Research Projects

Current Research Projects

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Crosstalk between Glioma Cells and Immune Cells in the Tumor Microenvironment Therapeutic Implications

Investigators: M. Castro, P. Lowenstein, M. Candolfi
Sponsor: National Institutes of HealthNational Institute of Neurological Disorders and Stroke

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults; in spite of improved standard of care (surgery, chemotherapy, and radiation), less than 5% of the patients survive more than 5 years post-diagnosis. Thus, new treatments for GBM are needed. We and others have shown that immune suppressive cells infiltrate the GBM microenvironment, i.e., regulatory T cells and myeloid derived suppressor cells (MDSC). MDSCs are characterized by co-expression of the myeloid cell-lineage differentiation antigen GR1 and CD11b. Expansion of MDSCs can be triggered by factors produced by the tumor cells themselves, which stimulate myelopoiesis and inhibit the differentiation of mature myeloid cells. Our preliminary data demonstrates that GBM cells in vitro and in vivo express ligands for the receptor for advanced glycation end products (RAGE), i.e., S100 calcium binding protein A8 (S100A8) and S100A9, which have been shown to play a critical role in mediating the expansion of MDSCs in several cancer models. The hypothesis we wish to test is that GBM-derived factors play a major role in activating signaling pathways on MDCS leading to immune suppression and glioma progression. The discovery that GBM-derived ligands play a critical role in brain cancer progression and hamper effective anti-tumor immune responses by promoting the expansion and activation of MDSCs, will lead to a paradigm shift in the development of novel immune therapeutic approaches for GBM aimed at inhibiting MDSCs expansion and activation. The overarching goal of this proposal is to elucidate the role of MDSCs in glioma progression and their impact on the development of novel immune therapeutics. Thus, SA 1 will test the hypothesis that GBM-derived ligands signaling via the RAGE pathway induce the expansion and migration of MDSCs into the tumor microenvironment. In SA2, we will test the hypothesis that glioma-induced MDSCs inhibit anti-tumor effector T cells’ functions in vitro and in vivo. In SA3 we will test the hypothesis that blocking the RAGE signaling pathway in vivo will hinder brain tumor progression and enhance T cell mediated anti-GBM immunity. These results will pave the way for novel anti-GBM immune therapeutic strategies aimed at manipulating the expansion and activation of MDSCs; leading to inhibition of tumor progression and enabling the induction of effective anti-tumor immunity in response to immune therapeutics. 

Aims: 

  • Specific Aim 1: To test the hypothesis that GBM-derived RAGE-ligands induce the expansion and migration of myeloid derived suppressor cells into the tumor microenvironment.
  • Specific Aim 2: To test the hypothesis that glioma-induced MDSCs inhibit anti-tumor effector T cells’ functions: implications for tumor progression.
  • Specific Aim 3: To test the hypothesis that blocking the RAGE signaling pathway in vivo will inhibit brain tumor progression and enhance T cell mediated anti-tumor immunity.

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Gene Therapy and the Brain:  Neuroimmune Interactions

Investigators: M. Castro, P. Lowenstein
Sponsor: National Institutes of HealthNational Institute of Neurological Disorders and Stroke

Gene therapy provides exciting new approaches to treat numerous incurable neurodegenerative disorders such as Parkinson's disease, multiple sclerosis, or brain cancer. Unfortunately the immune response to therapeutic vectors remains a major obstacle to the clinical realization of gene therapy. If primed, the immune system eliminates expression of therapeutic transgenes from the brain, curtailing gene therapy's efficacy. We will investigate immune regulation of transgene expression mediated by clinically effective first generation adenoviral vectors, and novel high capacity 'gutless' adenoviral vectors in a clinically relevant model. In this model animals will be pre-immunized to adenovirus to mimic immune status in the majority of human patients that were exposed to adenovirus before receiving gene therapy. In this proposal we will test the hypothesis that the immune system eliminates expression of therapeutic transgenes from the brain, primarily through cytotoxic, and secondarily, through non-cytotoxic mechanisms. To address these issues, we developed a specific method to differentiate immune system-induced brain cell death from selective down-regulation of vector-mediated transgene expression. This method is based on transgenic mice containing a floxed beta-gal construct, and viral vectors expressing Cre under pancellular and cell type specific promoters. Infected cells thus express a gene marker from their genomes, expression of which will be regulated independently of expression from the viral vector's genome. Preliminary experiments using our new method demonstrate that the immune system utilizes both mechanisms, namely it can eliminate expression of therapeutic transgenes from the brain by [1] direct cytotoxicity of transduced cells; and, [2] functional inhibition of transgene expression. Herein we will test specific hypothesis concerning the immune mechanisms that eliminate brain transgene expression, in both males and females, and in two mouse strains; one that displays TH1 biased immune responses (C57BI/6), and another one that displays a TH2 bias (DBA/2J). As a result of this work we will make available enhanced and safer gene therapy approaches, and more efficient clinical treatment paradigms. The results from this proposal will have a direct impact on experimental and clinical gene therapy, as well as make major contributions to understanding how the immune system eliminates or regulates gene expression in virally infected brain cells. 

