NIH Grant Abstracts

National Institutes of Health grant abstracts

NIH Principal Investigators at Legacy: 

Boison, Detlev, PhD
Bottlang, Michael, PhD
Burgoyne, Claude F., MD
Demirel, Shaban, PhD
Downs, J. Crawford, PhD
Fortune, Brad, OD, PhD
Gardiner, Stuart, PhD
Lusardi, Theresa A., PhD
Mansberger, Steven L., MD, MPH
Meller, Robert, DPhil
Ochoa, Jose L., MD, PhD
Simon, Roger P., MD
Wackym, P. Ashley, MD
Wang, Lin, MD
Xiong, Zhigang, MD, PhD
Zhou, An, PhD

PI:  Michael Bottlang, PhD
R21AR053958  "Evaluating and improving an emergent technology for fixation of bone fractures"

DESCRIPTION (provided by applicant): Each year, 6.2 million extremity fractures are treated in the US at a cost in excess of $13 billion. Even today, 5-8% of these fractures experience complications in fixation and healing, which presents a significant secondary, but preventable, public health issue. Locked plating is thought to be one of the most important, present-day advancements in fracture fixation. It preserves periosteal blood supply and may improve fixation in osteoporotic bone. As such, it provides a tremendous opportunity to improve the healing rate in problematic high-energy fractures and osteoporotic fractures alike.

By providing a potential solution to a pressing clinical challenge, locked plating technology has gained rapid and widespread acceptance despite the virtual absence of supportive data. This enthusiasm is in stark contrast to the 13-21% failure rates found in recent clinical reports on locked plates. The majority of these clinical complications manifest as delayed unions or non-unions. The likely reason for these complications is the high stiffness of locked plate constructs, which can suppress the interfragmentary motion required for secondary bone healing. Unlike conventional plates, locked plates rely on secondary bone healing by callus formation.

As a solution to this problem inherent to locked plating, we have explored a novel strategy, termed Far Cortical Locking (FCL), capable of reducing the stiffness of locked plate constructs while retaining sufficient fixation strength. The proposed in vivo study will investigate for the first time, how locked plating affects fracture healing in an ovine fracture model. In addition, we will test if stiffness-reduced FCL fixation can enhance fracture healing.

Specific Aim 1 will compare periosteum-sparing locked plating and conventional plating to determine if biology-preserving locked plates can improve fracture healing. Specific Aim 2 will compare locked plating and FCL plating to determine if stiffness-reduced FCL constructs can improve fracture healing. Specific Aim 3 will compare FCL plating to standard conventional plating to evaluate the combined benefit of biology-preservation and reduced stiffness provided by FCL plating. This proposed exploratory and developmental research constitutes an urgent first step toward evaluation and optimization of locked plating technology on a scientific basis. Results will have direct clinical implications by providing a long-overdue evaluation of readily adopted locked plating technology. Furthermore, results may direct the future evolution of locked plate technology by providing a working solution to an inherent problem associated with contemporary locked plates.  

PI: Detlev Boison, PhD
R01NS058780 "Adenosine-releasing brain implants for epilepsy therapy" 

DESCRIPTION (provided by applicant): Suppression of pharmacoresistant seizures in mesial temporal lobe epilepsy (MTLE) remains a major challenge in epilepsy therapy. Adenosine is an endogenous neuromodulator with potent anticonvulsant properties and thus highly suited to meet this therapeutic need. However, due to peripheral side effects of adenosine, a local mode is needed to supplement adenosine to the brain.

We aim to develop biocompatible scaffolds to deliver the endogenous seizure suppressor adenosine to suppress seizures in a rat model of MTLE. The rationale for this approach is based on the following findings from our labs: (1) Deficits of adenosinergic neuromodulation, in particular upregulation of the main adenosine removing enzyme adenosine kinase, contribute to epileptogenesis and seizures. (2) Augmentation of adenosine is sufficient to suppress pharmacoresistant seizures. (3) Local delivery of approx. 200 ng adenosine per day by brain implants of adenosine releasing cells is sufficient to provide complete seizure suppression in a model of MTLE. (4) Stem cell derived brain implants engineered to release adenosine retard epileptogenesis. (5) The combination of biopolymers and engineered stem cells promotes the release of adenosine. (6) Polymer scaffolds and cell encapsulation matrices are available, which permit cell replacement and sustained local drug delivery.

Our CENTRAL HYPOTHESIS is that local brain implants based on a combination of slow degrading biopolymers with adenosine and/or cells engineered to release adenosine are needed for clinical applications aimed at suppressing seizures in pharmacoresistant MTLE. The model system to address this hypothesis will consist of a novel protein-based polymer system, silk fibroin, in combination with adenosine and adenosine-producing cells, to be tested in an animal model of epilepsy. Our SPECIFIC AIMS are: Aim 1. Engineer biocompatible polymers for the local therapeutic delivery of adenosine. Aim 2. Develop a polymer/cell based system for the sustained delivery of adenosine. 

PI:  Detlev Boison, PhD
R21NS057475 "Adenosine kinase as novel therapeutic target to prevent acquired epilepsy"

DESCRIPTION (provided by applicant): Approximately 30% of all epilepsies are symptomatic and traumatic brain injury (TBI) is estimated to cause 20% of all symptomatic epilepsies. Thus, it is estimated that in the United States, at least 0.5 million surviving individuals live with posttraumatic epilepsy (PTE). Increased extracellular adenosine as an acute response to brain injury is known to provide seizure suppression and neuroprotection. However, astrogliosis associated with acute injury results in increased adenosine kinase (ADK), the key regulator of ambient adenosine levels. Upregulation of the astroglial based kinase ADK leads to deficits in the adenosinergic inhibitory feedback-system and thus promotes seizures.

Astrogliosis is not only a hallmark of many types of epilepsy, but also a consequence of TBI. Since TBI can lead to subsequent epileptogenesis, it is important to understand how astrogliosis may contribute to epileptogenesis. We aim to investigate how ADK is regulated in response to TBI and how these findings can be translated into applications to prevent epileptogenesis. The rationale for these studies is derived from the following previous findings from our lab: (1) Deficits of the adenosinergic system, in particular upregulation of ADK during astrogliosis, contribute to epileptogenesis and seizures. (2) Pharmacological blockade or RNAi-mediated downregulation of ADK effectively suppress seizures.

Our CENTRAL HYPOTHESIS is that, as an astrogliotic response to injury, upregulation of ADK occurs as a general phenomenon and is a cause for epileptogenesis after TBI. Consequently, local therapeutic downregulation of ADK after TBI is expected to prevent subsequent epileptogenesis. We will therefore monitor astrogliosis, upregulation of ADK and the development of seizures in a novel rat model of TBI. In a therapeutic approach, downregulation of ADK expression with lentiviral RNAi is expected to prevent epileptogenesis after TBI. The SPECIFIC AIMS of this project are: Aim 1. Investigate astrogliosis, ADK-expression, and seizures in TBI-model of PTE. Aim 2. Prevent PTE by local therapeutic intervention. PUBLIC HEALTH RELEVANCE: We plan to interfere with a response of the injured brain to reduce the abundance of the endogenous neuroprotective modulator adenosine. A gene therapy approach will be used to prevent the local reduction of adenosine after injury. Thus, new therapeutic strategies to prevent epileptogenesis after traumatic brain injury become feasible.

PI:  Detlev Boison, PhD
R21NS057538 "Adenosine kinase as therapeutic target to induce and maintain ischemic tolerance"

DESCRIPTION (provided by applicant): Acute brain injury can result in neuroprotection and tolerance to subsequent injury. However, the mechanisms of this endogenous neuroprotection are incompletely known. As the increase in adenosine following acute seizures is both neuroprotective and antiepileptic, adenosine may also provide neuroprotection and tolerance in ischemia. The elevation of adenosine following acute seizures is due to downregulation of adenosine kinase (ADK), the key enzyme of adenosine metabolism. Thus, the adenosine- ADK system may be a candidate as an endogenous tolerance effector.

We aim to investigate how ADK is regulated in response to ischemic brain injury and how these findings can be translated into applications to prevent damage to the injured brain. The rationale for these studies is derived from the following previous findings from our lab: (1) ADK is rapidly and transiently downregulated as an acute response to both injurious seizures and transient focal cerebral ischemia. (2) Upregulation of ADK renders the brain more vulnerable to ischemic cell loss. (3) Intrastriatal implants of adenosine releasing stem cells protect the brain from subsequent ischemia. (4) Pharmacological blockade or RNAi-mediated downregulation of ADK effectively suppress seizures and seizure-induced injury.

Our CENTRAL HYPOTHESIS is that the acute and transient downregulation of ADK after an insult is a general phenomenon of injury and responsible for the induction of ischemic tolerance and that augmentation and prolongation of this beneficial ADK-response is neuroprotective. We will investigate ADK expression in rodent models of ischemia. In a therapeutic approach ischemic tolerance will be induced by inhibiting ADK expression by lentiviral RNAi. The SPECIFIC AIMS of this project are: Aim 1. Study the involvement of the adenosine system in the induction of ischemic tolerance. Aim 2. Induce tolerance by therapeutic downregulation of ADK. PUBLIC HEALTH RELEVANCE: We plan to investigate novel mechanisms and strategies to prevent cell loss after stroke. Therapeutically, augmentation of the brain's endogenous neuroprotective adenosine system is expected to induce tolerance to ischemic brain injury. Our findings may be translated into effective new therapies for the prevention of brain damage after stroke.

PI:  Detlev Boison, PhD
R01MH083973 "Adenosine and schizophrenia: mechanisms and therapies"

 
DESCRIPTION (provided by applicant):  Schizophrenia (SZ) is a debilitating mental illness with tremendous human, social and financial costs to society. Unfortunately, existing treatments are unsatisfactory and current development remains stagnant due to poor understanding of the biological bases of the disease. Two perspectives have emphasized disturbances in two neurochemical messengers in the brain -dopamine and glutamate, in relation to disparate SZ- symptoms. This grant will examine a third messenger -adenosine (ADO), as a potential link uniting the dopamine and glutamate hypotheses of SZ. ADO can regulate both dopamine and glutamate neurotransmission via receptors with opposing actions (A1 vs. A2A adenosine receptors). ADO is therefore uniquely positioned as an upstream coordinator/regulator between these two neurotransmitter systems. Hence, ADO-based treatment may be an attractive alternative with dual corrective actions on the glutamate and dopamine systems, thereby achieving effective control over selected SZ symptoms.

