Facial Pain Research Foundation 2016 Research Progress
Compiled and submitted April, 2017 by Douglas K. Anderson, Ph.D., Eminent Scholar Professor and Chair Emeritus, Department of Neuroscience, University of Florida College of Medicine; Director of Research and Trustee, Facial Pain Research Foundation
Trigeminal Neuralgia (TN), which is considered to be is one of the most painful afflictions known to humans and is one example of neuropathic pain which is pain caused by injury to or malfunctioning of the nervous system. Despite the debilitating effects chronic pain has on the activities of daily living of individuals with painful conditions like TN and on the cost these afflictions add to our health care system (greater than $100 billion/year), research funding from federal agencies, primarily the National Institutes of Health (NIH) and pharmaceutical companies continues to be insufficient.
This inadequacy of funding from traditional sources has led the growing realization that discovering the root cause of and treatment for TN and other neuropathic pain conditions is going to have to be accomplished with funding from the private sector. To this end, The Facial Pain Research Foundation (FPRF) was created in January, 2011 to provide the critical elements that are necessary and sufficient to study these facial pain conditions.
Consequently, the sole mission of the FPRF is “…to establish a well-funded translational (i.e., fundamental discovery to clinical application) research continuum that is dedicated to identifying the mechanisms underlying neuropathic facial pain and to develop novel new therapeutic strategies that will permanently stop the pain of TN and related neuropathic pain syndromes.”
Since its inception a little over six years ago,the FPRF has raised $3,345,021 to support five separate and distinct research projects. The FPRF used three fundamental criteria in choosing which proposals would be considered for funding: (1) the projects were novel and unique; (2) the investigator(s) responsible for each project are among the leading researchers in their respective fields; and (3) all of the projects were not “cut from the same cloth”, i.e., there was significant diversification in the research strategies among the projects.
It is important to note that these FPRF funds have also served in a multiplier capacity in that some of our investigators have used FPRF support as seed funding to generate the necessary preliminary data which contributed to these investigators acquiring large grants from the NIH. The purpose of this yearly update is to briefly summarize these five projects and to highlight the progress that each achieved in 2016.
Investigating Protein-Lipid Interactions in Peripheral Nerve Myelin
Lucia Notterpek, Ph.D., Professor and Chair, Department of Neuroscience, University of Florida College of Medicine
Project 1: Cholesterol homeostasis in peripheral nerve myelin
During the past year, Dr. Notterpek and her research team have made excellent progress on their cholesterol homeostasis project. As a result, various team members have given presentations on their findings at several national meetings. Dr. Notterpek continues herinvestigation of the proteins and lipids that make up peripheral nerve myelin. For these studies, she used (and continues to use) a genetic animal model that allows for the examination of compression induced peripheral neuropathies. This model was created by deleting the peripheral myelin protein 22 (PMP22) gene which causes abnormalities in the myelin membrane, that makes it predisposed to compression-induced degeneration. Dr. Notterpek found that a normal functioning PMP22 is responsible for forming specific microdomains (clusters of lipids and proteins in membranes called lipid rafts) in the Schwann cell membrane, that are critical for myelin stability. These microdomains are enriched in cholesterol and the membrane localization appears to depend on the correct expression of PMP22.
To corroborate these findings, they have inserted mutated amino acids in the PMP22 protein that have been predicted to be critical for cholesterol binding and are now in the midst of examining the properties and functionability of these mutated amino acid carrying PMP22 proteins in living cells. During their study of the PMP22-deficient mice, they detected systemic alterations in lipid metabolism in neuropathic animals which were associated with pathology in the liver. Further study produced preliminary data which suggested that this liver pathology caused an inflammatory response that could amplify the nerve pathology through circulating macrophages. To test if the liver and neuro-pathologies were linked, for six weeks they fed rats a diet high in “bad fats” that is known to induce cardiovascular disease in rodents. Exposure of the animals to this high fat diet indeed led to circulatory lipid abnormalities, and enhanced liver pathology, including inflammation. As expected, this bad high fat diet also enhanced the neuropathology, including motor and myelin defects.
