Neurology

1. Senior Research Staff

Susan Bowyer, Ph.D. Jieli Chen, M.D. Michael Chopp, Ph.D., Vice Chair for Research Stanton B. Elias, M.D., Chair James Ewing, Ph.D. Feng Jiang, Pharm.D. Hao Jiang, Ph.D.

Quan Jiang, Ph.D. Robert Knight, Ph.D. Yi Li, M.D. Stephen Robinson, Ph.D. Rhonna Shatz, D.O. Norman Tepley, Ph.D. Zheng Gang Zhang, M.D., Ph.D.

2. Research Summaries

Principal Investigator: Chen, Jieli, M.D.
Title: Neurorestorative Therapy of Stroke with Statins (NIH R01 NS04768201A1)

(HMG)-CoA reductase inhibitors (statins) are widely and effectively employed to control cholesterol in humans. This class of cholesterol reducing agents may also be highly effective in reducing neurological deficits after stroke. Statins administered to rats one or more days after stroke significantly enhance brain plasticity, and improve neurological function. The following specific aims and associated hypotheses seek to develop statin restorative therapy in young and old rats in a model of middle cerebral artery occlusion (MCAo), and to investigate the molecular sequelae that activate statin mediated alterations of ischemic brain. Three aims are proposed: Aim 1 will determine the effects of statin (simvastatin) treatment on functional recovery in old and young adult rats after stroke. The hypothesis to be tested is that treatment of stroke with simvastatin initiated at one day after stroke onset improves neurological functional recovery in old and adult young rats. Aim 2 will measure the changes in brain evoked by statin treatment of stroke that promote therapeutic benefit. We hypothesize that simvastatin induces angiogenesis, synaptogenesis, neurogenesis and neuronal migration in old and young rats after stroke. In Aim 3 will employ molecular factors and inhibitors, genetically modified mice and an array of novel technologies to investigate molecular mechanisms that promote therapeutic benefit of statin in vitro. The effects of simvastatin on regulation of small G-protein, activation phosphatidylinositol 3-kinase (PI3K)/AKT/endothelial nitric oxide synthase (eNOS) and the Ras/extracellular signal-regulated kinase (ERK) pathways, expression of vascular endothelial growth factor (VEGF) and associated receptors will be measured in cultured mouse brain-derived endothelial cells, primary cortical neurons, and in adult subventricular zone (SVZ) explant culture. The hypotheses to be investigated are that:

The long-term goal of this application is to translate our finding of therapeutic benefit after treatment of experimental stroke with statin to the patient.

Principal Investigator: Chopp, Michael, Ph.D.
Center for Stroke Research (NIH P01 NS23393-17)

The applicants propose a highly integrated application focused on preclinical and clinical studies to investigate and develop treatment of stroke with an anti-platelet aggregation agent alone, and in combination with thrombolysis using recombinant tissue plasminogen activator (rtPA). Permeating this Program is the development and application of MRI to enhance the management of the stroke patient. Three interdependent Projects and two Cores constitute this grant application. Project 1, Anti-Platelet Aggregation Therapy for Embolic Stroke, will investigate the mechanisms promoting secondary thrombosis after embolic stroke and treatment with rtPA in rat, and will test, in a controlled experimental model, treatment of embolic stroke with an antibody against the GPIIb/IIIa receptor. This receptor binds the platelet to fibrin and is responsible for platelet aggregation and therefore, platelet mediated thrombosis. This project leads into Project 2, MR Assessment of Transient Cerebral Ischemia, which develops and applies a multi-parameter MRI model to experimental embolic stroke in rats. The goals of this Project are to develop and test the application of the multi-parameter MRI model to identify candidates for therapy and to exclude candidates from therapy after embolic stroke. In addition, the MRI response to thrombolysis with rtPA and rtPA in combination with an antagonist to platelet aggregation will be tested. Projects 1 and 2 form the preclinical support for a Phase II Pilot Clinical Trial of treatment of stroke with an anti-platelet aggregation agent, abciximab. This Project will test activity of treatment of the stroke patient with abciximab and will identify an MRI based surrogate marker for activity and accrue MR data to select patients for anti-platelet aggregation therapy. Core A is an Administrative and Biostatistical Core. Core B, the MRI core, services all three Projects. The Program Project provides an integrated highly coherent effort to enhance management and therapeutic intervention in the treatment of acute stroke.

Principal Investigator: Chopp, Michael, Ph.D.
Photodynamic Therapy: Basic Science Studies - Project 5

We are investigating ways to enhance the therapeutic efficacy of  treatment of glioma with photodynamic therapy (PDT).  In PDT a light sensitive non-toxic drug is injected into a vein this drug is selectively taken up by tumor. When laser light illuminates the tumor the drug becomes toxic and destroys the tumor. Our work has shown that certain tumor cells survive this treatment and that the tumor regrows. However, preliminary data from our laboratory has indicated that an antiangiogenic agent, an agent which inhibits the formation of new blood vessels, used in conjunction with PDT for the treatment of glioma greatly increases tumor kill and extends survival. Our studies will therefore develop this combination therapy for the treatment of glioma.

Principal Investigator: Chopp, Michael, M.D.
PPG Project Core A (NIH P01 NS23393-17)

The Administrative and Biostatistics Core will: promote high quality collaborative research in cerebral vascular disease by providing a supportive academic environment in which investigators can carry out scientific projects, facilitate the interaction of projects and cores, oversee the administration and financial aspects of the knowledge towards an understanding of the mechanisms and management of cerebrovascular disease. The role of the Biostatistics Core in the Stroke Project is to support the clinical and laboratory based research. This includes a high-level data management, collaboration with investigators in the design and conduct of research studies, statistical modeling, hypothesis formulation and data analyses. There are four major activities: 1) consultation and collaboration in the planning, conduct, analysis and reporting of the pilot clinical trial; 2) consultation and collaboration with research investigators in developing an MRI based model to stage ischemic tissue and to integrate MRI measurements into the evaluation of therapeutic intervention with a platelet aggregation inhibitor and thrombolysis with rtPA; 3) evaluation of the prognostic significance of risk factors and MRI biological markers, based on the presence/absence of quantitative levels to identify non-response subjects, 4) coordination on project conduct, responsibility for data management and data quality.