Our central hypothesis is that subtle disturbances in adenosinergic neuromodulation can give rise to selected behavioral endophenotypes implicated in SZ; thus corresponding corrective interventions targeting at the ADO system should confer therapeutic potential against such SZ endophenotypes, and thereby validate our hypothesis. Our hypothesis will be tested by three specific aims. First, we will characterize the emergence of selected SZ-related endophenotypes as well as their opposing phenotypes in transgenic mice with either over- or under- expression of brain ADO achieved by genetic manipulation of adenosine kinase. Second, we aim to identify the molecular mechanisms of adenosine-based modulation of dopaminergic and glutamatergic neurotransmission. This will be achieved by behavioral and biochemical examination of A1R and A2AR knockout mice. Third, we aim to dissect the brain regions in which ADO-dopamine interactions and ADO-glutamate interactions contribute to the regulation of specific SZ-related endophenotypes.

To achieve this, ADO will locally be modified by transplantation of ADO-secreting stem cells and by focal infusion of drugs acting on ADO-receptors. The expected outcomes of this project include: (i) the biological validation of a novel neurochemical theory of SZ, and (ii) the feasibility-test of a novel ADO-based strategy to produce behavioral adjustment with therapeutic potential. PUBLIC HEALTH RELEVANCE: Schizophrenia is a devastating mental disorder to the individual and society alike, yet the efficacy of current drug treatment remains poor, and the development of novel drugs is limited to either blocking dopamine or enhancing glutamate neurotransmission. This proposal will examine a novel drug target for schizophrenia-therapy -adenosine: Given adenosine's unique position to interact in parallel with dopamine and glutamate neurotransmission, adenosine-modulating drugs are hypothesized to confer therapeutic potential against multiple SZ symptoms.

PI:  Detlev Boison, PhD
R01NS061844 "Astrocyte dysfunction in epileptogenesis:  the role of adenosine"

 
DESCRIPTION (provided by applicant): This grant  studies the hypothesis that astrogliosis and resulting dysfunction of adenosine-based neuromodulation is a mechanistic cause for the development of epilepsy (i.e. epileptogenesis). This is of importance, since to date no effective prophylaxis for epilepsy is available. This proposal will explore status epilepticus (SE)- triggered epileptogenesis and dysfunction of the endogenous adenosine-based seizure control system in mice in search for an astrocyte-based mechanism of epileptogenesis and thus may provide a foundation for the development of novel antiepileptogenic therapies. The proposal is based on the following findings: (i) Adenosine kinase (ADK) is the key enzyme for the regulation of adenosine; (ii) In adult brain, ADK is expressed in astrocytes; (iii) Astrogliosis is a hallmark of epileptogenesis; (iv) ADK is over-expressed within epileptic astrogliotic hippocampus; (v) Augmentation of adenosine by implants of adenosine releasing cells prevents kindled seizures. (vi) Transgenic overexpression of ADK increases seizure susceptibility; (vii) Local reduction of ADK in transgenic mice prevents epileptogenesis.

Our CENTRAL HYPOTHESIS is that an epileptogenesis triggering event (e.g. SE) induces astrogliosis with resultant regional upregulation of ADK as a necessary component of epileptogenesis and that reconstitution of brain adenosine by stem cell derived brain implants can prevent such epileptogenesis. The model system to address this hypothesis consists of intraamygdaloid application of kainic acid (KA) to initiate epileptogenesis selectively in the CA3 area of the hippocampal formation of mice and to transplant ADK-deficient adenosine releasing embryonic stem (ES) cells into the epileptogenic region. SPECIFIC AIMS: In Aim 1 we will study the causal, temporal, and spatial relations of astrogliosis, upregulation of ADK and seizures in a mouse model of CA3-selective epileptogenesis. In Aim 2 we will use a panel of different Adk-transgenic mice, in which we can molecularly separate cell-type specific functions of ADK expression from astrogliosis, to study both mechanisms independently. In Aim 3 we will use ADK-deficient ES cell-derived intrahippocampal implants in a therapeutic approach to prevent epileptogenesis. The expected outcome of these studies is to define astrocytic ADK as a target for the prevention of epileptic seizures and to translate these findings into a novel stem cell based treatment approach.

PUBLIC HEALTH RELEVANCE: Currently, no therapy is available to prevent the development of epilepsy. This proposal studies a defective function of the brain's own adenosine-based seizure control system as a mechanistic cause for epilepsy and translates these findings into a novel approach to prevent epilepsy by implanting adenosine releasing stem cells. 

PI:  Theresa Lusardi, PhD
F32NS054257  "Sensitization in primary and secondary brain injury"

DESCRIPTION (provided by applicant): The 1.5 million new cases per year of traumatic brain injury (TBI) are a significant cause of morbidity in the US. Treatment is complicated when the primary mechanical event is followed by secondary insults, resulting in reduced oxygen to the brain and exacerbation of injury.

This proposal tests the hypothesis that sublethal mechanical injury sensitizes the brain to secondary injury from mild hypoxia. Using cultured organotypic hippocampal slices and a novel in vitro TBI model, the contributions of mechanical injury (acceleration) and hypoxia can be studied (Aim 1). The delay between acceleration and oxygen glucose deprivation (OGD) and the duration of OGD will define the temporal profile of secondary injury. Sensitization by acceleration will be quantified by LDH release, and injury regionally identified with PI labeling, with cell subtype determined by immunocytochemistry.

Mechanisms of, and protection against, sensitization will be investigated. Methods include measurement of [Ca2+]i (Aim 2), and electrophysiologic measurements of field evoked post synaptic potentials and long-term potentiation (Aim 3). The goal is to identify novel mechanisms responsible for sensitization, expected to be critical in establishing novel neuroprotective strategies. 

PI: Trevor Lujan, PhD
1R41AR059433-01 "A cost-effective bioreactor to advance functional tissue engineering of cartilage"


DESCRIPTION (provided by applicant): Osteoarthritis (OA) is the leading cause of chronic disability in the United States.  A clinical goal in the treatment and prevention of OA is to develop replacement cartilage using tissue engineering (TE) technologies.  Although TE cartilage presently lacks the mechanical stability of native cartilage, studies have demonstrated that mechanical stability can be enhanced with specific chemical and mechanical stimuli. To speed the discovery of optimal stimulation protocols, research platforms need to be available that enable fast, clear and reliable communication of functional outcomes (i.e material properties).  Towards this goal, we introduce a six-chamber bioreactor that combines the efficiency of batch testing with the accuracy normally reserved for dedicated single-specimen material test systems. This system is therefore capable of mapping functional development of six individual specimens exposed to highly-specific mechanical stimulation protocols.  To remain cost-effective and portable, the bioreactor leverages system redundancies to eliminate hardware.  The specific aim of this study is to test the bioreactor's capacity to deliver accurate mechanical stimulations and material property evaluations in all six test chambers.  The effect of loading conditions and specimen geometry on accurate mechanical stimulation will be quantified using external sensors.  The viscoelastic material properties of soft TE scaffolds and stiff cartilage plugs will be characterized in both the six-chamber bioreactor and a conventional single-stage testing device.  Results between the bioreactor and the model testing system will be statistically compared. If validation of the bioreactor is successful, we envision this product will provide an economical and reliable research platform that fosters TE technology transfer.  

PI:  Robert Meller, DPhil
R21NS054023 "Rapid ischemic tolerance:  ubiquitin-mediated structural reorganization"

DESCRIPTION (provided by applicant): Tolerance, the cellular response to mild stress, which protects against a toxic stress, is a conserved feature of many organisms. Most research has focused on long-term tolerance, which occurs 24-72 hours following preconditioning and is mediated by changes in gene expression. In contrast we have focused on short-term ischemic tolerance, which is mediated by rapid biochemical events and occurs 1 hour following the preconditioning.

The identification of the mechanisms of short-term tolerance may identify novel rapid acting therapeutic targets to treat acute ischemic episodes (stroke). Our studies have suggested the ubiquitin-proteasome system, which regulates protein degradation, as being responsible for rapid ischemic tolerance. Using a proteomics screen we identified 2 candidate proteins, fascin and myristilated alanine-rich C kinase substrate (MARCKS), which are selectively ubiquitinated and degraded following preconditioning ischemia. The degradation of these candidate proteins results in the reorganization of the actin cytoskeleton, and may reduce NMDA receptor function, which is implicated in excitotoxic cell injury following ischemia. Our central hypothesis is the degradation of fascin and MARCKS following preconditioning ischemia, results in brief cytoskeleton re-arrangement and disruption of NMDA receptor function thereby protecting the brain from harmful ischemia.

Our hypothesis will be tested using an in vitro model of ischemia. In Aim 1, Investigate the effect of preconditioning ischemia on cytoskeletal protein degradation by the ubiquitin-proteasome system, we will investigate the role of the ubiquitin-proteasome system on the rapid degradation of our two candidate proteins, fascin and MARCKS, following preconditioning ischemia. In the Aim 2, Investigate changes in postsynaptic structural reorganization in ischemic tolerance, we will study the effect of preconditioning ischemia on the reorganization of the postsynaptic membrane. Experiments will address the effect of preconditioning ischemia on the structure of dendrites. We will determine the relevance of actin reorganization on ischemic tolerance by using actin stabilizing/ destabilizing compounds. Using co- immunoprecipitation we will investigate actin-NMDA receptor interactions following preconditioning. Understanding how preconditioning remodels synaptic structure and function may help identify harmful vs. protective mechanisms induced by brief ischemia in the brain, and give a clearer understanding of brain cell death. We believe that the rapid and selective degradation of specific brain proteins induces a protective state and may reveal suitable targets for pharmacological therapeutics. Indeed, the long-term aim of these studies is to discover endogenous protective mechanisms that can be translated into effective rapid acting neuroprotective agents for stroke.

PI:  Robert Meller, DPhil
R01NS059588 "Rapid ischemic tolerance:  Synaptic re-organization and reduced excitotoxicity"

DESCRIPTION (provided by applicant): We propose to identify molecular and cellular mechanisms that regulate the endogenous neuroprotective phenomenon of rapid ischemic tolerance. Our prior studies have shown that rapid ischemic tolerance is mediated by selective protein degradation via the ubiquitin-proteasome system. Using proteomics we have identified a pattern of ubiquitinated proteins in rapid tolerance: many of the proteins are involved with the regulation or function of the post synaptic density. Following preconditioning ischemia protein degradation of key post-synaptic structural elements results in rearrangement of actin filaments and the retraction of dendritic spines. These changes in neuronal morphology following preconditioning ischemia result in altered NMDA cell signaling and decreased NMDA-induced neurotoxicity in tolerant cells. Therefore further understanding the mechanisms of rapid ischemic tolerance may identify morphological and cell signaling at the synapse, and reveal novel targets for neuroprotective therapies. Our central hypothesis for this proposal is thus: - Rapid ischemic tolerance following preconditioning ischemia results in a) actin cytoskeletal re-arrangement and synaptic reorganization leading to b) the disruption of NMDA receptor anchoring to the cytoskeleton, c) thus altering NMDA receptor function with resultant protection from harmful ischemia. These events occur rapidly- within one hour- and have relevance for acute stroke therapy. This project with test our hypothesis and well as investigating whether morphological re-organization is a generalized response to preconditioning agents. The project utilizes both in vivo and in vitro models of ischemia.

