are critically involved in the regulation of nociception and the production of analgesia. Insights gain from this work will guide the development of new, more effective pharmacotherapies or cognitive approaches for the relief of persistent pain.

DESCRIPTION (Adapted From The Applicant's Abstract): Recent studies have revealed an unexpected plasticity of afferent pain pathways following neuropathic or inflammatory injury, and concomitant changes in the efficacy of opioid analgesics. Previous investigations of the effects of injury limited their examination to neurons in the periphery and the spinal cord. Consequently, very little is known about the effects of injury at the supraspinal level and, in particular, about the effects of inflammatory nociception on the pain modulatory pathways that originate in the pons or medulla. Neurons in NRM and NGCp alpha of the ventromedial medulla that project to the spinal cord comprise one of the important, common efferent pathways for the modulation of nociception, and are also implicated in the production of antinociception by opioid receptor agonists. Recent studies in this laboratory indicate that the antinociceptive effects of DAMGO, a mu opioid receptor agonist, are enhanced after microinjection in the NRM or NGCp alpha of rats with monoarthritis induced by intraplantar injection of CFA. However, the mechanism for this enhancement is unknown. This application proposes to use neuroanatomical and molecular neuroanatomical techniques to identify the mechanisms responsible for this enhancement. Its specific aims are to: Use in situ hybridization histochemistry to identify alterations in the expression of mRNA for the endogenous opioid peptides and their receptors as a function of time after injury. Use immunocytochemistry to identify corresponding alterations in the number and distribution of neurons immunoreactive for these peptides and their receptors. These experiments will provide new insights into the effects of inflammatory nociception on brainstem neurons that comprise an important efferent pathway for the modulation of nociception and a key site of action for opioid analgesics. Moreover, the findings of these experiments will be strengthened by our ability to relate these alterations to the results of ongoing pharmacological investigations of the effects of opioid receptor agonists and antagonists in the ventromedial medulla after inflammatory injury.

DESCRIPTION (provided by applicant): This application seeks renewal of support for an interdisciplinary program of training in pain research at the University of Iowa. The Program has been highly successful in terms of increasing collaborations among trainers and mentoring pre- and postdoctoral trainees of exemplary quality to independent funding. The program will continue its focus on the cellular, molecular, pharmacological, physiological and anatomical mechanisms that subserve acute and chronic pain after injury. The 18 program faculty are NIH-funded investigators with complementary expertise in the fields of molecular, cellular and systems neurobiology. The trainers comprise a balanced representation of basic and physician scientists, as well as bench and patient-based research. Each trainer has a strong record of training both predoctoral and postdoctoral fellows and will also mentor a subset of junior trainers of great promise. Support is requested for two pre-doctoral trainees and two postdoctoral trainees for this award period. In line with our goal to facilitate translational research, the postdoctoral positions will be primarily targeted for M.D., M.D. Ph. D., R. N. Ph. D., P. T. Ph. D. or D. P. T. trainees. These individuals will particularly benefit from a period of protected time to learn the elements of research and/or to restart their research programs in advance of a faculty level appointment. A cadre of nurse scientists with established clinical and translational research programs in pain has been recruited to strengthen translational aspects of the training program. The training program provides a highly structured and diverse program of didactic coursework, including monthly seminars, weekly journal clubs and bi-weekly work - in - progress meetings, that is coupled with research training in a highly collaborative and interactive environment. All courses, as well as mechanisms of interaction and trainee evaluation are established. The overall goal of the training program is to provide our trainees with (1) an individualized curriculum that provide a solid knowledge base appropriate to their career plans; (2) rigorous training in the selements of scientific investigation including the formulation of research hypotheses, experimental design and analysis; (3) mastery of two to three research techniques; (4) opportunities to develop their verbal communication skills; (5) experience in the construction of manuscripts and grant proposals;and (6) the ability to interact and collaborate with basic and clinical investigators both within and outside the instition who are actively engaged in research. Trainees in this program will acquire the knowledge, experience, and skill sets necessary for successful transition to an independent research career in academia, industry of government.