With these intriguing preliminary results as a foundation, this past February, Dr. Notterpek submitted a large NIH grant proposal titled “Dysregulated lipid metabolism, a disease modifier in PMP22-dependent neuropathies.” The overall goal of this project is to (a) provide data to gain a better understanding of the heterogeneity in clinical presentation among hereditary neuropathic patients and (b) recognize the vulnerability of certain individual to compression induced neuropathies, such as in trigeminal neuralgia. They hypothesize that dyslipidemia is a disease- modifying comorbidity in neuropathic patients.
Current efforts are focused on assembling these results into a major manuscript, which will include some of the molecular mechanisms underlying the observed pathologies. The unexpected dysregulation of hepatic cholesterol metabolism with the induction of fatty liver and enhanced inflammation in neuropathic animals has opened a new field for future studies for Dr. Notterpek and her team which has clear translational significance. They also have a second manuscript planned, which will use molecular and cellular approaches in combination with electrophysiology to dissect how changes in lipids, particularly cholesterol, affect the biological properties of myelin.
Presentations in poster format:
Ye Zhou, Sooyeon Lee, Alex I. Fethiere, Hagai Tavori, Sergio Fazio, Angela Corona, Gary E. Landreth, Lucia Notterpek. Altered cholesterol trafficking in mice deficient in peripheral myelin protein 22
Ye Zhou, Alex I. Fethiere, Elliott Soto, Lucia Notterpek. Cholesterol homeostasis in neuropathic Schwann cells International Peripheral Nerve meeting July 2017
International Peripheral Nerve Meeting July, 2017
Zhou Y, Tavori H, Lee S, Al Salihi M, Fazio S, Notterpek L. Dysregulated lipid metabolism in the absence of peripheral myelin protein 22 (pmp22).
Project 2: Cholesterol homeostasis in peripheral nerve myelin, with a focus on statins
Myelin is an insulating sheath around nerves that promotes the electrical conduction of signals. It is composed of certain proteins and lipids, principally cholesterol, which helps stabilize the membrane. Most individuals diagnosed with the facial pain condition termed trigeminal neuralgia, have a localized region on their trigeminal nerve with evidence of varying degrees of demyelination.
Although the cause(s) for having this localized myelin damage is not known, the close proximity of the Superior Cerebellar Artery (with its strong pulsations) to the trigeminal nerve could be the trigger for or a substantial contributor to the evolution of this lesion with the subsequent development of debilitating pain in susceptible individuals. In addition, this demyelinating lesion could involve the use of cholesterol reducing drugs, such as statins. A survey conducted by the Instructional Management Systems (IMS) found that there are nearly 15 million Americans are currently taking a statin medication who after two years of continued use showed evidence suggesting that neuropathies could occur. Moreover, continued use of a statin increased the risk of developing Type 2 diabetes2. With serious side effects surrounding the use of statins, it is of great importance to understand the pathophysiology of this medication and its influence on myelin damage and diabetes.
Dr. Notterpek hypothesizes that in certain individuals, statins alter the stability of myelin making it prone to localized compression-induced demyelination. If correct, this linkage could explain the increasing rise of trigeminal neuropathy and other neuropathies in patients taking cholesterol-lowering medication. Statins inhibit the cholesterol-synthesizing enzyme, HMG-CoA reductase, which reduces the level of cholesterol synthesized by the liver. It can also cross the blood-brain barrier and is capable of inhibiting remyelination by blocking glial progenitor differentiation3—a reversible side effect once the subjects discontinue the use of statins.
To test their hypothesis of a statin-induced weakening of peripheral nerve myelin, they propose to use their laboratory tool kit to experimentally manipulate (elevate or reduce) the levels of cholesterol in glial cells from mice that are prone to the development of compression induced demyelinating neuropathy. In collaboration with neurologists, Dr. Notterpek proposes to nudge this project towards clinical application starting with this first step of performing a retrospective clinical data analysis to see if tghey can find any evidence of a link between the lipid and cholesterol profile of an individual and the development of trigeminal neuralgia or other demyelinating diseases.