The proposal has 4 aims. SPECIFIC AIM ONE: Test the hypothesis that proteins regulating actin cytoskeleton reorganization mediate neuroprotection following preconditioning ischemia. Specifically the effect of preconditioning ischemia on WAVE-1, CYFIP, and actin related protein 2/3 (Arp2/3) complex protein levels and interactions. In addition both pharmacological and viral transfer mediated peptide inhibitors of the Arp2/3 complex and upstream regulatory proteins will be investigated for their role in rapid ischemic tolerance.

SPECIFIC AIM TWO: Test the hypothesis that preconditioning ischemia induces a change in NMDA receptor function. We will investigate the effect of preconditioning on NMDA receptor-mediated electrophysiological responses, calcium signaling, nitric oxide synthesis and cyclic AMP response element binding protein (CREB) phosphorylation. In addition we will investigate the effect of reconditioning on NMDA receptor interactions with, and activation of, the tyrosine kinases src and pyk.
SPECIFIC AIM THREE: Test the hypothesis that dendritic spine loss, actin re-organization and NMDA protection is a common phenotype following pharmacological as well as ischemic preconditioning. We will test whether two pharmacological inducers of rapid tolerance (adenosine and diazoxide) have a protective phenotype similar to rapid ischemic tolerance. Specifically we will determine the effect of adenosine and diazoxide on actin filaments reorganization, dendritic spine density and tolerance to NMDA excitotoxicity.

SPECIFIC AIM FOUR: Test the hypothesis that preconditioning ischemia induces synaptic structural re-organization in an in vivo model ischemia. To determine the therapeutic potential of our observations we will determine, in a focal model of ischemia, whether preconditioning induces changes to dendrite morphology, actin organization, NMDA receptor scaffolding and NMDA mediated excitotoxicity. The rapid and selective degradation of specific brain proteins induces a protective state and may reveal suitable targets for pharmacological therapeutics. Indeed, the long-term aim of these studies is to discover endogenous protective mechanisms that can be translated into effective rapid-acting, but long lasting, neuroprotective agents for stroke or where ischemia may be predicted for example, heart bypass surgery.

PUBLIC HEALTH RELEVANCE: Our novel observations suggest that the morphology of neurons change as part of a protective mechanism following brief ischemia (stroke). It is our aim to investigate these mechanisms to help develop new therapeutics for stroke, or for circumstances where ischemia can be predicted, such as heart bypass surgery. 

PI:  Julie A. Saugstad, PhD
R01NS050221 "Neuroprotection by novel regulators of mGluR signaling"

DESCRIPTION (provided by applicant): Glutamate activates ionotropic glutamate receptors (iGluRs) that mediate fast synaptic transmission, and metabotropic glutamate receptors (mGluRs) that modulate cell excitability. The iGluRs gate extracellular sodium and calcium entry into the cell, while the Group I mGluRs (1, 5) lead to the release of calcium from intracellular stores. Brain injury such as stroke or ischemia leads to increased extracellular glutamate, uncontrolled activation of iGluRs and mGluRs, and the toxic accumulation of intracellular calcium that is an essential initiator of cell death.

Thus therapeutic strategies for the treatment of brain ischemia have focused on the use of iGluR and Group I mGluR antagonists. While iGluR antagonists are neuroprotective in modeled ischemia studies, likely due to inhibition of intracellular calcium accumulation, these compounds have failed in clinical trials. Similarly, Group I mGluR antagonists are neuroprotective in modeled ischemia studies, likely due to inhibition of intracellular calcium accumulation, yet delivery of most mGluR-selective compounds to the brain is difficult. We have begun to explore alternative neuroprotective strategies for brain ischemia using proteomics to identify novel proteins or peptides that modulate Group I mGluR signaling via interactions at the pharmacologically accessible extracellular amino terminal domain. Our first proteomic studies focused on the Group I mGluR subtype, mGluRS, and revealed a novel interaction with a recently cloned extracellular protein that promotes cell survival, ADNP (activity-dependent neuroprotective protein). ADNP contains an eight amino acid peptide sequence (NAPVSIPQ; NAP) that was shown to be the smallest active element of ADNP that can induce neuroprotection. Preclinical experiments show that NAP has potent neuroprotective, memory enhancing and neurotrophic properties. However, the mechanisms that underlie neuroprotection by NAP or ADNP are not known.

Our preliminary data suggest that one mechanism of neuroprotection by NAP and ADNP is to regulate Group I mGluR signaling. The research studies proposed herein are important because 1) they begin to delineate the mechanisms of neuroprotection by NAP, an exogenous peptide, and ADNP, an endogenous protein, 2) they provide evidence for the novel regulation of mGluR signaling by peptide or protein interactions at the extracellular domain, and 3) they offer new approaches for therapeutic intervention in brain ischemia.

PI:  Julie A. Saugstad, PhD
R21NS054220 "Role of microRNAs in ischemic tolerance"  

DESCRIPTION (provided by applicant): Ischemic tolerance is a phenomenon whereby a sub-lethal ischemic injury, preconditioning, induces endogenous protective mechanisms that lessen the impact of a subsequent, more severe ischemic insult. We have used modeled ischemic tolerance in rat and mouse, both in vivo and in vitro, to describe multiple effectors of neuroprotection in these endogenous systems. Recently, we used DNA microarray to examine changes in gene expression in mouse brains subjected to preconditioning induced by a brief duration of ischemia, injurious ischemia, or a tolerant brain (preconditioned, then challenged with injurious ischemia). These experiments revealed that injurious ischemia up-regulated gene expression, while ischemic tolerance resulted in significant down-regulation of gene expression.

Thus, the signature of ischemic tolerance is that of suppressed gene expression. Elucidating the molecular mechanism(s) that underlie this focused transcriptional suppression is our goal. Eukaryotic gene expression is regulated by microRNAs, a newly identified class of small non-protein coding RNAs that regulate mRNA translation and chromatin activity in mammalian cells. Based on the signature of ischemic tolerance, a suppression of gene expression, we hypothesize that ischemic tolerance leads to regulated microRNA expression that in turn leads to the protected phenotype. In support of this hypothesis, our preliminary data show that distinct subsets of microRNAs are regulated in preconditioned, ischemic, and tolerant mouse brain.

To fully examine a role for microRNAs in ischemic tolerance, we propose the following specific aims: 1) establish the expression profile of microRNAs in preconditioned, ischemic, and tolerant mouse brain by microarray analysis, 2) confirm changes in, and examine the temporal expression of, microRNAs regulated in preconditioned, ischemic, and tolerant mouse brain, and 3) examine the effect of regulated microRNAs on target protein expression and cell survival. These studies are among the first to examine the regulated expression of miRNAs in response to modeled ischemia, and given the current development of short, interference RNAs as novel therapeutic agents for a number of diseases including macular degeneration, asthma, diabetes, cancer, Huntington's, and Hepatitis C infection, these studies may provide rationale for the development of similar strategies for the treatment or prevention of stroke and brain ischemia.

PI:  Julie A. Saugstad, PhD
R01NS064270  "Role of microRNAs in ischemic tolerance"

DESCRIPTION (provided by applicant): Ischemic preconditioning, induced by a sub-lethal duration of ischemia, triggers endogenous responses that protect the brain against a subsequent, severe ischemic insult, a phenomenon known as "tolerance". Ischemic tolerance requires new protein synthesis, involves genomic reorganization, and is transient. Our long-term objective is to elucidate the molecular mechanisms by which the preconditioning stimulus induces tolerance. Our studies support the conceptual framework that preconditioning regulates the interaction between mRNAs and RISCs leading to increased translation of the mRNAs, particularly those that function as transcriptional regulators that can reprogram the genome and attenuate responses to ischemic injury, resulting in tolerance.

We will the following aims to test specific mechanisms of ischemic preconditioning-induced tolerance, including (1) the molecular mechanisms of ischemic preconditioning-induced regulation of RISCs, (2) the regulation of RISC-bound RNAs by ischemic preconditioning, (3) the regulation of the nuclear proteome and transcription rates by ischemic preconditioning, and (4) to overall test of the conceptual framework: tolerance by regulation of miRNAs and mRNAs. These studies are directly related to the mission of NIH and NINDS in that ischemic brain injuries are among the most common and important causes of disability and death worldwide. Clinical evidence suggests that endogenous preconditioning triggered by a transient ischemic attack is present in the human brain. While not without challenges, promising strategies to elicit endogenous brain protection are under clinical development. Thus, we will use a combination of biochemical, molecular, and proteomic studies to examine these distinct and novel mechanisms of in vivo ischemic preconditioning on the induction of tolerance. Our goal is to provide evidence for miRNAs as effectors of endogenous neuroprotection that will translate into novel strategies for the treatment or prevention of ischemic brain injury. 
 
PUBLIC HEALTH RELEVANCE: Ischemic brain injuries due to stroke or cardiac arrest are common and prominent causes of disability and death worldwide. Yet, there is evidence to suggest that a transient ischemic attack can actually protect the human brain from a subsequent, more severe ischemic attack. As we can model this protection in laboratory studies, this research will improve public health through the identification of novel mechanisms that contribute to this protection, and their translation into clinical strategies for the treatment or prevention of ischemic brain injury.  

PI: Roger P. Simon, MD
R01NS024728 "Cell excitation in ischemic brain injury"  

DESCRIPTION (provided by applicant): Drug therapy designed in the laboratory to protect ischemic brain has not translated to humans. For this reason attention has been focused on molecular mechanisms by which the brain can induce endogenous neuroprotective strategies. Such neuroprotection occurs when the brain is "preconditioned" by exposure to brief ischemic stress resulting in "tolerance" to subsequent severe ischemia. Such ischemic tolerance produces robust neuroprotection through protein-synthesis dependent mechanisms, which require 24 hours to evolve. These gene-based mechanisms of tolerance were the focus during our previous grant period.

We now propose to investigate the cellular mechanisms involved in rapid ischemic tolerance, a protective mechanism, well know in the heart, which is protein synthesis independent, and inducible with in an hour, a time frame likely to have substantial clinical potential. We offer novel preliminary data describing the cell biology producing rapid ischemic tolerance in brain, resulting from the action of the constitutive anti apoptotic protein Bcl-w. We demonstrate its neuroprotective effects, the mechanism by which its function is rapidly potentiated in ischemia, offer experiments showing additional mechanisms to further regulate Bcl-w function and show a hitherto unknown function of an anti-apoptotic bcl-2 family member protein, that of modulating GABA channel currents which are additionally neuroprotective.