DESCRIPTION (provided by applicant): Loss of GABA-mediated inhibition in the spinal cord is proposed to contribute to the development and maintenance of neuropathic pain. However, the supporting data are surprisingly weak. Further, these studies have not considered the complex molecular biology of GABA receptors, and how subunit composition and pharmacology change in disease states and under conditions of repetitive activity as occurs after nerve injury. We hypothesize that nerve injury causes immediate, as well as long-term changes in the expression, distribution and subunit composition of GABAA or GABAB receptors in the spinal cord. We propose to characterize these changes in the context of defined presynaptic and postsynaptic elements of the afferent pain pathways and a well-characterized model of spinal nerve ligation that exhibits time-dependent changes in neuropathic pain behaviors. Of particular importance are the changes induced in the expression and molecular composition of these receptors on uninjured, and on injured primary afferent neurons and on dorsal horn neurons as a function of time after nerve injury. Changes in the expression and subunit composition of GABAA and GABAB receptor subunits in the spinal cord dorsal horn, and in ipsilateral and contralateral L4 and L5 DRG of ligated and sham rats will be determined by Western blot. Two-color indirect immunofluorescence methods will be used with stereological measurements to examine the distribution of GABAA and GABAB receptor subunits on different populations of immunohistochemically-identified primary afferent neurons in the L4 and L5 DRG of ligated and sham-operated rats. Immunohistochemical methods will also be used in conjunction with retrograde labeling of spinothalamic and spinoparabrachial neurons in ligated and sham rats to determine whether the distribution and composition of postsynaptic GABAA and GABAB receptors in the spinal cord dorsal horn is also altered after nerve injury. Time points for analysis will range 7 to 140 days after injury. These studies will provide new information about the distribution of GABAA and GABAB receptors on identified populations of primary afferent neurons and dorsal horn neurons in the naive animal. They will describe how the distribution and molecular composition of these receptors changes as a function of time after nerve injury and provide new insights into the molecular and neuroanatomical bases of inhibitorv svnaptic transmission in the spinal cord.

DESCRIPTION (provided by applicant): Loss of GABA-mediated inhibition in the spinal cord is proposed to contribute to the development and maintenance of neuropathic pain. However, the supporting data are surprisingly weak. Further, these studies have not considered the complex molecular biology of GABA receptors, and how subunit composition and pharmacology change in disease states and under conditions of repetitive activity as occurs after nerve injury. We hypothesize that nerve injury causes immediate, as well as long-term changes in the expression, distribution and subunit composition of GABAA or GABAB receptors in the spinal cord. We propose to characterize these changes in the context of defined presynaptic and postsynaptic elements of the afferent pain pathways and a well-characterized model of spinal nerve ligation that exhibits time-dependent changes in neuropathic pain behaviors. Of particular importance are the changes induced in the expression and molecular composition of these receptors on uninjured, and on injured primary afferent neurons and on dorsal horn neurons as a function of time after nerve injury. Changes in the expression and subunit composition of GABAA and GABAB receptor subunits in the spinal cord dorsal horn, and in ipsilateral and contralateral L4 and L5 DRG of ligated and sham rats will be determined by Western blot. Two-color indirect immunofluorescence methods will be used with stereological measurements to examine the distribution of GABAA and GABAB receptor subunits on different populations of immunohistochemically-identified primary afferent neurons in the L4 and L5 DRG of ligated and sham-operated rats. Immunohistochemical methods will also be used in conjunction with retrograde labeling of spinothalamic and spinoparabrachial neurons in ligated and sham rats to determine whether the distribution and composition of postsynaptic GABAA and GABAB receptors in the spinal cord dorsal horn is also altered after nerve injury. Time points for analysis will range 7 to 140 days after injury. These studies will provide new information about the distribution of GABAA and GABAB receptors on identified populations of primary afferent neurons and dorsal horn neurons in the naive animal. They will describe how the distribution and molecular composition of these receptors changes as a function of time after nerve injury and provide new insights into the molecular and neuroanatomical bases of inhibitorv svnaptic transmission in the spinal cord.