Principal Investigator: Chopp, Michael, Ph.D.
Treatment for Neural Injury (NIH P01 NS04234502)

The underlying hypothesis of this Program Project application is that bone marrow stromal cells (MSCs) delivered to brain via an intravenous route can be employed to improve functional outcome after neural injury, specifically, stroke and traumatic brain injury. Three complementary projects and two supporting cores are proposed: Project 1 Treatment of Stroke with MSCs; Project 2/Treatment of Traumatic Brain Injury with MSCs; Project 3/Analysis of MSC Interaction with Tissue. Core A provides the administrative and biostatistical support for the Program Project, and Core B provides the outcome measures of function and behavior after stroke and trauma, measures of cellular and molecular responses to injury and treatment, and the preparation of cells to be employed for treatment. Projects 1 and 2, will determine the optimal means of applying MSC therapy to experimental models in the rat and the mouse of stroke (young and old animals, male, female) and traumatic brain injury (young male), respectively, with safety as an overriding consideration. The hypothesis to be tested is that MSCs in brain evoke the production of trophic factors that alter injured brain to promote functional benefit. Marrow stromal ceils administered to animals intravenously find their way to ischemic or damaged cerebral tissue and foster functional improvement. Thus, under the clinically relevant conditions of intravenous administration, Projects 1 (stroke) and 2 (traumatic brain injury) will optimize and define the boundaries of therapeutic intervention, measure specific neurotrophic factors and structural and morphological changes in treated brain and clarify how the injured brain responds to MSC treatment. Project 3, will employ antibodies, and genetically modified mice and an array of novel technologies to investigate the mechanisms by which treatment of stroke and trauma with MSCs provides functional improvement. A specific set of neurotrophic factors i.e. VEGF, bFGF and BDNF are identified (in Projects I and 2) as key mediators of MSC therapeutic benefit. In Project 3, these factors are manipulated in the MSC treated mouse to determine their roles in MSC therapy of stroke and trauma, with an emphasis on how these factors induced in injured tissue by MSC treatment, promote plasticity and neuroprotection. The long-term goal of this Program Project application is to translate our finding of therapeutic benefit after treatment of experimental stroke and traumatic brain injury with MSCs to the patient.

Principal Investigator: Chopp, Michael, M.D.
PPG Project Core A: Biostatistical (NIH P01 NSO4234502)

This core will: promote high quality collaborative research by providing a supportive academic environment in which investigators can carry out scientific projects, facilitate the interaction of projects and cores, oversee the administration and financial aspects of the Program Project, interact with core facilities effectively, foster new research projects, and contribute new knowledge towards an understanding of marrow stromal cell therapy of stroke and traumatic brain injury. The role of the Biostatistics Core is to support the preclinical and laboratory based research. This includes a high-level data management, collaboration with investigators in the design and conduct of research studies, statistical modeling, hypothesis formulation and data analyses.

Principal Investigator: Gautam, Subhash, Ph.D.
PPG Project Core B: Outcome Measures and Cell Preparation (NIH P01 NSO4234502)

Core B provides the cells to be employed for treatment as well as the essential outcome measurements for all constituent projects of the Program Project. Neurologic function and behavior are the primary outcome measures for Project 1 (Treatment of Stroke with MSCs) Project 2 (Treatment of Traumatic Brain Injury with MSCs) and are extensively employed in Project 3 (Analysis of MSC Interaction with Tissue). In this core, we describe the sets of functional tests to be employed in evaluating response to MSC therapy. Included in the battery of tests are, motor and somatosensory tests, and neurological scales. Projects 1-3 also address the fate of the injected MSCs, the factors produced by these cells and their effect on cerebral tissue that promote improved functional outcome, and the effects of MSCs on the endogenous parenchymal cells. This core provides all the resources required for cellular identification, measurement and identification of trophic factors, and microscopy. All three projects employ marrow stromal cells. Core B, provides the facilities and personnel dedicated to the production and characterization of these cells. Focusing resources needed for the component projects in Core B, allows for efficient and effective utilization of resources essential for all three projects.

Prinicpal Investigator: Jiang, Feng, Pharm. D.
Glioma-Treatment with PDT and Anti-Angiogenic Agents (NIH R01 CA10048601A2)

We propose to test a novel hypothesis, that angiogenesis in non-tumor tissue induced independently of tumor, contributes to the growth of tumor. Thus if a therapeutic intervention to treat tumor induces angiogenesis in adjacent non-tumor tissue, this angiogenesis increases the likelihood of tumor regrowth from residual tumor cells. We propose to test this hypothesis in a model of human glioma in the nude mouse. The anti tumor treatment employed is photodynamic therapy (PDT) using Photofrin as the photosensitizer. In specific aim A, the angiogenic response of normal brain to PDT is tested and the effects of implantation of tumor in PDT induced angiogenic brain are analyzed. As a logical consequence of experiments performed in specific Aim A, we test the effects of combination treatment of U87 human glioma in the mouse brain using PDT and antiangiogenic agents in specific aim B. We expect that the combination treatment will significantly reduce the rate of tumor regrowth and thereby extend the life of animals compare to the individual treatments. The anti-angiogenic agents employed are antibodies against the vescular endothelial growth factor receptors (VEGFR1 and VEGFR2). If we are correct, that endogenous brain angiogenesis contributes to tumor regret, and then our study has important implications for the treatment of brain tumor using most forms of anti tumor agents, and the use of a combination anti-angiogenic and direct tumoricidal treatment may translate into improved therapeutic opportunity.