The postdoc (Dr. Al Salihi) for this project started work on this project January 1, 2017 and is making good progress in learning the literature and experimental techniques.
1. The Link between Nerve Damage and Statin Drugs. (n.d.). Retrieved March 02, 2016, from http://articles.mercola.com/sites/articles/archive/2012/01/25/nerve-damage-with-cholesterol- meds.aspx
2. Hitman, G. A. (2014). Statins and diabetes. Diabet. Med. Diabetic Medicine, 31(6), 639-639. Retrieved March 2, 2016, from http://www.ncbi.nlm.nih.gov/pubmed/26892999
3. Miron, V. E., Zehntner, S. P., Kuhlmann, T., Ludwin, S. K., Owens, T., Kennedy, T. E., Antel, J. P. (2009). Statin Therapy Inhibits Remyelination in the Central Nervous System. The American Journal of Pathology, 174(5), 1880-1890. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2671276/
Mapping Towards a Cure: Identification of Neurophysiologic Signatures of Trigeminal Neuralgia Pain
John K. Neubert, D.D.S., Ph.D. (Project Coordinator, Animal modeling)
Mingzhou Ding, Ph.D. (Human imaging)
Marcelo Febo, Ph.D. (Animal imaging)
Robert M. Caudle, Ph.D. (Therapeutics)
University of Florida
Andrew H. Ahn, M.D., Ph.D., Eli Lilly Company, IN (Consultant)
John K, Neubert, D.D.S., Ph.D. continues to lead an impressive team of co-investigators (Mingzhou Ding, Ph.D., Robert Caudle, Ph.D., Marcelo Febo, Ph.D., Todd Golde, M.D., Ph.D. at the University of Florida (UF) and Evelyn F. and William L. McKnight Brain Institute (MBI) of the University of Florida. This talented group represents experts in the areas of orofacial pain, functional imaging, pharmacology, and viral vector therapeutics that is focused on identifying the neurophysiological signature of TN pain and provide promising new therapies.
This is a complex, translational project whose purpose is to determine if the neurobiology of TN in rats replicates or is very similar to the neurobiology of TN in humans. Using state of the art magnetic resonance (MR) scanners, the UF team is attempting to identify and compare in both TN patients and rats, the individual imaging patterns caused by the pain induced activation of different areas in the brain and spinal cord. The specific areas of the brain and spinal cord that “light up” or are activated by TN pain are called “signature” centers of activity. These signature centers will be the targets for treatments with any new therapies their team has identified and/or developed. In 2016, Dr. Neubert’s team made significant progress towards completing the Phase One.
Objectives of this project as described below.
The recruitment goal for the human imaging study was originally 25-30 subjects. To date, Dr. Neubert’s group has recruiting 16 subjects with 11 new subjects scanned last year (6 were diagnosed with TN1, 2 with TN2, and 3 with TN1/TN2). Working in a world-class environment at UF and the MBI allows the investigators to benefit from advances in techniques that were not available at the conception of the study. The Ding laboratory is now able to study free-water content in the brain of TN subjects to provide a potential biomarker for neuroinflammation. Dr. Neubert’s team has discovered promising regions of pain induced brain activation that warrant further investigation. In particular, a region of the brain (insula) involved as a gateway/relay station for pain processing in the brain show differences in free water content between TN subjects that had surgery as compared to those without surgery (Figs 1, 2). Collectively, these findings are important because they may provide insight into better diagnosing TN as well as providing new areas for the application of novel therapies. While preliminary, these results were sufficiently encouraging for them to increase their recruitment by additional 30-40 TN subjects. The FPRF and UF MBI have generously provided the additional funds needed to image these subjects in hopes of reaching their target in 2018.
Dr. Febo’s group has completed approximately 50 high-resolution fMRI scans of rats with various orofacial pain conditions and treated them with different therapeutic agents. Data presented in Figure 3A shows that nerve injury (CCI) significantly increased connectivity in the ventral tegmental area (VTA) as compared with rats receiving just a sham (control) procedure. This finding could cause Dr. Febo to focus on the VTA as a target for treatments. Note that the data generated by this study is both voluminous and complex and, thus, is very time consuming to analyze and interpret. Dr. Febo’s group is in the midst of processing this large data set. In a pilot study with Dr. Golde’s group, Dr. Neubert’s team has injected virus-vectors that can either turn-on or turn-off the pain system. Fig. 4 demonstrates that treatment with a substance called AAV-DREADDs to the trigeminal nerve can significantly increase orofacial pain. This is important because this is a critical step for developing a reliable orofacial pain model.