Aims: 

  • Specific Aim 1: Will test the hypothesis that pre-existing anti-Adv immunity eliminates transgene expression from brain cells transduced with fg-RAdv, primarily through cytotoxic, and secondarily, non-cytotoxic mechanisms.
  • Specific Aim 2: Will test the hypothesis that pre-existing anti-Adv immune responses can reduce transgene expression from brain cells transduced with novel generation HC-Adv, primarily through cytotoxic, and secondarily, non-cytotoxic mechanisms, but to a lower extent than the elimination seen for fg-RAdv.
  • Specific Aim 3: Will test the hypothesis that anti-Adv specific CD4+ and/or CD8+T cells, but not an auto-immune response against the endogenous myelin protein MOG, eliminates transgene expression from brain cells transduced with fg-RAdv, and partially reduces transgene expression from brain cells transduced with novel HC-Adv.

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Mechanisms of Glioma Growth and Invasion Novel Therapeutic Strategies

Investigators: M. Castro, P. Lowenstein
Sponsor: National Institutes of HealthNational Institute of Neurological Disorders and Stroke

High grade gliomas are uniformly lethal, and resistant to surgery, chemotherapy and radiotherapy. The precise cellular and molecular mechanisms by which glioma cells disperse through the brain and grow to form macroscopic symptomatic tumor masses remains poorly understood. Herein we propose to test novel cellular, molecular and mechanistic hypotheses concerning glioma growth, and how to translate this knowledge into new anti-glioma therapeutics. Preliminary work from my laboratory, using confocal, electron and multiphoton microscopy has shown that glioma cells and human glioma stem cells disperse through the brain in vivo by traveling preferentially along the perivascular compartment, a potential migration network surrounding the brain microvasculature. As glioma cells move throughout the perivascular network they dislodge glial endfeet from blood vessels and compromise adjacent brain tissue; this is later replaced by tumor cells. We have also generated preliminary data that a glycan binding protein, galectin-1, is essential for this growth mechanism. Down regulation of galectin-1 abolishes glioma growth in the brain in vivo, without affecting growth in vitro. These new data have several clinical consequences: (i) lymph drains from the brain through the perivascular compartment; its obstruction by gliomas would contribute to glioma-induced edema; (ii) human glioma tumors grow to large size before causing symptoms; glioma cell replacement of atrophied brain tissue could explain protracted and indolent tumor growth, and the delayed changes in total brain volume; (iii) inhibition of galectin-1 could represent a novel treatment of human gliomas. This proposal will (I) test the hypothesis that rodent and human glioma cells, and glioma stem cells grow preferentially along the perivascular space; (II) test the hypothesis that galectin-1 mediates glioma perivascular invasion and growth, and that inhibition of galectin-1 can be used as a novel therapeutic strategy; and (III) test the hypothesis that inhibition of galectin-1 will enhance specific anti-glioma immune responses. By progressing from glioma pathophysiology to molecular mechanisms of glioma migration to experimental therapeutics, we aim for our work to lead to novel early phase clinical translational trials for the treatment of human gliomas. Of note, our first clinical trial for gene therapy of human gliomas is approaching the start of patient recruitment (it was approved by FDA on 4/7/11 [IND 14574] and very recently by the University of Michigan IBC and IRB). Therefore, our laboratory is in a strong and realistic position to guide our research towards the translational implementation of novel clinical trials for this currently deadly human cancer.

Aims: 

  • Specific Aim 1: To test the hypothesis that rodent and human glioma cells and glioma stem cells grow preferentially along the network of the perivascular space.
  • Specific Aim 2: To test the hypothesis that galectin‐1 mediates glioma perivascular invasion and growth, and that inhibition of galectin‐1 can be used as a novel therapeutic strategy.
  • Specific Aim 3: To test the hypothesis that inhibition of galectin‐1 will enhance specific anti‐glioma immune responses.