Prinicpal Investigator: Jiang, Hao, Ph.D.
Resveratrol-Induced Apoptotic Cell Death in Glioma (NIH R21AT003436-01A2)

Glioma is a malignant brain tumor in adults, difficult to treat and resistant to conventional radiotherapy and chemotherapy. Several major signaling cascades have been implicated in the development of gliomas, which include over-expression and activation of growth factors and their receptors (i.e. EGFR), over-expression and activation of intracellular signaling proteins (i.e. PI3-K/Akt) and deregulation of cell proliferation and the cell cycle. Resveratrol (trans-3,4',5-trihydroxystilbene) is a polyphenolic compound highly enriched in grapes, peanuts, red wine and a variety of plant and food sources. Resveratrol has been reported to have anti-inflammatory and antioxidant properties and acts as a cancer chemopreventive agent, inhibiting different steps of the carcinogenesis process, such as tumor initiation, promotion and progression. However, the signaling mechanisms mediated by resveratrol are not well defined in vivo. Our published in vitro studies show that resveratrol suppresses cell proliferation and induces apoptotic cell death in human U251 glioma cells in a dose- and time-dependent manner. Activation of caspases and suppression of phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (Akt)-mediated signaling pathways are involved in this process. In the current study, we investigate the effect of resveratrol on the suppression of tumor growth and its underlying molecular mechanism in vivo using a U251 intracerebral glioma model in nude mice. We hypothesize that resveratrol suppresses the development of glioma in vivo through the down-regulation of expression and/or suppression of the activation of EGFR- and PI3-K/Akt-mediated signaling pathways. Specifically, the effect of resveratrol on the tumor growth will be determined and the regulation of EGFR/PI3-K/Akt-mediated signaling pathways by resveratrol will be examined. Natural products, such as resveratrol, usually have little or no toxic effects, are low cost and can be consumed orally. Development of potential preventive or adjunctive therapies using such products will be of great benefit for the treatment of glioma, as well as other human diseases. Glioma is a malignant brain tumor in adults, which is difficult to treat and resistant to conventional radiotherapy and chemotherapy. Resveratrol is a polyphenolic compound highly enriched in grapes, peanuts, red wine and a variety of plant and food sources. Resveratrol has anti-inflammatory and antioxidant properties and acts as a cancer chemopreventive agent. Our in vitro studies show that resveratrol suppresses cell proliferation and induces apoptotic cell death in glioma cells. Therefore, we seek to test the effect of resveratrol in vivo using a well-established animal model of glioma. By elucidating the molecular mechanisms underlying the therapeutic effects of resveratrol, this study would facilitate the development of therapeutic agents using natural products and greatly improve the treatment of brain tumors.

Prinicpal Investigator: Jiang, Quan, Ph.D.
In Vivo MR Evaluation of Cell Therapy for Stroke (NIH RO1 NS48349)

Treatment of stroke with bone marrow stromal cells (MSCs) significantly improves functional recovery in experimental stroke. However, little is known about temporal and spatial profiles of migration of transplanted MSCs and the effects of these cells on host cerebral tissue leading to functional recovery. We propose to develop noninvasive in vivo magnetic resonance (MR) methodology for tracking transplanted MSCs and investigating their effects on the host brain. In Specific Aim 1, we will first optimize methodology for magnetic contrast agent labeling and in vivo three dimensional magnetic resonance (MRI) monitoring of transplanted MSCs in the host brain. Using the optimized methodology, we will then examine the effects of different routes (intravenous vs intracisternal) of transplantation on survival, migration, and distribution patterns of transplanted cells in brain subjected to stroke. In Specific Aim 2, dynamic effects of transplanted MSCs on the host brain angiogenesis and functional recovery after stroke will be noninvasively measured by means of MR. Neurological outcome will be measured using a battery of behavioral tests. Correlation between changes in MR measurements and dynamic functional improvements will be analyzed in the same animal. In addition, changes in MR measurements of angiogenesis resulting from MSC transplantation will be verified using three dimensional laser scanning confocal microscopy in combination with immuno-histochemistry. With these novel approaches, we expect that noninvasive MR measurements will simultaneously detect the migration and distribution of transplanted cells and the effects of these cells on the host brain tissue, and that changes in MR measurements will correlate to neurological function. Therefore, MR could potentially provide important noninvasive measurements for developing a successful cell therapy for stroke. After completing our studies, we expect to demonstrate that MR tracking magnetic labeled cells is a valid new technique for studying cell therapy for stroke, which will lead to optimization of cell transplantation protocols and improved management of stroke.

Principal Investigator: Jiang, Quan, Ph.D.
PPG Project 2: MR Assessment of Transient Cerebral Ischemia (NIH P01 NS23393-17)

The objective of this project is to develop magnetic resonance imaging (MRI) as a tool to non-invasively evaluate and monitor recovery in stroke patients. Our studies during the current finding period indicate that certain agents can enhance functional recovery after stroke in experimental animals based on histological and functional assessments. A prime candidate among restorative agents is sildenafil, a phosphodiesterase type-5 inhibitor, which has been widely used in humans to increase cyclic guanosine monophosphate (cGMP). Our data show that sildenafil significantly improves funcational recovery in both young and aged animals after stroke. Hence, we will employ sildenafil in the proposed studies. We have also generated preliminary imaging data, which indicate that certain MRI methods may be sensitive to post-stroke tissue remodeling (vascular and neuronal) events that are thought to influence eventual outcome. The studies proposed in this current application expand on this theme by combining and applying these novel imaging, histological and restorative treatment strategies to assess post-stroke vascular (Aim 1) and neuronal (Aim 2) remodeling in both young and aged animals with and without restorative therapy. MRI will be used to identify, characterize and define tissue-remodeling events that contribute to improvement of late-term functional outcome after stroke. These MRI assessments will be compared and correlated to corresponding measures of tissue remodeling determined from 3D laser scanning confocal microscopy and immunohistochemistry. Finally, Aim 3 will study the relationship between the various MRI and histological indices of neuronal and vascular remodeling and will relate these data to functional outcome as measured by a battery of behavioral test. The proposed studies are novel and innovative, moving our research away from acute diagnostics and neuroprotective therapeutic approaches for stroke treatment and toward a relatively unexplored area of late-term restorative therapy that promotes brain tissue remodeling events that ultimately improve functional outcome. If successful, these studies are expected to generate ground breaking information about post-stroke brain tissue remodeling that will be useful for monitoring stroke recovery and may lead to optimization of treatment protocols that would benefit all stroke patients.