As with the first year of this project Dr. Neubert’s team continues to have issues regarding subject enrollment and staffing difficulties. To help increase enrollment, the FPRF has sent out emails to thousands of potential subjects. Additionally, they have agreed to help provide need-based travel funds for subjects to come to Gainesville, FL to participate in the study.
Plans for year 3:
Recruit more subjects to scan – plan to expand recruitment with travel support
- Study 50-100 animals using different trigeminal injury models
- Evaluate compounds from Dr. Golde’s and Dr. Caudle’s laboratories for pain relief and image the brains of these animals using MRI
- Establish the ability to do EEG & MRI studies in these animals
Cell Replacement Therapy as a Treatment for Injury-Induced Neuropathic Pain
Allan Basbaum, Ph.D., FRS, Professor and Chair, Department of Anatomy, University of California San Francisco
As described in a previous report, Dr. Basbaum’s laboratory has developed a powerful new approach to reversing the abnormal hyperexcitability that underlies many neuropathic pain conditions including trigeminal neuralgia. Although there are many explanations for the hyperexcitability that occurs after nerve injury, Dr. Basbaum’s laboratory has focused on the loss of inhibitory controls interposed between the peripheral nerve fiber input and the central nervous system circuits that transmit that input to the brain where pain is perceived. There is now considerable evidence that the loss of inhibitory control results from a significantly reduced function of a population of nerve cells that synthesize the inhibitory neurotransmitter, GABA. With these findings confirmed, Dr. Basbaum’s group took on the task of developing a strategy for rebuilding those lost or dysfunctional circuits. They chose a transplant paradigm and, to date, have documented the survivability and functionality of precursors of GABA nerve cells that have been transplanted into the spinal cord of a mouse model of neuropathic pain created with a hindlimb peripheral nerve injury. Results reported in a series of publications from Dr. Basbaum’s laboratory established that these nerve cells integrate incredibly well into host spinal cord circuits and restore inhibition, the result of which is a near complete loss of the hyperexcitability of spinal cord neurons and most importantly of the mechanical hypersensitivity that occurs in this neuropathic pain model.
Building upon these studies, Dr. Basbaum and his team have now turned their attention to a model of trigeminal neuropathic pain. In this model, there is a partial nerve injury of a major branch of the trigeminal nerve, namely the infraorbital nerve. Within days of the injury, the mouse develops hypersensitivity of the face region innervated by the infraorbital nerve. It is of particular interest that the test of hypersensitivity involves a very powerful behavioral model recently developed by John Neubert, D.D.S., Ph.D. of the University of Florida, who is another grantee of the FPRF. Following upon the methods used to treat hypersensitivity of the spinal cord after hindlimb peripheral nerve injury, Dr. Basbaum’s team of investigators transplanted GABA precursor cells into the trigeminal nucleus caudalis, which is the brainstem nucleus that receives input from the infraorbital nerve. The behavior of the mice was followed for weeks after the transplant and once again the transplants reversed the mechanical hypersensitivity created by this model. To what extent damage to the infraorbital nerve models human trigeminal neuralgia remains to be determined. Nevertheless, it is significant that Dr. Basbaum’s group has shown that these GABA precursor cell transplants can also reduce both the mechanical and thermal hypersensitivity that occurs after chemotherapy treatment. Thus, a very important finding arising from Dr. Basbaum’s experiments is that his findings are consistent with there being a common etiology underlying nerve injury-induced neuropathic pain conditions.
In ongoing studies, Dr. Basbaum’s laboratory, in collaboration with scientists at Neurona, are evaluating the utility of transplanting human embryonic stem cells that have been modified to take on properties of GABAergic nerve cells. Those studies are critical first steps in the development of an approach to treatment clinical pain conditions. Hopefully, Dr. Basbaum’s next report will bring encouraging news on that front.