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A Non-randomized, Open-label Dose-finding Trial of Combined Cytotoxic and Immune Stimulatory Strategy for the Treatment of Resectable Primary Malignant Glioma – Clinical Trial

Investigators: M. Castro, P. Lowenstein
Sponsor: Phase I – The Road to Curing Cancer

Highly malignant grade IV glioblastoma multiforme tumors are uniformly fatal even when treated with the best available current standard of care, i.e., surgical excision followed by radiotherapy and temozolomide chemotherapy. Treatment in the best high complexity medical centers puts survival at 18-21 months, while centers of less medical complexity put survival at 12-14 months post-diagnosis. Thus, there is a great need for new treatments for highly malignant brain tumors. Over the last fifteen years we have developed a new approach for the treatment of human malignant brain tumors based on gene therapy. Using genetically engineered adenoviral vectors our strategy comprises the use of two viral vectors in combination. One vector expresses the thymidine kinase of HSV-1, while the second vectors express the cytokine Flt3L. The thymidine kinase of HSV-1 will metabolize the systemically administered prodrug (Valtrex) into a phosphorylated derivative that will kill dividing cells. Flt3L, as we have shown earlier in multiple publications, will attract immune cells, and especially dendritic cells to the brain tumor microenvironment. This strategy kick-starts a systemic immune response against brain tumors, which is even able to treat a second tumor implanted into the second hemisphere at a later stage, or recognize a neo-antigen never seen before by the immune system. Substantial efficacy studies in many different experimental models of brain tumors support the move of this therapeutic strategy into early phase clinical trials. With more than ten years of background research published in some of the most important scientific journals, such as Nature Medicine, PLoS Medicine, J. Exp. Med, amongst others, we intend to test this therapeutic approach in human patients suffering from high grade malignant brain tumors. For the clinical trial patients will have their gliomas resected, and viral vectors will be injected into the resection cavity; the total trial will treat 18 patients divided into 6 cohorts of increasing viral vector doses. Following surgery, patients will be treated with radiotherapy and temozolomide, the standard of care for this disease. They will then receive oral Valtrex to activate the tumor cell killing of HSV-1 TK. Patients will then be followed by standard clinical procedures. As this is an early Phase I clinical trial, we will closely monitor the existence of any untoward toxic side effects of the therapy. Long term survival of the patients will also be followed. Over the last few years we held pre-IND discussions with the FDA and then went forward towards implementing early phase clinical trials. Viral vectors of GMP grade were generated at the facility at Baylor College of Medicine, in Houston, TX, and toxicity studies with such vectors were done at Explora, in San Diego, CA. A full IND application was submitted to the FDA in 2010 and the IND #14574 was granted by FDA on 4-7-11. After my move to The University of Michigan, Ann Arbor, our application was approved by the UM IBC, and the UM IRB. We are now in the last steps of finalizing all preparatory work together with clinician-scientists from Neurosurgery, Neurology, Radiation Oncology, Pharmacy, Human Applications Laboratory, etc. towards starting this first Phase I clinical trial for the combined treatment of human glioblastoma grade IV with gene therapy using adenoviral vectors expressing HSV1-TK, and Flt3L.

Aims: 

The Grant funds shall be used for the following purpose(s): A non-randomized, open-label dose-finding trial of combined cytotoxic and immune­ stimulatory strategy for the treatment of resesctable primary malignant glioma. The Project shall be conducted and managed by Doctors Pedro Lowenstein and Maria Castro.

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Mechanisms of Brain Injury after Intraventricular Hemorrhage

Investigators: G. Xi, R.F. Keep, Y. Hua
Sponsor: National Institutes of Health

Goals: The purpose of our project is to investigate the mechanisms involved in thrombin-induced brain edema. If our hypotheses are correct, these experiments may lead to novel methods of treating intracerebral hemorrhage.

Spontaneous ICH is a common and often fatal stroke subtype. If the patient survives the ictus, the resulting hematoma within the brain parenchyma triggers a series of events leading to secondary insults and severe neurological deficits.