Thus, we offer a new description of an endogenous neuroprotective cell biology strategy for ischemic brain which is rapidly effective. To further define this biology we offer the following aims: Aim 1: Investigate the role of Bcl-w in rapid ischemic tolerance Aim 2: Investigate the effect of over expression of Bcl-w on ischemia-induced cell death Aim 3: Investigate the role of 14-3-3 and CKs (casein kinases )in regulating Bcl-w function Aim 4: Investigate the role of Bcl-w-induced enhancement of GABA mediated currents in rapid ischemic preconditioning In order to investigate these aims we will use a variety of approaches, including immunoprecipitation, knockout mice deficient in the Bcl-w gene, HIV TAT-based protein transduction vectors for delivery of Bcl-w to neurons, electrophysiological recordings from neurons and site directed mutagenesis studies. Experiments will be predominantly performed using in vitro ischemia models using cortical neuronal cultures, and key observations replicated using in vivo focal ischemia models. As such, understanding the endogenous mechanisms that regulate rapid ischemic tolerance may lead to the identification of novel therapeutic targets for the treatment of stroke. 

PI: Roger P. Simon, MD
P01NS035965 "Molecular mechanisms of Ischemia"

DESCRIPTION (provided by applicant):   The central theme of this program project is that potent endogenous mechanisms of neuroprotection are encoded in the genome and that the expression of a subset of these genes helps to determine whether cells survive ischemia. The scientific goals are to identify and characterize these genes and the neuroprotective pathways through which their protein products operate. The rationale for this approach is the understanding that the brain's response to injury is an active process that involves new protein synthesis. Identifying gene products that are endogenous neuroprotectants would contribute significantly to our understanding of the pathophysiology of ischemic neuronal injury and would point the way toward new therapeutic approaches to stroke and to related disorders, such as traumatic brain injury. For example, the discovery of a network of transcription factors and target genes that regulate ischemic tolerance in brain would advance pharmacologic efforts to mimic this effect.

We will focus on in vivo and in vitro systems wherein endogenous neuroprotection has been induced and the brain has been made tolerant to subsequent ischemic injury (ischemic preconditioning and tolerance). The strategy for discovering neuroprotective genes in ischemia is to use mouse models of ischemic tolerance and microarray analysis to identify genes that are transcriptionally regulated in tolerance (Project 1). Identified genes will then be studied in vitro in models of ischemia and tolerance to characterize and confirm neuroprotective function (Project 2). Finally, gene products that are neuroprotective will be investigated by increasing or reducing their expression in mice in vivo, using pharmacologic and genetic approaches (Project 3). A Genomics Core (Core A) will provide Affymetrix microarray analysis to each project. Our collaborators at Pacific Northwest National Laboratory's Supercomputer and Bioinformatics Division will employ network analysis of gene clusters via conditional probability approaches and functional assignment of unknown genes using analysis of sequence similarities. The Administrative Core (Core B) will coordinate manuscripts, computer connections, data sharing, speaker travel, grants management, and statistical consultation for the interacting laboratories, as well as scientific consultation through internal and external advisory boards.

PI: Roger P. Simon, MD
R01NS050610 "Acid-sensing ion channels and ischemic brain injury"  

DESCRIPTION (provided by applicant): Brain ischemia is characterized by a marked, rapid fall in pH, which is assumed to be injurious although through multiple or uncertain mechanisms. The recent discovery in brain of Acid Sensing Ion Channels (ASICs), which are ubiquitous and function physiologically in synaptic neurotransmission, offers a diffuse, membrane based, receptor gated ion channel system, which will respond to the pathologic pH fall in brain ischemia. These H+ receptor gated channels are Na+ channels, a portion of which is also Ca2+ permeable. Acid sensitivity and Ca2+ permeability suggest a role in ischemic brain injury. Using patch clamp techniques and Ca2+ imaging of native neurons in cortical cultures we show the pH sensitivity of these channels and acid induced Ca2+ uptake. Both the acid induced channel currents and Ca2+ uptake are greatly potentiated in the setting of modeled ischemia (Oxygen Glucose Deprivation-OGD or NaCN).

Thus, these channels respond to both acidosis and "ischemia" in a mutually potentiating manner. The channel current and Ca2+ uptake are glutamate independent, are inhibited by ASIC pharmacologic blockade and by specific blockade of the ASIC 1a channel subunit. Thus, our preliminary studies support new cellular and molecular mechanisms mediating ischemic-acidosis induced brain injury, which we offer to dissect with the following Specific Aims: Single cell recording and transfection of individual ASIC subunit cDNAs will show that: 1) Ca2+ permeable ASICs produce cell injury in modeled ischemia via a subunit specific mechanism. Using rat and mouse global ischemia models and ASIC1a & ASIC2a knockout mice, we propose that: 2) Blockade of ASICs protects against ischemic brain injury in vivo. With molecular techniques including site directed mutagenesis we will describe endogenous Zn2+ modulation of Ca2+permeable ASICs: 3) Characterize high-affinity Zn2+ modulation of ASIC currents in acidosis and ischemia -induced cell injury. These experiments will describe new, glutamate independent, mechanisms of ischemic brain injury and the central role of ischemic-acidosis. ASIC blockade will offer new and potent potential therapy for stroke.        

PI: Roger P. Simon, MD
R01NS039016 "Molecular mechanisms of seizure-induced brain injury"  

DESCRIPTION (provided by applicant): Optimal post-seizure therapy requires anticonvulsant and neuroprotectant therapeutic strategies, as both prolonged seizures (status epilepticus) and repetitive seizures occurring over time (epilepsy) cause neuronal death in brain and cognitive decline. Our data challenge assumptions on the relevant molecular pathways for such neuronal loss, revealing that Death Receptors of the extrinsic pathway are preferentially activated following seizures, which then trigger endoplasmic reticulum (ER) dysfunction. This extrinsic pathway activation occurs before any (intrinsic) mitochondria-linked cell death pathways become activated. We show protection from extrinsic pathway activation by ER-resident anti-apoptotic Bcl-w, which functions through effects on calcium regulation, attenuation of ER based pro-apoptotic Bcl-2 family protein function and inhibition of the integral ER membrane BAP31 complex.

The SPECIFIC AIMS of this project are: Aim 1. Determine the significance and mechanism of death receptor complex activation as a cause of ER dysfunction and neuronal death following seizures. Aim 2. Determine how Bcl-w protects against seizure-induced neuronal death via effects on the extrinsic pathway target: Aim 3. Show the relationship between Extrinsic and Intrinsic pathway activation by seizures. To accomplish these aims we have developed a mouse model of seizure-induced brain injury, not previously available, with continual EEC seizure monitoring, permitting electrographic seizure quantitation and distinction between injurious and non-injurious seizure types. We also use an in vitro seizure model permitting single cell, calcium imaging and culture studies. With mouse modeling we will use knockouts for down regulation of apoptosis modulatory genes under study and provide up-regulation of protective gene products with adenoassociated viruses and TAT fusion proteins. We determine the effect of pharmacological and molecular manipulation of the extrinsic cell death pathway on seizure induced cell damage. Finally, using continuous video-EEC monitoring, we will investigate how prevention of neuronal damage following seizures effects the development of an epileptic phenotype.    

PI:  Roger P. Simon, MD
R01NS047622 "Molecular determinants of epileptic brain injury"  

DESCRIPTION (provided by applicant): Neurons die following certain brief seizures. Such neuronal death may contribute to cognitive decline in patients with poorly managed seizures, or contribute to more severe seizures in epileptic brain. Brief seizures activate a coordinated molecular pathway within the hippocampus that involves dimerization interactions of pro- and antiapoptotic members of the Bcl-2 gene family and their satellite regulators, protein kinase B and 14-3-3 proteins. Pharmacological interventions confirm this pathway drives as much as half of cell death after seizures.

Our preliminary data reveals that expression and interaction of two cell death regulators, Bcl-w and Bim, are fundamentally critical to whether seizures cause neuronal death. Seizure-induced activation of forkhead transcription factors drive Bim overexpression, which quenches Bcl-w, an endogenous molecular brake on cell death. In turn, mice deficient in the Bcl-w gene exhibit a lowered threshold for injury following seizures, despite reactive upregulation of protective genes.

Our central hypothesis is: Bim and Bcl-w regulate the majority of neuronal death after brief seizures. The specific aims of this project are: 1. Characterize the expression and interactions of cell death regulators Bcl-w and Bim following brief seizures and in long-term epilepsy. 2. Investigate the effects of manipulating Bcl-w expression on seizure-induced damage and epileptogenesis. 3. Demonstrate the in vivo functional significance of Bcl-w and Bim by examining seizure-induced neuronal damage in mice deficient in each gene. 4. Determine the consequence of Bim and Bcl-w gene deletions on the generation of an epileptic phenotype. These studies will identify potent regulatory sites in the molecular pathways by which neurons die following .brief, electrographically defined seizures, thereby offering novel, focused neuroprotective targets beyond anticonvulsants for treating at-risk epilepsy patients.

PI: P. Ashley Wackym, MD
R01DC02971 "Molecular Basis of Vestibular Efferent Function"

DESCRIPTION: The long-term objective of this ongoing research program is to understand the projection of the vestibular efferent neurons to the vestibular periphery in the context of the fundamental molecular sensory organization of the receptors and the vestibular primary afferents.  Based on data resulting from studies performed during the PI's K08, R29 and R01 Awards, the diversity and organization of the efferent receptors and second messengers is far more complex than our original hypothetical model predicted.  During the current term of the grant, four questions are addressed: 1) what are the topographic distributions of these expressed genes and proteins in the rat and human vestibular end-organs and primary afferent ganglia?; 2) what are the full length cDNA sequences encoding the mRNAs subserving the efferent/afferent interaction in the vestibular periphery?; 3) what is the chromosome location and the genomic sequence for each of these genes and how are they processed to yield these cDNAs?; and 4) what changes occur in these genes during the aging process?

The studies proposed are either hypothesis testing or hypothesis generating in nature.  Specific Aim 1 will determine the topographical distributions in the vestibular epithelia and Scarpa's ganglia of specific muscarinic AChR subtypes and nicotinic AChR subunits, purinergic receptors, and second messenger/transduction proteins using in situ hybridization histochemistry in the rat vestibular periphery and immunohistochemistry in the rat and human vestibular periphery.  Specific Aim 2 will characterize the full length cDNAs identified in the initial funding period which may be responsible for the efferent/afferent interaction in the rat vestibular periphery and using radiation hybrid mapping, determine the chromosomal locations for each of these genes in the vestibular periphery.  Specific Aim 3 will explore how the expression of the genes potentially subserving the efferent/afferent interaction changes during the process of aging using microarray expression profiling.

After a detailed molecular understanding of this efferent/afferent interaction, experimental studies can be performed to induce changes in this system.  These studies will provide a model for determining the role of the efferent and afferent pathways during vestibular compensation, after vestibular injury, or during specific vestibular disorders such as Meniere disease or migraine associated vertigo (benign positional vertigo of childhood).  In addition, molecular characterization of these neurotransmitter and receptor distributions may lead to advances in the pharmacotherapy of patients with vestibular dysfunction.