DESCRIPTION (provided by applicant): Our understanding of the mechanisms by which inflammatory injury leads to sustained changes in the function and properties of pain modulatory neurons in the rostral ventromedial medulla (RVM) remains rudimentary. Substance P (SubP) contributes to central sensitization after injury, yet surprisingly little is known about its role in the modulation of nociception by the RVM, where it also exists in high concentrations. Pilot data indicate that SubP has both antinociceptive and pronociceptive actions in the RVM in the uninjured state, but acts to mediate and sustain hyperalgesia after inflammatory injury. These data support four related hypotheses. 1) SubP exerts both pronociceptive and antinociceptive effects in the uninjured state by time-dependent activation of pain inhibitory and pain facilitatory bulbospinal pathways. Behavioral pharmacological studies will establish the dose-dependence and duration of SubP effects in the RVM of rats in the uninjured state, and confirm the role of neurokinin-1 (NK1) receptors. The bulbospinal pathways that mediate the effects of SubP will be determined by challenge with spinally-administered receptor antagonists before and at various times after SubP injection. 2) SubP is released in the RVM in response to inflammatory injury, where it plays a pronociceptive role in the development of hyperalgesia. The role of endogenous SubP released in the RVM after injury will be assessed by NK1 receptor internalization and by microinjection of NK receptor antagonists in the RVM of rats with acute and persistent hyperalgesia induced by complete Freund's adjuvant (CFA). 3) SubP effects reflect the expression of the NK1 receptor by specific types of RVM neurons, a pattern that may change after injury. Tract tracing and immunohistochemistry will identify RVM neurons that express NK1 receptors by their neurotransmitter content and projections to the spinal cord and DLPT. Subsequent studies will determine whether this expression changes after CFA, and will identify the types of RVM neurons in which NK1 receptor internalization (indicative of SubP release) occurs after injury. 4) SubP acts at specific populations of spinally-projecting RVM neurons and, in CFA-treated rats, enhances excitatory inputs to specific types of RVM neurons to mediate hyperalgesia. Whole-cell patch clamp recording from RVM neurons, coupled with retrograde labeling and immunohistochemical staining, will identify which types of RVM neurons express functional NK1 receptors and determine how the actions of SubP change after CFA. Extracellular recordings will determine the effect of SubP on ON, OFF and NEUTRAL cells and its role in the sensitization of these neurons after CFA. These studies will provide a mechanistic framework in which the antinociceptive and pronociceptive effects of SubP are related to specific populations of RVM neurons. These data in turn may enable us to identify their function (pro- vs antinociceptive). Collectively, these results will advance our understanding of the means and mechanisms by which peripheral inflammatory injury alters the responses and function of critical brainstem pain modulatory systems, and inform a more rationale development of centrally-acting analgesics for the relief of persistent pain. PUBLIC HEALTH RELEVANCE Persistent pain of an inflammatory nature, such as that associated with arthritis or soft tissue injury, exacts a significant financial, emotional and physical toll on its sufferers. The results of these studies will identify how persistent pain changes the function of brainstem pathways that are critically involved in the regulation of nociception and the production of analgesia. Insights gain from this work will guide the development of new, more effective pharmacotherapies or cognitive approaches for the relief of persistent pain.