Brain edema plays an important role in the secondary brain injury following ICH. It is the long-term goal of our laboratory to identify the mechanisms involved in brain edema formation following ICH. Our previous studies indicate that edema formation following ICH may involve several phases. An early phase involves the clotting cascade and thrombin production. Although our data indicate that thrombin inhibition is a therapeutic target, concerns over rebleeding may limit the utility of this approach. It is, therefore, important to understand the downstream mechanisms activated by thrombin. Our preliminary studies have found:

  • Thrombin activity increases immediately after ICH
  • Complement cascade is activated in the brain following ICH and thrombin injection
  • Thrombin upregulates protease-activated receptors
  • Thrombin increases brain TNF-alpha levels
  • Thrombin potentiates iron-mediated damage.

We will determine whether:

  • Thrombin formation following ICH activates the complement cascade in the brain resulting in blood-brain barrier disruption and edema formation. These experiments will employ complement inhibitors as well as C3 and C5 deficient mice.
  • Thrombin causes brain edema through activation of PARs. These experiments will employ PAR-l, 3 and -4 agonists and antagonists as well as PAR-1 knockout mice
  • Thrombin exacerbates brain edema induced by iron.

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Endothelial Preconditioning and Ischemic Brain Injury

Investigators: R.F. Keep, S.R. Ennis, Andjelkovic-Zochowska, J. Xiang
Sponsor: National Institutes of Health

Goals: These experiments should provide information on endogenous defense mechanisms that protect the cerebral endothelium from ischemic injury and may be therapeutic targets; the role of endothelial preconditioning in the effects of IPC on the brain; and the role of endothelial injury in the overall effects of ischemia on the brain.

Ischemic preconditioning has proved to be one of the most effective methods of reducing ischemic brain damage in animal models of stroke. Attention has focused on the mechanisms by which neurons may be protected by such preconditioning. However, we have found that IPC also protects the endothelium, which forms the blood-brain barrier, from ischemic damage. “Ischemic” preconditioning also protects cerebral endothelial cells in vitro from oxygen glucose deprivation induced injury indicating that there can be direct preconditioning of the endothelium. Damage to the cerebral endothelium may potentiate ischemic brain injury in a number of ways; determining mechanisms to reduce endothelial damage is particularly pertinent at the moment considering the role of endothelial injury (hemorrhagic transformation) in limiting the use of tissue plasminogen activator for the treatment of ischemic stroke.

We will examine the mechanisms involved in cerebral endothelial preconditioning both in vivo (rat middle cerebral artery occlusion) and in vitro (primary cultures of rat cerebral microvessel endothelial cells). The in vitro experiments will facilitate exploration of the mechanisms involved in preconditioning while assuring that the preconditioning acts directly on the endothelium. The in vivo experiments will ensure that the mechanisms elucidated in vitro also occur in the whole animal as well as allowing an assessment of the effects of preconditioning on other parameters (such as blood flow, capillary morphology and infarction).

Overall, the research project has three main goals to determine:

  • The time course of preconditioning and the extent of its effects on the endothelium
  • The events that can trigger endothelial preconditioning
  • Which mechanisms are triggered to protect the endothelium.

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Mechanisms of Thrombin-Induced Tolerance to Brain Injury

Investigators: G. Xi, R.F. Keep, Y. Hua, T. Schallert  
Sponsor: National Institutes of Health

Goals: The purpose of our project is to investigate the mechanisms involved in thrombin preconditioning. An examination of TPC will also help our understanding of ischemic preconditioning since preliminary data suggests that TPC is a component of that phenomenon. TPC seems to be receptor mediated, greatly facilitating analysis compared to ischemic preconditioning. The long-term goal of these studies is to find mechanisms to limit brain injury after ICH.

Intracerebral hemorrhage is a common and often fatal subtype of stroke and produces severe neurologic deficits in survivors. Brain injury after ICH appears to involve several phases, including an early phase involving the clotting cascade and thrombin production and a later phase involving erythrocyte lysis (breakdown) and hemoglobin toxicity. Although high concentrations of thrombin cause brain edema and cell death, low concentrations are neuroprotective. Thus, we have found that prior treatment with a low dose of thrombin attenuates the brain edema induced by thrombin or hemorrhage, and significantly reduces the infarct size in a rat middle cerebral artery occlusion model. We have termed this “phenomenon thrombin preconditioning.” In our recent studies, we have found that TPC not only reduces brain edema induced by high dose thrombin, but also attenuates edema induced by lysed erythrocytes and FeCl2. TPC appear to involve upregulation and increased transferrin, activation of thrombin receptor, HIF-1 transferrin receptor and ferritin levels.