The William H. and Leila A. Cilker Genetics Research Program To Find a Cure for Trigeminal Neuralgia (“Finding the Genes that Predispose to Trigeminal Neuralgia”)
Marshall Devor, Ph.D. (Project Coordinator). Professor and Chair, Department of Cell and Animal Biology, Institute of Life Sciences, Hebrew University of Jerusalem
Kim J. Burchiel, M.D. (Phenotyping and Sample Collection) Professor Department of Neurosurgery, Oregon Health and Science University
Ze’ev Seltzer, Ph.D. (Genomics) Professor of Pain Genetics, Faculty of Dentistry, Professor of Physiology, Faculty of Medicine, University of Toronto
Scott R. Diehl, Ph.D. (Genomics Consultant) Professor, Department of Oral Biology, Rutgers School of Dental Medicine, Professor of Health Informatics, Rutgers School of Health Related Professions, Rutgers University
The remarkable progress made in genetic analysis in the past two decades of the Human Genome Project has greatly enhanced our capacity to discover genes associated with different diseases, to reveal their function, and to develop therapeutic and/or preventive approaches precisely targeted at the specific disease. This project asks the question: Are some individuals genetically predisposed to develop trigeminal neuralgia (TN) pain? This question emanates from research which shows that although 17% of mature adults have a TN lesion (usually a vascular compression of the trigeminal nerve close to where it exits the brain) only a very small minority (0.01-0.2%) of these individuals actually experience TN pain. This disconnect between lesion and pain is an important clue pointing toward a genetic predilection for TN. Other research shows that more than one third of patients with TN do not have vascular compression of the nerve, and the rare patients with bilateral TN do not generally have vascular compression of the nerve on both sides. Further, it appears that younger patients (less than 45 years of age) are four times more likely to be women, than men. All of these new findings point to the possibility of a genetic predisposition for TN. This project was conceived with the assumption that the risk for having classical TN has a genetic basis and the study’s primary objective is identifying genetic mutation(s) (aka, sequence variant(s) or polymorphism(s) associated with the development of TN pain. To achieve this objective, DNA has now been collected from over 800 TN patients.
Analysis of the whole genome has now been carried out on 500 of these patients using the latest in genetic technology and compared to matched reference controls to determine if there are candidate sequence variant(s) or polymorphism(s) present in the genome of the TN patients that are not found in the controls. Such sequence variants might cause TN pain by directly affecting the function of the proteins that these genes encode, or by altering other aspects of gene expression. Either way, assuming that genetics is indeed the basis for developing TN, there is a high probability that this project will reveal the genes and ultimately the pathophysiological mechanisms that cause the lesions to be painful. With this knowledge molecular targets for treatments that will cure the pain can be identified not only of TN but perhaps also for a variety of other facial and segmental neuropathic pain conditions. To date, nine centers, including two international centers, have actively acquired DNA samples via saliva in patients with TN1. At the inception of this study, it was not clear that in such a relatively small sample, whether we would be able to identify genes that predisposed persons to the development of TN. Remarkably, our analysis of our first 500 patients has now identified around a half dozen gene segments that appear to be related TN, and all of these genes do seem to be related to nervous system structure or function.
Given the goals that have been achieved thus far, this team of investigators has put forward a strong case for doubling the number of DNA samples analyzed from 500 to 1000. At present, we have only 150 more specimens to collect to attain a sample size of 1000, and this should be achievable within the next six months. In the past few years, even since the beginning of our study, the technology for gene detection has advanced significantly. Our plan is to process all 1000 specimens using a new technology to both improve the resolution of our gene analysis, and to replicate our findings from our first set of patients. The investigators are very gratified with the notable progress they have made thus far and are increasingly confident that they will be able to identify the genes that predispose individuals to the development of TN.