Principal Investigator: Knight, Robert, Ph.D.A Seven Tesla MRI System for Small Animals MRI/MRS (NIH 1S10RR022603-01A2) 

We propose to purchase a new 7 Tesla superconducting magnet and gradient set to upgrade our existing small animal magnetic resonance imaging (MRI) and spectroscopy (MRS) facilities. Our present system relies on a magnet that is 18 years old and is heavily used by 7 NIH-funded grants for physiological measurements in small animal models of stroke, tumor, intracerebral hemorrhage and epilepsy. The aim of the present request is to purchase and install a new magnet and gradient set onto our present system to bring it up to current standards so that we can serve our present grants and remain competitive as a laboratory. Henry Ford Health System (HFHS) will meet the remainder of any costs associated with this system upgrade (total $927,000) above the funds provided by the Shared Instrument Grant award ($500,000). The NMR laboratory is a HFHS Core Facility, and has been an NIH/NINCDS stroke center (NINCDS 1 PO-1 NS 23393) since 1986. Historically, this facility has been strongly focused on cerebral ischemia and stroke. In recent years, cerebral tumor has gained increasing importance in our time commitments. New initiatives in diffusion tensor imaging, vascular physiology, molecular imaging, MR tracking of precursor cells in a variety of organ systems, intracerebral hemorrhage and epilepsy are currently underway at HFHS. These proposals have led us to conclude that our present system will not be capable of meeting the present and steadily increasing demands for various types of noninvasive small-animal anatomical and physiological evaluations and must either be replaced or upgraded. The most cost effective way to accomplish this will be to replace the magnet and gradient set. This Shared Instrumentation Grant will partially supply the funds to bring the present MRI/MRS system up to current standards, with HFHS supplying the rest.

Principal Investigator: Li, Yi, M.D.
PPG Project 1: Treatment of Stroke with MSCs (NIH P01 NSO4234502)

Stroke is the number three cause of death and a leading cause of serious, long-term disability. Treatment of stroke is restricted to thrombolysis within a three-hour window after ictus. There are also no treatments for stroke specifically designed to promote functional recovery. Preliminary data demonstrate that intravenously (iv) injected bone marrow stromal cells (MSCs) one day or one week after onset of symptoms reduces functional deficits associated with cerebral ischemia. This therapeutic benefit lasts for months. Intravenously injected MSCs enter ischemic brain and therein promote increased growth factor expression, e.g., BDNF, bFGF, VEGF. tn tight of these findings, two primary specific aims are proposed. First, MSC therapy is developed preclinically. Aim 1: To select effective doses and to test the safety and toxicity of iv administration of MSCs in young adult and old rodents subjected to stroke. The major hypotheses being tested in this aim are: MSCs injected iv selectively enter lesioned ischemic brain and improve neurological functional recovery; treatment of stroke within a range of MSC doses is safe and effective; therapeutic benefits are dependent on cellular dose, therapeutic window, and animal age. In Aim 2, the molecular and cellular mechanisms responsible for the therapeutic effect of MSC treatment of stroke are investigated. Aim 2: To provide insight into the mechanisms relating MSC treatment to functional recovery, to measure: levels of growth and trophic factor expression (BDNF, bFGF, VEGF) in the ischemic brain, the spatial and temporal profiles and the phenotypic fate of MSCs in ischemic brain, and the response of endogenous proliferating cells in brain to MSC administration. The corresponding major hypotheses tested are: that growth and trophic factor expressions are enhanced in compromised brain by MSC administration; MSCs administered iv are mostly distributed in the boundary zone of the ischemic lesion; with time, the neural phenotype of MSCs increase; the growth and trophic factors produced in the ischemic brain with resident MSCs alter the compromised brain, e.g. MSCs enhance proliferation of endogenous cells in the ischemic brain. A series of experiments are therefore designed to measure: MSC phenotypic changes, cell fusion and the long-term status of resident MSCs, and the proliferation of cells within select regions of the ischemic brain. The goals of this application are to develop MSC therapy as an effective and safe treatment of stroke. The long-term benefits of MSCs reside in an ability to harvest and amplify a patient's MSCs and to re-inject them iv to treat stroke and possibly other forms of central nervous system disorders.

Principal Investigator: Mahmood, Asim, M.D.
PPG Project 2: Treatment of Traumatic Brain Injury with MSCs (NIH P01 NSO4234502)

This project is designed to investigate the effects of intravenous transplantation of bone marrow stromal cells on brain after traumatic brain injury. Traumatic brain injury (TBI) is an important cause of human morbidity and as many as 50,000 Americans are killed and an equal number are disabled by head trauma each year. Currently, treatment of TBI primarily consists of evacuating mass lesions and providing an optimal milieu for the brain to recover. Preliminary data indicate that bone marrow stromal cells (MSCs) induce functional benefit in animals subjected to TBI. In this application, functional response of young male rats and mice subjected to TBI after intravenous (i.v.) administration of MSCs will be measured, and effective doses of MSCs will be determined. A battery of functional outcome measurements will be performed to test for enhanced recovery resulting from MSC treatment (Specific Aim 1). We propose, that therapeutic benefit from MSC treatment of TBI derives from the production of growth factors in brain by the interaction of MSCs with injured brain. To support this hypothesis, growth factors produced in brain in response to different doses of MSCs will be measured for different degrees of injury in rats, and a temporal profile of growth factor production will be measured and related to functional response (Specific Aim 2). The MSC enhancement of these growth factors may affect endogenous cell proliferation and their phenotype in the subventdcular zone (SVZ) and the hippocampal formation in animals subjected to TBI. Of basic interest, is the fate of the MSCs in brain after TBI, whether they are modified and express over the long-term markers of other cell types. To address these questions, the temporal profile for cell phenotype of exogenous MSCs in injured brain and endogenous cell proliferation and phenotype in the SVZ and hippocampal formation will be measured in rats subjected to TBI and treated with MSCs. Cell phenotype and cell proliferation measurements will also be performed in mice receiving MSCs from transgenic GFP mice. If intravenous transplantation of marrow stromal cells succeeds in improving functional outcome, a new avenue will be opened for further development of therapeutic interventions to improve outcome after traumatic brain injury.