DESCRIPTION (provided by applicant): The virtual slide scanner and quantitative software is absolutely essential in allowing pathology truth to define imaging truth. We are funded through NIH grants to undertake this important task for lung and prostate cancer in the human. The proposed instrumentation, the Olympus VS120 slide-scanning microscope with VisioMorph newCAST software is critical for the completion of several very important NIH funded research projects and NIH funded educational grants at the University of Iowa. Some of the immediate applications include distinguishing malignant from benign lung nodules, 3D reconstruction imaging of mouse, ferret and pig lungs as a baseline for animal models in cystic fibrosis animals, defining the development of cystic fibrosis phenotype in the pancreas. The successful completion of Dr. Henry's research will lead to identification of patients at risk for lethal prostte cancer at stages early enough for effective treatment. Dr. Hammond seeks clarification of the role of inflammation in chronic pain leading to enhanced therapies for a higher quality of life for chronic pain patients. Dr. Dunnwald and Dr. Murray are analyzing the role of the protein Irf6 in immune cells associated with wound healing. Dr. Bonthius will test the efficacy of gene therapy against Alexander's disease, a disease with devastating and lethal affects on infants and children. The virtual slide scanner will also support the microscopy component of a current National Library of Medicine funded educational research and resource proposal that is aimed at increasing the efficiency and cost effectiveness of laboratory test ordering in clinical medicin. Updating and expanding the Virtual Slidebox for medical, dental and graduate students. The Virtual Slidebox increases accessibility, student satisfaction and efficiency of learning. This database is also used to assess the diagnostic competency of resident physicians. Enhanced medical student and resident education will continue to improve the lives and health of countless patients.

DESCRIPTION (provided by applicant): Chronic pain is a significant issue in health care delivery, affecting 116 million Americans at a conservative cost of $560 billion. This application seeks renewal of support for an interdisciplinary training program in pain research at the University of Iowa. The program has been highly successful in terms of increasing collaborations among trainers and mentoring pre- and postdoctoral trainees of exemplary quality to independent funding and careers. The program will continue to study the cellular, molecular, pharmacological, physiological and anatomical mechanisms that subserve acute and chronic pain while further strengthening the clinical and translational work begun during the previous award. The 16 core program faculty are NIH-funded investigators with complementary expertise in the fields of molecular, cellular and systems neurobiology. They comprise a balanced representation of basic and physician scientists, as well as bench and patient-based research. Each trainer has a strong record of training both predoctoral and postdoctoral fellows and will also mentor a subset of junior trainers of great promise. Support is requested for two predoctoral and two postdoctoral trainees for this award period. In line with our goal to strengthen translational research, the postdoctoral positions will be primarily targeted for MD, MD/PhD, PT/PhD or DPT trainees, but will also accept PhD trainees with a strong interest in translational research. These individuals will benefit from a period of protected time to learn the elements of research and/or to restart their research programs in advance of a faculty level appointment. Two nurse scientists continue as adjunct trainers to pursue collaborations in clinical and translational research programs developed in the last award period. In the next funding period, the program proposes to build a stronger relationship with members of the Institute for Clinical and Translational Science who have expertise in patient-centered outcomes research and comparative effectiveness research. This approach will recruit new faculty to the training program and increase attractiveness to clinicians. The training program provides a highly structured and diverse program of didactic coursework, including monthly seminars, weekly journal clubs and biweekly work-in-progress meetings, that is coupled with research training in a highly collaborative and inter-active environment. All courses, as well as mechanisms of interaction and trainee evaluation are established. The overall goal of the training program is to provide our trainees with (1) an individualized curriculum that provide a solid knowledge base appropriate to their career plans; (2) rigorous training in the elements of scientific investigation including the formulation of research hypotheses, experimental design and analysis; (3) mastery of two to three research techniques; (4) opportunities to develop their verbal communication skills; (5) experience in the construction of manuscripts and grant proposals; and (6) the ability to interact and collaborate with basic and clinical investigators boh within and outside the institution who are actively engaged in research. Trainees in this program will acquire the knowledge, experience, and skill sets necessary for successful transition to an independent research career in academia, industry or government.