This suggests that thrombin release during an ICH might induce protective mechanisms against factors released upon clot lysis. This data has led us to the following specific aims:

  • To determine whether TPC reduces hemorrhagic brain injury caused by lysis of erythrocytes
  • To determine whether protease-activated receptors play a key role in TPC
  • To determine whether TPC modulates iron transport and storage protein levels in the brain, which then affects iron homeostasis after ICH.

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Thrombin-mediated Ischemic Brain Injury

Investigators: Guohua Xi, M.D.
Sponsor: American Heart Association Grant-in-Aid

Goals: The research project seeks to investigate the role of thrombin in brain injury after ischemic stroke. The long-term goal of our studies is to identify pathophysiologic mechanisms underlying brain injury from stroke, so that effective therapies can be developed to prevent secondary injury.

Every year, cerebral ischemia affects about 600,000 people in the United States. We will determine the effects of thrombin production on ischemic brain injury. Clarification of the mechanisms of brain injury after cerebral ischemia will help develop new therapeutic strategies for stroke.

Stroke ranks high as a cause of death and significant neurologic impairment with no effective treatment except thrombolytic recanalization with tissue plasminogen activator within three hours. Many different mechanisms have been implicated in secondary ischemic brain injury. Recent studies and our preliminary data indicate that thrombin is an important factor in ischemic injury. Thrombin receptors are upregulated in brain slices after oxygen-glucose deprivation. Hirudin, a thrombin inhibitor, reduces infarct volume in gerbils. In addition, our preliminary studies indicate that thrombin activity is increased in the ischemic core with an increase of expression of prothrombin mRNA. Furthermore, anti-thrombin treatment attenuates neurological deficits after cerebral ischemia with reperfusion, whereas exogenous thrombin exacerbates ischemic injury. After ischemia, the cerebral vasculature becomes permeable to plasma protein within several hours. Blood-brain barrier disruption then allows large molecules such as prothrombin entry into the brain parenchyma which may cause secondary damage following ischemia. In addition, prothrombin is also generated within the brain itself.

The specific aims of this project are:

  • To determine whether focal cerebral ischemia stimulates prothrombin and factor X synthesis in the brain and whether thrombin production increases in the ischemic area
  • To determine whether thrombin inhibitors in the brain parenchyma are changed after focal cerebral ischemia and whether focal ischemia induces upregulation of protease-activated receptors
  • To determine whether thrombin inhibition attenuates neurological deficits, infarct volume, brain edema, cerebral blood flow reduction and blood-brain barrier disruption following focal cerebral ischemia.

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Delayed Neurodegeneration After Intracerebral Hemorrhage

Investigators: G. Xi, R.F. Keep, Y. Hua
Sponsor: National Institutes of Health

Goals: The purpose of our project is to investigate the mechanisms of delayed neurodegeneration after ICH. The long-term goal of our studies is to limit hemorrhagic brain injury. If our hypotheses are correct, these experiments may lead to novel therapies for intracerebral hemorrhage by either limiting iron overload or attenuating oxidative brain injury.

Spontaneous ICH is a common and often fatal stroke subtype. If the patient survives the ictus, the resulting hematoma within brain parenchyma triggers a series of events leading to secondary insults and severe neurological deficits. Although the hematoma in human gradually resolves within several months, restoration of function is graded and usually incomplete. The neurological deficits in ICH patients are permanent and disabling.

To understand the underlying mechanisms of ICH-induced brain injury and to evaluate therapeutic interventions a number of animal models of ICH have been developed. A reproducible rat ICH model, involving infusion of autologous blood into the caudate, has been used extensively to study mechanisms of brain injury and, in particular, early edema formation. It has been difficult to find correlates of the long-term effects of human ICH. Recently, however, we have developed behavioral tests that can detect prolonged neurological deficits and we, and others, have found that there is delayed brain atrophy in animal models of ICH. The mechanisms involved in this prolonged and delayed brain injury after ICH are as yet unknown, but our preliminary data suggest a role for iron overload and oxidative stress.

We propose to test the following hypotheses:

  • To determine whether iron overload in the brain after red blood cell lysis plays a key role in brain atrophy and prolonged neurological deficits after ICH
  • To determine whether iron overload aggravates oxidative stress which contributes to delayed neurodegeneration after ICH.