Principal Investigator: Shatz, Rhonna, D.O.
Genetic Risk Factors for Alzheimer’s Disease (5R01 NS030914-11, subcontract)

This collaborative study between Henry Ford Hospital (HFH) and Johns Hopkins University (JHU) is a five-year case-control study of Alzheimer’s disease (AD) among African-Americans (AA) to identify new genetic, vascular, and environmental risk factors for AD in this ethnic group.  AD is a neurodegenerative disorder of heterogeneous etiologies; both genetic and environmental factors contribute to the expression of the disease. The aims of this study are to recruit 500 AA patients with possible or probable AD at the HFH Neurology Clinic and 600 frequency-matched healthy patients as our control group.  All 500 AD cases and a subset of 200 controls will undergo a complete neuropsychological, medical, laboratory (for vascular markers) and MRI evaluations. All cases and controls will be genotyped for 10 vascular candidate genes at the JHU Genetics Laboratory.  The associations between SNP alleles or haplotypes in candidate genes and AD will be investigated among AD cases vs. controls, stratified by vascular disease groups.  Through statistical modeling, an exploratory analysis will be made regarding the relationship between genetic factors, their influence on intermediate biomarkers, and dementia outcome.  Genetic and environmental risk factors elucidated through this research can be targeted for future AD prevention.

Principal Investigator: Tepley, Norman, Ph.D.
Development of Hardware and Software for Clinical MEG (National Institute of Neurological Disorders and Stroke 5R01 NS030914-11)

The ongoing objective of our research, since the establishment of the HFH MEG lab, has been to develop hardware, software, and techniques to expand the utility of Magnetoencephalography (MEG), both as a clinical diagnostic tool and as a modality for basic neuroscience studies.  With the proliferation of MEG systems both in the U.S. and worldwide, and the availability of CPT codes for some clinical MEG studies, the development of new and more effective clinical applications to enable users to more fully exploit these costly systems has become our highest priority.  Accordingly, we are developing new analytical tools and demonstrating that MEG data contains much more clinically useful information than can be found using only the usual dipole techniques.  For example, the integration of our Multi Resolution FOCUSS with Principal Component Analysis (PCA) or singular Value Decomposition (SVD) shows promise of enhanced computational efficiency and the ability to identify regions of high brain coherence indicative of epileptic tissue.  During the proposed grant, these new techniques will be further developed and applied to neurologic and learning disorders.  In Specific Aim One, interoperative source confirmation will be compared to results of our mapping techniques.  DC-MEG will be used to monitor interictal cortical excitability in migraine patients and how this excitability responds to anti-migraine medications.  Determination of focal secondary generalized epileptic activity may be used to predict successful surgical resections.  In Specific Aim Two we proposed to continue the development of new analytical tools and to make them available to the MEG community on the internet as they become validated.  In particular, we will add new functionality to our “MEG Tools” software suite by incorporating ICA, Frequency Analysis, and MEG co-registration with Diffusion Tensor Imaging (DTI).  In Specific Aim Three, our new techniques will be applied to imaging differences in cortical activation between normal readers and individuals with learning disorders during reading tasks.  These techniques will be used to map various cortical areas activated by auditory and visual lexical stimuli, and to differentiate the responses of normal and reading disabled individuals.   The exquisite temporal and spatial resolution of MEG, enhanced by these new imaging techniques will provide clinicians and researchers new noninvasive methods to investigate pathological and normal neurological functioning.

Principal Investigator: Zhang, Zheng Gang, M.D., Ph.D.
PPG Project 3: Analysis of MSC Interaction with Tissue (NIH P01 NSO4234502)

Stroke and traumatic brain injury are major causes of morbidity and disability and functional recovery is slow and uncertain. Our preliminary data demonstrate that intravenously transplanted bone marrow stromal cells (MSCs) migrate to the stroked and injured brain tissue and improve functional recovery, This application is concerned with elucidating the cellular and molecular mechanisms responsible for the beneficial effect of MSC therapy on stroke and traumatic brain injury. The major hypothesis to be tested is that the MSCs delivered to the brain after stroke and traumatic brain injury enhance angiogenesis, promote axonal and dendritic sprouting and synapse formation, reduce cell death in the boundary regions of injured brain, and promote neurogenesis in the subventricular zone and dentate gyrus and thereby reduce neurological deficits, in addition, we propose to test the hypothesis that therapeutic benefit derives from MSC-induced increase in brain of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and brain-derived neurotrophic factor (BDNF). This hypothesis will be tested by blocking receptors and using gone knock-out mice. Specifically, by blocking VEGF receptor 2, we will examine the effect of VEGF on MSC-enhanced angiogenesis. Using bFGF-/- and BDNF-/-mice, we will examine the effects of bFGF and BDNF on MSC-induced axonal and dendritic sprouting and synapse formation and neurogenesis. Angiogenesis, neurogenesis and axonal and dendritic sprouting and synapse formation will be measured using Laser Scanning Confocal Microscopy, and a novel software program for three dimensional image analysis. We believe that these are unique and fresh approaches that may provide fundamental insights into the cellular and molecular mechanisms underlying the therapeutic benefit provided by MSC cellular therapy of stroke and traumatic brain injury.

Principal Investigator: Zhang, Zheng Gang, M.D.
PPG Project 1: Anti-Platelet Aggregation THerapy for Embolic Stroke (NIH P01 NS23393-17)

Treatment of stroke with a selective inhibitor of phosphodiesterase type 5 (PDE5), e.g. sildenafil, significantly promotes functional recovery concomitant with significant increases of neurogenesis and angiogenesis when the treatment is initiated 1 or 7 days after stroke onset. In this application, we seek to elucidate fundamental mechanisms of neurogenesis, neuroblast migration and the coupling of neurogenesis and angiogenesis after stroke with and without treatment with sildenafil as the neurorestorative agent. Our hypotheses are that sildenafil mediated increases of cGMP levels in stroke brain: 1) promote proliferation of neural progenitor cells via shortening the length of the cell cycle which results from a decreased expression of cyclin-dependent kinase inhibitors; 2) enhance neuroblast migration towards the ischemic boundary regions via increasing expression of stromal-derived factor-1a (SDF-1a), CXCR4 and matrix metalloproteinases (MMPs); 3) foster angiogenesis which generates a permissive niche to promote neurogenesis and to guide neuroblast migration towards the ischemic boundary regions; and 4) the PI3K/Akt signaling pathway mediates sildenafil-enhanced neurogenesis and neuroblast migration. The proposed experiments have been designed to test these hypotheses. We will investigate whether sildenafil amplifies cell cycle kinetics and the orientation of cell division that regulate the proliferation and differentiation of neural progenitor and stem cells in the subventricular zone. Using retroviral vector, siRNA, and time-lapse microscopy, we will delve into the molecular mechanisms, e.g. SDF-1/CXCR4 and MMPs, which promote neuroblast migration in the ischemic brain after treatment with sildenafil. To examine the effects of sildenafil-induced angiogenesis and neurogenesis and neuroblast migration, we will measure the interaction between angiogenesis and neurogenesis and proteins secreted by the endothelial cells. By blocking SDF-1a and MMPs, we will examine whether SDF-1a and MMPs secreted by endothelial cells mediate neuroblast migration. Using specific inhibators, we will investigate whether blocking the PI3K/Akt signaling pathway attenuates the effects of sildenafil on neurogenesis, neuroblast migration and angiogenesis. These basic scientific studies will elucidate the mechanisms how the brain remodels itself after stroke and how CGMP amplifies this process in the adult brain, which may lead to novel methods to facilitate brain remodeling during stroke recovery.