DESCRIPTION (provided by applicant): The aim of this training program is to continue to produce physician-scientists who will advance understanding of human health and disease and enhance delivery of effective clinical care. The Iowa Medical Student Research Program achieves this goal through fostering innovation, professional scientific interactions, mentorship, training in research ethics and communication and development of a critical approach to one's own and peers' scientific work. The University of Iowa Carver College of Medicine (CCOM) has a long history supporting medical student research with a successful and growing research- training program that began over 40 years ago. The NIH has continuously funded the program since 1980. Under the auspices of the training grant, students are given the opportunity to be involved in a research project of their choosing for 12 weeks in the summer preceding or following the first year of medical school. Students choose a mentor from any of 20 clinical and 6 basic science departments in the Carver College of Medicine, or the Colleges of Public Health, Pharmacy, or Liberal Arts and Sciences. Over 150 faculty mentors with ideas for short-term projects are listed on a searchable online database from which students may sort options by department, type of research or by keyword. Students are required to submit a proposal, which is competitively reviewed by the Research Committee of the CCOM. Student applications are rank- ordered by the score received and those with the best scores are chosen for the 32 NIH training stipends with another 50 stipends funded by the CCOM, individual departments and other sources. The program exposes the students to the entire research process, from writing a research proposal, to working with their mentor to submit their research accomplishments in the form of an abstract and presentation of their research at the annual Medical Student Research Day. This experience permits students to acquire skills such as application of scientific methods, critical evaluation of previous related experimental work, and statistical approaches to data analysis. At the same time, they acquire knowledge and develop skills related to a specific research project while working closely with a faculty role model and mentor. The program has led to a substantial number of student presentations at national meetings and numerous publications in peer-reviewed journals. There has been steady interest in continuing involvement in research throughout medical school on the Research Distinction Track, an honors program for CCOM students with a sustained commitment to research. Graduates repeatedly cite the summer research fellowship experience as the basis for their choice of specialty or decision to conduct basic, clinical or translational research as faculty in academic medical centers. The long-term impact of this program is the nationwide dissemination of a cadre of physicians, who developed an appreciation for team-based science early in their career, equipping them to make evidence-based decisions and advance healthcare quality.

? DESCRIPTION (provided by applicant): This is a new application from the University of Iowa (U Iowa) for an R25 Post-baccalaureate Research Education Program (PREP). It draws upon the collective talent and commitment of >40 experienced faculty to provide an exceptional experience to 10 talented participants each year who are members of under- represented (UR) groups. This application is based on an innovative employment initiative undertaken two years ago that enabled recent college graduates with career aspirations in the health professions and biomedical sciences to secure research positions in laboratories for 1-2 years without competing for positions with career research staff. The program has been a great success. With this experience, we are now positioned to develop a program specific to individuals interested in the PhD or MD/PhD. This mechanism will provide the financial support, infrastructure, resources, and programmatic activities necessary to enhance the qualifications and experiences of its participants, making them highly competitive for top-tier graduate programs - including those at U Iowa. The program includes an introductory class in fundamental laboratory skills, followed by two 3-week research rotations, selection of a laboratory and identification of a research problem, and submission of an individual development plan that is revisited quarterly. Participants will attend monthly career development workshops, weekly scientific seminars and laboratory meetings, present updates on their research at quarterly work-in-progress meetings, and present their research at multiple local and one national meeting. They will take one graduate level course in the fall and spring semester, and will be provided a GRE preparatory course. Each participant will have a research committee comprised of their mentor, a faculty member from the Advisory Committee, and another faculty member so that knowledgeable letters of recommendation can be written on their behalf. Participants will be carefully mentored through the graduate school application and interview processes. In order to maximize the mentoring experience and foster learning environments that are conducive to inclusive excellence, mentors will participate in professional development seminars that will focus on cross-cultural mentoring. In addition, the institution will cover the costs of visits by five gradute program directors each year to highlight the program, showcase its participants and develop pipelines to top programs. The participants will be integrated into the activities of first-year graduate students. Metrics of success for the initial 5 years include the number of participants who apply for and are accepted in a graduate program, that author or co-author a publication, the number of invitations received to interview and the quality of those programs, and the number who eventually secure independent funding by appointment to T32 or F31 mechanisms. Mentors, participants and programs into which the participants matriculate will be surveyed annually for feedback and as a means to track the career paths of our participants. The program will undergo continual assessment so that it can adapt to the needs of the participants.