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Does Surgical Debulking of Pituitary Adenomas Improve Responsiveness to Octreotide Long-acting Release in the Treatment of Acromegaly

Investigator: W.F. Chandler
Sponsor: Novartis

Goals: Until now, no randomized studies have been done to assess whether this surgical debulking improves the efficacy of therapy with medical therapy, including somatostatin analogs. In this study, we will investigate whether surgical debulking increases the efficacy of a long-acting depot somatostatin preparation, Sandostatin LAR Depot, in achieving biochemical normalization.

Acromegaly, a rare, debilitating disease associated with increased morbidity and mortality, is caused usually by a growth hormone-secreting pituitary tumor. Excess GH leads, in turn, to elevated levels of insulin-like growth factor I (IGF-I), which is responsible for many of the biological manifestations of the disease.

The goals of treatment of acromegaly include amelioration of the signs and symptoms of the disease, reduction of tumor mass, preservation of pituitary function, and normalization of GH and IGF-I. None of the currently available treatment options, including surgical removal of the tumor, radiation therapy of the pituitary gland and drug therapy, are fully effective in curing patients. Conventional radiation therapy takes more than 10 years to lower the GH level, surgery provides a permanent cure for about 40 percent of subjects, primary medical therapy with somatostatin analogs results in IGF-I normalization in about 60 percent of patients and dopaminergic agonists are effective in less than 20 percent of individuals. Thus, a significant number of individuals have persistently elevated GH and IGF-I level with any single treatment modality.

It is generally assumed (and supported by the limited available data) that individuals with lower initial GH levels are more responsive to medical therapy. To optimize utilization of combination therapy, it is necessary to determine whether lowering GH with surgery will render non-curved patients more responsive to medical therapy. 

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Effect of Spinal Cord Stimulation on Cerebral Blood Flow

Investigators: O. Sagher
Sponsor: National Institutes of Health

Goals: We will utilize an in vivo model of SCS and CBF measurement in rats in order to obtain information about the magnitude, time course and spatial characteristics of CBF changes induced by SCS. Using this model, we will also investigate the effects of SCS on stroke induced by focal cerebral ischemia. An understanding of the mechanisms involved in the cerebrovascular effects of SCS has direct ramifications in the treatment of conditions where cerebral blood flow is compromised, such as stroke, cerebral vasospasm and traumatic brain injury.

Spinal cord stimulation, a widely used modality for the treatment of chronic pain, is also known to have a significant vasodilatory effect on peripheral vasculature. Interest in the vascular effects of SCS has resulted in its use to treat both peripheral limb ischemia and angina. Similar effects of SCS on cerebral blood flow also have been suggested. The significance, character and underlying mechanisms of the cerebrovascular effects of SCS, however, remain unclear.

Our study delineates the cerebrovascular effects of SCS in an animal model and examines the mechanism(s) that may be involved. We also will examine the feasibility of using SCS in the prevention of cerebral infarction. Specifically, we will test the following central hypotheses:

  • SCS significantly enhances global cerebral blood flow
  • Cerebrovascular effects of SCS are related to changes in sympathetic tone
  • Cerebrovascular effects of SCS involve activation of brainstem/cerebellar vasomotor centers
  • Trigeminovascular innervation of cerebral vasculature mediates SCS effects on CBF
  • SCS has a neuroprotective effect in the setting of focal cerebral ischemia.

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Role of PEPT2 in Peptide/Mimetic Disposition-Dynamics

Investigators: D. Smith, F.C. Brosius, R.F. Keep
Sponsor: National Institutes of Health

Goals: By combining membrane, cellular, immunolocalization and whole animal studies in wild type and PEPT2 null mice, the studies will greatly advance our understanding of the in vivo role, significance and vectorial transport of peptide substrates and drugs by PEPT2 (as opposed to PEPT1, PHT1, PHT2 and other potential transporters). These studies may also provide rare insight into the variability of peptide/mimetic kinetics and response in those human subjects with genetic polymorphisms (e.g., hPEPT2 R57H). Finally, the studies may have important implications for the design, delivery and targeting of peptide-based pharmaceuticals to the brain for severe CNS disorders (e.g., Alzheimer’s disease, AIDS dementia, stroke, epilepsy, schizophrenia, infection and cancer).