Principal Investigator: Zhang, Zheng Gang, M.D.
Treatment of Acute Embolic Stroke with Statins and rt-PA (NIH 2R01HL064766-05A2)

Approximately 80-90% of human cerebral ischemic events are caused by thromboembolism. There is a compelling need to develop acute therapeutic interventions for the treatment of stroke. We present preliminary data indicating that atorvastatin, a hydroxymethylglutaryl coenzyme A reductase inhibitor, extends the therapeutic window of thrombolysis in a rat model of embolic stroke by activation of the PI3- K/Akt signaling pathway. However, the atorvastatin mediated therapeutic effect is independent of eNOS and lipid levels. The goals of this application are to determine the efficacy of statins in combination with thrombolysis for treatment of acute ischemic stroke and to investigate mechanisms underlying the therapeutic effects of atorvastatin as an adjuvant agent to recombinant human tissue plasminogen activator (rht-PA). Our hypotheses are: 1) Treatment of stroke with atorvastatin in combination with rht-PA extends the therapeutic window for acute stroke; 2) Atorvastatin activates the PI3-K/Akt signaling transduction pathway in cerebral endothelial cells, which decreases cerebral microvascular thrombosis and blood brain barrier (BBB) leakage by negatively regulating endothelial cell genes that promote thrombogenicity, vascular permeability, and inflammation; 3) Activation of the PI3-K/Akt cell survival pathway in neurons by atorvastatin attenuates ischemic neuronal damage exacerbated by delayed treatment of rht-PA. The proposed experiments have been designed to test these hypotheses. Using Magnetic Resonance Imaging (MRI) and 3D laser scanning confocal microscopy (LSCM) techniques, we will first investigate the effects of short-term, high-dose atorvastatin on cerebral vascular patency and integrity including cerebral blood flow (CBF) and BBB leakage, and the neurotoxic effects of tPA. Using laser capture microdissection in combination with real time PCR, Western blot analysis and specific inhibitors which block PI3-K/Akt activation, we will then delve into the mechanisms by which the PI3K/Akt signaling pathway mediates expression of endothelial cell genes involved in thrombosis and BBB leakage, and expression of neuronal genes engaged in the neurotoxic effects of tPA. These studies will lead to a comprehensive understanding of mechanisms underlying the therapeutic effects of statins on extending the window of thrombolysis for acute ischemic stroke and may provide a novel and useful treatment strategy for human ischemic stroke.

1. Senior Research Staff

Chaya Brodie, Ph.D. Kost Elisevich, M.D. Dunyue Lu, M.D. Asim Mahmood, M.D.

Thomas Mikkelsen, M.D. Sandra Rempel, Ph.D. Mark Rosenblum, M.D., Chair Donald Seyfried, M.D.

2. Research Summaries

Principal Investigator: Brodie, Chaya, Ph.D. 
Regulation of Glioma Cell Apoptosis in PKCDelta (NIH-NCI R01CA109196)

PKCdelta is a key enzyme in the regulation of cell apoptosis in various cellular systems. PKCdelta acts as a pro or anti-apoptotic kinase depending on the specific cell type and apoptotic stimulus, however the mechanisms underlying its diverse effects are not understood. In this proposal we seek to understand the molecular mechanisms involved in the regulation of cell apoptosis by PKCdelta focusing on gliomas as a cellular system. Gliomas exhibit deregulated cell apoptosis due to altered apoptotic pathways that favor cell survival. In a recent study we demonstrated that PKCdelta expression is reciprocally correlated with the degree of malignancy in gliomas. Moreover, PKCdelta regulates the apoptosis of these cells in a stimulus-specific manner. The main hypothesis of this proposal is that PKCdelta is a major regulator of glioma cell apoptosis and that the pro and anti-apoptotic effects of PKCdelta in glioma cells are determined by its activation, phosphorylation on distinct tyrosine residues and by its subcellular localization. To test this hypothesis we will employ different apoptotic stimuli and will first examine the role of PKCdelta activity in its pro and anti-apoptotic effects by using a PKCdelta inhibitory peptide, a PKCdelta KD mutant and siRNAs directed against PKCdelta mRNA. Tyrosine phosphorylation of PKCdelta is often induced by apoptotic stimuli. Therefore, the tyrosine phosphorylation of PKCdelta will be studied in response to the various apoptotic stimuli employed in this study. The specific tyrosine residues and the tyrosine kinases involved in their phosphorylation will be identified and their role in the PKCdelta apoptotic effects will be determined. The translocation of PKCdelta in response to the different apoptotic stimuli will be examined using GFP-tagged PKCdelta and the role of PKCdelta tyrosine phosphorylation in the translocation of PKCdelta will be explored using PKCdelta tyrosine mutants. The role of PKCdelta localization in its apoptotic effects will be studied using a PKCdelta mutant in which the NLS was mutated and by employing vectors targeting PKCdelta to the nucleus, ER, cytosol and mitochondria. Finally, the cleavage of PKCdelta and the roles of the cleaved regulatory and catalytic fragments will be studied for their effects on the apoptotic function of PKCdelta. To identify proteins and signaling pathways that mediate the pro and anti-apoptotic effects of PKCdelta the effect of PKCdelta on the levels, activation and phosphorylation of different apoptosis-related proteins and on the activation of signaling pathways associated with cell apoptosis and survival (members of the MAP kinase family and Akt) will be determined. The roles of tyrosine phosphorylation and subcellular localization of PKCdelta in the activation of these proteins will be then evaluated. Another important factor in delineating the function of PKCdelta in glioma cell apoptosis is identifying its binding partners. This will be done using GST-PKCdelta and GST-PKCdelta mutants fusion proteins to identify PKCdelta binding proteins in glioma cells stimulated with different apoptotic stimuli and by screening glioma cDNA libraries using the yeast two hybrid system. The results of this study will enhance our understanding of the factors contributing to the divergent effects of PKCdelta on cell apoptosis and of the role of PKCdelta in the regulation of glioma cell apoptosis. A better understanding of the molecular mechanisms underlying the diverse effects of PKCdelta in gliomas may enable us to predict the tumor response to therapy based on its molecular profile and may lead to novel approaches for altering the sensitivity of gliomas to specific therapeutic agents.