PI: Lin Wang, MD
R01EY019939 "Dynamic and Static Autoregulation Impairment in the Optic Nerve Head of Glaucoma"


DESCRIPTION (provided by applicant): Glaucoma is a disease characterized by irreversible damage of optic nerve affecting millions of Americans. Yet details of the underlying disease mechanism are still unclear. While recognizing the crucial role of intraocular pressure (IOP), autoregulation (AR) dysfunction has been proposed as a cause of circulatory aberrations in the optic nerve head (ONH) associated with glaucomatous optic neuropathy. Autoregulation in the normal ONH initiated by an ocular perfusion pressure change contains two phases: an initial dynamic phase (dAR) when vascular smooth muscles dilate and contract to adjust the vascular resistance in an attempt to return blood flow (BF) to its original level; and a later steady-state phase (sAR) when dynamic BF changes have equilibrated to a steady level. Due primarily to methodological limitations, most previous studies in human and experimental glaucoma have assessed only sAR and failed to detect AR dysfunction in the ONH. With a modified laser speckle flowgraphy device (LSFG) and newly established methods for measuring the dAR and sAR, this proposal will test the following central hypothesis: Chronic IOP elevation induces AR dysfunction in the ONH, which importantly contributes to the pathophysiology of glaucomatous ONH damage. This hypothesis will be tested in three Specific Aims. Specific Aim 1: To test the hypothesis that ONH dAR abnormalities develop early in the monkey experimental glaucoma model, that they precede sAR and bBF alterations and that they progress in parallel with clinical measures of ONH and RNFL structural disruption. Specific Aim 2: To test the hypothesis that the ONH AR abnormalities occurring in the monkey model of experimental glaucoma are a primary result of exposure to chronic IOP elevation rather than a secondary result of neurodegeneration. Specifically, we will test the prediction that AR abnormalities will not develop in two alternative, non-IOP-related, axonal injury models - optic nerve transection and intra-retinal laser axotomy. Specific Aim 3: the ONH tissues obtained from the animals studied in Specific Aims 1 and 2 will be used to carry out two postmortem histological studies: 3A) To assess regional BF in the monkey ONH using a state-of-the-art microsphere method and compare these measurements with the LSFG bBF estimates obtained immediately prior to sacrifice; and 3B) To test the hypothesis that AR dysfunction detected in vivo by LSFG is associated with derangement of the relationship between ONH astrocytes, lamina cribrosacytes and the blood vessels within the underlying laminar beams and peripapillary scleral beam insertions. In a follow up R01 proposal we expect to extend our investigation in: 1) clinical application by modifying the current techniques of dAR analysis to noninvasively so as to elicit a controlled ONH dAR response. 2) Characterization of ONH astrocytes and lamina cribrosacyte role in ONH AR using 3D electron microscopic reconstructions and fresh ONH tissue slice preparations. 3) To test the hypothesis of age-related alterations between astrocytes and capillary endothelial/pericytes in AR. 
 
PUBLIC HEALTH RELEVANCE: This project seeks evidence of autoregulation (AR) dysfunction in the optic nerve head and its role in pathogenesis of glaucoma as well as to develop and assess a method for its clinical detection. These developments will make the detection of AR dysfunction in human ocular hypertensive and glaucoma patients a credible goal and the presence of AR dysfunction a therapeutic target for the treatment of the disease. 

PI: Zhigang Xiong, MD, PhD
R01NS047506 "Acid-sensing channels as novel target for brain ischemia"  

DESCRIPTION (provided by applicant): Brain acidosis is a common feature in acute neurological diseases particularly in ischemia, and has been assumed to play an important role in the pathology of neuronal injury. However, the cellular and molecular mechanisms underlying acidosis-induced injury remain uncertain, multifactorial and vague. We have substantial preliminary data demonstrating that activation of newly described acid-sensing ion channels (ASICs), members of Degenerin/EnaC superfamily, and subsequent Ca2+ entry through these channels are largely responsible for acidosis-induced, glutamate receptor-independent neuronal injury. In cultured mouse cortical neurons, lowering pH activates amiloride-sensitive ASIC currents.

In the majority of these neurons, ASICs are also permeable to Ca2+, and activation of these channels induces increases in the concentration of intracellular Ca2+([Ca2+]i). Activation of ASICs by brief incubation of neurons with acidic solutions induces time-dependent cell injury in the presence of the blockers for both voltage-gated Ca2+ channels and the glutamate receptors. This acid-induced injury is, however, inhibited by the blockers of ASICs, and by reducing the extracellular [Ca2+]. Acid treatment of COS-7 cells that lack functional ASICs does not induce significant cell injury. Similar to the primary cultured neurons, acid treatment induces injury in organotypic brain slices, and the injury of brain slices is inhibited by the blockers of ASICs.

Preliminary in vivo studies also demonstrate that intraventricular injection of ASIC1 blocker reduced the infarction volume, and knockout of the ASIC1 gene protects the mouse brain from ischemic injury. Furthermore, our preliminary studies demonstrate that ischemic treatment and metabolic inhibition dramatically potentiate the ASIC currents. This potentiation of ASICs in turn increases acidosis-induced neuronal injury. Our overall objective is to investigate the pathological role of ASICs in the central nervous system and to test the hypothesis that activation of ASICs with subsequent Ca2+ entry is largely responsible for acidosis-mediated, glutamate-independent ischemic brain injury. Specific Aims 
Aim 1. Ca2+ -permeability of acid-sensing ion channels in CNS neurons 
Aim 2. Specific subunit configurations are responsible for acidosis-induced neuronal injury 
Aim 3. Potentiation of ASIC currents by hypoxia/ischemia 
Aim 4. Neuroprotective role of ASIC blockers or ASIC gene knockout in an in vivo model of brain ischemia and the organotypic brain slices. 

PI:  Zhigang Xiong, MD, PhD
R01NS049470 "A novel cation channel in excitatory neuronal injury"  

DESCRIPTION (provided by applicant): Calcium is one of the most important ions in the central nervous system, essential for the regulation of neuronal excitability, synaptic transmission, neuronal development and differentiation. Alterations of Ca2+ homeostasis have been shown to be involved in the pathology of various neurological diseases/disorders. Accumulation of intracellular Ca2+ ([Ca2+]i), for example, has been recognized as a central pathological feature in brain ischemia. Along with an increase in [Ca2+]i, ischemia also causes a dramatic decrease in the concentration of extracellular Ca2+ ([Ca2+]e).

Although it is well-known that the increase of [Ca2+]j is critical for excitatory neuronal injury, it is not clear whether the alteration of [Ca2+]e plays any role in the pathology of brain ischemia. We have previously demonstrated that lowering [Ca2+]e to the level commonly seen in brain ischemia strongly depolarized and excited cultured hippocampal neurons through activation of a non-selective cation channel. This channel has electrophysiological properties and pharmacological profiles different from known channels, suggesting activation of a novel Ca2+-sensitive ion channel. Our preliminary study also demonstrated that activation of this channel caused an increase in [Ca2+]i and potentiated NMDA-receptor mediated membrane responses as well as neuronal injury.

We therefore hypothesize that decreases of [Ca2+]e to the level seen in brain ischemia activates a distinct Ca2+-sensing non-selective cation (csNSC) channel. Activation of these channels induces membrane depolarization and neuronal excitation, which contributes to excitotoxicity either directly, or indirectly through potentiation of NMDA receptor mediated responses. Our objective is to provide additional evidence that csNSC is indeed a distinct new channel and to investigate its pathological role in hypoxic/ischemic neuronal injury. In addition to cultured neurons, we will characterize detailed electrophysiological properties of the csNSC channel in acutely dissociated mature neurons and the neurons in brain slices, develop a pharmacological profile and search for a specific channel blocker. We will define the Ca2+-permeability of the csNSC channel, and determine whether ischemic treatment enhances the Ca2+-permeability. Since NMDA channels play a critical role in excitatory neuronal injury, we will characterize detailed interaction of csNSC channels with NMDA receptor-mediated membrane responses and neuronal injury. Using in vitro ischemic models, we will determine whether preventing the activation of csNSC channels protects neurons from ischemic injury. Specific Aims are: 1. Provide further evidence that lowering [Ca2+]e to the level seen in brain ischemia activates a distinct nonselective cation channel in the central nervous system. 2. Demonstrate that csNSC channels are Ca2+-permeable. 3. Demonstrate potentiation of NMDA channel function by csNSC activation. 4; Determine the potential role of csNSC channels in ischemic neuronal injury.  

PI:  An Zhou, PhD
R01NS046560 "Brain ischemia attenuates Neuropeptide biosynthesis"  

DESCRIPTION (provided by applicant): Neuropeptides play crucial roles in maintaining normal function of the brain and the brain's response to stresses, e.g. ischemia. Virtually all known neuropeptides are processed from larger precursors by the action of a set of processing enzymes in the secretory pathway. The biological function of precursor forms may differ profoundly from that of final processed forms to include even a switch from pro-apoptotic rather than anti-apoptotic function. Little is known about how the biosynthetic processing of neuropeptides may be affected by ischemic stress in the brain. Our preliminary studies indicate that ischemia causes attenuation in the activation of key neuropeptide processing enzymes and an accumulation of certain neuropeptides in precursor forms.

Our hypothesis is: ischemia has adverse effects on the functional status of the neuropeptide processing system, thus impairing the ability of brain cells to produce certain protective peptide factors. This ischemia-induced attenuation of neuropeptide processing contributes to post-ischemia cell death in the brain. In the proposed study, we will first examine changes in the expression levels of several key neuropeptide processing enzymes and their enzymatic activities in ischemic rat brains. Then, we will determine the levels and molecular forms of neuropeptides that are known to have modulatory roles in the brain after ischemia and to require the action of processing enzymes investigated in this study for proper processing. Using in vitro ischemia models in cultured cells, we will investigate how ischemic stress may influence the activation and maturation processes of the processing enzymes and alter the production and secretion of neuropeptides. In parallel, we will analyze peptides secreted from ischemic cells using a quantitative proteomic approach. Finally, we will investigate if animals with specific deficiencies in neuropeptide processing may be more vulnerable to ischemic stress and determine if peptides secreted from processing-deficient neuronal cells may cause or exaggerate ischemic cells death. The proposed study will offer a novel mechanism concerning how ischemic stroke may damage the brain by attenuating neuropeptide processing. Our long-term goal is to elucidate the molecular mechanisms of neuropeptide processing-mediated stress response of the brain.