Novel Ways to Deliver Compounds That Can Eliminate the Pain Of Trigeminal Neuralgia
Wolfgang Liedtke M.D., Ph.D., Professor Department of Neurology, Duke University
In 2015, Dr. Wolfgang Liedtke approached the Facial Pain Research Foundation (FPRF) to acquire funding to develop novel techniques for direct anatomic targeting of specific components of the trigeminal system. The primary advantage of this approach is with delivery of the drug directly to the lesioned area, only a small fraction of the normal systemic dose of the drug will be needed thereby eliminating the unwanted side effects associated with systemic or whole body delivery of the drug. After review by two knowledgeable scientists, Dr. Liedtke’s proposal was recommended for funding to the Facial Pain Research Foundation’s (FPRF) Board of Trustees (BOT) and subsequently was awarded a one year $50,000 seed grant to initiate his program.
To develop his unique delivery system, Dr. Liedtke proposes to combine material science with cell engineering to create a way that will provide focused delivery of molecules or drugs that have previously shown the capacity to block or eliminate pain in other models and/or primitive models of TN pain. As a consequence, the objective of this approach is to deliver molecules that have displayed the capacity to shut down transmission of the aberrant neuronal signals arising from injured site(s) on the trigeminal system that are responsible for producing the pain of TN. These little assemblies are designed to be placed around peripheral trigeminal nerve branches and around the trigeminal nerve root at the point where this root exits the brainstem. The experiments described in this proposal are based on previous work by Dr. Liedtke and collaborators using a specifically-created kind of carbon nanomaterial, i.e., a few-walled carbon nanotubes with high electrical conductance that has been highly purified to the point that it is devoid of any neurotoxic contaminating by-products. This particular type of carbon nanomaterial is known to lower intercellular chloride ion levels by “turning on”, in neurons, the gene KCC2. Activation of KCC2 will cause this gene to increase production of the protein for which it codes. The function of this KCC2 coded protein is to enhance removal of the chloride ion from neurons. Lowering intracellular chloride levels in CNS neurons will amplify the inhibitory capacity of the inhibitory neurotransmitters, GABA and glycine. In an earlier study, Dr. Liedtke and co-workers applied these ultra-pure, few-walled carbon nanotubes to cultured cortical neurons and showed that direct contact of the carbon fibers with the cultured neurons in increased levels of KCC2 in these cells causing an enhanced efflux of chloride from them. Thus, in addition to serving as a topical drug delivery vehicle, the carbon nanotubes themselves may have pain inhibiting properties.
Dr. Liedtke proposes to develop another kind of topical drug delivery vehicle. This device will be will be made of immunologically inert biocompatible polymers designed to hold human fibroblast-like cells. Using gene therapy techniques, i.e., remove the native or viral DNA from a wild-type virus and replace it in the now empty protein coat with a therapeutic gene (DNA) which now can be used as a vital vector to deliver the therapeutic gene to animals or humans where they infect target cells and produce the protein product of the therapeutic gene. In Dr. Liedtke’s case, the wild-type viruses native DNA will be replaced with genes harvested from fibroblasts (which are readily obtainable from skin) that code for pain inhibiting substances like GABA or glycine effectively turning them into anti-pain generating pumps. The biocompatible polymer devices will prevent the cells from dispersing but will be made sufficiently porous to allow the pain inhibiting molecules to escape.
In 2016, Dr. Liedtke was able to demonstrate engineered cells that secrete an endogenous hormone-like substance which is known to combat pain, when injected into the cerebrospinal fluid space of mice reduced specific, measurable pain behaviors in animals that can be evoked by the application of noxious stimuli to neural structures innervated by the trigeminal nerve. In regards to translational medical use of carbon nanotubes, Dr. Liedtke made significant progress toward demonstrating that he can adapt the carbon nanotubes so that they can be used for wrapping of nerves such as trigeminal nerve root that connects brainstem and trigeminal ganglion, also peripheral branches of the trigeminal nerve between trigeminal ganglion and innervated target tissue such as e.g. the infra-orbital nerve. His future plans include using these two approaches to deliver substances to the cisternal trigeminal nerve root in an attempt to create a valid pre-clinical model of TN which can then be used to obtain the necessary and sufficient pre-clinical data to move these two technologies towards clinical studies.