Principal Investigator: Mahmood, Asim, M.D.
Treatment of Traumatic Brain Injury with Simvastatin (NIH R01 NS052280-02)

Principal Investigator:  Mahmood, Asim, M.D.
Traumatic Brain Injury and Marrow Stromal Cells (NIH 5R01 NS042259)

Principal Investigator: Mikkelsen, Tom, M.D.
New Applications in Brain Tumor Therapy (NIH R01 CA62432)

The HFH Hermelin Brain Tumor Center has participated in the New Applications in Brain Tumor Therapy [ John Hopkins University ] (NABTT) consortium since its inception as a research and clinical trials group in 1993. We have been a productive and integral member contributing to its success, as demonstrated by our accrual and data quality awards. We continue to aggressively pursue state-of-the art surgical, radiation and medical therapies for our patients, to improve methods for longitudinal follow-up using neuroimaging, and pursue active research in the laboratory for new diagnostic, therapeutic and prognostic tools. These efforts speak to the ultimate goal of our group and NABTT Consortium: improving the outcome of brain tumor patients.

Principal Investigator: Rempel, Sandra A., Ph.D.
The Role of SPARC in Glioma Invasion (NIH R01 CA086997)

SPARC is a protein that is secreted by the cell into the surrounding matrix. There it is capable of interacting with other proteins important in regulating the ability of a cell to attach to or move through the matrix. In addition, SPARC can affect cell division, tending to slow the growth of normal cells. SPARC is present during fetal brain development, but its expression is greatly reduced in normal adult brain tissue. When we compared normal brain tissue with brain tumor tissue, we found that SPARC was present in much higher levels in all grades of astrocytomas. Furthermore, it was present in the tumor cells that invaded adjacent brain tissue. This suggests that SPARC may be inappropriately turned on in tumor cells and that it may actually help tumor cells invade the brain. Therefore, the focus of our research has been to determine whether SPARC contributes to brain tumor invasion, and if so, how it works. We have found that SPARC promotes brain tumor invasion by slowing tumor cell growth, altering tumor cell adhesion to the brain matrix proteins, and increasing the amount of digestive enzymes released by the tumor cells. These combined functions appear to work together to clear space in the adjacent brain and allow the tumor cell to change from a dividing cell into an invading cell. Our present work is focused on understanding how SPARC is performing all these functions and determining whether we can inhibit invasion if we interfere with SPARC's functions. If we can inhibit SPARC function, then we may impact the ability of the tumor to invade. Any impact on tumor cell invasion would be of benefit to brain tumor patients who succumb quickly to these highly infiltrative tumors.

Spontaneous intracerebral hemorrhage (ICH) affects approximately 75,000 people in the U.S. every year, yet current treatment modalities are limited and most of the patients either die or are left with significant neurological morbidity. Our study is designed to investigate the use of a statin drugs, simvastatin and atorvastatin, after experimental ICH in the rat. Preliminary work in the ICH model as well as in animal models of ischemic stroke and traumatic brain injury has suggested significant improvement in neurological outcome with statin medication, with postulated mechanisms of neurogenesis, angiogenesis, improved blood flow, decreased cerebral ischemia, and growth factor regulation in the region of brain injury. The goals of this revised study are to provide preclinical evidence of the benefit of statin drugs after ICH and to delineate the underlying mechanisms of action so that this type of medication, which is already in widespread clinical use for lowering cholesterol, can have application to patients suffering from ICH. We will compare the effects of simvastatin and atorvastatin on neurological recovery in rats after the autologous blood injection method of ICH using established behavioral measurements after various times of survival. Dose response testing and therapeutic response testing will be obtained to determine the dose and time window of greatest benefit from simvastatin after ICH. The mechanisms by which simvastatin and atorvastatin have their beneficial effects will be studied by measuring neurogenesis and changes in the cellular environment, including markers of new neuronal connections, secretion of growth factors, proliferation of endothelial cells and neovascularization in the perihematoma region. Local effects from the hematoma such as cerebral edema and altered cerebral blood flow will be measured by MR imaging, and the results of statin treatment on these parameters will be calculated. Since there normally is significant loss of cerebral tissue around the ICH in humans and in the animal model, preservation or restoration of the cerebral tissue in the region of the ICH by administration of simvastatin will be measured using both histological and MR imaging techniques. This study will demonstrate the efficacy of statin treatment of ICH and provide insight into the multifaceted restorative effects initiated by statins, with the ultimate objective of translating our pre-clinical studies to the ICH patient.

1. Senior Research Staff

Paul Edwards, M.D., Chair
Nauman Imami, M.D.
Michael Ober, M.D.
A. Guillermo Scicli, Ph.D.

2. Research Summaries

Principal Investigator: Imami, Nauman, M.D.
Ocular Hypertension Treatment Study (NIH SU1 OEY010330)

This is an application to become a participating clinical center in the Ocular Hypertension Treatment Study (OHTS). This proposal provides complete documentation of the ability of Henry Ford Hospital to screen large numbers of ocular hypertensive patients and to enroll at least 50 eligible patients over a 24-month period. Documentation is provided of the capabilities of the proposed investigators and their staff for performance of the study in accord with the details of the OHTS Manual of Procedures, the nature and extent of their commitment to Henry Ford Hospital, and a list of eye care providers in the area who will refer patients screened for enrollment and randomization to treatment in the OHTS clinical trial.