PI:  An Zhou, PhD
R21EY017345 "Neuropeptide processing and ischemic retina injury" 

DESCRIPTION (provided by applicant): Retinal ischemia has been implicated in a number of retinal disorders. One category of molecules that can regulate the retina's response to ischemia is neuropeptides. An ischemia-induced increase in neuropeptide expression has been considered an endogenous protective mechanism in the retina. It is poorly understood, however, why ischemic retinal injury still occurs regardless of an increased expression in protective neuropeptides. Neuropeptides are initially synthesized as large precursors that are processed into smaller, active forms by the action of a set of processing enzymes. Our parallel studies on ischemic brains have revealed that brain ischemia has an adverse effect on the biosynthetic activation steps of key neuropeptide processing enzymes, resulting in an accumulation of neuropeptide precursors in ischemic brains. Animals null of a neuropeptide processing enzyme are more sensitive to ischemic stress.

These findings have directed us to investigate if a similar, ischemia-induced blockade in neuropeptide processing may also occur in the retina, and if this may be a mechanism of ischemic retinal injury. Results of our preliminary studies on retina ganglion cells (RGC) support such notions. Little is known about the molecular mechanisms of neuropeptide processing in the retina. The specific aims of this proposal are: 1) To establish the potential involvement of proprotein converse 1 and 2 (PC1 and PC2, respectively) in retinal neuropeptide processing. These two critical enzymes process many neuropeptides in the brain including those that are also found in the retina. Studies under this aim include examination of the presence and developmental changes of PC1 and PC2 in the retina; characterization of biosynthetic activation steps of PC1 and PC2 in RGC and their potential roles in neuropeptide processing; and a quantitative proteomic comparison of neuropeptide profiles in the retinas of wild type, PC1-null and PC2-null mice. 2) To investigate how retinal ischemia may alter the activation steps of PC1 and PC2 and the production of neuropeptides by retinal cells, using both in vivo and in vitro ischemia models. The response of PC1-null and PC2-null mice to retina ischemia will also be investigated. Our long-term goal is to obtain a thorough understanding of neuropeptide processing in the retina and its role in regulating the retina's response to injurious stresses. Ultimately, such knowledge will help development of new therapeutic strategies and targets for treatment of retinal diseases.

PI:  Jose Ochoa, MD, PhD
R01NS048932 "Primary C nociceptors and C sympathetics in CRPS" 

DESCRIPTION (provided by applicant): Chronic neuropathic pains and hyperalgesias/allodynias following peripheral nerve or tissue insults, whether associated with nerve pathology (CRPS II) or not (CRPS I), curse patients, elude therapies and puzzle researchers. Hyperfunction of peripheral pain receptors (nociceptors) is an accepted cause of abnormal painful input in neuropathy (CRPSII) and a hypothetical source of secondary upheaval of pain-signaling neurons in the spinal cord, in CRPS I. Either peripheral or central mechanisms might account for spontaneous pains and for mechanical and thermal hyperalgesias/allodynias. Abnormal interaction between peripheral sympathetic and nociceptor neurons, in skin or muscle, also remains a theoretical mechanism of neuropathic pain and hyperalgesias.

Over the past three decades our group has regularly contributed factual scientific knowledge about fine structure, pathology, and normal and abnormal sensory function of nociceptors and efferent function of sympathetic units, in patients and volunteers. We remain engaged in clinical and research studies on neuropathic pains and recently developed robust methods that allow direct and simultaneous functional and biophysical characterization and testing of multiple subtypes of nociceptors, thermal (cold) receptors and sympathetic units, while observing possible nerve fiber interactions. This is achieved through microneurography and automated latency tracking (Qtrac) in awake humans.

These approaches, complemented by in vitro studies on DRG neuron somata from CRPS patients, will be used to test 3 hypotheses for the origin of pain in CRPS: 1) Irritable primary nociceptor neurons, of different subtypes, evoke spontaneous pains and hyperalgesias/allodynias in CRPS patients via ectopic nerve impulse activity, caused by abnormal behavior of particular ion channels in nociceptor membranes. 2) Sympathetic efferent discharge, acting at axonal and/or soma sites, may increase primary cutaneous or deep tissue nociceptor activity in CRPS, thus generating pains. 3) Cold hyperalgesia/allodynia, a prominent symptom in CRPS, is due to (a) sensitization of specific noxious low- temperature membrane receptors in nociceptor afferents, (b) ectopic expression of non-noxious cooling receptors in nociceptor afferents, (c) central release of noxious cold-signaling input, or (d) secondary central sensitization. Results of this project will prove, or question, pertinent hypothetical mechanisms of neuropathic pains and will facilitate mechanism-targeted therapy, while protecting CRPS patients from iatrogenic harm.  

PI:  Claude Burgoyne, MD
R01EY011610  "IOP-related force and failure in the optic nerve head"

DESCRIPTION (provided by applicant):  In glaucoma, there is no science to predict what level of intraocular pressure (IOP) will be safe for a given optic nerve head (ONH). The goal of this project is to identify the clinically important components of optic nerve head (ONH) susceptibility to glaucomatous damage using basic engineering principles.

The proposed biomechanical studies test the following hypotheses within high-resolution, digital, three dimensional (3D) reconstructions (1.5 x 1.5 x 1.5 urn voxel) and finite element models (FE Models) of the normal and glaucomatous ONH: 1) The distribution of stress (force/cross-sectional area) and strain (local deformation) within the lamina cribrosa and peripapillary scleral of the Normal ONH predicts the sites of connective tissue damage in Early Experimental Glaucoma; 2) At all levels of IOP, strains within the remaining connective tissues of Early and Moderate but not Severe Glaucoma eyes are higher than in Normal eyes; 3) A predictable pattern of fixed (permanent) deformation of the ONH connective tissues underlies the onset and progression of glaucomatous cupping in Early, Moderate and Severe Glaucoma but is not present in the optic neuropathies of Early and Late ONH Ischemia and Optic Nerve Transection; 4) the ONH connective tissues of Old eyes are hardened compared to those of Young eyes and this difference in ONH connective tissue stiffness affects the clinical behavior of the Aged, Glaucomatous ONH.

The Specific Aims and Objectives are: 1) To expand our continuum and micro FE Modeling of the ONH tissues to Severe Glaucoma and to the Young and Very Old Normal ONH; 2) To perform 3D histomorphometry within the digital 3D geometries of Normal Young and Very Old, Early, Moderate and Severe Glaucomatous, Early and Late Ischemic and Optic Nerve Transection ONH so as to characterize and compare the 3D patterns of connective tissue architecture and damage; 4) To test the hypothesis that the ONH connective tissues of the Aged ONH are less susceptible to deformation than those of the Young ONH and that this difference in susceptibility affects the clinical appearance and behavior of the Aged ONH at the onset of glaucomatous damage as seperately defined by structural and functional criteria. The methodology includes longitudinal ONH surface change detection using Heidelburg Retinal Tomographic Change Analysis; multifocal Electroretinogram (mfERG) testing; high-resolution, 3D reconstruction and 3D histomorphetry of the ONH neural and connective tissues; automated optic nerve axon counting; 3D material properties testing of intact posterior scleral shells; and continuum and micro FE modeling of the ONH neural and connective tissues. 

PI:  Claude Burgoyne, MD
R01EY021281  "Optic nerve head SDOCT imaging in glaucoma"


DESCRIPTION (provided by applicant): The primary goal of this project is to test three hypotheses regarding glaucomatous damage to the visual system. First, that clinically detectable neural, glial and connective tissue alterations occur deep in the optic nerve head (ONH) at a very early stage in the pathophysiology of glaucomatous damage to the visual system. Second, that the location and magnitude of the earliest of these ONH changes, detectable in vivo by spectral domain optical coherence tomography (SDOCT), predict the specific locations of subsequent alterations of the peripapillary retinal nerve fiber layer (RNFL) and orbital optic nerve axon loss. Third, that ONH connective tissue structural stiffness is altered both by age and glaucomatous damage and that it underlies the clinical appearance of the glaucomatous optic disc, specifically by influencing the "depth" of glaucomatous ONH structural change or "cupping". Until now, all animal models of glaucoma have been studied in isolation from human glaucoma. A second goal is to demonstrate that our hypotheses and techniques have evolved to a point where both can be simultaneously tested in monkeys (Specific Aim 1) and humans (Specific Aim 2).  Aim 1 is to characterize the onset and progression of SDOCT ONH structural change within pre- and post- laser SDOCT ONH data sets from both eyes of 70 unilateral experimental glaucoma (EG) monkeys. Aim 2 is to characterize the onset and progression of ONH structural change within longitudinal SDOCT ONH data sets from 250 human ocular hypertensive and early glaucoma patients. The methodology includes: longitudinal Heidelberg Spectralis 870 and 1060 nm SDOCT ONH image acquisition in monkeys (870 nm only in humans); their visualization, delineation and quantification within custom Multiview software; and in monkeys only, post- mortem 3D histomorphometric ONH reconstruction and quantification, co-localized, eye-specific comparison of SDOCT ONH and 3D histomorphometric reconstructions and regionally-aligned orbital optic nerve axon damage map generation using our custom Axonmaster axon counting software.  The expected outcomes are: 1) deep ONH structural change will occur before and predict subsequent ONH surface and RNFL change during the onset and progression of glaucomatous damage in both monkey and human eyes; 2) early ONH structural change will co-localize to orbital optic nerve axon loss in monkey eyes and precede RNFL alterations in both monkey and human hypertensive eyes; 3) in monkeys, younger ONHs will be "more compliant" and older ONHs will be "stiffer" when normal, and both will demonstrate transient hypercompliance followed by progressive stiffening as glaucomatous damage progresses; 4) younger monkey and human ONHs will demonstrate a "deeper" form of "cupping" than older ONHs; 5) in younger compared to older eyes, the onset and progression of structural change ("cupping") will include a larger connective tissue component; and 6) 1060 nm SDOCT imaging will improve visualization of deep monkey ONH imaging targets compared to the existing clinical standard (870 nm imaging). 

PUBLIC HEALTH RELEVANCE: The clinical detection of the onset and progression of glaucomatous damage to the optic nerve head (ONH) is central to the care of every glaucoma patient. We propose to use 870 nm and 1060 nm Heidelberg Spectralis Spectral Domain Optical Coherence Tomography (SDOCT) to characterize the onset and progression of ONH structural change within pre and post- laser SDOCT ONH data sets from both eyes of 70 unilateral experimental glaucoma (EG) monkeys and 250 ocular hypertensive and early glaucoma patients. In this project we will translate 11 years of NIH-funded, post-mortem monkey work to an in-vivo imaging modality that will be shown to have important and novel clinical care applications in humans. 

PI:  J. Crawford Downs, PhD
R21EY018152  "3D reconstruction of optic nerve heads" 

DESCRIPTION (provided by applicant): Elevated intraocular pressure (IOP) has long been assumed to play a causative role in glaucomatous damage to the optic nerve head (ONH). It is still unclear, however, how IOP triggers the cascade of events that lead to retinal ganglion cell death. It has been proposed that the elevated IOP can 1) exert a direct mechanical strain on the connective tissues and axons in the optic nerve head, and 2) impair the blood supply to the ONH, resulting in hypoxia of the ONH tissues and subsequent cell death.