Cellular, molecular and physiological studies have made major contributions toward a mechanistic understanding of PEPT2 structure, function and localization. However, these experimental approaches are often limited by an in vitro design and lack of blood supply, overlapping substrate specificities and contribution of multiple transport systems, some of which are unknown at the time of study. As a result, it is difficult, if not impossible, to define the function of a single specific gene product and its significance in relation to other possible proteins that are present in the tissue or organ of interest. Our laboratory has recently developed PEPT2-deficient mice, a unique resource in which to validate in vitro mechanistic studies with in vivo whole animal experiments. With this in mind, the long-term objectives of this competing renewal application are to define the physiological and pharmacological roles and relevance of PEPT2, a nutrient and drug transporter.

Our working hypothesis is that PEPT2 is the primary transporter responsible for the disposition and dynamics of peptides and peptide-like drugs within the body, particularly the brain and kidney. To test this hypothesis, the following specific aims are proposed: 

  • To define the role and relative importance of PEPT2 in affecting the invivo pharmacokinetics, tissue distribution and systemic exposure of peptides and peptide-like drugs
  • To determine the regional influence of PEPT2 on peptide/mimetic efflux from cerebrospinal fluid and its influence on pharmacologic response in the brain
  • To characterize the cellular localization and functional activity of PEPT2 in the brain parenchyma of developing mice.

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Stem Cell-Based Gene Transfer for Hurler's Disease

Investigator: J. Heth, M.D.
Sponsor: William and Ella Owens Medical Research Foundation

Goals: This research proposes to explore two methods to improve distribution of the gene and the enzyme within the central nervous system to treat Hurler’s disease.

Hurler’s disease is a prototypical lysosomal storage disease. Patients inherit the syndrome in autosomal recessive fashion and lack the enzyme -L-iduronidase. A reproducible, durable, low-risk method to treat the CNS in Hurler’s disease and the LSDs has not been found. One of the primary difficulties lies in distributing either the enzyme or its gene within the CNS. Stem cells, viral-mediated gene transfer and the peripheral administration of recombinant iduronidase show promise in the treatment of Hurler’s disease.

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Carotid Occlusion Surgery Study

Investigator: B.G. Thompson, M.D.
Sponsor: National Institutes of Health

Goal: This clinical trial will require an intensive and continuing effort to identify and enroll 930 clinically eligible subjects and will require the participation of qualified neurosurgeons, clinical neurologists, PET investigators and trained clinical coordinators.

Participation in this trial will test the hypothesis that surgical anastomosis of the superficial temporal artery to the middle cerebral artery when added to the best medical therapy can reduce by 40 percent – despite perioperative stroke and death – subsequent ipsilateral ischemic stroke (fatal and non-fatal) at two years in subjects with recently (within 120 days) symptomatic internal carotid artery occlusion, and ipsilateral increased oxygen extraction fraction as measured by positron emission tomography. This hypothesis will be tested by conducting a randomized, non-blinded, controlled trial in 372 patients randomized equally to surgical or non-surgical treatment. 

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Intracerebral Hemorrhage in Aging Rats

Investigator: Y. Hua, Ph.D.
Sponsor: American Heart Association

Goal: Clarification of the mechanisms of brain injury after ICH in the aging brain should help develop new therapeutic strategies for hemorrhagic brain injury. It also may give insight into how aging may affect other forms of brain injury.

Intracerebral hemorrhage is a common and often fatal subtype of stroke and produces severe neurological deficits in survivors. Edema following ICH contributes to these outcomes causing both acute herniation-related death and long-term neurological deficits. To understand the underlying mechanism of ICH-induced brain injury and to evaluate therapeutic interventions, a number of animal models of ICH have been developed. A reproducible rat ICH model, involving infusion of autologous blood into the caudate, has been used extensively to study mechanisms of brain injury and neurological deficits.

ICH is mostly a disease of the elderly, but most current experimental ICH models have used young animals. Age is an important factor affecting brain injury in cerebral ischemia, which may also affect brain injury after ICH. Our previous studies that complement system activated and system depletion complement system reduce the brain edemafter ICH. Our preliminary data showed that microglia activation, brain edema and neurological deficits were greater in the 18-month-old rats than those in the three-month-old rats. Microglia plays important role in activation of complement by synthesis and release complement components. It is the long-term goal of our laboratory to study the mechanisms of brain injury after ICH.

The study will:

  • Determine whether aging rats have more severe blood-brain barrier (BBB) permeability  disruption, brain edema, brain atrophy and neurological deficits after ICH compare to those in the young rats
  • Determine whether microglia inhibition reduces BBB disruption, brain edema, brain atrophy and neurological deficits in aging animals after ICH
  • Determine whether complement depletion reduces BBB disruption, brain edema, brain atrophy and neurological deficits in aging animals after ICH.