Principal Investigator: Ober, Michael, M.D.
The Age-related Eye Disease Study II – A Multi-Center, Randomized Trial of Lutein, Zeaxanthin, and Omega 3 Fatty Acids in Age-Related Macular Degeneration (NIH P50CA101451)

This phase III multi-center clinical trial is structured as a double-masked, randomized, placebo-controlled trial evaluating the efficacy of lutein, zeaxanthin, CHA and EPA in reducing the risk of disease progression in 4,000 participants with ARED categories 3 or 4 age-related macular degeneration (AMD).  Up to 60 clinical centers will recruit and treat participants in this 7-year study. 

1. Senior Research Staff

2. Research Summaries

Principal Investigator: Bey, Michael, Ph.D.
Shoulder Function After Rotator Cuff Repair (1 R01 AR051912-01A1)

Rotator cuff tears are a common shoulder injury, affecting 30-40% of individuals over age 60 and significantly impacting function and quality of life.  Treatment strategies vary widely in invasiveness and cost, and there is significant controversy regarding the optimal treatment strategy.  Consequently, shoulder function after rotator cuff surgery varies tremendously, with at least 30% of patients experiencing long-term shoulder disability and worker’s compensation claims exceeding $2 billion per year in the U.S. alone.  It is believed that the rotator cuff contributes to shoulder strength and provides dynamic glenohumeral joint stability, but accurate measures of in-vivo glenohumeral joint stability do not exist.  This study will use a unique, accurate biplane x-ray system to non-invasively measure dynamic glenohumeral joint stability in the repaired and contralateral shoulders of patients having rotator cuff repair surgery.  These measurements, along with measures of shoulder strength, will be recorded at 3, 12, and 24 months post-surgery.  In addition, dynamic glenohumeral joint stability and shoulder strength will be measured in a control population with no history of shoulder injury or shoulder surgery.The long-term goal of this research program is to develop treatment techniques that restore and maintain shoulder function for patients with rotator cuff tears.  The following specific aims will be investigated: 1) determine if rotator cuff surgery restores and maintains dynamic joint stability, 2) determine the relationship between shoulder strength and dynamic joint stability, and 3) determine if dynamic joint stability is predictive of clinical outcome.  The central hypothesis is that dynamic joint stability is not completely restored by rotator cuff surgery, thus compromising shoulder function and potentially leading to long-term shoulder disability.  This study will provide data that are fundamental to our understanding of rotator cuff function and the effect of rotator cuff surgery on shoulder function.  This study and subsequent studies will provide data necessary to form a basis for evaluating surgical procedures and rehabilitation protocols for patients with rotator cuff tears.  As the population ages and stays active in later years, normal shoulder function will be critical to maintaining a healthy, active lifestyle.  Improving the efficacy of rotator cuff surgery and rehabilitation will also reduce both direct healthcare costs and secondary costs resulting from diminished productivity.

Principal Investigator: Bey, Michael, Ph.D.
Dynamic Knee Stability after ACL Reconstruction (NIH-NIAMS R01AR046387)

Principal Investigator: Les, Clifford M., Ph.D.
Degradation and Recovery of Bone: OVX and Treatment (R01AR050562)

Age-related bone fracture is only partially explained by the reduction in bone mass that universally occurs with aging. The failure to predict fracture comes in part from a) the loss of geometrical information when projection x-ray methods are used to measure bone mass, b) ignorance of sufficiently detailed information about the failure properties of bone, and also c) ignorance of detailed in vivo loads on bones (Fracture risk is related to the ratio of load to strength, rather than to bone strength alone). A fourth barrier to accurately predicting fracture risk is that changes in bone tissue mechanical properties caused by age or disease related changes in the collagen, non-collagenous proteins or water content of bone are not detectable to xray methods. As a result of the invisibility of soft tissue changes to x-ray detection, there are age and menopause related changes in tissue material properties (in bone quality) that are invisible, not understood and that unpredictably increase bone fracture risk. Our data support the novel working hypotheses: 1) The viscoelastic properties of bone affect the fracture toughness and apparent strength of bone tissue, 2) The viscoelastic properties of ovine bone degrade after ovariectomy, causing a decrease in the strength and fracture toughness of bone tissue for rates of loading associated with falls. The main goal of the proposed work is to determine the onset time of viscoelastic property degradation and whether cortical bone can recover normal viscoelastic and strength properties with estrogen replacement.

Principal Investigator: Yeni, Yener, Ph.D.
Tissue Stress Variability & Strength in Vertebral Bone (R01AR04934302)

A great percentage of the elderly population is at the risk of a vertebral fracture due to osteoporosis. Fractures of thoracic 12 and lumbar 1 constitute the majority of these vertebral fractures. External factors, such as loading, being aside, bone quality is the major determinant of a fracture. The measure of bone quality that is relevant to fracture is bone strength. However, current measures of bone quality, such as bone mineral density, are only surrogates of bone strength and have variable diagnostic success. New technologies with potentially high diagnostic value that are under development make use of the observation that bone stiffness and strength are highly correlated, i. e., how much load the bone can carry before fracture is related to how flexible the bone is under a given load. How these two apparent properties are related through bone's microstructural organization and to what extent bone stiffness is related to bone strength are very fundamental questions of bone biology. We believe that the close relationship between the apparent strength and apparent stiffness of cancellous bone is a result of an adaptation where tissue (trabeculae) stress magnitude and variability is controlled through microstructural organization such that the average and the scatter of the tissue stresses are correlated. This project will determine the relationships between bone microstructure, tissue stress distributions and bone apparent properties at a tissue and organ level for T4-L5 vertebrae from human spines using microcomputed tomography, large-scale finite element analysis and mechanical testing. The results of this project will reveal: (i) whether greater levels of damaging stresses are generated in the tissue from T12-L1 junction independent of external factors, (ii) whether the association between tissue stresses and bone apparent properties is sensitive to the choice of within-patient and between-patient samples, (iii) microstructural features of bone that give rise to damaging forms of stresses in the tissue, and (iv) whether vertebral bone strength at the organ level can be predicted by stiffness measurements using the concepts introduced for cancellous bone tissue. The long-term goal of this research is to enhance our ability to diagnose and develop strategies for prevention of bone disease and failure through a better understanding of the stress-regulated structure of vertebral bone.