Using three-dimensional (3D) reconstructions of the ONH, and principles of biomechanical engineering, we have studied the mechanical effects of elevated IOP in glaucoma. However, the relationship between the mechanical and vascular effects of elevated IOP is still unclear. What is the role of the vasculature and ischemia in the development and progression of the disease? Is ONH blood flow related to IOP-induced deformation of the ONH connective tissues? Are the robustness of the ONH connective tissues and the vasculature the key to understanding individual susceptibility to glaucoma? To answer these questions, methods for determining the relationship between IOP-induced deformation of ONH connective tissues and ONH blood flow are needed. We propose to develop a system that constitutes the essential first step in quantifying ocular perfusion, an important risk factor for glaucomatous damage. While many additional variables will need to be considered, the ability to isolate, quantify, and model co-localized vascular and connective tissue structures within three- dimensional (3D) reconstructions of the ONH will be a powerful advance in studies of ONH susceptibility. In a preliminary test, both the ONH connective tissue and retinal vasculature were revealed by spontaneous and acquired fluorescence, respectively. The proposed system will provide simultaneous, co-localized, high- resolution, 3D reconstructions of the vascular and connective tissue structures of the ONH.

We also propose to quantify and correlate the regional changes in connective tissue deformation, capillary patency, and vascular volume in contralateral eyes perfusion-fixed at acute IOPs of 10 and 45 mm Hg, respectively. Within the proposed 3D reconstructions, we can perform geometric quantification and biomechanical modeling of the coupled connective tissue and vascular systems to predict IOP-induced changes in ONH blood flow. Elevated intraocular pressure (IOP) has long been assumed to play a causative role in glaucomatous damage to the optic nerve head (ONH), but the interplay between IOP-related connective tissue deformation and ONH blood flow is unclear. We propose to develop a system that will generate simultaneous, co-localized, high- resolution, three-dimensional (3D) reconstructions of the vascular and connective tissue structures of the ONH. Using these 3D reconstructions of the ONH and principles of biomechanical engineering, we can model the mechanical and vascular effects of elevated IOP in glaucoma. 

PI:  Shaban Demirel, PhD
R01EY019674 "Predicting the rate of progression in glaucoma"


DESCRIPTION (provided by applicant): Glaucoma is one of the leading causes of treatable blindness in the world. However, the manner and rate of disease progression varies markedly between patients, and so predicting future progression and hence the prognosis for an individual is challenging. Much of the previous work in this field has concentrated on determining whether a patient with certain risk factors will develop glaucoma. There has been less attention paid to the likelihood that a patient will suffer significant loss of vision as a result of their glaucoma, or how soon vision loss may manifest. The overall goal of this project is to improve the ability to predict the future course of an individual's disease, and so enable the clinician to adapt their management strategy accordingly. Better knowledge of glaucomatous pathophysiology will also be gained, with meaningful implications for developing new methods of diagnosis and treatment strategies. The primary tool used in this project will be longitudinal data collected biannually from over 250 subjects with early or suspected glaucoma, and 50 normal subjects. For the majority of the glaucoma subjects, up to ten years of prior information is available and will also be used. The first part of the project aims to improve understanding of the structure-function relationship in glaucoma. Three non-competing hypotheses will be tested that seek to explain the weak observed correlation between functional change (to the patient's visual field) and structural change (to the optic nerve head and/or retinal nerve fiber layer). The first experiment will determine the true underlying correlation after taking into account the variability inherent in the testing procedures. The second experiment will compare changes to the optic nerve head surface topography against subsurface changes and changes to the retinal nerve fiber layer, to determine whether there is temporal dissociation caused by surface change not driven by a loss of neural tissue. The third experiment will use a sampling-limited test of retinal ganglion cell density, to determine whether there is temporal dissociation caused by dysfunctional retinal ganglion cells that are still present in the retina but have altered response characteristics. Results from these three experiments will provide new insights into the disease process, with possible implications for future development of testing and treatment strategies. The second part of the project aims to use this new information and advanced analysis techniques (including regression trees) to better predict the future rate of functional change on a per-eye basis. First, subsequent change will be predicted based on test results at one time point. Second, improved predictions will be made when a series of test results are available. The fundamental motivation for this project is to enable a clinician to prevent severe visual disability or blindness in an ocular hypertensive or glaucoma patient, by identifying a rapid progression rate or a high likelihood for rapid progression at the earliest stages of the disease.

PIs:  J. Crawford Downs, PhD and Christopher A. Girkin, MD, (Univ of Alabama Birmingham)
R01EY018926 "Age-related changes in optic nerve head structure and biomechanics"  

DESCRIPTION (provided by applicant):  Elevated intraocular pressure (IOP) has long been assumed to play a causative role in glaucomatous damage to the optic nerve head (ONH). Patient age is among the most important risk factors for the onset and progression of glaucomatous damage, regardless of the stage of glaucoma or the level of intraocular pressure (IOP) at which it has occurred. It is still unclear, however, how IOP triggers the cascade of events that lead to retinal ganglion cell death. We hypothesize that age-related alterations in ONH biomechanics contribute importantly to the increased susceptibility of the aged ONH in humans.  
 
Using three-dimensional (3D) reconstructions of the ONH, and principles of biomechanical engineering, we have studied the mechanical effects of elevated IOP in glaucoma. However, the relationship between patient age and the mechanical effects of elevated IOP is still unclear. How is the ONH altered as in the older patient that increases its susceptibility to IOP? Is the robustness of the ONH connective tissues the key to understanding individual susceptibility to glaucoma? What role does the structural stiffness of the lamina cribrosa and peripapillary sclera play in the increased age-related risk for glaucomatous progression? To answer these questions, we will use novel methods to elucidate the relationship between age and the IOP- induced deformation of ONH connective tissues are needed.  
 
By "ONH biomechanics" we mean the interactions between IOP and connective tissue structural stiffness (the combination of tissue architecture and material properties) in the ONH and peripapillary sclera. The immediate goals of this project are to characterize age-related differences in ONH biomechanics and elucidate their effects on ONH susceptibility. Our long-term goal is to develop clinical diagnostics and interventions designed to manage each important biomechanical risk factor in the development and progression of glaucoma. To accomplish our immediate goals, we will build digital three-dimensional reconstructions of young and old human ONH tissues, quantify the ONH connective tissue architecture within each reconstruction, and build computational finite element models of the ONH connective tissues to estimate their biomechanical response to normal and elevated levels of IOP. We will also correlate the age-related variations in ONH architecture, tissue stiffness, and biomechanical behavior with the increased susceptibility and clinical behavior of the aged ONH.

PUBLIC HEALTH RELEVANCE. Elevated intraocular pressure (IOP) has long been assumed to play a causative role in glaucomatous damage to the optic nerve head (ONH), and older patients have higher risk of development and progression of the disease. We propose to measure the age-related differences in ONH structure and IOP-induced biomechanical response. Then, using the principles of biomechanical engineering, we will use these data to create computational models of the age-related mechanical effects of elevated IOP on the ONH to elucidate the link between advancing age and glaucomatous susceptibility.  

PIs:  J. Crawford Downs, PhD and Christopher A. Girkin, MD, (Univ of Alabama Birmingham)
R01EY019333  "Racial Variations in Optic Nerve Head Structure and Biomechanics"

DESCRIPTION (provided by applicant): Elevated intraocular pressure (IOP) has long been assumed to play a causative role in glaucomatous damage to the optic nerve head (ONH). There is compelling evidence to suggest that patients of African descent are at much greater risk for the onset and progression of glaucomatous damage at elevated levels of IOP.

In this proposal, we test the hypothesis that racial differences in optic nerve head (ONH) biomechanics importantly contribute to this difference in risk. It is still unclear, however, how IOP triggers the cascade of events that lead to retinal ganglion cell death. Using three-dimensional (3D) reconstructions of the ONH, and principles of biomechanical engineering, we have studied the mechanical effects of elevated IOP in glaucoma. However, the relationship between African ancestry and the mechanical effects of elevated IOP is still unclear.

How do racial variations in ONH structure and biomechanics increase its susceptibility to IOP? Is the robustness of the ONH connective tissues the key to understanding individual susceptibility to glaucoma? What role does the structural stiffness of the lamina cribrosa and peripapillary sclera play in the increased risk for glaucomatous progression in patients of African descent? To answer these questions, we will use novel methods to elucidate the relationship between ancestry and the IOP-induced deformation of ONH connective tissues. By "ONH biomechanics" we mean the interactions between IOP and connective tissue structural stiffness (the combination of tissue architecture and material properties) in the ONH and peripapillary sclera.

The immediate goals of this project are to characterize racial variations in ONH biomechanics and elucidate their effects on ONH susceptibility. Our long-term goal is to develop clinical diagnostics and interventions designed to manage each important biomechanical risk factor in the development and progression of glaucoma. To accomplish our immediate goals, we will build digital three-dimensional reconstructions of human ONH tissues from donors of African and European descent, quantify the ONH connective tissue architecture within each reconstruction, and build computational finite element models of the ONH connective tissues to estimate their biomechanical response to normal and elevated levels of IOP. PUBLIC HEALTH RELEVANCE Elevated intraocular pressure (IOP) has long been assumed to play a causative role in glaucomatous damage to the optic nerve head (ONH), and patients of African descent have higher risk of development and progression of the disease. We propose to measure the racial variation in ONH structure and IOP-induced biomechanical response. Then, using the principles of biomechanical engineering, we will use these data to create computational models of the age-related mechanical effects of elevated IOP on the ONH to elucidate the link between African ancestry and glaucomatous susceptibility.

PI:  George A. "Jack" Cioffi, MD
U10EY011610  "Ocular Hypertension Treatment Study (OHTS)"

This is an application to become a participating Clinical Center in the        
Ocular Hypertension treatment Study (OHTS).  The complete details of the       
OHTS rationale, design, and methods are contained in the OHTS Manual of        
Procedures.

This proposal provides complete documentation of the ability of Devers Eye     
Institute, in conjunction with Oregon Medical Eye Clinic and Oregon Eye        
Care, 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 the performance of the study in accord with the details    
of the OHTS Manual of Procedures, the nature and extent of their commitment    
to Devers Eye Institute, and a list of two organizations/sixteen               
practitioners in the area who will refer patients screened for enrollment      
and randomization to treatment in the OHTS clinical trial.