Chemotherapy is frequently accompanied by peripheral neuropathies so painful and debilitating that it may be necessary to reduce the dose of agent, delay one or more cycles of chemotherapy, or even cease treatment. Furthermore, the peripheral neuropathies can worsen after treatment has ended and may not resolve with time. Both the dose-limiting nature and the persistence of chemotherapy-induced peripheral neuropathies (CIPN) are significant health care problems. There are few - if any - evidence-based therapies for the relief of CIPN. The long term goal of this research team is to identify a new strategy to alleviated CIPN in cancer patients, resulting in a meaningful improvement in their quality of life and the ability to sustain better and longer treatment. Here we propose a phase II clinical trial of the efficacy of nicotinamide riboside (NR), a naturally occurring vitamin precursor of NAD+, as a therapeutic agent. This proposal is based on the hypothesis that paclitaxel injures primary afferent neurons by reducing NAD+ in these neurons and their fibers. Our preclinical data demonstrate that paclitaxel does indeed decrease levels of NAD+ in dorsal root ganglia and that NR protects neuronal NAD+ in the face of intense chemotherapy treatment. Moreover, pre- treatment with NR protects against, and post-treatment with NR reverses, the mechanical hypersensitivity and the aversive dimension of pain in a rodent model of CIPN induced by paclitaxel. The specific aim of this proposal is to evaluate if daily dosing of NR prevents progressive worsening of sensory neuropathy in patients undergoing adjuvant or neo-adjuvant therapy with paclitaxel or nab-paclitaxel for the treatment of stage 1-3 breast cancer. The primary outcome measure is the change in the grade of sensory neuropathy between the baseline assessment and the 8th day of NR treatment after the final (i.e.12th) taxane infusion as determined by the CTCAE questionnaire. Secondary outcome measures are incidence of dose reduction, actual vs planned cumulative dose of paclitaxel administered, percentage of patients with dose reduction, and patient-reported function. This trial will be an open-label study in which all patients receive NR. Comparisons will be made to historical data on severity and progression in this population. Additional analyses will confirm that NR increases levels of NAD+ or its metabolite NAAD in blood, and does not interfere with the pharmaco- kinetics of paclitaxel. This phase II clinical trial will provide preliminary data as to whether NR treatment prevents a progressive worsening of CIPN and enables patients to complete the chemotherapy treatment without a reduction in dose of paclitaxel. It will also generate preliminary data correlating efficacy to an increase in NAD+ levels. This work will drive large Phase III studies of NR in the treatment of CIPN, and the discovery of a new pharmacotherapy for a significant unmet need in oncology. The findings have great potential to transform the practice of oncology with a therapy that results in a meaningful improvement in the patient's quality of life and the ability to complete optimal chemotherapy treatment regimens.

Two highly significant and challenging health care problems converge in the comorbidity of chronic pain and smoking. Chronic pain affects ~116 million persons. Among those with chronic pain, the prevalence of smoking (~50%) is more than twice that in the general population (~21%). This association is alarming. Not only is smoking the leading preventable cause of mortality, but clinical studies clearly demonstrate that smoking exacerbates both the intensity and associated impairments of chronic pain. Moreover, smoking rates are positively correlated with pain severity and its resulting functional impairment. Clinical observations suggest that chronic pain and nicotine use are intertwined in a positive feedback loop. Individuals smoke to relieve their pain, smoking exacerbates the pain, and individuals in turn continue to smoke or smoke more in an effort to alleviate the pain. In addition, persistent pain may diminish the rewarding properties of nicotine. With the rapid acceptance of ?vaping? nicotine as a ?safer? alternative to smoking, the comorbidity of nicotine use and chronic pain will persist as a significant health care problem. Systematic studies of the mechanistic basis for the comorbidity of nicotine use and chronic pain are lacking in the preclinical literature. This explora- tory R21 proposal seeks to test the hypothesis that chronic exposure to nicotine enhances the somatosensory components of pain, exacerbates on-going pain and increases the averseness of noxious stimulation following peripheral injury. To do so, it will use a new method for chronic, intermittent administration of nicotine by inhalation that mimics the pharmacokinetics of nicotine in smokers. It will determine how prior intermittent exposure to nicotine alters the organism's response to peripheral nerve or inflammatory injury using a battery of tests that assess heat hyperalgesia, mechanical hypersensitivity, on-going pain and the aversiveness of noxious stimulation. These measures will be made in the period immediately before injury, shortly after injury (acute pain) and again two weeks later (chronification of pain). In a complementary aim, autoradiography will be used to quantitate ?4?2 and ?7 nicotinic acetylcholine receptors, and to determine how chronic nicotine, chronic pain and the combination of chronic nicotine and chronic alter these receptors. The dorsal horn, as well as cortical, midbrain, pontine, and medullary nuclei implicated in reward or in pain modulation will be surveyed. These studies will be conducted in male and female rats to investigate potential sex differences given that the prevalence of chronic pain is greater in women and women find nicotine more rewarding. Successful interventions against chronic pain in those who smoke or vape nicotine will require a much better understanding of the pharmacological and physiological bases for the feedforward relationship. Validation of a preclinical model of concomitant chronic pain and nicotine use is a necessary first step, and will provide foundational data to drive additional mechanistic studies. Insights gained from this work will also guide new approaches to abrogate the chronification of pain in other populations as well.