 

 

Obesity and Hypertension: The Role of 5HT2C Receptors

Investigators: Richard Keep, Ph.D., Steven R. Ennis Ph.D., Roger Grekin, M.D.
Sponsor: National Institutes of Health

Hypertension related to obesity is a major cause of morbidity and mortality in the U.S. A better understanding of the basic mechanisms linking these two factors will have major therapeutic relevance.

Acute intracerebroventricular infusions of 5HT2 receptor agonists activate the sympathetic nervous system and increase blood pressure. Preliminary data indicate that activation of a subset of the 5HT2 receptors in the brain, the 5HT2C receptors, is a contributory factor to deoxycorticosterone-salt induced hypertension. The central 5HT2C receptors are also involved in reducing food intake and body weight, 5HT2C receptor knock-out mice becoming obese. This suggests that 5HT2C receptor activation could be a link between obesity and hypertension. Specifically, we will examine the hypothesis that obesity causes 5HT2C receptor activation to reduce body weight but that chronic activation of this system causes hypertension.

This overall hypothesis is supported by preliminary data that hypertension induced by intracerebroventricular infusions of leptin, a hormone involved in reducing body weight, is blocked by 5HT2C receptor antagonists. Leptin is secreted by adipose tissue, is transported into brain at the blood-brain and blood-CSF barriers and has hypothalamic actions. The choroid plexus (the site of the blood-CSF barrier) have the highest densities of leptin and 5HT2C receptors suggesting that the choroid plexus may be one site of interaction between leptin and 5HT. We will examine whether 5HT2C receptor activation modulates blood to brain leptin transport as a feedback mechanism.

This project has four specific aims:

  • To determine whether chronic 5HT2C receptor activation can induce hypertension
  • To examine whether 5HT2C receptor activation is the cause of hypertension in rats fed a high-fat diet
  • To determine whether leptin activates 5HT2C receptors to produce a pressor effect
  • To examine whether 5HT2C receptor activation alters blood to brain leptin transport.

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Blood-Brain Barrier Transport and Ischemic Brain Injury

Investigators: Richard F. Keep, Ph.D., Steven R. Ennis, Ph.D.
Sponsor: National Institutes of Health

Because of its juxtaposition to blood, the cerebral endothelium (which forms the blood-brain barrier) has been thought to be relatively resistant to the effects of cerebral ischemia. However, examination of taurine, glutamine and myo-inositol influx into brain (all Na+-dependent processes) indicate a marked early (< 1 hour) reduction in transport during focal cerebral ischemia suggesting that endothelial cell injury could play a role in primary, rather than secondary, ischemic brain damage. This may be particularly the case if efflux from as well as influx into brain are affected since those efflux systems are involved in controlling the concentration of potentially toxic factors in the brain extracellular space. This research project, therefore, has two major goals:

  • To determine whether energy-dependent efflux from brain to blood is inhibited during cerebral ischemia 
  • To examine whether changes in influx and efflux transport mechanisms at the BBB contribute to ischemic brain damage.

The cerebral volume of distribution reached by [3H]vinblastine (a P-glycoprotein substrate) and p-[3H] aminohippuric acid (PAH, an organic acid transporter substrate) will be determined following middle cerebral artery occlusion in rat and mouse (Specific Aim 1). Whether an increased volume of distribution with ischemia reflects a change in influx or an alteration in efflux at the BBB will then be determined, the latter by examining the effect of cerebral ischemia in the absence of BBB P-glycoprotein (the mdr1a knock out mouse) or during probenecid-induced inhibition of organic acid transport. Specific Aim 2 will examine the mechanism by which ischemia inhibits efflux, by examining PAH, L-glutamate and methyl aminoisobutyric acid efflux (an A-System amino acid transporter substrate) uptake into choroid plexus using ventriculo-cisternal perfusion. Specific Aim 3 will determine the effect of altering specific transporters at the BBB on ischemic brain injury and will examine whether drugs known to ameliorate the effect of reperfusion on BBB disruption actually have these effects by altering transport during ischemia.

As well as determining whether early BBB dysfunction should be an alternate therapeutic target early during cerebral ischemia, the finding that there is an inhibition of energy-dependent efflux at the BBB during ischemia has major implications for drug delivery to the injured brain. P-glycoprotein and the organic acid transporter both play a major role in limiting the access of some drugs to the brain.
 
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