PI:  Brad Fortune, OD, PhD
R01EY019327  "Axonal Cytoskeletal Changes in Experimental Glaucoma"

DESCRIPTION (provided by applicant): This project proposes that prior to retinal ganglion cell (RGC) death in glaucoma, and before permanent loss of vision, there exists a stage of RGC dysfunction characterized by degradation of axonal microtubules (MTs). Emerging evidence suggests that MT degradation can occur initially without substantial changes in axonal caliber. Therefore, it is proposed that early stage RGC dysfunction involving MT degradation should be preferentially detectable by scanning laser polarimetry (SLP) of the retinal nerve fiber layer (RNFL) prior to changes in RNFL thickness. This is because the fundamental optical principle of SLP is based on detecting phase retardance of polarized light, which is due to the optical property birefringence produced in the RNFL by the long, thin cylindrical MTs. Preliminary studies demonstrate that RNFL retardance declines prior to, and faster than RNFL thickness in several different experimental models of RGC injury, including experimental glaucoma (EG). Clinical detection of axonal MT disruption by SLP, in the absence of RNFL thickness changes, might represent an early and potentially reversible phase of glaucomatous damage and provide a clinically detectable marker for therapeutic adjustment. Thus the central hypothesis of this proposal is that disruption of MTs within the axons of the peripapillary RNFL is an early indicator of glaucomatous damage, preceding both changes in axonal caliber and physical loss of those axons. Predictions arising from this hypothesis are tested in three Specific Aims using a non-human primate (NHP) model of EG. Specific Aim 1: To test the prediction that peripapillary RNFL retardance will decline prior to RNFL thickness changes measured by spectral domain optical coherence tomography (sd-OCT) and prior to optic nerve head (ONH) surface changes measured by confocal scanning laser tomography (CSLT) in NHP eyes with EG; Specific Aim 2: To test the predictions that histological evidence of peripapillary RNFL MT disruption will be more pronounced than histologically-defined RNFL thickness changes and retrobulbar optic nerve axon loss; Specific Aim 3: To test the prediction that RGC functional abnormalities are associated with the intermediate stage of RGC degeneration characterized by abnormal axonal MTs. To achieve these Aims, EG will be induced via laser photocoagulation of the trabecular meshwork to cause moderate, unilateral chronic IOP elevation in 24 NHPs. Weekly measurements of peripapillary RNFL retardance, RNFL thickness and ONH surface topography will be made in both eyes of each NHP using SLP, sd-OCT and CSLT, respectively, during a 4-week pre-laser baseline period and for up to 8 months after onset of EG (Aim 1). For each parameter, statistically significant change is defined as any change exceeding the baseline intersession variability for each individual eye, twice confirmed. Once each animal progresses to its endpoint, it is sacrificed for histological data collection and analysis (Aim 2). During each week of in vivo structural testing for Aim 1, RGC function will also be assessed in both eyes using three proven forms of electroretinography (Aim 3). PUBLIC HEALTH RELEVANCE: Glaucoma is one of the most common causes of blindness in the United States and around the world. It is a chronic disease with no known cure, but prospective longitudinal trials have found that treatment to lower intraocular pressure decreases the rate of progressive vision loss. Thus, early diagnosis enables timely therapeutic intervention and reduces the overall impact of glaucoma. However, achieving a timely diagnosis requires clinical detection of the onset and progression of glaucomatous damage to the optic nerve head (ONH) and retinal nerve fiber layer (RNFL), which remain a central challenge in the clinical care of every glaucoma patient. This project evaluates and advances clinical tools for detecting early damage and progression of glaucoma.  

PI: Brad Fortune, OD, PhD
1R21EY021311 "Imaging retinal astrocytes, ganglion cells and axonal transport in vivo"


DESCRIPTION (provided by applicant): Astrocytes are a major class of glia in the vertebrate retina. They are located primarily within the innermost retinal layers; their processes surround retinal ganglion cell (RGC) axons and axon bundles as well as all blood vessels. Because of this anatomical relationship, and a variety of physiological evidence, astrocytes are thought to have a major role in the mechanisms of retinal blood flow autoregulation, i.e. the maintenance of nearly constant blood flow in response to variations of ocular perfusion pressure. Astrocytes are also thought to play an important role in the pathophysiology of many ocular diseases by responding to a variety of insults such as ischemia, increased intraocular pressure and neuronal degeneration in a manner that has been characterized as gliosis. Hence, the ability to image astrocytes in vivo could help to elucidate aspects of disease pathophysiology. Similarly, there is evidence to suggest that RGC axonal cytoskeletal components, specifically microtubules, are disrupted during the earliest stages of response to experimental injuries such as axotomy and experimental glaucoma. This disruption is significant because microtubules are the "tracks" upon which axonal transport is driven. Thus, if microtubule abnormalities develop early in response to injury, the resultant axonal transport disruption could exacerbate the injury and inhibit protective or rescue responses from achieving full potential. The overall goal of this R21 project is to develop the methods for imaging retinal astrocytes, RGCs, their axons and axonal transport in vivo. The specific objectives are as follows: Specific Aim 1: To establish methodologies for in vivo visualization of retinal astrocytes, RGCs, their axons and active axonal transport in the rat eye. To evaluate the optimal concentration, follow-up duration and persistence of in vivo markers as well as perform histopathological studies to corroborate in vivo observations. Specific Aim 2: To evaluate potential toxicity of in vivo astrocyte markers and axonal transport tracers using sensitive measures of retinal function (electroretinography, ERG) and retinal structure (spectral domain optical coherence tomography, SDOCT), so as to assess potential for use in primate experimental models. Specific Aim 3: To evaluate the sensitivity of our newly developed methods by comparing the impact of four unilateral experimental injury models (intravitreal injection of nocodazole/colchicine to disrupt axonal microtubules and inhibit active axonal transport; acute elevation of intraocular pressure; chronic elevation of intraocular pressure; and optic nerve crush) with results obtained in bilateral control eyes. The novel methods developed in this proposal will make possible in future proposals, studies about the onset of astrocyte abnormalities and RGC axonal transport abnormalities and comparisons of those phenomena to the course of RGC and axonal degeneration in experimental models of RGC injury. 
 
PUBLIC HEALTH RELEVANCE: Glaucoma is one of the most common causes of blindness in the United States and around the world. It is a chronic disease with no known cure. Though prospective longitudinal trials have found that treatment to lower intraocular pressure decreases the rate of progressive vision loss, some individuals continue to lose vision despite successful therapy to lower their intraocular pressure. Thus, a more thorough understanding of the events leading to damage and vision loss in glaucoma is required. The goal of this project is to develop methods for evaluating two groups of cells and aspects of their function in the living eye using specialized imaging techniques. 

PI: Stuart Gardiner, PhD
1R01EY020922 "Functional Testing for Glaucoma"


DESCRIPTION (provided by applicant): Glaucoma is a leading cause of blindness both in the US and worldwide. The long-term purpose of this project is to improve functional testing in glaucoma. Assessment and follow-up of patients currently relies on automated perimetry to provide functional testing of the visual field. However, the ability to assess progression and/or response to treatment using perimetry is hampered by high variability, especially in areas of moderate or severe glaucomatous damage. Recent findings by our laboratory and others have advanced our understanding of perimetry by challenging key assumptions about the test. This proposal aims to use these advances to explain and reduce the test variability. This will improve the accuracy, efficiency and utility of current functional testing, giving immediate impact in both research and clinical settings, and laying groundwork for the next generation of instruments and algorithms. The first Specific Aim is to produce an accurate and physiologically justified measure of the Effective Dynamic Range (EDR) of perimetry. It is postulated that the very high contrast stimuli used by perimetry in glaucomatous defects saturate the response of the visual system. The resultant nonlinearity in the contrast-response function would cause the detection probability to asymptote below 100%, explaining the high variability in sensitivities in damaged areas. The limit of the EDR will be defined as the contrast beyond which response linearity cannot be assumed. This will be measured by collecting frequency-of-seeing curves in subjects with moderate or advanced glaucoma. The same technique will be used to determine whether the EDR is extended by use of an increased stimulus size. The second Specific Aim is to derive and test a spatial filter to reduce the variability. This will be the first filter to be based both on sensitivities at other locations in the visual field and on the structure of the optic nerve head. The third Specific Aim is to assess the potential utility of using a linear scale for sensitivity, rather than the current logarithmic decibel scale. First, an efficient linear-scaled thresholding algorithm will be derived and tested, to determine whether it will reduce variability both between tests and in the structure-function relation. Second, linear-scaled global indices of the central visual field will be examined, to determine whether they offer improved prognostic value compared with current decibel-scaled indices when assessing progression. The three aims are complementary. It is anticipated that by combining these aims, variability in perimetry will be better understood, and significantly reduced. Such an improvement in a test as commonly performed as perimetry will significantly impact future clinical practice.  

PI: Steven Mansberger, MD, MPH
K23WY015501  "The Northwest tribal vision project"

This application proposes a training plan to develop Steven L. Mansberger, M.D., M.P.H. into an
independent clinician/scientist specializing in ophthalmic epidemiology and biostatistics. Dr. Mansberger is a board certified ophthalmologist with subspecialty training in glaucoma. He has prior research experience involving screening for glaucoma, the relationship between optic disc pathology and visual function, and ocular blood flow. Devers Eye Institute, Oregon Health Science University, and the Northwest Portland Area Indian Health Board have extensive experience with research and education and are especially well suited as training sites.

This training program will consist of advanced coursework in biostatistics and epidemiology, multi-disciplinary conferences, and mentored research. Mentored research will be conducted under the direction of a mentorship committee composed of Thomas Becker, M.D., Ph.D., Interim Chairman of the Department of Public Health and Preventive Medicine at OHSU, Chris Johnson, Ph.D., Director of Diagnostic Research at Discoveries In Sight/Devers Eye Institute, and Francine Romero, Ph.D., M.P.H., Principal Investigator of the Northwest Tribal Health Research Center Project. George A. Cioffi, M.D., Anne L. Coleman, M.D., Ph.D., and Dongseok Choi, Ph.D. will serve as consultants on the project as committee members. Most epidemiological studies have used expert personnel, such as ophthalmologists or optometrists, to diagnose eye diseases as part of a prevalence survey.

However, newer methods of evaluating the visual field, retina, and optic nerve make it possible for non-MD, non-OD paraprofessionals (such as ophthalmic technicians) to perform a baseline examination and refer participants with abnormal results for a definitive exam by expert personnel. This staged protocol allows persons to be examined rapidly and with reduced expense. The specific aims of the research project are: 1) to determine if non-MD, non-OD paraprofessionals (such as ophthalmic technicians) performing newer methods of testing have good diagnostic precision and feasibility in comparison to an expert examiner; 2) to attain preliminary data regarding the age-specific prevalence of visual impairment, blindness, and ocular disease in a sample of Northwest American Indians and Alaskan Natives (AI/AN) aged 40 years and older. This project will then lead to a larger, more comprehensive survey of the prevalence and causes of visual impairment in AI/AN.

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