This project seeks to better understand the mechanisms that underlie painful peripheral neuropathy, and particularly to identify a new strategy to relieve chemotherapy-induced peripheral neuropathies (CIPN) for which there are no efficacious treatments. Indeed, CIPN may be so painful that it is necessary to reduce the dose of agent, delay chemotherapy, or even cease treatment. Furthermore, the peripheral neuropathies may not resolve with time. The dose-limiting nature and the persistence of CIPN are significant health care prob- lems. Much research in the field is driven by hypotheses that loss of intraepidermal nerve fibers (IENF), injury to primary afferent neurons and activation of glial cells and macrophage in the dorsal root ganglion (DRG) underlie taxane-induced CIPN, including a focus on mitochondrial dysfunction. Without an agent that can prevent mechanical hypersensitivity and the aversive dimension of pain, it is not possible to establish that these mechanisms are causative of CIPN or to test the hypothesis that mitochondrial dysfunction is an underly- ing mechanism. We propose to use nicotinamide riboside (NR), a natural product vitamin B3 precursor of NAD+ that can correct mitochondrial dysfunction, as an investigational tool. We show that a dose of NR that increases NAD+ levels can ameliorate the mechanical hypersensitivity and the aversive dimension of pain caused by paclitaxel in tumor-naïve female rats. In contrast, NR does not alleviate sensory neuropathy result- ing from frank nerve injury. This differential effect opens the door to rigorous comparative analyses of the mechanisms by which NR acts to alleviate sensory neuropathy. This proposal will first extend the findings with NR to paclitaxel-induced CIPN in tumor-bearing female rats. Second, it will confirm that NR does not suppress neuropathy in rats with SNI. Third, it will relate the differential actions of NR to injury specific changes in NAD+ biosynthetic enzymes that position DRG neurons in paclitaxel treated rats to make use of NR to correct defi- cits, whereas this pathway is not available after SNI, and support these studies with measurement of NAD+ in the DRG and distal nerve. Fourth, it will characterize the distribution of transcripts and protein for the biosyn- thetic enzymes among the different classes of DRG neurons and determine how they are altered by paclitaxel in tumor naïve and tumor-bearing rats, as well in SNI rats. Fifth, it will determine whether NR prevents the loss of IENF, loss of specific neuron populations, as well as activation of glial cells and macrophages in the DRG. The proposed studies will test the hypothesis that the protective effects of NR are mediated by NAD+ and link this to its protective effects on specific subpopulations of primary sensory neurons, the prevention of IEFN loss, and activation of macrophages and satellite cells in DRG. This work will increase our understanding of the mechanisms that underlie different types of painful sensory neuropathy, possibly guide the development of a new treatment to address a significant unmet need in oncology, and just as importantly provide a rationale as to why this natural product may not be a universal treatment for all types of sensory neuropathy.